Irina Tikhonova

Genetic diagnosis by whole exome capture and massively parallel DNA sequencing

Protein coding genes constitute only approximately 1% of the human genome but harbor 85% of the mutations with large effects on disease-related traits. Therefore, efficient strategies for selectively sequencing complete coding regions (i.e., “whole exome”) have the potential to contribute to the understanding of rare and common human diseases. Here we report a method for whole-exome sequencing coupling Roche/NimbleGen whole exome arrays to the Illumina DNA sequencing platform. We demonstrate the ability to capture approximately 95% of the targeted coding sequences with high sensitivity and specificity for detection of homozygous and heterozygous variants. We illustrate the utility of this approach by making an unanticipated genetic diagnosis of congenital chloride diarrhea in a patient referred with a suspected diagnosis of Bartter syndrome, a renal salt-wasting disease. The molecular diagnosis was based on the finding of a homozygous missense D652N mutation at a position in SLC26A3 (the known congenital chloride diarrhea locus) that is virtually completely conserved in orthologues and paralogues from invertebrates to humans, and clinical follow-up confirmed the diagnosis. To our knowledge, whole-exome (or genome) sequencing has not previously been used to make a genetic diagnosis. Five additional patients suspected to have Bartter syndrome but who did not have mutations in known genes for this disease had homozygous deleterious mutations in SLC26A3. These results demonstrate the clinical utility of whole-exome sequencing and have implications for disease gene discovery and clinical diagnosis.

De novo mutations in histone-modifying genes in congenital heart disease.

Congenital heart disease (CHD) is the most frequent birth defect, affecting 0.8% of live births. Many cases occur sporadically and impair reproductive fitness, suggesting a role for de novo mutations. Here we compare the incidence of de novo mutations in 362 severe CHD cases and 264 controls by analysing exome sequencing of parent–offspring trios. CHD cases show a significant excess of protein-altering de novo mutations in genes expressed in the developing heart, with an odds ratio of 7.5 for damaging (premature termination, frameshift, splice site) mutations. Similar odds ratios are seen across the main classes of severe CHD. We find a marked excess of de novo mutations in genes involved in the production, removal or reading of histone 3 lysine 4 (H3K4) methylation, or ubiquitination of H2BK120, which is required for H3K4 methylation. There are also two de novo mutations in SMAD2, which regulates H3K27 methylation in the embryonic left–right organizer. The combination of both activating (H3K4 methylation) and inactivating (H3K27 methylation) chromatin marks characterizes ‘poised’ promoters and enhancers, which regulate expression of key developmental genes. These findings implicate de novo point mutations in several hundreds of genes that collectively contribute to approximately 10% of severe CHD.

Performance comparison of one-color and two-color platforms within the MicroArray Quality Control (MAQC) project

Microarray-based expression profiling experiments typically use either a one-color or a two-color design to measure mRNA abundance. The validity of each approach has been amply demonstrated. Here we provide a simultaneous comparison of results from one- and two-color labeling designs, using two independent RNA samples from the Microarray Quality Control (MAQC) project, tested on each of three different microarray platforms. The data were evaluated in terms of reproducibility, specificity, sensitivity and accuracy to determine if the two approaches provide comparable results. For each of the three microarray platforms tested, the results show good agreement with high correlation coefficients and high concordance of differentially expressed gene lists within each platform. Cumulatively, these comparisons indicate that data quality is essentially equivalent between the one- and two-color approaches and strongly suggest that this variable need not be a primary factor in decisions regarding experimental microarray design.

Mutations in Kelch-like 3 and Cullin 3 cause hypertension and electrolyte abnormalities

Hypertension affects one billion people and is a principal reversible risk factor for cardiovascular disease. Pseudohypoaldosteronism type II (PHAII), a rare Mendelian syndrome featuring hypertension, hyperkalaemia and metabolic acidosis, has revealed previously unrecognized physiology orchestrating the balance between renal salt reabsorption and K+ and H+ excretion. Here we used exome sequencing to identify mutations in kelch-like 3 (KLHL3) or cullin 3 (CUL3) in PHAII patients from 41 unrelated families. KLHL3 mutations are either recessive or dominant, whereas CUL3 mutations are dominant and predominantly de novo. CUL3 and BTB-domain-containing kelch proteins such as KLHL3 are components of cullin–RING E3 ligase complexes that ubiquitinate substrates bound to kelch propeller domains. Dominant KLHL3 mutations are clustered in short segments within the kelch propeller and BTB domains implicated in substrate and cullin binding, respectively. Diverse CUL3 mutations all result in skipping of exon 9, producing an in-frame deletion. Because dominant KLHL3 and CUL3 mutations both phenocopy recessive loss-of-function KLHL3 mutations, they may abrogate ubiquitination of KLHL3 substrates. Disease features are reversed by thiazide diuretics, which inhibit the Na–Cl cotransporter in the distal nephron of the kidney; KLHL3 and CUL3 are expressed in this location, suggesting a mechanistic link between KLHL3 and CUL3 mutations, increased Na–Cl reabsorption, and disease pathogenesis. These findings demonstrate the utility of exome sequencing in disease gene identification despite the combined complexities of locus heterogeneity, mixed models of transmission and frequent de novo mutation, and establish a fundamental role for KLHL3 and CUL3 in blood pressure, K+ and pH homeostasis.

Genome-wide association study identifies susceptibility loci for IgA nephropathy

We carried out a genome-wide association study of IgA nephropathy, a major cause of kidney failure worldwide. We studied 1,194 cases and 902 controls of Chinese Han ancestry, with targeted follow up in Chinese and European cohorts comprising 1,950 cases and 1,920 controls. We identified three independent loci in the major histocompatibility complex, as well as a common deletion of CFHR1 and CFHR3 at chromosome 1q32 and a locus at chromosome 22q12 that each surpassed genome-wide significance (P values for association between 1.59 × 10−26 and 4.84 × 10−9 and minor allele odds ratios of 0.63–0.80). These five loci explain 4–7% of the disease variance and up to a tenfold variation in interindividual risk. Many of the alleles that protect against IgA nephropathy impart increased risk for other autoimmune or infectious diseases, and IgA nephropathy risk allele frequencies closely parallel the variation in disease prevalence among Asian, European and African populations, suggesting complex selective pressures.

De novo mutations in congenital heart disease with neurodevelopmental and other congenital anomalies

Putting both heart and brain at risk For reasons that are unclear, newborns with congenital heart disease (CHD) have a high risk of neurodevelopmental disabilities. Homsy et al. performed exome sequence analysis of 1200 CHD patients and their parents to identify spontaneously arising (de novo) mutations. Patients with both CHD and neurodevelopmental disorders had a much higher burden of damaging de novo mutations, particularly in genes with likely roles in both heart and brain development. Thus, clinical genotyping of patients with CHD may help to identify those at greatest risk of neurodevelopmental disabilities, allowing surveillance and early intervention. Science, this issue p. 1262 Genotyping of children with congenital heart disease may identify those at high risk of neurodevelopmental disorders. Congenital heart disease (CHD) patients have an increased prevalence of extracardiac congenital anomalies (CAs) and risk of neurodevelopmental disabilities (NDDs). Exome sequencing of 1213 CHD parent-offspring trios identified an excess of protein-damaging de novo mutations, especially in genes highly expressed in the developing heart and brain. These mutations accounted for 20% of patients with CHD, NDD, and CA but only 2% of patients with isolated CHD. Mutations altered genes involved in morphogenesis, chromatin modification, and transcriptional regulation, including multiple mutations in RBFOX2, a regulator of mRNA splicing. Genes mutated in other cohorts examined for NDD were enriched in CHD cases, particularly those with coexisting NDD. These findings reveal shared genetic contributions to CHD, NDD, and CA and provide opportunities for improved prognostic assessment and early therapeutic intervention in CHD patients.

Two locus inheritance of non-syndromic midline craniosynostosis via rare SMAD6 and common BMP2 alleles

Premature fusion of the cranial sutures (craniosynostosis), affecting 1 in 2000 newborns, is treated surgically in infancy to prevent adverse neurologic outcomes. To identify mutations contributing to common non-syndromic midline (sagittal and metopic) craniosynostosis, we performed exome sequencing of 132 parent-offspring trios and 59 additional probands. Thirteen probands (7%) had damaging de novo or rare transmitted mutations in SMAD6, an inhibitor of BMP – induced osteoblast differentiation (p<10−20). SMAD6 mutations nonetheless showed striking incomplete penetrance (<60%). Genotypes of a common variant near BMP2 that is strongly associated with midline craniosynostosis explained nearly all the phenotypic variation in these kindreds, with highly significant evidence of genetic interaction between these loci via both association and analysis of linkage. This epistatic interaction of rare and common variants defines the most frequent cause of midline craniosynostosis and has implications for the genetic basis of other diseases. DOI: http://dx.doi.org/10.7554/eLife.20125.001

Genetic diagnosis of neuroacanthocytosis disorders using exome sequencing.

Neuroacanthocytoses are neurodegenerative disorders marked by phenotypic and genetic heterogeneity. There are several associated genetic loci, and many defects, including gene deletions and insertions, and missense, nonsense, and splicing mutations, have been found spread over hundreds of kilobases of genomic DNA. In some cases, specific diagnosis is unclear, particularly in the early stages of disease or when there is an atypical presentation. Determination of the precise genetic defect allows assignment of the diagnosis and permits carrier detection and genetic counseling. The objective of this report was to utilize exome sequencing for genetic diagnosis in the neuroacanthocytosis syndromes. Genomic DNA from 2 patients with clinical features of chorea‐acanthocytosis was subjected to targeted exon capture. Captured DNA was subjected to ultrahigh throughput next‐generation sequencing. Sequencing data were assembled, filtered against known human variant genetic databases, and results were analyzed. Both patients were compound heterozygotes for mutations in the VPS13A gene, the gene associated with chorea‐acanthocytosis. Patient 1 had a 4‐bp deletion that removes the 5′ donor splice site of exon 58 and a nucleotide substitution that disrupts the 5′ donor splice site of exon 70. Patient 2 had a dinucleotide deletion in exon 16 and a dinucleotide insertion in exon 33. No mutations were identified in the XK, PANK2, or JPH3 gene loci. Exome sequencing is a valuable diagnostic tool in the neuroacanthocytosis syndromes. These studies may provide a better understanding of the function of the associated proteins and provide insight into the pathogenesis of these disorders.

Human Macrophage Response to L. (Viannia) panamensis: Microarray Evidence for an Early Inflammatory Response

Background Previous findings indicate that susceptibility to Leishmania (Viannia) panamensis infection of monocyte-derived macrophages from patients and asymptomatically infected individuals were associated with the adaptive immune response and clinical outcome. Methodology/Principal Findings To understand the basis for this difference we examined differential gene expression of human monocyte-derived macrophages following exposure to L. (V.) panamensis. Gene activation profiles were determined using macrophages from healthy volunteers cultured with or without stationary phase promastigotes of L. (V.) panamensis. Significant changes in expression (>1.5-fold change; p<0.05; up- or down-regulated) were identified at 0.5, 4 and 24 hours. mRNA abundance profiles varied over time, with the highest level of activation occurring at earlier time points (0.5 and 4 hrs). In contrast to observations for other Leishmania species, most significantly changed mRNAs were up- rather than down-regulated, especially at early time points. Up-regulated transcripts over the first 24 hours belonged to pathways involving eicosanoid metabolism, oxidative stress, activation of PKC through G protein coupled receptors, or mechanism of gene regulation by peroxisome proliferators via PPARα. Additionally, a marked activation of Toll-receptor mediated pathways was observed. Comparison with published microarray data from macrophages infected with L. (Leishmania) chagasi indicate differences in the regulation of genes involved in signaling, motility and the immune response. Conclusions Results show that the early (0.5 to 24 hours) human monocyte-derived macrophage response to L. (Viannia) panamensis is not quiescent, in contrast to published reports examining later response times (48–96 hours). Early macrophage responses are important for the developing cellular response at the site of infection. The kinetics and the mRNA abundance profiles induced by L. (Viannia) panamensis illustrate the dynamics of these interactions and the distinct biologic responses to different Leishmania species from the outset of infection within their primary host cell.

De novo mutations in inhibitors of Wnt, BMP, and Ras/ERK signaling pathways in non-syndromic midline craniosynostosis

Significance Craniosynostosis is a common congenital malformation resulting from premature fusion of the bones that comprise the cranial vault, requiring surgery in infancy to prevent adverse neurologic outcomes. Eighty-five percent of cases are non-syndromic and of unknown cause. By exome sequencing of families with non-syndromic midline craniosynostosis, we show that 5% of cases have de novo damaging mutations in negative regulators of the Wnt, bone morphogenetic protein (BMP), and Ras/ERK signaling pathways, developmental cascades that converge on common nuclear targets to promote bone formation. Another 5% have transmitted mutations in these pathways. Common variants near BMP2 show genetic interaction with these rare mutations. The results provide insight into pathophysiology and have immediate implications for the diagnosis and genetic counseling of families with craniosynostosis. Non-syndromic craniosynostosis (NSC) is a frequent congenital malformation in which one or more cranial sutures fuse prematurely. Mutations causing rare syndromic craniosynostoses in humans and engineered mouse models commonly increase signaling of the Wnt, bone morphogenetic protein (BMP), or Ras/ERK pathways, converging on shared nuclear targets that promote bone formation. In contrast, the genetics of NSC is largely unexplored. More than 95% of NSC is sporadic, suggesting a role for de novo mutations. Exome sequencing of 291 parent–offspring trios with midline NSC revealed 15 probands with heterozygous damaging de novo mutations in 12 negative regulators of Wnt, BMP, and Ras/ERK signaling (10.9-fold enrichment, P = 2.4 × 10−11). SMAD6 had 4 de novo and 14 transmitted mutations; no other gene had more than 1. Four familial NSC kindreds had mutations in genes previously implicated in syndromic disease. Collectively, these mutations contribute to 10% of probands. Mutations are predominantly loss-of-function, implicating haploinsufficiency as a frequent mechanism. A common risk variant near BMP2 increased the penetrance of SMAD6 mutations and was overtransmitted to patients with de novo mutations in other genes in these pathways, supporting a frequent two-locus pathogenesis. These findings implicate new genes in NSC and demonstrate related pathophysiology of common non-syndromic and rare syndromic craniosynostoses. These findings have implications for diagnosis, risk of recurrence, and risk of adverse neurodevelopmental outcomes. Finally, the use of pathways identified in rare syndromic disease to find genes accounting for non-syndromic cases may prove broadly relevant to understanding other congenital disorders featuring high locus heterogeneity.