ITPA gene variants protect against anaemia in patients treated for chronic hepatitis C.

Chronic infection with the hepatitis C virus (HCV) affects 170 million people worldwide and is an important cause of liver-related morbidity and mortality. The standard of care therapy combines pegylated interferon (pegIFN) alpha and ribavirin (RBV), and is associated with a range of treatment-limiting adverse effects. One of the most important of these is RBV-induced haemolytic anaemia, which affects most patients and is severe enough to require dose modification in up to 15% of patients. Here we show that genetic variants leading to inosine triphosphatase deficiency, a condition not thought to be clinically important, protect against haemolytic anaemia in hepatitis-C-infected patients receiving RBV.

A genome-wide association study of resistance to HIV infection in highly exposed uninfected individuals with hemophilia A.

Human genetic variation contributes to differences in susceptibility to HIV-1 infection. To search for novel host resistance factors, we performed a genome-wide association study (GWAS) in hemophilia patients highly exposed to potentially contaminated factor VIII infusions. Individuals with hemophilia A and a documented history of factor VIII infusions before the introduction of viral inactivation procedures (1979-1984) were recruited from 36 hemophilia treatment centers (HTCs), and their genome-wide genetic variants were compared with those from matched HIV-infected individuals. Homozygous carriers of known CCR5 resistance mutations were excluded. Single nucleotide polymorphisms (SNPs) and inferred copy number variants (CNVs) were tested using logistic regression. In addition, we performed a pathway enrichment analysis, a heritability analysis, and a search for epistatic interactions with CCR5 Δ32 heterozygosity. A total of 560 HIV-uninfected cases were recruited: 36 (6.4%) were homozygous for CCR5 Δ32 or m303. After quality control and SNP imputation, we tested 1 081 435 SNPs and 3686 CNVs for association with HIV-1 serostatus in 431 cases and 765 HIV-infected controls. No SNP or CNV reached genome-wide significance. The additional analyses did not reveal any strong genetic effect. Highly exposed, yet uninfected hemophiliacs form an ideal study group to investigate host resistance factors. Using a genome-wide approach, we did not detect any significant associations between SNPs and HIV-1 susceptibility, indicating that common genetic variants of major effect are unlikely to explain the observed resistance phenotype in this population.

Host genetics of HIV acquisition and viral control.

Since the discovery of HIV as the cause of AIDS, numerous insights have been gained from studies of its natural history and epidemiology. It has become clear that there are substantial interindividual differences in the risk of HIV acquisition and course of disease. Meanwhile, the field of human genetics has undergone a series of rapid transitions that have fundamentally altered the approach to studying HIV host genetics. We aim to describe the field as it has transitioned from the era of candidate-gene studies and the era of genome-wide association studies (GWAS) to its current state in the infancy of comprehensive sequencing. In some ways the field has come full circle, having evolved from being driven almost exclusively by our knowledge of immunology, to a bias-free GWAS approach, to a point where our ability to catalogue human variation far outstrips our ability to biologically interpret it.

Using ERDS to infer copy-number variants in high-coverage genomes.

Although there are many methods available for inferring copy-number variants (CNVs) from next-generation sequence data, there remains a need for a system that is computationally efficient but that retains good sensitivity and specificity across all types of CNVs. Here, we introduce a new method, estimation by read depth with single-nucleotide variants (ERDS), and use various approaches to compare its performance to other methods. We found that for common CNVs and high-coverage genomes, ERDS performs as well as the best method currently available (Genome STRiP), whereas for rare CNVs and high-coverage genomes, ERDS performs better than any available method. Importantly, ERDS accommodates both unique and highly amplified regions of the genome and does so without requiring separate alignments for calling CNVs and other variants. These comparisons show that for genomes sequenced at high coverage, ERDS provides a computationally convenient method that calls CNVs as well as or better than any currently available method.

Prioritizing genetic variants for causality on the basis of preferential linkage disequilibrium.

To date, the widely used genome-wide association studies (GWASs) of the human genome have reported thousands of variants that are significantly associated with various human traits. However, in the vast majority of these cases, the causal variants responsible for the observed associations remain unknown. In order to facilitate the identification of causal variants, we designed a simple computational method called the "preferential linkage disequilibrium (LD)" approach, which follows the variants discovered by GWASs to pinpoint the causal variants, even if they are rare compared with the discovery variants. The approach is based on the hypothesis that the GWAS-discovered variant is better at tagging the causal variants than are most other variants evaluated in the original GWAS. Applying the preferential LD approach to the GWAS signals of five human traits for which the causal variants are already known, we successfully placed the known causal variants among the top ten candidates in the majority of these cases. Application of this method to additional GWASs, including those of hepatitis C virus treatment response, plasma levels of clotting factors, and late-onset Alzheimer disease, has led to the identification of a number of promising candidate causal variants. This method represents a useful tool for delineating causal variants by bringing together GWAS signals and the rapidly accumulating variant data from next-generation sequencing.

Exome sequencing followed by large-scale genotyping suggests a limited role for moderately rare risk factors of strong effect in schizophrenia.

Schizophrenia is a severe psychiatric disorder with strong heritability and marked heterogeneity in symptoms, course, and treatment response. There is strong interest in identifying genetic risk factors that can help to elucidate the pathophysiology and that might result in the development of improved treatments. Linkage and genome-wide association studies (GWASs) suggest that the genetic basis of schizophrenia is heterogeneous. However, it remains unclear whether the underlying genetic variants are mostly moderately rare and can be identified by the genotyping of variants observed in sequenced cases in large follow-up cohorts or whether they will typically be much rarer and therefore more effectively identified by gene-based methods that seek to combine candidate variants. Here, we consider 166 persons who have schizophrenia or schizoaffective disorder and who have had either their genomes or their exomes sequenced to high coverage. From these data, we selected 5,155 variants that were further evaluated in an independent cohort of 2,617 cases and 1,800 controls. No single variant showed a study-wide significant association in the initial or follow-up cohorts. However, we identified a number of case-specific variants, some of which might be real risk factors for schizophrenia, and these can be readily interrogated in other data sets. Our results indicate that schizophrenia risk is unlikely to be predominantly influenced by variants just outside the range detectable by GWASs. Rather, multiple rarer genetic variants must contribute substantially to the predisposition to schizophrenia, suggesting that both very large sample sizes and gene-based association tests will be required for securely identifying genetic risk factors.

Mutation in TECPR2 reveals a role for autophagy in hereditary spastic paraparesis.

We studied five individuals from three Jewish Bukharian families affected by an apparently autosomal-recessive form of hereditary spastic paraparesis accompanied by severe intellectual disability, fluctuating central hypoventilation, gastresophageal reflux disease, wake apnea, areflexia, and unique dysmorphic features. Exome sequencing identified one homozygous variant shared among all affected individuals and absent in controls: a 1 bp frameshift TECPR2 deletion leading to a premature stop codon and predicting significant degradation of the protein. TECPR2 has been reported as a positive regulator of autophagy. We thus examined the autophagy-related fate of two key autophagic proteins, SQSTM1 (p62) and MAP1LC3B (LC3), in skin fibroblasts of an affected individual, as compared to a healthy control, and found that both protein levels were decreased and that there was a more pronounced decrease in the lipidated form of LC3 (LC3II). siRNA knockdown of TECPR2 showed similar changes, consistent with aberrant autophagy. Our results are strengthened by the fact that autophagy dysfunction has been implicated in a number of other neurodegenerative diseases. The discovered TECPR2 mutation implicates autophagy, a central intracellular mechanism, in spastic paraparesis.

Exome sequencing and functional validation in zebrafish identify GTDC2 mutations as a cause of Walker-Warburg syndrome.

Whole-exome sequencing (WES), which analyzes the coding sequence of most annotated genes in the human genome, is an ideal approach to studying fully penetrant autosomal-recessive diseases, and it has been very powerful in identifying disease-causing mutations even when enrollment of affected individuals is limited by reduced survival. In this study, we combined WES with homozygosity analysis of consanguineous pedigrees, which are informative even when a single affected individual is available, to identify genetic mutations responsible for Walker-Warburg syndrome (WWS), a genetically heterogeneous autosomal-recessive disorder that severely affects the development of the brain, eyes, and muscle. Mutations in seven genes are known to cause WWS and explain 50%-60% of cases, but multiple additional genes are expected to be mutated because unexplained cases show suggestive linkage to diverse loci. Using WES in consanguineous WWS-affected families, we found multiple deleterious mutations in GTDC2 (also known as AGO61). GTDC2's predicted role as an uncharacterized glycosyltransferase is consistent with the function of other genes that are known to be mutated in WWS and that are involved in the glycosylation of the transmembrane receptor dystroglycan. Therefore, to explore the role of GTDC2 loss of function during development, we used morpholino-mediated knockdown of its zebrafish ortholog, gtdc2. We found that gtdc2 knockdown in zebrafish replicates all WWS features (hydrocephalus, ocular defects, and muscular dystrophy), strongly suggesting that GTDC2 mutations cause WWS.

Exome sequencing followed by large-scale genotyping fails to identify single rare variants of large effect in idiopathic generalized epilepsy.

Idiopathic generalized epilepsy (IGE) is a complex disease with high heritability, but little is known about its genetic architecture. Rare copy-number variants have been found to explain nearly 3% of individuals with IGE; however, it remains unclear whether variants with moderate effect size and frequencies below what are reliably detected with genome-wide association studies contribute significantly to disease risk. In this study, we compare the exome sequences of 118 individuals with IGE and 242 controls of European ancestry by using next-generation sequencing. The exome-sequenced epilepsy cases include study subjects with two forms of IGE, including juvenile myoclonic epilepsy (n = 93) and absence epilepsy (n = 25). However, our discovery strategy did not assume common genetic control between the subtypes of IGE considered. In the sequence data, as expected, no variants were significantly associated with the IGE phenotype or more specific IGE diagnoses. We then selected 3,897 candidate epilepsy-susceptibility variants from the sequence data and genotyped them in a larger set of 878 individuals with IGE and 1,830 controls. Again, no variant achieved statistical significance. However, 1,935 variants were observed exclusively in cases either as heterozygous or homozygous genotypes. It is likely that this set of variants includes real risk factors. The lack of significant association evidence of single variants with disease in this two-stage approach emphasizes the high genetic heterogeneity of epilepsy disorders, suggests that the impact of any individual single-nucleotide variant in this disease is small, and indicates that gene-based approaches might be more successful for future sequencing studies of epilepsy predisposition.

Cold urticaria, immunodeficiency, and autoimmunity related to PLCG2 deletions.

BACKGROUND: Mendelian analysis of disorders of immune regulation can provide insight into molecular pathways associated with host defense and immune tolerance. METHODS: We identified three families with a dominantly inherited complex of cold-induced urticaria, antibody deficiency, and susceptibility to infection and autoimmunity. Immunophenotyping methods included flow cytometry, analysis of serum immunoglobulins and autoantibodies, lymphocyte stimulation, and enzymatic assays. Genetic studies included linkage analysis, targeted Sanger sequencing, and next-generation whole-genome sequencing. RESULTS: Cold urticaria occurred in all affected subjects. Other, variable manifestations included atopy, granulomatous rash, autoimmune thyroiditis, the presence of antinuclear antibodies, sinopulmonary infections, and common variable immunodeficiency. Levels of serum IgM and IgA and circulating natural killer cells and class-switched memory B cells were reduced. Linkage analysis showed a 7-Mb candidate interval on chromosome 16q in one family, overlapping by 3.5 Mb a disease-associated haplotype in a smaller family. This interval includes PLCG2, encoding phospholipase Cγ(2) (PLCγ(2)), a signaling molecule expressed in B cells, natural killer cells, and mast cells. Sequencing of complementary DNA revealed heterozygous transcripts lacking exon 19 in two families and lacking exons 20 through 22 in a third family. Genomic sequencing identified three distinct in-frame deletions that cosegregated with disease. These deletions, located within a region encoding an autoinhibitory domain, result in protein products with constitutive phospholipase activity. PLCG2-expressing cells had diminished cellular signaling at 37°C but enhanced signaling at subphysiologic temperatures. CONCLUSIONS: Genomic deletions in PLCG2 cause gain of PLCγ(2) function, leading to signaling abnormalities in multiple leukocyte subsets and a phenotype encompassing both excessive and deficient immune function. (Funded by the National Institutes of Health Intramural Research Programs and others.).