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Diabetes Mellitus (DM) is a complex disease with a significant impact in today’s world. Studies have emphasized the crucial role of genetics in DM, unraveling the distinction of monogenic diabetes from the most common types that have been recognized over the years, such as type 1 diabetes (T1DM) and type 2 diabetes (T2DM). A literature search was carried out to scrutinize the subtypes of maturity-onset diabetes of the young (MODY), as well as the connection between the recognized genetic and molecular mechanisms responsible for such phenotypes. Thus far, 14 subtypes of MODY have been identified. Here, the authors review the pathophysiological and molecular pathways in which monogenic diabetes genes are involved. Despite being estimated to affect approximately 2% of all T2DM patients in Europe, the exact prevalence of MODY is still unknown, enhancing the need for research focused on biomarkers. Due to its impact in personalized medicine, a follow-up of associated complications, and genetic implications for siblings and offspring of affected individuals, it is imperative to diagnose the monogenic forms of DM accurately. Currently, advances in the genetics field has allowed for the recognition of new DM subtypes, which until now were considered to be slight variations of the typical forms. New molecular insights can define therapeutic strategies, aiming for the prevention, correction, or at least delay of β-cell dysfunction. Thus, it is imperative to act in the close interaction between genetics and clinical manifestations to improve diagnosis and individualize treatment.

© Translational Lung Cancer Research. All rights reserved. This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/., Background: Liquid biopsy allows the identification of targetable cancer mutations in a minimally invasive manner. In patients with advanced non-small cell lung cancer (NSCLC), droplet digital PCR (ddPCR) is increasingly used to genotype the epidermal growth factor receptor (EGFR) gene in circulating cell-free DNA (cfDNA). However, the sensitivity of this method is still under debate. The aim of this study was to implement and assess the performance of a ddPCR assay for detecting the EGFR T790M mutation in liquid biopsies. Methods: A ddPCR assay was optimized to detect the EGFR T790M mutation in plasma samples from 77 patients with NSCLC in progression. Results: Our ddPCR assay enabled the detection and quantification of the EGFR T790M mutation at cfDNA allele frequency as low as 0.5%. The mutation was detected in 40 plasma samples, corresponding to a positivity rate of 52%. The number of mutant molecules per mL of plasma ranged from 1 to 6,000. A re-biopsy was analyzed for 12 patients that had a negative plasma test and the mutation was detected in 2 cases. A second liquid biopsy was performed for 6 patients and the mutation was detected in 3 cases. Conclusions: This study highlights the value of ddPCR to detect and quantify the EGFR T790M mutation in liquid biopsies in a real-world clinical setting. Our results suggest that repeated ddPCR tests in cfDNA may obviate tissue re-biopsy in patients unable to provide a tumor tissue sample suitable for molecular analysis., This work was supported by Fundação para a Ciência e Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior - Fundos do Orçamento de Estado (UID/BIM/50005/2019), and FCT/FEDER/POR Lisboa 2020, Programa Operacional Regional de Lisboa, PORTUGAL 2020 (LISBOA-01-0145-FEDER-016394). C.S. was a recipient of a FCT fellowship (SFRH/BDE/110544/2015).

Diffuse Idiopathic Skeletal Hyperostosis (DISH) is considered a metabolic condition, characterized by new bone formation affecting mainly at entheseal sites. Enthesitis and enthesopathies occur not only in the axial skeleton but also at some peripheral sites, and they resemble to some extent the enthesitis that is a cardinal feature in spondyloarthritis (SpA), which is an inflammatory disease. We review the possible non-metabolic mechanism such as inflammation that may also be involved at some stage and help promote new bone formation in DISH. We discuss supporting pathogenic mechanisms for a local inflammation at sites typically affected by this disease, and that is also supported by imaging studies that report some similarities between DISH and SpA. Local inflammation, either primary or secondary to metabolic derangements, may contribute to new bone formation in DISH. This new hypothesis is expected to stimulate further research in both the metabolic and inflammatory pathways in order to better understand the mechanisms that lead to new bone formation. This may lead to development of measures that will help in earlier detection and effective management before damage occurs.

Funding: The authors thank all the patients who participated in this research and made it possible. We also thank Isa Dutra (MSc), João Paulo Pinheiro (BsC) and Raquel Meneses (BsC) from Hospital Santo Espírito da Ilha Terceira, EPE, Epidemiology and Molecular Biology Service (SEEBMO), Angra do Heroísmo, Portugal, who performed the DNA extractions and Vânia Machado (MSc), who participated in the elaboration of the pedigrees. BP wassupported by “Fundo Regional para a Ciência e Tecnologia (FRCT)” (M3.1.2/F/023/2011) Objectives: DISH/CC is a poorly understood phenotype characterised by peripheral and axial enthesopathic calcifications, frequently fulfilling the radiological criteria for Diffuse Idiopathic Skeletal Hyperostosis (DISH, MIM 106400), and in some cases associated with Calcium Pyrophosphate Dihydrate (CPPD) Chondrocalcinosis (CC). The concurrence of DISH and CC suggests a shared pathogenic mechanism. In order to identify genetic variants for susceptibility we performed whole exome sequencing in four patients showing this phenotype. Materials and methods: Exome data were filtered in order to find a variant or a group of variants that could be associated with the DISH/CC phenotype. Variants of interest were subsequently confirmed by Sanger sequencing. Selected variants were screened in a cohort of 65 DISH/CC patients vs 118 controls from Azores. The statistical analysis was performed using PLINK V1.07. Results:We identified 21 genetic variants in 17 genes that were directly or indirectly related to mineralization, several are predicted to have a strong effect at a protein level. Phylogenetic analysis of altered amino acids indicates that these are either highly conserved in vertebrates or conserved in mammals. In case-control analyses, variant rs34473884 in PPP2R2D was significantly associated with the DISH/CC phenotype (p=0.028; OR=1.789, 95% CI= 1.060-3.021). Conclusion: The results of the present and preceding studies with the DISH/CC families suggests that the phenotype has a polygenic basis. The PPP2R2D gene could be involved in this phenotype in an as yet unknown way. publishersversion published

Background: Diabetes mellitus (DM) is a complex disease with significant impression in today's world. Aside from the most common types recognized over the years, such as type 1 diabetes (T1DM) and type 2 diabetes (T2DM), recent studies have emphasized the crucial role of genetics in DM, allowing the distinction of monogenic diabetes. Methods: Authors did a literature search with the purpose of highlighting and clarifying the subtypes of monogenic diabetes, as well as the accredited genetic entities responsible for such phenotypes. Results: The following subtypes were included in this literature review: maturity-onset diabetes of the young (MODY), neonatal diabetes mellitus (NDM) and maternally inherited diabetes and deafness (MIDD). So far, 14 subtypes of MODY have been identified, while three subtypes have been identified in NDM - transient, permanent, and syndromic. Discussion: Despite being estimated to affect approximately 2% of all the T2DM patients in Europe, the exact prevalence of MODY is still unknown, accentuating the need for research focused on biomarkers. Consequently, due to its impact in the course of treatment, follow-up of associated complications, and genetic implications for siblings and offspring of affected individuals, it is imperative to diagnose the monogenic forms of DM accurately. Conclusion: Currently, advances in the genetics field allowed the recognition of new DM subtypes, which until now, were considered slight variations of the typical forms. Thus, it is imperative to act in the close interaction between genetics and clinical manifestations, to facilitate diagnosis and individualize treatment.

Age at onset in spinocerebellar ataxia type 3 (SCA3/MJD) is incompletely explained by the size of the CAG tract at the ATXN3 gene, implying the existence of genetic modifiers. A role of inflammation in SCA3 has been postulated, involving altered cytokines levels; promoter variants leading to alterations in cytokines expression could influence onset. Using blood from 86 SCA3 patients and 106 controls, this work aimed to analyse promoter variation of four cytokines (IL1A, IL1B, IL6 and TNF) and to investigate the association between variants detected and their transcript levels, evaluated by quantitative PCR. Moreover, the effect of APOE isoforms, known to modulate cytokines, was investigated. Correlations between cytokine variants and onset were tested; the cumulative modifier effects of cytokines and APOE were analysed. Patients carrying the IL6*C allele had a significant earlier onset (4 years in average) than patients carrying the G allele, in agreement with lower mRNA levels produced by IL6*C carriers. The presence of APOE*ɛ2 allele seems to anticipate onset in average 10 years in patients carrying the IL6*C allele; a larger number of patients will be needed to confirm this result. These results highlight the pertinence of conducting further research on the role of cytokines as SCA3 modulators, pointing to the presence of shared mechanisms involving IL6 and APOE.

The ATP-binding cassette transporter 6 (ABCC6) gene encodes a cellular transmembrane protein transporter (MRP6) that is involved in the regulation of tissue calcification in mammals. Mutations in ABCC6 are associated with human ectopic calcification disorders. To gain insight into its evolution and involvement in tissue calcification we conducted a comparative analysis of the ABCC6 gene and the related gene ABCC1 from invertebrates to vertebrates where a bony endoskeleton first evolved. Taking into consideration the role of ABCC6 in ectopic calcification of human skin we analysed the involvement of both genes in the regeneration of scales, mineralized structures that develop in fish skin. The ABCC6 gene was only found in bony vertebrate genomes and was absent from Elasmobranchs, Agnatha and from invertebrates. In teleost fish the abcc6 gene duplicated but the two genes persisted only in some teleost genomes. Six disease causing amino acid mutations in human MRP6 are a normal feature of abcc6 in fish, suggesting they do not have a deleterious effect on the protein. After scale removal the abcc6 (5 and 10 days) and abcc1 (10 days) gene expression was up-regulated relative to the intact control skin and this coincided with a time of intense scale mineralization.

Objectives Ankylosing spondylitis (AS) is the most prevalent form of spondyloarthritis, with a known genetic association with the HLA-B27 molecule. The aim of this study was to assess the contribution of the HLA-G, HLA-E and HLA-F to AS susceptibility/protection in Portuguese patients with HLA-B27 AS and HLA-B27 unaffected controls. Methods High-resolution typing of HLA-G, HLA-E and HLA-F was performed in 228 patients with HLA-B27 AS and 244 HLA-B27 unaffected controls. Allelic, genotypic and haplotypic frequencies were compared between cohorts. To replicate the results, single nucleotide polymorphisms (SNPs) in HLA-E and HLA-F genes were typed in Australian cohorts. For further confirmation, a group of European-descent patients with AS and unaffected controls were genotyped for Major Histocompatibility Complex SNPs using the Illumina microarray. Results In the Portuguese population, no significant differences were found in HLA-G. For HLA-E, a significant difference was detected for the genotype HLA-E*01:01:01/01:03:01 (p=0.009; pc=0.009; OR=0.51), with a protection effect. For HLA-F, significant differences were detected in the allele HLA-F*01:01:02 (p=0.0049; pc=0.0098; OR=0.60) and corresponding SNP rs2075682 (p=0.0004; pc=0.0008; OR=0.53), suggesting protection and in the genotype HLA-F*01:01:01/01:03:01 (p=0.011; pc=0.043; OR=2.00), suggesting a susceptibility effect. Three G-E-F haplotypes with significant differences were detected but occur in a very small number of individuals. The only significant differences detected in the replication studies were for HLA-E rs1059510 in the Australians and for HLA-F rs1736924 in the European-descent cohorts. Conclusion Our results reveal suggestive AS protective and susceptibility effects from both HLA-E and HLA-F loci, however with population differences. To our knowledge, this is the first study showing association of HLA-F with AS.

Machado-Joseph disease (or spino- cerebellar ataxia type 3) is a late-onset polyglutamine neurodegenerative disorder caused by a mutation in the ATXN3 gene, which encodes for the ubiquitously expressed protein ataxin-3. Previous studies on cell and animal models have suggested that mutated ataxin-3 is involved in transcriptional dysregulation. Starting with a whole-transcriptome profiling of peripheral blood samples from patients and controls, we aimed to confirm abnormal expression profiles in Machado-Joseph disease and to identify promising up-regulated genes as potential candi- date biomarkers of disease status. The Illumina Human V4-HT12 array was used to measure transcriptome-wide gene expression in peripheral blood samples from 12 patients and 12 controls. Technical validation and valida- tion in an independent set of samples were performed by quantitative real-time polymerase chain reaction (PCR). Based on the results from the microarray, twenty six genes, found to be up-regulated in patients, were selected for technical validation by quantitative real-time PCR (validation rate of 81% for the up-regulation trend). Fourteen of these were further tested in an independent set of 42 patients and 35 controls; 10 genes maintained the up-regulation trend (FCGR3B, CSR2RA, CLC, TNFSF14, SLA, P2RY13, FPR2, SELPLG, YIPF6, and GPR96); FCGR3B, P2RY13, and SELPLG were signifi- cantly up-regulated in patients when compared with con- trols. Our findings support the hypothesis that mutated ataxin-3 is associated with transcription dysregulation, detectable in peripheral blood cells. Furthermore, this is the first report suggesting a pool of up-regulated genes in Machado-Joseph disease that may have the potential to be used for fine phenotyping of this disease. V C 2015 Inter- national Parkinson and Movement Disorder Society

Summary This study confirms for Madeira Island (Portugal) population the Type 1 Diabetes (T1D) susceptible and protective Human leucocyte antigens (HLA) markers previously reported in other populations and adds some local specificities. Among the strongest T1D HLA associations, stands out, as susceptible, the alleles DRB1*04:05 (OR = 7.3), DQB1*03:02 (OR = 6.1) and DQA1*03:03 (OR = 4.5), as well as the haplotypes DRB1*04:05-DQA1*03:03-DQB1*03:02 (OR = 100.9) and DRB1*04:04-DQA1*03:01-DQB1*03:02 (OR = 22.1), and DQB1*06:02 (OR = 0.07) and DRB1*15:01-DQA1*01:02-DQB1*06:02 (OR = 0.04) as protective. HLA-DQA1 positive for Arginine at position 52 (Arg52) (OR = 15.2) and HLA-DQB1 negative for Aspartic acid at the position 57 (Asp57) (OR = 9.0) alleles appear to be important genetic markers for T1D susceptibility, with higher odds ratio values than any single allele and than most of the haplotypes. Genotypes generated by the association of markers Arg52 DQA1 positive and Asp57 DQB1 negative increase T1D susceptibility much more than one would expected by a simple additive effect of those markers separately (OR = 26.9). This study also confirms an increased risk for DRB1*04/DRB1*03 heterozygote genotypes (OR = 16.8) and also a DRB1*04-DQA1*03:01-DQB1*03:02 haplotype susceptibility dependent on the DRB1*04 allele (DRB1*04:01, OR = 7.9; DRB1*04:02, OR = 3.2; DRB1*04:04, OR = 22.1).

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Em colaboração com a Rede Portuguesa de Laboratóros para o Diagnóstico da Gripe Introduction: Immune profile for influenza viruses is highly changeable over time. Serological studies can assess the prevalence of influenza, estimate the risk of infection, highlight asymptomatic infection rate and can also provide data on vaccine coverage. The aims of the study were to evaluate pre-existing cross-protection against influenza A(H3) drift viruses and to assess influenza immunity in the Portuguese population. Materials and methods: We developed a cross-sectional study based on a convenience sample of 626 sera collected during June 2014, covering all age groups, both gender and all administrative health regions of Portugal. Sera antibody titers for seasonal and new A(H3) drift influenza virus were evaluated by hemagglutination inhibition assay (HI). Seroprevalence to each seasonal influenza vaccine strain virus and to the new A(H3) drift circulating strain was estimated by age group, gender and region and compared with seasonal influenza-like illness (ILI) incidence rates before and after the study period. Results: Our findings suggest that seroprevalences of influenza A(H3) (39.9%; 95% CI: 36.2–43.8) and A(H1)pdm09 (29.7%; 95% CI: 26.3–33.4) antibodies were higher than for influenza B, in line with high ILI incidence rates for A(H3) followed by A(H1)pdm09, during 2013/2014 season. Low pre-existing crossprotection against new A(H3) drift viruses were observed in A(H3) seropositive individuals (46%). Both against influenza A(H1)pdm09 and A(H3) seroprotection was highest in younger than 14-years old. Protective antibodies against influenza B were highest in those older than 65 years old, especially for B/Yamagata lineage, 33.3% (95% CI: 25.7–41.9). Women showed a high seroprevalence to influenza, although without statistical significance, when compared to men. A significant decreasing trend in seroprotection from north to south regions of Portugal mainland was observed. Conclusions: Our results emphasize that low seroprotection increases the risk of influenza infection in the following winter season. Seroepidemiological studies can inform policy makers on the need for vaccination and additional preventive measures. This work was supported by the National Institute of Health Doutor Ricardo Jorge, IP Lisbon, Portugal. info:eu-repo/semantics/acceptedVersion

Familial hypercholesterolemia (FH) is an autosomal dominant disorder of the cholesterol metabolism, which constitutes a risk factor for coronary arterial disease (CAD). In the Azores Islands (Portugal), where mortality from CAD doubles its rate comparatively to the rest of the country and where a high frequency of dyslipidemia has been reported, the prevalence and distribution of FH remain unknown. The molecular characterization of a group of 33 possible cases of FH of Azorean background was undertaken in this study. A DNA array was initially used to search mutations in the LDLR, APOB and PCSK9 loci in 10 unrelated possible cases of FH. No mutations were detected in the array; after sequencing the full LDLR gene, 18 variants were identified, corresponding to two missense (c.806G > A; c.1171G > A) and sixteen synonymous alterations. Six of the synonymous variants which are consistently described in the literature as associated with altered cholesterol levels were used to build haplotypes. The most frequent haplotype corresponded to TTCGCC (45%), a "risk" haplotype, formed exclusively by alleles that were reported to increase cholesterol levels. Some of the variants detected in the full sequencing of the LDLR gene fell within the ligand-binding domain of this gene, defined by exons 2 to 6. To add information as to the role of such variants, these exons were sequenced in the remaining 23 possible FH cases. Two missense alterations (c.185C > T; c.806G > A) were found in this subset of possible FH cases. The missense alteration c.185C > T, identified in one individual, is novel for the Portuguese population. In silico analysis was not conclusive for this alteration, whose role will have to be further investigated. This study represents the first approach to the establishment of the mutational profile of FH in the Azores Islands. This work was supported by the project entitled “High prevalence pathologies in the Azores: genetic and biochemical markers” with reference (M2.1.2/I/026/2008) funded by SRCTE. M. R. receives a PhD fellowship (M3.1.2/F/006/2011) from Fundo Regional para a Ciência. T.C. receives a post-doctoral fellowships from Fundação para a Ciência e a Tecnologia (SFRH/BPD/38659/ 2007) and N. K. (M3.1.7/F/002/2008) and A. R. (M3.1.7/F/031/2011) both receives post-doctoral fellowships from Fundo Regional para a Ciência.

Objectives. To investigate the efficacy of infliximab in the treatment of severe calcium pyrophosphate deposition diseases (CPPD).Methods. Two patients with severe CPPD and diffuse idiopathic skeletal hyperostosis- (DISH-) like phenotype are described. Both patients were resistant to therapy with nonsteroidal anti-inflammatory drugs (NSAIDs). Both patients were treated with infliximab, a TNF-αreceptor antagonist, for nine years.Results. Treatment with infliximab resulted in major clinical and laboratory improvements without relevant side effects.Conclusions. These results suggest that infliximab may be an effective treatment of severe CPDD.

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine (polyQ) disorder for which the routine molecular testing is based on PCR and automated capillary electrophoresis. When only a normal allele is detected by standard PCR, the hypothesis of a failed amplification of the expanded allele must be raised. In such cases, complementary techniques such as Southern Blot or triplet repeat primed PCR (TP-PCR) have to be applied. For SCA3, TP-PCR is implemented in some diagnostic laboratories, but a tested protocol has yet to be published. The purpose of this study was to develop and test a TP-PCR protocol for SCA3. Sixty-five blood samples previously genotyped by standard PCR were used in the TP-PCR assay. Fourteen buccal swab samples were also analyzed to confirm the robustness of the technique. The reproducibility of the TP-PCR was evaluated by analyzing all samples in a second laboratory. The results obtained by TP-PCR confirmed the previous PCR results for 64 blood samples; in one sample an expanded allele, previously undetected by PCR, was identified. The results obtained for the buccal swab samples were totally concordant with those obtained for blood. Furthermore, the results obtained in the alternative laboratory were in full agreement with the results obtained in our study. The present TP-PCR protocol developed for SCA3 should constitute a reliable complementary technique to overcome the limitations of standard PCR.

This report describes a 32-year-old woman presenting since childhood with progressive calcium pyrophosphate disease (CPPD), characterized by severe arthropathy and chondrocalcinosis involving multiple peripheral joints and intervertebral disks. Because ANKH mutations have been previously described in familial CPPD, the proband’s DNA was assessed at this locus by direct sequencing of promoter and coding regions and revealed 3 sequence variants in ANKH. Sequences of exon 1 revealed a novel isolated nonsynonymous mutation (c.13 C>T), altering amino acid in codon 5 from proline to serine (CCG>TCG). Sequencing of parental DNA revealed an identical mutation in the proband’s father but not the mother. Subsequent clinical evaluation demonstrated extensive chondrocalcinosis and degenerative arthropathy in the proband’s father. In summary, we report a novel mutation, not previously described, in ANKH exon 1, wherein serine replaces proline, in a case of early-onset severe CPPD associated with metabolic abnormalities, with similar findings in the proband’s father.

Summary HLA class I diversity (loci A, B and C) was analysed in four populations, two from North Cameroon (Podokwo and Uldeme) and two from South Cameroon (Ewondo and Bamileke). Northern and southern Cameroon populations show a substantial genetic diversity in terms of haplotype sharing and genetic distances, even despite the low percentage of variance due to differences among populations evidenced by analysis of molecular variance. The signals of differentiation among populations are consistent with their linguistic affiliation, and support previous evidence, based on autosomal microsatellites and protein loci, which has shown that the complex pattern of genetic variation of Cameroon can in part be described by contrasting the northern and southern part of the country. Looking at our results in the more general framework of HLA diversity in sub-Saharan Africa, it turns out that the Podokwo and Uldeme show some genetic links to populations of the southern western branch of the Sahel corridor, while their high frequency of A*02 and C*04 alleles is congruent with previously hypothesised introgression of non-sub-Saharan alleles. On the other hand, signals of shared ancestry between the Bamileke and Ewondo and the Bantu speakers from central and southern Africa were detected.

Background: Atherosclerosis and thrombosis are the major manifestations underlying cardiovascular diseases (CVD), which are the leading cause of mortality and morbidity worldwide. Both result from an interaction between genetic and environmental risk factors. The goal of our study was to evaluate several polymorphisms identified as predisposing factors to atherosclerosis and thrombosis. Material and Methods: A series of 155 healthy unrelated individuals of Azorean origin were analyzed using the CVD StripAssay (ViennaLab Diagnostics, Austria) for the most established polymorphisms involved in blood coagulation (F2, F5, F13A1, FGB), fibrinolitic system (SERPINE1), platelet adhesion (ITGB3), homocysteine metabolism (MTHFR), reninangio-tensin system (ACE) and lipid metabolism (APOE). Results: No significant differences were observed in allelic frequencies when comparing our data to mainland Portugal. Group stratification according to the number of “increased” risk alleles, demonstrated that 116/155 (75%) individuals belong to the moderate risk group (5 - 10 risk alleles). Conclusions: Although acknowledging the fact that the allelic states at the analysed loci lack predictive value, the fact that a high frequency of individuals presents at least 5 risk alleles (124/155; 80%) is important for the establishment of the appropriate preventive measures in the Azorean population.

Human leukocyte antigen (HLA)-A locus polymorphisms were examined at high-resolution level, using sequence-based typing, in the four most representative Guinea-Bissau (Northwest Africa) ethnic groups: Balanta, Bijagós, Fula and Papel. Despite the Fula group having significant differences when compared with the other three ethnic groups, all four groups most likely received a genetic input from non sub-Saharans. The Bijagós and Papel groups showed similarities to neighboring populations from Mali and Senegal. The Balanta, despite their oral tradition of an East Africa origin, show affinities to Cameroon populations, highly influenced by Bantu migrations. These results are congruent with historical sources and other genetic studies that support the finding that the Guinea-Bissau genetic pool was influenced by several migrations from North Africa, Sahara and East Africa.

Introduction: The TGFB1 gene which encodes transforming growth factor beta 1, is a strong candidate for susceptibility to osteoporosis and several studies have reported associations between bone mineral density (BMD), osteoporotic fractures and polymorphisms of TGFB1, although these studies have yielded conflicting results. Methods: We investigated associations between TGFB1 polymorphisms and BMD and fracture in the GENOMOS study: a prospective multicenter study involving 10 European research studies including a total of 28,924 participants. Genotyping was conducted for known TGFB1 polymorphisms at the following sites: G- 1639-A (G- 800-A, rs1800468), C- 1348-T (C- 509-T, rs1800469), T29-C (Leu10Pro, rs1982073), G74-C (Arg25Pro, rs1800471) and C788-T (Thr263Ile, rs1800472). These polymorphisms were genotyped prospectively and methodology was standardized across research centers. Genotypes and haplotypes were related to BMD at the lumbar sine and femoral neck and fractures. Results: There were no significant differences in either women or men at either skeletal site for any of the examined polymorphisms with the possible exception of a weak association with reduced BMD (- 12 mg/cm2) in men with the T- 1348 allele (p < 0.05). None of the haplotypes was associated with BMD and none of the polymorphisms or haplotypes significantly affected overall risk of fractures, however, the odds ratio for incident vertebral fracture in carriers of the rare T788 allele was 1.64 (95% CI: 1.09-2.64), p < 0.05. Conclusions: This study indicates that polymorphic variation in the TGFB1 gene does not play a major role in regulating BMD or susceptibility to fractures. The weak associations we observed between the C- 1348-T and lumbar spine BMD in men and between C788-T and risk of incident vertebral fractures are of interest but could be chance findings and will need replication in future studies.

Recent studies have reported loss of function mutations in the LEMD3 gene, encoding an inner nuclear membrane protein that influences Smad signaling, as a cause of osteopoikilosis, Buschke-Ollendorff syndrome, and melorheostosis. We investigated LEMD3 in a three-generation family with osteopoikilosis from the Azores, an affected father and daughter from Ireland with osteopoikilosis (the daughter also had melorheostosis), and two other individuals from the UK with isolated melorheostosis. We found a novel C to T substitution at position 2032 bp (cDNA) in exon 8 of LEMD3, resulting in a premature stop codon at amino acid position 678. This mutation co-segregates with the osteopoikilosis phenotype in both the Azorean family and the Irish family. It was not detected in any of the six unaffected family members or in 342 healthy Caucasian individuals. No LEMD3 mutations were detected in the two patients with sporadic melorheostosis. The LEMD3 mutation reported was clearly the cause of osteopoikilosis in the two families but its relationship to melorheostosis in one of the family members is still unclear. Perhaps unsurprisingly in what is a segmental disease, we did not find LEMD3 mutations in peripheral-blood-derived DNA from the two other individuals with sporadic melorheostosis. The nature of the additional genetic and/or environmental influences required for the development of melorheostosis in those with osteopoikilosis requires further investigation.

BACKGROUND: The association between susceptibility to multiple sclerosis (MS) and HLA-DRB1*15 has been reported in various European populations. OBJECTIVE: To investigate the relationship between MS, HLA-DRB1*15 and other DRB1 alleles in a Portuguese population and their association with clinical course of MS. METHODS: The HLA-DRB1 alleles were analyzed by PCR-SSP in 248 MS patients and 282 healthy controls. In order to relate HLA-DRB1 alleles to disease aggressiveness, patients with relapsing remitting MS and secondary progressive MS were subdivided into 3 groups: 'benign' MS patients who maintain an Extended Disability Status Scale (EDSS) score of 3 after the same period and 'aggressive' MS those with EDSS>or=6 within 15 years of disease onset. RESULTS: As expected, a higher frequency of HLA-DRB1*15 was found in MS patients (29.8% vs 19.9%, odds ratio (OR)=1.72, 95% CI=1.15-2.56, p=0.008). The HLA-DRB1*03 allele was positively associated with MS in the overall patient population (22.6% vs 15.6%, OR=1.58, 95% CI=1.02-2.45). Concerning disease aggressiveness, HLA-DRB1*15 occurred more frequently in the group with benign disease (42.6% vs 19.9%, OR=2.99, 95% CI=1.56-5.72) and in the group with non-benign disease (34.1% vs 19.9%, OR=2.09, 95% CI=1.05-4.16) compared with controls. When time to reach an EDSS=3 or EDSS=6 was considered as end point, HLA-DRB1*15 negative patients were found to have a worse prognosis. CONCLUSIONS: In this population of Portuguese MS patients, the HLA-DRB1*15 allele is established as a genetic marker for susceptibility to MS and is also associated with a better outcome.

The polyglutamine spinocerebellar ataxias (SCAs) constitute a clinically and genetically heterogeneous group of rare late-onset neurodegenerative disorders, caused by CAG expansions in the coding region of the respective genes. Given their considerable clinical overlapping, differential diagnosis relies on molecular testing. Laboratory best practice guidelines for molecular genetic testing of the SCAs were released in 2010 by the European Molecular Genetics Quality Network, following the recognition of gross genotyping errors by some diagnostic laboratories. The main goal of this study was to verify the existence of inter-laboratorial consistency comparing genotypes for SCA1, SCA2, SCA3, SCA6 and SCA7 obtained by independent diagnostic laboratories. The individual impact of different methodological issues on the genotype for the several SCAs was also analysed. Four international collaborative diagnostic laboratories provided 79 samples and the respective SCA genotypes. Samples were genotyped in-house for all SCAs using an independent methodology; comparison of the allele size obtained with the one provided by the collaborative laboratories was performed. Globally, no significant differences were identified, a result which could be reflecting the fulfilment of recommendations for the molecular testing of SCAs and demonstrating an improvement in genotyping accuracy.

Gitelman syndrome (GS) is a rare autossomal recessive inherited tubulopathy, characterized by defective tubular reabsorption of magnesium and potassium, mostly caused by mutations in the SLC12A3 gene. The association of GS with chondrocalcinosis (CC) has been described in the literature as a typical example of hypomagnesemiainduced crystal deposition disease. We are presenting one case where the genetic cause for GS was identified in a proband with secondary early onset CC. A 60 years-old male patient with CC, hypomagnesemia and hypokalemia was identified in our hospital as a result of clinical and laboratory assessments. The clinical diagnosis of GS was performed and SLC12A3 gene was screened in the proband; variants detected were further searched in family members. The proband was homozygous for the S615L mutation; additionally, only one from the seven family members which were heterozygous presents CC. The presence of CC in two other individuals is most likely sporadic, in agreement with their advanced age.

Copyright © 2015 Helena Canhão et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited., Background: The aim of our work was to replicate, in a Southern European population, the association reported in Northern populations between PTPRC locus and response to anti-tumor necrosis factor (anti-TNF) treatment in rheumatoid arthritis (RA). We also looked at associations between five RA risk alleles and treatment response. Methods: We evaluated associations between anti-TNF treatment responses assessed by DAS28 change and by EULAR response at six months in 383 Portuguese patients. Univariate and multivariate linear and logistic regression analyses were performed. In a second step to confirm our findings, we pooled our population with 265 Spanish patients. Results: No association was found between PTPRC rs10919563 allele and anti-TNF treatment response, neither in Portuguese modeling for several clinical variables nor in the overall population combining Portuguese and Spanish patients. The minor allele for RA susceptibility, rs3761847 SNP in TRAF1/C5 region, was associated with a poor response in linear and logistic univariate and multivariate regression analyses. No association was observed with the other allellic variants. Results were confirmed in the pooled analysis. Conclusion: This study did not replicate the association between PTPRC and the response to anti-TNF treatment in our Southern European population. We found that TRAF1/C5 risk RA variants potentially influence anti-TNF treatment response., This work was supported by a grant from Harvard-Portugal Program HMSP-ICS/SAU-ICT/0002/2010. Daniel H. Solomon received support for this work from the NIH (K24-AR-055989). Elizabeth W. Karlson received support for this work from NIH (K24-AR-AR0524). Reuma.pt received unrestricted grants from Abbott, Bristol-Myers Squibb, Merck Sharp and Dohme, Pfizer, Roche, and UCB Pharma.

Copyright © 2015 Ana Filipa Mourão et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited., Introduction: This study aimed to assess the genetic determinants of poor outcome in Portuguese patients with juvenile idiopathic arthritis (JIA). Methods: Our study was conducted in Reuma.pt, the Rheumatic Diseases Portuguese Register, which includes patients with JIA. We collected prospectively patient and disease characteristics and a blood sample for DNA analysis. Poor prognosis was defined as CHAQ/HAQ >0.75 at the last visit and/or the treatment with biological therapy. A selected panel of single nucleotide polymorphisms (SNPs) associated with susceptibility was studied to verify if there was association with poor prognosis. Results: Of the 812 patients with JIA registered in Reuma.pt, 267 had a blood sample and registered information used to define "poor prognosis." In univariate analysis, we found significant associations with poor prognosis for allele A of TNFA1P3/20 rs6920220, allele G of TRAF1/C5 rs3761847, and allele G of PTPN2 rs7234029. In multivariate models, the associations with TRAF1/C5 (1.96 [1.17-3.3]) remained significant at the 5% level, while TNFA1P3/20 and PTPN2 were no longer significant. Nevertheless, none of associations found was significant after the Bonferroni correction was applied. Conclusion: Our study does not confirm the association between a panel of selected SNP and poor prognosis in Portuguese patients with JIA.

Objective. Twelve families that were multiply affected with diffuse idiopathic skeletal hyperostosis (DISH) and/or chondrocalcinosis, were identified on the island of Terceira, The Azores, potentially supporting the hypothesis that the 2 disorders share common etiopathogenic factors. The present study was undertaken to investigate this hypothesis. Methods. One hundred three individuals from 12 unrelated families were assessed. Probands were identified from patients attending the Rheumatic Diseases Clinic, Hospital de Santo Espirito, in The Azores. Family members were assessed by rheumatologists and radiologists. Radiographs of all family members were obtained, including radiographs of the dorsolumbar spine, pelvis, knees, elbows, and wrists, and all cases were screened for known features of chondrocalcinosis. Results. Ectopic calcifications were identified in 70 patients. The most frequent symptoms or findings were as follows: axial pain, elbow, knee and metacarpophalangeal (MCP) joint pain, swelling, and/or deformity, and radiographic enthesopathic changes. Elbow and MCP joint periarticular calcifications were observed in 35 and 5 patients, respectively, and chondrocalcinosis was identified in 12 patients. Fifteen patients had sacroiliac disease (ankylosis or sclerosis) on computed tomography scans. Fifty-two patients could be classified as having definite (17%), probable (26%), or possible (31%) DISH. Concomitant DISH and chondrocalcinosis was diagnosed in 12 patients. Pyrophosphate crystals were identified from knee effusions in 13 patients. The pattern of disease transmission was compatible with an autosomal-dominant monogenic disease. The mean age at which symptoms developed was 38 years. Conclusion. These families may represent a familial type of pyrophosphate arthropathy with a phenotype that includes peripheral and axial enthesopathic calcifications. The concurrence of DISH and chondrocalcinosis suggests a shared pathogenic mechanism in the 2 conditions.

KEYWORDS HLA B27; ankylosing spondylitis; review ABSTRACT The Spondyloarthritis are a group of diseases with a strong tendency for family agregation, which includes mainly Ankylosing Spondylitis (AS), Reiter's Syndrome / Reactive Arthritis (ReA), Enteropathic Spondylitis (Crohn's disease and Ulcerative Colitis), Psoriatic Arthropathy (PsA), and Undifferentiated Spondylitis (uSpA). The axial skeleton, mainly the sacroiliac joints, the peripheral joints - more frequently in the lower limbs, and the tendon insertions (enthesis), are particularly prone to an inflammatory process that may involve several targets at the same time or sequentially. Main symptoms depend on the stage of the disease or the SpA subset under examination, and their overlap is frequent. The most known subset of the SpA is Ankylosing Spondylitis, a chronic systemic inflammatory disease of the axial skeleton whose etiology is still unknown, affecting always the sacroiliac, and usually the apophyseal, costovertebral, and costotransverse joints of the spine. The symptoms begin in late adolescence or early adulthood, and chronic inflammatory back pain and stiffness are the most common and characteristic initial presenting complaints in adult-onset AS. The description in 1973 of a very strong association between HLA-B27, an immune response gene and AS permitted to consider this disease to have an autoimmune pathogenesis. The association of AS with HLA-B27 may be 95% to 99% according with the majority of authors but this proportion is inferior in Reiter's Syndrome and Psoriatic Arthritis.

One hundred and thirty unrelated Azorean individuals were randomly selected to study the frequencies of high-resolution HLA alleles and haplotypes in the Azorean (Terceira) population. HLA-A, -B, -Cw, -DRB1, -DQA1 and -DQB1 high-resolution genotyping was performed by polymerase chain reaction using commercial kits. HLA-E, -F and -G alleles, were genotyped by sequence-based typing. All loci were in HWE, showing no locus-level deviations. The genotype data is available in the Allele Frequencies Net Database under the population name "Azores Terceira Island" and the identifier (AFND112579).

Twelve families with exuberant and early-onset calcium pyrophosphate dehydrate chondrocalcinosis (CC) and diffuse idiopathic skeletal hyperostosis (DISH), hereafter designated DISH/CC, were identified in Terceira Island, the Azores, Portugal. Ninety-two (92) individuals from these families were selected for whole-genome-wide linkage analysis. An identity-by-descent (IBD) analysis was performed in 10 individuals from 5 of the investigated pedigrees. The chromosome area with the maximal logarithm of the odds score (1.32; P=0.007) was not identified using the IBD/identity-by-state (IBS) analysis; therefore, it was not investigated further. From the IBD/IBS analysis, two candidate genes, LEMD3 and RSPO4, were identified and sequenced. Nine genetic variants were identified in the RSPO4 gene; one regulatory variant (rs146447064) was significantly more frequent in control individuals than in DISH/CC patients (P=0.03). Four variants were identified in LEMD3, and the rs201930700 variant was further investigated using segregation analysis. None of the genetic variants in RSPO4 or LEMD3 segregated within the studied families. Therefore, although a major genetic effect was shown to determine DISH/CC occurrence within these families, the specific genetic variants involved were not identified.

Machado-Joseph disease (or spinocerebellar ataxia type 3) is a late-onset polyglutamine neurodegenerative disorder caused by a mutation in the ATXN3 gene, which encodes for the ubiquitously expressed protein ataxin-3. Previous studies on cell and animal models have suggested that mutated ataxin-3 is involved in transcriptional dysregulation. Starting with a whole-transcriptome profiling of peripheral blood samples from patients and controls, we aimed to confirm abnormal expression profiles in Machado-Joseph disease and to identify promising up-regulated genes as potential candidate biomarkers of disease status.The Illumina Human V4-HT12 array was used to measure transcriptome-wide gene expression in peripheral blood samples from 12 patients and 12 controls. Technical validation and validation in an independent set of samples were performed by quantitative real-time polymerase chain reaction (PCR).Based on the results from the microarray, twenty six genes, found to be up-regulated in patients, were selected for technical validation by quantitative real-time PCR (validation rate of 81% for the up-regulation trend). Fourteen of these were further tested in an independent set of 42 patients and 35 controls; 10 genes maintained the up-regulation trend (FCGR3B, CSR2RA, CLC, TNFSF14, SLA, P2RY13, FPR2, SELPLG, YIPF6, and GPR96); FCGR3B, P2RY13, and SELPLG were significantly up-regulated in patients when compared with controls.Our findings support the hypothesis that mutated ataxin-3 is associated with transcription dysregulation, detectable in peripheral blood cells. Furthermore, this is the first report suggesting a pool of up-regulated genes in Machado-Joseph disease that may have the potential to be used for fine phenotyping of this disease. © 2015 International Parkinson and Movement Disorder Society.

The HLA profile of the Azoreans has been compared with those of other world populations in order to provide additional information regarding the history of their origins. The allele frequencies, genetic distances between populations, correspondence analyses and most frequent haplotypes were calculated. Our results indicate that the Azorean population most likely contains an admixture of high-frequency Caucasoid, Mongoloid and, to a lesser degree, Negroid HLA genes. The middle Atlantic Azores Archipelago was officially colonized by the Portuguese after 1439 and historical records are concordant with the existence of Caucasoid and Negroid population. However, Mongoloid genes were not suspected, but the Oriental HLA haplotypes A24-B44-DR6-DQ1, A29-B21-DR7-DQ2 and A2-B50-DR7-DQ2 are the fourth, fifth and sixth most frequent ones in Azores. A correspondence analysis shows that the Azorean population is equidistant from Asian and European populations and genetic distances are in some cases closer to the Asian than to European ethnic groups, and never are significantly different; also, B*2707 subtype is found in Asians and Azoreans (but not in Europeans) and the same Machado-Joseph Disease founder haplotypes (Chr 14) are found in both Japanese and Azoreans. It is proposed that a Mongoloid population exists in Azores; whether, the arrival occurred prior to discovery is undetermined.

We have characterized HLA-B27 alleles in a sample of the population from the Azores (n=46) with the aim of investigating the contribution of different subtypes to ankylosing spondylitis (AS). The study was carried out using PCR-SSOP and in some samples genomic sequencing was conducted. Some significant new finding have arisen from this study. First, B*2705,B*2702,B*2703,B*2707 and B*2708 alleles were found to be represented in this population. The polymorphism of B27 alleles found in a sample of the population from the Azores is higher than the Caucasian groups described. B*2703 and B*2707 have not previously been described to be represented in Caucasians and this could indicate admixtures with different populations of the world. In addition, the B*2708 allele was found to be associated with AS in a large family from the Azores. This association has not been previously reported in either ethnic group and needs to be confirmed in other population studies. This is of considerable interest since has only been described as a rare subtype underrepresented in the British population and has not been previously found to be associated with AS. B*2708 carries the sequence specifying the Bw6 epitope in contrast to most B27 alleles which carry a Bw4 sequence. Differences in this region (residues 77-83) can alter the F-pocket and affect T-cell recognition. The importance that these molecular changes can play in the pathogenesis of AS is discussed.

Human leukocyte antigen (HLA)-A, HLA-B, and HLA-DRB1 polymorphisms were examined in Madeira Island populations. The data was obtained at high-resolution level, using sequence-based typing (SBT). The most frequent alleles at each loci were: A*020101 (24.6%), B*5101 (9.7%), B*440201 (9.2%), and DRB1*070101 (15.7%). The predominant three-loci haplotypes in Madeira were A*020101–B*510101–DRB1*130101 (2.7%) and A*010101–B*0801–DRB1*030101 (2.4%), previously found in north and central Portugal. The present study corroborates historical sources and other genetic studies that say Madeira were populated not only by Europeans, mostly Portuguese, but also sub-Saharan Africans due to slave trade. Comparison with other populations shows that Madeira experienced a stronger African influence due to slave trade than Portugal mainland and even the Azores archipelago. Despite this African genetic input, haplotype and allele frequencies were predominantly from European origin, mostly common to mainland Portugal.

H. Alves - INSA/Departamento Promoção da Saúde e Prevenção de Doenças Não Transmissíveis (Porto) OBJECTIVE: The association of non-MHC genes with AS has been recently suggested. We aimed to investigate the association of the ERAP1, IL23R and TNFSF15 regions and the susceptibility to and protection from AS in HLA-B27-positive individuals. METHODS: A total of 200 unrelated AS patients and 559 healthy unrelated subjects, all HLA-B27 positive, were tested. Twenty single nucleotide polymorphisms (SNPs) were investigated in and near IL23R (nine SNPs), in ERAP1 (five SNPs) and in TNFSF15 (six SNPs). RESULTS: ERAP1 rs30187 [odds ratio (OR) = 1.5, P = 4.7 × 10(-3)] had the strongest association with AS susceptibility. A protective effect was found in three of the ERAP1 SNPs: rs17482078 (OR = 0.7, P = 2.8 × 10(-2)), rs10050860 (OR = 0.7, P = 2.3 × 10(-2)), rs2287987 (OR = 0.6, P = 1.3 × 10(-2)). The ERAP1 haplotype rs17482078/rs10050860/rs30187/rs2287987-CCTT showed an association with AS susceptibility (P = 6.8 × 10(-3)) and a protective effect was identified in rs17482078/rs10050860/rs30187/rs2287987-TTCC (P = 3.1 × 10(-2)). Significant association with AS susceptibility was found in one IL23R marker (rs1004819, P = 4.3 × 10(-2), OR = 1.3). No associations were observed in the TNFSF15 region. CONCLUSION: The identification of a new protection haplotype in ERAP1 and the lack of association of the TNFSF15 region can provide new insights into the understanding of the mechanisms underlying the susceptibility to and protection from AS. This work was partially funded by a grant from the DRCT (Science and Technology Regional Board) from the Autonomous Government of Azores, Portugal (DRCT M2.1.2/I/014/2007).

Two unprecedented guaiane-type sesquiterpene glycosides (undulatumosides A and B) were isolated by bioassay-guided fractionation from the MeOH extract of Pittosporum undulatum fruits, along with six known compounds, including the guaiane isomers 5-guaien-11-ol and 4-guaien-11-ol. The structures of the compounds were established as 4-guaiene-11- O -β- d -(3′-angeloxy-6′-deoxy)-glucopyranoside and 1(5)-guaiene-11- O -β- d -(3′-angeloxy-6′-deoxy)-glucopyranoside by spectroscopic methods, including 1D and 2D homo- and heteronuclear NMR experiments (COSY, HSQC, HMBC and NOESY), and HR-mass spectrometry. P . undulatum is a highly invasive weed that often outcompetes other plants, yet its fruits have become a traditional anti-inflammatory medicine in Azores. Therefore, aiming to investigate the claimed properties, the in vitro anti-inflammatory activity of guaiane-type sesquiterpenes was evaluated by analyzing their inhibitory effects on chemical mediators released by the LPS activated RAW 264.7 murine macrophages cell line. In addition, the cytotoxicity of these compounds was also evaluated in this cell line. Undulatumoside A, 5-guaien-11-ol and 4-guaien-11-ol displayed anti-inflammatory activity with IC 50 values of 16.4, 8.1 and 7.2 μM, respectively, comparable to that of the positive control, indomethacin (IC 50 = 18.2 μM), with no cytotoxic effects (IC 50 ⩾ 198 μM). Furthermore, the same set of compounds was also assessed for anti-proliferative activity in lung large cell carcinoma COR-L23 and amelanotic melanoma C32 cells.

One hundred and twenty-seven unrelated Azorean individuals were randomly selected to study the gene frequencies of Killer-cell immunoglobulin-like receptors (KIR) in the Azorean (Terceira) population. KIR genotyping was performed by polymerase chain reaction using commercial sequence-specific oligonucleotide probe kits. All loci were in HWE, showing no locus-level deviations. The genotype data is available in the Allele Frequencies Net Database under the population name “Azores Terceira Island KIR”.

The association of human leukocyte antigen (HLA)-B27 with ankylosing spondylitis (AS) has been known for over 38 years. However, it is not the only gene associated with AS. The aim of this study was to confirm the association of HLA markers around HLA-DPA1/DPB1 region with AS in HLA-B27 positive populations. Five SNPs (rs422544, rs6914849, rs92777535, rs3128968 and rs2295119) from the HLA-DPA1/DPB1 region were genotyped in 340 individuals HLA-B27-positive from Portugal (137 AS patients and 203 healthy controls). Characterizations of HLA-DPA1/DPB1 alleles were also performed. rs422544 revealed a significant association with AS (P0.05) and sliding windows (SW) analysis showed association of some groups of adjacent SNPs within HLA-DPA1/DPB1 region with AS (P0.05). We also found association of the HLA-DPA1(∗)01:03 allele with AS (P0.05). This is the first study that confirms the association of HLA markers and haplotypes around HLA-DPA1 and HLA-DPB1 with AS.

Manuela Lima1,2, Jacome Bruges-Armas1,3 and Conceicao Bettencourt1,2,4 1Genetic and Arthritis Research Group, Institute for Molecular and Cell Biology (IBMC), University of Porto, Porto 2Center of Research in Natural Resources (CIRN) and Department of Biology, University of the Azores, Ponta Delgada 3Servico Especializado de Epidemiologia e Biologia Molecular, Hospital de Santo Espirito de Angra do Heroismo 4Laboratorio de Biologia Molecular, Instituto de Enfermedades Neurologicas, Fundacion Socio-Sanitaria de Castilla-La Mancha, Guadalajara 1,2,3Portugal 4Spain

Alternative splicing (AS) of pre-mRNA is an important regulatory mechanism that enables one gene to produce multiple mature transcripts and, therefore, multiple protein isoforms. Besides the information content of core splicing signals, additional cis-regulatory elements (splicing enhancers and silencers) are needed to precisely define exons. AS is well documented in ATXN3 gene, which encodes for ataxin-3 and, when mutated, is responsible for Machado–Joseph disease (MJD). By studying MJD patients and controls, we have previously identified 56 alternative transcript variants for this gene; some were predicted to encode “protective” ataxin-3 isoforms, making then pertinent to understand AS regulation. The present study aims to investigate the relationship between variation in ATXN3 cis-regulatory motifs and AS variants found for each individual. We have sequenced exonic and flanking intronic ATXN3 regions, in genomic DNA from MJD patients and controls previously studied. None of the 10 single nucleotide polymorphisms (SNPs) that were found was located in core splicing signals. In silico analysis showed those SNPs implied losses and gains of recognition motifs for splicing factors. Each particular allele was not directly reflected in alterations of the resulting splicing variants, indicating that AS cannot be determined solely by these cis-elements, but should result from a more complex mode of regulation.