Charcot-Marie-Tooth disease: genetic subtypes in the Sardinian population.

Charcot-Marie-Tooth disease (CMT) is characterised by great variability of genetic subtypes. This study aimed to assess the genetic subtypes of CMT disease in the Sardinian population. Genetic screening was performed for CMT cases (CMT1, CMT2, and hereditary neuropathy with susceptibility to pressure palsies [HNPP]). A total of 1,043 subjects (119 index cases) were evaluated. In CMT1 index cases (69/119; 58%), PMP22 duplication at 17p11.2 was the most frequent genetic diagnosis (60/69; 87%), followed by mutations in the GJB1 gene (5/69; 7.2%), in the SH3TC2 gene (3/69; 4.4%) and PMP22 Gly107Val point mutation (1/69; 1.4%). The CMT2 group (24/119; 20.1%) comprised 10/24 (41.6%) patients carrying MPZ gene Ser44Phe mutation, 6/24 (25%) with mutations in MFN2 and HSPB1, and 1/24 (4.2%) in GJB1 and LRSAM1. In the HNPP group (26/119; 21.9%), the majority of patients reported the PMP22 deletion (25/26; 96.2%). Further studies are needed to comprehend the overall picture of the disease in Mediterranean area.

The p.A382T TARDBP gene mutation in Sardinian patients affected by Parkinson's disease and other degenerative parkinsonisms.

Based on our previous finding of the p.A382T founder mutation in ALS patients with concomitant parkinsonism in the Sardinian population, we hypothesized that the same variant may underlie Parkinson's disease (PD) and/or other forms of degenerative parkinsonism on this Mediterranean island. We screened a cohort of 611 patients with PD (544 cases) and other forms of degenerative parkinsonism (67 cases) and 604 unrelated controls for the c.1144G > A (p.A382T) missense mutation of the TARDBP gene. The p.A382T mutation was identified in nine patients with parkinsonism. Of these, five (0.9 % of PD patients) presented a typical PD (two with familiar forms), while four patients (6.0 % of all other forms of parkinsonism) presented a peculiar clinical presentation quite different from classical atypical parkinsonism with an overlap of extrapyramidal-pyramidal-cognitive clinical signs. The mutation was found in eight Sardinian controls (1.3 %) consistent with a founder mutation in the island population. Our findings suggest that the clinical presentation of the p.A382T TARDBP gene mutation may include forms of parkinsonism in which the extrapyramidal signs are the crucial core of the disease at onset. These forms can present PSP or CBD-like clinical signs, with bulbar and/or extrabulbar pyramidal signs and cognitive impairment. No evidence of association has been found between TARDBP gene mutation and typical PD.

A review of the main genetic factors influencing the course of COVID-19 in Sardinia: the role of human leukocyte antigen-G.

Introduction: A large number of risk and protective factors have been identified during the SARS-CoV-2 pandemic which may influence the outcome of COVID-19. Among these, recent studies have explored the role of HLA-G molecules and their immunomodulatory effects in COVID-19, but there are very few reports exploring the genetic basis of these manifestations. The present study aims to investigate how host genetic factors, including HLA-G gene polymorphisms and sHLA-G, can affect SARS-CoV-2 infection. Materials and Methods: We compared the immune-genetic and phenotypic characteristics between COVID-19 patients (n = 381) with varying degrees of severity of the disease and 420 healthy controls from Sardinia (Italy). Results: HLA-G locus analysis showed that the extended haplotype HLA-G*01:01:01:01/UTR-1 was more prevalent in both COVID-19 patients and controls. In particular, this extended haplotype was more common among patients with mild symptoms than those with severe symptoms [22.7% vs 15.7%, OR = 0.634 (95% CI 0.440 - 0.913); P = 0.016]. Furthermore, the most significant HLA-G 3'UTR polymorphism (rs371194629) shows that the HLA-G 3'UTR Del/Del genotype frequency decreases gradually from 27.6% in paucisymptomatic patients to 15.9% in patients with severe symptoms (X2 = 7.095, P = 0.029), reaching the lowest frequency (7.0%) in ICU patients (X2 = 11.257, P = 0.004). However, no significant differences were observed for the soluble HLA-G levels in patients and controls. Finally, we showed that SARS-CoV-2 infection in the Sardinian population is also influenced by other genetic factors such as ß-thalassemia trait (rs11549407C>T in the HBB gene), KIR2DS2/HLA-C C1+ group combination and the HLA-B*58:01, C*07:01, DRB1*03:01 haplotype which exert a protective effect [P = 0.005, P = 0.001 and P = 0.026 respectively]. Conversely, the Neanderthal LZTFL1 gene variant (rs35044562A>G) shows a detrimental consequence on the disease course [P = 0.001]. However, by using a logistic regression model, HLA-G 3'UTR Del/Del genotype was independent from the other significant variables [ORM = 0.4 (95% CI 0.2 - 0.7), PM = 6.5 x 10-4]. Conclusion: Our results reveal novel genetic variants which could potentially serve as biomarkers for disease prognosis and treatment, highlighting the importance of considering genetic factors in the management of COVID-19 patients.

A Protective HLA Extended Haplotype Outweighs the Major COVID-19 Risk Factor Inherited From Neanderthals in the Sardinian Population.

Sardinia has one of the lowest incidences of hospitalization and related mortality in Europe and yet a very high frequency of the Neanderthal risk locus variant on chromosome 3 (rs35044562), considered to be a major risk factor for a severe SARS-CoV-2 disease course. We evaluated 358 SARS-CoV-2 patients and 314 healthy Sardinian controls. One hundred and twenty patients were asymptomatic, 90 were pauci-symptomatic, 108 presented a moderate disease course and 40 were severely ill. All patients were analyzed for the Neanderthal-derived genetic variants reported as being protective (rs1156361) or causative (rs35044562) for severe illness. The ß°39 C>T Thalassemia variant (rs11549407), HLA haplotypes, KIR genes, KIRs and their HLA class I ligand combinations were also investigated. Our findings revealed an increased risk for severe disease in Sardinian patients carrying the rs35044562 high risk variant [OR 5.32 (95% CI 2.53 - 12.01), p = 0.000]. Conversely, the protective effect of the HLA-A*02:01, B*18:01, DRB*03:01 three-loci extended haplotype in the Sardinian population was shown to efficiently contrast the high risk of a severe and devastating outcome of the infection predicted for carriers of the Neanderthal locus [OR 15.47 (95% CI 5.8 - 41.0), p < 0.0001]. This result suggests that the balance between risk and protective immunogenetic factors plays an important role in the evolution of COVID-19. A better understanding of these mechanisms may well turn out to be the biggest advantage in the race for the development of more efficient drugs and vaccines.

The double-sided of human leukocyte antigen-G molecules in type 1 autoimmune hepatitis.

The immunomodulatory effects of HLA-G expression and its role in cancers, human liver infections and liver transplantation are well documented, but so far, there are only a few reports addressing autoimmune liver diseases, particularly autoimmune hepatitis (AIH). Method and materials: We analyzed the genetic and phenotypic characteristics of HLA-G in 205 type 1 AIH patients (AIH-1) and a population of 210 healthy controls from Sardinia (Italy). Results: Analysis of the HLA-G locus showed no substantial differences in allele frequencies between patients and the healthy control population. The HLA-G UTR-1 haplotype was the most prevalent in both AIH-1 patients and controls (40.24% and 34.29%). Strong linkage was found between the HLA-G UTR-1 haplotype and HLA-DRB1*03:01 in AIH-1 patients but not controls (D' = 0.92 vs D' = 0.50 respectively; P = 1.3x10-8). Soluble HLA-G (sHLA-G) levels were significantly lower in AIH-1 patients compared to controls [13.9 (11.6 - 17.4) U/mL vs 21.3 (16.5 - 27.8) U/mL; P = 0.011]. Twenty-four patients with mild or moderate inflammatory involvement, as assessed from liver biopsy, showed much higher sHLA-G levels compared to the 28 patients with severe liver inflammation [33.5 (23.6 - 44.8) U/mL vs 8.8 (6.1 - 14.5) U/mL; P = 0.003]. Finally, immunohistochemistry analysis of 52 liver biopsies from AIH-1 patients did not show expression of HLA-G molecules in the liver parenchyma. However, a percentage of 69.2% (36/52) revealed widespread expression of HLA-G both in the cytoplasm and the membrane of plasma cells labeled with anti-HLA-G monoclonal antibodies. Conclusion: This study highlights the positive immunomodulatory effect of HLA-G molecules on the clinical course of AIH-1 and how this improvement closely correlates with plasma levels of sHLA-G. However, our results open the debate on the ambiguous role of HLA-G molecules expressed by plasma cells, which are pathognomonic features of AIH-1.

Heat shock protein 27 R127W mutation: evidence of a continuum between axonal Charcot-Marie-Tooth and distal hereditary motor neuropathy.

BACKGROUND: Heat shock protein 27 (HSP27) mutations have been reported to cause both Charcot-Marie-Tooth disease (CMT) type 2F and distal hereditary motor neuropathy (dHMN) although never previously in a single family. OBJECTIVE: To analyse clinical and electrophysiological findings obtained in a single large Sardinian family bearing the HSP27 R127W mutation. METHODS: Twenty-one members of a five generation Sardinian family have been studied, including thirteen members affected by peroneal muscular atrophy and proved heterozygous for the known HSP27 R127W mutation. Twelve patients and eight unaffected relatives were subjected to clinical examination. A standardised electrophysiological study was performed in eleven patients and six unaffected relatives. RESULTS: Mean age at onset (+/-SD) was 31.2+/-7.2 years. Mean age at investigation was 45.2+/-12.9 years and mean disease duration at the time of investigation was 14+/-12.9 years. According to current criteria for CMT2 and dHMN, of the 10 patients who had undergone both clinical and neurophysiological examination, five were diagnosed as CMT2, two as dHMN and a further two patients were labelled as an intermediate type. Finally, due to the presence of spastic paraplegia, the index patient did not meet established criteria for the diagnosis of CMT or dHMN. DISCUSSION: Findings obtained in the present study, broadening the spectrum of clinical manifestations of disorders associated with HSP27 mutations, support the hypothesis of a continuum between CMT2 and dHMN forms and suggest a possible common spectrum between these entities and several forms of CMT plus pyramidal features (HMSN V), providing important implications for molecular genetic testing.

Evolution of the phenotype in a family with an LMNA gene mutation presenting with isolated cardiac involvement.

The aim of this study is to report the evolution of a phenotype in members of a single family carrying the heterozygous exon 1 c.178 C/G, p.Arg 60 Gly LMNA gene mutation. All mutated family members underwent neurological and cardiological assessments for a period ranging from 10 to 20 years. At onset, 4 affected adult members presented a phenotype that required pacemaker implantation. Three subjects underwent cardiac transplantation leading to long-term survival in 2 of them. One of the 3 longest surviving relatives manifested late lipodystrophy, and the other 2 had lipodystrophy, insulin-resistant diabetes, and distal peripheral neuropathy. The findings demonstrate that the exon 1 c.178 C/G, p.Arg 60 Gly LMNA gene mutation is associated with a novel phenotype featuring cardiac involvement followed by late lipodystrophy, diabetes, and peripheral axonal neuropathy.

Assessing the Metabolomic Profile of Multiple Sclerosis Patients Treated with Interferon Beta 1a by 1H-NMR Spectroscopy.

Metabolomic research has emerged as a promising approach to identify potential biomarkers in multiple sclerosis (MS). The aim of the present study was to determine the effect of interferon beta (IFN ß) on the metabolome of MS patients to explore possible biomarkers of disease activity and therapeutic response. Twenty-one MS patients starting IFN ß therapy (Rebif® 44 µg; s.c. 3 times per week) were enrolled. Blood samples were obtained at baseline and after 6, 12, and 24 months of IFN ß treatment and were analyzed by high-resolution nuclear magnetic resonance spectroscopy. Changes in metabolites were analyzed. After IFN ß exposure, patients  were divided into responders and nonresponders according to the "no evidence of disease activity" (NEDA-3) definition (absence of relapses, disability progression, and magnetic resonance imaging activity), and samples obtained at baseline were analyzed to evaluate the presence of metabolic differences predictive of IFN ß response. The results of the investigation demonstrated differential distribution of baseline samples compared to those obtained during IFN ß exposure, particularly after 24 months of treatment (R2X = 0.812, R2Y = 0.797, Q2 = 0.613, p = 0.003). In addition, differences in the baseline metabolome between responder and nonresponder patients with respect to lactate, acetone, 3-OH-butyrate, tryptophan, citrate, lysine, and glucose levels were found (R2X = 0.442, R2Y = 0.768, Q2 = 0.532, p = 0.01). In conclusion, a metabolomic approach appears to be a promising, noninvasive tool that could potentially contribute to predicting the efficacy of MS therapies.

Genetic loci linked to type 1 diabetes and multiple sclerosis families in Sardinia.

BACKGROUND: The Mediterranean island of Sardinia has a strikingly high incidence of the autoimmune disorders Type 1 Diabetes (T1D) and Multiple Sclerosis (MS). Furthermore, the two diseases tend to be co-inherited in the same individuals and in the same families. These observations suggest that some unknown autoimmunity variant with relevant effect size could be fairly common in this founder population and could be detected using linkage analysis. METHODS: To search for T1D and MS loci as well as any that predispose to both diseases, we performed a whole genome linkage scan, sequentially genotyping 593 microsatellite marker loci in 954 individuals distributed in 175 Sardinian families. In total, 413 patients were studied; 285 with T1D, 116 with MS and 12 with both disorders. Model-free linkage analysis was performed on the genotyped samples using the Kong and Cox logarithm of odds (LOD) score statistic. RESULTS: In T1D, aside from the HLA locus, we found four regions showing a lod-score > or =1; 1p31.1, 6q26, 10q21.2 and 22q11.22. In MS we found three regions showing a lod-score > or =1; 1q42.2, 18p11.21 and 20p12.3. In the combined T1D-MS scan for shared autoimmunity loci, four regions showed a LOD >1, including 6q26, 10q21.2, 20p12.3 and 22q11.22. When we typed more markers in these intervals we obtained suggestive evidence of linkage in the T1D scan at 10q21.2 (LOD = 2.1), in the MS scan at 1q42.2 (LOD = 2.5) and at 18p11.22 (LOD = 2.6). When all T1D and MS families were analysed jointly we obtained suggestive evidence in two regions: at 10q21.1 (LOD score = 2.3) and at 20p12.3 (LOD score = 2.5). CONCLUSION: This suggestive evidence of linkage with T1D, MS and both diseases indicates critical chromosome intervals to be followed up in downstream association studies.

Phenotypic variability related to C9orf72 mutation in a large Sardinian kindred.

We investigated intrafamilial phenotypic variability in carriers of the C9orf72 mutation, analysing clinical, neuropsychological and imaging characteristics of various members from a large Sardinian kindred with FTD or ALS. We compared these with those of C9 + patients in our ALS and FTD cohorts. Results showed that three patients carried the C9orf72 mutation: two with ALS and one with FTD and Parkinsonism. C9 + patients in our bvFTD Sardinian cohort had a higher frequency of Parkinsonism than non-mutated patients (75% vs. 36.3%, p <0.02). Parkinsonism was present in 2.7% of our ALS cohort and 3.3% of the C9 + patients. The prevalence of Parkinsonism in C9 + patients in the bvFTD and ALS cohorts showed a statistically significant difference (p <0.006). In conclusion, Parkinsonism was frequently associated with FTD but not ALS in a large Sardinian family, a finding reflected in the wider C9orf72 associated Sardinian ALS and FTD populations.

Changes in GABA(A) receptor gene expression associated with selective alterations in receptor function and pharmacology after ethanol withdrawal.

Changes in the expression of subunits of the GABA type A (GABA(A)) receptor are implicated in the development of ethanol tolerance and dependence as well as in the central hyperexcitability associated with ethanol withdrawal. The impact of such changes on GABA(A) receptor function and pharmacological sensitivity was investigated with cultured rat hippocampal neurons exposed to ethanol for 5 d and then subjected to ethanol withdrawal. Both ethanol treatment and withdrawal were associated with a marked decrease in the maximal density of GABA-evoked Cl- currents, whereas the potency of GABA was unaffected. Ethanol exposure also reduced the modulatory efficacy of the benzodiazepine receptor agonists lorazepam, zolpidem, and zaleplon as well as that of the inverse agonists Ro 15-4513 and FG 7142, effects that were associated with a reduced abundance of mRNAs encoding the receptor subunits alpha1, alpha3, gamma2L, and gamma2S. Ethanol withdrawal restored the efficacy of lorazepam, but not that of low concentrations of zolpidem or zaleplon, to control values. Flumazenil, which was ineffective in control neurons, and Ro 15-4513 each potentiated the GABA response after ethanol withdrawal. These effects of withdrawal were accompanied by upregulation of the alpha2, alpha3, and alpha4 subunit mRNAs as well as of the alpha4 protein. Diazepam or gamma-hydroxybutyrate, but not baclofen, prevented the changes in both GABA(A) receptor pharmacology and subunit mRNA levels induced by ethanol withdrawal. Changes in GABA(A) receptor gene expression induced by prolonged exposure to and withdrawal of ethanol are thus associated with altered GABA(A) receptor function and pharmacological sensitivity.

Constructional apraxia in frontotemporal dementia associated with the C9orf72 mutation: broadening the clinical and neuropsychological phenotype.

In our study we analysed clinical and neuropsychological data in a cohort of 57 Sardinian patients with FTD (55 apparently unrelated and two belonging to the same family), who underwent genetic screening for the C9orf72 mutation. Eight out of 56 patients were found positive for the C9orf72 mutation representing 14% of the entire cohort and 31.6% of the familial cases (6/19). C9orf72 mutated patients differed from the other FTD cases of the cohort for a younger age of onset, higher frequency of familial history for FTD and higher prevalence of delusional psychotic symptoms and hallucinations. In the neuropsychological assessment, C9orf72 mutated patients differed from non-mutated for the high frequency of visuospatial dysfunction regarding constructional apraxia (p = 0.02). In conclusion, our study confirms that Sardinian FTD patients have peculiar genetic characteristics and that C9orf72 mutated patients have a distinctive clinical and neuropsychological profile that could help differentiate them from other FTD patients. In our cohort we found that constructional apraxia, rarely reported in FTD, can properly discriminate between C9orf72 mutated and non-mutated patients and contribute to broaden the neuropsychological profile in frontotemporal dementia associated with this mutation.

Clinical phenotypes and radiological findings in frontotemporal dementia related to TARDBP mutations.

It has been shown that different genes could be associated with distinctive clinical and radiological phenotypes of FTD. TARDBP gene has been described worldwide in few cases of FTD so its phenotype is still unclear. The objective is to study the clinical and radiological characteristics of TARDBP-related FTD. In the present study, we report clinical, neuropsychological and radiological features of five new Sardinian non-related cases of FTD carriers of the p.A382T TARDBP mutation. Furthermore, we reviewed non-related FTD cases with TARDBP mutations previously described in literature. The p.A382T missense mutation of TARDBP was present in the 21.7 % of familial cases of our FTD cohort (5/23) and in no one of the sporadic patients. 3 of 5 patients showed a temporal variant FTD and 4/5 a predominant temporal involvement at MRI. The review of the literature of FTD cases with TARDBP mutations showed that in 5 of 16 cases, the clinical phenotype was consistent with temporal variant of FTD or semantic dementia (31 %) and in 7 of 16 cases neuroimaging showed predominant temporal lobe involvement (43.7 %). Our study further supports the pathogenetic role of TARDBP mutations in pure FTD and in the full spectrum of FTD/ALS. The presence of a predominant temporal lobe involvement in a high percentage of FTD mutated patients with a peculiar clinical pattern could be useful in the differential diagnosis with other forms of dementia/FTD both sporadic and familial.

Role of interferon-beta in Mycobacterium avium subspecies paratuberculosis antibody response in Sardinian MS patients.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) is associated with MS in Sardinia. Because anti-MAP antibodies (Abs) were more frequent in interferon-beta treated patients, we hypothesize that interferon-beta could interact with the immune system. METHODS: Anti-MAP Abs were searched in the blood of 89 patients before commencing interferon-beta and after at least six months. RESULTS: Anti-MAP Abs were detected before and during treatment in 18.7% and 34.7% of patients, respectively. Twenty-three (20.5%) patients became positive during therapy, and 5 (4.4%) patients became negative (p=0.001). CONCLUSIONS: The study supports the hypothesis that interferon-beta could interact with the immune system, enhancing the immunological response against MAP.

C9ORF72 intermediate repeat expansion in patients affected by atypical parkinsonian syndromes or Parkinson's disease complicated by psychosis or dementia in a Sardinian population.

The hexanucleotide repeat expansion GGGGCC in the C9ORF72 gene larger than 30 repeats has been identified as a major genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent papers investigated the possible pathogenic role and associated clinical phenotypes of intermediate C9ORF72 repeat expansion ranging between 20 and 30 repeats. Some studies suggested its pathogenicity for typical Parkinson's disease (PD), atypical parkinsonian syndromes, FTD with/without parkinsonism, and ALS with/without parkinsonism or with/without dementia. In our study, we aimed to screen patients affected by atypical parkinsonian syndromes or PD complicated by psychosis or dementia for the presence of C9ORF72 repeat expansions, and in unrelated age- and sex-matched healthy controls. Consecutive unrelated patients with atypical parkinsonian syndromes and patients with PD complicated by psychosis or dementia were included in this study. Atypical parkinsonian syndromes were further divided into two groups: one with patients who met the criteria for the classic forms of atypical parkinsonism [multiple system atrophy (MSA), Lewy body disease (LBD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD)] ;and patients who did not meet the above criteria, named non-classical atypical parkinsonism with or without dementia. Ninety-two unrelated patients (48 men, 44 women) were enrolled. None of the patients was found to be carriers of C9ORF72 repeat expansions with more than 30 repeats. Intermediate 20-30 repeat expansions were detected in four female patients (4.3 %). Three of them presented clinical features of atypical parkinsonian syndromes, two with non-classical atypical parkinsonism and dementia FTD-like, and one with non-classical atypical parkinsonism without dementia. The other patient presented clinical features of typical PD complicated by psychosis. Among 121 control subjects, none presented long or short expansion for the C9ORF72 gene. Our findings seem to support the hypothesis that the hexanucleotide expansions of C9ORF72 gene with intermediate repetitions between 20 and 29 repetitions could be associated with typical PD with psychosis or dementia and atypical parkinsonisms with dementia (non-classical atypical parkinsonism with dementia FTD-like) or without dementia (non-classical atypical parkinsonism upper MND-like), although the causal relationship is still unclear. In these latter patients, parkinsonism, more or less levodopa responsive, constituted the symptomatological central core at onset.

Association of Mycobacterium avium subsp. paratuberculosis and SLC11A1 polymorphisms in Sardinian multiple sclerosis patients.

INTRODUCTION: Recent findings propose that Mycobacterium avium subsp. paratuberculosis (MAP) infection could act as risk factor in favoring multiple sclerosis (MS) progression. SLC11A1 is a gene associated with mycobacterial survival in the host and it may be involved in the induction and maintenance of autoimmune disease. METHODOLOGY: In this preliminary study, 100 MS patients and 100 healthy controls (HCs) from Sardinia were enrolled. Eight single nucleotide polymorphisms (SNPs) in the SLC11A gene were searched by PCR RFLP-genotyping. IS900 specie specific PCR was undertaken to search for MAP presence. Indirect ELISA was performed to asses if MS patients displayed a stronger humoral response against MAP2694 protein compared to the HCs. RESULTS: Only rs2276631 SNP was associated with MS. MAP DNA was detected in 23 out of 100 MS patients (23%) and in 7 out of 100 HCs (7%). A strong humoral response against MAP2694 protein was detected in 36% of MS patients and only in 3% of HCs. A correlation between ELISA sero-positivity and the rs2276631 SNP was also found. CONCLUSION: Our preliminary results suggest that the Sardinian population might be prone to develop autoimmune disease due to polymorphisms in immunomodulating the SLC11A1 gene, which is important in the immune response against intracellular bacteria such as MAP.

HLA-DRB1-DQB1 haplotypes confer susceptibility and resistance to multiple sclerosis in Sardinia.

INTRODUCTION: Genetic predisposition to multiple sclerosis (MS) in Sardinia (Italy) has been associated with five DRB1*-DQB1* haplotypes of the human leukocyte antigen (HLA). Given the complexity of these associations, an in-depth re-analysis was performed with the specific aims of confirming the haplotype associations; establishing the independence of the associated haplotypes; and assessing patients' genotypic risk of developing MS. METHODS AND RESULTS: A transmission disequilibrium test (TDT) of the DRB1*-DQB1* haplotypes in 943 trio families, confirmed a higher than expected transmission rate (over-transmission) of the *13:03-*03:01 (OR = 2.9, P = 7.6×10(-3)), *04:05-*03:01 (OR = 2.4, P = 4.4×10(-6)) and *03:01-*02:01 (OR = 2.1, P = 1.0×10(-15)) haplotype. In contrast, the *16:01-*05:02 (OR = 0.5, P = 5.4×10(-11)) and the *15:02-*06:01 (OR = 0.3, P = 1.5×10(-3)) haplotypes exhibited a lower than expected transmission rate (under-transmission). The independence of the transmission of each positively and negatively associated haplotype was confirmed relative to all positively associated haplotypes, and to the negatively associated *16:01-*05:02 haplotype. In patients, carriage of two predisposing haplotypes, or of protective haplotypes, respectively increased or decreased the patient's risk of developing MS. The risk of MS followed a multiplicative model of genotypes, which was, in order of decreasing ORs: *04:05-*0301/*03:01-*02:01 (OR = 4.5); *03:01-*02:01/*03:01-*02:01 (OR = 4.1); and the *16:01-*05:02/*16:01-*0502 (OR = 0.2) genotypes. Analysis of DRB1 and DQB1 protein chain residues showed that the Val/Gly residue at position 86 of the DRB1 chain was the only difference between the protective *16:01- *15:02 alleles and the predisposing *15:01 one. Similarly, the Ala/Val residue at position 38 of the DQB1 chain differentiated the positively associated *06:02 allele and the negatively associated *05:02, *06:01 alleles. CONCLUSIONS: These findings show that the association of specific, independent DRB1*-DQB1* haplotypes confers susceptibility or resistance to MS in the MS-prone Sardinian population. The data also supports a functional role for specific residues of the DRB1 and DQB1 proteins in predisposing patients to MS.

Vitamin D responsive elements within the HLA-DRB1 promoter region in Sardinian multiple sclerosis associated alleles.

Vitamin D response elements (VDREs) have been found in the promoter region of the MS-associated allele HLA-DRB1*15:01, suggesting that with low vitamin D availability VDREs are incapable of inducing *15:01 expression allowing in early life autoreactive T-cells to escape central thymic deletion. The Italian island of Sardinia exhibits a very high frequency of MS and high solar radiation exposure. We test the contribution of VDREs analysing the promoter region of the MS-associated DRB1 *04:05, *03:01, *13:01 and *15:01 and non-MS-associated *16:01, *01, *11, *07:01 alleles in a cohort of Sardinians (44 MS patients and 112 healthy subjects). Sequencing of the DRB1 promoter region revealed a homozygous canonical VDRE in all *15:01, *16:01, *11 and in 45/73 *03:01 and in heterozygous state in 28/73 *03:01 and all *01 alleles. A new mutated homozygous VDRE was found in all *13:03, *04:05 and *07:01 alleles. Functionality of mutated and canonical VDREs was assessed for its potential to modulate levels of DRB1 gene expression using an in vitro transactivation assay after stimulation with active vitamin D metabolite. Vitamin D failed to increase promoter activity of the *04:05 and *03:01 alleles carrying the new mutated VDRE, while the *16:01 and *03:01 alleles carrying the canonical VDRE sequence showed significantly increased transcriptional activity. The ability of VDR to bind the mutant VDRE in the DRB1 promoter was evaluated by EMSA. Efficient binding of VDR to the VDRE sequence found in the *16:01 and in the *15:01 allele reduced electrophoretic mobility when either an anti-VDR or an anti-RXR monoclonal antibody was added. Conversely, the Sardinian mutated VDRE sample showed very low affinity for the RXR/VDR heterodimer. These data seem to exclude a role of VDREs in the promoter region of the DRB1 gene in susceptibility to MS carried by DRB1* alleles in Sardinian patients.