Human Molecular Genetics - Advance Access

Quantitative trait loci predicting circulating sex steroid hormones in men from the NCI-Breast and Prostate Cancer Cohort Consortium (BPC3)

2009-07-02

Twin studies suggest a heritable component to circulating sex steroid hormones and sex hormone-binding globulin (SHBG). In the NCI-Breast and Prostate Cancer Cohort Consortium, 874 SNPs in 37 candidate genes in the sex steroid hormone pathway were examined in relation to circulating levels of SHBG (N=4,720), testosterone (N=4,678), 3-androstanediol-glucuronide (N=4,767), and 17β-estradiol (N=2,014) in Caucasian men. rs1799941 in SHBG is highly significantly associated with circulating levels of SHBG (p=4.52x10^–21), consistent with previous studies, and testosterone (p=7.54x10^–15), with mean difference of 26.9% and 14.3% respectively, comparing wildtype to homozygous variant carriers. Further noteworthy novel findings were observed between SNPs in ESR1 with testosterone levels (rs722208, mean difference=8.8%, p=7.37x10^–6) and SRD5A2 with 3-androstanediol-glucuronide (rs2208532, mean difference=11.8%, p=1.82x10^–6). Genetic variation in genes in the sex steroid hormone pathway is associated with differences in circulating SHBG and sex steroid hormones.

A genome-wide association study of northwestern Europeans involves the CNP signaling pathway in the etiology of human height variation

2009-07-01

Northwestern Europeans are among the tallest of human populations. The increase in body height in these people appears to have reached a plateau, suggesting the ubiquitous presence of an optimal environment in which genetic factors may have exerted a particularly strong influence on human growth. Therefore, we performed a genome-wide association study (GWAS) of body height using 2.2 million markers in 10,074 individuals from three Dutch and one German population-based cohorts. Upon genotyping the 12 most significantly height-associated single nucleotide polymorphisms (SNPs) from this GWAS in 6912 additional individuals of Dutch and Swedish origin, a genetic variant (rs6717918) on chromosome 2q37.1 was found to be associated with height at a genome-wide significance level (P_combined = 3.4 x 10^–9). Notably, a second SNP (rs6718438) located ~450 bp away and in strong LD (r² = 0.77) with rs6717918 was previously found to be suggestive of a height association in 29,820 individuals of mainly northwestern European ancestry, and the over-expression of a nearby natriuretic peptide precursor type C (NPPC) gene, has been associated with overgrowth and skeletal anomalies. We also found a SNP (rs10472828) located on 5p14 near the natriuretic peptide receptor 3 (NPR3) gene, encoding a receptor of the NPPC ligand, to be associated with body height (P_combined = 2.1 x 10^–7). Taken together, these results suggest that variation in the C-type natriuretic peptide (CNP) signaling pathway, involving the NPPC and NPR3 genes, plays an important role in determining human body height.

Identification of novel susceptibility loci for Guam neurodegenerative disease: Challenges of genome scans in genetic isolates

2009-06-30

Amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) is a fatal neurodegenerative disease found in the Chamorro people of Guam and other Pacific Island populations. The etiology is unknown, although both genetic and environmental factors appear important. To identify loci for ALS/PDC, we conducted both genome-wide linkage and association analyses, using ~400 microsatellite markers, in the largest sample assembled to date, comprising a nearly-complete sample of all living and previously-sampled deceased cases. A single, large, complex pedigree was ascertained from one village on Guam, with smaller families and a case-control sample ascertained from the rest of Guam by population-based neurological screening and archival review. We found significant evidence for two regions with novel ALS/PDC loci on chromosome 12, and supportive evidence for the involvement of the MAPT region on chromosome 17. D12S1617 on 12p gave the strongest evidence of linkage (maximum lod score, Z_max = 4.03) in our initial scan, with additional support in the complete case-control sample in the form of evidence of allelic association at this marker and another nearby marker. D12S79 on 12q also provided significant evidence of linkage (Z_max = 3.14) with support from flanking markers. Our results suggest that ALS/PDC may be influenced by as many as three loci, while illustrating challenges that are intrinsic in genetic analyses of isolated populations, as well as analytical strategies that are useful in this context. Elucidation of the genetic basis of ALS/PDC should improve our understanding of related neurodegenerative disorders including Alzheimer disease, Parkinson disease, frontotemporal dementia and ALS.

Direct and Indirect Roles of RECQL4 in Modulating Base Excision Repair Capacity

2009-06-29

RECQL4 is a human RecQ helicase which is mutated in approximately two-thirds of individuals with Rothmund-Thomson syndrome (RTS), a disease characterized at the cellular level by chromosomal instability. BLM and WRN are also human RecQ helicases, which are mutated in Werner and Bloom's syndrome, respectively, and associated with chromosomal instability as well as premature aging. Here we show that primary RTS and RECQL4 RNAi knockdown human fibroblasts accumulate more H₂O₂–induced DNA strand breaks than control cells, suggesting that RECQL4 may stimulate repair of H₂O₂–induced DNA damage. RTS primary fibroblasts also accumulate more XRCC1 foci than control cells in response to endogenous or induced oxidative stress and have a high basal level of endogenous formamidopyrimidines. In cells treated with H₂O_2, RECQL4 co-localizes with APE1, and FEN1, key participants in base excision repair. Biochemical experiments indicate that RECQL4 specifically stimulates the apurinic endonuclease activity of APE1, the DNA strand displacement activity of DNA polymerase β, and incision of a 1- or 10-nucleotide flap DNA substrate by Flap Endonuclease I. Additionally, RTS cells display an upregulation of BER pathway genes and fail to respond like normal cells to oxidative stress. The data herein support a model in which RECQL4 regulates both directly and indirectly base excision repair capacity.

Inhibition of Mitochondrial Fission Favours Mutant Over Wild-type Mitochondrial DNA

Malena, A., Loro, E., Di Re, M., Holt, I. J., Vergani, L. — 2009-06-27

Biased segregation of mitochondrial DNA variants has been widely documented, but little was known about its molecular basis. We set out to test the hypothesis that altering the balance between mitochondrial fusion and fission could influence the segregation of mutant and wild-type mtDNA variants, because it would modify the number of organelles per cell. Therefore human cells heteroplasmic for the pathological A3243G mitochondrial DNA mutation were transfected with constructs designed to silence Drp1 or hFis1, whose gene products are required for mitochondrial fission. Drp1 and hFis1 gene silencing were both associated with increased levels of mutant mitochondrial DNA. Thus, the extent of the mitochondrial reticular network appears to be an important factor in determining mutant load. The fact that the level of mutant and wild-type mitochondrial DNA can be manipulated by altering the expression of nuclear encoded factors involved in mitochondrial fission suggests new interventions for mitochondrial DNA disorders.

Protein kinase C{gamma}, a protein causative for dominant ataxia, negatively regulates nuclear import of recessive-ataxia-related aprataxin

2009-06-26

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant disease caused by mutations in the gene encoding protein kinase C (PKC). We report an SCA14 family with a novel deletion of a termination-codon-containing region, resulting in a missense change and a C-terminal 13-amino-acid extension with increased kinase activity. Notably, one patient with a severe phenotype is the first homozygote for the mutation causing SCA14. We show the novel molecular consequences of increased kinase activities of mutants: aprataxin (APTX), a DNA repair protein causative for autosomal recessive ataxia, was found to be a preferential substrate of mutant PKC, and phosphorylation inhibited its nuclear entry. The phosphorylated residue was Thr111, located adjacent to the nuclear localization signal, and disturbed interactions with importin , a nuclear import adaptor. Decreased nuclear APTX increased oxidative stress-induced DNA damage and cell death. Phosphorylation-resistant APTX, kinase inhibitors, and antioxidants may be therapeutic options for SCA14.

Fancm-deficient mice reveal unique features of Fanconi anemia complementation group M

2009-06-26

The Fanconi anemia (FA) core complex member FANCM remodels synthetic replication forks and recombination intermediates. Thus far, only one FA patient with FANCM mutations has been described, but the relevance of these mutations for the FA phenotype is uncertain. To provide further experimental access to the FA-M complementation group we have generated Fancm-deficient mice by deleting exon 2. FANCM deficiency caused hypogonadism in mice and hypersensitivity to cross-linking agents in MEFs, thus phenocopying other FA mouse models. However, Fancm^2/2 mice also showed unique features atypical for FA mice, including underrepresentation of female Fancm^2/2 mice and decreased overall and tumor-free survival. This increased cancer incidence may be correlated to the role of FANCM in the suppression of spontaneous sister chromatid exchanges as observed in MEFs. In addition, FANCM appeared to have a stimulatory rather than essential role in FANCD2 monoubiquitination. The FA-M mouse model presented here suggests that FANCM functions both inside and outside the FA core complex to maintain genome stability and to prevent tumorigenesis.

Common variants in KCNQ1 are associated with type 2 diabetes and impaired fasting glucose in a Chinese Han population

2009-06-25

Common variants in KCNQ1 have recently been reported to be associated with type 2 diabetes in East Asians. We aimed to examine whether these common variants (rs2074196, rs2237892, rs2237895 and rs2237897) were also associated with type 2 diabetes in a population-based cohort of 3,210 Chinese Hans and to explore the underlying mechanisms. The SNPs rs2237892, rs2237895 and rs2237897 were significantly associated with type 2 diabetes (OR: 1.33-1.36, P≤0.0009), IFG (OR: 1.16-1.19, P≤0.0193) and combined IFG/type 2 diabetes (OR: 1.23-1.24, P≤0.0004), and the corresponding population attributable risks of type 2 diabetes for the three SNPs were 32.5%, 18.8% and 35.8%, respectively. However, rs2074196 showed a weak but significant associations with IFG (OR 1.18[1.04-1.33], P=0.009) and combined IFG/type 2 diabetes (OR 1.17[1.05-1.30], P=0.0053), as well as a trend toward association with type 2 diabetes (OR 1.15[0.98-1.35], P=0.0882), suggesting a different pattern of association compared to the other three SNPs. The four SNPs were all significantly associated with HOMA-B (P≤0.042) while rs2237895 and rs22378897 also showed significant association with fasting glucose (P≤0.012). Notably, the associations with type 2 diabetes were markedly attenuated after adjusting for HOMA-B (OR_rs2237892 1.33[1.05-1.68], P=0.018; OR_rs2237895 1.24[1.00-1.54], P=0.0524; OR_rs2237897 1.22[0.98-1.53], P=0.09). Moreover, GCCC haplotype showed similar associations with type 2 diabetes (OR 1.48[1.17-1.85], P=0.0008), IFG (OR 1.32[1.10-1.57], P=0.0023), combined IFG/type 2 diabetes (OR 1.37[1.17-1.61], P=8.7x10^–5), and lower HOMA-B values (β=-4.41±1.62, P=0.006). These results suggest that KCNQ1 is a major type 2 diabetes gene in the Chinese Hans and it may confer type 2 diabetes risk by impaired β-cell function.

Harnessing the Information Contained Within Genome-wide Association Studies to Improve Individual Prediction of Complex Disease Risk

Evans, D. M., Visscher, P. M., Wray, N. R. — 2009-06-24

The current paradigm within genetic diagnostics is to test individuals only at loci known to affect risk of complex disease- yet the technology exists to genotype an individual at thousands of loci across the genome. We investigated whether information from genome-wide association studies could be harnessed to improve discrimination of complex disease affection status. We employed genome-wide data from the Wellcome Trust Case Control Consortium to test this hypothesis. Each disease cohort together with the same set of controls were split into two samples- a "Training Set", where thousands of SNPs that might predispose to disease risk were identified, and a "Prediction Set", where the discriminatory ability of these SNPs was assessed. Genome-wide scores consisting of, for example, the total number of risk alleles an individual carries were calculated for each individual in the prediction set. Case-control status was regressed on this score and the area under the receiver operator characteristic curve (AUC) estimated. In most cases, a liberal inclusion of SNPs in the genome-wide score improved AUC compared to a more stringent selection of top SNPs, but didn't perform as well as selection based upon established variants. The addition of genome-wide scores to known variant information produced only a limited increase in discriminative accuracy but was most effective for bipolar disorder, coronary heart disease and type II diabetes. We conclude that this small increase in discriminative accuracy is unlikely to be of diagnostic or predictive utility at the present time.

Common BMI-associated variants confer risk of extreme obesity

2009-06-24

To investigate the genetic architecture of severe obesity we performed a genome-wide association study of 775 cases and 3197 unascertained controls at approximately 550,000 markers across the autosomal genome. We found convincing association to the previously described locus including the FTO gene. We also found evidence of association at a further six of twelve other loci previously reported to influence body-mass index (BMI) in the general population and one of three associations to severe childhood and adult obesity, and that cases have a higher proportion of risk-conferring alleles than controls.. We found no evidence of homozygosity at any locus due to identity-by-descent associating with phenotype which would be indicative of rare, penetrant alleles, nor was there excess genome-wide homozygosity in cases relative to controls. Our results suggest that variants influencing BMI also contribute to severe obesity, a condition at the extreme of the phenotypic spectrum rather than a distinct condition.

The disease-protective complement factor H allotypic variant Ile62 shows increased binding affinity for C3b and enhanced cofactor activity

2009-06-23

Mutations and polymorphisms in the gene encoding factor H (CFH) have been associated with atypical haemolytic uraemic syndrome, dense deposit disease and age-related macular degeneration. The disease-predisposing CFH variants show a differential association with pathology that has been very useful to unravel critical events in the pathogenesis of one or other disease. In contrast, the fH-Ile₆₂ polymorphism confers strong protection to all three diseases. Using ELISA-based methods and surface plasmon resonance analyses we show here that the protective fH-Ile₆₂ variant binds more efficiently to C3b than fH-Val₆₂ and competes better with factor B in proconvertase formation. Functional analyses demonstrate an increased cofactor activity for fH-Ile₆₂ in the factor I-mediated cleavage of fluid phase and surface-bound C3b; however, the two fH variants show no differences in decay accelerating activity. From these data we conclude that the protective effect of the fH-Ile₆₂ variant is due to its better capacity to bind C3b, inhibit proconvertase formation and catalyse inactivation of fluid-phase and surface-bound C3b. This demonstration of the functional consequences of the fH-Ile₆₂ polymorphism provides relevant insights into the complement regulatory activities of fH that will be useful in disease prediction and future development of effective therapeutics for disorders caused by complement dysregulation.

A cell-autonomous role for WT1 in regulating Sry in vivo

Bradford, S. T., Wilhelm, D., Bandiera, R., Vidal, V., Schedl, A., Koopman, P. — 2009-06-23

Human patients with Frasier syndrome express reduced levels of the +KTS isoform of the developmental regulator WT1, and exhibit complete XY gonadal dysgenesis and male-to-female sex reversal. Mice with a targeted mutation that blocks production of this isoform show a reduction in Sry mRNA in the gonad, but the molecular and cellular basis of this reduction has not been established. Using immunofluorescence analysis, we found a significantly lower level of SRY protein per cell in XY Wt1(+KTS)-null mouse gonads. We also found a reduced number of SRY-expressing cells, correlating with a decrease in cell proliferation at and near the coelomic epithelium at 11.5 dpc. No reduction in somatic cell numbers was seen in XX Wt1(+KTS)-null gonads, indicating that the effect of WT1 on cell proliferation is mediated by Sry. Sertoli cell differentiation was blocked in in XY Wt1(+KTS)-null mouse gonads, as indicated by loss of SOX9 and Fgf9 expression, but addition of recombinant FGF9 to ex vivo gonad cultures rescued the mutant phenotype, as indicated by the induction of the Sertoli-cell specific marker AMH. Our data suggest that WT1(+KTS) is involved in the cell-autonomous regulation of Sry expression, which in turn influences cell proliferation and Sertoli cell differentiation via FGF9. Thus, sex reversal in Wt1(+KTS)-null mice and Frasier syndrome patients results from a failure of Sertoli cells to both fully differentiate and to reach sufficient numbers to direct testis development.

Mutation of SYNE-1, encoding an essential component of the nuclear lamina, is responsible for autosomal recessive arthrogryposis

2009-06-19

Arthrogryposis multiplex congenita (AMC) is a group of disorders characterized by congenital joint contractures caused by reduced fetal movements. AMC has an incidence of 1 in 3,000 newborns and is genetically heterogeneous. We describe an autosomal recessive form of myogenic AMC in a large consanguineous family. The disease is characterized by bilateral clubfoot, decreased fetal movements, delay in motor milestones, then progressive motor decline after the first decade. Genome wide linkage analysis revealed a single locus on chromosome 6q25 with Z_max=3.55 at =0.0 and homozygosity of the polymorphic markers at this locus in patients. Homozygous A to G nucleotide substitution of the conserved AG splice acceptor site at the junction of intron 136 and exon 137 of the SYNE-1 gene was found in patients. This mutation results in an aberrant retention of intron 136 of SYNE-1 RNA leading to premature stop codons and the lack of the C-terminal transmembrane domain KASH of nesprin-1, the SYNE-1 gene product. Mice lacking the KASH domain of nesprin-1 display a myopathic phenotype similar to that observed in patients. Altogether, these data strongly suggest that the splice site mutation of SYNE-1 gene found in the family is responsible for AMC. Recent reports have shown that mutations of the SYNE-1 gene might be responsible for autosomal recessive adult onset cerebellar ataxia. These data indicate that mutations of nesprin-1 which interacts with lamin A/C may lead to at least two distinct human disease phenotypes, myopathic or neurologic, a feature similar to that found in laminopathies.

Loss of positive allosteric interactions between neuronal nitric oxide synthase and phosphofructokinase contribute to defects in glycolysis and increased fatigability in muscular dystrophy

Wehling-Henricks, M., Oltmann, M., Rinaldi, C., Myung, K. H., Tidball, J. G. — 2009-06-19

Duchenne muscular dystrophy (DMD) involves a complex pathophysiology that is not easily explained by the loss of the protein dystrophin, the primary defect in DMD. Instead, many features of the pathology are attributable to the secondary loss of neuronal nitric oxide synthase (nNOS) from dystrophin-deficient muscle. In this investigation, we tested whether loss of nNOS contributes to the increased fatigability of mdx mice, a model of DMD. Our findings show that expression of a muscle-specific, nNOS transgene increases the endurance of mdx mice and enhances glycogen metabolism during treadmill-running, but did not affect vascular perfusion of muscles. We also find that the specific activity of phosphofructokinase (PFK; the rate limiting enzyme in glycolysis) is positively affected by nNOS in muscle; PFK specific activity is significantly reduced in mdx muscles and the muscles of nNOS null mutants, while significantly increased in nNOS transgenic muscles and muscles from mdx mice that express the nNOS transgene. PFK activity measured under allosteric conditions was significantly increased by nNOS, but unaffected by endothelial NOS or inducible NOS. The specific domain of nNOS that positively regulates PFK activity was assayed by cloning and expressing different domains of nNOS and assaying their effects on PFK activity. This approach yielded a polypeptide that included the FAD-binding domain of nNOS as the region of the molecule that promotes PFK activity. Smaller peptides in this domain were then synthesized and used in activity assays that showed a 36-amino acid peptide in the FAD-binding domain in which most of the positive allosteric activity of nNOS for PFK resides. Mapping this peptide onto the structure of nNOS shows that the peptide is exposed on the surface, readily available for binding. Collectively, these findings indicate that defects in glycolytic metabolism and increased fatigability in dystrophic muscle may be caused in part by the loss of positive allosteric interactions between nNOS and PFK.

Loss of Bloom syndrome protein destabilizes human gene cluster architecture

Killen, M. W., Stults, D. M., Adachi, N., Hanakahi, L., Pierce, A. J. — 2009-06-19

Bloom syndrome confers strong predisposition to malignancy in multiple tissue types. The BLM protein defective in the disease biochemically functions as a Holliday junction dissolvase and human cells lacking functional BLM show 10-fold elevated rates of sister chromatid exchange. Collectively, these phenomena suggest that dysregulated mitotic recombination drives the genomic instability underpinning the development of cancer in these individuals. Here we use physical analysis of the highly repeated, highly self-similar human ribosomal RNA gene clusters as sentinel biomarkers for dysregulated homologous recombination to demonstrate that loss of BLM protein function causes a striking increase in spontaneous molecular level genomic restructuring. Analysis of single-cell derived subclonal populations from wild-type human cell lines shows that gene cluster architecture is ordinarily very faithfully preserved under mitosis, but is so unstable in cell lines derived from Bloom syndrome patients as to make gene cluster architecture in different subclonal populations essentially unrecognizable one from another. Human cells defective in a different RecQ helicase, the WRN protein involved in the premature aging Werner syndrome, do not exhibit the gene cluster instability phenotype, indicating that the BLM protein specifically, rather than RecQ helicases generally, holds back this recombination mediated genomic instability. An ataxia-telangiectasia defective cell line also shows elevated rDNA gene cluster instability, although not to the extent of BLM defective cells. Genomic restructuring mediated by dysregulated recombination between the abundant low copy repeats in the human genome may prove to be an important additional mechanism of genomic instability driving the initiation and progression of human cancer.

CK2-Dependent Phosphorylation Determines Cellular Localization and Stability of Ataxin-3

Mueller, T., Breuer, P., Schmitt, I., Evert, B. O., Wullner, U. — 2009-06-19

The nuclear presence of the expanded disease proteins is of critical importance for the pathogeneses of polyglutamine diseases. Here we show that protein casein kinase 2 (CK2)-dependent phosphorylation controls the nuclear localization, aggregation, and stability of ataxin-3 (ATXN3), the disease protein in spinocerebellar ataxia type 3 (SCA3). Serine 340 and 352 within the third ubiquitin-interacting motif of ATXN3 were particularly important for nuclear localization of normal and expanded ATXN3 and mutation of these sites robustly reduced the formation of nuclear inclusions; a putative nuclear leader sequence was not required. ATXN3 associated with CK2 and pharmacological inhibition of CK2 decreased nuclear ATXN3 levels and the formation of nuclear inclusions. Moreover, we found that ATXN3 shifted to the nucleus upon thermal stress in a CK2-dependent manner, indicating a key role of CK2-mediated phosphorylation of ATXN3 in SCA3 pathophysiology.

Association of variants in the PCSK1 gene with obesity in the EPIC-Norfolk Study

Kilpelainen, T. O., Bingham, S. A, Khaw, K.-T., Wareham, N. J., Loos, R. J.F. — 2009-06-15

Recently, the rs6232 (N221D) and rs6235 (S690T) SNPs in the PCSK1 gene were associated with obesity in a meta-analysis comprising more than 13,000 individuals of European ancestry. Each additional minor allele of rs6232 or rs6235 was associated with a 1.34- or 1.22-fold increase in the risk of obesity, respectively. So far, only one relatively small study has aimed to replicate these findings, but could not confirm the association of the rs6235 SNP, and did not study the rs6232 variant. In the present study, we examined the associations of the rs6232 and rs6235 SNPs with obesity in a population-based cohort consisting of 20,249 individuals of European descent from Norfolk, UK. Logistic regression and generalized linear models were used to test the associations of the risk alleles with obesity and related quantitative traits, respectively. Neither of the SNPs was significantly associated with obesity, BMI, or waist circumference under the additive genetic model (P >0.05). However, we observed an interaction between rs6232 and age on the level of BMI (P = 0.010) and risk of obesity (P = 0.020). The rs6232 SNP was associated with BMI (P = 0.021) and obesity (P = 0.022) in the younger individuals (< median age (59 years)), but not among the older age group (P = 0.81 and P = 0.68 for BMI and obesity, respectively). In conclusion, our data suggest that the PCSK1 rs6232 and rs6235 SNPs are not major contributors to common obesity in the general population. However, the effect of rs6232 may be age-dependent.

Contiguous gene deletion of ELOVL7, ERCC8, and NDUFAF2 in a patient with a fatal multisystem disorder

2009-06-12

Contiguous gene syndromes affecting the mitochondrial oxidative phosphorylation system have been rarely reported. Here, we describe a patient with apparent mitochondrial encephalomyopathy accompanied by several unusual features, including dysmorphism and hepatopathy, caused by a homozygous triple gene deletion on chromosome 5. The deletion encompassed the NDUFAF2, ERCC8, and ELOVL7 genes, encoding complex I assembly factor 2 (also known as human B17.2L), a protein of the transcription-coupled nucleotide excision repair (TC-NER) machinery, and a putative elongase of very long-chain fatty acid synthesis, respectively. Detailed evaluation of cultured skin fibroblasts revealed disturbed complex I assembly, depolarization of the mitochondrial membrane, elevated cellular NAD(P)H level, increased superoxide production, and defective TC-NER. ELOVL7 mRNA was not detectable in these cells and no alterations in fatty acid synthesis were found. By means of baculoviral complementation we were able to restore the aberrations, thereby establishing causative links between genotype and cell-physiological phenotype. This first chromosomal microdeletion illustrates that beside primary defects in mitochondrial genes also additional genes possibly contribute to the disease phenotype, providing an additional explanation for the broad clinical symptoms associated with these disorders.

Impaired DNA replication prompts deletions within palindromic sequences, but does not induce translocations in human cells

2009-06-11

Palindromic regions are unstable and susceptible to deletion in prokaryotes and eukaryotes possibly due to stalled or slow replication. In the human genome, they also appear to become partially or completely deleted, while two palindromic AT-rich repeats (PATRR) contribute to known recurrent constitutional translocations. To explore the mechanism that causes the development of palindrome instabilities in humans, we compared the incidence of de novo translocations and deletions at PATRRs in human cells. Using a highly sensitive PCR assay that can detect single molecules, de novo deletions were detected neither in human somatic cells nor in sperm. However, deletions were detected at low frequency in cultured cell lines. Inhibition of DNA replication by administration of siRNA against the DNA polymerase alpha 1 (POLA1) gene or introduction of POLA inhibitors increased the frequency. This is in contrast to PATRR-mediated translocations that were never detected in similar conditions but were observed frequently in human sperm samples. Further, deletions were found to take place during both leading and lagging strand synthesis. Our data suggest that stalled or slow replication induces deletions within PATRRs, but that other mechanisms might contribute to PATRR-mediated recurrent translocations in humans.

Functional role for senataxin, defective in ataxia oculomotor apraxia type 2, in transcriptional regulation

2009-06-10

Ataxia oculomotor apraxia type 2 (AOA2) is an autosomal recessive neurodegenerative disorder characterized by cerebellar ataxia and oculomotor apraxia. The gene mutated in AOA2, SETX, encodes senataxin, a putative DNA/RNA helicase which shares high homology to the yeast Sen1p protein and has been shown to play a role in the response to oxidative stress. To investigate further the function of senataxin, we identified novel senataxin interacting proteins, the majority of which are involved in transcription and RNA processing, including RNA polymerase II. Binding of RNA polymerase II to candidate genes was significantly reduced in senataxin deficient cells and this was accompanied by decreased transcription of these genes, suggesting a role for senataxin in the regulation/modulation of transcription. RNA polymerase II-dependent transcription termination was defective in cells depleted of senataxin in keeping with the observed interaction of senataxin with poly(A) binding proteins 1 and 2. Splicing efficiency of specific mRNAs and alternate splice-site selection of both endogenous genes and artificial minigenes were altered in senataxin depleted cells. These data suggest that senataxin, similar to its yeast homolog Sen1p, plays a role in coordinating transcriptional events, in addition to its role in DNA repair.

Histone methylation is mechanistically linked to DNA methylation at imprinting control regions in mammals

Henckel, A., Nakabayashi, K., Sanz, L. A., Feil, R., Hata, K., Arnaud, P. — 2009-06-10

Mono-allelic expression of imprinted genes from either the paternal or the maternal allele is mediated by ‘imprinting control regions’ (ICRs), which are epigenetically marked in an allele-specific fashion. Although, in somatic cells, these epigenetic marks comprise both DNA methylation and histone methylation, the relationship between these two modifications in imprint acquisition and maintenance remains unclear. To address this important question, we analysed histone modifications at ICRs in mid-gestation embryos that were obtained from Dnmt3L-/- females, in which DNA methylation imprints at ICRs are not established during oogenesis. The absence of maternal DNA methylation imprints in these conceptuses led to a marked decrease and loss of allele-specificity of the repressive H3K9me3, H4K20me3 and H2A/H4R3me2 histone modifications, providing the first evidence of a mechanistic link between DNA and histone methylation at ICRs. The existence of this relationship was strengthened by the observation that when DNA methylation was still present at the Snrpn and Peg3 ICRs in some of the progeny of Dnmt3L-/- females, these ICRs were associated with the usual patterns of histone methylation. Combined, our data establish that DNA methylation is involved in the acquisition and/or maintenance of histone methylation at imprinting control regions.

Truncation and pathogenic mutations facilitate the formation of intracellular aggregates of TDP-43

Nonaka, T., Kametani, F., Arai, T., Akiyama, H., Hasegawa, M. — 2009-06-10

TAR DNA binding protein of 43 kDa (TDP-43) is a major component of the ubiquitin-positive inclusions found in the brain of patients with frontotemporal lobar degeneration (FTLD-U) and amyotrophic lateral sclerosis (ALS). Here, we report that expression of TDP-43 C-terminal fragments as green fluorescent protein (GFP) fusions in SH-SY5Y cells results in the formation of abnormally phosphorylated and ubiquitinated inclusions that are similar to those found in FTLD-U and ALS. Co-expression of DsRed-tagged full-length TDP-43 with GFP-tagged C-terminal fragments of TDP-43 causes formation of cytoplasmic inclusions positive for both GFP and DsRed. Cells with GFP and DsRed positive inclusions lack normal nuclear staining for endogenous TDP-43. These results suggest that GFP-tagged C-terminal fragments of TDP-43 are bound not only to transfected DsRed-full-length TDP-43 but also to endogenous TDP-43. Endogenous TDP-43 may be recruited to cytoplasmic aggregates of TDP-43 C-terminal fragments, which results in the failure of its nuclear localization and function. Interestingly, expression of GFP-tagged TDP-43 C-terminal fragments harboring pathogenic mutations that cause ALS significantly enhances the formation of inclusions. We also identified cleavage sites of TDP-43 C-terminal fragments deposited in the FTLD-U brains using mass spectrometric analyses. We propose that generation and aggregation of phosphorylated C-terminal fragments of TDP-43 play a primary role in the formation of inclusions and resultant loss of normal TDP-43 localization, leading to neuronal degeneration in TDP-43 proteinopathy.

Towards a functional classification of pathogenic FOXL2 mutations using transactivation reporter systems

2009-06-10

Mutations of FOXL2 are responsible for the Blepharophimosis-Ptotsis-Epicantus-inversus Syndrome (BPES), involving complex eyelid malformations often associated with premature ovarian failure (POF). Loss-of-function mutations are expected to lead to BPES associated with POF, whereas hypomorphic mutations would lead to BPES without ovarian dysfunction. However, multiple exceptions to the genotype-phenotype correlation have been described and missense mutations in the forkhead domain can lead to either type of BPES. This renders almost impossible the prediction of a POF condition from a given genotype. Moreover, no clear-cut correlation between nuclear and/or cytoplasmic aggregation or cytoplasmic retention of mutant FOXL2 forms and the BPES type has been established thus far. Here, we dissect the molecular and functional effects of 10 FOXL2 mutants, known to induce BPES associated with POF or not. We found a correlation between the transcriptional activity of FOXL2 variants on two different reporter promoters and the type of BPES. We used this functional classification framework to explore the behavior of 18 missense mutations leading to BPES of unknown type. The reporters used enabled us to assess the risk of POF associated with these mutations. Moreover, we document a previously overlooked correlation between subcellular mislocalization and aggregation of mutant FOXL2 and the type of BPES, known or predicted using our reporter assays. Thus, intranuclear aggregation and cytoplasmic mislocalization of mutant FOXL2 may be considered as loose predictors of ovarian dysfunction. The functional classification tool described here is a first step towards circumventing the lack of a clear-cut genotype-phenotype correlation in BPES.

Ciliary and centrosomal defects associated with mutation and depletion of the Meckel syndrome genes MKS1 and MKS3

2009-06-10

Meckel syndrome (MKS) is a lethal disorder characterized by renal cystic dysplasia, encephalocele, polydactyly and biliary dysgenesis. It is highly genetically heterogeneous with nine different genes implicated in this disorder. MKS is thought to be a ciliopathy because of the range of phenotypes and localization of some of the implicated proteins. However, limited data is available about the phenotypes associated with MKS1 and MKS3, and the published ciliary data is conflicting. Analysis of the wpk rat model of MKS3 revealed functional defects of the connecting cilium in the eye that resulted in lack of formation of the outer segment, while infertile wpk males developed spermatids with very short flagella that did not extend beyond the cell body. In wpk renal collecting duct cysts, cilia were generally longer than normal, with additional evidence of cells with multiple primary cilia and centrosome over-duplication. Kidney tissue and cells from MKS1 and MKS3 patients showed defects in centrosome and cilia number, including multi-ciliated respiratory-like epithelia, and longer cilia. Stable shRNA knockdown of Mks1 and Mks3 in IMCD3 cells induced multi-ciliated and multi-centrosomal phenotypes. These studies demonstrate that MKS1 and MKS3 are ciliopathies, with new cilia related eye and sperm phenotypes defined. MKS1 and MKS3 function is required for ciliary structure and function, including a role in regulating length and appropriate number through modulating centrosome duplication.

Functional interaction of mTOR complexes in regulating mammalian cell size and cell cycle

Rosner, M., Fuchs, C., Siegel, N., Valli, A., Hengstschlager, M. — 2009-06-08

Dysregulation of the mammalian target of rapamycin (mTOR) kinase pathway is centrally involved in a wide variety of cancers and human genetic diseases. In mammalian cells mTOR is part of two different kinase complexes, mTORC1 composed of mTOR, raptor and mLST8, and mTORC2 containing mTOR, rictor, Sin1 and mLST8. Whereas, mTORC1 is known to be a pivotal regulator of cell size and cell cycle control, the question whether the recently discovered mTORC2 complex is involved in these processes remained elusive. We report here that the mTORC1-mediated consequences on cell cycle and cell size are separable and do not involve effects on mTORC2 activity. However, we show that mTORC2 itself is a potent regulator of mammalian cell size and cell cycle via a mechanism involving the Akt/TSC2/Rheb cascade. Our data are of relevance for the understanding of the molecular development of the many human diseases caused by deregulation of upstream and downstream effectors of mTOR.

Disc1 regulates granule cell migration in the developing hippocampus

Meyer, K. D., Morris, J. A. — 2009-06-05

Schizophrenia is a severely debilitating psychiatric disease which is hypothesized to have its roots in neurodevelopment. Although the precise neuropathology underlying schizophrenia has remained elusive, there are consistent reports of abnormalities in several brain areas. Chief among these is the hippocampus, an area which has displayed both structural and functional abnormalities in many schizophrenic patients. In order to better understand how disruption of hippocampal development may contribute to the etiology of psychiatric disease, we investigated the function of a highly promising schizophrenia susceptibility gene, DISC1 (Disrupted-In-Schizophrenia 1), in the development of the hippocampus. DISC1 is strongly expressed in the hippocampus from its early development through adulthood and has been implicated in hippocampal structure and function in human studies. However, its precise role in the development of the hippocampus is not yet known. Here, we show that in utero electroporation of Disc1 shRNA into the developing mouse hippocampus hinders the migration of dentate gyrus granule cells. Intriguingly, Disc1 knockdown does not affect the migration of CA1 pyramidal neurons, suggesting that Disc1's role in regulating neuronal migration is spatially restricted within the hippocampus. These findings support the idea that DISC1 abnormalities that contribute to the onset of schizophrenia may do so through their influences on hippocampal development.

Expansion of the Parkinson Disease-Associated SNCA-Rep1 Allele Up-Regulates Human {alpha}-Synuclein in Transgenic Mouse Brain

2009-06-04

Alpha-synuclein (SNCA) gene has been implicated in the development of rare forms of familial Parkinson disease (PD). Recently it was shown that an increase in SNCA copy numbers leads to elevated levels of wild-type SNCA-mRNA and protein and is sufficient to cause early-onset, familial PD. A critical question concerning the molecular pathogenesis of PD is what contributory role, if any, is played by the SNCA gene in sporadic PD. Expansion of SNCA-Rep1, an upstream, polymorphic microsatellite of the SNCA gene, is associated with elevated risk for sporadic PD. However, whether SNCA-Rep1 is the causal variant and the underlying mechanism its effect is mediated by, remained elusive. We report here the effects of three distinct SNCA-Rep1 variants in the brains of 72 mice transgenic for the entire human SNCA locus. Human SNCA-mRNA and protein levels were increased 1.7- and 1.25-fold, respectively, in homozygotes for the expanded, PD risk-conferring allele compared with homozygotes for the shorter, protective allele. When adjusting for the total SNCA-protein concentration (endogenous-mouse and transgenic-human) expressed in each brain, the expanded risk allele contributed 2.6-fold more SNCA steady-state than the shorter allele. Furthermore, targeted deletion of Rep1 resulted in the lowest human SNCA-mRNA and protein concentrations in murine brain. In contrast, the Rep1 effect was not observed in blood lysates from the same mice. These results demonstrate that Rep1 regulates human SNCA expression by enhancing its transcription in the adult nervous system and suggest that homozygosity for the expanded Rep1 allele may mimic locus multiplication, thereby elevating PD risk.

Correction of tau mis-splicing caused by FTDP-17 MAPT mutations by spliceosome-mediated RNA trans-splicing

2009-06-04

Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by mutations in the MAPT gene, encoding the tau protein that accumulates in intraneuronal lesions in a number of neurodegenerative diseases. Several FTDP-17 mutations affect alternative splicing and result in excess exon 10 (E10) inclusion in tau mRNA. RNA reprogramming using spliceosome mediated RNA trans-splicing (SMaRT) could be a method of choice to correct aberrant E10 splicing resulting from FTDP-17 mutations. SMaRT creates a hybrid mRNA through a trans-splicing reaction between an endogenous target pre-mRNA and a pre-trans-splicing RNA molecule (PTM). However, FTDP-17 mutations affect the strength of cis-splicing elements and could favor cis-splicing over trans-splicing. Excess E10 inclusion in FTDP-17 can be caused by intronic mutations destabilizing a stem-loop protecting the 5' splice site at the E10/intron 10 junction. COS cells were transfected with a minigene containing the intronic +14 mutation, produce exclusively E10⁺ RNA. Generation of E10^– RNA was restored after co-transfection with a PTM designed to exclude E10. Similar results were obtained with a target containing the exonic N279K mutation strengthening a splicing enhancer within E10. Conversely, increase or decrease in E10 content was achieved by trans-splicing from a target carrying the 280K mutation, that weakens the same splicing enhancer. Thus E10 inclusion can be modulated by trans-splicing irrespective of the strength of the cis-splicing elements affected by FTDP-17 mutations. In conclusion, RNA trans-splicing could provide the basis of therapeutic strategies for impaired alternative splicing caused by pathogenic mutations in cis-acting splicing elements.

A Deletion of the HBII-85 Class of Small Nucleolar RNAs (snoRNAs) is Associated with Hyperphagia, Obesity and Hypogonadism

2009-06-04

Objective

Genetic studies in patients with severe early onset obesity have provided insights into the molecular and physiological pathways that regulate body weight in humans. We report a 19 year-old male with hyperphagia and severe obesity, mild learning difficulties and hypogonadism, in whom diagnostic tests for Prader-Willi syndrome (PWS) had been negative.

Research Design and Methods

We carried out detailed clinical and metabolic phenotyping of this patient and investigated the genetic basis of this obesity syndrome using Agilent 185 k array comparative genomic hybridisation (aCGH) and Affymetrix 6.0 genotyping arrays. The identified deletion was validated using multiplex ligation-dependent probe amplification (MLPA) and long range PCR, followed by breakpoint sequencing which enabled precise localisation of the deletion.

Results

We identified a ~187kb microdeletion at chromosome 15q11-13 that encompasses non-coding small nucleolar RNAs (including HBII-85 snoRNAs) which were not expressed in peripheral lymphocytes from the patient. Characterisation of the clinical phenotype revealed increased ad libitum food intake, normal basal metabolic rate when adjusted for fat-free mass, partial hypogonadotropic hypogonadism and growth failure.

Conclusions

We have identified a novel deletion on chromosome 15q11-13 in an individual with hyperphagia, obesity, hypogonadism and other features associated with PWS, which is normally caused by deficiency of several paternally expressed imprinted transcripts within chromosome 15q11-q13, a region that includes multiple protein-coding genes as well as several noncoding snoRNAs. These findings provide direct evidence for the role of a particular family of noncoding RNAs, the HBII-85 snoRNA cluster, in human energy homeostasis, growth and reproduction.

Golgi function and dysfunction in the first COG4-deficient CDG type II patient

2009-06-03

The Conserved Oligomeric Golgi (COG) complex is a hetero-octameric complex essential for normal glycosylation and intra-Golgi transport. An increasing number of Congenital Disorder of Glycosylation-type II (CDG-II) mutations are found in COG subunits indicating its importance in glycosylation. We report a new CDG-II patient harbouring a p.R729W missense mutation in COG4 combined with a submicroscopical deletion. The resulting downregulation of COG4 expression additionally affects expression or stability of other lobe A subunits. Despite this, full complex formation was maintained albeit to a lower extent as shown by glycerol gradient centrifugation. Moreover, our data indicate that subunits are present in a cytosolic pool and full complex formation assists tethering preceding membrane fusion. By extending this study to four other known COG-deficient patients, we now present the first comparative analysis on defects in transport, glycosylation and Golgi ultrastructure in these patients. The observed structural and biochemical abnormalities correlate with the severity of the mutation, with the COG4 mutant being the mildest. All together our results indicate that intact COG complexes are required to maintain Golgi dynamics and its associated functions. According to the current CDG nomenclature, this newly identified deficiency is designated CDG-IIj.

Partial loss of GATA factor Pannier impairs adult heart function in Drosophila

Qian, L., Bodmer, R. — 2009-06-03

The GATA transcription factor encoded by pannier (pnr) is a critical regulator of heart progenitor formation in Drosophila. Mutations in GATA4, the mammalian homologue of pnr, have also been implicated in causing human cardiac disease in a haploinsuficient manner. Mouse models of Gata4 loss-of-function and gain-of-function studies underscored the importance of Gata4 in regulating cardiac progenitor cells specification and differentiation. However, it is not known whether pnr/Gata4 is directly involved in establishing and maintaining adult heart physiology because of the lethality associated with defective heart function and redundancy among various GATA factors in vertebrates. Here, we took advantage of the Drosophila heart model to examine the function of pnr in adult heart physiology. We found that pnr heterozygous mutants result in defective cardiac performance in response to electrical pacing of the heart as well as in elevated arrhythmias. Adult-specific disruption of pnr function using a dominant-negative form pnrEnR revealed a cardiac autonomous requirement of pnr in regulating heart physiology. Moreover, we identified Tbx20/neuromancer (nmr) as a potential downstream mediator of pnr in regulating cardiac performance and rhythm regularity, based on the observation that overexpression of nmr genes, but not of tinman, partially rescues the adult defects in pnr mutants. We conclude that pnr is not only essential for early cardiac progenitor formation, along with tinman and T-box factors, but also plays an important role in establishing and/or maintaining proper heart function, which is partially through another key regulator Tbx20/nmr.

Discrimination of common and unique RNA-binding activities among Fragile-X mental retardation protein paralogs

Darnell, J. C., Fraser, C. E., Mostovetsky, O., Darnell, R. B. — 2009-06-01

Fragile-X mental retardation is caused by loss of function of a single gene encoding FMRP, an RNA binding protein that harbors three canonical RNA binding domains, two KH-type and one RGG box. Two autosomal paralogs of FMRP, FXR1P and FXR2P, are similar to FMRP in their overall structure, including the presence of putative RNA binding domains, but to what extent they provide functional redundancy with FMRP is unclear. While FMRP has been characterized as a polyribosome-associated regulator of translation, less is known about the functions of FXR1P and FXR2P. For example, FMRP binds intramolecular G-quadruplex and kissing complex RNA ligands (kcRNA) via the RGG box and KH2 domain, respectively, while the RNA ligands of FXR1P and FXR2P are unknown. Here we demonstrate that FXR1P and FXR2P KH2 domains bind kissing complex RNA ligands with the same affinity as the FMRP KH2 domain while other KH domains do not. RNA ligand recognition by this family is highly conserved, as the KH2 domain of the single Drosophila ortholog, dFMRP, also binds kcRNA. kcRNA was able to displace FXR1P and FXR2P from polyribosomes as it does for FMRP, and this displacement was FMRP-independent. This suggests that all three family members recognize the same binding site on RNA mediating their polyribosome association, and that they may be functionally redundant with regard to this aspect of translational control. In contrast, FMRP is unique in its ability to recognize G-quadruplexes, suggesting the FMRP RGG domain may play a non-redundant role in the pathophysiology of the disease.

Reciprocal imprinting of human GRB10 in placental trophoblast and brain: evolutionary conservation of reversed allelic expression

2009-06-01

Genomic imprinting may have evolved not only to regulate foetal growth and development, but also behaviour. The mouse Grb10 gene provides a remarkable model to explore this idea because it shows paternal expression in brain, whereas in the placenta and most other embryonic tissues, expression is from the maternal allele. To assess the biological relevance of this reciprocal pattern of imprinting, we explored its conservation in humans. As in mice, we find the human GRB10 gene to be paternally expressed in brain. Maternal-allele specific expression is conserved only in the placental villous trophoblasts, an essential part of the placenta involved in nutrient transfer. All other foetal tissues tested showed equal expression from both alleles. These data suggest that the maternal GRB10 expression in placenta is evolutionarily important, presumably in the control of foetal growth. As in the mouse, the maternal transcripts originate from several kilobases upstream of the imprinting control region (ICR) of the domain, from a promoter region at which we find no allelic chromatin differences. The brain-specific paternal expression from the ICR shows mechanistic similarities with the mouse as well. This conserved CpG island is DNA-methylated on the maternal allele and is marked on the paternal allele by developmentally regulated bivalent chromatin, with the presence of both H3 lysine-4 and H3 lysine-27 methylation. The strong conservation of the opposite allelic expression in placenta versus brain supports the hypothesis that GRB10 imprinting evolved to mediate diverse roles in mammalian growth and behaviour.

Serine racemase is associated with schizophrenia susceptibility in humans and in a mouse model

2009-05-30

Abnormal N-methyl-D-aspartate receptor (NMDAR) function has been implicated in the pathophysiology of schizophrenia. D-serine is an important NMDAR modulator, and to elucidate the role of the D-serine synthesis enzyme serine racemase (Srr) in schizophrenia, we identified and characterized mice with an ENU-induced mutation that results in a complete loss of Srr activity and dramatically reduced D-serine levels. Mutant mice displayed behaviors relevant to schizophrenia, including impairments in prepulse inhibition, sociability, and spatial discrimination. Behavioral deficits were exacerbated by an NMDAR antagonist and ameliorated by D-serine or the atypical antipsychotic clozapine. Expression profiling revealed that the Srr mutation influenced several genes that have been linked to schizophrenia and cognitive ability. Transcript levels altered by the Srr mutation were also normalized by D-serine or clozapine treatment. Furthermore, analysis of SRR genetic variants in humans identified a robust association with schizophrenia. This study demonstrates that aberrant Srr function and diminished D-serine may contribute to schizophrenia pathogenesis.

Variation in aggregation propensities among ALS-associated variants of SOD1: Correlation to human disease

Prudencio, M., Hart, P. J., Borchelt, D. R., Andersen, P. M. — 2009-05-30

To date 146 different mutations in superoxide dismutase 1 (SOD1) have been identified in patients with familial amyotrophic lateral sclerosis (ALS). The mean age of disease onset in patients inheriting mutations in SOD1 is 45-47 years of age. However, although the length of disease duration is highly variable, there are examples of consistent disease durations associated with specific mutations (e. g. A4V,<2 years). In the present study, we have used a large set of data from SOD1-associated ALS pedigrees to identify correlations between disease features and biochemical/biophysical properties of more than 30 different variants of mutant SOD1. Using a reliable cell culture assay we show that all ALS-associated mutations in SOD1 increase the inherent aggregation propensity of the protein. However, the relative propensity to do so varied considerably among mutants. We were not able to explain the variation in aggregation rates by differences in known protein properties such as enzyme activity, protein thermostability, mutation position, or degree of change in protein charge. Similarly, we were not able to explain variability in the duration of disease in SOD1-associated ALS pedigrees by these properties. However, we find that the majority of pedigrees in which patients exhibit reproducibly short disease durations are associated with mutations that show a high inherent propensity to induce aggregation of SOD1.

VEGF gene polymorphisms as prognostic markers for ocular manifestations in pseudoxanthoma elasticum

Zarbock, R., Hendig, D., Szliska, C., Kleesiek, K., Gotting, C. — 2009-05-30

Pseudoxanthoma elasticum (PXE) is a heritable disorder affecting the skin, eyes and cardiovascular system. It is caused by mutations in the ABCC6 gene and its clinical picture is highly variable. PXE often leads to severe visual impairment due to the development of choroidal neovascularisation (CNV). CNV in PXE-associated retinopathy is believed to be mediated by the action of vascular endothelial growth factor (VEGF). The objective of the present study was to evaluate a possible impact of variations in the VEGFA gene on ocular manifestations of PXE. For this purpose, we evaluated the distribution of ten single nucleotide polymorphisms (SNPs) in the promoter and coding region of the VEGFA gene in DNA samples from 163 German patients affected by PXE and in 163 healthy control subjects. Haplotype analysis of SNPs c.-1540A>C, c.-460C>T, c.-152G>A, c.405C>G, c.674C>T, c.1032C>T, c.4618C>T and c.5092C>A revealed that the haplotype CTGGCCCC was associated with PXE (OR 2.05, 95% CI 1.33 – 3.15, P_corrected = 0.01). Furthermore, five SNPs showed significant association with severe retinopathy. The most significant single SNP association was c.-460C>T (OR 3.83, 95% CI 2.01 – 7.31, P_corrected = 0.0003). Logistic regression analysis identified the c.-460T and the c.674C allele as independent risk factors for development of severe retinopathy. Our findings suggest an involvement of VEGF in the pathogenesis of ocular PXE manifestations. VEGF gene polymorphisms might prove useful as prognostic markers for the development of PXE-associated retinopathy and permit earlier therapeutic intervention in order to prevent loss of central vision, one of the most devastating consequences of this disease.

Parkin promotes intracellular A{beta}1-42 clearance

Burns, M. P., Zhang, L., Rebeck, G. W., Querfurth, H. W., Moussa, C. E-H. — 2009-05-29

Alzheimer's disease and Parkinson's disease are common neurodegenerative diseases that may share some underlying mechanisms of pathogenesis. Aβ_1-42 fragments are found intracellularly, and extracellularly as amyloid plaques, in Alzheimer's disease and in dementia with Lewy Bodies. Parkin is an E3-ubiquitin ligase involved in proteasomal degradation of intracellular proteins. Mutations in parkin, which result in loss of parkin function, lead to early onset Parkinsonism. Here we tested whether the ubiquitin ligase activity of parkin could lead to reduction in intracellular human Aβ_1-42. Lentiviral constructs encoding either human parkin or human Aβ_1-42 were used to infect M17 neuroblastoma cells. Parkin expression resulted in reduction of intracellular human Aβ_1-42 levels and protected against its toxicity in M17 cells. Co-injection of lentiviral constructs into control rat primary motor cortex demonstrated that parkin co-expression reduced human Aβ_1-42 levels and Aβ_1-42-induced neuronal degeneration in vivo. Parkin increased proteasomal activity, and proteasomal inhibition blocked the effects of parkin on reducing Aβ_1-42 levels. Incubation of Aβ_1-42 cell lysates with ubiquitin, in the presence of parkin, demonstrated the generation of Aβ-ubiquitin complexes. These data indicate that parkin promotes ubiquitination and proteasomal degradation of intracellular Aβ_1-42 and demonstrate a protective effect in neurodegenerative diseases with Aβ deposits.

Mitochondrial abnormalities, energy deficit and oxidative stress are features of calpain 3 deficiency in skeletal muscle

2009-05-29

Mutations in the non-lysosomal cysteine protease calpain-3 cause autosomal recessive limb girdle muscular dystrophy. Pathological mechanisms occurring in this disease have not yet been elucidated. Here, we report both morphological and biochemical evidence of mitochondrial abnormalities in calpain-3 knockout (C3KO) muscles including irregular ultrastructure and distribution of mitochondria. The morphological abnormalities in C3KO muscles are associated with reduced in vivo mitochondrial ATP production as measured by ³¹P Magnetic Resonance Spectroscopy. Mitochondrial abnormalities in C3KO muscles also correlate with the presence of oxidative stress; increased protein modification by oxygen free radicals and an elevated concentration of the anti-oxidative enzyme Mn-superoxide dismutase were observed in C3KO muscles. Previously we identified a number of mitochondrial proteins involved in β-oxidation of fatty acids as potential substrates for calpain-3. In order to determine if the mitochondrial abnormalities resulted from loss of direct regulation of mitochondrial proteins by calpain-3, we validated the potential substrates that were identified in previous proteomic studies. This analysis showed that the β-oxidation enzyme, VLCAD, is cleaved by calpain-3 in vitro, but we were not able to confirm that VLCAD is an in vivo substrate for calpain-3. However, the activity of VLCAD was decreased in C3KO mitochondrial fractions compared with wild type, a finding that likely reflects a general mitochondrial dysfunction. Taken together, these data suggest that mitochondrial abnormalities leading to oxidative stress and energy deficit are important pathological features of calpainopathy and possibly represent secondary effects of the absence of calpain-3.

DNMT3B interacts with constitutive centromere protein CENP-C to modulate DNA methylation and the histone code at centromeric regions

2009-05-29

DNA methylation is an epigenetically imposed mark of transcriptional repression that is essential for maintenance of chromatin structure and genomic stability. Genome-wide methylation patterns are mediated by the combined action of three DNA methyltransferases: DNMT1, DNMT3A, and DNMT3B. Compelling links exist between DNMT3B and chromosome stability as emphasized by the mitotic defects that are a hallmark of ICF syndrome, a disease arising from germline mutations in DNMT3B. Centromeric and pericentromeric regions are essential for chromosome condensation and the fidelity of segregation. Centromere regions contain distinct epigenetic marks, including dense DNA hypermethylation, yet the mechanisms by which DNA methylation is targeted to these regions remains largely unknown. In the present study, we used a yeast two-hybrid screen and identified a novel interaction between DNMT3B and constitutive centromere protein CENP-C. CENP-C is itself essential for mitosis. We confirm this interaction in mammalian cells and map the domains responsible. Using siRNA knock downs, bisulfite genomic sequencing, and ChIP, we demonstrate for the first time that CENP-C recruits DNA methylation and DNMT3B to both centromeric and pericentromeric satellite repeats and that CENP-C and DNMT3B regulate the histone code in these regions, including marks characteristic of centromeric chromatin. Finally, we demonstrate that loss of CENP-C or DNMT3B leads to elevated chromosome misalignment and segregation defects during mitosis and increased transcription of centromeric repeats. Taken together, our data reveal a novel mechanism by which DNA methylation is targeted to discrete regions of the genome and contributes to chromosomal stability.

Functional complementation studies identify candidate genes and common genetic variants associated with ovarian cancer survival

2009-05-29

Inhibition of myostatin does not ameliorate disease features of severe SMA mice

2009-05-28

There is currently no treatment for the inherited motor neuron disease, spinal muscular atrophy (SMA). Severe SMA causes lower motor neuron loss, impaired myofiber development, profound muscle weakness, and early mortality. Myostatin is a TGF-β family member that inhibits muscle growth. Loss or blockade of myostatin signaling increases muscle mass and improves muscle strength in mouse models of primary muscle disease and in the motor neuron disease, amyotrophic lateral sclerosis. In this study, we evaluated the effects of blocking myostatin signaling in severe SMA mice (hSMN2/delta7SMN/mSmn^-/-) by two independent strategies: 1) transgenic overexpression of the myostatin inhibitor follistatin and 2) post-natal administration of a soluble activin receptor IIB (ActRIIB-Fc). SMA mice overexpressing follistatin showed little increase in muscle mass and no improvement in motor function or survival. SMA mice treated with ActRIIB-Fc showed minimal improvement in motor function, and no extension of survival compared to vehicle-treated mice. Together these results suggest that inhibition of myostatin may not be a promising therapeutic strategy in severe forms of SMA.

Segregation of expression of mPeriod gene homologs in neurons and glia: Possible divergent roles of mPeriod1 and mPeriod2 in the brain

2009-05-28

The suprachiasmatic nuclei (SCN) of the mammalian hypothalamus function as the master circadian clock, coordinating the timing of diverse cell populations and organ systems. Dysregulation of clock timing is linked to a broad range of human conditions, including obesity, cardiovascular disease and a wide spectrum of neurological disorders. Aberrant regulation of expression of the PERIOD genes has been associated with improper cell division and human cancers, while the autosomal dominant disorder familial advanced sleep phase syndrome (FASPS) has been mapped to a single missense mutation within the critical clock gene hPERIOD2. An essential tool to begin to dissect the inherent molecular timing process is the clock gene reporter. Here, we functionally characterize two new mouse transgenic clock reporters, mPeriod1-Venus and mPeriod2-DsRED. Venus and DsRED are fluorescent proteins that can be used to monitor transcription in individual cells in real-time (1, 2). Imaging of the SCN revealed oscillations, as well as light inducibility, in Venus and DsRED expression. Rhythmic Venus and DsRED expression was observed in distinct SCN cell populations, suggesting the existence of discrete cellular SCN clocks. Outside of the SCN, mPeriod1-Venus expression was broadly expressed in neuronal and non-neuronal populations. Conversely, mPeriod2-DsRED was expressed in glial populations and progenitor cells of the dentate gyrus; limited expression was detected in neurons. This distinct expression pattern of the two reporters reveals that the CNS possesses mechanistically distinct subpopulations of neuronal and non-neuronal cellular clocks. These novel mouse models will facilitate our understanding of clock timing and its role in human diseases.

Aberrant de novo methylation of the p16INK4A CpG island is initiated post gene silencing in association with chromatin remodeling and mimics nucleosome positioning

2009-05-28

Changes in the epigenetic landscape are widespread in neoplasia, with de novo methylation and histone repressive marks commonly enriched in CpG island associated promoter regions. DNA hypermethylation and histone repression correlate with gene silencing, however the dynamics of this process are still largely unclear. The tumour suppressor gene p16^INK4A is inactivated in association with CpG island methylation during neoplastic progression in a variety of cancers, including breast cancer. Here we investigated the temporal progression of DNA methylation and histone remodelling in the p16^INK4A CpG island in primary human mammary epithelial cell (HMEC) strains during selection, as a model for early breast cancer. Silencing of p16^INK4A has been previously shown to be necessary before HMECs can escape from selection. Here we demonstrate that gene silencing occurs prior to de novo methylation and histone remodelling. An increase in DNA methylation was associated with a rapid loss of both histone H3K27 trimethylation and H3K9 acetylation and a gradual gain of H3K9 dimethylation. Interestingly, we found that regional-specific "seeding" methylation occurs early after post-selection and that the de novo methylation pattern observed in HMECs correlates with the apparent footprint of nucleosomes across the p16^INK4A CpG island. Our results demonstrate for the first time that p16^INK4A gene silencing is a precursor to epigenetic suppression and that subsequent de novo methylation initially occurs in nucleosome-free regions across the p16^INK4A CpG island and this is associated with a dynamic change in histone modifications.

HnRNP U mediates the long-range regulation of Shh expression during limb development

Zhao, J., Ding, J., Li, Y., Ren, K., Sha, J., Zhu, M., Gao, X. — 2009-05-28

Transcriptional modulation may be mediated by cis-regulatory elements distant from their target genes. Mutations in a conserved locus about 1 Mb upstream of the Shh coding region often affect Shh expression and are associated with preaxial polydactyly (PPD) defects. To understand the molecular mechanism, we analyzed a novel mouse PPD model with a T to A point mutation in this distant locus. A core element of mutation (CEM) with putative enhancer activity was identified by promoter activity assay and shown to contain a matrix attachment region (MAR). HnRNP U preferentially bound to the mutant but not the wildtype CEM. Interestingly, HnRNP U also bound to the 5’ UTR of the Shh gene, which was not located in the nuclear matrix in wildtype embryonic cells, as indicated by chromatin immunoprecipitation. We propose that the 5’ UTR of Shh was pulled into the nuclear matrix by HnRNP U when the CEM was mutated, and consequently affected Shh expression. Therefore, distant cis-elements may modulate gene expression by altering HnRNP U's affinity for certain mediator proteins and nuclear relocation.

In vivo and in vitro effects of two novel gamma actin (ACTG1) mutations that cause DFNA20/26 hearing impairment

2009-05-28

Here we report the functional assessment of two novel deafness-associated -actin mutants, K118 N and E241K, in a spectrum of different situations with increasing biological complexity by combining biochemical and cell biological analysis in yeast and mammalian cells. Our in vivo experiments showed that while the K118N had a very mild effect on yeast behaviour, the phenotype caused by the E241K mutation was very severe and characterized by a highly compromised ability to grow on glycerol as a carbon source, an aberrant multi-vacuolar pattern and the deposition of thick F-actin bundles randomly in the cell. The latter feature is consistent with the highly unusual spontaneous tendency of the E241K mutant to form bundles in vitro, although this propensity to bundle was neutralized by tropomyosin and the E241K filament bundles were hypersensitive to severing in the presence of cofilin. In transiently transfected NIH3T3 cells both mutant actins were normally incorporated into cytoskeleton structures, although cytoplasmic aggregates were also observed indicating an element of abnormality caused by the mutations in vivo. Interestingly, gene-gun mediated expression of these mutants in cochlear hair cells results in no gross alteration in cytoskeletal structures or the morphology of stereocilia. Our results provide a more complete picture of the biological consequences of deafness-associated -actin mutants and support the hypothesis that the postlingual and progressive nature of the DFNA20/26 hearing loss is the result of a progressive deterioration of the hair cell cytoskeleton over time.

Somatic expansion of the Huntington's disease CAG repeat in the brain is associated with an earlier age of disease onset

2009-05-23

The age of onset of Huntington's disease (HD) is determined primarily by the length of the HD CAG repeat mutation, but is also influenced by other modifying factors. Delineating these modifiers is a critical step towards developing validated therapeutic targets in HD patients. The HD CAG repeat is somatically unstable, undergoing progressive length increases over time, particularly in brain regions that are the targets of neurodegeneration. Here, we have explored the hypothesis that somatic instability of the HD CAG repeat is itself a modifier of disease. Using small pool-PCR, we quantified somatic instability in the cortex region of the brain from a cohort of HD individuals exhibiting phenotypic extremes of young and old disease onset as predicted by the length of their constitutive HD CAG repeat lengths. After accounting for constitutive repeat length, somatic instability was found to be a significant predictor of onset age, with larger repeat length gains associated with earlier disease onset. These data are consistent with the hypothesis that somatic HD CAG repeat length expansions in target tissues contribute to the HD pathogenic process, and support pursuing factors that modify somatic instability as viable therapeutic targets.

Uromodulin mutations causing Familial Juvenile Hyperuricaemic Nephropathy lead to protein maturation defects and retention in the endoplasmic reticulum

2009-05-22

Familial Juvenile Hyperuricaemic Nephropathy (FJHN), an autosomal dominant disorder, is caused by mutations in the UMOD gene, which encodes Uromodulin, a glycosylphosphatidylinositol (GPI)-anchored protein that is expressed in the thick ascending limb of the loop of Henle, and excreted in the urine. Uromodulin contains 3 epidermal growth factor-like (EGF) domains, a cysteine-rich region which includes a domain of eight cysteines (D8C), and a zona pellucida (ZP) domain. Over 90% of UMOD mutations are missense, and 62% alter a cysteine residue, implicating a role for protein misfolding in the disease. We investigated 20 Northern European FJHN probands for UMOD mutations. Wild-type and mutant Uromodulins were functionally studied by expression in HeLa cells and the use of Western blot analysis and confocal microscopy. Six different UMOD missense mutations (Cys32Trp, Arg185Gly, Asp196Asn, Cys217Trp, Cys223Arg, and Gly488Arg) were identified. Patients with UMOD mutations were phenotypically similar to those without UMOD mutations. The mutant Uromodulins had significantly delayed maturation, retention in the endoplasmic reticulum (ER) and reduced expression at the plasma membrane. However, Gly488Arg, which is the only mutation we identified in the ZP domain, was found to be associated with milder in vitro abnormalities and to be the only mutant Uromodulin detected in conditioned medium from transfected cells, indicating that the severity of the mutant phenotypes may depend on their location within the protein. Thus, FJHN-causing Uromodulin mutants are retained in the ER, with impaired intracellular maturation and trafficking, thereby indicating mechanisms whereby Uromodulin mutants may cause the phenotype of FJHN.

Impaired PGC-1{alpha} function in muscle in Huntington's disease

2009-05-21

We investigated the role of PPAR coactivator 1 (PGC-1) in muscle dysfunction in Huntington's Disease (HD). We observed reduced PGC-1 and target genes expression in muscle of HD transgenic mice. We produced chronic energy deprivation in HD mice by administering the catabolic stressor β-guanidinopropionic acid (GPA), a creatine analog that reduces ATP levels, activates AMP kinase (AMPK), which in turn activates PGC-1. Treatment with GPA resulted in increased expression of AMPK, PGC-1 target genes, genes for oxidative phosphorylation, electron transport chain and mitochondrial biogenesis, increased oxidative muscle fibers, numbers of mitochondria, and motor performance in wild type, but not in HD mice. In muscle biopsies from HD patients, there was decreased PGC-1, PGC-1β and oxidative fibers. Oxygen consumption, PGC-1, NRF1, and response to GPA were significantly reduced in myoblasts from HD patients. Knockdown of mutant huntingtin resulted in increased PGC-1 expression in HD myoblast. Lastly, adenoviral mediated delivery of PGC-1 resulted increased expression of PGC-1 and markers for oxidative muscle fibers and reversal of blunted response for GPA in HD mice. These findings show that impaired function of PGC-1 plays a critical role in muscle dysfunction in HD, and that treatment with agents to enhance PGC-1 function could exert therapeutic benefits. Furthermore, muscle may provide a readily accessible tissue in which to monitor therapeutic interventions.

A variant in the gene FUT9 is associated with susceptibility to placental malaria infection

2009-05-21

Malaria in pregnancy forms a substantial part of the worldwide burden of malaria, with an estimated annual death toll of up to 200,000 infants, as well as increased maternal morbidity and mortality. Studies of genetic susceptibility to malaria have so far focused on infant malaria, with only a few studies investigating the genetic basis of placental malaria, focusing only on a limited number of candidate genes. The aim of this study therefore was to identify novel host genetic factors involved in placental malaria infection. To this end we carried out a nested case-control study on 180 Mozambican pregnant women with placental malaria infection, and 180 controls within an intervention trial of malaria prevention. We genotyped 880 SNPs in a set of 64 functionally related genes involved in glycosylation and innate immunity. A SNP located in the gene FUT9, rs3811070, was significantly associated with placental malaria infection (OR = 2.31, permutation p-value = 0.028). Haplotypic analysis revealed a similarly strong association of a common haplotype of four SNPs including rs3811070. FUT9 codes for a fucosyl-transferase that is catalyzing the last step in the biosynthesis of the Lewis-x antigen, which forms part of the Lewis blood group-related antigens. These results therefore suggest an involvement of this antigen in the pathogenesis of placental malaria infection.

Epigenomic Profiling Indicates a Role for DNA Methylation in Early Postnatal Liver Development

2009-05-20

The question of whether DNA methylation contributes to the stabilization of gene expression patterns in differentiated mammalian tissues remains controversial. Using genome-wide methylation profiling, we screened 3,757 gene promoters for changes in methylation during postnatal liver development to test the hypothesis that developmental changes in methylation and expression are temporally correlated. We identified 31 genes that gained methylation and 111 that lost methylation from embryonic day 17.5 to postnatal day 21. Promoters undergoing methylation changes in postnatal liver tended not to be associated with CpG islands. At most genes studied, developmental changes in promoter methylation were associated with expression changes, suggesting both that transcriptional inactivity attracts de novo methylation, and that transcriptional activity can override DNA methylation and successively induce developmental hypomethylation. These in vivo data clearly indicate a role for DNA methylation in mammalian differentiation, and provide the novel insight that critical windows in mammalian developmental epigenetics extend well beyond early embryonic development.

Natural Mutations of the anti-Mullerian Hormone type II Receptor Found in Persistent Mullerian Duct Syndrome affect Ligand Binding, Signal Transduction and Cellular Transport

2009-05-20

The Anti-Müllerian hormone type II receptor (AMHRII) is the primary receptor for Anti-Müllerian Hormone (AMH), a protein produced by Sertoli cells and responsible for regression of the Müllerian duct in males. AMHRII is a membrane protein containing an N-terminal extracellular domain that binds AMH, a transmembrane domain, and an intracellular domain with serine/threonine kinase activity. Mutations in the AMHRII gene lead to persistent Müllerian duct syndrome in human males. In this report, we have investigated the effects of ten AMHRII mutations, namely four mutations in the extracellular domain and six in the intracellular domain. Molecular models of the extra- and intracellular domains are presented and provide insight into how the structure and function of eight of the mutant receptors, which are still expressed at the cell surface, are affected by their mutations. Interestingly, two soluble receptors truncated upstream of the transmembrane domain are not secreted, unless the transforming growth factor beta type II receptor signal sequence is substituted for the endogenous one. This shows that the AMHRII signal sequence is defective and suggests that AMHRII uses its transmembrane domain instead of its signal sequence to translocate to the endoplasmic reticulum, a characteristic of type III membrane proteins.

The transcription co-factor CITED2 functions during sex determination and early gonad development

Buaas, F. W., Val, P., Swain, A. — 2009-05-20

The early bi-potential mammalian gonad requires the expression of a Y-linked gene, Sry, during a brief window of time to ensure proper testis development. WT1 and its direct target gene Sf1 function during sex determination as well as in the specified testes and ovaries. We have previously shown that the transcription co-factor CITED2 interacts with WT1 to stimulate the expression of Sf1 in the adrenogonadal primordium to ensure adrenal development. We now show through genetic interactions and expression analyses that Cited2 acts in the gonad with Wt1 and Sf1 to increase the expression of Sry levels to attain a critical threshold to efficiently initiate testis development. Reducing the gene dosage of Wt1 or Sf1 in Cited2 mutant gonads was sufficient to produce partial XY sex reversal while full sex reversal was attained in mutants containing a hypomorphic Sry^POS allele. A direct correlation was observed between XY sex reversal and reduced expression levels of Sry and Sf1 during sex determination, which indicated that Sry is a downstream target of the CITED2/WT1/SF1 regulatory pathway. Our results provide in vivo evidence for the identification of the first transcription co-factor to function during mammalian sex determination, as part of the WT1/SF1 regulatory mechanism. This highlights the gene dosage sensitivity of the pathway's effect on Sry levels and embryonic gonad development.

Germline and somatic cancer-associated mutations in the ATP-binding motifs of PTEN influence its subcellular localization and tumor suppressive function

Lobo, G. P., Waite, K. A., Planchon, S. M., Romigh, T., Nassif, N. T., Eng, C. — 2009-05-20

Germline and somatic PTEN mutations are found in Cowden syndrome and multiple sporadic malignancies, respectively. PTEN function appears to be modulated by subcellular compartmentalization, and mislocalization may affect function. We have shown that cellular ATP levels affect nuclear PTEN levels. Here, we examined the ATP-binding capabilities of PTEN and functional consequences, relevant to cancer-associated mutations. PTEN mutation analysis of Cowden syndrome patients and sporadic colorectal carcinomas and comparative-amino-acid analysis were utilized to identify mutations in ATP-binding motifs. The ability of wild-type or mutant PTEN to bind ATP was assessed by ATP-agarose-binding assays. Subcellular fractionation, Western blotting, confocal microscopy and growth assays were used to determine relative nuclear-cytoplasmic localization and function. Somatic colorectal carcinoma-derived PTEN missense mutations were associated with nuclear mislocalization. These mutations altered cellular proliferation, apoptosis and anchorage-dependent growth. Examination of PTEN's amino acid sequence revealed these mutations resided in previously undescribed ATP-binding motifs (c.60-73; c.122-136). In contrast to wild-type PTEN, both cancer-associated somatic and germline-derived PTEN missense mutations, which lie within the ATP-binding motifs, result in mutant-PTEN that does not bind ATP efficiently. We also show that Cowden syndrome patients with germline ATP-binding motif-mutations had nuclear PTEN mislocalization. Of four unrelated patients with functional germline ATP-binding domain mutations, all three female patients had breast cancers. Germline and somatic mutations within PTEN's ATP-binding domain play important pathogenic roles in both heritable and sporadic carcinogenesis by PTEN nuclear mislocalization resulting in altered signaling and growth. Manipulation of ATP may represent novel therapies in tumors with such PTEN alterations.

Diet-induced hepatocellular carcinoma in genetically-predisposed mice

2009-05-19

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide, with ~70% of cases resulting from hepatitis B and C viral infections, aflatoxin exposure, chronic alcohol use or genetic liver diseases. The remaining ~30% of cases are associated with obesity, type 2 diabetes and related metabolic diseases, although a direct link between these pathologies and HCCs has not been established. We tested the long-term effects of high-fat and low-fat diets on males of two inbred strains of mice and discovered that C57BL/6J but not A/J males were susceptible to NASH (non-alcoholic steatohepatitis) and HCC on a high-fat but not low-fat diet. This strain-diet interaction represents an important model for genetically-controlled, diet-induced HCC. Susceptible mice showed morphological characteristics of NASH (steatosis, hepatitis, fibrosis and cirrhosis), dysplasia, and HCC. mRNA profiles of HCCs versus tumor-free liver showed involvement of two signaling networks, one centered on Myc and the other on NFB, similar to signaling described for the two major classes of HCC in humans. miRNA profiles revealed dramatically increased expression of a cluster of miRNAs on the X chromosome without amplification of the chromosomal segment. A switch from high-fat to low-fat diet reversed these outcomes, with switched C57BL/6J males being lean rather than obese and without evidence for NASH or HCCs at the end of the study. A similar diet modification may have important implications for prevention of HCCs in humans.

Linkage and Linkage Disequilibrium Scan for Autism Loci in an Extended Pedigree from Finland

2009-05-19

Population isolates, such as Finland, have proved beneficial in mapping rare causative genetic variants due to a limited number of founders resulting in reduced genetic heterogeneity and extensive linkage disequilibrium. We have here used this special opportunity to identify rare alleles in autism by genealogically tracing 20 autism families into one extended pedigree with verified genealogical links reaching back to the 17^th century. In this unique pedigree we performed a dense microsatellite marker genome-wide scan of linkage and linkage disequilibrium, and followed initial findings with extensive fine-mapping. We identified a putative autism susceptibility locus at 19p13.3, and obtained further evidence for previously identified loci at 1q23 and 15q11-13. Most promising candidate genes were TLE2 and TLE6 genes clustered at 19p13 and ATP1A2 at 1q23.

Human ISD11 is essential for both iron-sulfur cluster assembly and maintenance of normal cellular iron homeostasis

Shi, Y., Ghosh, M., Tong, W.-H., Rouault, T. A. — 2009-05-18

The LYR family consists of proteins of diverse functions that contain the conserved tripeptide ‘LYR’ near the N-terminus, and it includes Isd11, which was previously observed to have an important role in iron-sulfur (FeS) cluster biogenesis in S. cerevisiae. Here we have cloned and characterized human ISD11 and shown that human ISD11 forms a stable complex in vivo with the human cysteine desulfurase, ISCS, which generates the inorganic sulfur needed for Fe-S protein biogenesis. Similar to ISCS, we have found that ISD11 localizes to the mitochondrial compartment, as expected, but also to the nucleus of mammalian cells. Using RNA interference techniques, we have shown that suppression of human ISD11 inactivated mitochondrial and cytosolic aconitases. In addition, ISD11 suppression activated IRE-binding activity of iron regulatory protein 1 (IRP1), increased protein levels of iron regulatory protein 2 (IRP2), and resulted in abnormal punctate ferric iron accumulations in cells. These results indicate that ISD11 is important in biogenesis of iron-sulfur clusters in mammalian cells, and its loss disrupts normal mitochondrial and cytosolic iron homeostasis.

INTRODUCING THE HUMAN LEIGH SYNDROM MUTATION T9176G INTO SACCHAROMYCES CEREVIAE MITOCHONDRIAL DNA LEADS TO SEVERE DEFECTS IN THE INCORPORATION OF Atp6p INTO THE ATP SYTNHASE AND IN THE MITOCHONDRIAL MORPHOLOGY

Kucharczyk, R., Salin, B., di Rago, J.-P. — 2009-05-18

The Leigh syndrome is a severe neurological disorder that has been associated with mutations affecting the mitochondrial energy transducing system. One of these mutations, T9176G, has been localized in the mitochondrial ATP6 gene encoding the Atp6p (or a) subunit of the ATP synthase. This mutation converts a highly conserved leucine residue into arginine within a presumed trans-membrane -helical segment, at position 217 of Atp6p. The T9176G mutation was previously shown to severely reduce the rate of mitochondrial ATP production in cultured human cells containing high loads of this mutation. However the underlying mechanism responsible for the impaired ATP production is still unknown. To better understand how T9176G affects the ATP synthase we have created and analyzed the properties of a yeast strain bearing an equivalent of this mutation. We show that incorporation of Atp6p within the ATP synthase was almost completely prevented in the modified yeast. Based on previous partial biochemical characterization of human T9176G cells, it is likely that this mutation similarly affects the human ATP synthase instead of causing a block in the rotary mechanism of this enzyme as it had been suggested. Interestingly, the T9176G yeast exhibits important anomalies in mitochondrial morphology, an observation which indicates that the pathogenicity of T9176G may not be limited to a bioenergetic deficiency.

Segmental Duplications Mediate Novel, Clinically Relevant Chromosome Rearrangements

2009-05-14

Copy number studies have led to an explosion in the discovery of new segmental duplication-mediated deletions and duplications. We have analyzed copy number changes in 2,419 patients referred for clinical array CGH studies. Twenty-three percent of the abnormal copy number changes we found are immediately flanked by segmental duplications ≥10 kb in size and ≥95% identical in direct orientation, consistent with deletions and duplications generated by nonallelic homologous recombination (NAHR). Here we describe copy number changes in five previously unreported loci with genomic organization characteristic of NAHR-mediated gains and losses; namely, 2q11.2, 7q36.1, 17q23, 2q13, and 7q11.21. Deletions and duplications of 2q11.2, deletions of 7q36.1, and deletions of 17q23 are interpreted as pathogenic based on their genomic size, gene content, de novo inheritance, and absence from control populations. The clinical significance of 2q13 deletions and duplications is still emerging, as these imbalances are also found in phenotypically normal family members and control individuals. Deletion of 7q11.21 is a benign copy number change well represented in control populations and copy number variation databases. Here we discuss the genetic factors that can modify the phenotypic expression of such gains and losses, which likely play a role in these and other recurrent genomic disorders.

Predisposition to relapsing nephrotic syndrome by a nephrin mutation that interferes with assembly of functioning microdomains

2009-05-14

Minimal change disease (MCD) is the most common cause of nephrotic syndrome (NS) and is characterized by only minor morphological alterations in podocytes. A subtype of MCD arises from mutations in nephrin, a major component of the slit diaphragm (SD). Idiopathic MCD is a complex trait where interactions of genetic and immunological factors are implicated. However, the pathogenic mechanisms remain unclear. Here we studied the molecular basis for familial NS characterized by frequent relapses and minimal-change histology. Our previous mutational analysis revealed that the two affected children were compound heterozygotes for nephrin variants C265R and V822M (Kidney Int., 2008). When heterologously expressed, these variants exhibited normal metabolic half-life and raft binding. C265R exhibited substantial ER retention, reflecting an intracellular trafficking defect. By contrast, V822M was able to reach the plasma membrane but was restricted in lateral diffusion as well as trafficking at the cell surface. Clustering of V822M failed to evoke a maximum tyrosine-phosphorylation and actin reorganization, suggesting the inability to assemble into functioning membrane microdomains.

Our results suggest that C265R and V822M compose a dysfunctional SD complex due to their mixed defects comprising reduced cell surface targeting and ineffective assembly of signaling microdomains. The defective SD likely confers a susceptibility to immunogenic stimuli and predisposes to a relapsing phenotype.

Risk for nicotine dependence and lung cancer is conferred by mRNA expression levels and amino acid change in CHRNA5

2009-05-14

Nicotine dependence risk and lung cancer risk are associated with variants in a region of chromosome 15 encompassing genes encoding the nicotinic receptor subunits CHRNA5, CHRNA3 and CHRNB4. To identify potential biologic mechanisms that underlie this risk we tested for cis-acting eQTLs for CHRNA5, CHRNA3 and CHRNB4 in human brain. Using gene expression and disease association studies we provide evidence that both nicotine dependence risk and lung cancer risk are influenced by functional variation in CHRNA5. We demonstrated that the risk allele of rs16969968 primarily occurs on the low mRNA expression allele of CHRNA5. The non-risk allele at rs16969968 occurs on both high and low expression alleles tagged by rs588765 within CHRNA5. When the non-risk allele occurs on the background of low mRNA expression of CHRNA5, the risk for nicotine dependence and lung cancer is significantly lower compared to those with the higher mRNA expression. Together these variants identify three levels of risk associated with CHRNA5. We conclude that there are at least two distinct mechanisms conferring risk for nicotine dependence and lung cancer: altered receptor function caused by a D398 N amino acid variant in CHRNA5 (rs16969968) and variability in CHRNA5 mRNA expression.

The A2A adenosine receptor rescues the urea cycle deficiency of Huntington's disease by enhancing the activity of the ubiquitin-proteasome system

2009-05-14

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion in the Huntingtin (Htt) gene. The resultant mutant Htt protein (mHtt) forms aggregates in the brain and several peripheral tissues (e.g., the liver), and causes devastating neuronal degeneration. Metabolic defects resulting from Htt aggregates in peripheral tissues also contribute to HD pathogenesis. Simultaneous improvement of defects in both the CNS and peripheral tissues is thus the most effective therapeutic strategy, and is highly desirable. We earlier showed that an agonist of the A_2A adenosine receptor (A_2A receptor), CGS21680 (CGS), attenuates neuronal symptoms of HD. We found herein that the A_2A receptor also exists in the liver, and that CGS ameliorated the urea cycle deficiency by reducing mHtt aggregates in the liver. By suppressing aggregate formation, CGS slowed the hijacking of a crucial transcription factor (HSF1) and two protein chaperons (Hsp27 and Hsp70) into hepatic Htt aggregates. Moreover, the abnormally high levels of high-molecular-mass ubiquitin conjugates in the liver of a HD mouse model (R6/2) were also ameliorated by CGS. The protective effect of CGS against mHtt-induced aggregate formation was reproduced in two cells lines, and was prevented by an antagonist of the A_2A receptor and a protein kinase A (PKA) inhibitor. Most importantly, the mHtt-induced suppression of proteasome activity was also normalized by CGS through PKA. Our findings reveal a novel therapeutic pathway of A_2A receptors in HD and further strengthen the concept that the A_2A receptor can be a drug target in treating HD.

Loss of connexin43 mediated gap junctional coupling in the mesenchyme of limb buds leads to altered expression of morphogens in mice

2009-05-12

Mutations in the GJA1 gene coding for connexin43 (Cx43) cause oculodentodigital dysplasia (ODDD) a pleiotropic human disorder with characteristic morphologic anomalies of face, teeth, bones and digits. Interdigital webbings, also called syndactylies, are a characteristic phenotype of this disease showing high intra- and interfamilial penetrance. Therefore, we decided to study the molecular basis of syndactylies caused by Cx43 mutations. In order to reveal the impact of Cx43 mediated gap junctional coupling, we used mice expressing the human point mutation Cx43G138R and, in addition, "knock-out" mice lacking Cx43. Both conditional mouse models developed syndactylies as a consequence of disturbed interdigital apoptosis, which we show to be due to reduced expression of two key morphogens: sonic hedgehog (Shh) and bone morphogenic protein 2 (Bmp2). Diminished levels of Bmp2 and subsequent up-regulation of fibroblast growth factors (Fgfs) lead to an insufficient induction of interdigital apoptosis. Interestingly, the reduction of Shh expression in Cx43 mutants begins on embryonic day 10.5 indicating a disturbance of the Fgf/Shh regulatory feedback loop, and confirming the recently published observation that gap junctions can relay Fgf signals to neighboring cells.

Thus, Cx43 mediated gap junctional coupling in the mesenchyme of limb buds after ED11 is essential to maintain Shh expression, which regulates the downstream signaling of Bmp2. Besides diminished interdigital apoptosis the decreased expression of Bmp2 in Cx43 mutants may also be involved in other morphological alterations in patients suffering from ODDD.

DNA hypomethylation restricted to the murine forebrain induces cortical degeneration and impairs postnatal neuronal maturation

2009-05-10

DNA methylation is a major epigenetic factor regulating genome reprogramming, cell differentiation, developmental gene expression. To understand the role DNA methylation in CNS neurons, we generated conditional Dnmt1 mutant mice that possess ~90% hypomethylated cortical and hippocampal cells in the dorsal forebrain from E13.5 on. The mutant mice were viable with a normal lifespan, but displayed severe neuronal cell death between E14.5 to 3-weeks postnatally. Accompanied with the striking cortical and hippocampal degeneration, adult mutant mice exhibited neurobehavioral defects in learning and memory in adulthood. Unexpectedly, a fraction of Dnmt1-/- cortical neurons survived throughout postnatal development, so that the residual cortex in mutant mice contained 20-30% of hypomethylated neurons across the life. Hypomethylated excitatory neurons exhibited multiple defects in postnatal maturation including abnormal dendritic arborization and impaired neuronal excitability. The mutant phenotypes are coupled with deregulation of those genes involved in neuronal layer-specification, cell death, and the function of ion channels. Our results suggest that DNA methylation, through its role in modulating neuronal gene expression, plays multiple roles in regulating cell survival and neuronal maturation in the CNS.

Pathogenic mechanisms of tooth agenesis linked to paired domain mutations in human PAX9

2009-05-09

Mutations in the paired-domain transcription factor PAX9 are associated with non-syndromic tooth agenesis that preferentially affects posterior dentition. Of the eighteen mutations identified to date, eight are phenotypically well-characterized missense mutations within the DNA-binding paired domain. We determined the structural and functional consequences of these paired domain missense mutations and correlated our findings with the associated dental phenotype variations. In vitro testing included subcellular localization, protein-protein interactions between MSX1 and mutant PAX9 proteins, binding of PAX9 mutants to a DNA consensus site and transcriptional activation from the Pax9 effector promoters Bmp4 and Msx1 with and without MSX1 as co-activator. All mutant PAX9 proteins were localized in the nucleus of transfected cells and physically interacted with MSX1 protein. Three of the mutants retained the ability to bind the consensus paired domain recognition sequence; the others were unable or only partly able to interact with this DNA fragment and also showed a similarly impaired capability for activation of transcription from the Msx1 and Bmp4 promoters. For seven of the eight mutants the degree of loss of DNA-binding and promoter activation correlated quite well with the severity of the tooth agenesis pattern seen in vivo. One of the mutants however showed neither reduction in DNA-binding nor decrease in transactivation; instead, a loss of responsiveness to synergism with MSX1 in target promoter activation and a dominant negative effect when expressed together with wildtype PAX9 could be observed. Our structure-based studies, which modeled DNA binding and subdomain stability, were able to predict functional consequences quite reliably.

Vascular defects in a mouse model of hypotrichosis-lymphedema-telangiectasia syndrome indicate a role for SOX18 in blood vessel maturation

Downes, M., Francois, M., Ferguson, C., Parton, R. G., Koopman, P. — 2009-05-09

Mutations in the transcription factor gene SOX18 cause vascular, lymphatic and hair follicle defects in humans with dominant and recessive forms of hypotrichosis-lymphedema-telangiectasia (HLT) syndrome. Here, we clarify the role of SOX18 in the vascular dysfunction in HLT by ultrastructural, immunofluorescence, molecular and functional analysis of vascular anomalies in embryos of the naturally occurring Sox18-mutant mouse strain ragged-opossum (Ra^Op). Early genesis and patterning of vasculature was unimpaired in Ra^Op embryos, but surface capillaries became enlarged from 12.5 dpc and embryos developed massive surface haemorrhage by 14.5 dpc. Large focal breaches in the endothelial barrier were observed, in addition to endothelial hyperplasia associated with impaired pericyte recruitment to the microvasculature. Expression of the genes encoding the endothelial factors MMP7, IL7R and N-cadherin was reduced in Ra^Op embryos, suggesting these are downstream targets of SOX18. Together our results indicate that vascular anomalies in HLT arise from defects in regulation of genes required for the acquisition of structural integrity during microvascular maturation.

A common genetic variant in the 15q24 nicotinic acetylcholine receptor gene cluster (CHRNA5-CHRNA3-CHRNB4) is associated with a reduced ability of women to quit smoking in pregnancy

2009-05-09

Maternal smoking during pregnancy is associated with low birth weight and adverse pregnancy outcomes. Women are more likely to quit smoking during pregnancy than at any other time in their lives, but some pregnant women continue to smoke. A recent genome-wide association study demonstrated an association between a common polymorphism (rs1051730) in the nicotinic acetylcholine receptor gene cluster, (CHRNA5-CHRNA3-CHRNB4), and both smoking quantity and nicotine dependence. We aimed to test whether the same polymorphism that predisposes to greater cigarette consumption would also reduce the likelihood of smoking cessation in pregnancy.

We studied 7845 pregnant women of European descent from the South-West of England. Using 2474 women who smoked regularly immediately pre-pregnancy, we analyzed the association between the rs1051730 risk allele and both smoking cessation during pregnancy and smoking quantity.

Each additional copy of the risk allele was associated with a 1.27-fold higher odds [95%CI: 1.11-1.45] of continued smoking during pregnancy (P=0.0006). Adjustment for pre-pregnancy smoking quantity weakened, but did not remove this association (odds ratio, OR: 1.20 [95%CI:1.03-1.39]; P=0.018). The same risk allele was also associated with heavier smoking before pregnancy and in the first, but not the last, trimester (OR for smoking 10+ cigarettes/day versus 1-9/day in first trimester=1.30 [95%CI: 1.13-1.50]; P=0.0003).

To conclude, we have found strong evidence of association between the rs1051730 variant and an increased likelihood of continued smoking in pregnancy, and have confirmed the previously observed association with smoking quantity. Our data support the role of genetic factors in influencing smoking cessation during pregnancy.

Promoter and intron 1 polymorphisms of COL1A1 interact to regulate transcription and susceptibility to osteoporosis

Jin, H., van't Hof, R. J., Albagha, O. M. E., Ralston, S. H. — 2009-05-09

Three polymorphisms (-1997G/T; -1663IndelT and +1245G/T) have been identified in the 5’ flank of COL1A1 gene that are associated with osteoporosis but the underlying mechanism is unclear. Here we investigated the functional effects of these variants on COL1A1 transcription. Transcription was with 2-fold higher with the osteoporosis-associated G-del-T haplotype compared with the common G-Ins-G haplotype. Gel shift assays showed that the region surrounding the -1663IndelT polymorphism recognised a complex of proteins essential for osteoblast differentiation and function including Nmp4 and Osterix and the osteoporosis-associated -1663delT allele had increased binding affinity for this complex. Chromatin immunoprecipitation assays confirmed that the region flanking -1663insdelT bound a complex of proteins including Osterix and Nmp4 and also showed evidence of recruitment of Nmp4 to the Sp1 binding site in intron 1. Further studies showed that haplotype G-del-T had higher binding affinity for RNA polymerase II, consistent with increased transcription of the G-del-T allele and there was a significant inverse association between carriage of G-del-T and BMD in a cohort of 3270 Caucasian women. We conclude that common polymorphic variants in the 5’ flank of COLIA1 regulate transcription by affecting DNA - protein interactions and that increased levels of transcription correlate with reduced BMD values in vivo. This is consistent with a model whereby increased COL1A1 transcription predisposes to osteoporosis, probably by increasing production of the alpha 1 chain and disrupting the normal ratio of collagen type 1 alpha 1 and alpha 2 chains.

Positional identification of variants of Adamts16 linked to inherited hypertension

2009-05-07

A previously reported blood pressure (BP) quantitative trait locus on rat Chromosome 1 was isolated in a short congenic segment spanning 804.6kb. The 804.6kb region contained only two genes, LOC306664 and LOC306665. LOC306664 is predicted to translate into A Disintegrin-like Metalloproteinase with Thrombospondin Motifs-16 (Adamts16). LOC306665 is a novel gene. All predicted exons of both LOC306664 and LOC306665 were sequenced. Nonsynonymous variants were identified in only one of these genes, LOC306664. These variants were naturally existing polymorphisms among inbred, outbred and wild rats. The full-length rat transcript of Adamts16 was detected in multiple tissues. Similar to ADAMTS16 in humans, expression of Adamts16 was prominent in the kidney. Renal transcriptome analysis suggested that a network of genes related to BP was differential between congenic and S rats. These genes were also differentially expressed between kidney cell lines with or without knock-down of Adamts16. Adamts16 is conserved between rats and humans. It is a candidate gene within the homologous region on human Chromosome 5, which is linked to systolic and diastolic BP in the Quebec Family Study. Multiple variants, including an Ala to Pro variant in codon 90 (rs2086310) of human ADAMTS16, were associated with human resting Systolic BP (SBP). Replication study in GenNet, confirmed the association of two variants of ADAMTS16 with SBP, including rs2086310. Overall, our report represents a high resolution positional cloning and translational study for Adamts16 as a candidate gene controlling BP.

PDK1 signaling in oocytes controls reproductive aging and lifespan by manipulating the survival of primordial follicles

2009-05-07

The molecular mechanisms that control reproductive aging and menopausal age in females are poorly understood. Here, we provide genetic evidence that 3-phosphoinositide-dependent protein kinase-1 (PDK1) signaling in oocytes preserves reproductive lifespan by maintaining the survival of ovarian primordial follicles. In mice lacking the PDK1-encoding gene Pdk1 in oocytes, the majority of primordial follicles are depleted around the onset of sexual maturity, causing premature ovarian failure (POF) during early adulthood. We further showed that suppressed PDK1–Akt–p70 S6 kinase 1 (S6K1)–ribosomal protein S6 (rpS6) signaling in oocytes appears to be responsible for the loss of primordial follicles, and mice lacking the Rps6 gene in oocytes show POF similar to that in Pdk1-deficient mice. In combination with our earlier finding that phosphatase and tensin homolog deleted on chromosome ten (PTEN) in oocytes suppresses follicular activation, we have now pinpointed the molecular network involving phosphatidylinositol 3 kinase (PI3K)/PTEN–PDK1 signaling in oocytes that controls the survival, loss, and activation of primordial follicles, which together determine reproductive aging and the length of reproductive life in females. Underactivation or overactivation of this signaling pathway in oocytes is shown to cause pathological conditions in the ovary, including POF and infertility.

Functional interaction of DYX1C1 with estrogen receptors suggests involvement of hormonal pathways in dyslexia

2009-05-07

Dyslexia, or specific reading disability, is the unexpected failure in learning to read and write when intelligence and senses are normal. One of the susceptibility genes, DYX1C1, has been implicated in neuronal migration, but little is known about its interactions and functions. As DYX1C1 was suggested to interact with the U-box protein CHIP (Carboxy terminus of Hsc70-Interacting Protein), which also participate in the degradation of estrogen receptors alpha (ER) and beta (ERβ), we hypothesized that the effects of DYX1C1 might be at least in part mediated through regulation of estrogen receptors. ERs have shown to be important in brain development and cognitive functions. Indeed, we show that DYX1C1 interacts with both ERs in the presence of 17β-estradiol, as determined by co-localization, co-immunoprecipitation and proximity ligation assays. Protein levels of endogenous ER or exogenous ERβ were reduced upon over-expression of DYX1C1, resulting in decreased transcriptional responses to 17β-estradiol. Furthermore, we detected in vivo complexes of DYX1C1 with ER or ERβat endogenous levels along neurites of primary rat hippocampal neurons. Taken together, our data suggest that DYX1C1 is involved in the regulation of ER and ERβ, and may thus affect the brain development and regulate cognitive functions. These findings provide novel insights into the function of DYX1C1 and link neuronal migration and developmental dyslexia to the estrogen signaling effects in the brain.

Defective pulmonary vascular remodeling in Smad8 mutant mice

Huang, Z., Wang, D., Ihida-Stansbury, K., Lloyd Jones, P., Martin, J. F. — 2009-05-05

Pulmonary artery hypertension (PAH), a progressive, lethal condition that results in pathologic changes in the pulmonary arterial tree, eventually leads to right heart failure. Work identifying mutations in the Type II Bone morphogenetic protein (Bmp) receptor, BmpRII, in families with PAH has implicated Bmp-signaling in the pathogenesis of PAH. However, the effectors downstream of BmpRII in PAH remain unclear since BmpRII signals via Smad-dependent and independent mechanisms. We investigated Smad8 function, a divergent receptor regulated Smad downstream of Bmp-signaling, using gene targeting in mice. We show that Smad8 loss of function in adults resulted in characteristic changes in distal pulmonary arteries including medial thickening and smooth muscle hyperplasia that is observed in patients with PAH. Smad8 mutant pulmonary vasculature had upregulated Activin/Tgfβ signaling and pathologic remodeling with aberrant Prx1 and Tenascin-C expression. A subset of Smad8 mutants had pulmonary adenomas uncovering a function for Smad8 in normal growth control. These findings implicate Smad8 in both pulmonary hypertension and lung tumorigenesis and support Smad8 as a candidate gene for PAH in humans.

Otoferlin interacts with myosin VI: implications for maintenance of the basolateral synaptic structure of the inner hair cell

2009-05-05

Otoferlin has been proposed to be the Ca²⁺ sensor in hair cell exocytosis, compensating for the classical synaptic fusion proteins synaptotagmin-1 and synaptotagmin-2. In the present study, yeast two-hybrid assays reveal myosin VI as a novel otoferlin binding partner. Co-immunoprecipitation assay and co-expression suggest an interaction of both proteins within the basolateral part of inner hair cells (IHCs). Comparison of otoferlin mutants and myosin VI mutant mice indicate non-complementary and complementary roles of myosin VI and otoferlin for synaptic maturation: (1) IHCs from otoferlin mutant mice exhibited a decoupling of CtBP2/RIBEYE and Ca_V1.3 and severe reduction of exocytosis. (2) Myosin VI mutant IHCs failed to transport BK channels to the membrane of the apical cell regions, and the exocytotic Ca²⁺ efficiency did not mature. (3) Otoferlin and myosin VI mutant IHCs showed a reduced basolateral synaptic surface area and altered active zone topography. Membrane infoldings in otoferlin mutant IHCs indicated disturbed transport of endocytotic membranes and link the above morphological changes to a complementary role of otoferlin and myosin VI in transport of intracellular compartments to the basolateral IHC membrane.

Unravelling the genetic basis of variable clinical expression in neurofibromatosis 1

2009-05-05

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder which displays considerable inter- and intra-familial variability in phenotypic expression. To evaluate the genetic component of variable expressivity in NF1, we examined the phenotypic correlations between affected relatives in 750 NF1 patients from 275 multiplex families collected through the NF-France Network. Twelve NF1-related clinical features, including five quantitative traits (number of café-au-lait spots of small size and of large size, and number of cutaneous, subcutaneous, and plexiform neurofibromas) and seven binary ones, were scored. All clinical features studied, with the exception of neoplasms, showed significant familial aggregation after adjusting for age and sex. For most of them, patterns of familial correlations indicated a strong genetic component with no apparent influence of the constitutional NF1 mutation. Heritability estimates of the five quantitative traits ranged from 0.26 to 0.62. Moreover, we investigated for the first time the role of the normal NF1 allele in the variable expression of NF1 through a family-based association study. Nine tag SNPs in NF1 were genotyped in 1,132 individuals from 313 NF1 families. No significant deviations of transmission of any of the NF1 variants to affected offspring was found for any of the twelve clinical features examined, based on single marker or haplotype analysis. Taken together, our results provided evidence that genetic modifiers, unlinked to the NF1 locus, contribute to the variable expressivity of the disease.

CADASIL mutations enhance spontaneous multimerization of NOTCH3

2009-05-05

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic cause of stroke and vascular dementia. Disease-causing mutations invariably affect cysteine residues within epidermal growth factor-like repeat domains in the extracellular domain of the NOTCH3 receptor (N3^ECD). The biochemical and histopathological hallmark of CADASIL is accumulation of N3^ECD at the cell surface of vascular smooth muscle cells which degenerate over the course of the disease. The molecular mechanisms leading to N3^ECD accumulation remain unknown. Here we show that both wild-type and CADASIL-mutated N3^ECD spontaneously form oligomers and higher order multimers in vitro and that multimerization is mediated by disulfide bonds. Using single-molecule analysis techniques ("scanning for intensely fluorescent targets", SIFT) we demonstrate that CADASIL-associated mutations significantly enhance multimerization compared to wild-type. Taken together, our results for the first time provide experimental evidence for N3 self-association and strongly argue for a neomorphic effect of CADASIL mutations in disease pathogenesis.

Usher Syndrome IIIA Gene Clarin-1 is Essential for Hair Cell Function and Associated Neural Activation

2009-05-03

Usher syndrome 3A (USH3A) is an autosomal recessive disorder characterized by progressive loss of hearing and vision due to mutation in the clarin-1 (CLRN1) gene. Lack of an animal model has hindered our ability to understand the function of CLRN1 and the pathophysiology associated with USH3A. Here we report for the first time a mouse model for ear disease in USH3A. Detailed evaluation of inner ear phenotype in the Clrn1 knockout mouse (Clrn1^–/-) coupled with expression pattern of Clrn1 in the inner ear are presented here. Clrn1 was expressed as early as embryonic day 16.5 in the auditory and vestibular hair cells and associated ganglionic neurons, with its expression being higher in outer hair cells (OHCs) than inner hair cells (IHCs). Clrn1^–/- mice showed early-onset hearing loss that rapidly progressed to severe levels. Two to three weeks after birth (P14-P21), Clrn1^–/- mice showed elevated auditory-evoked brainstem response (ABR) thresholds and prolonged peak and interpeak latencies. By P21 ~70% of Clrn1^–/- mice had no detectable ABR and by P30 these mice were deaf. Distortion product otoacoustic emissions were not recordable from Clrn1^–/- mice. Vestibular function in Clrn1^–/- mice mirrored the cochlear phenotype, although it deteriorated more gradually than cochlear function. Disorganization of OHC stereocilia was seen as early as P2 and by P21 OHC loss was observed. In sum, hair cell dysfunction and prolonged peak latencies in vestibular and cochlear evoked potentials in Clrn1^–/- mice strongly indicate that Clrn1 is necessary for hair cell function and associated neural activation.

AIP-1 ameliorates {beta}-amyloid peptide toxicity in a Caenorhabditis elegans Alzheimer's disease model

Hassan, W. M., Merin, D. A., Fonte, V., Link, C. D. — 2009-05-03

Multiple neurodegenerative diseases are causally linked to aggregation-prone proteins. Cellular mechanisms involving protein turnover may be key defense mechanisms against aggregating protein disorders. We have used a transgenic Caenorhabditis elegans (C. elegans) Alzheimer's disease model to identify cellular responses to proteotoxicity resulting from expression of the human beta amyloid peptide (Aβ). We show upregulation of aip-1 in Aβ-expressing animals. Mammalian homologues of AIP-1 have been shown to associate with, and regulate the function of, the 26S proteasome, leading us to hypothesize that induction of AIP-1 may be a protective cellular response directed toward modulating proteasomal function in response to toxic protein aggregation. Using our transgenic model, we show that overexpression of AIP-1 protected against, while RNAi knockdown of AIP-1 exacerbated Aβ toxicity. AIP-1 overexpression also reduced accumulation of Aβ in this model, which is consistent with AIP-1 enhancing protein degradation. Transgenic expression of one of the two human aip-1 homologues (AIRAPL), but not the other (AIRAP), suppressed Aβ toxicity in C. elegans, which advocates the biological relevance of the data to human biology. Interestingly, AIRAPL and AIP-1 contain a predicted farnesylation site, which is absent from AIRAP. This farnesylation site was shown by others to be essential for an AIP-1 prolongevity function. Consistent with this, we show that an AIP-1 mutant lacking the predicted farnesylation site failed to protect against Aβ toxicity. Our results implicate AIP-1 in the regulation of protein turnover and protection against Aβ toxicity and point at AIRAPL as the functional mammalian homologue of AIP-1.

This article was published online in error and has been removed by the publisher.

2007-05-03