Variants Of Significance Research Articles

Understanding variants of unknown significance and classification of genomic alterations.

Despite recent efforts to issue clinical guidelines outlining strategies to define the pathogenicity of genomic variants, there is currently no standardized framework for which to make these assertions. This review does not present a step-by-step methodology, but rather takes a holistic approach to discuss many aspects which should be taken into consideration when determining variant pathogenicity. Categorization should be curated to reflect relevant findings within the scope of the specific medical context. Functional characterization should evaluate all available information, including results from literature reviews, different classes of genomic data repositories, and applicable computational predictive algorithms. This article further proposes a multidimensional view to infer pathogenic status from many genomic measurements across multiple axes. Notably, tumor suppressors and oncogenes exhibit fundamentally different biology which helps refine the importance of effects on splicing, mutation interactions, copy number thresholds, rearrangement annotations, germline status, and genome-wide signatures. Understanding these relevant datapoints with thoughtful perspective could aid in the reclassification of variants of unknown significance (VUS), which are ambiguously understood and currently have uncertain clinical implications. Ongoing assessments of VUS examining these relevant biological axes could lead to more accurate classification of variant pathogenicity interpretation in diagnostic oncology.

Dual white matter pathology in fetal holoprosencephaly featuring concurrent malformative and destructive features: A case series.

Holoprosencephaly (HPE) is a classic brain malformation involving defective forebrain induction and patterning. Cases of HPE bearing white matter abnormalities have not been well documented, with only rare cases exhibiting hypoxic-ischemic damage. However, neuroradiologic studies of HPE using diffusion tensor imaging have suggested the presence of white matter architectural disarray. Described in this case series are the clinicopathologic features of 8 fetuses with HPE who underwent autopsy at BC Children's Hospital. All 8 cases exhibited subacute to chronic, periventricular leukomalacia (PVL)-like white matter pathology, with 7 of 8 cases also demonstrating aberrant white matter tracts, one of which manifested as a discreet bundle crossing the midline within the ventral aspects of the fused deep gray nuclei. In 6 of these 7 cases, the PVL-like pathology resided within this aberrant white matter tract. Original workup, alongside an additional HPE-focused next-generation sequencing panel identified a likely etiologic cause for the HPE in 4 cases, with an additional 2 cases exhibiting a variant of unknown significance in genes previously suggested to be involved in HPE. Despite our in-depth clinicopathologic and molecular review, no unifying etiology was definitively identified among our series of fetal HPE bearing this unusual pattern of white matter pathology.

Clinical and Genetic Characterization of a Cohort of Small-for-Gestational-Age Patients: Cost-Effectiveness of Whole-Exome Sequencing and Effectiveness of Treatment with GH.

Background/Objectives: Develop a clinical and genetic characterization, in a group of small-for-gestational-age (SGA) patients who did not experience catch-up growth Methods: In an ambispective cohort study with (SGA) patients. These patients received one treatment with growth hormone (GH) over 14 years. This study analyzes their response to treatment and conducts a genetic analysis in order to identify cases with specific phenotypic and auxological characteristics, defined as presenting two or more dysmorphic traits and/or a stature below -3 SDS (standard deviation score). Whole-exome sequencing (WES) was performed on selected patients. Results: Forty-four SGA patients were examined, with an average age of 6.4 (2.49) years and an initial size of -3.3 SDS. The pubertal growth was 24.1 (5.2) cm in boys and 14.7 (4.3) cm in girls. WES in 11 SGA patients revealed conclusive genetic variants in eight, including two pathogenic ACAN variants, one 15q26.2-q26.3 deletion, and four variants of uncertain significance in other genes. Conclusions: Treatment with GH in SGA patients was shown to be effective, with a similar response in the group with positive genetic results and in the group who did not undergo a genetic study. Genetic testing based on auxological and clinical criteria proved highly cost-effective.

An integrated clinical approach to children at genetic risk for neurodevelopmental and psychiatric conditions: interdisciplinary collaboration and research infrastructure

BackgroundA sizeable proportion of pathogenic genetic variants identified in young children tested for congenital differences are associated with neurodevelopmental psychiatric disorders (NPD). In this growing group, a genetic diagnosis often precedes the emergence of diagnosable developmental concerns. Here, we describe DAGSY (Developmental Assessment of Genetically Susceptible Youth), a novel interdisciplinary ‘genetic-diagnosis-first’ clinic integrating psychiatric, psychological and genetic expertise, and report our first observations and feedback from families and referring clinicians.MethodsWe retrieved data on referral sources and indications, genetic and NPD diagnoses and recommendations for children seen at DAGSY between 2018 and 2022. Through a survey, we obtained feedback from twenty families and eleven referring clinicians.Results159 children (mean age 10.2 years, 57.2% males) completed an interdisciplinary (psychiatry, psychology, genetic counselling) DAGSY assessment during this period. Of these, 69.8% had a pathogenic microdeletion or microduplication, 21.5% a sequence-level variant, 4.4% a chromosomal disorder, and 4.4% a variant of unknown significance with emerging evidence of pathogenicity. One in four children did not have a prior NPD diagnosis, and referral to DAGSY was motivated by their genetic vulnerability alone. Following assessment, 76.7% received at least one new NPD diagnosis, most frequently intellectual disability (24.5%), anxiety (20.7%), autism spectrum (18.9%) and specific learning (16.4%) disorder. Both families and clinicians responding to our survey expressed satisfaction, but also highlighted some areas for potential improvement.ConclusionsDAGSY addresses an unmet clinical need for children identified with genetic variants that confer increased vulnerability for NPD and provides a crucial platform for research in this area. DAGSY can serve as a model for interdisciplinary clinics integrating child psychiatry, psychology and genetics, addressing both clinical and research needs for this emerging population.

Genome-wide association study of major anxiety disorders in 122,341 European-ancestry cases identifies 58 loci and highlights GABAergic signaling.

The major anxiety disorders (ANX; including generalized anxiety disorder, panic disorder, and phobias) are highly prevalent, often onset early, persist throughout life, and cause substantial global disability. Although distinct in their clinical presentations, they likely represent differential expressions of a dysregulated threat-response system. Here we present a genome-wide association meta-analysis comprising 122,341 European ancestry ANX cases and 729,881 controls. We identified 58 independent genome-wide significant ANX risk variants and 66 genes with robust biological support. In an independent sample of 1,175,012 self-report ANX cases and 1,956,379 controls, 51 of the 58 associated variants were replicated. As predicted by twin studies, we found substantial genetic correlation between ANX and depression, neuroticism, and other internalizing phenotypes. Follow-up analyses demonstrated enrichment in all major brain regions and highlighted GABAergic signaling as one potential mechanism underlying ANX genetic risk. These results advance our understanding of the genetic architecture of ANX and prioritize genes for functional follow-up studies.

Pathogenic Mutations Conferring α1-Antitrypsin Deficiency Are Identified Accurately by a Novel Multiplexed Molecular Assay

BackgroundAlpha-1 antitrypsin deficiency (AATD) is a common, under-diagnosed disease caused by mutations in the polymorphic SERPINA1 gene. AATD is often causes chronic obstructive pulmonary disease (COPD), other respiratory ailments and/or severe liver disease. AATD under-diagnosis is associated with the lack of a quick, high precision test for SERPINA1 variants. Research QuestionCan a rapid and more accurate molecular diagnostic assay be developed that identifies AATD disease-associated mutations and outperforms current limited methodology? Study Design and MethodsWe developed a multiplexed PCR assay that that uses mass spectrometry to detect 20 pathogenic SERPINA1 mutations, two normal M allele variants and an additional variant of unknown significance, as an accessible frontline genetic test for AATD. Blood samples from 177 patients with AATD indication, 176 presumed normal genotype patients and 10 buccal swabs or blood spots were tested to validate the assay. Additionally, 760 whole blood samples from AATD indication patients were evaluated to identify AATD-associated mutations. ResultsThe novel genotyping assay described here accurately detected 23 SERPINA1 single nucleotide polymorphisms (SNPs) (“AAT SNP23”). Of 177 AATD samples, 96% had abnormal SNPs, whereas 9.1% of the 176 presumed normal samples had abnormal SNPs. AAT SNP23 genotypes correlated well with known serum AAT levels. This genotyping assay was more accurate and streamlined than phenotyping isoelectric focusing assay used to identify AATD variants. For clinical testing, serum AAT protein level determination and AAT SNP23 genotyping assay were performed. AAT SNP23 was successfully implemented using AATD indication patient samples to evaluate the most common SERPINA1 mutations indicative of AATD. AAT SNP23 has allowed for quick and accurate AAT genotyping of patients. InterpretationWe developed a novel, multiplexed genotyping assay that rapidly and accurately identifies multiple AATD-associated SERPINA1 SNPs. This assay is useful to quickly diagnose AATD in patients with pulmonary and/or hepatic diseases of unknown etiology.

P-551 Application of simultaneous PGT-M/PGT-A using whole genome sequencing and haplotyping for monogenetic disorders, multiple variants, low penetrance, and de novo disorders

Abstract Study question Is the concurrent use of high-resolution PGT-A and genome-wide haplotyping with disease-specific assays for PGT-M effective in an unselected patient population at-risk for monogenetic disorders? Summary answer PGT-A/PGT-M is an effective treatment for couples/patients burdened with genetic diseases, that provides optimal family planning, prevents vertical transmission, and reduces personal and societal encumbrance What is known already Societal awareness of genetic testing expanded the use of carrier screening, diagnostic genetic tests, and higher in vitro fertilization (IVF) utilization has increased demand for preimplantation genetic testing for monogenetic disorders (PGT-M), as an alternative to invasive prenatal tests. The scope of PGT-M testing expands beyond common severe childhood disorders, to include serious and mild late onset disorders, cancer predisposition, variants of unknown significance, de novo mutations, and variants in multiple genes. This highlights novel ethical dilemmas, necessitating adjustment of generic genome wide approaches to complement, or eventually replace, targeted disease-specific PGT-M assays. Study design, size, duration This is a retrospective study of 359 couples at-risk for monogenetic disorders referred to the CReATe Fertility Centre, Toronto, Canada. Our objective was to evaluate the efficiency of concurrent use of genome wide haplotyping and disease specific assays for PGT-M and high resolution PGT-A in an unselected patient population at risk for monogenetic disorders, multiple and de novo variants, low penetrance and variants of unknown significance. Participants/materials, setting, methods All patients received comprehensive genetic counselling. For each couple/patient, DNA from gamete providers, related family member/s and whole-genome amplified (WGA)DNA from a single trophectoderm(TE) biopsy were analyzed. Whole-genome SNP-array (Karyomapping, Illumina, CA) and STR-analysis were used to obtain parental haplotypes and Sanger-sequencing was used for direct variant analysis. PGT-A was performed using Illumina platform(NextSeq 550). NxClinical (BioDiscovery) and BlueFuse Multi(Illumina) software were used for data analysis. Main results and the role of chance 1826 embryos, from 235 IVF cycles were analyzed for 88 unique genes (14 cancer predisposing, 42 autosomal recessive and 32 dominant genes) and a total of 282 unique variants. The most frequent genes tested were BRCA1/2 -21% of all cases, followed by HBB-8.7%, CFTR-8.7% and CYP21A2-8.7%. PGT-M for 11 cases was done for a de novo variant detected in a sibling and in 2 cases for a parental mosaic variant. Multiple genes and VUS were analyzed in 12 and 6 cases, respectively. PGT-A analysis showed 31.6% aneuploidy, 9.6% mosaicism and 58.8% euploidy. Genome wide haplotyping was used for euploid and mosaic embryos with an average case call rate of 95%, and average case Allele Drop Out (ADO) rate of 2.7%. Recombination events in 3% of cases prevented assignment of haplotype. Direct mutation testing was obtained for all samples and was 100% concordant with the predicted haplotypes. In cases with no predicted haplotype, direct testing established the PGT-M diagnosis. The distribution for both recessive and dominant disorders was in Mendelian order. To date 66 live births confirmed the results from PGT-M diagnosis. Limitations, reasons for caution Ethical considerations and extensive genetic counselling are prerequisites for PGT-A/M testing implementation. Simultaneous screening for embryo aneuploidy and monogenetic disorders optimizes patient treatment and family planning, but may not be available in all jurisdictions. Although the data was collected prospectively, retrospective design is a limitation. Wider implications of the findings Simultaneous PGT-A and PGT-M with direct variant testing provides optimal embryo prioritization for transfer and significantly reduces time to a healthy pregnancy in couples at risk for inherited disorders. Karyomapping is effective for generic haplotyping, with a low risk of undetected recombination events in the genomic region of interest. Trial registration number not applicable

P-084 Exome sequencing reveals the novel genetic mutations associated with teratospermia

Abstract Study question This research aims to explore the molecular mechanisms of genetic variations in the small acrosome sperm subtype within teratospermia and its correlation with male infertility. Summary answer Novel gene mutations associated with teratospermia were identified, providing important insights into the genetic mechanisms underlying this condition. What is known already Male infertility is a substantial challenge in reproductive medicine, impacting a significant proportion of couples globally. Teratospermia, marked by abnormal sperm morphology, is a major factor in male reproductive disorders. Unraveling the genetic foundation of teratospermia is essential for comprehending the intricate molecular mechanisms governing male fertility. Study design, size, duration This study uses exome sequencing to investigate the genetic landscape of male infertility, particularly focusing on teratospermia. Through a comprehensive examination of the exome, our goal is to uncover novel gene mutations and understand the molecular mechanisms connecting genetic changes to abnormal sperm morphology and, consequently, male infertility. Participants/materials, setting, methods Sperm DNA was extracted from 55 patients with a confirmed diagnosis of teratospermia for this study. Subsequently, comprehensive exome sequencing was performed on the extracted sperm DNA to investigate potential genetic variations associated with teratospermia.Further functional studies were conducted to unveil the potential roles of mutated genes in the development of the reproductive system and spermogenesis. Main results and the role of chance Within the known genes associated with spermatogenic dysfunction, we identified one pathogenic/likely pathogenic variant and four variants of unknown significance, all of which have not been previously reported.Cell phenotype experiments revealed that mutations in CCDC62 and KCNU1 have specific effects on the proliferation and apoptosis of GC-2 cells. Limitations, reasons for caution Further molecular experiments and pathway mechanism studies are required to enhance our understanding of the observed effects and provide a more comprehensive interpretation of the findings. Wider implications of the findings This study unveils novel genes and pathways in teratospermia, informing diagnosis, prognosis, and potential treatments for male infertility. Exploring the genetic landscape enhances our comprehension of the complex interplay between genetics and male fertility, guiding personalized approaches in reproductive medicine. Trial registration number not applicable

Temporal dynamics and persistence of resistance genes to broad spectrum antibiotics in an urban community

The constantly evolving and growing global health crisis, antimicrobial resistance (AMR), endangers progress in medicine, food production, and life expectancy. Limited data on population-level prevalence, including seasonal and temporal variations, hampers accurate risk assessment for AMR transmission, despite its paramount importance on both global and national fronts. In this study, we used quantitative real-time PCR (q-PCR) to analyze 123 antibiotic resistance genes (ARGs) and 13 mobile genetic elements (MGEs) in wastewater of a selected urban community. Sampling was conducted monthly over a 5-month period (December 2021–April 2022) to assess resistance diversity, temporal dynamics, co-abundance of ARGs, MGEs, and resistance mechanisms. Approximately 50% of the tested ARG subtypes were consistently detected in all months, with frequency ranging from 52 to 61% with maximum absolute abundance in the winter months (December and January). In co-abundance analysis, specific genes were clustered into modules, highlighting shared distribution patterns and functional associations among the ARGs and MGEs. Detected clinically significant genes (ndm-1 and cfiA) and other variants (blaoxy, aph, aacC, tet-35, tet M, tet-32) are capable of imparting resistance to 3rd and 4th generation (gen) β-lactam, aminoglycoside, tetracycline, and multidrug classes. These contribute significantly to core/persistent resistance. This study deepens our comprehension of temporal/seasonal fluctuations in ARG and MGE distribution, providing valuable evidence to guide AMR control policies and promote responsible antibiotic/antimicrobial use for preserving effectiveness.

P-537 Linking genetic variation to infertility and embryonic genomic instability. Lessons from a database of whole genomes from targeted populations: challenges, and future directions

Abstract Study question Can novel genomic analysis methods be developed to identify genetic variants that predispose to specific types of infertility and genomic instability in embryos? Summary answer Whole genome analysis and the creation of a genomic variation database of highly targeted populations yielded several genomic links to human infertility and genomic instability. What is known already Infertility is a phenotypic manifestation that originates from highly variable causes, like repeated miscarriage (RM), repeated implantation failure (RIF) or various male and female factors. Specific genetic variations can predispose carriers to distinct types of infertility however quantifiable genetic risks are difficult to be established clinically due to the high amount of variables that exist in any relevant infertility studies. In addition, the combination of the maternal and paternal genetics further complicates the interpretation of genomics results especially when genetic instability in embryos are studied. Several studies have indicated multi[le risk variants however few have managed to establish causal relationships. Study design, size, duration The causes of infertility are extremely variable, which presents a challenge in the design and analysis of genome sequencing studies that involve large populations of participants with similar characteristics. In this study a targeted approach highly selected populations allows more detailed examination of the whole genome of the participants and the different subgroups that exist among them. The participants were recruited using specific selection criteria and samples were collected over a period of 5 years. Participants/materials, setting, methods Couples presenting with infertility and went through preimplantation genetic testing for aneuploidy (PGT- A) were invited to participate. Selection criteria were applied: i) referred for PGT-A after >3 assisted reproduction failed cycles, ii) Normal karyotypes, iii) Female age less than 37, iv) Embryonic instability in their subsequent PGT-A cycles in more than 40% of the produced embryos. After whole genome sequencing (WGS) and variant calling, a bespoke genomic SQL database was constructed for genomic analysis. Main results and the role of chance From 118 individuals that consented to participate 28 individuals with repeated implantation failure (RIF) and 8 individuals RIF/RM passed the selection criteria and their DNA was processed with WGS using a short read platform. In addition, WGS Validation was performed using genomes with known mutations. After the WGS quality control and validation. In total 50 genomes were processed and produced variant results. A novel MySQL database was successfully constructed and validated. Variant z scores were calculated for each participant and the significantly flagged variants were pulled and analysed and comparison with other bespoke and publicly available databases was performed. Ethnicity bias in novel variations detected and corrected. Several smaller cohorts were identified with similar genomic characteristics and accumulation of specific sets of polymorphisms, that were associated with certain types of infertility and preimplantation embryo abnormalities. Of particular interest were variants in synaptonemal complex genes associated with gamete failure, meiotic errors and instability in embryos. Structural variations were significant for male factor infertility. The WGS approach also revealed novel intronic and extragenic variants that would not be usually picked up by other methods like exome sequencing but can play a vital role in gene expression. Limitations, reasons for caution As with all genomic studies relating to infertility, association does not mean causation. Further functional analysis would be required to establish definitive causal pathways based on significant risk variants. In addition, although our study is highly targeted more genomic data can enhance to validation of these results. Wider implications of the findings This highly targeted study using a personalised approach to genome analysis combined with WGS can lead to protocols that determine the cumulative clinical risk for infertility, by revelling the accumulation of specific sets of polymorphisms, intronic and exonic, can be associated with certain types of infertility and preimplantation embryo abnormalities. Trial registration number not applicable

Multisite Validation of a Functional Assay to Adjudicate SCN5A Brugada Syndrome-Associated Variants.

Brugada syndrome is an inheritable arrhythmia condition that is associated with rare, loss-of-function variants in SCN5A. Interpreting the pathogenicity of SCN5A missense variants is challenging, and ≈79% of SCN5A missense variants in ClinVar are currently classified as variants of uncertain significance. Automated patch clamp technology enables high-throughput functional studies of ion channel variants and can provide evidence for variant reclassification. An in vitro SCN5A-Brugada syndrome automated patch clamp assay was independently performed at Vanderbilt University Medical Center and Victor Chang Cardiac Research Institute. The assay was calibrated according to ClinGen Sequence Variant Interpretation recommendations using high-confidence variant controls (n=49). Normal and abnormal ranges of function were established based on the distribution of benign variant assay results. Odds of pathogenicity values were derived from the experimental results according to ClinGen Sequence Variant Interpretation recommendations. The calibrated assay was then used to study SCN5A variants of uncertain significance observed in 4 families with Brugada syndrome and other arrhythmia phenotypes associated with SCN5A loss-of-function. Variant channel parameters generated independently at the 2 research sites showed strong correlations, including peak INa density (R2=0.86). The assay accurately distinguished benign controls (24/25 concordant variants) from pathogenic controls (23/24 concordant variants). Odds of pathogenicity values were 0.042 for normal function and 24.0 for abnormal function, corresponding to strong evidence for both American College of Medical Genetics and Genomics/Association for Molecular Pathology benign and pathogenic functional criteria (BS3 and PS3, respectively). Application of the assay to 4 clinical SCN5A variants of uncertain significance revealed loss-of-function for 3/4 variants, enabling reclassification to likely pathogenic. This validated high-throughput assay provides clinical-grade functional evidence to aid the classification of current and future SCN5A-Brugada syndrome variants of uncertain significance.

Reduction of sacsin levels in peripheral blood mononuclear cells as a diagnostic tool for spastic ataxia of Charlevoix-Saguenay.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay is a rare neurodegenerative disease caused by biallelic variants in the SACS gene encoding for sacsin. More than 200 pathogenic variants have been identified to date, most of which are missense. It is likely that the prevalence of autosomal recessive spastic ataxia of Charlevoix-Saguenay is underestimated due to the lack of an efficient diagnostic tool able to validate variants of uncertain significance. We have previously shown that sacsin is almost absent in fibroblasts of patients with autosomal recessive spastic ataxia of Charlevoix-Saguenay regardless of the type of SACS variant, because sacsin carrying missense variants is cotranslationally degraded. In this work, we aimed to establish the pathogenicity of SACS variants by quantifying sacsin protein in blood samples, with relevant implications for autosomal recessive spastic ataxia of Charlevoix-Saguenay diagnosis. We developed a protocol to assess sacsin protein levels by western blot using small amounts of peripheral blood mononuclear cells, which can be propagated in culture and cryopreserved. The study involves eight patients with autosomal recessive spastic ataxia of Charlevoix-Saguenay (including a novel case) carrying variants of different types and positions along the SACS gene and two parents who are carriers of heterozygous missense variants. We show that patients with autosomal recessive spastic ataxia of Charlevoix-Saguenay (carrying either missense or truncating variants) almost completely lacked sacsin in peripheral blood mononuclear cells. Moreover, both carriers of a SACS missense variant showed 50% reduction in sacsin protein levels compared to controls. We also describe a patient with uniparental isodisomy carrying a homozygous nonsense variant near the 3' end of the SACS gene. This resulted in a stable sacsin protein lacking the last 202 amino acids, probably due to escape of nonsense-mediated decay of mRNA. In conclusion, we have optimized a minimally invasive diagnostic tool for autosomal recessive spastic ataxia of Charlevoix-Saguenay in blood samples based on sacsin protein level assessment. Indeed, our results provide definite evidence that sacsin carrying missense pathogenic variants undergoes cotranslational degradation. The quantitative reduction in sacsin levels in the case of missense variants of uncertain significance allows defining them as pathogenic variants, something which cannot be predicted bioinformatically with high certainty.

Identifying Novel Germline Mutations and Copy Number Variations in Patients With SCLC

IntroductionSCLC has traditionally been considered to arise from toxic exposure factors, such as smoking. Recent evidence has revealed that germline mutations may also affect the development of SCLC; however, these alterations remain understudied. We sought to identify novel germline mutations in SCLC including germline copy number variations (CNVs) in our cohort of patients. MethodsWe designed a custom hybrid-capture gene panel to evaluate germline alterations in 192 cancer-predisposition and frequently mutated genes in SCLC. We applied this panel to germline analysis of a treatment-naive cohort of 67 patients with SCLC at our institution. Subsequently, we annotated the variants using the American College of Medical Genetics criteria and further classified variants of uncertain significance using a set of in silico tools, including DeepMind AlphaMissense, MutationTaster, SIFT, and Polyphen2. ResultsWe identified American College of Medical Genetics pathogenic/likely pathogenic alterations in seven of 67 patients. Five (71%) were novel alterations (BCORL1, FANCC, ATR, and BBC3) and a novel CNV (SLFN11) with two (29%) previously described mutations (CHEK1 and BRIP1). We also identified 191 variants of uncertain significance in 60 of 67 patients, of which, depending on the in silico tool, 5% to 14% were predicted to be pathogenic. Patients with SCLC with the seven pathogenic alterations were observed to have a numerically longer overall survival (hazard ratio = 0.50) and progression-free survival (hazard ratio = 0.45) though not statistically significant compared with the remaining cohort. ConclusionsOur study identifies novel germline alterations, including a CNV, and provides additional evidence that germline factors could be important contributing factors to the development of SCLC.

Integration of validated functional evidence to support the pathogenicity of KCNH2 variants

Functional investigation of genetic variants found in long QT syndrome can provide evidence that is needed to confirm the genetic diagnosis and establish the cause of the condition. We performed functional assessment to determine the z-score, using a clinically calibrated automated patch clamp assay, for 2 missense KCNH2 variants found in 2 families that have been diagnosed with long QT syndrome. These variants are currently classified as variant of uncertain significance in ClinVar. The z-scores for homozygous and heterozygous NM_000238.4:c.1819A>T p.(Ile607Phe) from family 1 were –5.16 and –3.97, respectively, and for heterozygous NM_000238.4:c.1832A>G p.(Tyr611Cys) from family 2 was –6.63. The z-scores for these variants are consistent with severe loss-of-function phenotypes. We have established the genetic cause of the long QT syndrome in these 2 families by providing validated functional evidence that supports the pathogenicity of p.(Ile607Phe) and p.(Tyr611Cys). Clinical diagnosis of long QT syndrome has been very successful in providing adequate clinical management for patients. However, functional assessment of variants found in these patients by using variant-specific z-scores can further enhance the current clinical management of patients with long QT syndrome through shared decision making based on validated experimental evidence.

The genetic spectrum of NF1 variants in 10 unrelated Chinese families with neurofibromatosis type 1.

To investigate the clinical and genetic features in a cohort of Chinese families with neurofibromatosis type 1 (NF1). The clinical information of 21 patients with NF1 in 10 families was retrospectively analyzed. To broaden the genetic spectrum of NF1, multiplex ligation-dependent probe amplification analysis was performed first, followed by the whole-exome sequencing, in order to identify pathogenic or potentially pathogenic variants of NF1 gene in 10 unrelated Chinese families. Nine different NF1 variants were identified in all 10 families. Of these, 7 were known pathogenic variants and included the exon 1 deletion, exons 1-58 deletion, c.5401C>T (p.Q1801*), c.2291-2A>C, c.484C>T (p.Q162*), c.4922G>A (p.W1641*) and c.1019_1020del (p.S340Cfs*25). The 2 novel variants were c.5197T>C (p.S1733P) and c.783_797delinsC (p.K261Nfs*25). The p.S1733P variant was classified as a variant of uncertain significance, while p.K261Nfs*25 was classified as pathogenic. Hence, the positive detection rate of NF1 variants was 100% (10/10). While the truncating variants were responsible for 60.0% (6/10) of the cases, the splicing variant was responsible for 10% (1/10) of the cases. We identified 2 novel heterozygous variants (c.5197T>C and c.783_797delinsC) in the NF1 gene, which broadens the genetic spectrum of the NF1 gene.

Detection of an 8p23.1 Inversion Using High-Resolution Optical Genome Mapping

Abstract Objective To evaluate the performance of optical genome mapping (OGM) in identifying an inversion located in the short arm of chromosome 8 (8p, 8p23.1), flanked by regions of complex segmental duplication (SD), using the GRCh38 and telomere-to-telomere (T2T) genome references. Methods We investigated a couple suspected of carrying the 8p23.1 inversion due to a terminal deletion combined with an interstitial duplication of 8p found in their abortus. OGM was performed on both individuals. The data were mapped to the current GRCh38 and the updated T2T genome references, respectively. Results The 8p23.1 inversion was observed in the female when mapping OGM data to the T2T assembly. In contrast, under the GRCh38 reference, the orientation between the suspected breakpoints within the SD regions could not be distinguished. Additional variants of uncertain significance were also identified in both individuals. Conclusion Our findings highlight the superiority of the T2T reference in recognizing structural variations involving SD regions. The enhanced SV detection using the T2T reference may contribute to a better understanding of genome instability and human diseases.

Molecular and Clinical Features of Congenital Hypothyroidism Due to Multiple DUOX2 Variants.

Background: DUOX2 is one of the major causative genes of congenital hypothyroidism (CH). Still, the mutation spectrum and clinical outcomes of biallelic DUOX2 variants are not fully understood. This study aimed to elucidate the molecular features and long-term clinical manifestations of CH caused by multiple pathogenic DUOX2 variants. Methods: A total of 255 patients with CH were screened for rare variants of 11 known causative genes. DUOX2 variants were classified according to their protein structure and residual activity. In vitro assays were performed for several variants of unknown functions. Clinical analyses were conducted for patients with multiple pathogenic variants of DUOX2 but not of other genes. Results: We identified 24 pathogenic variants of DUOX2, together with two benign variants and seven variants of uncertain significance, in 63 patients. The pathogenic variants included three missense substitutions and one frameshift variant that have not yet been linked to CH. Twenty-one patients carried multiple pathogenic DUOX2 variants without any other pathogenic gene variants. Three of the 21 patients harbored homozygous variants. Family analysis, long-read amplicon sequencing, and haplotype phasing confirmed compound heterozygosity of the DUOX2 variants in 14 patients, whereas the allelic positions of the variants in the remaining four patients could not be determined. Of the 21 patients, 19 were treated with levothyroxine; their ages at drug withdrawal ranged from 9 months to 21.4 years. Three patients required retreatment after drug-free intervals of 6 months, 8 months, and 10 years. There were no differences in clinical severity among patients with DUOX2 amorphic/amorphic, amorphic/hypomorphic, and hypomorphic/hypomorphic variants. Conclusions: These results broaden the mutational spectrum of DUOX2. Furthermore, our data imply that patients with multiple pathogenic DUOX2 variants typically exhibit transient CH without significant genotype-phenotype correlations. Most importantly, this study demonstrated for the first time that these patients are at risk of developing recurrent hypothyroidism after a long drug-free interval.

The Yield of Genetic Testing in Management of Nephrolithiasis

ObjectiveTo describe the yield and indications for performing genetic testing in patients with nephrolithiasis. Genetic testing for nephrolithiasis is becoming more accessible and rapid due to technologic advances. This study aimed to determine the diagnostic yield of genetic testing in a cohort of high-risk stone formers and to identify 24-hour urine characteristics to prompt genetic screening. Materials and MethodsWe retrospectively identified patients who underwent genetic testing for nephrolithiasis from 2020-22 at a single institution using a custom PerkinElmer™ genomics panel for nephrolithiasis. We compared characteristics of patients with and without genetic abnormalities. We used receiver operator characteristic (ROC) analysis to identify candidate thresholds for genetic testing. ResultsFourteen of 36 patients (39%) who underwent genetic testing had identifiable mutations. Five patients (14%) had known pathogenic mutations, including genes for primary hyperoxaluria (PH2 and PH3), cystinuria, and enamel renal syndrome. The remaining mutations were variants of uncertain significance. Of the 14 patients with identified mutations, only 6 had concordant 24-hour urine abnormalities, including 3/5 with known pathogenic mutations. In patients with urine oxalate ≥40mg/day, 3/29 (10.3%) had PH2 or PH3. ROC analysis showed that an oxalate threshold of ≥80mg/day may have promising screening characteristics. ConclusionsGenetic testing for nephrolithiasis remains controversial due to unknown yield and the time and energy required to discuss results with patients. This preliminary report describes the yield and identifies clinical factors and a potential cut-off that may assist clinicians in deciding when genetic profiling should be pursued.

Expanding the genetics and phenotypes of ocular congenital cranial dysinnervation disorders

PurposeTo identify genetic etiologies and genotype/phenotype associations for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs). MethodsWe coupled phenotyping with exome or genome sequencing of 467 probands (550 affected and 1108 total individuals) with genetically unsolved oCCDDs, integrating analyses of pedigrees, human and animal model phenotypes, and de novo variants to identify rare candidate single-nucleotide variants, insertion/deletions, and structural variants disrupting protein-coding regions. Prioritized variants were classified for pathogenicity and evaluated for genotype/phenotype correlations. ResultsAnalyses elucidated phenotypic subgroups, identified pathogenic/likely pathogenic variant(s) in 43 of 467 probands (9.2%), and prioritized variants of uncertain significance in 70 of 467 additional probands (15.0%). These included known and novel variants in established oCCDD genes, genes associated with syndromes that sometimes include oCCDDs (eg, MYH10, KIF21B, TGFBR2, and TUBB6), genes that fit the syndromic component of the phenotype but had no prior oCCDD association (eg, CDK13, TGFB2), genes with no reported association with oCCDDs or the syndromic phenotypes (eg, TUBA4A, KIF5C, CTNNA1, KLB, and FGF21), and genes associated with oCCDD phenocopies that had resulted in misdiagnoses. ConclusionThis study suggests that unsolved oCCDDs are clinically and genetically heterogeneous disorders often overlapping other Mendelian conditions and nominates many candidates for future replication and functional studies.

Peripheral T Cell Development and Immunophenotyping of Twins with Heterozygous FOXN1 Mutations.

The transcription factor FOXN1 plays an established role in thymic epithelial development to mediate selection of maturing thymocytes. Patients with heterozygous loss-of-function FOXN1 variants are associated with T cell lymphopenia at birth and low TCR excision circles that can ultimately recover. Although CD4+ T cell reconstitution in these patients is not completely understood, a lower proportion of naive T cells in adults has suggested a role for homeostatic proliferation. In this study, we present an immunophenotyping study of fraternal twins with low TCR excision circles at birth. Targeted primary immunodeficiency testing revealed a heterozygous variant of uncertain significance in FOXN1 (c.1205del, p.Pro402Leufs*148). We present the immune phenotypes of these two patients, as well as their father who carries the same FOXN1 variant, to demonstrate an evolving immune environment over time. While FOXN1 haploinsufficiency may contribute to thymic defects and T cell lymphopenia, we characterized the transcriptional activity and DNA binding of the heterozygous FOXN1 variant in 293T cells and found the FOXN1 variant to have different effects across several target genes. These data suggest multiple mechanisms for similar FOXN1 variants pathogenicity that may be mutation specific. Increased understanding of how these variants drive transcriptional regulation to impact immune cell populations will guide the potential need for therapeutics, risk for infection or autoimmunity over time, and help inform clinical decisions for other variants that might arise.