CASR gene activating mutations in two families with autosomal dominant hypocalcemia.

BACKGROUND: Autosomal dominant hypocalcemia (ADH) is an endocrine disorder caused by activating mutations of the calcium-sensing receptor (CASR) gene which plays a major role in maintaining calcium homeostasis. Biochemical features of ADH are hypocalcemia and hypercalciuria with inappropriately low levels of parathyroid hormone (PTH). We report on two four-generation families affected by ADH. AIM: To identify mutations of CASR gene in subjects affected by familial idiopathic hypoparathyroidism. To perform functional assays of identified CASR variants by transient transfection on HEK293 cells. RESULTS: We identified two CASR variants (Q681R and P221L): the Q681R variant was novel while the P221L had been previously published. Functional assays on the Q681R variant showed that it did not alter the whole expression nor the correct plasmamembrane localization, but enhanced the signaling function, increasing the sensitivity of the receptor as compared to the WT. CONCLUSIONS: We report two activating CASR mutations in two families affected by ADH and the functional assays performed on the novel variant Q681R. Our work enlarged the spectrum of mutations of the CASR and contributed to a better elucidation of the protein function.

High RAD51 mRNA expression characterize estrogen receptor-positive/progesteron receptor-negative breast cancer and is associated with patient's outcome.

Mutations in DNA double-strand breaks (DSB) repair genes are involved in the pathogenesis of hereditary mammary tumors, it is, however, still unclear whether defects in this pathway may play a role in sporadic breast cancer. In this study, we initially determined mRNA expression of 15 DSB related genes by reverse transcription quantitative polymerase chain reaction in paired normal tissue and cancer specimen from 20 breast cancer cases to classify them into homogeneous clusters. G22P1/ku70, ATR and RAD51 genes were differentially expressed in the three branches recognized by clustering analysis. In particular, a breast cancer subgroup characterized by high RAD51 mRNA levels and estrogen receptor (ER)-positive/progesteron receptor (PR)-negative phenotype was identified. This result was confirmed by the analysis of G22P1/ku70, ATR and RAD51 mRNA levels on paired normal and tumor specimens from an extended breast cancer cohort (n = 75). RAD51 mRNA levels were inversely associated with PR status (p = 0.02) and the highest levels were, indeed, detected in ER-positive/PR-negative tumors (p = 0.03). RAD51 immunostaining of a tissue microarray confirmed the inverse relationship between high RAD51 expression and negative PR status (p = 0.002), as well as, the association with ER-positive/PR-negative phenotype (p = 0.003). Interestingly, the analysis of microarray expression data from 295 breast cancers indicate that RAD51 increased mRNA expression is associated with higher risk of tumor relapse, distant metastases and worst overall survival (p = 0.015, p = 0.009 and p = 0.013 respectively). Our results suggest that RAD51 expression determination could contribute to a better molecular classification of mammary tumors and may represent a novel tool for evaluating postoperative adjuvant therapy for breast cancer patients.

Large rearrangements detected by MLPA, point mutations, and survey of the frequency of mutations within the SLC3A1 and SLC7A9 genes in a cohort of 172 cystinuric Italian patients.

Cystinuria is a rare inherited disorder characterized by defective renal reabsorption of cystine and the dibasic amino acids. SLC3A1 and SLC7A9 have been identified as responsible genes. The large majority of the more than 200 mutations so far identified in the two genes are point mutations, while only few alleles carrying gross genomic alterations have been reported. We screened 39 cystinuric patients for large rearrangements, by two home-made multiplex ligation-dependent probe amplification (MLPA) assays. MLPA analysis led to the identification of 6 different alleles in SLC3A1 and 2 in SLC7A9 accounting for a total of 25 copy number changes, 11 in SLC3A1 and 14 in SLC7A9. Three large rearrangements in SLC3A1, deletion of exons 2-4 (E2_E4del), deletion of exons 5-6 (E5_E6del) and duplication of exons 8-9 (E8_E9dup) are novel. A complete SLC7A9 gene deletion was found in three patients. In addition, we report the identification of three novel point mutations in SLC7A9 (p.G105E, p.R250K, c.1416_1417insAC), the frequency and the occurrence of cystinuria mutations in a cohort of 172 Italian patients. In conclusion, we developed a reliable and robust MLPA analytic method for SLC3A1 and SLC7A9 genes that represents an optimal complement to DNA sequence analysis in patients with cystinuria, enabling the screening for deletions and duplications.

Twenty-four novel mutations identified in a cohort of 85 patients by direct sequencing of the SLC3A1 and SLC7A9 cystinuria genes.

Mutations in the SLC3A1 and SLC7A9 genes cause cystinuria (OMIM 220100), an autosomal recessive disorder of amino acid transport and reabsorption in the proximal renal tubule and in the epithelial cells of the gastrointestinal tract. In an attempt to characterize the molecular defect in the SLC3A1 and SLC7A9 genes, we analyzed a cohort of 85 unrelated subjects clinically diagnosed as affected by cystinuria on the basis of stone formation, prevalently of Italian and Greek origin. Analysis of all coding region and exon-intron junctions of the SLC3A1 and SLC7A9 genes by using direct sequencing method allowed us to identify 62 different mutations in 83 out of 85 patients accounting for 90.5% of all affected chromosomes. Twenty-four out of 62 are novel mutations, 9 in SLC3A1 and 15 in SLC7A9. In conclusion, this report expands the spectrum of SLC3A1 and SLC7A9 mutations and confirms the heterogeneity of this disorder.

Indirect CFTR mutation identification by PCR/OLA anomalous electropherograms.

Mutations of CFTR gene are responsible for cystic fibrosis (CF) and other clinical conditions such as congenital absence of the vas deferens (CAVD), chronic pancreatitis (IP), and idiopathic disseminated bronchiectasis (DBE) classified as CFTR-related disorders. The PCR/OLA assay is designed to detect 31 known mutations including the 24 most common CF mutations worldwide, as identified by the CF Consortium. In order to define the CFTR genotype a series of 1812 individuals from central-southern Italy with and without CF manifestations were screened by using the PCR/OLA assay. Here we report the description of five cases of anomalous electropherograms obtained after PCR/OLA analysis, that led to the identification, in the homozygous state, of two point mutations (D110H and S589N) not included in the assay test panel, a large gene deletion (CFTRdel14b_17b), and an exonic polymorphism (c.4002A > G). Haplotype and real time PCR analysis were also performed in the subject carrying the large CFTR deletion. The study demonstrates that the PCR/OLA assay, besides being an efficient and user-friendly method to screen known mutations in the CFTR gene, may also function as a mutation/polymorphism-scanning assay, at least for certain nucleotide changes located in some critical regions of the gene.

Mutation analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the cationic trypsinogen (PRSS1) gene, and the serine protease inhibitor, Kazal type 1 (SPINK1) gene in patients with alcoholic chronic pancreatitis.

Susceptibility to alcoholic chronic pancreatitis (ACP) could be genetically determined. Mutations in cationic trypsinogen (PRSS1), cystic fibrosis transmembrane conductance regulator (CFTR), and serine protease inhibitor, Kazal type 1 (SPINK1) genes have been variably associated with both the hereditary and the idiopathic form of chronic pancreatitis (CP). Our aim was to analyze the three genes in ACP patients. Mutational screening was performed in 45 unrelated ACP patients and 34 patients with alcoholic liver disease (ALD). No mutation of PRSS1 was found in ACP and ALD patients. Three mutations of CFTR were detected in four ACP patients with a prevalence (8.9%) not significantly different from that observed (3.0%) in ALD patients and from that expected (3.2%) in our geographical area. Neither compound heterozygotes for CFTR nor trans-heterozygotes for CFTR/SPINK1 were found. One ACP patient (2.2%) was found to carry the most common mutation (N34S) of SPINK1 compared to none of the ALD patients (P=NS). In five other patients (two with ACP and three with ALD) other rare variants, including P55S, were found. In contrast with the hereditary and the idiopathic forms of CP, in which mutations of PRSS1, CFTR, and SPINK1 genes may occur, ACP is still a "gene(s)-orphan" disease. The supposed genetic susceptibility to ACP relies on other yet unknown gene(s) which could affect the alcohol metabolism or modulate the pancreatic inflammatory response to alcohol abuse.

20 novel point mutations and one large deletion in EXT1 and EXT2 genes: report of diagnostic screening in a large Italian cohort of patients affected by hereditary multiple exostosis.

BACKGROUND: Hereditary multiple exostosis represents the most frequent bone tumor disease in humans. It consists of cartilage deformities affecting the juxta-ephyseal region of long bones. Usually benign, exostosis could degenerate in malignant chondrosarcoma form in less than 5% of the cases. Being caused by mutations in the predicted tumor suppressor genes, EXT1 (chr 8q23-q24) and EXT2 (chr 11p11-p12) genes, HMEs are usually inherited with an autosomal dominant pattern, although "de novo" cases are not infrequent. AIM: Here we present our genetic diagnostic report on the largest Southern Italy cohort of HME patients consisting of 90 subjects recruited over the last 5years. RESULTS: Molecular screening performed by direct sequencing of both EXT1 and EXT2 genes, by MLPA and Array CGH analyses led to the identification of 66 mutations (56 different occurrences) and one large EXT2 deletion out of 90 patients (74.4%). The total of 21 mutations (20 different occurrences, 33.3%) and the EXT2 gene deletion were novel. In agreement with literature data, EXT1 gene mutations were scattered along all the protein sequence, while EXT2 lesions fell in the first part of the protein. Conservation, damaging prediction and 3-D modeling, in-silico, analyses, performed on three novel missense variants, confirmed that at least in two cases the novel aminoacidic changes could alter the structure stability causing a strong protein misfolding. CONCLUSIONS: Here we present 20 novel EXT1/EXT2 mutations and one large EXT2 deletion identified in the largest Southern Italy cohort of patients affected by hereditary multiple exostosis.