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 β°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.

IntroductionA 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 MethodsWe 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).ResultsHLA-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 β-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].ConclusionOur 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.

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 materialsWe 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).ResultsAnalysis 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.ConclusionThis 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.

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.

Background: Multiple Sclerosis (MS) is a chronic multifactorial disease of the central nervous system, specifically characterized by inflammatory demyelination and primary neurode-generation. Recently, a possible role for the TARDBP Ala382Thr mutation in inducing neurode-generation in MS has been hypothesized, considering its confirmed role in other neurodegenerative diseases. The aim of the present study was to explore if this mutation plays a role in inducing or enhancing the brain atrophy in MS. Methods: The study included a group of MS patients carrying the TARDBP Ala382Thr mutation, genetically tested at the MS Centre of the University of Cagliari. Mutated MS patients were age, sex, disease course and EDSS-matched with MS patients without mutation, randomly selected from the genetic database. Recruited patients underwent a brain MRI with a 1.5 Tesla Siemens scanner. Volumes of Whole Brain (WB), White Matter (WM) and Grey Matter (GM) were estimated with SIENAX. The difference in brain volumes was assessed with independent samples t-test. Results: Lower WM volumes resulted significantly associated to mutated group (688.68 ml +/- 36.55 vs. 713,22 ml +/- 27.34; p 0.03). No difference in WB and GM volumes was reported between the two groups. Discussion and Conclusion: Despite this mutation does not play a major role in MS pathogenesis, it may contribute in enhancing the brain atrophy, especially for WM. However, additional studies in other MS populations are needed to better know this issue.

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.

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.

Background: Amongst Sardinians the human leukocyte antigen (HLA) DRB1-DQB1 haplotypes *15:02-*06:01, *16: 01-*05:02, *14:01-4-*05:03 are protective for multiple sclerosis (MS), while *13:03-*03:01, *04:05-*03:01, *03:01-*02:01, *15:01-*06:02 and Mycobacterium avium subspecies paratubercolosis ( MAP) are predisposing factors. We studied the correlation between MAP and HLA.Methods: Five hundred thirty-one patients were searched for anti-MAP2694 antibodies, DRB1-DQB1 genotyping was performed. The haplotypes were classified as predisposing, neutral or protective.Results: Anti-MAP2694 were found in 23 % of subjects carrying one protective HLA versus 32 % without (p = 0.04).Conclusions: We showed a lower frequency of Abs in patients with protective HLA. These haplotypes could have a protective role for both MS and MAP.

"Progressive apraxia of speech in a patient with a C9orf72 mutation." Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, 17(7-8), pp. 608–609

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.

Multiple sclerosis (MS) is a chronic disease of the central nervous system characterized by inflammation and accompanied and followed by neurodegeneration. Missense mutations of the TAR DNA Binding Protein gene (TARDBP) located in the chromosome 1p36.22 region, and the hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9orf72) are pathogenic in other neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Assuming that TARDBP Ala382Thr mutation and C9orf72 expansion may underlie MS, we evaluated their frequency in a large cohort of MS patients and controls from Sardinia, an island characterized by a very high frequency of MS and an unusual genetic background. Genomic DNA was extracted from peripheral blood and analyzed for the presence of a TARDBP Ala382Thr mutation and C9orf72 expansion. Difference in the frequency of these mutations between MS patients and controls was calculated using the χ2 test with a standard 2 × 2 table. The Ala382Thr mutation in its heterozygous state was found in 27/1833 patients (1.4%) and 20/1475 controls (1.3%), whereas C9orf72 pathogenic repeat expansion was found in 6/1014 MS patients (0.6%) and 2/333 controls (0.6%). Individuals carrying the mutations did not present with other neurodegenerative conditions and any differences were reported between groups. TARDBP Ala382Thr mutation and C9orf72 expansion do not play a major role in MS pathogenesis in the Sardinian population. Further analyses on larger samples of MS patients from other populations are needed to better define the possible role of these genes in the complex interplay between neuroinflammation and neurodegeneration in MS.

Multiple sclerosis (MS) is a complex autoimmune disease originated from the interplay between genetic and environmental factors. An overlap of clinical and neuroradiological parameters has been described between MS and an adult-onset neurodegenerative disorder, the fragile-X-associated tremor/ataxia syndrome (FXTAS). This syndrome is caused by a trinucleotide premutation expansion of a CGG sequence in the 55–200 repeat range, which is located in the fragile-X mental retardation 1 (FMR1) gene. Female premutation carriers have an increased propensity for immune-mediated disorders. Recently, a case of co-occurrence of MS and FXTAS was reported. Assuming that the premutation expansion may play a role in the MS susceptibility, we evaluated its frequency in a cohort of MS patients from Sardinia, an island characterized by a very high frequency of MS. Nuclear DNA was extracted by standard methods, purified with bisulfite treatment and then amplified twice by PCR with specific primers. Microsatellite analysis was performed and emizogotic subjects were sequenced. Clinical data of patients were also collected. Only 1/755 MS patients exhibited the premutation expansion with a heterozygosis pattern (30/58). No pathogenic repeat expansions (>200 repeats) were found in the entire cohort. Repeats labeled as the gray zone (45–60 repeats) were observed in 15/755 patients. No specific clinical features concerning disease course, disease activity, and disability were reported for these patients. Our results do not support a possible role for premutation or gray zone alleles in MS Sardinian patients. Further studies are needed to better understand the relationship between FXTAS and MS.