Sandra D’Alfonso

Kallikrein genes are associated with lupus and glomerular basement membrane-specific antibody-induced nephritis in mice and humans.

Immune-mediated nephritis contributes to disease in systemic lupus erythematosus, Goodpasture syndrome (caused by antibodies specific for glomerular basement membrane [anti-GBM antibodies]), and spontaneous lupus nephritis. Inbred mouse strains differ in susceptibility to anti-GBM antibody-induced and spontaneous lupus nephritis. This study sought to clarify the genetic and molecular factors that maybe responsible for enhanced immune-mediated renal disease in these models. When the kidneys of 3 mouse strains sensitive to anti-GBM antibody-induced nephritis were compared with those of 2 control strains using microarray analysis, one-fifth of the underexpressed genes belonged to the kallikrein gene family,which encodes serine esterases. Mouse strains that upregulated renal and urinary kallikreins exhibited less evidence of disease. Antagonizing the kallikrein pathway augmented disease, while agonists dampened the severity of anti-GBM antibody-induced nephritis. In addition, nephritis-sensitive mouse strains had kallikrein haplotypes that were distinct from those of control strains, including several regulatory polymorphisms,some of which were associated with functional consequences. Indeed, increased susceptibility to anti-GBM antibody-induced nephritis and spontaneous lupus nephritis was achieved by breeding mice with a genetic interval harboring the kallikrein genes onto a disease-resistant background. Finally, both human SLE and spontaneous lupus nephritis were found to be associated with kallikrein genes, particularly KLK1 and the KLK3 promoter, when DNA SNPs from independent cohorts of SLE patients and controls were compared. Collectively, these studies suggest that kallikreins are protective disease-associated genes in anti-GBM antibody-induced nephritis and lupus.

A loss-of-function variant of PTPN22 is associated with reduced risk of systemic lupus erythematosus

A gain-of-function R620W polymorphism in the PTPN22 gene, encoding the lymphoid tyrosine phosphatase LYP, has recently emerged as an important risk factor for human autoimmunity. Here we report that another missense substitution (R263Q) within the catalytic domain of LYP leads to reduced phosphatase activity. High-resolution structural analysis revealed the molecular basis for this loss of function. Furthermore, the Q263 variant conferred protection against human systemic lupus erythematosus, reinforcing the proposal that inhibition of LYP activity could be beneficial in human autoimmunity.

Genetics of multiple sclerosis: linkage and association studies.

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system caused by an interplay of environmental and genetic factors. The only genetic region that has been clearly demonstrated by linkage and association studies to contribute to MS genetic susceptibility is the human leukocyte antigen (HLA) system. The majority of HLA population studies in MS have focused on Caucasians of Northern European descent, where the predisposition to disease has been consistently associated with the class II DRBl*1501-DQAl*0102-DQBl*0602 haplotype. Apositive association with DR4 was detected in Sardinians and in other Mediterranean populations. Moreover DR1, DR7, DR11 have been found to be protective in several populations.Systematic searches aimed at identifying non-HLA susceptibility genes were undertaken in several populations by means of linkage studies with microsatellite markers distributed across the whole genome. The conclusion of these studies was that there is no major MS locus, and genetic susceptibility to the disease is most likely explained by the presence of different genes each conferring a small contribution to the overall familial aggregation.The involvement of several candidate genes was tested by association studies, utilizing either a population-based (case control) or a family-based (transmission disequilibrium test) approach. Candidate genes were selected mainly on the basis of their involvement in the autoimmune pathogenesis and include immunorelevant molecules such as cytokines, cytokine receptors, immunoglobulin, T cell receptor subunits and myelin antigens. With the notable exception of HLA, association studies met only modest success. This failure may result from the small size of the tested samples and the small number of markers considered for each gene. New tools for large scale screening are needed to identify genetic determinants with a low phenotypic effect. Large collaborative studies are planned to screen several thousands of patients with MS with several thousands of genetic markers. The tests are increasingly based on the DNA pooling procedure.

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.

Prolactin and prolactin receptor gene polymorphisms in multiple sclerosis and systemic lupus erythematosus.

Genes encoding for prolactin (PRL) and its receptor (PRLR) are possible candidates for multiple sclerosis (MS) and systemic lupus erythematosus (SLE) susceptibility. In fact: (1) a prolactin secretion dysfunction has been described in several autoimmune diseases including SLE and MS and their animal models; (2) both PRL and PRLR are structurally related to members of the cytokine/hematopoietin family and have a role in the regulation of the immune response; and (3) both PRL and PRLR genes map in genomic regions that showed linkage with autoimmunity. Prolactin maps on chromosome 6p, about 11-kb telomeric to HLA-DRB1 and PRLR in 5p12-13, which revealed evidence of linkage with MS in different populations. To evaluate a possible role of these two genes in SLE and MS we performed an association study of 19 PRL and PRLR single nucleotide polymorphisms (SNPs). These were directly searched by DHPLC in a panel of SLE and MS patients and selected from databases and the literature. The SNP allele frequencies were determined on patient and control DNA pools by primer-extension genotyping and HPLC analysis. Moreover a panel of HLA typed SLE and control individuals were individually genotyped for the PRL G-1149T polymorphism previously described to be associated with SLE. No statistically significant difference in the allele distribution was observed for any of the tested variations.

No association of DPP6 with amyotrophic lateral sclerosis in an Italian population.

We have attempted to replicate a recently reported association of polymorphism rs10260404, in the Dipeptidyl-peptidase 6 gene (DPP6), with susceptibility to amyotrophic lateral sclerosis (ALS) in a large independent Italian cohort of 904 cases and 1036 controls. Minor allele frequency was 0.38 in cases and 0.39 in controls and no evidence of association with ALS was observed (P=0.638). Our negative results agree with those recently reported in additional Polish and Italian cohorts.

Polymorphisms in the genes coding for iron binding and transporting proteins are associated with disability, severity, and early progression in multiple sclerosis

BackgroundIron involvement/imbalance is strongly suspected in multiple sclerosis (MS) etiopathogenesis, but its role is quite debated. Iron deposits encircle the veins in brain MS lesions, increasing local metal concentrations in brain parenchyma as documented by magnetic resonance imaging and histochemical studies. Conversely, systemic iron overload is not always observed. We explored the role of common single nucleotide polymorphisms (SNPs) in the main iron homeostasis genes in MS patients.MethodsBy the pyrosequencing technique, we investigated 414 MS cases [Relapsing-remitting (RR), n=273; Progressive, n=141, of which: Secondary (SP), n=103 and Primary (PP), n=38], and 414 matched healthy controls. Five SNPs in 4 genes were assessed: hemochromatosis (HFE: C282Y, H63D), ferroportin (FPN1: -8CG), hepcidin (HEPC: -582AG), and transferrin (TF: P570S).ResultsThe FPN1-8GG genotype was overrepresented in the whole MS population (OR=4.38; 95%CI, 1.89-10.1; P<0.0001) and a similar risk was found among patients with progressive forms. Conversely, the HEPC -582GG genotype was overrepresented only in progressive forms (OR=2.53; 95%CI, 1.34-4.78; P=0.006) so that SP and PP versus RR yielded significant outputs (P=0.009). For almost all SNPs, MS disability score (EDSS), severity score (MSSS), as well as progression index (PI) showed a significant increase when comparing homozygotes versus individuals carrying other genotypes: HEPC -582GG (EDSS, 4.24±2.87 vs 2.78±2.1; P=0.003; MSSS, 5.6±3.06 vs 3.79±2.6; P=0.001); FPN1-8GG (PI, 1.11±2.01 vs 0.6±1.31; P=0.01; MSSS, 5.08±2.98 vs 3.85±2.8; P=0.01); HFE 63DD (PI, 1.63±2.6 vs 0.6±0.86; P=0.009). Finally, HEPC -582G-carriers had a significantly higher chance to switch into the progressive form (HR=3.55; 1.83-6.84; log-rank P=0.00006).ConclusionsPolymorphisms in the genes coding for iron binding and transporting proteins, in the presence of local iron overload, might be responsible for suboptimal iron handling. This might account for the significant variability peculiar to MS phenotypes, particularly affecting MS risk and progression paving the way for personalized pharmacogenetic applications in the clinical practice.

Association study of a new polymorphism in the PECAM-1 gene in multiple sclerosis

Genetic polymorphisms of immunorelevant genes may modulate occurrence or clinical features of multifactorial diseases. PECAM-1 is an adhesion molecule crucial for transmigration of cells from blood to tissues, but its genetic contribution to multifactorial diseases has never been investigated. We have identified and characterized a tetranucleotide repeat polymorphism within the third intron of PECAM-1. In a cohort of healthy controls (HC), we found 10 alleles. An assessment of the association of this polymorphism with multiple sclerosis (MS) showed similar allele and genotype frequencies in HC and MS patients as well as in MS patients differing for the gravity of their disease course. We conclude that although potentially able to affect organ-specific autoimmune diseases, this new PECAM-1 polymorphism, does not seem to contribute to the genetic background of MS.

TUBA4A gene analysis in sporadic amyotrophic lateral sclerosis: identification of novel mutations

Viviana Pensato • Cinzia Tiloca • Lucia Corrado • Cinzia Bertolin • Valentina Sardone • Roberto Del Bo • Daniela Calini • Jessica Mandrioli • Giuseppe Lauria • Letizia Mazzini • Giorgia Querin • Mauro Ceroni • Roberto Cantello • Stefania Corti • Barbara Castellotti • Giulia Solda • Stefano Duga • Giacomo P. Comi • Cristina Cereda • Gianni Soraru • Sandra D’Alfonso • Franco Taroni • Christopher E. Shaw • John E. Landers • Nicola Ticozzi • Antonia Ratti • Cinzia Gellera • Vincenzo Silani • The SLAGEN Consortium

Analysis of genes, pathways and networks involved in disease severity and age at onset in primary-progressive multiple sclerosis

Background: The role of genetic factors in influencing the clinical expression of multiple sclerosis (MS) is unclear. Objective: The objective of this paper is to identify genes, pathways and networks implicated in age at onset (AAO) and severity, measured using the Multiple Sclerosis Severity Score (MSSS), of primary-progressive MS (PPMS). Methods: We conducted a genome-wide association study (GWAS) of 470 PPMS patients of Italian origin:. Allelic association of 296,589 SNPs with AAO and MSSS was calculated. Pathway and network analyses were also conducted using different tools. Results: No single association signal exceeded genome-wide significance in AAO and MSSS analyses. Nominally associated genes to AAO and MSSS were enriched in both traits for 10 pathways, including: “oxidative phosphorylation” (FDRAAO=9*10−4; FDRMSSS=3.0*10−2), “citrate (TCA) cycle” (FDRAAO=1.6*10−2; FDRMSSS=3.2*10−3), and “B cell receptor signaling” (FDRAAO=3.1*10−2; FDRMSSS=2.2*10−3). In addition, an enrichment of “chemokine signaling pathway” (FDR=9*10−4) for AAO and of “leukocyte transendothelial migration” (FDR=2.4*10−3) for MSSS trait was observed, among others. Network analysis revealed that p53 and CREB1 were central hubs for AAO and MSSS traits, respectively. Conclusions: Despite the fact that no major effect signals emerged in the present GWAS, our data suggest that genetic variants acting in the context of oxidative stress and immune dysfunction could modulate the onset and severity of PPMS.