Macarena Gomez-Lira

Mutations in the SPINK1 gene in idiopathic pancreatitis Italian patients

Idiopathic chronic and acute recurrent pancreatitis (IP) have been associated with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Mutations in the serine protease inhibitor Kazal 1 (SPINK1) have been described in some idiopathic chronic patients and it has been suggested that mutations in this gene could be responsible for a loss of trypsin inhibitor function. In this study, the 5′UTR region, and the four exons and exon–intron boundaries of the SPINK1 gene in 32 IP patients have been analyzed. Three IP patients (9.3%) and one control/100 carried the N34S mutation of the SPINK1 gene (Fishers exact test, P=0.044). No other mutation that could be associated with an altered function of the SPINK1 protein was observed. The N34S mutation was present in two patients who carried the CFTR-IVS8 5T variant and in one who carried the L997F variant in the CFTR gene. The association of SPINK1 with CFTR gene mutations in IP patients is statistically significant (3/32 IP cases and 0/100 control individuals carrying mutations in both genes; Fishers exact test P=0.01).

CD45 and multiple sclerosis: the exon 4 C77G polymorphism (additional studies and meta-analysis) and new markers

We re-evaluated the association with multiple sclerosis (MS) of the C77G splicing regulatory variation in the CD45 gene and screened for new mutations the three alternatively spliced exons (#4, 5 and 6). No association with C77G was detected in two groups of patients (total=448) and controls (total=559) from Northern and Southern Italy. When excluding the first published study indicating a positive association, a meta-analysis of the five further studies conducted to date (including the present one) led to a non-significant combined odds ratio (OR) of 1.11. None of the four newly identified nucleotide substitutions, namely C77T (Pro59Pro) in exon 4, G69C (Asp121His) in exon 5, T127A (Ile187Asn) and A138G (Thr191Ala) in exon 6, was significantly associated to MS.

A base substitution at IVS-19 3'-end splice junction causes exon 20 skipping in pro alpha 2(I) collagen mRNA and produces mild osteogenesis imperfecta.

Molecular investigations on a young patient and her family were undertaken to identify the molecular defect responsible for a mild form of osteogenesis imperfecta (OI) with blue sclerae, dentinogenesis imperfecta and joint laxity. Analysis of collagenous proteins from the probands fibroblasts showed the presence of two populations of α2(I) chains, one normal and one migrating faster on SDS gels, thereby suggesting deletion of amino acid sequences. The faster migrating chains were retained mainly in the cell layer and not found in the extracellular matrix deposited by cultured fibroblasts. Chemical cleavage of mismatch (CCM) analysis on the patients proα2(I) mRNA:normal cDNA heteroduplexes localized the molecular defect. cDNA sequencing revealed a deletion of exon 20 (54 bp) in about half of the molecules. Genomic DNA sequencing revealed heterozygosity for a G-to-C transversion of the last nucleotide of intron 19, which changed the 3′ consensus splicing site. As a consequence proα2(I)mRNA was abnormally spliced from the last codon of exon 19 to the first codon of exon 21. To our knowledge, this is the first acceptor site mutation so far described in an OI patient. Restriction analysis indicated that the mutation was present also in three other affected family members. The full sequence of COL1A2 introns 19 and 20 are reported.

Molecular genetic characterization of two metachromatic leukodystrophy patients who carry the T799G mutation and show different phenotypes; description of a novel null-type mutation

Metachromatic leukodystrophy (MLD) is an autosomal recessive storage disease caused by deficiency of the lysosomal enzyme, arylsulfatase A. Two common mutations causing MLD have been characterized and correlations between phenotype and genotype have been established. A third common mutation, T799G, has also been identified in European MLD patients, and is associated with the late-onset forms of the disease. We report the molecular analysis of two Italian MLD patients, with juvenile and adult onset of the disease, respectively, who carried the T799G mutation in compound heterozygosity with different null mutations. A novel rapid mutation detection method is demonstrated for patient screening. One patient has a novel mutation, a T556C transition that results in the substitution of Pro for Leu at codon 135, and produces no enzymatic activity in transfection experiments.

Myelin oligodendrocyte glycoprotein polymorphisms and multiple sclerosis.

A detailed analysis of the coding sequences of myelin oligodendrocyte glycoprotei (MOG) gene was performed in multiple sclerosis (MS) patients and in control individuals and three new polymorphisms are described: T636C, nt 571+77C-->T (IVS 4), and nt 710-44A-->G (IVS 6). Screening studies demonstrated that T636C was present in three MS patients and in no control individual and that polymorphisms nt 571+77C-->T (IVS 4), and nt 710-44A-->G (IVS 6), were present with no significant frequency differences in MS patients and control individuals. No mutations were found after sequencing the coding sequences of the extracellular domain of MOG gene in 20 MS patients and 20 control individuals. Screening studies were also performed for known polymorphisms: G15A, Val142Leu, nt 571+68A-->G (IVS 4), and 571+92C-->G (IVS 4). Polymorphism Val 142 Leu, which is linked to nt 571+68A-->G (IVS 4), resulted under-represented in MS patients.

Common CFTR haplotypes and susceptibility to chronic pancreatitis and congenital bilateral absence of the vas deferens

CFTR mutations enhance susceptibility for idiopathic chronic pancreatitis (ICP) and congenital bilateral absence of the vas deferens (CBAVD); however, it is unknown why CFTR heterozygotes are at increased disease risk. We recently showed that common CFTR variants are associated with aberrantly spliced transcripts. Here, we genotyped for common CFTR variants and tested for associations in two ICP (ICP‐A: 126 patients, 319 controls; ICP‐B: 666 patients, 1,181 controls) and a CBAVD population (305 patients, 319 controls). Haplotype H10 (TG11‐T7‐470V) conferred protection (ICP‐A: OR 0.19, P<0.0001; ICP‐B: OR 0.78, P = 0.06; CBAVD OR 0.08, P<0.001), whereas haplotype H3 (TG10‐T7‐470M) increased disease risk (ICP‐A: OR 8.34, P = 0.003; ICP‐B: OR 1.88, P = 0.007; CBAVD: OR 5.67, P = 0.01). The risk of heterozygous CFTR mutations carriers for ICP (OR 2.44, P<0.001) and CBAVD (OR 14.73, P<0.001) was fully abrogated by the H10/H10 genotype. Similarly, ICP risk of heterozygous p.Asn34Ser SPINK1 mutation carriers (OR 10.34, P<0.001) was compensated by H10/H10. Thus, common CFTR haplotypes modulate ICP and CBAVD susceptibility alone and in heterozygous CFTR and p.Asn34Ser mutation carriers. Determination of these haplotypes helps to stratify carriers into high‐ and low‐risk subjects, providing helpful information for genetic counseling. Hum Mutat 32:1–9, 2011.

Myelin oligodendrocyte glycoprotein (MOG) polymorphisms and adrenoleukodystrophy.

We describe four novel sequence variants in the Myelin Oligodendrocyte Glycoprotein (MOG) gene. A total of six sequence variants of the MOG gene were identified in eleven out of 44 ALD patients investigated: G15A, CTC repeat in exon 1, Val142Leu, Val145Ile, 551+68A-->G and 551+77C-->T. Screening studies demonstrated that all these polymorphisms are present in 50 unaffected control male individuals of the same population and in the different phenotypes of ALD patients, indicating that they do not contribute to phenotype variability in ALD.

Polymorphism -2604G>A variants in TLR4 promoter are associated with different gene expression level in peripheral blood of atherosclerotic patients

Toll-like receptor-4 (TLR4) is a primary receptor of the innate immune reaction and compelling evidence demonstrates its involvement in the pathogenesis of atherosclerosis and stroke. TLR4 is constitutively expressed on monocytes and endothelial cells; it is highly expressed in atherosclerotic plaques and in peripheral blood of patients after ischemic stroke. Polymorphisms in the promoter region that alter the transcriptional regulation of this gene may represent genetic risk factors involved in the predisposition to atherosclerotic disease. In this study we investigated the effect on TLR4 gene expression of three polymorphisms in the upstream regulatory region at positions −1607T>C/rs10759932, −2026A>G/rs1927914 and −2604G>A/rs10759931 in peripheral blood of atherosclerotic patients. RNA from individuals homozygous for the −2604A allele showed a lower expression of the gene when compared to patients carrying the counterparts GG+GA. Electrophoretic mobility shift assays showed differences in the electrophoretic mobility of the DNA–nuclear protein complexes formed by the G>A variants, suggesting that the two alleles differ in their binding affinity to transcriptional factors.

Association of microRNA 146a polymorphism rs2910164 and the risk of melanoma in an Italian population.

Association of microRNA 146a polymorphism rs2910164 and the risk of melanoma in an Italian population Macarena Gomez-Lira, Silvia Ferronato, Elisa Orlandi, Anna Dal Molin, Giovanni Malerba, Simona Frigerio, Monica Rodolfo* and Maria Grazia Romanelli* Department of Life and Reproduction Sciences, Section of Biology and Genetics, University of Verona, Verona, Italy; Unit of Immunotherapy, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy Correspondence: Macarena Gomez-Lira, Department of Life and Reproduction Sciences, Section of Biology and Genetics, University of Verona, Strada Le Grazie, 8. 37134 Verona, Italy, Tel.: +39 0458027674, Fax: 0458027180, e-mail: macarena.gomezlira@univr.it *Rodolfo M and Romanelli MG, are both co-senior authors

Cyclooxygenase 2, toll-like receptor 4 and interleukin 1β mRNA expression in atherosclerotic plaques of type 2 diabetic patients.

Objectives and designInflammation has a prominent role in the development of atherosclerosis. Type 2 diabetes could contribute to atherosclerosis development by promoting inflammation. This status might accelerate changes in intrinsic vascular wall cells and favor plaque formation. Cyclooxygenase 2 (COX-2) is highly expressed in atherosclerotic plaques. COX-2 gene expression is promoted through activation of toll-like receptor 4 (TLR4) and pro-inflammatory cytokine interleukin 1β (IL1-β). Aim of this study is to investigate whether expression profiles of pro-inflammatory genes such as COX-2, TLR4 and IL1-β in atherosclerotic plaques are altered in type 2 diabetes (T2D).MethodsTotal RNA was isolated from plaques of atherosclerotic patients and expression of COX-2, TLR4, IL1-β analyzed using real-time PCR. Histological analysis was performed on sections of the plaque to establish the degree of instability.ResultsStatistically significant differences in mRNA expression of COX-2 and IL1-β were found in plaques of T2D compared with non-T2D patients. A multi-variable linear regression model suggests that COX-2 mRNA expression is affected by T2D pathology and IL1-β mRNA expression in atherosclerotic plaques.ConclusionsOur results support the hypothesis that T2D pathology contributes in vivo to increase the inflammatory process associated with the atherosclerotic plaque formation, as shown by an increment of COX-2 and IL1-β mRNA expression.