James R. Priest

De Novo and Rare Variants at Multiple Loci Support the Oligogenic Origins of Atrioventricular Septal Heart Defects.

Congenital heart disease (CHD) has a complex genetic etiology, and recent studies suggest that high penetrance de novo mutations may account for only a small fraction of disease. In a multi-institutional cohort surveyed by exome sequencing, combining analysis of 987 individuals (discovery cohort of 59 affected trios and 59 control trios, and a replication cohort of 100 affected singletons and 533 unaffected singletons) we observe variation at novel and known loci related to a specific cardiac malformation the atrioventricular septal defect (AVSD). In a primary analysis, by combining developmental coexpression networks with inheritance modeling, we identify a de novo mutation in the DNA binding domain of NR1D2 (p.R175W). We show that p.R175W changes the transcriptional activity of Nr1d2 using an in vitro transactivation model in HUVEC cells. Finally, we demonstrate previously unrecognized cardiovascular malformations in the Nr1d2tm1-Dgen knockout mouse. In secondary analyses we map genetic variation to protein-interaction networks suggesting a role for two collagen genes in AVSD, which we corroborate by burden testing in a second replication cohort of 100 AVSDs and 533 controls (p = 8.37e-08). Finally, we apply a rare-disease inheritance model to identify variation in genes previously associated with CHD (ZFPM2, NSD1, NOTCH1, VCAN, and MYH6), cardiac malformations in mouse models (ADAM17, CHRD, IFT140, PTPRJ, RYR1 and ATE1), and hypomorphic alleles of genes causing syndromic CHD (EHMT1, SRCAP, BBS2, NOTCH2, and KMT2D) in 14 of 59 trios, greatly exceeding variation in control trios without CHD (p = 9.60e-06). In total, 32% of trios carried at least one putatively disease-associated variant across 19 loci,suggesting that inherited and de novo variation across a heterogeneous group of loci may contribute to disease risk.

Rare copy number variants in isolated sporadic and syndromic atrioventricular septal defects

Atrioventricular septal defects (AVSDs) are a frequent but not universal component of Down syndrome (DS), while AVSDs in otherwise normal individuals have no well‐defined genetic basis. The contribution of copy number variation (CNV) to specific congenital heart disease (CHD) phenotypes including AVSD is unknown. We hypothesized that de novo CNVs on chromosome 21 might cause isolated sporadic AVSDs, and separately that CNVs throughout the genome might constitute an additional genetic risk factor for AVSD in patients with DS. We utilized a custom oligonucleotide arrays targeted to CNV hotspots that are flanked by large duplicated segments of high sequence identity. We assayed 29 euploid and 50 DS individuals with AVSD, and compared to general population controls. In patients with isolated‐sporadic AVSD we identified two large unique deletions outside of chromosome 21 not seen in the expanded set of 8,635 controls, each overlapping with larger deletions associated with similar CHD reported in the DECIPHER database. There was a small duplication in one patient with DS and AVSD. We conclude that isolated sporadic AVSDs may be occasionally associated with large de novo genomic structural variation outside of chromosome 21. The absence of CNVs on chromosome 21 in patients with isolated sporadic AVSD suggests that sub‐chromosomal duplications or deletions of greater than 150 kbp on chromosome 21 do not cause sporadic AVSDs. Large CNVs do not appear to be an additive risk factor for AVSD in the DS population.

Molecular Diagnosis of Long-QT syndrome at 10 Days of Life by Rapid Whole Genome Sequencing

BACKGROUND The advent of clinical next generation sequencing is rapidly changing the landscape of rare disease medicine. Molecular diagnosis of long QT syndrome (LQTS) can affect clinical management, including risk stratification and selection of pharmacotherapy on the basis of the type of ion channel affected, but results from the current gene panel testing requires 4-16 weeks before return to clinicians. OBJECTIVE A term female infant presented with 2:1 atrioventricular block and ventricular arrhythmias consistent with perinatal LQTS, requiring aggressive treatment including epicardial pacemaker and cardioverter-defibrillator implantation and sympathectomy on day of life 2. We sought to provide a rapid molecular diagnosis for the optimization of treatment strategies. METHODS We performed Clinical Laboratory Improvement Amendments-certified rapid whole genome sequencing (WGS) with a speed-optimized bioinformatics platform to achieve molecular diagnosis at 10 days of life. RESULTS We detected a known pathogenic variant in KCNH2 that was demonstrated to be paternally inherited by follow-up genotyping. The unbiased assessment of the entire catalog of human genes provided by WGS revealed a maternally inherited variant of unknown significance in a novel gene. CONCLUSION Rapid clinical WGS provides faster and more comprehensive diagnostic information at 10 days of life than does standard gene panel testing. In selected clinical scenarios such as perinatal LQTS, rapid WGS can provide more timely and clinically actionable information than can a standard commercial test.

Prepregnancy Diabetes and Offspring Risk of Congenital Heart Disease: A Nationwide Cohort Study.

Background— Maternal diabetes mellitus is associated with an increased risk of offspring congenital heart defects (CHD); however, the causal mechanism is poorly understood. We further investigated this association in a Danish nationwide cohort. Methods and Results— In a national cohort study, we identified 2 025 727 persons born from 1978 to 2011; among them were 7296 (0.36%) persons exposed to maternal pregestational diabetes mellitus. Pregestational diabetes mellitus was identified by using the National Patient Register and individual-level information on all prescriptions filled in Danish pharmacies. Persons with CHD (n=16 325) were assigned to embryologically related cardiac phenotypes. The CHD prevalence in the offspring of mothers with pregestational diabetes mellitus was 318 per 10 000 live births (n=232) in comparison with a baseline risk of 80 per 10 000; the adjusted relative risk for CHD was 4.00 (95% confidence interval, 3.51–4.53). The association was not modified by year of birth, maternal age at diabetes onset, or diabetes duration, and CHD risks associated with type 1 (insulin-dependent) and type 2 (insulin-independent) diabetes mellitus did not differ significantly. Persons born to women with previous acute diabetes complications had a higher CHD risk than those exposed to maternal diabetes mellitus without complications (relative risk, 7.62; 95% confidence interval, 5.23–10.6, and relative risk, 3.49; 95% confidence interval, 2.91–4.13, respectively; P=0.0004). All specific CHD phenotypes were associated with maternal pregestational diabetes mellitus (relative risk range, 2.74–13.8). Conclusions— The profoundly increased CHD risk conferred by maternal pregestational diabetes mellitus neither changed over time nor differed by diabetes subtype. The association with acute pregestational diabetes complications was particularly strong, suggesting a role for glucose in the causal pathway.

Activating mutations affecting the Dbl homology domain of SOS2 cause Noonan syndrome

The RASopathies constitute a family of autosomal‐dominant disorders whose major features include facial dysmorphism, cardiac defects, reduced postnatal growth, variable cognitive deficits, ectodermal and skeletal anomalies, and susceptibility to certain malignancies. Noonan syndrome (NS), the commonest RASopathy, is genetically heterogeneous and caused by functional dysregulation of signal transducers and regulatory proteins with roles in the RAS/extracellular signal‐regulated kinase (ERK) signal transduction pathway. Mutations in known disease genes account for approximately 80% of affected individuals. Here, we report that missense mutations altering Son of Sevenless, Drosophila, homolog 2 (SOS2), which encodes a RAS guanine nucleotide exchange factor, occur in a small percentage of subjects with NS. Four missense mutations were identified in five unrelated sporadic cases and families transmitting NS. Disease‐causing mutations affected three conserved residues located in the Dbl homology (DH) domain, of which two are directly involved in the intramolecular binding network maintaining SOS2 in its autoinhibited conformation. All mutations were found to promote enhanced signaling from RAS to ERK. Similar to NS‐causing SOS1 mutations, the phenotype associated with SOS2 defects is characterized by normal development and growth, as well as marked ectodermal involvement. Unlike SOS1 mutations, however, those in SOS2 are restricted to the DH domain.

Relationships of the Location and Content of Rounds to Specialty, Institution, Patient-Census, and Team Size

Objective Existing observational data describing rounds in teaching hospitals are 15 years old, predate duty-hour regulations, are limited to one institution, and do not include pediatrics. We sought to evaluate the effect of medical specialty, institution, patient-census, and team participants upon time at the bedside and education occurring on rounds. Methods and Participants Between December of 2007 and October of 2008 we performed 51 observations at Lucile Packard Childrens Hospital, Seattle Childrens Hospital, Stanford University Hospital, and the University of Washington Medical Center of 35 attending physicians. We recorded minutes spent on rounds in three location and seven activity categories, members of the care team, and patient-census. Results Results presented are means. Pediatric rounds had more participants (8.2 vs. 4.1 physicians, p<.001; 11.9 vs. 2.4 non-physicians, p<.001) who spent more minutes in hallways (96.9 min vs. 35.2 min, p<.001), fewer minutes at the bedside (14.6 vs. 38.2 min, p = .01) than internal medicine rounds. Multivariate regression modeling revealed that minutes at the bedside per patient was negatively associated with pediatrics (−2.77 adjusted bedside minutes; 95% CI −4.61 to −0.93; p<.001) but positively associated with the number of non-physician participants (0.12 adjusted bedside minutes per non physician participant; 95% CI 0.07 to 0.17; p = <.001). Education minutes on rounds was positively associated with the presence of an attending physician (2.70 adjusted education minutes; 95% CI 1.27 to 4.12; p<.001) and with one institution (1.39 adjusted education minutes; 95% CI 0.26 to 2.53; p = .02). Conclusions Pediatricians spent less time at the bedside on rounds than internal medicine physicians due to reasons other than patient-census or the number of participants in rounds. Compared to historical data, internal medicine rounds were spent more at the bedside engaged in patient care and communication, and less upon educational activities.

Brucellosis and Sacroiliitis: A Common Presentation of an Uncommon Pathogen

Musculoskeletal problems are the most common chief complaint in ambulatory medicine across all specialties, and back pain is one of the top 10 problems encountered by the general practitioner.1,2 The differential diagnosis of lower back pain is exhaustive, but a history significant for constitutional symptoms or unusual exposures should prompt a work-up for an infectious cause. We describe the case of a 25-year-old man with a Brucella abortus sacroiliitis and possible orchiitis after consumption of unpasteurized cheese imported from El Salvador. The patient was successfully treated with gentamycin, rifampin, and doxycycline. Though the presentations of brucellosis are myriad, osteoarticular involvement of the axial skeleton is the most common presentation of this zoonotic infection.3 In the United States brucellosis is rarely encountered and is typically limited to people who are exposed during travel to endemic areas. Here we review briefly the epidemiology and presentation of a Brucella infection and current recommendations for treatment.

Early somatic mosaicism is a rare cause of long-QT syndrome

Significance Most genetic studies and clinical genetic testing do not look for the possibility of mosaic variation. The genetic form of long-QT syndrome (LQTS) can result in life-threatening arrhythmias, but 30% of patients do not have a genetic diagnosis. We performed deep characterization of a mosaic variant in an infant with perinatal LQTS and developed a computational model showing how abnormal cellular repolarization in only 8% of heart cells may cause arrhythmia. Finally we looked at the prevalence of mosaicism among patients with LQTS; in a population of 7,500 individuals we found evidence of pathogenic early somatic mosaicism in approximately 0.17% of LQTS patients without a genetic diagnosis. Together these data establish an unreported mechanism for LQTS and other genetic arrhythmias. Somatic mosaicism, the occurrence and propagation of genetic variation in cell lineages after fertilization, is increasingly recognized to play a causal role in a variety of human diseases. We investigated the case of life-threatening arrhythmia in a 10-day-old infant with long QT syndrome (LQTS). Rapid genome sequencing suggested a variant in the sodium channel NaV1.5 encoded by SCN5A, NM_000335:c.5284G > T predicting p.(V1762L), but read depth was insufficient to be diagnostic. Exome sequencing of the trio confirmed read ratios inconsistent with Mendelian inheritance only in the proband. Genotyping of single circulating leukocytes demonstrated the mutation in the genomes of 8% of patient cells, and RNA sequencing of cardiac tissue from the infant confirmed the expression of the mutant allele at mosaic ratios. Heterologous expression of the mutant channel revealed significantly delayed sodium current with a dominant negative effect. To investigate the mechanism by which mosaicism might cause arrhythmia, we built a finite element simulation model incorporating Purkinje fiber activation. This model confirmed the pathogenic consequences of cardiac cellular mosaicism and, under the presenting conditions of this case, recapitulated 2:1 AV block and arrhythmia. To investigate the extent to which mosaicism might explain undiagnosed arrhythmia, we studied 7,500 affected probands undergoing commercial gene-panel testing. Four individuals with pathogenic variants arising from early somatic mutation events were found. Here we establish cardiac mosaicism as a causal mechanism for LQTS and present methods by which the general phenomenon, likely to be relevant for all genetic diseases, can be detected through single-cell analysis and next-generation sequencing.

Sequence to Medical Phenotypes: A Framework for Interpretation of Human Whole Genome DNA Sequence Data

Abstract High throughput sequencing has facilitated a precipitous drop in the cost of genomic sequencing, prompting predictions of a revolution in medicine via genetic personalization of diagnostic and therapeutic strategies. There are significant barriers to realizing this goal that are related to the difficult task of interpreting personal genetic variation. A comprehensive, widely accessible application for interpretation of whole genome sequence data is needed. Here, we present a series of methods for identification of genetic variants and genotypes with clinical associations, phasing genetic data and using Mendelian inheritance for quality control, and providing predictive genetic information about risk for rare disease phenotypes and response to pharmacological therapy in single individuals and father-mother-child trios. We demonstrate application of these methods for disease and drug response prognostication in whole genome sequence data from twelve unrelated adults, and for disease gene discovery in one father-mother-child trio with apparently simplex congenital ventricular arrhythmia. In doing so we identify clinically actionable inherited disease risk and drug response genotypes in pre-symptomatic individuals. We also nominate a new candidate gene in congenital arrhythmia, ATP2B4, and provide experimental evidence of a regulatory role for variants discovered using this framework.

Maternal Midpregnancy Glucose Levels and Risk of Congenital Heart Disease in Offspring.

IMPORTANCE There is a well-described association between maternal diabetes mellitus and risk of congenital heart disease (CHD) in offspring. Although the clinical diagnoses of type 2 diabetes or gestational diabetes are strong risk factors for CHD, subclinical abnormalities of glucose and insulin metabolism are common within the general population and could also confer risk for CHD. We hypothesize that continuous measures of blood analytes related to maternal diabetes are related to odds of cardiac malformations. OBJECTIVE To explore the potential association of 2 different CHD phenotypes in offspring with maternal midpregnancy measures of glucose and insulin. DESIGN, SETTING, AND PARTICIPANTS Case-control study from a population-based cohort of 277 pregnant women in southern and central California carrying infants with tetralogy of Fallot (TOF) (n = 55), dextrotransposition of the great arteries (dTGA) (n = 42), or healthy infants without CHD (n = 180). Serum samples were collected from 2003 through 2007. The analysis was conducted from March through June 2015. MAIN OUTCOMES AND MEASURES Blood analytes related to maternal glucose metabolism were measured from random nonfasting second-trimester blood samples. We measured serum insulin levels by a validated radioimmunoassay, and we measured glucose levels. Multivariable logistic regression models estimated the association between these levels and case status. RESULTS Serum glucose values were elevated in the maternal samples for offspring with TOF (median, 97.0 mg/dL [to convert to millimoles per liter, multiply by 0.0555]) relative to controls (median, 91.5 mg/dL) (P = .01, Wilcoxon rank sum test), a phenomenon not observed in the maternal samples for offspring with dTGA (median, 90.0 mg/dL) relative to controls (P = .18, Wilcoxon rank sum test). Serum insulin levels were significantly different between controls (median, 18.8 μIU/mL [to convert to picomoles per liter, multiply by 6.945]) and maternal samples for offspring with dTGA (median, 13.1 μIU/mL; P = .048, Wilcoxon rank sum test) but not with TOF (median, 14.3 μIU/mL; P = .35, Wilcoxon rank sum test). Relative to maternal blood glucose levels of infants without cardiac malformations, we observed that maternal blood glucose levels in models including insulin were strongly associated with odds of TOF (adjusted odds ratio = 7.54; 95% CI, 2.30-24.69) but not with dTGA (adjusted odds ratio = 1.16; 95% CI, 0.28-4.79). CONCLUSIONS AND RELEVANCE These results represent a direct correlation of glucose as a continuous variable to odds of specific cardiac malformations. The association between serum glucose and odds of TOF indicates the need for additional epidemiological and mechanistic investigations into the risk conferred by insulin signaling and glucose metabolism during early pregnancy.