BRCA1, a crucial tumor suppressor gene, has several splicing isoforms, including Δ9–11, Δ11, and Δ11q, which lack exon 11, coding for significant portions of the protein. These isoforms are naturally ...present in both normal and cancerous cells, exhibiting altered activity compared to the full-length BRCA1. Despite this, the impact on cancer risk of the germline intronic variants promoting the exclusive expression of these Δ11 isoforms remains uncertain. Consequently, they are classified as variants of uncertain significance (VUS), posing challenges for traditional genetic classification methods due to their rarity and complexity. Our research utilizes a yeast-based functional assay, previously validated for assessing missense BRCA1 variants, to compare the activity of the Δ11 splicing isoforms with known pathogenic missense variants. This approach allows us to elucidate the functional implications of these isoforms and determine whether their exclusive expression could contribute to increased cancer risk. By doing so, we aim to provide insights into the pathogenic potential of intronic VUS-generating BRCA1 splicing isoforms and improve the classification of BRCA1 variants.
BRCA1 (breast cancer 1, early onset) missense mutations have been detected in familial breast and ovarian cancers, but the role of these variants in cancer predisposition is often difficult to ...ascertain. In this work, the molecular mechanisms affected in human cells by two BRCA1 missense variants, M1775R and A1789T, both located in the second BRCT (BRCA1 C Terminus) domain, have been investigated. Both these variants were isolated from familial breast cancer patients and the study of their effect on yeast cell transcriptome has previously provided interesting clues to their possible role in the pathogenesis of breast cancer.
We compared by Human Whole Genome Microarrays the expression profiles of HeLa cells transfected with one or the other variant and HeLa cells transfected with BRCA1 wild-type. Microarray data analysis was performed by three comparisons: M1775R versus wild-type (M1775RvsWT-contrast), A1789T versus wild-type (A1789TvsWT-contrast) and the mutated BRCT domain versus wild-type (MutvsWT-contrast), considering the two variants as a single mutation of BRCT domain.
201 differentially expressed genes were found in M1775RvsWT-contrast, 313 in A1789TvsWT-contrast and 173 in MutvsWT-contrast. Most of these genes mapped in pathways deregulated in cancer, such as cell cycle progression and DNA damage response and repair.
Our results represent the first molecular evidence of the pathogenetic role of M1775R, already proposed by functional studies, and give support to a similar role for A1789T that we first hypothesized based on the yeast cell experiments. This is in line with the very recently suggested role of BRCT domain as the main effector of BRCA1 tumor suppressor activity.
Germline mutations in the BRCA1 gene have been reported to increase the lifetime risk of developing breast and/or ovarian cancer (BOC). By new sequencing technologies, numerous variants of uncertain ...significance (VUS) are identified. It is mandatory to develop new tools to evaluate their functional impact and pathogenicity. As the expression of pathogenic BRCA1 variants in
increases the frequency of intra- and inter-chromosomal homologous recombination (HR), and gene reversion (GR), we validated the two HR and the GR assays by testing 23 benign and 23 pathogenic variants and compared the results with those that were obtained in the small colony phenotype (SCP) assay, an additional yeast-based assay, that was validated previously. We demonstrated that they scored high accuracy, sensitivity, and sensibility. By using a classifier that was based on majority of voting, we have integrated data from HR, GR, and SCP assays and developed a reliable method, named yBRCA1, with high sensitivity to obtain an accurate VUS functional classification (benign or pathogenic). The classification of BRCA1 variants, important for assessing the risk of developing BOC, is often difficult to establish with genetic methods because they occur rarely in the population. This study provides a new tool to get insights on the functional impact of the BRCA1 variants.
Fertility remains a challenge for men with 5α-reductase-2 deficiency. Such a diagnosis was made in 2 adult brothers who are compound heterozygous for the 5α-reductase type 2 gene (SRD5A2; c.308G>C; ...c.689A>C). They were born with ambiguous genitalia and the male sex was assigned. Both brothers underwent reconstructive genital surgery during pediatric age and had spontaneous virilization at puberty. The older brother experienced natural conception, while the younger had a son by assisted reproductive technology. Other family members were demonstrated to be compound heterozygous or heterozygous for the same genetic variants. The older brother is the third man with 5α-reductase-2 deficiency and spontaneous paternity. The little series of men with 5α-reductase-2 deficiency and documented spontaneous or assisted paternity is reviewed. In conclusion, the possibility of fatherhood is a main indication for male sex assignment in patients with 5α-reductase-2 deficiency.
Approximately 30–50% of hereditary breast and ovarian cancer (HBOC) is due to the presence of germline pathogenic variants in the BRCA1 (OMIM 113705) and BRCA2 (OMIM 600185) onco-suppressor genes, ...which are involved in DNA damage response. Women who carry pathogenic BRCA1 variants are particularly likely to develop breast cancer (BC) and ovarian cancer (OC), with a 45–79 percent and 39–48 percent chance, respectively. The BRCA1 c.4096+1G>A variant has been frequently ascertained in Tuscany, Italy, and it has also been detected in other Italian regions and other countries. Its pathogenetic status has been repeatedly changed from a variant of uncertain significance, to pathogenic, to likely pathogenic. In our study, 48 subjects (38 of whom are carriers) from 27 families were genotyped with the Illumina OncoArray Infinium platform (533,531 SNPs); a 20 Mb region (24.6 cM) around BRCA1, including 4130 SNPs (21 inside BRCA1) was selected for haplotype analysis. We used a phylogenetic method to estimate the time to the most recent common ancestor (MRCA) of BRCA1 c.4096+1G>A founder pathogenic variant. This analysis suggests that the MRCA lived about 155 generations ago—around 3000 years ago.
Blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES) is due to heterozygous FOXL2 intragenic mutations in about 70% of the patients, whereas total or partial gene deletions account for a ...minority of cases. Alteration of FOXL2 regulatory elements has been rarely described in patients with BPES. In this study, a prepubertal girl with BPES due to a 197-kb de novo deletion of the regulatory elements upstream of FOXL2 is reported . This girl presented with additional clinical features such as a soft cleft palate and microcephaly; thus, this copy number variant might have other somatic effects. The present deletion encompasses 2 coding genes ( MRPS22 and COPB2 ), whose homozygous mutations have been associated with microcephaly. In our case, the sequences of the non-deleted allele were normal, ruling out a compound genetic defect. Normal levels of new biomarkers of ovarian reserve (anti-müllerian hormone, inhibin B) likely indicate an early diagnosis of type 2 BPES, but an evolutive gonadal damage will be excluded only by long-term follow-up. Additional reports of microdeletions upstream of FOXL2 are needed to better define the underlying genetic mechanism and the related phenotypic spectrum; the ability of the new hormonal markers to predict ovarian function in adolescence and adulthood should be confirmed.
Multiple Endocrine Neoplasia type 1 is a rare genetic syndrome mainly caused by mutations of
gene and characterized by a combination of several endocrine and non-endocrine manifestations. The ...objective of this study was to describe cutaneous lesions and other non-endocrine manifestations of
in a cohort of patients with familial (F) and sporadic (S)
, compare the prevalence of these manifestations between the two cohorts, and investigate the correlation with
mutation status.
We collected phenotypic and genotypic data of 185 patients with F-
and S-
followed from 1997 to 2022. The associations between F-
and S-
or
mutation-positive and mutation-negative patients and non-endocrine manifestations were determined using chi-square or Fisher's exact tests or multivariate exact logistic regression analyses.
The prevalence of angiofibromas was significantly higher in F-
than in S-
in both the whole (p < 0.001) and index case (p = 0.003) cohorts. The prevalence of lipomas was also significantly higher in F-
than in S-
(p = 0.009) and in
mutation-positive than in
mutation-negative (p = 0.01) index cases. In the whole cohort, the prevalence of lipomas was significantly higher in
mutation-positive compared to
mutation-negative patients (OR = 2.7, p = 0.02) and in F-
than in S-
(p = 0.03), only after adjustment for age. No significant differences were observed for the other non-endocrine manifestations between the two cohorts. Hibernoma and collagenoma were each present in one patient (0.5%) and meningioma and neuroblastoma in 2.7% and 0.5%, respectively. Gastric leiomyoma was present in 1.1% of the patients and uterine leiomyoma in 14% of women. Thyroid cancer, breast cancer, lung cancer, basal cell carcinoma, melanoma, and colorectal cancer were present in 4.9%, 2.7%, 1.6%, 1.6%, 2.2%, and 0.5% of the whole series, respectively.
We found a significantly higher prevalence of angiofibromas and lipomas in F-
compared with S-
and in
mutation-positive compared to
mutation-negative patients. In patients with one major endocrine manifestation of
, the presence of cutaneous lesions might suggest the diagnosis of
and a possible indication for genetic screening.
With the progress of sequencing technologies, an ever-increasing number of variants of unknown functional and clinical significance (VUS) have been identified in both coding and non-coding regions of ...the main Breast Cancer (BC) predisposition genes. The aim of this study is to identify a mutational profile of coding and intron-exon junction regions of 12 moderate penetrance genes (ATM, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53) in a cohort of 450 Italian patients with Hereditary Breast/Ovarian Cancer Syndrome, wild type for germline mutation in BRCA1/2 genes. The analysis was extended to 5′UTR and 3′UTR of all the genes listed above and to the BRCA1 and BRCA2 known regulatory regions in a subset of 120 patients. The screening was performed through NGS target resequencing on the Illumina platform MiSeq. 8.7% of the patients analyzed is carriers of class 5/4 coding variants in the ATM (3.6%), BRIP1 (1.6%), CHEK2 (1.8%), PALB2 (0.7%), RAD51C (0.4%), RAD51D (0.4%), and TP53 (0.2%) genes, while variants of uncertain pathological significance (VUSs)/class 3 were identified in 9.1% of the samples. In intron-exon junctions and in regulatory regions, variants were detected respectively in 5.1% and in 32.5% of the cases analyzed. The average age of disease onset of 44.4 in non-coding variant carriers is absolutely similar to the average age of disease onset in coding variant carriers for each proband’s group with the same cancer type. Furthermore, there is not a statistically significant difference in the proportion of cases with a tumor onset under age of 40 between the two groups, but the presence of multiple non-coding variants in the same patient may affect the aggressiveness of the tumor and it is worth underlining that 25% of patients with an aggressive tumor are carriers of a PTEN 3′UTR-variant. This data provides initial information on how important it might be to extend mutational screening to the regulatory regions in clinical practice.
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