Putative Pathogenic Variants Research Articles

Screening for mitochondrial tRNA variants in 200 patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a common autoimmune disease with unknown etiology. Recently, a growing number of evidence suggested that mitochondrial dysfunctions played active roles in the pathogenesis of SLE, but its detailed mechanism remains largely undetermined. The aim of this study was to analysis the frequencies of mitochondrial tRNA (mt-tRNA) variants in Chinese individuals with SLE. We carried out a mutational screening of mt-tRNA variants in a cohort of 200 patients with SLE and 200 control subjects by PCR-Sanger sequencing. The potential pathogenicity of mt-tRNA variants were evaluated by phylogenetic conservation and haplogroup analyses. In addition, trans-mitochondrial cybrid cell lines were established, mitochondrial functions including ATP, reactive oxygen species (ROS), mitochondrial DNA (mtDNA) copy number, mitochondrial membrane potential (MMP), superoxide dismutase (SOD) and mt-RNA transcription were analyzed in cybrids with and without these putative pathogenic mt-tRNA variants. We identified five possible pathogenic variants: tRNAVal G1606A, tRNALeu(UUR) A3243G, tRNAIle A4295G, tRNAGly T9997C, and tRNAThr A15924G that only found in SLE patients but were absent in controls. Interestingly, these variants were located at extremely conserved nucleotides of the corresponding tRNAs and may alter tRNAs' structure and function. Furthermore, cells carrying these tRNA variants had much lower levels of ATP, mtDNA copy number, MMP and SOD than controls, by contrast, the levels of ROS increased significantly (p<0.05 for all). Furthermore, a significantly reductions in mt-ND1, ND2, ND3, ND5 and A6 mRNA expression were observed in cells with these mt-tRNA variants, while compared with controls. Thus, failures in tRNAs metabolism caused by these variants would impair mitochondrial translation, and subsequently lead to mitochondrial dysfunction that was involved in the progression and pathogenesis of SLE. Our study suggested that mt-tRNA variants were important causes for SLE, screening for mt-tRNA pathogenic variants was recommended for early detection and prevention for this disorder.

Four putative pathogenic ARHGAP29 variants in patients with non-syndromic orofacial clefts (NsOFC).

The pathophysiological basis of non-syndromic orofacial cleft (NsOFC) is still largely unclear. However, exome sequencing (ES) has led to identify several causative genes, often with reduced penetrance. Among these, the Rho GTPase activating protein 29 (ARHGAP29) has been previously implicated in 7 families with NsOFC. We investigated a cohort of 224 NsOFCs for which no genetic pathogenic variant had been identified by diagnostic testing. We used ES and bioinformatic variant filtering and identified four novel putative pathogenic variants in ARHGAP29 in four families. One was a missense variant leading to the substitution of the first methionine with threonine, two were heterozygous frameshift variants leading to a premature termination codon, and one was a nonsense variant. All variants were predicted to result in loss of function, either through mRNA decay, truncated ARHGAP29, or abnormal N-terminal initiation of translation of ARHGAP29. The truncated ARHGAP29 proteins would lack the important RhoGAP domain. The variants were either absent or rare in the control population databases, and the loss of intolerance score (pLI) of ARHGAP29 is 1.0, suggesting that ARHGAP29 haploinsufficiency is not tolerated. Phenotypes ranged from microform cleft lip (CL) to complete bilateral cleft lip and palate (CLP), with one unaffected mutation carrier. These results extend the mutational spectrum of ARHGAP29 and show that it is an important gene underlying variable NsOFC phenotypes. ARHGAP29 should be included in diagnostic genetic testing for NsOFC, especially familial cases, as it may be mutated in ∼4% of them (4/97 in our cohort) with high penetrance (89%).

Galectin-3 zebrafish model: identification and pharmacological modulation of arrhythmogenic cardiomyopathy-related phenotypes

Abstract Background Arrhythmogenic Cardiomyopathy (ACM) is rare hereditary cardiac condition characterized by the substitution of ventricular myocardium with fibro-fatty tissue leading to high risk of life-threatening arrhythmias and sudden cardiac death, particularly among young individuals and athletes. Based on genotype-phenotype correlation, the spectrum of the ACM phenotype was shown to be broader than previously believed. Approximately 40% of ACM patients carry one or more putative pathogenic variants in genes encoding desmosomal proteins. Recently LGALS3 gene involvement in ACM early pathogenesis has been advocated. Aim To achieve a comprehensive understanding of LGALS3 role in the pathogenesis of AC. Methods This stable mutant line was generated by CRISPR/Cas9 strategy and then deeply characterized. Phenotyping included heart activity characterization using a recently published software specific for zebrafish, the pyHeart4Fish. Gene expression analysis was carried out by using RNAseq, Real Time PCR and signaling pathway reporter zebrafish lines. Immunofluorescence analysis were performed using and antibody against L-plastin, a leukocyte marker for the detection of inflammatory cells and Acridine Orange/Ethidium Bromide staining to check the possible presence of apoptotic and necrotic events. Results Morphological investigation revealed notable structural abnormalities such as pericardial effusion and/or hemopericardium. The heart activity analysis detected a diminished ventricle contractility and ejection fraction, with bradycardia and occurrence of arrhythmia events. Rna analysis highlighted the dysregulation of Wnt/β-catenin signaling pathway. Moreover, the L-plastin inflammatory marker revealed a significant infiltration of inflammatory cells in the heart as well as an elevated number of apoptotic and necrotic cells (figure 1). The pharmacological treatment, partially rescued a subset of AC-related cardiac phenotypes observed, including heart contractility, bradycardia and arrhythmia. Conclusion Our findings strongly validate our lgals3a zebrafish mutant as an effective model to elucidate its role in ACM related phenotype. This model accurately replicates the cardiac abnormalities and dysregulation in cell signaling that are characteristic of ACM. Further, observed cell death and inflammation and the partial recovery of cardiac phenotypes through pharmacological intervention, underscores the potential of targeting the Wnt/β-catenin signaling pathway as future perspective for ACM.Figure 1

Whole-Exome Sequencing Improves Understanding of Inherited Retinal Dystrophies in Korean Patients.

Retinitis pigmentosa (RP) encompasses a diverse range of hereditary, degenerative retinal ailments, presenting notable obstacles to molecular genetic diagnoses due to the intricate array of variants in different genes involved. This study enrolled 21 probands and their families who have been diagnosed with nonsyndromic RP but without a previous molecular diagnosis. We employed whole-exome sequencing (WES) to detect possible harmful gene variations in individuals with unknown-cause RP at the molecular level. WES allowed the identification of ten potential disease-causing variants in eight different genes. In 8 out of the total 21 patients, this method successfully identified the underlying molecular causes, such as putative pathogenic variants in genes including CRB1, KLHL7, PDE6B, RDH12, RP1, RPE65, USH2A, and RHO. A novel variant was identified in one of these genes, specifically PDE6B, providing valuable information on prospective targets for future enhanced gene therapeutic approaches.

Structural Variants and Implicated Processes Associated with Familial Tourette Syndrome.

Gilles de la Tourette syndrome (GTS) is a neurodevelopmental psychiatric disorder with complex and elusive etiology with a significant role of genetic factors. The aim of this study was to identify structural variants that could be associated with familial GTS. The study group comprised 17 multiplex families with 80 patients. Structural variants were identified from whole-genome sequencing data and followed by co-segregation and bioinformatic analyses. The localization of these variants was used to select candidate genes and create gene sets, which were subsequently processed in gene ontology and pathway enrichment analysis. Seventy putative pathogenic variants shared among affected individuals within one family but not present in the control group were identified. Only four private or rare deletions were exonic in LDLRAD4, B2M, USH2A, and ZNF765 genes. Notably, the USH2A gene is involved in cochlear development and sensory perception of sound, a process that was associated previously with familial GTS. In addition, two rare variants and three not present in the control group were co-segregating with the disease in two families, and uncommon insertions in GOLM1 and DISC1 were co-segregating in three families each. Enrichment analysis showed that identified structural variants affected synaptic vesicle endocytosis, cell leading-edge organization, and signaling for neurite outgrowth. The results further support the involvement of the regulation of neurotransmission, neuronal migration, and sound-sensing in GTS.

Upstream open reading frame-introducing variants in patients with primary familial brain calcification.

More than 50% of patients with primary familial brain calcification (PFBC), a rare neurological disorder, remain genetically unexplained. While some causative genes are yet to be identified, variants in non-coding regions of known genes may represent a source of missed diagnoses. We hypothesized that 5'-Untranslated Region (UTR) variants introducing an AUG codon may initiate mRNA translation and result in a loss of function in some of the PFBC genes. After reannotation of exome sequencing data of 113 unrelated PFBC probands, we identified two upstream AUG-introducing variants in the 5'UTR of PDGFB. One, NM_002608.4:c.-373C>G, segregated with PFBC in the family. It was predicted to create an upstream open reading frame (ORF). The other one, NM_002608.4:c.-318C>T, was found in a simplex case. It was predicted to result in an ORF overlapping the natural ORF with a frameshift. In a GFP reporter assay, both variants were associated with a dramatic decrease in GFP levels, and, after restoring the reading frame with the GFP sequence, the c.-318C>T variant was associated with a strong initiation of translation as measured by western blotting. Overall, we found upstream AUG-introducing variants in the 5'UTR of PDGFB in 2/113 (1.7%) undiagnosed PFBC cases. Such variants thus represent a source of putative pathogenic variants.

Gastric and duodenal cancer in individuals with Lynch syndrome: a nationwide cohort study

Lynch syndrome increases the risk of gastric cancer (GC) and duodenal cancer (DC), particularly in individuals with MLH1 and MSH2 pathogenic variants (PVs). To provide further insight into whether, and from what age, esophagogastroduodenoscopy (EGD) surveillance may be beneficial, we evaluated the cumulative incidence and tumour characteristics of GC and DC in a large nationwide cohort of Dutch individuals with LS. For this retrospective nationwide cohort study, clinical data of individuals with LS registered at the Dutch Hereditary Cancer Registry were matched with pathology reports filed by the Dutch Pathology registry. All individuals registered between Jan 1, 1989 and Dec 31, 2021 with proven or putative PVs in one of the mismatch repair genes were included. Cumulative incidences of GC and DC were estimated for high-risk (MLH1, MSH2 and EpCAM) and low-risk (MSH6 and PMS2) PVs using competing risk methodology (Fine and Gray method) with death due to other causes as competing risk. Among 1002 individuals with high-risk and 765 individuals with low-risk PVs, 29GCs (1.6%) and 39 DCs (2.2%) were diagnosed. Cumulative incidence of GC and DC under the age of 50 was very low (≤1%) for all individuals. At age 70 and 75, cumulative incidence of GC was 3% [95% CI 1%-5%] and 5% [3%-8%] for high-risk PVs and 1% [0%-2%] and 1% [0%-2%] for low-risk PVs (p=0.006). For DC, cumulative incidence at age 70 and 75 was 5% [3%-7%] and 6% [3%-8%] in high-risk, 1% [0%-1%] and 2% [0%-4%] in low-risk PVs, respectively (p=0.01). Primary tumour resection was performed in 62% (18/29) of GCs and 77% (30/39) of DC cases. Early-stage GC, defined as TNM stage I, was found in 32% (9/28) of GCs. Early-stage DC, defined as TNM stage I-IIa, was found in 39% (14/36) of DCs. Individuals with MLH1, MSH2, and EpCAM PVs have an increased risk of developing GC and DC at the age of 70 years, but this risk is very low before the age of 50 years. The age of onset of surveillance, the yield of GC and DC during EGD surveillance, and its cost-effectiveness should be subject of future studies. None.

Putative Pathogenic Variants of ABCG5 and ABCG8 of Sitosterolemia in Patients With Hyper-Low-Density Lipoprotein Cholesterolemia.

Sitosterolemia is a rare autosomal recessive disease caused by the deleterious variants of adenosine 5'-triphosphate (ATP)-binding cassette sub-family G member 5 (ABCG5) or ATP-binding cassette sub-family G member 8 (ABCG8). There are only few data on the pathogenicity of ABCG5 and ABCG8. This study aimed to propose a scheme for determining variant pathogenicity and to catalog the putative pathogenic variants in sitosterolemia. This study enrolled 377 consecutive Japanese patients with hyper-low-density lipoprotein cholesterolemia (mean age: 46.5±19.8 years, with 192 men) who have targeted-sequenced data on ABCG5 or ABCG8 (among 21 Mendelian lipid genes for any dyslipidemias) and serum sitosterol levels at Kanazawa University Hospital from 2016 to 2021. Serum sitosterol levels were divided by 0.79 in patients treated with ezetimibe, accounting for the average reduction with this drug. ABCG5 or ABCG8 variants were defined as putative pathogenic if associated with serum sitosterol levels ≥5 µg/mL or homozygous if associated with serum sitosterol levels ≥10 µg/mL. Twenty-three ABCG5 or ABCG8 variants (16 missense, 2 nonsense, 2 frameshift, 2 deletion, and 1 splice mutation) were identified. Based on our definition, 11 putative pathogenic variants (median sitosterol level: 10.1 [6.5-17.1] µg/mL) were found in 36 individuals and 12 benign variants (median sitosterol: 3.5 [2.5-4.1] µg/mL) in 14 individuals. The scheme proposed for assessing the pathogenicity of genetic variations (ABCG5 and ABCG8) is useful. Using this scheme, 11 putative pathogenic, and 12 benign variants in ABCG5 or ABCG were classified.

Combined bioinformatic and splicing analysis of likely benign intronic and synonymous variants reveals evidence for pathogenicity

PurposeClinical variant analysis pipelines likely have poor sensitivity to the effects on splicing from variants beyond 10 to 20 bases of exon-intron boundaries. Here, we demonstrate the value of SpliceAI to inform curation of rare variants previously classified as benign/likely benign (B/LB) under current guidelines. MethodsExome sequencing data from 576 pediatric cancer patients enrolled in the Texas KidsCanSeq study were filtered for intronic or synonymous variants absent from population databases, predicted to alter splicing via SpliceAI (>0.20), and scored >10 by combined annotation-dependent depletion. Rare synonymous or intronic B/LB variants in 61 genes submitted to ClinVar were also evaluated and RNA further assessed in monocyte-derived messenger RNA and/or an in vitro splice reporter assay in HEK-293T cells. ResultsSpliceAI-supplemented analysis of the KidsCanSeq cohort revealed a DICER1 intronic variant that resulted in missplicing in RNA from a proband with a personal and family history of pleuropulmonary blastoma but negative clinical exome and panel reports. Analysis of 34,188 B/LB ClinVar variants yielded 18 variants predicted to cause disrupted reading frames. Assessment of 8 variants (DICER1 n = 4, CDH1 n = 2, PALB2 n = 2) by in vitro splicing assay demonstrated abnormal splice products (mean 66%; range 6% to 100%). When available, phenotypic information from submitting laboratories demonstrated DICER1-associated tumors in 2 families (1 variant) and breast cancer in 3 families (2 PALB2 variants). ConclusionIncorporation of SpliceAI in variant curation pipelines may improve classification of B/LB intronic and synonymous variants and highlight putative pathogenic variants for functional assays and RNA analysis, thereby increasing diagnostic yield for rare diseases.

NEXN Gene in Cardiomyopathies and Sudden Cardiac Deaths: Prevalence, Phenotypic Expression, and Prognosis.

Few clinical data are available on NEXN mutation carriers, and the gene's involvement in cardiomyopathies or sudden death has not been fully established. Our objectives were to assess the prevalence of putative pathogenic variants in NEXN and to describe the phenotype and prognosis of patients carrying the variants. DNA samples from consecutive patients with cardiomyopathy or sudden cardiac death/sudden infant death syndrome/idiopathic ventricular fibrillation were sequenced with a custom panel of genes. Index cases carrying at least one putative pathogenic variant in the NEXN gene were selected. Of the 9516 index patients sequenced, 31 were carriers of a putative pathogenic variant in NEXN only, including 2 with double variants and 29 with a single variant. Of the 29 unrelated probands with a single variant (16 males; median age at diagnosis, 32.0 [26.0-49.0] years), 21 presented with dilated cardiomyopathy (prevalence, 0.33%), and 3 presented with hypertrophic cardiomyopathy (prevalence, 0.14%). Three patients had idiopathic ventricular fibrillation, and there were 2 cases of sudden infant death syndrome (prevalence, 0.46%). For patients with dilated cardiomyopathy, the median left ventricle ejection fraction was 37.5% (26.25-50.0) at diagnosis and improved with treatment in 13 (61.9%). Over a median follow-up period of 6.0 years, we recorded 3 severe arrhythmic events and 2 severe hemodynamic events. Putative pathogenic NEXN variants were mainly associated with dilated cardiomyopathy; in these individuals, the prognosis appeared to be relatively good. However, severe and early onset phenotypes were also observed-especially in patients with double NEXN variants. We also detected NEXN variants in patients with hypertrophic cardiomyopathy and sudden infant death syndrome/idiopathic ventricular fibrillation, although a causal link could not be established.

Abstract 18674: Deep Phenotyping Arrhythmogenic MVP With Imaging and Genotyping Reveals Important Shared Genetic Etiology With Arrhythmia and Cardiomyopathy Genes

Introduction: Mitral valve prolapse (MVP) has a high population prevalence 2-4% with an arrhythmogenic subtype presenting with premature ventricular complexes (PVC) ventricular tachycardia (VT) and sudden death. There is a continued search for markers of MVP to help risk stratify patients. Hypothesis: Arrhythmogenic MVP shares genetic etiology with Mendelian causes of arrhythmia and cardiomyopathy Methods: Consecutive adult patients with arrhythmogenic MVP patients prospectively evaluated Results: A total of 33 cases (age 55±15 years, female 51.5%) presented with history of hypertension (33%), diabetes (3%), coronary disease (12%), atrial fibrillation (45%), bileaflet MVP (58%) and syncope (18%). ECG findings include inverted T-waves in inferior leads in 6%, pleomorphic PVC in 6%, Ventricular couplets in 51.5%, non-sustained VT in 73% and sustained VT in 33%; High PVC burden was seen in 55% of cases with burden 15±14%. Echocardiography noted LV ejection fraction of 55±8%, mitral regurgitations in 70% (severe 18%, moderate 27%, mild 24%), mitral annular disjunction in 39%, and Pickelhaube sign in 9% of cases. Cardiac magnetic resonance showed LVESV 113±119mm, LVEDV 117±70mm, and late Gadolinium enhancement in 48% with highest prevalence in basal inferolateral segment. PET-CT performed in 17 cases and showed fluorodeoxyglucose uptake in 24% cases, mostly in basal inferolateral segment as well. Genetic testing was performed in 23 cases and reported a putative pathogenic variant in 21.7%: FLNC (Exemplar case, Figure 1 ) cause of dilated cardiomyopathy, TBX5 cause of Holt-Oram syndrome, DSP cause of arrhythmogenic cardiomyopathy , TTN causes of dilated cardiomyopathy, MTO1 cause of mitochondrial hypertrophic cardiomyopathy. Conclusion: Full evaluation of arrhythmogenic MVP with multimodality imaging and genetic testing reveals high burden of fibrosis, inflammation, and candidate pathogenic variants.

Genetic assessment of pathogenic germline alterations in lysosomal genes among Asian patients with pancreatic ductal adenocarcinoma

BackgroundLysosomes are closely linked to autophagic activity, which plays a vital role in pancreatic ductal adenocarcinoma (PDAC) biology. The survival of PDAC patients is still poor, and the identification of novel genetic factors for prognosis and treatment is highly required to prevent PDAC-related deaths. This study investigated the germline variants related to lysosomal dysfunction in patients with PDAC and to analyze whether they contribute to the development of PDAC.MethodsThe germline putative pathogenic variants (PPV) in genes involved in lysosomal storage disease (LSD) was compared between patients with PDAC (n = 418) and healthy controls (n = 845) using targeted panel and whole-exome sequencing. Furthermore, pancreatic organoids from wild-type and KrasG12D mice were used to evaluate the effect of lysosomal dysfunction on PDAC development. RNA sequencing (RNA-seq) analysis was performed with established PDAC patient-derived organoids (PDOs) according to the PPV status.ResultsThe PPV in LSD-related genes was higher in patients with PDAC than in healthy controls (8.13 vs. 4.26%, Log2 OR = 1.65, P = 3.08 × 10–3). The PPV carriers of LSD-related genes with PDAC were significantly younger than the non-carriers (mean age 61.5 vs. 65.3 years, P = 0.031). We further studied a variant of the lysosomal enzyme, galactosylceramidase (GALC), which was the most frequently detected LSD variant in our cohort. Autophagolysosomal activity was hampered when GALC was downregulated, which was accompanied by paradoxically elevated autophagic flux. Furthermore, the number of proliferating Ki-67+ cells increased significantly in pancreatic organoids derived from Galc knockout KrasG12D mice. Moreover, GALC PPV carriers tended to show drug resistance in both PDAC cell line and PDAC PDO, and RNA-seq analysis revealed that various metabolism and gene repair pathways were upregulated in PDAC PDOs harboring a GALC variant.ConclusionsGenetically defined lysosomal dysfunction is frequently observed in patients with young-onset PDAC. This might contribute to PDAC development by altering metabolism and impairing autophagolysosomal activity, which could be potentially implicated in therapeutic applications for PDAC.

Rules and mechanisms governing G protein coupling selectivity of GPCRs

G protein-coupled receptors (GPCRs) convert extracellular stimuli into intracellular signaling by coupling to heterotrimeric G proteins of four classes: Gi/o, Gq, Gs, and G12/13. However, our understanding of the G protein selectivity of GPCRs is incomplete. Here, we quantitatively measure the enzymatic activity of GPCRs in living cells and reveal the G protein selectivity of 124 GPCRs with the exact rank order of their G protein preference. Using this information, we establish a classification of GPCRs by functional selectivity, discover the existence of a G12/13-coupled receptor, G15-coupled receptors, and a variety of subclasses for Gi/o-, Gq-, and Gs-coupled receptors, culminating in development of the predictive algorithm of G protein selectivity. We further identify the structural determinants of G protein selectivity, allowing us to synthesize non-existent GPCRs with de novo G protein selectivity and efficiently identify putative pathogenic variants.

Genetic Screening of a Hungarian Cohort with Focal Dystonia Identified Several Novel Putative Pathogenic Gene Variants.

Dystonia is a rare movement disorder which is characterized by sustained or intermittent muscle contractions causing abnormal and often repetitive movements, postures, or both. The two most common forms of adult-onset focal dystonia are cervical dystonia (CD) and benign essential blepharospasm (BSP). A total of 121 patients (CD, 74; BSP, 47) were included in the study. The average age of the patients was 64 years. For the next-generation sequencing (NGS) approach, 30 genes were selected on the basis of a thorough search of the scientific literature. Assessment of 30 CD- and BSP-associated genes from 121 patients revealed a total of 209 different heterozygous variants in 24 genes. Established clinical and genetic validity was determined for nine heterozygous variations (three likely pathogenic and six variants of uncertain significance). Detailed genetic examination is an important part of the work-up for focal dystonia forms. To our knowledge, our investigation is the first such study to be carried out in the Middle-European region.

Family-base rare variant association analysis in Saudi Arabian hydrocephalus subjects using whole exome sequencing.

Hydrocephalus is a highly heterogeneous multifactorial disease that arises from genetic and environmental factors. Familial genetic studies of hydrocephalus have elucidated four robustly associated hydrocephalus associated loci. This study aims to identify potential genetic causation in cases of hydrocephalus, with or without spina bifida and Dandy Walker Syndrome (DWS), using family-based rare variant association analysis of whole exome sequencing. We performed whole exome sequencing in 143 individuals across 48 families where at least one offspring was affected with hydrocephalus (N.=27), with hydrocephalus with spina bifida (N.=21) and with DWS (N.=3), using Illumina HiSeq 2500 instrument. No pathogenic or putative pathogenic single-nucleotide variants were evident in the four known hydrocephalus loci in our subjects. However, after examining 73 known hydrocephalus genes previously identified from literature, we identified three potentially impactful variants from the cohort. Using a gene panel comprising variants in known neural tube defects loci, we identified a total of 1024 potentially deleterious variants, of which 797 were missense variants and 191 were frameshift variants, 36 were stop gain/loss variants. A small portion of our family pedigree analyses yielded putative genetic signals which may be responsible for hydrocephaly elated phenotypes, however the low diagnostic yield may be due to lack of capture of genetic variants in the exonic regions i.e. structural variants may only be evident from whole genome sequencing. We identified three potentially impactful variants from our cohort in 73 known hydrocephalus genes previously identified in literature.

DHX37 and NR5A1 Variants Identified in Patients with 46,XY Partial Gonadal Dysgenesis.

The group of disorders known as 46,XY gonadal dysgenesis (GD) is characterized by anomalies in testis determination, including complete and partial GD (PGD) and testicular regression syndrome (TRS). Several genes are known to be involved in sex development pathways, however approximately 50% of all cases remain elusive. Recent studies have identified variants in DHX37, a gene encoding a putative RNA helicase essential in ribosome biogenesis and previously associated with neurodevelopmental disorders, as a cause of PGD and TRS. To investigate the potential role of DHX37 in disorders of sexual development (DSD), 25 individuals with 46,XY DSD were analyzed and putative pathogenic variants were found in four of them. WES analyses were performed on these patients. In DHX37, the variant p.(Arg308Gln), recurrent associated with DSD, was identified in one patient; the p.(Leu467Val), predicted to be deleterious, was found together with an NR5A1 loss-of-function variant in patient 2; and, the p.(Val999Met) was identified in two unrelated patients, one of whom (patient 3) also carried a pathogenic NR5A1 variant. For both patients carrying DHX37 and NR5A1 pathogenic variants, a digenic inheritance is suggested. Our findings support the importance of DHX37 variants as a cause of disorders of sex development, implying a role in testis development.

Abstract 4178: Lysosomal storage dysfunction as a risk factor in pancreatic cancer

Abstract Background: Lysosome is closely linked to autophagy activity which has an important role in pancreatic adenocarcinoma (PDAC). We investigated whether lysosome storage dysfunction (LSD) contributes to PDAC development. Materials and Methods: Comparison of germline putative pathogenic variants (PPV) in genes involved in lysosome functions was performed between PDAC patients (N=418) and healthy controls (N=745) using next generation sequencing. Using mouse pancreas organoid, consequences of GALC downregulation in PDAC development was evaluated. Transcriptome data analysis of human PDAC organoid according to PPV status was followed. Results: PPV in LSD related genes were enriched in PDAC patients compared to healthy controls (Log2OR = 1.65, P = 3.08 × 10−3). For limited stage PDAC patients, PPV carriers were diagnosed with PDAC in younger age compared to non-carriers (mean age 61.5 vs. 65.1 years, p=0.038). In mouse pancreas organoid, Ki-67 index significantly increased as GALC was downregulated. Altered autophagy activity with increased autophagy flux following GALC dysfunction was observed in both human cancer cell line and mouse organoid. mTOR downregulation was noted in mouse pancreas organoid. RNA sequencing analysis of human PDAC organoid revealed metabolism alteration related to LSD. Conclusion: Genetically defined lysosome dysfunction is frequently observed in young age onset PDACs. Lysosome dysfunction might contribute to PDAC development via altered metabolism and autophagy activity. Key words: lysosomal storage disease (LSD), germline, pancreatic ductal adenocarcinoma (PDAC) Citation Format: Hyemin Kim, Youngil Koh, Joo Kyung Park. Lysosomal storage dysfunction as a risk factor in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4178.

Perspectives on the implications of carrying putative pathogenic variants in the medulloblastoma predisposition genes ELP1 and GPR161

Recent genetic sequencing studies in large series’ of predominantly childhood medulloblastoma have implicated loss-of-function, predominantly truncating, variants in the ELP1 and GPR161 genes in causation of the MBSHH subtype specifically. The latter association, along with a report of an index case with some features of Gorlin syndrome has led to speculation that GPR161 may also cause Gorlin syndrome. We show that these genes are associated with relatively low absolute risks of medulloblastoma from extrapolating lifetime risks in the general population and odds ratios from the population database gnomAD. The projected risks are around 1 in 270–430 for ELP1 and 1 in 1600–2500 for GPR161. These risks do not suggest the need for MRI screening in infants with ELP1 or GPR161 variants as this is not currently recommended for PTCH1 where the risks are equivalent or higher. We also screened 27 PTCH1/SUFU pathogenic variant-negative patients with Gorlin syndrome for GPR161 and found no suspicious variants. Given the population frequencies of 0.0962% for GPR161 and 0.0687% for ELP1, neither of these genes can be a cause of Gorlin syndrome with an unexplained population frequency far lower at 0.0021%.

Whole exome sequencing in unexplained recurrent miscarriage families identified novel pathogenic genetic causes of euploid miscarriage.

Can whole exome sequencing (WES) followed by trio bioinformatics analysis identify novel pathogenic genetic causes of first trimester euploid miscarriage? We identified genetic variants in six candidate genes that indicated plausible underlying causes of first-trimester euploid miscarriage. Previous studies have identified several monogenic causes of Mendelian inheritance in euploid miscarriages. However, most of these studies are without trio analyses and lack cellular and animal models to validate the functional effect of putative pathogenic variants. Eight unexplained recurrent miscarriage (URM) couples and corresponding euploid miscarriages were included in our study for whole genome sequencing (WGS) and WES followed by trio bioinformatics analysis. Knock-in mice with Rry2 and Plxnb2 variants and immortalized human trophoblasts were utilized for functional study. Additional 113 unexplained miscarriages were included to identify the mutation prevalence of specific genes by multiplex PCR. Whole blood from URM couples and their <13 weeks gestation miscarriage products were both collected for WES, and all variants in selected genes were verified by Sanger sequencing. Different stage C57BL/6J wild-type mouse embryos were collected for immunofluorescence. Ryr2N1552S/+, Ryr2R137W/+, Plxnb2D1577E/+, and Plxnb2R465Q/+ point mutation mice were generated and backcrossed. Matrigel-coated transwell invasion assays and wound-healing assays were performed using HTR-8/SVneo cells transfected with PLXNB2 small-interfering RNA and negative control. Multiplex PCR was performed focusing on RYR2 and PLXNB2. Six novel candidate genes, including ATP2A2, NAP1L1, RYR2, NRK, PLXNB2, and SSPO, were identified. Immunofluorescence staining showed that ATP2A2, NAP1L1, RyR2, and PLXNB2 were widely expressed from the zygote to the blastocyst stage in mouse embryos. Although compound heterozygous mice with Rry2 and Plxnb2 variants did not show embryonic lethality, the number of pups per litter was significantly reduced when backcrossing Ryr2N1552S/+ ♂ with Ryr2R137W/+ ♀ or Plxnb2D1577E/+ ♂ with Plxnb2R465Q/+ ♀ (P < 0.05), which were in accordance with the sequencing results of Family 2 and Family 3, and the proportion of Ryr2N1552S/+ offspring was significantly lower when Ryr2N1552S/+ female mice were backcrossed with Ryr2R137W/+ male mice (P < 0.05). Moreover, siRNA-mediated PLXNB2 knockdown inhibited the migratory and invasive abilities of immortalized human trophoblasts. Besides, additional 10 variants of RYR2 and PLXNB2 were detected in 113 unexplained euploid miscarriages by multiplex PCR. The relatively small number of samples is a limitation of our study which may result in the identification of variants in unique candidate genes with no definitive although plausible causal effect. Larger cohorts are needed to replicate these findings and additional functional research is needed to confirm the pathogenic effects of these variants. Moreover, the sequencing coverage restricted the detection of low-level parental mosaic variants. For first-trimester euploid miscarriage, variants in unique genes may be underlying genetic etiologies and WES on trio could be an ideal model to identify potential genetic causes, which could facilitate individualized precise diagnostic and therapeutic regimens in the future. This study was supported by grants from the National Key Research and Development Program of China (2021YFC2700604), National Natural Science Foundation of China (31900492, 82101784, 82171648), Basic Science Center Program of the National Natural Science Foundation of China (31988101), Key Research and Development Program of Shandong Province (2021LCZX02), Natural Science Foundation of Shandong Province (ZR2020QH051), Natural Science Foundation of Jiangsu Province (BK20200223), Taishan Scholars Program for Young Experts of Shandong Province (tsqn201812154) and Young Scholars Program of Shandong University. The authors declare no conflicts of interest. N/A.

Genetic and clinical analysis of TP73 gene in amyotrophic lateral sclerosis patients from Chinese mainland.

TP73 was recently identified as a novel causative gene for amyotrophic lateral sclerosis (ALS). We aimed to determine the contribution of variations in TP73 in the Chinese ALS population and to further explore the genotype-phenotype correlations. We screened rare, putative pathogenic TP73 mutations in a large Chinese ALS cohort and performed association analysis of both rare and common TP73 variations between cases and controls. Of the 985 ALS patients studied, six rare, heterozygous putative pathogenic variants in TP73 were identified among six unrelated sALS patients. Exon 14 of TP73 might be a mutant hotspot in our cohort. Patients with ALS with only rare, putative pathogenic TP73 mutations exhibited a characteristic clinical profile. Patients harboring multiple mutations in TP73 and other ALS-related genes displayed a significantly earlier onset of ALS. Association analysis revealed that rare TP73 variants in the untranslated regions (UTRs) were enriched among ALS patients; meanwhile, two common variants in the exon-intron boundary were discovered to be associated with ALS. We demonstrate that TP73 variations also have contributed to ALS in the Asian population and broaden the genotypic and phenotypic spectrum of TP73 variants in the ALS-frontotemporal dementia (FTD) spectrum. Furthermore, our findings first suggest that TP73 is not only a causative gene, but also exerts a disease-modifying effect. These results may contribute to a better understanding of the molecular mechanism of ALS.