A method to comprehensively identify germline SNVs, INDELs and CNVs from whole exome sequencing data of BRCA1/2 negative breast cancer patients.

In the rapidly evolving field of genomics, understanding the genetic basis of complex diseases like breast cancer, particularly its familial/hereditary forms, is crucial. Current methods often examine genomic variants-such as Single Nucleotide Variants (SNVs), insertions/deletions (Indels), and Copy Number Variations (CNVs)-separately, lacking an integrated approach. Here, we introduced a robust, flexible methodology for a comprehensive variants' analysis using Whole Exome Sequencing (WES) data. Our approach uniquely combines meticulous validation with an effective variant filtering strategy. By reanalyzing two germline WES datasets from BRCA1/2 negative breast cancer patients, we demonstrated our tool's efficiency and adaptability, uncovering both known and novel variants. This contributed new insights for potential diagnostic, preventive, and therapeutic strategies. Our method stands out for its comprehensive inclusion of key genomic variants in a unified analysis, and its practical resolution of technical challenges, offering a pioneering solution in genomic research. This tool presents a breakthrough in providing detailed insights into the genetic alterations in genomes, with significant implications for understanding and managing hereditary breast cancer.

The Diverse Genomic Landscape of Diamond-Blackfan Anemia: Two Novel Variants and a Mini-Review.

Diamond-Blackfan anemia (DBA) is a ribosomopathy characterized by bone marrow erythroid hypoplasia, which typically presents with severe anemia within the first months of life. DBA is typically attributed to a heterozygous mutation in a ribosomal protein (RP) gene along with a defect in the ribosomal RNA (rRNA) maturation or levels. Besides classic DBA, DBA-like disease has been described with variations in 16 genes (primarily in GATA1, followed by ADA2 alias CECR1, HEATR3, and TSR2). To date, more than a thousand variants have been reported in RP genes. Splice variants represent 6% of identifiable genetic defects in DBA, while their prevalence is 14.3% when focusing on pathogenic and likely pathogenic (P/LP) variants, thus highlighting the impact of such alterations in RP translation and, subsequently, in ribosome levels. We hereby present two cases with novel pathogenic splice variants in RPS17 and RPS26. Associations of DBA-related variants with specific phenotypic features and malignancies and the molecular consequences of pathogenic variations for each DBA-related gene are discussed. The determinants of the spontaneous remission, cancer development, variable expression of the same variants between families, and selectivity of RP defects towards the erythroid lineage remain to be elucidated.

Identifying novel regulatory effects for clinically relevant genes through the study of the Greek population.

BACKGROUND: Expression quantitative trait loci (eQTL) studies provide insights into regulatory mechanisms underlying disease risk. Expanding studies of gene regulation to underexplored populations and to medically relevant tissues offers potential to reveal yet unknown regulatory variants and to better understand disease mechanisms. Here, we performed eQTL mapping in subcutaneous (S) and visceral (V) adipose tissue from 106 Greek individuals (Greek Metabolic study, GM) and compared our findings to those from the Genotype-Tissue Expression (GTEx) resource. RESULTS: We identified 1,930 and 1,515 eGenes in S and V respectively, over 13% of which are not observed in GTEx adipose tissue, and that do not arise due to different ancestry. We report additional context-specific regulatory effects in genes of clinical interest (e.g. oncogene ST7) and in genes regulating responses to environmental stimuli (e.g. MIR21, SNX33). We suggest that a fraction of the reported differences across populations is due to environmental effects on gene expression, driving context-specific eQTLs, and suggest that environmental effects can determine the penetrance of disease variants thus shaping disease risk. We report that over half of GM eQTLs colocalize with GWAS SNPs and of these colocalizations 41% are not detected in GTEx. We also highlight the clinical relevance of S adipose tissue by revealing that inflammatory processes are upregulated in individuals with obesity, not only in V, but also in S tissue. CONCLUSIONS: By focusing on an understudied population, our results provide further candidate genes for investigation regarding their role in adipose tissue biology and their contribution to disease risk and pathogenesis.

A Novel Variant in the TP53 Gene Causing Li-Fraumeni Syndrome.

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer syndrome associated with germline pathogenic variants in the tumor protein p53 (TP53) gene and elevated risk of a broad range of early-onset malignancies. Patients with LFS are at risk of a second and third primary tumor. A 15-month-old girl consulted for clitoromegaly and pubic hair. Adrenal ultrasound detected a large left adrenal tumor. Left total adrenalectomy confirmed adrenocortical carcinoma. Family history revealed multiple highly malignant neoplasms at an early age across five generations, and a genetic dominant trait seemed probable. Whole-genome sequencing was performed. Multiple members of the family were found positive for a novel likely pathogenic variant (c. 892delGinsTTT, p. Glu298PhefsX48, NM_000546.6) in the TP53 gene, causing the loss of normal protein function through non-sense-mediated mRNA decay. According to the PSV1 supporting criteria and the Auto PVS1 online tool this frameshift variant: hg19/17-7577045-TC-TAAA:NM_000546.6 has a very strong, definitive clinical validity for LFS with autosomal dominant inheritance. Proper guidance resulted in timely diagnosis of a second tumor (primary osteosarcoma) in the index case and in the early detection of breast and cervical cancer in her young mother. Patients with cancer predisposition syndromes like LFS require close multidisciplinary cancer surveillance and appropriate referral to expert centers.

Rare variant enrichment analysis supports GREB1L as a contributory driver gene in the etiology of Mayer-Rokitansky-Küster-Hauser syndrome.

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is characterized by aplasia of the female reproductive tract; the syndrome can include renal anomalies, absence or dysgenesis, and skeletal anomalies. While functional models have elucidated several candidate genes, only WNT4 (MIM: 603490) variants have been definitively associated with a subtype of MRKH with hyperandrogenism (MIM: 158330). DNA from 148 clinically diagnosed MRKH probands across 144 unrelated families and available family members from North America, Europe, and South America were exome sequenced (ES) and by family-based genomics analyzed for rare likely deleterious variants. A replication cohort consisting of 442 Han Chinese individuals with MRKH was used to further reproduce GREB1L findings in diverse genetic backgrounds. Proband and OMIM phenotypes annotated using the Human Phenotype Ontology were analyzed to quantitatively delineate the phenotypic spectrum associated with GREB1L variant alleles found in our MRKH cohort and those previously published. This study reports 18 novel GREB1L variant alleles, 16 within a multiethnic MRKH cohort and two within a congenital scoliosis cohort. Cohort-wide analyses for a burden of rare variants within a single gene identified likely damaging variants in GREB1L (MIM: 617782), a known disease gene for renal hypoplasia and uterine abnormalities (MIM: 617805), in 16 of 590 MRKH probands. GREB1L variant alleles, including a CNV null allele, were found in 8 MRKH type 1 probands and 8 MRKH type II probands. This study used quantitative phenotypic analyses in a worldwide multiethnic cohort to identify and strengthen the association of GREB1L to isolated uterine agenesis (MRKH type I) and syndromic MRKH type II.

Perturbations of genes essential for Müllerian duct and Wölffian duct development in Mayer-Rokitansky-Küster-Hauser syndrome.

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is associated with congenital absence of the uterus, cervix, and the upper part of the vagina; it is a sex-limited trait. Disrupted development of the Müllerian ducts (MD)/Wölffian ducts (WD) through multifactorial mechanisms has been proposed to underlie MRKHS. In this study, exome sequencing (ES) was performed on a Chinese discovery cohort (442 affected subjects and 941 female control subjects) and a replication MRKHS cohort (150 affected subjects of mixed ethnicity from North America, South America, and Europe). Phenotypic follow-up of the female reproductive system was performed on an additional cohort of PAX8-associated congenital hypothyroidism (CH) (n = 5, Chinese). By analyzing 19 candidate genes essential for MD/WD development, we identified 12 likely gene-disrupting (LGD) variants in 7 genes: PAX8 (n = 4), BMP4 (n = 2), BMP7 (n = 2), TBX6 (n = 1), HOXA10 (n = 1), EMX2 (n = 1), and WNT9B (n = 1), while LGD variants in these genes were not detected in control samples (p = 1.27E-06). Interestingly, a sex-limited penetrance with paternal inheritance was observed in multiple families. One additional PAX8 LGD variant from the replication cohort and two missense variants from both cohorts were revealed to cause loss-of-function of the protein. From the PAX8-associated CH cohort, we identified one individual presenting a syndromic condition characterized by CH and MRKHS (CH-MRKHS). Our study demonstrates the comprehensive utilization of knowledge from developmental biology toward elucidating genetic perturbations, i.e., rare pathogenic alleles involving the same loci, contributing to human birth defects.

Exome Sequencing in BRCA1- and BRCA2-Negative Greek Families Identifies MDM1 and NBEAL1 as Candidate Risk Genes for Hereditary Breast Cancer.

Approximately 10% of breast cancer (BC) cases are hereditary BC (HBC), with HBC most commonly encountered in the context of hereditary breast and ovarian cancer (HBOC) syndrome. Although thousands of loss-of-function (LoF) alleles in over 20 genes have been associated with HBC susceptibility, the genetic etiology of approximately 50% of cases remains unexplained, even when polygenic risk models are considered. We focused on one of the least-studied European populations and applied whole-exome sequencing (WES) to 52 individuals from 17 Greek HBOC families, in which at least one patient was negative for known HBC risk variants. Initial screening revealed pathogenic variants in known cancer genes, including BARD1:p.Trp91* detected in a cancer-free individual, and MEN1:p.Glu260Lys detected in a BC patient. Gene- and variant-based approaches were applied to exome data to identify candidate risk variants outside of known risk genes. Findings were verified in a collection of Canadian HBOC patients of European ancestry (FBRCAX), in an independent group of Canadian BC patients (CHUM-BC) and controls (CARTaGENE), as well as in individuals from The Cancer Genome Atlas (TCGA) and the UK Biobank (UKB). Rare LoF variants were uncovered in MDM1 and NBEAL1 in Greek and Canadian HBOC patients. We also report prioritized missense variants SETBP1:c.4129G > C and C7orf34:c.248C > T. These variants comprise promising candidates whose role in cancer pathogenicity needs to be explored further.

Prevalence of BRCA1 mutations among 403 women with triple-negative breast cancer: implications for genetic screening selection criteria: a Hellenic Cooperative Oncology Group Study.

In spite the close association of the triple-negative breast cancer immunophenotype with hereditary breast cancers and the BRCA1 pathway, there is a lack of population studies that determine the frequency of BRCA1 mutations among triple-negative breast cancer patients. To address this, we have screened a large sample of 403 women diagnosed with triple-negative invasive breast cancer, independently of their age or family history, for germline BRCA1 mutations. Median age at diagnosis was 50 years (range 20-83). The overall prevalence of triple-negative cases among the initial patient group with invasive breast cancer was 8%. BRCA1 was screened by direct DNA sequencing in all patients, including all exons where a mutation was previously found in the Greek population (exons 5, 11, 12, 16, 20, 21, 22, 23, 24-77% of the BRCA1 coding region), including diagnostic PCRs to detect the three Greek founder large genomic rearrangements. Sixty-five deleterious BRCA1 mutations were identified among the 403 triple-negative breast cancer patients (16%). Median age of onset for mutation carriers was 39 years. Among a total of 106 women with early-onset triple-negative breast cancer (<40 years), 38 (36%) had a BRCA1 mutation, while 27% of women with triple-negative breast cancer diagnosed before 50 years (56/208) had a BRCA1 mutation. A mutation was found in 48% (50/105) of the triple-negative breast cancer patients with family history of breast or ovarian cancer. It is noteworthy, however, that of the 65 carriers, 15 (23%) had no reported family history of related cancers. All but one of the carriers had grade III tumors (98%). These results indicate that women with early-onset triple-negative breast cancer, and ideally all triple-negative breast cancer patients, are candidates for BRCA1 genetic testing even in the absence of a family history of breast or ovarian cancer.

Molecular comparison of single cell MDA products derived from different cell types.

The quality of DNA obtained from single cells for molecular analysis is primarily dependent on cell type and cell lysis. Multiple displacement amplification (MDA) amplifies the DNA isothermally with the use of Phi29 polymerase and random hexamer primers. The efficiency and accuracy of MDA was assessed on different cell types (buccal cells, lymphocytes, fibroblasts) using two multiplex PCR reactions that have been applied in clinical preimplantation genetic diagnosis cases (DM triplex-DM1, APOC2, Dl9S112 and CF triplex-DF508del, IVS8CA, IVS17TA). These results were compared using the DM triplex with MDA products from single blastomeres. Cells were lysed using a modified protocol excluding dithiothreitol in the alkaline lysis buffer. The MDA amplification efficiency for buccal cells was 82.0% (41/50) compared with 96.0% (48/50) for lymphocytes and 100% (20/20) for fibroblasts. The average allele dropout (ADO) rates were 31.0% for buccal cells, 20.8% for lymphocytes and 20.0% for fibroblasts with high inter-locus variation across all cell types (5.0-45.5%). Overall, MDA on single lymphocytes and fibroblasts lysed using the modified protocol produced DNA of sufficient quantity and quality for subsequent molecular analysis by PCR and gave results comparable with MDA products from blastomeres, in contrast to buccal cells.

Women's perception of information and experiences of nuchal translucency screening in Greece.

OBJECTIVE: First trimester ultrasound (US) screening has become part of antenatal care. The aim of this study was to explore pregnant women's perceptions of the information given and experiences of first trimester nuchal translucency (NT) screening and to identify the background factors in a woman's biography that influence her information needs and experiences of NT screening. METHODS: This study was a descriptive, prospective survey which involved collecting information from the participants by using a questionnaire and took place in a public hospital in Athens. The sample consisted of 510 consecutive, unselected pregnant women of gestational age between 11 and 14 weeks, who had attended a nuchal translucency (NT) screening. RESULTS: The majority of women felt that they had received either a limited or intermediate amount of information about the US screening and stated that they would have liked to receive more information. However, most of them (98%) answered that their expectations were fulfilled. 82% felt that the US examination was a positive experience, while 16% felt that US examination was a stressful experience. Furthermore, higher rates of positive experiences were observed among women with a higher educational level and greater satisfaction with the information received (chi(2)((d.f.=6)) = 29.411, p = 0.000, chi(2)((d.f.=3)) = 30.171, p = 0.000 respectively). Most women had wished to undertake NT screening to ensure that the fetus was healthy and without any defects and believed that a US examination should be performed at every antenatal visit. CONCLUSION: This study shows that most women lack information, specifically about the purposes and the diagnostic limitations of NT screening. Health professionals should therefore dedicate more time to discussing with women, and provide appropriate and understandable information tailored to the educational level of women and should emphasize the indications, purposes and limitations of US screening.