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The gene content of the X and Y chromosomes has dramatically diverged during evolution. The ensuing dosage imbalance within the genome of males and females has led to unique chromosome-wide regulatory mechanisms with significant and sex-specific impacts on X-linked gene expression. X inactivation or silencing of most genes on one X chromosome chosen at random in females profoundly affects the manifestation of X-linked diseases, as males inherit a single maternal allele, while females express maternal and paternal alleles in a mosaic manner. An additional complication is the existence of genes that escape X inactivation and thus are ubiquitously expressed from both alleles in females. The mosaic nature of X-linked gene expression and the potential for escape can vary between individuals, tissues, cell types and stages of life. Our understanding of the specialized nature of X-linked genes and of the multilayer epigenetic regulation that influence their expression throughout the organism has been helped by molecular studies conducted by tissue-specific and single-cell-specific approaches. In turn, the definition of molecular events that control X silencing has helped develop new approaches for the treatment of some X-linked disorders. This review focuses on the peculiarities of the X chromosome genetic content and epigenetic regulation in shaping the manifestation of congenital and acquired X-linked disorders in a sex-specific manner.

The Forkhead Box C1 (FOXC1) gene encodes a forkhead/winged helix transcription factor involved in embryonic development. Mutations in this gene cause dysgenesis of the anterior segment of the eye, most commonly Axenfeld-Rieger syndrome (ARS), often with other systemic features. The developmental mechanisms and pathways regulated by FOXC1 remain largely unknown. There are two conserved orthologs of FOXC1 in zebrafish, foxc1a and foxc1b. To further examine the role of FOXC1 in vertebrates, we generated foxc1a and foxc1b single knockout zebrafish lines and bred them to obtain various allelic combinations. Three genotypes demonstrated visible phenotypes: foxc1a−/− single homozygous and foxc1−/− double knockout homozygous embryos presented with similar characteristics comprised of severe global vascular defects and early lethality, as well as microphthalmia, periocular edema and absence of the anterior chamber of the eye; additionally, fish with heterozygous loss of foxc1a combined with homozygosity for foxc1b (foxc1a+/−;foxc1b−/−) demonstrated craniofacial defects, heart anomalies and scoliosis. All other single and combined genotypes appeared normal. Analysis of foxc1 expression detected a significant increase in foxc1a levels in homozygous and heterozygous mutant eyes, suggesting a mechanism for foxc1a upregulation when its function is compromised; interestingly, the expression of another ARS-associated gene, pitx2, was responsive to the estimated level of wild-type Foxc1a, indicating a possible role for this protein in the regulation of pitx2 expression. Altogether, our results support a conserved role for foxc1 in the formation of many organs, consistent with the features observed in human patients, and highlight the importance of correct FOXC1/foxc1 dosage for vertebrate development.

Autosomal dominant polycystic kidney disease (ADPKD) is among the most common monogenic disorders mainly associated with PKD1/PC1 mutations. We show herein that renal regulation in Pc1 dosage-reduced and -increased mouse models converge toward stimulation of c-Myc expression along with β-catenin, delineating c-Myc as a key Pkd1 node in cystogenesis. Enhanced renal c-Myc-induced ADPKD in SBM transgenic mice lead conversely to striking upregulation of Pkd1/Pc1 expression and β-catenin activation, lending credence for reciprocal crosstalk between c-Myc and Pc1. In adult SBM kidneys, c-Myc is strongly enriched on Pkd1 promoter with RNA pol II, consistent with Pkd1 upregulation during cystogenesis. Similar c-Myc direct binding at birth uncovers an equivalent role on Pkd1 regulation during renal developmental program. Concurrent with enriched c-Myc binding, recruitment of active chromatin modifying co-factors by c-Myc at the Pkd1 regulatory region probably opens chromatin to stimulate transcription. A similar transcriptional activation by c-Myc is also likely operant on endogenous human PKD1 gene from our transactivation analysis in response to human c-MYC upregulation. Genetic ablation of c-Myc in Pc1-reduced and -increased mouse models significantly attenuates cyst growth, proliferation and PKD progression. Our study determined a dual role for c-Myc, as a major contributor in Pc1-induced cystogenesis and in a feed-forward regulatory Pkd1–c-Myc loop mechanism that may also prevail in human ADPKD.

The recent generation of induced pluripotent stem cells (iPSCs) from a patient with Parkinson's disease (PD) resulting from triplication of the α-synuclein (SNCA) gene locus allows unprecedented opportunities to explore its contribution to the molecular pathogenesis of PD. We used the double-nicking CRISPR/Cas9 system to conduct site-specific mutagenesis of SNCA in these cells, generating an isogenic iPSC line with normalized SNCA gene dosage. Comparative gene expression analysis of neuronal derivatives from these iPSCs revealed an ER stress phenotype, marked by induction of the IRE1α/XBP1 axis of the unfolded protein response (UPR) and culminating in terminal UPR activation. Neuropathological analysis of post-mortem brain tissue demonstrated that pIRE1α is expressed in PD brains within neurons containing elevated levels of α-synuclein or Lewy bodies. Having used this pair of isogenic iPSCs to define this phenotype, these cells can be further applied in UPR-targeted drug discovery towards the development of disease-modifying therapeutics.

Deoxyguanosine kinase (DGUOK) provides guanosine and adenosine nucleotides for mitochondrial DNA (mtDNA) replication, and its deficiency in humans leads to hepatocerebral mtDNA depletion syndrome or to isolated hepatic disease. There are poor treatment options for DGUOK deficiency and the aim of this study was to generate a model for further studies of the disease that could reveal novel treatment strategies. We report a Dguok-deficient mouse strain that, similar to humans, is most severely affected in the liver. The Dguok complete knockout mice (Dguok−/−) were born normal, but began to lose weight at week 6. A change of fur color from black to blueish grey started at week 16 and was complete at week 20. The movements and behavior were indistinguishable compared to wild-type (wt) mice. A decrease of mtDNA copy number occurred in multiple tissues, with the liver being the most severely affected. The mtDNA-encoded protein cytochrome c oxidase was much lower in Dguok−/− liver tissue than in the wt, whereas the expression of the nuclear-encoded succinate dehydrogenase complex subunit A was unaffected. Histopathology showed severe alterations and immunohistochemistry showed signs of both oxidative stress and regeneration in Dguok−/− liver. The subcutaneous fat layer was undetectable in Dguok−/−, which, in addition to gene expression analysis, indicated an altered lipid metabolism. We conclude that Dguok has a major role for the synthesis of deoxyribonucleotides for mtDNA replication particularly in the liver, similar to the human disorder. Our data also show a catabolic lipid metabolism in liver tissue of Dguok−/−.

A common null polymorphism (R577X) in ACTN3 causes α-actinin-3 deficiency in ∼ 18% of the global population. There is no associated disease phenotype, but α-actinin-3 deficiency is detrimental to sprint and power performance in both elite athletes and the general population. However, despite considerable investigation to date, the functional consequences of heterozygosity for ACTN3 are unclear. A subset of studies have shown an intermediate phenotype in 577RX individuals, suggesting dose-dependency of α-actinin-3, while others have shown no difference between 577RR and RX genotypes. Here, we investigate the effects of α-actinin-3 expression level by comparing the muscle phenotypes of Actn3(+/-) (HET) mice to Actn3(+/+) [wild-type (WT)] and Actn3(-/-) [knockout (KO)] littermates. We show reduction in α-actinin-3 mRNA and protein in HET muscle compared with WT, which is associated with dose-dependent up-regulation of α-actinin-2, z-band alternatively spliced PDZ-motif and myotilin at the Z-line, and an incremental shift towards oxidative metabolism. While there is no difference in force generation, HET mice have an intermediate endurance capacity compared with WT and KO. The R577X polymorphism is associated with changes in ACTN3 expression consistent with an additive model in the human genotype-tissue expression cohort, but does not influence any other muscle transcripts, including ACTN2. Overall, ACTN3 influences sarcomeric composition in a dose-dependent fashion in mouse skeletal muscle, which translates directly to function. Variance in fibre type between biopsies likely masks this phenomenon in human skeletal muscle, but we suggest that an additive model is the most appropriate for use in testing ACTN3 genotype associations.

Identifying dosage sensitive genes is a key to understand the mechanisms underlying intellectual disability in Down syndrome (DS). The Dp(17Abcg1-Cbs)1Yah DS mouse model (Dp1Yah) show cognitive phenotype and needs to be investigated to identify the main genetic driver. Here, we report that, in the Dp1Yah mice, 3 copies of the Cystathionine-beta-synthase gene (Cbs)are necessary to observe a deficit in the novel object recognition (NOR) paradigm. Moreover, the overexpression ofCbsalone is sufficient to induce NOR deficit. Accordingly targeting the overexpression of human CBS, specifically in Camk2a-expressing neurons, leads to impaired objects discrimination. Altogether this shows thatCbsoverdosage is involved in DS learning and memory phenotypes. In order to go further, we identified compounds that interfere with the phenotypical consequence of CBS overdosage in yeast. Pharmacological intervention in the Tg(CBS) with one selected compound restored memory in the novel object recognition. In addition, using a genetic approach, we demonstrated an epistatic interaction betweenCbsandDyrk1a, another human chromosome 21 gene encoding the dual-specificity tyrosine phosphorylation-regulated kinase 1a and an already known target for DS therapeutic intervention. Further analysis using proteomic approaches highlighted several pathways, including synaptic transmission, cell projection morphogenesis, and actin cytoskeleton, that are affected by DYRK1A and CBS overexpression. Overall we demonstrated that CBS overdosage underpins the DS-related recognition memory deficit and that bothCBSandDYRK1Ainteract to control accurate memory processes in DS. In addition, our study establishes CBS as an intervention point for treating intellectual deficiencies linked to DS.SIGNIFICANT STATEMENTHere, we investigated a region homologous to Hsa21 and located on mouse chromosome 17. We demonstrated using three independent genetic approaches that the overdosage of the Cystathionine-beta-synthase gene (Cbs) gene, encoded in the segment, is necessary and sufficient to induce deficit in novel object recognition (NR).In addition, we identified compounds that interfere with the phenotypical consequence of CBS overdosage in yeast and in mouse transgenic lines. Then we analyzed the relation between Cbs overdosage and the consequence of DYRK1a overexpression, a main driver of another region homologous to Hsa21 and we demonstrated that an epistatic interaction exist betweenCbsandDyrk1aaffecting different pathways, including synaptic transmission, cell projection morphogenesis, and actin cytoskeleton.

DYT1 dystonia, the most common inherited form of primary dystonia, is a neurodevelopmental disease caused by a dominant mutation in TOR1A. This mutation ('ΔE') removes a single glutamic acid from the encoded protein, torsinA. The effects of this mutation, at the molecular and circuit levels, and the reasons for its neurodevelopmental onset, remain incompletely understood. To uniquely address key questions of disease pathogenesis, we generated a conditional Tor1a knock-in allele that is converted from wild-type to DYT1 mutant ('induced' ΔE: Tor1a(i-ΔE)), following Cre recombination. We used this model to perform a gene dosage study exploring the effects of the ΔE mutation at the molecular, neuropathological and organismal levels. These analyses demonstrated that ΔE-torsinA is a hypomorphic allele and showed no evidence for any gain-of-function toxic properties. The unique capabilities of this model also enabled us to test a circuit-level hypothesis of DYT1 dystonia, which predicts that expression of the DYT1 genotype (Tor1a(ΔE/+)) selectively within hindbrain structures will produce an overtly dystonic animal. In contrast to this prediction, we find no effect of this anatomic-specific expression of the DYT1 genotype, a finding that has important implications for the interpretation of the human and mouse diffusion tensor-imaging studies upon which it is based. These studies advance understanding of the molecular effects of the ΔE mutation, challenge current concepts of the circuit dysfunction that characterize the disease and establish a powerful tool that will be valuable for future studies of disease pathophysiology.

Trisomy 21 causes skeletal alterations in individuals with Down syndrome (DS), but the causative trisomic gene and a therapeutic approach to rescue these abnormalities are unknown. Individuals with DS display skeletal alterations including reduced bone mineral density, modified bone structure and distinctive facial features. Due to peripheral skeletal anomalies and extended longevity, individuals with DS are increasingly more susceptible to bone fractures. Understanding the genetic and developmental origin of DS skeletal abnormalities would facilitate the development of therapies to rescue these and other deficiencies associated with DS. DYRK1A is found in three copies in individuals with DS and Ts65Dn DS mice and has been hypothesized to be involved in many Trisomy 21 phenotypes including skeletal abnormalities. Return of Dyrk1a copy number to normal levels in Ts65Dn mice rescued the appendicular bone abnormalities, suggesting that appropriate levels of DYRK1A expression are critical for the development and maintenance of the DS appendicular skeleton. Therapy using the DYRK1A inhibitor epigallocatechin-3-gallate improved Ts65Dn skeletal phenotypes. These outcomes suggest that the osteopenic phenotype associated with DS may be rescued postnatally by targeting trisomic Dyrk1a.

Genomic disorders are the clinical conditions manifested by submicroscopic genomic rearrangements including copy number variants (CNVs). The CNVs can be identified by array-based comparative genomic hybridization (aCGH), the most commonly used technology for molecular diagnostics of genomic disorders. However, clinical aCGH only informs CNVs in the probe-interrogated regions. Neither orientational information nor the resulting genomic rearrangement structure is provided, which is a key to uncovering mutational and pathogenic mechanisms underlying genomic disorders. Long-range polymerase chain reaction (PCR) is a traditional approach to obtain CNV breakpoint junction, but this method is inefficient when challenged by structural complexity such as often found at the PLP1 locus in association with Pelizaeus-Merzbacher disease (PMD). Here we introduced 'capture and single-molecule real-time sequencing' (cap-SMRT-seq) and newly developed 'asymmetry linker-mediated nested PCR walking' (ALN-walking) for CNV breakpoint sequencing in 49 subjects with PMD-associated CNVs. Remarkably, 29 (94%) of the 31 CNV breakpoint junctions unobtainable by conventional long-range PCR were resolved by cap-SMRT-seq and ALN-walking. Notably, unexpected CNV complexities, including inter-chromosomal rearrangements that cannot be resolved by aCGH, were revealed by efficient breakpoint sequencing. These sequence-based structures of PMD-associated CNVs further support the role of DNA replicative mechanisms in CNV mutagenesis, and facilitate genotype-phenotype correlation studies. Intriguingly, the lengths of gained segments by CNVs are strongly correlated with clinical severity in PMD, potentially reflecting the functional contribution of other dosage-sensitive genes besides PLP1. Our study provides new efficient experimental approaches (especially ALN-walking) for CNV breakpoint sequencing and highlights their importance in uncovering CNV mutagenesis and pathogenesis in genomic disorders.

Williams-Beuren syndrome is a developmental multisystemic disorder caused by a recurrent 1.55-1.83 Mb heterozygous deletion on human chromosome band 7q11.23. Through chromosomal engineering with the cre-loxP system, we have generated mice with an almost complete deletion (CD) of the conserved syntenic region on chromosome 5G2. Heterozygous CD mice were viable, fertile and had a normal lifespan, while homozygotes were early embryonic lethal. Transcript levels of most deleted genes were reduced 50% in several tissues, consistent with gene dosage. Heterozygous mutant mice showed postnatal growth delay with reduced body weight and craniofacial abnormalities such as small mandible. The cardiovascular phenotype was only manifested with borderline hypertension, mildly increased arterial wall thickness and cardiac hypertrophy. The neurobehavioral phenotype revealed impairments in motor coordination, increased startle response to acoustic stimuli and hypersociability. Mutant mice showed a general reduction in brain weight. Cellular and histological abnormalities were present in the amygdala, cortex and hippocampus, including increased proportion of immature neurons. In summary, these mice recapitulate most crucial phenotypes of the human disorder, provide novel insights into the pathophysiological mechanisms of the disease such as the neural substrates of the behavioral manifestations, and will be valuable to evaluate novel therapeutic approaches., This work was supported by the Spanish Ministry of Ecomomy and Competitivity to V.C. (grant SAF2012-40036) and to L.P.J. (FIS PM002512 and SAF2004-06382), the European AnEuploidy project to L.P.J., M.D. and Y.H. The Rare Diseases CIBER (CIBERER) Fellowship supported M.S-P. and C.B.

The hereditary spastic paraplegias (HSPs) are a heterogeneous group of motorneuron diseases characterized by progressive spasticity and paresis of the lower limbs. Mutations in Spastic Gait 4 (SPG4), encoding spastin, are the most frequent cause of HSP. To understand how mutations in SPG4 affect human neurons, we generated human induced pluripotent stem cells (hiPSCs) from fibroblasts of two patients carrying a c.1684C>T nonsense mutation and from two controls. These SPG4 and control hiPSCs were able to differentiate into neurons and glia at comparable efficiency. All known spastin isoforms were reduced in SPG4 neuronal cells. The complexity of SPG4 neurites was decreased, which was paralleled by an imbalance of axonal transport with less retrograde movement. Prominent neurite swellings with disrupted microtubules were present in SPG4 neurons at an ultrastructural level. While some of these swellings contain acetylated and detyrosinated tubulin, these tubulin modifications were unchanged in total cell lysates of SPG4 neurons. Upregulation of another microtubule-severing protein, p60 katanin, may partially compensate for microtubuli dynamics in SPG4 neurons. Overexpression of the M1 or M87 spastin isoforms restored neurite length, branching, numbers of primary neurites and reduced swellings in SPG4 neuronal cells. We conclude that neurite complexity and maintenance in HSP patient-derived neurons are critically sensitive to spastin gene dosage. Our data show that elevation of single spastin isoform levels is sufficient to restore neurite complexity and reduce neurite swellings in patient cells. Furthermore, our human model offers an ideal platform for pharmacological screenings with the goal to restore physiological spastin levels in SPG4 patients.

Coarctation of the aorta (CoA) and hypoplastic left heart syndrome (HLHS) have been reported in rare individuals with large terminal deletions of chromosome 15q26. However, no single gene important for left ventricular outflow tract (LVOT) development has been identified in this region. Using array-comparative genomic hybridization, we identified two half-siblings with CoA with a 2.2 Mb deletion on 15q26.2, inherited from their mother, who was mosaic for this deletion. This interval contains an evolutionary conserved, protein-coding gene, MCTP2 (multiple C2-domains with two transmembrane regions 2). Using gene-specific array screening in 146 individuals with non-syndromic LVOT obstructive defects, another individual with HLHS and CoA was found to have a de novo 41 kb intragenic duplication within MCTP2, predicted to result in premature truncation, p.F697X. Alteration of Mctp2 gene expression in Xenopus laevis embryos by morpholino knockdown and mRNA overexpression resulted in the failure of proper OT development, confirming the functional importance of this dosage-sensitive gene for cardiogenesis. Our results identify MCTP2 as a novel genetic cause of CoA and related cardiac malformations.

Niemann–Pick disease type C (NP-C) is a rare, autosomal-recessive, progressive neurological disease caused by mutations in either the NPC1 gene (in 95% of cases) or the NPC2 gene. This observational, multicentre genetic screening study evaluated the frequency and phenotypes of NP-C in consecutive adult patients with neurological and psychiatric symptoms. Diagnostic testing for NP-C involved NPC1 and NPC2 exonic gene sequencing and gene dosage analysis. When available, results of filipin staining, plasma cholestane-3β,5α,6β-triol assays and measurements of relevant sphingolipids were also collected. NPC1 and NPC2 gene sequencing was completed in 250/256 patients from 30 psychiatric and neurological reference centres across the EU and USA [median (range) age 38 (18–90) years]. Three patients had a confirmed diagnosis of NP-C; two based on gene sequencing alone (two known causal disease alleles) and one based on gene sequencing and positive filipin staining. A further 12 patients displayed either single mutant NP-C alleles (8 with NPC1 mutations and 3 with NPC2 mutations) or a known causal disease mutation and an unclassified NPC1 allele variant (1 patient). Notably, high plasma cholestane-3β,5α,6β-triol levels were observed for all NP-C cases (n = 3). Overall, the frequency of NP-C patients in this study [1.2% (95% CI; 0.3%, 3.5%)] suggests that there may be an underdiagnosed pool of NP-C patients among adults who share common neurological and psychiatric symptoms.

Environmental factors including ionizing radiation and chemical agents have been known to be able to induce DNA rearrangements and cause genomic structural variations (SVs); however, the roles of intrinsic characteristics of the human genome, such as regional genome architecture, in SV formation and the potential mechanisms underlying genomic instability remain to be further elucidated. Recently, locus-specific observations showed that 'self-chain' (SC), a group of short low-copy repeats (LCRs) in the human genome, can induce autism-associated SV mutations of the MECP2 and NRXN1 genes. In this study, we conducted a genome-wide analysis to investigate SCs and their potential roles in genomic SV formation. Utilizing a vast amount of human SV data, we observed a significant biased distribution of human germline SV breakpoints to SC regions. Notably, the breakpoint distribution pattern is different between SV types across deletion, duplication, inversion and insertion. Our observations were coincident with a mechanism of SC-induced DNA replicative errors, whereas SC may sporadically be used as substrates of nonallelic homologous recombination (NAHR). This contention was further supported by our consistent findings in somatic SV mutations of cancer genomes, suggesting a general mechanism of SC-induced genome instability in human germ and somatic cells.

The nonsense-mediated mRNA decay (NMD) pathway functions not only to degrade transcripts containing premature termination codons (PTC), but also to regulate the transcriptome. UPF3B and RBM8A, important components of NMD, have been implicated in various forms of intellectual disability (ID) and Thrombocytopenia with Absent Radius (TAR) syndrome, which is also associated with ID. To gauge the contribution of other NMD factors to ID, we performed a comprehensive search for copy number variants (CNVs) of 18 NMD genes among individuals with ID and/or congenital anomalies. We identified 11 cases with heterozygous deletions of the genomic region encompassing UPF2, which encodes for a direct interacting protein of UPF3B. Using RNA-Seq, we showed that the genome-wide consequence of reduced expression of UPF2 is similar to that seen in patients with UPF3B mutations. Out of the 1009 genes found deregulated in patients with UPF2 deletions by at least 2-fold, majority (95%) were deregulated similarly in patients with UPF3B mutations. This supports the major role of deletion of UPF2 in ID. Furthermore, we found that four other NMD genes, UPF3A, SMG6, EIF4A3 and RNPS1 are frequently deleted and/or duplicated in the patients. We postulate that dosage imbalances of these NMD genes are likely to be the causes or act as predisposing factors for neuro-developmental disorders. Our findings further emphasize the importance of NMD pathway(s) in learning and memory.

Traditionally, genetic disorders have been classified as either Mendelian diseases or complex diseases. This nosology has greatly benefited genetic counseling and the development of gene mapping strategies. However, based on two well-established databases, we identified that 54% (524 of 968) of the Mendelian disease genes were also involved in complex diseases, and this kind of genes has not been systematically analyzed. Here, we classified human genes into five categories: Mendelian and complex disease (MC) genes, Mendelian but not complex disease (MNC) genes, complex but not Mendelian disease (CNM) genes, essential genes and OTHER genes. First, we found that MC genes were associated with more diseases and phenotypes, and were involved in more complex protein-protein interaction network than MNC or CNM genes on average. Secondly, MC genes encoded the longest proteins and had the highest transcript count among all gene categories. Especially, tissue specificity of MC genes was much higher than that of any other gene categories (P < 7.5 × 10(-5)), although their expression level was similar to that of essential genes. Thirdly, evidences from different aspects supported that MC genes have been subjected to both purifying and positive selection. Interestingly, functions of some human disease genes might be different from those of their orthologous genes in non-primate mammalians since they were even less conserved than OTHER genes. The significant over-representation of copy number variations (CNVs) in CNM genes suggested the important roles of CNVs in complex diseases. In brief, our study not only revealed the characteristics of MC genes, but also provided new insights into the other four gene categories.

Myelin insulates axons in the peripheral nervous system to allow rapid propagation of action potentials, and proper myelination requires the precise regulation of genes encoding myelin proteins, including PMP22. The correct gene dosage of PMP22 is critical; a duplication of PMP22 is the most common cause of the peripheral neuropathy Charcot-Marie-Tooth Disease (CMT) (classified as type 1A), while a deletion of PMP22 leads to another peripheral neuropathy, hereditary neuropathy with liability to pressure palsies. Recently, duplications upstream of PMP22, but not containing the gene itself, were reported in patients with CMT1A like symptoms, suggesting that this region contains regulators of PMP22. Using chromatin immunoprecipitation analysis of two transcription factors known to upregulate PMP22—EGR2 and SOX10—we found several enhancers in this upstream region that contain open chromatin and direct reporter gene expression in tissue culture and in vivo in zebrafish. These studies provide a novel means to identify critical regulatory elements in genes that are required for myelination, and elucidate the functional significance of non-coding genomic rearrangements.

In complex organisms, different tissues express different genes, which ultimately shape the function and phenotype of each tissue. An important goal of modern biology is to understand how some genes are turned on and off in specific tissues and how the numbers of different gene expression products are determined. These aspects are named 'expression breadth' (or 'tissue specificity') and 'expression level', respectively. Here, we show that we can predict substantial amount of variation in levels and breadths of gene expression using genomic information of each gene. Interestingly, many genomic traits are correlated with both aspects of gene expression in similar directions, suggesting shared molecular pathways. However, to elucidate distinctive molecular mechanisms governing gene expression levels and breadths, we need to identify the relative significance of each genomic trait on these two aspects of gene expression. To this end, we developed a novel multivariate multiple regression method. Using this new method, we show that gene compactness (in particular, the mean size of exons), codon usage bias and non-synonymous rates have a stronger influence on expression levels compared with their effects on expression breadths. In contrast, the propensity of promoter DNA methylation is a stronger indicator of expression breadths than of expression levels. Interestingly, intron DNA methylation exhibits an opposite pattern to the promoter DNA methylation in the human genome, suggesting that DNA methylation may play multiple roles depending upon its genomic targets. Furthermore, synonymous rates have stronger associations with expression breadths than with expression levels in the human genome. These findings provide clues toward distinctive molecular mechanisms regulating different aspects of gene expression.

CHARGE is a multiple congenital anomaly disorder and a common cause of pubertal defects, olfactory dysfunction, growth delays, deaf-blindness, balance disorders and congenital heart malformations. Mutations in CHD7, the gene encoding chromodomain helicase DNA binding protein 7, are present in 60-80% of individuals with the CHARGE syndrome. Mutations in CHD7 have also been reported in the Kallmann syndrome (olfactory dysfunction, delayed puberty and hypogonadotropic hypogonadism). CHD7 is a positive regulator of neural stem cell proliferation and olfactory sensory neuron formation in the olfactory epithelium, suggesting that the loss of CHD7 might also disrupt development of other neural populations. Here we report that female Chd7(Gt/+) mice have delays in vaginal opening and estrus onset, and erratic estrus cycles. Chd7(Gt/+) mice also have decreased circulating levels of luteinizing hormone and follicle-stimulating hormone but apparently normal responsiveness to gonadotropin-releasing hormone (GnRH) agonist and antagonist treatment. GnRH neurons in the adult Chd7(Gt/+) hypothalamus and embryonic nasal region are diminished, and there is decreased cellular proliferation in the embryonic olfactory placode. Expression levels of GnRH1 and Otx2 in the hypothalamus and GnRHR in the pituitary are significantly reduced in adult Chd7(Gt/+) mice. Additionally, Chd7 mutant embryos have CHD7 dosage-dependent reductions in expression levels of Fgfr1, Bmp4 and Otx2 in the olfactory placode. Together, these data suggest that CHD7 has critical roles in the development and maintenance of GnRH neurons for regulating puberty and reproduction.

Common fragile sites (cFS) represent chromosomal regions that are prone to breakage after partial inhibition of DNA synthesis. Activation of cFS is associated with various forms of DNA instability in cancer cells, and is thought to be an initiating event in the generation of DNA damage in early-stage tumorigenesis. Only a few cFS have been fully characterized despite the growing interest in cFS instability in cancer genomes. In this study, six-color fluorescence in situ hybridization revealed that FRA2C consists of two cFS spanning 747 kb FRA2Ctel and 746 kb FRA2Ccen at 2p24.3 and 2p24.2, respectively. Both cFS are separated by a 2.8 Mb non-fragile region containing MYCN. Fine-tiling array comparative genomic hybridization of MYCN amplicons from neuroblastoma (NB) cell lines and primary tumors revealed that 56.5% of the amplicons cluster in FRA2C. MYCN amplicons are either organized as double minutes or as homogeneously stained regions in addition to the single copy of MYCN retained at 2p24. We suggest that MYCN amplicons arise from extra replication rounds of unbroken DNA secondary structures that accumulate at FRA2C. This hypothesis implicates cFS in high-level gene amplification in cancer cells. Complex genomic rearrangements, including deletions, duplications and translocations, which originate from double-strand breaks, were detected at FRA2C in different cancers. These data propose a dual role for cFS in the generation of gross chromosomal rearrangements either after DNA breakage or by inducing extra replication rounds, and provide new insights into the highly recombinogenic nature of cFS in the human cancer genome.

Huntington's disease (HD) is an inherited, progressive and ultimately fatal neurodegenerative disorder that is characterized by psychiatric, cognitive and motor symptoms. Among the pathways implicated in HD are those involving mitogen-activated protein kinase signaling and particularly the Ras-extracellular signal-regulated kinase (ERK) cascade. Studies in both cells and animal models suggest that ERK activation might provide a novel therapeutic target for the treatment of HD but compounds that specifically activate ERK are few. To test the hypothesis that pharmaceutical activation of ERK might be protective for HD, a polyphenol, fisetin, which was previously shown to activate the Ras-ERK cascade, was tested in three different models of HD: PC12 cells expressing mutant Httex1 under the control of an inducible promoter, Drosophila expressing mutant Httex1 and the R6/2 mouse model of HD. The results indicate that fisetin can reduce the impact of mutant huntingtin in each of these disease models. Prompted by this observation, we determined that the related polyphenol, resveratrol, also activates ERK and is protective in HD models. Notably, although more than a dozen small molecule inhibitors of ERK activation are in clinical trials, very few small molecule activators of ERK signaling are reported. Thus, fisetin, resveratrol and related compounds might be useful for the treatment of HD by virtue of their unique ability to activate ERK.

The ETV6/RUNX1 fusion gene, present in 25% of B-lineage childhood acute lymphoblastic leukemia (ALL), is thought to represent an initiating event, which requires additional genetic changes for leukemia development. To identify additional genetic alterations, 24 ETV6/RUNX1-positive ALLs were analyzed using 500K single nucleotide polymorphism arrays. The results were combined with previously published data sets, allowing us to ascertain genomic copy number aberrations (CNAs) in 164 cases. In total, 45 recurrent CNAs were identified with an average number of 3.5 recurrent changes per case (range 0-13). Twenty-six percent of cases displayed a set of recurrent CNAs identical to that of other cases in the data set. The majority (74%), however, displayed a unique pattern of recurrent CNAs, indicating a large heterogeneity within this ALL subtype. As previously demonstrated, alterations targeting genes involved in B-cell development were common (present in 28% of cases). However, the combined analysis also identified alterations affecting nuclear hormone response (24%) to be a characteristic feature of ETV6/RUNX1-positive ALL. Studying the correlation pattern of the CNAs allowed us to highlight significant positive and negative correlations between specific aberrations. Furthermore, oncogenetic tree models identified ETV6, CDKN2A/B, PAX5, del(6q) and +16 as possible early events in the leukemogenic process.

Copy number variations (CNVs) are universal genetic variations, and their association with disease has been increasingly recognized. We designed high-density microarrays for CNVs, and detected 3000–4000 CNVs (4– 6% of the genomic sequence) per population that included CNVs previously missed because of smaller sizes and residing in segmental duplications. The patterns of CNVs across individuals were surprisingly simple at the kilo-base scale, suggesting the applicability of a simple genetic analysis for these genetic loci. We utilized the probabilistic theory to determine integer copy numbers of CNVs and employed a recently developed phasing tool to estimate the population frequencies of integer copy number alleles and CNV–SNP haplotypes. The results showed a tendency toward a lower frequency of CNV alleles and that most of our CNVs were explained only by zero-, one- and two-copy alleles. Using the estimated population frequencies, we found several CNV regions with exceptionally high population differentiation. Investigation of CNV–SNP linkage disequilibrium (LD) for 500–900 bi- and multi-allelic CNVs per population revealed that previous conflicting reports on bi-allelic LD were unexpectedly consistent and explained by an LD increase correlated with deletion-allele frequencies. Typically, the bi-allelic LD was lower than SNP–SNP LD, whereas the multi-allelic LD was somewhat stronger than the bi-allelic LD. After further investigation of tag SNPs for CNVs, we conclude that the customary tagging strategy for disease association studies can be applicable for common deletion CNVs, but direct interrogation is needed for other types of CNVs.

The discovery of 'high-risk' de novo copy number variants (CNVs) associated with neuropsychiatric disorders such as schizophrenia offers the opportunity to translate these findings into useful tools for clinical geneticists. However, this will require estimation of penetrance for these variants, which has not yet been properly considered. To facilitate this process, we estimated the penetrance of CNVs associated with schizophrenia, at 15q13.3, 1q21.1, 15q11.2, 17p12, 2p16.3, 16p13.1 and 16p11.2 with a novel Bayesian method applied to pooled data from published case-control studies. For these CNVs, penetrance for schizophrenia was between 2 and 7.4%, which contrasts with the much higher penetrance for schizophrenia of the 22q11.2 deletions found in velo-cardio-facial syndrome. The highest penetrance was for 15q13.3 deletion (6-9% in individual studies) and the lowest was for 15q11.2 (2%). CNVs confer much higher risk for schizophrenia than common variants, but their penetrance is substantially lower than Mendelian disorders or other syndromic conditions. Since these CNVs predispose to multiple disorders, including epilepsy, autism and intellectual impairment, penetrance estimates will also need to take into account diagnostic specificity, and their overall penetrance for any neuropsychiatric disorder is likely to be much higher. Thus, although CNVs are still far from being clinically useful or relevant to genetic counselling for specific disorders, their detection may hold an important clinical value in predicting negative developmental outcomes.

A peculiar form of hepatocerebral mtDNA depletion syndrome is caused by mutations in the MPV17 gene, which encodes a small hydrophobic protein of unknown function located in the mitochondrial inner membrane. In order to define the molecular basis of MPV17 variants associated with the human disorder, we have previously taken advantage of S. cerevisiae as a model system thanks to the presence of an MPV17 ortholog gene, SYM1. We demonstrate here that the SYM1 gene product is essential to maintain OXPHOS, glycogen storage, mitochondrial morphology and mtDNA stability in stressing conditions such as high temperature and ethanol-dependent growth. To gain insight into the molecular basis of the Sym1-less phenotype, we identified and characterized multicopy suppressor genes and metabolic suppressor compounds. Our results suggest that (i) metabolic impairment and mtDNA instability occur independently from each other as a consequence of SYM1 ablation; (ii) ablation of Sym1 causes depletion of glycogen storage, possibly due to defective anaplerotic flux of tricarboxylic acid (TCA) cycle intermediates to the cytosol; (iii) flattening of mitochondrial cristae in Sym1-defective organelles suggests a role for Sym1 in the structural preservation of the inner mitochondrial membrane, which could in turn control mtDNA maintenance and stability.

The fine-scale structure of the majority of copy number variation (CNV) regions remains unknown. The killer immunoglobulin receptor (KIR) gene complex exhibits significant CNV. The evolutionary plasticity of the KIRs and their broad biomedical relevance makes it important to understand how these immune receptors evolve. In this paper, we describe haplotype re-arrangement creating novel loci at the KIR complex. We completely sequenced, after fosmid cloning, two rare contracted haplotypes. Evidence of frequent hybrid KIR genes in samples from many populations suggested that re-arrangements may be frequent and selectively advantageous. We propose mechanisms for formation of novel hybrid KIR genes, facilitated by protrusive non-B DNA structures at transposon recombination sites. The heightened propensity to generate novel hybrid KIR receptors may provide a proactive evolutionary measure, to militate against pathogen evasion or subversion. We propose that CNV in KIR is an evolutionary strategy, which KIR typing for disease association must take into account.

Copy number variants (CNVs) are genomic segments which are duplicated or deleted among different individuals. CNVs have been implicated in both Mendelian and complex traits, including immune and behavioral disorders, but the study of the mechanisms by which CNVs influence gene expression and clinical phenotypes in humans is complicated by the limited access to tissues and by population heterogeneity. We now report studies of the effect of 19 CNVs on gene expression and metabolic traits in a mouse intercross between strains C57BL/6J and C3H/HeJ. We found that 83% of genes predicted to occur within CNVs were differentially expressed. The expression of most CNV genes was correlated with copy number, but we also observed evidence that gene expression was altered in genes flanking CNVs, suggesting that CNVs may contain regulatory elements for these genes. Several CNVs mapped to hotspots, genomic regions influencing expression of tens or hundreds of genes. Several metabolic traits including cholesterol, triglycerides, glucose and body weight mapped to three CNVs in the genome, in mouse chromosomes 1, 4 and 17. Predicted CNV genes, such as Itlna, Defcr-1, Trim12 and Trim34 were highly correlated with these traits. Our results suggest that CNVs have a significant impact on gene expression and that CNVs may be playing a role in the mechanisms underlying metabolic traits in mice.

One of the causes of chronic pancreatitis is the duplication and triplication of a approximately 605 kb segment containing the trypsinogen locus. Employing array-comparative genomic hybridization, we fully characterized the triplication copy number mutation (CNM) and found it to be part of a complex rearrangement that also contains a triplicated approximately 137 kb segment and 21 bp sequence tract. This triplication allele therefore constitutes a gain of two tandemly arranged composite duplication blocks, each comprising a copy of the approximately 605 kb segment, a copy of the inverted approximately 137 kb segment and a copy of the inverted 21 bp sequence tract. As such, it represents the first characterization of a human complex triplication CNM at the DNA sequence level. All triplications and duplications identified were found to arise from a common founder chromosome. A two-step process is proposed for the generation of this highly unusual triplication CNM. Thus, the first composite duplication block is envisaged to have been generated by break-induced serial replication slippage during mitosis. This duplication would have provided the sequence homology required to promote non-allelic homologous recombination (NAHR) during meiosis which would then, in a second step, have generated the complex triplication allele. Our data provide support for the view that many human germline copy number variants arise through replication-based mechanisms during the premeiotic mitotic divisions of germ cells. The low copy repeats thereby generated could then serve to promote NAHR during meiosis, giving rise to amplified DNA sequences which would themselves predispose to further recombinational events during both mitosis and meiosis.

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

Copy number studies have led to an explosion in the discovery of new segmental duplication-mediated deletions and duplications. We have analyzed copy number changes in 2419 patients referred for clinical array comparative genomic hybridization studies. Twenty-three percent of the abnormal copy number changes we found are immediately flanked by segmental duplicationsor =10 kb in size andor =95% identical in direct orientation, consistent with deletions and duplications generated by non-allelic homologous recombination. Here, we describe copy number changes in five previously unreported loci with genomic organization characteristic of NAHR-mediated gains and losses; namely, 2q11.2, 7q36.1, 17q23, 2q13 and 7q11.21. Deletions and duplications of 2q11.2, deletions of 7q36.1 and deletions of 17q23 are interpreted as pathogenic based on their genomic size, gene content, de novo inheritance and absence from control populations. The clinical significance of 2q13 deletions and duplications is still emerging, as these imbalances are also found in phenotypically normal family members and control individuals. Deletion of 7q11.21 is a benign copy number change well represented in control populations and copy number variation databases. Here, we discuss the genetic factors that can modify the phenotypic expression of such gains and losses, which likely play a role in these and other recurrent genomic disorders.

Nance-Horan syndrome (NHS) is an X-linked developmental disorder characterized by congenital cataract, dental anomalies, facial dysmorphism and, in some cases, mental retardation. Protein truncation mutations in a novel gene (NHS) have been identified in patients with this syndrome. We previously mapped X-linked congenital cataract (CXN) in one family to an interval on chromosome Xp22.13 which encompasses the NHS locus; however, no mutations were identified in the NHS gene. In this study, we show that NHS and X-linked cataract are allelic diseases. Two CXN families, which were negative for mutations in the NHS gene, were further analysed using array comparative genomic hybridization. CXN was found to be caused by novel copy number variations: a complex duplication–triplication re-arrangement and an intragenic deletion, predicted to result in altered transcriptional regulation of the NHS gene. Furthermore, we also describe the clinical and molecular analysis of seven families diagnosed with NHS, identifying four novel protein truncation mutations and a novel large deletion encompassing the majority of the NHS gene, all leading to no functional protein. We therefore show that different mechanisms, aberrant transcription of the NHS gene or no functional NHS protein, lead to different diseases. Our data highlight the importance of copy number variation and non-recurrent re-arrangements leading to different severity of disease and describe the potential mechanisms involved.

Altered gene expression in tumors can be caused by copy number alterations to DNA or mutation affecting coding or regulatory regions of genes. However, epigenetic events may also influence gene expression. Malignant cells can show major disruptions in DNA methylation profiles, which are manifested as aberrant hypermethylation or as hypomethylation of gene promoters, as well as global genomic hypomethylation. In this study we performed integrative whole-genome analysis of DNA copy number, promoter methylation and gene expression using 10 osteosarcomas. We identified significant changes including: hypomethylation, gain, and overexpression of histone cluster 2 genes at chromosome 1q21.1-q21.3; loss of chromosome 8p21.2-p21.3 and underexpression of DOCK5 and TNFRSF10A/D genes; and amplification-related overexpression of RUNX2 at chromosome 6p12.3-p21.1. Amplification and overexpression of RUNX2 could disrupt G2/M cell cycle checkpoints, and downstream osteosarcoma-specific changes, such as failure of bone differentiation and genomic polyploidization. Failure of DOCK5-signaling, together with p53 and TNFRSF10A/D-related cell cycle and death pathways, may play a critical role in abrogating apoptosis. Our analyses show that the RUNX2 interactome may be constitutively activated in osteosarcoma, and that the downstream intracellular pathways are strongly associated with the regulation of osteoblast differentiation and control of cell cycle and apoptosis in osteosarcoma.

Autism spectrum disorders (ASDs) constitute a group of severe neurodevelopmental conditions with complex multifactorial etiology. In order to explore the hypothesis that submicroscopic genomic rearrangements underlie some ASD cases, we have analyzed 96 Spanish patients with idiopathic ASD after extensive clinical and laboratory screening, by array comparative genomic hybridization (aCGH) using a homemade bacterial artificial chromosome (BAC) array. Only 13 of the 238 detected copy number alterations, ranging in size from 89 kb to 2.4 Mb, were present specifically in the autistic population (12 out of 96 individuals, 12.5%). Following validation by additional molecular techniques, we have characterized these novel candidate regions containing 24 different genes including alterations in two previously reported regions of chromosome 7 associated with the ASD phenotype. Some of the genes located in ASD-specific copy number variants act in common pathways, most notably the phosphatidylinositol signaling and the glutamatergic synapse, both known to be affected in several genetic syndromes related with autism and previously associated with ASD. Our work supports the idea that the functional alteration of genes in related neuronal networks is involved in the etiology of the ASD phenotype and confirms a significant diagnostic yield for aCGH, which should probably be included in the diagnostic workup of idiopathic ASD.

International audience; The molecular mechanisms that lead to the cognitive defects characteristic of Down syndrome (DS), the most frequent cause of mental retardation, have remained elusive. Here we use a transgenic DS mouse model (152F7 line) to show that DYRK1A gene dosage imbalance deregulates chromosomal clusters of genes located near neuron-restrictive silencer factor (REST/NRSF) binding sites. We found that Dyrk1a binds the SWI/SNF complex known to interact with REST/NRSF. The mutation of a REST/NRSF binding site in the promoter of the REST/NRSF target gene L1cam modifies the transcriptional effect of Dyrk1a-dosage imbalance on L1cam. Dyrk1a dosage imbalance perturbs Rest/Nrsf levels with decreased Rest/Nrsf expression in embryonic neurons and increased expression in adult neurons. Using transcriptome analysis of embryonic brain subregions of transgenic 152F7 mouse line, we identified a coordinated deregulation of multiple genes that are responsible for dendritic growth impairment present in DS. Similarly, Dyrk1a overexpression in primary mouse cortical neurons induced severe reduction of the dendritic growth and dendritic complexity. We propose that DYRK1A overexpression-related neuronal gene deregulation via disturbance of REST/NRSF levels, and the REST/NRSF-SWI/SNF chromatin remodelling complex, significantly contributes to the neural phenotypic changes that characterize DS.

The search for oncogenes is becoming increasingly important in cancer genetics because they are suitable targets for therapeutic intervention. To identify novel oncogenes, activated by gene amplification, we analyzed cDNA microarrays by high-resolution comparative genome hybridization and compared DNA copy number and mRNA expression levels in lung cancer cell lines. We identified several amplicons (5p13, 6p22-21, 11q13, 17q21 and 19q13) that had a concomitant increase in gene expression. These regions were also found to be amplified in lung primary tumours. We mapped the boundaries and measured expression levels of genes within the chromosome 6p amplicon. The Sry-HMG box gene SOX4 (sex-determining region Y box 4), which encodes a transcription factor involved in embryonic cell differentiation, was overexpressed by a factor of 10 in cells with amplification relative to normal cells. SOX4 expression was also stronger in a fraction of lung primary tumours and lung cancer cell lines and was associated with the presence of gene amplification. We also found variants of SOX4 in lung primary tumours and cancer cell lines, including a somatic mutation that introduced a premature stop codon (S395X) at the serine-rich C-terminal domain. Although none of the variants increased the transactivation ability of SOX4, overexpression of the wildtype and of the non-truncated variants in NIH3T3 cells significantly increased the transforming ability of the weakly oncogenic RHOA-Q63L. In conclusion, our results show that, in lung cancer, SOX4 is overexpressed due to gene amplification and provide evidence of oncogenic properties of SOX4. © The Author 2009. Published by Oxford University Press. All rights reserved., The work was supported by grants from the Spanish Ministerio de Educacion (SAF2005-00626) and Instituto de Salud Carlos III (RTICC), to M.S.-C., and Spanish Ministerio de Educacion (SAF2007-60242 and CSD2007-0017), Junta de Castilla y Leon (Consejeria de Educacion, CSI-14A08 and GR-15) to P.A.L. Fellowships to M.S.-G. and S.D.C. were granted by the CSIC and Comunidad Autónoma de Madrid, respectively.

Amyotrophic lateral sclerosis (ALS) is an unrelenting neurodegenerative condition characterized by adult-onset loss of motor neurons. Genetic risk factors have been implicated in ALS susceptibility. Copy number variants (CNVs) account for more inter-individual genetic variation than SNPs and have the capacity to alter gene dose and phenotype. We sought to identify the contribution both of commonly polymorphic CNVs and rare ALS-specific CNVs to sporadic ALS (SALS). Using high-density genome-wide data from 408 Irish individuals and 868 Dutch individuals and the QuantiSNP CNV-detection algorithm, we showed that no common CNV locus is significantly associated with ALS risk. However, we identified 39 recurrent CNV loci and 16 replicated ALS-specific gene dose alterations that occur exclusively in patients with ALS and do not occur in more than 11 000 previously identified CNVs in the Database of Genomic Variation. Ataxin genes and the hereditary haemochromatosis locus were implicated along with ENSG00000176605, an uncharacterized gene on chromosome 14. Our data support the hypothesis that multiple rare CNVs may contribute risk for SALS. Future work should seek to profile the contribution of CNVs located in regions not covered on the present SNP platforms.

Haploinsufficiency of the transcription co-activator EYA1 causes branchio–oto–renal syndrome, congenital birth defects that account for as many as 2% of profoundly deaf children; however, the underlying cause for its dosage requirement and its specific role in sensory cell development of the inner ear are unknown. Here, an allelic series of Eya1 were generated to study the basis of Eya1 dosage requirements for sensory organ development. Our results show different threshold requirements for the level of Eya1 in different regions of the inner ear. Short and disorganized hair cell sterocilia was observed in wild-type/null heterozygous or hypomorphic/hypomorphic homozygous cochleae. Patterning and gene-marker analyses indicate that in Eya1 hypomorphic/null heterozygous mice, a reduction of Eya1 expression to 21% of normal level causes an absence of cochlear and vestibular sensory formation. Eya1 is initially expressed in the progenitors throughout the epithelium of all six sensory regions, and later on during sensory cell differentiation, its expression becomes restricted to the differentiating hair cells. We provide genetic evidence that Eya1 activity, in a concentration-dependent manner, plays a key role in the regulation of genes known to be important for sensory development. Furthermore, we show that Eya1 co-localizes with Sox2 in the sensory progenitors and both proteins physically interact. Together, our results indicate that Eya1 appears to be upstream of very early events during the sensory organ development, hair cell differentiation and inner-ear patterning. These results also provide a molecular mechanism for understanding how hypomorphic levels of EYA1 cause inner-ear defects in humans.

22q11.2 Deletion Syndrome (22q11.2DS) is a common microdeletion syndrome with congenital and late-onset features. Testing for the genomic content of copy number variations (CNVs) may help elucidate the 22q11.2 deletion mechanism and the variable clinical expression of the syndrome including the high (25%) risk for schizophrenia. We used genome-wide microarrays to assess CNV content and the parental origin of 22q11.2 deletions in a cohort of 100 adults with 22q11.2DS (44 with schizophrenia) and controls. 22q11.2DS subjects with schizophrenia failed to exhibit de novo CNVs or any excess of novel inherited CNVs outside the 22q11.2 region. There were no significant effects of parental origin of the 22q11.2 deletion, deletion length, parental age or family history on expression of schizophrenia. There was no evidence for a general increase of de novo CNVs in 22q11.2DS. A novel finding was the relative paucity of males with de novo 22q11.2 deletions of paternal origin (P = 0.019). The Y chromosome may play a mediating role in the mechanism of 22q11.2 deletion events during spermatogenesis, resulting in the previously observed excess of maternal de novo 22q11.2 deletions. Hemizygosity of the 22q11.2 region appears to be the major CNV-related risk factor for schizophrenia in 22q11.2DS. The results reinforce the need for further efforts to identify specific molecular mechanisms underlying this expression and to identify the 1% of patients with schizophrenia who carry 22q11.2 deletions.

Retinoblastoma clinical observations revealed the role of tumor suppressor genes in human cancer, Knudson's 'two-hit' model of cancer induction. We now demonstrate that loss of both RB1 tumor suppressor gene alleles initiates quiescent RB1(-/-) retinomas with low level genomic instability and high expression of the senescence-associated proteins p16(INK4a) and p130. Although retinomas can remain unchanged throughout life, highly proliferative, clonal and aneuploid retinoblastomas commonly emerge, exhibiting altered gene copy number and expression of oncogenes (MYCN, E2F3, DEK, KIF14 and MDM4) and tumor suppressor genes (CDH11, p75(NTR)) and reduced expression of p16(INK4a) and p130. We suggest that RB1 inactivation in developing retina induces genomic instability, but senescence can block transformation at the stage of retinoma. However, stable retinoma is rarely clinically observed because progressive genomic instability commonly leads to highly proliferative retinoblastoma.

Segmental duplications or low-copy repeats (LCRs) constitute approximately 5% of the sequenced portion of the human genome and are associated with many human congenital anomaly disorders. The low-copy repeats on chromosome 22q11.2 (LCR22s) mediate chromosomal rearrangements resulting in deletions, duplications and translocations. The evolutionary mechanisms leading to LCR22 formation is unknown. Four genes, USP18, BCR, GGTLA and GGT, map adjacent to the LCR22s and pseudogene copies are located within them. It has been hypothesized that gene duplication occurred during primate evolution, followed by recombination events, forming pseudogene copies. We investigated whether gene duplication could be detected in non-human hominoid species. FISH mapping was performed using probes to the four functional gene loci. There was evidence for a single copy in humans but additional copies in hominoid species. We then compared LCR22 copy number using LCR22 FISH probes. Lineage specific LCR22 variation was detected in the hominoid species supporting the hypothesis. To independently validate initial findings, real time PCR, and screening of gorilla BAC library filters were performed. This was compared to array comparative genome hybridization data available. The most striking finding was a dramatic amplification of LCR22s in the gorilla. The LCR22s localized to the telomeric or subtelomeric bands of gorilla chromosomes. The most parsimonious explanation is that the LCR22s became amplified by inter-chromosomal recombination between telomeric bands. In summary, our results are consistent with a lineage specific coupling between gene and LCR22 duplication events. The LCR22s thus serve as an important model for evolution of genome variation.

Somatic copy number alterations (SCNAs) play an important role in carcinogenesis. However, the impact of genomic architecture on the global patterns of SCNAs in cancer genomes remains elusive. In this work, we conducted multiple linear regression (MLR) analyses of the pooled SCNA data from The Cancer Genome Atlas (TCGA) Pan-Cancer project. We performed MLR analyses for 11 individual cancer types and three different kinds of SCNAs--amplifications and deletions, telomere-bound and interstitial SCNAs and local SCNAs. Our MLR model explains >30% of the pooled SCNA breakpoint variation, with the explanatory power ranging from 13 to 32% for different cancer types and SCNA types. In addition to confirming previously identified features [e.g. long interspersed element-1 (L1) and short interspersed nuclear elements], we also identified several novel informative features, including distance to telomere, distance to centromere and low-complexity repeats. The results of the MLR analyses were additionally confirmed on an independent SCNA data set obtained from the catalogue of somatic mutations in cancer database. Using a rare-event logistic regression model and an extremely randomized tree classifier, we revealed that genomic features are informative for defining common SCNA breakpoint hotspots. Our findings shed light on the molecular mechanisms of SCNA generation in cancer.

Genetic abnormalities frequently give rise to a mental retardation phenotype. Recent advances in resolution of comparative genomic hybridization and genomic sequence annotation has identified new syndromes at chromosome 3q29 and 9q34. The finding of a significant number of copy number polymorphisms in the genome in the normal population, means that assigning pathogenicity to deletions and duplications in patients with mental retardation can be difficult but has been identified for duplications of MECP2 and L1CAM. Novel autosomal genes that cause mental retardation have been identified recently including CC2D1A identified by homozygosity mapping. Several new genes and pathways have been identified in the field of X-linked mental retardation but many more still await identification. Analysis of families where only a single male is affected reveals that the chance of this being due to a single X-linked gene abnormality is significantly less than would be expected if the excess of males in the population is entirely due to X-linked disease. Recent identification of novel X-linked mental retardation genes has identified components of the post-synaptic density and multiple zinc finger transcription factors as disease causing suggesting new mechanisms of disease causation. The first therapeutic treatments of animal models of mental retardation have been reported, a Drosophila model of Fragile X syndrome has been treated with lithium or metabotropic glutamate receptor (mGluR) antagonists and a mouse model of NF1 has been treated with the HMG-CoA reductase inhibitor lavastatin, which improves the learning and memory skills in these models.