Frameshift Mutation In Exon Research Articles

Novel mutations in the exon 5, intron 2 and 3′ UTR regions of IL-12B gene were observed in clinically proven tuberculosis patients of south India

Interleukin-12 (IL-12) is formed by the interaction of IL-12p35 and IL-12p40 expressed independently from IL-12A and IL-12B genes. This interleukin plays prominent role in the T-helper type-1 (Th1) response against intracellular pathogens. Variations in IL-12B gene causes disruption of various activities one of them is suppression of Th1 response and is one of the characteristic features observed in patients with active tuberculosis. Hence, in the present study IL-12B gene status was evaluated in 50 new sputum smear-positive pulmonary tuberculosis patients (NSP-PTB) as identified by Ziehl-Nielsen (ZN) staining and 50 apparently healthy control subjects (HCS) who were sputum smear-negative. The sequence analysis showed novel missense mutations p.Ser205Ile, p.Leu206Glu, p.Pro207Ser, p.Glu209Lys, p.Val210Ser, p.(Ser205_Cys327delinsIleGlu) and p.(Lys217_Leu218delinsIle) were found in exon 5 of the IL-12B gene in nine patients resulting formation of inactive IL-12 and three patients showed novel frame shift mutations p.(Asn222Leufs∗23) in exon 5 of causing the formation of truncated protein. Several mutations were noted in intron 2 of the IL-12B gene in 5 patients and in 13 patients mutations were observed in 3′ UTR region. All together 30/50 patients (60%) showed mutations in IL-12B gene. Decreased levels of interferon-gamma (IFN-γ) and IL-12 as determined by ELISA and flow cytometry were observed in the peripheral blood mononuclear cell culture supernatants in TB patients having mutations compared with control subjects. Further, in silico analysis revealed due to frame shift mutations in exon 5 at Asn222 resulted in deletion of functional fibronectin type-III (FN3) domain which leads to formation of inactive IL-12 in these patients.

Correction: Recessive coding and regulatory mutations in FBLIM1 underlie the pathogenesis of chronic recurrent multifocal osteomyelitis (CRMO)

[This corrects the article DOI: 10.1371/journal.pone.0169687.].

A novel, complex RUNX2 gene mutation causes cleidocranial dysplasia

BackgroundHaploinsufficiency of the runt-related transcription factor 2 (RUNX2) gene is known to cause cleidocranial dysplasia (CCD). Here, we investigated a complex, heterozygous RUNX2 gene mutation in a Chinese family with CCD and the pathogenesis associated with the variations.MethodsGenomic DNA extracted from peripheral venous blood was taken from the proband, her parents and 3 siblings, and 150 normal controls. Analysis of their respective RUNX2 gene sequences was performed by PCR amplification and Sanger sequencing. Pathogenesis associated with RUNX2 mutations was investigated by performing bioinformatics, real-time PCR, western blot analysis, and subcellular localization studies.ResultsWe identified 2 complex heterozygous mutations involving a c.398–399 insACAGCAGCAGCAGCA insertion and a c.411–412 insG frameshift mutation in exon 3 of the RUNX2 gene. The frameshift mutation changed the structure of the RUNX2 protein while did not affect its expression at the mRNA level. Transfection of HEK293T cells with a plasmid expressing the RUNX2 variant decreased the molecular weight of the variant RUNX2 protein, compared with that of the wild-type protein. Subcellular localization assays showed both nuclear and cytoplasmic localization for the mutant protein, while the wild-type protein localized to the nucleus.ConclusionsOur findings demonstrated that the novel c.398–399insACAGCAGCAGCAGCA mutation occurred alongside the c.411–412insG frameshift mutation, which resulted in RUNX2 truncation. RUNX2 haploinsufficiency was associated with CCD pathogenesis. These results extend the known mutational spectrum of the RUNX2 gene and suggest a functional role of the novel mutation in CCD pathogenesis.

Metastatic Pancreatic Gastrinoma, Severe Primary Hyperparathyroidism, And Adrenal Cushing Syndrome In A Patient With Multiple Endocrine Neoplasia Type 1: An Unusual Presentation And Review Of The Literature

ABSTRACT Objective: Multiple endocrine neoplasia type 1 (MEN1) has an estimated incidence in the general population of 0.01 to 2.5 cases per 1,000 individuals. Here we report on the case of a 36-year-old female patient with some uncommon aspects of MEN1. Methods: The patient was admitted with primary hyperparathyroidism (PHPT) and suspicion of parathyroid carcinoma, demanding additional exams. Abdominal resonance imaging revealed a left adrenal mass, three pancreatic tumors, and hepatic lesions. Diagnostic work-up revealed hypergastrinemia and autonomous production of corticosteroids. Results: The patient was subjected to multiple surgeries: subtotal parathyroidectomy, left adrenalectomy, subtotal pancreatectomy associated with enucleation of a tumor lesion in the pancreatic head, and resection of a hepatic metastasis. The patient's DNA sequencing analysis revealed a frameshift mutation in exon 3 of the MEN1 gene. Her daughter was found to be affected by the same mutation. Conclusion: This case presents t...

Genetic or CD40L-Mediated Loss of Iκbα Is Associated with Resistance to the Dual SYK/JAK Inhibitor Cerdulatinib in DLBCL Cell Lines

Abnormal upregulation of NFκB activity is observed in a variety of B cell malignancies, resulting in proliferative and survival signals that contribute to tumor progression. Under normal resting conditions, NFκB is negatively regulated principally via its physical association with IκB (inhibitor of NFκB) family members, thereby inhibiting nuclear transport or access to DNA. In B cells, NFκB is typically activated via various external stimuli (e.g., ligation of the B cell antigen receptor (BCR), toll-like receptors, cytokine receptors, CD40), leading to IκB kinase complex-dependent phosphorylation of IκB members and targeting them for ubiquitination and degradation. In some cases, the need for external stimuli is diminished or completely circumvented by mutations to critical regulators of NFκB, as has been described in the context of activating mutations to CD79A/B, MYD88, and CARD11, as well as inactivation of negative regulators such as A20 and IκB family members (reviewed by Staudt, 2010). Each of these mutations has been observed clinically in patients with B cell malignancies (Wilson et al, 2012; Norenberg et al, 2015; Mansouri et al, 2015), and can impact the anti-tumor activity of selective BCR pathway inhibition (Davis et al, 2010; Wilson et al, 2012) in part via induction of autocrine cytokine stimulation leading to JAK/STAT-dependent up-regulation of MCL1 (Lam et al, 2008).We previously reported that cerdulatinib, a small molecule kinase inhibitor that dually targets SYK and the JAK family members JAK1, JAK3, and TYK2, maintained anti-tumor activity in DLBCL cell lines bearing mutations to CARD11, MYD88, and A20 (Ma et al, 2015). The majority of DLBCL cell lines exhibit various degrees of reliance on SYK and JAK signaling for survival, however in a screen of 15 DLBCL cell lines we found 3 that were completely resistant to cerdulatinib and are described here. In one of the cerdulatinib-resistant cell lines, RCK8, next generation sequencing revealed bi-allelic inactivation of the IκBα gene. One allele carries a frameshift mutation in exon 1 resulting in the generation of a premature stop codon, and the second allele is a nonsense mutation in exon 3 at Gln154, also leading to a stop codon. In accord with a previous report (Kalaitzidis et al, 2002), the cell line lacks IκBα protein expression. We therefore proceeded to explore the possibility that loss of IκBα was responsible for resistance to cerdulatinib.Consistent with the loss of IκBα, the RCK8 cell line exhibited enhanced basal NFκB activity. Genetic re-introduction of wild type IκBα led to rapid suppression of NFκB, and ultimately cell cycle arrest and cell death, indicating that the cell line was dependent upon loss of this gene for survival. Associated with suppression of NFκB was decreased phosphorylation of cellular pAKT S473 and pERK Y202, but not of pSTAT3 Y705. We then attempted to knock down IκBα in cerdulatinib-sensitive cell lines using siRNA to determine if resistance to SYK/JAK inhibition could be generated. None of the DLBCL cell lines tested (n=4) could tolerate IκBα gene knock down, suggesting an additional mutation in RCK8 enables survival under conditions of homozygous loss of IκBα. Ligation of CD40 leads to a transient down-regulation of IκBα at the protein level (Oeckinghaus and Ghosh, 2009). We therefore examined the effect of CD40L on multiple DLBCL cell lines and found that IκBα was maximally suppressed within 30-60 minutes post CD40 stimulation, returning to pre-treatment levels by 2-4 hours. In contrast, the impact on NFκB activation was much longer, and 5 of 7 cerdulatinib-sensitive cell lines tested were made resistant by incubation with CD40L. Associated with this resistance was not only induction of NFκB, but also pERK Y204, pAKT S473, and pSTAT3 Y705. Interestingly, whereas the CD40L-induced NFκB activation was not inhibited by cerdulatinib, the other signaling events were, despite the generation of resistance.Loss of IkB family members has been described in the context of Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, and chronic lymphocytic leukemia (Cabannes et al, 1999; Norenberg et al, 2015; Mansouri et al, 2015). Here we demonstrate that loss of IκBα in multiple DLBCL cell lines generates resistance to cerdulatinib. We will be exploring the clinical relevance of these in vitro observations in cell lines as part of an ongoing phase II trial of cerdulatinib in patients with relapsed/refractory B cell malignancies. DisclosuresCoffey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Feng:Portola Pharmaceuticals: Employment, Equity Ownership, Research Funding. Wang:Portola Pharmaceuticals: Honoraria, Research Funding. Michelson:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Pandey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Curnutte:3-V Biosciences: Equity Ownership; Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding; Sea Lane Biotechnologies: Consultancy. Conley:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding.

FANCN Hypomorphic Mutation Retains BRCA1 Binding Domain

Introduction: Fanconi anemia (FA) is a recessive genetic disease that can result in bone marrow failure, morphological defects and cancer predisposition. Patients with mutations in the FA gene, FANCN/PALB2 (partner and localizer of BRCA2) typically have more severe disease with earlier onset of cancer and bone marrow failure. FANCN is known to promote homologous recombination (HR) and contains an N-terminal BRCA1 binding domain, and a C-terminal BRCA2 binding domain. To further investigate the function of FANCN, we used recombinant adeno-associated virus (rAAV)-mediated gene targeting techniques to make a conditional null FANCN cell line. We targeted early exons 2 and 3 and found that removal of the conditional allele resulted in spontaneous chromosomal breaks and rearrangements that ultimately lead to chromosomal catastrophe within 48 hours. Due to the essential function of FANCN in genome stability, we were curious how FANCN patients can survive with biallelic loss of function mutations in FANCN.Methods: A hypomorphic FANCN cell line was created using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated 9 (Cas9)-mediated gene targeting of FANCN in exon 5. In addition, we complemented the FANCN conditional null cell line with four different FANCN cDNAs containing patient mutations with premature stop codons that completely eliminate or disrupt the N-terminal BRCA2 binding domain. These FANCN mutant cell lines have been investigated for expression of truncated protein and will be tested in their ability to bind and form foci with BRCA1 and BRCA2. Finally, these FANCN mutant cell lines will be evaluated in their sensitivity to mitomycin C (MMC), PARP inhibitors, replication stress, and in their ability to repair DNA.Results: We have determined that FANCN is essential for maintaining genomic stability and cellular viability. However, contrary to the lethality of deleting exons 2 and 3, the biallelic frameshift mutations in exon 5 resulted in a viable FANCN mutant cell line. These hypomorphic FANCN exon 5 frameshift mutations truncate the C-terminal BRCA2 binding domain, and this cell line has a growth defect and MMC sensitivity. Furthermore, we have demonstrated that the patient mutation complemented cell lines are expressing truncated FANCN proteins that will be functionally characterized.Conclusions: We are beginning to unveil important and separate functional domains of FANCN. For example, the FANCN N-terminus with the BRCA1 binding domain serves an essential function in viability and genomic stability, whereas the FANCN C-terminus with the BRCA2 binding domain servers an essential role in cell proliferation and MMC resistance. This research is expected to continue to advance our mechanistic understanding of FANCN and how these FANCN patient mutations result in disease. In addition, these novel FANCN cell lines provide a means for screening potential new therapeutics for the treatment of Fanconi anemia. DisclosuresHendrickson:Horizon Discovery , Humanetics and Intellia Therapeutics: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties: licensing fees, Research Funding.

Novel ELN mutation in a family with supravalvular aortic stenosis and intracranial aneurysm

Pathogenic germline mutations in ELN can be detected in patients with supravalvular aortic stenosis. The mutation might occur de novo or be inherited following an autosomal dominant pattern of inheritance. In this report we describe a three-generation family suffering from supravalvular aortic stenosis, various other arterial stenoses, sudden death, and intracranial aneurysms. A frameshift mutation in exon 12, not described before, was detected in the affected family members. This report emphasises the importance of family history, genetic counselling, and demonstrates the great variability in the phenotype within a single SVAS family.

Abstract 19455: A Novel Caveolin-1 Frameshift Mutation Shows Critical Regulation of Mitochondrial Structure, Function and Extracellular Transport

Introduction: Caveolins act as molecular scaffolds for signal transduction. A number of caveolin-1 (Cav-1) mutations are associated with pulmonary hypertension with one recent frameshift mutation in exon 3 (c.478_479delTT) resulting in progeroid-like phenotype, lipodystrophy, and pulmonary hypertension. The cellular etiology of these diverse phenotypes is unknown. We have recently shown a critical relationship between caveolins in regulation of mitochondrial function. Hypothesis: As mitochondria are a key to cellular homeostasis, we hypothesize that a Cav-1 mutation (CM) can alter mitochondrial structure and function. Methods and Results: We obtained fibroblasts from a patient containing the CM as well as controls and utilized a variety of microscopy, cell biology, and biochemical techniques to study the role of Cav-1 in mitochondrial regulation. Transmission electron microscopy revealed dysmorphic nuclei, enlarged and increased number of endoplasmic reticulum, and increased number of smaller, swollen, and rounded mitochondria compared to normal cells. Mitochondrial DNA content was increased in CM cells with immunoblots showing decreased mitochondrial fission protein DRP1. Mitotracker staining showed large pools of aggregated mitochondria and DCFDA assay showed 3-fold increase in production of reactive oxygen species in CM cells compared to normal cells (p<0.0001, n=8). Seahorse metabolic flux assay showed lower maximal respiration rates in CM cells compared to normal cells (p=0.0035, n=24). Mitochondrial complex II activity was impaired as addition of succinate failed to evoke any response to ADP in CM cells compared to normal cells. Live 3D microscopy with mitotracker staining showed extracellular mitochondria being transported along structured nanotubes between normal cell; however, impaired extracellular mitochondrial cargo arrangement and movement was observed in cells with the CM. Rescue experiments with Cav-1 overexpression resulted in improved mitochondrial respiration in CM cells. Conclusion: Together the results indicate that Cav-1 may be a critical regulator of mitochondrial structure, function, and transport and mutations in Cav-1 may explain the mitochondrial etiology of a variety of diseases.

Detection ofCALRMutation in Clonal and Nonclonal Hematologic Diseases Using Fragment Analysis and Next-Generation Sequencing

To describe three methods used to screen for frameshift mutations in exon 9 of the CALR gene. Genomic DNA from 47 patients was extracted from peripheral blood and bone marrow using the EZ1 DNA Blood Kit (Qiagen, Valencia, CA) and quantified by the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, San Diego, CA). After clinical history, cytogenetics, and molecular tests, patients were diagnosed with either clonal or nonclonal hematologic diseases. CALR screening was primarily performed using fragment analysis polymerase chain reaction, then next-generation sequencing and Sanger sequencing. Among the 18 patients diagnosed with clonal diseases, one had acute myeloid leukemia (positive for trisomy 8), and 17 had myeloproliferative neoplasms (MPNs), including chronic myeloid leukemia (CML), essential thrombocythemia (ET), primary myelofibrosis (PMF), and polycythemia vera (PV). Patients with CML were positive for the BCR-ABL1 fusion. Ten patients were positive for JAK2 (PMF, n = 1; ET, n = 2; PV, n = 7), and three were CALR positive (ET, n = 1; PMF, n = 2). Patients diagnosed with a nonclonal disease were negative for JAK2, BCR-ABL, and CALR mutations. Screening for CALR mutations is essential in BCR-ABL-negative MPNs since it not only provides valuable diagnostic and prognostic information but also identifies potential treatment targets. Since this study describes the importance of screening for known and novel biomarkers, we described in detail three methods that could be easily integrated into a clinical laboratory.

F24 - Triple-negative breast cancer and BRCA mutation: looking at the future

Background: More than 75% of breast carcinomas that develop in BRCA mutation carriers (BRCA+) are triple-negative breast cancer (TNBC). Despite higher prevalence, it is controversial whether BRCA+ have lower survival. The aim of this study was to compare disease free survival (DFS) and overall survival (OS) in TNBC patients with and without deleterious BRCA1/2 mutations. Patients and methods: A total of 116 women affected by TNBC were selected to our Institution for genetic counseling and underwent BRCA genetic testing between 2002 and 2015; 13 patients with a BRCA variant of uncertain significance (VUS) were excluded from the statistical analysis. Associations between clinical features and outcomes were evaluated using univariate and multivariate Cox analysis. Results: Overall, 103 women were included, 55 (47%) were BRCA+ (BRCA1 n = 48, BRCA2 n = 7) and 48 cases wild-type (BRCA-). Recurrent mutations were: 2 frameshift mutations in exon 11 in 5 cases with 3901delT and 4 with 962del4 Stop 297 respectively. Six patients presented exon 5 frameshift mutation (300T > G) and 5 women presented large genomic rearrangement (Del 1 and 2). Median age was 42 years in BRCA+ and 48 years in BRCA-. The two patients groups were comparable for clinical features and prognostic factors. Median follow-up was 6.2 years (range 0.5-22.8), the 5-year RFS rates were 79% and 72% (P = 0.12) in BRCA+ and BRCA- and 5-year OS rates was 83% in both groups. No significant prognostic difference was evidenced in DFS (p = 0.54) and OS (p = 0.24). A better DFS was associated to early stage and absence of lymphovascular invasion; these findings were confirmed to multivariate analysis (p = 0.01, HR 3.02 CI 1.28-7.11 and p = 0.02, HR 3.01 CI 1.23-7.38). The OS was correlated only to early stage (p = 0.04, HR 0.33 CI 0.09-0.96). Conclusion: Given the 47% prevalence of deleterious BRCA1 mutations, referral for genetic testing should be considered in TNBC. The 4 detected deleterious BRCA1 mutations seem recurrent in our Country. TNBC prognosis is similar between BRCA+ and sporadic disease. These findings parallel previous literature data showing limited clinical impact of BRCA status as a whole in TNBC survival. Study limitation include small number of events which may have reduced statistical power. Further studies are required to find prognostic/predictive factors in order to guide therapy in TNBC BRCA positive and negative.

Novel FBN1 mutations are responsible for cardiovascular manifestations of Marfan syndrome.

The fibrillin-1 (FBN1) gene mutations result in Marfan syndrome (MFS) and have a variety of phenotypic variations. This disease is involved in the skeletal, ocular and cardiovascular system. Here we analyzed genotype-phenotype correlation in two Chinese families with MFS. Two patients with thoracic aortic aneurysms and dissections were diagnosed as MFS according to the revised Ghent criteria. Peripheral blood samples were collected and genomic DNAs were isolated from available cases, namely, patient-1 and his daughter and son, and patient-2 and his parents. According to the next-generation sequencing results, the mutations in FBN1 were confirmed by direct sequencing. A heterozygous frameshift mutation in exon 12 of FBN1 was found in the proband-1 and his daughter. They showed cardiovascular phenotype thoracic aortic aneurysms and dissections, a life-threatening vascular disease, and atrial septal defect respectively. One de novo missense mutation in exon 50 of FBN1 was identified only in the patient-2, showing aortic root aneurysm and aortic root dilatation. Intriguingly, two novel mutations mainly caused the cardiovascular complications in affected family members. No meaningful mutations were found in these two patients by screening all exons of 428 genes related with cardiovascular disease. The high incidence of cardiovascular manifestations might be associated with the two novel mutations in exon 12 and 50 of FBN1.

GCK mutations in Chinese MODY2 patients: a family pedigree report and review of Chinese literature.

Maturity-onset diabetes of the young type 2 (MODY2) is caused by mutations in the glucokinase (GCK) gene and is rare in the Chinese population. We report three Chinese families with MODY2 and the sequencing of the GCK gene. Three unrelated Chinese families with MODY2 and their pedigrees were investigated. In Family 1, the proband was a 7-year-old girl with impaired fasting glucose (IFG) and impaired glucose tolerance (IGT). Her mother and maternal grandfather had IFG. In Family 2, the proband was a boy who had diabetes mellitus at 11 years. His sister had IFG. His father and grandmother had diabetes mellitus at 22 and 25 years, respectively. In Family 3, the proband was a boy who had IFG and IGT at 12 years. His sister had diabetes mellitus at 8 years. His father and grandfather had IFG and/or IGT. The GCK gene was directly sequenced. Diabetes mellitus or IFG/IGT was found among three consecutive generations in three families. One novel nonsense heterozygous mutation in exon 5 (c.556 C>T, p.Arg 186 stop) was detected in Family 1. Another novel frameshift mutation in exon 4 (c.367-374dupTTCGACTA, p.Ile 126 fs) was found in Family 2. A previously reported, a missense heterozygous mutation in exon 5 (c.571 C>T, p.Arg 191Trp) was detected in Family 3. The thorough investigation of three Chinese families with MODY2 revealed two novel mutations and one known mutation. GCK gene sequencing helps in MODY2, especially when there is uncertain IFG or IGT.

Amelioration of junctional epidermolysis bullosa due to exon skipping

Mutations in the COL17A1 gene lead to the genetic blistering disorder junctional epidermolysis bullosa generalized intermediate type (JEB-gen-intermed). Antisense oligonucleotide-mediated exon skipping is a strategy that aims to skip the mutation-containing exon and thereby produce a smaller but functional protein. COL17A1 is an interesting candidate, as 53 of the 55 exons (96%) can be skipped without disturbing the reading frame. Information on the functionality of the shortened protein product is important in order to obtain support for this therapeutic strategy. Here we report a patient with JEB-gen-intermed with amelioration of the phenotype due to exon 49 skipping by two distinct mechanisms - premature termination codon-induced exon skipping and revertant mosaicism - both of which induced skipping of the same exon. The patient was compound heterozygous for two inherited COL17A1 mutations, a frameshift mutation in exon 18 (c.1490_1491delinsT, p.Ala497Valfs*23) and a nonsense mutation in exon 49 (c.3487G>T, p.Glu1163Ter). Upon clinical examination, skin patches were found that were resistant to blister formation. In these patches, naturally corrected cells were present that harboured an additional splice-site mutation, c.3419-1G>T, resulting in skipping of the mutation-containing exon 49. This natural gene therapy phenomenon shows that type XVII collagen with residues 1140-1169 deleted is largely functional. In addition, in affected skin cells a low level of exon 49 skipping was observed. Our results support the notion that skipping of a mutated in-frame exon in COL17A1 ameliorates the phenotype.

A Distinct Genotype of XP Complementation Group A: Surprisingly Mild Phenotype Highly Prevalent in Northern India/Pakistan/Afghanistan

A Distinct Genotype of XP Complementation Group A: Surprisingly Mild Phenotype Highly Prevalent in Northern India/Pakistan/Afghanistan

MG-100 Review of a large family with schwannomatosis identifies an expanded phenotype

Background Schwannomatosis and atypical teratoid rhabdoid tumour syndrome (ATRT) are autosomal dominant disorders associated with mutations in the SMARCB1 gene on chromosome 22q, but with different age at onset and tumour predisposition. Missense mutations in the first and last exons of SMARCB1 typically cause multiple, benign schwannomas of the peripheral and spinal nerves (average age 28.3y), whereas, large deletion and frameshift mutations in exons 2–8 typically cause malignant rhabdoid tumours (before age 3y). Recently, families with both types of tumours have been described including a Newfoundland and Labrador (NL) family identified through a proband with ‘glioma’ and her brother with multiple spinal cord schwannomas. Objectives We were interested in determining the age-at-onset, type and number of tumours for NL family members in order to develop a clinical screening program. Design/method We obtained family history, and reviewed clinical records and genetic test results for 18 consenting members of this family with ‘schwannomatosis’ and a SMARCB1 missense mutation in exon 9. Results Seven family members had from 1–26 peripheral schwannomas, four had multiple spinal schwannomas, one had an ocular schwannoma, the proband (who died at 52y) had a high grade glioma with rhabdoid features, a five year old had an ATRT, and two obligate carriers in their 70s had no identified tumours. Conclusions The spectrum of tumours and age at onset was broader than expected. We recommend a complete clinical examination and, for adults, full-body MRI at diagnosis, annual clinical review, and repeat imaging to review any new pain, ‘lumps’ or dysfunction.

Novel CREB3L3 Nonsense Mutation in a Family With Dominant Hypertriglyceridemia.

Cyclic AMP responsive element-binding protein 3-like 3 (CREB3L3) is a novel candidate gene for dominant hypertriglyceridemia. To date, only 4 kindred with dominant hypertriglyceridemia have been found to be carriers of 2 nonsense mutations in CREB3L3 gene (245fs and W46X). We investigated a family in which hypertriglyceridemia displayed an autosomal dominant pattern of inheritance. The proband was a 49-year-old woman with high plasma triglycerides (≤1300 mg/dL; 14.68 mmol/L). Her father had a history of moderate hypertriglyceridemia, and her 51-year-old brother had triglycerides levels as high as 1600 mg/dL (18.06 mmol/L). To identify the causal mutation in this family, we analyzed the candidate genes of recessive and dominant forms of primary hypertriglyceridemia by direct sequencing. The sequencing of CREB3L3 gene led to the discovery of a novel minute frame shift mutation in exon 3 of CREB3L3 gene, predicted to result in the formation of a truncated protein devoid of function (c.359delG-p.K120fsX20). Heterozygosity for the c.359delG mutation resulted in a severe phenotype occurring later in life in the proband and her brother and a good response to diet and a hypotriglyceridemic treatment. The same mutation was detected in a 13-year-old daughter who to date is normotriglyceridemic. We have identified a novel pathogenic mutation in CREB3L3 gene in a family with dominant hypertriglyceridemia with a variable pattern of penetrance.

Multiple Structural and Epigenetic Defects in the Human Leukocyte Antigen Class I Antigen Presentation Pathway in a Recurrent Metastatic Melanoma Following Immunotherapy

Scant information is available about the molecular basis of multiple HLA class I antigen-processing machinery defects in malignant cells, although this information contributes to our understanding of the molecular immunoescape mechanisms utilized by tumor cells and may suggest strategies to counteract them. In the present study we reveal a combination of IFN-γ-irreversible structural and epigenetic defects in HLA class I antigen-processing machinery in a recurrent melanoma metastasis after immunotherapy. These defects include loss of tapasin and one HLA haplotype as well as selective silencing of HLA-A3 gene responsiveness to IFN-γ. Tapasin loss is caused by a germ-line frameshift mutation in exon 3 (TAPBP(684delA)) along with a somatic loss of the other gene copy. Selective silencing of HLA-A3 gene and its IFN-γ responsiveness is associated with promoter CpG methylation nearby site-α and TATA box, reversible after DNA methyltransferase 1 depletion. This treatment combined with tapasin reconstitution and IFN-γ stimulation restored the highest level of HLA class I expression and its ability to elicit cytotoxic T cell responses. These results represent a novel tumor immune evasion mechanism through impairing multiple components at various levels in the HLA class I antigen presentation pathway. These findings may suggest a rational design of combinatorial cancer immunotherapy harnessing DNA demethylation and IFN-γ response.

A novel frameshift MSX1 mutation in a Saudi family with autosomal dominant premolar and third molar agenesis

ObjectivesIn this study, the aim was to investigate a consanguineous Saudi family with non-syndromic premolars and third molars agenesis and to identify the causal mutation(s) using whole exome sequencing. DesignFamily phenotype and family pedigree were constructed from clinical and radiographic examinations. Whole exome sequencing was performed in two affected members of the Saudi family using the SureSelect Human all Exon 50 Mb kit (Agilent Technologies, Inc., Santa Clara, CA) and then sequenced on an Illumina HiSeq. SNP and indel calling were performed using samtools version 0.18 and were annotated using the software ANNOVAR. ResultsThe family pedigree showed that the inheritance was autosomal dominant. Whole exome sequencing revealed that the affected members in this family were heterozygous with a novel frameshift mutation in exon 2 of the MSX1 gene, (NM_002448:c.750_751insACCGGCTGCC, p.F251PfsX92). ConclusionsThe novel MSX1 frameshift mutation was linked to a family with moderate to severe tooth agenesis phenotype affecting second premolars and third molars in both arches. This expands the genotype–phenotype of MSX1 associated conditions.

Identification of Two Cases of Ciliopathy-Associated Diabetes and Their Mutation Analysis Using Whole Exome Sequencing.

BackgroundAlström syndrome and Bardet-Biedl syndrome are autosomal recessively inherited ciliopathies with common characteristics of obesity, diabetes, and blindness. Alström syndrome is caused by a mutation in the ALMS1 gene, and Bardet-Biedl syndrome is caused by mutations in BBS1-16 genes. Herein we report genetically confirmed cases of Alström syndrome and Bardet-Biedl syndrome in Korea using whole exome sequencing.MethodsExome capture was done using SureSelect Human All Exon Kit V4+UTRs (Agilent Technologies). HiSeq2000 system (Illumina) was used for massive parallel sequencing. Sanger sequencing was used for genotype confirmation and familial cosegregation analysis.ResultsA 21-year old Korean woman was clinically diagnosed with Alström syndrome. She had diabetes, blindness, obesity, severe insulin resistance, and hearing loss. Whole exome sequencing revealed a nonsense mutation in exon 10 of ALMS1 (c.8776C>T, p.R2926X) and a seven base-pair deletion resulting in frameshift mutation in exon 8 (c.6410_6416del, p.2137_2139del). A 24-year-old Korean man had Bardet-Biedl syndrome with diabetes, blindness, obesity, and a history of polydactyly. Whole exome sequencing revealed a nonsynonymous mutation in exon 11 of the BBS1 gene (c.1061A>G, p.E354G) and mutation at the normal splicing recognition site of exon 7 of the BBS1 gene (c.519-1G>T).ConclusionWe found novel compound heterozygous mutations of Alström syndrome and Bardet-Biedl syndrome using whole exome sequencing. The whole exome sequencing successfully identified novel genetic variants of ciliopathy-associated diabetes.

Netherton Syndrome in a Neonate with Possible Growth Hormone Deficiency and Transient Hyperaldosteronism.

Netherton syndrome, a rare autosomal recessive genetic disorder, is classified as an ichthyosiform syndrome. In this report we present the case of a neonate with erythroderma shortly after birth, accompanied by severe hypernatremia, recurrent infections, transient hyperaldosteronism, and signs of growth hormone (GH) deficiency. DNA molecular analysis in the SPINK5 gene revealed heterozygosity in our index patient for 238insG and 2468delA frameshift mutations in exons 4 and 26, respectively, in the maternal allele and 1431-12G>A splice-site mutation in intron 15 in the paternal allele as well as the missense variation E420K in homozygous state. Combination of the identified mutations along with transient hyperaldosteronism and possible GH deficiency have not been described before. Accordingly, the importance of early multidisciplinary approach is highlighted, in order to reach accurate diagnosis, initiate prompt treatment, and ensure survival with fewer disease complications.