Human Molecular Genetics - recent issues

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Tue, 17 Nov 2009 06:54:15 PST

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Tue, 17 Nov 2009 06:54:15 PST

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Tue, 17 Nov 2009 06:54:15 PST

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Tue, 17 Nov 2009 06:54:15 PST

The mtDNA nt7778 G/T polymorphism affects autoimmune diseases and reproductive performance in the mouse

Tue, 17 Nov 2009 06:54:15 PST

Mitochondria are organelles of all nucleated cells, and variations in mtDNA sequence affect a wide spectrum of human diseases. However, animal models for mtDNA-associated diseases are rare, making it challenging to explore mechanisms underlying the contribution of mitochondria. Here, we identify a polymorphism in the mitochondrial genome, G-to-T at position 7778, which results in an aspartic acid-to-tyrosine (D-Y) substitution in the fifth amino acid of the highly conserved N-terminus of ATP synthase 8 (ATP8). Using a series of conplastic strains we show that this polymorphism increases susceptibility to multiple autoimmune diseases, including collagen-induced arthritis, autoimmune diabetes, nephritis and autoimmune pancreatitis. In addition, it impairs reproductive performance in females, but only in the MRL/MpJ strain. We also demonstrate that the mtAtp8 polymorphism alters mitochondrial performance, increasing H₂O₂ production and affecting mitochondrial structure. Functional analysis reveals that the polymorphism increase the CD4 T cell adaptive potential to an oxidative phosphorylation impaired condition. Our findings provide direct experimental evidence for the role of mitochondria in autoimmunity and reproduction.

AIRE activated tissue specific genes have histone modifications associated with inactive chromatin

Tue, 17 Nov 2009 06:54:15 PST

The Autoimmune Regulator (AIRE) protein is expressed in thymic medullary epithelial cells, where it promotes the ectopic expression of tissue-restricted antigens needed for efficient negative selection of developing thymocytes. Mutations in AIRE cause APECED syndrome, which is characterized by a breakdown of self-tolerance. The molecular mechanism by which AIRE increases the expression of a variety of different genes remains unknown. Here, we studied AIRE-regulated genes using whole genome expression analysis and chromatin immunoprecipitation. We show that AIRE preferentially activates genes that are tissue-specific and characterized by low levels of initial expression in stably transfected HEK293 cell model and mouse thymic medullary epithelial cells. In addition, the AIRE-regulated genes lack active chromatin marks, such as histone H3 trimethylation (H3K4me3) and acetylation (AcH3), on their promoters. We also show that during activation by AIRE, the target genes acquire histone H3 modifications associated with transcription and RNA polymerase II. In conclusion, our data show that AIRE is able to promote ectopic gene expression from chromatin associated with histone modifications characteristic to inactive genes.

Nephrocystin-1 and nephrocystin-4 are required for epithelial morphogenesis and associate with PALS1/PATJ and Par6

Tue, 17 Nov 2009 06:54:15 PST

Nephronophthisis (NPH) is an autosomal recessive disorder characterized by renal fibrosis, tubular basement membrane disruption and corticomedullary cyst formation leading to end-stage renal failure. The disease is caused by mutations in NPHP1-9 genes, which encode the nephrocystins, proteins localized to cell–cell junctions and centrosome/primary cilia. Here, we show that nephrocystin mRNA expression is dramatically increased during cell polarization, and shRNA-mediated knockdown of either NPHP1 or NPHP4 in MDCK cells resulted in delayed tight junction (TJ) formation, abnormal cilia formation and disorganized multi-lumen structures when grown in a three-dimensional collagen matrix. Some of these phenotypes are similar to those reported for cells depleted of the TJ proteins PALS1 or Par3, and interestingly, we demonstrate a physical interaction between these nephrocystins and PALS1 as well as their partners PATJ and Par6 and show their partial co-localization in human renal tubules. Taken together, these results demonstrate that the nephrocystins play an essential role in epithelial cell organization, suggesting a plausible mechanism by which the in vivo histopathologic features of NPH might develop.

Multilocus methylation analysis in a large cohort of 11p15-related foetal growth disorders (Russell Silver and Beckwith Wiedemann syndromes) reveals simultaneous loss of methylation at paternal and maternal imprinted loci

Tue, 17 Nov 2009 06:54:15 PST

Genomic imprinting plays an important role in mammalian development. Loss of imprinting (LOI) through loss (LOM) or gain (GOM) of methylation is involved in many human disorders and cancers. The imprinted 11p15 region is crucial for the control of foetal growth and LOI at this locus is implicated in two clinically opposite disorders: Beckwith Wiedemann syndrome (BWS) with foetal overgrowth associated with an enhanced tumour risk and Russell–Silver syndrome (RSS) with intrauterine and postnatal growth restriction. So far, only a few studies have assessed multilocus LOM in human imprinting diseases. To investigate multilocus LOI syndrome, we studied the methylation status of five maternally and two paternally methylated loci in a large series (n = 167) of patients with 11p15-related foetal growth disorders. We found that 9.5% of RSS and 24% of BWS patients showed multilocus LOM at regions other than ICR1 and ICR2 11p15, respectively. Moreover, over two third of multilocus LOM RSS patients also had LOM at a second paternally methylated locus, DLK1/GTL2 IG-DMR. No additional clinical features due to LOM of other loci were found suggesting an (epi)dominant effect of the 11p15 LOM on the clinical phenotype for this series of patients. Surprisingly, four patients displayed LOM at both ICR1 and ICR2 11p15. Three of them had a RSS and one a BWS phenotype. Our results show for the first time that multilocus LOM can also concern RSS patients. Moreover, LOM can involve both paternally and maternally methylated loci in the same patient.

EKV mutant connexin 31 associated cell death is mediated by ER stress

Tattersall, D., Scott, C. A., Gray, C., Zicha, D., Kelsell, D. P. — Tue, 17 Nov 2009 06:54:15 PST

The epidermis expresses a number of connexin (Cx) proteins that are implicated in gap junction-mediated cell communication. Distinct dominantly inherited mutations in Cx31 cause the skin disease erythrokeratoderma variabilis (EKV) and hearing loss with or without neuropathy. Functional studies reveal tissue-specific effects of these Cx31 disease-associated mutations. The Cx31 mutants (R42P)Cx31, (C86S)Cx31 and (G12D)Cx31 are associated with EKV and the mutant (66delD)Cx31 with peripheral neuropathy and hearing loss, however the mechanisms of pathogenesis remain to be elucidated. Expression of (R42P)Cx31, (C86S)Cx31 and (G12D)Cx31 in vitro, but not (WT)Cx31 or (66delD)Cx31, cause elevated levels of cell-type specific cell death. Previous studies suggest that Cx-associated cell death may be related to abnormal ‘leaky’ hemichannels but we produced direct evidence against that being the major mechanism. Additionally, our immunocytochemistry showed upregulation of components of the unfolded protein response (UPR) in cells expressing the EKV-associated Cx31 mutants but not (WT)Cx31 or (66delD)Cx31. We conclude that the endoplasmic reticulum (ER) stress leading to the UPR is the main mechanism of mutant Cx31-associated cell death. These results indicate that, in vivo, ER stress may lead to abnormal keratinocyte differentiation and hyperproliferation in EKV patient skin.

Expression of the familial Mediterranean fever gene is regulated by nonsense-mediated decay

Grandemange, S., Soler, S., Touitou, I. — Tue, 17 Nov 2009 06:54:15 PST

Mutations in the MEditerranean FeVer (MEFV) gene are responsible for familial Mediterranean fever (FMF), a recessively inherited auto-inflammatory disease. Cases of dominant inheritance and phenotype–genotype heterogeneity have been reported; however, the underlying molecular mechanism is not currently understood. The FMF protein named pyrin or marenostrin (P/M) is thought to be involved in regulating innate immunity but its function remains subject to controversy. Recent studies postulate that a defect in MEFV expression regulation may play a role in FMF physiopathology. Our group, along with others, has identified several alternatively spliced MEFV transcripts in leukocytes. Since alternative splicing and nonsense-mediated decay (NMD) pathways are usually coupled in the post-transcriptional regulation of gene expression, we hypothesized that NMD could contribute to the regulation of the MEFV gene. To address this issue, we examined the effect of indirect and direct inhibition of NMD on expression of the MEFV transcripts in THP1, monocyte and neutrophil cells. We showed that MEFV is the first auto-inflammatory gene regulated by NMD in both a cell- and transcript-specific manner. These results and preliminary western-blot analyses suggest the possible translation of alternatively spliced MEFV transcripts into several P/M variants according to cell type and inflammatory state. Our results introduce the novel hypothesis that variation of NMD efficiency could play an important role in FMF physiopathology as a potent phenotypic modifier.

A new mouse model for the trisomy of the Abcg1-U2af1 region reveals the complexity of the combinatorial genetic code of down syndrome

Tue, 17 Nov 2009 06:54:15 PST

Mental retardation in Down syndrome (DS), the most frequent trisomy in humans, varies from moderate to severe. Several studies both in human and based on mouse models identified some regions of human chromosome 21 (Hsa21) as linked to cognitive deficits. However, other intervals such as the telomeric region of Hsa21 may contribute to the DS phenotype but their role has not yet been investigated in detail. Here we show that the trisomy of the 12 genes, found in the 0.59 Mb (Abcg1–U2af1) Hsa21 sub-telomeric region, in mice (Ts1Yah) produced defects in novel object recognition, open-field and Y-maze tests, similar to other DS models, but induces an improvement of the hippocampal-dependent spatial memory in the Morris water maze along with enhanced and longer lasting long-term potentiation in vivo in the hippocampus. Overall, we demonstrate the contribution of the Abcg1–U2af1 genetic region to cognitive defect in working and short-term recognition memory in DS models. Increase in copy number of the Abcg1–U2af1 interval leads to an unexpected gain of cognitive function in spatial learning. Expression analysis pinpoints several genes, such as Ndufv3, Wdr4, Pknox1 and Cbs, as candidates whose overexpression in the hippocampus might facilitate learning and memory in Ts1Yah mice. Our work unravels the complexity of combinatorial genetic code modulating different aspect of mental retardation in DS patients. It establishes definitely the contribution of the Abcg1–U2af1 orthologous region to the DS etiology and suggests new modulatory pathways for learning and memory.

Impaired cone function and cone degeneration resulting from CNGB3 deficiency: down-regulation of CNGA3 biosynthesis as a potential mechanism

Tue, 17 Nov 2009 06:54:15 PST

The cone cyclic nucleotide-gated (CNG) channel is essential for central and color vision and visual acuity. This channel is composed of two structurally related subunits, CNGA3 and CNGB3; CNGA3 is the ion-conducting subunit, whereas CNGB3 is a modulatory subunit. Mutations in both subunits are associated with achromatopsia and progressive cone dystrophy, with mutations in CNGB3 alone accounting for 50% of all known cases of achromatopsia. However, the molecular mechanisms underlying cone diseases that result from CNGB3 deficiency are unknown. This study investigated the role of CNGB3 in cones, using CNGB3^–/– mice. Cone dysfunction was apparent at the earliest time point examined (post-natal day 30) in CNGB3^–/– mice. When compared with wild-type (WT) controls: photopic electroretingraphic (ERG) responses were decreased by ~75%, whereas scotopic ERG responses were unchanged; visual acuity was decreased by ~20%, whereas contrast sensitivity was unchanged; cone density was reduced by ~40%; photoreceptor apoptosis was detected; and outer segment disorganization was observed in some cones. Notably, CNGA3 protein and mRNA levels were significantly decreased in CNGB3^–/– mice; in contrast, mRNA levels of S-opsin, Gnat2 and Pde6c were unchanged, relative to WT mice. Hence, we show that loss of CNGB3 reduces biosynthesis of CNGA3 and impairs cone CNG channel function. We suggest that down-regulation of CNGA3 contributes to the pathogenic mechanism by which CNGB3 mutations lead to human cone disease.

Chromosomal and genetic alterations in human hepatocellular adenomas associated with type Ia glycogen storage disease

Tue, 17 Nov 2009 06:54:15 PST

Hepatocellular adenoma (HCA) is a frequent long-term complication of glycogen storage disease type I (GSD I) and malignant transformation to hepatocellular carcinoma (HCC) is known to occur in some cases. However, the molecular pathogenesis of tumor development in GSD I is unclear. This study was conducted to systematically investigate chromosomal and genetic alterations in HCA associated with GSD I. Genome-wide SNP analysis and mutation detection of target genes was performed in ten GSD Ia-associated HCA and seven general population HCA cases for comparison. Chromosomal aberrations were detected in 60% of the GSD Ia HCA and 57% of general population HCA. Intriguingly, simultaneous gain of chromosome 6p and loss of 6q were only seen in GSD Ia HCA (three cases) with one additional GSD I patient showing submicroscopic 6q14.1 deletion. The sizes of GSD Ia adenomas with chromosome 6 aberrations were larger than the sizes of adenomas without the changes (P = 0.012). Expression of IGF2R and LATS1 candidate tumor suppressor genes at 6q was reduced in more than 50% of GSD Ia HCA that were examined (n = 7). None of the GSD Ia HCA had biallelic mutations in the HNF1A gene. These findings give the first insight into the distinct genomic and genetic characteristics of HCA associated with GSD Ia. These results strongly suggest that chromosome 6 alterations could be an early event in the liver tumorigenesis in GSD I, and may be in general population. These results also suggest an interesting relationship between GSD Ia HCA and steps to HCC transformation.

Skeletal dysplasias due to filamin A mutations result from a gain-of-function mechanism distinct from allelic neurological disorders

Clark, A. R., Sawyer, G. M., Robertson, S. P., Sutherland-Smith, A. J. — Tue, 17 Nov 2009 06:54:15 PST

Filamin A (FLNA) crosslinks F-actin and binds proteins consistent with roles integrating cell signalling and the cytoskeleton. FLNA missense mutations are associated with the otopalatodigital syndrome (OPD) spectrum of skeletal disorders, clustering in discrete domains. One cluster is found in the second calponin homology domain of the FLNA actin-binding domain (ABD), implicating this region as essential for mediating correct function. Here we show that OPD (FLNA E254K) fibroblast lysates have equivalent concentrations of FLNA compared with controls and that recombinant FLNA E254K ABD has increased in vitro F-actin binding (K_d 13 µm) compared with wild type (WT; K_d 48 µm). These observations are consistent with a gain-of-function mechanism for OPD. We have determined the crystal structures of the WT and E254K FLNA ABDs at 2.3 Å resolution, revealing that they adopt similar closed conformations. The E254K mutation removes a conserved salt bridge but does not disrupt the ABD structure. The solution structures are also equivalent as determined by circular dichroism spectroscopy, but differential scanning fluorimetry denaturation showed reduced stability (decreased T_m of 5.6°C) for E254K relative to WT. Ex vivo characterization of E254K OPD patient fibroblasts revealed they have similar motility and adhesion as control cells, implying that many core functions mediated by FLNA are unaffected, consistent with OPD only affecting specific tissues despite FLNA being widely expressed. These data provide the first biochemical evidence for a gain-of-function mechanism for the OPD disorders, and mechanistically distinguishes them from the loss-of-function phenotypes that manifest as disorders of neuronal migration.

Adenosine deamination in human transcripts generates novel microRNA binding sites

Tue, 17 Nov 2009 06:54:15 PST

Animals regulate gene expression at multiple levels, contributing to the complexity of the proteome. Among these regulatory events are post-transcriptional gene silencing, mediated by small non-coding RNAs (e.g. microRNAs), and adenosine-to-inosine (A-to-I) editing, generated by adenosine deaminases that act on double-stranded RNA (ADAR). Recent data suggest that these regulatory processes are connected at a fundamental level. A-to-I editing can affect Drosha processing or directly alter the microRNA (miRNA) sequences responsible for mRNA targeting. Here, we analyzed the previously reported adenosine deaminations occurring in human cDNAs, and asked if there was a relationship between A-to-I editing events in the mRNA 3' untranslated regions (UTRs) and mRNA:miRNA binding. We find significant correlations between A-to-I editing and changes in miRNA complementarities. In all, over 3000 of the 12 723 distinct adenosine deaminations assessed were found to form 7-mer complementarities (known as seed matches) to a subset of human miRNAs. In 200 of the ESTs, we also noted editing within a specific 13 nucleotide motif. Strikingly, deamination of this motif simultaneously creates seed matches to three (otherwise unrelated) miRNAs. Our results suggest the creation of miRNA regulatory sites as a novel function for ADAR activity. Consequently, many miRNA target sites may only be identifiable through examining expressed sequences.

Epigenetic profiling of somatic tissues from human autopsy specimens identifies tissue- and individual-specific DNA methylation patterns

Tue, 17 Nov 2009 06:54:15 PST

DNA methylation is known to be associated with cell differentiation, aging, disease and cancer. There exists an expanding base of knowledge regarding tissue-specific DNA methylation, but we have little information about person-specific DNA methylation. Here, we analyze the DNA methylation patterns of multiple tissues from multiple individuals using a high-throughput quantitative assay of genome-wide DNA methylation, namely the Illumina GoldenGate BeadArray. DNA methylation patterns were largely conserved across 11 different tissues (r = 0.852) and across six individuals (r = 0.829), and we found that DNA was highly methylated in non-CpG islands and/or CpG sites that are not occupied by either H3K4me3 or H3K27me3 (P < 0.05). Finally, we found that the Illumina GoldenGate assay features a large number of probes (265/1505 probes, 17.6%) that contain single-nucleotide polymorphisms, which may interfere with DNA methylation analyses in genome-wide studies.

Identification of a microRNA signature associated with progression of leukoplakia to oral carcinoma

Tue, 17 Nov 2009 06:54:15 PST

MicroRNAs (miRs) are non-coding RNA molecules involved in cancer initiation and progression. Deregulated miR expression has been implicated in cancer; however, there are no studies implicating an miR signature associated with progression in oral squamous cell carcinoma (OSCC). Although OSCC may develop from oral leukoplakia, clinical and histological assessments have limited prognostic value in predicting which leukoplakic lesions will progress. Our aim was to quantify miR expression changes in leukoplakia and same-site OSCC and to identify an miR signature associated with progression. We examined miR expression changes in 43 sequential progressive samples from 12 patients and four non-progressive leukoplakias from four different patients, using TaqMan Low Density Arrays. The findings were validated using quantitative RT-PCR in an independent cohort of 52 progressive dysplasias and OSCCs, and five non-progressive dysplasias. Global miR expression profiles distinguished progressive leukoplakia/OSCC from non-progressive leukoplakias/normal tissues. One hundred and nine miRs were highly expressed exclusively in progressive leukoplakia and invasive OSCC. miR-21, miR-181b and miR-345 expressions were consistently increased and associated with increases in lesion severity during progression. Over-expression of miR-21, miR-181b and miR-345 may play an important role in malignant transformation. Our study provides the first evidence of an miR signature potentially useful for identifying leukoplakias at risk of malignant transformation.

Selective neuronal requirement for huntingtin in the developing zebrafish

Tue, 17 Nov 2009 06:54:15 PST

Huntington's disease shares a common molecular basis with eight other neurodegenerative diseases, expansion of an existing polyglutamine tract. In each case, this repeat tract occurs within otherwise unrelated proteins. These proteins show widespread and overlapping patterns of expression in the brain and yet the diseases are distinguished by neurodegeneration in a specific subset of neurons that are most sensitive to the mutation. It has therefore been proposed that expansion of the polyglutamine region in these genes may result in perturbation of the normal function of the respective proteins, and that this perturbation in some way contributes to the neuronal specificity of these diseases. The normal functions of these proteins have therefore become a focus for investigation as potential pathogenic pathways. We have used synthetic antisense morpholinos to inhibit the translation of huntingtin mRNA during early zebrafish development and have previously reported the effects of huntingtin reduction on iron transport and homeostasis. Here we report an analysis of the effects of huntingtin loss-of-function on the developing nervous system, observing distinct defects in morphology of neuromasts, olfactory placode and branchial arches. The potential common origins of these defects were explored, revealing impaired formation of the anterior-most region of the neural plate as indicated by reduced pre-placodal and telencephalic gene expression with no effect on mid- or hindbrain formation. These investigations demonstrate a specific ‘rate-limiting’ role for huntingtin in formation of the telencephalon and the pre-placodal region, and differing levels of requirement for huntingtin function in specific nerve cell types.

Preventing Ataxin-3 protein cleavage mitigates degeneration in a Drosophila model of SCA3

Jung, J., Xu, K., Lessing, D., Bonini, N. M. — Tue, 17 Nov 2009 06:54:15 PST

Protein cleavage is a common feature in human neurodegenerative disease. Ataxin-3 protein with an expanded polyglutamine (polyQ) repeat causes spinocerebellar ataxia type-3 (SCA3), also called Machado–Joseph disease, and is cleaved in mammalian cells, transgenic mice and SCA3 patient brain tissue. However, the pathological significance of Ataxin-3 cleavage has not been carefully examined. To gain insight into the significance of Ataxin-3 cleavage, we developed a Drosophila SL2 cell-based model as well as transgenic fly models. Our data indicate that Ataxin-3 protein cleavage is conserved in the fly and may be caspase-dependent as reported previously. Importantly, comparison of flies expressing either wild-type or caspase-site mutant proteins indicates that Ataxin-3 cleavage enhances neuronal loss in vivo. This genetic in vivo confirmation of the pathological role of Ataxin-3 cleavage indicates that therapies targeting Ataxin-3 cleavage might slow disease progression in SCA3 patients.

Detecting natural selection by empirical comparison to random regions of the genome

Tue, 17 Nov 2009 06:54:15 PST

Historical episodes of natural selection can skew the frequencies of genetic variants, leaving a signature that can persist for many tens or even hundreds of thousands of years. However, formal tests for selection based on allele frequency skew require strong assumptions about demographic history and mutation, which are rarely well understood. Here, we develop an empirical approach to test for signals of selection that compares patterns of genetic variation at a candidate locus with matched random regions of the genome collected in the same way. We apply this approach to four genes that have been implicated in syndromes of impaired neurological development, comparing the pattern of variation in our re-sequencing data with a large-scale, genomic data set that provides an empirical null distribution. We confirm a previously reported signal at FOXP2, and find a novel signal of selection centered at AHI1, a gene that is involved in motor and behavior abnormalities. The locus is marked by many high frequency derived alleles in non-Africans that are of low frequency in Africans, suggesting that selection at this or a closely neighboring gene occurred in the ancestral population of non-Africans. Our study also provides a prototype for how empirical scans for ancient selection can be carried out once many genomes are sequenced.

Defective autophagy in neurons and astrocytes from mice deficient in PI(3,5)P2

Ferguson, C. J., Lenk, G. M., Meisler, M. H. — Tue, 17 Nov 2009 06:54:15 PST

Mutations affecting the conversion of PI3P to the signaling lipid PI(3,5)P₂ result in spongiform degeneration of mouse brain and are associated with the human disorders Charcot–Marie–Tooth disease and amyotrophic lateral sclerosis (ALS). We now report accumulation of the proteins LC3-II, p62 and LAMP-2 in neurons and astrocytes of mice with mutations in two components of the PI(3,5)P₂ regulatory complex, Fig4 and Vac14. Cytoplasmic inclusion bodies containing p62 and ubiquinated proteins are present in regions of the mutant brain that undergo degeneration. Co-localization of p62 and LAMP-2 in affected cells indicates that formation or recycling of the autolysosome is impaired. These results establish a role for PI(3,5)P₂ in autophagy in the mammalian central nervous system (CNS) and demonstrate that mutations affecting PI(3,5)P₂ can contribute to inclusion body disease.

FOXE1 association with both isolated cleft lip with or without cleft palate, and isolated cleft palate

Tue, 17 Nov 2009 06:54:15 PST

Nonsyndromic orofacial clefts are a common complex birth defect caused by genetic and environmental factors and/or their interactions. A previous genome-wide linkage scan discovered a novel locus for cleft lip with or without cleft palate (CL/P) at 9q22–q33. To identify the etiologic gene, we undertook an iterative and complementary fine mapping strategy using family-based CL/P samples from Colombia, USA and the Philippines. Candidate genes within 9q22–q33 were sequenced, revealing 32 new variants. Concurrently, 397 SNPs spanning the 9q22–q33 2-LOD-unit interval were tested for association. Significant SNP and haplotype association signals (P = 1.45E – 08) narrowed the interval to a 200 kb region containing: FOXE1, C9ORF156 and HEMGN. Association results were replicated in CL/P families of European descent and when all populations were combined the two most associated SNPs, rs3758249 (P = 5.01E – 13) and rs4460498 (P = 6.51E – 12), were located inside a 70 kb high linkage disequilibrium block containing FOXE1. Association signals for Caucasians and Asians clustered 5' and 3' of FOXE1, respectively. Isolated cleft palate (CP) was also associated, indicating that FOXE1 plays a role in two phenotypes thought to be genetically distinct. Foxe1 expression was found in the epithelium undergoing fusion between the medial nasal and maxillary processes. Mutation screens of FOXE1 identified two family-specific missense mutations at highly conserved amino acids. These data indicate that FOXE1 is a major gene for CL/P and provides new insights for improved counseling and genetic interaction studies.

Genetic association of FOXO1A and FOXO3A with longevity trait in Han Chinese populations

Tue, 17 Nov 2009 06:54:15 PST

FOXO1A and FOXO3A are two members of the FoxO family. FOXO3A has recently been linked to human longevity in Japanese, German and Italian populations. Here we tested the genetic contribution of FOXO1A and FOXO3A to the longevity phenotype in Han Chinese population. Six tagging SNPs from FOXO1A and FOXO3A were selected and genotyped in 1817 centenarians and younger individuals. Two SNPs of FOXO1A were found to be associated with longevity in women (P = 0.01–0.005), whereas all three SNPs of FOXO3A were associated with longevity in both genders (P = 0.005–0.001). One SNP from FOXO1A was found not to be associated with longevity. In haplotype association tests, the OR (95% CI) for haplotypes TTG and CCG of FOXO1A in association with female longevity were 0.72 (0.58–0.90) and 1.38 (1.08–1.76), P = 0.0033 and 0.0063, respectively. The haplotypes of FOXO3A were associated with longevity in men [GTC: OR (95% CI) = 0.67 (0.51–0.86), P = 0.0014; CGT: OR (95% CI) = 1.48 (1.12–1.94), P = 0.0035] and in women [GTC: OR (95% CI) = 0.75 (0.60–0.94), P = 0.0094; CGT: OR (95% CI) = 1.47 (1.16–1.86), P = 0.0009]. The haplotype association tests were validated by permutation analysis. The association of FOXO1A with female longevity was replicated in 700 centenarians and younger individuals that were sampled geographically different from the original population. Thus, we demonstrate that, unlike FOXO3A, FOXO1A is more closely associated with human female longevity, suggesting that the genetic contribution to longevity trait may be affected by genders.

Depletion of mitochondrial DNA in fibroblast cultures from patients with POLG1 mutations is a consequence of catalytic mutations

Tue, 17 Nov 2009 06:54:15 PST

NLRP7 mutations in women with diploid androgenetic and triploid moles: a proposed mechanism for mole formation

Tue, 17 Nov 2009 06:54:15 PST

Contents Page

Wed, 04 Nov 2009 06:02:11 PST

Cover Page

Wed, 04 Nov 2009 06:02:11 PST

Editorial Board

Wed, 04 Nov 2009 06:02:11 PST

Subscription Page

Wed, 04 Nov 2009 06:02:11 PST

Ku70 regulates Bax-mediated pathogenesis in laminin-{alpha}2-deficient human muscle cells and mouse models of congenital muscular dystrophy

Vishnudas, V. K., Miller, J. B. — Wed, 04 Nov 2009 06:02:11 PST

The severely debilitating disease Congenital Muscular Dystrophy Type 1A (MDC1A) is caused by mutations in the gene encoding laminin-2. Bax-mediated muscle cell death is a significant contributor to the severe neuromuscular pathology seen in the Lama2-null mouse model of MDC1A. To extend our understanding of pathogenesis due to laminin-2-deficiency, we have now analyzed molecular mechanisms of Bax regulation in normal and laminin-2-deficient muscles and cells, including myogenic cells obtained from patients with a clinical diagnosis of MDC1A. In mouse myogenic cells, we found that, as in non-muscle cells, Bax co-immunoprecipitated with the multifunctional protein Ku70. In addition, cell permeable pentapeptides designed from Ku70, termed Bax-inhibiting peptides (BIPs), inhibited staurosporine-induced Bax translocation and cell death in mouse myogenic cells. We also found that acetylation of Ku70, which can inhibit binding to Bax and can be an indicator of increased susceptibility to cell death, was more abundant in Lama2-null than in normal mouse muscles. Furthermore, myotubes formed in culture from human laminin-2-deficient patient myoblasts produced high levels of activated caspase-3 when grown on poly-l-lysine, but not when grown on a laminin-2-containing substrate or when treated with BIPs. Finally, cytoplasmic Ku70 in human laminin-2-deficient myotubes was both reduced in amount and more highly acetylated than in normal myotubes. Increased susceptibility to cell death thus appears to be an intrinsic property of human laminin-2-deficient myotubes. These results identify Ku70 as a regulator of Bax-mediated pathogenesis and a therapeutic target in laminin-2-deficiency.

Lowe syndrome patient fibroblasts display Ocrl1-specific cell migration defects that cannot be rescued by the homologous Inpp5b phosphatase

Coon, B. G., Mukherjee, D., Hanna, C. B., Riese, D. J., Lowe, M., Aguilar, R. C. — Wed, 04 Nov 2009 06:02:11 PST

The Lowe syndrome (LS) is a life-threatening, developmental disease characterized by mental retardation, cataracts and renal failure. Although this human illness has been linked to defective function of the phosphatidylinositol 5-phosphatase, Ocrl1 (Oculo-Cerebro-Renal syndrome of Lowe protein 1), the mechanism by which this enzyme deficiency triggers the disease is not clear. Ocrl1 is known to localize mainly to the Golgi apparatus and endosomes, however it translocates to plasma membrane ruffles upon cell stimulation with growth factors. The functional implications of this inducible translocation to the plasma membrane are presently unknown. Here we show that Ocrl1 is required for proper cell migration, spreading and fluid-phase uptake in both established cell lines and human dermal fibroblasts. We found that primary fibroblasts from two patients diagnosed with LS displayed defects in these cellular processes. Importantly, these abnormalities were suppressed by expressing wild-type Ocrl1 but not by a phosphatase-deficient mutant. Interestingly, the homologous human PI-5-phosphatase, Inpp5b, was unable to complement the Ocrl1-dependent cell migration defect. Further, Ocrl1 variants that cannot bind the endocytic adaptor AP2 or clathrin, like Inpp5b, were less apt to rescue the migration phenotype. However, no defect in membrane recruitment of AP2/clathrin or in transferrin endocytosis by patient cells was detected. Collectively, our results suggest that Ocrl1, but not Inpp5b, is involved in ruffle-mediated membrane remodeling. Our results provide new elements for understanding how Ocrl1 deficiency leads to the abnormalities associated with the LS.

X11{beta} rescues memory and long-term potentiation deficits in Alzheimer's disease APPswe Tg2576 mice

Wed, 04 Nov 2009 06:02:11 PST

Increased production and deposition of amyloid β-protein (Aβ) are believed to be key pathogenic events in Alzheimer's disease. As such, routes for lowering cerebral Aβ levels represent potential therapeutic targets for Alzheimer's disease. X11β is a neuronal adaptor protein that binds to the intracellular domain of the amyloid precursor protein (APP). Overexpression of X11β inhibits Aβ production in a number of experimental systems. However, whether these changes to APP processing and Aβ production induced by X11β overexpression also induce beneficial effects to memory and synaptic plasticity are not known. We report here that X11β-mediated reduction in cerebral Aβ is associated with normalization of both cognition and in vivo long-term potentiation in aged APPswe Tg2576 transgenic mice that model the amyloid pathology of Alzheimer's disease. Overexpression of X11β itself has no detectable adverse effects upon mouse behaviour. These findings support the notion that modulation of X11β function represents a therapeutic target for Aβ-mediated neuronal dysfunction in Alzheimer's disease.

Common fragile sites are characterized by histone hypoacetylation

Wed, 04 Nov 2009 06:02:12 PST

Common fragile sites (CFSs) represent large, highly unstable regions of the human genome. CFS sequences are sensitive to perturbation of replication; however, the molecular basis for the instability at CFSs is poorly understood. We hypothesized that a unique epigenetic pattern may underlie the unusual sensitivity of CFSs to replication interference. To examine this hypothesis, we analyzed chromatin modification patterns within the six human CFSs with the highest levels of breakage, and their surrounding non-fragile regions (NCFSs). Chromatin at most of the CFSs analyzed has significantly less histone acetylation than that of their surrounding NCFSs. Trichostatin A and/or 5-azadeoxycytidine treatment reduced chromosome breakage at CFSs. Furthermore, chromatin at the most commonly expressed CFS, the FRA3B, is more resistant to micrococcal nuclease than that of the flanking non-fragile sequences. These results demonstrate that histone hypoacetylation is a characteristic epigenetic pattern of CFSs, and chromatin within CFSs might be relatively more compact than that of the NCFSs, indicating a role for chromatin conformation in genomic instability at CFSs. Moreover, lack of histone acetylation at CFSs may contribute to the defective response to replication stress characteristic of CFSs, leading to the genetic instability characteristic of this regions.

An allergy-associated polymorphism in a novel regulatory element enhances IL13 expression

Kiesler, P., Shakya, A., Tantin, D., Vercelli, D. — Wed, 04 Nov 2009 06:02:12 PST

IL-13 is a central effector of Th2-mediated allergic inflammation and is critical for the induction of IgE synthesis. Common IL13 variants are associated with allergy phenotypes in populations of distinct ethnic background. We recently demonstrated that IL13 expression by human CD4⁺ T cells is paralleled by extensive IL13 locus remodeling, which results in the appearance of multiple DNase I hypersensitive sites. Among these, HS4 in the distal promoter is constitutive in both naïve and polarized Th1 and Th2 cells, and spans a common single nucleotide polymorphism, IL13-1512A>C (rs1881457), strongly associated with total serum IgE levels. We recently characterized HS4 as a novel cis-acting element that upregulates IL13 transcription in activated human and murine T cells. Here we show that IL13-1512A>C is a functional polymorphism that significantly enhances HS4-dependent IL13 expression by creating a binding site for the transcription factor Oct-1. Of note, endogenous Oct-1 was preferentially recruited to the IL13-1512C risk allele in primary CD4⁺ T cells from IL13-1512A>C heterozygous subjects. Moreover, the IL13-1512C allele was overexpressed in transfected Th2 cells from Oct1^+/+ mice, but not from Oct1^+/– mice, demonstrating that increased activity was exquisitely dependent on physiologic levels of Oct-1. Our results illustrate how a functional variant in a regulatory element enhances transcription of an allergy-associated gene, thereby modulating disease susceptibility.

Functional analysis of 5-lipoxygenase promoter repeat variants

Wed, 04 Nov 2009 06:02:12 PST

Variants of a hexanucleotide repeat polymorphism in the promoter of the 5-lipoxygenase (5-LO) gene have been associated with cardiovascular disease traits in humans, which may be due, at least in part, to differential expression of the at-risk alleles. To more fully characterize these variants, we carried out gene expression and DNA methylation studies in primary leukocytes from healthy individuals carrying various 5-LO promoter alleles. Regardless of genotype, 5-LO and 5-LO-activating protein (FLAP) gene expression was higher in granulocytes compared with monocytes and lymphocytes, whereas leukotriene A₄ hydrolase (LTA4H) expression was higher in monocytes. In all three leukocyte populations, 5-LO mRNA levels were positively correlated with those of FLAP and LTA4H, with the highest correlation observed in granulocytes. In lymphocytes, individuals homozygous for the shorter 3 and 4 repeat alleles had between 20–35% higher 5-LO, FLAP and LTA4H expression compared with homozygous carriers of the wild-type 5 repeat allele (P = 0.03–0.0001). DNA methylation analysis of four CpG islands in a 1500 bp region encompassing the 5-LO promoter and the first ~100 bp of intron 1 revealed relatively low overall DNA methylation across all genotypes and leukocyte populations. However, analysis of the promoter repeats themselves demonstrated that, regardless of cell population, the 4 allele was methylated approximately twice as much as the 3 allele (P < 0.0001). Our results demonstrate that, in lymphocytes, the shorter repeat alleles of the 5-LO promoter lead to higher gene expression, which may be regulated through differential DNA methylation of the CpGs located within these repeats.

The ocular albinism type 1 (OA1) G-protein-coupled receptor functions with MART-1 at early stages of melanogenesis to control melanosome identity and composition

Giordano, F., Bonetti, C., Surace, E. M., Marigo, V., Raposo, G. — Wed, 04 Nov 2009 06:02:12 PST

OA1 (GPR143; GPCR, G-protein-coupled receptor), the protein product of the ocular albinism type 1 gene, encodes a pigment-cell-specific GPCR that localizes intracellularly to melanosomes. OA1 mutations result in ocular albinism due to alterations in melanosome formation, suggesting that OA1 is a key player in the biogenesis of melanosomes. To address the function of OA1 in melanosome biogenesis, we have used siRNA inactivation and combined morphological and biochemical methods to investigate melanosome ultrastructure, melanosomal protein localization and expression in human pigmented melanocytic cells. OA1 loss of function leads to decreased pigmentation and causes formation of enlarged aberrant premelanosomes harboring disorganized fibrillar structures and displaying proteins of mature melanosomes and lysosomes at their membrane. Moreover, we show that OA1 interacts biochemically with the premelanosomal protein MART-1. Inactivation of MART-1 by siRNA leads to a decreased stability of OA1 and is accompanied by similar defects in premelanosome biogenesis and composition. These data show for the first time that melanosome composition and identity are regulated at early stages by OA1 and that MART-1 likely acts as an escort protein for this GPCR.

Pathogenic NAP57 mutations decrease ribonucleoprotein assembly in dyskeratosis congenita

Grozdanov, P. N., Fernandez-Fuentes, N., Fiser, A., Meier, U. T. — Wed, 04 Nov 2009 06:02:12 PST

X-linked dyskeratosis congenita (DC) is a rare bone marrow failure syndrome caused by mostly missense mutations in the pseudouridine synthase NAP57 (dyskerin/Cbf5). As part of H/ACA ribonucleoproteins (RNPs), NAP57 is important for the biogenesis of ribosomes, spliceosomal small nuclear RNPs, microRNAs and the telomerase RNP. DC mutations concentrate in the N- and C-termini of NAP57 but not in its central catalytic domain raising questions as to their impact. We demonstrate that the N- and C-termini together form the binding surface for the H/ACA RNP assembly factor SHQ1 and that DC mutations modulate the interaction between the two proteins. Pinpointing impaired interaction between NAP57 and SHQ1 as a potential molecular basis for X-linked DC has implications for therapeutic approaches, e.g. by targeting the NAP57–SHQ1 interface with small molecules.

Mutant SOD1 in neuronal mitochondria causes toxicity and mitochondrial dynamics abnormalities

Wed, 04 Nov 2009 06:02:12 PST

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by motor neuron degeneration. Mutations in Cu,Zn-superoxide dismutase (SOD1) are responsible for 20% of familial ALS cases via a toxic gain of function. In mutant SOD1 transgenic mice, mitochondria of spinal motor neurons develop abnormal morphology, bioenergetic defects and degeneration, which are presumably implicated in disease pathogenesis. SOD1 is mostly a cytosolic protein, but a substantial portion is associated with organelles, including mitochondria, where it localizes predominantly in the intermembrane space (IMS). However, whether mitochondrial mutant SOD1 contributes to disease pathogenesis remains to be elucidated. We have generated NSC34 motor neuronal cell lines expressing wild-type or mutant SOD1 containing a cleavable IMS targeting signal to directly investigate the pathogenic role of mutant SOD1 in mitochondria. We show that mitochondrially-targeted SOD1 localizes to the IMS, where it is enzymatically active. We prove that mutant IMS-targeted SOD1 causes neuronal toxicity under metabolic and oxidative stress conditions. Furthermore, we demonstrate for the first time neurite mitochondrial fragmentation and impaired mitochondrial dynamics in motor neurons expressing IMS mutant SOD1. These defects are associated with impaired maintenance of neuritic processes. Our findings demonstrate that mutant SOD1 localized in the IMS is sufficient to determine mitochondrial abnormalities and neuronal toxicity, and contributes to ALS pathogenesis.

A mouse model for Meckel syndrome reveals Mks1 is required for ciliogenesis and Hedgehog signaling

Weatherbee, S. D., Niswander, L. A., Anderson, K. V. — Wed, 04 Nov 2009 06:02:12 PST

Meckel syndrome (MKS) is a rare autosomal recessive disease causing perinatal lethality associated with a complex syndrome that includes occipital meningoencephalocele, hepatic biliary ductal plate malformation, postaxial polydactyly and polycystic kidneys. The gene mutated in type 1 MKS encodes a protein associated with the base of the cilium in vertebrates and nematodes. However, shRNA knockdown studies in cell culture have reported conflicting results on the role of Mks1 in ciliogenesis. Here we show that loss of function of mouse Mks1 results in an accurate model of human MKS, with structural abnormalities in the neural tube, biliary duct, limb patterning, bone development and the kidney that mirror the human syndrome. In contrast to cell culture studies, loss of Mks1 in vivo does not interfere with apical localization of epithelial basal bodies but rather leads to defective cilia formation in most, but not all, tissues. Analysis of patterning in the neural tube and the limb demonstrates altered Hedgehog (Hh) pathway signaling underlies some MKS defects, although both tissues show an expansion of the domain of response to Shh signaling, unlike the phenotypes seen in other mutants with cilia loss. Other defects in the skull, lung, rib cage and long bones are likely to be the result of the disruption of Hh signaling, and the basis of defects in the liver and kidney require further analysis. Thus the disruption of Hh signaling can explain many, but not all, of the defects caused by loss of Mks1.

Interaction between environmental and genetic factors modulates schizophrenic endophenotypes in the Snap-25 mouse mutant blind-drunk

Oliver, P. L., Davies, K. E. — Wed, 04 Nov 2009 06:02:12 PST

To understand the pathophysiology of neuropsychiatric disorders such as schizophrenia requires consideration of multiple genetic and non-genetic factors. However, very little is known about the consequences of combining models of synaptic dysfunction with controlled environmental manipulations. Therefore, to generate new insights into gene–environment interactions and complex behaviour, we examined the influence of variable prenatal stress (PNS) on two mouse lines with mutations in synaptosomal-associated protein of 25 kDa (Snap-25): the blind-drunk (Bdr) point mutant and heterozygous Snap-25 knockout mice. Neonatal development was analysed in addition to an assessment of adult behavioural phenotypes relevant to the psychotic, cognitive and negative aspects of schizophrenia. These data show that PNS influenced specific anxiety-related behaviour in all animals. In addition, sensorimotor gating deficits previously noted in Bdr mutants were markedly enhanced by PNS; significantly, these effects could be reversed with the application of anti-psychotic drugs. Moreover, social interaction abnormalities were observed only in Bdr animals from stressed dams but not in wild-type littermates or mutants from non-stressed mothers. These results show for the first time that combining a synaptic mouse point mutant with a controlled prenatal stressor paradigm produces both modified and previously unseen phenotypes, generating new insights into the interactions between genetics and the environment relevant to the study of psychiatric disease.

CDKL5 influences RNA splicing activity by its association to the nuclear speckle molecular machinery

Wed, 04 Nov 2009 06:02:12 PST

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been shown to cause severe neurodevelopmental disorders including infantile spasms, encephalopathy, West-syndrome and an early-onset variant of Rett syndrome. CDKL5 is a serine/threonine kinase whose involvement in Rett syndrome can be inferred by its ability to directly bind and mediate phosphorylation of MeCP2. However, it remains to be elucidated how CDKL5 exerts its function. Here, we report that CDKL5 localizes to specific nuclear foci referred to as nuclear speckles in both cell lines and tissues. These sub-nuclear structures are traditionally considered as storage/modification sites of pre-mRNA splicing factors. Interestingly, we provide evidence that CDKL5 regulates the dynamic behaviour of nuclear speckles. Indeed, CDKL5 overexpression leads to nuclear speckle disassembly, and this event is strictly dependent on its kinase activity. Conversely, its down-regulation affects nuclear speckle morphology leading to abnormally large and uneven speckles. Similar results were obtained for primary adult fibroblasts isolated from CDKL5-mutated patients. Altogether, these findings indicate that CDKL5 controls nuclear speckle morphology probably by regulating the phosphorylation state of splicing regulatory proteins. Nuclear speckles are dynamic sites that can continuously supply splicing factors to active transcription sites, where splicing occurs. Notably, we proved that CDKL5 influences alternative splicing, at least as proved in heterologous minigene assays. In conclusion, we provide evidence that CDKL5 is involved indirectly in pre-mRNA processing, by controlling splicing factor dynamics. These findings identify a biological process whose disregulation might affect neuronal maturation and activity in CDKL5-related disorders.

Missense mutations in the SH3TC2 protein causing Charcot-Marie-Tooth disease type 4C affect its localization in the plasma membrane and endocytic pathway

Wed, 04 Nov 2009 06:02:12 PST

Mutations in SH3TC2 (KIAA1985) cause Charcot-Marie-Tooth disease (CMT) type 4C, a demyelinating inherited neuropathy characterized by early-onset and scoliosis. Here we demonstrate that the SH3TC2 protein is present in several components of the endocytic pathway including early endosomes, late endosomes and clathrin-coated vesicles close to the trans-Golgi network and in the plasma membrane. Myristoylation of SH3TC2 in glycine 2 is necessary but not sufficient for the proper location of the protein in the cell membranes. In addition to myristoylation, correct anchoring also needs the presence of SH3 and TPR domains. Mutations that cause a stop codon and produce premature truncations that remove most of the TPR domains are expressed as the wild-type protein. In contrast, missense mutations in or around the region of the first-TPR domain are absent from early endosomes, reduced in plasma membrane and late endosomes and are variably present in clathrin-coated vesicles. Our findings suggest that the endocytic and membrane trafficking pathway is involved in the pathogenesis of CMT4C disease. We postulate that missense mutations of SH3TC2 could impair communication between the Schwann cell and the axon causing an abnormal myelin formation.

Myosin VI is required for the proper maturation and function of inner hair cell ribbon synapses

Wed, 04 Nov 2009 06:02:12 PST

The ribbon synapses of auditory inner hair cells (IHCs) undergo morphological and electrophysiological transitions during cochlear development. Here we report that myosin VI (Myo6), an actin-based motor protein involved in genetic forms of deafness, is necessary for some of these changes to occur. By using post-embedding immunogold electron microscopy, we showed that Myo6 is present at the IHC synaptic active zone. In Snell's waltzer mutant mice, which lack Myo6, IHC ionic currents and ribbon synapse maturation proceeded normally until at least post-natal day 6. In adult mutant mice, however, the IHCs displayed immature potassium currents and still fired action potentials, as normally only observed in immature IHCs. In addition, the number of ribbons per IHC was reduced by 30%, and 30% of the remaining ribbons were morphologically immature. Ca²⁺-dependent exocytosis probed by capacitance measurement was markedly reduced despite normal Ca²⁺ currents and the large proportion of morphologically mature synapses, which suggests additional defects, such as loose Ca²⁺-exocytosis coupling or inefficient vesicular supply. Finally, we provide evidence that Myo6 and otoferlin, a putative Ca²⁺ sensor of synaptic exocytosis also involved in a genetic form of deafness, interact at the IHC ribbon synapse, and we suggest that this interaction is involved in the recycling of synaptic vesicles. Our findings thus uncover essential roles for Myo6 at the IHC ribbon synapse, in addition to that proposed in membrane turnover and anchoring at the apical surface of the hair cells.

GIGYF2 gene disruption in mice results in neurodegeneration and altered insulin-like growth factor signaling

Wed, 04 Nov 2009 06:02:12 PST

Grb10-Interacting GYF Protein 2 (GIGYF2) was initially identified through its interaction with Grb10, an adapter protein that binds activated IGF-I and insulin receptors. The GIGYF2 gene maps to human chromosome 2q37 within a region linked to familial Parkinson's disease (PARK11 locus), and association of GIGYF2 mutations with Parkinson's disease has been described in some but not other recent publications. This study investigated the consequences of Gigyf2 gene disruption in mice. Gigyf2 null mice undergo apparently normal embryonic development, but fail to feed and die within the first 2 post-natal days. Heterozygous Gigyf2^+/– mice survive to adulthood with no evident metabolic or growth defects. At 12–15 months of age, the Gigyf2^+/– mice begin to exhibit motor dysfunction manifested as decreased balance time on a rotating horizontal rod. This is associated with histopathological evidence of neurodegeneration and rare intracytoplasmic Lewy body-like inclusions in spinal anterior horn motor neurons. There are -synuclein positive neuritic plaques in the brainstem and cerebellum, but no abnormalities in the substantia nigra. Primary cultured embryo fibroblasts from Gigyf2 null mice exhibit decreased IGF-I-stimulated IGF-I receptor tyrosine phosphorylation and augmented ERK1/2 phosphorylation. These data provide further evidence for an important role of GIGYF2 in age-related neurodegeneration and IGF pathway signaling.

Pharmacological activation of PPAR{beta}/{delta} stimulates utrophin A expression in skeletal muscle fibers and restores sarcolemmal integrity in mature mdx mice

Wed, 04 Nov 2009 06:02:12 PST

A therapeutic strategy to treat Duchenne muscular dystrophy (DMD) involves identifying compounds that can elevate utrophin A expression in muscle fibers of affected patients. The dystrophin homologue utrophin A can functionally substitute for dystrophin when its levels are enhanced in the mdx mouse model of DMD. Utrophin A expression in skeletal muscle is regulated by mechanisms that promote the slow myofiber program. Since activation of peroxisome proliferator-activated receptor (PPAR) β/ promotes the slow oxidative phenotype in skeletal muscle, we initiated studies to determine whether pharmacological activation of PPARβ/ provides functional benefits to the mdx mouse. GW501516, a PPARβ/ agonist, was found to stimulate utrophin A mRNA levels in C2C12 muscle cells through an element in the utrophin A promoter. Expression of PPARβ/ was greater in skeletal muscles of mdx versus wild-type mice. We treated 5–7-week-old mdx mice with GW501516 for 4 weeks. This treatment increased the percentage of muscle fibers expressing slower myosin heavy chain isoforms and stimulated utrophin A mRNA levels leading to its increased expression at the sarcolemma. Expression of 1-syntrophin and β-dystroglycan was restored to the sarcolemma. Improvement of mdx sarcolemmal integrity was evidenced by decreased intracellular IgM staining and decreased in vivo Evans blue dye (EBD) uptake. GW501516 treatment also conferred protection against eccentric contraction (ECC)-induced damage of mdx skeletal muscles, as shown by a decreased contraction-induced force drop and reduction of dye uptake during ECC. These results demonstrate that pharmacological activation of PPARβ/ might provide functional benefits to DMD patients through enhancement of utrophin A expression.

A genome-wide study of common SNPs and CNVs in cognitive performance in the CANTAB

Wed, 04 Nov 2009 06:02:12 PST

Psychiatric disorders such as schizophrenia are commonly accompanied by cognitive impairments that are treatment resistant and crucial to functional outcome. There has been great interest in studying cognitive measures as endophenotypes for psychiatric disorders, with the hope that their genetic basis will be clearer. To investigate this, we performed a genome-wide association study involving 11 cognitive phenotypes from the Cambridge Neuropsychological Test Automated Battery. We showed these measures to be heritable by comparing the correlation in 100 monozygotic and 100 dizygotic twin pairs. The full battery was tested in ~750 subjects, and for spatial and verbal recognition memory, we investigated a further 500 individuals to search for smaller genetic effects. We were unable to find any genome-wide significant associations with either SNPs or common copy number variants. Nor could we formally replicate any polymorphism that has been previously associated with cognition, although we found a weak signal of lower than expected P-values for variants in a set of 10 candidate genes. We additionally investigated SNPs in genomic loci that have been shown to harbor rare variants that associate with neuropsychiatric disorders, to see if they showed any suggestion of association when considered as a separate set. Only NRXN1 showed evidence of significant association with cognition. These results suggest that common genetic variation does not strongly influence cognition in healthy subjects and that cognitive measures do not represent a more tractable genetic trait than clinical endpoints such as schizophrenia. We discuss a possible role for rare variation in cognitive genomics.

Genetic variation in GPR133 is associated with height: genome wide association study in the self-contained population of Sorbs

Wed, 04 Nov 2009 06:02:12 PST

Recently, associations of several common genetic variants with height have been reported in different populations. We attempted to identify further variants associated with adult height in a self-contained population (the Sorbs in Eastern Germany) as discovery set. We performed a genome wide association study (GWAS) (~390 000 genetic polymorphisms, Affymetrix gene arrays) on adult height in 929 Sorbian individuals. Subsequently, the best SNPs (P < 0.001) were taken forward to a meta-analysis together with two independent cohorts [Diabetes Genetics Initiative, British 1958 Birth Cohort, (58BC, publicly available)]. Furthermore, we genotyped our best signal for replication in two additional German cohorts (Leipzig, n = 1044 and Berlin, n = 1728). In the primary Sorbian GWAS, we identified 5 loci with a P-value < 10^–5 and 455 SNPs with P-value < 0.001. In the meta-analysis on those 455 SNPs, only two variants in GPR133 (rs1569019 and rs1976930; in LD with each other) retained a P-value at or below 10^–6 and were associated with height in the three cohorts individually. Upon replication, the SNP rs1569019 showed significant effects on height in the Leipzig cohort (P = 0.004, beta = 1.166) and in 577 men of the Berlin cohort (P = 0.049, beta = 1.127) though not in women. The combined analysis of all five cohorts (n = 6,687) resulted in a P-value of 4.7 x 10^–8 (beta = 0.949). In conclusion, our GWAS suggests novel loci influencing height. In view of the robust replication in five different cohorts, we propose GPR133 to be a novel gene associated with adult height.

Genetic evidence for a role of adiponutrin in the metabolism of apolipoprotein B-containing lipoproteins

Wed, 04 Nov 2009 06:02:12 PST

Adiponutrin (PNPLA3) is a predominantly liver-expressed transmembrane protein with phospholipase activity that is regulated by fasting and feeding. Recent genome-wide association studies identified PNPLA3 to be associated with hepatic fat content and liver function, thus pointing to a possible involvement in the hepatic lipoprotein metabolism. The aim of this study was to examine the association between two common variants in the adiponutrin gene and parameters of lipoprotein metabolism in 23 274 participants from eight independent West-Eurasian study populations including six population-based studies [Bruneck (n = 800), KORA S3/F3 (n = 1644), KORA S4/F4 (n = 1814), CoLaus (n = 5435), SHIP (n = 4012), Rotterdam (n = 5967)], the SAPHIR Study as a healthy working population (n = 1738) and the Utah Obesity Case-Control Study including a group of 1037 severely obese individuals (average BMI 46 kg/m²) and 827 controls from the same geographical region of Utah. We observed a strong additive association of a common non-synonymous variant within adiponutrin (rs738409) with age-, gender-, and alanine-aminotransferase-adjusted lipoprotein concentrations: each copy of the minor allele decreased levels of total cholesterol on average by 2.43 mg/dl (P = 8.87 x 10^–7), non-HDL cholesterol levels by 2.35 mg/dl (P = 2.27 x 10^–6) and LDL cholesterol levels by 1.48 mg/dl (P = 7.99 x 10^–4). These associations remained significant after correction for multiple testing. We did not observe clear evidence for associations with HDL cholesterol or triglyceride concentrations. In conclusion, our study suggests that adiponutrin is involved in the metabolism of apoB-containing lipoproteins.

Genome-wide significant predictors of metabolites in the one-carbon metabolism pathway

Wed, 04 Nov 2009 06:02:12 PST

Low plasma B-vitamin levels and elevated homocysteine have been associated with cancer, cardiovascular disease and neurodegenerative disorders. Common variants in FUT2 on chromosome 19q13 were associated with plasma vitamin B₁₂ levels among women in a genome-wide association study in the Nurses’ Health Study (NHS) NCI-Cancer Genetic Markers of Susceptibility (CGEMS) project. To identify additional loci associated with plasma vitamin B₁₂, homocysteine, folate and vitamin B₆ (active form pyridoxal 5'-phosphate, PLP), we conducted a meta-analysis of three GWA scans (total n = 4763, consisting of 1658 women in NHS-CGEMS, 1647 women in Framingham-SNP-Health Association Resource (SHARe) and 1458 men in SHARe). On chromosome 19q13, we confirm the association of plasma vitamin B₁₂ with rs602662 and rs492602 (P-value = 1.83 x 10^–15 and 1.30 x 10^–14, respectively) in strong linkage disequilibrium (LD) with rs601338 (P = 6.92 x 10^–15), the FUT2 W143X nonsense mutation. We identified additional genome-wide significant loci for plasma vitamin B₁₂ on chromosomes 6p21 (P = 4.05 x 10^–08), 10p12 (P-value=2.87 x 10^–9) and 11q11 (P-value=2.25 x 10^–10) in genes with biological relevance. We confirm the association of the well-studied functional candidate SNP 5,10-methylene tetrahydrofolate reductase (MTHFR) Ala222Val (dbSNP ID: rs1801133; P-value=1.27 x 10^–8), on chromosome 1p36 with plasma homocysteine and identify an additional genome-wide significant locus on chromosome 9q22 (P-value=2.06 x 10^–8) associated with plasma homocysteine. We also identified genome-wide associations with variants on chromosome 1p36 with plasma PLP (P-value=1.40 x 10^–15). Genome-wide significant loci were not identified for plasma folate. These data reveal new biological candidates and confirm prior candidate genes for plasma homocysteine, plasma vitamin B₁₂ and plasma PLP.

An increased frequency of the 5A allele in the promoter region of the MMP3 gene is associated with abdominal aortic aneurysms

Wed, 04 Nov 2009 06:02:12 PST

Contents Page

Fri, 23 Oct 2009 09:16:41 PDT

Cover Page

Fri, 23 Oct 2009 09:16:41 PDT

Editorial Board

Fri, 23 Oct 2009 09:16:41 PDT

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Fri, 23 Oct 2009 09:16:41 PDT

Imprinting regulates mammalian snoRNA-encoding chromatin decondensation and neuronal nucleolar size

Leung, K. N., Vallero, R. O., DuBose, A. J., Resnick, J. L., LaSalle, J. M. — Fri, 23 Oct 2009 09:16:41 PDT

Imprinting, non-coding RNA and chromatin organization are modes of epigenetic regulation that modulate gene expression and are necessary for mammalian neurodevelopment. The only two known mammalian clusters of genes encoding small nucleolar RNAs (snoRNAs), SNRPN through UBE3A(15q11–q13/7qC) and GTL2(14q32.2/12qF1), are neuronally expressed, localized to imprinted loci and involved in at least five neurodevelopmental disorders. Deficiency of the paternal 15q11–q13 snoRNA HBII-85 locus is necessary to cause the neurodevelopmental disorder Prader–Willi syndrome (PWS). Here we show epigenetically regulated chromatin decondensation at snoRNA clusters in human and mouse brain. An 8-fold allele-specific decondensation of snoRNA chromatin was developmentally regulated specifically in maturing neurons, correlating with HBII-85 nucleolar accumulation and increased nucleolar size. Reciprocal mouse models revealed a genetic and epigenetic requirement of the 35 kb imprinting center (IC) at the Snrpn–Ube3a locus for transcriptionally regulated chromatin decondensation. PWS human brain and IC deletion mouse Purkinje neurons showed significantly decreased nucleolar size, demonstrating the essential role of the 15q11–q13 HBII-85 locus in neuronal nucleolar maturation. These results are relevant to understanding the molecular pathogenesis of multiple human neurodevelopmental disorders, including PWS and some causes of autism.

Knock-down of PQBP1 impairs anxiety-related cognition in mouse

Ito, H., Yoshimura, N., Kurosawa, M., Ishii, S., Nukina, N., Okazawa, H. — Fri, 23 Oct 2009 09:16:41 PDT

PQBP1 (polyglutamine tract-binding protein 1) is a causative gene for a relatively frequent X-linked syndromic and non-syndromic mental retardation (MR). To analyze behavioral abnormalities of these patients from molecular basis, we developed a knock-down (KD) mouse model. The KD mice possess a transgene expressing 498 bp double-strand RNA that is endogenously cleaved to siRNA suppressing PQBP1 efficiently. After confirming that PQBP1 is selectively suppressed to nearly 50% of the control mice, we performed behavioral analyses of PQBP1-KD mice. The KD mice possessed normal ability in ordinary memory tests including water-maze test, whereas they showed abnormal anxiety-related behavior in light/dark exploration test and open-field test and showed obvious declines of anxiety-related cognition in the repetitive elevated plus maze or novel object recognition test. Correspondingly, we found c-fos upregulation and histone H3 acetylation after behavior tests were declined in neurons of amygdala, prefrontal cortex and hippocampus. Furthermore, we found that 4-phenylbutyric acid, an HDAC inhibitor, efficiently improved expression of these genes and rescued the abnormal phenotypes in adult PQBP1-KD mice. These results suggested that PQBP1 dysfunction in regulating gene expression might underlie the abnormal behavior and cognition of PQBP1-KD mice and that the recovery of expression of such PQBP1 target genes might improve the symptoms in adult patients.

The interval between Ins2 and Ascl2 is dispensable for imprinting centre function in the murine Beckwith-Wiedemann region

Fri, 23 Oct 2009 09:16:41 PDT

Imprinted genes are commonly clustered in domains across the mammalian genome, suggesting a degree of coregulation via long-range coordination of their monoallelic transcription. The distal end of mouse chromosome 7 (Chr 7) contains two clusters of imprinted genes within a ~1 Mb domain. This region is conserved on human 11p15.5 where it is implicated in the Beckwith–Wiedemann syndrome. In both species, imprinted regulation requires two critical cis-acting imprinting centres, carrying different germline epigenetic marks and mediating imprinted expression in the proximal and distal sub-domains. The clusters are separated by a region containing the gene for tyrosine hydroxylase (Th) as well as a high density of short repeats and retrotransposons in the mouse. We have used the Cre-loxP recombination system in vivo to engineer an interstitial deletion of this ~280-kb intervening region previously proposed to participate in the imprinting mechanism or to act as a boundary between the two sub-domains. The deletion allele, Del^7AI, is silent with respect to epigenetic marking at the two flanking imprinting centres. Reciprocal inheritance of Del^7AI demonstrates that the deleted region, which represents more than a quarter of the previously defined imprinted domain, is associated with intrauterine growth restriction in maternal heterozygotes. In homozygotes, the deficiency behaves as a Th null allele and can be rescued pharmacologically by bypassing the metabolic requirement for TH in utero. Our results show that the deleted interval is not required for normal imprinting on distal Chr 7 and uncover a new imprinted growth phenotype.

Gp78, an ER associated E3, promotes SOD1 and ataxin-3 degradation

Ying, Z., Wang, H., Fan, H., Zhu, X., Zhou, J., Fei, E., Wang, G. — Fri, 23 Oct 2009 09:16:41 PDT

Superoxide dismutase-1 (SOD1) and ataxin-3 are two neurodegenerative disease proteins in association with familial amyotrophic lateral sclerosis and Machado–Joseph disease/spinocerebellar ataxia type 3. Both normal and mutant types of SOD1 and ataxin-3 are degraded by the proteasome. It was recently reported that these two proteins are associated with the endoplasmic reticulum (ER). Mammalian gp78 is an E3 ubiquitin ligase involved in ER-associated degradation (ERAD). Here, we show that gp78 interacts with both SOD1 and ataxin-3. Overexpression of gp78 promotes the ubiquitination and degradation of these two proteins, whereas knockdown of gp78 stabilizes them. Moreover, gp78 represses aggregate formation of mutant SOD1 and protect cells against mutant SOD1-induced cell death. Furthermore, gp78 is increased in cells transfected with these two mutant proteins as well as in ALS mice. Thus, our results suggest that gp78 functions in the regulation of SOD1 and ataxin-3 to target them for ERAD.

Reversibility of symptoms in a conditional mouse model of spinocerebellar ataxia type 3

Fri, 23 Oct 2009 09:16:41 PDT

Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of a CAG repeat tract that affects the MJD1 gene which encodes the ataxin-3 protein. In order to analyze whether symptoms caused by ataxin-3 with an expanded repeat are reversible in vivo, we generated a conditional mouse model of SCA3 using the Tet-Off system. We used a full-length human ataxin-3 cDNA with 77 repeats in order to generate the responder mouse line. After crossbreeding with a PrP promoter mouse line, double transgenic mice developed a progressive neurological phenotype characterized by neuronal dysfunction in the cerebellum, reduced anxiety, hyperactivity, impaired Rotarod performance and lower body weight gain. When ataxin-3 expression was turned off in symptomatic mice in an early disease state, the transgenic mice were indistinguishable from negative controls after 5 months of treatment. These results show that reducing the production of pathogenic ataxin-3 indeed may be a promising approach to treat SCA3, provided that such treatment is applied before irreversible damage has taken place and that it is continued for a sufficiently long time.

Expression quantitative trait loci detected in cell lines are often present in primary tissues

Bullaughey, K., Chavarria, C. I., Coop, G., Gilad, Y. — Fri, 23 Oct 2009 09:16:41 PDT

Expression quantitative trait loci (eQTL) mapping is a powerful tool for identifying genetic regulatory variation. However, at present, most eQTLs in humans were identified using gene expression data from cell lines, and it remains unknown whether these eQTLs also have a regulatory function in other expression contexts, such as human primary tissues. Here we investigate this question using a targeted strategy. Specifically, we selected a subset of large-effect eQTLs identified in the HapMap lymphoblastoid cell lines, and examined the association of these eQTLs with gene expression levels across individuals in five human primary tissues (heart, kidney, liver, lung and testes). We show that genotypes at the eQTLs we selected are often predictive of variation in gene expression levels in one or more of the five primary tissues. The genotype effects in the primary tissues are consistently in the same direction as the effects inferred in the cell lines. Additionally, a number of the eQTLs we tested are found in more than one of the tissues. Our results indicate that functional studies in cell lines may uncover a substantial amount of genetic variation that affects gene expression levels in human primary tissues.

Stra13 regulates oxidative stress mediated skeletal muscle degeneration

Fri, 23 Oct 2009 09:16:41 PDT

Duchenne Muscular Dystrophy (DMD), caused by loss of dystrophin is characterized by progressive muscle cell necrosis. However, the mechanisms leading to muscle degeneration in DMD are poorly understood. Here, we demonstrate that Stra13 protects muscle cells from oxidative damage, and its absence leads to muscle necrosis in response to injury in Stra13-deficient mice. Interestingly, Stra13–/– mutants express elevated levels of TNF, reduced levels of heme-oxygenase-1, and display apparent signs of oxidative stress prior to muscle death. Moreover, Stra13–/– muscle cells exhibit an increased sensitivity to pro-oxidants, and conversely, Stra13 overexpression provides resistance to oxidative damage. Consistently, treatment with anti-oxidant N-acetylcysteine ameliorates muscle necrosis in Stra13–/– mice. We also demonstrate that Stra13 expression is elevated in muscles from dystrophin-deficient (mdx) mice, and mdx/Stra13–/– double mutants exhibit an early onset of muscle degeneration. Our studies underscore the importance of oxidative stress-mediated muscle degeneration in muscular dystrophy, and reveal the contribution of Stra13 in maintenance of muscle integrity.

Parkin selectively alters the intrinsic threshold for mitochondrial cytochrome c release

Fri, 23 Oct 2009 09:16:41 PDT

Autosomal-recessive mutations in the Parkin gene are the second most common cause of familial Parkinson's disease (PD). Parkin deficiency leads to the premature demise of the catecholaminergic neurons of the ventral midbrain in familial PD. Thus, a better understanding of parkin function may elucidate molecular aspects of their selective vulnerability in idiopathic PD. Numerous lines of evidence suggest a mitochondrial function for parkin and a protective effect of ectopic parkin expression. Since mitochondria play a critical role in cell survival/cell death through regulated cytochrome c release and control of apoptosis, we sought direct evidence of parkin function in this pathway. Mitochondria were isolated from cells expressing either excess levels of human parkin or shRNA directed against endogenous parkin and then treated with peptides corresponding to the active Bcl-2 homology 3 (BH3) domains of pro-apoptotic proteins and the threshold for cytochrome c release was analyzed. Data obtained from both rodent and human neuroblastoma cell lines showed that the expression levels of parkin were inversely correlated with cytochrome c release. Parkin was found associated with isolated mitochondria, but its binding per se was not sufficient to inhibit cytochrome c release. In addition, pathogenic parkin mutants failed to influence cytochrome c release. Furthermore, PINK1 expression had no effect on cytochrome c release, suggesting a divergent function for this autosomal recessive PD-linked gene. In summary, these data demonstrate a specific autonomous effect of parkin on mitochondrial mechanisms governing cytochrome c release and apoptosis, which may be relevant to the selective vulnerability of certain neuronal populations in PD.

RPGRIP1 is essential for normal rod photoreceptor outer segment elaboration and morphogenesis

Fri, 23 Oct 2009 09:16:41 PDT

The function of the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1) gene is currently not known. However, mutations within the gene lead to Leber Congenital Amaurosis and autosomal recessive retinitis pigmentosa in human patients. In a previously described knockout mouse model of the long splice variant of Rpgrip1, herein referred to as Rpgrip1^tm1Tili mice, mislocalization of key outer segment proteins and dysmorphogenesis of outer segment discs preceded subsequent photoreceptor degeneration. In this report, we describe a new mouse model carrying a splice acceptor site mutation in Rpgrip1, herein referred to as Rpgrip1^nmf247 that is phenotypically distinct from Rpgrip1^tm1Tili mice. Photoreceptor degeneration in homozygous Rpgrip1^nmf247 mice is earlier in onset and more severe when compared with Rpgrip1^tm1Tili mice. Also, ultrastructural studies reveal that whereas Rpgrip1^nmf247 mutants have a normal structure and number of connecting cilia, unlike Rpgrip1^tm1Tili mice, they do not elaborate rod outer segments (OS). Therefore, in addition to its role in OS disc morphogenesis, RPGRIP1 is essential for rod OS formation. Our study indicates the absence of multiple Rpgrip1 isoforms in Rpgrip1^nmf247 mice, suggesting different isoforms may play different roles in photoreceptors and underscores the importance of considering splice variants when generating targeted null mutations.

Pluripotency can be rapidly and efficiently induced in human amniotic fluid-derived cells

Fri, 23 Oct 2009 09:16:41 PDT

Direct reprogramming of human somatic cells into pluripotency has broad implications in generating patient-specific induced pluripotent stem (iPS) cells for disease modeling and cellular replacement therapies. However, the low efficiency and safety issues associated with generation of human iPS cells have limited their usage in clinical settings. Cell types can significantly influence reprogramming efficiency and kinetics. To date, human iPS cells have been obtained only from a few cell types. Here, we report for the first time rapid and efficient generation of iPS cells from human amniotic fluid-derived cells (hAFDCs) via ectopic expression of four human factors: OCT4/SOX2/KLF4/C-MYC. Significantly, typical single iPS cell colonies can be picked up 6 days after viral infection with high efficiency. Eight iPS cell lines have been derived. They can be continuously propagated in vitro and express pluripotency markers such as AKP, OCT4, SOX2, SSEA4, TRA-1-60 and TRA-1-81, maintaining the normal karyotype. Transgenes are completely inactivated and the endogenous OCT4 promoter is adequately demethylated in the established iPS cell lines. Moreover, various cells and tissues from all three germ layers are found in embryoid bodies and teratomas, respectively. In addition, microarray analysis demonstrates a high correlation coefficient between hAFDC-iPS cells and human embryonic stem cells, but a low correlation coefficient between hAFDCs and hAFDC-iPS cells. Taken together, these data identify an ideal human somatic cell resource for rapid and efficient generation of iPS cells, allowing us to establish human iPS cells using more advanced approaches and possibly to establish disease- or patient-specific iPS cells.

Deficiency in COG5 causes a moderate form of congenital disorders of glycosylation

Fri, 23 Oct 2009 09:16:41 PDT

The conserved oligomeric Golgi (COG) complex is a tethering factor composed of eight subunits that is involved in the retrograde transport of intra-Golgi components. Deficient biosynthesis of COG subunits leads to alterations of protein trafficking along the secretory pathway and thereby to severe diseases in humans. Since the COG complex affects the localization of several Golgi glycosyltransferase enzymes, COG deficiency also leads to defective protein glycosylation, thereby explaining the classification of COG deficiencies as forms of congenital disorders of glycosylation (CDG). To date, mutations in COG1, COG4, COG7 and COG8 genes have been associated with diseases, which range from severe multi-organ disorders to moderate forms of neurological impairment. In the present study, we describe a new type of COG deficiency related to a splicing mutation in the COG5 gene. Sequence analysis in the patient identified a homozygous intronic substitution (c.1669-15T>C) leading to exon skipping and severely reduced expression of the COG5 protein. This defect was associated with a mild psychomotor retardation with delayed motor and language development. Analysis of different serum glycoproteins revealed a CDG phenotype with typical undersialylation of N- and O-glycans. Retrograde Golgi-to-endoplasmic reticulum trafficking was markedly delayed in the patient's fibroblast upon brefeldin-A treatment, which is a hallmark of COG deficiency. This trafficking delay could be restored to normal values by expressing a wild-type COG5 cDNA in the patient cells. This case demonstrates that COG deficiency and thereby CDG must be taken into consideration even in children presenting mild neurological impairments.

ALX4 dysfunction disrupts craniofacial and epidermal development

Fri, 23 Oct 2009 09:16:41 PDT

Genetic control of craniofacial morphogenesis requires a complex interaction of numerous genes encoding factors essential for patterning and differentiation. We present two Turkish families with a new autosomal recessive frontofacial dysostosis syndrome characterized by total alopecia, a large skull defect, coronal craniosynostosis, hypertelorism, severely depressed nasal bridge and ridge, bifid nasal tip, hypogonadism, callosal body agenesis and mental retardation. Using homozygosity mapping, we mapped the entity to chromosome 11p11.2–q12.3 and subsequently identified a homozygous c.793C->T nonsense mutation in the human ortholog of the mouse aristaless-like homeobox 4 (ALX4) gene. This mutation is predicted to result in a premature stop codon (p.R265X) of ALX4 truncating 146 amino acids of the protein including a part of the highly conserved homeodomain and the C-terminal paired tail domain. Although the RNA is stable and not degraded by nonsense-mediated RNA decay, the mutant protein is likely to be non-functional. In a skin biopsy of an affected individual, we observed a hypomorphic interfollicular epidermis with reduced suprabasal layers associated with impaired interfollicular epidermal differentiation. Hair follicle-like structures were present but showed altered differentiation. Our data indicate that ALX4 plays a critical role both in craniofacial development as in skin and hair follicle development in human.

Mice defective in Trpm6 show embryonic mortality and neural tube defects

Fri, 23 Oct 2009 09:16:41 PDT

The syndrome of hypomagnesemia with secondary hypocalcemia is caused by defective TRPM6. This protein is an ion channel that also contains a kinase in its C-terminus. It is usually diagnosed in childhood and, without treatment with supplemental Mg, affected children suffer from mental retardation, seizures and retarded development. We developed a mouse lacking Trpm6 in order to understand in greater detail the function of this protein. In contrast to our expectations, Trpm6^–/– mice almost never survived to weaning. Many mice died by embryonic day 12.5. Most that survived to term had neural tube defects consisting of both exencephaly and spina bifida occulta, an unusual combination. Feeding dams a high Mg diet marginally improved offspring survival to weaning. The few Trpm6^–/– mice that survived were fertile but matings between Trpm6^–/– mice produced no viable pregnancies. Trpm6^+/– mice had normal electrolytes except for modestly low plasma [Mg]. In addition, some Trpm6^+/– mice died prematurely. Absence of Trpm6 produces an apparently different phenotype in mice than in humans. The presence of neural tube defects identifies a previously unsuspected role of Trpm6 in effecting neural tube closure. This genetic defect produces one of very few mouse models of spina bifida occulta. These results point to a critical role of Trpm6 in development and suggest an important role in neural tube closure.

Transplantation directs oocyte maturation from embryonic stem cells and provides a therapeutic strategy for female infertility

Fri, 23 Oct 2009 09:16:41 PDT

Ten to 15% of couples are infertile, with the most common causes being linked to the production of few or no oocytes or sperm. Yet, our understanding of human germ cell development is poor, at least in part due to the inaccessibility of early stages to genetic and developmental studies. Embryonic stem cells (ESCs) provide an in vitro system to study oocyte development and potentially treat female infertility. However, most studies of ESC differentiation to oocytes have not documented fundamental properties of endogenous development, making it difficult to determine the physiologic relevance of differentiated germ cells. Here, we sought to establish fundamental parameters of oocyte development during ESC differentiation to explore suitability for basic developmental genetic applications using the mouse as a model prior to translating to the human system. We demonstrate a timeline of definitive germ cell differentiation from ESCs in vitro that initially parallels endogenous oocyte development in vivo by single-cell expression profiling and analysis of functional milestones including responsiveness to defined maturation media, shared genetic requirement of Dazl, and entry into meiosis. However, ESC-derived oocyte maturation ultimately fails in vitro. To overcome this obstacle, we transplant ESC-derived oocytes into an ovarian niche to direct their functional maturation and, thereby, present rigorous evidence of oocyte physiologic relevance and a potential therapeutic strategy for infertility.

Leucine-rich repeat kinase 2 interacts with Parkin, DJ-1 and PINK-1 in a Drosophila melanogaster model of Parkinson's disease

Fri, 23 Oct 2009 09:16:41 PDT

Mutations in the LRRK2 gene are the most common genetic cause of familial Parkinson's disease (PD). However, its physiological and pathological functions are unknown. Therefore, we generated several independent Drosophila lines carrying WT or mutant human LRRK2 (mutations in kinase, COR or LRR domains, resp.). Ectopic expression of WT or mutant LRRK2 in dopaminergic neurons caused their significant loss accompanied by complex age-dependent changes in locomotor activity. Overall, the ubiquitous expression of LRRK2 increased lifespan and fertility of the flies. However, these flies were more sensitive to rotenone. LRRK2 expression in the eye exacerbated retinal degeneration. Importantly, in double transgenic flies, various indices of the eye and dopaminergic survival were modified in a complex fashion by a concomitant expression of PINK1, DJ-1 or Parkin. This evidence suggests a genetic interaction between these PD-relevant genes.

A fusion peptide directs enhanced systemic dystrophin exon skipping and functional restoration in dystrophin-deficient mdx mice

Fri, 23 Oct 2009 09:16:41 PDT

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the synthesis of dystrophin protein. Antisense oligonucleotides (AOs) targeted to trigger excision of an exon bearing a mutant premature stop codon in the DMD transcript have been shown to skip the mutated exon and partially restore functional dystrophin protein in dystrophin-deficient mdx mice. To fully exploit the therapeutic potential of this method requires highly efficient systemic AO delivery to multiple muscle groups, to modify the disease process and restore muscle function. While systemic delivery of naked AOs in DMD animal models requires high doses and is of relatively poor efficiency, we and others have recently shown that short arginine-rich peptide-AO conjugates can dramatically improve in vivo DMD splice correction. Here we report for the first time that a chimeric fusion peptide (B-MSP-PMO) consisting of a muscle-targeting heptapeptide (MSP) fused to an arginine-rich cell-penetrating peptide (B-peptide) and conjugated to a morpholino oligomer (PMO) AO directs highly efficient systemic dystrophin splice correction in mdx mice. With very low systemic doses, we demonstrate that B-MSP-PMO restores high-level, uniform dystrophin protein expression in multiple peripheral muscle groups, yielding functional correction and improvement of the mdx dystrophic phenotype. Our data demonstrate proof-of-concept for this chimeric peptide approach in DMD splice correction therapy and is likely to have broad application.

Identification of brain transcriptional variation reproduced in peripheral blood: an approach for mapping brain expression traits

Fri, 23 Oct 2009 09:16:41 PDT

Genome-wide gene expression studies may provide substantial insight into gene activities and biological pathways differing between tissues and individuals. We investigated such gene expression variation by analyzing expression profiles in brain tissues derived from eight different brain regions and from blood in 12 monkeys from a biomedically important non-human primate model, the vervet (Chlorocebus aethiops sabaeus). We characterized brain regional differences in gene expression, focusing on transcripts for which inter-individual variation of expression in brain correlates well with variation in blood from the same individuals. Using stringent criteria, we identified 29 transcripts whose expression is measurable, stable, replicable, variable between individuals, relevant to brain function and heritable. Polymorphisms identified in probe regions could, in a minority of transcripts, confound the interpretation of the observed inter-individual variation. The high heritability of levels of these transcripts in a large vervet pedigree validated our approach of focusing on transcripts that showed higher inter-individual compared with intra-individual variation. These selected transcripts are candidate expression Quantitative Trait Loci, differentially regulating transcript levels in the brain among individuals. Given the high degree of conservation of tissue expression profiles between vervets and humans, our findings may facilitate the understanding of regional and individual transcriptional variation and its genetic mechanisms in humans. The approach employed here—utilizing higher quality tissue and more precise dissection of brain regions than is usually possible in humans—may therefore provide a powerful means to investigate variation in gene expression relevant to complex brain related traits, including human neuropsychiatric diseases.

Loss of Tsc1, but not Pten, in renal tubular cells causes polycystic kidney disease by activating mTORC1

Zhou, J., Brugarolas, J., Parada, L. F. — Fri, 23 Oct 2009 09:16:41 PDT

Tuberous sclerosis complex (TSC) is a genetic disorder linked to mutations of either the TSC1 or TSC2 gene, which encode proteins that form a complex to negatively regulate mammalian target of rapamycin complex 1 (mTORC1). Clinically, a small percentage of TSC patients develop severe infantile polycystic kidney disease (PKD), which is believed to be caused by deletion of the contiguous TSC2 and PKD1 genes on human chromosome 16. Recent studies have implicated the TSC/mTORC1 signaling pathway in PKD, but how dysfunction of the TSC/mTORC1 pathway induces PKD is not clear. We report a PKD mouse model created by knocking out Tsc1 in a subset of renal tubular cells. Extensive renal cyst formation in these mice is accompanied by broadly elevated mTORC1 activity in both cell autonomous and non-cell autonomous compartments. Furthermore, cyst development requires mTORC1 activation, as low dosage of rapamycin administration effectively blocks cyst formation. Interestingly, disruption of Pten, an upstream regulator of TSC1/TSC2, in the same cells, does not lead to PKD seemingly due to limited activation of mTORC1, suggesting that PTEN may not be a major upstream regulator of TSC/mTORC1 during early postnatal kidney development.

Common variants in LSP1, 2q35 and 8q24 and breast cancer risk for BRCA1 and BRCA2 mutation carriers

Fri, 23 Oct 2009 09:16:41 PDT

Genome-wide association studies of breast cancer have identified multiple single nucleotide polymorphisms (SNPs) that are associated with increased breast cancer risks in the general population. In a previous study, we demonstrated that the minor alleles at three of these SNPs, in FGFR2, TNRC9 and MAP3K1, also confer increased risks of breast cancer for BRCA1 or BRCA2 mutation carriers. Three additional SNPs rs3817198 at LSP1, rs13387042 at 2q35 and rs13281615 at 8q24 have since been reported to be associated with breast cancer in the general population, and in this study we evaluated their association with breast cancer risk in 9442 BRCA1 and 5665 BRCA2 mutation carriers from 33 study centres. The minor allele of rs3817198 was associated with increased breast cancer risk only for BRCA2 mutation carriers [hazard ratio (HR) = 1.16, 95% CI: 1.07–1.25, P-trend = 2.8 x 10^–4]. The best fit for the association of SNP rs13387042 at 2q35 with breast cancer risk was a dominant model for both BRCA1 and BRCA2 mutation carriers (BRCA1: HR = 1.14, 95% CI: 1.04–1.25, P = 0.0047; BRCA2: HR = 1.18 95% CI: 1.04–1.33, P = 0.0079). SNP rs13281615 at 8q24 was not associated with breast cancer for either BRCA1 or BRCA2 mutation carriers, but the estimated association for BRCA2 mutation carriers (per-allele HR = 1.06, 95% CI: 0.98–1.14) was consistent with odds ratio estimates derived from population-based case–control studies. The LSP1 and 2q35 SNPs appear to interact multiplicatively on breast cancer risk for BRCA2 mutation carriers. There was no evidence that the associations vary by mutation type depending on whether the mutated protein is predicted to be stable or not.

A non-synonymous variant in ADH1B is strongly associated with prenatal alcohol use in a European sample of pregnant women

Fri, 23 Oct 2009 09:16:41 PDT

Pregnant women are advised to abstain from alcohol despite insufficient evidence on the fetal consequences of moderate prenatal alcohol use. Mendelian randomization could help distinguish causal effects from artifacts due to residual confounding and measurement errors; however, polymorphisms reliably associated with alcohol phenotypes are needed. We aimed to test whether alcohol dehydrogenase (ADH) gene variants were associated with alcohol use before and during pregnancy. Ten variants in four ADH genes were genotyped in women from South-West England. Phenotypes of interest were quantity and patterns of alcohol consumption before and during pregnancy, including quitting alcohol following pregnancy recognition. We tested single-locus associations between genotypes and phenotypes with regression models. We used Bayesian models (multi-locus) to take account of linkage disequilibrium and reanalyzed the data with further exclusions following two conservative definitions of ‘white ethnicity’ based on the woman's reported parental ethnicity or a set of ancestry-informative genetic markers. Single-locus analyses on 7410 women of white/European background showed strong associations for rs1229984 (ADH1B). Rare allele carriers consumed less alcohol before pregnancy [odds ratio (OR) = 0.69; 95% confidence interval (CI): 0.56–0.86, P = 0.001], were less likely to have ‘binged’ during pregnancy (OR = 0.55, 95% CI: 0.38–0.78, P = 0.0009), and more likely to have abstained in the first trimester of gestation (adjusted OR = 1.42, 95% CI: 1.12–1.80, P = 0.004). Multi-locus models confirmed these results. Sensitivity analyses did not suggest the presence of residual population stratification. We confirmed the established association of rs1229984 with reduced alcohol consumption over the life-course, contributing new evidence of an effect before and during pregnancy.