Daniel J. Dykas

A common variant in the patatin-like phospholipase 3 gene (PNPLA3) is associated with fatty liver disease in obese children and adolescents†‡

The genetic factors associated with susceptibility to nonalcoholic fatty liver disease (NAFLD) in pediatric obesity remain largely unknown. Recently, a nonsynonymous single‐nucleotide polymorphism (rs738409), in the patatin‐like phospholipase 3 gene (PNPLA3) has been associated with hepatic steatosis in adults. In a multiethnic group of 85 obese youths, we genotyped the PNLPA3 single‐nucleotide polymorphism, measured hepatic fat content by magnetic resonance imaging and insulin sensitivity by the insulin clamp. Because PNPLA3 might affect adipogenesis/lipogenesis, we explored the putative association with the distribution of adipose cell size and the expression of some adipogenic/lipogenic genes in a subset of subjects who underwent a subcutaneous fat biopsy. Steatosis was present in 41% of Caucasians, 23% of African Americans, and 66% of Hispanics. The frequency of PNPLA3(rs738409) G allele was 0.324 in Caucasians, 0.183 in African Americans, and 0.483 in Hispanics. The prevalence of the G allele was higher in subjects showing hepatic steatosis. Surprisingly, subjects carrying the G allele showed comparable hepatic glucose production rates, peripheral glucose disposal rate, and glycerol turnover as the CC homozygotes. Carriers of the G allele showed smaller adipocytes than those with CC genotype (P = 0.005). Although the expression of PNPLA3, PNPLA2, PPARγ2(peroxisome proliferator‐activated receptor gamma 2), SREBP1c(sterol regulatory element binding protein 1c), and ACACA(acetyl coenzyme A carboxylase) was not different between genotypes, carriers of the G allele showed lower leptin (LEP)(P = 0.03) and sirtuin 1 (SIRT1) expression (P = 0.04). Conclusion: A common variant of the PNPLA3 gene confers susceptibility to hepatic steatosis in obese youths without increasing the level of hepatic and peripheral insulin resistance. The rs738409 PNPLA3 G allele is associated with morphological changes in adipocyte cell size. (HEPATOLOGY 2010.)

Variant in the glucokinase regulatory protein (GCKR) gene is associated with fatty liver in obese children and adolescents

Recently, the single nucleotide polymorphism (SNP) identified as rs1260326, in the glucokinase regulatory protein (GCKR), was associated with hypertriglyceridemia in adults. Because accumulation of triglycerides in hepatocytes represents the hallmark of steatosis, we aimed to investigate whether this variant might be associated with fatty liver (hepatic fat content, HFF%). Moreover, because recently rs738409 in the PNPLA3 and rs2854116 in the APOC3 were associated with fatty liver, we explored how the GCKR SNP and these two variants jointly influence hepatosteatosis. We studied 455 obese children and adolescents (181 Caucasians, 139 African Americans, and 135 Hispanics). All underwent an oral glucose tolerance test and fasting lipoprotein subclasses measurement by proton nuclear magnetic resonance. A subset of 142 children underwent a fast gradient magnetic resonance imaging to measure the HFF%. The rs1260326 was associated with elevated triglycerides (Caucasians P = 0.00014; African Americans P = 0.00417), large very low‐density lipoprotein (VLDL) (Caucasians P = 0.001; African Americans, P = 0.03), and with fatty liver (Caucasians P = 0.034; African Americans P = 0.00002; and Hispanics P = 0.016). The PNPLA3, but not the APOC3 rs2854116 SNP, was associated with fatty liver but not with triglyceride levels. There was a joint effect between the PNPLA3 and GCKR SNPs, explaining 32% of HFF% variance in Caucasians (P = 0.00161), 39.0% in African Americans (P = 0.00000496), and 15% in Hispanics (P = 0.00342). Conclusion: The rs1260326 in GCKR is associated with hepatic fat accumulation along with large VLDL and triglyceride levels. GCKR and PNPLA3 act together to convey susceptibility to fatty liver in obese youths. (Hepatology 2012)

Routine Genetic Testing for Thoracic Aortic Aneurysm and Dissection in a Clinical Setting

BACKGROUND Hereditary factors play an important etiologic role in thoracic aortic aneurysm and dissection (TAAD), with a number of genes proven to predispose to this condition. We initiated a clinical program for routine genetic testing of individuals for TAAD by whole exome sequencing (WES). Here we present our initial results. METHODS The WES was performed in 102 patients (mean age 56.8 years; range 13 to 83; 70 males [68.6%]) with TAAD. The following 21-gene panel was tested by WES: ACTA2, ADAMTS10, COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, ELN, FBLN4, FLNA, FBN1, FBN2, MYH11, MYLK, NOTCH1, PRKG1, SLC2A10, SMAD3, TGFB2, TGFBR1, TGFBR2. RESULTS Seventy-four patients (72.5%) had no medically important genetic alterations. Four patients (3.9%) had a deleterious mutation identified in the FBN1, COL5A1, MYLK, and FLNA genes. Twenty-two (21.6%) previously unreported suspicious variants of unknown significance were identified in 1 or more of the following genes: FBN1 (n = 5); MYH11 (n = 4); ACTA2 (n = 2); COL1A1 (n = 2); TGFBR1 (n = 2); COL3A1 (n = 1); COL5A1 (n = 1); COL5A2 (n = 1); FLNA (n = 1); NOTCH1 (n = 1); PRKG1 (n = 1); and TGFBR3 (n = 1). Identified mutations had implications for clinical management. CONCLUSIONS Routine genetic screening of patients with TAAD provides information that enables genetically personalized care and permits identification of novel mutations responsible for aortic pathology. Analysis of large data sets of variants of unknown significance that include associated clinical features will help define the mutational spectrum of known genes underlying this phenotype and potential identify new candidate loci.

Clinical testing for the nevoid basal cell carcinoma syndrome in a DNA diagnostic laboratory.

Purpose: This study determines which clinical features predict positive test results among samples submitted for DNA-based diagnostic nevoid basal cell carcinoma syndrome (NBCCS) testing, and further defines the mutational spectrum of the PTCH gene.Methods: DNA was extracted from peripheral blood leukocytes, and polymerase chain reaction products from exons 1 to 23 of the PTCH gene were directly sequenced. Pedigree phenotypic information was obtained by written questionnaire.Results: Among 106 presumably unrelated pedigrees, 44 independent mutations were found in 47 families. There were 11 nonsense mutations; 1 in-frame deletion; 17 deletions, 6 insertions, and 1 deletion-insertion that generated frameshifts; 5 splice-site mutations; 1 in-frame duplication; and 2 presumptive missense mutations. Twenty-seven of 46 pedigrees (58.7%) with two or more typical radiographic or pathologic features of NBCCS tested positive for PTCH mutations. Of these, 26 had jaw cysts in combination with other characteristics or neoplasms including basal cell carcinomas, palmar pits, skeletal abnormalities, ocular abnormalities, medulloblastomas, cardiac or ovarian fibromas, calcification of the falx cerebri, polydactyly, cleft lip and/or palate, and agenesis of the corpus callosum or other central nervous system malformations. None of the 13 pedigrees solely affected by multiple or early-onset basal cell carcinomas and none of the four pedigrees with jaw cysts alone had PTCH mutations.Conclusions: Pedigrees with multiple features of NBCCS were most likely to test positive for PTCH mutations. Pedigrees with multiple or early-onset basal cell carcinomas without other features of the disease did not test positive for PTCH mutations.

Application of Whole Exome Sequencing in the Clinical Diagnosis and Management of Inherited Cardiovascular Diseases in Adults.

Background— With the advent of high throughput sequencing, the identification of genetic causes of cardiovascular disease (CVD) has become an integral part of medical diagnosis and management and at the forefront of personalized medicine in this field. The use of whole exome sequencing for clinical diagnosis, risk stratification, and management of inherited CVD has not been previously evaluated. Methods and Results— We analyzed the results of whole exome sequencing in first 200 adult patients with inherited CVD, who underwent genetic testing at the Yale Program for Cardiovascular Genetics. Genetic diagnosis was reached and reported with a success rate of 26.5% (53 of 200 patients). This compares to 18% (36 of 200) that would have been diagnosed using commercially available genetic panels (P=0.04). Whole exome sequencing was particularly useful for clinical diagnosis in patients with aborted sudden cardiac death, in whom the primary insult for the presence of both depressed cardiac function and prolonged QT had remained unknown. The analysis of the remaining cases using genome annotation and disease segregation led to the discovery of novel candidate genes in another 14% of the cases. Conclusions— Whole exome sequencing is an exceptionally valuable screening tool for its capability to establish the clinical diagnosis of inherited CVDs, particularly for poorly defined cases of sudden cardiac death. By presenting novel candidate genes and their potential disease associations, we also provide evidence for the use of this genetic tool for the identification of novel CVD genes. Creation and sharing of exome databases across centers of care should facilitate the discovery of unknown CVD genes.

Whole-exome sequencing in evaluation of patients with venous thromboembolism

Genetics play a significant role in venous thromboembolism (VTE), yet current clinical laboratory-based testing identifies a known heritable thrombophilia (factor V Leiden, prothrombin gene mutation G20210A, or a deficiency of protein C, protein S, or antithrombin) in only a minority of VTE patients. We hypothesized that a substantial number of VTE patients could have lesser-known thrombophilia mutations. To test this hypothesis, we performed whole-exome sequencing (WES) in 64 patients with VTE, focusing our analysis on a novel 55-gene extended thrombophilia panel that we compiled. Our extended thrombophilia panel identified a probable disease-causing genetic variant or variant of unknown significance in 39 of 64 study patients (60.9%), compared with 6 of 237 control patients without VTE (2.5%) (P < .0001). Clinical laboratory-based thrombophilia testing identified a heritable thrombophilia in only 14 of 54 study patients (25.9%). The majority of WES variants were either associated with thrombosis based on prior reports in the literature or predicted to affect protein structure based on protein modeling performed as part of this study. Variants were found in major thrombophilia genes, various SERPIN genes, and highly conserved areas of other genes with established or potential roles in coagulation or fibrinolysis. Ten patients (15.6%) had >1 variant. Sanger sequencing performed in family members of 4 study patients with and without VTE showed generally concordant results with thrombotic history. WES and extended thrombophilia testing are promising tools for improving our understanding of VTE pathogenesis and identifying inherited thrombophilias.

The rs626283 Variant in the MBOAT7 Gene is Associated with Insulin Resistance and Fatty Liver in Caucasian Obese Youth

Objectives:Non alcoholic fatty liver disease (NAFLD) is a leading cause of liver damage in childhood, its occurrence is influenced by genetic and environmental factors. Recently, the rs626283 polymorphism in the MBOAT7 gene has been found to be associated with alcoholic liver disease and NAFLD in adults.Methods:In a multiethnic cohort of obese children and adolescents we genotyped the rs626283 polymorphism in the MBOAT7 gene, evaluated insulin sensitivity by an oral glucose tolerance test, and measured the intra-hepatic fat content (HFF%) by magnetic resonance imaging.Results:In Caucasian youth, the minor allele (C) was associated with HFF% in (P=0.003), fasting insulin (P=0.03), area under the curve of glucose (P=0.03), and lower degree of whole-body insulin sensitivity (P=0.01) independent of age, gender, and body mass index z-score. A partial correlation showed that the association between the rs626283 variant and insulin resistance was driven by the presence of hepatic steatosis (P=0.009). However, there was no association between the rs626283 and hepatic steatosis among Hispanic and African American children and youth. The association between the rs626283 in the MBOAT7 gene among Caucasians was independent of the PNPLA3 rs738409, GCKR 1260326, and TM6SF2 rs58542926 (P=0.01). The four polymorphisms combined explained~19% of the HFF% in Caucasian obese children and adolescents.Conclusions:The rs626283 variant in the MBOAT7 gene is associated with NAFLD and may affect glucose metabolism by modulating intra-hepatic fat content in Caucasian obese children and adolescents.

Single Amino Acid Deletion in MYH11 Segregating in a Family with TAAD

Single Amino Acid Deletion in MYH11 Segregating in a Family with TAAD Background Seven genes have been identified as causative in the development of thoracic aortic aneurysms (TAA) and dissections (TAAD). In this study, we identify a single amino acid deletion in MYH11 gene which segregates with disease in a large TAAD family. Methods We identified five members in one family who had a history of TAA or TAAD. Blood samples from fifteen members of this family were collected for whole exome sequencing (WES) analysis and mutation analysis. Thirteen members of this family underwent echocardiography and cardiac pulse wave velocity (PWV) measurement. Results WES analysis revealed a mutation of myosin heavy chain 11 (MYH11) – a three base-pair deletion of leucine at position 1264 - that segregated with the disease phenotype in this family. PWV was not correlated with mutation or disease status. Conclusion Deletion of leucine at position 1264 in MYH11 appears to play an important role in the development of familial TAA and TAAD.

PCR Based Diagnosis of Fragile X Syndrome

Fragile X syndrome, a common cause of mental retardation in males and milder learning problems in females, is related to CGG trinucleotide repeat expansion in the FMR1 gene. Premutations are a cause of premature ovarian failure in females and adult-onset syndrome of ataxia, tremor, and cognitive decline (FXTAS) in males. Clinical testing for premutations can be achieved by PCR amplification of the CGG repeats in a denaturing buffer and size fractionation on an ethidium bromide–stained gel. Full mutations are detected by Southern blotting of the PCR products and probing with a CGG oligonucleotide.