Genes,mutations, and human inherited disease at the dawn of the age of personalized genomics

The number of reported germline mutations in human nuclear genes, either underlying or associated with inherited disease, has now exceeded 100,000 in more than 3,700 different genes. The availability of these data has both revolutionized the study of the morbid anatomy of the human genome and facilitated “personalized genomics.” With ～300 new “inherited disease genes” (and ～10,000 new mutations) being identified annually, it is pertinent to ask how many “inherited disease genes” there are in the human genome, how many mutations reside within them, and where such lesions are likely to be located? To address these questions, it is necessary not only to reconsider how we define human genes but also to explore notions of gene “essentiality” and “dispensability.” Answers to these questions are now emerging from recent novel insights into genome structure and function and through complete genome sequence information derived from multiple individual human genomes. However, a change in focus toward screening functional genomic elements as opposed to genes sensu stricto will be required if we are to capitalize fully on recent technical and conceptual advances and identify new types of disease‐associated mutation within noncoding regions remote from the genes whose function they disrupt. Hum Mutat 31:631–655, 2010. © 2010 Wiley‐Liss, Inc.     

Triangulation of the human, chimpanzee, and Neanderthal genome sequences identifies potentially compensated mutations

Triangulation of the human, chimpanzee, and Neanderthal genome sequences with respect to 44,348 disease‐causing or disease‐associated missense mutations and 1,712 putative regulatory mutations listed in the Human Gene Mutation Database was employed to identify genetic variants that are apparently pathogenic in humans but which may represent a “compensated” wild‐type state in at least one of the other two species. Of 122 such “potentially compensated mutations” (PCMs) identified, 88 were deemed “ancestral” on the basis that the reported wild‐type Neanderthal nucleotide was identical to that of the chimpanzee. Another 33 PCMs were deemed to be “derived” in that the Neanderthal wild‐type nucleotide matched the human but not the chimpanzee wild‐type. For the remaining PCM, all three wild‐type states were found to differ. Whereas a derived PCM would require compensation only in the chimpanzee, ancestral PCMs are useful as a means to identify sites of possible adaptive differences between modern humans on the one hand, and Neanderthals and chimpanzees on the other. Ancestral PCMs considered to be disease‐causing in humans were identified in two Neanderthal genes (DUOX2, MAMLD1). Because the underlying mutations are known to give rise to recessive conditions in human, it is possible that they may also have been of pathological significance in Neanderthals. Hum Mutat 31:1–8, 2010. © 2010 Wiley‐Liss, Inc.     

Breast cancer,consanguinity, and lethal tumor genes: simulation of BRCA1/2 prevalence over 40 generations

Marriage between biological relatives is a social custom with a long history in many parts of the world. Today, hundreds of millions of individuals live in consanguineous families. The offspring of consanguineous parents are more likely to have the same two alleles (homozygosity) by descent. In consanguineous family with BRCA1/2 gene mutations, an offspring is more likely to be BRCA1/2 homozygous. The consequences of BRCA1/2 mutation homozygosity in humans are unknown. In knockout mice, BRCA1 or BRCA2 homozygotes die as embryos. Because tumor suppressor genes are conserved and less species-specific than other genes, human BRCA1/2 homozygotes are likely to be biologically non-viable and are unknown to exist. Among the conceptuses of consanguineous couples, there are excess deaths (abortions, stillbirths, perinatal and early-childhood deaths) as well as a decreased risk of breast cancer, especially in younger females. It has been suggested that, in part, the excess deaths are due to BRCA1/2 and other still undiscovered tumor gene homozygotes. To examine the consequences of the long-term practice of consanguineous marriage on the prevalence of lethal cancer genes, we simulated, by computer, the mating of non-consanguineous and consanguineous populations over 40 generations. The program was developed in Basic for a Macintosh computer. The input comprised the rates of consanguineous marriage types and the output parameter was the rate of heterozygotes (carriers) in each generation. The combined prevalence of BRCA1/2 mutation of 1% was used as a starting reference point. Absence of spontaneous mutations and gene flow were assumed. In a randomly mating population, the BRCA1/2 carrier rate decreases on average 0.0035% every 25 years. In a highly consanguineous population, the carrier rate decreases on average 0.022% every 25 years, or six times faster than in a non-consanguineous population. There is a worldwide trend of decreasing breast cancer incidence with an increasing consanguinity rate. In conclusion, the BRCA1/2 and possibly other undiscovered tumor gene carrier rates are significantly lower in consanguineous than in non-consanguineous populations. Gene frequency in a population depends on the rate of inbreeding and length of consanguineous practices. A drift phenomenon may exert a major effect on the carrier rate. Consanguinity may explain part of the worldwide variation of breast cancer incidence.     

Long contiguous stretches of homozygosity in the human genome

Single nucleotide polymorphisms (SNPs) are the most common sequence variation in the human genome; they have been successfully used in mapping disease genes and more recently in studying population genetics and cancer genetics. In a population-based association study using high-density oligonucleotide arrays for whole-genome SNP genotyping, we discovered that in the genomes of unrelated Han Chinese, 34 out of 515 (6.6%) individuals contained long contiguous stretches of homozygosity (LCSHs), ranging in the size from 2.94 to 26.27 Mbp (10.22+/-5.95 Mbp). Four out of four (100%) Taiwan aborigines also demonstrated this genetic characteristic. The number of LCSH regions increased markedly in the offspring of consanguineous marriages. LCSH was also detected in Caucasian samples (11/42; 26.2%) and African American samples (2/42; 4.76%). A total of 26 LCSH regions were recurrently detected among Han Chinese, Taiwan aborigines, and Caucasians. DNA copy number determination by hybridization intensity analysis and real-time quantitative PCR (qPCR) excluded deletion as the cause of LCSH. Our results suggest that LCSHs are common in the human genome of the outbred population and this genetic characteristic could have a significant impact on population genetics and disease gene studies.     

Natural human knockouts and Mendelian disorders: deep phenotyping in Italian isolates

Whole genome sequencing (WGS) allows the identification of human knockouts (HKOs), individuals in whom loss of function (LoF) variants disrupt both alleles of a given gene. HKOs are a valuable model for understanding the consequences of genes function loss. Naturally occurring biallelic LoF variants tend to be significantly enriched in “genetic isolates,” making these populations specifically suited for HKO studies. In this work, a meticulous WGS data analysis combined with an in-depth phenotypic assessment of 947 individuals from three Italian genetic isolates led to the identification of ten biallelic LoF variants in ten OMIM genes associated with known autosomal recessive diseases. Notably, only a minority of the identified HKOs (C7, F12, and GPR68 genes) displayed the expected phenotype. For most of the genes, instead, (ACADSB, FANCL, GRK1, LGI4, MPO, PGAM2, and RP1L1), the carriers showed none or few of the signs and symptoms typically associated with the related diseases. Of particular interest is a case presenting with a FANCL biallelic LoF variant and a positive diepoxybutane test but lacking a full Fanconi anemia phenotypic spectrum. Identifying KO subjects displaying expected phenotypes suggests that the lack of correct genetic diagnoses may lead to inappropriate and delayed treatment. In contrast, the presence of HKOs with phenotypes deviating from the expected patterns underlines how LoF variants may be responsible for broader phenotypic spectra. Overall, these results highlight the importance of in-depth phenotypical characterization to understand the role of LoF variants and the advantage of studying these variants in genetic isolates.Subject terms: Genetics research, DNA sequencing, Rare variants     

Effect of inbreeding on intellectual disability revisited by trio sequencing

In outbred Western populations, most individuals with intellectual disability (ID) are sporadic cases, dominant de novo mutations (DNM) are frequent, and autosomal recessive ID (ARID) is very rare. Because of the high rate of parental consanguinity, which raises the risk for ARID and other recessive disorders, the prevalence of ID is significantly higher in near- and middle-east countries. Indeed, homozygosity mapping and sequencing in consanguineous families have already identified a plethora of ARID genes, but because of the design of these studies, DNMs could not be systematically assessed, and the proportion of cases that are potentially preventable by avoiding consanguineous marriages or through carrier testing is hitherto unknown. This prompted us to perform whole-exome sequencing in 100 sporadic ID patients from Iran and their healthy consanguineous parents. In 61 patients, we identified apparently causative changes in known ID genes. Of these, 44 were homozygous recessive and 17 dominant DNMs. Assuming that the DNM rate is stable, these results suggest that parental consanguinity raises the ID risk about 3.6-fold, and about 4.1 to 4.25-fold for children of first-cousin unions. These results do not rhyme with recent opinions that consanguinity-related health risks are generally small and have been “overstated” in the past.     

Closely spaced multiple mutations as potential signatures of transient hypermutability in human genes

Data from diverse organisms suggests that transient hypermutability is a general mutational mechanism with the potential to generate multiple synchronous mutations, a phenomenon probably best exemplified by closely spaced multiple mutations (CSMMs). Here we have attempted to extend the concept of transient hypermutability from somatic cells to the germline, using human inherited disease‐causing multiple mutations as a model system. Employing stringent criteria for data inclusion, we have retrospectively identified numerous potential examples of pathogenic CSMMs that exhibit marked similarities to the CSMMs reported in other systems. These examples include (1) eight multiple mutations, each comprising three or more components within a sequence tract of <100 bp; (2) three possible instances of “mutation showers”; and (3) numerous highly informative “homocoordinate” mutations. Using the proportion of CpG substitution as a crude indicator of the relative likelihood of transient hypermutability, we present evidence to suggest that CSMMs comprising at least one pair of mutations separated by ≤100 bp may constitute signatures of transient hypermutability in human genes. Although this analysis extends the generality of the concept of transient hypermutability and provides new insights into what may be considered a novel mechanism of mutagenesis underlying human inherited disease, it has raised serious concerns regarding current practices in mutation screening.Hum Mutat 30:1–14, 2009. © 2009 Wiley‐Liss, Inc.     

Knockout of human muscle genes revealed by large scale whole-exome studies

Large scale whole-exome sequence studies have revealed that a number of individuals from different populations have predicted loss-of-function of different genes due to nonsense, frameshift, or canonical splice-site mutations. Surprisingly, many of these mutations do not apparently show the deleterious phenotypic consequences expected from gene knockout. These homozygous null mutations, when confirmed, can provide insight into human gene function and suggest novel approaches to correct gene dysfunction, as the lack of the expected disease phenotype may reflect the existence of modifier genes that reveal potential therapeutic targets. Human knockouts complement the information derived from mouse knockouts, which are not always good models of human disease. We have examined human knockout datasets searching for genes expressed exclusively or predominantly in striated muscle. A number of well-known muscle genes was found in one or more datasets, including genes coding for sarcomeric myosins, components of the sarcomeric cytoskeleton, sarcoplasmic reticulum and plasma membrane, and enzymes involved in muscle metabolism. The surprising absence of phenotype in some of these human knockouts is critically discussed, focusing on the comparison with the corresponding mouse knockouts.     

Maternally inherited mitochondrial DNA disease in consanguineous families

Mitochondrial respiratory chain disease represents one of the most common inborn errors of metabolism and is genetically heterogeneous, with biochemical defects arising from mutations in the mitochondrial genome (mtDNA) or the nuclear genome. As such, inheritance of mitochondrial respiratory chain disease can either follow dominant or recessive autosomal (Mendelian) inheritance patterns, the strictly matrilineal inheritance observed with mtDNA point mutations or X-linked inheritance. Parental consanguinity in respiratory chain disease is often assumed to infer an autosomal recessive inheritance pattern, and the analysis of mtDNA may be overlooked in the pursuit of a presumed nuclear genetic defect. We report the histochemical, biochemical and molecular genetic investigations of two patients with suspected mitochondrial disease who, despite being born to consanguineous first-cousin parents, were found to harbour well-characterised pathogenic mtDNA mutations, both of which were maternally transmitted. Our findings highlight that any diagnostic algorithm for the investigation of mitochondrial respiratory chain disease must include a full and complete analysis of the entire coding sequence of the mitochondrial genome in a clinically relevant tissue. An autosomal basis for respiratory chain disease should not be assumed in consanguineous families and that 'maternally inherited consanguineous' mitochondrial disease may thus be going undiagnosed.     

The natural history of Huntington disease: Possible role of “aging genes”

In this paper we consider a model in which genetic factors that control aging also modify expression of the Huntington disease (HD) gene. Significant correlation coefficients were obtained for age-at-onset (AO) and age-at-death (AD) between affected parents and their affected offspring. However, more relevant to our hypothesis, the correlations between mean AD in normal sibs and AD in the affected parent (r =.57) and mean AD in their affected sibs (r =.54) are also significant. When onset is used instead of death for affected individuals the coefficients are.46 and.52, respectively. Also, AD in the normal parent is significantly correlated with AD in his affected (r =.39) and normal (r =.36) offspring. Current genetic theories on aging and related trends in HD are discussed. Our results and evidence from gerontological studies support the hypothesis that HD gene carriers with “superior” aging genes manifest symptoms later in life and have increased longevity over those with “inferior” aging genes.     

Homozygosity mapping in a family presenting with schizophrenia, epilepsy and hearing impairment

Homozygosity mapping within consanguineous families is a powerful method of localising genes for autosomal recessive disease. We investigated a family from Punjab, Pakistan, a region where consanguineous marriages are frequent. The parents have no detectable clinical disorders. However, five out of six children present with schizophrenia, epilepsy or hearing impairment either alone or in combination. This unusual phenotype in several offspring of first cousins is strongly suggestive of a rare, Mendelian recessive disorder. Two genome-wide scans initially using low-density microsatellites, and subsequently high-density SNP markers were used to map homozygous-by-descent regions in affected individuals. Candidate genes within these loci were subsequently screened for mutations. Homozygosity analysis and inbreeding coefficients were investigated to give an estimate of consanguinity. Two putative disease loci were mapped to 22q12.3-q13.3 and 2p24.3. The candidate locus on chromosome 2p24 overlaps with a deafness locus, DFNB47, linked to autosomal recessive hearing impairment, while positive findings reported for affective psychosis and schizophrenia cluster in a region of 4-5 cM on 22q13.1 within our second candidate locus. Sequence analysis of three candidate genes (KCNF1 (2p); ATF4, CACNG2 (22q)) did not reveal any exonic mutations. Inbreeding coefficients calculated for each family member support a very high degree of ancestral and recent inbreeding. The screening of other candidate genes located within these newly identified disease intervals on Chr2p24.3 and 22q12.3-q13.3 may lead to the discovery of causative variants, and consequent disrupted molecular pathways associated with this rare phenotype.     

Synergistic interaction of heterozygous deletions impairs performance and confers susceptibility to disease at all ages

Rare recessive conditions are more common in the offspring of cousin unions than in the general population even in communities in which less than 0.5% of all marriages are between first cousins. It is shown that if the human genome has 30,000 genes in the haploid set then (Y+X)6/(Y)5 < 19, where X is the mean number of new mutations entering the human genome per generation and Y is the mean number of heterozygous deletions in the germ line of adults. This places an upper limit on X of 1.3. Functions specifying the frequency of homozygous deletions in the offspring of cousin and sibling unions are also derived in terms of X and Y. The best estimates with these three measures are that Y is between 6 and 10, and X between 0.5 and 1.3. Data on mutation rates in bacteria, worms, yeast, flies and mice are consistent with these estimates. It is argued that selection against heterozygous deletions must occur in order to prevent progressive build up in the genome; furthermore, it is postulated that the selection must be based on synergistic interaction. If heterozygous deletions interact synergistically to degrade biological performance then this has implications for human fertility, for polygenic disease, for the genetic basis of polygenic traits such as intelligence and the association between birth weight and adult disease (Barker's phenomenon) and the links between status and health inequality. The concept proposed is that a large set of genes specify each aspect of biological capability; the genes specify a redundant system so that one or two deletions will have minimal effect; but several deletions will interact synergistically to degrade performance and cause disease.     

Interlocus gene conversion events introduce deleterious mutations into at least 1% of human genes associated with inherited disease

Establishing the molecular basis of DNA mutations that cause inherited disease is of fundamental importance to understanding the origin, nature, and clinical sequelae of genetic disorders in humans. The majority of disease-associated mutations constitute single-base substitutions and short deletions and/or insertions resulting from DNA replication errors and the repair of damaged bases. However, pathological mutations can also be introduced by nonreciprocal recombination events between paralogous sequences, a phenomenon known as interlocus gene conversion (IGC). IGC events have thus far been linked to pathology in more than 20 human genes. However, the large number of duplicated gene sequences in the human genome implies that many more disease-associated mutations could originate via IGC. Here, we have used a genome-wide computational approach to identify disease-associated mutations derived from IGC events. Our approach revealed hundreds of known pathological mutations that could have been caused by IGC. Further, we identified several dozen high-confidence cases of inherited disease mutations resulting from IGC in ～1% of all genes analyzed. About half of the donor sequences associated with such mutations are functional paralogous genes, suggesting that epistatic interactions or differential expression patterns will determine the impact upon fitness of specific substitutions between duplicated genes. In addition, we identified thousands of hitherto undescribed and potentially deleterious mutations that could arise via IGC. Our findings reveal the extent of the impact of interlocus gene conversion upon the spectrum of human inherited disease.     

Meiotic recombination favors the spreading of deleterious mutations in human populations

Although mutations that are detrimental to the fitness of organisms are expected to be rapidly purged from populations by natural selection, some disease-causing mutations are present at high frequencies in human populations. Several nonexclusive hypotheses have been proposed to account for this apparent paradox (high new mutation rate, genetic drift, overdominance, or recent changes in selective pressure). However, the factors ultimately responsible for the presence at high frequency of disease-causing mutations are still contentious. Here we establish the existence of an additional process that contributes to the spreading of deleterious mutations: GC-biased gene conversion (gBGC), a process associated with recombination that tends to favor the transmission of GC-alleles over AT-alleles. We show that the spectrum of amino acid-altering polymorphisms in human populations exhibits the footprints of gBGC. This pattern cannot be explained in terms of selection and is evident with all nonsynonymous mutations, including those predicted to be detrimental to protein structure and function, and those implicated in human genetic disease. We present simulations to illustrate the conditions under which gBGC can extend the persistence time of deleterious mutations in a finite population. These results indicate that gBGC meiotic drive contributes to the spreading of deleterious mutations in human populations.     

Postzygotic single‐nucleotide mosaicisms contribute to the etiology of autism spectrum disorder and autistic traits and the origin of mutations

The roles and characteristics of postzygotic single‐nucleotide mosaicisms (pSNMs) in autism spectrum disorders (ASDs) remain unclear. In this study of the whole exomes of 2,361 families in the Simons Simplex Collection, we identified 1,248 putative pSNMs in children and 285 de novo SNPs in children with detectable parental mosaicism. Ultra‐deep amplicon resequencing suggested a validation rate of 51%. Analyses of validated pSNMs revealed that missense/loss‐of‐function (LoF) pSNMs with a high mutant allele fraction (MAF≥ 0.2) contributed to ASD diagnoses (P = 0.022, odds ratio [OR] = 5.25), whereas missense/LoF pSNMs with a low MAF (MAF<0.2) contributed to autistic traits in male non‐ASD siblings (P = 0.033). LoF pSNMs in parents were less likely to be transmitted to offspring than neutral pSNMs (P = 0.037), and missense/LoF pSNMs in parents with a low MAF were transmitted more to probands than to siblings (P = 0.016, OR = 1.45). We estimated that pSNMs in probands or de novo mutations inherited from parental pSNMs increased the risk of ASD by approximately 6%. Adding pSNMs into the transmission and de novo association test model revealed 13 new ASD risk genes. These results expand the existing repertoire of genes involved in ASD and shed new light on the contribution of genomic mosaicisms to ASD diagnoses and autistic traits.     

Mutation rate: A simple concept has become complex

The factors that cause new mutations or affect the rate at which they occur have important implications for many areas of genetics. But recent work on phenomena such as premeiotic mutations, which yield a cluster of identical new mutants at the same time, led us to realize that researchers are using the term “mutation rate” in different, and sometimes contradictory, ways. One premeiotic genetic change may ultimately yield several new mutant offspring, but should this be considered one new mutation or many? The way the data are handled in analyses can have a significant effect on the results. How, then, does one handle clusters in the estimation of mutation rates? We explore this question and propose that geneticists begin to distinguish clearly between three different phenomena that to this point have been given the same name: the initial prerepair “genetic damage rate,” the postrepair “mutational event rate,” and the observed “mutation rate” as it is expressed in the proportion of new mutant offspring. We believe that all new mutant offspring should be counted when estimating mutation rate, irrespective of when in the developmental cycle it is believed that the initial mutational event occurred. Environ. Mol. Mutagen. 32: 292–300, 1998 © 1998 Wiley-Liss, Inc.     

Revealing the human mutome

Chen JM, Férec C, Cooper DN. Revealing the human mutome. The number of known mutations in human nuclear genes, underlying or associated with human inherited disease, has now exceeded 100,000 in more than 3700 different genes (Human Gene Mutation Database). However, for a variety of reasons, this figure is likely to represent only a small proportion of the clinically relevant genetic variants that remain to be identified in the human genome (the ‘mutome’). With the advent of next‐generation sequencing, we are currently witnessing a revolution in medical genetics. In particular, whole‐genome sequencing (WGS) has the potential to identify all disease‐causing or disease‐associated DNA variants in a given individual. Here, we use examples of recent advances in our understanding of mutational/pathogenic mechanisms to guide our thinking about possible locations outwith gene‐coding sequences for those disease‐causing or disease‐associated variants that are likely so often to have been overlooked because of the inadequacy of current mutation screening protocols. Such considerations are important not only for improving mutation‐screening strategies but also for enhancing the interpretation of findings derived from genome‐wide association studies, whole‐exome sequencing and WGS. An improved understanding of the human mutome will not only lead to the development of improved diagnostic testing procedures but should also improve our understanding of human genome biology.     

Simple and Efficient Identification of Rare Recessive Pathologically Important Sequence Variants from Next Generation Exome Sequence Data

Massively parallel (“next generation”) DNA sequencing (NGS) has quickly become the method of choice for seeking pathogenic mutations in rare uncharacterized monogenic diseases. Typically, before DNA sequencing, protein‐coding regions are enriched from patient genomic DNA, representing either the entire genome (“exome sequencing”) or selected mapped candidate loci. Sequence variants, identified as differences between the patient's and the human genome reference sequences, are then filtered according to various quality parameters. Changes are screened against datasets of known polymorphisms, such as dbSNP and the 1000 Genomes Project, in the effort to narrow the list of candidate causative variants. An increasing number of commercial services now offer to both generate and align NGS data to a reference genome. This potentially allows small groups with limited computing infrastructure and informatics skills to utilize this technology. However, the capability to effectively filter and assess sequence variants is still an important bottleneck in the identification of deleterious sequence variants in both research and diagnostic settings. We have developed an approach to this problem comprising a user‐friendly suite of programs that can interactively analyze, filter and screen data from enrichment‐capture NGS data. These programs (“Agile Suite”) are particularly suitable for small‐scale gene discovery or for diagnostic analysis.     

UVEOGENE: An SNP database for investigations on genetic factors associated with uveitis and their relationship with other systemic autoimmune diseases

Uveitis is an intraocular inflammatory disease which can lead to serious visual impairment. Genetic factors have been shown to be involved in its development. However, few databases have focused on the information of associations between single nucleotide polymorphisms (SNPs) and uveitis. To discover the exact genetic background of uveitis, we developed an SNP database specific for uveitis, “UVEOGENE,” which includes 370 genes and 918 SNPs covering 14 uveitis entities and 40 populations from 286 PubMed English‐language papers. Stratification analyses by gender, HLA status, and different clinical features were also extracted from the publications. As a result, 371 associations were judged as “statistically significant.” These associations were also shared with Global Variome shared Leiden Open Variation Database (LOVD) ( https://databases.lovd.nl/shared/genes ). Based on these associations, we investigated the genetic relationship among three widely studied uveitis entities including Behcet's disease (BD), Vogt–Koyanagi–Harada (VKH) disease, and acute anterior uveitis (AAU). Furthermore, “UVEOGENE” can be used as a reliable and informative resource to identify similarities as well as differences in the genetic susceptibility among uveitis and other autoimmune diseases. UVEOGENE is freely accessible at http://www.uvogene.com .