Identification of suitable mRNAs and microRNAs as reference genes for expression analyses in skin cells under sex hormone exposure.

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 17ß-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.

Expression of TLR4-PTGE2 signaling genes in atherosclerotic carotid plaques and peripheral blood.

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.

PTCH1 gene haplotype association with basal cell carcinoma after transplantation.

BACKGROUND: Basal cell carcinoma (BCC) is 10 times more frequent in organ transplant recipients (OTRs) than in the general population. Factors in OTRs conferring increased susceptibility to BCC include ultraviolet radiation exposure, immunosuppression, viral infections such as human papillomavirus, phototype and genetic predisposition. The PTCH1 gene is a negative regulator of the hedgehog pathway, that provides mitogenic signals to basal cells in skin. PTCH1 gene mutations cause naevoid BCC syndrome, and contribute to the development of sporadic BCC and other types of cancers. Associations have been reported between PTCH1 polymorphisms and BCC susceptibility in nontransplanted individuals. OBJECTIVES: To search for novel common polymorphisms in the proximal 5' regulatory region upstream of PTCH1 gene exon 1B, and to investigate the possible association of PTCH1 polymorphisms and haplotypes with BCC risk after organ transplantation. METHODS: Three PTCH1 single nucleotide polymorphisms (rs2297086, rs2066836 and rs357564) were analysed by restriction fragment length polymorphism analysis in 161 northern Italian OTRs (56 BCC cases and 105 controls). Two regions of the PTCH1 gene promoter were screened by heteroduplex analysis in 30 cases and 30 controls. RESULTS: Single locus analysis showed no significant association. Haplotype T(1686)-T(3944) appeared to confer a significantly higher risk for BCC development (odds ratio 2.98, 95% confidence interval 2.55-3.48; P = 0.001). Two novel rare polymorphisms were identified at positions 176 and 179 of the 5'UTR. Two novel alleles of the -4 (CGG)(n) microsatellite were identified. No association of this microsatellite with BCC was observed. CONCLUSIONS: Haplotypes containing T(1686)-T(3944) alleles were shown to be associated with an increased BCC risk in our study population. These data appear to be of great interest for further investigations in a larger group of transplant individuals. Our results do not support the hypothesis that common polymorphisms in the proximal 5' regulatory region of the PTCH1 gene could represent an important risk factor for BCC after organ transplantation.

Two new highly polymorphic markers in the 3' UTR region of the PLA2G7 gene.

We described two new highly polymorphic markers located 31 bp downstream of the last nucleotide of exon 12 in the 3' UTR region of the gene PLA2G7: 1344 +31TG(n) AG(m). Eight and 14 alleles were observed for the AG and TG repeats, respectively. These two markers have the highest heterozygosity until now reported for PLA2G7 gene.

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.

Prevalence of dementia and apolipoprotein e genotype distribution in the elderly of buttapietra, verona province, Italy.

We investigated the prevalence of dementia and the apolipoprotein E (APOE) genotype distribution in the elderly of Buttapietra, a village near Verona, Italy. All residents over the age of 74 (n = 238), including those who were institutionalized, were studied using a direct-contact, single-phase design. The overall prevalence of dementia, clinically defined by DSM-III-R criteria, was 15.8 cases per 100 population, with age-specific figures increasing steeply with advancing age in both sexes. Alzheimer's disease (AD) was the most frequent dementing disorder (43%). APOE genotyping was determined after DNA amplification by restriction isotyping. We found that the epsilon4 allele and the epsilon3/epsilon4 genotype were associated with all types of dementia, although only the association of epsilon3/epsilon4 with AD reached statistical significance (odds ratio 4.5, 95% confidence interval 1.3-16.1). However, as reported in other Mediterranean countries, the frequency of the epsilon4 allele in our population was low (8.9%), suggesting that the population-attributable risk for AD, at least for elderly individuals (> or =75 years), could be small.

Analysis of the entire coding region of the cystic fibrosis transmembrane regulator gene in idiopathic pancreatitis.

Many Cystic Fibrosis (CF) carriers have been detected testing some subjects with chronic pancreatitis for a limited number of mutations. The aim of this study was to find out if some subjects with pancreatitis and a CFTR mutation actually carry another, undetected mutation. We screened for 18 CFTR mutations plus the CFTR intron 8 poly(T) tract length a population of 67 patients suffering from idiopathic either acute, or recurrent acute, or chronic pancreatitis. Three of them were diagnosed as affected by CF. Among the others, a subset of 14 (8 CFTR mutation carriers, 4 5T carriers, and 2 sweat chloride borderliners) was selected and analyzed by denaturing gradient gel electrophoresis. Six possibly CF-related mutations were detected: L997F and 3878delG were found in two of the subjects already carrying another mutation, S1235R and L997F in one patient carrying the 5T, and L997F and D614G in the two patients with borderline sweat chloride. Among the 14 selected cases a total of 11 patients carried at least one mutation, and three of them were compound heterozygotes. Though it is debatable whether these three individuals can be considered affected by CF, their pancreatitis is possibly a clinical manifestation of some CFTR-related disease. Hum Mutat 18:166, 2001.

Osteogenesis imperfecta at the beginning of bone and joint decade.

Osteogenesis imperfecta (OI), or brittle bone disease, is a heritable disorder characterized by increased bone fragility. Four different types of the disease are commonly distinguished, ranging from a mild condition (type I) to a lethal one (type II). Types III and IV are the severe forms surviving the neonatal period. In most cases, there is a reduction in the production of normal type I collagen or the synthesis of abnormal collagen as a result of mutations in the type I collagen genes. These classic forms of OI are described in this review. There are instances, however, where alterations in bone matrix components, other than type I collagen, are the basic abnormalities of the OI. Recently, three such discrete types have been identified by histomorphometric evaluation (types V and VI) and linkage analysis (Rhizomelic OI). They provide evidence for the as yet poorly understood complexity of the phenotype-genotype correlation in OI. We also discuss bisphosphonates treatment as well as fracture management and surgical correction of deformities observed in the patients with OI. However, ultimately, strengthening bone in OI will involve steps to correct the underlying genetic mutations that are responsible for this disorder. Thus, we also describe different genetic therapeutic approaches that have been tested either on OI cells or on available OI murine models.

A novel 4-bp deletion creates a premature stop codon and dramatically decreases HEXB mRNA levels in a severe case of Sandhoff disease.

We present the molecular genetic analysis of an infantile-onset Sandhoff disease patient. Genomic DNA amplification, heteroduplex analysis, cloning and sequencing revealed a 4-bp deletion in exon 4 (497 DeltaAGTT). The result is a frameshift mutation that leads to a stop codon in exon 5. This mutation is associated with a dramatic decrease of HEXB mRNA levels.

CFTR and cationic trypsinogen mutations in idiopathic pancreatitis and neonatal hypertrypsinemia.

BACKGROUND/AIMS: The CFTR gene has been shown to be involved in sporadic idiopathic pancreatitis (IP) and neonatal hypertrypsinemia with normal sweat chloride test (NHNST). The cationic trypsinogen gene (Try4) is responsible for hereditary pancreatitis. The aim of the present study was to find a correlation between mutations in the two genes and the two phenotypes. METHODS: Analysis of some known gene mutations and complete gene screening by denaturing gradient gel electrophoresis and DNA sequencing were undertaken. Thirty-two sporadic IP patients were investigated for the CFTR study, while 13 sporadic IP patients plus 4 hereditary pancreatitis families (24 tested individuals) were examined for the Try4 study. Fifty neonates with NHNST were investigated for the study of both genes. RESULTS: CFTR mutations were more frequently observed in sporadic IP cases with a common cystic fibrosis mutation or borderline sweat chloride than in cases with a negative sweat test. Try4 mutations were found in 1 out of the 13 sporadic IP cases tested. CONCLUSIONS: The CFTR gene may be involved in IP and NHNST, while the Try4 gene may be involved in IP, but not in NHNST, in this limited series of observations.

Adult-onset MLD: a gene mutation with isolated polyneuropathy.

A 22-year-old man presented with recurrent ulnar mononeuropathies and diffusely slow nerve conduction velocities. Arylsulfatase A (ASA) activity from leukocytes and fibroblasts was reduced, and urinary sulfatides were increased. Sural nerve biopsy revealed a reduction in myelinated fibers and Schwann cell inclusions. Results of studies of CNS integrity, including cranial MRI, evoked potentials, and neuropsychologic tests, were normal. Molecular genetic analyses revealed a novel homozygous missense mutation (Thr286Pro) in the ASA gene.

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.

Two novel frameshift mutations in the adrenoleukodystrophy gene in Italian patients.

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.

Two novel missense mutations causing adrenoleukodystrophy in Italian patients.

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.

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

Four new cases of lethal osteogenesis imperfecta due to glycine substitutions in COL1A1 and genes. Mutations in brief no. 152. Online.

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 case of lethal OI. Two glycine substitutions within the COL1A1 gene (G478S, G994D) and two glycine substitutions within the COLIA2 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 hereteroduplex nucleic acids. Subsequent reverse transcription PCR amplification, cloning and sequencing, led to mutation identification. The aminoacid substitutions were due to two G-->A transitions in COL1A1(cases 1,2), to a G-->T transversion in COL1A2 (case 3), and to two contiguous point mutations in COL1A2 (case 4). All five nucleotide changes appeared to be fresh mutations. COLIA1(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.

A novel mutation which represents the fifth non-pathogenic polymorphism in the coding sequence of the arylsulfatase A gene.

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 a polymorphic site for the restriction endonuclease N/aIII.

A common beta hexosaminidase gene mutation in adult Sandhoff disease patients.

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.