Patterns and rates of exonic de novo mutations in autism spectrum disorders

Autism spectrum disorders (ASD) are believed to have genetic and environmental origins, yet in only a modest fraction of individuals can specific causes be identified. To identify further genetic risk factors, here we assess the role of de novo mutations in ASD by sequencing the exomes of ASD cases and their parents (n = 175 trios). Fewer than half of the cases (46.3%) carry a missense or nonsense de novo variant, and the overall rate of mutation is only modestly higher than the expected rate. In contrast, the proteins encoded by genes that harboured de novo missense or nonsense mutations showed a higher degree of connectivity among themselves and to previous ASD genes as indexed by protein-protein interaction screens. The small increase in the rate of de novo events, when taken together with the protein interaction results, are consistent with an important but limited role for de novo point mutations in ASD, similar to that documented for de novo copy number variants. Genetic models incorporating these data indicate that most of the observed de novo events are unconnected to ASD; those that do confer risk are distributed across many genes and are incompletely penetrant (that is, not necessarily sufficient for disease). Our results support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5- to 20-fold. Despite the challenge posed by such models, results from de novo events and a large parallel case-control study provide strong evidence in favour of CHD8 and KATNAL2 as genuine autism risk factors.     

Rate of de novo mutations and the importance of father's age to disease risk

Mutations generate sequence diversity and provide a substrate for selection. The rate of de novo mutations is therefore of major importance to evolution. Here we conduct a study of genome-wide mutation rates by sequencing the entire genomes of 78 Icelandic parent-offspring trios at high coverage. We show that in our samples, with an average father's age of 29.7, the average de novo mutation rate is 1.20?×?10(-8) per nucleotide per generation. Most notably, the diversity in mutation rate of single nucleotide polymorphisms is dominated by the age of the father at conception of the child. The effect is an increase of about two mutations per year. An exponential model estimates paternal mutations doubling every 16.5?years. After accounting for random Poisson variation, father's age is estimated to explain nearly all of the remaining variation in the de novo mutation counts. These observations shed light on the importance of the father's age on the risk of diseases such as schizophrenia and autism.     

De novo mutations revealed by whole-exome sequencing are strongly associated with autism

Multiple studies have confirmed the contribution of rare de novo copy number variations to the risk for autism spectrum disorders. But whereas de novo single nucleotide variants have been identified in affected individuals, their contribution to risk has yet to be clarified. Specifically, the frequency and distribution of these mutations have not been well characterized in matched unaffected controls, and such data are vital to the interpretation of de novo coding mutations observed in probands. Here we show, using whole-exome sequencing of 928 individuals, including 200 phenotypically discordant sibling pairs, that highly disruptive (nonsense and splice-site) de novo mutations in brain-expressed genes are associated with autism spectrum disorders and carry large effects. On the basis of mutation rates in unaffected individuals, we demonstrate that multiple independent de novo single nucleotide variants in the same gene among unrelated probands reliably identifies risk alleles, providing a clear path forward for gene discovery. Among a total of 279 identified de novo coding mutations, there is a single instance in probands, and none in siblings, in which two independent nonsense variants disrupt the same gene, SCN2A (sodium channel, voltage-gated, type II, α subunit), a result that is highly unlikely by chance.     

A map of human genome variation from population-scale sequencing

The 1000 Genomes Project aims to provide a deep characterization of human genome sequence variation as a foundation for investigating the relationship between genotype and phenotype. Here we present results of the pilot phase of the project, designed to develop and compare different strategies for genome-wide sequencing with high-throughput platforms. We undertook three projects: low-coverage whole-genome sequencing of 179 individuals from four populations; high-coverage sequencing of two mother-father-child trios; and exon-targeted sequencing of 697 individuals from seven populations. We describe the location, allele frequency and local haplotype structure of approximately 15 million single nucleotide polymorphisms, 1 million short insertions and deletions, and 20,000 structural variants, most of which were previously undescribed. We show that, because we have catalogued the vast majority of common variation, over 95% of the currently accessible variants found in any individual are present in this data set. On average, each person is found to carry approximately 250 to 300 loss-of-function variants in annotated genes and 50 to 100 variants previously implicated in inherited disorders. We demonstrate how these results can be used to inform association and functional studies. From the two trios, we directly estimate the rate of de novo germline base substitution mutations to be approximately 10(-8) per base pair per generation. We explore the data with regard to signatures of natural selection, and identify a marked reduction of genetic variation in the neighbourhood of genes, due to selection at linked sites. These methods and public data will support the next phase of human genetic research.     

Large recurrent microdeletions associated with schizophrenia

Reduced fecundity, associated with severe mental disorders, places negative selection pressure on risk alleles and may explain, in part, why common variants have not been found that confer risk of disorders such as autism, schizophrenia and mental retardation. Thus, rare variants may account for a larger fraction of the overall genetic risk than previously assumed. In contrast to rare single nucleotide mutations, rare copy number variations (CNVs) can be detected using genome-wide single nucleotide polymorphism arrays. This has led to the identification of CNVs associated with mental retardation and autism. In a genome-wide search for CNVs associating with schizophrenia, we used a population-based sample to identify de novo CNVs by analysing 9,878 transmissions from parents to offspring. The 66 de novo CNVs identified were tested for association in a sample of 1,433 schizophrenia cases and 33,250 controls. Three deletions at 1q21.1, 15q11.2 and 15q13.3 showing nominal association with schizophrenia in the first sample (phase I) were followed up in a second sample of 3,285 cases and 7,951 controls (phase II). All three deletions significantly associate with schizophrenia and related psychoses in the combined sample. The identification of these rare, recurrent risk variants, having occurred independently in multiple founders and being subject to negative selection, is important in itself. CNV analysis may also point the way to the identification of additional and more prevalent risk variants in genes and pathways involved in schizophrenia.     

Genetic imprinting suggested by maternal heterodisomy in nondeletion Prader-Willi syndrome

Prader-Willi syndrome (PWS) is the most common form of dysmorphic genetic obesity associated with mental retardation. About 60% of cases have a cytological deletion of chromosome 15q11q13 (refs 2, 3). These deletions occur de novo exclusively on the paternal chromosome. By contrast, Angelman syndrome (AS) is a very different clinical disorder and is also associated with deletions of region 15q11q13 (refs 6-8), indistinguishable from those in PWS except that they occur de novo on the maternal chromosome. The parental origin of the affected chromosomes 15 in these disorders could, therefore, be a contributory factor in determining their clinical phenotypes. We have now used cloned DNA markers specific for the 15q11q13 subregion to determine the parental origin of chromosome 15 in PWS individuals not having cytogenetic deletions; these individuals account for almost all of the remaining 40% of PWS cases. Probands in two families displayed maternal uniparental disomy for chromosome 15q11q13. This is the first demonstration that maternal heterodisomy--the presence of two different chromosome 15s derived from the mother--can be associated with a human genetic disease. The absence of a paternal contribution of genes in region 15q11q13, as found in PWS deletion cases, rather than a mutation in a specific gene(s) in this region may result in expression of the clinical phenotype. Thus, we conclude that a gene or genes in region 15q11q13 must be inherited from each parent for normal human development.     

Proto-genes and de novo gene birth

Novel protein-coding genes can arise either through re-organization of pre-existing genes or de novo. Processes involving re-organization of pre-existing genes, notably after gene duplication, have been extensively described. In contrast, de novo gene birth remains poorly understood, mainly because translation of sequences devoid of genes, or 'non-genic' sequences, is expected to produce insignificant polypeptides rather than proteins with specific biological functions. Here we formalize an evolutionary model according to which functional genes evolve de novo through transitory proto-genes generated by widespread translational activity in non-genic sequences. Testing this model at the genome scale in Saccharomyces cerevisiae, we detect translation of hundreds of short species-specific open reading frames (ORFs) located in non-genic sequences. These translation events seem to provide adaptive potential, as suggested by their differential regulation upon stress and by signatures of retention by natural selection. In line with our model, we establish that S. cerevisiae ORFs can be placed within an evolutionary continuum ranging from non-genic sequences to genes. We identify ~1,900 candidate proto-genes among S. cerevisiae ORFs and find that de novo gene birth from such a reservoir may be more prevalent than sporadic gene duplication. Our work illustrates that evolution exploits seemingly dispensable sequences to generate adaptive functional innovation.     

Paternal origin of new mutations in von Recklinghausen neurofibromatosis

Von Recklinghausen neurofibromatosis (NF-1) is a common autosomal dominant disorder. The estimated new mutation rate (1 x 10(-4] is one of the highest for a human disorder. Here we report that in 12 of 14 families we have analysed, the new mutation is of paternal origin. This result is similar to that recently obtained for retinoblastoma. In other genetic disorders that show a bias towards paternal origin of new mutations, there is a marked increase in the incidence of mutations with paternal age, consistent with the mutations arising from replication errors in mitosis of spermatogonial stem cells. In retinoblastoma and NF-1, however, such paternal age effects are slight or absent. The mechanism or timing of germline mutation could therefore be different in the two cases.     

Uniparental paternal disomy in a genetic cancer-predisposing syndrome.

The 11p15.5 region of human chromosome 11 seems to contain a locus or loci involved in congenital overgrowth anomalies as well as in the genesis of many tumours associated with the Beckwith-Wiedemann syndrome (BWS). Given the unusual differential parental allele involvement in the different aetiological forms of BWS and the loss of maternal alleles in associated tumours, we have now used 11p15.5 markers to determine the parental origin of chromosome 11 in eight sporadic cases of BWS. Probands in three informative families had uniparental paternal disomy for region 11p15.5. Further, an overall greatly increased frequency of homozygosity for several 11p15.5 markers in 21 sporadic BWS patients suggests that isodisomy probably accounts for an even higher proportion of BWS sporadic cases. This demonstrates that uniparental paternal disomy can be associated with a genetic cancer-predisposing syndrome.     

耳聋基因GJB2的突变分析及其与临床相关性的研究

为了研究苏州地区耳聋人群中GJB2基因突变的频率、突变类型及这些突变与耳聋发生的相关性,收集苏州地区散发性耳聋患者样本106例,家系32个(父母和患儿),听力正常对照组样本100例,PCR-直接测序法分析GJB2基因的突变,PCR-RFLP方法对明确有235delC突变的家系作进一步分析.结果显示:106例散发性耳聋患者中发现了11种变异,235delC是最主要的突变,其中18例为235delC纯合性缺失.235delC纯合突变导致常染色体隐性遗传性耳聋,患者多为双耳重深度耳聋(双耳大于90 dB),发病年龄早;235delc和其他位于不同染色体上的突变形成双重杂合性突变时,也可以导致耳聋.PCR-RFLP方法可在明确235delC突变的患者家系中进行基因诊断.     

Preferential mutation of paternally derived RB gene as the initial event in sporadic osteosarcoma

Successive loss of function of both alleles of the retinoblastoma susceptibility gene (RB) on human chromosome 13 seems to be critical in the development of retinoblastoma and osteosarcoma. In cases where the tumour is familial and susceptibility is inherited, a mutation in one of the alleles is carried in the germline. We have recently shown that cytogenetically visible germline mutations are usually in the paternally derived gene. Such a bias would not be expected for sporadic (non-familial) tumours, where both mutations occur in somatic tissue, but there has been some indication of a bias towards initial somatic mutation in the paternally derived gene on chromosome 11 in sporadic Wilms tumour. We have now examined 13 sporadic osteosarcomas and find evidence which indicates that in 12 cases the initial mutation was in the paternal gene, suggesting the involvement of germinal imprinting in producing the differential susceptibility of the two genes to mutation.     

Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two most common non-Hodgkin lymphomas (NHLs). Here we sequenced tumour and matched normal DNA from 13 DLBCL cases and one FL case to identify genes with mutations in B-cell NHL. We analysed RNA-seq data from these and another 113 NHLs to identify genes with candidate mutations, and then re-sequenced tumour and matched normal DNA from these cases to confirm 109 genes with multiple somatic mutations. Genes with roles in histone modification were frequent targets of somatic mutation. For example, 32% of DLBCL and 89% of FL cases had somatic mutations in MLL2, which encodes a histone methyltransferase, and 11.4% and 13.4% of DLBCL and FL cases, respectively, had mutations in MEF2B, a calcium-regulated gene that cooperates with CREBBP and EP300 in acetylating histones. Our analysis suggests a previously unappreciated disruption of chromatin biology in lymphomagenesis.     

Recurrent mutations in haemophilia A give evidence for CpG mutation hotspots

Haemophilia A is a common disorder of blood coagulation caused by a deficiency of factor VIII. It is inherited as an X-linked recessive trait, and one-third of all cases are thought to result from de novo mutations. The clinical severity of haemophilia A varies markedly among different families and a subset of the patients with severe disease develop antibodies against factor VIII, called inhibitors. Because of this heterogeneity, it is likely that many different molecular lesions result in haemophilia A. Indeed, of the nine mutations described to date, all appear to be unique changes. However in this study of 83 patients with haemophilia A we have identified two different point mutations, one in exon 18 and one in exon 22, that have recurred independently in unrelated families. Each mutation produces a nonsense codon by a change of CG to TG, and each occurred de novo on the X-chromosome donated by the maternal grandfather. These observations strongly support the view that CpG dinucleotides are mutation hotspots.     

Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder characterized by features reminiscent of marked premature ageing. Here, we present evidence of mutations in lamin A (LMNA) as the cause of this disorder. The HGPS gene was initially localized to chromosome 1q by observing two cases of uniparental isodisomy of 1q-the inheritance of both copies of this material from one parent-and one case with a 6-megabase paternal interstitial deletion. Sequencing of LMNA, located in this interval and previously implicated in several other heritable disorders, revealed that 18 out of 20 classical cases of HGPS harboured an identical de novo (that is, newly arisen and not inherited) single-base substitution, G608G(GGC > GGT), within exon 11. One additional case was identified with a different substitution within the same codon. Both of these mutations result in activation of a cryptic splice site within exon 11, resulting in production of a protein product that deletes 50 amino acids near the carboxy terminus. Immunofluorescence of HGPS fibroblasts with antibodies directed against lamin A revealed that many cells show visible abnormalities of the nuclear membrane. The discovery of the molecular basis of this disease may shed light on the general phenomenon of human ageing.     

A Met-to-Val mutation in the skeletal muscle Na+ channel alpha-subunit in hyperkalaemic periodic paralysis.

HYPERKALAEMIC periodic paralysis (HYPP) is an autosomal dominant disease that results in episodic electrical inexcitability and paralysis of skeletal muscle. Electrophysiological data indicate that tetrodotoxin-sensitive sodium channels from muscle cells of HYPP-affected individuals show abnormal inactivation. Genetic analysis of nine HYPP families has shown tight linkage between the adult skeletal muscle sodium channel alpha-subunit gene on chromosome 17q and the disease (lod score, z = 24; recombination frequency 0 = 0), strongly suggesting that mutations of the alpha-subunit gene cause HYPP. We sequenced the alpha-subunit coding region isolated from muscle biopsies from affected (familial HYPP) and control individuals by cross-species polymerase chain reaction-mediated complementary DNA cloning. We have identified an A----G substitution in the patient's messenger RNA that causes a Met----Val change in a highly conserved region of the alpha-subunit, predicted to be in a transmembrane domain. This same change was found in a sporadic case of HYPP as a new mutation. We have therefore discovered a voltage-gated channel mutation responsible for a human genetic disease.     

基于EST-SSR标记鉴定猴耳环自由授粉的全同胞子代

【目的】猴耳环是重要的药用树种,具有较好的工业应用价值。利用EST-SSR标记对猴耳环自然群体1株母树的自由授粉子代进行父本鉴定,从而确定全同胞子代,为基于全同胞家系的后续研究提供材料基础。【方法】以自然群体中母树ELS31自由授粉产生的1 489株子代及该群体内挂果的38株候选父本为材料,利用15个EST-SSR标记检测子代多样性和标记多态性,基于最大似然法鉴定各子代的父本,母本父本均相同的子代即构成全同胞家系,并估算群体内花粉传播距离,检验各父本对应的全同胞家系的多样性,通过卡方检验判断标记是否合乎预期的孟德尔分离比。【结果】15个EST-SSR标记的引物(对)共扩增出89个等位片段。子代群体期望杂合度(H_e)为0.525,表明群体多样性为中等水平;基于自然群体18株无亲缘关系的单株估算的标记平均多态性信息量(PIC)为0.736,表明标记多态性高。在1 489株自由授粉的子代中,确定了857株子代(57.6%)的34株父本。子代数最多的10个父本产生的子代数为26~184株,其他24个父本仅共产生子代139株。未发现自交子代,表明猴耳环是异交物种,自交的可能性极低。对子代数量20株以上的10个父本对应的全同胞家系观测杂合度(H_o)为0.502~0.693,H_e为0.417~0.544,各家系均是H_o大于H_e,表明存在一定程度的杂合子过剩。花粉传播的范围为10.0~559.1 m,平均119.2 m,但主要传播距离在150 m以内。716株子代(83.5%)的父本(10株)与母树距离在150 m以内。距离最远的父本ELS01 (559.1 m)和ELS02 (552.2 m)分别仅产生了9和12株子代。15个标记在子代20株以上的部分或全部全同胞家系中均有不同程度的偏分离,平均每家系的偏分离标记数为8.6个;偏分离最严重的是标记ARCeSSR141,在父本ELS30的全同胞家系中卡方(χ²)值为164.55。【结论】基于15个EST-SSR标记鉴定了猴耳环自然群体1株母本的1 489株自由授粉子代的父本,获得了子代20株以上的10个父本的全同胞家系。这为后续遗传测定、遗传图谱构建和数量性状位点定位等研究提供了材料基础。