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Aims: The miR-17-92 cluster and the HDAC9 gene are involved in inflammatory, apoptotic, and angiogenic processes that are activated in the vulnerable carotid plaque. The aim of this research was to determine whether expression of one or more of the miRs of the miR-17-92 cluster and/or HDAC9 expression could represent biomarkers for patients with unstable atherosclerotic carotid plaques. Materials and Methods: Plasma levels of miRs and HDAC9 expression in peripheral blood were analyzed by real-time PCR in patients with histologically classified stable or unstable plaques. Results: No differences were observed between the two groups. Discussion and Conclusions: Levels of the miR-17-92 cluster in plasma and HDAC9 gene expression in peripheral blood cannot be considered appropriate biomarkers to identify patients with unstable plaques at risk of rupture.

Quantitative RT-PCR is the most accurate technique for the study of gene expression profiles, however, to ensure the accuracy of qPCR results, suitable reference genes are necessary for data normalization. Hormones influence the development and function of skin cells, regulating the expression of genes and miRNAs. Nevertheless, the stability of reference genes after sex hormone treatment has not been thoroughly investigated. In this study, we evaluated the expression of a set of candidate mRNAs and microRNsA (miRNA) as reference genes in keratinocytes (HaCaT cells), primary human fibroblasts and a melanoma cell line (LM-36 cells) under testosterone or 17β-estradiol treatment. Two algorithms, namely geNorm, Best-Keeper, and the comparative ΔCt method were used to evaluate the expression stability of the candidate reference genes. The comprehensive ranking showed that TBP and miR-191-5p are the most stable expressed genes across all cultured cells under hormone treatment. Furthermore, we observed that GAPDH, HPRT1 and U6 snRNA expression may be altered by hormone exposure, thus, these genes are not recommended as reference genes. In conclusion, the present study provides, to the best of our knowledge, the first evaluation of expressed mRNA(s) and miRNA(s) as reference genes in three different types of skin cells under the stimulation of sex hormones.

Toll-like receptor 4 (TLR4)/prostaglandine synthetase 2 (PTGS2) signaling plays a relevant role in atherosclerotic plaque vulnerability. The purpose of this study was to check the gene expression of 6 genes participating to TLR4/PTGS2 signaling (TLR4, PTGS2, ACSL4, PTGER3, PTGER4, and EPRAP) in carotid plaques and blood samples from the same individual and to evaluate these genes as biomarker of plaque progression. We investigated differential gene expression by qRT-PCR in 62 atherosclerotic patients’ carotid plaques and corresponding blood sample. A very weak or no correlation was observed in the overall population or analyzing asymptomatic patients. These analyzed genes are most likely not suitable for inclusion in the clinical routine as biomarkers of plaque instability.

Inflammation has a key role and translates the effects of many known risk factors for the disease in atherosclerotic vulnerable plaques. Aiming to look into the elements that induce the development of either a vulnerable or stable atherosclerotic plaque, and considering that inflammation has a central role in the progression of lesions, we analyzed the expression of genes involved in the ACE/TLR4/PTGS2 signaling in carotid plaques of symptomatic and asymptomatic patients. Patients with internal carotid artery stenosis undergoing carotid endarterectomy at Verona University Hospital were included in this study. A total of 71 patients was considered for gene expression analysis (29 atherothrombotic stroke patients and 42 asymptomatic patients). Total RNA was extracted from the excised plaques and expression of PTGS2, ACE, TLR4, PTGER4, PTGER3, EPRAP and ACSL4 genes was analyzed by real-time PCR. The correlation between the pair of genes was studied by Spearman coefficient. From the analyzed genes, we did not observe any individual difference in gene expression but the network of co-expressed genes suggests a different activation of pathways in the two groups of plaques.

The genetics of melanoma is complex and, in addition to environmental influences, numerous genes are involved or contribute toward melanoma predisposition. In this study, we evaluated the possible interaction between miR-146a and one of its putative targets ribonuclease L (RNASEL) in the risk of sporadic melanoma. Polymorphisms rs2910164 in miR-146a and rs486907 in the RNASEL gene have both independently been associated with the risk of different cancers, and an interaction between them has been observed in nonmelanoma skin cancer. Polymorphisms rs2910164 G/C and rs486907 A/G were genotyped by restriction fragment length polymorphism analysis in 304 sporadic melanoma patients and 314 control individuals. Genotype distribution between cases and controls for each of the two polymorphisms was compared using Fisher's exact test. Epistasis between the two polymorphisms was tested by a logistic regression model. In the present study, we observed a sex-specific effect of the miR-146a rs2910164 C allele restricted to individuals carrying the RNASEL rs486907 A allele as well. Men carrying this allelic combination have the highest risk of melanoma, whereas it seems to have no effect or even an opposite relationship to melanoma risk in the female population. The results reported in the present study suggest a sex-specific interaction between miR-146a and RNASEL genes in melanoma skin cancer susceptibility, and could account for possible discordant results in association studies when stratification according to sex is not performed.

A variant located at the end of HDAC9 gene within clusters of DNAse I sensitivity zones and histone modification hotspots has been associated with large vessel stroke and could be linked to plaque instability. The aim of the study is to define if an altered expression of HDAC9, TWIST1 and FERD3L genes could be involved in plaque vulnerability. Histological classification and gene expression analysis were performed in 6 stable and 16 unstable plaques obtained from asymptomatic patients undergoing endarterectomy. Gene expression was analysed by real-time PCR. TWIST1 gene expression resulted higher in stable plaques (P

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.

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 T553C [corrected] transition that results in the substitution of Pro for Leu at codon 135, and produces no enzymatic activity in transfection experiments.

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.

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.

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 (Fisher's 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; Fisher's exact test P=0.01).

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.

The authors present two new missense mutations in exon 1 of the adrenoleukodystrophy (ALD) gene. The first, a C813T transition, results in the substitution Pro143 Ser in the third putative transmembrane domain of the adrenoleukodystrophy protein (ALDP) in an adult onset case. The second, a de novo C709T transition, results in a substitution Ser 108 Leu between the second and the third putative transmembrane segments, in a childhood case.

Two novel frameshift adrenoleukodystrophy mutations in two families were identified: a complex dinucleotide deletion/tetranucleotide insertion at 1116 TC--GAGA (codon 244 [serine]) and an AG deletion at nucleotide 1462 (codon 359 [glutamic acid]). Both mutations are predicted to cause premature termination of protein synthesis. The patients were affected by childhood cerebral adrenoleukodystrophy and by adrenomyeloneuropathy with mild Addison disease, respectively.

A novel mutation, a C→T transition at nucleotide 455 of the coding sequence of the ARSA gene, was found in a control individual during the search for metachromatic leukodystrophy mutations. Its distribution in three different populations was examined. The frequency of the T allele was 0·058, 0·025 and 0·033, in Italian, German and Greek populations, respectively. The mutation results in no amino acid substitution and can be identified as it creates a polymorphic site for the restriction endonuclease Nla III.

beta-Hexosaminidase gene mutations were analyzed in two adult-onset Sandhoff disease Italian patients by PCR analysis of a common known mutation (delta 5') and by heteroduplex analysis of genomic and RT-PCR DNA fragments, covering the whole gene. The patients' genotypes were delta 5'/C1214%, and G890A/C1214T, respectively. As mutation C1214T (Pro405Leu) is also present in the other two late-onset cases so far described, we suggest that C1214T is a common mutation in this type of Sandhoff disease. Mutation G890A (Cys297Tyr) is a novel mutation which presumably causes altered processing of the pro beta chain.

Perinatal lethal osteogenesis imperfecta is the result of heterozygous mutations of the COL1A1 and COL1A2 genes. Here we describe the molecular defects responsible for four cases of lethal OI. Two glycine substitutions within the COL1A1 gene (G478S, G994D) and two glycine substitutions within the COL1A2 gene (G319V, G697C) were identified. The mutation sites were localized in proalpha2(I)and proalpha2(I)mRNA molecules, respectively, by chemical cleavage of mismatch in heteroduplex nucleic acids. Subsequent reverse transcription- PCR amplification, cloning and sequencing, led to mutation identification. The amino acid substitutions were due to two GA transitions in COL1A1(cases 1,2), to a GT transversion in COL1A2 (case 3), and to two contiguous point mutations in COL1A2 (case 4). All five nucleotide changes appeared to be fresh mutations. COL1A1(accession number Z74615) and COL1A2 (accession number Z74616) wild type coding sequences (cDNA) were deduced from the EMBL DNA sequence database. The mutations described here can also be found in the human type I collagen mutation database at the web site: http://www.le.ac.uk/genetics/collagen. Hum Mutat 12;71–72, 1998. © 1998 Wiley-Liss, Inc.