Truncating Variants Research Articles

Expanding Genotype-Phenotype Correlation of CLCNKA and CLCNKB Variants Linked to Hearing Loss.

The ClC-K channels CLCNKA and CLCNKB are crucial for the transepithelial transport processes required for sufficient urinary concentrations and sensory mechanoelectrical transduction in the cochlea. Loss-of-function alleles in these channels are associated with various clinical phenotypes, ranging from hypokalemic alkalosis to sensorineural hearing loss (SNHL) accompanied by severe renal conditions, i.e., Bartter's syndrome. Using a stepwise genetic approach encompassing whole-genome sequencing (WGS), we identified one family with compound heterozygous variants in the ClC-K channels, specifically a truncating variant in CLCNKA in trans with a contiguous deletion of CLCNKA and CLCNKB. Breakpoint PCR and Sanger sequencing elucidated the breakpoint junctions derived from WGS, and allele-specific droplet digital PCR confirmed one copy loss of the CLCNKA_CLCNKB contiguous deletion. The proband that harbors the CLCNKA_CLCNKB variants is characterized by SNHL without hypokalemic alkalosis and renal anomalies, suggesting a distinct phenotype in the ClC-K channels in whom SNHL predominantly occurs. These results expanded genotypes and phenotypes associated with ClC-K channels, including the disease entities associated with non-syndromic hearing loss. Repeated identification of deletions across various extents of CLCNKA_CLCNKB suggests a mutational hotspot allele, highlighting the need for an in-depth analysis of the CLCNKA_CLCNKB intergenic region, especially in undiagnosed SNHL patients with a single hit in CLCNKA.

Identification of pathogenic SORL1 genetic variants in Alzheimer’s disease patients

AbstractBackgroundAn estimated ∼2‐3% of early‐onset Alzheimer’s disease (AD) patients carry a predicted rare damaging variant in the SORL1 gene, which encodes the SORLA receptor protein. While protein‐truncating variants (loss‐of‐function, LOF) occur almost exclusively in AD patients, the large majority of damaging variants are missense variants. However, the effect of individual missense variants on AD risk varies widely, warranting a variant pathogenicity screen.MethodsWe prioritized genetic variants in SORL1 by investigating to what extent they lead to familial disease when they occur in protein homologs. We used a sample of 18,959 non‐related AD cases from the ADES/ADSP studies to investigate the age‐at‐onset in carriers of a prioritized SORL1 variant relative to WT SORL1 in context of APOE genotype. Second, since functional SORLA is cleaved at the neuronal membrane into soluble SORLA (sSORLA) we used Western Blot and proteomics to test whether SORL1 variants affected the level of sSORLA in CSF.ResultsCompared to SORL1 wild type AD patients, carriers of prioritized SORL1 variants had an expedited age at onset similar to carriers of LoF variants: of those homozygous for APOE‐ε4 who carried a prioritized SORL1 variant, 80% had AD by age 61 (95%CI 60‐NA, n = 6) compared to 73 for WT‐SORL1 carriers (95%CI 72‐73, n = 1091). Of those heterozygous for APOE‐ε4 who carried a prioritized SORL1 variant, 80% had AD by age 71 (95%CI 68‐76, n = 62) compared to age 78 WT‐SORL1 carriers (95%CI 78‐78, n = 4816). Of those negative for the APOE‐ε4 allele with a SORL1 variant, 80% had AD by 83 years (95%CI 78‐90, n = 54) compared to 85 years for WT‐SORL1 carriers (95%CI 85‐85, n = 5773). These effects were absent for non‐prioritized missense variants. Second, using WB we found that sSORLA levels in carriers of pathogenic missense variants were reduced to ∼50% relative to carriers of benign SORL1 variants, while levels of truncating SORL1 variants were reduced to ∼65%.DiscussionPrioritizing SORL1 missense variants according to variant‐effects in protein homologs leads to variants that expedite the age‐at‐onset independent of APOE background. Carriers of pathogenic missense or truncating variants have low CSF‐sSORLA levels, which may serve as a biomarker for impaired SORL1.

Neuromuscular and cardiovascular phenotypes in paediatric titinopathies: a multisite retrospective study

BackgroundPathogenic variants in TTN cause a spectrum of autosomal dominant and recessive cardiovascular, skeletal muscle and cardioskeletal disease with symptom onset across the lifespan. The aim of this study was...

Molecular consequences of PQBP1 deficiency, involved in the X-linked Renpenning syndrome.

Mutations in the PQBP1 gene (polyglutamine-binding protein-1) are responsible for a syndromic X-linked form of neurodevelopmental disorder (XL-NDD) with intellectual disability (ID), named Renpenning syndrome. PQBP1 encodes a protein involved in transcriptional and post-transcriptional regulation of gene expression. To investigate the consequences of PQBP1 loss, we used RNA interference to knock-down (KD) PQBP1 in human neural stem cells (hNSC). We observed a decrease of cell proliferation, as well as the deregulation of the expression of 58 genes, comprising genes encoding proteins associated with neurodegenerative diseases, playing a role in mRNA regulation or involved in innate immunity. We also observed an enrichment of genes involved in other forms of NDD (CELF2, APC2, etc). In particular, we identified an increase of a non-canonical isoform of another XL-NDD gene, UPF3B, an actor of nonsense mRNA mediated decay (NMD). This isoform encodes a shorter protein (UPF3B_S) deprived from the domains binding NMD effectors, however no notable change in NMD was observed after PQBP1-KD in fibroblasts containing a premature termination codon. We showed that short non-canonical and long canonical UPF3B isoforms have different interactomes, suggesting they could play distinct roles. The link between PQBP1 loss and increase of UPF3B_S expression was confirmed in mRNA obtained from patients with pathogenic variants in PQBP1, particularly pronounced for truncating variants and missense variants located in the C-terminal domain. We therefore used it as a molecular marker of Renpenning syndrome, to test the pathogenicity of variants of uncertain clinical significance identified in PQPB1 in individuals with NDD, using patient blood mRNA and HeLa cells expressing wild-type or mutant PQBP1 cDNA. We showed that these different approaches were efficient to prove a functional effect of variants in the C-terminal domain of the protein. In conclusion, our study provided information on the pathological mechanisms involved in Renpenning syndrome, but also allowed the identification of a biomarker of PQBP1 deficiency useful to test variant effect.

Genetic Complementation Studies Reveal That Many Disease-Associated DDX41 Variants Do Not Cause Loss of Protein Function

Germline variants in DDX41 are the most common cause of inherited predisposition to Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). DDX41 codes for an RNA helicase with many described functions, and thus the precise role of these variants in disease is uncertain. Approximately 2/3 of disease-associated DDX41 variants cause truncation of the DDX41 protein due to frameshift, loss of a translation start, or splice alterations, while the other 1/3 of DDX41 variants are missense. The effect of these missense variants on DDX41 function is largely unknown and thus they are typically categorized as variants of unknown significance (VUSs). Importantly, 30-50% of MDS/AML patients with either truncating or missense DDX41 variants acquire a somatic mutation in the other allele of DDX41, and this mutation has strong preference for the amino acid substitution R525H. Our previous work demonstrated that the R525H mutation causes loss of DDX41 function. To better understand how germline variants affect DDX41 function, we designed a genetic complementation strategy in immortalized hematopoietic progenitor cells with inducible deletion of DDX41. We screened six of the most common missense VUSs by expressing them from a lentivirus in DDX41-proficient cells and then inducing deletion of endogenous DDX41 to determine the effect of each VUS on proliferation and survival of the cells. Empty vector-transduced DDX41-deleted cells undergo cell-cycle arrest and apoptosis within 3-4 days. In contrast, viral expression of wild-type DDX41 rescues the survival and proliferation of the cells, while expression of the R525H mutant fails to rescue the cells. We screened the Y33H, M155H, G173R, R219H, P258L, and I396T variants of DDX41 in this assay, and all six of the mutants successfully rescued the survival and proliferation of DDX41-deleted cells long-term. This indicates that the mutant proteins coded for by these variants are functional. To test the effect of commonly observed truncating variants in the same assay, we expressed M1I, Q52fs, and D140Gfs variants of DDX41. Surprisingly, we saw complete rescue of cell proliferation and survival by the M1I and Q52fs variants. In contrast, the D140Gfs variant failed to rescue the cells. These data indicate that frameshift at D140 or later causes loss of DDX41 function. It is likely that translation from alternative methionine residues located at M127 or M132 of DDX41 accounts for the rescue of the cells by truncating variants affecting sites upstream of M127. In support of this theory, we targeted DDX41 for CRISPR-mediated knockout in MOLM13 cells and found that guide RNAs targeting exons 1-4, which are upstream of M127, produced homozygous frameshift mutations in proliferating cells from single-cell clones. However, guides targeting exon 6, which is downstream of M127 and M132, did not produce any surviving cells with homozygous frameshift mutations, despite screening 96 clones. This finding indicates that short DDX41 isoforms expressed from alternative start sites are functional and can support cell proliferation when full-length protein is lost. Collectively, these data raise important questions about the precise role of DDX41 variants in hematologic malignancy. Data from recently published patient cohorts indicates that patients with missense DDX41 variants have similar patient characteristics to those with truncating variants and thus these aberrations are thought to cause disease by similar mechanisms. While these cell line models may not fully recapitulate the pathogenic mechanism by which DDX41 variants cause MDS/AML predisposition, the results of this study demonstrate that the germline variants are not complete loss-of-function alleles in all cases and more complex mechanisms may be involved.

Truncating SPEN Mutations Highlight BN2-Subtype DLBCL with Aggressive Biology and Features of Immune Evasion

Introduction: Molecular classifications have been developed to characterize the heterogeneity of diffuse large B cell lymphoma (DLBCL), with activation of BCL6 and Notch signaling pathways acting as driving pathogenic features in C1/BN2 subtype tumors. Among C1/BN2 defining variants are truncating/inactivating SPEN mutations. SPEN regulates the Notch signaling pathway by initiating formation of a repressive complex that facilitates transcriptional repression of Notch target genes. Our group has identified SPEN mutations enriched in DLBCL cases failing to achieve event-free status at 24 months (EFS24). This contrasted with generally favorable outcomes in C1/BN2 tumors, suggesting a potential high-risk molecular feature with unexplored significance. Presenting an opportunity to expand underlying mechanisms and risk stratification in C1/BN2 tumors, we aimed to use a functional genomic approach, combined with clinical data, to characterize the alternative biology stemming from SPEN truncating mutations. Methods: FFPE tumor biopsies from newly diagnosed DLBCL cases (N=444) enrolled in the Mayo Clinic/Iowa Lymphoma Molecular Epidemiology Resource (MER) program were analyzed by WES (n=404) and RNA-seq (n=321). Biologic and clinical variables were annotated for all cases. CRISPR/Cas9 introduced truncating mutations to the endogenous SPEN locus in OCI-LY3 & SU-DHL-2 ABC-type human cell lines to establish models of SPEN depletion. Functional in vitro analysis utilized standard methods for RNA-seq, cell proliferation, western blot, and flow cytometry. Results: WES identified 29/404 ndDLBCL (7.2%) harboring SPEN mutations; n=17 missense and n=12 nonsense or frameshift insertions/deletions (truncating variants - [trunc]), the later considered as the focal point of our study. Against all cases, SPEN trunc cases displayed enrichment for high-risk factors including non-GCB COO (p=0.06, OR=3.7, 95% CI [0.9, 22.2]) and primary refractory disease (PTR; p=0.02, OR=5.0, 95% CI [1.05, 19.9]), and demonstrated inferior OS (p=0.06, HR=2.2), where a majority of clinical progression events occurred within 12 months of diagnosis. Leveraging RNA-seq, we observed 11 of 11 SPEN trunc cases exhibiting a double-hit negative gene expression signature, 9/11 cases with inflammatory or depleted microenvironment (LME), and 8/11 with a signature associating with intermediate/high risk of EFS24 failure. Applying the LymphGen algorithm, 5/12 SPEN trunc cases received the BN2 classification, observing co-occurring mutations with CD70 (p<0.01, OR=9.68), BRCA2 (p=0.03, OR=5.84), TET2 (p=0.02, OR=4.91), IRF4 (p=0.02, OR=7.27), and PTPRD (p<0.01, OR=8.31) genes, and were mutually exclusive with ARID1A, BCL2, EZH2, FAS, HLA-B, IRF8, and TMEM30A mutations ( Figure 1A). Isolating SPEN trunc against BN2 cases without SPEN variants (n=14), despite similar clustering of non-GCB COO (p=0.7), PTR was exclusively observed in SPEN trunc cases, accompanied again by inferior OS (p=0.07, HR=4.2; Figure 1B). In the OCI-LY3 SPEN depletion model, GSEA analysis from RNA-seq revealed activation of E2F (NES=2.7; p=9.4E-20) and MYC target genes (NES=2.3; p=4.1E-6), while both OCI-LY3 and SU-DHL-2 models featured depletion of antigen presentation genes (NES= -1.8, p=5.7E-4; NES= -2.2, p=3.8E-7, respectively). A decrease of MHC class II proteins (HLA-DR, HLA-DP) was confirmed by flow cytometry in both models (p<0.01). Hypothesizing loss of SPEN enhances reliance on the BCL6-HDAC3 axis featured in BN2 tumors, we treated both SPEN depletion models with a class-I HDAC inhibitor highly selective for HDAC3, BRD-3308, and indeed observed a 1.4 and 2.4-fold increase in sensitivity compared to WT cells (p=0.07, p<0.01, respectively). Significance: We find SPEN trunc mutations in poor acting DLBCL cases with clinical and biologic characteristics that diverge from C1/BN2 biology. The enhanced rate of clinical progression highlights urgency to better characterize these tumors. Our data suggests loss of SPEN regulatory mechanisms may act cooperatively with C1/BN2 biology to facilitate tumor immune evasion and drive progression through Notch-mediated pathways. Importantly, in vitro evidence shows SPEN deficient lymphoma cells are susceptible to targeted therapeutics against dependent pathways, providing rationale for advancing individualized care strategies for affected patients.

BAX Mutated Clonal Hematopoiesis Arises Following Treatment with the BCL2 Inhibitor Class of Therapeutics across a Range of Hematological and Non-Hematological Neoplasms

Clonal hematopoiesis (CH) is an age-related phenomenon characterized by the expansion of genomically mutated hematopoietic clones with a selective fitness advantage. Venetoclax, a selective BCL2 inhibitor, has established efficacy in hematological malignancies such as chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). Our previous studies described the emergence of deleterious BAX variants within the myeloid compartment of patients (pts) receiving venetoclax for CLL (Blombery, Blood2022). Whether this phenomenon is specific to venetoclax and the CLL disease context is currently unknown. We therefore aimed to investigate the prevalence and pattern of BAX-mutatedCH across different hematological and non-hematological neoplasms in response to BCL2 inhibitor therapy (both venetoclax and BGB-11417). We first examined pts with AML in complete morphologic remission after venetoclax exposure in both the newly diagnosed (CAVEAT; Chua, JCO 2020) and early relapse (VALDAC; Tiong, ASH 2022) settings using unique molecular index-based error-corrected next generation sequencing with a sensitivity of 0.5% (QIAseq targeted DNA panel). BAX variants were detected in remission samples in 11/20 (55%) and 9/26 (35%) pts in these cohorts, respectively, after a median of 9- and 4-months post venetoclax. This represented a significant higher incidence compared to remission samples from pts with newly diagnosed AML post conventional cytotoxic chemotherapy without venetoclax (6/87 [7%]; adjusted p<0.01 for both pairwise comparisons). Having observed the increased frequency of BAX-mutated CH in remission in myeloid (AML) and lymphoid (CLL) neoplasms, we then investigated whether BAX variants may be acquired in response to BCL2 inhibitor therapy outside the context of hematological neoplasm. We assessed a cohort of pts with metastatic breast cancer who received venetoclax in combination with tamoxifen (mBEP; Lok, Cancer Discover 2019), and detected BAX variants from peripheral blood samples in 5/16 (31%) pts (median age 66) after a median duration of 20 (range 13-44) months on venetoclax. In contrast, no BAX variants were detected in a similarly aged control cohort of 50 pts (median age 62) with breast cancer without exposure to venetoclax (p=0.01). The rates of any CH-related mutation were 75% and 50% pts, respectively (p=0.09). No pt developed MDS or AML during follow up in the mBEP cohort. Finally, to explore whether emergence of BAX-mutated CH is specific to venetoclax as an agent, we studied a cohort of 9 pts with CLL who had received BGB-11417, a more potent and selective BCL2 inhibitor; median treatment duration 13 months. Notably, we observed the emergence of BAX variants in 2/9 (22%) pts in this cohort. The characteristics of the BAX variants (n=88) in 28 pts (AML, CLL or breast cancer) are summarized in Figure 1; 3 variants (in 3 pts) were detectable in pre-treatment samples. Over half of the missense (n=22) or in-frame variants (n=1) were clustered in the C-terminus of the BAX protein, while truncating variants (n=65) were observed throughout the gene. The median number of BAX variants per pt was 2, with 32% of pts having ≥3 variants (max=13). The median VAF was 1.4%, and only 16% of variants had a VAF >5%. The number and VAF of BAX variants did not significantly correlate with the duration of therapy. Among 12 pts with AML who had serial testing (median inter-test interval 10 months), BAX variants (n=24) did not significantly change in VAF (median Δ 0.7%) with time after initial acquisition (Pearson's coefficient 0.17). To clarify the compartments affected by BAX variants we performed single-cell combined DNA/protein sequencing (Tapestri) of a sample with multiple BAX variants and demonstrated their presence in multiple independent clones with myeloid-biased lineage but also within the natural killer cell compartment ( Figure 2). In summary, the emergence of BAX-mutated CH appears to be a generalized phenomenon of adaptive hematopoiesis that occurs across both hematological and non-hematological malignancy settings as well being observed with other BCL2 inhibitors consistent with a therapeutic class effect. Further research is needed to understand the contribution of this phenomenon to the subsequent development of clinically relevant hematological abnormalities and its implications for future therapeutic resistance.

PAM variants in patients with thyrotrophinomas, cyclical Cushing's disease and prolactinomas.

Germline loss-of-function variants in PAM, encoding peptidylglycine α-amidating monooxygenase (PAM), were recently discovered to be enriched in conditions of pathological pituitary hypersecretion, specifically: somatotrophinoma, corticotrophinoma, and prolactinoma. PAM is the sole enzyme responsible for C-terminal amidation of peptides, and plays a role in the biosynthesis and regulation of multiple hormones, including proopiomelanocortin (POMC). We performed exome sequencing of germline and tumour DNA from 29 individuals with functioning pituitary adenomas (12 prolactinomas, 10 thyrotrophinomas, 7 cyclical Cushing's disease). An unfiltered analysis was undertaken of all PAM variants with population prevalence <5%. We identified five coding, non-synonymous PAM variants of interest amongst seven individuals (six germline, one somatic). The five variants comprised four missense variants and one truncating variant, all heterozygous. Each variant had some evidence of pathogenicity based on population prevalence, conservation scores, in silico predictions and/or prior functional studies. The yield of predicted deleterious PAM variants was thus 7/29 (24%). The variants predominated in individuals with thyrotrophinomas (4/10, 40%) and cyclical Cushing's disease (2/7, 29%), compared to prolactinomas (1/12, 8%). This is the second study to demonstrate a high yield of suspected loss-of-function, predominantly germline, PAM variants in individuals with pathological pituitary hypersecretion. We have extended the association with corticotrophinoma to include the specific clinical entity of cyclical Cushing's disease and demonstrated a novel association between PAM variants and thyrotrophinoma. PAM variants might act as risk alleles for pituitary adenoma formation, with a possible genotype-phenotype relationship between truncating variants and altered temporal secretion of cortisol.

The Advantages of Next-Generation Sequencing Molecular Classification in Endometrial Cancer Diagnosis

Endometrial cancer (EC) is the most frequent gynecological cancer. The ESGO/ESTRO/ESP 2020 guidelines identify prognostic groups based on morpho-molecular characteristics. This study aims to evaluate the clinical applicability of NGS analysis to define an appropriate risk class and to improve the diagnostic and prognostic stratification of ECs. Cases of serous carcinoma (OHEC) and high- (HGEC) and low-grade (LGEC) endometrioid carcinoma diagnosed with the morphological and immunohistochemical (IHC) protocols were considered. After a standardized pre-analytical phase, tumor DNA was semi-automatically extracted and analyzed using NGS with a panel of 14 genes. A total of 63 cases were considered. NGS analysis was successful in 60 cases; all of these were classified according to the new diagnostic algorithm. The molecular risk classification showed a good correlation with the morphological (k = 0.8). The study showed that the protocols of the pre-analytical and analytical phases used are robust and can lead to molecular results that fall within the standards required, which can be used in clinical practice for more precise diagnostic–therapeutic management of patients. The implementation of the classification is particularly relevant for better prognostic stratification of HGECs. In addition, the identification of a suspicious VUS in POLE questions the classification of truncating variants.

ERG Is a New Predisposition Gene for Bone Marrow Failure and Hematological Malignancy

There remain gaps in our knowledge of hereditary and sporadic causes of hematological malignancy (HM) and bone marrow failure (BMF) that prevent optimal diagnosis, disease surveillance and treatment. Here we report the discovery of ERG as a novel predisposition gene for BMF and HM. ERG is a known oncogene, typically via gene-fusions, leading to dysregulated ERG overexpression in blood and solid cancers. We identified a germline ERG ETS domain variant p.Y373C segregating with thrombocytopenia in a mother, who progressed to AML (27 yr) and then therapy-related MDS (35 yr), and in her 2 sons. All three showed copy neutral loss of heterozygosity of all or part of chromosome 21q, including the ERG locus, with the oldest son showing at least 2 somatic genetic rescue (SGR) events. The possibility of causal RUNX1 variants were ruled out, with the smallest somatic cnLOH event beginning within the RUNX1 gene, but not encompassing the RUNT domain where the majority of pathogenic missense variants are located. ERG, a highly constrained gene (LOEUF &amp;lt;0.33), is critical for definitive hematopoiesis, adult hematopoietic stem cell (HSC) function and platelet maintenance. An identical corresponding heterozygous germline variant (p.Y343C) in ERG’s closest gene by homology, FLI1, causes platelet-type bleeding disorder-21 (BDPLT21, OMIM #617443). Through global collaborations, we have identified 15 heterozygous variants in the ERG gene, 13 of which are missense and 2 truncating variants, in 17 individuals with cytopenia and/or HM (mainly myeloid) or lymphedema (Table). Onset of hematological symptoms ranged from birth to 38 years for truncating and constrained ETS domain variants. Of these 15 variants, 12 have been confirmed germline including 2 de novo. Only 4 meiotic transmissions are observed. None of the missense variants in the highly conserved ETS domain of ERG which mediates DNA binding, protein-protein interactions and nuclear localization, are present in gnomAD. We have functionally characterized 19 ERG variants, 12 potentially pathogenic, 1 known mouse pathogenic variant and 3 population controls demonstrating that most ETS domain missense variants display loss-of-function (LOF) characteristics disrupting transcriptional transactivation (Figure), DNA-binding and/or nuclear localization in vitro. Robust preliminary data from ex vivo models of ERG overexpression in mouse fetal liver cells in tissue culture (cytokine-independence), a mouse transplant assay and previous germline mutant Erg mouse models are concordant with ETS domain missense variants being LOF compared to wildtype ERG and benign controls. Together, these data provide clinical, in vitro and ex vivo functional studies implicating LOF variants in hematological disease predisposition. LOF ERG mutations also occur in sporadic cases of HM. Recently, as part of a Genomics England Research Consortium population study, 4 truncating ERG variants were described in 7 individuals across 4 families with 3 meiotic transmissions and a de novo case with primary lymphedema (1) and we add 2 novel missense variants here. One patient showed SGR across the ERG locus in blood. Blood phenotypes were not described. Our results demonstrate that germline ERG variants predispose to diverse cytopenia, BMF and HM in both children and adults. In our family mentioned above, the mother received an unrelated alloHSCT due to t-MDS while her 2 sons with cytopenias continue to be monitored. The natural history of this new syndrome will require careful identification of germline lesions with additional longitudinal studies in more patients and families needed. This ERG syndrome parallels GATA2 deficiency syndrome (HM and lymphedema) and RUNX1 Familial Platelet disorder-myeloid malignancy (thrombocytopenia and HM). Like the well-known disease genes GATA2 and RUNX1, ERG is also a member of the transcription factor heptad involved in HSC maintenance and differentiation. ERG adds to a growing list of genes whose unregulated expression contributes to HM and other cancers. Identification of causal germline ERG variants like those outlined in this study, has direct clinical implications for patient and family management including diagnosis, counselling, surveillance and treatment strategies such as selection of bone marrow transplant donors and potential for targeted therapies including gene and cell therapy. Reference 1. Greene D et al. Nat.Med. 29:679-688 2023

CCDC65, encoding a component of the axonemal Nexin-Dynein regulatory complex, is required for sperm flagellum structure in humans.

Sperm flagella share an evolutionary conserved microtubule-based structure with motile cilia expressed at the surface of several cell types, such as the airways epithelial cells. As a result, male infertility can be observed as an isolated condition or a syndromic trait, illustrated by Primary Cilia Dyskinesia (PCD). We report two unrelated patients showing multiple morphological abnormalities of the sperm flagella (MMAF) and carrying distinct homozygous truncating variants in the PCD-associated gene CCDC65. We characterized one of the identified variants (c.1208del; p.Asn403Ilefs*9), which induces the near absence of CCDC65 protein in patient sperm. In Chlamydomonas, CCDC65 ortholog (DRC2, FAP250) is a component of the Nexin-Dynein Regulatory complex (N-DRC), which interconnects microtubule doublets and coordinates dynein arms activity. In sperm cells from the patient, we also show the loss of GAS8, another component of the N-DRC, supporting a structural/functional link between the two proteins. Our work indicates that, similarly to ciliary axoneme, CCDC65 is required for sperm flagellum structure. Importantly, our work provides first evidence that mutations in the PCD-associated gene CCDC65 also cause asthenozoospermia.

Prevalence of BRCA1 and BRCA2 germline variants in an unselected pancreatic cancer patient cohort in Pakistan

BackgroundBRCA1 and BRCA2 (BRCA1/2) are the most frequently investigated genes among Caucasian pancreatic cancer patients, whereas limited reports are available among Asians. We aimed to investigate the prevalence of BRCA1/2 germline variants in Pakistani pancreatic cancer patients.MethodsOne hundred and fifty unselected and prospectively enrolled pancreatic cancer patients were comprehensively screened for BRCA1/2 germline variants using denaturing high-performance liquid chromatography and high-resolution melting analyses, followed by DNA sequencing of the variant fragments. The novel variants were analyzed for their pathogenic effect using in-silico tools. Potentially functional variants were further screened in 200 cancer-free controls.ResultsProtein truncating variant was detected in BRCA2 only, with a prevalence of 0.7% (1/150). A frameshift BRCA2 variant (p.Asp946Ilefs*14) was identified in a 71-year-old male patient of Pathan ethnicity, with a family history of abdominal cancer. Additionally, we found a novel variant in BRCA2 (p.Glu2650Gln), two previously reported variants in BRCA1 (p.Thr293Ser) and BRCA2 (p.Ile2296Leu) and a recurrent nonsense variant in BRCA2 (p.Lys3326Ter). These variants were classified as variants of uncertain significance (VUS). It is noteworthy that none of these VUS carriers had a family history of pancreatic or other cancers.ConclusionsIn this first study, BRCA1/2 pathogenic variant is identified with a low frequency in pancreatic cancer patients from Pakistan. Comprehensive multigene panel testing is recommended in the Pakistani pancreatic cancer patients to enhance genetic understanding in this population.

Insights on Titin cardiomyopathy: from truncating variants site to phenotypic expression and outcomes

Abstract Background Titin truncating variants (TTNtv) represent the most prevalent genotype underlying dilated cardiomyopathy (DCM). Recent molecular studies provided initial evidence about distinct pathogenic mechanisms of different protein truncating sites (A-band vs non-A-band), possibly related to the presence or absence of truncated peptides. Yet, clinical studies on TTNtv-DCM failed to demonstrate any relevant clinical difference according to the band affected. We hypothesized this lack of distinction could be due to the selection bias of including only DCM patients. Thus, we studied the clinical phenotypes and outcomes of our entire population of TTNtv carriers, regardless of phenotypic expression at onset. Methods We retrospectively analysed a population of pathogenic TTNtv carriers, enrolled in our referral centre. TTNtv were classified according to their site (Z disk, I band, A band, A-M junction, M band) and further grouped for analysis in A-band and non-A band. Kaplan-Meier curves were estimated for the primary composite endpoint of all-cause mortality and heart transplant (HTx); while cumulative incidence functions were estimated for the two secondary endpoints (i) heart failure-related death, heart transplantation, or destination left ventricular assist device implantation (LVAD) and (ii) first major ventricular arrhythmia (MVA, defined as sudden death or life-threatening ventricular arrhythmia), compared by Gray’s test. Analyses were then repeated adding an independent cohort of TTVtv carriers from another tertiary centre. Results 161 TTNtv carriers were included in the study (79% A-band, 21% non-A band), 63% were probands. The most prevalent phenotype was DCM (92%). Mean age at diagnosis was 43±16 years and 68% were males. There was no difference between groups in the primary composite outcome nor in the secondary composite endpoint of heart failure-related death, HTx or destination LVAD. Instead, MVA were significantly more frequent in the non-A-band group, in which 45% (n=15) of TTNtv carriers experienced MVA as compared to 17% (n=20) of subjects in the A-band group (p=0.001). Interestingly, we observed a tendency in MVA occurring at a higher mean left ventricular ejection fraction (LVEF) in the non-A-band group as compared to the A-band group (42±13% vs 35±9%, respectively). Furthermore, there was no significant association between MVA and LVEF at the event in the non-A group, while this association was significant in A-band (p=0.03). When an external population of 22 TTNtv carriers (95% DCM, 77% A-band) was added for analysis, this secondary outcome was confirmed (p&amp;lt;0.001). Conclusions Among TTNtv carriers referred to two tertiary centres, those with a mutation in the non-A band seem characterized by higher arrhythmic risk, occurring independently from EF. This novel finding highlights the importance of considering the site of TTN truncating variants to guide the most appropriate clinical management for each patient.Fig 1.Secondary Outcome, arrhythmic risk

Arrhythmogenic cardiomyopathy phenotype associated with the pathogenic founder variant c.1211dup in PKP2

Abstract Background Arrhythmogenic cardiomyopathy results in life-threatening ventricular arrhythmia and heart failure. Phenotypic features vary according to genetic aetiology. Genotype-phenotype research is therefore crucial for patient counselling and treatment decisions such as implantable cardioverter defibrillator therapy. By collecting data from patient records we characterized the clinical phenotype associated with variant c.1211dup (p.Val406Serfs*4) in the plakophilin-2 gene (PKP2), common in the Dutch population. Purpose We aimed to investigate the incidence of ventricular arrhythmia and heart failure in families carrying the PKP2 c.1211dup variant. Furthermore, we aimed to determine the founder status of this variant, and compare phenotype severity with other truncating PKP2 variants. Methods Clinical data were collected retrospectively from medical records of 106 PKP2 c.1211dup heterozygous carriers (&amp;gt;85% of known carriers in The Netherlands) in a Castor EDC database. Using occurrence of ventricular arrhythmia and heart failure, event-free survival was compared between males and females, and probands and family members, using log-rank tests. Using data from a central curated arrhythmogenic cardiomyopathy database, c.1211dup was compared to three other truncating PKP2 variants (c.235C&amp;gt;T (p.Arg79*), c.397C&amp;gt;T (p.Gln133*) and c.2489+1G&amp;gt;A (p.?)) on ventricular arrhythmia-free survival by a Cox proportional hazards model, corrected for sex and proband status. Results Forty-seven carriers (44%) were diagnosed with arrhythmogenic cardiomyopathy at 41 years on average. As shown in figure 1, 29 participants (27%) experienced episodes of ventricular arrhythmia and 12 (11%) developed heart failure, with male carriers showing significantly higher risks than females on both endpoints (p&amp;lt;0.05). Based on available magnetic resonance imagery and echocardiographic data, 46% of carriers showed either right ventricular dilatation and/or dysfunction, and a substantial minority of 37% showed some form of left ventricular involvement. Both geographical distribution of carriers (figure 2) and haplotype analysis suggested PKP2 c.1211dup to be a founder variant originating from the South-Western coast of the Netherlands. Finally, a Cox proportional hazards model suggested significant differences in ventricular arrhythmia-free survival between four PKP2 truncating founder variants, including c.1211dup. Conclusions The PKP2 c.1211dup variant is a Dutch founder variant associated with a typical right ventricular-dominant arrhythmogenic cardiomyopathy phenotype, but also notable left ventricular involvement. Males were most susceptible to the frequent ventricular arrhythmia, and less frequent heart failure was observed in general. Finally, c.1211dup possibly leads to a more severe phenotype than other Dutch PKP2 founder variants.Phenotype onset stratified by sexGeographic distribution carriers

Clinical genetic testing in arrhythmogenic mitral valve prolapse syndrome highlights a potential overlap with familial dilated cardiomyopathy

Abstract Background/Introduction Arrhythmogenic mitral valve prolapse syndrome (AMVPS) is a female-predominant sudden cardiac death (SCD)-predisposing disorder characterized clinically by myxomatous mitral valve prolapse (MVP), inferolateral T-wave inversions and complex ventricular ectopy. However, insights into the pathogenesis and potential genetic basis of AMVPS are currently lacking. Purpose Considering recent case reports that suggest a potential role for genetic cardiomyopathies in the pathogenesis of AMVPS, we sought to determine the yield of pathogenic/likely pathogenic (P/LP) genetic variants in strong evidence cardiomyopathy-susceptibility genes amongst AMVPS patients who underwent clinical genetic testing. Methods In this retrospective study, the medical records of 4,907 patients referred to our cardiovascular genetics clinic was used to identify those patients with a clinical phenotype consistent with AMVPS. After exclusion of individuals with severe mitral valve regurgitation, pertinent clinical, electrocardiographic, imaging and genetic testing data was extracted from the electronic record. For those AMVPS that underwent a form of genetic testing that encompassed 23 Clinical Genome Resource-defined strong/definitive evidence cardiomyopathy-susceptibility genes, ultra-rare (minor allele frequency ≤ 0.00005 in the Genome Aggregation Database [n=141,456 individuals]) non-synonymous variants identified within these genes were adjudicated independently as P, LP or variant of uncertain significance with the assistance of Varsome. The frequency of P/LP variants was then compared to a previously published cohort of 973 ostensibly healthy control exomes. Results Overall, 56 unrelated patients with suspected AMVPS were identified (82% female; average age at presentation of 31 ± 13 years; 32% with a family history of MVP and/or SCD). Of these, 24/56 (43%) underwent a form of clinical genetic testing that encompassed the pre-defined panel of cardiomyopathy-susceptibility genes. In comparison to control exomes (n=973), P/LP variants in strong/definitive evidence cardiomyopathy-susceptibility genes were over-represented in AMVPS patients [4/24 (16.7%) vs 20/973 (2.1%); p=0.002]. Interestingly, this enrichment was driven in large part by an excess number of A-band-localizing P/LP truncating variants in TTN-encoded titin in AMVPS cases [3/24 (12.5%) versus 4/973 (0.4%); p=0.0004]. Conclusion This study provides further evidence that a subset of AMVPS may arise secondary to a "two-hit" model whereby an underlying cardiomyopathic genetic substrate is exacerbated by the mechanical force generated by genetically unrelated myxomatous MVP resulting in maladaptive and ventricular arrhythmia-predisposing fibrosis within the inferobasal left ventricle and posteromedial papillary muscles. Larger studies are needed to better understand the potential role of genetic testing in the diagnosis, risk-stratification and management of patients with suspected AMVPS.

Shared and distinguishing phenotypic characteristics of filamin c and desmoplakin cardiomyopathy

Abstract Background Rare truncating variants in the genes Filamin C (FLNC) and Desmoplakin (DSP) are increasingly recognised causes of Dilated Cardiomyopathy and Arrhythmogenic Cardiomyopathy (1,2). A direct comparison of both genotypes is needed to aid in subclassification and genotype-specific risk modelling. Purpose We sought to identify shared and distinguishing clinical characteristics and imaging phenotypes between FLNC and DSP cardiomyopathy. Methods 41 patients with ‘pathogenic’ or ‘likely pathogenic’ FLNC or DSP variants by American College of Medical Genetics (ACMG) criteria were identified from a tertiary centre cardiovascular genetics laboratory. Clinical, genetic and imaging data were compiled. Continuous variables are presented as median [IQR] or mean±SD and analysed by nonparametric Mann Whitney U or t-test. Categorical variables were analysed by Chi-Squared or Fisher’s exact test. Results FLNC and DSP cohorts included a similar proportion of patients with truncating variants (91% vs 100%) and female sex (12/21, 57% vs 13/20, 65%). FLNC patients were younger (median age 30 [13-51] vs 57 [41-65] years, p&amp;lt;0.0008) and more likely to present with sustained ventricular arrhythmia (5/21 24% vs 3/20, 15%) (Table 1). Acute myocarditis episodes were exclusively linked to DSP cardiomyopathy (4/20, 20%) with no association to FLNC (0/21, 0%, p&amp;lt;0.05). FLNC cardiomyopathy patients had normal left ventricular (LV) systolic function (LVEF 57±9% SD vs 46±13% DSP, p&amp;lt;0.03) and normal left ventricular cavity side (mean indexed LV end diastolic volume (EDV) 85±12 vs 112±26ml/m2 DSP, p&amp;lt;0.02) (Table 2). Both FLNC and DSP cause exclusive left ventricular or biventricular late gadolinium enhancement (75% Vs 82%) on cardiac magnetic resonance imaging (MRI). Fibrosis was predominantly in a subepicardial distribution in both cohorts (66% FLNC vs 57% DSP). In FLNC, circumferential fibrosis was the most frequent pattern of distribution (5/9, 55%), whereas in DSP inferior and lateral wall fibrosis was the predominant feature (8/14, 57%). There were no cases of isolated RV involvement and no cases fulfilled ‘definite’ ARVC taskforce diagnostic criteria. Over a follow up of median 45months, there was no significant difference in the proportion of FLNC and DSP patients experiencing a composite end point of LVAD implantation, heart transplant, aborted sudden cardiac death or mortality (7/21, 33% FLNC Vs 7/20, 29% DSP p&amp;gt;0.99). Conclusion Normal systolic function with marked circumferential fibrosis is the predominant feature of FLNC cardiomyopathy whereas mild LV dilatation, mild systolic impairment and inferolateral fibrosis are more frequent in DSP cardiomyopathy. Myocarditis episodes are a distinguishing feature of DSP cardiomyopathy, suggesting a different underlying molecular mechanism driven by inflammation. The overall clinical trajectory of FLNC and DSP cardiomyopathy patients are comparable.

The role of TBX20 truncating variants in dilated and left ventricular non-compaction cardiomyopathy

Abstract Background The genetic cause of dilated cardiomyopathy (DCM) remains unexplained in a considerable proportion of cases. TBX20, the T-Box transcription factor 20 gene, has been linked to cardiac septal defects. Although only a few case reports have described an association between TBX20 and DCM/left ventricular non-compaction (LVNC), there are no case-control studies available, and TBX20 is still considered a gene with limited evidence for these phenotypes. Objectives This study sought to investigate the relationship between truncating variants in TBX20 (TBX20tv) and the development of DCM and LVNC. Methods TBX20 was sequenced using massive parallel sequencing in 7,463 unrelated probands with DCM/LVNC and 22,773 probands with other diagnoses (e.g., hypertrophic or arrhythmogenic cardiomyopathy, channelopathies, or aortic diseases) as internal controls. It was tested for enrichment and cosegregation of TBX20tv in DCM/LVNC, and clinical characteristics and outcomes in carriers were analyzed. Results Twenty-four probands with 22 TBX20tv were identified. Variant frequencies were significantly higher in patients with DCM/LVNC (24/7,463; 0.29%) than in internal controls (2/22,773; 0.008%) or controls from the gnomAD database (4/124,098; 0.003%), with an OR of 70.18 (CI95%: 9.48 to 519.8; p &amp;lt;0.0001) and 95.61 (CI95%: 33.06-276.55; p &amp;lt;0.0001), respectively. Cosegregation was studied in 21 families, identifying 57 carriers, of whom 43 (75.4%) had cardiac involvement. A combined LODs score of 4.41 was obtained, which is indicative of very strong segregation. Regarding clinical characteristics, the mean age at diagnosis was 37 years old, and 50% of carriers were female. Forty-six percent developed left-ventricular dysfunction, which was moderate to severe in half of the cases. Three carriers received a heart transplant, and another carrier died due to heart failure. Congenital heart defects were present in 16 patients (28.1%). Valvular involvement was the most frequent congenital abnormality (5 cases had bicuspid aortic valve, and another 5 had mitral valve defects), followed by heart septal defects (3 atrial and 3 ventricular); coronary anomalies (2) and aortic coarctation (2) were also detected. In some patients, these defects presented as complex congenital heart disease. Conclusions TBX20tv are associated with the development of dilated and non-compaction cardiomyopathy, and the presence of congenital heart defects is also common. Although some carriers, particularly younger ones, may not present the phenotype, nearly half of them experience ventricular dysfunction, and significant cardiac events, such as cardiac death or transplantation, are not uncommon. Therefore, the TBX20 gene should be routinely included in genetic testing panels for DCM and LVNC.

Genetic and phenotypic characterization of Nexilin (NEXN) related cardiomyopathy

Abstract Background Clinical and genetic features of Nexilin (NEXN) related cardiomyopathies (CMPs) are largely uncharacterized. At present, the strength of the gene-disease association for NEXN is currently moderate for Dilated Cardiomyopathy (DCM), limited for Hypertrophic Cardiomyopathy (HCM), and unevaluated for Arrhythmogenic Cardiomyopathy (ACM). More evidence is needed to establish a definite genotype-phenotype association. Purpose We sought to investigate 1) the causative role of NEXN gene in CMPs, and 2) its phenotypic expression and prognostic profile. Methods All patients carrying NEXN variants, recruited in 11 international referral centres, were classified according to reported phenotype and genetic variant. Variants were considered rare if below GnomAD Popmax Filtering Allele Frequency of 8x10-5. Burden enrichment testing of rare NEXN variants was performed in our case-cohort of patients with DCM/ACM (n=3053), in comparison with the GnomAD population of non-Finnish Europeans (NFE). Clinical phenotypes and outcomes of patients carrying validated variants were described. A previously established cohort of patients with Titin-related CMP was used for prognostic comparison. Results Data from 87 patients were collected. Forty patients were excluded due to being carriers of non-rare NEXN variants (n=22) and/or pathogenic variants in other genes (n=18). Of the remaining patients, only 2 had HCM diagnosis, precluding any further analysis on this phenotype. NEXN truncating variants (tv) resulted significantly enriched in DCM/ACM disease-cohort with a prevalence of 0.39% vs 0.09% in GnomAD NFE (p=0.0001), whereas non-truncating ones were not. Patients carrying NEXNtv (n=16), plus a further patient carrying a homozygous p.G650del variant were thus considered affected by NEXN-related CMP (NEXN-CMP). Out of 17 patients with NEXN-CMP, 82% were probands, 53% were males and median age at diagnosis was 46 (IQR 35-55). At baseline, the most prevalent phenotypes were DCM (53%) and ACM (41%) and NYHA I class was common (71%). Left ventricular (LV) indexed End Diastolic Volume was mildly increased (75mL, IQR 43-87) and ejection fraction mildly reduced (LVEF 41%±15). Myocardial fibrosis was reported for up to 67% of patients, median ventricular ectopic beats were 515/24h (IQR 43-5071). During a median follow of 42 months, 8 patients were implanted with ICD, 1 patient died and 3 (21%) had malignant ventricular arrythmias (MVA). When compared to a cohort of patients with TTN-CMP, patients with NEXN-CMP showed MVA earlier, and with higher LVEF. Conclusions NEXNtv showed significant disease association with DCM/ACM phenotypes. NEXN-CMP seems to be characterized by mild LV dilation and dysfunction, uncommon heart failure, but frequent myocardial fibrosis and ventricular arrythmias. These findings from the largest cohort of NEXN variants carriers strongly contribute to defining the causative role of this rare genotype and its related phenotype.

Exome Sequencing Reveals Novel Germline Variants in Breast Cancer Patients in the Southernmost Region of Thailand.

Germline carriers of pathogenic variants in cancer susceptibility genes are at an increased risk of breast cancer (BC). We characterized germline variants in a cohort of 151 patients diagnosed with epithelial BC in the southernmost region of Thailand, where the predominant ethnicity differs from that of the rest of the country. Whole exome sequencing was used to identify and subsequently filter variants present in 26 genes known to be associated with cancer predisposition. Of the 151 individuals assessed, 23, corresponding to 15.2% of the sample, exhibited the presence of one or more pathogenic or likely pathogenic variants associated with BC susceptibility. We identified novel germline truncating variants in BRIP1, CHEK2, MSH6, PALB2, and PTEN and annotated variants of uncertain significance (VUSs), both novel and previously documented. Therefore, it is advisable to use genetic testing as an additional risk screening method for BC in this area.

Mechanomics study of cardiomyocytes with filamin c truncations

Abstract Background Filamin C (FLNC) is expressed in cardiomyocytes, where it binds to actin and localizes to Z-discs, sarcolemma, and intercalated discs. Although FLNC truncation variants (FLNCtv) are an established cause of arrhythmias and heart failure (arrhythmogenic cardiomyopathy), the underlying mechanisms, in particular changes in mechanical properties of cardiomyocytes, are mostly unknown. Purpose In this study, we applied a biophysical approach to investigate the interaction between altered mechanical properties and biological response in human induced pluripotent stem cells–derived cardiomyocytes (hiPSC-CMs) carrying FLNCtv. Methods CRISPR/Cas9 genome-edited FLNC-/- hiPSC-CMs, a homozygous condition that is lethal in animal models, and FLNC+/- hiPSC-CMs, recapitulating the heterozygous human disease, were analyzed. Wild-type FLNC hiPSC-CMs were used as controls. Atomic Force microscopy (AFM) was used to perform single-cell force spectroscopy (SCFS) microindentation, to evaluate passive and dynamic mechanical properties. From the loading curves, two cell Young’s Moduli were calculated: E1, related to the compression of the plasma membrane and actin cortex, and E2, including the rest of the inner cell layers (cytoskeleton and nucleus). Cell adhesion was assessed using the retraction curve of the SCFS. Data were processed in terms of adhesion force (the highest value of the force exerted by the cell-surface contact to the cantilever during retraction) and work of adhesion (integrating the area under the retraction curve). Finally, FLNC-/- hiPSC-CMs were incubated with 1 µM Crenolanib (a PDGFRA signaling inhibitor) for 3 days, and the microindentation was performed again using DMSO-only-treated FLNC-/- cells as controls. Results The AFM indentation curve for the cells analyzed showed significant differences. Both elastic contributions (E1 and E2) show that wild-type FLNC iPSC-CMs are stiffer than both mutant FLNC+/- and FLNC-/- iPSC-CMs (see Figure upper panel). The adhesion showed the same trend as the elasticity: mutant FLNC iPSC-CMs showed a progressive decrease of work of adhesion as well as max adhesion force from the FLNC+/- to the FLNC-/- models. Rescue of the latter with Crenolanib showed to moderately increase their stiffness. Moreover, a qualitative analysis of the beating traces showed that FLNC+/- but mostly FLNC-/- display "irregular" small peaks, resembling those of delayed after depolarizations (see Figure lower panel). Conclusions We found that FLNCtv causes a decrease in cell stiffness which declines progressively from the heterozygous status, which is associated with FLNC haploinsufficiency, to the homozygous status, which lacks FLNC. This indicates a progressive softening of the cardiomyocytes, suggesting a damaged cytoskeleton. Crenolanib rescue suggests that the inhibition of PDGFRA signaling could partially restore such damage (see Figure upper panel - right corner).