Clinical and Molecular Genetic Analysis of Cases with Ectodermal Dysplasia.

INTRODUCTION: Ectodermal dysplasias are a group of >200 clinically and congenitally heterogeneous disorders characterized by abnormal development in the ectodermal structures, such as hair, nails, teeth, and sweat glands. We report here the clinical and molecular genetic analysis of five Greek families with different types of ectodermal dysplasia (ED). SUBJECTS: The study involved 15 individuals from 5 Greek families that included 8 ED patients, 5 carriers of recessive X-linked or autosomal ED, and 2 healthy relatives. After genetic counseling, the parents signed an informed consent form before subsequent genetic testing. METHODS: Genomic DNA was isolated from white blood cells of all studied individuals. The search for mutations was realized in patients' DNA samples using next-generation sequencing (NGS) gene panel, whole exome sequencing (WES), chromosomal microarray analysis (CMA), and multiplex ligation-dependent probe amplification (MLPA) technique. RESULTS: The clinical diagnosis of common X-linked recessive hypohidrotic ectodermal dysplasia (HED) was suspected in five male patients with partial anodontia of baby and permanent teeth, hypohidrosis, and thin hair from three families. All HED patients were hemizygous for deletions in the EDA1 gene (Xq13.1): three related patients had a 20 bp deletion, one had a 19 bp deletion, and one had a 180 bp deletion. A female patient had the rare autosomal dominant syndrome of ankyloblepharon-ectodermal dysplasia-cleft lip/palate (AEC) caused by heterozygous missense mutation in the TP63 gene (3q28) that appeared de novo. Two siblings with hypotrichosis and hypodontia, a female and a male, had two pathogenic mutations in compound heterozygosity in the TSPEAR gene (21q22.3); therefore they presented with ectodermal dysplasia type 14 (ECTD14). CONCLUSION: Clinical and molecular genetic analysis may set an accurate diagnosis of different types of ED. In the reported families, genetic diagnosis and genetic counselling assisted the parents to view their children's condition realistically and to cooperate with the specialists who will contribute to the best possible treatment for their children.

Study on the admission levels of circulating cell-free DNA in patients with acute myocardial infarction using different quantification methods.

Circulating cell-free DNA (cf-DNA) is present in human biological fluids, mainly in plasma and serum, originating from cell death, a process that massively takes place during acute myocardial infarction (AMI). In the present study, cf-DNA was assessed by different quantification techniques, in order to determine its levels in patients admitted with AMI. A total of 130 subjects were included in the study: 80 ST elevation myocardial infarction (STEMI) patients and 50 healthy controls. Cf-DNA extracted from plasma was analyzed by: a) Qubit 3.0 with single (ss) and double (ds) stranded DNA assay kits, b) NanoDrop and c) quantitative PCR (qPCR). Cf-DNA levels were recorded elevated in AMI patients compared to those of healthy individuals. Specifically, Qubit 3.0 ss-DNA kit provided the highest cf-DNA concentration values for all the samples analyzed in comparison with ds-DNA assay kit and NanoDrop, approaching the values obtained by qPCR. Cf-DNA augments in massive cell death settings, including AMI, proposing that the quantification of its levels by novel methodologies could contribute to patient diagnosis and clinical management.

Characterization of the c.793-1G > A splicing variant in CHEK2 gene as pathogenic: a case report.

BACKGROUND: CHEK2 is involved in the DNA damage repair response Fanconi anemia (FA)-BRCA pathway. An increased risk for breast and other cancers has been documented in individuals who carry a single pathogenic CHEK2 variant. As for other genes involved in cancer predisposition, different types of pathogenic variants have been observed, including single nucleotide variations, short insertions/deletions, large genomic rearrangements and splicing variants. Splicing variants occurring in the splicing acceptor or donor site result in alternative mature mRNA produced and can cause intron retention, exon skipping, or creation of alternative 3' and 5' splice site. Thus, the pathogenicity of this type of alterations should always be explored experimentally and their effect in the mRNA and consequently the protein produced, should be defined. The aim of this study was the delineation of the effect of a splicing variant in the CHEK2 gene. CASE PRESENTATION: A healthy 28-year-old woman with a family history of breast and ovarian cancer was referred for genetic testing. The variant c.793-1G > A (rs730881687) was identified by Next Generation Sequencing (NGS) using a solution-based capture method, targeting 33 cancer predisposition genes (SeqCap EZ Probe library, Roche NimbleGen). Experimental analysis in patient-derived leukocytes using RT-PCR of mRNA followed by cDNA sequencing revealed the deletion of one base from the alternative transcript created (r.793del). This resulted in a frameshift leading to premature termination codon within exon 7 (p.(Asp265Thrfs*10)). CONCLUSIONS: This finding suggests that the CHEK2 splicing variant c.793-1G > A is a deleterious variant. Our case shows that RNA analysis is a valuable tool for uncharacterized splice site variants in individuals referred for testing and facilitates their personalized management.

Analysis of hereditary cancer syndromes by using a panel of genes: novel and multiple pathogenic mutations.

BACKGROUND: Hereditary cancer predisposition syndromes are responsible for approximately 5-10% of all diagnosed cancer cases. In the past, single-gene analysis of specific high risk genes was used for the determination of the genetic cause of cancer heritability in certain families. The application of Next Generation Sequencing (NGS) technology has facilitated multigene panel analysis and is widely used in clinical practice, for the identification of individuals with cancer predisposing gene variants. The purpose of this study was to investigate the extent and nature of variants in genes implicated in hereditary cancer predisposition in individuals referred for testing in our laboratory. METHODS: In total, 1197 individuals from Greece, Romania and Turkey were referred to our laboratory for genetic testing in the past 4 years. The majority of referrals included individuals with personal of family history of breast and/or ovarian cancer. The analysis of genes involved in hereditary cancer predisposition was performed using a NGS approach. Genomic DNA was enriched for targeted regions of 36 genes and sequencing was carried out using the Illumina NGS technology. The presence of large genomic rearrangements (LGRs) was investigated by computational analysis and Multiplex Ligation-dependent Probe Amplification (MLPA). RESULTS: A pathogenic variant was identified in 264 of 1197 individuals (22.1%) analyzed while a variant of uncertain significance (VUS) was identified in 34.8% of cases. Clinically significant variants were identified in 29 of the 36 genes analyzed. Concerning the mutation distribution among individuals with positive findings, 43.6% were located in the BRCA1/2 genes whereas 21.6, 19.9, and 15.0% in other high, moderate and low risk genes respectively. Notably, 25 of the 264 positive individuals (9.5%) carried clinically significant variants in two different genes and 6.1% had a LGR. CONCLUSIONS: In our cohort, analysis of all the genes in the panel allowed the identification of 4.3 and 8.1% additional pathogenic variants in other high or moderate/low risk genes, respectively, enabling personalized management decisions for these individuals and supporting the clinical significance of multigene panel analysis in hereditary cancer predisposition.

Gender Specificity of a Genetic Variant of Androgen Receptor and Risk of Coronary Artery Disease.

BACKGROUND: Androgens are known to influence the risk of developing cardiovascular diseases. This study aims at investigating the possible association between G1733A polymorphism in the coding region of androgen receptor (AR) gene and premature coronary artery disease (CAD). METHODS: A total of 460 Greek subjects were investigated for the G1733A polymorphism. The patient group consisted of 250 CAD individuals, aged less than 58 years, while 210 healthy individuals served as controls. Genotyping was performed using the PCR-RFLP method. RESULTS: Significant differences in genotype distribution (P = 0.0067) and allele frequencies (P = 0.0060) have been observed between patients and controls in the women's subgroup. Conversely, the genotype/allele frequencies were similar between patients and controls in the subgroup of men. CONCLUSION: We may conclude that the G1733A polymorphism of AR gene could be a useful genetic marker for the assessment of a woman's risk for CAD in our Caucasian Greek population.

Neuroendocrine Breast Tumors: Could Multigene Assays Help in Guiding Treatment Decisions? Case Presentation.

BACKGROUND/AIM: Breast cancer remains the most prevalent type of cancer among women worldwide, and it remains the primary cause of cancer-related deaths in this demographic. Neuroendocrine breast cancer (NBC), an uncommon subtype comprising less than 1% of cases, typically occurs in older women and displays as a slow-growing, low-grade condition. NBC exhibits distinct histological patterns and immunohistochemical markers. Given the limited data on NBC, assays are required that will provide information on molecular profiling and assist in clinical decision making. The aim of the study was to investigate whether a modern Multigene Assay (MGA) could assist on treatment planning of NBC patients. CASE REPORT: A cohort of four patients was analyzed using a MGA. The presented cases featured young, pre-menopausal women with clear NBC, lacking family history. All were lymph node-negative, with robust expression of neuroendocrine markers. Despite high hormone receptor expression, all tumors were poorly differentiated with elevated Ki67 levels. Oncotype DX analysis indicated a need for chemotherapy in three cases and not in one. This underscores the heterogeneity within NBC, emphasizing the importance of personalized treatment decisions. CONCLUSION: While NBC is rare and lacks extensive studies, the use of multigene assays like Oncotype DX may play a pivotal role in treatment planning, especially in cases with varying histological parameters.

Germline Co-deletion of CDKN2A and CDKN2B Genes in Pleomorphic Xanthoastrocytoma: Case Report.

BACKGROUND/AIM: Gliomas are highly heterogeneous malignancies originating from diverse cell types within the brain. Although their precise etiology is frequently unknown, risk factors, such as chemical exposure, radiation, and specific uncommon genetic disorders have been identified. Diagnosis typically entails imaging tests, such as magnetic resonance imaging and computed tomography, complemented by a biopsy for confirmation, which may be further validated through genetic testing. CASE REPORT: Next-generation sequencing technology revealed germline co-deletion deletion of cyclin-dependent kinase inhibitor 2 A and B genes (CDKN2A and CDKN2B) in a patient diagnosed with pleomorphic xanthoastrocytoma based on the tumor's molecular characteristics. Following this result, we performed focused genetic analysis with use of multiplex ligation-dependent probe amplification technology for the mother that revealed the same co-deletion. Moreover, due to the father's neuroendocrine pancreatic cancer, application of the NGS technology detected a pathogenic variant in the BRCA1-interacting helicase 1 (BRIP1) gene. Comprehensive multi-gene testing conducted within the familial context, marked by a varied spectrum of cancer type, revealed a constellation of genetic predispositions. CONCLUSION: This case study underscores the critical importance of molecular testing for tumor characterization and highlights the pivotal role of genetic testing in facilitating early intervention and screening for at-risk family members. Furthermore, the identification of germline co-deletions in cancer lays the foundation for the development of targeted therapeutic strategies aimed at restoring normal cellular regulation and improving patient management.

Microsatellite Instability Is Insufficiently Used as a Biomarker for Lynch Syndrome Testing in Clinical Practice.

PURPOSE: The pan-cancer presence of microsatellite instability (MSI)-positive tumors demonstrates its clinical utility as an agnostic biomarker for identifying immunotherapy-eligible patients. Additionally, MSI is a hallmark of Lynch syndrome (LS), the most prevalent cancer susceptibility syndrome among patients with colorectal and endometrial cancer. Therefore, MSI-high results should inform germline genetic testing for cancer-predisposing genes. However, in clinical practice, such analysis is frequently disregarded. METHODS: A next-generation sequencing (NGS)-based technique was used for MSI analysis in 4,553 patients with various tumor types. Upon request, somatic BRAF gene analysis was conducted. In addition, hereditary testing of cancer-associated genes was performed in MSI-high cases using a capture-based NGS protocol. MLH1 promoter methylation analysis was conducted retrospectively in patients with colorectal and endometrial cancer to further investigate the origin of MSI at the tumor level. RESULTS: The MSI positivity rate for the entire cohort was 5.27%. Endometrial, gastric, colorectal, urinary tract, and prostate cancers showed the highest proportion of MSI-high cases (15.69%, 8.54%, 7.40%, 4.55%, and 3.19%, respectively). A minority of 45 patients (22.73%) among the MSI-high cases underwent germline testing to determine whether the mismatch repair pathway deficiency was inherited. 24.44% of those who performed the genetic test carried a pathogenic variant in an LS-associated gene. Three MSI-high individuals had non-LS gene alterations, including BRCA1, BRCA2, and CDKN2A pathogenic variants, indicating the presence of non-LS-associated gene alterations among MSI-high patients. CONCLUSION: Although MSI analysis is routinely performed in clinical practice, as many as 77% of MSI-high patients do not undergo LS genetic testing, despite international guidelines strongly recommending it. BRAF and MLH1 methylation analysis could shed light on the somatic origin of MSI in 42.50% of the MSI-high patients; however, MLH1 analysis is barely ever requested in clinical practice.

The Clinical and Genetic Landscape of Hereditary Cancer: Experience from a Single Clinical Diagnostic Laboratory.

BACKGROUND/AIM: The application of next-generation sequencing (NGS) technology in the genetic investigation of hereditary cancer is important for clinical surveillance, therapeutic approach, and reducing the risk of developing new malignancies. The aim of the study was to explore genetic predisposition in individuals referred for hereditary cancer. MATERIALS AND METHODS: A total of 8,261 individuals were referred for multigene genetic testing, during the period 2020-2023, in the laboratory, and underwent multigene genetic testing using NGS. Among the examined individuals, 56.17% were diagnosed with breast cancer, 6.77% with ovarian cancer, 2.88% with colorectal cancer, 1.91% with prostate cancer, 6.43% were healthy with a significant family history of cancer, while 3.06% had a different type of cancer and 0.21% had not provided any information. Additionally, in 85 women with breast cancer we performed whole exome sequencing analysis. RESULTS: 20% of the examined individuals carried a pathogenic variant. Specifically, 54.8% of the patients had a pathogenic variant in a clinically significant gene (BRCA1, BRCA2, PALB2, RAD51C, PMS2, CDKN2A, MLH1, MSH2, TP53, MSH6, APC, RAD51D, PTEN, RET, CDH1, MEN1, and VHL). Among the different types of pathogenic variants detected, a significant percentage (6.52%) represented copy number variation (CNV). With WES analysis, the following findings were detected: CTC1: c.880C>T, p.(Gln294*); MLH3: c.405del, p.(Asp136Metfs*2), PPM1D: c.1426_1430del, p.(Glu476Leufs*3), and SDHB: c.395A>G, p.(His132Arg). CONCLUSION: Comprehensive multigene genetic testing is necessary for appropriate clinical management of pathogenic variants' carriers. Additionally, the information obtained is important for determining the risk of malignancy development in family members of the examined individuals.

Reclassification of Splicing Gene Variants in Hereditary Cancer: Cases Report and Literature Review.

Alternative splicing (AS), a crucial cellular process, is a source of transcriptomic expansion and protein variability. Its contribution to cancer development and progression among a vast repertoire of human diseases, is highlighted lately and is under extensive investigation. In this review, the relative recent aspects of AS as a hallmark of cancer are described. In parallel, the importance of the identification of splicing-related variants through next-generation sequencing technologies is discussed. Cancer therapy and the management of patients and their families can highly benefit by the classification of these variants.

A novel deletion of exon 4 in the Ectodysplasin A gene associated with X-linked hypohidrotic ectodermal dysplasia.

OBJECTIVE: Identify the disease-causing mutation in a patient with features of X-linked hypohidrotic ectodermal dysplasia, which is a genetic disorder characterized by hypodontia, hypohidrosis and hypotrichosis. It is caused by mutations in Ectodysplasin A gene, which encodes ectodysplasin A, a member of the tumor necrosis factor superfamily. DESIGN: Genetic analysis, was performed using chromosomal microarray analysis, whole exome sequencing and multiplex ligation-dependent probe amplification analysis in a 4-year-old boy with hypohidrotic ectodermal dysplasia features. Moreover, the boy's parents were tested for clinically significant findings identified in order to elucidate the pattern of inheritance of the finding detected in the proband. RESULTS: A novel deletion of entire exon 4 in Ectodysplasin A gene identified in the 4-year-old patient. This deletion was found in heterozygous state in the mother of the proband and was not detected in his father. RNA analysis revealed an in-frame deletion r.527_706del, p.(176_236del) in exon 4 of the Ectodysplasin A gene. CONCLUSION: We identified a novel gross deletion in the Ectodysplasin A gene in a male patient with X-linked hypohidrotic ectodermal dysplasia. Clinical and molecular genetic analysis are crucial to set an accurate diagnosis in patients with hypohidrotic ectodermal dysplasia. These results highlight the importance of the collagen domain of Ectodysplasin A, encoded by exon 4, for its function in vivo.

Only 32.3% of Breast Cancer Families with Pathogenic Variants in Cancer Genes Utilized Cascade Genetic Testing.

BACKGROUND: Hereditary cancer predisposition syndromes are responsible for approximately 5-10% of all diagnosed cancer cases. In order to identify individuals at risk in a cost-efficient manner, family members of individuals carrying pathogenic alterations are tested only for the specific variant that was identified in their carrier relative. The purpose of this study was to investigate the clinical use and implementation of cascade family testing (CFT) in families of breast cancer patients with pathogenic/likely pathogenic variants (PVs/LPVs) in cancer-related predisposition genes. METHODS: Germline sequencing was carried out with NGS technology using a 52-gene panel, and cascade testing was performed by Sanger sequencing or MLPA. RESULTS: In a cohort of 1785 breast cancer patients (families), 20.3% were found to have PVs/LPVs. Specifically, 52.2%, 25.1%, and 22.7% of patients had positive findings in high-, intermediate-, and low-penetrance breast cancer susceptibility genes, respectively. Although CFT was recommended to all families, only 117 families (32.3%) agreed to proceed with genetic testing. Among the first-degree relatives who underwent CFT, 70.3% were female, and 108 of 121 (89.3%) were cancer free. Additionally, 42.7%, 36.7%, and 20.6% were offspring, siblings, and parents of the subject, respectively. Our data suggest that CFT was mostly undertaken (104/117, 88.8%) in families with positive findings in high-risk genes. CONCLUSIONS: Cascade family testing can be a powerful tool for primary cancer prevention by identifying at-risk family members. It is of utmost importance to implement genetic counseling approaches leading to increased awareness and communication of genetic testing results.

Copy Number Variations (CNVs) Account for 10.8% of Pathogenic Variants in Patients Referred for Hereditary Cancer Testing.

BACKGROUND/AIM: Germline copy number variation (CNV) is a type of genetic variant that predisposes significantly to inherited cancers. Today, next-generation sequencing (NGS) technologies have contributed to multi gene panel analysis in clinical practice. MATERIALS AND METHODS: A total of 2,163 patients were screened for cancer susceptibility, using a solution-based capture method. A panel of 52 genes was used for targeted NGS. The capture-based approach enables computational analysis of CNVs from NGS data. We studied the performance of the CNV module of the commercial software suite SeqPilot (JSI Medical Systems) and of the non-commercial tool panelcn.MOPS. Additionally, we tested the performance of digital multiplex ligation-dependent probe amplification (digitalMLPA). RESULTS: Pathogenic/likely pathogenic variants (P/LP) were identified in 464 samples (21.5%). CNV accounts for 10.8% (50/464) of pathogenic variants, referring to deletion/duplication of one or more exons of a gene. In patients with breast and ovarian cancer, CNVs accounted for 10.2% and 6.8% of pathogenic variants, respectively. In colorectal cancer patients, CNV accounted for 28.6% of pathogenic/likely pathogenic variants. CONCLUSION: In silico CNV detection tools provide a viable and cost-effective method to identify CNVs from NGS experiments. CNVs constitute a substantial percentage of P/LP variants, since they represent up to one of every ten P/LP findings identified by NGS multigene analysis; therefore, their evaluation is highly recommended to improve the diagnostic yield of hereditary cancer analysis.

Importance of multigene panel test in patients with consanguineous marriage and family history of breast cancer.

Next-generation sequencing (NGS) technology is used to evaluate hereditary cancer risks of patients worldwide; however, information concerning the germline multigene mutational spectrum among patients with breast cancer (BC) with consanguineous marriage (CM) is limited. Therefore, this prospective study aimed to determine the molecular characteristics of patients with BC who were tested with multigene hereditary cancer predisposition NGS panel and to show the effect of CM on cancer-related genes. Patients with BC with or without CM and family history (FH) of BC treated in our breast center were selected according to The National Comprehensive Cancer Network (NCCN) criteria for hereditary BC. In these patients, the analysis of a panel of 33 genes involved in hereditary cancer predisposition was performed after genetic counseling by using NGS. The pathogenic variant (PV) and the variant of uncertain significance (VUS) were found to be 15.8 and 47.4%, respectively. PVs were identified in 10/33 genes in 34 patients; 38.2% in BRCA1/2 genes; 6, 24, and 14% in other high, moderate and low-risk genes, respectively. The CM rate was 17.7% among the 215 patients with BC. The PV rate was 13.2% in patients with CM and 16.4% in patients without CM (P=0.80). When PV and VUS were evaluated together, the PV+VUS ratio was significantly higher in patients with CM and FH of BC than patients without CM and FH of BC (88.2 vs. 63.3%, P=0.045). Analysis of multigene panel provided 9.76% additional PVs in moderate/low-risk genes. The PV rate was similar in patients with BC with or without CM. A high PV+VUS ratio in patients with CM and FH of BC suggests that genes whose importance are unknown are likely to be pathogenic genes later.

Genetic Predisposition to Male Breast Cancer: A Case Series.

BACKGROUND/AIM: Male breast cancer (MBC) is a very rare disorder affecting approximately 1 in 833 men. Genetic predisposition is one of the most important risk factors of MBC with BRCA2 being the most commonly mutated gene in males diagnosed with breast cancer. However, a large part of MBC heritability is still unexplained. This study sought to add to the data already available on the genetics of MBC. MATERIALS AND METHODS: Our study initially involved comprehensive analysis of BRCA1 and BRCA2, followed by analysis of 43 genes implicated in cancer predisposition in a series of 100 Greek patients diagnosed with MBC between 1995-2015. RESULTS: Pathogenic variants were identified in 13 patients, with BRCA2 being the most commonly affected gene, followed by BRCA1, RAD50, RAD51B, and MSH3. CONCLUSION: In agreement with previous reports, BRCA2 is the most important genetic factor of MBC predisposition, while the remaining known cancer predisposition genes are each very rarely involved, rendering conclusions as to their cumulative effect difficult to draw.

Revisiting the Implications of Positive Germline Testing Results Using Multi-gene Panels in Breast Cancer Patients.

BACKGROUND/AIM: The use of multi-gene panels for germline testing in breast cancer enables the estimation of cancer risk and guides risk-reducing management options. The aim of this study was to present data that demonstrate the different levels of actionability for multi-gene panels used in genetic testing of breast cancer patients and their family members. MATERIALS AND METHODS: We performed an analysis in our clinical database to identify breast cancer patients undergoing genetic testing. We reviewed positive results in respect of risk estimation and management, cascade family testing, secondary findings and information for treatment decision-making. RESULTS: A total of 415 positive test reports were identified with 57.1%, 18.1%, 10.8% and 13.5% of individuals having pathogenic/likely pathogenic variants in high, moderate, low and with insufficient evidence for breast cancer risk genes, respectively. Six point seven percent of individuals were double heterozygotes. CONCLUSION: Germline findings in 92% of individuals are linked to evidence-based treatment information and risk estimates for predisposition to breast and/or other cancer types. The use of germline findings for treatment decision making expands the indication of genetic testing to include individuals that could benefit from targeted treatments.

Genetic variation in the CYP17 gene and recurrent spontaneous abortions.

PURPOSE: The CYP17 gene encodes the enzyme cytochrome P450c17α, which functions at key steps in the synthesis process of human sex steroid hormones. A T/C polymorphism in the 5' promoter region of the CYP17 gene has been described previously. Serum levels of androgens and estrogens have been shown to be elevated in individuals who carry the C substitution (Α2 allele). We hypothesized that variability in genes that control the sex hormone (estrogens, testosterone) biosynthesis might affect the pregnancy outcome. In the present study, we investigated the possible association between the T/C polymorphism of the promoter of CYP17 gene and the risk of recurrent spontaneous abortions in the Greek population. METHODS: In the prospective case-control study, 148 patients and 134 healthy controls were studied. Women who had at least three unexplained spontaneous abortions before 20 weeks of gestation were included in the patient group. The PCR-RFLP method was used to genotype the subjects. RESULTS: The frequencies of A1A1, A1A2, A2A2 genotypes were 0.34, 0.52, 0.14, respectively, in the patient group and 0.32, 0.47, 0.21, respectively, in the control group. The allele frequencies were 0.595 and 0.405 for A1 and A2, in the patient group and 0.555 and 0.445 for A1 and A2, respectively, in the control group. The data between the two groups were analyzed by χ(2) test. Our results showed that there are no significant differences in genotype (P = 0.3883) or in allele frequencies (P = 0.3800) between the patient and the control group. CONCLUSION: The T/C promoter polymorphism of the CYP17 gene is not associated with the risk for recurrent spontaneous abortions in our Caucasian population.

miRNA polymorphisms and risk of premature coronary artery disease.

OBJECTIVE: Several microRNA (miRNA) polymorphisms have been associated with susceptibility to specific health disorders, including cardiovascular diseases. The aim of the present study was to investigate whether four well-studied miRNA polymorphisms in non-Caucasian populations, namely miR146a G>C (rs2910164), miR149 C>T (rs2292832), miR196a2 C>T (rs11614913) and miR499 A>G (rs3746444), contribute to the risk for the development of premature Coronary Artery Disease (CAD) in the Greek population. METHODS: We used a case-control study to examine these associations in 400 individuals: 200 CAD patients [including a subgroup of myocardial infraction (MI) patients] and 200 healthy controls, all of Greek origin. MiRNA polymorphisms were genotyped using three different assays: Polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP), High resolution Melting (HRM) and Sanger sequencing. RESULTS: Two of these polymorphisms, miR196a2 C>T (rs11614913) and miR499 A>G (rs3746444) were found to be strongly associated with increased risk for CAD (p=0.0388 and p=0.0013, respectively) and for MI (p=0.0281 and p=0.0273, respectively). Furthermore, miR146C-miR149C-miR196T-miR499G allele combination appeared to be significantly related to CAD (p=0.0185) and MI (p=0.0337) prevalence. CONCLUSIONS: Our results suggest that at least two of the studied polymorphisms, miR196a2 C>T (rs11614913) and miR499 A>G (rs3746444), as well as the miR146C-miR149C-miR196T-miR499G allele combination could represent useful biomarkers of CAD and/or MI susceptibility in the Greek population. These special genetic characteristics, in combination with environmental factors and personal habits, might contribute to CAD and/or MI prevalence.

Clinical Utility of Functional RNA Analysis for the Reclassification of Splicing Gene Variants in Hereditary Cancer.

BACKGROUND: Classification of splicing variants (SVs) in genes associated with hereditary cancer is often challenging. The aim of this study was to investigate the occurrence of SVs in hereditary cancer genes and the clinical utility of RNA analysis. MATERIAL AND METHODS: 1518 individuals were tested for cancer predisposition, using a Next Generation Sequencing (NGS) panel of 36 genes. Splicing variant analysis was performed using RT-PCR and Sanger Sequencing. RESULTS: In total, 34 different SVs were identified, 53% of which were classified as pathogenic or likely pathogenic. The remaining 16 variants were initially classified as Variant of Uncertain Significance (VUS). RNA analysis was performed for 3 novel variants. CONCLUSION: The RNA analysis assisted in the reclassification of 20% of splicing variants from VUS to pathogenic. RNA analysis is essential in the case of uncharacterized splicing variants, for proper classification and personalized management of these patients.

Report of a germline double heterozygote in MSH2 and PALB2.

BACKGROUND: Carriers with pathogenic variants in MSH2 have increased risk to develop colorectal, endometrium, ovarian, and other types of cancer. The PALB2 is associated with breast, ovarian, pancreatic, and prostate cancer. We describe the case of a 42-year-old female diagnosed with endometrial cancer at the age of 42 years with a strong family history of colorectal cancer, which was referred to our private diagnostic laboratory for genetic testing. METHODS: In this study, we performed next-generation sequencing (NGS) using an amplicon based 26 genes panel. The presence of multi-exonic copy number variations (CNVs) was investigated by computational analysis and Multiplex Ligation-dependent Probe Amplification (MLPA). RESULTS: A gross deletion of the genomic region encompassing exons 11-16 of the MSH2 and the loss-of-function variant c.757_758delCT, p.(Leu253Ilefs*3) in the PALB2 were identified in the proband. CONCLUSIONS: Multigene analysis using NGS technology allows the identification of pathogenic variants in genes that would normally not be tested based on the patient diagnosis. In our case these results explained not only the personal and/or family history of cancer but also allowed the surveillance for prevention of other cancer types. Moreover, the detection of large genomic rearrangements should be routinely included in hereditary cancer testing.