A trans-specific polymorphism in ZC3HAV1 is maintained by long-standing balancing selection and may confer susceptibility to multiple sclerosis.

The human ZC3HAV1 gene encodes an antiviral protein. The longest splicing isoform of ZC3HAV1 contains a C-terminal PARP-like domain, which has evolved under positive selection in primates. We analyzed the evolutionary history of this same domain in humans and in Pan troglodytes. We identified two variants that segregate in both humans and chimpanzees; one of them (rs3735007) does not occur at a hypermutable site and accounts for a nonsynonymous substitution (Thr851Ile). The probability that the two trans-specific polymorphisms have occurred independently in the two lineages was estimated to be low (P = 0.0054), suggesting that at least one of them has arisen before speciation and has been maintained by selection. Population genetic analyses in humans indicated that the region surrounding the shared variants displays strong evidences of long-standing balancing selection. Selection signatures were also observed in a chimpanzee population sample. Inspection of 1000 Genomes data confirmed these findings but indicated that search for selection signatures using low-coverage whole-genome data may need masking of repetitive sequences. A case-control study of more than 1,000 individuals from mainland Italy indicated that the Thr851Ile SNP is significantly associated with susceptibility to multiple sclerosis (MS) (odds ratio [OR] = 1.47, 95% confidence intervals [CI]: 1.08-1.99, P = 0.011). This finding was confirmed in a larger sample of 4,416 Sardinians cases/controls (OR = 1.18, 95% CI: 1.037-1.344, P = 0.011), but not in a population from Belgium. We provide one of the first instances of human/chimpanzee trans-specific coding variant located outside the major histocompatibility complex region. The selective pressure is likely to be virus driven; in modern populations, this variant associates with susceptibility to MS, possibly via the interaction with environmental factors.

Burden of rare coding variants in an Italian cohort of familial multiple sclerosis.

BACKGROUND: Multiple Sclerosis (MS) is a chronic inflammatory and neurodegenerative demyelinating disease of the central nervous system. It is a complex and heterogeneous disease caused by a combination of genetic and environmental factors, and it can cluster in families. OBJECTIVE: to evaluate at gene-level the aggregate contribution of predicted damaging low-frequency and rare variants to MS risk in multiplex families. METHODS: We performed whole exome sequencing (WES) in 28 multiplex MS families with at least 3 MS cases (81 affected and 42 unaffected relatives) and 38 unrelated healthy controls. A gene-based burden test was then performed, focusing on two sets of candidate genes: i) literature-driven selection and ii) data-driven selection. RESULTS: We identified 11 genes enriched with predicted damaging low-frequency and rare variants in MS compared to healthy individuals. Among them, UBR2 and DST were the two genes with the strongest enrichment (p = 5 × 10-4 and 3 × 10-4, respectively); interestingly enough the association signal in UBR2 is driven by rs62414610, which was present in 25% of analysed families. CONCLUSION: Despite limitations, this is one of the first studies evaluating the aggregate contribution of predicted damaging low-frequency and rare variants in MS families using WES data. A replication effort in independent cohorts is warranted to validate our findings and to evaluate the role of identified genes in MS pathogenesis.

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 non-synonymous SNP within membrane metalloendopeptidase-like 1 (MMEL1) is associated with multiple sclerosis.

Several single-nucleotide polymorphism (SNP) genome-wide association studies (GWASs) have been completed in multiple sclerosis (MS). Follow-up studies of the variants with the most promising rankings, especially when supplemented by informed candidate gene selection, have proven to be extremely successful. In this study we report the results of a multi-stage replication analysis of the putatively associated SNPs identified in the Wellcome Trust Case Control Consortium non-synonymous SNP (nsSNP) screen. In total, the replication sample consisted of 3444 patients and 2595 controls. A combined analysis of the nsSNP screen and replication data provides evidence implicating a novel additional locus, rs3748816 in membrane metalloendopeptidase-like 1 (MMEL1; odds ratio=1.16, P=3.54 × 10⁻6) in MS susceptibility.

A bioinformatics approach to ascertaining the rarity of HLA alleles.

A project of the 15th International Histocompatibility Workshop examined the rarity of human leukocyte antigen (HLA) alleles. A section was constructed in the website, www.allelefrequencies.net to contain this data from different sources. A mechanism to search the data was implemented for use by any individual.

Mutations in the lamin B1 gene are not present in multiple sclerosis.

BACKGROUND: Whole gene duplication of the lamin B1 gene (LMNB1), encoding for a protein of the nuclear lamina, causes an adult-onset autosomal dominant leukodystrophy (ADLD). Clinical features of ADLD (onset in adult life, dysautonomic symptoms, followed by pyramidal and cerebellar dysfunctions) partially resemble those of multiple sclerosis (MS), particularly the primary-progressive form. Our aim was to test whether LMNB1 gene mutations were present amongst patients with a diagnosis of MS. METHODS: One hundred eighty-two MS patients were screened for copy number variations of the LMNB1 gene using a qPCR assay. Point mutations in the LMNB1 gene were searched by denaturing high-performance liquid chromatography and direct sequencing in a subgroup of 16 patients with familial MS. RESULTS: No duplication/deletion of the lamin B1 gene was found amongst MS patients, and no point mutation was identified in the familial cases. CONCLUSION: Our work indicates that lamin B1 defects are probably not responsible for signs and symptoms resembling multiple sclerosis.

BDNF Val66Met polymorphism is associated with cognitive impairment in Italian patients with Parkinson's disease.

BACKGROUND AND PURPOSE: A possible association between Parkinson's disease (PD) and the polymorphism of Brain Derived Neurotrophic Factor (BDNF) G196A (Val66Met) has been suggested by different studies that nevertheless yielded-contrasting result. The purpose of this study was to analyze such possible association in a cohort of Italian PD patients. METHODS: The BDNF polymorphisms were analyzed in 294 Italian patients with PD; results were compared to those obtained in 233 age- and sex-matched healthy controls (HC) enrolled from two tertiary centres in Italy. Polymorphisms were determined by Restriction Fragment Length Polymorphism (RFLP) analysis; correlations between BDNF G196A polymorphism, and cognitive function were established by sub analyzing the results upon dividing PD patients based on their Mini Mental State Examination (MMSE) score. RESULTS: Univariate analysis showed a highly significant correlation between the BDNF(AA) genotype and a MMSE score < or =24. Hence, the distribution of this genotype in PD individuals with a MMSE score < or =24 was significantly increased compared to PD patients with an MMSE score >24 and HC (P < 0.001 in both cases). Multivariate analyses showed that BDNF (AA) genotype was associated to a sixfold risk of cognitive impairment. CONCLUSIONS: The BDNF(AA) homozygote genotype is over-represented in PD patients compared with normal individuals; this genotype was significantly correlated to cognitive impairment, age and disease severity. These results, although preliminary, could be important in establishing novel diagnostic and therapeutic approaches to PD.

HLA-A*01 is associated with late onset of Alzheimer's disease in Italian patients.

In this study, the distribution of HLA-A alleles was analyzed in Italian Alzheimer's Disease (AD)patients. Interaction between HLA alleles, APOE genotypes, age of onset, and gender were also analyzed. The results were compared to those obtained in healthy controls (HC). One hundred-seventy-three AD patients and 258 age-and-sex-matched healthy controls were enrolled in the study. AD patients were classified according to age at the onset of disease using quartiles of the distribution. HLA-A genotyping was performed by PCR-SSP; APOE genotyping was performed by RFLP. A correlation between late disease onset and HLA-A*01 was observed. Thus, HLA-A*01, calculated as number of alleles, was significantly more present in patients with age of onset > 74.0 years than in HC (20% vs 10.5%; p=0.014); the distribution of this allele was skewed also in patients 68.1-74 years of age (16.3%), even if the difference did not reach statistical significance. The relative risk ratio (RRR) of AD onset calculated by a multinomial logistic regression adjusted for sex and presence of APOE-4 confirmed a significant association of HLA-A*01 with AD onset > 74.0 years of age (RRR=2.2; 95%CI: 1.1-4.6; p=0.033). A high RRR (2.04) was also present in patients 68.1-74 years (p=0.064). Lower age of disease onset did not correlate with HLA-A*01. Data herein suggest that the presence of HLA-A*01 results in delayed AD development, even in patients carrying APOE-4. These results could offer new insights into the etiopathogenesis of Alzheimer's disease.

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.

Variations of the perforin gene in patients with multiple sclerosis.

Perforin is involved in cell-mediated cytotoxicity and mutations of its gene (PRF1) cause familial hemophagocytic lymphohistiocytosis (FLH2). PRF1 sequencing in 190 patients with multiple sclerosis and 268 controls detected two FLH2-associated variations (A91V, N252S) in both groups and six novel mutations (C999T, G1065A, G1428A, A1620G, G719A, C1069T) in patients. All together, carriers of these variations were more frequent in patients than in controls (phenotype frequency: 17 vs 9%, P=0.0166; odds ratio (OR)=2.06, 95% confidence interval (CI): 1.13-3.77). Although A91V was the most frequent variation and displayed a trend of association with multiple sclerosis (MS) in the first population of patients and controls (frequency of the 91V allele: 0.076 vs 0.043, P=0.044), we used it as a marker to confirm PRF1 involvement in MS and assessed its frequency in a second population of 966 patients and 1520 controls. Frequency of the 91V allele was significantly higher in patients than in controls also in the second population (0.075 vs 0.058%, P=0.019). In the combined cohorts of 1156 patients and 1788 controls, presence of the 91V allele in single or double dose conferred an OR=1.38 (95% CI=1.10-1.74). These data suggest that A91V and possibly other perforin variations indicate susceptibility to MS.

HLA-DRB1 polymorphisms distribution in chronic dysimmune polyneuropathy.

The frequency of HLA-DRB1*15 polymorphism, which is strongly associated to multiple sclerosis, was investigated in 84 adult patients with chronic dysimmune polyneuropathy and 272 healthy controls. No significant differences were detected between cases and controls and, among patients, according to gender, peripheral nerve antigen antibody seropositivity, and electrophysiological features. A trend towards an increase of HLA-DRB1*11 in anti-MAG neuropathy was detected.

Association between SNAP-25 gene polymorphisms and cognition in autism: functional consequences and potential therapeutic strategies.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is involved in different neuropsychiatric disorders, including schizophrenia and attention-deficit/hyperactivity disorder. Consistently, SNAP-25 polymorphisms in humans are associated with hyperactivity and/or with low cognitive scores. We analysed five SNAP-25 gene polymorphisms (rs363050, rs363039, rs363043, rs3746544 and rs1051312) in 46 autistic children trying to correlate them with Childhood Autism Rating Scale and electroencephalogram (EEG) abnormalities. The functional effects of rs363050 single-nucleotide polymorphism (SNP) on the gene transcriptional activity, by means of the luciferase reporter gene, were evaluated. To investigate the functional consequences that SNAP-25 reduction may have in children, the behaviour and EEG of SNAP-25(+/-) adolescent mice (SNAP-25(+/+)) were studied. Significant association of SNAP-25 polymorphism with decreasing cognitive scores was observed. Analysis of transcriptional activity revealed that SNP rs363050 encompasses a regulatory element, leading to protein expression decrease. Reduction of SNAP-25 levels in adolescent mice was associated with hyperactivity, cognitive and social impairment and an abnormal EEG, characterized by the occurrence of frequent spikes. Both EEG abnormalities and behavioural deficits were rescued by repeated exposure for 21 days to sodium salt valproate (VLP). A partial recovery of SNAP-25 expression content in SNAP-25(+/-) hippocampi was also observed by means of western blotting. A reduced expression of SNAP-25 is responsible for the cognitive deficits in children affected by autism spectrum disorders, as presumably occurring in the presence of rs363050(G) allele, and for behavioural and EEG alterations in adolescent mice. VLP treatment could result in novel therapeutic strategies.

Immune responses to antigens of human endogenous retroviruses in patients with acute or stable multiple sclerosis.

A possible role for human endogenous retroviruses (HERV) in the pathogenesis of MS was investigated by analyzing HERV peptides-stimulated proliferation and cytokine production in MS patients with acute (AMS) or stable (SMS) disease. HERV peptides specific-proliferation and type 1 cytokine production by peripheral blood mononuclear cells was observed in AMS but not in SMS individuals, in whom a type 2 cytokine profile dominates. HERV peptides-stimulated immune responses were modified by changes in disease expression; mediated by CD4+ T lymphocytes; and not related to HLA class II molecules. These data suggest the possibility of a pathogenic role for HERV and HERV-specific immune responses in MS.

[Polymorphism of length of tetranucleotide repeat from the 5'-side from the myelin basic protein gene in multiple sclerosis in Russians]. / Polimorphizm dliny tetranukleotidnogo povtora s 5'-storny ot gena osnovnogo belka mielina pri rasseiannom skleroze u Russkikh.

The myelin basic protein gene (MBP) can confer the susceptibility to multiple sclerosis, because its protein product is the main protein component of myelin of the central nervous system and a potential autoimmune antigen in the disease. A possible association of multiple sclerosis with alleles and genotypes of a microsatellite repeat (TGGA)n, located to the 5' side from the first exon of MBP in ethnic Russians (126 patients with reliable multiple sclerosis and 142 healthy controls from Central Russia) was analyzed using the case-control method. Upon separation of the tetranucleotide repeat site amplification products in 1.5% agarose gel, one can see two distinct bands that can be analyzed as two allele groups (A and B). The distribution of allele A and B group frequencies as well as frequency of allele group B and genotype A/A reliably differs in multiple sclerosis patients and healthy controls. Alleles A and the A/A genotype are associated with the development of multiple sclerosis. We also analyzed the association of multiple sclerosis with combined bearing of alleles and genotypes A and B of MBP and groups of alleles of the DRB1 gene of the major histocompatibility complex that correspond to serospecificities DR1-DR18. The comparison of subgroups of multiple sclerosis patients and healthy individuals, formed on the basis of the DRB1 phenotype, has shown a reliable increase in the frequency of allele B in healthy individuals and the genotype A/A frequency in patients, only among DR4- and DR5-positive individuals. No reliable difference was found in the MBP allele and genotype distribution between multiple sclerosis patients and healthy individuals in combined groups of (DR4,DR5)-negative individuals, i.e., no carriers of any phenotype except DR4 and DR5 were revealed. Thus, MBP or some other nearby gene is involved in the multiple sclerosis development in Russians, predominantly (or exclusively) among DR4 and DR5 carriers. In this case, without stratification of analyzed individuals by the MBP alleles, multiple sclerosis is reliably associated only with DR2(15), but not of DR4 and DR5 alleles of DRB1. The results obtained are in favor of the genetic heterogeneity of multiple sclerosis, and suggest the possibility of epistatic interactions between the MBP and DRB1 genes.

Serum auto antibodies presence in multiple sclerosis patients treated with beta-interferon 1a and 1b.

To verify the possible effect of IFN-beta treatment on auto antibodies development in multiple sclerosis (MS) we studied 69 MS patients before and during the treatment with IFN-beta 1b (n=35) and IFN-beta 1a (n=20) for 27 and 12 months respectively, and, as controls, 14 untreated MS patients. The serum, collected every 3 months from all the patients, was investigated for the presence of antinuclear (ANA), anti-smooth muscle (ASMA), anti-mitochondrial (AMA), anti-native DNA (nDNA) anti-cardiolipin (aCL), anti-parietal cells (APCA), anti-microsomal (AMC) and anti-tireoglobulin (ATG) antibodies. Among the IFN-beta 1b-treated MS patients an increase of the frequency and of the level of ANA, AMC and ATG was observed. ASMA and ANA antibodies were already present in about 45% of the MS patients before the treatment and fluctuated over the time. In one patient the treatment was interrupted after 6 months because of the occurrence of high ASMA level and of an autoimmune hepatitis. The data obtained in the smaller number of MS patients treated with IFN-beta 1a were very similar. No increase in aCL level was observed during both the IFN treatments. Our results indicate that the treatment with IFN-beta induces an increase of AMC and ATG antibodies in MS patients and confirm that, although rare, autoimmune diseases could be observed. The possible effect of these auto antibodies on the treatment efficacy and on MS clinical course need to be further investigated.

Endothelin and nitric oxide levels in cerebrospinal fluid of patients with multiple sclerosis.

In order to investigate the potential role of endothelins (ETs) and nitric oxide (NO) in the pathogenesis of multiple sclerosis (MS) we evaluated the levels of these vasoactive mediators in cerebrospinal fluid (CSF) of relapsing remitting MS patients and in a group of subjects with other neurological diseases (OND) and in a control group of subjects without neurological disease. Eighty patients affected from clinically diagnosed MS were selected, 44 of them were studied during an acute clinical attack and 36 in a stable phase. The OND group included 21 subjects affected by degenerative non inflammatory (n=9) and inflammatory (n=12) neurological disease while the control group included 22 subjects with cancer of the prostate (n=11) and with bladder disease (n=11). ET levels were significantly increased in CSF of relapsing remitting MS patients with an acute clinical attack in comparison with those in a stable phase, the OND group and the control group. Moreover significant differences were observed among the four groups with regard to the NO levels: MS patients in a stable and acute phase like OND group have high levels of NO compared to the control group. Since the blood-brain barrier index values did not differ significantly between the three groups, the data of this study suggest an important role for NO and ET in cerebral microcirculation in MS patients.

Molecular evidences for a role of HSV-1 in multiple sclerosis clinical acute attack.

To verify the possible role of human herpesviruses as triggering or aggravating factors in relapsing-remitting multiple sclerosis (RRMS) clinical acute attack, we studied the prevalence of some herpesviruses in the peripheral blood mononuclear cells (PBMCs) collected from 22 MS patients during an MS relapse and in a stable phase and from 18 healthy controls (HC). DNA belonging to Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), Human cytomegalovirus (HCMV), Epstein-Barr virus (EBV) and Human Herpes virus 6 (HHV-6) has been searched by specific nested polymerase chain reaction (n-PCR). EBV and HHV6 DNA has been detected with high frequency in acute and stable MS and in healthy controls without significant differences. HCMV DNA was observed both in acute and stable MS but not in HC, and, more interestingly, HSV-1 DNA was only found in 13% of acute MS, while both stable MS and healthy controls were negative. On the basis of these results we focused on HSV-1, and to confirm them and to demonstrate that HSV-1 is actively replicating in MS patients during clinical relapse, we searched both messenger RNA (mRNA) and DNA of HSV-1 in the PBMCs of 15 acute MS patients and 15 healthy controls. We found HSV-1 mRNA and DNA in a significant number of acute MS patients but not in the control group. On the whole these data indicate that HSV-1 reactivate in the peripheral blood of MS patients during clinical acute attack and probably play a role in the triggering of MS relapses.

Late-onset cerebellar ataxia with hypogonadism and muscle coenzyme Q10 deficiency.

Two brothers had late-onset progressive ataxia, cerebellar atrophy, and hypergonadotropic hypogonadism associated with coenzyme Q10 (CoQ10) deficiency in skeletal muscle. Both patients improved on high-dose CoQ10 supplementation, stressing the importance of CoQ10 deficiency in the differential diagnosis of cerebellar ataxia, even when onset is late.

A polymorphism in the repetitive (TGGA)n sequence 5' to the human myelin basic protein gene in Italian multiple sclerosis patients.

Human myelin basic protein (hMBP) gene is one of the candidate genes in the complex mosaic of multiple sclerosis (MS) susceptibility. In this study we verified the distribution of the polymorphism of the region 5' flanking the first exon of the hMBP gene, in 97 relapsing remitting, 74 primary progressive Italian MS patients, and in 236 healthy controls, using polymerase chain reaction (PCR) and gel electrophoresis analysis in this region from 1116 - 1540 nt. Three different band patterns were observed: one homozygote with a 354 bp long fragment, one homozygote with 424 bp long fragment and one heterozygote with both bands present. The short fragment was statistically more frequent in RRMS patients than in HC (P<0.05). The long fragment was more present in HC. Similarly the short homozygous pattern (354 bp/354 bp) was significantly higher in the RRMS patients versus the healthy controls (P<0.01). The sequence analysis of the hMBP alleles showed that while the long fragments matched the prototype sequence completely, all the short fragments showed a deletion of 70 bp from nt 1177 to nt 1247, which explains the short 354 bp allele detected by PCR. Moreover two single mismatches in positions 1386 (T-->C) and 1431 (G-->A), were present only in the short hMBP fragment.

Myelin basic protein gene is associated with MS in DR4- and DR5-positive Italians and Russians.

BACKGROUND: The myelin basic protein (MBP) gene may confer genetic susceptibility to multiple sclerosis (MS). The association of MS with alleles of the (TGGA)n variable number tandem repeat (VNTR) 5' to the MBP gene is the subject of conflicting reports. OBJECTIVE: To study possible MS association with VNTR alleles of MBP gene in ethnic Italians and ethnic Russians. METHODS: Two hundred sixty-nine unrelated patients with definite MS and 385 unrelated healthy control subjects from Italy and Russia were genotyped for the MBP VNTR region and for the human leukocyte antigen (HLA) class II DRB1 gene. The phenotype, allele, and genotype frequencies for two groups of MBP alleles were determined. Patients and control subjects were stratified according to HLA-DRB1 phenotypes. RESULTS: The distribution of MBP alleles and genotypes in the two ethnic groups, including both MS patients and control subjects, was very similar. When MS patients and healthy control subjects were stratified according to HLA-DRB1 phenotypes, a significant association of MS with MBP alleles was found only in the DR4- and DR5-positive subgroups. A significant association with MBP alleles was also observed in the nonstratified groups, owing mainly to the contribution of the DR4- and DR5-positive individuals. CONCLUSION: Polymorphism of the MBP or another gene in its vicinity appears to contribute to the etiology of MS for the subgroups of DR4- and DR5-positive Italians and Russians.