Evans, Jared M.

Background— The molecular underpinnings of hypoplastic left heart are poorly understood. Staged surgical palliation has dramatically improved survival, yet eventual failure of the systemic right ventricle necessitates cardiac transplantation in a subset of patients. We sought to identify genetic determinants of hypoplastic left heart with latent right ventricular dysfunction in individuals with a Fontan circulation. Methods and Results— Evaluation of cardiac structure and function by echocardiography in patients with hypoplastic left heart and their first-degree relatives identified 5 individuals with right ventricular ejection fraction ≤40% after Fontan operation. Whole genome sequencing was performed on DNA from 21 family members, filtering for genetic variants with allele frequency <1% predicted to alter protein structure or expression. Secondary family-based filtering for de novo and recessive variants revealed rare inherited missense mutations on both paternal and maternal alleles of MYH6 , encoding myosin heavy chain 6, in 2 patients who developed right ventricular dysfunction 3 to 11 years postoperatively. Parents and siblings who were heterozygous carriers had normal echocardiograms. Protein modeling of the 4 highly conserved amino acid substitutions, residing in both head and tail domains, predicted perturbation of protein structure and function. Conclusions— In contrast to dominant MYH6 mutations with variable penetrance identified in other congenital heart defects and dilated cardiomyopathy, this study reveals compound heterozygosity for recessive MYH6 mutations in patients with hypoplastic left heart and reduced systemic right ventricular ejection fraction. These findings implicate a shared molecular basis for the developmental arrest and latent myopathy of left and right ventricles, respectively.

Background— Long-QT syndrome (LQTS) may result in syncope, seizures, or sudden cardiac arrest. Although 16 LQTS-susceptibility genes have been discovered, 20% to 25% of LQTS remains genetically elusive. Methods and Results— We performed whole-exome sequencing child–parent trio analysis followed by recessive and sporadic inheritance modeling and disease-network candidate analysis gene ranking to identify a novel underlying genetic mechanism for LQTS. Subsequent mutational analysis of the candidate gene was performed with polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing on a cohort of 33 additional unrelated patients with genetically elusive LQTS. After whole-exome sequencing and variant filtration, a homozygous p.D18fs*13 TRDN -encoded triadin frameshift mutation was discovered in a 10-year-old female patient with LQTS with a QTc of 500 milliseconds who experienced recurrent exertion-induced syncope/cardiac arrest beginning at 1 year of age. Subsequent mutational analysis of TRDN revealed either homozygous or compound heterozygous frameshift mutations in 4 of 33 unrelated cases of LQTS (12%). All 5 TRDN-null patients displayed extensive T-wave inversions in precordial leads V 1 through V 4 , with either persistent or transient QT prolongation and severe disease expression of exercise-induced cardiac arrest in early childhood (≤3 years of age) and required aggressive therapy. The overall yield of TRDN mutations was significantly greater in patients ≤10 years of age (5 of 10, 50%) compared with older patients (0 of 24, 0%; P =0.0009). Conclusions— We identified TRDN as a novel underlying genetic basis for recessively inherited LQTS. All TRDN-null patients had strikingly similar phenotypes. Given the recurrent nature of potential lethal arrhythmias, patients fitting this phenotypic profile should undergo cardiac TRDN genetic testing.

Idiopathic dilated cardiomyopathy is a heritable, genetically heterogeneous disorder characterized by progressive heart failure. Dilated cardiomyopathy typically exhibits autosomal dominant inheritance, yet frequently remains clinically silent until adulthood. We sought to discover the molecular basis of idiopathic, non‐syndromic dilated cardiomyopathy in a one‐month‐old male presenting with severe heart failure. Previous comprehensive testing of blood, urine, and skin biopsy specimen was negative for metabolic, mitochondrial, storage, and infectious etiologies. Ophthalmologic examination was normal. Chromosomal microarray and commercial dilated cardiomyopathy gene panel testing failed to identify a causative mutation. Parental screening echocardiograms revealed no evidence of clinically silent dilated cardiomyopathy. Whole exome sequencing was carried out on the family trio on a research basis, filtering for rare, deleterious, recessive and de novo genetic variants. Pathogenic compound heterozygous truncating mutations were identified in ALMS1, diagnostic of Alström syndrome and prompting disclosure of genetic findings. Alström syndrome is a known cause for dilated cardiomyopathy in children yet delayed and mis‐diagnosis are common owing to its rarity and age‐dependent emergence of multisystem clinical manifestations. At six months of age the patient ultimately developed bilateral nystagmus and hyperopia, features characteristic of the syndrome. Early diagnosis is guiding clinical monitoring of other organ systems and allowing for presymptomatic intervention. Furthermore, recognition of recessive inheritance as the mechanism for sporadic disease has informed family planning. This case highlights a limitation of standard gene testing panels for pediatric dilated cardiomyopathy and exemplifies the potential for whole exome sequencing to solve a diagnostic dilemma and enable personalized care. © 2015 Wiley Periodicals, Inc.

Abstract Aims Exome sequencing was used to genotype comprehensively a Takotsubo (stress) cardiomyopathy (TC) cohort, enabling investigation of a vast 486 gene network for adrenergic signalling. Methods and results Twenty-eight TC subjects, including a mother–daughter pair and five recurrent cases, underwent whole-exome sequencing. Frequencies of 17 common, functional adrenergic polymorphisms were statistically similar to those of population controls. Filtering for rare, predicted-deleterious, catecholamine/adrenergic signalling variants revealed heterozygosity in 55 genes in TC cases and 59 genes in healthy controls. Overall allele burden was similar and did not discriminate clinical variables among TC subjects, but gene identities were largely cohort specific, and TC cases were enriched for variants within functional domains (68% vs. 48%, P = 0.031). Two-thirds of TC cases carried more than one filtered adrenergic pathway variant, and 11 genes harboured a variant in ≥2 cases. The mother–daughter pair shared missense variants in highly conserved functional domains of ADH5, CACNG1, EPHA4, and PRKCA. An adrenergic pathway-independent analysis of the cohort exposed no common gene for TC. Conclusions Overall, these data support genetic heterogeneity in TC susceptibility and a likely polygenic basis, conferring a cumulative effect on adrenergic pathway dysregulation in a subset of individual subjects. Study of larger cohorts and non-coding regulatory regions is warranted to define genetic risk factors for TC further.

AbstractImproving the effectiveness of adipose‐tissue derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed characterization of mechanisms supporting cell proliferation and multi‐potency. We investigated the molecular phenotype of AMSCs that were either actively proliferating in platelet lysate or in a basal non‐proliferative state. Flow cytometry combined with high‐throughput RNA sequencing (RNASeq) and RT‐qPCR analyses validate that AMSCs express classic mesenchymal cell surface markers (e.g., CD44, CD73/NT5E, CD90/THY1, and CD105/ENG). Expression of CD90 is selectively elevated at confluence. Self‐renewing AMSCs express a standard cell cycle program that successively mediates DNA replication, chromatin packaging, cyto‐architectural enlargement, and mitotic division. Confluent AMSCs preferentially express genes involved in extracellular matrix (ECM) formation and cellular communication. For example, cell cycle‐related biomarkers (e.g., cyclins E2 and B2, transcription factor E2F1) and histone‐related genes (e.g., H4, HINFP, NPAT) are elevated in proliferating AMSCs, while ECM genes are strongly upregulated (>10‐fold) in quiescent AMSCs. AMSCs also express pluripotency genes (e.g., POU5F1, NANOG, KLF4) and early mesenchymal markers (e.g., NES, ACTA2) consistent with their multipotent phenotype. Strikingly, AMSCs modulate expression of WNT signaling components and switch production of WNT ligands (from WNT5A/WNT5B/WNT7B to WNT2/WNT2B), while upregulating WNT‐related genes (WISP2, SFRP2, and SFRP4). Furthermore, post‐proliferative AMSCs spontaneously express fibroblastic, osteogenic, chondrogenic, and adipogenic biomarkers when maintained in confluent cultures. Our findings validate the biological properties of self‐renewing and multi‐potent AMSCs by providing high‐resolution quality control data that support their clinical versatility. J. Cell. Biochem. 115: 1816–1828, 2014. © 2014 Wiley Periodicals, Inc.

Abstract Motivation: Exome sequencing (exome-seq) data, which are typically used for calling exonic mutations, have also been utilized in detecting DNA copy number variations (CNVs). Despite the existence of several CNV detection tools, there is still a great need for a sensitive and an accurate CNV-calling algorithm with built-in QC steps, and does not require a paired reference for each sample. Results: We developed a novel method named PatternCNV, which (i) accounts for the read coverage variations between exons while leveraging the consistencies of this variability across different samples; (ii) reduces alignment BAM files to WIG format and therefore greatly accelerates computation; (iii) incorporates multiple QC measures designed to identify outlier samples and batch effects; and (iv) provides a variety of visualization options including chromosome, gene and exon-level views of CNVs, along with a tabular summarization of the exon-level CNVs. Compared with other CNV-calling algorithms using data from a lymphoma exome-seq study, PatternCNV has higher sensitivity and specificity. Availability and implementation: The software for PatternCNV is implemented using Perl and R, and can be used in Mac or Linux environments. Software and user manual are available at http://bioinformaticstools.mayo.edu/research/patterncnv/ , and R package at https://github.com/topsoil/patternCNV/ . Contact: Asmann.Yan@mayo.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Background— Long QT syndrome (LQTS) is the most common cardiac channelopathy with 15 elucidated LQTS-susceptibility genes. Approximately 20% of LQTS cases remain genetically elusive. Methods and Results— We combined whole-exome sequencing and bioinformatic/systems biology to identify the pathogenic substrate responsible for nonsyndromic, genotype-negative, autosomal dominant LQTS in a multigenerational pedigree, and we established the spectrum and prevalence of variants in the elucidated gene among a cohort of 102 unrelated patients with “genotype-negative/phenotype-positive” LQTS. Whole-exome sequencing was used on 3 members within a genotype-negative/phenotype-positive family. Genomic triangulation combined with bioinformatic tools and ranking algorithms led to the identification of a CACNA1C mutation. This mutation, Pro857Arg-CACNA1C, cosegregated with the disease within the pedigree, was ranked by 3 disease-network algorithms as the most probable LQTS-susceptibility gene and involves a conserved residue localizing to the proline, gltamic acid, serine, and threonine (PEST) domain in the II-III linker. Functional studies reveal that Pro857Arg-CACNA1C leads to a gain of function with increased I Ca,L and increased surface membrane expression of the channel compared to wild type. Subsequent mutational analysis identified 3 additional variants within CACNA1C in our cohort of 102 unrelated cases of genotype-negative/phenotype-positive LQTS. Two of these variants also involve conserved residues within Ca v 1.2’s PEST domain. Conclusions— This study provides evidence that coupling whole-exome sequencing and bioinformatic/systems biology is an effective strategy for the identification of potential disease-causing genes/mutations. The identification of a functional CACNA1C mutation cosegregating with disease in a single pedigree suggests that CACNA1C perturbations may underlie autosomal dominant LQTS in the absence of Timothy syndrome.