Frameshift Deletion Research Articles

P3567Genetic screening for monogenic hypertension in hypertensive individuals in a clinical setting

Abstract Objectives Monogenic hypertension describes a series of hypertension syndromes inherited by Mendelian law and present with complex phenotypes. Methods 1179 cases with monogenic hypertension potential were evaluated by sequencing 37 causative genes. Pathogenic variants were classified by using American College of Medical Genetics guidelines. Additionally, 49 variants of unknown significance were selected to receive functional analysis. The yield of combined genetic and functional analysis was evaluated. Results 21 deleterious variants were identified in 33 of 1179 (2.80%). Functional analysis for 49 unknown significant variants showed 32 variants harbored by 61 individuals led to abnormally expressed protein levels. Overall, combining genetic screening with functional analysis promoted diagnostic yield to 8.73%. The main etiology established was primary aldosteronism, with CACNA1H harboring the greatest mutation burden. Logistic regression analysis showed hypertension complicated with special manifestations had the strongest correlation with disease causing variants detection (p=0.03). Sequencing Results Summary Number of variants Number of individuals* Percentage† Individuals with no variant 0 524 44.44% Individuals with variants identified 592 655 55.56% Individuals with single contributing variant 297 480 40.71% Individuals with two or multiple contributing variants 295 175 14.84% Number of variants identified Pathogenic and likely pathogenic variants 21 33 2.80% Variants of unknown significance 570 634 53.77% Benign or likely benign variants 1 1 0.08% Type of variant Frameshift deletion 8 15 1.27% Frameshift insertion 5 5 0.42% Nonframeshift deletion 10 10 0.85% Nonframeshift insertion 6 12 1.02% Nonsynonymous SNV 546 607 51.48% Stopgain SNV 18 30 2.54% WES, whole-exome sequencing. *The statistics in this table was based on 1179 individuals. †The percentage was calculated by the number of individuals in each category. A flow chart of this study. Conclusion Our findings demonstrate an enhanced diagnostic ability by combining genetic analysis with functional evaluation and enables targeted treatment and prevention of hypertension. Acknowledgement/Funding This work was supported by National Basic Research Program of China (973 Program, 2014CB542300, 2014CB542302).

Evaluation of the contribution of germline variants in BRCA1 and BRCA2 to uveal and cutaneous melanoma.

Germline mutations of BRCA1 and BRCA2 predispose individuals to a high risk of breast and ovarian cancer, and elevated risk of other cancers, including those of the pancreas and prostate. BRCA2 mutation carriers may have increased risk of uveal melanoma (UM) and cutaneous melanoma (CM), but associations with these cancers in BRCA1 mutation carriers have been mixed. Here, we further assessed whether UM and CM are associated with BRCA1 or BRCA2 by assessing the presence, segregation and reported/predicted pathogenicity of rare germline mutations (variant allele frequency < 0.01) in families with multiple members affected by these cancers. Whole-genome or exome sequencing was performed on 160 CM and/or UM families from Australia, the Netherlands, Denmark and Sweden. Between one and five cases were sequenced from each family, totalling 307 individuals. Sanger sequencing was performed to validate BRCA1 and BRCA2 germline variants and to assess carrier status in other available family members. A nonsense and a frameshift mutation were identified in BRCA1, both resulting in premature truncation of the protein (the first at p.Q516 and the second at codon 91, after the introduction of seven amino acids due to a frameshift deletion). These variants co-segregated with CM in individuals who consented for testing and were present in individuals with pancreatic, prostate and breast cancer in the respective families. In addition, 33 rare missense mutations (variant allele frequency ranging from 0.00782 to 0.000001 in the aggregated ExAC data) were identified in 34 families. Examining the previously reported evidence of functional consequence of these variants revealed all had been classified as either benign or of unknown consequence. Seeking further evidence of an association between BRCA1 variants and melanoma, we examined two whole-genome/exome sequenced collections of sporadic CM patients (total N = 763). We identified one individual with a deleterious BRCA1 variant, however, this allele was lost (with the wild-type allele remaining) in the corresponding CM, indicating that defective BRCA1 was not a driver of tumorigenesis in this instance. Although this is the first time that deleterious BRCA1 mutations have been described in high-density CM families, we conclude that there is an insufficient burden of evidence to state that the increased familial CM or UM susceptibility is because of these variants. In addition, in conjunction with other studies, we conclude that the previously described association between BRCA2 mutations and UM susceptibility represents a rare source of increased risk.

Clinical characteristics and mutation Spectrum of NF1 in 12 Chinese families with orbital/periorbital plexiform Neurofibromatosis type 1

BackgroundOrbital/periorbital plexiform neurofibroma (OPPN) can compromise physical appearance and visual function. However, the clinical characteristics and NF1 mutation landscape in patients with heritable OPPN have not been reported.MethodsThe medical charts of 26 Chinese patients with OPPN from 12 families were reviewed. Mutation analysis of the entire coding region and flanking splice sites of the NF1 gene was performed using next-generation sequencing (NGS). Novel NF1 mutations were confirmed by Sanger sequencing.ResultsCompared to the parental generation, a significantly larger proportion of OPPN patients in the successive generation presented with earlier onset (p = 0.001), amblyopia (p = 0.034), motility disorders (p = 0.009) and bony orbital expansion (p = 0.019). Six novel NF1 mutations were identified in 11 (91.67%) families, including 6 (42.9%) single-base substitutions, 4 (28.5%) splicing mutations, 3 (21.4%) frameshift deletions, and 1 (7.14%) intron mutation.ConclusionsThe successive generation of OPPN patients presented with earlier onset and exhibited more severe ocular signs than did their parents or grandparents. Special attention should be paid to successive generations of OPPN patients. Considering that 6 mutations were novel, comprehensive NF1 mutation analysis is required or necessary or proposed for genetic counselling.

Novel mutations in actionable breast cancer genes by targeted sequencing in an ethnically homogenous cohort

BackgroundGenetic testing is becoming an essential tool for breast cancer (BC) diagnosis and treatment pathway, and particularly important for early detection and cancer prevention. The purpose of this study was to explore the diagnostic yield of targeted sequencing of the high priority BC genes.MethodsWe have utilized a cost-effective targeted sequencing approach of high priority actionable BC genes (BRCA1, BRCA2, ERBB2 and TP53) in a homogeneous patient cohort from Bangladesh (n = 52) by using tumor and blood samples.ResultsBlood derived targeted sequencing revealed 25.58% (11/43) clinically relevant mutations (both pathogenic and variants of uncertain significance (VUS)), with 13.95% (6/43) of samples carrying a pathogenic mutations. We have identified and validated five novel pathogenic germline mutations in this cohort, comprising of two frameshift deletions in BRCA2, and missense mutations in BRCA1, BRCA2 and ERBB2 gene respectively. Furthermore, we have identified three pathogenic mutations and a VUS within three tumor samples, including a sample carrying pathogenic mutations impacting both TP53 (c.322dupG; a novel frameshift insertion) and BRCA1 genes (c.116G > A). 22% of tissue samples had a clinically relevant TP53 mutation. Although the cohort is small, we have found pathogenic mutations to be enriched in BRCA2 (9.30%, 4/43) compare to BRCA1 (4.65%, 2/43). The frequency of germline VUS mutations found to be similar in both BRCA1 (4.65%; 2/43) and BRCA2 (4.65%; 2/43) compared to ERBB2 (2.32%; 1/43).ConclusionsThis is the first genetic study of BC predisposition genes in this population, implies that genetic screening through targeted sequencing can detect clinically significant and actionable BC-relevant mutations.

Deleterious de novo variants of X-linked ZC4H2 in females cause a variable phenotype with neurogenic arthrogryposis multiplex congenita.

Pathogenic variants in the X-linked gene ZC4H2, which encodes a zinc-finger protein, cause an infrequently described syndromic form of arthrogryposis multiplex congenita (AMC) with central and peripheral nervous system involvement. We present genetic and detailed phenotypic information on 23 newly identified families and simplex cases that include 19 affected females from 18 families and 14 affected males from nine families. Of note, the 15 females with deleterious de novo ZC4H2 variants presented with phenotypes ranging from mild to severe, and their clinical features overlapped with those seen in affected males. By contrast, of the nine carrier females with inherited ZC4H2 missense variants that were deleterious in affected male relatives, four were symptomatic. We also compared clinical phenotypes with previously published cases of both sexes and provide an overview on 48 males and 57 females from 42 families. The spectrum of ZC4H2 defects comprises novel and recurrent mostly inherited missense variants in affected males, and de novo splicing, frameshift, nonsense, and partial ZC4H2 deletions in affected females. Pathogenicity of two newly identified missense variants was further supported by studies in zebrafish. We propose ZC4H2 as a good candidate for early genetic testing of males and females with a clinical suspicion of fetal hypo-/akinesia and/or (neurogenic) AMC.

Review of 52 cases with Hailey-Hailey disease identified 25 novel mutations in Chinese Han population.

Hailey-Hailey disease (HHD) is a rare autosomal dominant inherited keratosis caused by mutations in ATP2C1. The aim of our study was to identify and analyze the features of the mutations in HHD. We examined 52 Chinese Han cases which were diagnosed as HHD based on their clinical and histological findings. Genomic DNA polymerase chain reaction and direct sequencing of ATP2C1 were performed from peripheral blood samples of the patients and 100 unrelated healthy controls. Twenty-five novel mutations and 14 recurrent mutations were identified, including 11 (28.2%) missense mutations, nine (23.1%) frame-shift deletion mutations, eight (20.5%) nonsense mutations, seven (17.9%) splicing mutations and four (10.3%) frame-shift insertion mutations. Together with ours, all 209 mutations showed a uniform distribution without hotspots or clusters. In addition, there is no specific genotype-phenotype correlation in HHD. Our findings update the spectrum of mutations in ATP2C1.

T-Cell Receptor Gene Therapy for Human Papillomavirus–Associated Epithelial Cancers: A First-in-Human, Phase I/II Study

PURPOSEGenetically engineered T-cell therapy is an emerging treatment of hematologic cancers with potential utility in epithelial cancers. We investigated T-cell therapy for the treatment of metastatic human papillomavirus (HPV)–associated epithelial cancers.METHODSThis phase I/II, single-center trial enrolled patients with metastatic HPV16-positive cancer from any primary tumor site who had received prior platinum-based therapy. Treatment consisted of autologous genetically engineered T cells expressing a T-cell receptor directed against HPV16 E6 (E6 T-cell receptor T cells), a conditioning regimen, and systemic aldesleukin.RESULTSTwelve patients were treated in the study. No dose-limiting toxicities were observed in the phase I portion. Two patients, both in the highest-dose cohort, experienced objective tumor responses. A patient with three lung metastases experienced complete regression of one tumor and partial regression of two tumors, which were subsequently resected; she has no evidence of disease 3 years after treatment. All patients demonstrated high levels of peripheral blood engraftment with E6 T-cell receptor T cells 1 month after treatment (median, 30%; range, 4% to 53%). One patient’s resistant tumor demonstrated a frameshift deletion in interferon gamma receptor 1, which mediates response to interferon gamma, an essential molecule for T-cell–mediated antitumor activity. Another patient’s resistant tumor demonstrated loss of HLA-A*02:01, the antigen presentation molecule required for this therapy. A tumor from a patient who responded to treatment did not demonstrate genetic defects in interferon gamma response or antigen presentation.CONCLUSIONEngineered T cells can induce regression of epithelial cancer. Tumor resistance was observed in the context of T-cell programmed death-1 expression and defects in interferon gamma and antigen presentation pathway components. These findings have important implications for development of cellular therapy in epithelial cancers.

Genome-Wide Analysis of Artificial Mutations Induced by Ethyl Methanesulfonate in the Eggplant (Solanum melongena L.).

Whole-genome sequences of four EMS (ethyl methanesulfonate)-induced eggplant mutants were analyzed to identify genome-wide mutations. In total, 173.01 GB of paired-end reads were obtained for four EMS-induced mutants and (WT) wild type and 1,076,010 SNPs (single nucleotide polymorphisms) and 183,421 indels were identified. The most common mutation type was C/G to T/A transitions followed by A/T to G/C transitions. The mean densities were one SNP per 1.3 to 2.6 Mb. The effect of mutations on gene function was annotated and only 7.2% were determined to be deleterious. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis showed 10 and 11 genes, which were nonsynonymous mutation or frameshift deletion in 48-5 and L6-5 involved in the anthocyanin biosynthesis or flavone and flavonol biosynthesis. QRT-PCR results showed that only the Sme2.5_06210.1_g00004.1, which was annotated as UFGT (Flavonoid galactosidase transferase), expression significantly decreased in the L6-5 mutant compared with the WT. Also, the Sme2.5_06210.1_g00004.1 expression was lower in the colorless eggplant compared with colorful eggplant in the natural eggplant cultivar. These results suggest that Sme2.5_06210.1_g00004.1 may play a key role in eggplant anthocyanin synthesis.

Genetic basis and timing of a major mating system shift in Capsella.

A crucial step in the transition from outcrossing to self-fertilization is the loss of genetic self-incompatibility (SI). In the Brassicaceae, SI involves the interaction of female and male specificity components, encoded by the genes SRK and SCR at the self-incompatibility locus (S-locus). Theory predicts that S-linked mutations, and especially dominant mutations in SCR, are likely to contribute to loss of SI. However, few studies have investigated the contribution of dominant mutations to loss of SI in wild plant species. Here, we investigate the genetic basis of loss of SI in the self-fertilizing crucifer species Capsella orientalis, by combining genetic mapping, long-read sequencing of complete S-haplotypes, gene expression analyses and controlled crosses. We show that loss of SI in C.orientalis occurred <2.6Mya and maps as a dominant trait to the S-locus. We identify a fixed frameshift deletion in the male specificity gene SCR and confirm loss of male SI specificity. We further identify an S-linked small RNA that is predicted to cause dominance of self-compatibility. Our results agree with predictions on the contribution of dominant S-linked mutations to loss of SI, and thus provide new insights into the molecular basis of mating system transitions.

Signatures of Relaxed Selection in the CYP8B1 Gene of Birds and Mammals.

The CYP8B1 gene is known to catalyse reactions that determine the ratio of primary bile salts and the loss of this gene has recently been linked to lack of cholic acid in the bile of naked-mole rats, elephants and manatees using forward genomics approaches. We screened the CYP8B1 gene sequence of more than 200 species and test for relaxation of selection along each terminal branch. The need for retaining a functional copy of the CYP8B1 gene is established by the presence of a conserved open reading frame across most species screened in this study. Interestingly, the dietary switch from bovid to cetacean species is accompanied by an exceptional ten amino acid extension at the C-terminal end through a single base frame-shift deletion. We also verify that the coding frame disrupting mutations previously reported in the elephant are correct, are shared by extinct Elephantimorpha species and coincide with the dietary switch to herbivory. Relaxation of selection in the CYP8B1 gene of the wombat (Vombatus ursinus) also corresponds to drastic change in diet. In summary, our forward genomics-based screen of bird and mammal species identifies recurrent changes in the selection landscape of the CYP8B1 gene concomitant with a change in dietary lipid content.

Adhesion G-protein coupled receptor 56 is required for 3T3-L1 adipogenesis.

Obesity-associated conditions represent major global health and financial burdens and understanding processes regulating adipogenesis could lead to novel intervention strategies. This study shows that adhesion G-protein coupled receptor 56 (GPR56) gene transcripts are reduced in abdominal visceral white adipose tissue derived from obese Zucker rats versus lean controls. Immunostaining in 3T3-L1 preadipocytes reveals both mitotic cell restricted surface and low level general expression patterns of Gpr56. Gpr56 transcripts are differentially expressed in 3T3-L1 cells during adipogenesis. Transient knockdown (KD) of Gpr56 in 3T3-L1 cells dramatically inhibits differentiation through reducing the accumulation of both neutral cellular lipids (56%) and production of established adipogenesis Pparγ 2 (60-80%), C/ebpα (40-78%) mediator, and Ap2 (56-80%) marker proteins. Furthermore, genome editing of Gpr56 in 3T3-L1 cells created CW2.2.4 and RM4.2.5.5 clones (Gpr56 -/- cells) with compound heterozygous deletion frameshift mutations which abolish adipogenesis. Genome edited cells have sustained levels of the adipogenesis inhibitor β-catenin, reduced proliferation, reduced adhesion, altered profiles, and or abundance of extracellular matrix component gene transcripts for fibronectin, types I, III, and IV collagens and loss of actin stress fibers. β-catenin KD alone is insufficient to restore adipogenesis in Gpr56 -/- cells. Together these data show that Gpr56 is required for adipogenesis in 3T3-L1 cells. This report is the first demonstration that Gpr56 participates in regulation of the adipogenesis developmental program. Modulation of the levels of this protein and/or its biological activity may represent a novel target for development of therapeutic agents for the treatment of obesity.

A novel frame-shift deletion in FANCF gene causing autosomal recessive Fanconi anemia: a case report

BackgroundFanconi anemia (FA) is a heterogeneous genetic disorder characterized by congenital anomalies, early-onset bone marrow failure, and a high predisposition to cancers. Up to know, different genes involved in the DNA repair pathway, mainly FANCA genes, have been identified to be affected in patients with FA.Case presentationHere, we report clinical, laboratory and genetic findings in a 3.5-year-old Iranian female patient, a product of a consanguineous marriage, who was suspicious of FA, observed with short stature, microcephaly, skin hyperpigmentation, anemia, thrombocytopenia and hypo cellular bone marrow. Therefore, Next Generation Sequencing was performed to identify the genetic cause of the disease in this patient. Results revealed a novel, private, homozygous frameshift mutation in the FANCF gene (NM_022725: c. 534delG, p. G178 fs) which was confirmed by Sanger sequencing in the proband.ConclusionSuch studies may help uncover the exact pathomechanisms of this disorder and establish the genotype-phenotype correlations by identification of more mutations in this gene. It is the first report of a mutation in the FANCF gene in Iranian patients with Fanconi anemia. This new mutation correlates with a hematological problem (pancytopenia), short stature, and microcephaly and skin hyperpigmentation. Until now, no evidence of malignancy was detected.

Abstract 4243: Genetic variants frequently detected in Egyptian breast cancer tumors: Comprehensive cancer panel by ion torrent DNA sequencing technology

Abstract Breast cancer is the leading cause of cancer-related death in women worldwide. In Egypt, it is the most common cancer among females and its incidence is progressively increasing with a great tendency to occur with advanced stages in younger ages. Due to the heterogeneity of breast cancer, it was classified into different subtypes, each exhibits a unique gene mutation profile, based on biological characteristics and on gene expression pattern. Therefore, further research is needed to investigate the unknown genetic mutations involved in the progression of that disease. This study aimed to sequence 409 exons of tumor suppressor genes and oncogenes to identify the frequency of the detected genetic mutations in breast cancer using Ion Comprehensive Cancer Panel. Forty-eight tissue samples of various breast cancer subtypes were collected from National Cancer Institute (NCI) outpatient clinic, Cairo University. Analysis revealed 191 exonic and splicing variants. In this paper, we will address the most frequently detected Egyptian genetic variants (in 31.25 % of cases or more) as well as other deleterious variants commonly associated with breast cancer. Most of the detected genetic variants were checked in 1000g, dbSNP and Exac All databases. Other variants were found at known hotspot sites. We reported fifty-one somatic and germline mutations in thirty-two genes; AKAP9, BUB1B, RPS6KA2, AURKB, FANCA, RNF213, FGFR4, KAT6B, NLRP1, KAT6A, PER1, ERBB4, IL6ST, PIK3CA, P53, AURKA, WRN, PALB2, PTEN, GATA3, AKT1, ERBB2 and KRAS. Only KAT6B incurred non-frameshift deletion and only GATA3 had frameshift insertion while KAT6A, ERBB4 and PTEN had frameshift deletion. All the identified variants were detected with different frequencies in each breast cancer subtype. Each sample harbored at least four mutations and the maximum number of mutations per sample was twelve. The current data showed that gene panels analyzed by Next-Generation Sequencing (NGS) identifies large number of germline and somatic mutations that is crucial for understanding cancer predisposition and developing personalized or combination therapies that efficiently target the individual breast cancer-specific mutations. Key words: Breast cancer, Somatic mutations, Germline mutations, Ion torrent sequencing, Targeted sequencing. Note: This abstract was not presented at the meeting. Citation Format: Abdel-Rahman N. Zekri, Osman Mansour, Samah A. Loutfy, Mohamed M. Hafez, M. Gomaa, Abeer Bahnassy, Mai M. Lotfy, Amira S. Youssef, Ola S. Ahmed, Mohammed Abouelhouda, Auhood Nassar. Genetic variants frequently detected in Egyptian breast cancer tumors: Comprehensive cancer panel by ion torrent DNA sequencing technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4243.

Abstract 311: Diverse biological mechanisms drive resistance to Lorlatinib in ALK-rearranged Lung Cancer

Abstract Background: ALK rearrangements occur in 3-6% of patients (pts) with lung adenocarcinoma. Lorlatinib, is a novel third generation ALK tyrosine kinase inhibitor (TKI) with proven efficacy for patients previously treated with second generation ALK TKI. Methods: The MATCH-R study is a prospective single-institution trial aiming to identify mechanisms of resistance to targeted agents and immunotherapy in pts with advanced cancer (NCT02517892). Patients that achieve an initial partial or complete response or stability of disease for at least 6 months with selected agents are included upon disease progression. Tumor biopsies are performed and serial blood samples are collected. Extensive molecular profiling with panel next-generation sequencing (NGS), whole exome sequencing (WES) and RNA sequencing (RNAseq) is performed on tumor samples. Patient-derived xenografts (PDX) in NOD scid gamma (NSG) or nude mice and patient-derived cell lines are developed. We report mechanisms of resistance in a cohort of pts with ALK-rearranged lung cancer treated with lorlatinib. Results: From June 29th 2015 to November 15th 2018, 113 pts treated with a TKI were included in the MATCH-R study, of which 14 (12%) received treatment with ALK TKI, 6 pts treated with lorlatinib and with adequate tumor biopsies for molecular analysis were included. Tumor types studied were lung adenocarcinoma (n=4), anaplastic thyroid carcinoma (ATC, n=1) and myofibroblastic inflammatory tumor (MIT, n=1). An NF2 frame-shift deletion was detected by NGS in the ATC sample and a TNIK Q674 missense mutation was detected in the MIT sample. In the four pts with lung cancer treated with lorlatinib, we identified novel ALK G1202R/F1174L compound mutations from the tumor biopsy in one case and characterized them with Ba/F3 models (ctDNA analysis will be presented). Induction of epithelial mesenchymal transition (EMT) with lorlatinib exposure was responsible for resistance in one patient-derived model and susceptible to combined ALK/SRC inhibition. This cell line also had ALK C1156Y/G1269A compound mutations, not contributing to lorlatinib resistance. In a third case, double deleterious events in NF2 were identified in temporo-spatial distinct tumor biopsies on progression to lorlatinib. We further validated the effect of these events in patient-derived cell lines developed from two different biopsies. Downstream mTOR pathway activation conferred resistance to lorlatinib, and was reversible with mTOR inhibitors. We performed NF2 knockout in H3122 cells using Crispr-Cas9 gene editing to validate these findings. The resistance mechanism to lorlatinib treatment is yet to be elucidated in one patient-derived model. Conclusions: Mechanisms of resistance to lorlatinib can be diverse and complex, involving compound mutations, EMT and bypass activation. The present evidence could provide new insights for the development of tailored treatments for patients. Citation Format: Gonzalo Recondo, Laura Mezquita, David Planchard, Anas Gazzah, Francesco Facchinetti, Ludovic Bigot, Ahsan Z. Rizvi, Jean-Paul Thiery, Jean-Yves Scoazec, Rosa L. Frias, Tony Sourisseau, Linda Mahjoubi, Justine Galissant, Aurelie Abou-Lovergne, Gilles Vassal, Rastislav Bahleda, Antoine Hollebecque, Claudio Nicotra, Maud Ngocamus, Stefan Michiels, Ludovic Lacroix, Catherine Richon, Nathalie Auger, Thierry De Baere, Frederic Deschamps, Eric Solary, Ken A. Olaussen, Eric Angevin, Alexander Eggermont, Fabrice André, Christophe Massard, Jean-Charles Soria, Benjamin Besse, Luc Friboulet. Diverse biological mechanisms drive resistance to Lorlatinib in ALK-rearranged Lung Cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 311.

Distinctive genetic variation of long‐segment Hirschsprung's disease in Taiwan

Hirschsprung's disease (HSCR) is a congenital disorder with the absence of myenteric and submucosal ganglion cells within distal gut. Due to multigenic inheritance and interactions, we employed next-generation sequencing (NGS) to investigate genetic backgrounds of long-segment HSCR (L-HSCR) in Taiwan. Genomic DNA extracted from peripheral blood of L-HSCR patients was subjected to capture-based NGS, based on a 31-gene panel. The variants with allele frequency <0.05 and predicted by computational methods as deleterious were further validated by Sanger sequencing in patients and their family as well to tell de novo from inherited variants. Between 2015/04 and 2018/05, this study enrolled 23 L-HSCR patients, including 15 (65.2%) sporadic cases and 8 (34.8%) familial patients in 4 different families. Six sporadic and seven familial cases showed possible harmful variants across eight different genes, accounting for an overall detection rate of 56.5%. These variants mainly resided in SEMA3C, followed by RET, NRG1, and NTRK1. Three sporadic and 2 familial cases exhibited strong pathogenic variants as a deletional frameshift or stop codon in RET, L1CAM or NRG1. In a HSCR family, the father passed on a pathogenic RET frameshift to two daughters; however, only one developed HSCR. Using NGS, we disclosed deleterious mutations such as a frameshift or stop codon in either familial or sporadic patients. Our cases with isolated L-HSCR or even total colonic aganglionosis appeared to exhibit complex patterns of inheritance and incomplete penetrance even in families with the same genetic variants, reflecting the possible effects of environmental factors and genetic modifiers.

An undergraduate laboratory module that uses the CRISPR/Cas9 system to generate frameshift mutations in yeast.

The CRISPR/Cas9 system is a powerful tool for gene editing and it has become increasingly important for biology students to understand this emerging technique. Most CRISPR laboratory teaching modules use complex metazoan systems or mammalian cell culture which can be expensive. Here, we present a lab module that engages students in learning the fundamentals of CRISPR/Cas9 methodology using the simple and inexpensive model system, Saccharomyces cerevisiae. Students use CRISPR/Cas9 and nonhomologous end joining to generate frameshift insertion and deletion mutations in the CAN1 gene, which are easily selected for using media plates that have canavanine. DNA sequencing is also performed to determine what type of mutation occurred in gene-edited cells. This easy to implement set of experiments has been run as both a 5-week and a shorter 3-week lab module. Learning assessments demonstrate increased understanding in CRISPR-related concepts as well as increased confidence using molecular techniques. Thus, this CRISPR/Cas9 lab module can be added to an existing Genetics, Microbiology, or Molecular Biology lab course to help undergraduate students learn current gene editing techniques with limited effort and cost. © 2019 International Union of Biochemistry and Molecular Biology, 47(5):573-580, 2019.

Genetic and phenotypic characteristics of SCN1A-related epilepsy in Chinese children.

SCN1A gene mutations are associated with epilepsy and neurodevelopmental disorders. This study aimed to explore the genotype and phenotype spectrum of SCN1A gene related epilepsy. Epileptic patients who were treated in the Children's Hospital of Chongqing Medical University from January 2015 to July 2018 and identified as having SCN1A mutations by targeted next-generation sequencing were included. Clinical manifestations of all patients were analyzed retrospectively. A total of 24 patients with SCN1A mutations were identified. The age of epilepsy onset ranged from 2 months to 2 years and 9 months. Multiple seizure types were observed. A total of 13 (54.2%) patients had three or more types of seizures. Overall, 16 (66.7%) patients had status epilepticus, 11 (45.8%) patients had fever sensitivity, and nine (37.5%) patients had seizures after vaccination. Moreover, 15 (62.5%) patients showed varying degrees of cognitive and motor development retardation. In addition, two patients had mutations inherited from one of their parents and 22 (91.7%) patients had de novo mutations. The following SCN1A mutation types were identified: missense (16 patients, 66.7%), nonsense (four patients, 16.7%), splice site (one patient), frameshift (one patient), and large deletions (two patients). Overall, 23 of the patients received antiepileptic therapy, of which eight (33.3%) patients had no decrease in seizures and 11 (45.8%) patients had more than 50% decrease in seizure frequency. Three patients had poor response to antiepileptic drug therapy before attempting ketogenic diet, after which seizure frequency decreased by 50%. A total of 10 (41.7%) patients had used sodium channel blockers before accurate diagnosis, all of whom showed ineffective or even aggravated seizure response. SCN1A mutations are associated with a spectrum of seizure-related disorders, ranging from a relatively mild form of febrile seizures to a more severe epileptic encephalopathy known as Dravet syndrome. Early diagnosis of SCN1A mutation-associated epilepsy can aid in appropriate choice of antiepileptic drugs for treatment and reducing adverse sequelae.

PS1308 TARGETING T‐CELL RECEPTOR SIGNALING PATHWAY BY DASATINIB IN RELAPSED/REFRACTORY ANGIOIMMUNOBLASTIC T‐CELL LYMPHOMA

Background:Angioimmunoblastic T‐cell lymphoma (AITL) is an intractable T‐cell lymphoma. The recurrent hotspot (p.Gly17Val) mutations in RHOAencoding a small GTPase, together with the loss‐of‐function mutations in TET2encoding an epigenetic regulator, are the genetic hallmark in AITL. We previously identified that the p. Gly17Val RHOA mutant hyperactivates T‐cell receptor (TCR) signaling through direct binding to VAV1, a component of the TCR pathway. VAV1mutations were also found in RHOA‐negative AITL cases. The phosphorylation of VAV1 induced by the RHOA binding or its own amino acid changes was effectively inhibited by dasatinib at low concentrations. Dasatinib also prolonged the survival of AITL model mice (ASH 2017).Aims:Here, we performed a phase I clinical trial of dasatinib monotherapy in relapsed/refractory AITL patients.Methods:Five patients (one male and four females; 51–72 y/o; median, 65 y/o) being diagnosed with AITL were enrolled. Written informed consent was obtained from all the patients.Results:The median number of prior chemotherapies was 2 (range, 1–5). Two patients (Patient 1 and 2) were refractory to the latest chemotherapies. One patient (Patient 3) relapsed after the autologous transplantation. The performance status was 0‐ 2 (Table 1). Dasatinib was started at a dose of 100 mg/body q.d. and continued until days 10–78 (median day 58). Patient 2 withdrew the consent on day 10 and thus the response was not evaluable. In the remaining four patients, dasatinib was continued until day 54 or later and the response evaluation was possible. The trial was ultimately discontinued due to further treatment with allogeneic hematopoietic stem cell transplantation, disease progression, or severe adverse event in these four patients. Grade3/4 adverse drug reactions were observed in one case (Patient 5). The other adverse events were attributable to exacerbation of the diseases. There were no new safety concerns other than reported in BCR‐ABL‐positive chronic myelogenous leukemia/acute lymphoblastic leukemia. The maximum response was partial responses (PR) in all these four patients. The response at the discontinuation of trial was PR in two (Patient 1 and 5) and progressive disease (PD) in two (Patient 3 and 4).Targeted sequencing using a panel for 427 genes identified 34 candidate mutations including 22 nonsilent single nucleotide variants (SNVs), 2 nonsense mutations, 3 frameshift deletions, 2 frameshift insertions, 2 nonframeshift deletions, and one nonframeshift insertion. Two TET2 mutations were found in 4 samples (PAT1, PAT2, PAT4, and PAT5). The Gly17Val RHOA mutations were found by targeted sequencing in 2 samples (PAT3 and PAT4). VAV1 mutations were found in 2 samples (PAT2 and PAT5). Additionally one tandem duplication involving VAV1 locus was also identified by manual inspection of Integral Genome Viewer (IGV) in PAT5.Summary/Conclusion:AITL is highly dependent on TCR signaling, and dasatinib appears to be a promising candidate treatment modality in AITL.

PB1659 WHOLE EXOME SEQUENCING IN ADULT PATIENT WITH PHILADELPHIA / BCR‐ABL1 POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA

Background:BCR‐ABL1 gene fusion is detected at approximately 20% of adult acute lymphoblastic leukemia (ALL). Patients with Philadephia (Ph) / BCR‐ABL1 positive are a high risk group that associated with poor prognosis and short duration remission. Patients showed markedly improved with introduction of tyrosine kinase inhibitor combined with chemotherapy regimen but a small fraction of these patients remain refractory and poor response to treatment.Aims:The aim of this study was to determine the somatic mutations profile using whole exome sequencing of patient with Ph‐positive ALL.Methods:DNA from matched tumour‐normal samples for four patients with Ph‐positive ALL was extracted from peripheral blood or bone marrow leukemia cells by QIAamp DNA Mini kit. The exome library was enriched using the Nextera Rapid Capture Expended Enrichement kit (Illumina, San Diego, CA) according to the manufacturer's protocol and were subsequently sequenced on Illumina HiSeq 2500 system. Somatic mutations were separated from germline variants using Genome Analaysis Toolkits (GATK) best practices filtering strategy.Results:We identified a total of 14,804 intragenic somatic mutations (range from 2,948 to 4,165 in each). Of these 760 variants were passed through the variant caller. Among them 462 were single nucleotide variants (SNVs) and 298 were insertion/deletions (InDels). Total of 48 common variants were observed but failed Mutect caller. Among SNVs, 110 variants were nonsynonymous, 46 synonymous, 6 stopgain, 3 unknown and majority 297 (64.3%) null variants. A total of 53 nonsynonymous SNVs and 14 InDeLs (8 were frameshift deletion, 5 frameshift insertion and one stopgain) were identified as possible pathogeneic variants. The identified mutation genes in this study mostly also found in other malignancies disease and two alteration genes MCM7 and STAT5 were found in disease progression of CML.Summary/Conclusion:We have sequenced the whole coding sequence of four Ph / BCR‐ABL1 positive ALLs and corresponding normal samples that allows us to overview of candidate gene in the coding regions that driving Ph‐positive ALL development and as well as enhanced our understanding of the candidate genes in diagnosis testing and prognostic prediction of adult ALLs patient.

PS997 A NEW BIOMARKER OF RESPONSE TO 5‐AZACITIDINE THERAPY IN MDS AND AML PATIENTS: SIRPB1

Background:Myelodisplastic syndromes (MDS) and Acute Myeloid Leukemia (AML) are a group of diseases of the elderly that initiates in a hematopoietic stem cell and are characterized by clonal hematopoiesis and uncertain prognosis, mostly due to cytogenetic background. In both diseases, 5‐Azacitidine (5‐Aza) has been successful, inducing prolonged survival and delayed AML evolution.Aims:To identify the genes mostly predictive of treatment response, we use high‐throughput genomic analysis (SNP arrays and/or NGS‐RNA‐seq and/or NGS‐WES and/or GEP) in azacitidine‐sensitive and resistant MDS/AML patients.Methods:NGS‐WES or RNA seq HiSeq 2000 (Illumina) was positively done in 35/214 AML samples (16%), GEP (GeneChip Human Transcriptome Array 2.0, Affymetrix Inc.) was performed in 65/214 AML samples (30%). SNPs arrays (CytoScan HD Array, Affymetrix Inc.) in 125/214 AML samples (58%) and 18/32 MDS samples (56%) at diagnosis, then analyzed by Chromosome Analysis Suite (ChAS) v1.2 (Affymetrix Inc.), Nexus Copy Number™ v7.5 (BioDiscovery) and GeneGo MetaCore™ software.Results:We treated 246 adult pts with MDS or AML: 214 were AML and 32 were MDS with a median age of 59 and 70 years, respectively. 45 were treated with 5‐Aza (32 MDS/13 AML), while 201 AML with conventional chemotherapy. 45 MDS/AML were treated with at least one complete cycle of 5‐Aza (75 mg/sqm/daily). SNP arrays was done in 22/45 (49%), 13 pts were defined “insensitive/resistant”, ie. never achieving clinical complete remission (CCR) and 9 were defined “sensitive”, ie. all of them obtaining CCR. Copy Number Alterations (CNAs) ranged from loss or gain of complete chr arms to focal deletions and gains targeting one or few genes involving macroscopic (&gt;1.5 Mbps), submicroscopic genomic intervals (50 Kbps‐1.5 Mbps) and LOH (&gt;5 Mbps) events. Macroscopic CNAs affecting a complete chromosome or its arms were detected in 5 of 22 pts (23%), while classical cytogenetic was able to detect only two cases of trisomy 8 (9%), suggesting superiority of SNPs array for CNAs identifications. Microscopic CNAs abnormalities were detected in all of the patients affecting all the chromosomes. Chromosomic aberrations disease‐related are more statistically frequent on pts “insensitive” versus pts. “sensitive” (64% vs. 35%) (p≤0.01). Moreover we found that from the median of chromosomic alterations lenghts (in kbp) the group of “insensitive” MDS/AML patients to 5‐Aza present more gains and losses than “sensitive” ones. By Nexus Copy Number software, we identify 137 genes highly differentially gain (SIRPB1 and KIT with p ≤ 0.05) or loss (SIRPB1, LCE1C, BCAS1, EXD3 with p ≤ 0.05) or LOH between “insensitive” versus “sensitive” to 5‐Aza (p ≤ 0.05). Among these, we focused on SIRPB1 (cytoband 20p13, 56Kbps), since it was loss on 14/22 (64%) “insensitive” pts (p = 0,023) and gain on 7/22 (70%) “sensitive” ones with a significantly (p = 0,0324), respectively. SIRPB1 common deletion region goes from 1571 to 1598 (27 kbps) and common amplified region goes from 1561 to 1591 (30 kbps). By NGS‐WES we analyzed 35/214 (16%) AML samples at diagnosis, finding mutations in SF3B1, NPM1, CBL, RUNX1, BCOR, KIT, GATA2, IDH2, KDM6A, KIAA1324L, PRIM2, RRN3, APOBR and again in SIRPB1 an heterozigosity frameshift deletion (c. 388delC; p. H130fs) in exone 2 in a AML pts with normal karyotype. By GEP we further analyzed 48/214 (22%) AML samples for SIRPB1 in order to correlate and confirm the expression or loss of expression of these genes, in correlation with 5‐Aza response.Summary/Conclusion:We conclude that SIRPB1 is a promising marker of response to 5‐Aza treatment in MDS and AML.