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1.  Haploinsufficiency of DNA Damage Response Genes and their Potential Influence in Human Genomic Disorders 
Current Genomics  2008;9(3):137-146.
Genomic disorders are a clinically diverse group of conditions caused by gain, loss or re-orientation of a genomic region containing dosage-sensitive genes. One class of genomic disorder is caused by hemizygous deletions resulting in haploinsufficiency of a single or, more usually, several genes. For example, the heterozygous contiguous gene deletion on chromosome 22q11.2 causing DiGeorge syndrome involves at least 20-30 genes. Determining how the copy number variation (CNV) affects human variation and contributes to the aetiology and progression of various genomic disorders represents important questions for the future. Here, I will discuss the functional significance of one form of CNV, haploinsufficiency (i.e. loss of a gene copy), of DNA damage response components and its association with certain genomic disorders. There is increasing evidence that haploinsufficiency for certain genes encoding key players in the cells response to DNA damage, particularly those of the Ataxia Telangiectasia and Rad3-related (ATR)-pathway, has a functional impact. I will review this evidence and present examples of some well known clinically similar genomic disorders that have recently been shown to be defective in the ATR-dependent DNA damage response. Finally, I will discuss the potential implications of a haploinsufficiency-induced defective DNA damage response for the clinical management of certain human genomic disorders.
PMCID: PMC2679649  PMID: 19440510
DNA damage response; ATR; haploinsufficiency; genomic disorders.
2.  Novel Interactions between Actin and the Proteasome Revealed by Complex Haploinsufficiency 
PLoS Genetics  2011;7(9):e1002288.
Saccharomyces cerevisiae has been a powerful model for uncovering the landscape of binary gene interactions through whole-genome screening. Complex heterozygous interactions are potentially important to human genetic disease as loss-of-function alleles are common in human genomes. We have been using complex haploinsufficiency (CHI) screening with the actin gene to identify genes related to actin function and as a model to determine the prevalence of CHI interactions in eukaryotic genomes. Previous CHI screening between actin and null alleles for non-essential genes uncovered ∼240 deleterious CHI interactions. In this report, we have extended CHI screening to null alleles for essential genes by mating a query strain to sporulations of heterozygous knock-out strains. Using an act1Δ query, knock-outs of 60 essential genes were found to be CHI with actin. Enriched in this collection were functional categories found in the previous screen against non-essential genes, including genes involved in cytoskeleton function and chaperone complexes that fold actin and tubulin. Novel to this screen was the identification of genes for components of the TFIID transcription complex and for the proteasome. We investigated a potential role for the proteasome in regulating the actin cytoskeleton and found that the proteasome physically associates with actin filaments in vitro and that some conditional mutations in proteasome genes have gross defects in actin organization. Whole-genome screening with actin as a query has confirmed that CHI interactions are important phenotypic drivers. Furthermore, CHI screening is another genetic tool to uncover novel functional connections. Here we report a previously unappreciated role for the proteasome in affecting actin organization and function.
Author Summary
Individuals inherit two copies of each gene, one from each parent, and frequently the two copies are different from each other. Sometimes one copy is completely defective, but since there is one normal copy there may be no negative consequences. Our research is focused on understanding the consequence of inheriting one bad copy for two or more different genes. Geneticists refer to inheriting one bad copy of a gene as a haploinsufficiency and inheriting one bad copy of multiple genes as a complex haploinsufficiency. Using yeast as a model system, we have addressed, in a systematic way, what occurs if a cell inherits one bad copy of the gene called actin and one bad copy of one of the ∼1,000 essential genes in the yeast genome. We discovered that complex haploinsufficiency between actin and one of 60 different essential genes leads to reduced cell viability. These 60 genes are highly enriched for certain functional groups, including those involved in protein degradation and gene expression. It is expected that approaches such as these in model organisms will be applicable to understanding the potential for deleterious complex haploinsufficient interactions in the human genome.
PMCID: PMC3178594  PMID: 21966278
3.  MYBL2 is a sub-haploinsufficient tumor suppressor gene in myeloid malignancy 
eLife  2013;2:e00825.
A common deleted region (CDR) in both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN) affects the long arm of chromosome 20 and has been predicted to harbor a tumor suppressor gene. Here we show that MYBL2, a gene within the 20q CDR, is expressed at sharply reduced levels in CD34+ cells from most MDS cases (65%; n = 26), whether or not they harbor 20q abnormalities. In a murine competitive reconstitution model, Mybl2 knockdown by RNAi to 20–30% of normal levels in multipotent hematopoietic progenitors resulted in clonal dominance of these ‘sub-haploinsufficient’ cells, which was reflected in all blood cell lineages. By 6 months post-transplantation, the reconstituted mice had developed a clonal myeloproliferative/myelodysplastic disorder originating from the cells with aberrantly reduced Mybl2 expression. We conclude that downregulation of MYBL2 activity below levels predicted by classical haploinsufficiency underlies the clonal expansion of hematopoietic progenitors in a large fraction of human myeloid malignancies.
eLife digest
Blood cells are produced within bone marrow by specialized stem cells and progenitor cells. Abnormalities in this process lead to a group of diseases known as myeloid malignancies, which include acute myeloid leukaemia—in which the bone marrow produces abnormal white blood cells—and myelodysplastic syndromes, which are caused by too few mature blood cells being produced.
Many individuals affected by these disorders possess a shortened form of chromosome 20 that lacks a number of genes. This deletion is only ever seen in one of their two copies of the chromosome—suggesting that at least some of these genes are essential for survival—but the identity of the gene(s) that are associated with the increased risk of myeloid malignancies is unknown.
Now, Heinrichs et al. have uncovered a key tumor suppressor among those genes frequently lost on chromosome 20. The gene, which is called MYBL2, encodes a transcription factor that helps to control the cell division cycle. Myeloid malignancy patients lacking one copy of this gene showed levels of MYBL2 expression that were less than 50% of those in healthy individuals. This suggests that additional mechanisms must be acting to reduce expression of their remaining copy of the gene. Surprisingly, MYBL2 levels were also reduced in myeloid malignancy patients who possessed two intact copies of chromosome 20, indicating that loss of a single copy represents only one mechanism to reduce MYBL2 expression, i.e., the ‘tip-of-the-iceberg’. Hence, this finding reveals a more general role for MYBL2 as it indicates that more patients are likely to be affected by altered expression of this gene.
To confirm their findings from studies in patients, Heinrichs et al. used gene silencing techniques to reduce the expression of MYBL2 in mice and showed that this induced symptoms of myeloid malignancies in the animals. Moreover, injection of modified cells from these animals into healthy mice also induced symptoms in the recipients. The modified cells are able to expand more robustly than normal cells, and this dominance induced by downregulation of the tumor suppressor increases the risk of malignancy.
In addition to revealing a new tumor suppressor gene and its contribution to myeloid malignancies, the study by Heinrichs et al. highlights the importance of gene dosage in mediating the effects of tumor suppressors.
PMCID: PMC3713455  PMID: 23878725
Myelodysplastic Syndromes; MYBL2; 20q CDR; Human; Mouse
4.  Haploinsufficiency and the sex chromosomes from yeasts to humans 
BMC Biology  2011;9:15.
Haploinsufficient (HI) genes are those for which a reduction in copy number in a diploid from two to one results in significantly reduced fitness. Haploinsufficiency is increasingly implicated in human disease, and so predicting this phenotype could provide insights into the genetic mechanisms behind many human diseases, including some cancers.
In the present work we show that orthologues of Saccharomyces cerevisiae HI genes are preferentially retained across the kingdom Fungi, and that the HI genes of S. cerevisiae can be used to predict haploinsufficiency in humans. Our HI gene predictions confirm known associations between haploinsufficiency and genetic disease, and predict several further disorders in which the phenotype may be relevant. Haploinsufficiency is also clearly relevant to the gene-dosage imbalances inherent in eukaryotic sex-determination systems. In S. cerevisiae, HI genes are over-represented on chromosome III, the chromosome that determines yeast's mating type. This may be a device to select against the loss of one copy of chromosome III from a diploid. We found that orthologues of S. cerevisiae HI genes are also over-represented on the mating-type chromosomes of other yeasts and filamentous fungi. In animals with heterogametic sex determination, accumulation of HI genes on the sex chromosomes would compromise fitness in both sexes, given X chromosome inactivation in females. We found that orthologues of S. cerevisiae HI genes are significantly under-represented on the X chromosomes of mammals and of Caenorhabditis elegans. There is no X inactivation in Drosophila melanogaster (increased expression of X in the male is used instead) and, in this species, we found no depletion of orthologues to yeast HI genes on the sex chromosomes.
A special relationship between HI genes and the sex/mating-type chromosome extends from S. cerevisiae to Homo sapiens, with the microbe being a useful model for species throughout the evolutionary range. Furthermore, haploinsufficiency in yeast can predict the phenotype in higher organisms.
PMCID: PMC3058074  PMID: 21356089
5.  Deregulated FGF and homeotic gene expression underlies cerebellar vermis hypoplasia in CHARGE syndrome 
eLife  2013;2:e01305.
Mutations in CHD7 are the major cause of CHARGE syndrome, an autosomal dominant disorder with an estimated prevalence of 1/15,000. We have little understanding of the disruptions in the developmental programme that underpin brain defects associated with this syndrome. Using mouse models, we show that Chd7 haploinsufficiency results in reduced Fgf8 expression in the isthmus organiser (IsO), an embryonic signalling centre that directs early cerebellar development. Consistent with this observation, Chd7 and Fgf8 loss-of-function alleles interact during cerebellar development. CHD7 associates with Otx2 and Gbx2 regulatory elements and altered expression of these homeobox genes implicates CHD7 in the maintenance of cerebellar identity during embryogenesis. Finally, we report cerebellar vermis hypoplasia in 35% of CHARGE syndrome patients with a proven CHD7 mutation. These observations provide key insights into the molecular aetiology of cerebellar defects in CHARGE syndrome and link reduced FGF signalling to cerebellar vermis hypoplasia in a human syndrome.
eLife digest
CHARGE syndrome is a rare genetic condition that causes various developmental abnormalities, including heart defects, deafness and neurological defects. In most cases, it is caused by mutations in a human gene called CHD7. CHD7 is known to control the expression of other genes during embryonic development, but the molecular mechanisms by which mutations in CHD7 lead to the neural defects found in CHARGE syndrome are unclear.
During embryonic development, the neural tube—the precursor to the nervous system—is divided into segments, which give rise to different neural structures. The r1 segment, for example, forms the cerebellum, and the secretion of a protein called FGF8 (short for fibroblast growth factor 8) by a nearby structure called the isthmus organiser has an important role in this process. Since a reduction in FGF8 causes defects similar to those found in CHARGE syndrome, Yu et al. decided to investigate if the FGF signalling pathway was involved in this syndrome.
Mice should have two working copies of the Chd7 gene, and mice that lack one of these suffer from symptoms similar to those of humans with CHARGE syndrome. Yu et al. examined the embryos of these mice and found that the isthmus organiser produced less FGF8. Embryos with no working copies of the gene completely lost the r1 segment. The loss of this segment appeared to be caused by changes in the expression of homeobox genes (the genes that determine the identity of brain segments).
Embryos that did not have any working copies of the Chd7 gene died early in development, which made further studies impossible. However, embryos that had one working copy of the Chd7 gene survived, and Yu et al. took advantage of this to study the effects of reduced FGF8 expression on these mice. These experiments showed that mice with just one working copy of the Fgf8 gene and one working copy of the Chd7 gene had a small cerebellar vermis. This part of the cerebellum is known to be very sensitive to changes in FGF8 signalling. Yu et al. then used an MRI scanner to look at the cerebellar vermis in patients with CHARGE syndrome, and found that more than half of the patients had abnormal cerebella.
In addition to confirming that studies on mouse embryos can provide insights into human disease, the work of Yu et al. add defects in the cerebellar vermis to the list of developmental abnormalities associated with CHARGE syndrome. The next step will be to test if any mutations in the human FGF8 gene can contribute to cerebellar defects in CHARGE syndrome, and to investigate if any other developmental defects in CHARGE syndrome are associated with abnormal FGF8 levels.
PMCID: PMC3870572  PMID: 24368733
cerebellum; CHARGE syndrome; CHD7; FGF8; OTX2; GBX2; Human; Mouse
6.  Evidence of perturbations of cell cycle and DNA repair pathways as a consequence of human and murine NF1-haploinsufficiency 
BMC Genomics  2010;11:194.
Neurofibromatosis type 1 (NF1) is a common monogenic tumor-predisposition disorder that arises secondary to mutations in the tumor suppressor gene NF1. Haploinsufficiency of NF1 fosters a permissive tumorigenic environment through changes in signalling between cells, however the intracellular mechanisms for this tumor-promoting effect are less clear. Most primary human NF1+/- cells are a challenge to obtain, however lymphoblastoid cell lines (LCLs) have been collected from large NF1 kindreds. We hypothesized that the genetic effects of NF1-haploinsufficiency may be discerned by comparison of genome-wide transcriptional profiling in somatic, non-tumor cells (LCLs) from NF1-affected and -unaffected individuals. As a cross-species filter for heterogeneity, we compared the results from two human kindreds to whole-genome transcriptional profiling in spleen-derived B lymphocytes from age- and gender-matched Nf1+/- and wild-type mice, and used gene set enrichment analysis (GSEA), Onto-Express, Pathway-Express and MetaCore tools to identify genes perturbed in NF1-haploinsufficiency.
We observed moderate expression of NF1 in human LCLs and of Nf1 in CD19+ mouse B lymphocytes. Using the t test to evaluate individual transcripts, we observed modest expression differences in the transcriptome in NF1-haploinsufficient LCLs and Nf1-haploinsuffiicient mouse B lymphocytes. However, GSEA, Onto-Express, Pathway-Express and MetaCore analyses identified genes that control cell cycle, DNA replication and repair, transcription and translation, and immune response as the most perturbed in NF1-haploinsufficient conditions in both human and mouse.
Haploinsufficiency arises when loss of one allele of a gene is sufficient to give rise to disease. Haploinsufficiency has traditionally been viewed as a passive state. Our observations of perturbed, up-regulated cell cycle and DNA repair pathways may functionally contribute to NF1-haploinsufficiency as an "active state" that ultimately promotes the loss of the wild-type allele.
PMCID: PMC2858150  PMID: 20307317
7.  Molecular Mechanisms Generating and Stabilizing Terminal 22q13 Deletions in 44 Subjects with Phelan/McDermid Syndrome 
PLoS Genetics  2011;7(7):e1002173.
In this study, we used deletions at 22q13, which represent a substantial source of human pathology (Phelan/McDermid syndrome), as a model for investigating the molecular mechanisms of terminal deletions that are currently poorly understood. We characterized at the molecular level the genomic rearrangement in 44 unrelated patients with 22q13 monosomy resulting from simple terminal deletions (72%), ring chromosomes (14%), and unbalanced translocations (7%). We also discovered interstitial deletions between 17–74 kb in 9% of the patients. Haploinsufficiency of the SHANK3 gene, confirmed in all rearrangements, is very likely the cause of the major neurological features associated with PMS. SHANK3 mutations can also result in language and/or social interaction disabilities. We determined the breakpoint junctions in 29 cases, providing a realistic snapshot of the variety of mechanisms driving non-recurrent deletion and repair at chromosome ends. De novo telomere synthesis and telomere capture are used to repair terminal deletions; non-homologous end-joining or microhomology-mediated break-induced replication is probably involved in ring 22 formation and translocations; non-homologous end-joining and fork stalling and template switching prevail in cases with interstitial 22q13.3. For the first time, we also demonstrated that distinct stabilizing events of the same terminal deletion can occur in different early embryonic cells, proving that terminal deletions can be repaired by multistep healing events and supporting the recent hypothesis that rare pathogenic germline rearrangements may have mitotic origin. Finally, the progressive clinical deterioration observed throughout the longitudinal medical history of three subjects over forty years supports the hypothesis of a role for SHANK3 haploinsufficiency in neurological deterioration, in addition to its involvement in the neurobehavioral phenotype of PMS.
Author Summary
Terminal chromosome deletions are among the most commonly observed rearrangements detected by cytogenetics and may result in several well-known genetic syndromes. We used 22q13 deletions to study how these types of chromosome abnormalities arise. Children with Phelan/McDermid syndrome, caused by deletion of the terminal portion of chromosome 22, experience developmental delay, absent or severely delayed speech, and frequent behavioral problems. Lack of one copy of SHANK3, a key gene for the correct development and organization of brain synapses, is very likely the basis of the syndrome's major neurological features. Deletion of additional genes probably causes more complex phenotypes in subjects with larger deletions. We also studied patients who only lack a portion of SHANK3 and demonstrated that small, hard-to-detect deletions of this gene may cause substantial clinical problems. Until now, the 22q distal deletion had been only diagnosed in very young people. We studied a large group of patients of different ages and discovered that all adult patients face progressive cognitive decline. Our study demonstrates that deletion of the terminal portion of chromosome 22, a prototype for terminal deletions in human chromosomes, can occur in several ways. Mosaic deletions of different size can also form in early embryogenesis.
PMCID: PMC3136441  PMID: 21779178
8.  Secretory Pathway Stress Responses as Possible Mechanisms of Disease Involving Golgi Ca2+ Pump Dysfunction 
Biofactors (Oxford, England)  2011;37(3):150-158.
In mammalian tissues, uptake of Ca2+ and Mn2+ by Golgi membranes is mediated by the secretory pathway Ca2+- ATPases, SPCA1 and SPCA2, encoded by the ATP2C1 and ATP2C2 genes. Loss of one copy of the ATP2C1 gene, which causes SPCA1 haploinsufficiency, leads to squamous cell tumors of keratinized epithelia in mice and to Hailey-Hailey Disease, an acantholytic skin disease, in humans. Although the disease phenotypes resulting from SPCA1 haploinsufficiency in mice and humans are quite different, each species-specific phenotype is remarkably similar to those arising as a result of null mutations in one copy of the ATP2A2 gene, encoding SERCA2, the endoplasmic reticulum (ER) Ca2+ pump. SERCA2 haploinsufficiency, like SPCA1 haploinsufficiency, causes squamous cell tumors in mice and Darier’s Disease, also an acantholytic skin disease, in humans. The phenotypic similarities between SPCA1 and SERCA2 haploinsufficiency in the two species, and the general functions of the two pumps in consecutive compartments of the secretory pathway, suggest that the underlying disease mechanisms are similar. In this review we discuss evidence supporting the view that chronic Golgi stress and/or ER stress resulting from Ca2+ pump haploinsufficiencies leads to activation of cellular stress responses in keratinocytes, with the predominance of pro-apoptotic pathways (though not necessarily apoptosis itself) leading to acantholytic skin disease in humans and the predominance of pro-survival pathways leading to tumors in mice.
PMCID: PMC3338190  PMID: 21674634
secretory pathway stress; Golgi stress; endoplasmic reticulum stress; acantholysis; Darier disease; cornification; unfolded protein response
9.  Network modeling of the transcriptional effects of copy number aberrations in glioblastoma 
DNA copy number aberrations (CNAs) are a characteristic feature of cancer genomes. In this work, Rebecka Jörnsten, Sven Nelander and colleagues combine network modeling and experimental methods to analyze the systems-level effects of CNAs in glioblastoma.
We introduce a modeling approach termed EPoC (Endogenous Perturbation analysis of Cancer), enabling the construction of global, gene-level models that causally connect gene copy number with expression in glioblastoma.On the basis of the resulting model, we predict genes that are likely to be disease-driving and validate selected predictions experimentally. We also demonstrate that further analysis of the network model by sparse singular value decomposition allows stratification of patients with glioblastoma into short-term and long-term survivors, introducing decomposed network models as a useful principle for biomarker discovery.Finally, in systematic comparisons, we demonstrate that EPoC is computationally efficient and yields more consistent results than mRNA-only methods, standard eQTL methods, and two recent multivariate methods for genotype–mRNA coupling.
Gains and losses of chromosomal material (DNA copy number aberrations; CNAs) are a characteristic feature of cancer genomes. At the level of a single locus, it is well known that increased copy number (gene amplification) typically leads to increased gene expression, whereas decreased copy number (gene deletion) leads to decreased gene expression (Pollack et al, 2002; Lee et al, 2008; Nilsson et al, 2008). However, CNAs also affect the expression of genes located outside the amplified/deleted region itself via indirect mechanisms. To fully understand the action of CNAs, it is therefore necessary to analyze their action in a network context. Toward this goal, improved computational approaches will be important, if not essential.
To determine the global effects on transcription of CNAs in the brain tumor glioblastoma, we develop EPoC (Endogenous Perturbation analysis of Cancer), a computational technique capable of inferring sparse, causal network models by combining genome-wide, paired CNA- and mRNA-level data. EPoC aims to detect disease-driving copy number aberrations and their effect on target mRNA expression, and stratify patients into long-term and short-term survivors. Technically, EPoC relates CNA perturbations to mRNA responses by matrix equations, derived from a steady-state approximation of the transcriptional network. Patient prognostic scores are obtained from singular value decompositions of the network matrix. The models are constructed by solving a large-scale, regularized regression problem.
We apply EPoC to glioblastoma data from The Cancer Genome Atlas (TCGA) consortium (186 patients). The identified CNA-driven network comprises 10 672 genes, and contains a number of copy number-altered genes that control multiple downstream genes. Highly connected hub genes include well-known oncogenes and tumor supressor genes that are frequently deleted or amplified in glioblastoma, including EGFR, PDGFRA, CDKN2A and CDKN2B, confirming a clear association between these aberrations and transcriptional variability of these brain tumors. In addition, we identify a number of hub genes that have previously not been associated with glioblastoma, including interferon alpha 1 (IFNA1), myeloid/lymphoid or mixed-lineage leukemia translocated to 10 (MLLT10, a well-known leukemia gene), glutamate decarboxylase 2 GAD2, a postulated glutamate receptor GPR158 and Necdin (NDN). Furthermore, we demonstrate that the network model contains useful information on downstream target genes (including stem cell regulators), and possible drug targets.
We proceed to explore the validity of a small network region experimentally. Introducing experimental perturbations of NDN and other targets in four glioblastoma cell lines (T98G, U-87MG, U-343MG and U-373MG), we confirm several predicted mechanisms. We also demonstrate that the TCGA glioblastoma patients can be stratified into long-term and short-term survivors, using our proposed prognostic scores derived from a singular vector decomposition of the network model. Finally, we compare EPoC to existing methods for mRNA networks analysis and expression quantitative locus methods, and demonstrate that EPoC produces more consistent models between technically independent glioblastoma data sets, and that the EPoC models exhibit better overlap with known protein–protein interaction networks and pathway maps.
In summary, we conclude that large-scale integrative modeling reveals mechanistically and prognostically informative networks in human glioblastoma. Our approach operates at the gene level and our data support that individual hub genes can be identified in practice. Very large aberrations, however, cannot be fully resolved by the current modeling strategy.
DNA copy number aberrations (CNAs) are a hallmark of cancer genomes. However, little is known about how such changes affect global gene expression. We develop a modeling framework, EPoC (Endogenous Perturbation analysis of Cancer), to (1) detect disease-driving CNAs and their effect on target mRNA expression, and to (2) stratify cancer patients into long- and short-term survivors. Our method constructs causal network models of gene expression by combining genome-wide DNA- and RNA-level data. Prognostic scores are obtained from a singular value decomposition of the networks. By applying EPoC to glioblastoma data from The Cancer Genome Atlas consortium, we demonstrate that the resulting network models contain known disease-relevant hub genes, reveal interesting candidate hubs, and uncover predictors of patient survival. Targeted validations in four glioblastoma cell lines support selected predictions, and implicate the p53-interacting protein Necdin in suppressing glioblastoma cell growth. We conclude that large-scale network modeling of the effects of CNAs on gene expression may provide insights into the biology of human cancer. Free software in MATLAB and R is provided.
PMCID: PMC3101951  PMID: 21525872
cancer biology; cancer genomics; glioblastoma
10.  A Duplication CNV That Conveys Traits Reciprocal to Metabolic Syndrome and Protects against Diet-Induced Obesity in Mice and Men 
PLoS Genetics  2012;8(5):e1002713.
The functional contribution of CNV to human biology and disease pathophysiology has undergone limited exploration. Recent observations in humans indicate a tentative link between CNV and weight regulation. Smith-Magenis syndrome (SMS), manifesting obesity and hypercholesterolemia, results from a deletion CNV at 17p11.2, but is sometimes due to haploinsufficiency of a single gene, RAI1. The reciprocal duplication in 17p11.2 causes Potocki-Lupski syndrome (PTLS). We previously constructed mouse strains with a deletion, Df(11)17, or duplication, Dp(11)17, of the mouse genomic interval syntenic to the SMS/PTLS region. We demonstrate that Dp(11)17 is obesity-opposing; it conveys a highly penetrant, strain-independent phenotype of reduced weight, leaner body composition, lower TC/LDL, and increased insulin sensitivity that is not due to alteration in food intake or activity level. When fed with a high-fat diet, Dp(11)17/+ mice display much less weight gain and metabolic change than WT mice, demonstrating that the Dp(11)17 CNV protects against metabolic syndrome. Reciprocally, Df(11)17/+ mice with the deletion CNV have increased weight, higher fat content, decreased HDL, and reduced insulin sensitivity, manifesting a bona fide metabolic syndrome. These observations in the deficiency animal model are supported by human data from 76 SMS subjects. Further, studies on knockout/transgenic mice showed that the metabolic consequences of Dp(11)17 and Df(11)17 CNVs are not only due to dosage alterations of Rai1, the predominant dosage-sensitive gene for SMS and likely also PTLS. Our experiments in chromosome-engineered mouse CNV models for human genomic disorders demonstrate that a CNV can be causative for weight/metabolic phenotypes. Furthermore, we explored the biology underlying the contribution of CNV to the physiology of weight control and energy metabolism. The high penetrance, strain independence, and resistance to dietary influences associated with the CNVs in this study are features distinct from most SNP–associated metabolic traits and further highlight the potential importance of CNV in the etiology of both obesity and MetS as well as in the protection from these traits.
Author Summary
Genetic factors play a large role in obesity. However, despite recent technical progress in the search for genetic variants, the identities of causative and contributory genetic factors remain largely unknown. Whereas nucleotide sequence variation has been studied extensively with respect to its potential contribution to obesity, copy number variations (CNV), in which genes exist in abnormal numbers of copies mostly due to duplication or deletion, have only more recently been observed to be associated with human obesity. In this report, we utilize chromosome engineered mouse strains harboring a deletion or duplication CNV to address the potential functional impact of CNVs on weight control and metabolism. We show that the duplication CNV leads to lower body weight; it is also metabolically advantageous and protects from diet-induced obesity and metabolic syndrome (MetS). The deletion CNV causes a “mirror” phenotype with increased body weight and MetS–like phenotypes. Importantly, these effects manifest regardless of the genetic background and do not appear to be attributable to any single gene. These findings demonstrate experimentally that CNV can be causative for weight and metabolic phenotypes and highlight the potential relevance and importance of CNV in the etiology of obesity/MetS and the protection from these traits.
PMCID: PMC3359973  PMID: 22654670
11.  The role of noise and positive feedback in the onset of autosomal dominant diseases 
BMC Systems Biology  2010;4:93.
Autosomal dominant (AD) diseases result when a single mutant or non-functioning gene is present on an autosomal chromosome. These diseases often do not emerge at birth. There are presently two prevailing theories explaining the expression of AD diseases. One explanation originates from the Knudson two-hit theory of hereditary cancers, where loss of heterozygosity or occurrence of somatic mutations impairs the function of the wild-type copy. While these somatic second hits may be sufficient for stable disease states, it is often difficult to determine if their occurrence necessarily marks the initiation of disease progression. A more direct consequence of a heterozygous genetic background is haploinsufficiency, referring to a lack of sufficient gene function due to reduced wild-type gene copy number; however, haploinsufficiency can involve a variety of additional mechanisms, such as noise in gene expression or protein levels, injury and second hit mutations in other genes. In this study, we explore the possible contribution to the onset of autosomal dominant diseases from intrinsic factors, such as those determined by the structure of the molecular networks governing normal cellular physiology.
First, simple models of single gene insufficiency using the positive feedback loops that may be derived from a three-component network were studied by computer simulation using Bionet software. The network structure is shown to affect the dynamics considerably; some networks are relatively stable even when large stochastic variations in are present, while others exhibit switch-like dynamics. In the latter cases, once the network switches over to the disease state it remains in that state permanently. Model pathways for two autosomal dominant diseases, AD polycystic kidney disease and mature onset diabetes of youth (MODY) were simulated and the results are compared to known disease characteristics.
By identifying the intrinsic mechanisms involved in the onset of AD diseases, it may be possible to better assess risk factors as well as lead to potential new drug targets. To illustrate the applicability of this study of pathway dynamics, we simulated the primary pathways involved in two autosomal dominant diseases, Polycystic Kidney Disease (PKD) and mature onset diabetes of youth (MODY). Simulations demonstrate that some of the primary disease characteristics are consistent with the positive feedback - stochastic variation theory presented here. This has implications for new drug targets to control these diseases by blocking the positive feedback loop in the relevant pathways.
PMCID: PMC2902440  PMID: 20587063
12.  REEP1 mutation spectrum and genotype/phenotype correlation in hereditary spastic paraplegia type 31 
Brain : a journal of neurology  2008;131(Pt 4):1078-1086.
Mutations in the receptor expression enhancing protein 1 (REEP1) have recently been reported to cause autosomal dominant hereditary spastic paraplegia (HSP) type SPG31. In a large collaborative effort, we screened a sample of 535 unrelated HSP patients for REEP1 mutations and copy number variations. We identified 13 novel and 2 known REEP1 mutations in 16 familial and sporadic patients by direct sequencing analysis. Twelve out of 16 mutations were small insertions, deletions or splice site mutations. These changes would result in shifts of the open-reading-frame followed by premature termination of translation and haploinsufficiency. Interestingly, we identified two disease associated variations in the 3′-UTR of REEP1 that fell into highly conserved micro RNA binding sites. Copy number variation analysis in a subset of 133 HSP index patients revealed a large duplication of REEP1 that involved exons 2–7 in an Irish family. Clinically most SPG31 patients present with a pure spastic paraplegia; rare complicating features were restricted to symptoms or signs of peripheral nerve involvement. Interestingly, the distribution of age at onset suggested a bimodal pattern with the appearance of initial symptoms of disease either before the age of 20 years or after the age of 30 years. The overall mutation rate in our clinically heterogeneous sample was 3.0%; however, in the sub-sample of pure HSP REEP1 mutations accounted for 8.2% of all patients. These results firmly establish REEP1 as a relatively frequent autosomal dominant HSP gene for which genetic testing is warranted. We also establish haploinsufficiency as the main molecular genetic mechanism in SPG31, which should initiate and guide functional studies on REEP1 with a focus on loss-of-function mechanisms. Our results should be valid as a reference for mutation frequency, spectrum of REEP1 mutations, and clinical phenotypes associated with SPG31.
PMCID: PMC2841798  PMID: 18321925
hereditary spastic paraplegia; SPG31; REEP1; haploinsufficiency; micro RNA
13.  Backup without redundancy: genetic interactions reveal the cost of duplicate gene loss 
We show that genetic interaction profiles offer a powerful approach to elicit phenotypes that are far richer than is attainable using single gene deletions. This has allowed us to address the long-standing question of the role played by duplicate genes (paralogs) in robustness against deletion.We provide for the first time direct evidence that the capacity of some duplicates to cover for the loss of their paralogs can account for the observed difference in fitness between duplicate and singleton deletions mutants, but that the overall contribution of this effect to dispensability is small.More broadly, we demonstrate that paralogs possessing apparent backup capacity in some environments have in fact distinct and non-overlapping functions, and are unable to provide backup across a range of compromising conditions. This resolves the previous paradox of how backup genes conferring dispensability can nevertheless be independently maintained in the population.From a practical point of view, our findings suggest efficient strategies to elicit rich deletion phenotypes that should be highly relevant for the design of future phenotypic screens.
Much of our understanding of biological processes has been derived from the characterization of the functional consequence to an organism of altering one or more of its genes. Efforts to systematically evaluate the phenotypic effects of gene loss, however, have been hampered by the fact that the disruption of most genes has surprisingly modest effects on cell growth and viability. The high proportion of genes with no apparent deletion effect has wide-ranging practical and theoretical implications and has been the subject of considerable interest (Wagner, 2000, 2005; Giaever et al, 2002; Gu et al, 2003; Papp et al, 2004; Kafri et al, 2005). One factor that has been implicated as contributing to the high degree of dispensability is the abundance of closely related paralogs present in most genomes (Winzeler et al, 1999; Wagner, 2000; Giaever et al, 2002). Indeed, recent work in S. cerevisiae has shown that the existence of a paralog elsewhere in the genome significantly increases the chance that deletion of a given gene has little effect on growth (Gu et al, 2003). However, current analyses have been mostly correlative, and direct mechanistic evidence supporting or refuting the role of backup compensation in mutational robustness is still largely missing. Furthermore, backup between duplicates is not easily justified in evolutionary terms, in that a genuine ability to comprehensively cover for the loss of another gene is evolutionarily unstable (Brookfield, 1992).
Here, we exploit the recent availability of high-density quantitative genetic interaction profiles (EMAPs) to address these issues directly. To test whether SSL paralogs can account for the excess fitness of duplicates, we classified genes into fitness categories according to their deletion growth defect (Materials and methods). The subset of genes covered by our combined data set exhibits an over-representation of duplicate genes in the weak/no deletion phenotype (WNP) class similar to that reported previously (Gu et al, 2003) (Figure 1B). Strikingly, this difference corresponds to the number of WNP duplicates that have an SSL interaction with their corresponding paralog (Figure 1C). Our data thus provide direct evidence that it is indeed duplicate compensation that accounts for the observed difference in deletion growth defect between duplicates and singletons, at least for the genes covered by our data set.
Apart from the mechanism itself, the characteristic features of buffering duplicates have received considerable attention (Gu et al, 2003; Kafri et al, 2005; Wagner, 2005). Our data allowed us to unambiguously distinguish the subset of duplicates whose dispensability can be attributed to the existence of a backup paralog. The ability to identify backup duplicates directly put us in a position to study their features, and how they differ from other duplicates without buffering properties. In particular, we asked to what extent the observed buffering in rich media reflects functional similarity and a genuine ability to cover for the loss of a paralog in a broader range of conditions.
To assess the extent to which SSL duplicates can provide genuine backup under compromising conditions, we fist used genetic interaction profiles as a more stringent test for redundancy that assesses the effect of gene loss in the background of additional gene deletions. In contrast to the expectation that truly buffered duplicates should have few if any synthetic interactions, we find that the number is in fact substantial and often exceeds that of random genes and non-SSL duplicates (Figure 2B). Similarly, using a recent data set of sensitivity profiles of deletion strains to a range of agents and environments (Brown et al, 2006), we find that the deletion of SSL duplicates across a range of environments has on average no weaker (and in fact a slightly stronger) effect on cellular growth rate than that of non-SSL duplicates or random genes. Taken together, these findings suggest that the backup capacity of SSL duplicates is limited and not indicative of a comprehensive ability to cover for the loss of the paralogous partner.
We next tested the degree of functional similarity of buffering duplicates using similarity in genetic interaction as well as environmental sensitivity profiles as indicators of shared functionality (Tong et al, 2004; Schuldiner et al, 2005; Brown et al, 2006; Pan et al, 2006). In spite of their rich media buffering properties, we find that the interaction and sensitivity patterns of most SSL duplicates are divergent and are usually more similar to those of other, non-paralogous genes (Figure 2C and D; Supplementary Figure 10).
Lastly, in addition to our analysis of duplicate phenotypes, we used genetic interaction spectra as deletion phenotypes for generic genes whose single deletion in standard conditions has little measurable effect. As expected, genetic interactions provide a deletion phenotype for many more genes (80–90%) than single gene deletions in standard growth environments (Steinmetz et al, 2002), which yield a detectable growth defect only for 30–40% (Figure 4B). To assess whether these interactions reflect the cost of gene loss (gene importance), we asked if there is a relationship between the probability of a gene being retained between related species and its number of genetic interactions. Indeed, genetic interactivity exhibits a strong correlation with gene retention across related phyla (Figure 4C and Supplementary Figure 7), and predicts the likelihood of gene loss better than lethality/viability, quantitative growth deficiency or environmental specificity (Supplementary Figure 8). Thus, genetic interactions provide a cost of gene loss that effectively recapitulates evolutionary constraints. This is further supported by the observation that genetic interactions are significantly correlated with environmental sensitivity across a range of conditions. Thus, our findings suggest that for most genes there is a substantial cost of gene loss, even though this is often not reflected in single gene deletion tests carried out in standard conditions.
Many genes can be deleted with little phenotypic consequences. By what mechanism and to what extent the presence of duplicate genes in the genome contributes to this robustness against deletions has been the subject of considerable interest. Here, we exploit the availability of high-density genetic interaction maps to provide direct support for the role of backup compensation, where functionally overlapping duplicates cover for the loss of their paralog. However, we find that the overall contribution of duplicates to robustness against null mutations is low (∼25%). The ability to directly identify buffering paralogs allowed us to further study their properties, and how they differ from non-buffering duplicates. Using environmental sensitivity profiles as well as quantitative genetic interaction spectra as high-resolution phenotypes, we establish that even duplicate pairs with compensation capacity exhibit rich and typically non-overlapping deletion phenotypes, and are thus unable to comprehensively cover against loss of their paralog. Our findings reconcile the fact that duplicates can compensate for each other's loss under a limited number of conditions with the evolutionary instability of genes whose loss is not associated with a phenotypic penalty.
PMCID: PMC1847942  PMID: 17389874
duplication; evolution; genetic interactions; redundancy
14.  Gene Copy-Number Polymorphism Caused by Retrotransposition in Humans 
PLoS Genetics  2013;9(1):e1003242.
The era of whole-genome sequencing has revealed that gene copy-number changes caused by duplication and deletion events have important evolutionary, functional, and phenotypic consequences. Recent studies have therefore focused on revealing the extent of variation in copy-number within natural populations of humans and other species. These studies have found a large number of copy-number variants (CNVs) in humans, many of which have been shown to have clinical or evolutionary importance. For the most part, these studies have failed to detect an important class of gene copy-number polymorphism: gene duplications caused by retrotransposition, which result in a new intron-less copy of the parental gene being inserted into a random location in the genome. Here we describe a computational approach leveraging next-generation sequence data to detect gene copy-number variants caused by retrotransposition (retroCNVs), and we report the first genome-wide analysis of these variants in humans. We find that retroCNVs account for a substantial fraction of gene copy-number differences between any two individuals. Moreover, we show that these variants may often result in expressed chimeric transcripts, underscoring their potential for the evolution of novel gene functions. By locating the insertion sites of these duplicates, we are able to show that retroCNVs have had an important role in recent human adaptation, and we also uncover evidence that positive selection may currently be driving multiple retroCNVs toward fixation. Together these findings imply that retroCNVs are an especially important class of polymorphism, and that future studies of copy-number variation should search for these variants in order to illuminate their potential evolutionary and functional relevance.
Author Summary
Recent studies of human genetic variation have revealed that, in addition to differing at single nucleotide polymorphisms, individuals differ in copy-number at many regions of the genome. These copy-number variants (CNVs) are caused by duplication or deletion events and often affect functional sequences such as genes. Efforts to reveal the functional impact of CNVs have identified many variants increasing the risk of various disorders, and some that are adaptive. However, these studies mostly fail to detect gene duplications caused by retrotransposition, in which an mRNA transcript is reverse-transcribed and reinserted into the genome, yielding a new intron-less gene copy. Here we describe a method leveraging next-generation sequence data to accurately detect gene copy-number variants caused by retrotransposition, or retroCNVs, and apply this method to hundreds of whole-genome sequences from three different human subpopulations. We find that these variants account for a substantial number of gene copy-number differences between individuals, and that gene retrotransposition may often result in both deleterious and beneficial mutations. Indeed, we present evidence that two of these new gene duplications may be adaptive. These results imply that retroCNVs are an especially important class of CNV and should be included in future studies of human copy-number variation.
PMCID: PMC3554589  PMID: 23359205
15.  Rare Copy Number Variants Are a Common Cause of Short Stature 
PLoS Genetics  2013;9(3):e1003365.
Human growth has an estimated heritability of about 80%–90%. Nevertheless, the underlying cause of shortness of stature remains unknown in the majority of individuals. Genome-wide association studies (GWAS) showed that both common single nucleotide polymorphisms and copy number variants (CNVs) contribute to height variation under a polygenic model, although explaining only a small fraction of overall genetic variability in the general population. Under the hypothesis that severe forms of growth retardation might also be caused by major gene effects, we searched for rare CNVs in 200 families, 92 sporadic and 108 familial, with idiopathic short stature compared to 820 control individuals. Although similar in number, patients had overall significantly larger CNVs (p-value<1×10−7). In a gene-based analysis of all non-polymorphic CNVs>50 kb for gene function, tissue expression, and murine knock-out phenotypes, we identified 10 duplications and 10 deletions ranging in size from 109 kb to 14 Mb, of which 7 were de novo (p<0.03) and 13 inherited from the likewise affected parent but absent in controls. Patients with these likely disease causing 20 CNVs were smaller than the remaining group (p<0.01). Eleven (55%) of these CNVs either overlapped with known microaberration syndromes associated with short stature or contained GWAS loci for height. Haploinsufficiency (HI) score and further expression profiling suggested dosage sensitivity of major growth-related genes at these loci. Overall 10% of patients carried a disease-causing CNV indicating that, like in neurodevelopmental disorders, rare CNVs are a frequent cause of severe growth retardation.
Author Summary
With a frequency of 3%, shortness of stature is a common medical concern. Although family studies have clearly shown that gene defects play a pivotal role in the development of short stature, the underlying genetic variants involved remain unknown in about 80% of cases. In contrast to recent studies which aimed at the identification of common genetic variants to explain minor differences in the height variation in the general population, we targeted rare genomic variants where we expected a major gene effect on growth. By examining 200 patients clinically evaluated for short stature, we show that rare structural chromosomal aberrations (CNVs) are associated with shortness of stature in 10% of the cases. The identified CNVs were either de novo or segregated with short stature in the families and include genes that are functionally involved in growth regulation in humans or mice. We furthermore demonstrate an overlap of these CNVs with known microdeletion syndromes. Interestingly, 3 CNVs contain positions of common variants and confirm the localization of major growth-related genes. These findings are particularly important for identification of biological pathways leading to short stature, but also for further therapeutic approaches.
PMCID: PMC3597495  PMID: 23516380
16.  Metabolic modeling of endosymbiont genome reduction on a temporal scale 
This study explores the order in which individual metabolic genes are lost in an in silico evolutionary process leading from the metabolic network of Eschericia coli to that of the genome-reduced endosymbiont Buchnera aphidicola.
Simulating the reductive evolutionary process under several growth conditions, a remarkable correlation between in silico and phylogenetically reconstructed gene loss time is obtained.A gene's k-robustness (its depth of backups) is prime determinant of its loss time.In silico gene loss time is a better predictor of their actual loss times than genomic features and network properties.Simulating the reductive evolutionary process by the loss of large blocks followed by single-gene deletions, as known to occur in evolution, yields a remarkable correspondence with the phylogenetic reconstruction and the block loss reported in the literature.
An open fundamental challenge in Systems Biology is whether a genome-scale model can predict patterns of genome evolution by realistically accounting for the associated biochemical constraints. In this study, we explore the order in which individual genes are lost in an in silico evolutionary process, leading from the metabolic network of Eschericia coli to that of the endosymbiont Buchnera aphidicola.
To evaluate the in silico gene loss time, we repeated the reductive evolutionary process introduced by Pál et al (2006), denoting the in silico deletion time of a gene in a single run of the reductive evolutionary process as the number of genes deleted before its own deletion occurred. By comparing the in silico evaluations of the gene loss time to that obtained by a phylogenetic reconstruction (Figure 1), we could evaluate the ability of an in silico process to predict temporal patterns of genome reduction. Applying this procedure on a literature-based viable media, we obtained a mean Spearman's correlation of 0.46 (53% of the maximal correlation, empirical P-value <9.9e−4) between in silico and phylogenetically reconstructed loss times. In order to provide an upper bound on evolutionary necessity stemming from metabolic constraints, we searched the space of potential growth media and biomass functions via a simulated annealing search algorithm aimed at identifying an environment/biomass function that maximizes the target correlation between in silico and reconstructed loss times. Simulating the reductive evolutionary process under the growth conditions and biomass function obtained in this process, we managed to improve the correlation between in silico and reconstructed loss times to a mean Spearman's correlation of 0.54 (63% of the maximal correlation, empirical P-value <9.9e−4, Figure 3).
Examining the dependency of the predicted loss time of each gene on its intrinsic network-level properties we find a very strong inverse Spearman's correlation of −0.84 (empirical P-value <9.9e−4) between the order of gene loss predicted in silico and the k-robustness levels of the genes, the latter denoting the depth of their functional backups in the network (Deutscher et al, 2006). Moreover, in order to examine whether the relative loss time of a gene is influenced by its functional dependencies with other genes, we performed a flux-coupling analysis and identified pairs of reactions whose activities asymmetrically depend on each other, i.e., are directionally coupled (Burgard et al, 2004). We find that genes encoding reactions whose activity is needed for activating the other reaction (and not vice versa) have a tendency to be lost later, as one would expect (binomial P-value <1e−14).
To assess the scale of these results, we examined as a control how well genomic features and network properties predict the phylogenetically reconstructed gene loss times. We examined the dependency of the latter on several factors that are known be inversely correlated with the propensity of a gene to be lost (Brinza et al, 2009; Delmotte et al, 2006; Tamames et al, 2007), including the genes' mRNA levels, tAI values (Covert et al, 2004; Reis et al, 2004; Sharp and Li, 1987; Tuller et al, 2010a) and the number of partners the gene products have in a protein–protein interaction network. Remarkably, these genomic features yield considerably lower Spearman's correlation than that obtained by the in silico simulations. Moreover, multiply regressing the loss times from the phylogenetic reconstruction on the in silico gene loss time predictions and the genomic and network variables, we found that the (normalized) coefficient of the in silico predictions in the regression is much higher than those of the genomic features, further testifying to the considerable independent predictive power of the metabolic model.
Finally, simulating the evolutionary process as large block deletions at first followed by single-gene deletions as is thought to occur in evolution (Moran and Mira, 2001; van Ham et al, 2003), a remarkable correspondence with the phylogenetic reconstruction was found. Namely, we find that after a certain amount of genes are deleted from the genome, no further block deletions can occur due to the increasing density of essential genes. Notably, the maximum amount of genes that can be deleted in blocks (i.e., until no more blocks can be deleted) corresponds to the number of genes appearing in our phylogenetic reconstruction from the LCA (last common ancestor of Buchnera and E. coli) to the LCSA (last common symbiotic ancestor, nodes 1–3 in Figure 1A), as described in the literature.
A fundamental challenge in Systems Biology is whether a cell-scale metabolic model can predict patterns of genome evolution by realistically accounting for associated biochemical constraints. Here, we study the order in which genes are lost in an in silico evolutionary process, leading from the metabolic network of Eschericia coli to that of the endosymbiont Buchnera aphidicola. We examine how this order correlates with the order by which the genes were actually lost, as estimated from a phylogenetic reconstruction. By optimizing this correlation across the space of potential growth and biomass conditions, we compute an upper bound estimate on the model's prediction accuracy (R=0.54). The model's network-based predictive ability outperforms predictions obtained using genomic features of individual genes, reflecting the effect of selection imposed by metabolic stoichiometric constraints. Thus, while the timing of gene loss might be expected to be a completely stochastic evolutionary process, remarkably, we find that metabolic considerations, on their own, make a marked 40% contribution to determining when such losses occur.
PMCID: PMC3094061  PMID: 21451589
constraint-based modeling; endosymbiont; evolution; metabolism
17.  Interpretation of Genomic Variants Using a Unified Biological Network Approach 
PLoS Computational Biology  2013;9(3):e1002886.
The decreasing cost of sequencing is leading to a growing repertoire of personal genomes. However, we are lagging behind in understanding the functional consequences of the millions of variants obtained from sequencing. Global system-wide effects of variants in coding genes are particularly poorly understood. It is known that while variants in some genes can lead to diseases, complete disruption of other genes, called ‘loss-of-function tolerant’, is possible with no obvious effect. Here, we build a systems-based classifier to quantitatively estimate the global perturbation caused by deleterious mutations in each gene. We first survey the degree to which gene centrality in various individual networks and a unified ‘Multinet’ correlates with the tolerance to loss-of-function mutations and evolutionary conservation. We find that functionally significant and highly conserved genes tend to be more central in physical protein-protein and regulatory networks. However, this is not the case for metabolic pathways, where the highly central genes have more duplicated copies and are more tolerant to loss-of-function mutations. Integration of three-dimensional protein structures reveals that the correlation with centrality in the protein-protein interaction network is also seen in terms of the number of interaction interfaces used. Finally, combining all the network and evolutionary properties allows us to build a classifier distinguishing functionally essential and loss-of-function tolerant genes with higher accuracy (AUC = 0.91) than any individual property. Application of the classifier to the whole genome shows its strong potential for interpretation of variants involved in Mendelian diseases and in complex disorders probed by genome-wide association studies.
Author Summary
The number of personal genomes sequenced has grown rapidly over the last few years and is likely to grow further. In order to use the DNA sequence variants amongst individuals for personalized medicine, we need to understand the functional impact of these variants. Deleterious variants in genes can have a wide spectrum of global effects, ranging from fatal for essential genes to no obvious damaging effect for loss-of-function tolerant genes. The global effect of a gene mutation is largely governed by the diverse biological networks in which the gene participates. Since genes participate in many networks, no singular network captures the global picture of gene interactions. Here we integrate the diverse modes of gene interactions (regulatory, genetic, phosphorylation, signaling, metabolic and physical protein-protein interactions) to create a unified biological network. We then exploit the unique properties of loss-of-function tolerant and essential genes in this unified network to build a computational model that can predict global perturbation caused by deleterious mutations in all genes. Our model can distinguish between these two gene sets with high accuracy and we further show that it can be used for interpretation of variants involved in Mendelian diseases and in complex disorders probed by genome-wide association studies.
PMCID: PMC3591262  PMID: 23505346
18.  NFIA Haploinsufficiency Is Associated with a CNS Malformation Syndrome and Urinary Tract Defects 
PLoS Genetics  2007;3(5):e80.
Complex central nervous system (CNS) malformations frequently coexist with other developmental abnormalities, but whether the associated defects share a common genetic basis is often unclear. We describe five individuals who share phenotypically related CNS malformations and in some cases urinary tract defects, and also haploinsufficiency for the NFIA transcription factor gene due to chromosomal translocation or deletion. Two individuals have balanced translocations that disrupt NFIA. A third individual and two half-siblings in an unrelated family have interstitial microdeletions that include NFIA. All five individuals exhibit similar CNS malformations consisting of a thin, hypoplastic, or absent corpus callosum, and hydrocephalus or ventriculomegaly. The majority of these individuals also exhibit Chiari type I malformation, tethered spinal cord, and urinary tract defects that include vesicoureteral reflux. Other genes are also broken or deleted in all five individuals, and may contribute to the phenotype. However, the only common genetic defect is NFIA haploinsufficiency. In addition, previous analyses of Nfia−/− knockout mice indicate that Nfia deficiency also results in hydrocephalus and agenesis of the corpus callosum. Further investigation of the mouse Nfia+/− and Nfia−/− phenotypes now reveals that, at reduced penetrance, Nfia is also required in a dosage-sensitive manner for ureteral and renal development. Nfia is expressed in the developing ureter and metanephric mesenchyme, and Nfia+/− and Nfia−/− mice exhibit abnormalities of the ureteropelvic and ureterovesical junctions, as well as bifid and megaureter. Collectively, the mouse Nfia mutant phenotype and the common features among these five human cases indicate that NFIA haploinsufficiency contributes to a novel human CNS malformation syndrome that can also include ureteral and renal defects.
Author Summary
Central nervous system (CNS) and urinary tract abnormalities are common human malformations, but their variability and genetic complexity make it difficult to identify the responsible genes. Analysis of human chromosomal abnormalities associated with such disorders offers one approach to this problem. In five individuals described herein, a novel human syndrome that involves both CNS and urinary tract defects is associated with chromosomal disruption or deletion of NFIA, encoding a member of the Nuclear Factor I (NFI) family of transcription factors. This syndrome includes brain abnormalities (abnormal corpus callosum, hydrocephalus, ventriculomegaly, and Chiari type I malformation), spinal abnormalities (tethered spinal cord), and urinary tract abnormalities (vesicoureteral reflux). Nfia disruption in mice was already known to cause hydrocephalus and abnormal corpus callosum, and is now shown to exhibit renal defects and disturbed ureteral development. Other genes besides NFIA are also disrupted or deleted and may contribute to the observed phenotype. However, loss of one copy of NFIA is the only genetic defect common to all five patients. The authors thus provide evidence that genetic loss of NFIA contributes to a distinct CNS malformation syndrome with urinary tract defects of variable penetrance.
PMCID: PMC1877820  PMID: 17530927
19.  Gene Annotation and Drug Target Discovery in Candida albicans with a Tagged Transposon Mutant Collection 
PLoS Pathogens  2010;6(10):e1001140.
Candida albicans is the most common human fungal pathogen, causing infections that can be lethal in immunocompromised patients. Although Saccharomyces cerevisiae has been used as a model for C. albicans, it lacks C. albicans' diverse morphogenic forms and is primarily non-pathogenic. Comprehensive genetic analyses that have been instrumental for determining gene function in S. cerevisiae are hampered in C. albicans, due in part to limited resources to systematically assay phenotypes of loss-of-function alleles. Here, we constructed and screened a library of 3633 tagged heterozygous transposon disruption mutants, using them in a competitive growth assay to examine nutrient- and drug-dependent haploinsufficiency. We identified 269 genes that were haploinsufficient in four growth conditions, the majority of which were condition-specific. These screens identified two new genes necessary for filamentous growth as well as ten genes that function in essential processes. We also screened 57 chemically diverse compounds that more potently inhibited growth of C. albicans versus S. cerevisiae. For four of these compounds, we examined the genetic basis of this differential inhibition. Notably, Sec7p was identified as the target of brefeldin A in C. albicans screens, while S. cerevisiae screens with this compound failed to identify this target. We also uncovered a new C. albicans-specific target, Tfp1p, for the synthetic compound 0136-0228. These results highlight the value of haploinsufficiency screens directly in this pathogen for gene annotation and drug target identification.
Author Summary
Candida albicans is a normal inhabitant in our bodies, yet it can become pathogenic and cause infections that range from the superficial in healthy individuals to deadly in the immunocompromised. Comprehensive genetic analysis of C. albicans to identify mechanisms of virulence and new treatment strategies has been hampered by limited, publically accessible genomic resources. By combining the principles of Saccharomyces cerevisiae strain tagging with transposon mutagenesis to generate individually tagged mutants, we created the first entirely public resource that allows simultaneous measurement of strain fitness of ∼60% of the genome in a wide range of experimental treatments. By identifying genes that confer a fitness or growth defect when reduced in copy number, we uncovered genes whose protein products represent potential antifungal targets. Moreover, screening this strain collection with chemical compounds allowed us to identify anticandidal chemicals while concurrently gaining insight into their cellular mechanism of action. This resource, combined with straightforward screening methodology, provides powerful tools to generate hypotheses for functional annotation of the genome, and our results highlight the value of direct versus model-based pathogen studies.
PMCID: PMC2951378  PMID: 20949076
20.  FRA2A Is a CGG Repeat Expansion Associated with Silencing of AFF3 
PLoS Genetics  2014;10(4):e1004242.
Folate-sensitive fragile sites (FSFS) are a rare cytogenetically visible subset of dynamic mutations. Of the eight molecularly characterized FSFS, four are associated with intellectual disability (ID). Cytogenetic expression results from CGG tri-nucleotide-repeat expansion mutation associated with local CpG hypermethylation and transcriptional silencing. The best studied is the FRAXA site in the FMR1 gene, where large expansions cause fragile X syndrome, the most common inherited ID syndrome. Here we studied three families with FRA2A expression at 2q11 associated with a wide spectrum of neurodevelopmental phenotypes. We identified a polymorphic CGG repeat in a conserved, brain-active alternative promoter of the AFF3 gene, an autosomal homolog of the X-linked AFF2/FMR2 gene: Expansion of the AFF2 CGG repeat causes FRAXE ID. We found that FRA2A-expressing individuals have mosaic expansions of the AFF3 CGG repeat in the range of several hundred repeat units. Moreover, bisulfite sequencing and pyrosequencing both suggest AFF3 promoter hypermethylation. cSNP-analysis demonstrates monoallelic expression of the AFF3 gene in FRA2A carriers thus predicting that FRA2A expression results in functional haploinsufficiency for AFF3 at least in a subset of tissues. By whole-mount in situ hybridization the mouse AFF3 ortholog shows strong regional expression in the developing brain, somites and limb buds in 9.5–12.5dpc mouse embryos. Our data suggest that there may be an association between FRA2A and a delay in the acquisition of motor and language skills in the families studied here. However, additional cases are required to firmly establish a causal relationship.
Author Summary
Some human genetic diseases are caused by dynamic mutations, or expansions of a short repeated sequence in the genome that can be unstably passed on from generation to generation. A subset of these dynamic mutations known as fragile sites can be seen as a break or gap on the chromosome when cells are cultured under specific conditions. To date eight folate-sensitive fragile sites (FSFS) have been characterized, and all are due to CGG-repeat expansions within the 5′ UTR or promoter region of the respective gene. When the repeat expands in size, it becomes hypermethylated and the adjacent gene or genes are transcriptionally silenced. For at least four of the eight known fragile sites this silencing of the associated gene(s) lead to intellectual disability syndromes such as fragile X. In this work we describe molecular characterization of an autosomal FSFS called FRA2A on chromosome 2. As the molecular cause of FRA2A, we identify an expansion of a CGG repeat which subsequently results in silencing of the neighbouring gene AFF3. This gene is one of the four autosomal paralogss of the AFF2/FMR2 gene which, when mutated, is the cause of the FRAXE syndrome. We find that FRA2A expression is associated with highly variable developmental anomalies in the three FRA2A families studied.
PMCID: PMC3998887  PMID: 24763282
21.  A Dominant-Negative Mutation of Mouse Lmx1b Causes Glaucoma and Is Semi-lethal via LBD1-Mediated Dimerisation 
PLoS Genetics  2014;10(5):e1004359.
Mutations in the LIM-homeodomain transcription factor LMX1B cause nail-patella syndrome, an autosomal dominant pleiotrophic human disorder in which nail, patella and elbow dysplasia is associated with other skeletal abnormalities and variably nephropathy and glaucoma. It is thought to be a haploinsufficient disorder. Studies in the mouse have shown that during development Lmx1b controls limb dorsal-ventral patterning and is also required for kidney and eye development, midbrain-hindbrain boundary establishment and the specification of specific neuronal subtypes. Mice completely deficient for Lmx1b die at birth. In contrast to the situation in humans, heterozygous null mice do not have a mutant phenotype. Here we report a novel mouse mutant Icst, an N-ethyl-N-nitrosourea-induced missense substitution, V265D, in the homeodomain of LMX1B that abolishes DNA binding and thereby the ability to transactivate other genes. Although the homozygous phenotypic consequences of Icst and the null allele of Lmx1b are the same, heterozygous Icst elicits a phenotype whilst the null allele does not. Heterozygous Icst causes glaucomatous eye defects and is semi-lethal, probably due to kidney failure. We show that the null phenotype is rescued more effectively by an Lmx1b transgene than is Icst. Co-immunoprecipitation experiments show that both wild-type and Icst LMX1B are found in complexes with LIM domain binding protein 1 (LDB1), resulting in lower levels of functional LMX1B in Icst heterozygotes than null heterozygotes. We conclude that Icst is a dominant-negative allele of Lmx1b. These findings indicate a reassessment of whether nail-patella syndrome is always haploinsufficient. Furthermore, Icst is a rare example of a model of human glaucoma caused by mutation of the same gene in humans and mice.
Author Summary
Nail-patella syndrome is a human genetic disease caused by an inactivating mutation in one copy of a gene called LMX1B, with the amount of protein produced from the remaining copy of the gene not being enough for normal function. Patients with this disease have malformations of their nails, elbows and kneecaps. Some patients also develop kidney disease and glaucoma. LMX1B controls where and when other genes are expressed and it is important during development. Studies in mice have shown that complete absence of Lmx1b is lethal at birth. In contrast to humans, mice with only one copy of the gene are normal. Here we describe a new mutant mouse, Icst, which has a mutation in Lmx1b that abolishes the ability of the protein to bind near genes that it controls. Mice with one normal and one copy of Lmx1b with the Icst mutation have eye defects and some die shortly after birth probably due to kidney failure. Therefore having one functional and one mutant copy of Lmx1b is more detrimental than having a half dose of functional protein. The Icst mouse is a model of human glaucoma where mutation of the same gene causes glaucoma in humans and mice.
PMCID: PMC4014447  PMID: 24809698
22.  Network Hubs Buffer Environmental Variation in Saccharomyces cerevisiae 
PLoS Biology  2008;6(11):e264.
Regulatory and developmental systems produce phenotypes that are robust to environmental and genetic variation. A gene product that normally contributes to this robustness is termed a phenotypic capacitor. When a phenotypic capacitor fails, for example when challenged by a harsh environment or mutation, the system becomes less robust and thus produces greater phenotypic variation. A functional phenotypic capacitor provides a mechanism by which hidden polymorphism can accumulate, whereas its failure provides a mechanism by which evolutionary change might be promoted. The primary example to date of a phenotypic capacitor is Hsp90, a molecular chaperone that targets a large set of signal transduction proteins. In both Drosophila and Arabidopsis, compromised Hsp90 function results in pleiotropic phenotypic effects dependent on the underlying genotype. For some traits, Hsp90 also appears to buffer stochastic variation, yet the relationship between environmental and genetic buffering remains an important unresolved question. We previously used simulations of knockout mutations in transcriptional networks to predict that many gene products would act as phenotypic capacitors. To test this prediction, we use high-throughput morphological phenotyping of individual yeast cells from single-gene deletion strains to identify gene products that buffer environmental variation in Saccharomyces cerevisiae. We find more than 300 gene products that, when absent, increase morphological variation. Overrepresented among these capacitors are gene products that control chromosome organization and DNA integrity, RNA elongation, protein modification, cell cycle, and response to stimuli such as stress. Capacitors have a high number of synthetic-lethal interactions but knockouts of these genes do not tend to cause severe decreases in growth rate. Each capacitor can be classified based on whether or not it is encoded by a gene with a paralog in the genome. Capacitors with a duplicate are highly connected in the protein–protein interaction network and show considerable divergence in expression from their paralogs. In contrast, capacitors encoded by singleton genes are part of highly interconnected protein clusters whose other members also tend to affect phenotypic variability or fitness. These results suggest that buffering and release of variation is a widespread phenomenon that is caused by incomplete functional redundancy at multiple levels in the genetic architecture.
Author Summary
Most species maintain abundant genetic variation and experience a wide range of environmental conditions, yet phenotypic differences between individuals are usually small. This phenomenon, known as phenotypic robustness, presents an apparent contradiction: if biological systems are so resistant to variation, how do they diverge and adapt through evolutionary time? Here, we address this question by investigating the molecular mechanisms that underlie phenotypic robustness and how these mechanisms can be broken to produce phenotypic heterogeneity. We identify genes that contribute to phenotypic robustness in yeast by analyzing the variance of morphological phenotypes in a comprehensive collection of single-gene knockout strains. We find that ∼5% of yeast genes break phenotypic robustness when knocked out. The products of these genes tend to be involved in critical cellular processes, including maintaining DNA stability, processing RNA, modifying proteins, and responding to stressful environments. These genes tend to interact genetically with a large number of other genes, and their products tend to interact physically with a large number of other gene products. Our results suggest that loss of phenotypic robustness might be a common phenomenon during evolution that occurs when cellular networks are disrupted.
A genome-wide screen inSaccharomyces cerevisiae identifies over 300 gene products that buffer environmental variation--dubbed phenotypic capacitors--and function as hubs in protein-protein and synthetic-lethal interaction networks.
PMCID: PMC2577700  PMID: 18986213
23.  Convergence of Mutation and Epigenetic Alterations Identifies Common Genes in Cancer That Predict for Poor Prognosis  
PLoS Medicine  2008;5(5):e114.
The identification and characterization of tumor suppressor genes has enhanced our understanding of the biology of cancer and enabled the development of new diagnostic and therapeutic modalities. Whereas in past decades, a handful of tumor suppressors have been slowly identified using techniques such as linkage analysis, large-scale sequencing of the cancer genome has enabled the rapid identification of a large number of genes that are mutated in cancer. However, determining which of these many genes play key roles in cancer development has proven challenging. Specifically, recent sequencing of human breast and colon cancers has revealed a large number of somatic gene mutations, but virtually all are heterozygous, occur at low frequency, and are tumor-type specific. We hypothesize that key tumor suppressor genes in cancer may be subject to mutation or hypermethylation.
Methods and Findings
Here, we show that combined genetic and epigenetic analysis of these genes reveals many with a higher putative tumor suppressor status than would otherwise be appreciated. At least 36 of the 189 genes newly recognized to be mutated are targets of promoter CpG island hypermethylation, often in both colon and breast cancer cell lines. Analyses of primary tumors show that 18 of these genes are hypermethylated strictly in primary cancers and often with an incidence that is much higher than for the mutations and which is not restricted to a single tumor-type. In the identical breast cancer cell lines in which the mutations were identified, hypermethylation is usually, but not always, mutually exclusive from genetic changes for a given tumor, and there is a high incidence of concomitant loss of expression. Sixteen out of 18 (89%) of these genes map to loci deleted in human cancers. Lastly, and most importantly, the reduced expression of a subset of these genes strongly correlates with poor clinical outcome.
Using an unbiased genome-wide approach, our analysis has enabled the discovery of a number of clinically significant genes targeted by multiple modes of inactivation in breast and colon cancer. Importantly, we demonstrate that a subset of these genes predict strongly for poor clinical outcome. Our data define a set of genes that are targeted by both genetic and epigenetic events, predict for clinical prognosis, and are likely fundamentally important for cancer initiation or progression.
Stephen Baylin and colleagues show that a combined genetic and epigenetic analysis of breast and colon cancers identifies a number of clinically significant genes targeted by multiple modes of inactivation.
Editors' Summary
Cancer is one of the developed world's biggest killers—over half a million Americans die of cancer each year, for instance. As a result, there is great interest in understanding the genetic and environmental causes of cancer in order to improve cancer prevention, diagnosis, and treatment.
Cancer begins when cells begin to multiply out of control. DNA is the sequence of coded instructions—genes—for how to build and maintain the body. Certain “tumor suppressor” genes, for instance, help to prevent cancer by preventing tumors from developing, but changes that alter the DNA code sequence—mutations—can profoundly affect how a gene works. Modern techniques of genetic analysis have identified genes such as tumor suppressors that, when mutated, are linked to the development of certain cancers.
Why Was This Study Done?
However, in recent years, it has become increasingly apparent that mutations are neither necessary nor sufficient to explain every case of cancer. This has led researchers to look at so-called epigenetic factors, which also alter how a gene works without altering its DNA sequence. An example of this is “methylation,” which prevents a gene from being expressed—deactivates it—by a chemical tag. Methylation of genes is part of the normal functioning of DNA, but abnormal methylation has been linked with cancer, aging, and some rare birth abnormalities.
Previous analysis of DNA from breast and colon cancer cells had revealed 189 “candidate cancer genes”—mutated genes that were linked to the development of breast and colon cancer. However, it was not clear how those mutations gave rise to cancer, and individual mutations were present in only 5% to 15% of specific tumors. The authors of this study wanted to know whether epigenetic factors such as methylation contributed to causing the cancers.
What Did the Researchers Do and Find?
The researchers first identified 56 of the 189 candidate cancer genes as likely tumor suppressors and then determined that 36 of these genes were methylated and deactivated, often in both breast and colon (laboratory-grown) cancer cells. In nearly all cases, the methylated genes were not active but could be reactivated by being demethylated. They further showed that, in normal colon and breast tissue samples, 18 of the 36 genes were unmethylated and functioned normally, but in cells taken from breast and colon cancer tumors they were methylated.
In contrast to the genetic mutations, the 18 genes were frequently methylated across a range of tumor types, and eight genes were methylated in both the breast and colon cancers. The authors found by reviewing the genetics and epigenetics of those 18 genes in breast and colon cancer that they were either mutated, methylated, or both. A literature review showed that at least six of the 18 genes were known to have tumor suppressor properties, and the authors determined that 16 were located in parts of DNA known to be missing from cells taken from a range of cancer tumors.
Finally, the researchers analyzed data on cancer cases to show that methylation of these 18 genes was correlated with reduced function of these genes in tumors and with a greater likelihood that a cancer will be terminal or spread to other parts of the body.
What Do These Findings Mean?
The researchers considered only the 189 candidate cancer genes found in one previous study and not other genes identified elsewhere. They also did not consider the biological effects of the individual mutations found in those genes. Despite this, they have demonstrated that methylation of specific genes is likely to play a role in the development of breast and/or colon cancer cells either together with mutations or independently, most likely by turning off their tumor suppression function.
More broadly, however, the study adds to the evidence that future analysis of the role of genes in cancer should include epigenetic as well as genetic factors. In addition, the authors have also shown that a number of these genes may be useful for predicting clinical outcomes for a range of tumor types.
Additional Information.
Please access these Web sites via the online version of this summary at
A December 2006 PLoS Medicine Perspective article reviews the value of examining methylation as a factor in common cancers and its use for early detection
The Web site of the American Cancer Society has a wealth of information and resources on a variety of cancers, including breast and colon cancer is a nonprofit organization providing information about breast cancer on the Web, including research news
Cancer Research UK provides information on cancer research
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins publishes background information on the authors' research on methylation, setting out its potential for earlier diagnosis and better treatment of cancer
PMCID: PMC2429944  PMID: 18507500
24.  Ancestral Mutation in Telomerase Causes Defects in Repeat Addition Processivity and Manifests As Familial Pulmonary Fibrosis 
PLoS Genetics  2011;7(3):e1001352.
The telomerase reverse transcriptase synthesizes new telomeres onto chromosome ends by copying from a short template within its integral RNA component. During telomere synthesis, telomerase adds multiple short DNA repeats successively, a property known as repeat addition processivity. However, the consequences of defects in processivity on telomere length maintenance are not fully known. Germline mutations in telomerase cause haploinsufficiency in syndromes of telomere shortening, which most commonly manifest in the age-related disease idiopathic pulmonary fibrosis. We identified two pulmonary fibrosis families that share two non-synonymous substitutions in the catalytic domain of the telomerase reverse transcriptase gene hTERT: V791I and V867M. The two variants fell on the same hTERT allele and were associated with telomere shortening. Genealogy suggested that the pedigrees shared a single ancestor from the nineteenth century, and genetic studies confirmed the two families had a common founder. Functional studies indicated that, although the double mutant did not dramatically affect first repeat addition, hTERT V791I-V867M showed severe defects in telomere repeat addition processivity in vitro. Our data identify an ancestral mutation in telomerase with a novel loss-of-function mechanism. They indicate that telomere repeat addition processivity is a critical determinant of telomere length and telomere-mediated disease.
Author Summary
Mutations in the essential telomerase components cause a spectrum of diseases mediated by short telomeres. Most frequently, these disorders manifest in the lung in an age-related disease: idiopathic pulmonary fibrosis. Telomerase synthesizes telomere repeats using a specialized reverse transcriptase, hTERT, that copies from a short template within its intrinsic RNA. In order to add long telomere tracts, telomerase adds a single repeat followed by additional repeats successively. This property, known as repeat addition processivity, is unique to the telomerase polymerase. We identified two families that shared two unique variants in the catalytic domain of hTERT: V791I and V867M. The variants co-segregated, indicating they are on the same allele, and were associated with short telomeres. Family history suggested the two families may have a single ancestor, and genetic studies confirmed they had a common founder. Telomerase reconstitution indicated that, although the double mutant did not significantly affect telomerase's ability to add a single telomere repeat, hTERT 791I-867M had severe defects in repeat addition processivity. Our data identify an ancestral mutation in telomerase; this mutation possesses a unique loss-of-function mechanism. Defects in telomere addition processivity are important determinants of telomere length maintenance and of telomere-associated disease.
PMCID: PMC3069110  PMID: 21483807
25.  Genome-wide copy number variation analysis of a Branchio-Oto-Renal syndrome cohort identifies a recombination hotspot and implicates new candidate genes 
Human genetics  2013;132(12):10.1007/s00439-013-1338-8.
Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial arch anomalies, hearing loss and renal dysmorphology. Although haploinsufficiency of EYA1 and SIX1 are known to cause BOR, copy number variation analysis has only been performed on a limited number of BOR patients. In this study, we used high-resolution array-based comparative genomic hybridization (aCGH) on 32 BOR probands negative for coding-sequence and splice-site mutations in known BOR-causing genes to identify potential disease-causing genomic rearrangements. Of the >1,000 rare and novel copy number variants (CNVs) we identified, four were heterozygous deletions of EYA1 and several downstream genes that had nearly identical breakpoints associated with retroviral sequence blocks, suggesting that non-allelic homologous recombination seeded by this recombination hotspot is important in the pathogenesis of BOR. A different heterozygous deletion removing the last exon of EYA1 was identified in an additional proband. Thus in total 5 probands (14%) had deletions of all or part of EYA1. Using a novel disease-gene prioritization strategy that includes network analysis of genes associated with other deletions suggests that SHARPIN (Sipl1), FGF3 and the HOXA gene cluster may contribute to the pathogenesis of BOR.
PMCID: PMC3830662  PMID: 23851940
array CGH; EYA1; birth defects; Branchio-oto-renal syndrome; copy number variation

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