Congenital long QT syndrome (LQTS) is an inherited arrhythmia syndrome associated with sudden cardiac death. Accurate interpretation and classification of genetic variants in LQTS patients are crucial for effective management. All patients with LQTS with a positive genetic test over the past 18 years (2002-2020) in our single tertiary pediatric cardiac center were identified. Reevaluation of the reported variants in LQTS genes was conducted using the American College of Genetics and Genomics (ACMG) guideline after refinement by the US ClinGen SVI working group and guideline by Walsh et al. on genetic variant reclassification, under multidisciplinary input. Among the 59 variants identified. 18 variants (30.5%) were reclassified. A significant larger portion of variants of unknown significance (VUS) were reclassified compared to likely pathogenic (LP)/pathogenic (P) variants (57.7% vs 9.1%, p < 0.001). The rate of reclassification was significantly higher in the limited/disputed evidence group compared to the definite/moderate evidence group (p = 0.0006). All LP/P variants were downgraded in the limited/disputed evidence group (p = 0.0057). VUS upgrades are associated with VUS located in genes within the definite/moderate evidence group (p = 0.0403) and with VUS present in patients exhibiting higher corrected QT intervals (QTc) (p = 0.0445). A significant number of pediatric LQTS variants were reclassified, particularly for VUS. The strength of the gene-disease association of the genes influences the reclassification performance. The study provides important insights and guidance for pediatricians to seek for reclassification of \"outdated variants\" in order to facilitate contemporary precision medicine.

BACKGROUND: Lipoprotein lipase (LPL) plays a crucial role in triglyceride hydrolysis. Rare biallelic variants in the LPL gene leading to complete or near-complete loss of function cause autosomal recessive familial chylomicronemia syndrome. However, rare biallelic LPL variants resulting in significant but partial loss of function are rarely documented. This study reports a novel occurrence of such rare biallelic LPL variants in a Chinese patient with hypertriglyceridemia-induced acute pancreatitis (HTG-AP) during pregnancy and provides an in-depth functional characterization.
METHODS: The complete coding sequences and adjacent intronic regions of the LPL, APOC2, APOA5, LMF1, and GPIHBP1 genes were analyzed by Sanger sequencing. The aim was to identify rare variants, including nonsense, frameshift, missense, small in-frame deletions or insertions, and canonical splice site mutations. The functional impact of identified LPL missense variants on protein expression, secretion, and activity was assessed in HEK293T cells through single and co-transfection experiments, with and without heparin treatment.
RESULTS: Two rare LPL missense variants were identified in the patient: the previously reported c.809G > A (p.Arg270His) and a novel c.331G > C (p.Val111Leu). Genetic testing confirmed these variants were inherited biallelically. Functional analysis showed that the p.Arg270His variant resulted in a near-complete loss of LPL function due to effects on protein synthesis/stability, secretion, and enzymatic activity. In contrast, the p.Val111Leu variant retained approximately 32.3% of wild-type activity, without impacting protein synthesis, stability, or secretion. Co-transfection experiments indicated a combined activity level of 20.7%, suggesting no dominant negative interaction between the variants. The patient\'s post-heparin plasma LPL activity was about 35% of control levels.
CONCLUSIONS: This study presents a novel case of partial but significant loss-of-function biallelic LPL variants in a patient with HTG-AP during pregnancy. Our findings enhance the understanding of the nuanced relationship between LPL genotypes and clinical phenotypes, highlighting the importance of residual LPL function in disease manifestation and severity. Additionally, our study underscores the challenges in classifying partial loss-of-function variants in classical Mendelian disease genes according to the American College of Medical Genetics and Genomics (ACMG)\'s variant classification guidelines.

Inherited retinal diseases (IRDs) are a group of rare genetic eye conditions that cause blindness. Despite progress in identifying genes associated with IRDs, improvements are necessary for classifying rare autosomal dominant (AD) disorders. AD diseases are highly heterogenous, with causal variants being restricted to specific amino acid changes within certain protein domains, making AD conditions difficult to classify. Here, we aim to determine the top-performing in-silico tools for predicting the pathogenicity of AD IRD variants. We annotated variants from ClinVar and benchmarked 39 variant classifier tools on IRD genes, split by inheritance pattern. Using area-under-the-curve (AUC) analysis, we determined the top-performing tools and defined thresholds for variant pathogenicity. Top-performing tools were assessed using genome sequencing on a cohort of participants with IRDs of unknown etiology. MutScore achieved the highest accuracy within AD genes, yielding an AUC of 0.969. When filtering for AD gain-of-function and dominant negative variants, BayesDel had the highest accuracy with an AUC of 0.997. Five participants with variants in NR2E3, RHO, GUCA1A, and GUCY2D were confirmed to have dominantly inherited disease based on pedigree, phenotype, and segregation analysis. We identified two uncharacterized variants in GUCA1A (c.428T>A, p.Ile143Thr) and RHO (c.631C>G, p.His211Asp) in three participants. Our findings support using a multi-classifier approach comprised of new missense classifier tools to identify pathogenic variants in participants with AD IRDs. Our results provide a foundation for improved genetic diagnosis for people with IRDs.

The American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines recommend using variant enrichment among cases as \"strong\" evidence for pathogenicity per the PS4 criterion. However, quantitative support for PS4 thresholds from real-world Mendelian case-control cohorts is lacking.
To address this gap, we evaluated and established PS4 thresholds using data from the Chinese Deafness Genetics Consortium. A total of 9,050 variants from 13,845 patients with hearing loss (HL) and 6,570 ancestry-matched controls were analyzed. Positive likelihood ratio and local positive likelihood ratio values were calculated to determine the thresholds corresponding to each strength of evidence across three variant subsets.
In subset 1, consisting of variants present in both cases and controls with an allele frequency (AF) in cases ≥ 0.0005, an odds ratio (OR) ≥ 6 achieved strong evidence, while OR ≥ 3 represented moderate evidence. For subset 2, which encompassed variants present in both cases and controls with a case AF < 0.0005, and subset 3, comprising variants found only in cases and absent from controls, we defined the PS4_Supporting threshold (OR > 2.27 or allele count ≥ 3) and the PS4_Moderate threshold (allele count ≥ 6), respectively. Reanalysis applying the adjusted PS4 criteria changed the classification of 15 variants and enabled diagnosis of an additional four patients.
Our study quantified evidence strength thresholds for variant enrichment in genetic HL cases, highlighting the importance of defining disease/gene-specific thresholds to improve the precision and accuracy of clinical genetic testing.

PRSS1 was the first reported chronic pancreatitis (CP) gene. The existence of both gain-of-function (GoF) and gain-of-proteotoxicity (GoP) pathological PRSS1 variants, together with the fact that PRSS1 variants have been identified in CP subtypes spanning the range from monogenic to multifactorial, has made the classification of PRSS1 variants very challenging.
All currently reported PRSS1 variants (derived primarily from two databases) were manually reviewed with respect to their clinical genetics, functional analysis and population allele frequency. They were classified by variant type and pathological mechanism within the framework of our recently proposed ACMG/AMP guidelines-based seven-category system.
The total number of distinct germline PRSS1 variants included for analysis was 100, comprising 3 copy number variants (CNVs), 12 5\' and 3\' variants, 19 intronic variants, 5 nonsense variants, 1 frameshift deletion variant, 6 synonymous variants, 1 in-frame duplication, 3 gene conversions and 50 missense variants. Based upon a combination of clinical genetic and functional analysis, population data and in silico analysis, we classified 26 variants (all 3 CNVs, the in-frame duplication, all 3 gene conversions and 19 missense) as \"pathogenic\", 3 variants (missense) as \"likely pathogenic\", 5 variants (four missense and one promoter) as \"predisposing\", 13 variants (all missense) as \"unknown significance\", 2 variants (missense) as \"likely benign\", and all remaining 51 variants as \"benign\".
We describe an expert classification of the 100 PRSS1 variants reported to date. The results have immediate implications for reclassifying many ClinVar-registered PRSS1 variants as well as providing optimal guidelines/standards for reporting PRSS1 variants.

Curated databases of genetic variants assist clinicians and researchers in interpreting genetic variation. Yet, these databases contain some misclassified variants. It is unclear whether variant misclassification is abating as these databases rapidly grow and implement new guidelines.
Using archives of ClinVar and HGMD, we investigated how variant misclassification has changed over 6 years, across different ancestry groups. We considered inborn errors of metabolism (IEMs) screened in newborns as a model system because these disorders are often highly penetrant with neonatal phenotypes. We used samples from the 1000 Genomes Project (1KGP) to identify individuals with genotypes that were classified by the databases as pathogenic. Due to the rarity of IEMs, nearly all such classified pathogenic genotypes indicate likely variant misclassification in ClinVar or HGMD.
While the false-positive rates of both ClinVar and HGMD have improved over time, HGMD variants currently imply two orders of magnitude more affected individuals in 1KGP than ClinVar variants. We observed that African ancestry individuals have a significantly increased chance of being incorrectly indicated to be affected by a screened IEM when HGMD variants are used. However, this bias affecting genomes of African ancestry was no longer significant once common variants were removed in accordance with recent variant classification guidelines. We discovered that ClinVar variants classified as Pathogenic or Likely Pathogenic are reclassified sixfold more often than DM or DM? variants in HGMD, which has likely resulted in ClinVar\'s lower false-positive rate.
Considering misclassified variants that have since been reclassified reveals our increasing understanding of rare genetic variation. We found that variant classification guidelines and allele frequency databases comprising genetically diverse samples are important factors in reclassification. We also discovered that ClinVar variants common in European and South Asian individuals were more likely to be reclassified to a lower confidence category, perhaps due to an increased chance of these variants being classified by multiple submitters. We discuss features for variant classification databases that would support their continued improvement.

ATP1A2 gene mutation has been indicated to cause alternating hemiplegia of childhood (AHC); however, limited evidence supports this relationship so far.
We reported two Chinese patients with de novo ATP1A2 variants (c.970G>A and c.889G>A). Both patients presented with episodes of alternating hemiplegia, seizures and mild developmental delay. Brain magnetic resonance imaging revealed abnormal signals in both patients.
The new genetic evidence we reported here strengthened the gene-disease relationship, and the gene curation level between ATP1A2 and AHC became \"Moderate\" following the ClinGen Standard Operation Procedure. Consequently, the two variants can be reclassified as likely pathogenic.

TMEM127 is a poorly known tumor suppressor gene associated with pheochromocytomas, paragangliomas, and renal carcinomas. Our incomplete understanding of TMEM127 function has limited our ability to predict variant pathogenicity.
To better understand the function of the transmembrane protein TMEM127 we undertook cellular and molecular evaluation of patient-derived germline variants.
Subcellular localization and steady-state levels of tumor-associated, transiently expressed TMEM127 variants were compared to the wild-type protein using immunofluorescence and immunoblot analysis, respectively, in cells genetically modified to lack endogenous TMEM127. Membrane topology and endocytic mechanisms were also assessed.
We identified 3 subgroups of mutations and determined that 71% of the variants studied are pathogenic or likely pathogenic through loss of membrane-binding ability, stability, and/or internalization capability. Investigation into an N-terminal cluster of missense variants uncovered a previously unrecognized transmembrane domain, indicating that TMEM127 is a 4- transmembrane, not a 3-transmembrane domain-containing protein. Additionally, a C-terminal variant with predominant plasma membrane localization revealed an atypical, extended acidic, dileucine-based motif required for TMEM127 internalization through clathrin-mediated endocytosis.
We characterized the functional deficits of several germline TMEM127 variants and identified novel structure-function features of TMEM127. These findings will assist in determining pathogenicity of TMEM127 variants and will help guide future studies investigating the cellular role of TMEM127.

Pathogenic variants in GJB2 are the most common cause of autosomal recessive sensorineural hearing loss. The classification of c.101T>C/p.Met34Thr and c.109G>A/p.Val37Ile in GJB2 are controversial. Therefore, an expert consensus is required for the interpretation of these two variants.
The ClinGen Hearing Loss Expert Panel collected published data and shared unpublished information from contributing laboratories and clinics regarding the two variants. Functional, computational, allelic, and segregation data were also obtained. Case-control statistical analyses were performed.
The panel reviewed the synthesized information, and classified the p.Met34Thr and p.Val37Ile variants utilizing professional variant interpretation guidelines and professional judgment. We found that p.Met34Thr and p.Val37Ile are significantly overrepresented in hearing loss patients, compared with population controls. Individuals homozygous or compound heterozygous for p.Met34Thr or p.Val37Ile typically manifest mild to moderate hearing loss. Several other types of evidence also support pathogenic roles for these two variants.
Resolving controversies in variant classification requires coordinated effort among a panel of international multi-institutional experts to share data, standardize classification guidelines, review evidence, and reach a consensus. We concluded that p.Met34Thr and p.Val37Ile variants in GJB2 are pathogenic for autosomal recessive nonsyndromic hearing loss with variable expressivity and incomplete penetrance.

Variants with known or possible pathogenicity located in genes that are unrelated to primary disease conditions are defined as secondary findings. Secondary findings are not the primary targets of whole exome and genome sequencing (WES/WGS) assay but can be of great practical value in early disease prevention and intervention. The driving force for this study was to investigate the impact of racial difference and disease background on secondary findings. Here, we analyzed secondary findings frequencies in 421 whole exome-sequenced Chinese children who are phenotypically normal or bear congenital heart diseases/juvenile obesity. In total, 421 WES datasets were processed for potential deleterious variant screening. A reference gene list was defined according to the American College of Medical Genetics and Genomics (ACMG) recommendations for reporting secondary findings v2.0 (ACMG SF v2.0). The variant classification was performed according to the evidence-based guidelines recommended by the joint consensus of the ACMG and the Association for Molecular Pathology (AMP).
Among the 421 WES datasets, we identified 11 known/expected pathogenic variants in 12 individuals, accounting for 2.85% of our samples, which is much higher than the reported frequency in a Caucasian population. In conclusion, secondary findings are not so rare in Chinese children, which means that we should pay more attention to the clinical interpretation of sequencing results.