MBL2 and MASP2 gene polymorphisms in patients with hepatocellular carcinoma.

The pathogenesis of hepatocellular carcinoma (HCC) is not fully understood, but the majority of patients with HCC are associated with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. Mannan-binding lectin (MBL) is a collectin that can act directly as opsonine or activate MBL-associated serine proteases (MASPs) thus initiating the antibody-independent pathway of the complement system. In our study, we analysed two MBL2 and MASP2 functional polymorphisms (MBL2 allele A/0 and MASP2 D120G) as well as MASP2 polymorphism (Y371D) responsible for an amino acidic change in the protein in 215 HCC patients (HBV-infected, HCV-infected, HBV/HCV co-infected and patients with HCC with no viral infection) and 164 healthy controls to give new insights regarding the role of these two molecules in HCC and viral infection pathogenesis. No significant association was found between MBL2 or MASP2 alleles or genotypes, neither comparing the total patients with HCC and healthy controls nor between the different groups of HCC subjects divided for type of viral infection. Also, dividing the total HCC patients group into low MBL producer (A0 and 00 genotypes) and normal producer (AA genotype) and comparing MASP2 polymorphisms in these two groups, no significant differences were found. Our data do not seem to suggest a role for MBL2 and MASP2 polymorphisms in HCC susceptibility either for HBV-HCV infection-dependent HCC or for HCC raised as a consequence of exposure to different risk factors.

X-chromosome inactivation analysis in a female carrier of FOXP3 mutation.

Immune 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.

Single-tube genotyping of MBL-2 polymorphisms using melting temperature analysis.

Recently several authors correlated MBL-2 gene polymorphisms with different pathologies and there is a growing interest for MBL-2 genotyping in a large number of individuals. We have developed a single-tube, rapid, economic, and fully automated melting temperature analysis screening method, based on ABI 7700 Sequence Detection System technology and SYBR Green I chemistry, for the detection of three polymorphisms (exon 1, codons 52, 54, 57) in the MBL-2 gene. We also developed an electronic sheet for the automatic calling of different genotypes, based on the analysis of the first derivative of ABI 7700 raw data.

Quantitative in situ detection of high-risk human papillomavirus in cytological specimens by SYBR Green I fluorescent labeling.

In this study we developed an in situ protocol for quantitative detection of high-risk human papillomavirus (HPV), based on direct in situ polymerase chain reaction (PCR) with SYBR Green I labeling and GeneAmp 5700 Sequence Detection System technology. This protocol was applied on cytological specimens of patients with cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma (SCC). We performed direct in situ quantitative PCR on cell smears, uninfected human skin fibroblasts, Hela and Caski cells. After in situ amplification, slides were counterstained with propidium iodide and analyzed under a fluorescent microscope in order to localize high-risk HPV and verify preservation of morphology. After PCR optimization, we obtained the following results. The Hela cells showed values ranging from 15 to 33 copies of high-risk HPV per cell, the Caski cell line from 220 to 300 high-risk HPV copies per cell and the cell smear (both CIN and SCC) around 20-35 copies of high-risk HPV per cell. No high-risk HPV amplification was detected in uninfected human fibroblasts, healthy controls, non-amplification control, and non-specific primer control. A positive intranuclear high-risk HPV amplification was detected in cell smears from 20 patients with CIN and 10 with SCC. In conclusion, our in situ quantitative protocol for high-risk HPV detection on cell smears combines both quantitative data and in situ localization of the target, with preservation of morphology. For this reason it could be used as a rapid screening tool when both morphological and quantitative results are requested on the same slide.

Molecular analysis of uromodulin and SAH genes, positional candidates for autosomal dominant medullary cystic kidney disease linked to 16p12.

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.

Detection of AGXT bgene mutations by denaturing high-performance liquid chromatography for diagnosis of hyperoxaluria type 1.

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 I 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.

ALS with variable phenotypes in a six-generation family caused by leu144phe mutation in the SOD1 gene.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder. The mutations of Cu/Zn superoxide dismutase gene (SOD1) are responsible for familial ALS. We investigated a large family of Istro-Rumanian origin characterized by an autosomal dominant ALS occurring in 18 cases (three of which are still alive) throughout six generations. METHODS: Clinical data were available for nine patients from the 2nd generation onward, among which one contained the neuropathological details. The mean age at onset of the disease (+/-SD) was 57.3+/-8.9 years (range 49-72), while the duration of the disease spanned over a length of time equal to 4.9+/-1.96 years (range 1.5-7). The analysis of the coding region of SOD1 was done by PCR and direct sequencing. The SOD1 activity was measured by using the red and mononuclear cells belonging to three of the patients. RESULTS: The leu144phe mutation of SOD1 was identified in four patients while a normal sequence was found in five healthy related subjects. The molecular defect was responsible for a decrease in SOD1 activity. Most of patients in this family presented clinical manifestations of ALS (in particular, the lower limb onset variant) not as severe as typical ALS caused by other SOD1 mutations. However, one patient suffering from hyperthyroidism for 17 years, showed an early onset and a rapidly progressing ALS coupled with dementia. CONCLUSIONS: We described a large family with a relatively not severe phenotype of ALS (due to a leu144phe SOD1 mutation) that was compromised in one patient by a concomitant hyperthyroidism.

Effects of cAMP on intercellular coupling and osteoblast differentiation.

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 Ca(2+) 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.

Polymorphisms in the promoter region and at codon 54 of the MBL2 gene are not associated with IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN) occurs sporadically in unrelated individuals. Several different polymorphic genes have been investigated in recent years in order to demonstrate their possible association with IgAN. Three recent, different studies with conflicting conclusions have discussed the role of the mannose binding lectin (MBL), a serum lectin involved in natural immunity, in the IgAN pathogenesis by examination of MBL deposits in biopsies. In the present study we investigated several polymorphisms of the MBL gene located in the promoter region and in the first exon. METHODS: MBL polymorphism detection was performed in 22 Italian patients with familial IgA nephropathy and in 138 Italian patients with the sporadic form of the disease. The polymorphisms in the MBL2 promoter region and in the exon 1 were investigated, respectively, by direct sequencing and by amplification refractory mutation system-polymerase chain reaction on genomic DNA collected from peripheral blood. Seventy-four unrelated healthy subjects matched for ethnic origin were used as controls. RESULTS: Allelic and genotypic frequencies of the polymorphisms at position -550, -328, -221 and at codon 54 did not show any differences between patients and controls. Similar frequency distributions of these polymorphisms were also found in the subgroups of IgAN patients subdivided according to the clinical manifestations and the progression of the disease. CONCLUSIONS: This study indicates that the analysed polymorphisms of the MBL gene do not appear to be primarily involved in the susceptibility and severity of IgAN.

Detection of MRP1 mRNA in human tumors and tumor cell lines by in situ RT-PCR.

The detection of the multridrug resistance-associated proteins is becoming increasingly important in assessing tumor sensitivity to treatment. In this work we describe a new, rapid, sensitive, and robust method for the detection of MRP1 expression based on direct RT-in situ-PCR technology and fluorochrome-modified (dCTP(Cy3)) nucleotides. MRP1 expression was found in both placenta (BeWo) and liver (Hep G2)-derived tumor cell line as well as in small cell lung carcinoma. In liver-derived cells, MRP1 expression was detected by RT-in situ-PCR but not by in situ hybridization, suggesting a higher sensitivity of in situ amplification for the low level of expression in Hep G2 cells. RT solution PCR confirmed the presence of MRP1 in BeWo and Hep G2 cells, although the level of the gene expression was lower in liver cells. This method represents a viable alternative to conventional immunohistochemistry, and may be useful in the evaluation of MRP1 expression in different tissue or cell lines.

Polymorphisms in the MBL2 promoter correlated with risk of HIV-1 vertical transmission and AIDS progression.

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.