Skip Navigation


Human Molecular Genetics Advance Access originally published online on June 25, 2007
Human Molecular Genetics 2007 16(17):2040-2052; doi:10.1093/hmg/ddm152
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrowOA All Versions of this Article:
16/17/2040    most recent
ddm152v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Google Scholar
Right arrow Articles by Besson, V.
Right arrow Articles by Hérault, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Besson, V.
Right arrow Articles by Hérault, Y.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

©2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Modeling the monosomy for the telomeric part of human chromosome 21 reveals haploinsufficient genes modulating the inflammatory and airway responses

Vanessa Besson1, Véronique Brault1, Arnaud Duchon1, Dieudonné Togbe1, Jean-Charles Bizot2, Valérie F.J. Quesniaux1, Bernard Ryffel1 and Yann Hérault1,*

1 Institut de Transgenose, Molecular Immunology and Embryology, UMR6218, CNRS, Université Orléans, 3B rue de la Férollerie, Orléans, Cedex 2 45071, France and 2 Key-Obs SA, 45150 Orléans, France

* To whom correspondence should be addressed. Tel/Fax: +33 238257930; Email: herault{at}cnrs-orleans.fr

Received May 11, 2007; Accepted June 14, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Monosomy 21 is a rare human disease due to gene dosage errors disturbing a variety of physiological and morphological systems including brain, skeletal, immune and respiratory functions. Most of the human condition corresponds to partial or mosaic monosomy suggesting that Monosomy 21 may be lethal. In order to search for dosage-sensitive genes involved in the human pathology, we generated by chromosomal engineering a monosomic mouse for the Prmt2–Col6a1 interval corresponding to the most telomeric part of human chromosome 21. Haploinsufficiency of the 13 genes, located in the 0.5 Mb genetic interval and conserved in man and mouse, caused apparently no morphological defect as observed in patients. However, monosomic mice displayed an enhanced inflammatory response after local intranasal lipopolysaccharide administration with enhanced recruitment of neutrophils and secretion of cytokines such as tumor necrosis factor-{alpha} (TNF-{alpha}), IL-1ß, IL-12p70 and IFN-{gamma} in the lung as well increased TNF-{alpha} production after systemic administration. Further analysis demonstrates that monosomic macrophages were involved and that a few genes, Prmt2, Pcnt2, Mcm3ap and Lss located in the region were candidate for the inflammatory response. Altogether, these results demonstrate the existence of dosage-sensitive genes in the Prmt2–Col6a1 region that control the inflammation and the lung function. Furthermore, they point out that similar partial Monosomies 21 in human might have eluded the diagnosis due to the very specific defects observed in this murine model.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Human chromosome 21 is often associated with the aneuploidy named Down syndrome or Trisomy 21. Although very rare, the Monosomy 21 is the second form of aneuploidy linked to human chromosome 21 (HSA21 for Homo sapiens chromosome 21). This disease is associated with several phenotypes, including mental retardation, facial dysmorphism, cardiac anomalies, microcephaly and respiratory complications (1–4). The study of Monosomy 21 is undeniably relevant and complementary to the analysis of Trisomy. Indeed locating regions of chromosome 21 and/or pathways that are affected by variation of gene dosage will definitely help to understand the physiopathology of the Down syndrome but also the consequence of more subtle variations in the expression or the function of genes from the HSA21.

Complete Monosomy 21 is almost not compatible with life and mostly partial cases have been described in the literature (1–8). Thus, complementary to human genetic approaches, mouse models have been developed to identify the dosage-sensitive genes from the HSA21 that are involved in induction of the features associated with Monosomy 21 (9–11). In the mouse genome, homologous regions to HSA21 are mapped on three distinct mouse chromosomes. From 21cen to 21qter, about 23.2 Mb are homologous to mouse chromosome 16 (MMU for Mus musculus), 1.1 Mb to MMU17 and 2.3 Mb to MMU10. Two monosomic mouse models carrying deletions of the MMU16 have been isolated that display several deficits (9–11). However, the contribution of additional regions, homologous to HSA21, to the monosomy remains unclear. Thus, extra monosomic models are crucial to evaluate the contribution of conserved genes to the patient phenotypes and to unravel dosage-sensitive genes.

To this aim, we focused our attention to the distal part of HSA21 in between the COL6A1 and PRMT2 genes that is homologous to a region located on MMU10. The region extends through 0.5 Mb and contains 13 genes, among which nine have known functions. First, the protein arginine methyltransferase 2 (PRMT2) is known to act as a negative regulator of the NF-{kappa}B pathway and could participate to the regulation of chromatin (12,13). S100b encodes a calcium-binding protein involved in various biological processes including brain function, inflammation and p53 trafficking (14–16). Pericentrin (PCNT), a major component of the centriole, is expressed during development and is located at the basis of the primary cilia (17,18). MCM3-associated protein, MCM3AP, could interact with the glucocorticoid receptor (19) and is implicated in the proliferation of B lymphocytes in germinal cell center in T cell-dependent antigen response or during lymphomagenesis (20–22); Col6a1 and Col6a2 are members of the collagen family and are associated with Bethlem myopathy and Ullrich congenital muscular dystrophy (23–26). Lanosterol synthase (Lss) mutations have been found in the Shumiya cataract rats where it should control cholesterol levels in cataractous lenses and thus indirectly the proliferation of lens epithelial cells (27). Also linked to human diseases, FTCD (for Formiminotransferase cyclodeaminase) plays a role in type 2 autoimmune hepatitis (28) and glutamate formiminotransferase deficiency (29).

Using chromosomal engineering [for review see (30)] we created a new mouse model of Monosomy 21, Ms1Yah (Ms for Monosomy), by the controlled deletion of the Prmt2–Col6a1 region. Phenotypic analysis of the Ms1Yah mice revealed no gross morphological and behavioral abnormality. However, the monosomic mice showed an impaired airway response and an increased inflammatory response after local lipopolysaccharide (LPS) action. Further investigations implicated macrophages (M{Phi}) as an important cellular compartment for the increased production of pro-inflammatory cytokines. Expression profiling and candidate gene analysis pinpoint Prmt2, Pcnt2, Mcm3ap and Lss as dosage-sensitive genes, which likely participate to the defects observed in Ms1Yah mice. In addition, this new model is of relevance for the study of genes function in monosomy and open perspectives on the consequence of gene copy number variation on human health.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Generation of monosomic mice for the 0.5 Mb Prmt2–Col6a1 region
The deletion of the Prmt2–Col6a1 interval was generated in vivo by chromosomal engineering (30). Briefly, two vectors were selected from the 3'-Hprt and 5'-Hprt libraries (31) to introduce loxP sites in the same relative orientation once integrated in the genome at the Prmt2 and Col6a1 loci (Fig. 1A). The targeting experiments were carried out successively in HM-1 ES cells (32) and confirmed by various restriction enzymes and specific probes (Fig. 1). We derived a cis(Prmt2–Col6a1)tm1Yah/+ mouse line (cis, Fig. 1B) from the ES cells with the two integrated loxP sites in a cis configuration. By breeding these mice with the Tg(CMVCRE)1Pcn line, we recovered 73% of mice with the parental genotype and 27% with a mosaic profile, carrying the two alleles Prmt2tm1Yah and Col6a1tm1Yah plus the deletion Del(Prmt2–Col6a1)Yah out of 101 animals analyzed (Del, Fig. 1B). An additional cross of the mosaic animals with B6 mice was necessary to obtain six individuals out of 18 segregating the deletion and to establish the Del(Prmt2–Col6a1)Yah mouse line that was renamed Ms1Yah after confirmation by FISH analyses (Fig. 1C). We also observed a normal Mendelian segregation ratio, with 48% Ms1Yah animals out of 73 animals recovered from heterozygous outcrosses at the third round of backcross showing that the viability of the Ms1Yah monosomic individuals was not affected during the backcrossing procedure on the B6 genetic background. Intercrosses were also carried out in between monosomic mice but no nullisomic mice were recovered at birth out of 150 animals generated (73 adult animals and 77 embryos aged from 3.5 to 12.5 dpc). Thus, even if monosomic mice were viable, nullisomy of the corresponding Prmt2–Col6a1 interval corresponded to a lethal embryonic condition.


Figure 1
View larger version (32K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. Generating a 0.5 Mb deletion between the Col6a1 and Prmt2 loci. (A) The 0.5 Mb targeted region, defined by the Prmt2 and Col6a1 genes, contains 13 genes as shown from a capture derived from the UCSC genome browser (http://genome.ucsc.edu). The targeting vectors containing a loxP site (green arrow), a selectable antibioti c resistance gene (puro, neo) and part of the Hprt gene (3'- or 5'-Hprt, black arrows) were integrated successively in the Prmt2 locus (Prmt2tm1Yah) and in the 5'-part of the Col6a1 gene (Col6a1tm1Yah). (B) The Prmt2tm1Yah allele was checked by Southern analysis with probe A, showing an additional XbaI fragment of 7.5 kb compared with the wild-type allele (wt, 9.6 kb), whereas the Col6a1tm1Yah locus was confirmed using the BglII enzyme and a second probe B. Similarly, targeted alleles were detected using the BglII enzyme and probe C, located inside targeting vectors. After Cre-mediated recombination, only the Del(Prmt2–Col6a1)Yah deleted (Del) allele produced a restriction fragment of 10 kb in the same conditions. B, BglII; X, XbaI; puro, puromycin; neo, neomycin. (C) Interphase FISH analysis with BAC probes that map in the region of the deletion (red) and outside (green). The wild-type (2n) showed two red and two green adjacent signals, whereas nuclei from Ms1Yah $showed two red and only one green signal due to the deletion of the Prmt2–Col6a1 region.

 
Loss of one copy of the Prmt2–Col6a1 region does not affect the general morphology, behavior and hematology
The phenotype of the monosomic model was analyzed for a set of parameters of morphology, behavior and hematology. Ms1Yah mice were viable, fertile and did not show overt phenotype upon observation. Moreover, the weight and growth of monosomic mice (n = 10; 16.8 ± 1.2 g) were similar to euploid animals (n = 10; 17 ± 1.5 g) matched for the sex (five males and five females), the age (129 ± 5 dpp) and the genetic background B6129N3. Further, no physical or anatomical change of the organs was observed upon necropsy examination suggesting that the Ms1Yah mice were suffering no dysmorphology.

Monosomic and control littermates (n = 10) were subjected to a series of tests evaluating balance, muscular tonus, aggressiveness and motor function. No significant alteration was found in terms of negative geotaxis righting reflex (Fig. 2A), locomotor horizontal (running distance; Fig. 2B) and vertical (number of rears; Fig. 2C) activities in the open-field session and spontaneous alternation behavior in the Y-maze (data not shown). Ms1Yah mice had no balance defect, a similar locomotor capacity and no spatial working memory impairment, compared with control animals showing that the mice had no major behavioral defect.


Figure 2
View larger version (29K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. Phenotypic characterization of Ms1Yah mice. (A–C) Behavioral studies did not reveal any abnormality between the two groups. (A) Monosomic (n = 10) succeeded in the negative geotaxis test like control animals (n = 10). Moreover, no difference was noted regarding the running distance (B) and the number of rears (C) performed by monosomic (n = 10) and control littermate mice (n = 10) in the open-field session, suggesting no locomotor defect in Ms1Yah mice. (D) Pulmonary airway resistance in monosomic mice. Whereas an intranasal administration of the control saline (NaCl) solution did not affect the Penh in wild-type (2n, n = 3) and mutant mice (Ms1Yah, n = 3), the response to LPS (10 µg) was strongly attenuated in monosomic animals (Ms1Yah LPS, n = 5) compared with control littermates (2n LPS, n = 6) treated in the same conditions.

 
The hematological analysis revealed only two slight differences between monosomic and control groups affecting the hematocrit and the red cell distribution (Fisher-Student's t-test; P < 0.05), however, the biological significance is not clear (Supplementary Material, Table S1). Taken together, we conclude that the aneuploidy of the Pmrt2–Col6a1 region did not induce dramatic consequence on morphology, behavior and blood homeostasis.

Changes in lung function and inflammatory response of monosomic mice towards lipopolysaccharide-induced distress
We then investigated the pulmonary and inflammatory responses after an LPS challenge of the airways. Intranasal endotoxin exposure of the airways leads to a complex response of the respiratory function, local inflammatory response, but also a vascular response that are not fully understood. To monitor lung function, we used the parameter of enhanced pause (Penh) that reflects the resistance of the airways. In addition, we followed the recruitment of inflammatory cells and secretion of cytokines and chemokines to trigger the inflammatory response mediated through the innate immune system by the TLR4 and the MyD88/NF-{kappa}B signaling pathway (33,34). First, we studied the influence of LPS on the pulmonary function of the monosomic mice. As shown in Figure 2E, the instillation of saline solution did not affect respiratory function as assessed by Penh in wild-type and mutant mice, whereas an intranasal administration of LPS (10 µg) induced a respiratory response characterized by an increase of the Penh values within 90–120 min, which lasted 3–4 h and decreased slowly, in the control mice. Surprisingly, this response was reduced in Ms1Yah mice when compared with wild-type control (Fig. 2E), showing that monosomic mice are less sensitive to the functional airway distress induced by LPS administration than euploid animals.

We then analyzed the innate immune response in terms of inflammatory cell recruitment and of cytokines production 24 h after LPS application, in the bronchoalveolar lavage fluids (BALF) from monosomic and control individuals. First we found that neutrophil counts were higher in the BALF of mutant animals in response to LPS (Fig. 3A) when compared with wild-type. Similarly, the MPO activity (Fig. 3B), an enzyme specifically expressed in neutrophils, was enhanced in lungs of mutants when compared with control littermates (Fig. 3B). Taken together, Ms1Yah mice show an enhanced recruitment of inflammatory cells, both in the lungs and in the alveolar space in comparison with euploid mice. Further, several cytokines such as tumor necrosis factor-{alpha} (TNF-{alpha}), IL-1ß, IL-6, IL-12p70, IFN-{gamma}, IL-10 and chemokines such as CXCL1 and CCL2, were measured in the BALF 24 h after LPS treatment. LPS significantly increased the concentrations of proinflammatory cytokines TNF-{alpha}, IL-1ß, IL-6, IL-12p70 and IFN-{gamma} in the BALF of monosomic mice when compared with control mice, whereas there was only trend for increased secretion of IL-10, CXCL1 and CCL2 (Fig. 3C). Therefore, consistent with the augmented cell recruitment, TNF-{alpha}, IL-1ß, IL-6, IL-12p70 and IFN-{gamma} production was increased in the monosomic mice treated with LPS.


Figure 3
View larger version (37K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3. Characterization of the inflammatory response induced by LPS in lungs of monosomic mice. (A) The recruitment of neutrophils (open bars), was significantly enhanced in the BALF of mutant individuals (Ms1 LPS, n = 5) versus wild-type (2n LPS, n = 6; Student's t-test, **P < 0.01) at 24 h in response to LPS. (B–D). The monosomic and the 2n control groups are represented, respectively, by closed and open bars. (B) After the LPS stimulation, a higher activity of the myeloperoxydase (MPO, A460 nm) enzyme was observed in the lung of monosomic (n = 5) than in those of the control group (n = 6; Student's t-test, **P < 0.01), whereas no difference appeared following administration of a saline solution (NaCl; n = 3 in each group). (C) Level of cytokines and chemokines was measured in the BALF of the Ms1Yah and euploid mice stimulated or not by the LPS (n = 5 and n = 3 for both groups, respectively, at 24 h). In response to LPS, monosomic mice showed an enhanced concentration of TNF-{alpha} (Student's t-test, **P < 0.01), IL1ß (**P < 0.01), IL6 (*P < 0.05), IL12p70 (*P < 0.05) and IFN{gamma} (*P < 0.05) compared with the one detected in euploid individuals. However, the concentrations of IL10, CXCL1 and CCL2 appeared significantly unchanged between groups. (D) In response to 100 µg of LPS administered intraperitoneally, the concentration of TNF{alpha} at 90 min was significantly increased in the serum of mutant (n = 6) versus euploid mice (n = 6; Student's t-test, **P < 0.05). The monosomic and wild-type animals, treated with PBS, did not reveal any difference (n = 3 in both the groups). All the results are expressed by mean ± SEM.

 
To test whether an LPS-induced endotoxic shock could lead to a general increase of inflammatory response in Ms1Yah mice, an intraperitoneal injection of 100 µg LPS was carried out in control and mutant animals. As for the local response in the airway, systemic LPS administration led to increased TNF-{alpha} concentration in the serum of monosomic animals that was higher when compared with control littermate mice (Fig. 3D). Hence, the enhanced secretion of proinflammatory cytokines, such as TNF-{alpha}, in response to LPS, was not restricted to the lung, but was also observed after systemic endotoxin stimulation in Ms1Yah mice.

Lipopolysaccharide-induced tumor necrosis factor-{alpha} and nitrite secretions are increased in monosomic bone marrow-derived macrophages
Macrophages (M{Phi}) and dendritic cells (DC) play a key role in the host defense against microorganisms and in the response towards endotoxins. Therefore, we next asked whether they could contribute directly to the enhanced secretion of cytokines seen after LPS treatment of Ms1Yah mice. Bone marrow-derived M{Phi}s and DCs from monosomic Ms1Yah and control mice were compared for their response towards LPS by measuring the production of TNF-{alpha} and nitric oxide (NO) in vitro. We found no difference between the levels of TNF-{alpha} (Fig. 4A) and nitrites (Fig. 4B) secreted by monosomic and control DC, in response to LPS and to bacterial lipoprotein (BLP), agonists of the TLR4 and TLR2 pathway, respectively. In contrast, LPS-stimulated M{Phi} derived from Ms1Yah mice produced a slightly higher amount of TNF-{alpha} than those derived from control animals (Fig. 4C). In addition, the concentration of NO was significantly enhanced in monosomic M{Phi}, in response to LPS and to BLP (Fig. 4D). Thus, M{Phi}s appeared as one cellular compartment contributing to the enhanced inflammatory response towards LPS seen in the monosomic model.


Figure 4
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4. Production of TNF-{alpha} and nitrites in bone marrow-derived dendritic cells and macrophages of monosomic individuals. The monosomic group is represented by closed bars and the control littermates by open bars. No difference was observed for the secretion of TNF-{alpha} (A) and nitrites (NO; B) in the supernatant of dendritic cells cultured from Ms1Yah (n = 2) and euploid animals (n = 2), in response to LPS or to BLP at 24 h. On the contrary, in response to LPS, macrophages derived from mutant mice showed a secretion of TNF-{alpha} (C, n = 8) and nitrites (D, n = 6) significantly higher than controls (Student's t-test, *P < 0.05 and **P < 0.01, respectively). Moreover, in response to BLP, monosomic macrophages produced more of nitrites (D) than control macrophages (n = 6; Student's t-test, *P < 0.05). (E) After LPS stimulation, monosomic macrophages showed clearly a higher secretion of bioactive TNF-{alpha} than the one obtained from wild-type macrophages (n = 6; Student's t-test, **P < 0.01). The results are expressed by mean ± SEM.

 
Furthermore, we controlled the bioactivity of TNF-{alpha} secreted in the supernatants of monosomic M{Phi} in the WEHI cytotoxicity assay (35). The data confirmed our previous results and showed a markedly higher concentration of bioactive TNF-{alpha} secreted by monosomic M{Phi} than in controls stimulated by LPS (Fig. 4E). Therefore. at least one dosage-sensitive gene located in the Prmt2–Col6a1 genetic region seems to contribute to the regulation of proinflammatory cytokine production in M{Phi} stimulated by LPS.

Genes located in the Prmt2–Col6a1 region are differentially expressed in lungs and macrophages before and after lipopolysaccharide treatment
To investigate which of the 13 genes present in the deleted Prmt2–Col6a1 region could be responsible for the attenuated airway response and the enhanced inflammation of Ms1Yah mice to LPS treatment, we compared the expression levels of these genes between Ms1Yah and wild-type mice in both lungs and M{Phi}. In addition, gene expression profiles were also evaluated with and without LPS induction to check which gene expression was affected by the LPS treatment. Levels of gene expression were assessed in total RNA extracts by quantitative real-time PCR (QRT-PCR) and normalized using the GeNorm method (36). The relative expression level of the genes located in the deleted interval, or from the neighboring zone, or used as control for standardization is shown in Figure 5.


Figure 5
View larger version (47K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5. Comparison of monosomic/euploid ratios of expression obtained by quantitative real-time PCR (QRT-PCR) for the 13 genes of the Prmt2–Col6a1 region, two neighboring genes, Slc5a4a and Pcbp3 and housekeeping genes used for the normalization in lungs (A) and macrophages (B) with (LPS) and without (PBS) LPS induction. The monosomic group is represented by closed bars and the control littermates by open bars. Data are represented as mean (2–{delta}{delta}Ct) ± SEM and are representative of n = 4 (except for monosomic in M{Phi} where n = 6). All the tested samples were performed in triplicate and experiments were done twice. Asterisks indicate statistically significant differences between monosomic and wild-type controls (bars spanning the two wild-type and monosomic groups) or between LPS-treated and untreated samples (bar over the LPS-treated wild-type sample to compare wild-type LPS-treated to wild-type PBS controls (PBS); bar over the LPS-treated monosomic sample to compare monosomic LPS-treated to monosomic vehicle control treated). U test, *P < 0.05.

 
In lungs, no statistically relevant change was observed for both neighboring genes, Slc5a4a and Pcbp3 in control conditions (PBS) while expression levels of S100b, Dip2, Pcnt2, Q9D5N2 and Q9D0B9 varied significantly between Ms1Yah and euploid mice, as a consequence of gene dosage and independently of the LPS stimulation (Fig. 5; U test P < 0.05). No expression was detected for Lss, C21orf56 and Ftcd. In addition, three genes Prmt2, Mcm3ap and Col6a2 that were not much affected by gene dosage in control condition (PBS), were significantly decreased in Ms1Yah lungs after LPS stimulation [U test P = 0.051 (PBS) and 0.012 (LPS) for Prmt2; P = 0.061 (PBS) and 0.031 (LPS) for Mcm3ap; P = 0.061 (PBS) and 0.030 (LPS) for Col6a2]. Unexpectedly, C21Orf57 displayed a significant downregulation in monosomic compared with wild-type lungs in steady-state condition, but was upregulated after LPS treatment, abolishing the gene dosage effect. Thus, 10 genes of the Prmt2–Col6a1 region were expressed in lungs with various expression patterns. Five genes S100b, Dip2, Pcnt2, Q9D5N2 and Q9D0B9, were found to be dosage-sensitive in treated and non-treated lungs, while for the others, Prmt2, Mcm3ap, Col6a2 and C21orf57, the influence of genetic dosage on expression was only apparent after the LPS action.

A significant dosage effect was observed for Prmt2, Pcnt2, Mcm3ap and Lss in monosomic M{Phi} compared with wild-type, independent of LPS, while Dip2 and C21orf57 expression levels did not vary notably as well as the genes from the flanking regions. No transcript was detected for S100b, Q9D5N2, Q9D0B9, C21orf56, Ftcd, Col6a2 and Col6a1. In addition, Lss was strongly downregulated by LPS in wild-type M{Phi}. From the six genes of the region expressed in M{Phi}, four were dosage sensitive with either no change (Prmt2, Pcnt2, Mcm3ap) or an additional downregulation (Lss) after LPS treatment. Overall, the response to LPS either in lungs or in M{Phi} is complex. Several genes of the Prmt2–Col6a1 region behave as dosage sensitive in the lung or in M{Phi} and likely participate to the phenotypes observed in Ms1Yah mice.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
We report here the generation and the phenotypic analysis of the Ms1Yah mouse, a monosomic model for the 0.5 Mb Prmt2–Col6a1 region. This genetic interval is homologous to the most telomeric part of HSA21 and overlaps 13 genes located on the MMU10. The model was generated through in vivo chromosomal engineering using a mouse line, named cis(Prmt2tm1Yah;Col6a1tm1Yah)/+ carrying two loxP sites in a cis configuration, and a general CRE deleter strain, the Tg(CMVCRE)1pcn. From such a cross, only mosaic animals were recovered, but once crossed with wild-type mice, we obtained in the next generation 33% of individuals carrying the expected monosomy. Even though megabase deletions could be recovered by increasing the number of mice generated from such mosaic individuals (37,38), improvement of the in vivo strategy, such as new CRE deleter strains, should be tested in the future to induce more frequently large rearrangements in the mouse genome.

The detailed phenotypic analysis of the Ms1Yah monosomic model revealed no dramatic change in morphology, hematology and general behavior, even though more detailed analysis has to be done for craniofacial alteration. As a conclusion, the genes located in the Prmt2–Col6a1 region are not sufficient to induce major phenotypes associated with human Monosomy 21. Nevertheless, we cannot rule out the possibility that they could act in synergy with genes located in other region homologous to HSA21 that are known to induce severe alterations in human patients (1–3). Accordingly, deletions encompassing the Prmt2–Col6a1 region in human have been described without correlated phenotypic anomalies in a few complex cases of monosomy for the distal part of 21q22.3 with ring chromosome or additional rearrangements (5,6). Interestingly nullisomic animals were never observed after implantation. Thus, it seems likely that homozygosity for the deletion of at least one of the genes located in the region dramatically affects the early division of the embryos. Of note in this regard is Pcnt2 which encodes a major component of the centrosome and of the spindle pole body during mitosis.

The challenge of the respiratory function by LPS revealed a reduced response of monosomic Ms1Yah mice. In addition, the induced inflammatory response was stronger with the recruitment of more activated neutrophils and the enhanced secretion of proinflammatory signals. This confirms that the LPS-induced stress effect on the lung and the inflammatory responses may depend on different cell types in lungs (33,39). Investigation of the cellular mediator of the inflammatory response towards LPS in Ms1Yah mice pinpoints that the enhanced secretion of proinflammatory cytokines, such as TNF-{alpha}, was partly recapitulated in monosomic M{Phi}. Furthermore, systemic injection of LPS leads to a similar overproduction of TNF-{alpha} in the plasma. This is the first report showing a correlation between lung and inflammatory functions defect and the Prmt2–Col6a1 genetic interval.

Somehow at least one gene deleted in the monosomic mice, is a dosage-sensitive gene involved in the control of proinflammatory cytokines production stimulated by LPS in M{Phi}s and mediated by TLR4. In our QRT-PCR experiment analysis, such a dose effect on transcriptional level was detected for almost nine genes in lungs among the 10 expressed and five genes in M{Phi} out of the six transcribed. For those genes, we observed a two-fold decrease in monosomic mice compared with controls. We confirmed that whereas the expression level variation is only due to the Prmt2–Col6a1 deletion, since no change has been noticed for the two neighboring genes, Slc5a4a and Pcbp3 in both tissues studied. These results are consistent with data on gene dosage defect caused by different forms of aneuploidy: for example, monosomy results in a 0.5-fold expression of the gene dosage imbalance, whereas trisomy in a 1.5-fold increased (40–46). Considering the two analyzed tissues in monosomic mice, we found that the mRNA levels of genes, such as Dip2 and C21orf57, were no more sensitive to gene dosage in M{Phi}, whereas their expression was reduced in the lung. A similar tissue-dependent effect has already been described by Lyle et al. (2004) who found a significant overexpression of the Ets2 gene in the heart of Ts65Dn animals, whereas the same gene was not sensitive to the dose-effect in the liver of affected 30-day-old mice. Somehow the decrease in copy number of Dip2 and C21Orf57 was compensated in monosomic M{Phi}. Interestingly in the lungs, the major effect of the monosomy is a reduction of gene expression level suggesting that gene copy number has a major effect on expression even in a tissue with such a level of cellular complexity. Only C21orf57, which was sensitive to dose effect in lungs, was upregulated after LPS stimulation of monosomic and euploid mice. These results emphasize that dosage compensation is a tissue-dependent mechanism that might be restricted to a small set of genes (2 out of 13 in this study), specific cell type and could be partly modulated by the physiological conditions.

Compensatory effect is a general phenomenon for the X chromosome genes in several species such as invertebrates and vertebrates (47,48). Nevertheless, similar dosage compensations have been observed in aneuploid conditions. For example, the expression of the Igf2r imprinted gene is compensated in the segmental mouse Trisomy 17 (49), but also some genes found in Down syndrome mouse models behave similarly with the same expression level even if there are three copies of the gene (43,44). Whereas the expected values in transcriptional gene expression should be lesser than 0.5-fold in the case of monosomy, we and others (43,44) have obtained several evidences proving that a change in copy number does not always result in a lower or higher expression of the affected genes. Thus, the dosage effect on gene expression is undoubtedly dependent on the tissue, the cell type analyzed and the physiological conditions.

The attenuated airway and the enhanced immune responses of Ms1Yah mice to LPS treatment is a consequence of the dosage imbalance due to genes deleted in the Prmt2–Col6a1 region. Among the haploid genes of the monosomic mice, almost 10 genes out of 13 appeared as dosage-sensitive genes in the lungs, whereas only Pcnt2, Mcm3ap, Lss and Prmt2 were dosage-sensitive in M{Phi}. Nothing is known about the function of Q9D5N2, Dip2, C21Orf57 and Q9D0B9, whereas Lss mutation induces embryonic lethality and/or cataract in the Shumiya rat model (27) and Mcm3ap is involved in the maintenance and the generation of B cells in germinal centers (20). However, a series of reports shows that Pcnt2, S100b and Prmt2 are clearly involved in the lung function or the inflammatory response (see below).

Pcnt2 is located at the base of the motile cilia of epithelial cells as well as of the primary cilia of other cells (18). Thus, it could play a role in cell migration of leucocytes or in the motility of the cilia found at the apical membrane of the bronchiolar epithelial cells. In case of motile cilia dysfunction, such as in primary ciliary dyskinesia (PCD; OMIM242650), respiratory dysfunction, including bronchiectasis and abnormal mucociliary clearance are observed (50,51). Downregulation of Pcnt2 expression in lungs of Ms1Yah mice could lead to a defect in the motile cilia that would certainly alter the lung function. As a consequence a change in the mucociliary transport would modify the exposure time to LPS and thus the inflammatory response.

Interestingly, S100b is a member of a large family of calcium-binding proteins of the EF-hand type, involved in cell growth, contraction, motility, differentiation, transcription and secretory pathways (52). S100b protein members have a role in inflammation exerting regulatory effects on the secretion of pro-inflammatory signaling molecules such as IL-1ß and NO in glia cells (53–56) or peritoneal M{Phi}s (57). S100b, like HMGB1, is released from the cytoplasm of cells ongoing unscheduled death and belongs to the damage-associated molecular pattern (58). It binds to the receptor for advanced glycation end products (RAGE) that regulates sepsis (59) and is expressed in lung tissue (60) as S100b (61,62). Thus, the decreased level of S100b–RAGE interactions in Ms1Yah mice would likely contribute to their altered response towards LPS.

Prmt2 codes for the protein arginine N-methyltransferase 2, which is overexpressed in alveolar type 2 cells under hypoxic conditions (63), and known to inhibit the NF-{kappa}B activity by increasing nuclear I{kappa}B-{alpha} in mouse fibroblasts (12). Here, we showed that Prmt2 is expressed and dosage-sensitive in both LPS-treated lungs and M{Phi}. Thus, the decrease of such a NF-{kappa}B inhibitor in lungs or in M{Phi}s would participate to the enhanced production of cytokines under the control of the TLR4/NF-{kappa}B signaling pathway. Therefore, the phenotype of lung and inflammatory dysfunctions are probably due to the haploinsufficiency of several genes such as Pcnt2, S100b and Prmt2. Nevertheless, we suggest that downregulation of Prmt2 would contribute to an increased production of proinflammatory cytokines by downregulating an inhibitory control pathway of NF-{kappa}B.

The Ms1Yah line represents the third model of human Monosomy 21 after the Ms1Rhr that corresponds to a deletion of the Cbr1–Orf9 genes on MMU16 (10) and the Ms1Cje from Sod1 to the telomere of MMU16 (9). Contrary to the severe phenotypes described in the Ms1Rhr (11), the deletion of Prmt2–Col6a1 region results in a subtle phenotype. Somehow in human similar partial Monosomy 21 would lead to an increased sensibility to lung inflammation and potentially to asthma. Interestingly, a susceptibility loci for asthma has been located in the telomeric part of HSA21 in the hispanic population (64). Accordingly, large-scale deletions of genomic DNA, and more generally variation of gene copy number, notably in telomeric regions (65), not only contribute to common genetic variation in healthy individuals but would play a role in the genetics of complex traits, human syndromes and susceptibility to diseases.

The recent global genome-wide survey of copy number variation (CNV) (66) highlights the presence of at least 1500 variable regions among 270 HapMap samples that covers 12% of our genome and hundred of genes whose copy number varies dramatically among the human population. Similarly, CNV were found in different strains of mice. Hence, the greatest source of genetic variability in the human and mouse species might not depend only on single nucleotide polymorphisms (SNPs) but rather more on the variation of copy numbers (67–69). Lying within these copy number polymorphic regions are genes associated with the environmental response such as the chemosensation, drug detoxification, immunity and inflammation. Stranger et al. (70) showed that CNVs were associated with 17.7% of the total detected genetic variation in gene expression and thus might participate in complex phenotypes. Hence, deletions appear to be common variations in the human, but also and maybe more often associated with susceptibility to diseases and complex traits. On the opposite, such events might also contribute simply to polymorphic variation with no apparent phenotypic or only benign consequences, depending on whether or not dosage-sensitive genes are affected by the rearrangement. According to our results, the Prmt2–Col6a1 region is an example of such a variation that does not induce a drastic phenotype but subtle physiological consequences that might be unnoticed in the human population. Among those CNV already found in human, was identified CNP1359 which encompasses the Prmt2, S100b and the 5'-part of Dip2 genes (66). Thus the work presented here is of relevance to study the effect of the CNP1359 on human physiology. Consequently, it is of importance to pursue the effort of CNV surveys and of the use of mouse models to ascertain the phenotypic consequence of CNV.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Generating deletion and mouse strains
The targeting vectors for Prmt2 and Col6a1 were isolated, respectively, from the 3'-Hprt and 5'-Hprt libraries (31), with loxP sites in the same relative orientation and inserted by homologous recombination in HM-1 Hprt-deficient ES cells (32). ES cell clones carrying the two loxP sites in the Prmt2 and Col6a1 loci in a cis configuration were named cis(Prmt2-Col6a1)tm1Yah/+ and were injected into C57BL/6J (B6) blastocysts to generate chimera. These animals were crossed with B6 mice to obtain the corresponding mouse line. The monosomic mice, called the Ms1Yah, were generated in vivo by crossing the cis(Prmt2-Col6a1)tm1Yah/+ line with a general deleter strain Tg(CMVCRE)1Pcn (71). All the animals used in the following experiments were derived from the Ms1Yah line with at least three backcrosses on B6. They were bred under SPF conditions and were treated in compliance with animal welfare policies from the French Ministry of Agriculture (law 87 848 and YH accreditation 45–31).

Southern blot analysis
Mice and ES cells were genotyped by Southern blot analysis in standard conditions. Briefly, 10 µg of genomic tail or ES cell DNA extracts were digested with the appropriate enzyme and separated by electrophoresis through 0.8% agarose gel. The digested DNA was transferred to Hybond nylon membrane (GE Healthcare, Chalfont St Giles, UK) and hybridized with a specific DIG-labeling probe (Roche, Mannheim, Germany). Autoradiography was performed using Kodak Biomax XAR-Films (Kodak, Chalon-sur-Saone, France).

Fluorescence in situ hybridization (FISH)
Interphase nuclei were recovered by affixing of a frozen/defrosted sample of kidney on a slide. Mouse BAC clones were chosen to be located inside (RPCI24-445L22) or outside (RPCI24-247G13) the deletion (http://bacpac.chori.org/mmouse24.htm). One microgram of mouse BAC DNA was used to generate DNA probes labeled by nick translation with DIG-dUTP (for 445L22) and biotin-dUTP (for 247G13). Detection was achieved by the use of both antidigoxigenin-rhodamine and avidin-fluorescein antibodies (Roche, Mannheim, Germany). The slide was mounted with vectashield medium containing DAPI 4',6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). Images were analyzed by SmartCapture2F (Digital Scientific, Cambridge, UK) using a Zeiss AxioPlanII microscope equipped with a cooled camera (Photometrics, Tuscon, USA).

Behavioral analysis
Groups of age-, sex- (n = 10) and genetic background (B6129N3)-matched monosomic and wild-type littermate mice were subjected to a series of tests as described previously (72,73), following the recommendations from the standard operating procedures developed by the Eumorphia network (http://www.eumorphia.org). First, adult animals were scored for the negative geotaxis to evaluate their balance and their locomotor activity in an open-field session (Actitrack, Panlab, SL, Spain). During the 30 min test, the distance traveled (in meters) and the number of rears were recorded. Then, spatial working memory performance was assessed by recording spontaneous alternation behavior in a transparent Y-maze (74). Each animal was tested in a single 5-min session, in the course of which it was placed in the central platform and allowed free exploration of the maze. The series of arm entries were scored to define alternation as successive entries into the three arms, in overlapping triplet sets. The percent alternation was calculated as the ratio of actual to possible alternations (defined as the total number of arm entries – 2) x 100. All the results were expressed as mean ± SEM (standard error of the mean).

Hematological studies
Monosomic (eight males and six females) and control littermate mice (seven males and eight females) matched for the age (116 ± 2 dpp) were anesthetized by inhalation of isoflurane. Blood samples were collected into Microtainer tubes containing heparin solution that were slowly agitated to avoid coagulation. Two samples of 150 µl of blood were used to test twice 17 hematological parameters measured by Technicon H1E (Bayer).

Airways resistance, bronchoalveolar lavage fluids and histological analysis of lungs
Animals were treated by intranasal instillation with either isotonic saline solution or 10 µg of LPS (Escherichia coli, serotype O55B5, 10 µg, Sigma–Aldrich, St Louis, USA) in deep anesthesia. Airway response was investigated over a period of 6 h after treatment using whole-body plethysmography and measurements of the Penh, a dimensionless parameter that accounts for the respiratory profile, taken in consideration the period of expiration and the variations of pressure measured in a closed chamber during the respiratory cycle (EMKA Technologies, Paris, France). Twenty-four hours after stimulation, mice were euthanized and BALFs were analyzed for cell composition and cytokines quantification as described in (33,75). An aliquot was stained with Turk's solution and analyzed to determine cellular content. After centrifugation on microscopic slides, air-dried preparations were fixed and stained with Diff-Quick (Merz & Dade) using a May-Grünwald-Giemsa coloration. Two hundred cells were counted twice for the determination of the differential counts of each cell type in the BALF. Part of the lung was stored at –20°C for the myeloperoxidase (MPO) assay. After the BALF experiments, lungs were removed, fixed and processed for histological analysis.

Measurement of cytokines, nitrites and myeloperoxidase assay
Concentrations of cytokines in the sera of BALF were determined by the ELISA method, according to the manufacturer's instructions (Duoset, R&D Systems Inc., Minneapolis, USA) or using a SearchLight® Multiplex Assay (Perbio, Aalst, Belgium). Nitrites were dosed from supernatants by using the Griess method. Briefly, 50 µl of the supernatant was mixed with 25 µl of Griess1 (1% sulfamide in 2.5% phosphoric acid, Sigma–Aldrich) solution and 25 µl of Griess2 (0.1% N-dichloric naphthylenediamine in 2.5% phosphoric acid, Sigma–Aldrich). After 30 min at 37°C under agitation, the activity was quantified by absorbance at 540 nm. The MPO activity in the lung tissue was evaluated by absorbance at 460 nm as described (33).

Bone marrow-derived cell culture, stimulation and measurement of the bioactive tumor necrosis factor-{alpha}
Murine bone marrow cells were isolated from femurs and were differentiated into M{Phi} (76) and into myeloid dendritic cells [BMDC; (77)]. For the experiment, the cells were plated in 96-wells plates at 105 cells/well in medium containing 0.25% FCS, and stimulated with LPS (at 100 ng/ml; Sigma–Aldrich) and bacterial lipoprotein (BLP, Pam3CSK4 at 500 ng/ml). Supernatants were harvested after 24 h and analyzed directly for cytokines quantification. The M{Phi} experiment was performed with eight monosomic and control mice matched for sex (four males and four females in each group), age (95 ± 6 dpp) and genetic background. The BMDC experiment was done with two monosomic and two wild-type females aged 91 days and maintained on a B6129N5 genetic background. Evaluation of the bioactive TNF-{alpha} was assessed on M{Phi} supernatants, using WEHI 164-based bioassay (35). In brief, sample dilutions were incubated in 96-well cell culture plates with 104 WEHI 164 cells per well. After 48 h of incubation at 37°C and 5% CO2, the cell death was assessed by using the methylthiazoletetrazolium method. The absorbance was measured at 610 nm and the results were compared with a standard curve generated with murine TNF and expressed as picograms per milliliter of TNF.

Endotoxic shock induced by administration of lipopolysaccharide
Intraperitoneal injections of 100 µg LPS (E. coli, serotype O111:B4, Sigma–Aldrich) or saline control solution were used to evaluate the systemic inflammatory response in mice. Mice were euthanized 90 min after the injection. The blood was collected through the femoral vein and centrifuged at 3000g for 15 min. The serum was collected and stored at –20°C for TNF-{alpha} assay.

RNA extraction and quantitative RT-PCR analysis
Total RNA was extracted 24 h after LPS stimulation from the M{Phi} using the RNAeasyR mini-kit (Qiagen) and from the lung of mice using Trizol reagent (Invitrogen, Carlsbad, USA). Then, it was treated with the Turbo DNA free TM kit (Ambion, Austin, USA). cDNA synthesis was performed using the AbsoluteTM 2-step QRT-PCR SYBR Green Kit (ABgene, Epsom, UK). A series of primer pairs (supplemental data available upon request) were designed to span intron–exon junctions in order to determine the relative expression of the 13 genes encompassed in the deleted region plus one neighboring gene on each side, namely Slc5a4a and Pcbp3 (Fig. 1). Each primer pair was tested successfully with the efficiency ranging from 92 to 104%. The QPCR was performed with 15 ng of cDNA and 200 nM of each primer in a 15 µl final reaction in a Stratagene Mx4000 with a standard amplification procedure. In parallel, similar experiments were carried out for the seven following housekeeping genes, Actb, ß2m, Gadp, Pgk1, Rpl13a, Tbp, Tubb4, used for normalization through the Genorm procedure (36). Transcript levels were measured for each sample and normalized using the Genorm approach (36) in order to correct the variations of the amount of source RNA in the starting material.

Statistical analysis
Statistical analysis was performed using either the parametric Fischer Student's t-test when applicable or the non-parametric Wilcoxon Mann–Whitney's U test via the Statgraphics software (Centurion XV, Sigma plus, Levallois Perret). Values are presented as mean ± SEM and the significant threshold was P < 0.05 or otherwise indicated.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We thank members of the laboratory for helpful discussion, Candice Hackney, Elodie Desale and Karine Vallon for taking care of the animals, Alexandre Herpin for his expert technical assistance in the behavioral studies, S. Briault for his help with the FISH experiments and members of the Eumorphia consortium for their recommendations regarding the phenotype analysis (www.eumorphia.org). The Ms1Yah mutant mice are available for distribution through the EMMA network (‘European Mouse Mutant Archive’, www.emmanet.org). This work was funded by grants from the CNRS, the Fondation Jerome Lejeune and the AnEUploidy project (LSHG-CT-2006–037627) supported by the European commission under FP6. ‘Funding to pay the Open Access publication charges for this article was provided by the Aneuploidy project.’

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

  1. Yao G., Chen X.N., Flores-Sarnat L., Barlow G.M., Palka G., Moeschler J.B., McGillivray B., Morse R.P., Korenberg J.R. Deletion of chromosome 21 disturbs human brain morphogenesis. Genet. Med. (2006) 8:1–7.[ISI][Medline]

  2. Ehling D., Kennerknecht I., Junge A., Prager B., Exeler R., Behre B., Horst J., Schmitt-John T., Bartsch O., Wirth J. Mild phenotype in two unrelated patients with a partial deletion of 21q22.2-q22.3 defined by FISH and molecular studies. Am. J. Med. Genet. A (2004) 131:265–272.[Medline]

  3. Chettouh Z., Croquette M.F., Delobel B., Gilgenkrants S., Leonard C., Maunoury C., Prieur M., Rethore M.O., Sinet P.M., Chery M., et al. Molecular mapping of 21 features associated with partial Monosomy 21: involvement of the APP–SOD1 region. Am. J. Hum. Genet. (1995) 57:62–71.[ISI][Medline]

  4. Chen C.P., Lin S.P., Chern S.R., Lee C.C., Huang J.K., Wang W., Liao Y.W. De novo satellite 21q associated with corpus callosum dysgenesis, colpocephaly, a concealed penis, congenital heart defects, and developmental delay. Genet. Couns. (2004) 15:437–442.[ISI][Medline]

  5. Pangalos C., Theophile D., Sinet P.M., Marks A., Stamboulieh-Abazis D., Chettouh Z., Prieur M., Verellen C., Rethore M.O., Lejeune J., et al. No significant effect of monosomy for distal 21q22.3 on the Down syndrome phenotype in ‘mirror’ duplications of chromosome 21. Am. J. Hum. Genet. (1992) 51:1240–1250.[ISI][Medline]

  6. McGinniss M.J., Kazazian H.H. Jr, Stetten G., Petersen M.B., Boman H., Engel E., Greenberg F., Hertz J.M., Johnson A., Laca Z., et al. Mechanisms of ring chromosome formation in 11 cases of human ring chromosome 21. Am. J. Hum. Genet. (1992) 50:15–28.[ISI][Medline]

  7. Yao X.L., Jenkins E.C. Translocation 4p;21q identified by FISH in a case previously described as ‘presumptive Monosomy 21’. Am. J. Med. Genet. (1994) 52:491–492.[CrossRef][ISI][Medline]

  8. Riegel M., Hargreaves P., Baumer A., Guc-Scekic M., Ignjatovic M., Schinzel A. Unbalanced 18q/21q translocation in a patient previously reported as Monosomy 21. Eur. J. Med. Genet. (2005) 48:167–174.[CrossRef][ISI][Medline]

  9. Olson L.E., Roper R.J., Baxter L.L., Carlson E.J., Epstein C.J., Reeves R.H. Down syndrome mouse models Ts65Dn, Ts1Cje, and Ms1Cje/Ts65Dn exhibit variable severity of cerebellar phenotypes. Dev. Dyn. (2004) 230:581–589.[CrossRef][ISI][Medline]

  10. Olson L.E., Richtsmeier J.T., Leszl J., Reeves R.H. A chromosome 21 critical region does not cause specific Down syndrome phenotypes. Science (2004) 306:687–690.[Abstract/Free Full Text]

  11. Olson L.E., Roper R.J., Sengstaken C.L., Peterson E.A., Aquino V., Galdzicki Z., Siarey R., Pletnikov M., Moran T.H., Reeves R.H. Trisomy for the Down syndrome ‘critical region’ is necessary but not sufficient for brain phenotypes of trisomic mice. Hum. Mol. Genet. (2007) 16:774–782.[Abstract/Free Full Text]

  12. Ganesh L., Yoshimoto T., Moorthy N.C., Akahata W., Boehm M., Nabel E.G., Nabel G.J. Protein methyltransferase 2 inhibits NF-kappaB function and promotes apoptosis. Mol. Cell. Biol. (2006) 26:3864–3874.[Abstract/Free Full Text]

  13. Yoshimoto T., Boehm M., Olive M., Crook M.F., San H., Langenickel T., Nabel E.G. The arginine methyltransferase PRMT2 binds RB and regulates E2F function. Exp. Cell Res. (2006) 312:2040–2053.[CrossRef][ISI][Medline]

  14. Nishiyama H., Knopfel T., Endo S., Itohara S. Glial protein S100B modulates long-term neuronal synaptic plasticity. Proc. Natl Acad. Sci. USA (2002) 99:4037–4042.[Abstract/Free Full Text]

  15. Fernandez-Fernandez M.R., Veprintsev D.B., Fersht A.R. Proteins of the S100 family regulate the oligomerization of p53 tumor suppressor. Proc. Natl Acad. Sci. USA (2005) 102:4735–4740.[Abstract/Free Full Text]

  16. Sen J., Belli A. S100B in neuropathologic states: the CRP of the brain? J. Neurosci. Res. (2007) 85:1373–1380.[CrossRef][ISI][Medline]

  17. Salisbury J.L. Primary cilia: putting sensors together. Curr. Biol. (2004) 14:R765–R767.[CrossRef][ISI][Medline]

  18. Miyoshi K., Onishi K., Asanuma M., Miyazaki I., Diaz-Corrales F.J., Ogawa N. Embryonic expression of pericentrin suggests universal roles in ciliogenesis. Dev. Genes Evol. (2006) 216:537–542.[CrossRef][ISI][Medline]

  19. Osman W., Laine S., Zilliacus J. Functional interaction between the glucocorticoid receptor and GANP/MCM3AP. Biochem. Biophys. Res. Commun. (2006) 348:1239–1244.[CrossRef][ISI][Medline]

  20. Kuwahara K., Fujimura S., Takahashi Y., Nakagata N., Takemori T., Aizawa S., Sakaguchi N. Germinal center-associated nuclear protein contributes to affinity maturation of B cell antigen receptor in T cell-dependent responses. Proc. Natl Acad. Sci. USA (2004) 101:1010–1015.[Abstract/Free Full Text]

  21. Sakaguchi N., Fujimura S., Kuwahara K. Involvement of GANP in B cell activation in T cell-dependent antigen response. Dev. Immunol. (2002) 9:169–172.[CrossRef][Medline]

  22. Fujimura S., Xing Y., Takeya M., Yamashita Y., Ohshima K., Kuwahara K., Sakaguchi N. Increased expression of germinal center-associated nuclear protein RNA-primase is associated with lymphomagenesis. Cancer Res. (2005) 65:5925–5934.[Abstract/Free Full Text]

  23. Bonaldo P., Braghetta P., Zanetti M., Piccolo S., Volpin D., Bressan G.M. Collagen VI deficiency induces early onset myopathy in the mouse: an animal model for Bethlem myopathy. Hum. Mol. Genet. (1998) 7:2135–2140.[Abstract/Free Full Text]

  24. Lampe A.K., Bushby K.M. Collagen VI related muscle disorders. J. Med. Genet. (2005) 42:673–685.[Abstract/Free Full Text]

  25. Giusti B., Lucarini L., Pietroni V., Lucioli S., Bandinelli B., Sabatelli P., Squarzoni S., Petrini S., Gartioux C., Talim B., et al. Dominant and recessive COL6A1 mutations in Ullrich scleroatonic muscular dystrophy. Ann. Neurol. (2005) 58:400–410.[CrossRef][ISI][Medline]

  26. Pepe G., Lucarini L., Zhang R.Z., Pan T.C., Giusti B., Quijano-Roy S., Gartioux C., Bushby K.M., Guicheney P., Chu M.L. COL6A1 genomic deletions in Bethlem myopathy and Ullrich muscular dystrophy. Ann. Neurol. (2006) 59:190–195.[CrossRef][ISI][Medline]

  27. Mori M., Li G., Abe I., Nakayama J., Guo Z., Sawashita J., Ugawa T., Nishizono S., Serikawa T., Higuchi K., et al. Lanosterol synthase mutations cause cholesterol deficiency-associated cataracts in the Shumiya cataract rat. J. Clin. Invest. (2006) 116:395–404.[CrossRef][ISI][Medline]

  28. Lapierre P., Hajoui O., Homberg J.C., Alvarez F. Formiminotransferase cyclodeaminase is an organ-specific autoantigen recognized by sera of patients with autoimmune hepatitis. Gastroenterology (1999) 116:643–649.[CrossRef][ISI][Medline]

  29. Hilton J.F., Christensen K.E., Watkins D., Raby B.A., Renaud Y., de la Luna S., Estivill X., MacKenzie R.E., Hudson T.J., Rosenblatt D.S. The molecular basis of glutamate formiminotransferase deficiency. Hum. Mutat. (2003) 22:67–73.[CrossRef][ISI][Medline]

  30. Brault V., Pereira P., Duchon A., Herault Y. Modeling chromosomes in mouse to explore the function of genes, genomic disorders, and chromosomal organization. PLoS Genet. (2006) 2:e86.[CrossRef][Medline]

  31. Zheng B., Mills A.A., Bradley A. A system for rapid generation of coat color-tagged knockouts and defined chromosomal rearrangements in mice. Nucleic Acids Res. (1999) 27:2354–2360.[Abstract/Free Full Text]

  32. Magin T.M., McWhir J., Melton D.W. A new mouse embryonic stem cell line with good germ line contribution and gene targeting frequency. Nucleic Acids Res. (1992) 20:3795–3796.[Free Full Text]

  33. Noulin N., Quesniaux V.F., Schnyder-Candrian S., Schnyder B., Maillet I., Robert T., Vargaftig B.B., Ryffel B., Couillin I. Both hemopoietic and resident cells are required for MyD88-dependent pulmonary inflammatory response to inhaled endotoxin. J. Immunol. (2005) 175:6861–6869.[Abstract/Free Full Text]

  34. Takeda K., Akira S. Toll-like receptors in innate immunity. Int. Immunol. (2005) 17:1–14.[Abstract/Free Full Text]

  35. Espevik T., Nissen-Meyer J. A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic factor/tumor necrosis factor from human monocytes. J. Immunol. Methods (1986) 95:99–105.[CrossRef][ISI][Medline]

  36. Vandesompele J., De Preter K., Pattyn F., Poppe B., Van Roy N., De Paepe A., Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. (2002) 3:0034.1–0034.11.

  37. Merscher S., Funke B., Epstein J.A., Heyer J., Puech A., Lu M.M., Xavier R.J., Demay M.B., Russell R.G., Factor S., et al. TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell (2001) 104:619–629.[CrossRef][ISI][Medline]

  38. Spitz F., Herkenne C., Morris M.A., Duboule D. Inversion-induced disruption of the Hoxd cluster leads to the partition of regulatory landscapes. Nat. Genet. (2005) 37:889–893.[CrossRef][ISI][Medline]

  39. Andonegui G., Bonder C.S., Green F., Mullaly S.C., Zbytnuik L., Raharjo E., Kubes P. Endothelium-derived Toll-like receptor-4 is the key molecule in LPS-induced neutrophil sequestration into lungs. J. Clin. Invest. (2003) 111:1011–1020.[CrossRef][ISI][Medline]

  40. Mao R., Zielke C.L., Zielke H.R., Pevsner J. Global up-regulation of chromosome 21 gene expression in the developing Down syndrome brain. Genomics (2003) 81:457–467.[CrossRef][ISI][Medline]

  41. Mao R., Wang X., Spitznagel E.L. Jr, Frelin L.P., Ting J.C., Ding H., Kim J.W., Ruczinski I., Downey T.J., Pevsner J. Primary and secondary transcriptional effects in the developing human Down syndrome brain and heart. Genome Biol. (2005) 6:R107.[CrossRef][Medline]

  42. Amano K., Sago H., Uchikawa C., Suzuki T., Kotliarova S.E., Nukina N., Epstein C.J., Yamakawa K. Dosage-dependent over-expression of genes in the trisomic region of Ts1Cje mouse model for Down syndrome. Hum. Mol. Genet. (2004) 13:1333–1340.[Abstract/Free Full Text]

  43. Lyle R., Gehrig C., Neergaard-Henrichsen C., Deutsch S., Antonarakis S.E. Gene expression from the aneuploid chromosome in a trisomy mouse model of Down syndrome. Genome Res. (2004) 14:1268–1274.[Abstract/Free Full Text]