Functional characterization of 2,832 JAG1 variants supports reclassification for Alagille syndrome and improves guidance for clinical variant interpretation.

Pathogenic variants in the JAG1 gene are a primary cause of the multi-system disorder Alagille syndrome. Although variant detection rates are high for this disease, there is uncertainty associated with the classification of missense variants that leads to reduced diagnostic yield. Consequently, up to 85% of reported JAG1 missense variants have uncertain or conflicting classifications. We generated a library of 2,832 JAG1 nucleotide variants within exons 1-7, a region with a high number of reported missense variants, and designed a high-throughput assay to measure JAG1 membrane expression, a requirement for normal function. After calibration using a set of 175 known or predicted pathogenic and benign variants included within the variant library, 486 variants were characterized as functionally abnormal (n = 277 abnormal and n = 209 likely abnormal), of which 439 (90.3%) were missense. We identified divergent membrane expression occurring at specific residues, indicating that loss of the wild-type residue itself does not drive pathogenicity, a finding supported by structural modeling data and with broad implications for clinical variant classification both for Alagille syndrome and globally across other disease genes. Of 144 uncertain variants reported in patients undergoing clinical or research testing, 27 had functionally abnormal membrane expression, and inclusion of our data resulted in the reclassification of 26 to likely pathogenic. Functional evidence augments the classification of genomic variants, reducing uncertainty and improving diagnostics. Inclusion of this repository of functional evidence during JAG1 variant reclassification will significantly affect resolution of variant pathogenicity, making a critical impact on the molecular diagnosis of Alagille syndrome.

Genome sequencing increases diagnostic yield in clinically diagnosed Alagille syndrome patients with previously negative test results.

PURPOSE: Detection of all major classes of genomic variants in a single test would decrease cost and increase the efficiency of genomic diagnostics. Genome sequencing (GS) has the potential to provide this level of comprehensive detection. We sought to demonstrate the utility of GS in the molecular diagnosis of 18 patients with clinically defined Alagille syndrome (ALGS), who had a negative or inconclusive result by standard-of-care testing. METHODS: We performed GS on 16 pathogenic variant-negative probands and two probands with inconclusive results (of 406 ALGS probands) and analyzed the data for sequence, copy-number, and structural variants in JAG1 and NOTCH2. RESULTS: GS identified four novel pathogenic alterations including a copy-neutral inversion, a partial deletion, and a promoter variant in JAG1, and a partial NOTCH2 deletion, for an additional diagnostic yield of 0.9%. Furthermore, GS resolved two complex rearrangements, resulting in identification of a pathogenic variant in 97.5% (n = 396/406) of patients after GS. CONCLUSION: GS provided an increased diagnostic yield for individuals with clinically defined ALGS who had prior negative or incomplete genetic testing by other methods. Our results show that GS can detect all major classes of variants and has potential to become a single first-tier diagnostic test for Mendelian disorders.

Impaired Redox and Protein Homeostasis as Risk Factors and Therapeutic Targets in Toxin-Induced Biliary Atresia.

BACKGROUND & AIMS: Extrahepatic biliary atresia (BA) is a pediatric liver disease with no approved medical therapy. Recent studies using human samples and experimental modeling suggest that glutathione redox metabolism and heterogeneity play a role in disease pathogenesis. We sought to dissect the mechanistic basis of liver redox variation and explore how other stress responses affect cholangiocyte injury in BA. METHODS: We performed quantitative in situ hepatic glutathione redox mapping in zebrafish larvae carrying targeted mutations in glutathione metabolism genes and correlated these findings with sensitivity to the plant-derived BA-linked toxin biliatresone. We also determined whether genetic disruption of HSP90 protein quality control pathway genes implicated in human BA altered biliatresone toxicity in zebrafish and human cholangiocytes. An in vivo screening of a known drug library was performed to identify novel modifiers of cholangiocyte injury in the zebrafish experimental BA model, with subsequent validation. RESULTS: Glutathione metabolism gene mutations caused regionally distinct changes in the redox potential of cholangiocytes that differentially sensitized them to biliatresone. Disruption of human BA-implicated HSP90 pathway genes sensitized zebrafish and human cholangiocytes to biliatresone-induced injury independent of glutathione. Phosphodiesterase-5 inhibitors and other cyclic guanosine monophosphate signaling activators worked synergistically with the glutathione precursor N-acetylcysteine in preventing biliatresone-induced injury in zebrafish and human cholangiocytes. Phosphodiesterase-5 inhibitors enhanced proteasomal degradation and required intact HSP90 chaperone. CONCLUSION: Regional variation in glutathione metabolism underlies sensitivity to the biliary toxin biliatresone and may account for the reported association between BA transplant-free survival and glutathione metabolism gene expression. Human BA can be causatively linked to genetic modulation of protein quality control. Combined treatment with N-acetylcysteine and cyclic guanosine monophosphate signaling enhancers warrants further investigation as therapy for BA.

Thermally Stable Low-Loss Polymer Dielectrics Enabled by Attaching Cross-Linkable Antioxidant to Polypropylene.

Polymer dielectrics with low-loss and high-temperature tolerance are extremely desirable as electrical energy storage materials for advanced electronics and electrical power applications. They can allow fast switching rates during power conversion and therefore achieve high power densities without thermal issues. Here, we explore polypropylene (PP), the state of the art dielectric polymer, and present an innovative approach to substantially improve the thermal stability and concurrently reduce the dielectric loss of PP. In particular, cross-linkable antioxidant groups, hindered phenol (HP), are incorporated into PP via well-controlled chemical synthesis. The grafted HP can simultaneously serve as radical scavenger and cross-linker, thereby constraining thermally decomposed radicals and charge transport in the synthesized PP-HP copolymer. As a result, the upper-temperature limit of PP-HP is greatly extended to 190 °C and the electrical loss is even gradually reduced upon thermal annealing. The copolymer after heating under 190 °C exhibits better dielectric properties than the PP without any thermal treatment. The experimental results indicate that the PP-HP copolymers are promising materials for high-temperature, low-loss, and high-voltage dielectric applications.

Protein-elongating mutations in MYH11 are implicated in a dominantly inherited smooth muscle dysmotility syndrome with severe esophageal, gastric, and intestinal disease.

Gastrointestinal motility disorders include a spectrum of mild to severe clinical phenotypes that are caused by smooth muscle dysfunction. We investigated the genetic etiology of severe esophageal, gastric, and colonic dysmotility in two unrelated families with autosomal dominant disease presentation. Using exome sequencing, we identified a 2 base pair insertion at the end of the myosin heavy chain 11 (MYH11) gene in all affected members of Family 1 [NM_001040113:c.5819_5820insCA(p.Gln1941Asnfs*91)] and a 1 base pair deletion at the same genetic locus in Proband 2 [NM_001040113:c.5819del(p.Pro1940Hisfs*91)]. Both variants are predicted to result in a similarly elongated protein product. Heterozygous dominant negative MYH11 pathogenic variants have been associated with thoracic aortic aneurysm and dissection while biallelic null alleles have been associated with megacystis microcolon intestinal hypoperistalsis syndrome. This report highlights heterozygous protein-elongating MYH11 variants affecting the SM2 isoforms of MYH11 as a cause for severe gastrointestinal dysmotility, and we hypothesize that the mechanistic pathogenesis of this disease, dominant hypercontractile loss-of-function, is distinct from those implicated in other diseases involving MYH11 dysfunction.

Alagille syndrome mutation update: Comprehensive overview of JAG1 and NOTCH2 mutation frequencies and insight into missense variant classification.

Alagille syndrome is an autosomal dominant disease with a known molecular etiology of dysfunctional Notch signaling caused primarily by pathogenic variants in JAGGED1 (JAG1), but also by variants in NOTCH2. The majority of JAG1 variants result in loss of function, however disease has also been attributed to lesser understood missense variants. Conversely, the majority of NOTCH2 variants are missense, though fewer of these variants have been described. In addition, there is a small group of patients with a clear clinical phenotype in the absence of a pathogenic variant. Here, we catalog our single-center study, which includes 401 probands and 111 affected family members amassed over a 27-year period, to provide updated mutation frequencies in JAG1 and NOTCH2 as well as functional validation of nine missense variants. Combining our cohort of 86 novel JAG1 and three novel NOTCH2 variants with previously published data (totaling 713 variants), we present the most comprehensive pathogenic variant overview for Alagille syndrome. Using this data set, we developed new guidance to help with the classification of JAG1 missense variants. Finally, we report clinically consistent cases for which a molecular etiology has not been identified and discuss the potential for next generation sequencing methodologies in novel variant discovery.

Compound heterozygous mutations in NEK8 in siblings with end-stage renal disease with hepatic and cardiac anomalies.

We studied two brothers who presented in the newborn period with cardiac, renal, and hepatic anomalies that were initially suggestive of ALGS, although no mutations in JAG1 or NOTCH2 were identified. Exome sequencing demonstrated compound heterozygous mutations in the NEK8 gene (Never in mitosis A-related Kinase 8), a ciliary kinase indispensable for cardiac and renal development based on murine studies. The mutations included a c.2069_2070insC variant (p.Ter693LeufsTer86), and a c.1043C>T variant (p.Thr348Met) in the highly conserved RCC1 (Regulation of Chromosome Condensation 1) domain. The RCC1 domain is crucial for localization of the NEK8 protein to the centrosomes and cilia. Mutations in NEK8 have been previously reported in three fetuses (from a single family) with renal-hepatic-pancreatic dysplasia 2 (RHPD2), similar to Ivemark syndrome, and in three individuals with nephronophthisis (NPHP9). This is the third report of disease-causing mutations in the NEK8 gene in humans and only the second describing multi-organ involvement. The clinical features we describe differ from those in the previously published report in that (1) a pancreatic phenotype was not observed in the individuals reported here, (2) there were more prominent cardiac findings, (consistent with observations in murine models), and (3) we observed bile duct hypoplasia rather than ductal plate malformation. The patients reported here expand our understanding of the NEK8-associated phenotype. Our findings highlight the variable phenotypic expressivity and the spectrum of clinical manifestations due to mutations in the NEK8 gene.

Germline gain-of-function mutations in AFF4 cause a developmental syndrome functionally linking the super elongation complex and cohesin.

Transcriptional elongation is critical for gene expression regulation during embryogenesis. The super elongation complex (SEC) governs this process by mobilizing paused RNA polymerase II (RNAP2). Using exome sequencing, we discovered missense mutations in AFF4, a core component of the SEC, in three unrelated probands with a new syndrome that phenotypically overlaps Cornelia de Lange syndrome (CdLS) that we have named CHOPS syndrome (C for cognitive impairment and coarse facies, H for heart defects, O for obesity, P for pulmonary involvement and S for short stature and skeletal dysplasia). Transcriptome and chromatin immunoprecipitation sequencing (ChIP-seq) analyses demonstrated similar alterations of genome-wide binding of AFF4, cohesin and RNAP2 in CdLS and CHOPS syndrome. Direct molecular interaction of the SEC, cohesin and RNAP2 was demonstrated. These data support a common molecular pathogenesis for CHOPS syndrome and CdLS caused by disturbance of transcriptional elongation due to alterations in genome-wide binding of AFF4 and cohesin.

Heterozygous deletion of FOXA2 segregates with disease in a family with heterotaxy, panhypopituitarism, and biliary atresia.

Biliary atresia (BA) is a pediatric cholangiopathy with unknown etiology occurring in isolated and syndromic forms. Laterality defects affecting the cardiovascular and gastrointestinal systems are the most common features present in syndromic BA. Most cases are sporadic, although reports of familial cases have led to the hypothesis of genetic susceptibility in some patients. We identified a child with BA, malrotation, and interrupted inferior vena cava whose father presented with situs inversus, polysplenia, panhypopituitarism, and mildly dysmorphic facial features. Chromosomal microarray analysis demonstrated a 277 kb heterozygous deletion on chromosome 20, which included a single gene, FOXA2, in the proband and her father. This deletion was confirmed to be de novo in the father. The proband and her father share a common diagnosis of heterotaxy, but they also each presented with a variety of other issues. Further genetic screening revealed that the proband carried an additional protein-altering polymorphism (rs1904589; p.His165Arg) in the NODAL gene that is not present in the father, and this variant has been shown to decrease expression of the gene. As FOXA2 can be a regulator of NODAL expression, we propose that haploinsufficiency for FOXA2 combined with a decreased expression of NODAL is the likely cause for syndromic BA in this proband.

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, α1S 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.

Postoperative electroencephalographic seizures are associated with deficits in executive function and social behaviors at 4 years of age following cardiac surgery in infancy.

OBJECTIVE: The occurrence of an electroencephalographic (EEG) seizure after surgery for complex congenital heart defects has been associated with worse neurodevelopmental (ND) outcomes. We previously identified postoperative seizures documented by 48-hour EEG monitoring in 11% of 178 neonates and infants. Evaluation at 1 year of age did not identify an adverse effect of an EEG seizure on ND outcomes. The current study was undertaken to determine if testing in the preschool period would identify deficits that become apparent as children develop. METHODS: The ND outcomes assessed at 4 years of age included cognition, language, attention, impulsivity, executive function, behavior problems, academic achievement, and visual and fine motor skills. RESULTS: Developmental evaluations were performed in 132 (87%) of 151 survivors. For the entire cohort, the Full-Scale IQ was 95.0 ± 18.5. IQ was 95.1 ± 18.7 for patients without a history of seizure and 93.6 ± 16.7 for those with a history of seizure. After covariate adjustment, occurrence of an EEG seizure was associated with worse executive function (P = .037) and impaired social interactions/restricted behavior (P = .05). Seizures were not significantly associated with worse performance for cognition, language, attention, impulsivity, academic achievement, or motor skills (all P > .1). CONCLUSIONS: The occurrence of a postoperative seizure is a biomarker of brain injury. This study confirms that postoperative EEG seizures are associated with worse ND outcomes, characterized by impairments of executive function and a higher prevalence of deficits in social interactions and repetitive/restricted behaviors in preschool survivors of cardiac surgery in infancy. However, EEG seizures were not associated with worse cognitive, language, or motor skills.

Large-scale gene-centric meta-analysis across 32 studies identifies multiple lipid loci.

Genome-wide association studies (GWASs) have identified many SNPs underlying variations in plasma-lipid levels. We explore whether additional loci associated with plasma-lipid phenotypes, such as high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and triglycerides (TGs), can be identified by a dense gene-centric approach. Our meta-analysis of 32 studies in 66,240 individuals of European ancestry was based on the custom ∼50,000 SNP genotyping array (the ITMAT-Broad-CARe array) covering ∼2,000 candidate genes. SNP-lipid associations were replicated either in a cohort comprising an additional 24,736 samples or within the Global Lipid Genetic Consortium. We identified four, six, ten, and four unreported SNPs in established lipid genes for HDL-C, LDL-C, TC, and TGs, respectively. We also identified several lipid-related SNPs in previously unreported genes: DGAT2, HCAR2, GPIHBP1, PPARG, and FTO for HDL-C; SOCS3, APOH, SPTY2D1, BRCA2, and VLDLR for LDL-C; SOCS3, UGT1A1, BRCA2, UBE3B, FCGR2A, CHUK, and INSIG2 for TC; and SERPINF2, C4B, GCK, GATA4, INSR, and LPAL2 for TGs. The proportion of explained phenotypic variance in the subset of studies providing individual-level data was 9.9% for HDL-C, 9.5% for LDL-C, 10.3% for TC, and 8.0% for TGs. This large meta-analysis of lipid phenotypes with the use of a dense gene-centric approach identified multiple SNPs not previously described in established lipid genes and several previously unknown loci. The explained phenotypic variance from this approach was comparable to that from a meta-analysis of GWAS data, suggesting that a focused genotyping approach can further increase the understanding of heritability of plasma lipids.

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.

Endoscopic sinus surgery in chronic rhinosinusitis and nasal polyposis: a comparative study.

Nasal polyposis are common presentations in patients of chronic rhinosinusitis and are considered to be associated with more severe forms of disease with poor treatment outcome. The presentation and treatment outcome after endoscopic sinus surgery in patients of chronic rhinosinusitis and nasal polyposis have been analysed in this study. A prospective analysis of 90 patients of chronic rhinosinusitis who were classified into two groups depending on presence and absence of nasal polyps was performed in the study. The two groups were evaluated using subjective (patient complaints) and objective (computed tomography scan and endoscopy scores) criteria. Preoperative data were compared with data obtained 12 months post endoscopic sinus surgery. The study included 38 patients of chronic rhinosinusitis and 52 patients of nasal polyps. The patients of nasal polyp group presented with increased severity of symptoms of nasal blockage, nasal discharge and reduced sense of smell as compared to the chronic rhinosinusitis group who had significantly higher presentation of headache and facial pain. The preoperative CT scan revealed significantly higher bilateral disease with increased involvement of multiple sinuses in nasal polyp group. Post endoscopic sinus surgery both the groups showed significant improvement in their symptoms with the nasal polyp group demonstrating reduction in improvement on 1 year follow up. In our study we have found the patients with chronic rhinosinusitis and nasal polyp have varied severity of symptoms with the nasal polyp group having higher nasal symptoms and increased severity as compared to chronic rhinosinusitis group. Though the universal rationale of management by adequate drainage and ventilation of sinus is similar in both groups, there is a reduction in both objective and subjective scores during 1 year follow up in the nasal polyp group.

J. Maxwell Chamberlain Memorial Paper for congenital heart surgery. 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.

Genome-wide association study of plasma lipoprotein(a) levels identifies multiple genes on chromosome 6q.

Plasma lipoprotein(a) (Lp[a]) level is an independent risk factor of cardiovascular disease that is under strong genetic control. We conducted a genome-wide association study of plasma Lp(a) in 386 members of a founder population that adheres to a communal lifestyle, proscribes cigarette smoking, and prepares and eats meals communally. We identified associations with 77 single nucleotide polymorphisms (SNPs) spanning 12.5 Mb on chromosome 6q26-q27 that met criteria for genome-wide significance (P