Patients' Disalignment in Two Different Healthcare Settings.

Among the important bulk of research devoted to medical consultations, one recurrently discussed issue has been that of patients' alignment with practitioners' recommendations. If this question has not always been formulated in terms of alignment, all the studied cases deal with how patients comply, or not, with practitioners' first actions. They show that social actions such as suggestions, proposals, offers, etc. are not unilaterally offered by practitioners to patients, but frequently discussed and negotiated. This may result in patients being more willing to comply with jointly achieved solutions. In this paper, we will fill in some more details of this picture by focusing on interactional resources used by patients to show their disalignment toward less investigated types of first actions (i.e. non-medication recommendations, home remedies, proposals or suggestions to accomplish certain activities), thereby acknowledging the central role played by patients in two different healthcare settings (general and mental health). We will also compare how linguistic and cultural diversity are handled when patients and practitioners communicate directly as well as when communication is interpreter-mediated, thereby problematizing the presence of an interpreter who needs to grasp the variety of resources used by patients in order to render both their disalignment and practitioners' responses to it. Taking into account audio- and video-recorded naturally occurring data collected in Italy and France, we will additionally show the relevance of multimodal analysis for a better understanding of the resources involved, as well as of the dynamics of interpreter-mediated communication in healthcare.

Investigating the Causal Effect of Brain Expression of CCL2, NFKB1, MAPK14, TNFRSF1A, CXCL10 Genes on Multiple Sclerosis: A Two-Sample Mendelian Randomization Approach.

Multiple Sclerosis (MS) exhibits considerable heterogeneity in phenotypic expression, course, prognosis and response to therapy. This suggests this disease involves multiple, as yet poorly understood, causal mechanisms. In this work we assessed the possible causal link between gene expression level of five selected genes related to the pro-inflammatory NF-κB signaling pathway (i.e., CCL2, NFKB1, MAPK14, TNFRSF1A, CXCL10) in ten different brain tissues (i.e., cerebellum, frontal cortex, hippocampus, medulla, occipital cortex, putamen, substantia nigra, thalamus, temporal cortex and intralobular white matter) and MS. We adopted a two-stage Mendelian Randomization (MR) approach for the estimation of the causal effects of interest, based on summary-level data from 20 multiplex Sardinian families and data provided by the United Kingdom Brain Expression Consortium (UKBEC). Through Radial-MR and Cochrane's Q statistics we identified and removed genetic variants which are most likely to be invalid instruments. To estimate the total causal effect, univariable MR was carried out separately for each gene and brain region. We used Inverse-Variance Weighted estimator (IVW) as main analysis and MR-Egger Regression estimator (MR-ER) and Weighted Median Estimator (WME) as sensitivity analysis. As these genes belong to the same pathway and thus they can be closely related, we also estimated their direct causal effects by applying IVW and MR-ER within a multivariable MR (MVMR) approach using set of genetic instruments specific and common (composite) to each multiple exposures represented by the expression of the candidate genes. Univariate MR analysis showed a significant positive total causal effect for CCL2 and NFKB1 respectively in medulla and cerebellum. MVMR showed a direct positive causal effect for NFKB1 and TNFRSF1A, and a direct negative causal effect for CCL2 in cerebellum; while in medulla we observed a direct positive causal effect for CCL2. Since in general we observed a different magnitude for the gene specific causal effect we hypothesize that in cerebellum and medulla the effect of each gene expression is direct but also mediated by the others. These results confirm the importance of the involvement of NF-κB signaling pathway in brain tissue for the development of the disease and improve our understanding in the pathogenesis of MS.

Acid sensing ion channel 2: A new potential player in the pathophysiology of multiple sclerosis.

Acid-sensing ion channels (ASICs) are proton-gated channels involved in multiple biological functions such as: pain modulation, mechanosensation, neurotransmission, and neurodegeneration. Earlier, we described the genetic association, within the Nuoro population, between Multiple Sclerosis (MS) and rs28936, located in ASIC2 3'UTR. Here we investigated the potential involvement of ASIC2 in MS inflammatory process. We induced experimental autoimmune encephalomyelitis (EAE) in wild-type (WT), knockout Asic1-/- and Asic2-/- mice and observed a significant reduction of clinical score in Asic1-/- mice and a significant reduction in the clinical score in Asic2-/- mice in a limited time window (i.e., at days 20-23 after immunization). Immunohistochemistry confirmed the reduction in adaptive immune cell infiltrates in the spinal cord of EAE Asic1-/- mice. Analysis of mechanical allodynia, showed a significant higher pain threshold in Asic2-/- mice under physiological conditions, before immunization, as compared to WT mice and Asic1-/- . A significant reduction in pain threshold was observed in all three strains of mice after immunization. More importantly, analysis of human autoptic brain tissue in MS and control samples showed an increase of ASIC2 mRNA in MS samples. Subsequently, in vitro luciferase reporter gene assays, showed that ASIC2 expression is under possible miRNA regulation, in a rs28936 allele-specific manner. Taken together, these findings suggest a potential role of ASIC2 in the pathophysiology of MS.

Percutaneous Endoscopic Transgastric Jejunostomy (PEG-J) Tube Placement for Levodopa-Carbidopa Intrajejunal Gel Therapy in the Interventional Radiology Suite: A Long-term Follow-up.

BACKGROUND: Percutaneous endoscopic gastrojejunostomy (PEG) and radiologically inserted gastrojejunostomy (RIG) are both safe and effective techniques for gastrojejunal tube placement. The authors compared these 2 procedures in patients with advanced Parkinson's disease (PD) who required the continuous intrajejunal delivery of a levodopa/carbidopa gel suspension (LCIG). METHODS: Outcomes were retrospectively collated from 30 PEG and 12 RIG procedures performed at 2 centers in patients with advanced PD for the delivery of LCIG. RESULTS: Baseline clinical characteristics, incidence of early severe adverse events, late major complications, dropout, and the mean time-lapse of tube replacements were comparable in the PEG and RIG groups. CONCLUSION: The current results suggest that, in patients with PD, the RIG technique is as safe and effective as the endoscopic procedure, and it can be considered a valid option for patients who require LCIG when the endoscopic procedure is not available or unfeasible.

Investigating multiple sclerosis genetic susceptibility on the founder population of east-central Sardinia via association and linkage analysis of immune-related loci.

BACKGROUND: A wealth of single-nucleotide polymorphisms (SNPs) responsible for multiple sclerosis (MS) susceptibility have been identified; however, they explain only a fraction of MS heritability. OBJECTIVES: We contributed to discovery of new MS susceptibility SNPs by studying a founder population with high MS prevalence. METHODS: We analyzed ImmunoChip data from 15 multiplex families and 94 unrelated controls from the Nuoro Province, Sardinia, Italy. We tested each SNP for both association and linkage with MS, the linkage being explored in terms of identity-by-descent (IBD) sharing excess and using gene dropping to compute a corresponding empirical p-value. By targeting regions that are both associated and in linkage with MS, we increase chances of identifying interesting genomic regions. RESULTS: We identified 486 MS-associated (p < 1 × 10-4) and 18,426 MS-linked (p < 0.05) SNPs. A total of 111 loci were both linked and associated with MS, 18 of them pointing to 14 non-major histocompatibility complex (MHC) genes, and 93 of them located in the MHC region. CONCLUSION: We discovered new suggestive signals and confirmed some previously identified ones. We believe this to represent a significant step toward an understanding of the genetic basis of MS.

Posterior Reversible Encephalopathy Syndrome with Bilateral Independent Epileptic Foci Precipitated By Guillain-Barrè Syndrome.

We report the case of a 56-year-old woman who developed status epilepticus (SE) related to independent occipital foci as clinical manifestation of posterior reversible encephalopathy syndrome (PRES) in the background of Guillain-Barrè syndrome (GBS). SE resulted from a series of focal seizures clinically characterized by left- and rightward deviations of the head and consequent oculoclonic movements. Electroencephalography recorded independent seizure activity in both occipital regions with alternate involvement of the two cerebral hemispheres. The epileptic foci corresponded topographically to parenchymal abnormalities of PRES in the occipital lobes. The manifestation of bilateral, independent occipital seizures with alternate deviations of the head and oculoclonic movements, previously not reported in patients with PRES, highlights the acute epileptogenicity of the cerebral lesions in this syndrome. Despite the variable clinical expression of seizures due to occipital damage in PRES, the development of independent seizure activity in both occipital lobes might represent a distinctive epileptic phenomenon of this encephalopathy.

TBK1 is associated with ALS and ALS-FTD in Sardinian patients.

Recently, mutations in the TANK-binding kinase 1 (TBK1) gene were identified as a cause for amyotrophic lateral sclerosis (ALS) with or without comorbid frontotemporal dementia. We have assessed the frequency and clinical characteristics of TBK1 mutations in a cohort of ALS patients of Sardinian ancestry. Whole-exome sequencing was performed on Hiseq2000 platform (Illumina). Genome analysis Toolkit was used to align and to code variants according to Human Genome (UCSC hg19). Mutation was confirmed with Sanger sequence. In our screening of 186 Sardinian ALS cases, we found 3 (1.6%) patients carrying 3 distinct novel genetic variants: a nonsynonymous SNV c.1150C>T leading to a p.Arg384Thr change in exon 9; a nonsynonymous SNV c.1331G>A causes a p.Arg444Gln change in exon 11; and a frameshift deletion c.2070delG (p.Met690fs) at the exon 20 of the gene leading to a stop at 693 codon. The latter patients also carried missense mutation c.98C>T of the SQSTM1 gene causing a substitution of an arginine with a valine at the position 33 (p.Arg33Val). All variants were found to be deleterious according to in silico predictions. All cases were apparently sporadic and one of them showed frontotemporal dementia associated to ALS. These mutations were not found in 2 cohorts of 6780 ethnic-matched controls. We have found that TBK1 mutations account for 1.6% of Sardinian ALS cases. Our data support the notion that TBK1 is a novel ALS gene, providing important evidence complementary to the first descriptions.

ATXN2 is a modifier of phenotype in ALS patients of Sardinian ancestry.

Intermediate-length CAG expansions (encoding 27-33 glutamines, polyQ) of the Ataxin2 (ATXN2) gene represent a risk factor for amyotrophic lateral sclerosis (ALS). Recently, it has been proposed that ≥31 CAG expansions may influence ALS phenotype. We assessed whether ATXN2 intermediate-length polyQ expansions influence ALS phenotype in a series of 375 patients of Sardinian ancestry. Controls were 247 neurologically healthy subjects, resident in the study area, age- and gender-matched to cases. The frequency of ≥31 polyQ ATNX2 repeats was significantly more common in ALS cases (4 patients vs. no control, p = 0.0001). All patients with ≥31 polyQ repeats had a spinal onset versus 73.3% of patients with <31 polyQ repeats. Patients with an increased number of polyQ repeats have a shorter survival than those with <31 repeats (1.2 vs. 4.2 years, p = 0.035). In this large series of ALS patients of Sardinian ancestry, we have found that ≥31 polyQ repeats of the ATXN2 gene influenced patients' phenotype, being associated to a spinal onset and a significantly shorter survival.

A novel LIPE nonsense mutation found using exome sequencing in siblings with late-onset familial partial lipodystrophy.

BACKGROUND: Familial lipodystrophies are rare inherited disorders associated with redistribution of body fat and development of dyslipidemia, insulin resistance, and diabetes. We previously reported 2 siblings with unusual late-onset familial partial lipodystrophy in whom heretofore known causative genes had been excluded. We hypothesized they had a mutation in a novel lipodystrophy gene. METHODS: Our approach centred on whole exome sequencing of the patients' DNA, together with genetic linkage analysis and a bioinformatic prioritization analysis. All candidate variants were assessed in silico and available family members were genotyped to assess segregation of mutations. RESULTS: Our prioritization algorithm led us to a novel homozygous nonsense variant, namely p.Ala507fsTer563 in the hormone sensitive lipase gene encoding, an enzyme that is differentially expressed in adipocytes and steroidogenic tissues. Pathogenicity of the mutation was supported in bioinformatic analyses and variant cosegregation within the family. CONCLUSIONS: We have identified a novel nonsense mutation in hormone sensitive lipase gene, which likely explains the lipodystrophy phenotype observed in these patients.

Genetic architecture of ALS in Sardinia.

Conserved populations, such as Sardinians, displaying elevated rates of familial or sporadic amyotrophic lateral sclerosis (ALS) provide unique information on the genetics of the disease. Our aim was to describe the genetic profile of a consecutive series of ALS patients of Sardinian ancestry. All ALS patients of Sardinian ancestry, identified between 2008 and 2013 through the Italian ALS Genetic Consortium, were eligible to be included in the study. Patients and controls underwent the analysis of TARDBP, C9ORF72, SOD1, and FUS genes. Genetic mutations were identified in 155 out of 375 Sardinian ALS cases (41.3%), more commonly the p.A382T and p.G295S mutations of TARDBP and the GGGGCC hexanucleotide repeat expansion of C9ORF72. One patient had both p.G295S and p.A382T mutations of TARDBP and 8 carried both the heterozygous p.A382T mutation of TARDBP and a repeat expansion of C9ORF72. Patients carrying the p.A382T and the p.G295S mutations of TARDBP and the C9ORF72 repeat expansion shared distinct haplotypes across these loci. Patients with cooccurrence of C9ORF72 and TARDBP p.A382T missense mutation had a significantly lower age at onset and shorter survival. More than 40% of all cases on the island of Sardinia carry a mutation of an ALS-related gene, representing the highest percentage of ALS cases genetically explained outside of Scandinavia. Clinical phenotypes associated with different genetic mutations show some distinctive characteristics, but the heterogeneity between and among families carrying the same mutations implies that ALS manifestation is influenced by other genetic and nongenetic factors.

Amyotrophic lateral sclerosis in Sardinia, insular Italy, 1995-2009.

Recent genetic studies suggest a Sardinian type of amyotrophic lateral sclerosis (ALS). Thus, ALS incidence, prevalence and survival were investigated in a large population of Sardinians aimed to disclose population-specific patterns and their temporal changes. This is a population-based incidence and prevalence study in northern and central Sardinia, insular Italy (over 700,000 population). Incidence rates were computed for the time interval 1995-2009 and by quinquennia. Prevalence was computed for prevalence days 31 December 2004 and 2009. Onset-based survival for 1995-2009 is also reported. All ALS patients (El Escorial Criteria) in the study area were retrospectively included. The ALS crude incidence from 2005-2009 was 2.5 (95 % CIs: 0.1, 4.9), 3.4 in men and 1.6 in women. Onset occurred most often between the age of 65-74 years in men and 55-64 years in women. The ALS incidence tended to increase over the period 1995-2009. The mean age at onset was 61.7 years with no difference based on gender, varying significantly from 59.9 years in 1995-1999 to 63.9 years in 2005-2009. On December 31, 2009, the ALS crude prevalence was 10.8 per 100,000 (95 % CIs: 8.6, 13.1), 13.8 in men and 8.0 in women, whereas it was 6.3 per 100,000 (95 % CIs: 4.1, 8.6) on December 31, 2004 (M:F ratio of 0.95). Mean survival from onset was 37.0 months, with no difference based on gender, and a tendency to decrease during the period 1995-2009, in relation to type and age of onset. The population-based incidence and prevalence data of ALS in Sardinians indicate an increase of the disease occurrence over the past 40 years, providing support for a population-specific variant of ALS in Sardinia.

Clinical and molecular characterization of limb-girdle muscular dystrophy due to LAMA2 mutations.

INTRODUCTION: In this study we describe the clinical and molecular characteristics of limb-girdle muscular dystrophy (LGMD) due to LAMA2 mutations. METHODS: Five patients clinically diagnosed with LGMD and showing brain white matter hyperintensities on MRI were evaluated using laminin α2 genetic and protein testing. RESULTS: The patients had slowly progressive, mild muscular dystrophy with various degrees of CNS involvement. Epilepsy was observed in 2, and subtle symptoms of CNS involvement (mild deficit in executive functions and low IQ scores) were noted in 3 patients. Novel LAMA2 mutations were identified in all patients. The amount of laminin α2 protein in the muscle biopsies ranged from trace to about 50% compared with controls. CONCLUSIONS: This study represents the largest series of LGMD laminin α2-deficient patients and expands the clinical phenotype associated with LAMA2 mutations. The findings suggest that brain MRI could be included in the diagnostic work-up of patients with undiagnosed LGMD.

A patient carrying a homozygous p.A382T TARDBP missense mutation shows a syndrome including ALS, extrapyramidal symptoms, and FTD.

We have recently published data showing that a founder mutation of the TARDBP gene (p.A382T) accounts for approximately one third of amyotrophic lateral sclerosis (ALS) cases on the Mediterranean island of Sardinia (Chiò et al., 2011). In that report, we identified a 53-year-old man carrying a homozygous A382T missense mutation of the TARDBP gene with a complex neurological syndrome including amyotrophic lateral sclerosis, parkinsonian features, motor and vocal tics, and frontotemporal dementia (FTD). Due to the uniqueness of this case, here we provide a detailed clinical description, as well as neurophysiological, neuropsychological, and neuroimaging data for that case and his extended family.

Broadening the phenotype of TARDBP mutations: the TARDBP Ala382Thr mutation and Parkinson's disease in Sardinia.

Mutations in the TARDBP gene are a cause of autosomal dominant amyotrophic lateral sclerosis (ALS) and of frontotemporal lobar degeneration (FTLD), but they have not been found so far in patients with Parkinson's disease (PD). A founder TARDBP mutation (p.Ala382Thr) was recently identified as the cause of ~30% of ALS cases in Sardinia, a Mediterranean genetic isolate. We studied 327 consecutive Sardinian patients with clinically diagnosed PD (88 familial, 239 sporadic) and 578 Sardinian controls. One family with FTLD and parkinsonism was also included. The p.Ala382Thr heterozygous mutation was detected in eight unrelated PD patients (2.5%). The three patients from the FTLD/parkinsonism family also carried this mutation. Within the control group, there were three heterozygous mutation carriers. During follow-up, one of these individuals developed motoneuron disease and another, a rapidly progressive dementia; the third remains healthy at the age of 79 but two close relatives developed motoneuron disease and dementia. The eight PD patients carrying the p.Ala382Thr mutation had all sporadic disease presentation. Their average onset age was 70.0 years (SD 9.4, range 51-79), which is later but not significantly different from that of the patients who did not carry this mutation. In conclusion, we expand the clinical spectrum associated with TARDBP mutations to FTLD with parkinsonism without motoneuron disease and to clinically definite PD. The TDP-43 protein might be directly involved in a broader neurodegenerative spectrum, including not only motoneuron disease and FTLD but also PD.

Large proportion of amyotrophic lateral sclerosis cases in Sardinia due to a single founder mutation of the TARDBP gene.

OBJECTIVE: To perform an extensive screening for mutations of amyotrophic lateral sclerosis (ALS)-related genes in a consecutive cohort of Sardinian patients, a genetic isolate phylogenically distinct from other European populations. DESIGN: Population-based, prospective cohort study. PATIENTS: A total of 135 Sardinian patients with ALS and 156 healthy control subjects of Sardinian origin who were age- and sex-matched to patients. INTERVENTION: Patients underwent mutational analysis for SOD1, FUS, and TARDBP. RESULTS: Mutational screening of the entire cohort found that 39 patients (28.7%) carried the c.1144G>A (p.A382T) missense mutation of the TARDBP gene. Of these, 15 had familial ALS (belonging to 10 distinct pedigrees) and 24 had apparently sporadic ALS. None of the 156 age-, sex-, and ethnicity-matched controls carried the pathogenic variant. Genotype data obtained for 5 ALS cases carrying the p.A382T mutation found that they shared a 94-single-nucleotide polymorphism risk haplotype that spanned 663 Kb across the TARDBP locus on chromosome 1p36.22. Three patients with ALS who carry the p.A382T mutation developed extrapyramidal symptoms several years after their initial presentation with motor weakness. CONCLUSIONS: The TARDBP p.A382T missense mutation accounts for approximately one-third of all ALS cases in this island population. These patients share a large risk haplotype across the TARDBP locus, indicating that they have a common ancestor.

Amyotrophic lateral sclerosis-frontotemporal lobar dementia in 3 families with p.Ala382Thr TARDBP mutations.

BACKGROUND: TAR DNA-binding protein 43, encoded by the TARDBP gene, has been identified as the major pathological protein of frontotemporal lobar dementia (FTLD) with or without amyotrophic lateral sclerosis (ALS) and sporadic ALS. Subsequently, mutations in the TARDBP gene have been detected in 2% to 3% of patients with ALS (both familial and sporadic ALS). However, to our knowledge, there is only 1 description of 2 patients with FTLD and TARDBP gene mutations who later developed motor neuron disease. OBJECTIVE: To describe cognitive abnormalities in 3 Italian families with familial ALS and TARDBP gene mutations. DESIGN, SETTING, AND PARTICIPANTS: Genetic, neuropsychological, and neuroimaging analyses in 36 patients with familial non-superoxide dismutase 1 gene (SOD1) ALS and 280 healthy controls. Main Outcome Measure We identified 3 index cases of familial ALS carrying the p.Ala382Thr missense mutation of the TARDBP gene and with clinical, neuroimaging, and neuropsychological features of FTLD. RESULTS: The p.Ala382Thr missense mutation of the TARDBP gene was absent in the 280 controls. It was present in all affected members of the 3 families for whom DNA was available. All affected members of the 3 families developed FTLD after the onset of ALS, confirmed by neuropsychological testing and hypometabolism in frontal associative areas assessed with fludeoxyglucose F 18 positron emission tomography and computed tomography. CONCLUSIONS: Three apparently unrelated families with familial ALS carrying the p.Ala382Thr TARDBP missense mutation developed FTLD. In these families, FTLD cosegregates with ALS. Patients with ALS carrying TARDBP mutations may develop FTLD.

Association between protective and deleterious HLA alleles with multiple sclerosis in Central East Sardinia.

The human leukocyte antigen (HLA) complex on chromosome 6p21 has been unambiguously associated with multiple sclerosis (MS). The complex features of the HLA region, especially its high genic content, extreme polymorphism, and extensive linkage disequilibrium, has prevented to resolve the nature of HLA association in MS. We performed a family based association study on the isolated population of the Nuoro province (Sardinia) to clarify the role of HLA genes in MS. The main stage of our study involved an analysis of the ancestral haplotypes A2Cw7B58DR2DQ1 and A30Cw5B18DR3DQ2. On the basis of a multiplicative model, the effect of the first haplotype is protective with an odds ratio (OR) = 0.27 (95% confidence interval CI 0.13-0.57), while that of the second is deleterious, OR 1.78 (95% CI 1.26-2.50). We found both class I (A, Cw, B) and class II (DR, DQ) loci to have an effect on MS susceptibility, but we saw that they act independently from each other. We also performed an exploratory analysis on a set of 796 SNPs in the same HLA region. Our study supports the claim that Class I and Class II loci act independently on MS susceptibility and this has a biological explanation. Also, the analysis of SNPs suggests that there are other HLA genes involved in MS, but replication is needed. This opens up new perspective on the study of MS.

Partial status epilepticus related to independent occipital foci in posterior reversible encephalopathy syndrome (PRES).

Posterior reversible encephalopathy syndrome (PRES) is an acute disorder characterised by a variable association of neurologic symptoms with potentially reversible oedematous abnormalities mainly in the parieto-occipital regions of the brain. Despite the significant incidence of seizures, the EEG characteristics of epileptic disorders related to PRES have rarely been investigated. We report the case of an 85-year-old man who presented with generalised tonic-clonic seizures and prolonged disturbances of consciousness as clinical manifestations of PRES due to moderate exacerbation of chronic hypertension. An EEG performed during an alteration of mental function displayed a pattern of partial status epilepticus (SE) in both temporo-parieto-occipital regions. The seizure activity originated from two independent epileptic foci located in the occipital area of each hemisphere and could be related to the parenchymal abnormalities of PRES. The EEG pattern of partial SE related to independent occipital foci illustrates a distinctive seizure disorder that could be characteristic of PRES in adult patients.

Association between the ACCN1 gene and multiple sclerosis in Central East Sardinia.

Multiple genome screens have been performed to identify regions in linkage or association with Multiple Sclerosis (MS, OMIM 126200), but little overlap has been found among them. This may be, in part, due to a low statistical power to detect small genetic effects and to genetic heterogeneity within and among the studied populations. Motivated by these considerations, we studied a very special population, namely that of Nuoro, Sardinia, Italy. This is an isolated, old, and genetically homogeneous population with high prevalence of MS. Our study sample includes both nuclear families and unrelated cases and controls. A multi-stage study design was adopted. In the first stage, microsatellites were typed in the 17q11.2 region, previously independently found to be in linkage with MS. One significant association was found at microsatellite D17S798. Next, a bioinformatic screening of the region surrounding this marker highlighted an interesting candidate MS susceptibility gene: the Amiloride-sensitive Cation Channel Neuronal 1 (ACCN1) gene. In the second stage of the study, we resequenced the exons and the 3' untranslated (UTR) region of ACCN1, and investigated the MS association of Single Nucleotide Polymorphisms (SNPs) identified in that region. For this purpose, we developed a method of analysis where complete, phase-solved, posterior-weighted haplotype assignments are imputed for each study individual from incomplete, multi-locus, genotyping data. The imputed assignments provide an input to a number of proposed procedures for testing association at a microsatellite level or of a sequence of SNPs. These include a Mantel-Haenszel type test based on expected frequencies of pseudocase/pseudocontrol haplotypes, as well as permutation based tests, including a combination of permutation and weighted logistic regression analysis. Application of these methods allowed us to find a significant association between MS and the SNP rs28936 located in the 3' UTR segment of ACCN1 with p = 0.0004 (p = 0.002, after adjusting for multiple testing). This result is in tune with several recent experimental findings which suggest that ACCN1 may play an important role in the pathogenesis of MS.