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1.  Association of Common Variants in ERBB4 with Congenital Left Ventricular Outflow Tract Obstruction Defects 
The left ventricular outflow tract (LVOT) defects aortic valve stenosis (AVS), coarctation of the aorta (COA), and hypoplastic left heart syndrome (HLHS) represent an embryologically related group of congenital cardiovascular malformations. They are common and cause substantial morbidity and mortality. Prior evidence suggests a strong genetic component in their causation.
We selected NRG1, ERBB3, and ERBB4 of the epidermal growth factor receptor (EGFR) signaling pathway as candidate genes for investigation of association with LVOT defects based on the importance of this pathway in cardiac development and the phenotypes in knockout mouse models. Single nucleotide polymorphism (SNP) genotyping was performed on 343 affected case-parent trios of European ancestry.
We identified a specific haplotype in intron 3 of ERBB4 that was positively associated with the combined LVOT defects phenotype (p = 0.0005) and in each anatomic defect AVS, COA, and HLHS separately. Mutation screening of individuals with an LVOT defect failed to identify a coding sequence or splice site change in ERBB4. RT-PCR on lymphoblastoid cells from LVOT subjects did not show altered splice variant ratios among those homozygous for the associated haplotype.
These results suggest ERBB4 is associated with LVOT defects. Further replication will be required in separate cohorts to confirm the consistency of the observed association.
PMCID: PMC3736588  PMID: 21290564
genetics of cardiovascular disease; heart defects; congenital; congenital abnormalities; cardiovascular abnormalities; analysis; genetic association
2.  Neurodevelopmental disorders among individuals with duplication of 4p13 to 4p12 containing a GABAA receptor subunit gene cluster 
Recent studies have shown that certain copy number variations (CNV) are associated with a wide range of neurodevelopmental disorders, including autism spectrum disorders (ASD), bipolar disorder and intellectual disabilities. Implicated regions and genes have comprised a variety of post synaptic complex proteins and neurotransmitter receptors, including gamma-amino butyric acid A (GABAA). Clusters of GABAA receptor subunit genes are found on chromosomes 4p12, 5q34, 6q15 and 15q11-13. Maternally inherited 15q11-13 duplications among individuals with neurodevelopmental disorders are well described, but few case reports exist for the other regions. We describe a family with a 2.42 Mb duplication at chromosome 4p13 to 4p12, identified in the index case and other family members by oligonucleotide array comparative genomic hybridization, that contains 13 genes including a cluster of four GABAA receptor subunit genes. Fluorescent in-situ hybridization was used to confirm the duplication. The duplication segregates with a variety of neurodevelopmental disorders in this family, including ASD (index case), developmental delay, dyspraxia and ADHD (brother), global developmental delays (brother), learning disabilities (mother) and bipolar disorder (maternal grandmother). In addition, we identified and describe another individual unrelated to this family, with a similar duplication, who was diagnosed with ASD, ADHD and borderline intellectual disability. The 4p13 to 4p12 duplication appears to confer a susceptibility to a variety of neurodevelopmental disorders in these two families. We hypothesize that the duplication acts through a dosage effect of GABAA receptor subunit genes, adding evidence for alterations in the GABAergic system in the etiology of neurodevelopmental disorders.
PMCID: PMC3865421  PMID: 23695283
autism; bipolar disorder; chromosome disorders; DNA copy number variation; intellectual disabilities; GABAA
3.  Impact of Mendelian Inheritance in Cardiovascular Disease 
Cardiovascular disease is a leading cause of mortality worldwide. While the etiology for the majority of cardiovascular disease is presumed to be a combination of genetic and environmental factors, developments in our understanding of the basic biology of cardiac disorders have been greatly advanced through discoveries made studying heart diseases that exhibit Mendelian forms of inheritance. Most of these diseases primarily affect children and young adults and include cardiomyopathies, arrhythmias, aortic aneurysms and congenital heart defects. The discovery of the genetic etiologies for these diseases have had significant impact on our understanding of more complex forms of cardiovascular disease and in some cases led to novel diagnostic and treatment modalities. In this review, we will summarize these seminal genetic discoveries, highlighting a few that have resulted in significant impact on human disease, and discuss the potential utility of studying Mendelian-inherited heart disease with the development of new genetic technologies and our increased understanding of the human genome.
PMCID: PMC3489013  PMID: 20958326
4.  MCTP2 is a dosage-sensitive gene required for cardiac outflow tract development 
Human Molecular Genetics  2013;22(21):4339-4348.
Coarctation of the aorta (CoA) and hypoplastic left heart syndrome (HLHS) have been reported in rare individuals with large terminal deletions of chromosome 15q26. However, no single gene important for left ventricular outflow tract (LVOT) development has been identified in this region. Using array-comparative genomic hybridization, we identified two half-siblings with CoA with a 2.2 Mb deletion on 15q26.2, inherited from their mother, who was mosaic for this deletion. This interval contains an evolutionary conserved, protein-coding gene, MCTP2 (multiple C2-domains with two transmembrane regions 2). Using gene-specific array screening in 146 individuals with non-syndromic LVOT obstructive defects, another individual with HLHS and CoA was found to have a de novo 41 kb intragenic duplication within MCTP2, predicted to result in premature truncation, p.F697X. Alteration of Mctp2 gene expression in Xenopus laevis embryos by morpholino knockdown and mRNA overexpression resulted in the failure of proper OT development, confirming the functional importance of this dosage-sensitive gene for cardiogenesis. Our results identify MCTP2 as a novel genetic cause of CoA and related cardiac malformations.
PMCID: PMC3792692  PMID: 23773997
5.  Use of a targeted, combinatorial next-generation sequencing approach for the study of bicuspid aortic valve 
BMC Medical Genomics  2014;7:56.
Bicuspid aortic valve (BAV) is the most common type of congenital heart disease with a population prevalence of 1-2%. While BAV is known to be highly heritable, mutations in single genes (such as GATA5 and NOTCH1) have been reported in few human BAV cases. Traditional gene sequencing methods are time and labor intensive, while next-generation high throughput sequencing remains costly for large patient cohorts and requires extensive bioinformatics processing. Here we describe an approach to targeted multi-gene sequencing with combinatorial pooling of samples from BAV patients.
We studied a previously described cohort of 78 unrelated subjects with echocardiogram-identified BAV. Subjects were identified as having isolated BAV or BAV associated with coarctation of aorta (BAV-CoA). BAV cusp fusion morphology was defined as right-left cusp fusion, right non-coronary cusp fusion, or left non-coronary cusp fusion. Samples were combined into 19 pools using a uniquely overlapping combinatorial design; a given mutation could be attributed to a single individual on the basis of which pools contained the mutation. A custom gene capture of 97 candidate genes was sequenced on the Illumina HiSeq 2000. Multistep bioinformatics processing was performed for base calling, variant identification, and in-silico analysis of putative disease-causing variants.
Targeted capture identified 42 rare, non-synonymous, exonic variants involving 35 of the 97 candidate genes. Among these variants, in-silico analysis classified 33 of these variants as putative disease-causing changes. Sanger sequencing confirmed thirty-one of these variants, found among 16 individuals. There were no significant differences in variant burden among BAV fusion phenotypes or isolated BAV versus BAV-CoA. Pathway analysis suggests a role for the WNT signaling pathway in human BAV.
We successfully developed a pooling and targeted capture strategy that enabled rapid and cost effective next generation sequencing of target genes in a large patient cohort. This approach identified a large number of putative disease-causing variants in a cohort of patients with BAV, including variants in 26 genes not previously associated with human BAV. The data suggest that BAV heritability is complex and polygenic. Our pooling approach saved over $39,350 compared to an unpooled, targeted capture sequencing strategy.
PMCID: PMC4181662  PMID: 25260786
Bicuspid aortic valve; Genetics; Next-generation sequencing; Targeted capture; Combinatorial pooling
6.  Linkage analysis of left ventricular outflow tract malformations (aortic valve stenosis, coarctation of the aorta, hypoplastic left heart syndrome) 
The left ventricular outflow tract (LVOT) malformations aortic valve stenosis (AVS), coarctation of the aorta (CoA), and hypoplastic left heart syndrome (HLHS) are a significant cause of infant mortality. These three malformations are thought to share developmental pathogenetic mechanisms. A strong genetic component has been previously demonstrated, but the underlying genetic etiologies are unknown. Our objective was to identify genetic susceptibility loci for the broad phenotype of LVOT malformations. We genotyped 411 microsatellites spaced at an average of 10cM in 43 families constituting 289 individuals, with additional 5 cM spaced markers for fine mapping. A non-parametric linkage (NPL) analysis of the combined LVOT malformations gave three suggestive linkage peaks on chromosomes 16p12 (NPL scores [NPLS] of 2.52), 2p23 (NPLS= 2.41) and 10q21 (NPLS=2.14). Individually, suggestive peaks for AVS families occurred on chromosomes 16p12 (NPLS=2.64), 7q36 (NPLS=2.31) and 2p25 (NPLS=2.14); and for CoA families on chromosome 1q24 (NPLS=2.61), 6p23 (NPLS=2.29), 7p14 (NPLS=2.27), 10q11 (NPLS=1.98), and 2p15 (NPLS=2.02). Significant NPL scores in HLHS families were noted for chromosome 2p15 (NPLS=3.23), with additional suggestive peaks on 19q13 (NPLS=2.16) and 10q21 (NPLS=2.07). Overlapping linkage signals on 10q11 (AVS and CoA) and 16p12 (AVS, CoA, HLHS) led to higher NPL scores when all malformations were analyzed together. In conclusion, we report suggestive evidence for linkage to chromosomes 2p23, 10q21, and 16p12 for the LVOT malformations of AVS, CoA, and HLHS individually and in a combined analysis, with a significant peak on 2p15 for HLHS. Overlapping linkage peaks provides evidence for a common genetic etiology.
PMCID: PMC2916734  PMID: 19142209
Heart Defects; Congenital; Embryonic and Fetal Development; Hereditary disease; pediatrics; genetics
7.  An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge 
Brownstein, Catherine A | Beggs, Alan H | Homer, Nils | Merriman, Barry | Yu, Timothy W | Flannery, Katherine C | DeChene, Elizabeth T | Towne, Meghan C | Savage, Sarah K | Price, Emily N | Holm, Ingrid A | Luquette, Lovelace J | Lyon, Elaine | Majzoub, Joseph | Neupert, Peter | McCallie Jr, David | Szolovits, Peter | Willard, Huntington F | Mendelsohn, Nancy J | Temme, Renee | Finkel, Richard S | Yum, Sabrina W | Medne, Livija | Sunyaev, Shamil R | Adzhubey, Ivan | Cassa, Christopher A | de Bakker, Paul IW | Duzkale, Hatice | Dworzyński, Piotr | Fairbrother, William | Francioli, Laurent | Funke, Birgit H | Giovanni, Monica A | Handsaker, Robert E | Lage, Kasper | Lebo, Matthew S | Lek, Monkol | Leshchiner, Ignaty | MacArthur, Daniel G | McLaughlin, Heather M | Murray, Michael F | Pers, Tune H | Polak, Paz P | Raychaudhuri, Soumya | Rehm, Heidi L | Soemedi, Rachel | Stitziel, Nathan O | Vestecka, Sara | Supper, Jochen | Gugenmus, Claudia | Klocke, Bernward | Hahn, Alexander | Schubach, Max | Menzel, Mortiz | Biskup, Saskia | Freisinger, Peter | Deng, Mario | Braun, Martin | Perner, Sven | Smith, Richard JH | Andorf, Janeen L | Huang, Jian | Ryckman, Kelli | Sheffield, Val C | Stone, Edwin M | Bair, Thomas | Black-Ziegelbein, E Ann | Braun, Terry A | Darbro, Benjamin | DeLuca, Adam P | Kolbe, Diana L | Scheetz, Todd E | Shearer, Aiden E | Sompallae, Rama | Wang, Kai | Bassuk, Alexander G | Edens, Erik | Mathews, Katherine | Moore, Steven A | Shchelochkov, Oleg A | Trapane, Pamela | Bossler, Aaron | Campbell, Colleen A | Heusel, Jonathan W | Kwitek, Anne | Maga, Tara | Panzer, Karin | Wassink, Thomas | Van Daele, Douglas | Azaiez, Hela | Booth, Kevin | Meyer, Nic | Segal, Michael M | Williams, Marc S | Tromp, Gerard | White, Peter | Corsmeier, Donald | Fitzgerald-Butt, Sara | Herman, Gail | Lamb-Thrush, Devon | McBride, Kim L | Newsom, David | Pierson, Christopher R | Rakowsky, Alexander T | Maver, Aleš | Lovrečić, Luca | Palandačić, Anja | Peterlin, Borut | Torkamani, Ali | Wedell, Anna | Huss, Mikael | Alexeyenko, Andrey | Lindvall, Jessica M | Magnusson, Måns | Nilsson, Daniel | Stranneheim, Henrik | Taylan, Fulya | Gilissen, Christian | Hoischen, Alexander | van Bon, Bregje | Yntema, Helger | Nelen, Marcel | Zhang, Weidong | Sager, Jason | Zhang, Lu | Blair, Kathryn | Kural, Deniz | Cariaso, Michael | Lennon, Greg G | Javed, Asif | Agrawal, Saloni | Ng, Pauline C | Sandhu, Komal S | Krishna, Shuba | Veeramachaneni, Vamsi | Isakov, Ofer | Halperin, Eran | Friedman, Eitan | Shomron, Noam | Glusman, Gustavo | Roach, Jared C | Caballero, Juan | Cox, Hannah C | Mauldin, Denise | Ament, Seth A | Rowen, Lee | Richards, Daniel R | Lucas, F Anthony San | Gonzalez-Garay, Manuel L | Caskey, C Thomas | Bai, Yu | Huang, Ying | Fang, Fang | Zhang, Yan | Wang, Zhengyuan | Barrera, Jorge | Garcia-Lobo, Juan M | González-Lamuño, Domingo | Llorca, Javier | Rodriguez, Maria C | Varela, Ignacio | Reese, Martin G | De La Vega, Francisco M | Kiruluta, Edward | Cargill, Michele | Hart, Reece K | Sorenson, Jon M | Lyon, Gholson J | Stevenson, David A | Bray, Bruce E | Moore, Barry M | Eilbeck, Karen | Yandell, Mark | Zhao, Hongyu | Hou, Lin | Chen, Xiaowei | Yan, Xiting | Chen, Mengjie | Li, Cong | Yang, Can | Gunel, Murat | Li, Peining | Kong, Yong | Alexander, Austin C | Albertyn, Zayed I | Boycott, Kym M | Bulman, Dennis E | Gordon, Paul MK | Innes, A Micheil | Knoppers, Bartha M | Majewski, Jacek | Marshall, Christian R | Parboosingh, Jillian S | Sawyer, Sarah L | Samuels, Mark E | Schwartzentruber, Jeremy | Kohane, Isaac S | Margulies, David M
Genome Biology  2014;15(3):R53.
There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance.
A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization.
The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups.
PMCID: PMC4073084  PMID: 24667040
8.  Epidemiology of Noncomplex Left Ventricular Outflow Tract Obstruction Malformations (Aortic Valve Stenosis, Coarctation of the Aorta, Hypoplastic Left Heart Syndrome) in Texas, 1999 –2001 
The left ventricular outflow tract (LVOT) malformations aortic valve stenosis (AVS), coarctation of the aorta (CoA), and hypoplastic left heart syndrome (HLHS) contribute significantly to infant mortality due to birth defects. Previous epidemiology data showed rate differences between male and female and white and black ethnic groups. The Texas Birth Defects Registry, an active surveillance program, enables study in a large, diverse population including Hispanics.
Records of children up to 1 year old with AVS, CoA, and HLHS born in Texas from 1999 to 2001, were collected from the registry. Those including additional heart defects or a chromosomal anomaly were excluded. Multivariate analysis included: infant sex; United States–Mexico border county residence; and maternal age, race/ethnicity, birthplace, and education.
There were 910 cases among 1.08 million live births, of which 499 met inclusion criteria. Multivariate modeling of all LVOT malformations combined demonstrated lower prevalence rate ratios (PRRs) for black males (0.26) and Hispanic males (0.70). Similar results were found for CoA but not AVS or HLHS. Higher PRRs were noted for increased maternal age for LVOT (1.3 for 24–34 years; 1.7 for >34 years), AVS, and HLHS, but not CoA, and higher PRRs across all diagnoses for males (LVOT PRR, 2.4) were noted. CoA PRRs were higher in border county vs. non–border county residents (PRR, 2.1). Maternal education and birthplace were not significant factors.
There are rate differences for males among all 3 ethnic groups. Sex and ethnic differences suggest genetic etiologies, where the ethnic differences could be used to find susceptibility loci with mapping by admixture linkage disequilibrium. Increased CoA rates along the U.S.–Mexico border suggest environmental causes that will require further monitoring.
PMCID: PMC1361303  PMID: 16007587
congenital heart disease; Hispanic; Poisson distribution; regression analysis; prevalence; south-western United States; genetics
9.  Inheritance Analysis of Congenital Left Ventricular Outflow Tract Obstruction Malformations: Segregation, Multiplex Relative Risk, and Heritability 
The left ventricular outflow tract (LVOTO) malformations, aortic valve stenosis (AVS), coarctation of the aorta (COA), and hypoplastic left heart (HLH) constitute a mechanistically defined subgroup of congenital heart defects that have substantial evidence for a genetic component. Evidence from echocardiography studies has shown that bicuspid aortic valve (BAV) is found frequently in relatives of children with LVOTO defects. However, formal inheritance analysis has not been performed. We ascertained 124 families by an index case with AVS, COA, or HLH. A total of 413 relatives were enrolled in the study, of which 351 had detailed echocardiography exams for structural heart defects and measurements of a variety of aortic arch, left ventricle, and valve structures. LVOTO malformations were noted in 30 relatives (18 BAV, 5 HLH, 3 COA, and 3 AVS), along with significant congenital heart defects (CHD) in 2 others (32/413; 7.7%). Relative risk for first-degree relatives in this group was 36.9, with a heritability of 0.71–0.90. Formal segregation analysis suggests that one or more minor loci with rare dominant alleles may be operative in a subset of families. Multiplex relative risk analysis, which estimates number of loci, had the highest maximum likelihood score in a model with 2 loci (range of 1–6 in the lod-1 support interval). Heritability of several aortic arch measurements and aortic valve was significant. These data support a complex but most likely oligogenic pattern of inheritance. A combination of linkage and association study designs is likely to enable LVOTO risk gene identification. This data can also provide families with important information for screening asymptomatic relatives for potentially harmful cardiac defects.
PMCID: PMC1361302  PMID: 15690347
congenital heart defects; hypoplastic left heart syndrome; bicuspid aortic valve; cardiac development; echocardiography; genetics
10.  Successful Medical Therapy for Hypophosphatemic Rickets due to Mitochondrial Complex I Deficiency Induced de Toni-Debré-Fanconi Syndrome 
Case Reports in Pediatrics  2013;2013:354314.
Primary de Toni-Debré-Fanconi syndrome is a non-FGF23-mediated hypophosphatemic disorder due to a primary defect in renal proximal tubule cell function resulting in hyperphosphaturia, renal tubular acidosis, glycosuria, and generalized aminoaciduria. The orthopaedic sequela and response to treatment of this rare disorder are limited in the literature. Herein we report a long term followup of a 10-year-old female presenting at 1 year of age with rickets initially misdiagnosed as vitamin D deficiency rickets. She was referred to the metabolic bone and genetics clinics at 5 years of age with severe genu valgum deformities of 24 degrees and worsening rickets. She had polyuria, polydipsia, enuresis, and bone pain. Diagnosis of hypophosphatemic rickets due to de Toni-Debré-Fanconi syndrome was subsequently made. Respiratory chain enzyme analysis identified a complex I mitochondrial deficiency as the underlying cause. She was treated with phosphate (50–70 mg/kg/day), calcitriol (30 ng/kg/day), and sodium citrate with resolution of bone pain and normal growth. By 10 years of age, her genu valgus deformities were 4 degrees with healing of rickets. Her excellent orthopaedic outcome despite late proper medical therapy is likely due to the intrinsic renal tubular defect that is more responsive to combined alkali, phosphate, and calcitriol therapy.
PMCID: PMC3872385  PMID: 24386581
11.  Cardiac Teratogenicity in Mouse Maternal Phenylketonuria: Defining phenotype parameters and genetic background influences 
Molecular genetics and metabolism  2012;107(4):650-658.
Maternal phenylketonuria (MPKU) is a syndrome including cardiovascular malformations (CVMs), microcephaly, intellectual impairment, and small for gestational age, caused by in-utero exposure to elevated serum phenylalanine (Phe) due to PKU in the mother. It is becoming a public health concern as more women with PKU reach child bearing age. Although a mouse model of PKU, BTBR Pahenu2, has been available for 20 years, it has not been well utilized for studying MPKU. We used this model to delineate critical parameters in Phe cardiovascular teratogenicity and study the effect of genetic background. Dosing and timing experiments were performed with the BTBR Pahenu2 mouse. A dose response curve was noted, with CVM rates at maternal serum Phe levels <360 μM (control), 360 – 600 μM (low), 600 – 900 μM (mid), and >900μM (high) of 11.86%, 16.67%, 30.86%, and 46.67% respectively. A variety of CVMs were noted on the BTBR background, including double outlet right ventricle (DORV), aortic arch artery (AAA)abnormalities, and ventricular septal defects (VSDs). Timed exposure experiments identified a teratogenic window from embryonic day 8.5-13.5, with higher rates of conotruncal and valve defects occurring in early exposure time and persistent truncus arteriosus (PTA) and aortic arch branching abnormalities occurring with late exposure. Compared to the BTBR strain, N10+ Pahenu2 congenics on the C3H/HeJ background had higher rates of CVMs in general and propensity to left ventricular outflow tract (LVOT) malformations, while the C57B/L6 background had similar CVM rates but predominately AAA abnormalities. We have delineated key parameters of Phe cardiovascular teratogenicity, demonstrated the utility of this MPKU model on different mouse strains, and shown how genetic background profoundly affects the phenotype.
PMCID: PMC3504168  PMID: 22951387
12.  Novel X-linked glomerulopathy associated with a COL4A5 missense mutation in a noncollagenous interruption 
Kidney international  2010;79(1):120-127.
We report a novel COL4A5 mutation causing rapid progression to end stage renal disease in males despite the absence of clinical and biopsy findings associated with Alport syndrome. Affected males had proteinuria, variable hematuria, early progression to end stage renal disease; and renal biopsy findings which included global and segmental glomerulosclerosis, mesangial hypercellularity and basement membrane immune complex deposition.
Exon sequencing of the COL4A5 locus identified a thymine to guanine transversion at nucleotide 665, resulting in a phenylalanine to cysteine missense mutation at codon 222. This mutation was confirmed in 4 affected males and 4 female obligate carriers, but was absent in 6 asymptomatic male family members and 198 unrelated individuals. α5(IV) collagen staining in renal biopsies from affected males was normal.
The phenylalanine at position 222 is 100% conserved among vertebrates. This is the first description of a mutation in a non-collagenous interruption associated with severe renal disease, providing evidence for the importance of this structural motif. The range of phenotypes associated with COL4A5 mutations is more diverse than previously realized. COL4A5 mutation analysis should be considered when glomerulonephritis presents in an X-linked inheritance pattern, even with a distinct presentation from Alport syndrome.
PMCID: PMC3248803  PMID: 20881942
13.  NOTCH1 missense alleles associated with left ventricular outflow tract defects exhibit impaired receptor processing and defective EMT 
Biochimica et biophysica acta  2010;1812(1):121-129.
Notch signaling is essential for proper cardiac development. We recently identified missense variants in the NOTCH1 receptor in patients with diverse left ventricular outflow tract (LVOT) malformations (NOTCH1G661S and NOTCH1A683T) that reduce ligand-induced Notch signaling. Here, we examine the molecular mechanisms that contribute to reduced signaling and perturbed development. We find that NOTCH1A683T exhibits reduced S1 cleavage due to impaired trafficking through the endoplasmic reticulum (ER). This observation is consistent with improper localization of the variant receptor to the ER and decreased presentation at the cell surface. In contrast, the nearby mutation NOTCH1G661S exhibits reduced cell-surface presentation in the absence of overt folding or trafficking defects. To examine the implications of these variants in disease pathogenesis, we investigated their effect on epithelial-to-mesenchymal transition (EMT), a critical process for development of the outflow tract. We find that these LVOT-associated NOTCH1 alleles can contribute to defective EMT in endothelial cell lines through impaired induction of Snail and Hes family members. These data represent the first description of a molecular mechanism underlying NOTCH1 mutations in individuals with LVOT malformations, and have important implications regarding the functional contribution of these alleles to a complex set of developmental defects.
PMCID: PMC3180902  PMID: 20951801
Notch signaling; Left ventricular outflow tract; Bicuspid aortic valve; Epithelial to mesenchymal transition (EMT)
14.  Significant contributions of the extraembryonic membranes and maternal genotype to the placental pathology in heterozygous Nsdhl deficient female embryos 
Human Molecular Genetics  2009;19(2):364-373.
Mutations in the gene encoding the cholesterol biosynthetic enzyme NSDHL are associated with the X-linked male-lethal bare patches (Bpa) mouse. Mutant male embryos for several Nsdhl alleles die in midgestation with placental insufficiency. We examined here a possible role of the maternal genotype in such placental pathology. Pre-pregnancy plasma cholesterol levels were similar between wild-type (WT) and Bpa1H/+ dams fed a standard, cholesterol-free diet. However, there was a marked decrease in cholesterol levels between embryonic day (E)8.5 and E10.5 for both genotypes. Further, there was a significant lag between E11.5 and E13.5 (P = 0.0011) in the recovery of levels in Bpa1H/+ dams to their pre-pregnancy values. To investigate possible effects of the maternal genotype on fetal placentation, we generated transgenic mice that expressed human NSDHL and rescued the male lethality of the Bpa1H null allele. We then compared placenta area at E10.5 in WT and Bpa1H/+ female embryos where the mutant X chromosome was transmitted from a heterozygous mother or a rescued mutant father. In mutant conceptuses, placental areas were ∼50% less than WT. Surprisingly, expression of Nsdhl in trophoblast lineages of the placenta and yolk sac endoderm, which occurs only from the maternally inherited allele in a female embryo, had the largest effect on placental area (−0.681 mm2; P < 0.0001). The maternal genotype had a smaller effect, independent of the fetal genotype (−0.283 mm2; P = 0.024). These data demonstrate significant effects of the mother and fetal membranes on pregnancy outcome, with possible implications for cholesterol homeostasis during human pregnancy.
PMCID: PMC2796896  PMID: 19880419
15.  NOTCH1 mutations in individuals with left ventricular outflow tract malformations reduce ligand-induced signaling 
Human Molecular Genetics  2008;17(18):2886-2893.
Congenital aortic valve stenosis (AVS), coarctation of the aorta (COA) and hypoplastic left heart syndrome (HLHS) are congenital cardiovascular malformations that all involve the left ventricular outflow tract (LVOT). They are presumably caused by a similar developmental mechanism involving the developing endothelium. The exact etiology for most LVOT malformations is unknown, but a strong genetic component has been established. We demonstrate here that mutations in the gene NOTCH1, coding for a receptor in a developmentally important signaling pathway, are found across the spectrum of LVOT defects. We identify two specific mutations that reduce ligand (JAGGED1) induced NOTCH1 signaling. One of these mutations perturbs the S1 cleavage of the receptor in the Golgi. These findings suggest that the levels of NOTCH1 signaling are tightly regulated during cardiovascular development, and that relatively minor alterations may promote LVOT defects. These results also establish for the first time that AVS, COA and HLHS can share a common pathogenetic mechanism at the molecular level, explaining observations of these defects co-occurring within families.
PMCID: PMC2722892  PMID: 18593716
16.  Linkage analysis of left ventricular outflow tract malformations (aortic valve stenosis, coarctation of the aorta, and hypoplastic left heart syndrome) 
The left ventricular outflow tract (LVOT) malformations aortic valve stenosis (AVS), coarctation of the aorta (CoA), and hypoplastic left heart syndrome (HLHS) are significant causes of infant mortality. These three malformations are thought to share developmental pathogenetic mechanisms. A strong genetic component has been demonstrated earlier, but the underlying genetic etiologies are unknown. Our objective was to identify genetic susceptibility loci for the broad phenotype of LVOT malformations. We genotyped 411 microsatellites spaced at an average of 10 cM in 43 families constituting 289 individuals, with an additional 5 cM spaced markers for fine mapping. A non-parametric linkage (NPL) analysis of the combined LVOT malformations gave three suggestive linkage peaks on chromosomes 16p12 (NPL score (NPLS)=2.52), 2p23 (NPLS=2.41), and 10q21 (NPLS=2.14). Individually, suggestive peaks for AVS families occurred on chromosomes 16p12 (NPLS=2.64), 7q36 (NPLS=2.31), and 2p25 (NPLS=2.14); and for CoA families on chromosome 1q24 (NPLS=2.61), 6p23 (NPLS=2.29), 7p14 (NPLS=2.27), 10q11 (NPLS=1.98), and 2p15 (NPLS=2.02). Significant NPLS in HLHS families were noted for chromosome 2p15 (NPLS=3.23), with additional suggestive peaks on 19q13 (NPLS=2.16) and 10q21 (NPLS=2.07). Overlapping linkage signals on 10q11 (AVS and CoA) and 16p12 (AVS, CoA, and HLHS) led to higher NPL scores when all malformations were analyzed together. In conclusion, we report suggestive evidence for linkage to chromosomes 2p23, 10q21, and 16p12 for the LVOT malformations of AVS, CoA, and HLHS individually and in a combined analysis, with a significant peak on 2p15 for HLHS. Overlapping linkage peaks provide evidence for a common genetic etiology.
PMCID: PMC2916734  PMID: 19142209
heart defects; congenital; embryonic and fetal development; hereditary disease; pediatrics
17.  Echocardiographic Evaluation of Asymptomatic Parental and Sibling Cardiovascular Anomalies Associated With Congenital Left Ventricular Outflow Tract Lesions 
Pediatrics  2004;114(3):691-696.
Left ventricular outflow tract obstructive (LVOTO) malformations are a leading cause of infant mortality from birth defects. Genetic mechanisms are likely, and there may be a higher rate of asymptomatic LVOTO anomalies in relatives of affected children. This study sought to define the incidence of cardiac anomalies in first-degree relatives of children with congenital aortic valve stenosis (AVS), coarctation of the aorta (CoA), and hypoplastic left heart syndrome (HLHS).
A total of 113 probands with a nonsyndromic LVOTO malformation of AVS (n = 25), BAV (n = 3), CoA (n = 52), HLHS (n = 30), and aortic hypoplasia with mitral valve atresia (n = 2) were ascertained through chart review or enrolled at the time of diagnosis. Echocardiography was performed on 282 asymptomatic first-degree relatives.
Four studies had poor acoustic windows, leaving 278 studies for analysis. BAV were found in 13 (4.68%) first-degree relatives. The relative risk of BAV in the relatives was 5.05 (95% confidence interval: 2.2–11.7), and the broad sense heritability was 0.49, based on a general population frequency of 0.9%. BAV was more common in multiplex families compared with sporadic cases. An additional 32 relatives had anomalies of the aorta, aortic valve, left ventricle, or mitral valve.
The presence of an LVOTO lesion greatly increases the risk of identifying BAV in a parent or sibling, providing additional support for a complex genetic cause. The parents and siblings of affected patients should be screened by echocardiography as the presence of an asymptomatic BAV may carry a significant long-term health risk.
PMCID: PMC1361301  PMID: 15342840
congenital heart disease; genetics; recurrence risk; aortic valve; coarctation; hypoplastic left heart syndrome; CCVM, congenital cardiovascular malformation; LVOTO, left ventricular outflow tract obstruction; HLHS, hypoplastic left heart syndrome; AVS, aortic valve stenosis; CoA, coarctation of the aorta; BAV, bicuspid aortic valve; CI, confidence interval

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