Pathogenic Missense Variants Research Articles

Expanding the phenotypic and biochemical spectrum of NDUFAF3-related mitochondrial disease

Recessive variants in NDUFAF3 are a known cause of complex I (CI)-related mitochondrial disorders (MDs). The seven patients reported to date exhibited severe neurologic symptoms and lactic acidosis, followed by a fatal course and death during infancy in most cases.We present a 10-year-old patient with a neurodevelopmental disorder, progressive exercise intolerance, dystonia, basal ganglia abnormalities, and elevated lactate concentration in blood. Trio-exome sequencing revealed compound-heterozygosity for a pathogenic splice-site and a likely pathogenic missense variant in NDUFAF3. Spectrophotometric analysis of fibroblast-derived mitochondria demonstrated a relatively mild reduction of CI activity. Complexome analyses revealed severely reduced NDUFAF3 as well as CI in patient fibroblasts. Accumulation of early sub-assemblies of the membrane arm of CI associated with mitochondrial complex I intermediate assembly (MCIA) complex was observed. The most striking additional findings were both the unusual occurrence of free monomeric CI holding MCIA and other assembly factors.Here we discuss our patient in context of genotype, phenotype and metabolite data from previously reported NDUFAF3 cases. With the atypical presentation of our patient, we provide further insight into the phenotypic spectrum of NDUFAF3-related MDs. Complexome analysis in our patient confirms the previously defined role of NDUFAF3 within CI biogenesis, yet adds new aspects regarding the correct timing of both the association of soluble and membrane arm modules and CI-maturation as well as respiratory supercomplex formation.

Development of a novel prediction model based on protein structure for identifying RPE65-associated inherited retinal disease (IRDs) of missense variants.

This study aimed to develop a prediction model to classify RPE65-mediated inherited retinal disease (IRDs) based on protein secondary structure and to analyze phenotype-protein structure correlations of RPE65 missense variants in a Chinese cohort. Pathogenic or likely pathogenic missense variants of RPE65 were obtained from UniProt, ClinVar, and HGMD databases. The three-dimensional structure of RPE65 was retrieved from the Protein Data Bank (PDB) and modified with Pymol software. A novel prediction model was developed using LASSO regression and multivariate logistic regression to identify RPE65-associated IRDs. A total of 21 Chinese probands with RPE65 variants were collected to analyze phenotype-protein structure correlations of RPE65 missense variants. The study found that both pathogenic and population missense variants were associated with structural features of RPE65. Pathogenic variants were linked to sheet, β-sheet, strands, β-hairpins, Fe2+ (iron center), and active site cavity, while population variants were related to helix, loop, helices, and helix-helix interactions. The novel prediction model showed accuracy and confidence in predicting the disease type of RPE65 variants (AUC = 0.7531). The study identified 25 missense variants in Chinese patients, accounting for 72.4% of total mutations. A significant correlation was observed between clinical characteristics of RPE65-associated IRDs and changes in amino acid type, specifically for missense variants of F8 (H68Y, P419S). The study developed a novel prediction model based on the protein structure of RPE65 and investigated phenotype-protein structure correlations of RPE65 missense variants in a Chinese cohort. The findings provide insights into the precise diagnosis of RPE65-mutated IRDs.

Haploinsufficiency of A20 caused by a novel pathogenic missense variant of TNFAIP3 and successfully treated with anti-TNF and immunosuppressive therapies

Loss-of-function of protein A20, encoded by TNFAIP3, leads to an early-onset haploinsufficiency of A20 (HA20). This study reports one Chinese child with HA20 and explores the genetic etiology of TNFAIP3 variant. The patient exhibited transient recurrent episodes of fever, intermittent signs of arthritis, gastrointestinal symptoms and multiple colonic ulcers. Laboratory tests revealed elevated inflammatory indicators and mild to moderate anemia. Genetic analysis identified a heterozygous de novo variant in his TNFAIP3 gene (c.740C＞T, p. P247L), which had never been reported before. The novel missense variation was validated to be pathogenic through causing insufficient expression of A20, over-activation of NF-κB signaling pathway and elevated levels of proinflammatory cytokines in response to stimulation by lipopolysaccharide. A combination of oral corticosteroids, TNF-α inhibitors and thalidomide freed him from symptoms and abnormal inflammatory indicators. Furthermore, continual improvement of the patient's condition was observed during a follow-up period of five months. We demonstrate a case with a de novo missense variant resulting in a loss-of-function of TNFAIP3, which expands the clinical spectrum of HA20. Cytokine antagonists and immunosuppressants may be effective drugs.

Screening and evaluation of TBX20 and CITED2 mutations in children with congenital cardiac septal defects: Correlation with cardiac troponin T and caspase-3

Congenital cardiac septal defect (CCSD) is the main type of congenital heart disease and owns a very high mortality rate among newborns. CCSD is controlled by specific transcription factors, including T-box transcription factor 20 (TBX20) and Cbp/P300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 2 (CITED2) which are key molecular actors in heart development. Here, we screened for mutations in TBX20 and CITED2 genes in Egyptian children with CCSD and assessed their association with CCSD susceptibility and with cardiac troponin T (cTnT) and the apoptotic marker caspase-3 as biochemical markers for CCSD. Thirty unrelated newborns and children affected with CCSD and 30 matched healthy controls with no personal history of cardiac diseases were recruited. Selection criteria were children (<18 years) with any age diagnosed with CCSD using ECHO. Mutational analysis and genotyping were done using PCR-Sanger DNA sequencing technique. Serum cTnT and caspase-3 were analyzed using ELISA. Sequencing analysis identified 2 TBX20 variants (c.766T>C and c.39T>C) in the CCSD and control groups and 2 CITED2 variants (c.12T>C and c.9C>T) in one CCSD patient, while were absent in controls. In silico analysis identified TBX20 c.766T>C (rs3999941) as a missense (F256L) pathogenic variant and the other three variants as synonymous and benign. Compared with controls, TBX20 c.766T>C TC genotype and minor C allele were candidate high-risk factors for CCSD. Besides, serum cTnT and caspase-3 were dramatically elevated in CCSD children compared to controls. TBX20 c.766T>C TC genotype was associated with high cTnT in CCSD children. Conclusively, we advocate TBX20 c.766T>C variant as a potential genetic marker for CCSD which might associate with high cTnT levels. CITED2 genetic variants might have rare incidence among Egyptian CCSD children. Serum cTnT and caspase-3 are useful markers for ascertaining CCSD in children. These data could be exploited in prenatal genetic counseling, pre-implantation genotyping, and therapy of CCSD.

A novel, pathogenic COL4A1 missense variant involving thrombotic microangiopathic leukoencephalopathy in a neonate.

A novel, pathogenic COL4A1 missense variant involving thrombotic microangiopathic leukoencephalopathy in a neonate.

In vivo versus in silico assessment of potentially pathogenic missense variants in human reproductive genes

Infertility is a heterogeneous condition, with genetic causes thought to underlie a substantial fraction of cases. Genome sequencing is becoming increasingly important for genetic diagnosis of diseases including idiopathic infertility; however, most rare or minor alleles identified in patients are variants of uncertain significance (VUS). Interpreting the functional impacts of VUS is challenging but profoundly important for clinical management and genetic counseling. To determine the consequences of these variants in key fertility genes, we functionally evaluated 11 missense variants in the genes ANKRD31, BRDT, DMC1, EXO1, FKBP6, MCM9, M1AP, MEI1, MSH4 and SEPT12 by generating genome-edited mouse models. Nine variants were classified as deleterious by most functional prediction algorithms, and two disrupted a protein-protein interaction (PPI) in the yeast two hybrid (Y2H) assay. Though these genes are essential for normal meiosis or spermiogenesis in mice, only one variant, observed in the MCM9 gene of a male infertility patient, compromised fertility or gametogenesis in the mouse models. To explore the disconnect between predictions and outcomes, we compared pathogenicity calls of missense variants made by ten widely used algorithms to 1) those annotated in ClinVar and 2) those evaluated in mice. All the algorithms performed poorly in terms of predicting the effects of human missense variants modeled in mice. These studies emphasize caution in the genetic diagnoses of infertile patients based primarily on pathogenicity prediction algorithms and emphasize the need for alternative and efficient in vitro or in vivo functional validation models for more effective and accurate VUS description to either pathogenic or benign categories.

Multimorbidity due to novel pathogenic variants in the WFS1/RP1/NOD2 genes: autosomal dominant congenital lamellar cataract, retinitis pigmentosa and Crohn’s disease in a British family

BackgroundA five generation family has been analysed by whole exome sequencing (WES) for genetic associations with the multimorbidities of congenital cataract (CC), retinitis pigmentosa (RP) and Crohn’s disease (CD).MethodsWES was...

A recurrent homozygous LMNA missense variant p.Thr528Met causes atypical progeroid syndrome characterized by mandibuloacral dysostosis, severe muscular dystrophy, and skeletal deformities.

Atypical progeroid syndromes (APS) are premature aging syndromes caused by pathogenic LMNA missense variants, associated with unaltered expression levels of lamins A and C, without accumulation of wild-type or deleted prelamin A isoforms, as observed in Hutchinson-Gilford progeria syndrome (HGPS) or HGPS-like syndromes. A specific LMNA missense variant, (p.Thr528Met), was previously identified in a compound heterozygous state in patients affected by APS and severe familial partial lipodystrophy, whereas heterozygosity was recently identified in patients affected by Type 2 familial partial lipodystrophy. Here, we report four unrelated boys harboring homozygosity for the p.Thr528Met, variant who presented with strikingly homogeneous APS clinical features, including osteolysis of mandibles, distal clavicles and phalanges, congenital muscular dystrophy with elevated creatine kinase levels, and major skeletal deformities. Immunofluorescence analyses of patient-derived primary fibroblasts showed a high percentage of dysmorphic nuclei with nuclear blebs and typical honeycomb patterns devoid of lamin B1. Interestingly, in some protrusions emerin or LAP2α formed aberrant aggregates, suggesting pathophysiology-associated clues. These four cases further confirm that a specific LMNA variant can lead to the development of strikingly homogeneous clinical phenotypes, in these particular cases a premature aging phenotype with major musculoskeletal involvement linked to the homozygous p.Thr528Met variant.

Smooth muscle α-actin missense variant promotes atherosclerosis through modulation of intracellular cholesterol in smooth muscle cells.

The variant p.Arg149Cys in ACTA2, which encodes smooth muscle cell (SMC)-specific α-actin, predisposes to thoracic aortic disease and early onset coronary artery disease in individuals without cardiovascular risk factors. This study investigated how this variant drives increased atherosclerosis. Apoe-/- mice with and without the variant were fed a high-fat diet for 12 weeks, followed by evaluation of atherosclerotic plaque formation and single-cell transcriptomics analysis. SMCs explanted from Acta2R149C/+ and wildtype (WT) ascending aortas were used to investigate atherosclerosis-associated SMC phenotypic modulation. Hyperlipidemic Acta2R149C/+Apoe-/- mice have a 2.5-fold increase in atherosclerotic plaque burden compared to Apoe-/- mice with no differences in serum lipid levels. At the cellular level, misfolding of the R149C α-actin activates heat shock factor 1, which increases endogenous cholesterol biosynthesis and intracellular cholesterol levels through increased HMG-CoA reductase (HMG-CoAR) expression and activity. The increased cellular cholesterol in Acta2R149C/+ SMCs induces endoplasmic reticulum stress and activates PERK-ATF4-KLF4 signaling to drive atherosclerosis-associated phenotypic modulation in the absence of exogenous cholesterol, while WT cells require higher levels of exogenous cholesterol to drive phenotypic modulation. Treatment with the HMG-CoAR inhibitor pravastatin successfully reverses the increased atherosclerotic plaque burden in Acta2R149C/+Apoe-/- mice. These data establish a novel mechanism by which a pathogenic missense variant in a smooth muscle-specific contractile protein predisposes to atherosclerosis in individuals without hypercholesterolemia or other risk factors. The results emphasize the role of increased intracellular cholesterol levels in driving SMC phenotypic modulation and atherosclerotic plaque burden.

417-P: Functional Characterization of a Rare, Pathogenic Missense Variant Implicates FRAS1 as a Novel Gene for Risk of Progression to End-Stage Kidney Disease in Diabetes

Progressive renal decline is the central manifestation of diabetic nephropathy (DN) that ultimately leads to end-stage kidney disease (ESKD). Leveraging an innovative family-based approach that integrates electronic medical record data, a unique population-based genealogy resource, and next-generation sequencing, we recently identified a rare missense variant (p.R2516C, allele frequency=0.00002) in FRAS1, which encodes an extracellular matrix protein that regulates epidermal basement membrane adhesion and mediates glomerular integrity, in a family with diabetes and ESKD. While this rare missense variant is predicted to be deleterious (CADD score=32), whether this impacts FRAS1 function is unknown; here, we set out to assess the functional impact of this variant. To do so, we used CRISPR-Cas9 gene editing technology to generate 3 novel HEK293 cell lines (2 FRAS1 knock-in cell lines (a synonymous p.H2520H control cell line and a missense p.R2516C variant cell line) and a FRAS1 knock-out cell line) and performed in vitro cell migration and cell adhesion assays. Relative to the control cell line, both the FRAS1 knock-out cell line and the missense variant cell line displayed significantly inhibited cell migration (p = 1.54X10-13 and p = 5.05X10-5, respectively). Similarly, cell adhesion was significantly inhibited in cells in which FRAS1 was knocked out and in those carrying the p.R2516C variant compared to the control cell line (p = 6.72X10-9 and p = 6.63X10-6, respectively). Together, these data suggest that the FRAS1 p.R2516C variant negatively impacts FRAS1 function and further implicates this gene as having a potential role in the progression of ESKD in individuals with diabetes. Disclosure T.Chen: None. M.B.Neeley: None. J.M.Lazaro-guevara: None. M.Pezzolesi: None. C.A.Simeone: None. A.Rodan: None. W.L.Holland: Research Support; SymbioCellTech, LLC. M.G.Pezzolesi: Research Support; Renalytix. Funding National Institutes of Health (R01DK128641)

Study of The Molecular Nature of Congenital Cataracts in Patients from The Volga–Ural Region

Hereditary cataracts are characterized by significant clinical and genetic heterogeneity, which can pose challenges for early DNA diagnosis. To comprehensively address this problem, it is essential to investigate the epidemiology of the disease, perform population studies to determine the spectrum and frequencies of mutations in the responsible genes, and examine clinical and genetic correlations. Based on modern concepts, non-syndromic hereditary cataracts are predominantly caused by genetic disease forms associated with mutations in crystallin and connexin genes. Therefore, a comprehensive approach to studying hereditary cataracts is necessary for early diagnosis and improved treatment outcomes. The crystallin (CRYAA, CRYAB, CRYGC, CRYGD, and CRYBA1) and connexin (GJA8, GJA3) genes were analyzed in 45 unrelated families from the Volga–Ural Region (VUR) with hereditary congenital cataracts. Pathogenic and probably pathogenic nucleotide variants were identified in ten unrelated families, nine of which had cataracts in an autosomal dominant pattern of inheritance. Two previously undescribed likely pathogenic missense variants were identified in the CRYAA gene: c.253C &gt; T (p.L85F) in one family and c.291C &gt; G (p.H97Q) in two families. The known mutation c.272_274delGAG (p.G91del) was found in the CRYBA1 gene in one family, while no pathogenic variants were found in the CRYAB, CRYGC, or CRYGD genes in the examined patients. In the GJA8 gene, the known mutation c.68G &gt; C (p.R23T) was found in two families, and previously undescribed variants were identified in two other families: a c.133_142del deletion (p.W45Sfs*72) and a missense variant, c.179G &gt; A (p.G60D). In one patient with a recessive form of cataract, two compound-heterozygous variants were identified—a previously undescribed likely pathogenic missense variant, c.143A &gt; G (p.E48G), and a known variant with uncertain pathogenetic significance, c.741T &gt; G (p.I24M). Additionally, a previously undescribed deletion, c.del1126_1139 (p.D376Qfs*69), was identified in the GJA3 gene in one family. In all families where mutations were identified, cataracts were diagnosed either immediately after birth or during the first year of life. The clinical presentation of the cataracts varied depending on the type of lens opacity, resulting in various clinical forms. This information emphasizes the importance of early diagnosis and genetic testing for hereditary congenital cataracts to guide appropriate management and improve outcomes.

Structural and functional insights into a novel homozygous missense pathogenic variant in CUL7 identified in consanguineous Pakistani family

3M syndrome is a rare genetic familial disorder characterized by short stature, growth retardation, facial dysmorphism, skeletal abnormalities, fleshy protruding heels, and normal intelligence, caused by mutations in the CUL7, OBSL1 and CCDC8 genes. In the present study, a novel homozygous missense variant of CUL7 (NP_001161842.1, c.4493T > C, p.L1498P) has been identified in a consanguineous Pakistani family by whole exome sequencing. In silico structural evaluation, molecular docking and simulation studies of mutant CUL7 provides substantial evidence about its crucial role in the progression of discussed ailment. The newly discovered variant significantly altered the protein’s three dimensional structure, leading to abnormal interaction with binding proteins. This computational and experimental investigation provides useful information to drug developers for the synthesis of novel therapeutics against the discussed ailment. Communicated by Ramaswamy H. Sarma

#3166 RECOGNITION OF RENAL TUBULAR DYSGENESIS IN ADOLESCENT CKD BY BIALLELIC AGT VARIANTS REQUIRED BROAD GENETIC ANALYSIS

Abstract Background and Aims Autosomal recessive renal tubular dysgenesis (ARRTD) is a rare inherited disorder of renal (tubular) development, clinically characterized by fetal oligo-/anuria leading to oligohydramnion and Potter sequence, resulting in high mortality within the prenatal and neonatal period. Variants in genes encoding components of the renin-angiotensin system (RAS) are causative for this disorder. Herein, we report 2 European families with biallelic variants within AGT and aim to provide novel insides into disease understanding. Method The study was approved by our institutional ethics committee (approval number: 251_18 B). Clinical, histological and pedigree analysis were performed. Exome sequencing of a preselected gene panel (nephroma, 560 genes associated with renal disease) was analysed on the HiSeq System 2500 (Illumina) after enrichment by TWIST human core technology (TWIST Bioscience). Immunohistochemistry staining (IHC) and in-situ hybridization (ISH) for expression of renin were performed on kidney biopsy. In addition, renin expression was determined in primary tubular cells of the index patient by qPCR. Allele frequencies of heterozygous and biallelic predicted deleterious variants were determined by analysis of the Genomics England 100,000 Genomes Project. Results The first family was identified after transition from pediatric to adult nephrology at the University Hospital Erlangen. Initially, the male index patient of consanguineous Turkish descent presented with oligohydramnion in the prenatal period. Directly after birth (32nd gestational week) he suffered from profound hypotension, pulmonary hypoplasia and pulmonary stenosis, as well as third degree acute kidney injury leading to ICU treatment, but no need of hemodialysis. He survived the perinatal period while a decline in renal function was observed over time leading to CKD G3b at the age of 19 years. Exome sequencing revealed a previously reported pathogenic homozygous missense variant within AGT (c.1224G&amp;gt;A, p.Arg375Gln). Kidney biopsy at the age of 14 years revealed tubulointerstitial fibrosis, profound glomerulocystic changes with partly glomerulosclerosis and vasculopathic features. IHC and ISH showed recruitment and strong expression of renin in tubular cells and vessels, where cultured primary tubular cells also showed strong expression of renin. Circulating renin was profoundly elevated, with normal levels of aldosterone. The patient is not hypertensive and receives no antihypertensive medications. The family history revealed at least 2 miscarriages due to oligohydramnion. The second family was identified from the Genomics England 100,000 Genomes Project. The male index patient presented with prenatal bilateral hydronephrosis. He was delivered prematurely around the 35th gestational week. Both kidneys suffered from chronic bilateral urinary reflux leading to CKD G2 at the age of 8 years. Family history revealed one prior miscarriage. Genetic analysis revealed two compound heterozygous missense variants within AGT (c.842A&amp;gt;G, p.Tyr281Cys; c.151T&amp;gt;C, p.Cys51Arg). The heterozygous allele frequency in the Genomics England cohort for AGT predicted deleterious variants was very low. Therefore, we would predict a biallelic hit with the respective disease as an ultra-rare event. Reviewing the literature, to our knowledge 18 individuals are reported to date with different biallelic variants within AGT. From these cases only three survived the perinatal period. It is not reported if these individuals reached adulthood. Conclusion We hereby demonstrate two extremely rare cases, affected by biallelic variants within AGT, that survived the perinatal period and eventually led to chronic kidney disease. Since the phenotypes may be variable, a contemporary approach by broad genetic analysis is the only option to decode the genetic background in individuals with a suspected genetic disorder. Upregulation of renin in tubular cells by inactivation of AGT in the germline may drive tubulointerstitial fibrosis, which is a theoretical concern regarding ongoing targeted pharmacological approaches against AGT for arterial hypertension.

#3697 FAMILIAL PAPILLARY RENAL CELL CARCINOMA: A CASE REPORT

Abstract Background and Aims The incidence of Renal Cell Carcinoma (RCC) is the 9th commonest cancer worldwide. It originates from the renal tubular epithelium and to date has been subclassified into five different categories. Of which, papillary carcinoma is one subclassification and it includes two variants: sporadic and hereditary. This case report aims to outline a rare case of hereditary papillary RCC and the implications for the family involved. Method We performed a retrospective assessment of clinical and radiological characteristics of the index patient and affected relatives. Genetic diagnosis and management have been summarised. Results A 62-year-old male (II.1) was diagnosed with RCC after presenting with right-sided flank pain and multiple complex cystic lesions within both kidneys. A kidney biopsy revealed a papillary structure with foamy histocytes and clear cell-type epithelium clusters. Immunostaining of tumour cells demonstrated CK7 and P504S positivity consistent with RCC. Subsequently, he underwent bilateral nephrectomies and being rendered anephric, was commenced on haemodialysis. He underwent deceased donor renal transplantation four years later at the age of 66 (HLA 1:2:1, CMV -/-, cRF 35%) and remains dialysis independent six years on. After the diagnosis of the index patient, his son (III.1) was found to have multifocal papillary RCC, having also presented with flank pain, but this time in his 30s (Fig. 1). Patient III.1 underwent genetic testing which revealed a missense variant in the MET gene (NM_000245.4 c.3308G&amp;gt;A; p.Gly1103Glu). The variant affects a highly conserved residue in the Tyrosine Kinase domain of the MET protein. Pathogenic activating variants are associated with RCC. Identification of the same variant in the affected father with bilateral disease allowed classification of the variant as likely pathogenic according to the ACMG criteria [1]. The father has seven children, all of whom have now been tested. 5/7 of them have been found to carry the likely pathogenic MET missense variant. Of the 4 asymptomatic carriers, all are under surveillance by regular renal imaging and (one has had radiofrequency ablation). Conclusion The case outlines a rare form of hereditary RCC and the implications that detection of the genetic variant has meant in terms of early diagnosis of disease. Incidence of RCC is increasing worldwide. Hereditary papillary RCC is rare, it is typically inherited in an autosomal dominant pattern. Germline mutations in the MET proto-oncogene are located at 7q31, which encodes for the tyrosine kinase signaling pathway. Variants lead to uncontrolled activation of MET protein and aberrant cell growth [2]. Discussion Hereditary papillary RCC has an estimated incidence of &amp;lt; 1:1500000, its rarity highlighted by the fact that only ∼35 families have been reported worldwide in literature. In these cases it exhibits 100% penetrance, with patients developing renal cell carcinoma typically between their fifth and sixth decade [3]. We would like to acknowledge the family involved in this case report. As with many cancers, early diagnosis is helped by detection of a genetic variant, leading to improved long-term disease-free survival. In the case of metastatic papillary RCC 2-year survival is still estimated at 18% (3).

Breast cancer risk associated with missense variants in BRCA2.

10501 Background: Greater than 90% of missense variants in BRCA2 are classified as variants of uncertain significance (VUS). In addition, among the few known missense pathogenic variants (PVs) in BRCA2, breast cancer risk has been reported to be lower compared to pathogenic truncating variants (PTVs) (Li et al., 2022) . Herein, we aim to reclassify several missense variants in BRCA2 and investigate the associated breast cancer risk. Methods: A total of 450 BRCA2 missense variants in the C-terminal DNA binding domain (DBD) were evaluated for functional effect utilizing a previously validated homology-directed DNA double-strand repair (HDR) assay. The results of the functional evaluation along with clinical, structural, and in-silico data were applied to the rules-based ACMG/AMP criteria for variant classification to identify pathogenic/likely pathogenic (P/LP) variants and benign/likely benign (B/LB) variants. A pooled case-control analysis was performed comparing the frequency of reclassified P/LP missense variants among 82,372 women with breast cancer within the Ambry Genetics database as cases and 174,167 unaffected women within gnomAD, CARRIERS, and BRIDGES datasets as controls to evaluate associated breast cancer risk. Separate case-control analysis and comparisons with BRCA2 DBD PTVs, exon 11 PTVs, and known missense PV standards in ENIGMA were also performed. Results: From the 450 missense variants in the DBD, 137 (30.4%) were noted to be functionally deleterious and 313 (69.6%) were functionally neutral by the HDR assay. Incorporation of the functional and other data into an ACMG/AMP model led to the reclassification of 439 (97.6%) variants, with 131 as P/LP and 308 as B/LB variants. In a pooled case-control analysis, reclassified BRCA2 missense P/LP variants were associated with clinically significant increased risks of breast cancer (Odds Ratio (OR):5.6, 95%CI:3.9–8.0), whereas B/LB missense variants were not (OR:1.1, 95%CI:1.1–1.2). Similarly elevated risks of breast cancer were observed for BRCA2 PTVs in the DBD (OR:6.8, 95%CI:5.1–9.0) and missense PV standards in ENIGMA (OR:7.8, 95%CI:4.1–14.7). However, PTVs in exon 11 of BRCA2 were associated with a lower risk of breast cancer (OR:4.4, 95%CI:3.8–4.9) compared to PTVs in DBD, confirming the ovarian cancer cluster region effect in BRCA2. Conclusions: The study demonstrates that the HDR functional assay in conjunction with other clinical and genomic data can be a powerful tool that can aid in the reclassification of &gt; 95% of missense variants in BRCA2. In addition, missense PVs in DBD were noted to have a similarly elevated risk of breast cancer as PTVs. The reclassification of the BRCA2 missense VUS and identification of associated breast cancer risk in this study will significantly influence breast cancer risk assessment and management strategies among carriers of these missense variants.

Clinical features of PPP2 syndrome type R5D (Jordan's syndrome) to support standardization of care.

PPP2 syndrome type R5D, or Jordan's syndrome, is a neurodevelopmental disorder caused by pathogenic missense variants in PPP2R5D, a β-subunit of the Protein Phosphatase 2A (PP2A). The condition is characterized by global developmental delays, seizures, macrocephaly, ophthalmological abnormalities, hypotonia, attention disorder, social and sensory challenges often associated with autism, disordered sleep, and feeding difficulties. Among affected individuals, there is a broad spectrum of severity, and each person only has a subset of all associated symptoms. Some, but not all, of the clinical variability is due to differences in the PPP2R5D genotype. These suggested clinical care guidelines for the evaluation and treatment of individuals with PPP2 syndrome type R5D are based on data from 100 individuals reported in the literature and from an ongoing natural history study. As more data are available, particularly for adults and regarding treatment response, we anticipate that revisions to these guidelines will be made.

Rett-like Phenotypes in HNRNPH2-Related Neurodevelopmental Disorder.

Rett Syndrome (RTT) is a neurodevelopmental disorder with a prevalence of 1:10,000 to 15,000 females worldwide. Classic Rett Syndrome presents in early childhood with a period of developmental regression, loss of purposeful hand skills along with hand stereotypies, gait abnormalities, and loss of acquired speech. Atypical RTT is diagnosed when a child shows some but not all the phenotypes of classic RTT, along with additional supporting criteria. Over 95% of classic RTT cases are attributed to pathogenic variants in Methyl-CpG Binding Protein 2 (MECP2), though additional genes have been implicated in other RTT cases, particularly those with the atypical RTT clinical picture. Other genetic etiologies have emerged with similar clinical characteristics to RTT Syndrome. Our team has characterized HNRNPH2-related neurodevelopmental disorder (HNRNPH2-RNDD) in 33 individuals associated with de novo pathogenic missense variants in the X-linked HNRNPH2 gene, characterized by developmental delay, intellectual disability, seizures, autistic-like features, and motor abnormalities. We sought to further characterize RTT clinical features in this group of individuals by using caregiver report. Twenty-six caregivers completed electronic surveys, with only 3 individuals having previously received an atypical RTT diagnosis, and no individuals with a typical RTT diagnosis. Caregivers reported a high number of behaviors and/or phenotypes consistent with RTT, including the major criteria of the syndrome, such as regression of developmental skills and abnormal gait. Based on the survey results, 12 individuals could meet the diagnostic clinical criteria for atypical RTT Syndrome. In summary, individuals with HNRNPH2-RNDD exhibit clinical characteristics that overlap with those of RTT, and therefore, HNRNPH2-RNDD, should be considered on the differential diagnosis list with this clinical picture.

DARVIC: Dihedral angle-reliant variant impact classifier for functional prediction of missense VUS

BackgroundOf the large number of genetic variants identified, the functional impact for most of them remains unknown. Mutations in DNA damage repair genes such as MUTYH, which is involved in repairing A:8-oxoG mismatches caused by reactive oxygen species, can cause a higher risk of cancer. Mutations happening in other key genes such as TP53 also pose huge health threats and risk of cancer. The interpretation of genetic variants’ functional impact is a forefront issue that needs to be addressed. Many different in silico methods based on different principles have been developed and applied in interpreting genetic variants. However, a current challenge is that many existing methods tend to overpredict the pathogenicity of benign variants. A new approach is needed to tackle this issue to improve genetic variant interpretation through the use of in silico methods. MethodsIn this study, we developed another protein structural-based approach called Dihedral angle-reliant variant impact classifier (DARVIC) to predict the deleterious impact of the coding-changing missense variants. DARVIC uses Ramachandran's principle of protein stereochemistry as the theoretical foundation and uses molecular dynamics simulations coupled with a supervised machine learning algorithm XGBoost to determine the functional impact of missense variants on protein structural stability. ResultsWe characterized the features of dihedral angles in dynamic protein structures. We also tested the performance of DARVIC in MUTYH and TP53 missense variants and achieved satisfactory results in reflecting the functional impacts of the variants on protein structure. The method achieved a balanced accuracy of 84% in a functionally validated MUTYH dataset containing both benign and pathogenic missense variants, higher than other existing in silico methods. Along with that, DARVIC was able to predict 119 (47%) deleterious variants from a dataset of 254 MUTYH VUS. Further application of DARVIC to a functionally validated TP53 dataset had a balanced accuracy of 94%, topping other methods, demonstrating DARVIC's robustness. ConclusionDARVIC provides a valuable tool to predict the functional impacts of missense variants based on their effects on protein structural stability and motion. At its current state, DARVIC performed well in predicting the functional impact of the missense variants both in MUTYH and TP53. We expect its high potential to predict functional impact for the missense variants in other genes.

Delayed onset ADA-SCID presenting as secondary HLH in a patient with normal newborn screening

Severe combined immunodeficiency (SCID) is a rare genetic syndrome caused by aberrant immune cell development or function and is fatal without early identification and treatment. The Centers for Disease Control estimates the annual incidence of SCID to be one case per 40,000 to 100,000 live births. Newborn screening using T-cell receptor excision circles (TREC) allows for early detection and timely treatment of SCID. Adenosine deaminase (ADA) is a vital enzyme in the purine salvage pathway, and its deficiency results in disruption of T, B and NK cell development, accounting for 10–20% of SCID cases. The timing of ADA-SCID presentation ranges from birth to delayed onset disease. Importantly, without early recognition and treatment, delayed onset ADA-SCID results in accumulation of toxic metabolites causing immune dysfunction, recurrent infections, and subsequent organ damage. Recently, some groups have questioned whether traditional TREC newborn screening adequately identifies ADA-SCID, especially those cases with delayed onset. We present the case of a 2-year-old male with normal newborn screening but a history of recurrent otitis media and respiratory infections who developed multisystem organ failure and septicemia with Escherichia coli, candida, adenovirus and enterovirus despite broad-spectrum antimicrobials. The patient also had features of a hyperinflammatory syndrome with fever, pancytopenia, hypertriglyceridemia, and hyperferritinemia and was treated for hemophagocytic lymphohistiocytosis (HLH) with dexamethasone and plasma exchange followed by anakinra, etoposide, and emapalumab. The patient was maximally supported with mechanical ventilation, extracorporeal membrane oxygenation, and renal replacement therapy but had progressive clinical deterioration and ultimately expired. Autopsy was notable for thymic aplasia and erythrophagocytosis in the bone marrow and lymph nodes. Genetic testing revealed a heterozygous missense pathogenic variant in exon 7 in the ADA gene as well as novel heterozygous intronic variant of unknown significance in the ADA gene. Postmortem data suggests this patient had ADA-SCID with multi-pathogen infections and secondary HLH. This case potentially identifies a novel pathogenic variant in ADA missed by traditional TREC newborn screening and highlights the need to retain a high clinical suspicion of SCID in patients with a history of recurrent infections despite normal TREC screening.

Assessing variants of uncertain significance implicated in hearing loss using a comprehensive deafness proteome

Hearing loss is the leading sensory deficit, affecting ~ 5% of the population. It exhibits remarkable heterogeneity across 223 genes with 6328 pathogenic missense variants, making deafness-specific expertise a prerequisite for ascribing phenotypic consequences to genetic variants. Deafness-implicated variants are curated in the Deafness Variation Database (DVD) after classification by a genetic hearing loss expert panel and thorough informatics pipeline. However, seventy percent of the 128,167 missense variants in the DVD are “variants of uncertain significance” (VUS) due to insufficient evidence for classification. Here, we use the deep learning protein prediction algorithm, AlphaFold2, to curate structures for all DVD genes. We refine these structures with global optimization and the AMOEBA force field and use DDGun3D to predict folding free energy differences (∆∆GFold) for all DVD missense variants. We find that 5772 VUSs have a large, destabilizing ∆∆GFold that is consistent with pathogenic variants. When also filtered for CADD scores (> 25.7), we determine 3456 VUSs are likely pathogenic at a probability of 99.0%. Of the 224 genes in the DVD, 166 genes (74%) exhibit one or more missense variants predicted to cause a pathogenic change in protein folding stability. The VUSs prioritized here affect 119 patients (~ 3% of cases) sequenced by the OtoSCOPE targeted panel. Approximately half of these patients previously received an inconclusive report, and reclassification of these VUSs as pathogenic provides a new genetic diagnosis for six patients.