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.

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.

The HLA class I-CML association revisited taking into account the two forms of gene fusion in the Philadelphia chromosome. A multicenter study.

CML patients possess either a b3-a2 or a b2-a2 fusion between the BCR and ABL genes. Depending on the type of fusion, two different series of non-self potentially immunogenic peptides may be produced. If they are presented by HLA class I molecules and recognized by cytotoxic CD8 lymphocytes, individuals could be more susceptible or resistant to leukemic cells bearing one or the other form of fusion according to their HLA class I phenotype. To test this point, the frequencies of HLA-A and HLA-B alleles were compared between b3-a2 and the b2-a2 CML patients. In essence, no difference was found whose significance could withstand correction for multiple comparisons.

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.

Linkage analysis of multiple sclerosis with candidate region markers in Sardinian and Continental Italian families.

Previous genome screens in multiple sclerosis have shown some evidence of linkage in scattered chromosomal regions. Although in no case the evidence of each single study was compelling and although in general the linkage 'peaks' of the different studies did not coincide, some regions can be considered likely candidates for the presence of MS risk genes because of the clustering of MLS scores and homology with eae loci. We performed a linkage analysis of markers in these regions and of intragenic markers of some individual candidate genes (HLA-DRB1, CTLA-4, IL9, APOE, BCL2, TNFR2). For the first time, Southern European populations were targeted, namely Continental Italians and Sardinians. A total of 69 multiplex families were typed for 67 markers by a semi-automatic fluorescence-based assay. Results were analysed for linkage by two non-parametric tests: GENEHUNTER and SimIBD. In general, the linkage scores obtained were low, confirming the conclusion that no gene is playing a major role in the disease. However, some markers, in 2p11, 3q21.1, 7p15.2 and 22q13.1 stood out as promising since they showed higher scores with one or the other test. This stimulates further association analysis of a large number of simplex families from the same populations.

Xenoantisera to allotypic specificities of HLA-A and B antigens. Serological characterization.

By using the lysostrip and the blocking of cytotoxicity by Fab2 fragments, HLA-A and B antibodies were detected in sera from rabbits and goats immunized with human high-density lipoprotein (HDL)-associated HLA antigens isolated from sera and from cultured lymphoid cells. After appropriate absorptions, the xenoantisera became specific for HLA-A and B alloantigens, reacted with the same structures as alloantisera, and displayed the same patterns of cross-reactions as alloantisera; however, several xenoantisera contained antibodies cross-reactive with antigens coded by the A and B loci. Our data indicate that HDL-associated HLA antigens isolated from lymphoid cells and from serum efficiently elicit xenoantibodies to allotypic specificities of the human histocompatibility system.

Fine specificity and idiotype diversity of the murine anti-HLA-A2, A28 monoclonal antibodies CR11-351 and KS1.

The monoclonal antibodies (MoAb) CR11-351 and KS1 are secreted by hybridomas generated with splenocytes from BALB/c mice immunized with the cultured human B lymphoid cells LG-2 (HLA-A2,A2,B27,B27). The 2 monoclonal antibodies immunoprecipitated components with a superimposable 2-dimensional gel electrophoretic profile from the cultured B lymphoid cells LG-2 and inhibited the cytotoxicity of the anti-HLA-A2,A28 T cell clone MI#3 to a similar extent. In crossinhibition experiments, the MoAb CR11-351 and KS1 completely inhibited the binding of each other to lymphoid cells with the appropriate HLA phenotype. Testing with a panel of HLA-typed lymphoid cells showed that the MoAb CR11-351 and KS1 display the same serologic specificity, since both of them react with HLA-A2 and/or A28 antigens bearing cells. The 2 monoclonal antibodies recognize distinct, although spatially close, determinants, since only the MoAb CR11-351 displays differential reactivity with HLA-A2 variant cell lines and reacts with HLA-A9 bearing B lymphoid cells. Analysis of MoAb CR11-351 and KS1 with syngeneic polyclonal and monoclonal antiidiotypic antibodies detected no sharing of idiotopes between the 2 monoclonal antibodies. In view of their reactivity with distinct determinants, these results are in agreement with the concept that the antigenic specificity of an antibody controls at least in part the expression of its idiotypes.

The HLA-DR beta 16 allogenotype constitutes a risk factor for hypertrophic scarring.

Nineteen patients that had developed hypertrophic scars subsequent to thermal injury were typed for HLA class II allogenotypes with the restriction fragment length polymorphism technique. A significant association was found with DR beta 16 (pc = 1.45 x 10(-4); relative risk = 12.25). This finding adds evidence to other data suggesting that immunologic phenomena are involved in pathologic scarring. Moreover, the results presented here have allowed an identification of a genetically determined risk factor for hypertrophic scar formation located in the HLA region.

Characterization of a murine monoclonal antibody specific for human early lymphohemopoietic cells.

This paper summarizes the results of the characterization of A10, a murine monoclonal antibody which recognizes an epitope not restricted to cells of a definite lineage, but which seems to be specific for an early differentiation antigen present on precursors of circulating T and B cells. The structure recognized by the A10 monoclonal antibody, although strikingly structurally similar to the HLA-A,B,C complex, is immunologically different both from histocompatibility antigens and from beta 2 microglobulin. Furthermore, the distribution of the A10 antigen is analyzed in different cell and tissue samples, both in health and disease conditions.

Anti-HLA-B7,B27,Bw42,Bw54,Bw55,Bw56, Bw67,Bw73 monoclonal antibodies: specificity, idiotypes, and application for a double determinant immunoassay.

The monoclonal antibodies (MoAbs) KS3 and KS4 are secreted by hybridomas constructed with splenocytes from a BALB/c mouse sequentially immunized with the cultured lymphoid cells JKu and LG-2 which share only the HLA-B27 specificity. Serologic and immunochemical assays have shown that the two MoAbs recognize the same (or spatially close) determinant expressed by HLA-B7,B27,Bw42,Bw54,Bw55,Bw56,Bw67, and Bw73 alloantigens. This determinant is spatially close but distinct from those defined by the anti HLA-B27 monoclonal antibodies described in the literature. The syngeneic antiidiotypic MoAb T12-105 and T12-211 elicited with MoAb KS4 were shown to recognize idiotopes within the antigen combining site of MoAb KS3 and KS4. Neither idiotope was detected on the anti HLA class I and anti HLA class II monoclonal antibodies tested. The MoAb KS4 in combination with the anti human beta 2-microglobulin MoAb NAMB-1 was utilized to develop a double determinant immunoassay (DDIA). The latter represents a sensitive method to detect and quantitate HLA-B27 antigens in spent culture medium of lymphoid cell lines and in serum. Typing for HLA-B27 antigens with the DDIA of sera from HLA typed donors yielded results highly correlated with those of the conventional lymphocytotoxicity assay.

Gamma-IFN induces a differential expression of HLA-DR, DQ and DP antigens on peripheral blood myeloid leukemic blasts at various stages of differentiation.

Using specific monoclonal antibodies and the indirect immunofluorescence technique, peripheral blood myeloid leukemic blasts from 49 patients were studied for DR expression, 42 for DQ and nine for DP after four days of culture without and with gamma-IFN. The expression of class II molecules on untreated cells depended upon the stage of differentiation and was maximal for DR antigens and lower for DP, while DQ was present only in a small percentage of M2, M4 or M5 blasts. M3 blasts lacked both DR and DQ. Maximal increase of surface expression induced by gamma-IFN was observed for DR molecules independently from the differentiation stage. No significant modification was seen for DQ. Preliminary data concerning DP indicate an increased expression in some of the tested samples. Thus the results obtained on peripheral blood blasts parallel previous observations on leukemic cell lines.

CDKN2/p16 inactivation and p16 immunohistochemistry in astrocytic gliomas.

CDKN2/p16 inactivation is the most frequent alteration in the molecular regulation of G1-S transition. CDKN2/p16 homozygous deletions was studied in paraffin-embedded sections of 45 astrocytic tumours by multiplex PCR. Immunohistochemistry for p16 and proliferation marker Ki-67 MIB-1 was performed in adjacent sections; their labelling index (LI) have been calculated. CDKN2/p16 gene was not deleted in astrocytomas, while homozygous deletion was found in 26.7% anaplastic astrocytomas, and in 55.0% of glioblastomas. Analysis of CDKN2/p16 homozygous deletion in discrete areas of the same tumour, showed that the deletion occurred independently of the phenotypic aspect of the areas. Nevertheless a genotypic and phenotypic heterogeneity is present in few cases. p16 immunohistochemistry mostly corresponds to the genotypic pattern. No correlation was found between CDKN2/p16 homozygous deletion and MIB-1 LI.

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.

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.

p27/kip1 expression in human astrocytic gliomas.

In the cell cycle, p27/kip1 acts as an inhibitory protein of cyclin-cdk complexes. p27/kip1 immunohistochemistry was performed in 50 gliomas (15 astrocytomas, 15 anaplastic astrocytomas and 20 glioblastomas) by a polyclonal antiserum. In the same tumours, proliferation marker Ki-67 was studied by MIB-1 antibody. For both, a labelling index (LI) was calculated after counting at least 1000 cells at x1000 magnification. p27 is diffusely and strongly expressed in astrocytomas (LI mean = 44.4%), whereas positive nuclei decrease in number and in staining intensity in anaplastic astrocytomas (LI mean = 5.86%) and glioblastomas (LI mean = 2.1%). An inverse correlation has been found between MIB-1 LI and p27 LI calculated in the same areas. Interestingly, in malignant gliomas the absence of p27 was independent from any histological feature of differentiation or anaplasia. p27 expression is thus reduced in malignant gliomas as in other malignancies. Since mutations of p27/kip1 are extremely rare, posttranslational changes are hypothesised also in astrocytic gliomas.

Historical recombinant sites in extended HLA haplotypes.

Each ancestral or extended HLA haplotype contains a unique combination of alleles among which some may be entirely specific for that haplotype (haplospecific alleles). In the course of evolution many recombination events occurred which disrupted the original haplotypic combination. We analysed the sites of historical recombinations in four extended HLA haplotypes (B8-DR3; B18-DR3; B50-DR7 and B57-DR7) in 60 random Italian individuals selected through the presence of haplospecific alleles. In general the distribution of recombinations in each interval was similar for the four extended haplotypes and no haplospecific recombination "hot spot" could be detected. However some differences between the four haplotypes can be pointed out: a) only 48% fragmented B8-DR3 were found in contrast to 83% B18-DR3, 89% B50-DR7 and 88% B57-DR7; b) in the B8-DR3 haplotype recombinations fall preferentially in the B/TNF interval. In fact among 22 historical recombination events, 50% were mapped in this region; c) conversely, no recombination event was detected in the B/TNF interval among the 19 disrupted B18-DR3 haplotypes thus evidencing the presence of a putative recombination "cold spot".

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.

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.