Comparison of Variants of Uncertain Significance in Three Regions of the Human Glucokinase Protein Using In Vitro and In Silico Analyses.

There is a growing field of research focusing on the bioinformatic analysis of human genetic variation and the associated diseases. To study how well in vitro testing of purified proteins compares to bioinformatic variant prediction, we chose to analyze glucokinase (GCK) missense variations between residues 119-132, 257-262, and 412-427. These regions contained a large number of variants of uncertain significance (VUS) as well as a few pathogenic variants to use for comparison. We compared experimentally produced Vmax values from purified GCK variant proteins to predictive methods such as molecular dynamics simulation, ConSurf, iStable, the evolutionary model of variant effect (EVE), PredictSNP, and calculated binding energy. After determining which variants are pathogenic or benign based on experimental results or previous genetic studies, we found that ConSurf was the best at predicting pathogenicity. Interestingly, one VUS, D262N, showed an increase in activity and thus was difficult to interpret as pathogenic or benign. This study is an attempt to provide a framework for the utility of missense variant predictive programs.

Characterization of a novel heterozygous variant in the histidyl-tRNA synthetase gene associated with Charcot-Marie-Tooth disease type 2W.

Heterozygous pathogenic variants in the histidyl-tRNA synthetase (HARS) gene are associated with Charcot-Marie-Tooth (CMT) type 2W disease, classified as an axonal peripheral neuropathy. To date, at least 60 variants causing CMT symptoms have been identified in seven different aminoacyl-tRNA synthetases, with eight being found in the catalytic domain of HARS. The genetic data clearly show a causative role of aminoacyl-tRNA synthetases in CMT; however, the cellular mechanisms leading to pathology can vary widely and are unknown in the case of most identified variants. Here we describe a novel HARS variant, c.412T>C; p.Y138H, identified through a CMT gene panel in a patient with peripheral neuropathy. To determine the effect of p.Y138H we employed a humanized HARS yeast model and recombinant protein biochemistry, which identified a deficiency in protein dimerization and a growth defect which shows mild but significant improvement with histidine supplementation. This raises the potential for a clinical trial of histidine.

Prevalence and Reclassification of Genetic Variants in South African Populations with Breast Cancer.

Concurrent testing of numerous genes for hereditary breast cancer (BC) is available but can result in management difficulties. We evaluated use of an expanded BC gene panel in women of diverse South African ancestries and assessed use of African genomic data to reclassify variants of uncertain significance (VUS). A total of 331 women of White, Black African, or Mixed Ancestry with BC had a 9-gene panel test, with an additional 75 genes tested in those without a pathogenic/likely pathogenic (P/LP) variant. The proportion of VUS reclassified using ClinGen gene-specific allele frequency (AF) thresholds or an AF > 0.001 in nonguidelines genes in African genomic data was determined. The 9-gene panel identified 58 P/LP variants, but only two of the P/LP variants detected using the 75-gene panel were in confirmed BC genes, resulting in a total of 60 (18.1%) in all participants. P/LP variant prevalence was similar across ancestry groups, but VUS prevalence was higher in Black African and Mixed Ancestry than in White participants. In total, 611 VUS were detected, representing 324 distinct variants. 10.8% (9/83) of VUS met ClinGen AF thresholds in genomic data while 10.8% (26/240) in nonguideline genes had an AF > 0.001. Overall, 27.0% of VUS occurrences could potentially be reclassified using African genomic data. Thus, expanding the gene panel yielded few clinically actionable variants but many VUS, particularly in participants of Black African and Mixed Ancestry. However, use of African genomic data has the potential to reclassify a significant proportion of VUS.

A content analysis of parents' reflections on pathogenic and uncertain pediatric oncology germline sequencing results.

Germline genomic sequencing is increasingly integrated into pediatric cancer care, with pathogenic cancer-predisposing variants identified among 5-18% of affected children and variants of uncertain significance (VUS) in up to 70%. Given the potential medical implications for children and their families, parents' psychosocial responses to learning results are important to understand. Parents of children with cancer who learned their children's germline pathogenic or VUS results following paired tumor and germline genomic sequencing described their cognitive and affective responses to results in an open-ended write-in question after disclosure (M = 10 months post-disclosure; range = 1-28). Responses were coded and categorized using content analysis, then compared across results using chi-square and Fisher's exact test. Parents of children with pathogenic (n = 9), VUS (n = 52), and pathogenic plus VUS results (n = 9) described negative emotions, positive reactions, mixed emotions (i.e., positive and negative emotions), and neutral reactions. Negative emotions were described significantly more frequently with pathogenic results than VUS only (χ2 = 5.19; p = .02), with peace of mind and empowerment only described for those with VUS. Parents also described approach(es) to coping (e.g., faith, plan of action) and reactions specific to the uncertainty of VUS (e.g., disappointment at no explanation for cancer etiology). A subset with VUS described decreasing worry/distress with increased understanding of results, whereas others displayed misconceptions regarding VUS. Screening for emotional adjustment is warranted for parents of children with cancer receiving pathogenic germline results, and screening for understanding is warranted with VUS. Findings highlight the importance of pre-and posttest genetic counseling.

Functional pathogenicity of ESRRB variant of uncertain significance contributes to hearing loss (DFNB35).

Advances in next-generation sequencing technologies have led to elucidation of sensorineural hearing loss genetics and associated clinical impacts. However, studies on the functional pathogenicity of variants of uncertain significance (VUS), despite their close association with clinical phenotypes, are lacking. Here we identified compound heterozygous variants in ESRRB transcription factor gene linked to DFNB35, specifically a novel splicing variant (NM_004452.4(ESRRB): c.397 + 2T>G) in trans with a missense variant (NM_004452.4(ESRRB): c.1144C>T p.(Arg382Cys)) whose pathogenicity remains unclear. The splicing variant (c.397 + 2T>G) caused exon 4 skipping, leading to premature stop codon formation and nonsense-mediated decay. The p.(Arg382Cys) variant was classified as a VUS due to its particularly higher allele frequency among East Asian population despite disease-causing in-silico predictions. However, functional assays showed that p.(Arg382Cys) variant disrupted key intramolecular interactions, leading to protein instability. This variant also reduced transcriptional activity and altered expression of downstream target genes essential for inner ear function, suggesting genetic contribution to disease phenotype. This study expanded the phenotypic and genotypic spectrum of ESRRB in DFNB35 and revealed molecular mechanisms underlying ESRRB-associated DFNB35. These findings suggest that variants with high allele frequencies can also possess functional pathogenicity, providing a breakthrough for cases where VUS, previously unexplored, could be reinterpreted by elucidating their functional roles and disease-causing characteristics.

Functional characterization vs in silico prediction for TBX5 missense and splice variants in Holt-Oram syndrome.

PURPOSE: Predicting effects of genomic variants has become a real challenge in the diagnosis of rare human diseases. Holt-Oram syndrome (HOS) is an autosomal condition characterized by the association of radial and heart defects, due to variants in TBX5. Most variants are predicted to be truncating and result in haploinsufficiency. The pathogenicity of missense or splice variants is harder to demonstrate. METHODS: Fourteen TBX5 variants of uncertain significance (VUS) (5 missense, 9 splice) and 6 likely pathogenic missense variants were selected for functional testing, depending on the variant-type (immunolocalization, western blot, reporter assays, minigene splice assays and RT-PCR). Results were compared with in silico predictions. RESULTS: Functional tests allowed to reclassify 9/14 VUS in TBX5 as likely pathogenic, confirming their role in HOS. We demonstrated loss-of-function (n=8) or gain-of-function (n=1) for 9 of the 11 missense variants, whereas no functional impact was shown for the 2 variants: p.(Gly195Ala) and p.(Ser261Cys), as suggested by contradictory predictions of in silico approaches. Of 9 splice variants predicted to affect splicing by SpliceAI, we observed partial or complete exon skipping (n=6), intron retention (n=2) or exon shortening (n=1), inducing frame-shifting with premature stop codons. CONCLUSION: Bioinformatic and biological approaches are complementary, together with a good knowledge of clinical conditions, for accurate ACMG classification in human rare diseases.

Perinatal outcomes after a prenatal diagnosis of a fetal copy number variant: a retrospective population-based cohort study.

BACKGROUND: There are no established guidelines for the follow up of infants born after a prenatal diagnosis of a genomic copy number variant (CNV), despite their increased risk of developmental issues. The aims of this study were (i) to determine the perinatal outcomes of fetuses diagnosed with and without a CNV, and (ii) to establish a population-based paediatric cohort for long term developmental follow up. METHODS: An Australian state-wide research database was screened for pregnant individuals who had a prenatal chromosomal microarray (CMA) between 2013-2019 inclusive. Following linkage to laboratory records and clinical referrer details, hospital records were manually reviewed for study eligibility. Eligible participants were mother-child pairs where the pregnancy resulted in a livebirth, the mother was able to provide informed consent in English (did not require a translator) and the mother was the primary caregiver for the child at hospital discharge after birth. Research invitations were sent by registered post at an average of six years after the prenatal diagnostic test. Statistical analysis was performed in Stata17. RESULTS: Of 1832 prenatal records examined, 1364 (74.5%) mother-child pairs were eligible for recruitment into the follow up cohort. Of the 468 ineligible, 282 (60.3%) had 'no live pregnancy outcome' (209 terminations of pregnancy (TOP) and 73 miscarriages, stillbirths, and infant deaths), 157 (33.5%) required a translator, and 29 (6.2%) were excluded for other reasons. TOP rates varied by the type of fetal CNV detected: 49.3% (109/221) for pathogenic CNVs, 18.2% (58/319) for variants of uncertain significance and 3.3% (42/1292) where no clinically significant CNV was reported on CMA. Almost 77% of invitation letters were successfully delivered (1047/1364), and the subsequent participation rate in the follow up cohort was 19.2% (201/1047). CONCLUSIONS: This study provides Australia's first population-based data on perinatal outcomes following prenatal diagnostic testing with CMA. The relatively high rates of pregnancy loss for those with a prenatal diagnosis of a CNV presented a challenge for establishing a paediatric cohort to examine long term outcomes. Recruiting a mother-child cohort via prenatal ascertainment is a complex and resource-intensive process, but an important step in understanding the impact of a CNV diagnosis in pregnancy and beyond. TRIAL REGISTRATION: ACTRN12620000446965p; Registered on April 6, 2020.

CRISPR screening uncovers a long-range enhancer for ONECUT1 in pancreatic differentiation and links a diabetes risk variant.

Functional enhancer annotation is critical for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants. However, unbiased enhancer discovery in disease-relevant contexts remains challenging. To identify enhancers pertinent to diabetes, we conducted a CRISPR interference (CRISPRi) screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers identified, we focused on an enhancer we named ONECUT1e-664kb, ∼664 kb from the ONECUT1 promoter. Previous studies have linked ONECUT1 coding mutations to pancreatic hypoplasia and neonatal diabetes. We found that homozygous deletion of ONECUT1e-664kb in hPSCs leads to a near-complete loss of ONECUT1 expression and impaired pancreatic differentiation. ONECUT1e-664kb contains a type 2 diabetes-associated variant (rs528350911) disrupting a GATA motif. Introducing the risk variant into hPSCs reduced binding of key pancreatic transcription factors (GATA4, GATA6, and FOXA2), supporting its causal role in diabetes. This work highlights the utility of unbiased enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.

Significance of Variants of Uncertain Significance: The Human Cost of Genetic Uncertainty.

This piece narrates the journey of Maria (name of the mother has been altered to protect the family's privacy), a new mother confronting her newborn's unexpected diagnosis of very long chain acyl-CoA dehydrogenase (VLCAD) deficiency, despite undergoing proactive genetic carrier screening within a consanguineous marriage. It highlights the emotional and systemic challenges arising from the lack of diversity in genetic databases, which, in this case, failed to detect pathogenic variants in Maria and her husband. Maria's story sheds light on situations where a masked variant of uncertain significance (VUS) necessitates consultation with a trained genetics specialist and underscores the urgent need for a more equitable healthcare system.

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.

Clinical and Molecular Characterization of SMAD4 Splicing Variants in Patients with Juvenile Polyposis Syndrome.

Juvenile polyposis syndrome (JPS) is an inherited autosomal dominant condition that predisposes to the development of juvenile polyps throughout the gastrointestinal (GI) tract, and it poses an increased risk of GI malignancy. Germline causative variants were identified in the SMAD4 gene in a subset (20%) of JPS cases. Most SMAD4 germline genetic variants published to date are missense, nonsense, and frameshift mutations. SMAD4 germline alterations predicted to result in aberrant splicing have rarely been reported. Here, we report two unrelated Italian families harboring two different SMAD4 intronic variants, c.424+5G>A and c.425-9A>G, which are clinically associated with colorectal cancer and/or juvenile GI polyps. In silico prediction analysis, in vitro minigene assays, and RT-PCR showed that the identified variants lead to aberrant SMAD4 splicing via the exonization of intronic nucleotides, resulting in a premature stop codon. This is expected to cause the production of a truncated protein. This study expands the landscape of SMAD4 germline genetic variants associated with GI polyposis and/or cancer. Moreover, it emphasizes the importance of the functional characterization of SMAD4 splicing variants through RNA analysis, which can provide new insights into genetic disease variant interpretation, enabling tailored genetic counseling, management, and surveillance of patients with GI polyposis and/or cancer.

The Development and Evaluation of Novel Patient Educational Material for a Variant of Uncertain Significance (VUS) Result in Hereditary Cancer Genes.

A Variant of Uncertain Significance (VUS) is a difference in the DNA sequence with uncertain consequences for gene function. A VUS in a hereditary cancer gene should not change medical care, yet some patients undergo medical procedures based on their VUS result, highlighting the unmet educational needs among patients and healthcare providers. To address this need, we developed, evaluated, and refined novel educational materials to explain that while VUS results do not change medical care, it remains important to share any personal or family history of cancer with family members given that their personal and family medical history can guide their cancer risk management. We began by reviewing the prior literature and transcripts from interviews with six individuals with a VUS result to identify content and design considerations to incorporate into educational materials. We then gathered feedback to improve materials via a focus group of multidisciplinary experts and multiple rounds of semi-structured interviews with individuals with a VUS result. Themes for how to improve content, visuals, and usefulness were used to refine the materials. In the final round of interviews with an additional 10 individuals with a VUS result, materials were described as relatable, useful, factual, and easy to navigate, and also increased their understanding of cancer gene VUS results.

New insights into the landscape of ALPL gene variants in patients with hypophosphatasia from the Global HPP Registry.

Hypophosphatasia (HPP) is a rare, inherited metabolic disease characterized by low tissue-nonspecific alkaline phosphatase activity due to ALPL gene variants. We describe ALPL variants from the observational, prospective, multinational Global HPP Registry. Inclusion in the analysis required a diagnosis of HPP, low serum ALP activity, and ≥1 ALPL variant. Of 1176 patients enrolled as of September 2022, 814 met inclusion criteria in Europe (48.9%), North America (36.7%), Japan (10.2%), Australia (2.6%), and elsewhere (1.6%). Most patients (74.7%) had 1 ALPL variant; 25.3% had ≥2 variants. Nearly all patients (95.6%) had known disease-causing variants; 4.4% had variants of uncertain significance. Disease-causing variants were predominantly missense (770/1556 alleles). The most common variants were c.571G>A (102/1628 alleles), c.1250A>G (66/1628 alleles), and c.1559del (61/1628 alleles). Variant profiles were generally consistent, except in Japan, where a higher proportion of patients (68.7%) had ≥2 ALPL variants, likely because more had disease onset before age 6 months (53.0% vs. 10.1%-23.1% elsewhere). Frameshift mutations (61/164 alleles) and inframe deletions (7/164 alleles) were more common in Japan. Twenty-three novel variants were discovered, each in a single geographic region, predominantly Europe. Analyses confirmed previously known ALPL variants, identified novel variants, and characterized geographic variation in frequency and type of ALPL variants in a large population.

An ANXA11 P93S variant dysregulates TDP-43 and causes corticobasal syndrome.

INTRODUCTION: Variants of uncertain significance (VUS) surged with affordable genetic testing, posing challenges for determining pathogenicity. We examine the pathogenicity of a novel VUS P93S in Annexin A11 (ANXA11) - an amyotrophic lateral sclerosis/frontotemporal dementia-associated gene - in a corticobasal syndrome kindred. Established ANXA11 mutations cause ANXA11 aggregation, altered lysosomal-RNA granule co-trafficking, and transactive response DNA binding protein of 43 kDa (TDP-43) mis-localization. METHODS: We described the clinical presentation and explored the phenotypic diversity of ANXA11 variants. P93S's effect on ANXA11 function and TDP-43 biology was characterized in induced pluripotent stem cell-derived neurons alongside multiomic neuronal and microglial profiling. RESULTS: ANXA11 mutations were linked to corticobasal syndrome cases. P93S led to decreased lysosome colocalization, neuritic RNA, and nuclear TDP-43 with cryptic exon expression. Multiomic microglial signatures implicated immune dysregulation and interferon signaling pathways. DISCUSSION: This study establishes ANXA11 P93S pathogenicity, broadens the phenotypic spectrum of ANXA11 mutations, underscores neuronal and microglial dysfunction in ANXA11 pathophysiology, and demonstrates the potential of cellular models to determine variant pathogenicity. HIGHLIGHTS: ANXA11 P93S is a pathogenic variant. Corticobasal syndrome is part of the ANXA11 phenotypic spectrum. Hybridization chain reaction fluorescence in situ hybridization (HCR FISH) is a new tool for the detection of cryptic exons due to TDP-43-related loss of splicing regulation. Microglial ANXA11 and related immune pathways are important drivers of disease. Cellular models are powerful tools for adjudicating variants of uncertain significance.

Reclassification of Variants Following Renal Genetics Testing: Uncommon Yet Impactful for Diagnosis and Management.

Introduction: Genetic testing is increasingly utilized in nephrology practice, but limited real-world data exist on variant reclassification following renal genetics testing. Methods: A cohort of patients at the Cleveland Clinic Renal Genetics Clinic who underwent genetic testing through clinical laboratories was assessed with their clinical and laboratory data analyzed. Results: Between January 2019 and June 2023, 425 new patients with variable kidney disorders from 413 pedigrees completed genetic testing through 10 clinical laboratories, including 255 (60%) females with median (25th, 75th percentiles) age of 36 (22-54) years. Multigene panel was the most frequently used modality followed by single-gene testing, exome sequencing (ES), chromosomal microarray (CMA), and genome sequencing (GS). At initial report, 52% of patients had ≥1 variants of uncertain significance (VUS) with or without concurrent pathogenic variant(s). Twenty amendments were issued across 19 pedigrees involving 19 variants in 17 genes. The overall variant reclassification rate was 5%, with 63% being upgrades and 32% downgrades. Of the reclassified variants, 79% were initially reported as VUS. The median time-to-amendments from initial reports was 8.4 (4-27) months. Following the variant reclassifications, 60% of the patients received a new diagnosis or a change in diagnosis. Among these, 67% of patients received significant changes in clinical management. Conclusion: Variant reclassification following genetic testing is infrequent but important for diagnosis and management of patients with suspected genetic kidney disease. The majority of variant reclassifications involve VUS and are upgrades in clinically issued amended reports. Further studies are needed to investigate the predictors of such events.

Hormonally Silent Multifocal Pheochromocytoma in the Setting of a Well-Differentiated Neuroendocrine Tumor of the Appendix: A Case Report.

Pheochromocytomas (PCCs) and paragangliomas (PGLs) represent tumors arising from chromaffin cells of the adrenal medulla and extra-adrenal sympathetic paraganglia, respectively. PCCs commonly produce one or more catecholamines (epinephrine, norepinephrine, and dopamine), but rarely are they biochemically silent. PGLs on the other hand, generally do not produce catecholamines. They have the highest heritability of all adrenal tumors and are known to be associated with genetic mutations. Patients with hereditary tumors typically present at a younger age and with multifocal disease when compared to sporadic disease. Specific genetic mutations have been well established with hereditary syndromes involving PCC/PGLs. Further research has aimed to identify other mutations and delineate specific phenotypes associated with these mutations. A 34-year-old woman presented for evaluation following a laparoscopic appendectomy that identified a 4-cm well-differentiated neuroendocrine tumor on final pathology. Further work-up included a repeat CT scan followed by a Dotatate PET CT scan which revealed a large (7.3 x 5.8 cm) periaortic mass related to the left adrenal gland. Functional adrenal work-up was negative and her Chromogranin A level was 679 ng/mL. She did report intermittent chest tightness and palpitations but was otherwise asymptomatic. The patient subsequently underwent an exploratory laparotomy with left adrenalectomy and adjacent tumor resection as well as completion of right hemicolectomy with ileocolonic anastomosis. Surgical pathology revealed two distinct masses consistent with multifocal PCC. No residual tumor was found in the colectomy specimen and 24 lymph nodes were negative. She had an uneventful recovery and genetic testing showed a variant of uncertain significance for the POLE and VHL genes. She has received genetic counseling and will be enrolled in an appropriate surveillance protocol.

Novel characterization of CASK variant c.1963 A>G (p.Asn655Asp) through whole-exome sequencing in a monochorionic diamniotic twin fetus with significant brain anomalies: A case report.

Whole-exome sequencing is an evolving technology in perinatal diagnosis which allows identification of genetic etiologies that would otherwise go undetermined. In this case report, a 38-year-old Hispanic woman, G5P3013, with a monochorionic diamniotic twin gestation with one fetus displaying significant cranial abnormalities on prenatal ultrasound and magnetic resonance imaging (MRI) of the brain is presented. Fetal anomalies included bilateral ventriculomegaly, absent cavum septum pellucidum, and absent corpus callosum. Diagnostic amniocentesis with chromosome analysis, chromosomal microarray, alpha-fetoprotein, cytomegalovirus, toxoplasmosis, and parvovirus had normal results. Whole-exome sequencing for the anomalous fetus detected a de novo mosaic variant of uncertain significance (VUS) in the calcium/calmodulin dependent serine protein kinase (CASK) gene: c.1963 A > G (p.Asn655Asp). This variant was absent in the normal twin fetus, the mother, and the father. Pathogenic CASK gene mutations are associated with three syndromes: FG syndrome 4, intellectual developmental disorder and microcephaly with pontine and cerebellar hypoplasia (MICPCH), and intellectual developmental disorder with or without nystagmus. Whole-exome sequencing identified a potential etiology for the anomalies detected. The variant likely arose de novo and was the potential cause of the identified cranial abnormalities in one fetus of this monochorionic diamniotic twin gestation. Whole-exome sequencing may provide additional diagnostic utility when standard diagnostic testing is noncontributory.

Clinical interpretation of KCNH2 variants using a robust PS3/BS3 functional patch-clamp assay.

Long QT syndrome (LQTS), caused by the dysfunction of cardiac ion channels, increases the risk of sudden death in otherwise healthy young people. For many variants in LQTS genes, there is insufficient evidence to make a definitive genetic diagnosis. We have established a robust functional patch-clamp assay to facilitate classification of missense variants in KCNH2, one of the key LQTS genes. A curated set of 30 benign and 30 pathogenic missense variants were used to establish the range of normal and abnormal function. The extent to which variants reduced protein function was quantified using Z scores, the number of standard deviations from the mean of the normalized current density of the set of benign variant controls. A Z score of -2 defined the threshold for abnormal loss of function, which corresponds to 55% wild-type function. More extreme Z scores were observed for variants with a greater loss-of-function effect. We propose that the Z score for each variant can be used to inform the application and weighting of abnormal and normal functional evidence criteria (PS3 and BS3) within the American College of Medical Genetics and Genomics variant classification framework. The validity of this approach was demonstrated using a series of 18 KCNH2 missense variants detected in a childhood onset LQTS cohort, where the level of function assessed using our assay correlated to the Schwartz score (a scoring system used to quantify the probability of a clinical diagnosis of LQTS) and the length of the corrected QT (QTc) interval.

Expanding the genotype-phenotype spectrum in SCN8A-related disorders.

BACKGROUND: SCN8A-related disorders are a group of variable conditions caused by pathogenic variations in SCN8A. Online Mendelian Inheritance in Man (OMIM) terms them as developmental and epileptic encephalopathy 13, benign familial infantile seizures 5 or cognitive impairment with or without cerebellar ataxia. METHODS: In this study, we describe clinical and genetic results on eight individuals from six families with SCN8A pathogenic variants identified via exome sequencing. RESULTS: Clinical findings ranged from normal development with well-controlled epilepsy to significant developmental delay with treatment-resistant epilepsy. Three novel and three reported variants were observed in SCN8A. Electrophysiological analysis in transfected cells revealed a loss-of-function variant in Patient 4. CONCLUSIONS: This work expands the clinical and genotypic spectrum of SCN8A-related disorders and provides electrophysiological results on a novel loss-of-function SCN8A variant.

Release of reclassified VUS results of now deceased patients to family members: Practices and opinions.

Variants of uncertain significance (VUS) are commonly identified in genetic testing. The rate at which a VUS is reclassified depends on multiple factors. However, as the amount of time it might take for a VUS to be reclassified varies, some patients with a VUS genetic testing result might have passed away before the VUS is reclassified. A VUS that is reclassified after the patient's death has clinical implications for the deceased patient's family members. The disclosure of reclassified VUS results for a deceased patient has complex legal and ethical implications. There are no established guidelines on how the reclassified VUS result for a deceased patient should be disclosed to at-risk relatives. An online survey was sent to members of the National Society of Genetic Counselors (NSGCs) to elicit practices and opinions regarding this issue. A total of 153 (4%) NSGC members completed the survey. Thirty-seven (24.2%) respondents reported having received a reclassified VUS for a deceased patient. Respondents were more likely to attempt disclosure if the variant was reclassified as pathogenic (93.5%) versus benign (76.5%), although the difference did not reach statistical significance (p = 0.06). Respondents more often reported the impact on family members (85.5%) than the decedent's right to privacy (15.0%) as extremely important when considering disclosure to family members. A legal mechanism to allow disclosure to relatives was supported by 70.6% of respondents and 97.4% felt the issue was important enough to pursue if such a process was in place. Only 9.8% of respondents supported a legal requirement of consent before disclosing to family members when a VUS is reclassified after the patient has passed away. Our results indicate that there is no consensus for how these results should be handled and a mechanism for disclosure of reclassified results to family members is supported.