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1.  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
2.  Mineralocorticoid receptor phosphorylation regulates ligand binding and renal response to volume depletion and hyperkalemia 
Cell metabolism  2013;18(5):660-671.
Nuclear receptors are transcription factors that regulate diverse cellular processes. In canonical activation, ligand availability is sufficient to produce receptor binding, entraining downstream signaling. The mineralocorticoid receptor (MR) is normally activated by aldosterone, which is produced in both volume depletion and hyperkalemia, states that require different homeostatic responses. We report phosphorylation at S843 in the MR ligand-binding domain that prevents ligand binding and activation. In kidney, MRS843-P is found exclusively in intercalated cells of the distal nephron. In volume depletion, angiotensin II and WNK4 signaling decrease MRS843-P levels whereas hyperkalemia increases MRS843-P. Dephosphorylation of MRS843-P results in aldosterone-dependent increases of the intercalated cell apical proton pump and Cl−/HCO3− exchangers, increasing Cl− reabsorption and promoting increased plasma volume while inhibiting K+ secretion. These findings reveal a mechanism regulating nuclear hormone receptor activity and implicate selective MR activation in intercalated cells in the distinct adaptive responses to volume depletion and hyperkalemia.
PMCID: PMC3909709  PMID: 24206662
3.  Rare independent mutations in renal salt handling genes contribute to blood pressure variation 
Nature genetics  2008;40(5):592-599.
The effects of alleles in many genes are believed to contribute to common complex diseases such as hypertension. Whether risk alleles comprise a small number of common variants or many rare independent mutations at trait loci is largely unknown. We screened members of the Framingham Heart Study (FHS) for variation in three genes -SLC12A3 (NCCT), SLC12A1 (NKCC2) and KCNJ1 (ROMK)- causing rare recessive diseases featuring large reductions in blood pressure. Using comparative genomics, genetics, and biochemistry, we identified subjects with mutations proven or inferred to be functional. These mutations, all heterozygous and rare, produce clinically significant blood pressure reduction and protect from development of hypertension. Our findings implicate many rare alleles that alter renal salt handling in blood pressure variation in the general population, and identify alleles with health benefit that are nonetheless under purifying selection. These findings have implications for the genetic architecture of hypertension and other common complex traits.
PMCID: PMC3766631  PMID: 18391953
5.  An Incompletely Penetrant Novel Mutation in COL7A1 Causes Epidermolysis Bullosa Pruriginosa and Dominant Dystrophic Epidermolysis Bullosa Phenotypes in an Extended Kindred 
Pediatric dermatology  2012;29(6):725-731.
Epidermolysis bullosa pruriginosa (EBP) is a rare subtype of dystrophic epidermolysis bullosa (DEB) characterized by intense pruritus, nodular or lichenoid lesions, and violaceous linear scarring, most prominently on the extensor extremities. Remarkably, identical mutations in COL7A1, which encodes an anchoring fibril protein present at the dermal–epidermal junction, can cause both DEB and EBP with either autosomal dominant or recessive inheritance. We present one family with both dystrophic and pruriginosa phenotypes of epidermolysis bullosa. The proband is a 19-year-old Caucasian woman who initially presented in childhood with lichenoid papules affecting her extensor limbs and intense pruritus consistent with EBP. Her maternal grandmother saw a dermatologist for similar skin lesions that developed without any known triggers at age 47 and mostly resolved spontaneously after approximately 10 years. The proband’s younger brother developed a small crop of pruritic papules on his elbows, dorsal hands, knees, and ankles at age 13. Her second cousin once removed, however, reported a mild blistering disease without pruritus consistent with DEB. Genetic sequencing of the kindred revealed a single dominant novel intron 47 splice site donor G>A mutation, c.4668 + 1 G>A, which we predict leads to exon skipping. Incomplete penetrance is confirmed in her clinically unaffected mother, who carries the same dominant mutation. The wide diversity of clinical phenotypes with one underlying genotype demonstrates that COL7A1 mutations are incompletely penetrant and strongly suggests that other genetic and environmental factors influence clinical presentation.
PMCID: PMC3709244  PMID: 22515571
6.  Phosphoregulation of the Na-K-2Cl and K-Cl cotransporters by the WNK kinases 
Biochimica et biophysica acta  2010;1802(12):1150-1158.
Precise regulation of the intracellular concentration of chloride [Cl-]i is necessary for proper cell volume regulation, transepithelial transport, and GABA neurotransmission. The Na-K-2Cl (NKCCs) and K-Cl (KCCs) cotransporters, related SLC12A transporters mediating cellular chloride influx and efflux, respectively, are key determinants of [Cl-]i in numerous cell types, including red blood cells, epithelial cells, and neurons. A common "chloride/volume-sensitive kinase", or related system of kinases, has long been hypothesized to mediate the reciprocal but coordinated phosphoregulation of the NKCCs and the KCCs, but the identity of these kinase(s) has remained unknown. Recent evidence suggests the WNK (with no lysine = K) serine-threonine kinases directly or indirectly via the downstream Ste20-type kinases SPAK/OSR1, are critical components of this signaling pathway. Hypertonic stress (cell shrinkage), and possibly decreased [Cl-]i, triggers the phosphorylation and activation of specific WNKs, promoting NKCC activation and KCC inhibition via net transporter phosphorylation. Silencing WNK kinase activity can promote NKCC inhibition and KCC activation via net transporter dephosphorylation, revealing a dynamic ability of the WNKs to modulate [Cl-]. This pathway is essential for the defense of cell volume during osmotic perturbation, coordination of epithelial transport, and the gating of sensory information in the peripheral system. Commiserate with their importance in serving these critical roles in humans, mutations in WNKs underlie two different Mendelian diseases, pseudohypoaldosteronism type II (an inherited form of salt-sensitive hypertension), and hereditary sensory and autonomic neuropathy type 2. WNKs also regulate ion transport in lower multicellular organisms, including Caenorhabditis elegans, suggesting their functions are evolutionarily-conserved. An increased understanding of how the WNKs regulate the Na-K-2Cl and K-Cl cotransporters may provide novel opportunities for the selective modulation of these transporters, with ramifications for common human diseases like hypertension, sickle cell disease, neuropathic pain, and epilepsy.
PMCID: PMC3529164  PMID: 20637866
7.  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
8.  The DNA Methylome of Benign and Malignant Parathyroid Tumors 
Genes, chromosomes & cancer  2011;50(9):735-745.
The role of DNA methylation of CpG islands in parathyroid tumorigenesis has not been analyzed in an unbiased, systematic fashion. DNA was isolated from normal and pathologic parathyroid tissues, bisulphite modified and analyzed using the Infinium HumanMethylation27 BeadChip. Distinct hierarchical clustering of genes with altered DNA methylation profiles in normal and pathologic parathyroid tissue was evident. Comparing normal parathyroid tissue with parathyroid adenomas, 367 genes were significantly altered, while 175 genes significantly differed when comparing parathyroid carcinomas and normal parathyroid tissues. A comparison between parathyroid adenomas and parathyroid carcinomas identified 263 genes with significantly distinct methylation levels. Results were confirmed for certain genes in a validation cohort of 40 parathyroid adenomas by methylation-specific PCR. Genes of known or putative importance in the development of parathyroid tumors showed significant and frequent hypermethylation. DNA hypermethylation of CDKN2B, CDKN2A, WT1, SFRP1, SFRP2 and SFRP4 was associated with reduced gene expression in both benign and malignant parathyroid tumors. Treatment with 5-aza-2′-deoxycytidine of primary cell cultures restores expression of hypermethylated genes in benign and malignant parathyroid tumors. In conclusion, the unbiased, genome-wide study of the parathyroid tumor DNA methylome identified a number of genes with altered DNA methylation patterns of putative importance to benign and malignant parathyroid tumorigenesis.
PMCID: PMC3134609  PMID: 21638518
9.  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
10.  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
11.  A pilot genome-wide association study shows genomic variants enriched in the non-tumor cells of patients with well-differentiated neuroendocrine tumors of the ileum 
Endocrine-Related Cancer  2011;18(1):171-180.
Genetic studies of midgut carcinoid cancer have exclusively focused on genomic changes of the tumor cells. We investigated the role of constitutional genetic polymorphisms in predisposing individuals to ileal carcinoids. In all, 239 cases and 110 controls were collected from three institutions: the Uppsala University Hospital; the Dana-Farber Cancer Institute; and the MD Anderson Cancer Center, and were genotyped using microarrays assaying >300 000 single nucleotide polymorphisms. Association with rs2208059 in KIF16B approached statistical significance (Mantel-Haenszel odds ratio=2.42, P=4.16×10−7) at a Bonferroni-corrected level (<1.62×10−7). Using two computational algorithms, four copy-number variants (CNVs) were identified in multiple cases that were absent in study controls and markedly less frequent in ~1500 population-based controls. Of these four constitutional CNVs identified in blood-derived DNA, a 40 kb heterozygous deletion in Chr18q22.1 corresponded with a region frequently showing loss of heterozygosity (LOH) in ileal carcinoid tumor cells based on our meta-analysis of previously published cytogenetic studies (69.7% LOH, 95% confidence interval=60.0–77.9%). We analyzed the constitutional 40 kb deletion on chr18 in our study samples with a real-time quantitative PCR assay; 14/226 cases (6.19%) and 2/97 controls (2.06%) carried the CNV, although the exact boundaries of each deletion have not been determined. Given the small sample size, our findings warrant an independent cohort for a replication study. Owing to the rarity of this disease, we believe these results will provide a valuable resource for future work on this serious condition by allowing others to make efficient use of their samples in targeted studies.
PMCID: PMC3221459  PMID: 21139019
12.  NordicDB: a Nordic pool and portal for genome-wide control data 
European Journal of Human Genetics  2010;18(12):1322-1326.
A cost-efficient way to increase power in a genetic association study is to pool controls from different sources. The genotyping effort can then be directed to large case series. The Nordic Control database, NordicDB, has been set up as a unique resource in the Nordic area and the data are available for authorized users through the web portal ( The current version of NordicDB pools together high-density genome-wide SNP information from ∼5000 controls originating from Finnish, Swedish and Danish studies and shows country-specific allele frequencies for SNP markers. The genetic homogeneity of the samples was investigated using multidimensional scaling (MDS) analysis and pairwise allele frequency differences between the studies. The plot of the first two MDS components showed excellent resemblance to the geographical placement of the samples, with a clear NW–SE gradient. We advise researchers to assess the impact of population structure when incorporating NordicDB controls in association studies. This harmonized Nordic database presents a unique genome-wide resource for future genetic association studies in the Nordic countries.
PMCID: PMC3002853  PMID: 20664631
common controls; genome-wide data; Nordic Control Database; population stratification
13.  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.
PMCID: PMC3176340  PMID: 21254222
optimal pooling designs; rare variant detection; next-generation sequencing
14.  WNK2 Kinase Is a Novel Regulator of Essential Neuronal Cation-Chloride Cotransporters* 
The Journal of Biological Chemistry  2011;286(34):30171-30180.
NKCC1 and KCC2, related cation-chloride cotransporters (CCC), regulate cell volume and γ-aminobutyric acid (GABA)-ergic neurotranmission by modulating the intracellular concentration of chloride [Cl−]. These CCCs are oppositely regulated by serine-threonine phosphorylation, which activates NKCC1 but inhibits KCC2. The kinase(s) that performs this function in the nervous system are not known with certainty. WNK1 and WNK4, members of the WNK (with no lysine [K]) kinase family, either directly or via the downstream SPAK/OSR1 Ste20-type kinases, regulate the furosemide-sensitive NKCC2 and the thiazide-sensitive NCC, kidney-specific CCCs. What role the novel WNK2 kinase plays in this regulatory cascade, if any, is unknown. Here, we show that WNK2, unlike other WNKs, is not expressed in kidney; rather, it is a neuron-enriched kinase primarily expressed in neocortical pyramidal cells, thalamic relay cells, and cerebellar granule and Purkinje cells in both the developing and adult brain. Bumetanide-sensitive and Cl−-dependent 86Rb+ uptake assays in Xenopus laevis oocytes revealed that WNK2 promotes Cl− accumulation by reciprocally activating NKCC1 and inhibiting KCC2 in a kinase-dependent manner, effectively bypassing normal tonicity requirements for cotransporter regulation. TiO2 enrichment and tandem mass spectrometry studies demonstrate WNK2 forms a protein complex in the mammalian brain with SPAK, a known phosphoregulator of NKCC1. In this complex, SPAK is phosphorylated at Ser-383, a consensus WNK recognition site. These findings suggest a role for WNK2 in the regulation of CCCs in the mammalian brain, with implications for both cell volume regulation and/or GABAergic signaling.
PMCID: PMC3191056  PMID: 21733846
Brain; Chloride Transport; Neurons; Serine Threonine Protein Kinase; Sodium Transport
15.  Mitotic Recombination in Patients with Ichthyosis Causes Reversion of Dominant Mutations in KRT10 
Science (New York, N.Y.)  2010;330(6000):94-97.
Somatic loss of wild-type alleles can produce disease traits such as neoplasia. Conversely, somatic loss of disease-causing mutations can revert phenotypes, however these events are infrequently observed. We demonstrate that ichthyosis with confetti, a severe, sporadic skin disease, is associated with thousands of revertant clones of normal skin that arise from loss of heterozygosity on chromosome 17q via mitotic recombination. This enabled mapping and identification of disease-causing mutations in keratin 10 (KRT10); all result in frameshifts into the same alternative reading frame, producing an arginine-rich C-terminal peptide that redirects keratin 10 from the cytokeratin filament network to the nucleolus. The general rarity of spontaneous reversion and the specific absence of reversion of other dominant mutations in KRT10 implicate the frameshift peptide in the appearance of revertants. These results may have ramifications for reversion of other mutations.
PMCID: PMC3085938  PMID: 20798280
16.  A Cluster of Metabolic Defects Caused by Mutation in a Mitochondrial tRNA 
Science (New York, N.Y.)  2004;306(5699):1190-1194.
Hypertension and dyslipidemia are risk factors for atherosclerosis and occur together more often than expected by chance. Although this clustering suggests shared causation, unifying factors remain unknown. We describe a large kindred with a syndrome including hypertension, hypercholesterolemia, and hypomagnesemia. Each phenotype is transmitted on the maternal lineage with a pattern indicating mitochondrial inheritance. Analysis of the mitochondrial genome of the maternal lineage identified a homoplasmic mutation substituting cytidine for uridine immediately 5′ to the mitochondrial transfer RNAIle anticodon. Uridine at this position is nearly invariate among transfer RNAs because of its role in stabilizing the anticodon loop. Given the known loss of mitochondrial function with aging, these findings may have implications for the common clustering of these metabolic disorders.
PMCID: PMC3033655  PMID: 15498972
17.  Apolipoprotein C3 Gene Variants in Nonalcoholic Fatty Liver Disease 
The New England journal of medicine  2010;362(12):1082-1089.
Nonalcoholic fatty liver disease is associated with hepatic insulin resistance and type 2 diabetes mellitus. Whether this association has a genetic basis is unknown.
In 95 healthy Asian Indian men, a group known to have a high prevalence of non-alcoholic fatty liver disease, we genotyped two single-nucleotide polymorphisms (SNPs) in the gene encoding apolipoprotein C3 (APOC3) that are known to be associated with hypertriglyceridemia (rs2854116 [T-455C] and rs2854117 [C-482T]). Plasma apolipoprotein C3 concentrations, insulin sensitivity, and hepatic triglyceride content were measured. We also measured plasma triglyceride concentrations and retinyl fatty acid ester absorption as well as plasma triglyceride clearance after oral and intravenous fat-tolerance tests. Liver triglyceride content and APOC3 genotypes were also assessed in a group of 163 healthy non–Asian Indian men.
Carriers of the APOC3 variant alleles (C-482T, T-455C, or both) had a 30% increase in the fasting plasma apolipoprotein C3 concentration, as compared with the wild-type homozygotes. They also had a 60% increase in the fasting plasma triglyceride concentration, an increase by a factor of approximately two in the plasma triglyceride and retinyl fatty acid ester concentrations after an oral fat-tolerance test, and a 46% reduction in plasma triglyceride clearance. The prevalence of nonalcoholic fatty liver disease was 38% among variant-allele carriers and 0% among wild-type homozygotes (P<0.001). The subjects with nonalcoholic fatty liver disease had marked insulin resistance. A validation study involving non–Asian Indian men confirmed the association between APOC3 variant alleles and nonalcoholic fatty liver disease.
The polymorphisms C-482T and T-455C in APOC3 are associated with nonalcoholic fatty liver disease and insulin resistance.
PMCID: PMC2976042  PMID: 20335584
18.  LRP6 Mutation in a Family with Early Coronary Disease and Metabolic Risk Factors 
Science (New York, N.Y.)  2007;315(5816):1278-1282.
Coronary artery disease (CAD) is the leading cause of death worldwide and is commonly caused by a constellation of risk factors called the metabolic syndrome. We characterized a family with autosomal dominant early CAD, features of the metabolic syndrome (hyperlipidemia, hypertension, and diabetes), and osteoporosis. These traits showed genetic linkage to a short segment of chromosome 12p, in which we identified a missense mutation in LRP6, which encodes a co-receptor in the Wnt signaling pathway. The mutation, which substitutes cysteine for arginine at a highly conserved residue of an epidermal growth factor–like domain, impairs Wnt signaling in vitro. These results link a single gene defect in Wnt signaling to CAD and multiple cardiovascular risk factors.
PMCID: PMC2945222  PMID: 17332414
19.  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
20.  COL4A1 Mutation in Preterm Intraventricular Hemorrhage 
The Journal of pediatrics  2009;155(5):743-745.
Intraventricular hemorrhage is a common complication of preterm infants. Mutations in the type IV procollagen gene, COL4A1, are associated with cerebral small vessel disease with hemorrhage in adults and fetuses. We report a rare variant in COL4A1 associated with intraventricular hemorrhage in dizygotic preterm twins. These results expand the spectrum of diseases attributable to mutations in type IV procollagens.
PMCID: PMC2884156  PMID: 19840616
21.  Sites of Regulated Phosphorylation that Control K-Cl Cotransporter Activity 
Cell  2009;138(3):525-536.
Modulation of intracellular chloride concentration ([Cl−]i) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl− exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl−]I; however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3's intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl−]i and have implications for control of cell volume and neuronal function.
PMCID: PMC2811214  PMID: 19665974
22.  Accelerated Development of Collapsing Glomerulopathy in Mice Congenic for the HIVAN1 locus on chr 3A1-A3 
Kidney international  2008;75(4):366-372.
HIV-1 transgenic mice on the FVB/NJ background (TgFVB) represent a well-validated model of HIV-associated nephropathy (HIVAN). A mapping study between TgFVB and CAST/EiJ (CAST) strains previously demonstrated that this trait is influenced by a major susceptibility locus on Chr. 3A1-A3 (called HIVAN1), with CAST alleles associated with increased risk of disease. We introgressed a 50 Mb interval, encompassing the HIVAN1 locus from CAST into the TgFVB genome (TgFVB-HIVAN1CAST congenic mice). Compared to the TgFVB strain, TgFVB-HIVAN1CAST mice develop earlier onset of proteinuria, rapid progression to kidney failure and increased mortality. Prospective analysis of TgFVB-HIVAN1CAST mice demonstrated significantly greater histologic and biochemical evidence of glomerulopathy with one-third of mice developing global glomerulosclerosis by 6 weeks of age. An F2 cross between TgFVB and FVB-HIVAN1CAST demonstrated significant linkage (lod= 3.7, empiric p=0.001) to a 10 cM interval within the HIVAN1 region between D3Mit167 and D3Mit67, resulting in a 60% reduction of the original interval. These data independently confirm that a gene on chr3A1-A3 increases susceptibility to HIVAN, resulting in early onset and rapid progression of kidney disease. These mice represent a novel model for studying the development and progression of collapsing glomerulopathy.
PMCID: PMC2753461  PMID: 19092797
23.  Susceptibility loci for murine HIV-associated nephropathy encode trans-regulators of podocyte gene expression 
The Journal of Clinical Investigation  2009;119(5):1178-1188.
Multiple studies have linked podocyte gene variants to diverse sporadic nephropathies, including HIV-1–associated nephropathy (HIVAN). We previously used linkage analysis to identify a major HIVAN susceptibility locus in mouse, HIVAN1. We performed expression quantitative trait locus (eQTL) analysis of podocyte genes in HIV-1 transgenic mice to gain further insight into genetic susceptibility to HIVAN. In 2 independent crosses, we found that transcript levels of the podocyte gene nephrosis 2 homolog (Nphs2), were heritable and controlled by an ancestral cis-eQTL that conferred a 3-fold variation in expression and produced reactive changes in other podocyte genes. In addition, Nphs2 expression was controlled by 2 trans-eQTLs that localized to the nephropathy susceptibility intervals HIVAN1 and HIVAN2. Transregulation of podocyte genes was observed in the absence of HIV-1 or glomerulosclerosis, indicating that nephropathy susceptibility alleles induce latent perturbations in the podocyte expression network. Presence of the HIV-1 transgene interfered with transregulation, demonstrating effects of gene-environment interactions on disease. These data demonstrate that transcript levels of Nphs2 and related podocyte-expressed genes are networked and suggest that the genetic lesions introduced by HIVAN susceptibility alleles perturb this regulatory pathway and transcriptional responses to HIV-1, increasing susceptibility to nephropathy.
PMCID: PMC2673856  PMID: 19381020
24.  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
25.  Adenosine metabolism and murine strain–specific IL-4–induced inflammation, emphysema, and fibrosis 
Journal of Clinical Investigation  2006;116(5):1274-1283.
To define the factors that control the tissue effects of IL-4, we compared the effects of Tg IL-4 in Balb/c and C57BL/6 mice. In the former, IL-4 caused modest eosinophilic inflammation and mild airway fibrosis and did not shorten survival. In C57BL/6 mice, IL-4 caused profound eosinophilic inflammation, airway fibrosis, emphysematous alveolar destruction, and premature death. These differences could not be accounted for by changes in Th2 or Th1 cytokines, receptor components, STAT6 activation, MMPs, or cathepsins. In contrast, in C57BL/6 mice, alveolar remodeling was associated with decreased levels of tissue inhibitors of metalloproteinase 2, -3, and -4 and α1-antitrypsin, and fibrosis was associated with increased levels of total and bioactive TGF-β1. Impressive differences in adenosine metabolism were also appreciated, with increased tissue adenosine levels and A1, A2B, and A3 adenosine receptor expression and decreased adenosine deaminase (ADA) activity in C57BL/6 animals. Treatment with ADA also reduced the inflammation, fibrosis, and emphysematous destruction and improved the survival of C57BL/6 Tg animals. These studies demonstrate that genetic influences control IL-4 effector pathways in the murine lung. They also demonstrate that IL-4 has different effects on adenosine metabolism in Balb/c and C57BL/6 mice and that these differences contribute to the different responses that IL-4 induces in these inbred animals.
PMCID: PMC1451205  PMID: 16670768

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