Nonsyndromic sporadic thoracic aortic aneurysm (nssTAA) is characterized by diverse genetic variants that may vary in different populations. Our aim was to identify clinically relevant variants in genes implicated in hereditary aneurysms in Russian patients with nssTAA. Forty-one patients with nssTAA without dissection were analyzed. Using massive parallel sequencing, we searched for variants in exons of 53 known disease-causing genes. Patients were found to have no (likely) pathogenic variants in the genes of hereditary TAA. Six variants of uncertain significance (VUSs) were identified in four (9.8%) patients. Three VUSs [FBN1 c.7841C>T (p.Ala2614Val), COL3A1 c.2498A>T (p.Lys833Ile), and MYH11 c.4993C>T (p.Arg1665Cys)] are located in genes with \"definitive\" disease association (ClinGen). The remaining variants are in \"potentially diagnostic\" genes or genes with experimental evidence of disease association [NOTCH1 c.964G>A (p.Val322Met), COL4A5 c.953C>G (p.Pro318Arg), and PLOD3 c.833G>A (p.Gly278Asp)]. Russian patients with nssTAA without dissection examined in this study have ≥1 VUSs in six known genes of hereditary TAA (FBN1, COL3A1, MYH11, NOTCH1, COL4A5, or PLOD3). Experimental studies expanded genetic testing, and clinical examination of patients and first/second-degree relatives may shift VUSs to the pathogenic (benign) category or to a new class of rare \"predisposing\" low-penetrance variants causing the pathology if combined with other risk factors.

BACKGROUND: Genetic diagnostics support the diagnosis of hereditary arrhythmogenic diseases, but variants of uncertain significance (VUS) complicate matters, emphasising the need for regular reassessment. Our study aims to reanalyse rare variants in different genes in order to decrease VUS diagnoses and thus improve risk stratification and personalized treatment for patients with arrhythmogenic disorders.
METHODS: Genomic DNA was analysed using Sanger sequencing and next-generation sequencing (NGS). The Data was evaluated using various databases and in silico prediction tools and classified according to current ACMG standards by two independent experts.
RESULTS: We identified 53 VUS in 30 genes, of which 17 variants (32%) were reclassified. 13% each were downgraded to likely benign (LB) and benign (B) and 6% were upgraded to likely pathogenic (LP). Reclassifications mainly occurred among variants initially classified in 2017-2019, with rates ranging from 50 to 60%.
CONCLUSIONS: The results support the assumption that regular reclassification of VUS is important, as it provides new insights for genetic diagnostics, that benefit patients and guide therapeutic approach.

Most of endometrial cancers are sporadic, with 5% or less being attributed to inherited pathogenic germline mutations and mostly related to the Lynch syndrome. To our knowledge, this is the first study to investigate patterns and frequencies of germline mutations in patients with endometrial cancer in an Arab region. Consecutive patients with endometrial cancer (n = 130), regardless of their age and family history, were enrolled. Germline genetic testing, using an 84-gene panel, was performed on all. Almost half of the patient population (n = 64, 49.2%) was tested based on international guidelines, while the remaining patients (n = 66, 50.8%) were tested as part of an ongoing universal germline genetic testing program. Among the whole group, 18 (13.8%) patients had positive pathogenic or likely pathogenic (P/LP) germline variants. The most common variants encountered were in MLH1 (n = 4, 22.2%), PMS2 (n = 3, 16.7%), ATM, MSH2, MUTYH, and BRCA2 (n = 2, 11.1% each). In addition, three (2.3%) patients were found to have an increased risk allele of the APC gene. P/LP variants were more common among patients with carcinosarcoma and clear cell carcinoma, younger patients (age ≤ 50 years), and in patients with a non-metastatic disease. We conclude that germline genetic variants, mostly in genes related to the Lynch syndrome, are relatively common among Arab patients with endometrial cancer.

Germline TP53 pathogenic variants can lead to a cancer susceptibility syndrome known as Li-Fraumeni (LFS). Variants affecting its activity can drive tumorigenesis altering p53 pathways and their identification is crucial for assessing individual risk. This study explored the functional impact of TP53 missense variants on its transcription factor activity. We selected seven TP53 missense variants (c.129G > C, c.320A > G, c.417G > T, c.460G > A, c,522G > T, c.589G > A and c.997C > T) identified in Brazilian families at-risk for LFS. Variants were created through site-directed mutagenesis and transfected into SK-OV-3 cells to assess their transcription activation capabilities. Variants K139N and V197M displayed significantly reduced transactivation activity in a TP53-dependent luciferase reporter assay. Additionally, K139N negatively impacted CDKN1A and MDM2 expression and had a limited effect on GADD45A and PMAIP1 upon irradiation-induced DNA damage. Variant V197M demonstrated functional impact in all target genes evaluated and loss of Ser15 phosphorylation. K139N and V197M variants presented a reduction of p21 levels after irradiation. Our data show that K139N and V197M negatively impact p53 functions, supporting their classification as pathogenic variants. This underscores the significance of conducting functional studies on germline TP53 missense variants classified as variants of uncertain significance to ensure proper management of LFS-related cancer risks.

More than 450 genetic defects result in inborn errors of immunity (IEI). Their individual prevalence in specific cohorts is influenced by national characteristics and other factors. We present results of genetic testing conducted in 1809 Russian children with IEI. Genetic defects confirming IEI were found in 1112 out of 1809 (61.5%) probands. These defects included variants in 118 single genes (87.9% of patients) and aberrations in 6 chromosomes (11.8%). Notably, three patients harbored pathogenic variants in more than one IEI gene. Large deletions constituted 5% of all defects. Out of the 799 original variants, 350 (44%) have not been described previously. Rare genetic defects (10 or fewer patients per gene) were identified in 20% of the patients. Among 967 probands with germline variants, defects were inherited in an autosomal dominant manner in 29%, X-linked in 34%, and autosomal recessive in 37%. Four females with non-random X-inactivation exhibited symptoms of X-linked diseases (BTK, WAS, CYBB, IKBKG gene defects). Despite a relatively low rate of consanguinity in Russia, 47.9% of autosomal recessive gene defects were found in a homozygous state. Notably, 28% of these cases carried \"Slavic\" mutation of the NBN gene or known hot-spot mutations in other genes. The diversity of IEI genetic forms and the high frequency of newly described variants underscore the genetic heterogeneity within the Russian IEI group. The new variants identified in this extensive cohort will enrich genetic databases.

Developmental anomalies of the hearing organ, the cochlea, are diagnosed in approximately one-fourth of individuals with congenital deafness. Most patients with cochlear malformations remain etiologically undiagnosed due to insufficient knowledge about underlying genes or the inability to make conclusive interpretations of identified genetic variants. We used exome sequencing for genetic evaluation of hearing loss associated with cochlear malformations in three probands from unrelated families. We subsequently generated monoclonal induced pluripotent stem cell (iPSC) lines, bearing patient-specific knockins and knockouts using CRISPR/Cas9 to assess pathogenicity of candidate variants. We detected FGF3 (p.Arg165Gly) and GREB1L (p.Cys186Arg), variants of uncertain significance in two recognized genes for deafness, and PBXIP1(p.Trp574*) in a candidate gene. Upon differentiation of iPSCs towards inner ear organoids, we observed significant developmental aberrations in knockout lines compared to their isogenic controls. Patient-specific single nucleotide variants (SNVs) showed similar abnormalities as the knockout lines, functionally supporting their causality in the observed phenotype. Therefore, we present human inner ear organoids as a tool to rapidly validate the pathogenicity of DNA variants associated with cochlear malformations.

Replication-coupled gene editing using locked nucleic acid-modified single-stranded DNA oligonucleotides (LMOs) can genetically engineer mammalian cells with high precision at single nucleotide resolution. Based on this method, oligonucleotide-directed mutation screening (ODMS) was developed to determine whether variants of uncertain clinical significance of DNA mismatch repair (MMR) genes can cause Lynch syndrome. In ODMS, the appearance of 6-thioguanine-resistant colonies upon introduction of the variant is indicative for defective MMR and hence pathogenicity. Whereas mouse embryonic stem cells (mESCs) hemizygous for MMR genes were used previously, we now show that ODMS can also be applied in wild-type mESCs carrying two functional alleles of each MMR gene. 6-Thioguanine resistance can result from two possible events: first, the mutation is present in only one allele, which is indicative for dominant-negative activity of the variant; and second, both alleles contain the planned modification, which is indicative for a regular loss-of-function variant. Thus, ODMS in wild-type mESCs can discriminate fully disruptive and dominant-negative MMR variants. The feasibility of biallelic targeting suggests that the efficiency of LMO-mediated gene targeting at a nonselectable locus may be enriched in cells that had undergone a simultaneous selectable LMO targeting event. This turned out to be the case and provided a protocol to improve recovery of LMO-mediated gene modification events.

Accurate interpretation of human genetic data is critical for optimizing outcomes in the era of genomic medicine. Powerful methods for testing genetic variants for functional effects are allowing researchers to characterize thousands of variants across disease genes. Here, we review experimental tools enabling highly scalable assays of variants, focusing specifically on Saturation Genome Editing (SGE). We discuss examples of how this technique is being implemented for variant testing at scale and describe how SGE data for BRCA1 have been clinically validated and used to aid variant interpretation. The initial success at predicting variant pathogenicity with SGE has spurred efforts to expand this and related techniques to many more genes.

Lysosomal acid sphingomyelinase (ASM), a critical enzyme in lipid metabolism encoded by the SMPD1 gene, plays a crucial role in sphingomyelin hydrolysis in lysosomes. ASM deficiency leads to acid sphingomyelinase deficiency, a rare genetic disorder with diverse clinical manifestations, and the protein can be found mutated in other diseases. We employed a structure-based framework to comprehensively understand the functional implications of ASM variants, integrating pathogenicity predictions with molecular insights derived from a molecular dynamics simulation in a lysosomal membrane environment. Our analysis, encompassing over 400 variants, establishes a structural atlas of missense variants of lysosomal ASM, associating mechanistic indicators with pathogenic potential. Our study highlights variants that influence structural stability or exert local and long-range effects at functional sites. To validate our predictions, we compared them to available experimental data on residual catalytic activity in 135 ASM variants. Notably, our findings also suggest applications of the resulting data for identifying cases suited for enzyme replacement therapy. This comprehensive approach enhances the understanding of ASM variants and provides valuable insights for potential therapeutic interventions.

OBJECTIVE: Rare genetic variants in the PURA gene cause the PURA-related neurodevelopmental disorder (PURA-NDD), characterized by neonatal abnormalities and developmental delay. Using genome-wide DNA methylation analysis on patients with PURA variants, we aim to establish a PURA-NDD-specific methylation profile and provide further insights on the molecular basis of the PURA-NDD.
METHODS: Twenty three individuals (including 12 unpublished) carrying PURA variants were enrolled. We conducted the Illumina Infinium EPIC microarray analysis in 17 PURA-NDD individuals. In vitro experiments were performed to examine how PURA variants affect Pur-a expression.
RESULTS: Additional phenotypes in 12 newly identified patients were described in this study. Genome-wide DNA methylation analysis unveiled distinctive methylation profiles to PURA-NDD, and the established classifier can reclassify PURA variants of uncertain significance. Patients bearing PURA hapoloinsufficient and missense variants have comparable DNA methylation profiles, and cells expressing these PURA variants showed consistent Pur-a downregulation, suggesting a haploinsufficiency mechanism.
CONCLUSIONS: Patients with PURA-NDD exhibit a specific episignature, which has potential to aid identification and diagnosis of PURA-NDD patients and offer implications for further functional investigations.