Secreted protein acidic and rich in cysteine (SPARC) gene polymorphism association with hepatocellular carcinoma in Italian patients.

BACKGROUND AND AIM: Hepatocellular carcinoma (HCC) is a multifactorial disease driven by both genetic and epigenetic factors. Infection, inflammation and the immune response against hepatitis B virus and hepatitis C virus have been shown to play an important role in increasing cancer risk and promoting tumor development. In order to investigate the genetic component influencing HCC development, we analyzed 50 single nucleotide polymorphisms (SNP) spanning 34 different genes in 230 Italian patients affected by HCC and 230 controls. METHODS: Genes were selected on the basis of their known biological function and their possible involvement in the progression or in the susceptibility to HCC was considered. SNP genotyping was performed using allelic-specific fluorescent probes. RESULTS: For most SNP, no differences were identified between HCC patients and controls, with the exception of rs2304052, localized on the secreted protein acidic and rich in cysteine (SPARC) gene, which was significantly associated to the disease. The C allele was significantly more frequent in the HCC patients than in the healthy controls (23% vs 10%, corrected P < 0.001), as well as the CC genotype (13% vs 1%, corrected P < 0.001). CONCLUSION: Since the presence of the rs2304052 C allele is associated with an increased risk (odds ratio: 2.76) of developing hepatocarcinoma, our results allowed us to identify a SNP in the SPARC gene correlating to HCC susceptibility.

Genetic variant of C1GalT1 contributes to the susceptibility to IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN) is a common form of primary glomerulonephritis characterized by diffuse glomerular mesangial IgA1 deposition that leads to mesangial proliferation and chronic glomerular inflammation. Analyses of serum IgA1 from IgAN patients revealed an abnormal galactosylation of the O-linked carbohydrate moieties of IgA that may be a result of altered activity of core 1 beta1,3-galactosyltransferase (C1GalT1). To evaluate the association between C1GalT1 single nucleotide polymorphisms (SNPs) and IgAN, we performed a case control study on cohorts from the Italian population. METHODS: We sequenced C1GalT1 coding and promoter regions in 284 IgAN patients and 210 healthy controls. The functional role of 3' untranslated region (3'UTR) SNPs was studied using electrophoretic mobility shift assays and real-time quantitative PCR. RESULTS: We analyzed 8 SNPs in the C1GalT1 gene: 5 SNPs were in the promoter region and 3 SNPs in the 3'UTR. The allele 1365G in the 3'UTR was significantly more frequent in IgAN patients than in healthy controls. CONCLUSION: The 1365G allele and 1365G/G genotype seem to confer susceptibility to IgAN.

MBL2 genetic screening in patients with recurrent vaginal infections.

PROBLEM: Mannose-binding lectin (MBL) is an important component of the innate immunity, present at the mucosal level in vagina: a common pathogen's entry point. METHOD OF STUDY: We used a rapid genotyping method based on melting temperature assay to search for three single nucleotide polymorphisms (SNPs) located in the first exon of the MBL2 gene and we also measured MBL serum levels in patients with recurrent bacterial vaginosis (rBV) and recurrent vulvovaginal candidiasis (rVVC). RESULTS: Detected frequencies of MBL2 SNPs were comparable to the ones already reported for the Italian population and no significant differences were found between rVVC, rBV and controls. MBL serum levels did not show significant differences between the studied groups. CONCLUSION: No correlation for the screened mutations has been found neither in protecting nor in favoring the infection in rVVC and rBV patients. Our data demonstrate a lack of association between functional polymorphisms in the first exon of MBL2 gene, MBL deficiency, VVC and rBV.

Role of interferon-gamma gene polymorphisms in susceptibility to IgA nephropathy: a family-based association study.

T helper (h) lymphocytes in pathogenic immune response at mucosal effector site play a key role in IgA nephropathy (IgAN). We evaluated the impact of some Th1/Th2/Th3/T(R)-type, and of monocyte/macrophage cytokines on IgAN susceptibility with a family-based association study including 53 patients, 45 complete trios, 4 incomplete trios and 36 discordant siblings. Cytokine gene polymorphisms with a potential regulatory role on their production were investigated using the family-based association test (FBAT): IFNgamma intron-1 CA repeat at position 1349-1373; IL-13 -1055C/T; TGFbeta +915G/C; IL-10 5'-proximal and distal microsatellites; TNFalpha -308G/A, -238G/A. The FBAT multi-allelic analysis showed an association between IFNgamma polymorphism and susceptibility to IgAN (P=0.03). The bi-allelic analysis evidenced that the 13-CA repeat allele was preferentially transmitted to the affected individuals (P=0.006; Bonferroni P-value=0.04). The direct sequencing of IFNgamma amplicons showed a strict association between the 13-CA repeat allele and the A variant of the +874T/A single nucleotide polymorphism (SNP rs2430561) directly adjacent to the 5' end of the microsatellite. The in vitro production of IFNgamma evaluated in peripheral blood mononuclear cells from 10 genotyped patients demonstrated a correlation between the +874A allele and a lower production of IFNgamma (P=0.028 Mann-Whitney test). This SNP affects IFNgamma production lying within a binding site for the transcription factor NF-kappaB. No significant difference was observed in the 15 years renal survival between IgAN patients carrying different IFNgamma gene polymorphisms. This first family-based association study demonstrates that the +874A allele, strictly associated with IFNgamma 13-CA repeat allele, confers susceptibility to IgAN, without influencing renal survival.

Association of interferon-gamma +874A polymorphism with reduced long-term inflammatory response in haemodialysis patients.

BACKGROUND: We have studied the effects of interferon (IFN)-gamma allelic variations on expression levels of pro- and anti-inflammatory cytokines and on long-term inflammatory status in haemodialysis patients. METHODS: Genotyping was performed in 123 patients for single nucleotide polymorphisms in the first intron of the IFN-gamma gene (+874 T/A). They were prospectively followed for 2 years. Cytokine mRNA levels in whole blood cells (detected by real time (RT)-PCR technique) and serum C-reactive protein (CRP) concentrations were compared in patient groups with different IFN-gamma genotypes. Serum CRP was evaluated every month and inflammatory state was defined as percent of abnormal values (above 5 mg/l) over total determinations. Of the total, 102 patients survived and completed 24+/-1 monthly CRP determinations. The IFN-gamma +/-874 A/A, 'low-producer' genotype was associated with decreased (P<0.05) mRNA levels of IFN-gamma and of interleukin-6 and with a lower (P<0.05) frequency of CRP elevation (37+/-6%) than the +/-874 A/T and T/T, 'intermediate and high-producer' genotypes (59+/-6%, and 60+/-5%, respectively). The mRNA levels of tumor necrosis factor-alpha, IL-10 and of transforming growth factor-beta1 were not different in the three groups of patients. Pooled analysis in deceased (10+/-3 monthly CRP determinations) and survived patients confirmed the results obtained in the patients who completed the follow-up period. CONCLUSIONS: The 'low-producer' IFN-gamma +874 A/A genotype was associated with a preventive effect on long-term CRP elevation in haemodialysis patients possibly mediated by decreased gene expression of IFN-gamma and IL-6.

The IgA nephropathy Biobank. An important starting point for the genetic dissection of a complex trait.

BACKGROUND: IgA nephropathy (IgAN) or Berger's disease, is the most common glomerulonephritis in the world diagnosed in renal biopsied patients. The involvement of genetic factors in the pathogenesis of the IgAN is evidenced by ethnic and geographic variations in prevalence, familial clustering in isolated populations, familial aggregation and by the identification of a genetic linkage to locus IGAN1 mapped on 6q22-23. This study seems to imply a single major locus, but the hypothesis of multiple interacting loci or genetic heterogeneity cannot be ruled out. The organization of a multi-centre Biobank for the collection of biological samples and clinical data from IgAN patients and relatives is an important starting point for the identification of the disease susceptibility genes. DESCRIPTION: The IgAN Consortium organized a Biobank, recruiting IgAN patients and relatives following a common protocol. A website was constructed to allow scientific information to be shared between partners and to divulge obtained data (URL: http://www.igan.net). The electronic database, the core of the website includes data concerning the subjects enrolled. A search page gives open access to the database and allows groups of patients to be selected according to their clinical characteristics. DNA samples of IgAN patients and relatives belonging to 72 multiplex extended pedigrees were collected. Moreover, 159 trios (sons/daughters affected and healthy parents), 1068 patients with biopsy-proven IgAN and 1040 healthy subjects were included in the IgAN Consortium Biobank. Some valuable and statistically productive genetic studies have been launched within the 5th Framework Programme 1998-2002 of the European project No. QLG1-2000-00464 and preliminary data have been published in "Technology Marketplace" website: http://www.cordis.lu/marketplace. CONCLUSION: The first world IgAN Biobank with a readily accessible database has been constituted. The knowledge gained from the study of Mendelian diseases has shown that the genetic dissection of a complex trait is more powerful when combined linkage-based, association-based, and sequence-based approaches are performed. This Biobank continuously expanded contains a sample size of adequately matched IgAN patients and healthy subjects, extended multiplex pedigrees, parent-child trios, thus permitting the combined genetic approaches with collaborative studies.

Comparative genomic sequence analysis coupled to chromatin immunoprecipitation: a screening procedure applied to search for regulatory elements at the RET locus.

RET gene expression is characterized by high tissue and stage specificity during the development of neural crest derivatives and in the pathogenesis of inherited cancer syndromes and Hirschsprung disease. Identifying all elements contributing to its transcriptional regulation might provide new clues to clarify both developmental and pathogenic mechanisms. We previously demonstrated that chromatin acetylation affects RET transcription; therefore, we have set up a strategy based on analysis of sequences conserved among species at the RET locus, combined with the characterization of their chromatin structure, to identify new potential regulatory elements. The histone acetylation level was evaluated by the chromatin immunoprecipitation method applied to cells displaying different degrees of endogenous RET expression. Real-time quantitative PCR of immunoprecipitated DNA-protein complexes and transfection experiments, with constructs expressing a reporter gene in which the putative regulatory regions are inserted, indicate a correlation between histone acetylation and endogenous RET expression and highlight conserved sequences with potential regulatory roles. This paper presents a reliable screening procedure to unearth elements able to affect gene regulation at the transcriptional level in a large genomic region.

Uromodulin storage diseases: clinical aspects and mechanisms.

The recent discovery of mutations in the uromodulin gene ( UMOD ) in patients with medullary cystic kidney disease type 2 (MCKD2), familial juvenile hyperuricemic nephropathy (FJHN), and glomerulocystic kidney disease (GCKD) provides the opportunity for a revision of pathogenic aspects and puts forth the basis for a renewed classification. This review focuses on clinical, pathological, and cell biology advances in UMOD -related pathological states, including a review of the associated clinical conditions described to date in the literature. Overall, 31 UMOD mutations associated with MCKD2 and FJHN (205 patients) and 1 mutation associated with GCKD (3 patients) have been described, with a cluster at exons 4 and 5. Most are missense mutations causing a cysteine change in uromodulin sequence. No differences in clinical symptoms between carriers of cysteine versus polar residue changes have been observed; clinical phenotypes invariably are linked to classic MCKD2/FJHN. A common motif among all reports is that many overlapping symptoms between MCKD2 and FJHN are present, and a separation between these 2 entities seems unwarranted or redundant. Cell experiments with mutant variants indicated a delay in intracellular maturation and export dynamics, with consequent uromodulin storage within the endoplasmic reticulum (ER). Patchy uromodulin deposits in tubule cells were found by means of immunohistochemistry, and electron microscopy showed dense fibrillar material in the ER. Mass spectrometry showed only unmodified uromodulin in urine of patients with UMOD mutations. Lack of uromodulin function(s) is associated with impairments in tubular function, particularly the urine-concentrating process, determining water depletion and hyperuricemia. Intracellular uromodulin trapping within the ER probably has a major role in determining tubulointerstitial fibrosis and renal failure. We propose the definition of uromodulin storage diseases for conditions with proven UMOD mutations.

Primary hyperoxaluria: genotype-phenotype correlation.

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disorder caused by a deficiency of alanine-glyoxylate aminotransferase (AGT), which is encoded by a single copy gene (AGXT). Molecular diagnosis was used in conjunction with clinical, biochemical and enzymological data to evaluate genotype-phenotype correlation. Patients can present a severe form of PH1, an adult form and a mild to moderate decrease in renal function. Biochemical diagnosis is made by plasma, urine and dialyzate oxalate and glycolate assays, and by liver AGT activity and pyridoxine responsitivity. Molecular genetic diagnosis can be made using different techniques, for example, the single strand conformation polymorphism technique (SSCP), followed by the sequencing of the 11 AGXT exons. The disease is clinically and genetically classified as highly heterogeneous. Mutant alleles can be recognised in 80- 90% of chromosomes, depending on the techniques used. Mutations in exons 1, 2, 4 and 10 are more frequent in Italian patients. Normalized AGT activity seems to be lower in the severe form than in the adult form. Double heterozygous patients present a lower age at disease onset and they were more frequent in the more severe than in mild severe disease. The 444T>C mutation was more frequent in the severe form, while the opposite was observed for 630G>A. 630G>A mutation homozygotes had a higher AGT residual activity. The presence of allelic heterogeneity of the AGXT could be responsible, to some extent, for the phenotypic heterogeneity in PH1. Homozygous genotypes were more frequent than expected and were associated with a less severe form of the disease.

Effects of maturation on RNA transcription and protein expression of four MRP genes in human placenta and in BeWo cells.

The placenta is a multifunctional organ that protects the fetus from toxic compounds and the MRPs contribute to this function. The expression of MRP1, MRP2, MRP3, and MRP5 was compared in human placental tissue and in BeWo cells by real-time RT-PCR analysis; protein expression was assessed by Western blot. MRP1 and MRP3 were the most abundantly expressed genes in placenta but only MRP1 was highly expressed in the BeWo cells. Expression of MRP1 increased 4-fold in the third as compared with first trimester placental samples, and increased 20-fold with polarization of BeWo cells. MRP2, MRP3, and MRP5 were weakly expressed both in placenta and BeWo cells. Protein expression followed mRNA quantification for MRP1 and MRP5 but not for MRP2 and MRP3. These data indicated that MRP1 and MRP5 increase with trophoblast maturation, suggesting a particular role for these proteins in the organ functional development.

Variant mannose-binding lectin alleles are associated with celiac disease.

In this study, we investigated the role of mannose-binding lectin (MBL) in celiac disease, by performing genotype analysis for the three point mutations in the first exon of the gene in 117 Italian celiac patients (characterized by flat biopsy and positive for anti-endomysium antibody and human transglutaminase antibodies) and 130 pan-ethnic healthy controls. The frequency of homozygous mutant 0/ 0 was significantly higher in the 117 Italian celiac patients (0.13) than in the 130 pan-ethnic healthy controls (0.05; P=0.0405). An increased frequency of homozygous 0/0 allele was found among patients with celiac disease compared with controls. These results suggest an involvement of MBL in the pathophysiology of celiac disease.

Extracellular NAD+: a novel autocrine/paracrine signal in osteoblast physiology.

Intercellular communication allows co-ordination of cell metabolism and sensitivity to extracellular stimuli. In bone cells, paracrine stimulation and cell-to-cell coupling through gap junctions induce the formation of complex intercellular networks, which favours the intercellular exchange of nutrients and second messengers, ultimately controlling the process of bone remodelling. The importance of local factors in bone remodelling is known since many years. Bone cells secrete and respond to a variety signals, among which include prostaglandins, cytokines, growth factors, and ATP. We here report evidence that extracellular NAD(+) is a novel extracellular signal stimulating osteoblast differentiation. We found that HOBIT human osteoblastic cells, which are known to express ADP-ribosyl cyclase/CD38 activity, respond to micromolar concentrations of extracellular NAD(+) with oscillatory increases of the cytosolic Ca(2+) concentration. The initial Ca(2+) response was followed by a time-dependent inhibition of cell growth, the appearance of an epithelial morphology, and by an increase of alkaline phosphatase and osteocalcin expression. Under resting condition HOBIT cells release NAD(+) in the extracellular medium and the release is significantly potentiated by mechanical stimulation. Taken together these results point to NAD(+) as a novel autocrine/paracrine factor involved in stimulation and maintenance of the osteoblast differentiated phenotype.

AGXT gene mutations and their influence on clinical heterogeneity of type 1 primary hyperoxaluria.

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disorder that is caused by a deficiency of alanine: glyoxylate aminotransferase (AGT), which is encoded by a single copy gene (AGXT). Molecular diagnosis was used in conjunction with clinical, biochemical, and enzymological data to evaluate genotype-phenotype correlation. Twenty-three unrelated, Italian PH1 patients were studied, 20 of which were grouped according to severe form of PH1 (group A), adult form (group B), and mild to moderate decrease in renal function (group C). All 23 patients were analyzed by using the single-strand conformation polymorphism technique followed by the sequencing of the 11 AGXT exons. Relevant chemistries, including plasma, urine and dialyzate oxalate and glycolate assays, liver AGT activity, and pyridoxine responsiveness, were performed. Both mutant alleles were found in 21 out of 23 patients, and 13 different mutations were recognized in exons 1, 2, 4, and 10. Normalized AGT activity was lower in the severe form than in the adult form (P < 0.05). Double heterozygous patients presented a lower age at the onset of the disease (P = 0.025), and they were more frequent in group A (75%) than in the group B (14%; P = 0.0406). The T444C mutation was more frequent in the severe form (P < 0.05), and the opposite was observed for G630A (P < 0.05). G630A mutation homozygotes had a higher AGT residual activity (P = 0.00001). This study confirms the allelic heterogeneity of the AGXT, which could to some extent be responsible for the phenotypic heterogeneity in PH1.