Gastrokine 1 expression in patients with and without Helicobacter pylori infection.

BACKGROUND: To understand the molecular changes underlying Helicobacter pylori-related gastric diseases is mandatory to prevent gastric cancer. Proteomic technology is providing a rapid expansion of the basic knowledge, particularly in the discovery of new biomarkers involved in the tumourigenesis. AIM: To characterise changes in protein expression level of the gastric mucosa in H. pylori-infected patients. METHODS: The population enrolled comprised 41 dyspeptic patients. Proteins extracted from gastric mucosal specimens were analysed by 2-dimensional electrophoresis, sequenced by MALDI-TOF and identified by Edman's degradation. RESULTS: Twenty-one out of 41 patients had H. pylori infection of whom 17 had anti-CagA IgG antibodies. Several proteins were identified, of which Rho guanosine diphosphatase dissociation inhibitor alpha and heat shock protein 27 increased and glutathione transferase and antrum mucosa protein-18 decreased in H. pylori-positive in respect to H. pylori-negative patients. Interestingly, antrum mucosa protein-18, currently referred as gastrokine-1, showed two isoforms differing in the first N-terminal amino acid residue. Both gastrokine-1 isoforms were observed in the H. pylori-negative group whereas a lower expression or even absence of the gastrokine-1 basic isoform was found in a subgroup (7/21) of H. pylori-positive patients with moderate-severe gastritis. CONCLUSION: Our study demonstrated the presence of gastrokine-1 isoforms of which the basic isoform was reduced in a subset of patients with H. pylori infection.

Genetic prenatal RET testing and pregnancy management of multiple endocrine neoplasia Type II A (MEN2A): a case report.

Multiple endocrine neoplasia 2A (MEN 2A) is an inherited dominant syndrome characterised by medullary thyroid carcinoma, adrenal pheochromocytoma and hyperparathyroidism due to specific RET proto-oncogene mutations. Fertile MEN 2A women are at risk of complicated pregnancy because of unrecognised pheochromocytoma and transmission of RET mutation to the progeny. This condition may cause psychological distress in affected pregnant patients and their families. Here we describe the genetic prenatal testing, the pregnancy management and obstetric outcome in a MEN 2A patient with a right side adrenal hyperplasia and elevated calcitonin levels, a condition suspicious for possible recurrence of pheochromocytoma. We confirm that maternal or fetal complications are rare when MEN 2A diagnosis is made before pregnancy and an accurate monitoring is instituted. Furthermore, our results indicate that prenatal testing for RET mutations is highly recommended in making decisions and assuring parents on the lifelong risk of tumors. This will avoid the psychological distress that can further complicate the pregnancy of affected women.

Prenatal diagnosis of cystic fibrosis: a case of twin pregnancy diagnosis and a review of 5 years' experience.

We performed prenatal diagnoses for cystic fibrosis in 32 high risk (1:4) couples (including a dizygotic pregnancy). Chorionic villi sampling did not cause abortion or fetal malformation in any case. The preliminary analysis of 9 short tandem repeats always excluded maternal contamination of the DNA extracted from chorionic villi and confirmed paternity. Twenty-two prenatal diagnoses were made by direct analysis of the mutations. In seven cases diagnosis was made by the analysis of intragenic polymorphisms; in three cases, we analyzed two extragenic polymorphisms. The prenatal diagnosis (including genetic counselling) was completed within 24 h from the sampling. Seven prenatal diagnoses revealed an affected fetus; all couples opted for therapeutic abortion. In 17 cases the fetus was heterozygote, and in seven cases it was non carrier of mutated alleles. In the twin pregnancy, mutations were DeltaF508/N1303K. Direct analysis of the DNA extracted from the two independent samples of chorionic villi revealed one fetus non carrier of mutated alleles and the other a carrier of the N1303K mutation. Analysis of the HPRT locus predicted both the fetuses as males. Furthermore, the genotype of each fetus was defined after birth. The prenatal diagnosis with chorionic villi sampling plays a key role in the prevention of cystic fibrosis. The laboratories must be equipped for both the direct analysis of mutations and for the analysis of a large number of polymorphisms. The preliminary analysis of short tandem repeats is recommended both to exclude maternal contamination and to confirm parentage.

Severe liver impairment in a cystic fibrosis-affected child homozygous for the G542X mutation.

The clinical and laboratory findings of a cystic fibrosis (CF) patient homozygous for the G542X mutation are described. This is the first case, among the 7 G542X homozygous CF subjects described so far who shows severe liver involvement, associated pancreatic insufficiency, and moderate pulmonary expression of the disease, as demonstrated by laboratory and imaging data. This case adds to the conclusion that genotype/phenotype correlation in cystic fibrosis is more complex than formerly suspected.

Molecular epidemiology of cystic fibrosis mutations and haplotypes in southern Italy evaluated with an improved semiautomated robotic procedure.

We screened for 22 cystic fibrosis (CF) mutations in DNA from a first cohort of 69 CF patients from southern Italy using a semiautomated allele specific oligonucleotide (ASO) dot blot procedure based on two multiplex PCR amplifications. Seven mutations (delta F508, N1303K, G542X, 1717-1 G-->A, W1282X, 1148T, and R553X) identified 77.6% of CF chromosomes. Detection reached 79.8% with the 2183 AA-->G mutation analysed with the restriction generating PCR method. Thus, we included the 2183 AA-->G mutation in the ASO protocol and set up the conditions to amplify the gene regions that include the eight mutations in a single multiplex PCR reaction. With this method we tested the DNA of the first cohort of 69 CF patients, a second cohort of 63 CF patients, and 300 carrier relatives; we also performed 12 prenatal diagnoses. The results from the 132 CF patients showed differences in the distribution of CF mutations between the south and north of Italy. The XV2c, KM19, and intron 8 VNDR haplotypes suggested the presence, in CF chromosomes bearing undetected mutations, of a limited number of unknown mutations typical of southern Italy. Finally, for six of the eight mutations, we compared the ASO procedure with the methods based on restriction enzymes; the results obtained with the two procedures were identical for all the 57 chromosomes compared.

Clinical features of cystic fibrosis patients with rare genotypes.

We describe the clinical features of seven cystic fibrosis patients from southern Italy who bear rare genotypes: (1) a patient homozygous for the 2183 AA-->G mutation who was affected by a very early pulmonary form of cystic fibrosis, and five patients who were compound heterozygotes either for the 2183 AA-->G mutation or for the I148T mutation, in both instances with the delta F508 mutation; and (2) a patient homozygous for the early nonsense R553X mutation who showed only a moderately severe form of cystic fibrosis. Our results confirm that environmental or genetic factors unrelated to the CF disease contribute significantly to the development of the phenotype.

Molecular genetic characterization and prenatal diagnosis in a family with Hunter disease.

Hunter disease, Mucopolysaccharidosis type II, is an X-linked recessive lysosomal storage disorder caused by a deficiency in iduronate sulfatase activity. We studied at molecular level a Neapolitan family with the disease. We report, in patient, the delta 139 mutation on the third exon of the gene, on female family members, the DNA analysis that allowed to assess or exclude their carrier status and on fetal DNA from a pregnancy of patient's mother, a prenatal diagnosis that resulted negative.

Multiple control elements regulate transcription from the most distal promoter of human aldolase A gene.

Three major transcripts, differing in their 5'-untranslated regions, are produced from the human aldolase A gene by alternative usage of three promoters, designated distal, middle and proximal. We report that the genomic region (distal promoter) upstream from the first leader exon (exon L1) efficiently directs transcription of a reporter CAT gene after transient transfections in human hepatoma cells (Hep3B) in a fashion independent from the other two promoters of the same gene. The distal promoter region contains cis-acting elements that regulate transcription both positively and negatively.

In vivo activity of the most proximal promoter of the human aldolase A gene and analysis of transcriptional control elements.

The genomic region upstream from exon F (exon IV) of the human aldolase A gene has been studied for its ability to direct the transcription of a reporter gene in vivo. Transfection experiments in human hepatoma cells (Hep 3B) followed by CAT assay, and S1 mapping analysis, demonstrated that: (i) this region is able to drive CAT gene transcription; (ii) all the transcriptional control elements of this promoter are downstream from nucleotide -384 of the longer ubiquitous RNA start site and the sequences between -384 and -262 play a crucial role in transcriptional efficiency; (iii) initiation starting points for two mRNAs exist 61 bp apart. Gel retardation and footprinting assays demonstrated the presence of DNA-protein complexes mainly in the region between -384 and -262 and such ubiquitous binding factors as Sp1 and AP-1.

Human aldolase A gene. Structural organization and tissue-specific expression by multiple promoters and alternate mRNA processing.

The complete nucleotide sequence of the human aldolase A isoenzyme gene is reported. The cloned gene sequence, spanning 7530 bp, includes twelve exons and occurs as a single copy per haploid human genome. The structural organization of the gene is quite complex: eight exons containing the coding sequence are common to all mRNAs extracted from human and other mammalian sources; four additional exons are present in the 5' untranslated region, of these one is contained in the ubiquitous type of mRNA, the second is in the muscle-specific type of mRNA and the third and fourth are in a minor species of mRNA found in human liver tissue. Furthermore, the determined sequence includes 1000 nucleotides upstream from the first exon (exon I) in the 5' flanking region, and 400 nucleotides, which include the polyadenylation signal, downstream from the termination codon. S1-nuclease-protection analysis of the 5' end of mRNA extracted from human cultured fibroblasts, muscle and hepatoma cell lines indicates the existence of four different transcription-initiation sites. The latter are also supported by the presence of conventional sequences for eukaryotic promoters. Therefore, the four promoters on the same gene generate different tissue-specific transcripts, which share the translated sequence, but each has a unique 5' untranslated region as a result of differential mRNA processing. The nucleotide homology at the coding region and the intron-exon organization of the three human and mammalian aldolase A, B and C genes confirm that they arose from a common ancestral gene, and that aldolase B diverged first.

Human aldolase A gene: structural organization and expression in tumor cell lines.

The structural intron/exon organization of the cloned human aldolase A gene is reported. The gene is composed of twelve exons, including the coding and the 5' and 3' non-coding regions. Four mRNAs, different at the 5' non-coding region, are transcribed from this gene in a tissue-specific manner. The study of the expression of aldolase A gene in normal liver and neoplastic cell lines demonstrated the resurgence of foetal pattern of expression in all tumor liver cell lines and HeLa cells, thus supporting the foetalism theory for this gene system.

A new human species of aldolase A mRNA from fibroblasts.

A full-length cDNA aldolase A clone was isolated from a human fibroblast cDNA library and completely sequenced. Excluding the poly(A) tail, the clone covers 1095 base pairs (bp) of the coding region, plus 199 bp downstream for the termination codon and 146 bp upstream for the initiation codon, within a total of 1440 bp. Primer extension experiments performed with human cultured fibroblast mRNA indicate an elongated product of a further 40 bp. These results evaluated together with those obtained in a concurrent study concerning aldolase A mRNA isolated from human liver are direct evidence of aldolase A mRNA multiplicity in man. The data also suggest the existence in mammals of three different classes of aldolase A mRNA, which would account for tissue specificity and resurgence of foetal expression in tumors.