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Systematic screening using the SSCP technique followed by sequencing of bands with abnormal mobility derived from the AGXT exons of 15 unrelated Italian patients with primary hyperoxaluria type 1 (PH1) allowed us to characterize both the mutant alleles in each individual. Eight new mutations were identified: C155del, C156ins, G244T, C252T, GAG408ins, G468A, G588A and G1098del. This study demonstrates both the effectiveness of the screening strategy chosen to identify all the mutant alleles and the high degree of allelic heterogeneity in PH1.

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 444TC mutation was more frequent in the severe form, while the opposite was observed for 630GA. 630GA 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.

A new polymorphism

Innate immunity is the first line of defense against microorganisms in vertebrates and acts by providing an initial barrier to microorganisms and triggering adaptive immune responses (Ganz and Lehrer 1999; Oppenheim et al., 2002). Peptides such as b-defensins are an important component of this defense, both providing a broad spectrum of antimicrobial activity against bacteria, fungi, mycobacteria and several enveloped viruses (Ganz and Leher, 1994) and showing chemotactic activity by linking and activating CCR6 (Hoover DM et al. , 2002). b-defensins are small cationic peptides that vary in their expression patterns and spectrum of pathogen specificity (Ganz and Leher, 2002).

Background. The location of a second genetic locus for autosomal dominant medullary cystic kidney disease (ADMCKD) at chromosome 16p12 led us to further investigate the molecular analysis of the critical region where two genes coding for uromodulin and SA proteins with renal specific functions, UMOD and SAH, are localized. Methods: We characterized the intron-exon boundary sequences by screening phage and BAC DNA genomic clones for the development of new molecular tools functional to the mutation analysis of UMOD and SAH genes. Results: No consistent mutations for ADMCKD2 were found in the UMOD and SAH genes. We identified a silent polymorphism in the UMOD gene at codon C174 which co-segregates with the disease in the ADMCKD2 family. Conclusions: This study excludes the involvement of uromodulin and SAH genes in ADMCKD2, and provides new tools for their molecular analysis in other diseases.

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.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.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.The 1365G allele and 1365G/G genotype seem to confer susceptibility to IgAN.

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

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.

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.

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.

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.

Primary hyperoxaluria type 1 is an autosomal recessive disorder of glyoxylate metabolism, caused by a deficiency of alanine:glyoxylate aminotransferase, which is encoded by a single copy gene (AGXT). The aim of this research was to standardize denaturing high-performance liquid chromatography, a new, sensitive, relatively inexpensive, and automated technique, for the detection of AGXT mutation. Denaturing high-performance liquid chromatography was used to analyze in blind the AGXT gene in 20 unrelated Italian patients with primary hyperoxaluria type 1 previously studied by other standard methods (single-strand conformation polymorphism analysis and direct sequencing) and 50 controls. Denaturing high-performance liquid chromatography allowed us to identify 13 mutations and the polymorphism at position 154 in exon I of the AGXT gene. Hence the method is more sensitive and less time consuming than single-strand conformation polymorphism analysis for the detection of AGXT mutations, thus representing a useful and reliable tool for detecting the mutations responsible for primary hyperoxaluria type 1. The new technology could also be helpful in the search for healthy carriers of AGXT mutations amongst family members and their partners, and for screening of AGXT polymorphisms in patients with nephrolithiasis and healthy populations.

Bone-forming cells are organized in a multicellular network interconnected by gap junctions. Direct intercellular communication via gap junctions is an important component of bone homeostasis, coordinating cellular responses to external signals and promoting osteoblast differentiation. The cAMP pathway, a major intercellular signal transduction mechanism, regulates osteoblastic function and metabolism. We investigated the effects of this second messenger on junctional communication and on the expression of differentiation markers in human HOBIT osteoblastic cells. Increased levels of cAMP induce posttranslational modifications (i.e., phosphorylations) of connexin43 and enhancement of gap junction assembly, resulting in an increased junctional permeance to Lucifer yellow and to a positive modulation of intercellular Ca2+ waves. Increased intercellular communication, however, was accompanied by a parallel decrease of alkaline phosphatase activity and by an increase of osteocalcin expression. cAMP-dependent stimulation of cell-to-cell coupling induces a complex modulation of bone differentiation markers.

We investigated the polymorphisms of the promoter region of the MBL2 gene, which codifies for the Mannose-binding protein (MBP). The study population included 90 children with vertically acquired HIV-infection, further divided on the basis of the disease rate, 27 HIV exposed-uninfected children, and 74 healthy control subjects matched for ethnic origin to evaluate the MBP involvement in the risk of HIV-1 infection and to assess the role of the MBP promoter in AIDS progression. A region of 380 bp in the promoter of the MBL2 gene was analysed by PCR and direct sequencing of both DNA strands. We found that the polymorphism at position -550 influences the risk of HIV-infection and AIDS progression. Also a 6 bp deletion at position -328 was correlated with HIV-1 infection. This study indicates that the promoter of the MBL2 gene influences vertical transmission of HIV and the course of perinatal infection.

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.

Celiac disease (CD) is an autoimmune enteropathy triggered by ingestion of gluten in genetically susceptible subjects and represents one of the most frequently occurring, treatable, lifelong autoimmune disorders. Undetected or untreated CD may cause late more severe complications (Farrell and Kelly, 2002). So far, several factors have been identified as possible agents responsible for CD. There is a strong evidence that CD is associated with specific HLA haplotypes (HLADQA1* 0501, DQB1*0201 or DQA1*0301, DQB0302) (Sollid and Thorsby, 1993). Recently it has been demonstrated on Italian patients that polymorphisms of the first exon of MBL2 gene, which encodes for Mannose Binding Protein (MBP), could play a pathophysiological role in celiac disease (Boniotto et al., 2002). MBP is a serum protein involved in the natural or innate immune response. MBP acts as an ante-antibody and can enhance opsonisation, or can activate the classical pathway of the complement on bacteria, viruses and fungi (Sastry and Ezekowitz, 1993).

Gilbert syndrome (GS) is an inherited form of chronic mild unconjugated hyperbilirubinemia (1)(2)(3), although many patients do not have a clear family history (4). Hepatic glucuronidation of bilirubin is catalyzed by isoenzyme 1A1 of UDP-glucuronosyl transferase (UGT1A1). The majority of GS subjects were found to be homozygous for an extra TA in the TATA-box in the promoter region of UGT1A1 (5)(6)(7). Transcription of the (TA)7 allele is reduced by at least 70% compared with the wild-type (TA)6 allele. Because bilirubin UGT1A1 is the only enzyme with substantial bilirubin glucuronidating activity in humans (8), the presence of this extra TA in both alleles can explain the impaired conjugation of bilirubin found in Caucasoid GS patients (6). A previous study of a large population found that the prevalence of the “abnormal” bilirubin UGT1A1 allele was 35–40% (9), leading to an expected frequency of homozygotes of ∼16%; however, only 5% had increased serum concentrations of unconjugated bilirubin. Thus, a reduced expression of bilirubin UGT1A1, attributable to the (TA)7 abnormality in the promoter region, appears to be necessary, but not sufficient, for GS to be manifested clinically. To date, the TA polymorphism has been detected by PCR amplification of the TATA-box element and high resolution polyacrylamide gel electrophoresis (9) or by direct sequencing (6). Recently, a new technique for sensitive, relatively inexpensive and automated high-throughput screening of mutations, denaturing HPLC (DHPLC), was introduced (10)(11)(12)(13). In this report, we evaluate the feasibility of applying DHPLC for the detection of TATA-box variants in the promoter region of the UGT1A1 gene in subjects with GS. The UGT1A1 promoter was analyzed by both DHPLC and direct sequencing in 20 unrelated GS patients (16 males and 4 females; age range, 16–40 years) and 20 …

SUMMARYImmune dysregulation, polyendocrinopathy and enteropathy with X-linked inheritance (IPEX) is a serious disease arising from mutations in FOXP3. This gene codifies for a transcription factor whose dysfunction results in hyperactivation of T cells. It is not clear, however, why an intermediate phenotype is not seen in heterozygous females, who are completely healthy. In order to address this question, we investigated X-chromosome inactivation in peripheral blood lymphocytes from a heterozygous female with a child affected by IPEX. No preferential inactivation was shown in freshly sorted CD4+, CD8+, CD19+ cells or in IL-2 cultured CD4 and CD8 T cells, indicating that peripheral blood lymphocytes in these women are randomly selected. Moreover, only one single FOXP3 transcript was expressed by CD4 T cell clones analysed by RT-PCR, confirming that this gene is subject to X- inactivation. We hypothesize that hyper-activation of T cell in carriers of FOXP3 mutations is regulated by the presence of normal regulatory T cells.