Contribution of Previously Unrecognized RNA Splice-Altering Variants to Congenital Heart Disease.

BACKGROUND: Known genetic causes of congenital heart disease (CHD) explain <40% of CHD cases, and interpreting the clinical significance of variants with uncertain functional impact remains challenging. We aim to improve diagnostic classification of variants in patients with CHD by assessing the impact of noncanonical splice region variants on RNA splicing. METHODS: We tested de novo variants from trio studies of 2649 CHD probands and their parents, as well as rare (allele frequency, <2×10-6) variants from 4472 CHD probands in the Pediatric Cardiac Genetics Consortium through a combined computational and in vitro approach. RESULTS: We identified 53 de novo and 74 rare variants in CHD cases that alter splicing and thus are loss of function. Of these, 77 variants are in known dominant, recessive, and candidate CHD genes, including KMT2D and RBFOX2. In 1 case, we confirmed the variant's predicted impact on RNA splicing in RNA transcripts from the proband's cardiac tissue. Two probands were found to have 2 loss-of-function variants for recessive CHD genes HECTD1 and DYNC2H1. In addition, SpliceAI-a predictive algorithm for altered RNA splicing-has a positive predictive value of ≈93% in our cohort. CONCLUSIONS: Through assessment of RNA splicing, we identified a new loss-of-function variant within a CHD gene in 78 probands, of whom 69 (1.5%; n=4472) did not have a previously established genetic explanation for CHD. Identification of splice-altering variants improves diagnostic classification and genetic diagnoses for CHD. REGISTRATION: URL: https://clinicaltrials.gov; Unique identifier: NCT01196182.

Association between Multiple Myeloma and Ulcerative Colitis: A Cross-Sectional Analysis.

BACKGROUND AND AIMS: Multiple myeloma (MM) is a plasma cell dyscrasia that is common among patients with autoimmune diseases. However, the association between ulcerative colitis (UC) and multiple myeloma (MM) is yet to be established. We aimed to evaluate the prevalence of MM among patients with UC in the United States. METHODS: This cross-sectional cohort analysis used the National Inpatient Sample from 2015-2018 to assess the overall MM prevalence among patients with and without UC, and within specific demographic subgroups. Prevalences were compared using a logistic regression model controlling for sex and age. RESULTS: The crude prevalence of MM among patients with UC (n = 1750) compared with patients without UC (n = 366,265) was 0.44% vs. 0.37%, respectively. Patients with UC had increased overall odds of having MM (odds ratio (OR), 1.26). Males with UC had higher prevalence of MM (53.7% vs. 46.3%, respectively) than females. Patients with UC and MM were more likely to be African American than White (15.6% vs. 9.2%, respectively). Patients with UC age >64 had a higher prevalence of MM than those aged below 65 (70.9% vs. 29.1%, respectively). Patients with UC who were obese (BMI > 30) had a higher prevalence of MM than those who were non-obese (12.6% vs. 8.3%). CONCLUSIONS: Overall, UC appears to be associated with MM. This association can be particularly observed in specific demographic groups, such as obese, African American males, or patients >64 years of age. Thus, a high degree of clinical suspicion for MM is warranted, even with minimal symptomatology, in patients with UC, in particular among elder, obese, and African American males.

Pathogenesis of Cardiomyopathy Caused by Variants in ALPK3, an Essential Pseudokinase in the Cardiomyocyte Nucleus and Sarcomere.

BACKGROUND: ALPK3 encodes α-kinase 3, a muscle-specific protein of unknown function. ALPK3 loss-of-function variants cause cardiomyopathy with distinctive clinical manifestations in both children and adults, but the molecular functions of ALPK3 remain poorly understood. METHODS: We explored the putative kinase activity of ALPK3 and the consequences of damaging variants using isogenic human induced pluripotent stem cell-derived cardiomyocytes, mice, and human patient tissues. RESULTS: Multiple sequence alignment of all human α-kinase domains and their orthologs revealed 4 conserved residues that were variant only in ALPK3, demonstrating evolutionary divergence of the ALPK3 α-kinase domain sequence. Phosphoproteomic evaluation of both ALPK3 kinase domain inhibition and overexpression failed to detect significant changes in catalytic activity, establishing ALPK3 as a pseudokinase. Investigations into alternative functions revealed that ALPK3 colocalized with myomesin proteins (MYOM1, MYOM2) at both the nuclear envelope and the sarcomere M-band. ALPK3 loss-of-function variants caused myomesin proteins to mislocalize and also dysregulated several additional M-band proteins involved in sarcomere protein turnover, which ultimately impaired cardiomyocyte structure and function. CONCLUSIONS: ALPK3 is an essential cardiac pseudokinase that inserts in the nuclear envelope and the sarcomere M-band. Loss of ALPK3 causes mislocalization of myomesins, critical force-buffering proteins in cardiomyocytes, and also dysregulates M-band proteins necessary for sarcomere protein turnover. We conclude that ALPK3 cardiomyopathy induces ventricular dilatation caused by insufficient myomesin-mediated force buffering and hypertrophy by impairment of sarcomere proteostasis.

Neither cardiac mitochondrial DNA variation nor copy number contribute to congenital heart disease risk.

The well-established manifestation of mitochondrial mutations in functional cardiac disease (e.g., mitochondrial cardiomyopathy) prompted the hypothesis that mitochondrial DNA (mtDNA) sequence and/or copy number (mtDNAcn) variation contribute to cardiac defects in congenital heart disease (CHD). MtDNAcns were calculated and rare, non-synonymous mtDNA mutations were identified in 1,837 CHD-affected proband-parent trios, 116 CHD-affected singletons, and 114 paired cardiovascular tissue/blood samples. The variant allele fraction (VAF) of heteroplasmic variants in mitochondrial RNA from 257 CHD cardiovascular tissue samples was also calculated. On average, mtDNA from blood had 0.14 rare variants and 52.9 mtDNA copies per nuclear genome per proband. No variation with parental age at proband birth or CHD-affected proband age was seen. mtDNAcns in valve/vessel tissue (320 ± 70) were lower than in atrial tissue (1,080 ± 320, p = 6.8E-21), which were lower than in ventricle tissue (1,340 ± 280, p = 1.4E-4). The frequency of rare variants in CHD-affected individual DNA was indistinguishable from the frequency in an unaffected cohort, and proband mtDNAcns did not vary from those of CHD cohort parents. In both the CHD and the comparison cohorts, mtDNAcns were significantly correlated between mother-child, father-child, and mother-father. mtDNAcns among people with European (mean = 52.0), African (53.0), and Asian haplogroups (53.5) were calculated and were significantly different for European and Asian haplogroups (p = 2.6E-3). Variant heteroplasmic fraction (HF) in blood correlated well with paired cardiovascular tissue HF (r = 0.975) and RNA VAF (r = 0.953), which suggests blood HF is a reasonable proxy for HF in heart tissue. We conclude that mtDNA mutations and mtDNAcns are unlikely to contribute significantly to CHD risk.

GATA6 mutations in hiPSCs inform mechanisms for maldevelopment of the heart, pancreas, and diaphragm.

Damaging GATA6 variants cause cardiac outflow tract defects, sometimes with pancreatic and diaphragmic malformations. To define molecular mechanisms for these diverse developmental defects, we studied transcriptional and epigenetic responses to GATA6 loss of function (LoF) and missense variants during cardiomyocyte differentiation of isogenic human induced pluripotent stem cells. We show that GATA6 is a pioneer factor in cardiac development, regulating SMYD1 that activates HAND2, and KDR that with HAND2 orchestrates outflow tract formation. LoF variants perturbed cardiac genes and also endoderm lineage genes that direct PDX1 expression and pancreatic development. Remarkably, an exon 4 GATA6 missense variant, highly associated with extra-cardiac malformations, caused ectopic pioneer activities, profoundly diminishing GATA4, FOXA1/2, and PDX1 expression and increasing normal retinoic acid signaling that promotes diaphragm development. These aberrant epigenetic and transcriptional signatures illuminate the molecular mechanisms for cardiovascular malformations, pancreas and diaphragm dysgenesis that arise in patients with distinct GATA6 variants.

De Novo Damaging Variants, Clinical Phenotypes, and Post-Operative Outcomes in Congenital Heart Disease.

BACKGROUND: De novo genic and copy number variants are enriched in patients with congenital heart disease, particularly those with extra-cardiac anomalies. The impact of de novo damaging variants on outcomes following cardiac repair is unknown. METHODS: We studied 2517 patients with congenital heart disease who had undergone whole-exome sequencing as part of the CHD GENES study (Congenital Heart Disease Genetic Network). RESULTS: Two hundred ninety-four patients (11.7%) had clinically significant de novo variants. Patients with de novo damaging variants were 2.4 times more likely to have extra-cardiac anomalies (P=5.63×10-12). In 1268 patients (50.4%) who had surgical data available and underwent open-heart surgery exclusive of heart transplantation as their first operation, we analyzed transplant-free survival following the first operation. Median follow-up was 2.65 years. De novo variants were associated with worse transplant-free survival (hazard ratio, 3.51; P=5.33×10-04) and longer times to final extubation (hazard ratio, 0.74; P=0.005). As de novo variants had a significant interaction with extra-cardiac anomalies for transplant-free survival (P=0.003), de novo variants conveyed no additional risk for transplant-free survival for patients with these anomalies (adjusted hazard ratio, 1.96; P=0.06). By contrast, de novo variants in patients without extra-cardiac anomalies were associated with worse transplant-free survival during follow-up (hazard ratio, 11.21; P=1.61×10-05) than that of patients with no de novo variants. Using agnostic machine-learning algorithms, we identified de novo copy number variants at 15q25.2 and 15q11.2 as being associated with worse transplant-free survival and 15q25.2, 22q11.21, and 3p25.2 as being associated with prolonged time to final extubation. CONCLUSIONS: In patients with congenital heart disease undergoing open-heart surgery, de novo variants were associated with worse transplant-free survival and longer times on the ventilator. De novo variants were most strongly associated with adverse outcomes among patients without extra-cardiac anomalies, suggesting a benefit for preoperative genetic testing even when genetic abnormalities are not suspected during routine clinical practice. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01196182.

EM-mosaic detects mosaic point mutations that contribute to congenital heart disease.

BACKGROUND: The contribution of somatic mosaicism, or genetic mutations arising after oocyte fertilization, to congenital heart disease (CHD) is not well understood. Further, the relationship between mosaicism in blood and cardiovascular tissue has not been determined. METHODS: We developed a new computational method, EM-mosaic (Expectation-Maximization-based detection of mosaicism), to analyze mosaicism in exome sequences derived primarily from blood DNA of 2530 CHD proband-parent trios. To optimize this method, we measured mosaic detection power as a function of sequencing depth. In parallel, we analyzed our cohort using MosaicHunter, a Bayesian genotyping algorithm-based mosaic detection tool, and compared the two methods. The accuracy of these mosaic variant detection algorithms was assessed using an independent resequencing method. We then applied both methods to detect mosaicism in cardiac tissue-derived exome sequences of 66 participants for which matched blood and heart tissue was available. RESULTS: EM-mosaic detected 326 mosaic mutations in blood and/or cardiac tissue DNA. Of the 309 detected in blood DNA, 85/97 (88%) tested were independently confirmed, while 7/17 (41%) candidates of 17 detected in cardiac tissue were confirmed. MosaicHunter detected an additional 64 mosaics, of which 23/46 (50%) among 58 candidates from blood and 4/6 (67%) of 6 candidates from cardiac tissue confirmed. Twenty-five mosaic variants altered CHD-risk genes, affecting 1% of our cohort. Of these 25, 22/22 candidates tested were confirmed. Variants predicted as damaging had higher variant allele fraction than benign variants, suggesting a role in CHD. The estimated true frequency of mosaic variants above 10% mosaicism was 0.14/person in blood and 0.21/person in cardiac tissue. Analysis of 66 individuals with matched cardiac tissue available revealed both tissue-specific and shared mosaicism, with shared mosaics generally having higher allele fraction. CONCLUSIONS: We estimate that ~ 1% of CHD probands have a mosaic variant detectable in blood that could contribute to cardiac malformations, particularly those damaging variants with relatively higher allele fraction. Although blood is a readily available DNA source, cardiac tissues analyzed contributed ~ 5% of somatic mosaic variants identified, indicating the value of tissue mosaicism analyses.

Hypertrophic cardiomyopathy mutations in MYBPC3 dysregulate myosin.

The mechanisms by which truncating mutations in MYBPC3 (encoding cardiac myosin-binding protein C; cMyBPC) or myosin missense mutations cause hypercontractility and poor relaxation in hypertrophic cardiomyopathy (HCM) are incompletely understood. Using genetic and biochemical approaches, we explored how depletion of cMyBPC altered sarcomere function. We demonstrated that stepwise loss of cMyBPC resulted in reciprocal augmentation of myosin contractility. Direct attenuation of myosin function, via a damaging missense variant (F764L) that causes dilated cardiomyopathy (DCM), normalized the increased contractility from cMyBPC depletion. Depletion of cMyBPC also altered dynamic myosin conformations during relaxation, enhancing the myosin state that enables ATP hydrolysis and thin filament interactions while reducing the super relaxed conformation associated with energy conservation. MYK-461, a pharmacologic inhibitor of myosin ATPase, rescued relaxation deficits and restored normal contractility in mouse and human cardiomyocytes with MYBPC3 mutations. These data define dosage-dependent effects of cMyBPC on myosin that occur across the cardiac cycle as the pathophysiologic mechanisms by which MYBPC3 truncations cause HCM. Therapeutic strategies to attenuate cMyBPC activity may rescue depressed cardiac contractility in patients with DCM, whereas inhibiting myosin by MYK-461 should benefit the substantial proportion of patients with HCM with MYBPC3 mutations.

Droplet Digital PCR with EvaGreen Assay: Confirmational Analysis of Structural Variants.

DNA structural variants can be analyzed by droplet digital PCR (ddPCR), a water-oil microfluidics and fluorescence technology to quantify target nucleic acids with extreme precision and sensitivity. Traditional ddPCR uses expensive fluorescent oligonucleotide probes that require extensive optimization. Here we describe a variation of ddPCR using a DNA-binding dye (EvaGreen), whose properties allow target products to be effectively quantified at a significantly lower cost. © 2018 by John Wiley & Sons, Inc.

Robust identification of deletions in exome and genome sequence data based on clustering of Mendelian errors.

Multiple tools have been developed to identify copy number variants (CNVs) from whole exome (WES) and whole genome sequencing (WGS) data. Current tools such as XHMM for WES and CNVnator for WGS identify CNVs based on changes in read depth. For WGS, other methods to identify CNVs include utilizing discordant read pairs and split reads and genome-wide local assembly with tools such as Lumpy and SvABA, respectively. Here, we introduce a new method to identify deletion CNVs from WES and WGS trio data based on the clustering of Mendelian errors (MEs). Using our Mendelian Error Method (MEM), we identified 127 deletions (inherited and de novo) in 2,601 WES trios from the Pediatric Cardiac Genomics Consortium, with a validation rate of 88% by digital droplet PCR. MEM identified additional de novo deletions compared with XHMM, and a significant enrichment of 15q11.2 deletions compared with controls. In addition, MEM identified eight cases of uniparental disomy, sample switches, and DNA contamination. We applied MEM to WGS data from the Genome In A Bottle Ashkenazi trio and identified deletions with 97% specificity. MEM provides a robust, computationally inexpensive method for identifying deletions, and an orthogonal approach for verifying deletions called by other tools.

Robust identification of mosaic variants in congenital heart disease.

Mosaicism due to somatic mutations can cause multiple diseases including cancer, developmental and overgrowth syndromes, neurodevelopmental disorders, autoinflammatory diseases, and atrial fibrillation. With the increased use of next generation sequencing technology, multiple tools have been developed to identify low-frequency variants, specifically from matched tumor-normal tissues in cancer studies. To investigate whether mosaic variants are implicated in congenital heart disease (CHD), we developed a pipeline using the cancer somatic variant caller MuTect to identify mosaic variants in whole-exome sequencing (WES) data from a cohort of parent/affected child trios (n = 715) and a cohort of healthy individuals (n = 416). This is a novel application of the somatic variant caller designed for cancer to WES trio data. We identified two cases with mosaic KMT2D mutations that are likely pathogenic for CHD, but conclude that, overall, mosaicism detectable in peripheral blood or saliva does not account for a significant portion of CHD etiology.

Differential Potency of 2,6-Dimethylcyclohexanol Isomers for Positive Modulation of GABAA Receptor Currents.

GABAA receptors meet all of the pharmacological requirements necessary to be considered important targets for the action of general anesthetic agents in the mammalian brain. In the following patch-clamp study, the relative modulatory effects of 2,6-dimethylcyclohexanol diastereomers were investigated on human GABAA (α1ß3γ2s) receptor currents stably expressed in human embryonic kidney cells. Cis,cis-, trans,trans-, and cis,trans-isomers were isolated from commercially available 2,6-dimethylcyclohexanol and were tested for positive modulation of submaximal GABA responses. For example, the addition of 30 µM cis,cis-isomer resulted in an approximately 2- to 3-fold enhancement of the EC20 GABA current. Coapplications of 30 µM 2,6-dimethylcyclohexanol isomers produced a range of positive enhancements of control GABA responses with a rank order for positive modulation: cis,cis > trans,trans ≥ mixture of isomers > > cis,trans-isomer. In molecular modeling studies, the three cyclohexanol isomers bound with the highest binding energies to a pocket within transmembrane helices M1 and M2 of the ß3 subunit through hydrogen-bonding interactions with a glutamine at the 224 position and a tyrosine at the 220 position. The energies for binding to and hydrogen-bond lengths within this pocket corresponded with the relative potencies of the agents for positive modulation of GABAA receptor currents (cis,cis > trans,trans > cis,trans-2,6-dimethylcyclohexanol). In conclusion, the stereochemical configuration within the dimethylcyclohexanols is an important molecular feature in conferring positive modulation of GABAA receptor activity and for binding to the receptor, a consideration that needs to be taken into account when designing novel anesthetics with enhanced therapeutic indices.

MR imaging findings of ectopic pregnancy: a pictorial review.

Because of its lack of ionizing radiation and excellent soft-tissue contrast, magnetic resonance (MR) imaging is being increasingly used in the evaluation of acute abdominal pain in the pregnant patient. Roughly 2% of all pregnancies are ectopic. Although ectopic pregnancy is usually diagnosed on the basis of a combination of clinical, laboratory, and ultrasonographic findings, it occasionally is initially identified at MR imaging. Thus, it is imperative that the radiologist should be familiar with the variable appearance of ectopic pregnancy at MR imaging and should evaluate for ectopic pregnancy at any time when (a) a patient has positive results of a pregnancy test and (b) an intrauterine pregnancy is not definitively seen. Because of potential issues of fetal safety, a conservative approach should be used for MR imaging in pregnancy. An MR imaging protocol for the evaluation of possible appendicitis in pregnant women is detailed. Specific findings that can aid in the diagnosis of ectopic pregnancy are the lack of an intrauterine pregnancy, isolated hemoperitoneum, tubal masses, hematosalpinx, and interstitial masses. In the differential diagnosis of acute abdominal pain in pregnancy, consideration should be given to the more unusual forms of ectopic pregnancy, such as angular pregnancy, cornual pregnancy, and abdominal pregnancy. Potential mimics of ectopic pregnancy include placental abnormalities, ovarian neoplasms, and corpus luteum cysts.

Accuracy of cross-table lateral knee radiography for evaluation of joint effusions.

OBJECTIVE: The purpose of our study was to investigate the efficacy of cross-table lateral knee radiography in the diagnosis of knee effusions compared with an MRI reference standard, to evaluate reader experience in effusion assessment, and to establish a new threshold for suprapatellar pouch measurement for the diagnosis of effusion. MATERIALS AND METHODS: First- and third-year radiology residents and an attending musculoskeletal radiologist retrospectively assessed 108 cross-table lateral knee radiographs for qualitative grading of joint fluid and quantitative measurement of the suprapatellar pouch. Qualitative and quantitative evaluation of ipsilateral knee MRI examinations performed within 1 week of radiography was performed by two attending musculoskeletal radiologists as a reference standard. RESULTS: Qualitative visual grading of cross-table lateral radiographs had a sensitivity of 90-92%, specificity of 39-54%, and accuracy of 69-76% for joint effusion. Extrapolating from previous work showing 4 mL of fluid distends the suprapatellar pouch to 4 mm on midline sagittal MRI, the corresponding measurement on cross-table lateral radiographs was predicted to be 7 mm. Using this new criterion of effusion, sensitivity, specificity, and accuracy compared with an MR midline sagittal reference standard were 76%, 83%, and 81%, respectively. Historical data for overhead lateral radiographs had a sensitivity of 78%, specificity of 80%, and accuracy of 79%. CONCLUSION: Qualitative visual assessment of cross-table lateral knee radiographs is highly sensitive for the detection of joint effusion. By performing quantitative evaluation with a new 7-mm criterion for suprapatellar pouch measurement, sensitivity, specificity, and accuracy are equivalent to that of overhead lateral radiography.

Foreign body ingestion of blister pill pack causing small bowel obstruction.

We report a case of foreign body ingestion of a blister pill pack, causing small bowel obstruction. A 76-year-old woman on multiple medications presented with 3 days of progressive abdominal distention, nausea, and vomiting. A computed tomography (CT) scan demonstrated small bowel obstruction with a distinctive metallic foreign body in the distal ileum with associated wall thickening and mesenteric inflammatory changes. At exploratory laparotomy, an impacted, intact blister pill pack was removed from the distal ileum. The ingestion of blister pill packs has been associated with a range of clinical and imaging findings. To our knowledge, this is the only reported case of CT diagnosis of small bowel obstruction caused by blister pack ingestion. Early recognition of the imaging findings of an ingested blister pill pack is important to expedite appropriate management.