Anna Laura Putignano

Interleukin-10 promoter polymorphisms influence susceptibility to ulcerative colitis in a gender-specific manner

Objective. Pathological evidence supports a potential role of the pro- and anti-inflammatory cytokine network in the pathogenesis of inflammatory bowel disease (IBD). Moreover, associated studies suggest a possible involvement of cytokine-related genes in IBD susceptibility. In this study, we evaluated the effect of the anti-inflammatory interleukin-10 (IL10) gene on ulcerative colitis (UC). Material and methods. Two functional single nucleotide polymorphisms (−1082 G/A, −819 T/C) in the IL10 promoter in 203 Italian sporadic UC patients and 391 controls were determined using high-resolution melting analysis. Results. The frequency of the −1082A allele was significantly higher in the UC patients than in controls (p=0.00003); −1082 genotype frequencies were also significantly different between UC patients and controls (p=0.0001). Allele and genotype frequencies of −819 T/C were not significantly associated with UC. Furthermore, the frequencies of haplotypes −1082A/−819C and −1082A/−819T, which have been reported to have a lower promoter activity, were significantly higher in UC patients than in controls (p=0.0004). After gender stratification, we found a significant difference in the −1082A allele (p=0.00004) and genotype (p=0.0002) frequencies only between female UC patients and controls; the same result was obtained for the −1082A/−819C and −1082A/−819T haplotypes (p=0.0006). Conclusions. A gender effect is observed, with women of AG/AA IL10 genotypes and AC/AT haplotypes having a higher risk of developing UC at a younger age. This finding could be related to the previously documented lower IL10 production associated with the −1082A allele and to the IL10 down-regulating effect of estrogens.

FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor

Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer.

Susceptibility to Refractory Ulcerative Colitis Is Associated with Polymorphism in the hMLH1 Mismatch Repair Gene

The hMLH1 gene lies in the linkage susceptibility region to inflammatory bowel disease (IBD) on 3p21. A single nucleotide polymorphism, 655A>G, in exon 8 of the gene causes an I219V change in the MLH1 protein. To test whether hMLH1 may confer susceptibility to ulcerative colitis (UC), we investigated an association between the 655A>G polymorphism and the disease. DNA-based technologies were used to analyze the 655A>G polymorphism in 201 UC patients and 126 healthy ethnically matched controls. The comparison of the allelic frequencies of the 655A>G polymorphism in UC patients and healthy controls did not show significant differences. However, genotype frequencies at the hMLH1 655 position were found to be significantly different when patients with and without refractory UC were compared. This was mainly attributable to a higher level of homozygosity for the G allele in refractory UC patients. Almost 5 times as many (4.9 times) refractory UC patients carried the GG genotype compared with nonrefractory patients (P < 0.0001). The present study provides evidence that the hMLH1 gene is involved in genetic susceptibility to refractory UC. If confirmed by other studies, the GG genotype at position 655 of the hMLH1 gene may represent a useful predictive factor for the clinical management of UC patients.

Founder mutations account for the majority of BRCA1-attributable hereditary breast/ovarian cancer cases in a population from Tuscany, Central Italy

Background Germline mutations in the BRCA1 and BRCA2 tumour-suppressor genes predispose to early-onset breast and ovarian cancer. Although both genes display a highly heterogeneous mutation spectrum, a number of alterations recur in some populations. Only a limited number of founder mutations have been identified in the Italian population so far. Objective To investigate the spectrum of BRCA1/BRCA2 mutations in a set of families originary from the Central–Eastern part of Tuscany and to ascertain the presence of founder effects. We also wanted to approximate the age of the most frequent BRCA1 founder mutation. Results Overall, four distinct BRCA1 mutations accounted for a large fraction (72.7%) of BRCA1-attributable hereditary breast/ovarian cancer in families originary from this area. We identified common haplotypes for two newly recognised recurrent BRCA1 mutations, c.3228_3229delAG and c.3285delA. The c.3228_3229delAG mutation was estimated to have originated about 129 generations ago. Interestingly, male breast cancer cases were present in 3 out of 11 families with the c.3228_3229delAG mutation. Conclusions The observation that a high proportion of families with BRCA1 alterations from Central–Eastern Tuscany harbours a limited number of founder mutations can have significant impact on clinical management of at risk subjects from this area. In addition, the identification of a large set of families carrying an identical mutation that predisposes to breast and ovarian cancer provides unique opportunities to study the effect of other genetic and environmental factors on penetrance and disease phenotype.

Lack of association between TNF-α polymorphisms and Alzheimer’s disease in an Italian cohort

Tumor necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine that plays an important role in the inflammatory process that can be observed in Alzheimers disease (AD) brain. Different functional promoter polymorphisms within genes modulating inflammation have been demonstrated to elevate the AD risk; thus, we studied five common variations within the promoter region of the TNF-alpha gene in 609 subjects (253 AD patients and 356 controls). No positive associations were found, confirming the greater part of previous studies. Moreover, we also investigated the combined haplotypes of the five different polymorphisms without finding a positive association. Thus, the present investigation does not support the proposal that common nucleotide variations in the TNF-alpha gene can influence the development of AD at least in Italian population.

Characterization of an Italian Founder Mutation in the RING-Finger Domain of BRCA1

The identification of founder mutations in cancer predisposing genes is important to improve risk assessment in geographically defined populations, since it may provide specific targets resulting in cost-effective genetic testing. Here, we report the characterization of the BRCA1 c.190T>C (p.Cys64Arg) mutation, mapped to the RING-finger domain coding region, that we detected in 43 hereditary breast/ovarian cancer (HBOC) families, for the large part originating from the province of Bergamo (Northern Italy). Haplotype analysis was performed in 21 families, and led to the identification of a shared haplotype extending over three BRCA1-associated marker loci (0.4 cM). Using the DMLE+2.2 software program and regional population demographic data, we were able to estimate the age of the mutation to vary between 3,100 and 3,350 years old. Functional characterization of the mutation was carried out at both transcript and protein level. Reverse transcriptase-PCR analysis on lymphoblastoid cells revealed expression of full length mRNA from the mutant allele. A green fluorescent protein (GFP)-fragment reassembly assay showed that the p.Cys64Arg substitution prevents the binding of the BRCA1 protein to the interacting protein BARD1, in a similar way as proven deleterious mutations in the RING-domain. Overall, 55 of 83 (66%) female mutation carriers had a diagnosis of breast and/or ovarian cancer. Our observations indicate that the BRCA1 c.190T>C is a pathogenic founder mutation present in the Italian population. Further analyses will evaluate whether screening for this mutation can be suggested as an effective strategy for the rapid identification of at-risk individuals in the Bergamo area.

Germline mutations in MEN1 and BRCA1 genes in a woman with familial multiple endocrine neoplasia type 1 and inherited breast–ovarian cancer syndromes: a case report

The simultaneous occurrence of mutations in two different tumor suppressor genes in the same individual is a very rare event. Here we report the case of a woman in whom germline mutations in both MEN1 and BRCA1 were identified. The severity of MEN1-related biochemical and clinical findings did not significantly differ from that for other affected family members lacking the BRCA1 mutation, except for the development of an extremely large visceral lipoma; the proband has not developed any BRCA1-related malignancies. We explore genetic and molecular rationales for an association between these neoplastic processes.

Donor-Specific Anti-HLA Antibodies in Huntington's Disease Recipients of Human Fetal Striatal Grafts.

Fetal grafting in a human diseased brain was thought to be less immunogenic than other solid organ transplants, hence the minor impact on the efficacy of the transplant. How much prophylactic immune protection is required for neural allotransplantation is also debated. High-sensitive anti-HLA antibody screening in this field has never been reported. Sixteen patients with Huntingtons disease underwent human fetal striatal transplantation in the frame of an open-label observational trial, which is being carried out at Florence University. All patients had both brain hemispheres grafted in two separate robotic-stereotactic procedures. The trial started in February 2006 with the first graft to the first patient (R1). R16 was given his second graft on March 2011. All patients received triple immunosuppressive treatment. Pre- and posttransplant sera were analyzed for the presence of anti-HLA antibodies using the multiplexed microsphere-based suspension array Luminex xMAP technology. Median follow-up was 38.5 months (range 13-85). Six patients developed anti-HLA antibodies, which turned out to be donor specific. Alloimmunization occurred in a time window of 0–49 months after the first neurosurgical procedure. The immunogenic determinants were non-self-epitopes from mismatched HLA antigens. These determinants were both public epitopes shared by two or more HLA molecules and private epitopes unique to individual HLA molecules. One patient had non-donor-specific anti-HLA antibodies in her pretransplant serum sample, possibly due to previous sensitization events. Although the clinical significance of donor-specific antibodies is far from being established, particularly in the setting of neuronal transplantation, these findings underline the need of careful pre- and posttransplant immunogenetic evaluation of patients with intracerebral grafts.

Evaluation of a Next-Generation Sequencing Assay for BRCA1 and BRCA2 Mutation Detection

The efficiency of a novel targeted next-generation sequencing (NGS) test, the Devyser BRCA kit, for a comprehensive analysis of all 48 coding exons of the high-risk breast/ovarian cancer susceptibility genes BRCA1 and BRCA2 has been assessed. The new assay intended to detect nucleotide substitutions, small deletions/insertions, and large deletions/duplications. To document the false-negative and false-positive rates of the NGS assay in the hands of end users, 48 samples with previously identified 444 small variants and seven gross rearrangements were analyzed, showing 100% concordance with gold standards. Furthermore, all other 43 variants (42 single-nucleotide variation or insertion/deletion variation and one copy number variation, whose significance is or may be of clinical value), which were called by the NGS assay in a prospectively analyzed 179-sample set, were confirmed by Sanger sequencing or multiplex ligation probe amplification, according to their nature. We conclude that the Devyser BRCA kit performed satisfactorily for use in a clinical laboratory.

A novel PAX1 null homozygous mutation in autosomal recessive otofaciocervical syndrome associated with severe combined immunodeficiency

Otofaciocervical syndrome (OFCS) is a rare disorder characterized by facial anomalies, cup‐shaped low‐set ears, preauricular fistulas, hearing loss, branchial defects, skeletal anomalies, and mild intellectual disability. Autosomal dominant cases are caused by deletions or point mutations of EYA1. A single family with an autosomal recessive form of OFCS and a homozygous missense mutation in PAX1 gene has been described.