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author:("Choi, muris")
2.  Taurodontism, variations in tooth number, and misshapened crowns in Wnt10a null mice and human kindreds 
WNT10A is a signaling molecule involved in tooth development, and WNT10A defects are associated with tooth agenesis. We characterized Wnt10a null mice generated by the knockout mouse project (KOMP) and six families with WNT10A mutations, including a novel p.Arg104Cys defect, in the absence of EDA,EDAR, or EDARADD variations. Wnt10a null mice exhibited supernumerary mandibular fourth molars, and smaller molars with abnormal cusp patterning and root taurodontism. Wnt10a−/− incisors showed distinctive apical–lingual wedge-shaped defects. These findings spurred us to closely examine the dental phenotypes of our WNT10A families. WNT10A heterozygotes exhibited molar root taurodontism and mild tooth agenesis (with incomplete penetrance) in their permanent dentitions. Individuals with two defective WNT10A alleles showed severe tooth agenesis and had fewer cusps on their molars. The misshapened molar crowns and roots were consistent with the Wnt10a null phenotype and were not previously associated with WNT10A defects. The missing teeth contrasted with the presence of supplemental teeth in the Wnt10a null mice and demonstrated mammalian species differences in the roles of Wnt signaling in early tooth development. We conclude that molar crown and root dysmorphologies are caused by WNT10A defects and that the severity of the tooth agenesis correlates with the number of defective WNT10A alleles.
PMCID: PMC4299714  PMID: 25629078
Familial tooth agenesis; hypodontia; oligodontia, taurodontism
3.  Neural-Specific Deletion of Htra2 Causes Cerebellar Neurodegeneration and Defective Processing of Mitochondrial OPA1 
PLoS ONE  2014;9(12):e115789.
HTRA2, a serine protease in the intermembrane space, has important functions in mitochondrial stress signaling while its abnormal activity may contribute to the development of Parkinson’s disease. Mice with a missense or null mutation of Htra2 fail to thrive, suffer striatal neuronal loss, and a parkinsonian phenotype that leads to death at 30–40 days of age. While informative, these mouse models cannot separate neural contributions from systemic effects due to the complex phenotypes of HTRA2 deficiency. Hence, we developed mice carrying a Htra2-floxed allele to query the consequences of tissue-specific HTRA2 deficiency. We found that mice with neural-specific deletion of Htra2 exhibited atrophy of the thymus and spleen, cessation to gain weight past postnatal (P) day 18, neurological symptoms including ataxia and complete penetrance of premature death by P40. Histologically, increased apoptosis was detected in the cerebellum, and to a lesser degree in the striatum and the entorhinal cortex, from P25. Even earlier at P20, mitochondria in the cerebella already exhibited abnormal morphology, including swelling, vesiculation, and fragmentation of the cristae. Furthermore, the onset of these structural anomalies was accompanied by defective processing of OPA1, a key molecule for mitochondrial fusion and cristae remodeling, leading to depletion of the L-isoform. Together, these findings suggest that HTRA2 is essential for maintenance of the mitochondrial integrity in neurons. Without functional HTRA2, a lifespan as short as 40 days accumulates a large quantity of dysfunctional mitochondria that contributes to the demise of mutant mice.
PMCID: PMC4274161  PMID: 25531304
4.  Recurrent activating mutation in PRKACA in cortisol-producing adrenal tumors 
Nature genetics  2014;46(6):613-617.
Adrenal tumors autonomously producing cortisol cause Cushing syndrome1–4. Exome sequencing of 25 tumor-normal pairs revealed two groups. Eight tumors (including 3 carcinomas) had many somatic copy number variants (CNV+) with frequent deletion of CDC42 and CDKN2A, amplification of 5q31.2, and protein-altering mutations in TP53 and RB1. Seventeen (all adenomas) had no CNVs (CNV-), TP53 or RB1 mutations. Six of these had known gain of function mutations in CTNNB15,6 (beta-catenin) or GNAS7,8 (Gαs), Six others had somatic p.Leu206Arg mutations in PRKACA (protein kinase A (PKA) catalytic subunit). Further sequencing identified this mutation in 13 of 63 tumors (35% of adenomas with overt CS). PRKACA, GNAS and CTNNB1 mutations were mutually exclusive. Leu206 directly interacts with PKA’s regulatory subunit, PRKAR1A9,10. PRKACAL206R loses PRKAR1A binding, increasing phosphorylation of downstream targets. PKA activity induces cortisol production and cell proliferation11–15, providing a mechanism for tumor development. These findings define distinct mechanisms underlying adrenal cortisol-producing tumors.
PMCID: PMC4074779  PMID: 24747643
5.  A Form of the Metabolic Syndrome Associated with Mutations in DYRK1B 
The New England journal of medicine  2014;370(20):1909-1919.
Genetic analysis has been successful in identifying causative mutations for individual cardiovascular risk factors. Success has been more limited in mapping susceptibility genes for clusters of cardiovascular risk traits, such as those in the metabolic syndrome.
We identified three large families with coinheritance of early-onset coronary artery disease, central obesity, hypertension, and diabetes. We used linkage analysis and whole-exome sequencing to identify the disease-causing gene.
A founder mutation was identified in DYRK1B, substituting cysteine for arginine at position 102 in the highly conserved kinase-like domain. The mutation precisely cosegregated with the clinical syndrome in all the affected family members and was absent in unaffected family members and unrelated controls. Functional characterization of the disease gene revealed that nonmutant protein encoded by DYRK1B inhibits the SHH (sonic hedgehog) and Wnt signaling pathways and consequently enhances adipogenesis. Furthermore, DYRK1B promoted the expression of the key gluconeogenic enzyme glucose-6-phosphatase. The R102C allele showed gain-offunction activities by potentiating these effects. A second mutation, substituting proline for histidine 90, was found to cosegregate with a similar clinical syndrome in an ethnically distinct family.
These findings indicate a role for DYRK1B in adipogenesis and glucose homeostasis and associate its altered function with an inherited form of the metabolic syndrome. (Funded by the National Institutes of Health.)
PMCID: PMC4069260  PMID: 24827035
6.  Gene-environment interactions in severe intraventricular hemorrhage of preterm neonates 
Pediatric research  2013;75(0):241-250.
Intraventricular hemorrhage (IVH) of the preterm neonate is a complex developmental disorder, with contributions from both the environment and the genome. IVH, or hemorrhage into the germinal matrix of the developing brain with secondary periventricular infarction, occurs in that critical period of time before the 32nd – 33rd week post-conception and has been attributed to changes in cerebral blood flow to the immature germinal matrix microvasculature. Emerging data suggest that genes subserving coagulation, inflammatory and vascular pathways, and their interactions with environmental triggers may influence both the incidence and severity of cerebral injury and are the subject of this review.
Polymorphisms in the Factor V Leiden gene are associated with the atypical timing of IVH suggesting an as yet unknown environmental trigger. The methylenetetra-hydrofolate reeducates (MTHFR) variants render neonates more vulnerable to cerebral injury in the presence of perinatal hypoxia. The present study demonstrates that the MTHFR 677C>T polymorphism and low 5 minute Apgar score additively increase the risk of IVH. Finally, review of published preclinical data suggests the stressors of delivery result in hemorrhage in the presence of mutations in collagen 4A1 (COL4A1), a major structural protein of the developing cerebral vasculature. Maternal genetics and fetal environment may also play a role.
PMCID: PMC3946468  PMID: 24192699
7.  Rare deleterious mutations of the gene EFR3A in autism spectrum disorders 
Molecular Autism  2014;5:31.
Whole-exome sequencing studies in autism spectrum disorder (ASD) have identified de novo mutations in novel candidate genes, including the synaptic gene Eighty-five Requiring 3A (EFR3A). EFR3A is a critical component of a protein complex required for the synthesis of the phosphoinositide PtdIns4P, which has a variety of functions at the neural synapse. We hypothesized that deleterious mutations in EFR3A would be significantly associated with ASD.
We conducted a large case/control association study by deep resequencing and analysis of whole-exome data for coding and splice site variants in EFR3A. We determined the potential impact of these variants on protein structure and function by a variety of conservation measures and analysis of the Saccharomyces cerevisiae Efr3 crystal structure. We also analyzed the expression pattern of EFR3A in human brain tissue.
Rare nonsynonymous mutations in EFR3A were more common among cases (16 / 2,196 = 0.73%) than matched controls (12 / 3,389 = 0.35%) and were statistically more common at conserved nucleotides based on an experiment-wide significance threshold (P = 0.0077, permutation test). Crystal structure analysis revealed that mutations likely to be deleterious were also statistically more common in cases than controls (P = 0.017, Fisher exact test). Furthermore, EFR3A is expressed in cortical neurons, including pyramidal neurons, during human fetal brain development in a pattern consistent with ASD-related genes, and it is strongly co-expressed (P < 2.2 × 10−16, Wilcoxon test) with a module of genes significantly associated with ASD.
Rare deleterious mutations in EFR3A were found to be associated with ASD using an experiment-wide significance threshold. Synaptic phosphoinositide metabolism has been strongly implicated in syndromic forms of ASD. These data for EFR3A strengthen the evidence for the involvement of this pathway in idiopathic autism.
PMCID: PMC4032628  PMID: 24860643
Autism spectrum disorder; Genetics; Rare variants; EFR3A; Synapse; Phosphoinositide metabolism
8.  Multiple recurrent de novo copy number variations (CNVs), including duplications of the 7q11.23 Williams-Beuren syndrome region, are strongly associated with autism 
Neuron  2011;70(5):863-885.
Given prior evidence for the contribution of rare copy number variations (CNVs) to autism spectrum disorders (ASD), we studied these events in 4,457 individuals from 1,174 simplex families, composed of parents, a proband and, in most kindreds, an unaffected sibling. We find significant association of ASD with de novo duplications of 7q11.23, where the reciprocal deletion causes Williams-Beuren syndrome, featuring a highly social personality. We identify rare recurrent de novo CNVs at five additional regions including two novel ASD loci, 16p13.2 (including the genes USP7 and C16orf72) and Cadherin13, and implement a rigorous new approach to evaluating the statistical significance of these observations. Overall, we find large de novo CNVs carry substantial risk (OR=3.55; CI =2.16-7.46, p=6.9 × 10−6); estimate the presence of 130-234 distinct ASD-related CNV intervals across the genome; and, based on data from multiple studies, present compelling evidence for the association of rare de novo events at 7q11.23, 15q11.2-13.1, 16p11.2, and Neurexin1.
PMCID: PMC3939065  PMID: 21658581
9.  Somatic and germline CACNA1D calcium channel mutations in aldosterone-producing adenomas and primary aldosteronism 
Nature genetics  2013;45(9):1050-1054.
Adrenal aldosterone-producing adenomas (APAs) constitutively produce the salt-retaining hormone aldosterone and are a common cause of severe hypertension. Recurrent mutations in the potassium channel KCNJ5 that result in cell depolarization and Ca2+ influx cause ~40% of these tumors1. We found five somatic mutations (four altering glycine 403, one altering isoleucine 770) in CACNA1D, encoding a voltage-gated calcium channel, among 43 non-KCNJ5-mutant APAs. These mutations lie in S6 segments that line the channel pore. Both result in channel activation at less depolarized potentials, and glycine 403 mutations also impair channel inactivation. These effects are inferred to cause increased Ca2+ influx, the sufficient stimulus for aldosterone production and cell proliferation in adrenal glomerulosa2. Remarkably, we identified de novo mutations at the identical positions in two children with a previously undescribed syndrome featuring primary aldosteronism and neuromuscular abnormalities. These findings implicate gain of function Ca2+ channel mutations in aldosterone-producing adenomas and primary aldosteronism.
PMCID: PMC3876926  PMID: 23913001
10.  Mutations in DSTYK and Dominant Urinary Tract Malformations 
The New England journal of medicine  2013;369(7):10.1056/NEJMoa1214479.
Congenital abnormalities of the kidney and the urinary tract are the most common cause of pediatric kidney failure. These disorders are highly heterogeneous, and the etiologic factors are poorly understood.
We performed genomewide linkage analysis and whole-exome sequencing in a family with an autosomal dominant form of congenital abnormalities of the kidney or urinary tract (seven affected family members). We also performed a sequence analysis in 311 unrelated patients, as well as histologic and functional studies.
Linkage analysis identified five regions of the genome that were shared among all affected family members. Exome sequencing identified a single, rare, deleterious variant within these linkage intervals, a heterozygous splice-site mutation in the dual serine–threonine and tyrosine protein kinase gene (DSTYK). This variant, which resulted in aberrant splicing of messenger RNA, was present in all affected family members. Additional, independent DSTYK mutations, including nonsense and splice-site mutations, were detected in 7 of 311 unrelated patients. DSTYK is highly expressed in the maturing epithelia of all major organs, localizing to cell membranes. Knockdown in zebrafish resulted in developmental defects in multiple organs, which suggested loss of fibroblast growth factor (FGF) signaling. Consistent with this finding is the observation that DSTYK colocalizes with FGF receptors in the ureteric bud and metanephric mesenchyme. DSTYK knockdown in human embryonic kidney cells inhibited FGF-stimulated phosphorylation of extracellular-signal-regulated kinase (ERK), the principal signal downstream of receptor tyrosine kinases.
We detected independent DSTYK mutations in 2.3% of patients with congenital abnormalities of the kidney or urinary tract, a finding that suggests that DSTYK is a major determinant of human urinary tract development, downstream of FGF signaling. (Funded by the National Institutes of Health and others.)
PMCID: PMC3846391  PMID: 23862974
11.  Extended haplotype association study in Crohn’s disease identifies a novel, Ashkenazi Jewish-specific missense mutation in the NF-κB pathway gene, HEATR3 
Genes and immunity  2013;14(5):310-316.
The Ashkenazi Jewish population has a several-fold higher prevalence of Crohn’s disease compared to non-Jewish European ancestry populations and has a unique genetic history. Haplotype association is critical to Crohn’s disease etiology in this population, most notably at NOD2, in which three causal, uncommon, and conditionally independent NOD2 variants reside on a shared background haplotype. We present an analysis of extended haplotypes which showed significantly greater association to Crohn’s disease in the Ashkenazi Jewish population compared to a non-Jewish population (145 haplotypes and no haplotypes with P-value < 10−3, respectively). Two haplotype regions, one each on chromosomes 16 and 21, conferred increased disease risk within established Crohn’s disease loci. We performed exome sequencing of 55 Ashkenazi Jewish individuals and follow-up genotyping focused on variants in these two regions. We observed Ashkenazi Jewish-specific nominal association at R755C in TRPM2 on chromosome 21. Within the chromosome 16 region, R642S of HEATR3 and rs9922362 of BRD7 showed genome-wide significance. Expression studies of HEATR3 demonstrated a positive role in NOD2-mediated NF-κB signaling. The BRD7 signal showed conditional dependence with only the downstream rare Crohn’s disease-causal variants in NOD2, but not with the background haplotype; this elaborates NOD2 as a key illustration of synthetic association.
PMCID: PMC3785105  PMID: 23615072
haplotype association; Ashkenazi Jewish; Crohn’s disease; NF-κB signaling; synthetic association
12.  De novo mutations in histone modifying genes in congenital heart disease 
Nature  2013;498(7453):220-223.
Congenital heart disease (CHD) is the most frequent birth defect, affecting 0.8% of live births1. Many cases occur sporadically and impair reproductive fitness, suggesting a role for de novo mutations. By analysis of exome sequencing of parent-offspring trios, we compared the incidence of de novo mutations in 362 severe CHD cases and 264 controls. CHD cases showed 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 mutations. Similar odds ratios were seen across major classes of severe CHD. We found a marked excess of de novo mutations in genes involved in production, removal or reading of H3K4 methylation (H3K4me), or ubiquitination of H2BK120, which is required for H3K4 methylation2–4. There were also two de novo mutations in SMAD2; SMAD2 signaling in the embryonic left-right organizer induces demethylation of H3K27me5. H3K4me and H3K27me mark `poised' promoters and enhancers that regulate expression of key developmental genes6. These findings implicate de novo point mutations in several hundred genes that collectively contribute to ~10% of severe CHD.
PMCID: PMC3706629  PMID: 23665959
13.  ADCK4 mutations promote steroid-resistant nephrotic syndrome through CoQ10 biosynthesis disruption  
The Journal of Clinical Investigation  2013;123(12):5179-5189.
Identification of single-gene causes of steroid-resistant nephrotic syndrome (SRNS) has furthered the understanding of the pathogenesis of this disease. Here, using a combination of homozygosity mapping and whole human exome resequencing, we identified mutations in the aarF domain containing kinase 4 (ADCK4) gene in 15 individuals with SRNS from 8 unrelated families. ADCK4 was highly similar to ADCK3, which has been shown to participate in coenzyme Q10 (CoQ10) biosynthesis. Mutations in ADCK4 resulted in reduced CoQ10 levels and reduced mitochondrial respiratory enzyme activity in cells isolated from individuals with SRNS and transformed lymphoblasts. Knockdown of adck4 in zebrafish and Drosophila recapitulated nephrotic syndrome-associated phenotypes. Furthermore, ADCK4 was expressed in glomerular podocytes and partially localized to podocyte mitochondria and foot processes in rat kidneys and cultured human podocytes. In human podocytes, ADCK4 interacted with members of the CoQ10 biosynthesis pathway, including COQ6, which has been linked with SRNS and COQ7. Knockdown of ADCK4 in podocytes resulted in decreased migration, which was reversed by CoQ10 addition. Interestingly, a patient with SRNS with a homozygous ADCK4 frameshift mutation had partial remission following CoQ10 treatment. These data indicate that individuals with SRNS with mutations in ADCK4 or other genes that participate in CoQ10 biosynthesis may be treatable with CoQ10.
PMCID: PMC3859425  PMID: 24270420
14.  Recessive mutations in DGKE cause atypical hemolytic-uremic syndrome 
Nature genetics  2013;45(5):531-536.
Pathologic thrombosis is a major cause of mortality. Hemolytic-uremic syndrome (HUS) features episodes of small vessel thrombosis resulting in microangiopathic hemolytic anemia, thrombocytopenia and renal failure1. Atypical HUS (aHUS) can result from genetic or autoimmune factors2 that lead to pathologic complement cascade activation3. By exome sequencing we identify recessive mutations in DGKE (diacylglycerol kinase epsilon) that co-segregate with aHUS in 9 unrelated kindreds, defining a distinctive Mendelian disease. Affected patients present with aHUS before age 1, have persistent hypertension, hematuria and proteinuria (sometimes nephrotic range), and develop chronic kidney disease with age. DGKE is found in endothelium, platelets, and podocytes. Arachidonic acid-containing diacylglycerols (DAG) activate protein kinase C, which promotes thrombosis. DGKE normally inactivates DAG signaling. We infer that loss of DGKE function results in a pro-thrombotic state. These findings identify a new mechanism of pathologic thrombosis and kidney failure and have immediate implications for treatment of aHUS patients.
PMCID: PMC3719402  PMID: 23542698
15.  De novo mutations revealed by whole exome sequencing are strongly associated with autism 
Nature  2012;485(7397):237-241.
Multiple studies have confirmed the contribution of rare de novo copy number variations (CNVs) to the risk for Autism Spectrum Disorders (ASD).1-3 While de novo single nucleotide variants (SNVs) have been identified in affected individuals,4 their contribution to risk has yet to be clarified. Specifically, the frequency and distribution of these mutations has not been well characterized in matched unaffected controls, data that are vital to the interpretation of de novo coding mutations observed in probands. Here we show, via 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 ASD and carry large effects (OR=5.65; CI: 1.44-22.2; p=0.01 asymptotic test). Based on mutation rates in unaffected individuals, we demonstrate that multiple independent de novo SNVs 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, Alpha Subunit), a result that is highly unlikely by chance (p=0.005).
PMCID: PMC3667984  PMID: 22495306
16.  Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma 
Nature genetics  2012;44(9):1006-1014.
We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1P29S) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1P29S showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.
PMCID: PMC3432702  PMID: 22842228
17.  Familial Cortical Myoclonus with a Mutation in NOL3 
Annals of neurology  2012;72(2):175-183.
Myoclonus is characterized by sudden, brief involuntary movements and its presence is debilitating. We identified a family suffering from adult-onset, cortical myoclonus without associated seizures. We performed clinical, electrophysiological, and genetic studies to define this phenotype.
A large, four-generation family with history of myoclonus underwent careful questioning, examination, and electrophysiological testing. Thirty-five family members donated blood samples for genetic analysis, which included SNP mapping, microsatellite linkage, targeted massively parallel sequencing, and Sanger sequencing. In silico and in vitro experiments were performed to investigate functional significance of the mutation.
We identified 11 members of a Canadian Mennonite family suffering from adult-onset, slowly progressive, disabling, multifocal myoclonus. Somatosensory evoked potentials indicated a cortical origin of the myoclonus. There were no associated seizures. Some severely affected individuals developed signs of progressive cerebellar ataxia of variable severity late in the course of their illness. The phenotype was inherited in an autosomal dominant fashion. We demonstrated linkage to chromosome 16q21-22.1. We then sequenced all coding sequence in the critical region, identifying only a single co-segregating, novel, nonsynonymous mutation, which resides in the gene NOL3. Furthermore, this mutation was found to alter post-translational modification of NOL3 protein in vitro.
We propose that Familial Cortical Myoclonus (FCM) is a novel movement disorder that may be caused by mutation in NOL3. Further investigation of the role of NOL3 in neuronal physiology may shed light on neuronal membrane hyperexcitability and pathophysiology of myoclonus and related disorders.
PMCID: PMC3431191  PMID: 22926851
18.  On optimal pooling designs to identify rare variants through massive resequencing 
Genetic Epidemiology  2011;35(3):139-147.
The advent of next-generation sequencing technologies has facilitated the detection of rare variants. Despite the significant cost reduction, sequencing cost is still high for large-scale studies. In this article, we examine DNA pooling as a cost-effective strategy for rare variant detection. We consider the optimal number of individuals in a DNA pool to detect an allele with a specific minor allele frequency (MAF) under a given coverage depth and detection threshold. We found that the optimal number of individuals in a pool is indifferent to the MAF at the same coverage depth and detection threshold. In addition, when the individual contributions to each pool are equal, the total number of individuals across different pools required in an optimal design to detect a variant with a desired power is similar at different coverage depths. When the contributions are more variable, more individuals tend to be needed for higher coverage depths. Our study provides general guidelines on using DNA pooling for more cost-effective identifications of rare variants. Genet. Epidemiol. 35:139-147, 2011. © 2011 Wiley-Liss, Inc.
PMCID: PMC3176340  PMID: 21254222
optimal pooling designs; rare variant detection; next-generation sequencing
19.  Application of Whole Exome Sequencing to Identify Disease-Causing Variants in Inherited Human Diseases 
Genomics & Informatics  2012;10(4):214-219.
The recent advent of next-generation sequencing technologies has dramatically changed the nature of biomedical research. Human genetics is no exception-it has never been easier to interrogate human patient genomes at the nucleotide level to identify disease-associated variants. To further facilitate the efficiency of this approach, whole exome sequencing (WES) was first developed in 2009. Over the past three years, multiple groups have demonstrated the power of WES through robust disease-associated variant discoveries across a diverse spectrum of human diseases. Here, we review the application of WES to different types of inherited human diseases and discuss analytical challenges and possible solutions, with the aim of providing a practical guide for the effective use of this technology.
PMCID: PMC3543920  PMID: 23346032
discovery of disease-causing variants; inherited human disease; next-generation sequencing; whole exome sequencing
20.  Genome-wide Association Study Identifies Susceptibility Loci for IgA Nephropathy 
Nature genetics  2011;43(4):321-327.
We performed a genome-wide association study of IgA nephropathy (IgAN), a major cause of kidney failure worldwide. Discovery was in 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 (MHC), a common deletion of CFHR1 and CFHR3 at Chr. 1q32 and a locus at Chr. 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 10-fold variation in interindividual risk. Many of the IgAN–protective alleles impart increased risk of other autoimmune or infectious diseases, and IgAN risk allele frequencies closely parallel the variation in disease prevalence among Asian, European and African populations, suggesting complex selective pressures.
PMCID: PMC3412515  PMID: 21399633
21.  Mutations in Kelch-like 3 and Cullin 3 cause hypertension and electrolyte abnormalities 
Nature  2012;482(7383):98-102.
Hypertension affects one billion people and is a principal reversible risk factor for cardiovascular disease. A rare Mendelian syndrome, pseudohypoaldosteronism type II (PHAII), featuring hypertension, hyperkalemia, and metabolic acidosis, has revealed previously unrecognized physiology orchestrating the balance between renal salt reabsorption versus K+ and H+ excretion1. We used exome sequencing to identify mutations in Kelch-like 3 (KLHL3) or Cullin 3 (CUL3) in 41 PHAII kindreds. KLHL3 mutations are either recessive or dominant, while CUL3 mutations are dominant and predominantly de novo. CUL3 and BTB-Kelch proteins such as KLHL3 are components of Cullin/RING E3 ligase complexes (CRLs) that ubiquitinate substrates bound to Kelch propeller domains2–8. Dominant KLHL3 mutations are clustered in short segments within the Kelch propeller and BTB domains implicated in substrate9 and Cullin5 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 (NCC) in the distal nephron of the kidney; KLHL3 and CUL3 are expressed in this location, suggesting a mechanistic link between KLHL3/CUL3 mutations, increased Na-Cl reabsorption, and disease pathogenesis. These findings demonstrate the utility of exome sequencing in disease gene identification despite combined complexities of locus heterogeneity, mixed models of transmission, and frequent de novo mutation, and establish a fundamental role for KLHL3/CUL3 in blood pressure, K+, and pH homeostasis.
PMCID: PMC3278668  PMID: 22266938
22.  Comprehensive Re-Sequencing of Adrenal Aldosterone Producing Lesions Reveal Three Somatic Mutations near the KCNJ5 Potassium Channel Selectivity Filter 
PLoS ONE  2012;7(7):e41926.
Aldosterone producing lesions are a common cause of hypertension, but genetic alterations for tumorigenesis have been unclear. Recently, either of two recurrent somatic missense mutations (G151R or L168R) was found in the potassium channel KCNJ5 gene in aldosterone producing adenomas. These mutations alter the channel selectivity filter and result in Na+ conductance and cell depolarization, stimulating aldosterone production and cell proliferation. Because a similar mutation occurs in a Mendelian form of primary aldosteronism, these mutations appear to be sufficient for cell proliferation and aldosterone production. The prevalence and spectrum of KCNJ5 mutations in different entities of adrenocortical lesions remain to be defined.
Materials and Methods
The coding region and flanking intronic segments of KCNJ5 were subjected to Sanger DNA sequencing in 351 aldosterone producing lesions, from patients with primary aldosteronism and 130 other adrenocortical lesions. The specimens had been collected from 10 different worldwide referral centers.
G151R or L168R somatic mutations were identified in 47% of aldosterone producing adenomas, each with similar frequency. A previously unreported somatic mutation near the selectivity filter, E145Q, was observed twice. Somatic G151R or L168R mutations were also found in 40% of aldosterone producing adenomas associated with marked hyperplasia, but not in specimens with merely unilateral hyperplasia. Mutations were absent in 130 non-aldosterone secreting lesions.
KCNJ5 mutations were overrepresented in aldosterone producing adenomas from female compared to male patients (63 vs. 24%). Males with KCNJ5 mutations were significantly younger than those without (45 vs. 54, respectively; p<0.005) and their APAs with KCNJ5 mutations were larger than those without (27.1 mm vs. 17.1 mm; p<0.005).
Either of two somatic KCNJ5 mutations are highly prevalent and specific for aldosterone producing lesions. These findings provide new insight into the pathogenesis of primary aldosteronism.
PMCID: PMC3407065  PMID: 22848660
23.  Geographic Differences in Genetic Susceptibility to IgA Nephropathy: GWAS Replication Study and Geospatial Risk Analysis 
PLoS Genetics  2012;8(6):e1002765.
IgA nephropathy (IgAN), major cause of kidney failure worldwide, is common in Asians, moderately prevalent in Europeans, and rare in Africans. It is not known if these differences represent variation in genes, environment, or ascertainment. In a recent GWAS, we localized five IgAN susceptibility loci on Chr.6p21 (HLA-DQB1/DRB1, PSMB9/TAP1, and DPA1/DPB2 loci), Chr.1q32 (CFHR3/R1 locus), and Chr.22q12 (HORMAD2 locus). These IgAN loci are associated with risk of other immune-mediated disorders such as type I diabetes, multiple sclerosis, or inflammatory bowel disease. We tested association of these loci in eight new independent cohorts of Asian, European, and African-American ancestry (N = 4,789), followed by meta-analysis with risk-score modeling in 12 cohorts (N = 10,755) and geospatial analysis in 85 world populations. Four susceptibility loci robustly replicated and all five loci were genome-wide significant in the combined cohort (P = 5×10−32–3×10−10), with heterogeneity detected only at the PSMB9/TAP1 locus (I2 = 0.60). Conditional analyses identified two new independent risk alleles within the HLA-DQB1/DRB1 locus, defining multiple risk and protective haplotypes within this interval. We also detected a significant genetic interaction, whereby the odds ratio for the HORMAD2 protective allele was reversed in homozygotes for a CFHR3/R1 deletion (P = 2.5×10−4). A seven–SNP genetic risk score, which explained 4.7% of overall IgAN risk, increased sharply with Eastward and Northward distance from Africa (r = 0.30, P = 3×10−128). This model paralleled the known East–West gradient in disease risk. Moreover, the prediction of a South–North axis was confirmed by registry data showing that the prevalence of IgAN–attributable kidney failure is increased in Northern Europe, similar to multiple sclerosis and type I diabetes. Variation at IgAN susceptibility loci correlates with differences in disease prevalence among world populations. These findings inform genetic, biological, and epidemiological investigations of IgAN and permit cross-comparison with other complex traits that share genetic risk loci and geographic patterns with IgAN.
Author Summary
IgA nephropathy (IgAN) is the most common cause of kidney failure in Asia, has lower prevalence in Europe, and is very infrequent among populations of African ancestry. A long-standing question in the field is whether these differences represent variation in genes, environment, or ascertainment. In a recent genome-wide association study of 5,966 individuals, we identified five susceptibility loci for this trait. In this paper, we study the largest IgAN case-control cohort reported to date, composed of 10,775 individuals of European, Asian, and African-American ancestry. We confirm that all five loci are significant contributors to disease risk across this multi-ethnic cohort. In addition, we identify two novel independent susceptibility alleles within the HLA-DQB1/DRB1 locus and a new genetic interaction between loci on Chr.1p36 and Chr.22q22. We develop a seven–SNP genetic risk score that explains nearly 5% of variation in disease risk. In geospatial analysis of 85 world populations, the genetic risk score closely parallels worldwide patterns of disease prevalence. The genetic risk score also predicts an unsuspected Northward risk gradient in Europe. This genetic prediction is verified by examination of registry data demonstrating, similarly to other immune-mediated diseases such as multiple sclerosis and type I diabetes, a previously unrecognized increase in IgAN–attributable kidney failure in Northern European countries.
PMCID: PMC3380840  PMID: 22737082
24.  K+ Channel Mutations in Adrenal Aldosterone-Producing Adenomas and Hereditary Hypertension 
Science (New York, N.Y.)  2011;331(6018):768-772.
Endocrine tumors such as aldosterone-producing adrenal adenomas (APAs), a cause of severe hypertension, feature constitutive hormone production and unrestrained cell proliferation; the mechanisms linking these events are unknown. We identify two recurrent somatic mutations in and near the selectivity filter of the potassium (K+) channel KCNJ5 that are present in 8 of 22 human APAs studied. Both produce increased sodium (Na+) conductance and cell depolarization, which in adrenal glomerulosa cells produces calcium (Ca2+) entry, the signal for aldosterone production and cell proliferation. Similarly, we identify an inherited KCNJ5 mutation that produces increased Na+ conductance in a Mendelian form of severe aldosteronism and massive bilateral adrenal hyperplasia. These findings explain pathogenesis in a subset of patients with severe hypertension and implicate loss of K+ channel selectivity in constitutive cell proliferation and hormone production.
PMCID: PMC3371087  PMID: 21311022
25.  Recessive LAMC3 mutations cause malformations of occipital cortical development 
Nature Genetics  2011;43(6):590-594.
The biological basis for regional and inter-species differences in cerebral cortical morphology is poorly understood. We focused on consanguineous Turkish families with a single affected member with complex bilateral occipital cortical gyration abnormalities. By using whole-exome sequencing, we initially identified a homozygous 2-bp deletion in LAMC3, the laminin γ3 gene, leading to an immediate premature termination codon. In two other affected individuals with nearly identical phenotypes, we identified a homozygous nonsense mutation and a compound heterozygous mutation. In human but not mouse fetal brain, LAMC3 is enriched in postmitotic cortical plate neurons, localizing primarily to the somatodendritic compartment. LAMC3 expression peaks between late gestation and late infancy, paralleling the expression of molecules that are important in dendritogenesis and synapse formation. The discovery of the molecular basis of this unusual occipital malformation furthers our understanding of the complex biology underlying the formation of cortical gyrations.
PMCID: PMC3329933  PMID: 21572413

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