Ramakrishnan Rajagopalan

Exome sequencing reveals novel rare variants in the ryanodine receptor and calcium channel genes in malignant hyperthermia families.

Background: About half of malignant hyperthermia (MH) cases are associated with skeletal muscle ryanodine receptor 1 (RYR1) and calcium channel, voltage-dependent, L type, &agr;1S subunit (CACNA1S) gene mutations, leaving many with an unknown cause. The authors chose to apply a sequencing approach to uncover causal variants in unknown cases. Sequencing the exome, the protein-coding region of the genome, has power at low sample sizes and identified the cause of over a dozen Mendelian disorders. Methods: The authors considered four families with multiple MH cases lacking mutations in RYR1 and CACNA1S by Sanger sequencing of complementary DNA. Exome sequencing in two affecteds per family, chosen for maximum genetic distance, were compared. Variants were ranked by allele frequency, protein change, and measures of conservation among mammals to assess likelihood of causation. Finally, putative pathogenic mutations were genotyped in other family members to verify cosegregation with MH. Results: Exome sequencing revealed one rare RYR1 nonsynonymous variant in each of three families (Asp1056His, Val2627Met, Val4234Leu), and one CACNA1S variant (Thr1009Lys) in the fourth family. These were not seen in variant databases or in our control population sample of 5,379 exomes. Follow-up sequencing in other family members verified cosegregation of alleles with MH. Conclusions: The authors found that using both exome sequencing and allele frequency data from large sequencing efforts may aid genetic diagnosis of MH. In a sample selected by the authors, this technique was more sensitive for variant detection in known genes than Sanger sequencing of complementary DNA, and allows for the possibility of novel gene discovery.

Genetic Variation in LPAL2, LPA, and PLG Predicts Plasma Lipoprotein(a) Level and Carotid Artery Disease Risk

Background and Purpose— Lipoprotein(a) [Lp(a)] level is an established risk factor for coronary artery disease and has been implicated in carotid artery disease (CAAD). The relationship between genetic variation in the LPA gene region and CAAD risk remains unknown. Methods— We genotyped single nucleotide polymorphisms (SNPs) in the LPAL2, LPA, and PLG regions in 530 individuals with severe CAAD and 770 controls and kringle IV type 2 (KIV2) repeat length in a subset of 90 individuals. Results— Nine SNPs collectively accounted for 30% of the variance in Lp(a) level. Six SNPs were associated with Lp(a) level after accounting for KIV2 copy number, and the dominant KIV2 allele combined with these markers explained 60% of the variance in Lp(a) level. Five SNPs, including rs10455872, which had an odds ratio of 2.1 per minor allele and haplotypes formed by rs10455872, rs6919346, and rs3123629, were significant predictors of CAAD. After accounting for Lp(a) level, all evidence of CAAD-genotype association in the LPA region was eliminated. Conclusions— LPA region SNPs capture some but not all of the effect of KIV2 repeat length on Lp(a) level. There are associations between LPA region SNPs and CAAD that appear to be attributable to effects on Lp(a) level.

Analysis of recently identified dyslipidemia alleles reveals two loci that contribute to risk for carotid artery disease

BackgroundGenome-wide association studies have identified numerous single nucleotide polymorphisms (SNPs) affecting high density lipoprotein (HDL) or low density lipoprotein (LDL) cholesterol levels; these SNPs may contribute to the genetic basis of vascular diseases.ResultsWe assessed the impact of 34 SNPs at 23 loci on dyslipidemia, key lipid sub-phenotypes, and severe carotid artery disease (CAAD) in a case-control cohort. The effects of these SNPs on HDL and LDL were consistent with those previously reported, and we provide unbiased estimates of the percent variance in HDL (3.9%) and LDL (3.3%) explained by genetic risk scores. We assessed the effects of these SNPs on HDL subfractions, apolipoprotein A-1, LDL buoyancy, apolipoprotein B, and lipoprotein (a) and found that rs646776 predicts apolipoprotein B level while rs2075650 predicts LDL buoyancy. Finally, we tested the role of these SNPs in conferring risk for ultrasonographically documented CAAD stenosis status. We found that two loci, chromosome 1p13.3 near CELSR2 and PSRC1 which contains rs646776, and 19q13.2 near TOMM40 and APOE which contains rs2075650, harbor risk alleles for CAAD.ConclusionOur analysis of 34 SNPs contributing to dyslipidemia at 23 loci suggests that genetic variation in the 1p13.3 region may increase risk of CAAD by increasing LDL particle number, whereas variation in the 19q13.2 region may increase CAAD risk by promoting formation of smaller, denser LDL particles.

Genetic substructure of Kuwaiti population reveals migration history.

The State of Kuwait is characterized by settlers from Saudi Arabia, Iran, and other regions of the Arabian Peninsula. The settlements and subsequent admixtures have shaped the genetics of Kuwait. High prevalence of recessive disorders and metabolic syndromes (that increase risk of diabetes) is seen in the peninsula. Understanding the genetic structure of its population will aid studies designed to decipher the underlying causes of these disorders. In this study, we analyzed 572,366 SNP markers from 273 Kuwaiti natives genotyped using the illumina HumanOmniExpress BeadChip. Model-based clustering identified three genetic subgroups with different levels of admixture. A high level of concordance (Mantel test, p=0.0001 for 9999 repeats) was observed between the derived genetic clusters and the surname-based ancestries. Use of Human Genome Diversity Project (HGDP) data to understand admixtures in each group reveals the following: the first group (Kuwait P) is largely of West Asian ancestry, representing Persians with European admixture; the second group (Kuwait S) is predominantly of city-dwelling Saudi Arabian tribe ancestry, and the third group (Kuwait B) includes most of the tent-dwelling Bedouin surnames and is characterized by the presence of 17% African ancestry. Identity by Descent and Homozygosity analyses find Kuwait’s population to be heterogeneous (placed between populations that have large amount of ROH and the ones with low ROH) with Kuwait S as highly endogamous, and Kuwait B as diverse. Population differentiation FST estimates place Kuwait P near Asian populations, Kuwait S near Negev Bedouin tribes, and Kuwait B near the Mozabite population. FST distances between the groups are in the range of 0.005 to 0.008; distances of this magnitude are known to cause false positives in disease association studies. Results of analysis for genetic features such as linkage disequilibrium decay patterns conform to Kuwait’s geographical location at the nexus of Africa, Europe, and Asia.

Genetic and nongenetic sources of variation in phospholipid transfer protein activity.

Phospholipid transfer protein (PLTP) belongs to the lipid transfer/lipopolysaccharide-binding protein gene family. Expression of PLTP has been implicated in the development of atherosclerosis. We evaluated the effects of PLTP region tagging single nucleotide polymorphisms (SNPs) on the prediction of both carotid artery disease (CAAD) and PLTP activity. CAAD effects were evaluated in 442 Caucasian male subjects with severe CAAD and 497 vascular disease-free controls. SNP prediction of PLTP transfer activity was evaluated in both a subsample of 87 subjects enriched for an allele of interest and in a confirmation sample of 210 Caucasian males and females. Hemoglobin A1c or insulin level predicted 11–14% of age- and sex-adjusted PLTP activity. PLTP SNPs that predicted ∼11–30% of adjusted PLTP activity variance were identified in the two cohorts. For rs6065904, the allele that was associated with CAAD was also associated with elevated PLTP activity in both cohorts. SNPs associated with PLTP activity also predicted variation in LDL-cholesterol and LDL-B level only in the replication cohort. These results demonstrate that PLTP activity is strongly influenced by PLTP region polymorphisms and metabolic factors.

Deep Hypothermic circulatory arrest does not impair neurodevelopmental outcome in school-age children after infant cardiac surgery

BACKGROUND The purpose of this study was to assess deep hypothermic circulatory arrest (DHCA) as a modifier of neurodevelopmental (ND) outcomes in preschool children after cardiac surgery in infancy for repair of congenital heart defects (CHD). METHODS This is a planned analysis of infants enrolled in a prospective study of apolipoprotein E polymorphisms and ND outcome after cardiac surgery. The effect of DHCA was assessed in patients with single or biventricular CHD without aortic arch obstruction. Neurodevelopmental assessment at 4 years of age included cognition, language, attention, impulsivity, executive function, social competence, and visual-motor and fine-motor skills. Patient and procedural variables were evaluated in univariate and multivariate models. RESULTS Neurodevelopmental testing was completed in 238 of 307 eligible patients (78%). Deep hypothermic circulatory arrest was used at the discretion of the surgeon at least once in 92 infants (38.6%) with a median cumulative duration of 36 minutes (range, 1 to 132 minutes). By univariate analysis, DHCA patients were more likely to have single-ventricle CHD (p = 0.013), lower socioeconomic status (p < 0.001), a higher incidence of preoperative ventilation (p < 0.001), and were younger and smaller at the first surgery (p < 0.001). By multivariate analysis, use of DHCA was not predictive of worse performance for any ND outcome. CONCLUSIONS In this cohort of children undergoing repair of CHD in infancy, patients who underwent DHCA had risk factors associated with worse ND outcomes. Despite these, use of DHCA for repair of single-ventricle and biventricular CHD without aortic arch obstruction was not predictive of worse performance for any ND domain tested at 4 years of age.

Linkage and association of phospholipid transfer protein activity to LASS4

Phospholipid transfer protein activity (PLTPa) is associated with insulin levels and has been implicated in atherosclerotic disease in both mice and humans. Variation at the PLTP structural locus on chromosome 20 explains some, but not all, heritable variation in PLTPa. In order to detect quantitative trait loci (QTLs) elsewhere in the genome that affect PLTPa, we performed both oligogenic and single QTL linkage analysis on four large families (n = 227 with phenotype, n = 330 with genotype, n = 462 total), ascertained for familial combined hyperlipidemia. We detected evidence of linkage between PLTPa and chromosome 19p (lod = 3.2) for a single family and chromosome 2q (lod = 2.8) for all families. Inclusion of additional marker and exome sequence data in the analysis refined the linkage signal on chromosome 19 and implicated coding variation in LASS4, a gene regulated by leptin that is involved in ceramide synthesis. Association between PLTPa and LASS4 variation was replicated in the other three families (P = 0.02), adjusting for pedigree structure. To our knowledge, this is the first example for which exome data was used in families to identify a complex QTL that is not the structural locus.

Results of Genome-Wide Analyses on Neurodevelopmental Phenotypes at Four-Year Follow-Up following Cardiac Surgery in Infancy

Background Adverse neurodevelopmental sequelae are reported among children who undergo early cardiac surgery to repair congenital heart defects (CHD). APOE genotype has previously been determined to contribute to the prediction of these outcomes. Understanding further genetic causes for the development of poor neurobehavioral outcomes should enhance patient risk stratification and improve both prevention and treatment strategies. Methods We performed a prospective observational study of children who underwent cardiac surgery before six months of age; this included a neurodevelopmental evaluation between their fourth and fifth birthdays. Attention and behavioral skills were assessed through parental report utilizing the Attention Deficit-Hyperactivity Disorder-IV scale preschool edition (ADHD-IV), and Child Behavior Checklist (CBCL/1.5-5), respectively. Of the seven investigated, three neurodevelopmental phenotypes met genomic quality control criteria. Linear regression was performed to determine the effect of genome-wide genetic variation on these three neurodevelopmental measures in 316 subjects. Results This genome-wide association study identified single nucleotide polymorphisms (SNPs) associated with three neurobehavioral phenotypes in the postoperative children ADHD-IV Impulsivity/Hyperactivity, CBCL/1.5-5 PDPs, and CBCL/1.5-5 Total Problems. The most predictive SNPs for each phenotype were: a LGALS8 intronic SNP, rs4659682, associated with ADHD-IV Impulsivity (P = 1.03×10−6); a PCSK5 intronic SNP, rs2261722, associated with CBCL/1.5-5 PDPs (P = 1.11×10−6); and an intergenic SNP, rs11617488, 50 kb from FGF9, associated with CBCL/1.5-5 Total Problems (P = 3.47×10−7). 10 SNPs (3 for ADHD-IV Impulsivity, 5 for CBCL/1.5-5 PDPs, and 2 for CBCL/1.5-5 Total Problems) had p<10−5. Conclusions No SNPs met genome-wide significance for our three neurobehavioral phenotypes; however, 10 SNPs reached a threshold for suggestive significance (p<10−5). Given the unique nature of this cohort, larger studies and/or replication are not possible. Studies to further investigate the mechanisms through which these newly identified genes may influence neurodevelopment dysfunction are warranted.