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".

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.

Identification by denaturing high-performance liquid chromatography of numerous polymorphisms in a candidate region for multiple sclerosis susceptibility.

Genetic association analysis of candidate regions where evidence of linkage has accumulated is becoming a key issue in the study of complex diseases. A high density of markers, at least one per centimorgan, is required to improve the chances of observing linkage disequilibrium with disease alleles. A recently available single nucleotide polymorphism (SNP) map designed to cover the whole genome provides an average density of one marker per 2 cM. In the present study we show that the number of markers can be approximately doubled in a selected region, thus reaching a density suitable for association studies, by applying a completely automated technique for polymorphism detection, denaturing high-performance liquid chromatography (DHPLC). A systematic search for SNPs was performed in the region 5ptel-q13, where weak but convergent evidence for linkage with multiple sclerosis has accumulated. Screening for polymorphisms was performed on 124 sequence tagged sites (STSs) in the 3'UTR ends of expressed sequence tags totaling about 30,000 bp. Thirty SNPs in 28 STSs were found with less than 10% overlap with the markers already detected in the same region. The data confirm the validity of the approach using DHPLC on expressed gene sequences tagged by a set of standard commercially available primers.

The natural history of an HLA haplotype and its recombinants.

The presence of haplotype-specific recombination sites can be determined by analyzing the conservation of extended haplotypes in the population. This approach considers all meioses in the history of the population and requires the presence of characteristic markers that easily allow the identification of the haplotype or of its recombined segments. The recombination breakpoint can then be mapped by looking for shared alleles between haplotypes selected through the specific marker/s. We identified a rare perfect tandem duplication of a 145 base pair segment in the LTA promoter, which tags a B60 (B60D) haplotype. The duplication was detected in 16/90 B60+ Europeans, while absent in 101 B60+ Orientals. The conservation of the class I end and the extreme variability of the class II end suggested that the present-day B60D haplotypes originated from an ancestral haplotype by recombination events centromeric to the duplicated sequence. Through a fine mapping using markers of the HLA central region a preferential recombination site was localized in the 60 kilobase interval between TNFd,e, and D6S273/K11 Amicrosatellite loci (i.e., between LST1 and BAT3 genes). This site behaves as a potent recombination enhancer leading to fragmentation in most of the extant B60D haplotypes and can be considered responsible for their "instability". In the relatively recently founded Finnish population, where the LST1/BAT3 interval recombination has probably not yet had the chance to occur, a founder effect can explain the presence of a rare DP (DPB1(*)1601) allele in most B60D haplotypes in this population.

Reassessment of the specificity of lens opacities in myotonic dystrophy.

Cataract has been considered for a long time one of the major indicators of the presence of the mutated myotonic dystrophy (DM) gene in asymptomatic relatives of DM patients. However, some recent studies show that not all cases of cataract typical of DM are associated with the disease even in members of DM families. In order to determine the frequency of lens opacities characteristic of DM in the general population and to evaluate the specificity of lens anomalies for detection of the DM premutation, we screened a sample of 1,400 random individuals for the presence of 'myotonic cataract'. Ten individuals were found with the typical lens opacities and no neuromuscular signs of DM; molecular analysis of the DM mutation showed that they all carried two normal alleles. Our data allow to conclude that bilateral cortical iridescent and posterior cortical lens opacities cannot be considered a marker of the presence of the DM premutation in the general population.

Functional analysis of a new polymorphism in the human TNF alpha gene promoter.

In this paper the functional relevance of a TNFA promoter polymorphism, a G/A polymorphic sequence at position -238, was tested by analysing its influence on TNF alpha production upon in vitro stimulation of monocytes from 78 healthy, unrelated individuals by lipopolysaccharide (LPS) or after allogenic stimulation in a panel of 32 healthy individuals. All TNFA-A positive individuals were either DR3 or DR7 positive, confirming the previously reported strong linkage disequilibrium of the TNFA-A allele with the two extended haplotypes (B18, F1C30, DR3) and (B57, SC61, DR7). No individuals homozygous for the TNFA-A allele were present in the panel. The mean level of TNF alpha production was not significantly different in TNFA-G/G homozygous and in TNFA-A/G heterozygous individuals after LPS stimulation of monocytes (P = 0.35) or after allogenic stimulation (P = 0.7). After LPS and allogenic stimulation DR3 positive individuals had a higher mean TNF production. This could not be further differentiated by typing for TNF -283.

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.

Similar ectopic expression of ICAM-1 and HLA class II molecules in hypertrophic scars following thermal injury.

In previous studies we have shown that HLA Class II antigens are expressed by keratinocytes and fibroblasts in hypertrophic scars. Because of the potential role of immunological events in the pathogenesis of hypertrophic scars, in the present study we have tested hypertrophic scars for the expression of intercellular adhesion molecule-1 (ICAM-1), a molecule which plays an important role in immunological phenomena. Immunoperoxidase staining with anti-ICAM-1 MoAb of 10 hypertrophic scar samples detected this molecule on epidermal keratinocytes and on about 30 per cent of fibroblasts at the site of lymphoid infiltration. The expression of ICAM-1 in hypertrophic scars was similar to that of HLA Class II antigens. Since the concomitant expression of ICAM-1 and HLA Class II by keratinocytes is known to enhance their antigen-presenting properties, the present results support the possibility that immunological events play a role in the disruption of the normal processes of wound healing and tissue remodelling which result in hypertrophic scars.

Origin of a regressed myotonic dystrophy allele.

A new case of regression of the CTG copy number in the myotonic dystrophy allele was observed in a 7 year old boy. His affected father had an expanded allele of about 100 repeats in his lymphocyte DNA while the child showed a 60 repeat allele, of the same size as the present in the grandfather. Analysis of the father's sperm DNA allowed us to detect an expanded fragment of approximately the same size (62 repeats) as that present in the child's and grandfather's lymphocytes. This fragment was not detectable in the father's lymphocytes. Thus the regression is constitutive in the child, being already present in his father's germline. It is therefore likely that the regressed allele is present in all the tissues of the child, allowing a favourable prognosis.

HLA supratypes in an Italian population.

A supratype analysis of a North Italian population was performed, using 16 polymorphisms in the HLA region spanning the HLA-A-DP segment. Fourteen supratypes were identified, mostly corresponding to those found in other Caucasoid populations. The degree of their conservation both within the B-DR/DQ region and in the regions telomeric and centromeric from HLA-A and DP was evaluated and linkage disequilibria among several DR and DP alleles were identified. Notably, the degree of association with DP increased when the DR marker was part of a conserved B-DR/DQ supratype. These data are relevant to the definition of the genetic structure of the population and to the prediction of probabilities of histocompatibility matching between unrelated individuals.

Gametic association of HSP70-1 promoter region alleles and their inclusion in extended HLA haplotypes.

Gametic associations of a three-allele polymorphism of the HSP70-1 promoter region were analyzed in a random North Italian population, in 69 HLA homozygous cell lines and in 29 families in Boston, all typed for HLA class I, class II and complement alleles. Significant phenotypic associations were detected in the random population between HSP70-1 alleles and several HLA markers carried by extended haplotypes. The inclusion of HSP70-1 alleles in extended haplotypes, suggested by population analysis, was confirmed in genotyped cells, including 10W HLA homozygous cell lines and families, selected for the presence of the whole set of alleles reported for conserved extended haplotypes. Every tested extended haplotype was exclusively associated with a given HSP70-1 allele, except those carrying DR1. HSP70-1 C was included in both [HLA-B8, SC01, DR3] and [HLA-B18, F1C30, DR3] extended haplotypes, accounting for the previously observed strong association with DR3. In addition the same allele was found on the [HLA-B13, SC31, DR7], on the [HLA-B62, SB42, DR4] and on the [HLA-B60, SC02, DR13] extended haplotypes. The HSP70-1 A allele was carried by all DR4+ extended haplotypes except the one above cited. HSP70-1 B correlated with DR10, DQB1*0501 and BF*F. Thus the HSP70-1 promoter alleles provide new precisely located markers of extended haplotypes.

TNF production and hypertrophic scarring.

The percentage of TNF alpha- and beta-positive cells was analyzed in hypertrophic scar (N = 13), normotrophic scar (N = 7), and normal skin (N = 6) biopsies using monoclonal antibodies and immunoperoxidase staining of cryostat tissue sections. Samples were first characterized for infiltrating cells. In hypertrophic samples there was a significant increase in activated infiltrating cells, capable of producing TNF beta and IL-1 beta. In contrast, the percentage of TNF alpha-positive cells was significantly lower than that detected in normotrophic scars. In fact, in hypertrophic scar samples a positive staining with anti-TNF alpha mAb was restricted to 8% of tissue-infiltrating cells compared to 35.4% of the cells present in normotrophic scars; 12% of infiltrating cells were stained in normal skin sections. These results suggest that TNF alpha may be important for normal wound healing and that hypertrophic scarring might be partially a consequence of a low amount of TNF alpha.