Variants Of Undetermined Significance Research Articles

7655 Genetic Mysteries Unveiled: Unusual Mutation Unraveled In Hypophosphatasia

Abstract Disclosure: A. Sharma: None. P. Katikaneni: None. S. Tanveer: None. K. Selk: None. Introduction: Hypophosphatasia (HPP), an inherited dental-osseous metabolic illness, is caused by inactivating mutations in the gene encoding the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP), which leads to a decrease in TNSALP enzymatic activity. Patients with HPP typically experience dental and skeletal mineralization issues as their most common symptoms; musculoskeletal abnormalities, numerous fractures, tiredness, and migraines are less common clinical presentations. Case Report: Our 75-year-old male patient initially presented 1.5 years prior following a spontaneous T12 vertebral fracture. A bone biopsy was performed and did not reveal evidence of malignancy. Testing for SPEP/UPEP was not consistent with myeloma, and thyroid testing was also unrevealing. His alkaline phosphatase level was consistently <40 IU/L since 1999. An evaluation was done for low alkaline phosphatase and secondary reasons for bone loss. This testing did not reveal vitamin D deficiency, parathyroid disease, hypophosphatemia, laboratory evidence of celiac disease, hypogonadism, or hypomagnesemia. A vitamin b6 level was markedly elevated at 119 ng/mL (ref range 2.1-21.7) while not on supplementation. He had noticed multiple fractures as a child, but he had written them off as minor injuries. He could clearly remember breaking his right wrist and clavicle. As an adult, he had rib fractures and a foot fracture. He was unable to recollect losing his primary teeth. Almost all his teeth are crowns or implants despite taking exceptional care of them. He did not have joint hypermobility. Genetic testing revealed the patient was heterozygous for an ALPL gene variant, c.876_882delinsT. It was a variant of undetermined significance at that time. His daughter also was found to have a low alkaline phosphatase and the same genetic test result. She also has experienced dental carries and joint pain. Our patient initiated teriparatide due to the need for more urgent therapy for extensive spinal surgery. He was later transitioned to asfotase alfa therapy and has not had any further fractures at the time of this report. Discussion: Hypophosphatasia is a rare inborn disorder due to mutations in the tissue nonspecific alkaline phosphatase gene ALPL and is characterized by low ALP levels. Affected individuals will also have elevated vitamin b6 levels and urinary phosphoethanolamine (PEA) levels. Early diagnosis of HPP is necessary to prevent bone fracture pain and improve quality of life. Inaccurate assessments can miss diagnoses of parents and other family members and even lead to lost opportunities to treat a patient with a substantial illness burden. This case highlights the mutation c.876_882delinsT as a likely pathologic variant in the ALPL gene. It also supports that patients with HPP can also respond to teriparatide treatment. Presentation: 6/2/2024

Adult-onset leukoencephalopathy with vanishing white matter with compound heterozygous EIF2B3 gene variants

BackgroundLeukoencephalopathy with vanishing white matter (VWM) is an autosomal recessive disorder affecting the white matter of the brain. It typically manifests during childhood, with clinical features including sudden and severe neurological deterioration triggered by stressors such as febrile illness, minor head trauma, or stressful events. Adult-onset cases of VWM are exceptionally uncommon.Case presentationIn this case, we present an adult patient who exhibited late-onset progressive VWM characterized by ataxia, postural instability, cognitive impairment, and emotional disturbances. Comprehensive screening for endocrine, metabolic, tumor, and immunologic disorders yielded normal or negative results. Brain imaging revealed diffuse and confluent hyperintensity in the white matter on T2-weighted images, along with periventricular cavitations. Genetic testing confirmed the diagnosis of VWM, identifying two heterozygous variants in the eukaryotic translation initiation factor 2B subunit γ (EIF2B3) gene: a pathogenic variant, c.1037 T > C (p.I346T), and a variant of undetermined significance, c.22A > T (p.M8L). Upon a 2-year follow-up, the patient's symptoms deteriorated rapidly following a COVID-19 infection.ConclusionsIn conclusion, we have presented a case of classical adult-onset VWM. Since there are no cures or definitive treatments for the disease, it's extremely important to focus on early diagnosis and the prevention of stressors to avoid acute deterioration.

Tolerance and safety of adjuvant metronomic capecitabine in Indian patients with non-metastatic triple negative breast cancer (TNBC): An institutional experience.

e12530 Background: Triple-negative breast cancer (TNBC) subtype has a high risk of recurrence and overall poorer survival after standard treatment. SYSUCC-001 study presented in ASCO 2020 reported an increased disease-free survival after one year of metronomic capecitabine in operable TNBC patients after completion of local therapy. As per our clinical experience in metastatic breast cancer, many Indian patients are not able to tolerate capecitabine for longer duration. Hence, we wanted to assess the tolerance and safety of one year of daily dosing of capecitabine in our patients with Non-Metastatic TNBC. Methods: We performed a retrospective analysis of non-metastatic TNBC patients receiving adjuvant capecitabine 650 mg /m2 twice daily continuously for one year who took treatment from June 2020 to August 2022. The data extracted included patient socio-demographics, capecitabine information, side effects, and capecitabine treatment changes (dose reductions and dose interruptions). Statistical analysis was conducted using SPSS, version 24. Results: We analyzed 90 patients, out of which majority (62%) were stage II. Median age of the cohort was 48 years. Germline data was available for 64 patients, out which 8 patients were BRCA 1 positive and 8 patients had a variant of undetermined significance. Median duration of capecitabine in our patients was 230 days (IQR: 10 - 360). Twenty-six (28.8%) patients underwent dose reduction and 15 (16.6%) had reported grade 3 toxicity. Mean dose of capecitabine received was 535 mg / m2 twice daily. Treatment was interrupted due to toxicity in 20 (22%) of patients. The most common capecitabine-induced toxicity was hand foot syndrome, which was also the most common reason for dose reduction and treatment interruption. Dihydropyrimidine dehydrogenase (DPD) mutation status was known for 38 patients out of which only one (2.6%) was found to have DPD deficiency. Incidence of grade 3 toxicity was observed to be less in patients with age less than 50 years, ECOG performance status 0-1 and absence of comorbidities. Two-year disease free survival (DFS) and overall survival (OS) was estimated to be 79.3% and 80% respectively. Most common site of metastases was reported to be brain followed by lung. Conclusions: Continuous metronomic dosing of adjuvant capecitabine for 1 year is fairly well tolerated in Indian patients with non-metastatic TNBC, but may still require dose reduction in some patients due to toxicity.

Targeted sequencing for hereditary breast and ovarian cancer in BRCA1/2-negative families reveals complex genetic architecture and phenocopies

Approximately 20% of breast cancer cases are attributed to increased family risk; yet variation in BRCA1/2 can only explain 20-25% of cases. Historically, only single gene or single variant testing were common in at-risk family members, and further sequencing studies were rarely offered after negative results. In this study, we applied an efficient and inexpensive targeted sequencing approach to provide molecular diagnoses in 245 human samples representing 134 BRCA mutation negative (BRCAX) hereditary breast and ovarian cancer (HBOC) families recruited from 1973-2019 by Dr. Henry Lynch. Sequencing identified 391 variants, which were functionally annotated and ranked based on their predicted clinical impact. Known pathogenic CHEK2 breast cancer variants were identified in five BRCAX families in this study. While BRCAX was an inclusion criterion for this study, we still identified a pathogenic BRCA2 variant (p.Met192ValfsTer13) in one family. A portion of BRCAX families could be explained by other hereditary cancer syndromes that increase HBOC risk: Li-Fraumeni Syndrome (gene: TP53) and Lynch Syndrome (gene: MSH6). Interestingly, many families carried additional Variants of Undetermined Significance (VOUSs) that may further modify phenotypes of syndromic family members. Ten families carried more than one potential VOUS suggesting the presence of complex multi-variant families. Overall, nine BRCAX HBOC families in our study may be explained by known likely-pathogenic/pathogenic variants, and six families carried potential VOUSs, which require further functional testing. To address this, we developed a functional assay where we successfully re-classified one family’s PMS2 VOUS as benign.

Abstract PO4-08-10: Genetic mutations and associated rates of pathologic complete response among a diverse patient population with early stage triple negative breast cancer

Abstract Background: Combination treatment with chemotherapy and immunotherapy remains the standard of care for high risk, early-stage triple negative breast cancer (TNBC). Although the addition of immunotherapy improves rates of pathologic complete response (pCR) and event-free survival, approximately 50% of patients achieve pCR with neoadjuvant chemotherapy alone suggesting that some patients may safely avoid the potential toxicities of immunotherapy without compromising outcomes.1,2 Markers of immune activation including PD-L1 expression and tumor infiltrating lymphocytes have demonstrated prognostic significance; however, identifying a predictive biomarker to aid in selecting those patients most likely to benefit from immunotherapy remains an unmet need.3,4 Genetic mutations are suggestive of DNA susceptibility to damage and the role they may play as a potential marker of immune response is unclear.5,6 This study explores genetic analysis and mutation rates among a diverse cohort of patients with TNBC to identify patterns associated with and without pCR. Methods: We evaluated 105 women diagnosed with Stage I-III TNBC and treated with combination neoadjuvant chemotherapy and immunotherapy at Ochsner Cancer Center between December 2019 through June 2022. Exclusion criteria included ER+, PR+, HER2+, or unknown receptor status, no pembrolizumab in the neoadjuvant setting, no definitive surgery following neoadjuvant treatment, absence of documented race and age less than 18 years of age. Data was collected utilizing Epic Slicer Dicer program, in addition to chart review. Genetic analysis was performed using a variety of DNA-based next generation sequencing analysis platforms. Results: 91 patients met inclusion criteria. 17 patients were found to have pathogenic mutations (BRCA1/2, POLE and ATM), 38 patients had variants of undetermined significance (VUS) and 36 patients had neither. Among patients with pathogenic genetic mutations, 81.25% had a pCR following neoadjuvant treatment compared to 58.33% of those with a VUS and 47.06% of those with no known mutation (p=0.07). Before adjusting for age and stage, patients with a pathogenic mutation were 73% more likely to achieve pCR than those with no known genetic mutations (crude RR: 1.73, 95% CI: 1.13-2.65). After adjusting for age and stage, the relationship was not statistically significant (aRR: 1.15, 95% CI: 0.78-1.34). Among the 47 Black patients, 5 were found to have a pathogenic mutation and 29 had a VUS. 11 of the 39 White patients were found to have a pathogenic mutation while only 6 had a VUS. Across all genetic mutation categories, Black patients experienced lower rates of pCR than White patients. White patients with a known pathogenic mutation had the highest rate of pCR at 90.91%, while Black patients with no identified mutation had the lowest rate of pCR at 38.46%. The generalizability of these results may be limited due to small sample size. Conclusion: Our results demonstrating higher rates of genetic variants among patients achieving pCR regardless of race highlight the importance of better understanding how these variants correlate with tumor mutational burden (TMB) and further, the role this may play in predicting immune-response. Larger studies are needed to assess TMB as a predictive biomarker in early-stage TNBC as this may provide opportunities to further individualize treatment and minimize toxicity among this group of patients. Additionally, given differences in the prevalence of pathogenic mutations and VUS among Black patients, better understanding the relationship between mutation status and immune response is imperative to optimize outcomes in this high-risk population. Table. Citation Format: Caitlin Taylor, Melanie Sheen, Rabia Cattie, Victoria Chung, Melyssa Bratton, Meredith Lakey, Erin Biggs. Genetic mutations and associated rates of pathologic complete response among a diverse patient population with early stage triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-08-10.

In vitro data suggest a role for PMS2 Kozak sequence mutations in Lynch syndrome risk

Lynch syndrome (LS) is the most common hereditary cancer syndrome. Heterozygous loss-of-function variants in PMS2 are linked to LS. While these variants are not directly cancer causing, reduced PMS2 function results in the accumulation of somatic variants and increased cancer risk over time due to DNA mismatch repair dysfunction. It is reasonable that other types of genetic variation that impact the expression of PMS2 may also contribute to cancer risk. The Kozak sequence is a highly conserved translation initiation motif among higher eukaryotes and is defined as the nine base pairs upstream of the translation start codon through the first four bases of the translated sequence (5'-[GTT]GCATCCATGG-3'; human PMS2: NM_000535.7). While Kozak sequence variants in PMS2 have been reported in ClinVar in patients with suspected hereditary cancer, all variants upstream of the translation start site are currently classified as variants of undetermined significance (VUSs). We hypothesized that variants significantly disrupting the Kozak sequence of PMS2 would decrease PMS2 protein expression, contributing to increased cancer risk over time. Using a dual-luciferase reporter plasmid and site-directed mutagenesis, we generated the wild-type human PMS2 and the ClinVar VUSs within the PMS2 Kozak sequence. Besides the c.1A>C variant, which is already known to be pathogenic, we implicate six additional variants as American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) pathogenic supporting (PP) variants and classify ten as benign supporting (BP). In summary, we present a method developed for the classification of human PMS2 Kozak sequence variants that can contribute to the re-classification of VUSs identified in patients.

Diagnostic Approaches to Investigate JAK2-Unmutated Erythrocytosis Based on a Single Tertiary Center Experience.

Erythrocytosis is attributed to various clinical and molecular factors. Many cases of JAK2-unmutated erythrocytosis remain undiagnosed. We investigated the characteristics and causes of JAK2-unmutated erythrocytosis. We assessed the clinical and laboratory results of patients with erythrocytosis without JAK2 mutations and performed targeted next-generation sequencing (NGS) panels for somatic and germline mutations. In total, 117 patients with JAK2-unmutated erythrocytosis were included. The median hemoglobin and hematocrit levels were 17.9 g/dL and 53.4%, respectively. Erythropoietin levels were not below the reference range. Thrombotic events were reported in 17 patients (14.5%). Among JAK2-unmutated patients, 44 had undergone targeted panel sequencing consisting of myeloid neoplasm-related genes, and 16 had one or more reportable variants in ASXL1 (5/44), TET2, CALR, FLT3, and SH2B3 (2/44). Additional testing for germline causes revealed eight variants in seven genes in eight patients, including NF1, BPGM, EPAS1, PIEZO1, RHAG, SH2B3, and VHL genes. One NF1 pathogenic, one BPGM likely pathogenic, and six variants of undetermined significance were detected. Somatic and germline mutations were identified in 36.4% and 33.3 % of the JAK2-unmutated group; most variants had unknown clinical significance. Not all genetic causes have been identified; comprehensive diagnostic approaches are crucial for identifying the cause of erythrocytosis.

Transplant versus no transplant in myelodysplastic syndrome and acute myeloid leukemia with TP53 mutation; a referral center experience.

A remarkably high rate of post-transplant relapse in patients with TP53-mutated myelodysplastic syndrome/acute myeloid leukemia (MDS/AML) calls to question the utility of allogeneic stem cell transplant (HSCT). We, therefore, performed a retrospective analysis to compare the outcomes between HSCT (N = 38) versus non-HSCT (N = 45) approaches. Patients in the HSCT cohort were younger (median age 63 vs. 72) while patients in the non-HSCT cohort more commonly had complex karyotype with chromosome 17 aberrancy and 5q deletion (p < .01). A total of 69 TP53 variants including 64 pathogenic variants, and 5 variants of undetermined significance were detected. Nine patients (4 in HSCT and 5 in non-HSCT) had multi-hit TP53 variants. After induction: 57.9% versus 56.6% in the HSCT versus non-HSCT cohort achieved morphologic complete remission. Median time to HSCT was 6 months and median follow-up was 15.1 months for HSCT and 5.7 months for non-HSCT. Median disease-free survival (DFS) and overall survival (OS) were 11.7 and 15.9 months for HSCT, and 4.1 and 5.7 months for non-HSCT cohorts, respectively. Non-relapse mortality at 12 months was 22% versus 44% for HSCT versus non-HSCT. In the HSCT cohort, the rate of grade II-IV acute and chronic graft-versus-host disease (GVHD) was 55% and 18%, respectively. None of the patients from the non-HSCT cohort were alive while four patients from the HSCT cohort were alive, in remission, and without GVHD (GRFS) at the time of abstraction. Better treatment strategies for patients with TP53-mutated MDS/AML remain an area of unmet clinical need.

Heterogeneous Genetic Landscape of Chronic Idiopathic Neutropenia Revealed By Whole Exome Sequencing

Background: Adultpatients with persistent, unexplained neutropenia who do not fulfill the diagnostic criteria of any underlying disease are characterized as chronic idiopathic neutropenia (CIN) cases.It is an exclusion diagnosis that can be established after a thorough clinical/laboratory investigation for any underlying causes, including negative anti-neutrophil antibody testing, inconclusive bone marrow (BM) aspiration/biopsy and normal cytogenetics. CIN patients regardless of their absolute neutrophil counts (ANC) usually display a benign and uncomplicated clinical course. Although the pathogenesis of CIN remains largely unknown, we hypothesize that a constitutional/congenital background might exist, at least in some cases, that have potentially escaped diagnosis during childhood. Aim of the study: To investigate the genetic landscape of neutropenia in CIN by conducting whole exome sequencing (WES) analysis in a number of adult CIN patients. Methods: We have performed WES in 16 adult (median age 59 years, range 30-72 years) patients (11 females and 5 males) with CIN according to previously published criteria (Fioredda F, et al Hemasphere. 2023). The patients had median ANC 1.2 x 10 9/L (range 0.4-1.6 x 10 9/L) for prolonged period and unknown neutropenia onset in most cases. Genomic DNA was extracted from peripheral blood samples, coding fragment libraries were prepared, sequencing performed on Illumina NovaSeq 6000 System, and data subjected to an in-house pipeline for bioinformatic analysis for variant calling and annotations. Singleton analyses were performed focusing on genes associated with congenital defects of phagocytes, BM failure (BMF) and immune deficiency and dysregulation. Mean depth of coverage was 70x and the mean number of variants/sample was 22553. Results: WES analysis identified 10 pathogenic/likely pathogenic (P/LP) variants in 10 patients. The variants consisted of 4 frameshift mutations, 3 nonsense mutations, 1 missense mutation, 1 splice-site mutation and 1 splice-region mutation. Pathogenic variants in congenital neutropenia (CN)-related genes included only a heterozygous mutation in G6PC3:c.511C&amp;gt;T,p.Gln171Ter (previously detected in one of our CN patients; Nikolouzakis TK et al, Ann Hematol. 2022). Pathogenic variants in genes associated with BMF were detected in two genes, namely FANCM:c.1492C&amp;gt;T, p.Gln498Ter and CTC1:c.3058C&amp;gt;T,p.Gln1020Ter, respectively. The majority of the pathogenic variants were found in genes associated with immune deficiency and dysregulation. These involved: (i) a heterozygous mutation in DCLRE1C:c.1784dup,p.Ile596fs (associated with immunodeficiencies affecting cellular and humoral immunity); (ii) a LP mutation in ORAI1:c.132_133del,p. Arg45fs and a pathogenic variant in SPINK5:c.1302+4A&amp;gt;T (related to combined immunodeficiencies with associated or syndromic features); (iii) a pathogenic variant in MEFV:c.2080A&amp;gt;G, p.Met694Val and a LP variant in PSMG2:c.703-2A&amp;gt;T (both genes associated with autoinflammatory disorders); (iv) a LP variant in IRF7:c.1089dup,p.Pro364Alafs (associated with defects in intrinsic and innate immunity); and (v) a LP variant in PEPD:c.738_739insGT,p.Ser247fs (associated with immune dysregulation). Regarding variants of undetermined significance (VUS) an average of 4.5 VUS variants per patient were detected in genes related to CN ( HAX1, VPS13B, LYST), BMF ( GATA2, SAMD9, CXCR4) or immunodeficiency ( TCF3). However, none of these patients display clinical symptoms typical of these conditions. Specifically, the majority of the patients presented with mild/moderate neutropenia and rarely with severe neutropenia, they have relatively normal BM biopsies and do not display major symptoms or signs related to severe immunodeficiency or immune dysregulation syndromes. Conclusions: This study is the first attempt of WES analysis in adult CIN patients and shows that there is a heterogeneous genetic landscape underlying the disease. More studies are required so that the detected P/LP variants and VUS can be associated with the clinical phenotypes to more accurately interpret their potential contribution to the neutropenia phenotype.

Clinical and Molecular Spectrum of Somatic Mosaic States in DDX41 mutant Germline Predisposition Syndromes

Introduction: Germline DEAD-box RNA helicase-1 mutant (DDX41) predisposition syndrome ( DDX41-MT-GPS) is one of the most frequent hereditary predisposition syndromes in adults with myeloid neoplasms (MN-2-5%). Somatic mosaicism involving the unmutated DDX41 allele (e.g., R525H) as a potential mechanism for myeloid evolution has been reported in 39-50% of patients (pts). In addition, given the later age of onset, somatic mosaic states composed of age-related clonal hematopoiesis (ARCH) (e.g., DNMT3A, ASXL1, TET2, and TP53), with reported frequencies less than expected in MDS and AML, have been documented. We conducted this study to define the somatic mosaic landscape in DDX41-MT-GPS . Methods: After approval from the Mayo Clinic IRB the prospective GPS database was queried for pts with DDX41-MT GPS. Germline confirmation was conducted on skin fibroblast-, or hair follicle-derived DNA. Somatic CH testing was conducted using a targeted NGS panel as previously described. Given the high frequency of variants of undetermined significance (VUS), comparisons were made between pathogenic ( DDX41path) and DDX41VUS. Results: We identified 106 pts with DDX41-MT-GPS, of which 83 (78%) met criteria for MN, 16 (15%) for CH/CCUS (clonal cytopenias of undetermined significance), while 7 (6%) were unaffected carriers. Somatic mosaicism in DDX41-GPS patients without MN Sixteen DDX41-MT pts without MN met criteria for CH/CCUS ( DDX41path [n=4] and DDX41VUS [n=12]; median age 69 years (yrs) (range [R], 19-78) ( DDX41path [69 yrs] and DDX41VUS [67 yrs], p=0.85), with a male predilection (75%). Four (25%) pts had somatic DDX41 MT (R525H [n=3] and A376T [n=1]) with median variant allele frequency (VAF) 10.5% (R, 5-27); 2 (50%) pts with germline DDX41path had R525H (VAF; 5% and 13%) and 1 (8%) pt each with germline DDX41VUS had R525H (VAF 5%) and A376T (VAF 27%) somatic variants, respectively. Abnormal cytogenetics (CG) were observed in 2/13 evaluable (15%) pts ([+8 and del20q]) in the germline DDX41path group only (p= 0.05). ARCH co-mutations were observed in 11 (69%) pts, not significantly (NS) different between DDX41path (75%) and DDX41VUS (75%), p= 0.66. Individual co-mutations: DNMT3A (25% vs 16%, p= &amp;gt; 0.99), ASXL1 (25% vs 8%, p=0.71), TET2 (25% vs 8%, p= &amp;gt;0.99), and JAK2 (0% vs 8%, p= &amp;gt;0.99) were NS different between the two groups. Somatic mosaicism in DDX41-GPS patients with MN Eight-three pts met criteria for MN ( DDX41path [n=43] and DDX41VUS [n=40]; median age 67 yrs (range [R], 27-92), with a male predilection (58%). Fifty-four (65%) pts had MDS ( DDX41path [55%] and DDX41VUS [45%], p=0.55) and 29 (35%) had AML ( DDX41path [41%] and DDX41VUS [59%], p=0.65). Overall, 15 (18%) pts had somatic DDX41 MT; 12 (80%) pts with DDX41path and 3 (20%) pts with DDX41VUS (p= 0.003). The median VAF of somatic DDX41 MT was 12% (R, 5-37), NS different between DDX41path (10.5%) and DDX41VUS (12%) (p=0.08). The most common somatic DDX41 MT was R525H (66% [n=10]), significantly associated with DDX41path (9/10 [90%]), p=0.01. ARCH was observed in 17 (20%) pts, significantly higher in DDX41path (76%) compared to DDX41VUS (24%), p= 0.03. Abnormal CG were observed in 10/73 evaluable (14%) pts (-Y [n=2], del 20 [n=3], del 5q [n=2], del 7q [n=1], t (3;8) [n=1] and complex CG [n=1]), higher in DDX41path (70%) compared to DDX41VUS (30%) with p value &amp;gt;0.99. We compared the spectrum of CH between DDX41 MT CH/CCUS and MN pts ( Table 1) and found no difference in the proportion of pts with somatic DDX41 MT (p= 0.52) and ARCH/myeloid MT (p= 0.56). The individual occurrences of DNMT3A (p= 0.49), ASXL1 (p= 0.71), TET2 (p=0.59), JAK2 (p&amp;gt;0.99) and TP53 (p= 0.56) mutations were NS different between the two groups ( Figure 1). Conclusion: We define the somatic mosaic landscape in DDX41-MT GPS and demonstrate that somatic mutations involving the other allele, especially R525H, are seen in ~20% of pts (&amp;gt;80% of R525H present in MN). The frequency and composition of ARCH and myeloid driver mutations was lower than expected for the age of presentation, in comparison to de novo and secondary MDS/AML and occurred more frequently in DDX41path compared to DDX41VUS. Forty-two % (n=41), including 37% with MN, did not have somatic mosaicism, underscoring the fact that mechanisms of leukemia progression remain to be defined in subsets of affected pts.

Genetic testing and diagnostic strategies of fetal skeletal dysplasia: a preliminary study in Wuhan, China

BackgroundFetal skeletal dysplasia is a diverse group of degenerative diseases of bone and cartilage disorders that can lead to movement disorder and even death. This study aims to evaluate the diagnostic yield of sonographic examination and genetic testing for fetal skeletal dysplasia.MethodsFrom September 2015 to April 2021, the study investigated 24 cases with suspected short-limb fetuses, which were obtained from Tongji Hospital affiliated to Tongji Medical College of Huazhong University of Science and Technology. To identify the causative gene, multiple approaches (including karyotype analysis, copy number variations and whole exome sequencing) were performed on these fetuses. And further segregation analysis of the candidate variant was performed in parents by using Sanger sequencing.Results① Out of 24 cases, likely pathogenic variants in FGFR3, FBN2, COL1A2, CUL7 and DYNC2H1 were detected in 6 cases; pathogenic variants in FGFR3, IMPAD1 and GORAB were identified in other 6 cases; and variants in WNT1, FBN1, OBSL1, COL1A1, DYNC2H1 and NEK1, known as Variant of Undetermined Significance, were found in 4 cases. There were no variants detected in the rest 8 cases by the whole exome sequencing. ② Of 24 cases, 12 (50%) were found to carry variants (pathogenic or likely pathogenic) in seven genes with 12 variants. Four fetuses (16.7%) had variants of uncertain significance.ConclusionGenetic testing combining with ultrasound scanning enhances the accurate diagnosis of fatal skeletal dysplasia in utero, and then provides appropriate genetic counseling.

Pathogenicity and Function Analysis of Two Novel SLC4A11 Variants in Patients With Congenital Hereditary Endothelial Dystrophy.

The purpose of this study was to explore the pathogenicity and function of two novel SLC4A11 variants associated with congenital hereditary endothelial dystrophy (CHED) and to study the function of a SLC4A11 (K263R) mutant in vitro. Ophthalmic examinations were performed on a 28-year-old male proband with CHED. Whole-exome and Sanger sequencing were applied for mutation screening. Bioinformatics and pathogenicity analysis were performed. HEK293T cells were transfected with the plasmids of empty vector, wild-type SLC4A11, and SLC4A11 (K263R) mutant. The transfected cells were treated with SkQ1. Oxygen consumption, cellular reactive oxygen species (ROS) level, mitochondrial membrane potential, and apoptosis rate were measured. The proband had poor visual acuity with nystagmus since childhood. Corneal foggy opacity was evident in both eyes. Two novel SLC4A11 variants were detected. Sanger sequencing showed that the proband's father and sister carried c.1464-1G>T variant, and the proband's mother and sister carried c.788A>G (p.Lys263Arg) variant. Based on the American College of Medical Genetics (ACMG) guidelines, SLC4A11 c.1464-1G>T was pathogenic, whereas c.788A>G, p.K263R was a variant of undetermined significance. In vitro, SLC4A11 (K263R) variant increased ROS level and apoptosis rate. Decrease in mitochondrial membrane potential and oxygen consumption rate were remarkable. Furthermore, SkQ1 decreased ROS levels and apoptosis rate but increased mitochondrial membrane potential in the transfected cells. Two novel heterozygous pathogenic variants of the SLC4A11 gene were identified in a family with CHED. The missense variant SLC4A11 (K263R) caused mitochondrial dysfunction and increased apoptosis in mutant transfected cells. In addition, SkQ1 presented a protective effect suggesting the anti-oxidant might be a novel therapeutic drug. This study verified the pathogenicity of 2 novel variants in the SLC4A11 gene in a CHED family and found an anti-oxidant might be a new drug.

Reclassification of clinical exome data leads to significant clinical assessment changes in almost half of the patients

Purpose: With the global accumulation of genetic/clinical data, we are understanding the clinical significance of the reclassification of pathogenicity for gene variants. We hypothesized that this evolution in classification(s) may cause clinically-relevant discrepancies in the genetic risk assessment of subjects. In this study, we sought to reclassify the clinical exome sequence (CES) data of our patients to assess whether these changes would have clinical significance.&#x0D; Materials and Methods: The study included CES data of 23 cases diagnosed with cancer or familial cancer predisposition. The variants were first classified in 2020 and then reclassified a year after based on the ACMG database. Chart reviews were performed to record clinical history and interventions. &#x0D; Results: In the first classification of CES data, a total of 80 variants were identified as being not benign (26 likely pathogenic/pathogenic and 54 variants of undetermined significance (VUS)). The clinical significance of fifteen variants (19%) changed after reclassification in 10 patients (43%). The only upgraded variant was the c.9097 dup in exon 23 of BRCA2 gene (likely pathogenic to pathogenic). Fourteen variants were downgraded at reanalysis in 9 patients: from pathogenic to likely pathogenic (2 variants), pathogenic to VUS (2), likely pathogenic to VUS (4), and VUS to benign (6).&#x0D; Conclusion: Considering that the clinical significance of CES data changed due to reclassification in almost half of the studied patients, we believe genetic variant-related data should be assessed at regular intervals, regardless of follow-up status in the clinic.

Two Novel Variants of Undetermined Significance in the ABCC2 Gene Were Identified in a Patient with Dubin-Johnson Syndrome

Two Novel Variants of Undetermined Significance in the <i>ABCC2</i> Gene Were Identified in a Patient with Dubin-Johnson Syndrome

New insights into the correlation between genotype and cardiac phenotype in laminopathies

Abstract Funding Acknowledgements Type of funding sources: None. Background Genotype-phenotype correlation and risk stratification for heart failure progression in patients with cardiolaminopathies are poorly defined. Purpose The primary aim of the present study is to characterize and correlate phenotype with genotype and sex. The secondary aim is to test the two main existing prognostic scores (the Arbustini and the Whabi score), both developed to predict ventricular arrhythmic events in cardiolaminopathies, for both major ventricular arrhythmias (MVA) and end-stage heart failure (ESHF). Methods This is a retrospective, single-center study, including patients with lamin (LMNA) and/or emerin (EMD) genetic variants. Data were collected from the first medical contact (FMC) to the last follow-up (FU). Results 28 patients (57% male, 82% probands, 68% with a family history of sudden death) were included: 96% harbored LMNA heterozygous variants, 15% had non-missense variants, 11% had variant previously never described. Functionally, 50% were variant of undetermined significance (VUS, C3), the rest were either pathogenetic or likely pathogenic (C4/C5) variants. Median age at FMC was 41 years (IQR 30-55 years), median FU was 11 years (IQR 8-18 years). At FMC, all patients had cardiac involvement, 54% had muscle involvement. By the last FU, 54% developed atrial fibrillation, 43% atrioventricular block (AVB) &amp;gt; I degree, 50% carried an implantable cardioverter defibrillator (21% with resynchronization), 50% had suffered at least one episode of MVA, 25% developed ESHF: 11% died due to refractory HF, 14% underwent heart transplantation (mean age 43.5 ± 9 years). The age of onset of the different types of cardiac phenotype was not influenced by sex. The penetrance of atrioventricular (AV) conduction defects (Figure 1 Panel A) and of atrial tachyarrhythmias (Figure 1 Panel B) was higher and earlier in subjects with non-missense variants. Subjects with VUS, compared to those with pathogenetic/likely pathogenetic variants, showed a significantly lower and later penetrance of MVA (Figure 2 Panel A) and a later penetrance of non- sustained ventricular tachycardia (NSVT, Figure 2, Panel B), but not of AV conduction disturbances, atrial arrhythmias, or contractile dysfunction. Arbustini score at FMC proved to be a predictor of both MVA and ESHF, while Whabi score only predicted MVA. Conclusions In our series, the prevalence and the age of appearance of the different cardiac phenotypes of cardiolaminopathies were not influenced by sex, while the presence of VUS correlated with a lower risk of ventricular arrhythmias, but not of the other cardiac manifestations including contractile disfunction and heart failure. Our data suggest the importance of a close cardiac monitoring and of larger prospective studies even in patients with VUS. All patients developed a sever cardiac phenotype by the age of 60.

C27 RBM20 VARIANTS RELATED CARDIOMYOPATHY: AN ITALIAN CASE SERIES

Abstract Background RNA–binding motif protein 20 (RBM20) regulates post–transcriptional splicing, particularly in sarcomeric, but also in other cardiac genes crucial for homeostasis. RBM20 variants are a rare cause of cardiomyopathy (CMP), but genotype–phenotype correlation is still elusive, starting from the type of CMP (dilated, non–compacted, hypertrophic, and even restrictive phenotypes have been described), and ranging to sex–dependent and arrhythmic expression. Purpose: To describe cardiac involvement in 8 patients with RBM20 variants. Methods 189 patients (pts) underwent genetic analysis in our center (2019–2022) for suspected genetic CMP (TruSight Cardio panel, Illumina); 72% had a positive genetic report, including pathogenic/likely pathogenic (P/LP) or variants of undetermined significance (VUS). Among them, 9 (5%, 6 males and 3 females, from 6 families) carried a heterozygous RBM20 variant: 5 a sole P variant, 3 an RBM20–VUS associated to a second VUS in another CMP related gene, one an RBM20–VUS associated to a LP variant in BAG3; the last patient was excluded (figure with 8 pts). Results Most pts (75%) had a positive family history (FH) for sudden death. Mean age at diagnosis was significantly lower for P variant carriers compared to VUS carriers (25±15 vs 46±2 years, p=0.04); no other significant differences were found between the 2 groups. At diagnosis, 4/8 pts (50%) had NYHA class&amp;gt;2, mean LVEF was 34±18% and mean LV end diastolic volume 83±14 ml/m2; most pts (6/8, 75%) presented with a dilated phenotype, one with a non–compacted and one with a hypertrophic phenotype. Cardiac magnetic resonance, available in 6 pts, showed non–ischemic fibrosis in 3 (50%). At presentation, two pts had non–sustained VT (NSVT), one atrial flutter, one first degree AV block. The median follow–up was 2 years (IQR 2–10). Four pts (50%) underwent ICD implantation at 40 ± 13 years old. One female patient underwent heart transplant at the age of 13 and no one died from end stage HF; 3 pts had NSVT, one patient had atrial fibrillation (successfully treated with ablation), and none developed advanced AV blocks. Conclusions This is the first Italian case series of RMB20 variants related CMP. In our case series, RBM20 variants are associated with an early onset cardiac expression both in male and female pts, with LV dilated CMP and heart failure being the most common presentation. None of the pts presented with or developed advanced AV blocks, most suffered from NSVT.

P88 CARDIAC INVOLVEMENT IN FOUR NOVEL NESPRIN–1 GENETIC VARIANTS

Abstract Background Nesprins are proteins encoded by synaptic nuclear envelope (SYNE)–1 and –2 genes, that concur, together with lamin A/C and emerin, to nuclear envelope function. Almost 140 disease–related variants were identified, mostly resulting in central nervous system and skeletal muscle disorders; only a few were associated with cardiomyopathy, mostly with relatively late onset and no specific arrhythmic profile. Purpose To describe cardiac involvement in 4 novel SYNE1 variants. Methods 189 patients underwent genetic analysis in our center (2019–2022) for suspected genetic cardiomyopathy (TruSight Cardio panel, Illumina); 72% had a positive genetic report, including pathogenic/likely pathogenic or variants of undetermined significance (VUS). Among them, 4 (2%) males presented with a sole SYNE1 heterozygous VUS (figure). Results None had muscle or neurological involvement. Patients 1 and 2 had negative family history (FH) and underwent pacemaker implantation under 50 years old for high–grade atrioventricular block (AVB). Transthoracic echocardiography (TTE) and cardiac magnetic resonance (CMR) showed a mildly impaired left ventricular ejection fraction (LVEF) without oedema or fibrosis. During 3 and 6 years of follow–up (FU), several non–sustained episodes of fast ventricular tachycardia (VT) and of atrial tachycardia occurred. Patient 3 is a 55–year–old man with negative FH and a left bundle branch block (LBBB) known since the age of 45.In 2021, while playing football, he suffered a cardiac arrest due to ventricular fibrillation treated with two DC shocks; ECG showed first–degree AVB and LBBB; TTE and CMR revealed a mildly impaired LVEF without oedema or fibrosis. A subcutaneous ICD was implanted, which recorded 3 atrial fibrillation episodes in a 1–year FU. Patient 4 is a 49–year–old man, with FH of sudden death (mother’s brothers at 40 and 50 years) and of polycystic kidney, who underwent renal transplant. He had left anterior fascicular block and suffered 2 episodes of sustained monomorphic VT from the right ventricle treated with ablation. TTE and CMR showed mild LV concentric hypertrophy with normal LVEF, segmental abnormalities in right and LV strain, no fibrosis. Conclusions SYNE1 genetic variants were found to be associated with a complex cardiac phenotype, including early onset conduction disease, atrial and ventricular arrhythmias, and cardiomyopathy. Further studies are needed to determine the functional significance of the variants.

Germline genetic variants in pheochromocytoma/paraganglioma: single-center experience.

Pheochromocytoma and paragangliomas (PPGLs) are rare neuroendocrine tumors carrying 25-40% pathogenic germline gene variants (PGVs). We evaluated clinical, laboratory, and germline molecular profile of 115 patients with pathologic (14 patients were relatives from 8 different families recruited for genetic survey) confirmed PPGL followed in our institution. Patients with classic MEN2A/MEN2B phenotypes and at-risk relatives underwent direct analysis of RET proto-oncogene, and the remaining had samples submitted to complete next-generation sequencing aiming 23 PPGL-related genes: ATM, ATR, CDKN2A, EGLN1, FH, HRAS, KIF1B, KMT2D, MAX, MDH2, MERTK, MET, NF1, PIK3CA, RET, SDHA, SDHAF2, SDHB, SDHC, SDHD, TMEM127, TP53, and VHL. We also developed a clinical judgment score (CJS) to determine the probability of patients having a potentially hereditary disease. The resulting genetic landscape showed that 67 patients (58.3%) had variants in at least one gene: 34 (50.7%) had exclusively pathogenic or likely pathogenic variants, 13 (19.4%) had pathogenic or likely pathogenic variants and variant of undetermined significance (VUS), and 20 (29.8%) carried only VUS. PGVs were found in RET (n = 18; 38.3%), VHL (n = 10; 21.3%), SDHB and NF1 (n = 8; 17% each), and MAX, SDHD, TMEM127, and TP53 (n = 1; 2.1% each). Direct genetic testing disclosed 91.3% sensitivity, 81.2% specificity, and 76.4% and 93.3% positive predictive value (PPV) and negative predictive values (NPV), respectively. The CJS to identify patients who would not benefit from genetic testing had 75% sensitivity, 96.4% specificity, and 60% and 98.2% PPV and NPV, respectively. In summary, the landscape of PPGL germline gene variants from 115 Brazilian patients resulted in slightly higher prevalent pathogenic and likely pathogenic variants, especially in the RET gene. We suggest a CJS to identify PPGL patients who would not require initial genetic evaluation, improving test specificity and reducing costs.

A Rare Case of Hereditary Spastic Paraplegia 46 With Novel Compound Heterozygous GBA2 gene variants (P3-8.013)

<h3>Objective:</h3> To present a case of Hereditary Spastic Paraplegia (HSP) 46 with distinct imaging and clinical findings, with two novel variants in <i>GBA2</i>, previously classified as variants of undetermined significance (VUS). <h3>Background:</h3> HSP is an uncommon cause of lower extremity spasticity and weakness. There have been many genetic etiologies identified with various clinical features. HSP 46 is an autosomal recessive, slowly progressive form of HSP caused by biallelic pathogenic variants in <i>GBA2</i>. <h3>Design/Methods:</h3> NA (Case report) <h3>Results:</h3> A 21-year-old man presented with progressive lower extremity weakness and ambulatory dysfunction. Symptom onset was in second grade, when he was found to have difficulty walking and inversion of the right foot. His ambulatory dysfunction continued to progress. Extensive serologic testing was negative, as was MRI of his spinal cord. Brain MRI demonstrated thinning of the corpus collosum. Examination approximately 12 years after his initial presentation was notable for diffuse hyperreflexia, bilateral lower extremity spasticity, and bilateral Hoffmann and Babinski signs. He had excessive lower extremity circumduction in swing phase, hyperextension at the knees, and internal rotation at the hips; consistent with a scissoring gait. There were no cranial neuropathies, sensory deficits, or cerebellar ataxia. The family history was negative. Genetic testing via whole-exome sequencing demonstrated compound heterozygous <i>GBA2</i> variants, both classified as VUS: c.1820 A&gt;G (p.N607S) and c.472 G&gt;A (p.G158R). <h3>Conclusions:</h3> Considering the distinctive clinical features, MRI finding of dysgenesis of the corpus collosum, and absence of an alternative explanation, we propose that the GBA2 variants found in this patient may be causative. Functional studies and/or messenger ribonucleic acid sequencing may confirm our hypothesis. <b>Disclosure:</b> Dr. Gill has nothing to disclose. Dr. Gill has nothing to disclose. Madeline Williamson has nothing to disclose. Dr. Avila has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Alnylam Pharmaceuticals. Dr. Avila has received personal compensation in the range of $10,000-$49,999 for serving on a Speakers Bureau for Alnylam Pharmaceuticals. Dr. Avila has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Akcea Therapeutics.

Germline Mutations Landscape in a Cohort of the State of Minas Gerais, Brazil, in Patients Who Underwent Genetic Counseling for Gynecological and Breast Cancer.

The present study evaluated the profile of germline mutations present in patients who underwent genetic counseling for risk assessment for breast cancer (BC), ovarian cancer (OC), and endometrial cancer (EC) with a possible hereditary pattern. Medical records of 382 patients who underwent genetic counseling after signing an informed consent form were analyzed. A total of 55.76% of patients (213/382) were symptomatic (personal history of cancer), and 44.24% (169/382) were asymptomatic (absence of the disease). The variables analyzed were age, sex, place of birth, personal or family history of BC, OC, EC, as well as other types of cancer associated with hereditary syndromes. The Human Genome Variation Society (HGVS) nomenclature guidelines were used to name the variants, and their biological significance was determined by comparing 11 databases. We identified 53 distinct mutations: 29 pathogenic variants, 13 variants of undetermined significance (VUS), and 11 benign. The most frequent mutations were BRCA1 c.470_471delCT, BRCA1 c.4675 + 1G > T, and BRCA2 c.2T> G. Furthermore, 21 variants appear to have been described for the first time in Brazil. In addition to BRCA1/2 mutations, variants in other genes related to hereditary syndromes that predispose to gynecological cancers were found. This study allowed a deeper understanding of the main mutations identified in families in the state of Minas Gerais and demonstrates the need to assess the family history of non-gynecological cancer for risk assessment of BC, OC, and EC. Moreover, it is an effort that contributes to population studies to evaluate the cancer risk mutation profile in Brazil.