HLA-class I markers and multiple sclerosis susceptibility in the Italian population.

Previous studies reported an association with multiple sclerosis (MS) of distinct HLA-class I markers, namely HLA-A*02, HLA-Cw*05 and MOG-142L. In this work, we tested the association with MS of A*02 and Cw*05 in 1273 Italian MS patients and 1075 matched controls, which were previously analyzed for MOG-142, and explored the relationship among these three markers in modulating MS risk. HLA-A*02 conferred a statistically robust MS protection (odds ratio, OR=0.61; 95% confidence intervals, CI=0.51-0.72, P<10(-9)), which was independent of DRB1*15 and of any other DRB1* allele and remained similar after accounting for the other two analyzed class I markers. Conversely, the protective effect we previously observed for MOG-142L was secondary to its linkage disequilibrium with A*02. Cw*05 was not associated considering the whole sample, but its presence significantly enhanced the protection in the HLA-A*02-positive group, independently of DRB1: the OR conferred by A*02 in Cw*05-positive individuals (0.22, 95% CI=0.13-0.38) was significantly lower than in Cw*05-negative individuals (0.69, 95% CI=0.58-0.83) with a significant (P=4.94 x 10(-5)) multiplicative interaction between the two markers. In the absence of A*02, Cw*05 behaved as a risk factor, particularly in combination with DRB1*03 (OR=3.89, P=0.0006), indicating that Cw*05 might be a marker of protective or risk haplotypes, respectively.

A novel recessive splicing mutation in the POU1F1 gene causing combined pituitary hormone deficiency.

BACKGROUND: Mutations in the gene encoding the pituitary transcription factor POU1F1 (Pit-1, pituitary transcription factor-1) have been described in combined pituitary hormone deficiency (CPHD). AIM: The aim of this study was the characterisation of the molecular defect causing CPHD in a patient born to consanguineous parents. SUBJECT AND METHODS: The case of a 12.5-yr-old girl presenting with severe growth failure at diagnosis (-3 SD score at 3 months) and deficiency of GH, PRL, and TSH was investigated for the presence of POU1F1 gene mutations by denaturing high performance liquid chromatography analysis. RESULTS: A novel mutation adjacent to the IVS2 splicing acceptor site (IVS2-3insA) was identified in the patient at the homozygous state. Analysis of patient's lymphocyte mRNA and an in vitro splicing assay revealed the presence of 2 aberrant splicing products: a) deletion of the first 71 nucleotides of exon 3, altering the open reading frame and generating a premature stop codon, b) total exon 3 skipping resulting in an in frame deleted mRNA encoding a putative protein lacking part of the transactivation domain and of the POUspecific homeodomain. Notably, the patient's relatives heterozygous for the mutation had PRL levels under the normal range with no evident clinical symptoms. CONCLUSIONS: The IVS2- 3insAmutation, responsible for CPHD at the homozygous state, causes the presence of 2 aberrant splicing products encoding non-functional products. In the heterozygotes one normal allele might not guarantee a complete pituitary function.

A sequence variation in the MOG gene is involved in multiple sclerosis susceptibility in Italy.

Several studies suggest that the histocompatibility complex (HLA) class I region harbours genes modulating multiple sclerosis (MS) susceptibility independently from the effect of class II alleles. A candidate gene in this region is MOG, encoding the myelin oligodendrocyte glycoprotein. A significant association with the missense variation V142L (rs2857766) was previously reported in a small sample of 50 Italian MS patients. We confirmed this result in two independent Italian sample sets consisting of 878 MS patients and 890 matched controls (P=6.6 x 10(-4)) and 246 trio families (P=1.5 x 10(-3)). The comparison of genotype frequencies suggested a dominant-protective effect of L142. In the combined sample sets L142 conferred an odds ratio (OR)=0.70 (95% confidence interval (CI): 0.60-0.82) that remained similar after accounting for HLA-DRB1(*)15 carrier status. The association with MOG V142L was still significant after conditioning for all DRB1 alleles (P=0.035). Eleven additional single nucleotide polymorphisms in the MOG gene (namely -1077T/C, -910T/C, -875A/G, -93T/C, S5S, Indel L22, V145I, +814C/T, +900A/G, +1024A/T, +1059C/T), two microsatellites in the MOG 5' flanking (MOGCA) and 3' untranslated (MOGTAAA) regions and four microsatellites in the HLA-class I region, from HLA-B to HFE, (namely MIB, D6S265, D6S1683 and D6S2239) were tested by transmission disequilibrium test in 199 trio families. None of these polymorphisms or of their haplotypic combinations showed a significant transmission distortion, in the absence of V142L. In conclusion, MOG V142L, or an untested variant in tight-linkage disequilibrium with it, is an independent MS susceptibility-modulating factor in the HLA class I region.

A family-based study does not confirm the association of MYO9B with celiac disease in the Italian population.

Association between Myosin IXB (MYO9B) gene polymorphisms and celiac disease (CD) was recently detected by a case-control association study in the Dutch, but not confirmed in the British and Swedish/Norwegian populations. We tested the association between CD and the three most associated single nucleotide polymorphisms (SNPs) in the Dutch study by the transmission disequilibrium test in the Italian population. A total of 252 pediatric patients and 504 parents were genotyped. No transmission distortion was detected either for the single SNPs or for their haplotypic combinations. Control allele frequencies, calculated from untransmitted alleles, were significantly different from those of the Dutch control population. Conversely, allele frequencies were very similar in Italian, British, Swedish/Norwegian and Dutch patients. In conclusion, MYO9B is not involved in CD susceptibility in the Italian population. The difference with the Dutch result might be explained by an imperfect selection of the Dutch controls.

SOD1 gene mutations in Italian patients with Sporadic Amyotrophic Lateral Sclerosis (ALS).

Mutations in the SOD1 gene exons and exon/intron boundaries were searched in 66 sporadic and 4 familial Italian ALS cases consecutively referred to our centre from different Italian regions. A mutation was found in three sporadic cases (4.5%): a new nonsense mutation in exon 5 (K136X) in a patient with a rapid and severe disease course and two previously described missense nucleotide substitutions (N65S and A95T) in two patients with a mild disease course. Comparison of the clinical characteristics with previously reported patients carrying the same or similar mutations showed a remarkable genotype-phenotype correlation. No association was found with intronic sequence variations by comparing their frequency in the patients and in 181 matched controls.

Concordance, disease progression, and heritability of coeliac disease in Italian twins.

BACKGROUND AND AIMS: We adopted the twin method to disentangle the genetic and environmental components of susceptibility to coeliac disease (CD). We estimated disease concordance rate by zygosity and HLA genotypes, discordance times, progression rates to disease, and heritability. METHODS: We crosslinked the Italian Twin Registry with the membership lists of the Italian Coeliac Disease Association and recruited 23 monozygotic (MZ) and 50 dizygotic (DZ) twin pairs with at least one affected member. Zygosity was assigned by DNA fingerprinting, and HLA-DQ and DR alleles were genotyped. Disease status was ascertained by antiendomysial, anti-human tissue transglutaminase antibodies, and bowel biopsy. RESULTS: Concordance was significantly higher in MZ (83.3% probandwise, 71.4% pairwise) than in DZ (16.7% probandwise, 9.1% pairwise) pairs. Concordance was not affected by sex or HLA genotype of the co-twin and being MZ was significantly associated with the occurrence of CD (Cox adjusted hazard ratio 14.3 (95% confidence interval 4.0-50.3)). In 90% of concordant pairs the discordance time was

Two single-nucleotide polymorphisms in the 5' and 3' ends of the osteopontin gene contribute to susceptibility to systemic lupus erythematosus.

OBJECTIVE: To test the association of osteopontin (OPN) polymorphisms with systemic lupus erythematosus (SLE). METHODS: The coding 5' and 3' flanking regions of the OPN gene were scanned for polymorphisms by denaturing high-performance liquid chromatography. A case-control association study was performed in 394 Italian SLE patients and 479 matched controls. OPN serum levels were determined by enzyme-linked immunosorbent assay in 40 patients and 124 controls, and the mean levels were compared between the different OPN genotypes. RESULTS: Among the 13 detected single-nucleotide polymorphisms (SNPs), alleles -156G (frequency 0.714 versus 0.651; P = 0.006, corrected P [P(corr)] = 0.036) and +1239C (0.377 versus 0.297; P = 0.00094, P(corr) = 0.0056) were significantly increased in the SLE patients compared with the controls. The presence of the associated allele in single or double dose conferred an odds ratio (OR) of 2.35 (95% confidence interval [95% CI] 1.38-4.02) for SNP -156 and an OR of 1.57 (95% CI 1.16-2.13) for SNP +1239. These effects were independent of each other, i.e., not a consequence of linkage disequilibrium between the 2 alleles. The risk associated with a double dose of susceptibility alleles at both SNPs was 3.8-fold higher (95% CI 2.0-7.4) relative to the complete absence of susceptibility alleles. With regard to individual clinical and immunologic features, a significant association was seen between lymphadenopathy and -156 genotypes (overall P = 0.0011, P(corr) = 0.046). A significantly increased OPN serum level was detected in healthy individuals carrying +1239C (P = 0.002), which is indicative of an association between the SLE susceptibility allele and OPN levels. CONCLUSION: These data suggest the independent effect of a promoter (-156) and a 3'-untranslated region (+1239) SNP in SLE susceptibility. We can speculate that these sequence variants (or others in perfect linkage disequilibrium) create a predisposition to high production of OPN, and that this in turn may confer susceptibility to SLE.

Genetic defects in GH synthesis and secretion.

Growth hormone (GH) is a multifunctional hormone produced in the anterior pituitary that promotes postnatal growth of skeletal and soft tissues. GH secretion and release are complex phenomena depending on several intrinsic and extrinsic factors modulating the release of two hypothalamic hormones, GH releasing hormone and somatostatin. Any genetic or acquired disorder that impairs GH secretion or action causes a pathological phenotype characterized by harmonic short stature with isolated GH deficiency (IGHD) or combined pituitary hormone deficiency (CPHD). In this article we report current knowledge about the genetic basis of IGHD and CPHD.

Two new PROP1 gene mutations responsible for compound pituitary hormone deficiency.

Mutations in the Prophet of Pit-1 (Prop-1), a paired-like homeodomain transcription factor involved in the early embryonic pituitary development, have been reported as a cause of combined hormone deficiency (CPHD) involving growth hormone (GH), prolactin (PRL), thyroid-stimulating hormone (TSH), gonadotrophins and in some cases adrenocorticotrophic hormone (ACTH). We report two pre-pubertal siblings with short stature and deficiency of GH and TSH at presentation. Molecular analysis of the PROP1 gene revealed compound heterozygotes for two novel missense mutations of the PROP1 gene affecting the same amino acid (Arg71Cys and Arg71His) in the first alpha helix of the Prop-1 homeodomain.

Additional factor in some HLA DR3/DQ2 haplotypes confers a fourfold increased genetic risk of celiac disease.

Although HLA-DQ genes are the major celiac disease (CD) susceptibility genes, results from Finnish families suggest that not all DQ2-encoding haplotypes confer equal susceptibility to CD, implying the effect of other gene(s) in the HLA region. The aim of the present work was to extend and confirm the aforementioned results in a southern European population ( Italian) and to better localize the additional risk factor/s. The association of nine loci spanning the HLA region from DR to HFE, 4.5-Mb telomeric of HLA-A, was tested. The analysis was performed by comparing marker frequencies in DR3-DQ2 haplotypes transmitted and non-transmitted to the affected offspring in 156 Italian CD families selected for having at least one DR3-positive parent. The same analysis was performed independently in 101 Finnish CD families selected with the same criteria. Three alleles, MICA-A5.1, MICB-CA24 and MIB-350, all characteristic of the B8-DR3 extended haplotype, showed a significantly increased frequency in DR3 transmitted haplotypes in the Italian families. DR3 haplotypes carrying the combination of these alleles conferred an approximate fourfold increased CD risk. B8-DR3 transmitted haplotypes were significantly more conserved telomerically down to the MIC-Class I region. Similar results were seen in the Finnish families. The major conclusion that holds true in both populations is that, while DQ2 is an absolute requirement for the development of CD, the presence of an additional genetic factor within the MIC-Class I region confers an approximate 4-fold increased risk of the disease.

Association tests with systemic lupus erythematosus (SLE) of IL10 markers indicate a direct involvement of a CA repeat in the 5' regulatory region.

Many lines of evidence suggest that IL10 is a strong candidate gene for systemic lupus erythematosus (SLE) susceptibility. In our previously reported study an allele (IL10.G-140bp) of the microsatellite IL10.G located at position -1100 was significantly increased in Italian SLE patients in comparison with controls. Starting from this observation, we tested if sequence variations in the vicinity of IL10.G were more strongly associated with SLE. We performed a comprehensive association study including 26 SNPs (of which four were newly identified in the present study by DHPLC analysis) spanning 8.5 Kb of the 5' flanking and the transcribed region of the IL10 gene. The association study was performed by the DNA pool method on an extended panel of Italian patients (205) and controls (631). Haplotypic associations were studied by individual typing of seven selected markers surrounding IL10.G. Gene, genotype and haplotype frequencies were not significantly different in patients and controls. Thus the IL10.G microsatellite remains to date the only IL10 marker associated with SLE in our population. A meta-analysis of all published results indicates a possible direct role of the IL10.G repeat number in SLE susceptibility.

The IL12B gene does not confer susceptibility to coeliac disease.

Coeliac disease (CD) is a chronic inflammatory disorder where dietary gluten is not tolerated. In the lesion there are gluten reactive T cells predominantly secreting gamma-interferon. Both HLA and non-HLA genes contribute to CD susceptibility. Interleukin-12 (IL-12) regulates gamma-interferon production. The IL12B gene is located in a region (5q31.1-33.1) where there is evidence for linkage with CD. Allele 1 of an IL12B 3'UTR single-nucleotide polymorphism leads to increased expression of IL-12, and was recently implicated in susceptibility for type 1 diabetes (T1D). We found no evidence for association of allele 1 to CD by the transmission/disequilibrium test or case-control approach. No increased frequency was observed in patients belonging to families where the disease was linked to markers on chromosome 5q. Unlike T1D, allele 1 does not appear to confer susceptibility to CD.

Abnormal ubiquitination of axons in normally myelinated white matter in multiple sclerosis brain.

The hallmark of the lesions in multiple sclerosis (MS) is inflammatory demyelination with sparing of axons. Recent neuropathological and neuroradiological investigations show that structural changes of the axons occur, both in plaques and in the normal appearing white matter. A better understanding of the axonal damage in MS is important, since this may be responsible for permanent disability. We have investigated the immunoreactivity for ubiquitin, a sensitive method to detect axonal dystrophy and accumulation of abnormal proteins in pathological conditions of the nervous system, in the brains of six cases of MS (age range 39-66 years). Tissue blocks were fixed in formalin and embedded in paraffin. A panel of antibodies was used: anti-ubiquitin, anti-neurofilament (SMI-31 + SMI-32), anti-amyloid precursor protein and anti-PGP9.5. We focused our attention on chronic plaques, recognized by the absence of Luxol Fast Blue B-positive inclusions in macrophages. SMI-31 + SMI-32 showed the presence of a variable amount of axons within the plaques; the axonal network within the plaques was looser than outside. No ubiquitin reactivity was present in chronic plaques. In the normally myelinated white matter surrounding the plaques, a dense granular ubiquitin immunoreactivity was found both near and far from the plaque edge. No similar staining was found in control brains. Ubiquitination is the first step of a non-lysosomal degradation pathway of proteins. The present findings suggest a derangement of this proteolytic pathway in the axons outside the plaques, possibly as a consequence of chronic absence of myelin in the axonal segment inside the plaque. The spectrum of axonal changes in MS appears to be wider than expected and involves the apparently normal white matter.

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.

Existence of a genetic risk factor on chromosome 5q in Italian coeliac disease families.

Coeliac disease (CD) is a malabsorptive disorder of the small intestine resulting from ingestion of gluten. The HLA risk factors involved in CD are well known but do not explain the whole genetic susceptibility. Several regions of potential linkage on chromosomes 3q, 5q, 10q, 11q, 15q and 19q have already been reported in the literature. These six regions were analyzed with the Maximum Lod Score method on a dense set of markers. A new sample of 89 Italian sibpairs was available for study. There was no evidence for linkage for any of the regions tested, except for chromosome 5q. For this region, our data, as well as a sample of 93 sibpairs from our first genome screen (Greco et al. 1998), are compatible with the presence of a risk factor for CD with a moderate effect.

Determination of SNP allele frequencies in pooled DNAs by primer extension genotyping and denaturing high-performance liquid chromatography.

By testing DNA pools rather than single samples the number of tests for a case-control association study can be decreased to only two for each marker: one on the patient and one on the control pool. A fundamental requirement is that each pool represents the frequency of the markers in the corresponding population beyond the influence of experimental errors. Consequently the latter must be carefully determined. To this aim, we prepared pools of different size (49-402 individuals) with accurately quantified DNAs, estimated the allelic frequencies in the pools of two SNPs by primer extension genotyping followed by DHPLC analysis and compared them with the real frequencies determined in the single samples. Our data show that (1) the method is highly reproducible: the standard deviation of repeated determinations was +/-0.014; (2) the experimental error (i.e., the discrepancy between the estimated and real frequencies) was +/-0.013 (95% C.I.: 0.0098-0.0165). The magnitude of this error was not correlated to the pool size or to the type of SNP. The effect of the observed experimental error on the power of the association test was evaluated. We conclude that this method constitutes an efficient tool for high-throughput association screenings provided that the experimental error is low. We therefore recommend that before a pool is used for extensive association studies, its quality, i.e., the experimental error, is verified by determining the difference between estimated and real frequencies for at least one marker.

Distribution of activated caspase-3 in relation with apoptosis in human malignant gliomas.

Activated caspase-3has been immunohistochemically studied in 30glioblastomas. Its distribution has been compared with that of apoptotic nuclei demonstrated by terminal dUTP nick-end labeling (TUNEL) and morphology. The best procedure for the demonstration of caspase-3 requires formalin fixation, followed by Carnoy fixation, with microwave irradiation. The number of positive cells is lower than that of apoptotic nuclei shown by TUNEL technique, especially in perinecrotic pseudo-palisadings, and there are also qualitative variations. Positive staining occurs in nuclei, cytoplasms or in both cell compartments. The interpretation of Caspase-3 positive staining is based on its crucial position in the final pathway to apoptosis and on the mechanisms by which it cleaves cytoplasmic and nuclear proteins among which inhibitory/caspase-activated DNase system is included.

MICA and MICB microsatellite alleles in HLA extended haplotypes.

The present study is a contribution to the definition of the linkage disequilibrium relationship of MICA and MICB with adjacent loci and to the characterization of extended HLA haplotypes. These issues are of importance for the identification of disease associations and for a better definition of donor-recipient compatibility in bone-marrow grafts through the typing of haplospecific markers. The distribution of the five alleles of MICA and the 13 alleles of MICB microsatellites, located, respectively, in MICA transmembrane exon 5 and in MICB intron 1, was examined in 133 healthy Italian individuals previously typed for HLA class I, class II and complement loci and for the TNFa microsatellite. The MICB microsatellite was also analysed in 49 HTCLs for which MICA typing was already available. Very strong linkage disequilibria with HLA-B and TNFa were detected in the Italian population for both MICA and MICB microsatellite alleles, in spite of the high mutability rate of the larger MICB alleles. Some strong associations were also detected between MICB and DRB1. The strongest associations (P < 0.001, D' > 0.7) were those of MICA-A4 with HLA-B18, B27 and TNFa1, MICA-A5 with HLA-B35, B61 and B62, MICA-A5.1 with HLA-B7, B8, B13, B63 and MICB-CA24, MICA-A6 with HLA-B51, MICA-A9 with HLA-B39, B57 and TNFa2, MICB-CA14 with HLA-B14, B27 and TNFa1, MICB-CA15 with HLA-B52, TNFa4 and TNFa13, MICB-CA17 with HLA-B7 and TNFa11, MICB-CA18 with HLA-B13 and TNFa7, MICB-CA22 with HLA-B57, and MICB-CA24 with HLA-B8 and TNFa2. From pairwise associations in the random panel and results for the homozygous cell lines it was possible to deduce the MICA and MICB microsatellite alleles present in many of the well-known Caucasoid extended haplotypes.

An attempt of identifying MS-associated loci as a follow-up of a genomic linkage study in the Italian population.

Subsequent to a genomic linkage study on Sardinian and Continental Italian families, we considered the possibility that some of the tested microsatellite markers showed association to MS. Markers selected on the basis of the data obtained in the original set of 70 multiplex families were tested for MS association in an additional set of 154 simplex families. A limited set of markers were further tested on an additional set of 100 simplex families. The results indicate the presence of a putative MS gene in 19q13.13.

HLA class I in acute promyelocytic leukemia (APL): possible correlation with clinical outcome.

The majority of patients with acute promyelocytic leukemia (APL) possess either a bcr1 or a bcr3 type fusion between PML and RARalpha genes. The junction sequences may possibly be a target for immune response and influence susceptibility to the disease. In this case, HLA class I allele frequencies would be different between bcr1 and bcr3 patients. To test this hypothesis, we typed 102 APL patients for HLA-A, -B and -Cw alleles. The A*1, A*30, B*51, B*41, Cw*0602, and Cw*1701 alleles showed a different distribution between bcr1 and bcr3 patients, but in no case was this statistically significant after correction for the number of comparisons or was confirmed in an independent panel. Moreover, no difference was detected between bcr1 and bcr3 when HLA alleles were grouped according to their peptide binding specificities. Comparing HLA frequencies, clinical features at diagnosis and clinical outcome of the 64 patients homogeneously treated with all-trans retinoic acid and idarubicin (AIDA protocol) we observed a statistically significant association between HLA-B*13 and risk of relapse by univariate and multivariate regression analysis. Should this finding be confirmed in larger future studies, this observation would be of outmost importance in identifying patients at high risk of relapse in which more aggressive consolidation therapies should be used.