Premature Translational Stop Research Articles

A LINE-1 mediated deletion resulting in germline retinoblastoma predisposition.

Retinoblastoma is an ocular cancer associated with genomic variation in the RB1 gene. In individuals with bilateral retinoblastoma, a germline variant in RB1 is identified in virtually all cases. We describe herein an individual with bilateral retinoblastoma for whom multiple clinical lab assays performed by outside commercial laboratories failed to identify a germline RB1 variant. Paired tumor/normal exome sequencing, long-read whole genome sequencing, and long-read isoform sequencing was performed on a translational research basisultimately identified a germline likely de novo Long Interspersed Nuclear Element (LINE)-1 mediated deletion resulting in a premature stop of translation of RB1 as the underlying genetic cause of retinoblastoma in this individual. Based on these research findings, the LINE-1 mediated deletion was confirmed via Sanger sequencing in our clinical laboratory, and results were reported in thepatient'smedical record to allow for appropriate genetic counseling.

Abstract 14463: Beyond Sarcomere Mutations Plakophilin-2 and Familial Hypertrophic Cardiomyopathy

Background: Hypertrophic cardiomyopathy (HCM) is a genetically heterogeneous disorder with diverse clinical presentation, course and outcomes. It is commonly caused by mutations in sarcomeric genes. Non-sarcomeric gene mutations have been associated with HCM in certain metabolic and storage diseases. Pathogenic mutations in plakophilin-2 (PKP2) gene have been associated with enhanced desmosomal protein degradation and linked to arrhythmogenic right ventricular cardiomyopathy (ARVC), Brugada syndrome and dilated cardiomyopathy. We report an association between PKP2 gene mutation and familial HCM. Case presentation: A 65-year-old man with family history of HCM (maternal grandfather, 2 aunts and an uncle) presented with dyspnea on exertion (NYHA class II-III) and pedal edema. Echo showed severe LVH, mid-cavitary obstruction and LV apical aneurysm. Cardiac MRI confirmed findings (Figure:A and B) and additionally showed significant late gadolinium enhancement (Figure:C and D, >20% of LV myocardium). Extended ambulatory heart monitor detected frequent runs on non-sustained ventricular tachycardia. A primary prevention ICD was implanted. Genetic testing identified a pathogenic mutation [c.1237C>T (p.Arg413*)] in exon 5 of PKP2 gene resulting in premature translational stop signal and disrupted desmosomal protein plakophilin-2. Family screening (Figure:E) identified a sister with positive results who also had non-obstructive HCM (Figure:E). Discussion: We present a family with typical presentations of HCM and no phenotypic evidence of ARVC. This novel observation provides evidence for pleiotropic model of complex genotype-phenotype interactions through nonlinear networks. Further evidence would be needed to understand potential molecular basis of hypertrophic signaling in PKP2 gene mutation.

SAT509 The Clinical Significance of Cribriform Morular Thyroid Carcinoma

Abstract Disclosure: G. Soh: None. K. Sek: None. S. Yang: None. M. Nga: None. S. Mok: None. We present two young (aged below 30) female patients who were diagnosed with the rare cribriform morular thyroid carcinoma. Both patients presented with neck swellings and underwent total thyroidectomy for the thyroid malignancies. The histological diagnosis prompted subsequent genetic evaluation. Both patients demonstrated genotypic and phenotypic evidence of familial adenomatosis polyposis (FAP) syndrome. One of the patients had a mutation which resulted in a premature translational stop signal in the adenomatous polyposis coli (APC) gene. She was eventually diagnosed with adenocarcinoma in 2 colonic polyps and underwent a curative pan-proctocolectomy. The other patient was diagnosed clinically with FAP as she had multiple (30-40) colonic polyps which were tubular adenomas. She had a variant of unknown significance in the APC gene. She declined surgery and remained on colonoscopic surveillance. Both patients’ thyroid cancers are in clinical remission. While uncommon, diagnosing cribriform-morular thyroid carcinoma is significant as 25 to 30% of patients harbour genetic mutations in the APC gene on chromosome 5q21. This is associated with FAP syndrome, an autosomal dominant genetic syndrome characterized by young-onset colonic polyposis, usually of malignant potential. Additionally, these patients may exhibit features of Gardner’s syndrome, a subset of FAP syndrome in which patients can develop extra-colonic manifestations such as osteomas of the mandible and supernumerary teeth. Histologically, cribriform morular thyroid carcinoma are well circumscribed and show a variable combination of follicular, cribriform, papillary, trabecular, solid and morular patterns. There is nuclear and cytoplasmic staining for beta catenin, as the inactivating APC mutation causes a loss of beta catenin destruction complex, impeding beta catenin degradation and increasing its nuclear translocation. This is a key event in thyroid tumorigenesis. The diagnosis of cribriform morular thyroid carcinoma should alert the clinician about the possibility of FAP with consideration for further clinical and genetic evaluation to confirm the diagnosis. References1)Cameselle-Teijeiro, J. M., Peteiro-González, D., Caneiro-Gómez, J., Sánchez-Ares, M., Abdulkader, I., Eloy, C., Melo, M., Amendoeira, I., Soares, P., & Sobrinho-Simões, M. (2018). Cribriform-morular variant of thyroid carcinoma: A neoplasm with distinctive phenotype associated with the activation of the Wnt/β-catenin pathway. Modern Pathology, 31(8), 1168–1179. https://doi.org/10.1038/s41379-018-0070-2 2)Peiling Yang, S., & Ngeow, J. (2016). Familial non-medullary thyroid cancer: Unraveling the Genetic Maze. Endocrine-Related Cancer, 23(12). https://doi.org/10.1530/erc-16-0067 Presentation Date: Saturday, June 17, 2023

THU506 A Not-So-Simple Case Of Hyperparathyroidism

Abstract Disclosure: P.J. Bhatt: None. S. Rabiei: None. V. Kantorovich: None. Multiple endocrine neoplasia 1 (MEN-1) is a rare autosomal dominant disorder caused by an inactivating mutation of the MEN-1 tumor suppressor gene and is associated with tumors of the parathyroid (95%), endocrine pancreas (40-70%) and anterior pituitary (30-40%). Primary hyperparathyroidism is the most frequent and earliest expression of MEN1 syndrome with a typical onset at age 20-25 in inherited cases, reaching 100% penetrance after age 50. Current guidelines recommend genetic testing in all individuals with primary hyperparathyroidism diagnosed at a young age, with multiglandular disease, or with family history of primary hyperparathyroidism. A 58 year old female with a history of L. breast cancer, pulmonary embolisms, DM2, and recurrent nephrolithiasis was diagnosed with primary hyperparathyroidism at age 36 and had a parathyroidectomy with 3.5 gland removal and later a total thyroidectomy. She continued to be symptomatic and was found to have an ectopic parathyroid gland in her chest requiring parathyroid gland reimplantation 18 years later. After 3 years, she was admitted for a CHF exacerbation and a pancreatic tail lesion was incidentally discovered. There was no mention of pancreatic lesions prior to this reading. Family history was pertinent for a father with nephrolithiasis. Her daughter had recurrent nephrolithiasis, hyperparathyroidism and was found to have a MEN 1 variant of uncertain significance, prompting the patient to seek genetic counseling. 22 years after her initial diagnosis of hyperparathyroidism, she tested positive for a likely pathologic heterozygous variant mutation of the MEN1 gene called 1747C>T. At endocrinology follow-up, she was asymptomatic. Labs showed elevated Chromogranin A at 1,799 ng/ dL [<311] and gastrin 174pg/mL [</=100]. FSH, LH, prolactin, free alpha subunit, cortisol, and IGF-1 were normal. Further imaging confirmed a DOTATATE-avid 1.1cm nodular lesion in the pancreatic tail, concerning for a neuroendocrine tumor. MEN1 gene mutation is associated with MEN1 syndrome, familial isolated hyperparathyroidism and has an autosomal dominant predisposition to thyroid cancer and paraganglioma-pheochromocytoma syndrome. The 1747C>T sequence change creates a premature translational stop signal in the MEN1 gene and disrupts the NLS2 domain of the MEN1 protein, which is important for DNA binding and repression of cell proliferation. Upon diagnosis of multiglandular hyperparathyroidism, the patient should have been sent for genetic testing and instead, became an example of a multisystem failure to identify and provide necessary testing and treatment. Until her variant diagnosis, her pancreatic tumor was not identified as such. The main cause of mortality in MEN-1 patients is malignant spread of neuroendocrine tumors, hence early detection and prompt treatment are of the utmost importance to preserve quality of life and improve survival. Presentation: Thursday, June 15, 2023

Identification of a novel CNV at the APC gene in a Chinese family with familial adenomatous polyposis.

Introduction: Familial adenomatous polyposis (FAP) is the second most commonly inherited colorectal cancer (CRC) predisposition caused by germline mutations within the adenomatous polyposis coli (APC) gene. The molecular defects and clinical manifestations of two FAP families were analyzed, and individual prevention strategies suitable for mutation carriers in different families were proposed. Methods and results: The pathogenic gene mutations were identified among the two families using whole-exome sequencing and verified with Sanger sequencing or quantitative polymerase chain reaction (qPCR). One novel (GRCh37:Chr5: 112145676-112174368, del, 28,692bp) and a known (c.C847T:p.R283X) mutation in the APC gene were pathogenic mutations for FAP, according to the sequencing data and tumorigenesis pattern among the family members. The two mutations led to a premature translational stop signal, synthesizing an absent or disrupted protein product. Conclusion: Our findings expand the known germline mutation spectrum of the APC gene among the Chinese population. This reaffirms the importance of genetic testing in FAP. Genetic consultation and regular follow-ups are necessary for the individualized treatment of cancer-afflicted families with APC expression deficiency. Additional work is required to develop safe and effective chemotherapy and immunotherapy for FAP based on the mutation type.

Novel pathogenic variant in LMNA gene identified in a six-generation family causing atrial cardiomyopathy and associated right atrial conduction arrhythmias.

To characterize the cardiac phenotype associated with the novel pathogenic variant (c.1526del) of LMNA gene, which we identified in a large, six-generation family. A family tree was constructed. The clinical data of living and deceased family members were collected. DNA samples from 7 family members were analyzed for LMNA mutations using whole-exome high-throughput sequencing technology. The clinical presentation of pathogenic variant carriers was evaluated. In this six-generation family (n = 67), one member experienced sudden death at the age of 40-years-old. Three pathogenic variant carriers were identified to possess a novel heterozygous deletion mutation in LMNA gene (HGVS: NM_170707.4, c.1526del) located at exon 9 of LMNA chr1:156137145, which creates a premature translational stop signal (p.Pro509Leufs*39) in the LMNA gene and results in an mutant lamin A protein product. The main symptoms of the pathogenic variant carriers were palpitation, fatigue, and syncope, which typically occurred around 20-years-old. AV-conduction block and non-sustained ventricular tachycardia were the first signs of disease and would rapidly progress to atrial standstill around 30-years-old. Significant right atrial enlargement and bicuspid aortic valve malformation was also commonly seen in patients who carried this pathogenic variant. The pathogenic variant of c.1526del p.P509Lfs*39 was a frameshift deletion located at exon 9 of LMNA chr1:156137145 and causes severe right atrial enlargement, sick sinus syndrome, atrial standstill, ventricular tachycardia, and bicuspid aortic valve malformation. Our findings expand the phenotypic spectrum of novel LMNA gene mutations.

Normal LRBA expression in a patient with LRBA deficiency: Understanding the clinical test

An 11-year-old girl presented as a new immunology patient with a history of celiac disease, inflammatory bowel disease, asthma, chronic bilateral otitis, autoimmune hemolytic anemia, and chronic splenomegaly. In addition, she had generalized lymphadenopathies and chronic lung disease (bronchiectasis, ground glass infiltrates, and nodular opacities). Relevant laboratory findings included anemia, neutropenia, elevated ESR (62), positive ANAs (1:320), poor antibody responses and hypogammaglobulinemia, slightly elevated αβ+double negative T-cells (1.6%) and serum soluble CD25 levels (9560 pg/mL). Parents are consanguineous and genetic testing reveals a homozygous pathogenic variant in LRBA (c.5980C>T). This is a novel defect predicted to cause a premature translational stop signal (p. Arg1994*), resulting in an absent or disrupted protein. The patient responded favorably to abatacept and subcutaneous IgG replacement with resolution of lymphadenopathies, organomegaly and cytopenias, improvement of infections and gastrointestinal symptoms, and decrease in inflammatory markers and serum soluble CD25 levels. Unexpectedly, during her evaluation, flow cytometry to assess intracellular LRBA using a polyclonal rabbit antibody (Sigma-Aldrich) showed protein expression in 100% of T-cells and 99% of B-cells. Since the patient’s clinical and genetic findings and her response to abatacept indicated LRBA deficiency, we reassessed LRBA expression using a different assay with an anti-LRBA monoclonal antibody recognizing the protein N-terminal (Clone 2D4.1, Milipore). This assay showed essentially absent expression of LRBA in T-cells (1.7% in CD4+ and 11.5% in CD8+), B-cells (3.6%), and NK-cells (0.8%), fitting with both our genetic findings and clinical phenotype. Hence, assays detecting LRBA expression may have discrepant results and understanding the technicalities of how they are performed is critical for their interpretation and avoiding potential pitfalls. For instance, it is possible that epitopes targeted by a polyclonal antibody could still be identified on a mutated LRBA protein, yielding normal protein phenotyping levels in LRBA deficient patients. Presence of protein also does not equate to functional protein but a validated functional clinical test for LRBA is yet unavailable. In conclusion, this case underscores the importance of using genetic and functional data in the context of the clinical phenotype for IEI diagnosis.

A PATIENT WITH KABUKI SYNDROME AND HYPOGAMMAGLOBULINEMIA DEVELOPS BURKITT LYMPHOMA

<h3>Introduction</h3> Kabuki syndrome (KS) is a rare genetic disorder characterized by congenital abnormalities, autoimmunity, intellectual disability, distinctive facial abnormalities, and hypogammaglobulinemia. <h3>Case Description</h3> A 24-year-old male with KS and hypogammaglobulinemia presented to the emergency department (ED) with lower back pain and extremity paralysis. His previous genetic diagnosis of KS had a heterozygous novel variant causing a premature translational stop signal p.Pro235Alafs*6 in the KMT2D gene. The patient also has a history of congenital heart disease, autoimmune cytopenias, and longstanding hypogammaglobulinemia on/off replacement immunoglobulin with baseline IgG 300-400 mg/dL, low IgA 10-20 mg/dL, normal IgM 50-60 mg/dL, normal lymphocyte subsets, and absent protein and polysaccharide vaccine responses consistent with common variable immunodeficiency. On arrival at the ED, spinal imaging revealed a compression fracture and underlying mass. Right-axillary and bone marrow biopsies confirmed Burkitt lymphoma. He was treated with chemotherapy, radiation, and replacement immunoglobulin. He has suffered from pneumonia, adenovirus, ileus<b>,</b> heart failure, and stroke. Routine immunologic testing performed at time of diagnosis revealed normal complete blood cell count, mild transaminitis from baseline (AST/ALT 61/71 U/L), baseline immunoglobulins, and normal lymphocyte subsets. <h3>Discussion</h3> While KS is a well-known cause of hypogammaglobulinemia, the authors have identified only two other published cases of Burkitt lymphoma in KS. This case emphasizes the importance of oncological screening and that routine immunologic testing of lymphocyte subsets and immunoglobulins may not have abnormalities from baseline in patients with active lymphoma. A mild transaminitis, however, might be concerning for malignancy in these patients.

Cardiac Screening - Soccer

HISTORY: 18 year old male collegiate soccer player presented for routine physical for varsity soccer team. Unremarkable past medical history aside from an adenectomy as a child. Negative sickle cell disease screening and up to date on vaccinations. Screening EKG notable for a prolonged QT/QTc 520/510ms. He denied any cardiac symptoms, but noted a syncopal event at age 16, when he went from sitting to standing, associated with blurry vision and dizziness. At that time, he had quick return of consciousness and did not seek medical attention. Denied use use of any performance-enhancing drugs PHYSICAL EXAMINATION: General: athletic young man HEENT: midline trachea, no thyroid enlargement Respiratory: lungs clear to auscultation and percussion Cardiovascular: sinus bradycardia, no murmurs Abdomen: non-tender without organomegaly Extremities: no edema DIFFERENTIAL DIAGNOSIS: Congenital LQTS; Acquired LQTS; Bradyarrhythmia (AV block) TEST AND RESULTS: BMP and TSH normal; Repeat EKG significant for bradycardia, uncorrected QT 520ms, broad-based T wave. Echocardiogram with ejection fraction 62%, structurally normal. Wore a Zio patch: range of 33bpm-176bpm, average 61bpm. Predominantly sinus. Underwent stress testing, reached 81.2% predicted MHR, BP 178/75, no chest discomfort, normal ST segment response to stress. Referred to Genetic Counsellor for Diagnostic Testing. The pathogenic variant, c.1552CT (p.Arg518*), was identified in KCNQ1 (AD/AR arrhythmia conditions), as well as variants of uncertain significance in MYH6 and RBM20. FINAL WORKING DIAGNOSIS: Congenital long QT syndrome (LQTS) due to heterozygous mutation at KCNQ1, Exon 12, c.1552C >T (p.Arg518*). This pathogenic mutation causes a premature translational stop signal at codon 518 of KCNQ1 gene, this is known to cause LQTS. Heterozygous carriers with typically mild clinical manifestions. TREATMENT AND OUTCOMES: The athlete was started on nadolol 40mg tablet once daily, then uptitrated to 80mg shortly thereafter. A repeat EKG showed sinus bradycardia and a shorter QT 480ms. Repeat stress testing notable for a blunted heart rate response (as expected on a beta-blocker), QT of 380ms with no QT prolongation during maximal exercise. The athlete’s family purchased a defibrillator for home use, and he has been cleared to return to varsity athletics.

The pho1;2a'-m1.1 allele of Phosphate1 conditions misregulation of the phosphorus starvation response in maize (Zea mays ssp. mays L.).

Plant PHO1 proteins play a central role in the translocation and sensing of inorganic phosphate. The maize ( Zea mays ssp. mays ) genome encodes two co‐orthologs of the Arabidopsis PHO1 gene, designated ZmPho1;2a and ZmPho1;2b. Here, we report the characterization of the transposon footprint allele Zmpho1;2a′‐m1.1, which we refer to hereafter as pho1;2a. The pho1;2a allele is a stable derivative formed by excision of an Activator transposable element from the ZmPho1;2a gene. The pho1;2a allele contains an 8‐bp insertion at the point of transposon excision that disrupts the reading frame and is predicted to generate a premature translational stop. We show that the pho1;2a allele is linked to a dosage‐dependent reduction in Pho1;2a transcript accumulation and a mild reduction in seedling growth. Characterization of shoot and root transcriptomes under full nutrient, low nitrogen, low phosphorus, and combined low nitrogen and low phosphorus conditions identified 1100 differentially expressed genes between wild‐type plants and plants carrying the pho1;2a mutation. Of these 1100 genes, 966 were upregulated in plants carrying pho1;2a, indicating the wild‐type PHO1;2a to predominantly impact negative gene regulation. Gene set enrichment analysis of the pho1;2a‐misregulated genes revealed associations with phytohormone signaling and the phosphate starvation response. In roots, differential expression was broadly consistent across all nutrient conditions. In leaves, differential expression was largely specific to low phosphorus and combined low nitrogen and low phosphorus conditions. Of 276 genes upregulated in the leaves of pho1;2a mutants in the low phosphorus condition, 153 were themselves induced in wild‐type plants with respect to the full nutrient condition. Our observations suggest that Pho1;2a functions in the fine‐tuning of the transcriptional response to phosphate starvation through maintenance and/or sensing of plant phosphate status.

ASPP2κ Is Expressed In Human Colorectal Carcinoma And Promotes Chemotherapy Resistance And Tumorigenesis.

Alternative splicing is a common physiologic mechanism to generate numerous distinct gene products from one gene locus, which can result in unique gene products with differing important functional outcomes depending on cell context. Aberrant alternative splicing is a hallmark of cancer that can contribute to oncogenesis and aggressiveness of the disease as well as resistance to therapy. However, aberrant splicing might also result in novel targets for cancer therapy. ASPP2 is a haplo-insufficient tumor suppressor, that functions through both p53-dependent as well as p53-independent mechanisms to enhance cell death after stress. Interestingly, the common human tumor TP53 mutations result in a loss of the binding sites to ASPP2, leading to impaired induction of apoptosis. Vice versa, attenuation of ASPP2 has been described to be associated with high-risk disease, therapy failure and poor clinical outcome especially in tumors harboring the TP53 wildtype (WT) isoform. We have recently identified a novel, dominant-negative splicing variant of ASPP2, named ASPP2κ, with oncogenic potential. Exon-skipping results in a reading-frame shift with a premature translation stop, omitting most of the ASPP2 C-terminus - which harbors the p53-binding domain. Consequently, the ASPP2-p53 interaction is abrogated, which in part impacts on oncogenesis, aggressiveness of disease and response to therapy. Since ASPP2κ has been shown in hematologic malignancies to promote tumorigenesis, we further wished to determine if aberrant ASPP2κ expression plays a role in human solid tumors. In this report, we find that ASPP2κ is frequently expressed in human colorectal tumors (CRC). Using ASPP2κ overexpressing and interference CRC models, we demonstrate a functional role of ASPP2κ in contributing to oncogenesis and resistance to therapy in CRC by 1) enhancing proliferation, 2) promoting cell migration and, 3) conferring resistance to chemotherapy induced apoptosis. Our findings have far-reaching consequences for future diagnostic and therapeutic strategies for ASPP2κ expressing colorectal cancer patients and provide proof-of-principle to further explore ASPP2κ as potential predictive marker and target for therapy in clinical trials.

A homozygous nonsense mutation early in exon 5 of BRCA2 is associated with very severe Fanconi anemia

Fanconi anemia (FA) due to biallelic mutations in the BRCA2 gene is very rare and associated with an extremely high risk of early-onset of aggressive childhood malignancies, predominantly brain tumors, leukemia, and nephroblastoma. Here, we present a consanguineous family with three affected children of the D1 subtype of FA and describe the clinical consequences of the earliest known biallelic nonsense/stop-gain germ-line mutation in BRCA2, exon 5 c.469A>T, that leads to a premature stop of translation, p.Lys157*. The three patients were born with severe intrauterine growth restrictions and different degrees of congenital malformations. Altogether, they developed eight distinct malignancies and died within their first three years of life. Treatment with a reduced chemotherapy regimen was only performed in patient 2 for his first tumor, a nephroblastoma, which the patient tolerated well for eight months, until he developed myelodysplastic syndrome (MDS) and then acute myeloid leukemia (AML). Finally, the third patient experienced a hepatoblastoma, an unclassified brain tumor and MDS in parallel and died in her second year of life. Our report re-emphasizes the aggressiveness and fatality of the FA-D1 children with biallelic BRCA2 nonsense mutations, that are both located before exon 11, which contains binding domains for the RAD51 recombinase.

34P New pathogenic germline mutation in ATM gene in Khakass breast cancer patients

In Russia, more than 50,000 women are diagnosed with breast cancer every year. Russia is a multinational country - about 200 ethnic groups live on its territory. To date, there are a limited number of reports on inherited gene mutations associated with breast cancer among Khakass (Mongoloid indigenous people in Russia). The aim of this study was to assess the prevalence of mutations of breast cancer-associated genes in 27 Khakass women with breast cancer. Our study included 27 Khakass patients with breast cancer (range, 28 to 47 years). Genomic DNA isolated from blood samples was used to prepare libraries using a capture-based target enrichment kit, Hereditary Cancer Solution™ (SOPHiA GENETICS, Switzerland), covering 27 genes (ATM, APC, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, FAM175A, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PIK3CA, PMS2, PMS2CL, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53 and XRCC2). Next-generation sequencing (NGS) was performed using the Illumina NextSeq500 System (Illumina, USA). In our study, one pathogenic mutation was detected in ATM (rs780619951, NC_000011.10:g.108259022C>T) gene in two unrelated individuals with family history of cancer (prevalence of 7,4%, 2/27). As known, this sequence change creates a premature translational stop signal at codon 805 (p.Arg805*). It is also expected to result in an absent or disrupted protein product. This variant has been reported in individuals affected with ataxia-telangiectasia, as well as an individual with a personal or family history of breast or ovarian cancer. To the best of our knowledge, this report is the first to describe the pathogenic variant in the ATM gene (rs780619951) in two Khakass breast cancer patient with family history of cancer. The reported study was funded by RFBR according to research project 18-29-09046.

An intronic splice site alteration in combination with a large deletion affecting VPS13B (COH1) causes Cohen syndrome

Cohen syndrome (CS) is a rare, autosomal recessive disorder characterized by intellectual disability, postnatal microcephaly, facial abnormalities, abnormal truncal fat distribution, myopia, and pigmentary retinopathy. It is often considered an underdiagnosed condition, especially in children with developmental delay and intellectual disability. Here we report on four individuals from a large Jordanian family clinically diagnosed with CS. Using Trio Exome Sequencing (Trio-WES) and MLPA analyses we identified a maternally inherited novel intronic nucleotide substitution c.3446-23T>G leading to the activation of a cryptic splice site and a paternally inherited multi-exon deletion in VPS13B (previously termed COH1) in the index patient. Expression analysis showed a strong decrease of VPS13B mRNA levels and direct sequencing of cDNA confirmed splicing at a cryptic upstream splice acceptor site, resulting in the inclusion of 22 intronic bases. This extension results in a frameshift and a premature stop of translation (p.Gly1149Valfs*9). Segregation analysis revealed that three affected maternal cousins were homozygous for the intronic splice site variant. Our data show causality of both alterations and strongly suggest the expansion of the diagnostic strategy to search for intronic splice variants in molecularly unconfirmed patients affected by CS.

CHARGE syndrome associated with de novo (I1460Rfs*15) frameshift mutation of CHD7 gene in a patient with arteria lusoria and horseshoe kidney.

CHARGE syndrome is an autosomal dominant condition caused by mutations in the chromodomain helicase DNA binding protein 7 (CHD7) gene. The present study reported on the case of a 16-month-old female with plurimalformative syndrome, whose etiology was identified by clinical whole-exome sequencing (WES) analysis. Clinical and follow-up assessments identified multiple craniofacial dysmorphisms, congenital defects and functional symptoms, including dysphagia and Marcus Gunn jaw winking synkinesis. Trio-WES analysis was performed for the patient and their parents and the presence of CHARGE syndrome was further indicated using single-molecule real-time sequencing. A de novo pathogenic variant, c.4379_4380del (p.Ile1460Argfs*15), was identified in exon 19 of the CHD7 gene, which resulted in a premature translational stop signal. Trio-WES analysis was used for further investigation, indicating that neither of the patient's parents had the mutation and confirming its de novo nature. To the best of our knowledge, the case of the present study was the first reported case of CHARGE syndrome in Romania with congenital defects including an aberrant right subclavian artery and a horseshoe kidney. CHARGE syndrome was diagnosed in the patient based on the pathogenic mutation in the CHD7 gene. To the best of our knowledge, the present case report is the first to suggest that the CHD7 gene variant is associated with CHARGE syndrome.

Characterization of the Transcription Termination Factor Responsible for Polarity in Archaea

The absence of a nuclear compartment permits prokaryotic ribosomes to bind nascent RNAs to initiate translation before a transcript is completely synthesized. Transcription and translation are normally tightly coupled in prokaryotes (Archaea and Bacteria) and uncoupling translation from transcription serves as a regulatory signal, permitting premature transcription termination when translation fails or is inhibited. The resultant decrease in downstream gene expression, termed polarity, is a major regulatory pathway to ensure coordinated gene expression. While the importance and widespread use of polar repression is well documented, to date, the factor responsible for polarity in archaea cells has not been explicitly identified.In bacteria, the transcription termination factor rho is responsible for polarity, but there are no rho homologues encoded in archaeal genomes. FttA (Factor that terminates transcription in Archaea), a recently identified archaeal termination factor, appears to potentially fulfill the requirements to direct polarity in archaea, but other factors may be responsible. Our primary objective, employing an unbiased genetic selection, is to identify the transcription termination factor(s) responsible for polar repression in Archaea.We have established and will present our findings resultant from a three‐tiered genetic selection for spontaneous mutations that impact the efficiency of polar repression in archaeal cells. The selections were carried out in specialized strains of Thermococcus kodakarensis, a marine, hyperthermophilic archaeon with a facile genetic system and robust laboratory growth.Briefly, genomic modifications were introduced to place a non‐essential gene with a premature translation stop codon directly upstream and within the same operon of a reporter or essential (selection) gene. In wide‐type strains, the early stop codon permits a polarity factor to recognize uncoupling of transcription and translation and terminate transcription before productive expression of the downstream essential gene. However, when the activity of a polarity factor(s) is hindered, these selective genes can be expressed at high levels. For selection, the final genes of the tryptophan and histidine synthesis pathway were chosen, constructing auxotrophic mutant strains which can only develop in the absence of tryptophan and histidine if there is some disruption in polarity‐ allowing them to become prototrophic. The spontaneous mutations resultant in reduced polar repression are easily identified through whole‐genome sequencing and we present evidence for roles of multiple factors in mediating polarity in archaeal cells.

Lack of PDZD7 long isoform disrupts ankle-link complex and causes hearing loss in mice.

Usher syndrome (USH) is the most frequent form of combined hereditary deafness-blindness, characterized by hearing loss and retinitis pigmentosa, with or without vestibular dysfunction. PDZD7 is a PDZ domain-containing scaffold protein that was suggested to be a USH modifier and a contributor to digenic USH. In the inner ear hair cells, PDZD7 localizes at the ankle region of the stereocilia and constitutes the so-called ankle-link complex together with three other USH proteins Usherin, WHRN, and ADGRV1. PDZD7 gene is subjected to alternative splicing, which gives rise to two types of PDZD7 isoforms, namely the long and short isoforms. At present, little is known which specific isoform is involved in ankle-link formation and stereocilia development. In this work, we showed that PDZD7 long isoform, but not short isoforms, localizes at the ankle region of the stereocilia. Moreover, we established Pdzd7 mutant mice by introducing deletions into exon 14 of the Pdzd7 gene, which causes potential premature translational stop in the long isoform but leaves short isoforms unaffected. We found that lack of PDZD7 long isoform affects the localization of other ankle-link complex components in the stereocilia. Consequently, Pdzd7 mutant mice showed stereocilia development deficits and hearing loss as well as reduced mechanotransduction (MET) currents, suggesting that PDZD7 long isoform is indispensable for hair cells. Furthermore, by performing yeast two-hybrid screening, we identified a PDZD7 long isoform-specific binding partner PIP5K1C, which has been shown to play important roles in hearing and might participate in the function and/or transportation of PDZD7.

EP.23A novel LMNA mutation identified in an Argentinian patient with autosomal dominant Emery-Dreifuss muscular dystrophy phenotype

LMNA mutations result in a broad array of diseases, named laminopathies. The Emery-Dreifuss muscular dystrophy and the dilated cardiomyopathy with conduction defects phenotypes, result primarily from heterozygous missense mutations in the LMNA gene (often in a dominant negative fashion), whilst truncating mutations resulting in premature termination codon are less common, but also described. We report a 38-year-old man with slowly progressive weakness and wasting in an humeroperoneal distribution beginning in early childhood, accompanied by elbow and achilles tendon contractures. At the age of 37, an implantable cardioverter defibrillator was required as he developed severe cardiac conduction defects. Incipient structural myocardial changes were also evident in the cardiac nuclear magnetic resonance. His family history reveals the existence of a twin sister with similar weakness pattern and contractures, without conduction or structural cardiac abnormalities. Additionally, both his father and paternal uncle suffered from dilated cardiomyopathy, without muscular weakness, and passed away from sudden death at 45 and 67 years of age respectively. Suspecting a Laminopathy a target next generation panel for cardiomyopathy and skeletal muscle disease was performed. A novel pathogenic variant c.210del (p.Ser71Alafs*25), at heterozygous state, was identified in LMNA. This sequence change creates a premature translational stop signal in the LMNA gene. It is expected to result in an absent or disrupted protein product. Loss-of-function variants in LMNA are known to be pathogenic. This variant is not present in population databases, and has not been reported in the literature in individuals with LMNA-related conditions. We present a 38-year-old patient with autosomal dominant Emery-Dreifuss muscular dystrophy phenotype, with strong family history, and a novel truncating variant identified in LMNA. Functional protein studies would contribute to confirm the pathogenicity of this variant.

Expanding the clinical history associated with syndromic Klippel-Feil: A unique case of comorbidity with medulloblastoma

Klippel-Feil syndrome (KFS) is an exceedingly rare constitutional disorder in which a paucity of knowledge exists about the disease and its associated morbidity and mortality. We present a 4-year-old male with KFS, who notably was also diagnosed with large-cell anaplastic medulloblastoma. We evaluated the genetic basis of co-occurring KFS and medulloblastoma and the role of MYO18B as related to medulloblastoma. Constitutional and somatic variant and copy number analyses were performed from DNA-based exome studies, along with RNA-sequencing of tumor tissue, to elucidate the genetic etiology of the co-existing disease states. We identified novel constitutional compound heterozygous frameshift variants (NM_032608.5: p.Leu2257SerfsTer16 and p.Arg2220SerfsTer74) each encoding a premature stop of translation in MYO18B, consistent with a diagnosis of KFS. We did not identify any somatic variants of known relevance or disease-relevant therapeutic targets in the tumor. The somatic copy number profile was suggestive of Group 3γ medulloblastoma. Relative to pediatric brain tumors, medulloblastoma, particularly, Group 3, had increased gene expression of MYO18B. In summary, coexisting constitutional and somatic diagnoses in this patient enabled the elucidation of the genetic etiology of KFS and provided support for the role of MYO18B in tumor suppression.

406 An in vitro splicing assay reveals the pathogenicity of intronic variants in ABCC6, the gene at fault in pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused by inactivating mutations in the ABCC6 gene predominantly expressed in hepatocytes. Different types of mutations have been identified in ABCC6 including canonical splice site mutations affecting pre-mRNA splicing. Splicing variants in ABCC6 are often overlooked due to the unavailability of liver biopsies from patients with such variants to determine their consequences on splicing by transcriptional analysis. Thus, the interpretation of intronic variants purely relies on computational algorithms to predict their effects on splicing. Very often the prediction programs do not provide a consensus as to the effect of variants for disrupting normal splicing. Our recent ABCC6 mutation analysis of over 400 patients with PXE shows that this continues to be a problem: among 7 intronic variants in ABCC6 with conflicting results by current splice site prediction algorithms, 3 variants were found to affect splicing. These three intronic variants were analyzed in an in vitro functional splicing assay using expression of minigene constructs in transfected HepG2 cells. Total RNA was extracted and RT-PCR was performed to analyze the splice pattern followed by Sanger sequencing. Two of the variants, c.3883-46A>G and c.2070+5G>A, caused skipping of the adjacent exon, predicted to cause out-of-frame translation and premature stop of translation. One variant, c.3883-6G>A, created a new splice acceptor site resulting in a 4-bp retention from the intronic region and causing frame-shift of translation. The results provided evidence that intronic variants away from the canonical splice sites disrupted splicing, clearly demonstrating the presence of mutations beyond the canonical splice sites.