Decoding Cystic Fibrosis Phenotype

Cystic fibrosis (CF) is a monogenic autosomal recessive disease caused by mutations in transmembrane conductance regulator (CFTR) gene. The golden standard for the diagnosis of CF is sweat chloride testing (>60 mmol/L) together with the identification of two CFcausing variants of CFTR gene. Nevertheless, about 0.01% of patients with elevated sweat chloride and high clinical suspicion of CF do not carry any CF-causing variants. Here we present analysis of whole exome sequencing (WES) results for two patients with elevated sweat chloride levels and clinical presentation of CF in whom no CF-causing mutations were detected after CFTR gene whole coding region sequencing, and large insertion/deletion testing. Genomic DNA was extracted from whole blood, subjected to library preparation using DNA nanoball technology from BGI and sequenced on DNBSEQ-G400 (MGI). Produced fastq files were mapped to hg38 reference genome using BWA/SAM tools. VCF files were generated using GATK (BaseRecalibrator, HaplotypeCaller) and annotated with InterVar and AnnoVar tools. Filtering of detected variants for disease relevance was done using the following criteria: QC Filter, GnomAD Allele Frequency, Functional consequences and phenotype-genotype relationship. In both patients, similar number of variants predicted to impair protein function were detected (27 and 25). In two genes (CACNA1H and MUC5B) missense type variants were found in both patients and loss of function variants were found in 7 and 11 genes, respectively. Functional assessment of selected variants is underway. Bioinformatics analyses are a valuable tool enabling identification of underlining genetic bases of disease phenotype, important in the context of optimal patient management and targeted therapies.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Profiling Pre-Replication Complex Mutations in Cancer

The pre-replication complex (preRC) consists of 15 proteins that mark DNA replication initiation sites and regulate replication timing. Deficiency in preRC proteins results in genomic instability (re-replication) and developmental defects (Meier-Gorlin syndrome). Our aim was to assess the scope of preRC gene aberrations in cancer. Variations in preRC genes were studied using CBio Portal software and TCGA PanCancer dataset. The functional impact of detected variants was evaluated in silico by three different prediction tools: SIFT (sequence and evolutionary conservation - based), PolyPhen2 (protein sequence and structure – based) and MutPred2 (supervised learning method based on neural networks). No mutational hotspots were observed in any of the 15 preRC genes and no mutual exclusivity between mutations in preRC genes were detected. The highest alteration incidence in preRC genes was found in endometrial carcinoma and melanoma. The majority of the variations seen in preRC genes were non-synonymous. The functional assessment has shown that 253/1215 (21%) preRC gene mutations were predicted to be pathogenic with high confidence by 2/3 computational algorithms. None of the variants reached the high confidence pathogenicity score by all 3 prediction tool. In contrast, 49% of variants were predicted to be either benign by all three tools or benign by 2/3 or 1/3 tools, with the remaining 1/3 or 2/3, respectively, classifying them as low confidence pathogenic. These finding suggest that mutations in preRC proteins might be passenger mutations and that cancer cells can tolerate them. The future step is to see whether incidence of coding vs. noncoding preRC mutations correlates with Tumor Mutation Burden (TMB) and Genome Instability Index (GII) of cancer.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Influence of Cyp2c19∗2 Gene Variant on Therapeutic Response during Clopidogrel Treatment in Patients with Carotid Artery Stenosis

Background: Despite the proven clinical effect of oral antiplatelet drugs, a considerable number of patients do not have an adequate response to clopidogrel. The aim of our study was to determine the influence of CYP2C19*2 loss-of-function variant allele on clopidogrel responsiveness in patients with carotid artery stenosis. Methods: One hundred and twelve patients with carotid artery stenosis undergoing endarterectomy were included in this one-year prospective study. All of them received clopidogrel (75 mg daily) for at least 30 days after the intervention. They were followed from the moment of hospital admission. CYP2C19*2 genotyping was performed by TaqMan Assay. The influence of CYP2C19*2 variant allele on clopidogrel platelet reactivity was determined using multiple-electrode aggregometry (MEA). Results: Genotyping results showed that 82 (73.2%) patients were homozygous for wild type, 29 (25.9%) were heterozygous for the CYP2C19*2 allele and 1 (0.9%) was CYP2C19*2 homozygous. After 24 hours, among those with the wild type 29.3% were clopidogrel responders, and in those with the CYP2C19*2 alleles 10%. In the wild type group, 74.4% were clopidogrel responders after 7 days of taking the drug; 82.9% after 30 days of clopidogrel introduction, respectively. In patients with the CYP2C19*2 alleles the number of responders increased up to 46.7% after 7 days; 53.3% after 30 days of taking the drug, respectively. The risk for being a low-responder is higher for the patients heterozygous for the CYP2C19*2 allele vs. wild type (OR 4.250, 95% CI 1.695-10.658, P lt 0.01). Conclusions: The CYP2C19*2 loss-of-function variant allele has significant influence on clopidogrel response in patients with carotid artery stenosis undergoing endarterectomy

POTENTIAL NEW GENES INVOLVED IN CYSTIC FIBROSIS PHENOTYPE

Background: Cystic fibrosis (CF) is autosomal recessive disorder characterized by chronic respiratory problems and poor growth. CF is caused by defect in transmembrane conductance regulator (CFTR) protein. CF is diagnosed by sweat chloride analysis (>60 mmol/L) with the identification of two CF-causing variants of CFTR gene. With a longstanding history of CFTR gene analysis, our laboratory identified several patients with elevated sweat chloride and clinical manifestations of CF in whom no CF-causing mutations were detected after sequencing of whole coding region and testing for large insertion/deletion of CFTR gene. In order to elucidate genetic background of conditions that mimic CF we performed whole exome sequencing (WES) in two such patients. Methods: Library preparation was done using DNA nanoball technology. Produced fastq files were mapped to hg38. VCF files were generated using GATK and annotated with InterVar and AnnoVar tools. Variants filtering for disease relevance was done using the following criteria: QC, GnomAD Allele Frequency, Functional consequences and phenotype-genotype relationship. Results: CACNA1H and MUC5B genes were found to be impaired in both patients. Similar number of variants predicted to impair protein function were detected (27 and 25) in each patient. Loss of function variants were found in 7 and 11 genes, respectively. Conclusion: Further assessment of selected variants will clarify their functional effect and relevance for the patient’s clinical phenotype. WES analysis will help identify genetic aspects of disease and assist in optimal patient management in about 0.01% of patients with elevated sweat chloride and high clinical suspicion of CF that do not carry any CF-causing variants.ABSTRACT BOOK: “Genetic Diseases from Diagnostics to Prevention and Therapy” October 05-14th Balkan Congress of Human Genetics & 9th Rare Disease SEE Meeting 2023; Skopje, October 05-07, 202

Risk factors for clopidogrel high on-treatment platelet reactivity in patients with carotid artery stenosis undergoing endarterectomy

Objectives: A considerable number of patients do not achieve an adequate response to clopidogrel. Our study aimed to evaluate genetic and non-genetic factors as possible risks for clopidogrel high on-treatment platelet reactivity (HTPR) in patients (n=112) with carotid artery stenosis undergoing endarterectomy (CEA). Methods: Using multiple-electrode impedance aggregometry (MEA) the antiplatelet effectiveness of clopidogrel was measured after 24 h, 7 and 30 days of clopidogrel treatment, which was introduced after elective CEA at a dose of 75 mg daily, for at least 30 days. Results: HTPR was observed among 25% patients after clopidogrel therapy for 30 days. Further analysis showed that 53.3% of patients carrying the CYP2C19*2 gene variant had clopidogrel-HTPR, while in the wild type group there were 14.6% (p lt 0.001). Multivariate logistic regression analysis identified the CYP2C19*2 variant allele (OR 4.384; 95% CI 1.296-14.833, p=0.017) and high total cholesterol level (OR 2.090; 95% CI 1.263-3.459, p=0.004) as the only independent risk factors for clopidogrel-HTPR. Conclusion: The CYP2C19*2 gene variant and high total cholesterol level were major factors for clopidogrel-HTPR in patients with carotid artery stenosis undergoing CEA

Clopidogrel High On-Treatment Platelet Reactivity in Patients with Carotid Artery Stenosis Undergoing Endarterectomy. A Pilot Study

Objectives: A considerable number of patients do not achieve an adequate response to clopidogrel. Our study aimed to evaluate genetic and non-genetic factors as possible risks for clopidogrel high on-treatment platelet reactivity (HTPR) in patients (n=112) with carotid artery stenosis undergoing endarterectomy (CEA). Methods: Using multiple-electrode impedance aggregometry (MEA) the antiplatelet effectiveness of clopidogrel was measured after 24 h, 7 and 30 days of clopidogrel treatment, which was introduced after elective CEA at a dose of 75 mg daily, for at least 30 days. Results: HTPR was observed among 25% patients after clopidogrel therapy for 30 days. Further analysis showed that 53.3% of patients carrying the CYP2C19*2 gene variant had clopidogrel-HTPR, while in the wild type group there were 14.6% (p lt 0.001). Multivariate logistic regression analysis identified the CYP2C19*2 variant allele (OR 4.384; 95% CI 1.296-14.833, p=0.017) and high total cholesterol level (OR 2.090; 95% CI 1.263-3.459, p=0.004) as the only independent risk factors for clopidogrel-HTPR. Conclusion: The CYP2C19*2 gene variant and high total cholesterol level were major factors for clopidogrel-HTPR in patients with carotid artery stenosis undergoing CEA