[Intravenous folinic acid and vitamin B12 supplementation and homocysteine concentration in hemodialysis patients]. / Supplementazione e.v. di acido folinico e vitamina B12 e concentrazioni di omocisteina nei pazienti emodializzati.

INTRODUCTION: Hyperhomocysteinemia is one of the causes of the increased incidence of cardiovascular disease in uremia. Since homocysteine (Hcy) metabolism depends on the availability of folate and vitamin B12, we have measured the effects of chronic i.v. supplementation of folinic acid and vitamin B12 in a group of patients on maintenance hemodialysis. METHODS: We compared the blood concentration of total Hcy (tHcy), vitamin B12 and folate and the intraerythrocyte concentration of folate in a group of 27 hemodialysis patients (Treated group), given an i.v supplementation with folinic acid (0.9 mg) and Vitamin B12 (cyanocobalamine 1.5 mg and hydroxycobalamine 1.5 mg) three times per week at the end of each dialysis session with those measured in a similar group of 28 hemodialysis patients without supplementation (No Treatment group). The patients were also characterized for the thermolabile variant (mutation C667-->T) of the enzyme methylene-tetrahydrofolate reductase (tMTHFR). RESULTS: High plasma levels (< 11.7 micromol/L) of tHcy were observed in 54/55 patients. T patients had Hcy values significantly lower than NT ones (31.7+/-3.6 vs. 1.1+/-8.3micromol/L, p < 0.05). Serum vitamin B12 (1200 73.6 vs. 762+/-72.2 pmol/L, p < 0.001) and intraerythrocyte folate levels were also significantly higher in the T group (2176+/-127 vs. 1511+/-156, p < 0.005), while no significant difference was observed for serum folate. The distribution of tMTHFR genotypes was similar in the two groups. Homozygous patients showed higher levels of Hcy in comparison with wild type patients both in the whole population (62.32+/-15.9 vs.30.43+/-3.2, p < 0.05) and in the NT group (87.8+/-25.3 vs.36.8+/-13.1., p < 0.05), while no significant difference was observed among genotypes in the T group. CONCLUSIONS: Uremic patients on hemodialysis, when supplemented with even low i.v. dose of folinic acid and vitamin B12, show significantly lower plasma levels of tHcy than non-supplemented patients.

Folate supplementation in peritoneal dialysis patients with normal erythrocyte folate: effect on plasma homocysteine.

The possible role of folate supplementation in reducing hyperhomocysteinemia in dialysis patients has been reported in several recent papers. However, scant data are available for peritoneal dialysis patients; besides, none of these studies investigated either the role of intraerythrocyte folate concentration or the presence of side effects caused by folate administration. Sixty-six peritoneal dialysis patients with hyperhomocysteinemia (>15 micromol/l) and normal folate status (as assessed by erythrocyte folate level >600 nmol/l) were randomly allocated to receive either oral folate (5 mg/day) or no vitamin supplementation. After 2 months of therapy, patients were requested to answer a questionnaire investigating the occurrence of symptoms possibly related to folate supplementation. Twenty-nine treated patients and 30 untreated controls completed the study. In the treated patients, serum and erythrocyte folate increased significantly (p < 0.0001) (respectively from 10.6 +/- 4.9 to 237 +/- 231 nmol/l and from 1,201 +/- 297 to 2,881 +/- 294 nmol/l) to levels at the uppermost limit of detection by laboratory methods. Serum vitamin B(12) levels did not change. Plasma homocysteine levels decreased from 54 +/- 32 to 23 +/- 14 micromol/l after folate supplementation and remained unchanged in the control group. After 4 months of folate therapy, homocysteine concentration was within the normal range in 5 patients (17%) and below 30 micromol/l in the other 21 (72%). Folate therapy resulted in a decrease in homocysteine of more than 50% in 45% of the patients and decrease of more than 20% in a further 38%. No significant symptoms were reported. Thus, serum and erythrocyte folate increase confirms that normal folate levels are inadequate in dialysis patients, even if serum and erythrocyte levels before folate supplementation cannot predict the effect on homocysteine plasma levels.

Reduction of the homocysteine plasma concentration by intravenously administered folinic acid and vitamin B(12) in uraemic patients on maintenance haemodialysis.

BACKGROUND: Hyperhomocysteinaemia is an independent cardiovascular risk factor which can induce vascular lesions, thus contributing to the early development of atherosclerosis. Low-dose folic acid supplementation reduces the pretreatment homocysteine plasma levels by 25-35%. Recent studies report that higher intravenous or oral administration of the active form of folic acid reduces the homocysteine plasma concentration by nearly 70%. The reduction could also be influenced by the thermolabile variant of methylenetetrahydrofolate reductase (tMTHFR) and by the dialysis modality. METHODS: A cross-sectional clinical study was performed to evaluate the effect of a drug containing folinic acid and vitamin B(12) on the plasma homocysteine concentration and whether this variable could also be influenced by the presence of a genetic variant of the methionine pathway and the use of different dialysis modalities. The plasma homocysteine concentration was measured in 55 patients undergoing haemodialysis, 27 of whom have been treated intravenously for megaloblastic anaemia using a drug containing low concentrations of folinic acid and vitamin B(12) at the end of each dialysis session for 6 months. The presence of tMTHFR was sought by molecular analysis, and the role of the dialysis modality was also investigated. RESULTS: The patients given the folic acid treatment had lower homocysteine plasma levels than those not so treated. The plasma homocysteine concentration was significantly higher in the tMRHFR homozygotes than in the patients with a normal genotype, significantly lower in the treated than in the untreated homozygotes, and significantly higher in the untreated homozygotes than in the untreated subgroup with a normal genotype. The homocysteine level was also significantly lower in the patients who underwent convective haemodialysis than in those who received standard bicarbonate dialysis. CONCLUSIONS: A drug containing low concentrations of folinic acid combined with vitamin B(12) using an intermittent intravenous regimen is effective in reducing the homocysteine plasma concentration in uraemic patients. The homocysteine levels seem also to depend on genotype and dialysis modality.

Homocysteine, vitamin B12, serum and erythrocyte folate in peritoneal dialysis patients.

Plasma homocysteine (tHcy) is an important risk factor for atherosclerosis in dialysis patients. Few data were reported on the prevalence and severity of hyperhomocysteinemia in peritoneal dialysis (PD) patients. In addition, little attention was paid to the search of factors possibly involved in the pathogenesis of hyperhomocysteinemia in these patients. A cross-sectional study was performed in 107 stable PD patients. None of them was given folate or vitamin B12 supplementation before or during the study. Plasma tHcy, serum vitamin B12, serum and erythrocyte folate were measured by immunoenzymatic methods. Genetic analysis of the methylentetrahydrofolate-reductase thermolabile mutation (tMTHFR) was performed in 61 patients. 97% of patients had tHcy levels higher than normal. tHcy was not different between men and women, patients with or without malnutrition, with or without clinically evident atherosclerotic vasculopathy, with or without anemia. tHcy levels were significantly higher in homozygotes for the tMTHFR mutation than in patients carrying the wild type form. Significant univariate correlation was found between hyperhomocysteinemia and time since the start of dialysis, serum and erythrocyte folate and vitamin B12. The best fitted model equation was log tHcy = 108.53 + 0.1606 (duration of dialysis) -1.1053 (s-F) -0.7980 (age) 0.0215 (vitamin B12). Our results agree with those reported by other authors in hemodialysis patients. Despite the large number of PD patients with normal serum vitamin B12 and folate status, the relation between tHcy and vitamin B12 or folate suggests that the supplementation of these vitamins could be useful irrespective of their serum levels, especially in younger patients or in those treated for a long period of time with peritoneal dialysis.

Identification of a new member (ZNF183) of the Ring finger gene family in Xq24-25.

Four genes were mapped to the Xq24-25 region by searching the EST and the non-redundant database with short tracts of genomic sequences. These were random STSs present in the STS database or sequences derived from CpG islands (EagI-based STSs). One of the four matches corresponded to the full length transcript from the intronless glutamate dehydrogenase gene. The second was the human homolog of the bovine NADH ubiquinone oxidoreductase MWFE subunit gene (GDB symbol: NDUFA1). The other two, ZNF183 and ITBA4, were novel genes whose function cannot directly be inferred from their sequence analysis. However, a known motif, the C3HC4 Ring finger domain, shared by various tumor suppressors, DNA repair genes and cytokine receptor-associated molecules, is present at the C terminus of the ubiquitously expressed ZNF183 gene. ITBA4 is expressed at various levels in different tissues and is alternatively processed in brain. Similarity search did not detect any significant match in databases. These results, together with others previously reported by our laboratory, suggest that comparison of genomic and transcribed sequences which are continuously accumulating in databases, can provide 'virtual' mapping of a substantial number of ESTs to the specific genomic region which the STSs have been derived from.

Characterization and fine localization of two new genes in Xq28 using the genomic sequence/EST database screening approach.

Two new genes were identified and mapped by searching the EST databases with genomic sequences obtained from putative CpG islands of the rodent-human hybrid X3000. Previous mapping of these CpG islands in the proximity of the host cell factor (HCFC1) and GdX genes automatically localized these two new genes to Xq28 in the interval between the L1 cell adhesion molecule (L1CAM) and the glucose-6-phosphate dehydrogenase (G6PD) loci. Both genes are relatively short, contain an ORF of 261 and 105 amino acids, respectively, and are ubiquitously expressed. Combining sequencing of selected CpG islands, derived from hybrids containing small portions of the human genome, with an EST database search is an easy method of identifying and mapping new genes to specific regions of the genome.

Decreased affinity of apolipoprotein AII to high-density lipoprotein in patients with transthyretin-related amyloidosis (Met30, Gln89, Pro36, and Thr34).

We previously reported that patients with familial amyloidotic polyneuropathy (FAP) (Met30) showed low plasma apolipoprotein AII (apoAII) levels and apolipoprotein AII/AI (apoAII/AI) ratio as the progression of the disease while plasma levels of apoAI, B, CII, CIII, and E were all within normal ranges. In the present study, we investigated these apolipoproteins contained in high density lipoprotein (HDL) extracted from plasma of FAP (Met30), and the levels of apoAII gene expression in the liver from patients with FAP (Met30). Plasma apoAII levels and the apoAII/AI ratio in extracted HDL in all the FAP types examined decreased compared with those in normal control subjects. Levels of apoAII mRNA expression in the liver of FAP (Met30) patients did not show significant changes compared with those in control subjects. These results suggest that decreased affinity of apoAII to HDL could not be compensated by overproduction of apoAII in the liver of the FAP patients and as a result of this phenomenon, plasma apoAII levels might decrease as the progression of FAP.

Organization of the human CD40L gene: implications for molecular defects in X chromosome-linked hyper-IgM syndrome and prenatal diagnosis.

Recently, CD40L has been identified as the gene responsible for X chromosome-linked hyper-IgM syndrome (HIGM1). CD40L on activated T cells from HIGM1 patients fails to bind B-cell CD40 molecules, and subsequent analysis of CD40L transcripts by reverse transcription PCR demonstrated coding region mutations in these patients. This approach, however, is of limited use for prenatal diagnosis of HIGM1 in the early-gestation fetus. In this report, we have defined the genomic structure of the CD40L gene, which is composed of five exons and four intervening introns. With this information, we have defined at the genomic level the CD40L gene abnormalities for three previously described HIGM1 patients who demonstrated clustered deletions in the CD40L coding region. These different deletions arose from three distinct mechanisms, including (i) a splice donor mutation with exon skipping, (ii) a splice acceptor mutation with utilization of a cryptic splice site, and (iii) a deletion/insertion event with the creation of a new splice acceptor site. In addition, we have performed prenatal evaluation of an 11-week-old fetus at risk for HIGM1. CD40L genomic clones provide a starting point for further studies of the genetic elements that control CD40L expression. Our knowledge of the CD40L gene structure will prove useful for the identification of additional mutations in HIGM1 and for performing genetic counseling about this disease.

ZNF75: isolation of a cDNA clone of the KRAB zinc finger gene subfamily mapped in YACs 1 Mb telomeric of HPRT.

We have previously mapped a zinc finger genomic motif (ZNF75) to the Xq26 cytogenetic band by using a hybrid panel. Here, we report the isolation of the transcribed counterpart in a cDNA clone and its further localization. The cDNA clone, from a lung fibroblast library, is assembled from three exons, including a 289 amino acid (AA) long open reading frame containing a recently described motif, the Kruppel-associated box, 42 AA long, in exon 2. By comparison with other reported members of the subfamily, the exon-intron boundaries also appear to be very well conserved. Further analysis allowed us to map this gene 1 Mb downstream from the HPRT gene in the published YAC contig that extends across Xq26. Two other motifs, 87 and 78% homologous to ZNF75 at the amino acid level, were identified by PCR on total human DNA, but map outside Xq24-qter.

Mapping of two genes encoding isoforms of the actin binding protein ABP-280, a dystrophin like protein, to Xq28 and to chromosome 7.

ABP-280 is a ubiquitous actin binding protein present in the cytoskeleton of many different cell types. ABP-280 was mapped to distal Xq28, 50-60 kb downstream of the Green Colour Pigment (GCP) genes. To establish if ABP-280 may be a candidate for one of the muscle disease localized by linkage analysis to distal Xq28 we looked for alternative forms of ABP-280 mRNA. Several different ABP-280 mRNAs were indeed identified: two are X-linked and are produced by alternative splicing of a small exon of 24 nucleotides. At least one additional gene encoding a RNA more than 70% identical to ABP-280 in the 1700 bp sequenced has also been found. It was mapped to chromosome 7. While both forms of the X-linked ABP-280 are ubiquitous, the gene on chromosome 7 is highly expressed only in skeletal muscle and heart. The two genes were therefore excellent candidates for the X-linked and for the autosomal dominant form of the Emery-Dreifuss Muscular Dystrophy (EDMD) both of which have been described. So far, however we were unable to demonstrate mutations in the coding region or affecting the alternative splicing of the X-linked form of ABP-280, in several patients studied, and we think that it is quite unlikely that this is the gene responsible for EDMD.

Type 2 vasopressin receptor gene, the gene responsible nephrogenic diabetes insipidus, maps to Xq28 close to the LICAM gene.

Although long contigs have been assembled in Xq28, the interval between the anonymous probe St14 and the color vision locus is still incompletely defined. We report here that the recently cloned gene for type 2 vasopressin receptor (V2R) is physically linked to L1CAM using YACs and cosmids across about 180 kb of the region. Since it is known that L1CAM maps near the color pigment genes, this finding locates V2R in Xq28 in the area where nephrogenic diabetes insipidus (NDI) has been mapped by linkage analysis. The PFGE analysis of the clones positions V2R about 40 kb from the L1CAM gene in a region that appears to contain other unknown genes, since at least four putative CpG islands were identified by restriction analysis with rare cutter enzymes.

Isolation of a zinc finger motif (ZNF75) mapping on chromosome Xq26.

We report here the partial characterization of a new human zinc finger (ZNF75) gene of the Kruppel type mapping to the long arm of the X chromosome. A cosmid clone was isolated from a library specific to the Xq24-qter region by hybridization to a degenerate oligonucleotide representing the link between two contigous fingers of the C2H2 type. The sequence of the pertinent cosmid fragments demonstrated five consecutive zinc finger motifs, all pertaining to the Kruppel family. A reading frame starting at least 75 amino acids before the first zinc finger and ending 11 amino acids after the last one was identified; comparison with other ZF genes suggests that this genomic fragment represents the carboxy-terminal exon of the gene. Homology of approximately 55% in the zinc finger region was detected with many zinc finger genes including mouse Zfp-35 and human ZFN7 cDNA clones. Mapping using a panel of sematic cell hybrids and chromosomal in situ hybridization localized the gene to Xq26, in a region not previously known to contain zinc finger genes.

Methylation and sequence analysis around EagI sites: identification of 28 new CpG islands in XQ24-XQ28.

Thirty-two probes for CpG islands of the distal long arm of the human X chromosome have been identified. From a genomic library of DNA of the hamster-human cell hybrid X3000.1 digested with the rare cutter restriction enzyme EagI, 53 different human clones have been isolated and characterized by methylation and sequence analysis. The characteristic pattern of DNA methylation of CpG islands at the 5' end of genes of the X chromosome has been used to distinguish between EagI sites in CpG islands versus isolated EagI sites. The sequence analysis has confirmed and completed the characterization showing that sequences at the 5' end of known genes were among the clones defined CpG islands and that the non-CpG islands clones were mostly repetitive sequences with a non-methylated or variably methylated EagI site. Thus, since clones corresponding to repetitive sequences can be easily identified by sequencing, such libraries are a very good source of CpG islands. The methylation analysis of 28 different new probes allows to state that demethylation of CpG islands of the active X and methylation of those on the inactive X chromosome are the general rule. Moreover, the finding, in all instances, of methylation differences between male and female DNA is in very strong support of the notion that most genes of the distal long arm of the X chromosome are subject to X inactivation.

The nucleotide sequence of a CpG island demonstrates the presence of the first exon of the gene encoding the human lysosomal membrane protein lamp2 and assigns the gene to Xq24.

An EagI-EcoRI clone of human genomic DNA, p2-7, mapped to Xq24 has been sequenced. This analysis has confirmed the presence of a CpG island and has identified the first exon of the human LAMP2 gene, encoding a glycoprotein of the lysosomal membrane. Since the p2-7 clone corresponds to single-copy DNA, we can assign the human LAMP2 gene to Xq24.