Development of a comprehensive cardiovascular disease genetic risk assessment test.

Background: Despite monogenic and polygenic contributions to cardiovascular disease (CVD), genetic testing is not widely adopted, and current tests are limited by the breadth of surveyed conditions and interpretation burden. Methods: We developed a comprehensive clinical genome CVD test with semi-automated interpretation. Monogenic conditions and risk alleles were selected based on the strength of disease association and evidence for increased disease risk, respectively. Non-CVD secondary findings genes, pharmacogenomic (PGx) variants and CVD polygenic risk scores (PRS) were assessed for inclusion. Test performance was modeled using 2,594 genomes from the 1000 Genomes Project, and further investigated in 20 previously tested individuals. Results: The CVD genome test is composed of a panel of 215 CVD gene-disease pairs, 35 non-CVD secondary findings genes, 4 risk alleles or genotypes, 10 PGx genes and a PRS for coronary artery disease. Modeling of test performance using samples from the 1000 Genomes Project revealed ~6% of individuals with a monogenic finding in a CVD-associated gene, 6% with a risk allele finding, ~1% with a non-CVD secondary finding, and 93% with CVD-associated PGx variants. Assessment of blinded clinical samples showed complete concordance with prior testing. An average of 4 variants were reviewed per case, with interpretation and reporting time ranging from 9-96 min. Conclusions: A genome sequencing based CVD genetic risk assessment can provide comprehensive genetic disease and genetic risk information to patients with CVD. The semi-automated and limited interpretation burden suggest that this testing approach could be scaled to support population-level initiatives.

De novo variants in GABRA4 are associated with a neurological phenotype including developmental delay, behavioral abnormalities and epilepsy.

Nine out of 19 genes encoding GABAA receptor subunits have been linked to monogenic syndromes characterized by seizures and developmental disorders. Previously, we reported the de novo variant p.(Thr300Ile) in GABRA4 in a patient with epilepsy and neurodevelopmental abnormalities. However, no new cases have been reported since then. Through an international collaboration, we collected molecular and phenotype data of individuals carrying de novo variants in GABRA4. Patients and their parents were investigated either by exome or genome sequencing, followed by targeted Sanger sequencing in some cases. All variants within the transmembrane domain, including the previously reported p.(Thr300Ile) variant, were characterized in silico and analyzed by molecular dynamics (MD) simulation studies. We identified three novel de novo missense variants in GABRA4 (NM_000809.4): c.797 C > T, p.(Pro266Leu), c.899 C > A, p.(Thr300Asn), and c.634 G > A, p.(Val212Ile). The p.(Thr300Asn) variant impacts the same codon as the previously reported variant p.(Thr300Ile) and likely arose post-zygotically as evidenced by sequencing oral mucosal cells. Overlapping phenotypes among affected individuals included developmental delay (4/4), epileptiform EEG abnormalities (3/4), attention deficits (3/4), seizures (2/4), autistic features (2/4) and structural brain abnormalities (2/4). MD simulations of the three variants within the transmembrane domain of the receptor indicate that sub-microsecond scale dynamics differ between wild-type and mutated subunits. Taken together, our findings further corroborate an association between GABRA4 and a neurological phenotype including variable neurodevelopmental, behavioral and epileptic abnormalities.

Determining the characteristics of genetic disorders that predict inclusion in newborn genomic sequencing programs.

Over 30 international research studies and commercial laboratories are exploring the use of genomic sequencing to screen apparently healthy newborns for genetic disorders. These programs have individualized processes for determining which genes and genetic disorders are queried and reported in newborns. We compared lists of genes from 26 research and commercial newborn screening programs and found substantial heterogeneity among the genes included. A total of 1,750 genes were included in at least one newborn genome sequencing program, but only 74 genes were included on >80% of gene lists, 16 of which are not associated with conditions on the Recommended Uniform Screening Panel. We used a linear regression model to explore factors related to the inclusion of individual genes across programs, finding that a high evidence base as well as treatment efficacy were two of the most important factors for inclusion. We applied a machine learning model to predict how suitable a gene is for newborn sequencing. As knowledge about and treatments for genetic disorders expand, this model provides a dynamic tool to reassess genes for newborn screening implementation. This study highlights the complex landscape of gene list curation among genomic newborn screening programs and proposes an empirical path forward for determining the genes and disorders of highest priority for newborn screening programs.

A framework for the evaluation and reporting of incidental findings in clinical genomic testing.

Currently, there are no widely accepted recommendations in the genomics field guiding the return of incidental findings (IFs), defined here as unexpected results that are unrelated to the indication for testing. Consequently, reporting policies for IFs among laboratories offering genomic testing are variable and may lack transparency. Herein we describe a framework developed to guide the evaluation and return of IFs encountered in probands undergoing clinical genome sequencing (cGS). The framework prioritizes clinical significance and actionability of IFs and follows a stepwise approach with stopping points at which IFs may be recommended for return or not. Over 18 months, implementation of the framework in a clinical laboratory facilitated the return of actionable IFs in 37 of 720 (5.1%) individuals referred for cGS, which is reduced to 3.1% if glucose-6-phosphate dehydrogenase (G6PD) deficiency is excluded. This framework can serve as a model to standardize reporting of IFs identified during genomic testing.

Expert Panel Curation of 113 Primary Mitochondrial Disease Genes for the Leigh Syndrome Spectrum.

OBJECTIVE: Primary mitochondrial diseases (PMDs) are heterogeneous disorders caused by inherited mitochondrial dysfunction. Classically defined neuropathologically as subacute necrotizing encephalomyelopathy, Leigh syndrome spectrum (LSS) is the most frequent manifestation of PMD in children, but may also present in adults. A major challenge for accurate diagnosis of LSS in the genomic medicine era is establishing gene-disease relationships (GDRs) for this syndrome with >100 monogenic causes across both nuclear and mitochondrial genomes. METHODS: The Clinical Genome Resource (ClinGen) Mitochondrial Disease Gene Curation Expert Panel (GCEP), comprising 40 international PMD experts, met monthly for 4 years to review GDRs for LSS. The GCEP standardized gene curation for LSS by refining the phenotypic definition, modifying the ClinGen Gene-Disease Clinical Validity Curation Framework to improve interpretation for LSS, and establishing a scoring rubric for LSS. RESULTS: The GDR with LSS across the nuclear and mitochondrial genomes was classified as definitive for 31 of 114 GDRs curated (27%), moderate for 38 (33%), limited for 43 (38%), and disputed for 2 (2%). Ninety genes were associated with autosomal recessive inheritance, 16 were maternally inherited, 5 were autosomal dominant, and 3 were X-linked. INTERPRETATION: GDRs for LSS were established for genes across both nuclear and mitochondrial genomes. Establishing these GDRs will allow accurate variant interpretation, expedite genetic diagnosis of LSS, and facilitate precision medicine, multisystem organ surveillance, recurrence risk counseling, reproductive choice, natural history studies, and determination of eligibility for interventional clinical trials. ANN NEUROL 2023;94:696-712.

Reactive gene curation to support interpretation and reporting of a clinical genome test for rare disease: Experience from over 1,000 cases.

Current standards in clinical genetics recognize the need to establish the validity of gene-disease relationships as a first step in the interpretation of sequence variants. We describe our experience incorporating the ClinGen Gene-Disease Clinical Validity framework in our interpretation and reporting workflow for a clinical genome sequencing (cGS) test for individuals with rare and undiagnosed genetic diseases. This "reactive" gene curation is completed upon identification of candidate variants during active case analysis and within the test turn-around time by focusing on the most impactful evidence and taking advantage of the broad applicability of the framework to cover a wide range of disease areas. We demonstrate that reactive gene curation can be successfully implemented in support of cGS in a clinical laboratory environment, enabling robust clinical decision making and allowing all variants to be fully and appropriately considered and their clinical significance confidently interpreted.

The Gene Curation Coalition: A global effort to harmonize gene-disease evidence resources.

PURPOSE: Several groups and resources provide information that pertains to the validity of gene-disease relationships used in genomic medicine and research; however, universal standards and terminologies to define the evidence base for the role of a gene in disease and a single harmonized resource were lacking. To tackle this issue, the Gene Curation Coalition (GenCC) was formed. METHODS: The GenCC drafted harmonized definitions for differing levels of gene-disease validity on the basis of existing resources, and performed a modified Delphi survey with 3 rounds to narrow the list of terms. The GenCC also developed a unified database to display curated gene-disease validity assertions from its members. RESULTS: On the basis of 241 survey responses from the genetics community, a consensus term set was chosen for grading gene-disease validity and database submissions. As of December 2021, the database contained 15,241 gene-disease assertions on 4569 unique genes from 12 submitters. When comparing submissions to the database from distinct sources, conflicts in assertions of gene-disease validity ranged from 5.3% to 13.4%. CONCLUSION: Terminology standardization, sharing of gene-disease validity classifications, and resolution of curation conflicts will facilitate collaborations across international curation efforts and in turn, improve consistency in genetic testing and variant interpretation.

A clinical laboratory's experience using GeneMatcher-Building stronger gene-disease relationships.

The use of whole-genome sequencing (WGS) has accelerated the pace of gene discovery and highlighted the need for open and collaborative data sharing in the search for novel disease genes and variants. GeneMatcher (GM) is designed to facilitate connections between researchers, clinicians, health-care providers, and others to help in the identification of additional patients with variants in the same candidate disease genes. The Illumina Clinical Services Laboratory offers a WGS test for patients with suspected rare and undiagnosed genetic disease  and regularly submits potential candidate genes to GM to strengthen gene-disease relationships. We describe our experience with GM, including criteria for evaluation of candidate genes, and our workflow for the submission and review process. We have made 69 submissions, 36 of which are currently active. Ten percent of submissions have resulted in publications, with an additional 14 submissions part of ongoing collaborations and expected to result in a publication.

Copy-number variants in clinical genome sequencing: deployment and interpretation for rare and undiagnosed disease.

PURPOSE: Current diagnostic testing for genetic disorders involves serial use of specialized assays spanning multiple technologies. In principle, genome sequencing (GS) can detect all genomic pathogenic variant types on a single platform. Here we evaluate copy-number variant (CNV) calling as part of a clinically accredited GS test. METHODS: We performed analytical validation of CNV calling on 17 reference samples, compared the sensitivity of GS-based variants with those from a clinical microarray, and set a bound on precision using orthogonal technologies. We developed a protocol for family-based analysis of GS-based CNV calls, and deployed this across a clinical cohort of 79 rare and undiagnosed cases. RESULTS: We found that CNV calls from GS are at least as sensitive as those from microarrays, while only creating a modest increase in the number of variants interpreted (~10 CNVs per case). We identified clinically significant CNVs in 15% of the first 79 cases analyzed, all of which were confirmed by an orthogonal approach. The pipeline also enabled discovery of a uniparental disomy (UPD) and a 50% mosaic trisomy 14. Directed analysis of select CNVs enabled breakpoint level resolution of genomic rearrangements and phasing of de novo CNVs. CONCLUSION: Robust identification of CNVs by GS is possible within a clinical testing environment.

A detailed clinical and molecular survey of subjects with nonsyndromic USH2A retinopathy reveals an allelic hierarchy of disease-causing variants.

Defects in USH2A cause both isolated retinal disease and Usher syndrome (ie, retinal disease and deafness). To gain insights into isolated/nonsyndromic USH2A retinopathy, we screened USH2A in 186 probands with recessive retinal disease and no hearing complaint in childhood (discovery cohort) and in 84 probands with recessive retinal disease (replication cohort). Detailed phenotyping, including retinal imaging and audiological assessment, was performed in individuals with two likely disease-causing USH2A variants. Further genetic testing, including screening for a deep-intronic disease-causing variant and large deletions/duplications, was performed in those with one likely disease-causing change. Overall, 23 of 186 probands (discovery cohort) were found to harbour two likely disease-causing variants in USH2A. Some of these variants were predominantly associated with nonsyndromic retinal degeneration ('retinal disease-specific'); these included the common c.2276 G>T, p.(Cys759Phe) mutation and five additional variants: c.2802 T>G, p.(Cys934Trp); c.10073 G>A, p.(Cys3358Tyr); c.11156 G>A, p.(Arg3719His); c.12295-3 T>A; and c.12575 G>A, p.(Arg4192His). An allelic hierarchy was observed in the discovery cohort and confirmed in the replication cohort. In nonsyndromic USH2A disease, retinopathy was consistent with retinitis pigmentosa and the audiological phenotype was variable. USH2A retinopathy is a common cause of nonsyndromic recessive retinal degeneration and has a different mutational spectrum to that observed in Usher syndrome. The following model is proposed: the presence of at least one 'retinal disease-specific' USH2A allele in a patient with USH2A-related disease results in the preservation of normal hearing. Careful genotype-phenotype studies such as this will become increasingly important, especially now that high-throughput sequencing is widely used in the clinical setting.

Gain-of-function mutations in the calcium channel CACNA1C (Cav1.2) cause non-syndromic long-QT but not Timothy syndrome.

Gain-of-function mutations in CACNA1C, encoding the L-type Ca(2+) channel Cav1.2, cause Timothy syndrome (TS), a multi-systemic disorder with dysmorphic features, long-QT syndrome (LQTS) and autism spectrum disorders. TS patients have heterozygous mutations (G402S and G406R) located in the alternatively spliced exon 8, causing a gain-of-function by reduced voltage-dependence of inactivation. Screening 540 unrelated patients with non-syndromic forms of LQTS, we identified six functional relevant CACNA1C mutations in different regions of the channel. All these mutations caused a gain-of-function combining different mechanisms, including changes in current amplitude, rate of inactivation and voltage-dependence of activation or inactivation, similar as in TS. Computer simulations support the theory that the novel CACNA1C mutations prolong action potential duration. We conclude that genotype-negative LQTS patients should be investigated for mutations in CACNA1C, as a gain-of-function in Cav1.2 is likely to cause LQTS and only specific and rare mutations, i.e. in exon 8, cause the multi-systemic TS.

Systems genetics identifies Sestrin 3 as a regulator of a proconvulsant gene network in human epileptic hippocampus.

Gene-regulatory network analysis is a powerful approach to elucidate the molecular processes and pathways underlying complex disease. Here we employ systems genetics approaches to characterize the genetic regulation of pathophysiological pathways in human temporal lobe epilepsy (TLE). Using surgically acquired hippocampi from 129 TLE patients, we identify a gene-regulatory network genetically associated with epilepsy that contains a specialized, highly expressed transcriptional module encoding proconvulsive cytokines and Toll-like receptor signalling genes. RNA sequencing analysis in a mouse model of TLE using 100 epileptic and 100 control hippocampi shows the proconvulsive module is preserved across-species, specific to the epileptic hippocampus and upregulated in chronic epilepsy. In the TLE patients, we map the trans-acting genetic control of this proconvulsive module to Sestrin 3 (SESN3), and demonstrate that SESN3 positively regulates the module in macrophages, microglia and neurons. Morpholino-mediated Sesn3 knockdown in zebrafish confirms the regulation of the transcriptional module, and attenuates chemically induced behavioural seizures in vivo.

A genome-wide association study and biological pathway analysis of epilepsy prognosis in a prospective cohort of newly treated epilepsy.

We present the analysis of a prospective multicentre study to investigate genetic effects on the prognosis of newly treated epilepsy. Patients with a new clinical diagnosis of epilepsy requiring medication were recruited and followed up prospectively. The clinical outcome was defined as freedom from seizures for a minimum of 12 months in accordance with the consensus statement from the International League Against Epilepsy (ILAE). Genetic effects on remission of seizures after starting treatment were analysed with and without adjustment for significant clinical prognostic factors, and the results from each cohort were combined using a fixed-effects meta-analysis. After quality control (QC), we analysed 889 newly treated epilepsy patients using 472 450 genotyped and 6.9 × 10(6) imputed single-nucleotide polymorphisms. Suggestive evidence for association (defined as Pmeta < 5.0 × 10(-7)) with remission of seizures after starting treatment was observed at three loci: 6p12.2 (rs492146, Pmeta = 2.1 × 10(-7), OR[G] = 0.57), 9p23 (rs72700966, Pmeta = 3.1 × 10(-7), OR[C] = 2.70) and 15q13.2 (rs143536437, Pmeta = 3.2 × 10(-7), OR[C] = 1.92). Genes of biological interest at these loci include PTPRD and ARHGAP11B (encoding functions implicated in neuronal development) and GSTA4 (a phase II biotransformation enzyme). Pathway analysis using two independent methods implicated a number of pathways in the prognosis of epilepsy, including KEGG categories 'calcium signaling pathway' and 'phosphatidylinositol signaling pathway'. Through a series of power curves, we conclude that it is unlikely any single common variant explains >4.4% of the variation in the outcome of newly treated epilepsy.

Genome-wide methylation analyses of primary human leukocyte subsets identifies functionally important cell-type-specific hypomethylated regions.

DNA methylation is an important mechanism by which gene transcription and hence cellular function are regulated. Here, we provide detailed functional genome-wide methylome maps of 5 primary peripheral blood leukocyte subsets including T cells, B cells, monocytes/macrophages, and neutrophils obtained from healthy individuals. A comparison of these methylomes revealed highly specific cell-lineage and cell-subset methylation profiles. DNA hypomethylation is known to be permissive for gene expression and we identified cell-subset-specific hypomethylated regions (HMRs) that strongly correlate with gene transcription levels suggesting these HMRs may regulate corresponding cell functions. Single-nucleotide polymorphisms associated with immune-mediated disease in genome-wide association studies preferentially localized to these cell-specific regulatory HMRs, offering insight into pathogenesis by highlighting cell subsets in which specific epigenetic changes may drive disease. Our data provide a valuable reference tool for researchers aiming to investigate the role of DNA methylation in regulating primary leukocyte function in health and immune-mediated disease.

Non dominant-negative KCNJ2 gene mutations leading to Andersen-Tawil syndrome with an isolated cardiac phenotype.

Andersen-Tawil syndrome (ATS) is characterized by dysmorphic features, periodic paralyses and abnormal ventricular repolarization. After genotyping a large set of patients with congenital long-QT syndrome, we identified two novel, heterozygous KCNJ2 mutations (p.N318S, p.W322C) located in the C-terminus of the Kir2.1 subunit. These mutations have a different localization than classical ATS mutations which are mostly located at a potential interaction face with the slide helix or at the interface between the C-termini. Mutation carriers were without the key features of ATS, causing an isolated cardiac phenotype. While the N318S mutants regularly reached the plasma membrane, W322C mutants primarily resided in late endosomes. Co-expression of N318S or W322C with wild-type Kir2.1 reduced current amplitudes only by 20-25 %. This mild loss-of-function for the heteromeric channels resulted from defective channel trafficking (W322C) or gating (N318S). Strikingly, and in contrast to the majority of ATS mutations, neither mutant caused a dominant-negative suppression of wild-type Kir2.1, Kir2.2 and Kir2.3 currents. Thus, a mild reduction of native Kir2.x currents by non dominant-negative mutants may cause ATS with an isolated cardiac phenotype.

A genetic study of Wilson's disease in the United Kingdom.

Previous studies have failed to identify mutations in the Wilson's disease gene ATP7B in a significant number of clinically diagnosed cases. This has led to concerns about genetic heterogeneity for this condition but also suggested the presence of unusual mutational mechanisms. We now present our findings in 181 patients from the United Kingdom with clinically and biochemically confirmed Wilson's disease. A total of 116 different ATP7B mutations were detected, 32 of which are novel. The overall mutation detection frequency was 98%. The likelihood of mutations in genes other than ATP7B causing a Wilson's disease phenotype is therefore very low. We report the first cases with Wilson's disease due to segmental uniparental isodisomy as well as three patients with three ATP7B mutations and three families with Wilson's disease in two consecutive generations. We determined the genetic prevalence of Wilson's disease in the United Kingdom by sequencing the entire coding region and adjacent splice sites of ATP7B in 1000 control subjects. The frequency of all single nucleotide variants with in silico evidence of pathogenicity (Class 1 variant) was 0.056 or 0.040 if only those single nucleotide variants that had previously been reported as mutations in patients with Wilson's disease were included in the analysis (Class 2 variant). The frequency of heterozygote, putative or definite disease-associated ATP7B mutations was therefore considerably higher than the previously reported occurrence of 1:90 (or 0.011) for heterozygote ATP7B mutation carriers in the general population (P < 2.2 × 10(-16) for Class 1 variants or P < 5 × 10(-11) for Class 2 variants only). Subsequent exclusion of four Class 2 variants without additional in silico evidence of pathogenicity led to a further reduction of the mutation frequency to 0.024. Using this most conservative approach, the calculated frequency of individuals predicted to carry two mutant pathogenic ATP7B alleles is 1:7026 and thus still considerably higher than the typically reported prevalence of Wilson's disease of 1:30 000 (P = 0.00093). Our study provides strong evidence for monogenic inheritance of Wilson's disease. It also has major implications for ATP7B analysis in clinical practice, namely the need to consider unusual genetic mechanisms such as uniparental disomy or the possible presence of three ATP7B mutations. The marked discrepancy between the genetic prevalence and the number of clinically diagnosed cases of Wilson's disease may be due to both reduced penetrance of ATP7B mutations and failure to diagnose patients with this eminently treatable disorder.

A comparison of the whole genome approach of MeDIP-seq to the targeted approach of the Infinium HumanMethylation450 BeadChip(®) for methylome profiling.

DNA methylation is one of the most studied epigenetic marks in the human genome, with the result that the desire to map the human methylome has driven the development of several methods to map DNA methylation on a genomic scale. Our study presents the first comparison of two of these techniques - the targeted approach of the Infinium HumanMethylation450 BeadChip® with the immunoprecipitation and sequencing-based method, MeDIP-seq. Both methods were initially validated with respect to bisulfite sequencing as the gold standard and then assessed in terms of coverage, resolution and accuracy. The regions of the methylome that can be assayed by both methods and those that can only be assayed by one method were determined and the discovery of differentially methylated regions (DMRs) by both techniques was examined. Our results show that the Infinium HumanMethylation450 BeadChip® and MeDIP-seq show a good positive correlation (Spearman correlation of 0.68) on a genome-wide scale and can both be used successfully to determine differentially methylated loci in RefSeq genes, CpG islands, shores and shelves. MeDIP-seq however, allows a wider interrogation of methylated regions of the human genome, including thousands of non-RefSeq genes and repetitive elements, all of which may be of importance in disease. In our study MeDIP-seq allowed the detection of 15,709 differentially methylated regions, nearly twice as many as the array-based method (8070), which may result in a more comprehensive study of the methylome.

Exploration of signals of positive selection derived from genotype-based human genome scans using re-sequencing data.

We have investigated whether regions of the genome showing signs of positive selection in scans based on haplotype structure also show evidence of positive selection when sequence-based tests are applied, whether the target of selection can be localized more precisely, and whether such extra evidence can lead to increased biological insights. We used two tools: simulations under neutrality or selection, and experimental investigation of two regions identified by the HapMap2 project as putatively selected in human populations. Simulations suggested that neutral and selected regions should be readily distinguished and that it should be possible to localize the selected variant to within 40 kb at least half of the time. Re-sequencing of two ~300 kb regions (chr4:158Mb and chr10:22Mb) lacking known targets of selection in HapMap CHB individuals provided strong evidence for positive selection within each and suggested the micro-RNA gene hsa-miR-548c as the best candidate target in one region, and changes in regulation of the sperm protein gene SPAG6 in the other.

Comprehensive sequence analysis of nine Usher syndrome genes in the UK National Collaborative Usher Study.

BACKGROUND: Usher syndrome (USH) is an autosomal recessive disorder comprising retinitis pigmentosa, hearing loss and, in some cases, vestibular dysfunction. It is clinically and genetically heterogeneous with three distinctive clinical types (I-III) and nine Usher genes identified. This study is a comprehensive clinical and genetic analysis of 172 Usher patients and evaluates the contribution of digenic inheritance. METHODS: The genes MYO7A, USH1C, CDH23, PCDH15, USH1G, USH2A, GPR98, WHRN, CLRN1 and the candidate gene SLC4A7 were sequenced in 172 UK Usher patients, regardless of clinical type. RESULTS: No subject had definite mutations (nonsense, frameshift or consensus splice site mutations) in two different USH genes. Novel missense variants were classified UV1-4 (unclassified variant): UV4 is 'probably pathogenic', based on control frequency <0.23%, identification in trans to a pathogenic/probably pathogenic mutation and segregation with USH in only one family; and UV3 ('likely pathogenic') as above, but no information on phase. Overall 79% of identified pathogenic/UV4/UV3 variants were truncating and 21% were missense changes. MYO7A accounted for 53.2%, and USH1C for 14.9% of USH1 families (USH1C:c.496+1G>A being the most common USH1 mutation in the cohort). USH2A was responsible for 79.3% of USH2 families and GPR98 for only 6.6%. No mutations were found in USH1G, WHRN or SLC4A7. CONCLUSIONS: One or two pathogenic/likely pathogenic variants were identified in 86% of cases. No convincing cases of digenic inheritance were found. It is concluded that digenic inheritance does not make a significant contribution to Usher syndrome; the observation of multiple variants in different genes is likely to reflect polymorphic variation, rather than digenic effects.

Contrasting signals of positive selection in genes involved in human skin-color variation from tests based on SNP scans and resequencing.

BACKGROUND: Numerous genome-wide scans conducted by genotyping previously ascertained single-nucleotide polymorphisms (SNPs) have provided candidate signatures for positive selection in various regions of the human genome, including in genes involved in pigmentation traits. However, it is unclear how well the signatures discovered by such haplotype-based test statistics can be reproduced in tests based on full resequencing data. Four genes (oculocutaneous albinism II (OCA2), tyrosinase-related protein 1 (TYRP1), dopachrome tautomerase (DCT), and KIT ligand (KITLG)) implicated in human skin-color variation, have shown evidence for positive selection in Europeans and East Asians in previous SNP-scan data. In the current study, we resequenced 4.7 to 6.7 kb of DNA from each of these genes in Africans, Europeans, East Asians, and South Asians. RESULTS: Applying all commonly used neutrality-test statistics for allele frequency distribution to the newly generated sequence data provided conflicting results regarding evidence for positive selection. Previous haplotype-based findings could not be clearly confirmed. Although some tests were marginally significant for some populations and genes, none of them were significant after multiple-testing correction. Combined P values for each gene-population pair did not improve these results. Application of Approximate Bayesian Computation Markov chain Monte Carlo based to these sequence data using a simple forward simulator revealed broad posterior distributions of the selective parameters for all four genes, providing no support for positive selection. However, when we applied this approach to published sequence data on SLC45A2, another human pigmentation candidate gene, we could readily confirm evidence for positive selection, as previously detected with sequence-based and some haplotype-based tests. CONCLUSIONS: Overall, our data indicate that even genes that are strong biological candidates for positive selection and show reproducible signatures of positive selection in SNP scans do not always show the same replicability of selection signals in other tests, which should be considered in future studies on detecting positive selection in genetic data.