Comprehensive Genetic Analysis of RASopathy in the Era of Next-Generation Sequencing and Definition of a Novel Likely Pathogenic KRAS Variation

<b><i>Introduction:</i></b> Germline pathogenic variations of the genes encoding the components of the Ras-MAPK pathway are found to be responsible for RASopathies, a clinically and genetically heterogeneous group of diseases. In this study, we aimed to present the results of patients genetically investigated for RASopathy-related mutations in our Genetic Diagnosis Center. <b><i>Methods:</i></b> The results of 51 unrelated probands with RASopathy and 4 affected relatives (31 male, 24 female; mean age: 9.327 ± 8.214) were included in this study. Mutation screening was performed on DNA samples from peripheral blood of the patients either by Sanger sequencing of <i>PTPN11</i> hotspot regions (10/51 probands), or by a targeted amplicon next-generation sequencing panel (41/51 probands) covering the exonic regions of <i>BRAF</i>, <i>CBL</i>, <i>HRAS</i>, <i>KRAS</i>, <i>LZTR1</i>, <i>MAP2K1</i>, <i>MAP2K2</i>, <i>NF1</i>, <i>NRAS</i>, <i>PTPN11</i>, <i>RAF1</i>, <i>RASA2</i>, <i>RIT1</i>, <i>SHOC2</i>, <i>SOS1</i>, <i>SOS2</i>, <i>SPRED1</i>, and <i>KAT6B</i> genes. <b><i>Results:</i></b> Pathogenic/likely pathogenic variations found in 22 out of 51 probands (43.13%) and their 4 affected family members were located in <i>PTPN11</i>, <i>BRAF</i>, <i>KRAS</i>, <i>NF1</i>, <i>RAF1</i>, <i>SOS1</i>, and <i>SHOC2</i> genes. The c.148A&#x3e;C (p.Thr50Pro) variation in the <i>KRAS</i> gene was a novel variant detected in a sibling in our patient cohort. We found supportive evidence for the pathogenicity of the <i>NF1</i> gene c.5606G&#x3e;T (p.Gly1869Val) variation which we defined in an affected boy who inherited the mutation from his affected father. <b><i>Conclusion:</i></b> Although <i>PTPN11</i> is the most frequently mutated gene in our patient cohort, as in most previous reports, different mutation distribution among the other genes studied motivates the use of a next-generation sequencing gene panel including the possible responsible genes.

Multi-region exome sequencing reveals the intratumoral heterogeneity of surgically resected small cell lung cancer

Small cell lung cancer (SCLC) is a highly malignant tumor which is eventually refractory to any treatment. Intratumoral heterogeneity (ITH) may contribute to treatment failure. However, the extent of ITH in SCLC is still largely unknown. Here, we subject 120 tumor samples from 40 stage I-III SCLC patients to multi-regional whole-exome sequencing. The most common mutant genes are TP53 (88%) and RB1 (72%). We observe a medium level of mutational heterogeneity (0.30, range 0.0~0.98) and tumor mutational burden (TMB, 10.2 mutations/Mb, range 1.1~51.7). Our SCLC samples also exhibit somatic copy number variation (CNV) across all patients, with an average CNV ITH of 0.49 (range 0.02~0.99). In terms of mutation distribution, ITH, TMB, mutation clusters, and gene signatures, patients with combined SCLC behave roughly the same way as patients with pure SCLC. This condition also exists in smoking patients and patients with EGFR mutations. A higher TMB per cluster is associated with better disease-free survival while single-nucleotide variant ITH is linked to worse overall survival, and therefore these features may be used as prognostic biomarkers for SCLC. Together, these findings demonstrate the intratumoral genetic heterogeneity of surgically resected SCLC and provide insights into resistance to treatment.

Ciprofloxacin Induced Antibiotic Resistance in Salmonella Typhimurium Mutants and Genome Analysis

Salmonella species is an important food-borne pathogen. Ciprofloxacin is used for treatment of salmonellosis. The wild type of Salmonella Typhimurium was exposed to 10-fold of minimal inhibitory concentration (MIC) of ciprofloxacin. The survivors of the first exposure were regrown. This regrown population was exposed to similar ciprofloxacin treatment. After 20 repetitions of the antibiotic exposure, the survival population was evaluated for antibiotic resistance. The mutants had 100-fold more of the initial MIC. The mutants were of smaller size (0.7 ± 0.5 mm) with a lag phase of 70 min. These mutants were resistant to other antibiotics belonging to different classes but were sensitive to heat and osmotic stress. The whole genome sequencing (WGS) analysis of antibiotic resistant mutants revealed interesting mutation pattern. Total 40513 mutations were observed across the genome (total 3843 annotated genes). Relatively small percentage (5.2%) of mutations were non-synonymous. Four-fold more transitions were observed than transversions. Ratio of <1 transition vs transversion shows a positive selection for antibiotic resistant trait. Seven genes with over 50 mutations were observed. However, mutation distribution across the genome was relatively uniform. The important genes like dnaE, gyrA, iroC, metH and rpoB involved in antibiotic resistance had point mutations. The genome analysis showed that most affected pathways were transcription regulation, transmembrane transport, cell adhesion, pathogenesis, pilus assembly, oxidation-reduction mechanisms, phosphor-relay signal transduction and LPS biosynthesis. We propose that development resistance to ciprofloxacin is because of the mutations in these genes.

Impact of a frequent nearsplice SOD1 variant in amyotrophic lateral sclerosis: optimising SOD1 genetic screening for gene therapy opportunities

ObjectiveMutations in superoxide dismutase 1 gene (SOD1), encoding copper/zinc superoxide dismutase protein, are the second most frequent high penetrant genetic cause for amyotrophic lateral sclerosis (ALS) motor neuron disease in populations of European descent. More than 200 missense variants are reported along the SOD1 protein. To limit the production of these aberrant and deleterious SOD1 species, antisense oligonucleotide approaches have recently emerged and showed promising effects in clinical trials. To offer the possibility to any patient with SOD1-ALS to benefit of such a gene therapy, it is necessary to ascertain whether any variant of unknown significance (VUS), detected for example in SOD1 non-coding sequences, is pathogenic.MethodsWe analysed SOD1 mutation distribution after SOD1 sequencing in a large cohort of 470 French familial ALS (fALS) index cases.ResultsWe identified a total of 27 SOD1 variants in 38 families including two SOD1 variants located in nearsplice or intronic regions of the gene. The pathogenicity of the c.358–10T>G nearsplice SOD1 variant was corroborated based on its high frequency (as the second most frequent SOD1 variant) in French fALS, the segregation analysis confirmed in eight affected members of a large pedigree, the typical SOD1-related phenotype observed (with lower limb onset and prominent lower motor neuron involvement), and findings on postmortem tissues showing SOD1 misaccumulation.ConclusionsOur results highlighted nearsplice/intronic mutations in SOD1 are responsible for a significant portion of French fALS and suggested the systematic analysis of the SOD1 mRNA sequence could become the method of choice for SOD1 screening, not to miss these specific cases.

EPCO-39. DICER1 SYNDROME WITH PRIMITIVE NEUROECTODERMAL TUMOR FROM SCREENING MUTATIONS OF DICER1 GENE

BACKGROUND DICER1 syndrome is a rare disorder of tumor predisposition. The DICER1 gene encodes the Dicer protein of the ribonuclease Ⅲ family and plays a critical role in miRNA creation. Patients with DICER1 syndrome-related cancer commonly harbor an additional somatic variant in exon24 or 25 of DICER1 involving one of the hotspots including p.E1705, p.D1709, p.G1809, p.D1810, or p.E1813. Herein, we firstly report the presence of hotspot missence mutation in a patient with primitive neuroectodermal tumor (PNET). METHODS The mutations in DIRCER1 were searched in the tumor tissue or peripheral blood from 4,349 patients with different types of cancer via next generation sequencing. Among them, comprehensive genomic profiling (CGP) with 131 CNS cancer-related genes and 4 brain tumor-related chromosome structure variations was performed in 230 cases, and CGP with 539 cancer-related genes was administrated in 4,119 cases. The histopathology diagnosis of each case was confirmed via hematoxylin and eosin staining. RESULTS A number of 99 mutations in the exon and intron regions of DICER1 gene were observed from 88 patients in 4,349 cases. The ratio of somatic variants located on exon 24 and 25 where both encoded the RNase Ⅲb cleavage domain to all mutations was 9.09% (9/99). There were 3 and 6 somatic variants in the exon 24 and 25, respectively. Among the 9 mutations, 4 of them were p.E1813, one class of hotspots located on exon 25 leading to DICER1 syndrome, and the other 4 hotspots were not observed. Among 4 cases of DICER1 syndrom, pleuropulmenary blastoma was common, rhabdomyosarcomas arisen in two cases were also reported before, and the last one was patient with PNET. CONCLUSIONS In this study, we analyze the mutation distribution of DICER1 in cancer patients, report firstly a patient with PNET harbors a hotspot, and widen and deepen the understanding of DICER1 syndrome.

Genetic markers on the distribution of ancient marine hunters in Priokhotye

This is a review of studies on the genetic polymorphism of modern and ancient populations of the north of Asia and America, with the aim of reconstructing the history of migrations of ancient marine hunters in the Okhotsk Sea region. The data on mitochondrial DNA polymorphism and the “Arctic” mutation distribution – the rs80356779-A variant of the CPT1A gene – were analyzed. It is known that the “Arctic” variant of the CPT1A gene is widely distributed in modern populations of the Eskimos, Chukchis, Koryaks, and other peoples of the Okhotsk Sea region, whose economic structure is associated with marine hunting. According to paleogenomic data, the earliest cases of the “Arctic” variant of the CPT1A gene were found in the Greenland and Canadian Paleoeskimos (4 thousand years ago), among representatives of the Tokarev culture of the Northern Priokhotye (3 thousand years ago), and among the bearers of the culture of the late Jomon of Hokkaido (3.5–3.8 thousand years ago). The results of the analysis revealed several migration events associated with the spread of marine hunters in the Okhotsk Sea region. The latest migration, which left traces on bearers of the Epi-Jomon culture (2.0–2.5 thousand years ago), introduced the mitochondrial haplogroup G1b and the “Arctic” variant of the CPT1A gene from the north of Priokhotye to Hokkaido and neighboring territories of the Amur Region. Traces of earlier migration, which also brought the “Arctic” mutation, were recorded in the Hokkaido population of the late Jomon period (3.5–3.8 thousand years ago). A phylogenetic analysis of mitochondrial genomes belonging to the rare haplogroup C1a, found in populations of the Far East and Japan, but phylogenetically related to the C1-haplogroups of the Amerindians, was carried out. The results of the analysis showed that the divergence of mitochondrial lineages within the C1a haplogroup occurred in the range from 7.9 to 6.6 thousand years ago, and the age of the Japanese branch of the C1a haplogroup is approximately 5.2 thousand years. It is not yet known whether this migration is associated with the spread of the “Arctic” variant of the CPT1A gene or the presence of C1a haplotypes in the population of the Japanese islands marks another, earlier, episode of the migration history linking the populations of Northwest Pacific and North America.

The interplay of SARS-CoV-2 evolution and constraints imposed by the structure and functionality of its proteins

Fast evolution of the SARS-CoV-2 virus provides us with unique information about the patterns of genetic changes in a single pathogen in the timescale of months. This data is used extensively to track the phylodynamic of the pandemic’s spread and its split into distinct clades. Here we show that the patterns of SARS-CoV-2 virus mutations along its genome are closely correlated with the structural features of the coded proteins. We show that the foldability of proteins’ 3D structures and conservation of their functions are the universal factors driving evolutionary selection in protein-coding genes. Insights from the analysis of mutation distribution in the context of the SARS-CoV-2 proteins’ structures and functions have practical implications including evaluating potential antigen epitopes or selection of primers for PCR-based COVID-19 tests.

Genetic and functional insights into CDA-I prevalence and pathogenesis

BackgroundCongenital dyserythropoietic anaemia type I (CDA-I) is a hereditary anaemia caused by biallelic mutations in the widely expressed genes CDAN1 and C15orf41. Little is understood about either protein and it is unclear in which cellular pathways they participate.MethodsGenetic analysis of a cohort of patients with CDA-I identifies novel pathogenic variants in both known causative genes. We analyse the mutation distribution and the predicted structural positioning of amino acids affected in Codanin-1, the protein encoded by CDAN1. Using western blotting, immunoprecipitation and immunofluorescence, we determine the effect of particular mutations on both proteins and interrogate protein interaction, stability and subcellular localisation.ResultsWe identify six novel CDAN1 mutations and one novel mutation in C15orf41 and uncover evidence of further genetic heterogeneity in CDA-I. Additionally, population genetics suggests that CDA-I is more common than currently predicted. Mutations are enriched in six clusters in Codanin-1 and tend to affect buried residues. Many missense and in-frame mutations do not destabilise the entire protein. Rather C15orf41 relies on Codanin-1 for stability and both proteins, which are enriched in the nucleolus, interact to form an obligate complex in cells.ConclusionStability and interaction data suggest that C15orf41 may be the key determinant of CDA-I and offer insight into the mechanism underlying this disease. Both proteins share a common pathway likely to be present in a wide variety of cell types; however, nucleolar enrichment may provide a clue as to the erythroid specific nature of CDA-I. The surprisingly high predicted incidence of CDA-I suggests that better ascertainment would lead to improved patient care.

FAM120A-PTPN3 variations in neuroblastoma: Implications for poor prognosis and relapse.

e22512 Background: Neuroblastoma (NB), the most common tumor in infants, presents clinical unpredictability of relapse and treatment response. Staging and risk stratification depending on clinical examinations are often undertaken while molecular risk factors are less known. Relapse of high-risk NB remains the greatest clinical challenge. Thus, we conduct this study to investigate the molecular mechanism of NB relapse. Methods: Six patients with neuroblastoma who had both primary tumor and relapse samples were studied to identify key mutations in relapse. A cohort analysis of genetic profiles was then performed in order to find out gene mutations associated with the relapse samples. Mutation distribution and expression profiling in 127 patients from the national cancer institute database were used to identify relapse related gene networks. Kaplan-Meier analysis was performed for Overall survival (OS) on each mutation and Ingenuity pathway analysis (IPA) was consulted for their possible interactions. Gene manipulation in two neuroblastoma cell lines were used for verification. Results: We identified 40 potential mutations that associated with the relapse samples, including FAM120A and PTPN3. Mutation distribution in a larger NB population with 127 patients showed FAM120A mutation rate was 32.3% (41/127) and PTPN3 was 52.8% (67/127). Both had significant impact on OS. The median OS (mOS) with or without FAM120A mutation was 1527±214 days and 2300±139 days, respectively (p = 0.000). mOS with or without PTPN3 mutation was 1822±163 days and 2285±179 days, respectively (p = 0.037). Additionally, mOS in group lacking both mutations (42/127) was 2555±196 days, much higher than 1857±153 days, in those who had only one (62/127), or 1391±266 days, in those who had both (23/127). Furthermore, the prevalence of FAM120A mutation was at Chr.9:93543407 (61.0%, 25/41) and PTPN3 at Chr.9: 109457194 (61.2%, 41/67). Variant at Chr.9:93543407 had more significant impact on OS compared to other sites in FAM120A-mutation subgroup (p = 0.01). Expression of FAM120A was significantly down-regulated in the FAM120A-mutation subgroup compared to non-mutated (p = 0.01). IPA and in vitro assay showed these factors may have an additive effect possibly interacting through TRIM25, MYC and VIRMA. Conclusions: Our results suggest that variations in FAM120A and PTPN3 interact in neuroblastoma resulting in poor prognosis and cancer relapse, and may be a potential target for improvement of current treatment.

CDH1 Mutation Distribution and Type Suggests Genetic Differences between the Etiology of Orofacial Clefting and Gastric Cancer

Pathogenic variants in CDH1, encoding epithelial cadherin (E-cadherin), have been implicated in hereditary diffuse gastric cancer (HDGC), lobular breast cancer, and both syndromic and non-syndromic cleft lip/palate (CL/P). Despite the large number of CDH1 mutations described, the nature of the phenotypic consequence of such mutations is currently not able to be predicted, creating significant challenges for genetic counselling. This study collates the phenotype and molecular data for available CDH1 variants that have been classified, using the American College of Medical Genetics and Genomics criteria, as at least ‘likely pathogenic’, and correlates their molecular and structural characteristics to phenotype. We demonstrate that CDH1 variant type and location differ between HDGC and CL/P, and that there is clustering of CL/P variants within linker regions between the extracellular domains of the cadherin protein. While these differences do not provide for exact prediction of the phenotype for a given mutation, they may contribute to more accurate assessments of risk for HDGC or CL/P for individuals with specific CDH1 variants.