Inference of identity by descent in population isolates and optimal sequencing studies

In an isolated population, individuals are likely to share large genetic regions inherited from common ancestors. Identity by descent (IBD) can be inferred from SNP genotypes, which is useful in a number of applications, including identifying genetic variants influencing complex disease risk, and planning efficient cohort-sequencing strategies. We present ANCHAP – a method for detecting IBD in isolated populations. We compare accuracy of the method against other long-range and local phasing methods, using parent–offspring trios. In our experiments, we show that ANCHAP performs similarly as the other long-range method, but requires an order-of-magnitude less computational resources. A local phasing model is able to achieve similar sensitivity, but only at the cost of higher false discovery rates. In some regions of the genome, the studied individuals share haplotypes particularly often, which hints at the history of the populations studied. We demonstrate the method using SNP genotypes from three isolated island populations, as well as in a cohort of unrelated individuals. In samples from three isolated populations of around 1000 individual each, an average individual shares a haplotype at a genetic locus with 9–12 other individuals, compared with only 1 individual within the non-isolated population. We describe an application of ANCHAP to optimally choose samples in resequencing studies. We find that with sample sizes of 1000 individuals from an isolated population genotyped using a dense SNP array, and with 20% of these individuals sequenced, 65% of sequences of the unsequenced subjects can be partially inferred.     

Efficient approach to unique single-nucleotide polymorphism discovery

Single-nucleotide polymorphisms (SNPs) are the most frequently found DNA sequence variations in the human genome. It has been argued that a dense set of SNP markers can be used to identify genetic factors associated with complex disease traits. Because all high-throughput genotyping methods require precise sequence knowledge of the SNPs, any SNP discovery approach must involve both the determination of DNA sequence and allele frequencies. Furthermore, high-throughput genotyping also requires a genomic DNA amplification step, making it necessary to develop sequence-tagged sites (STSs) that amplify only the DNA fragment containing the SNP and nothing else from the rest of the genome. In this report, we demonstrate the utility of a SNP-screening approach that yields the DNA sequence and allele frequency information while screening out duplications with minimal cost and effort. Our approach is based on the use of a homozygous complete hydatidiform mole (CHM) as the reference. With this homozygous reference, one can identify and estimate the allele frequencies of common SNPs with a pooled DNA-sequencing approach (rather than having to sequence numerous individuals as is commonly done). More importantly, the CHM reference is preferable to a single individual reference because it reveals readily any duplicated regions of the genome amplified by the PCR assay before the duplicated sequences are found in GenBank. This approach reduces the cost of SNP discovery by 60% and eliminates the costly development of SNP markers that cannot be amplified uniquely from the genome.     

Novel presenilin 1 variant (P117A) causing Alzheimer's disease in the fourth decade of life

Over 160 rare genetic variants in presenilin 1 (PSEN1) are known to cause Alzheimer's disease (AD). In this study we screened a family with early-onset AD for mutations in PSEN1 using direct DNA sequencing. We identified a novel PSEN1 genetic variant which results in the substitution of a Proline with an Alanine at codon 117 (P117A). The P117A variant was present in all demented individuals and fifty percent of at risk individuals. This variant occurs at a site where three other disease-causing variants have been previously observed. In vitro functional studies demonstrate that the P117A variant results in an altered Abeta42/total Abeta ratio consistent with an AD causing mutation. The P117A variant is a novel mutation in PSEN1, which causes early-onset AD in an autosomal dominant manner.     

Exome sequencing can detect pathogenic mosaic mutations present at low allele frequencies

The development of next generation sequencing (NGS) has radically transformed the scientific landscape, making it possible to sequence the exome of any given individual in a cost-effective way. The power of this approach has been demonstrated by a number of groups who have identified pathogenic mutations in small pedigrees that have been resistant to traditional genetic mapping. Recently it has become clear that exome sequencing has great potential with respect to sporadic disease and the identification of de novo mutations. This is highlighted by studies reporting whole-exome sequencing of patient-parental trios affected by learning disability, autism and schizophrenia. It is widely anticipated that the introduction of this technique into a clinical setting will revolutionise genetic diagnosis. However, the sensitivity of NGS exome sequencing is currently unclear. Here, we describe the exome sequencing of DNA samples from a patient with double cortex syndrome and her parents, resulting in the detection of a mosaic splicing mutation in LIS1. This variant was found at an allele frequency of just 18%, demonstrating that NGS methods have the capacity to identify pathogenic mosaic mutations present at a low level.     

An integrated approach for classifying mitochondrial DNA variants: one clinical diagnostic laboratory’s experience

PurposeThe mitochondrial genome is highly polymorphic. A unique feature of deleterious mitochondrial DNA (mtDNA) mutations is heteroplasmy. Genetic background and variable penetrance also play roles in the pathogenicity for a mtDNA variant. Clinicians are increasingly interested in requesting mtDNA testing. However, interpretation of uncharacterized mtDNA variants is a great challenge. We suggest a stepwise interpretation procedure for clinical service.MethodsWe describe the algorithms used to interpret novel and rare mtDNA variants. mtDNA databases and in silico predictive algorithms are used to evaluate the pathogenic potential of novel and/or rare mtDNA variants.ResultsmtDNA variants can be classified into three categories: benign variants, unclassified variants, and deleterious mutations based on database search and in silico prediction. Targeted DNA sequence analysis of matrilineal relatives, heteroplasmy quantification, and functional studies are useful to classify mtDNA variants.ConclusionClinical significance of a novel or rare variant is critical in the diagnosis of the disease and counseling of the family. Based on the results from clinical, biochemical, and molecular genetic studies of multiple family members, proper interpretation of mtDNA variants is important for clinical laboratories and for patient care.     

Personal genome sequencing: current approaches and challenges

The revolution in DNA sequencing technologies has now made it feasible to determine the genome sequences of many individuals; i.e., “personal genomes.” Genome sequences of cells and tissues from both normal and disease states have been determined. Using current approaches, whole human genome sequences are not typically assembled and determined de novo, but, instead, variations relative to a reference sequence are identified. We discuss the current state of personal genome sequencing, the main steps involved in determining a genome sequence (i.e., identifying single-nucleotide polymorphisms [SNPs] and structural variations [SVs], assembling new sequences, and phasing haplotypes), and the challenges and performance metrics for evaluating the accuracy of the reconstruction. Finally, we consider the possible individual and societal benefits of personal genome sequences.     

Heterozygosity mapping for human dominant trait variants

Homozygosity mapping is a well‐known technique to identify runs of homozygous variants that are likely to harbor genes responsible for autosomal recessive disease, but a comparable method for autosomal dominant traits has been lacking. We developed an approach to map dominant disease genes based on heterozygosity frequencies of sequence variants in the immediate vicinity of a dominant trait. We demonstrate through theoretical analysis that DNA variants surrounding an inherited dominant disease variant tend to have increased heterozygosity compared with variants elsewhere in the genome. We confirm existence of this phenomenon in sequence data with known dominant pathogenic variants obtained on family members and in unrelated population controls. A computer‐based approach to estimating empirical significance levels associated with our test statistics shows genome‐wide p‐values smaller than 0.05 for many but not all of the individuals carrying a pathogenic variant.     

Whole genome sequencing and mutation rate analysis of trios with paternal dioxin exposure

2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin (TCDD) or dioxin, is commonly considered the most toxic man‐made substance. Dioxin exposure impacts human health and diseases, birth defects and teratogenesis were frequently observed in children of persons who have been exposed to dioxin. However, the impact of dioxin on human mutation rate in trios has not yet been elucidated at the whole genome level. To identify and characterize the genetic alterations in the individuals exposed to dioxin, we performed whole genome sequencing (WGS) of nine Vietnamese trios whose fathers were exposed to dioxin. In total, 846 de novo point mutations, 26 de novo insertions and deletions, 4 de novo structural variations, and 1 de novo copy number variation were identified. The number of point mutations and dioxin concentrations were positively correlated (P‐value < 0.05). Considering the substitution pattern, the number of A > T/T > A mutation and the dioxin concentration was positively correlated (P‐value < 0.05). Our analysis also identified one possible disease‐related mutation in LAMA5 in one trio. These findings suggested that dioxin exposure might affect father genomes of trios leading to de novo mutations in their children. Further analysis with larger sample sizes would be required to better clarify mutation rates and substitution patterns in trios caused by dioxin.     

Molecular Characterization of HCV 1b Intra-Familiar Infection Through Three Generations

Vertical transmission is an uncommon route of hepatitis C virus (HCV) infection. Little is known about the way of virus spread between relatives. Furthermore, the nucleotide sequence variability studies that can be used for the definition of cases of HCV transmission still need accurate standardization. In this study, we analyzed the HCV positive sera from subjects belonging to one family. Five out of seven individuals were positive both for anti-HCV and HCV-RNA. The epidemiological data, in our knowledge, excluded the possible risk of parenteral exposure to HCV for the members of the family. The genetic relatedness of the viruses infecting the members of this family was demonstrated by the phylogenetic analysis of sequences from E1 genome region. The analysis included the calculation of the genetic divergence specific index, based on the ratio of synonymous/non-synonymous mutations. By the analysis of this genome region, we demonstrated the occurrence of HCV transmission among family members. In 2 cases out of 3, Mother-to-Infant transmission was demonstrated that involved three generations of the family. Transmission by sexual route was absent. A method of sequence analysis of E1 HCV genome region is proposed as molecular approach for the definition of transmission cases of HCV.     

A novel homozygous missense variant in NECTIN4 (PVRL4) causing ectodermal dysplasia cutaneous syndactyly syndrome

Ectodermal dysplasia syndactyly syndrome 1 (EDSS1) is a rare form of ectodermal dysplasia including anomalies of hair, nails, and teeth along with bilateral cutaneous syndactyly of hands and feet. In the present report, we performed a clinical and genetic characterization of a consanguineous Pakistani family with four individuals affected by EDSS1. We performed exome sequencing using DNA of one affected individual. Exome data analysis identified a novel homozygous missense variant (c.242T>C; p.(Leu81Pro)) in NECTIN4 (PVRL4). Sanger sequencing validated this variant and confirmed its cosegregation with the disease phenotype in the family members. Thus, our report adds a novel variant to the NECTIN4 mutation spectrum and contributes to the NECTIN4‐related clinical characterization.     

Genetic mapping using fluorescent quantification of allele frequencies in pooled DNA loaded by solid support

An efficient and labour-saving method for fragment analysis in linkage studies using biotinylated primers and streptavidin-coated combs is presented. The level of streptavidin attached to the combs was used to control the amount of immobilised material. Thus, the need for titration of PCR products to fit the dynamic range of the sequencer was reduced. The method was used to investigate the possibility of quantitating allele frequencies in pools of DNA from family members with the autosomal dominant eye disorder Best's macular dystrophy. The method allowed the detection of one unique allele in a background of 39 other alleles. Using independent datasets, it was further found that the method was able to detect distorted allele frequencies in affected individuals of one family as compared to reference individuals, for markers located more than 30 cM from the disease locus. It was found that this procedure is a powerful alternative to conventional linkage analysis and the method may prove useful in a genome scan for genes involved in complex disorders.     

Evidence for an additional locus for split hand/foot malformation in chromosome region 8q21.11-q22.3

We identified a family where five members had nonsyndromic ectrodactyly. There were three known instances of nonpenetrance. Although four individuals had unilateral cleft hand, one individual had more severe, bilateral and asymmetric absence of the digits. None had foot abnormalities. After exclusion of linkage of SHFM in this family to five known loci, a genome wide scan was performed with DNA from 5 affected and 15 unaffected members of this family. Suggestive evidence for linkage of ectrodactyly to 8q was obtained on the basis of a maximum LOD score of 2.54 at theta (max) = 0 with GAAT1A4. Critical recombinants place the ectrodactyly gene in this family in a 16 cM (21 Mb) interval between D8S1143 and D8S556. Mutational analysis of two candidate genes (FZD6, GDF6) did not identify any mutations in affected members of this family. Our data indicate further genetic heterogeneity for ectrodactyly and suggest the presence of an additional SHFM locus in chromosome region 8q21.11-q22.3.     

Mutational analysis of candidate genes in psychiatric disorders

A genetic hypothesis for a disease presupposes the existence of variation in the DNA sequences of affected individuals. A series of techniques known together as “mutational analysis” can be applied towards identifying new sequence variations in selected genes. These techniques can screen a large series of individuals for mutations efficiently, so it is not necessary to determine the nucleotide sequence in every DNA sample. DNA samples suspected of harboring sequence variants are then sequenced. Denaturing gradient gel electrophoresis techniques, single stranded conformation polymorphism paradigms, and chemical cleavage of mismatches are 3 procedures widely used for the molecular screening of mutations today. We discuss each of these techniques for mutation screening. © 1993 Wiley-Liss, Inc.     

Analysis of germline mutation spectra at the Huntington's disease locus supports a mitotic [correction of amitotic] mutation mechanism [published erratum appears in Hum Mol Genet 1999 Apr;8(4):717]

Trinucleotide repeat disease alleles can undergo 'dynamic' mutations in which repeat number may change when a gene is transmitted from parent to offspring. By typing >3500 sperm, we determined the size distribution of Huntington's disease (HD) germline mutations produced by 26 individuals from the Venezuelan cohort with CAG/CTG repeat numbers ranging from 37 to 62. Both the mutation frequency and mean change in allele size increased with increasing somatic repeat number. The mutation frequencies averaged 82% and, for individuals with at least 50 repeats, 98%. The extraordinarily high mutation frequency levels are most consistent with a mutation process that occurs throughout germline mitotic divisions, rather than resulting from a single meiotic event. In several cases, the mean change in repeat number differed significantly among individuals with similar somatic allele sizes. This individual variation could not be attributed to age in a simple way or to ' cis ' sequences, suggesting the influence of genetic background or other factors. A familial effect is suggested in one family where both the father and son gave highly unusual spectra compared with other individuals matched for age and repeat number. A statistical model based on incomplete processing of Okazaki fragments during DNA replication was found to provide an excellent fit to the data but variation in parameter values among individuals suggests that the molecular mechanism might be more complex.      

The affected sib method. V. Testing the assumptions

The affected sib methods, which are used to make inferences about the genetic components of HLA associated diseases, have many underlying assumptions which may not always be realistic. These include no selective disadvantage of affected individuals, little or no recombination between the marker loci and the 'disease' locus, a single panmictic population, Mendelian segregation of the disease locus alleles and random distribution of individuals over environments. The effects of breaking these assumptions have been investigated. We have explicitly derived the haplotype sharing identity by descent (IBD) expectations for the cases of selection against affected individuals and recombination between the HLA marker loci and the 'disease' predisposing locus for affected sib trios (as was previously done for affected sib pairs). We have also derived, for both affected sib pairs and trios, the haplotype sharing expectations for non-random mating (positive assortative), admixture, meiotic drive (of disease allele carrying haplotypes), and a random versus shared environmental component for sibs. In order to assess the sensitivity of the affected sib methods to perturbations in the assumptions, the expectation spaces of haplotype sharing in affected sib pairs and sib trios under the single diallelic locus model with varying penetrances and allele frequencies are fully described. The effects on haplotype sharing and subsequent disease parameter estimation are different for each of the factors we have considered. The affected sib methods are found to be robust in many situations.     

Rapid mapping of zebrafish mutations with SNPs and oligonucleotide microarrays

Large-scale genetic screens in zebrafish have identified thousands of mutations in hundreds of essential genes. The genetic mapping of these mutations is necessary to link DNA sequences to the gene functions defined by mutant phenotypes. Here, we report two advances that will accelerate the mapping of zebrafish mutations: (1) The construction of a first generation single nucleotide polymorphism (SNP) map of the zebrafish genome comprising 2035 SNPs and 178 small insertions/deletions, and (2) the development of a method for mapping mutations in which hundreds of SNPs can be scored in parallel with an oligonucleotide microarray. We have demonstrated the utility of the microarray technique in crosses with haploid and diploid embryos by mapping two known mutations to their previously identified locations. We have also used this approach to localize four previously unmapped mutations. We expect that mapping with SNPs and oligonucleotide microarrays will accelerate the molecular analysis of zebrafish mutations.     

RIN2 and BBS7 variants as cause of a coincidental syndrome

BBS7 and RIN2 variants cause Bardet Biedl syndrome and RIN2 syndrome respectively. We investigated a consanguineous family in which five individuals manifested different phenotypes. Whole-exome sequencing analyses of the individual with multiple phenotypes revealed homozygosity for novel pathogenic variants in his DNA sample; a frameshift variant in RIN2 (c.1938delT) and a splice-site variant in BBS7 (c.1677-1G > A). Other affected individuals were homozygous for a variant in only one of either gene and consequently manifested phenotypes respective to one disorder. Our work shows that exome sequencing of the most severely affected individual can help in the identification of pathogenic variants in more than one involved genes in a particular family.     

Genotype calling and haplotyping in parent-offspring trios

Emerging sequencing technologies allow common and rare variants to be systematically assayed across the human genome in many individuals. In order to improve variant detection and genotype calling, raw sequence data are typically examined across many individuals. Here, we describe a method for genotype calling in settings where sequence data are available for unrelated individuals and parent-offspring trios and show that modeling trio information can greatly increase the accuracy of inferred genotypes and haplotypes, especially on low to modest depth sequencing data. Our method considers both linkage disequilibrium (LD) patterns and the constraints imposed by family structure when assigning individual genotypes and haplotypes. Using simulations, we show that trios provide higher genotype calling accuracy across the frequency spectrum, both overall and at hard-to-call heterozygous sites. In addition, trios provide greatly improved phasing accuracy—improving the accuracy of downstream analyses (such as genotype imputation) that rely on phased haplotypes. To further evaluate our approach, we analyzed data on the first 508 individuals sequenced by the SardiNIA sequencing project. Our results show that our method reduces the genotyping error rate by 50% compared with analysis using existing methods that ignore family structure. We anticipate our method will facilitate genotype calling and haplotype inference for many ongoing sequencing projects.In the past decade, genome-wide association studies (GWAS) have identified associations between thousands of common variants and a variety of complex traits and diseases (McCarthy et al. 2008; Hindorff et al. 2009). Next-generation sequencing technologies enable researchers to look beyond the common variants typically evaluated in these GWAS and systematically consider the contributions of rarer variants (Li and Leal 2008; Cirulli and Goldstein 2010). The ability to systematically examine these rare variants may improve our understanding of complex traits, by identifying the underlying biological mechanisms more completely and by improving our ability to predict individual outcomes (Manolio et al. 2009; Eichler et al. 2010).Next-generation sequencing can be used to study rare variation either by directly sequencing phenotyped individuals or by sequencing a reference set of individuals and then using genotype imputation to study association in phenotyped individuals. In the first case, it is of primary importance to obtain accurate genotypes for each of the studied individuals. In the second case, it is also important to obtain accurate haplotypes, since these are a key reagent for the imputation based analyses that follow. Since short reads from massively parallel technologies typically contain errors, sequencing depth is a key parameter: Some degree of redundancy is required to ensure adequate estimates of genotypes and haplotypes (Le and Durbin 2010; Li et al. 2010). However, we note that deep coverage can be achieved not only by sequencing a single sample deeply but also by combining information across individuals who share a particular haplotype (The 1000 Genomes Project Consortium 2010; Li et al. 2011).Most ongoing sequencing studies have focused on the analysis of unrelated samples. An example of the utility of sequencing related individuals is the work of Roach et al. (2010). By sequencing a nuclear family, including two children with Miller syndrome and their parents, they were able to identify the majority of sequencing errors and narrow their search for functional alleles. We reasoned that, by imposing Mendelian inheritance constraints and by checking for evidence of each variant across multiple related individuals, variant callers that directly examine parent-offspring trios would improve the quality of genotype and haplotype calls, particularly in cases where each individual is sequenced at low to modest depth (Le and Durbin 2010; Li et al. 2011).Here, we describe a new statistical method for estimating individual genotypes and haplotypes when next-generation sequence data are available on parent-offspring trios. We organize our paper as follows. First, we will describe how a hidden Markov model (HMM) designed for the analysis of sequence data in unrelated individuals can be extended to trios and parent offspring pairs in a computationally efficient manner. Second, we evaluate performance of the extended model in a variety of simulated data sets—varying sequencing depth, sequencing error rate, and sample size. Third, we evaluate our method in data from the ongoing SardiNIA sequencing project. Our results show that our method substantially outperforms existing approaches that ignore familial relatedness.