Blood and cerebellar abundance of ATXN3 splice variants in spinocerebellar ataxia type 3/Machado-Joseph disease.

Spinocerebellar ataxia type 3 (SCA3)/Machado-Joseph disease (MJD) is a heritable proteinopathy disorder, whose causative gene, ATXN3, undergoes alternative splicing. Ataxin-3 protein isoforms differ in their toxicity, suggesting that certain ATXN3 splice variants may be crucial in driving the selective toxicity in SCA3. Using RNA-seq datasets we identified and determined the abundance of annotated ATXN3 transcripts in blood (n = 60) and cerebellum (n = 12) of SCA3 subjects and controls. The reference transcript (ATXN3-251), translating into an ataxin-3 isoform harbouring three ubiquitin-interacting motifs (UIMs), showed the highest abundance in blood, while the most abundant transcript in the cerebellum (ATXN3-208) was of unclear function. Noteworthy, two of the four transcripts that encode full-length ataxin-3 isoforms but differ in the C-terminus were strongly related with tissue expression specificity: ATXN3-251 (3UIM) was expressed in blood 50-fold more than in the cerebellum, whereas ATXN3-214 (2UIM) was expressed in the cerebellum 20-fold more than in the blood. These findings shed light on ATXN3 alternative splicing, aiding in the comprehension of SCA3 pathogenesis and providing guidance in the design of future ATXN3 mRNA-lowering therapies.

Tissue-Specific Vulnerability to Apoptosis in Machado-Joseph Disease.

Machado-Joseph disease (MJD) is a dominant neurodegenerative disease caused by an expanded CAG repeat in the ATXN3 gene encoding the ataxin-3 protein. Several cellular processes, including transcription and apoptosis, are disrupted in MJD. To gain further insights into the extent of dysregulation of mitochondrial apoptosis in MJD and to evaluate if expression alterations of specific apoptosis genes/proteins can be used as transcriptional biomarkers of disease, the expression levels of BCL2, BAX and TP53 and the BCL2/BAX ratio (an indicator of susceptibility to apoptosis) were assessed in blood and post-mortem brain samples from MJD subjects and MJD transgenic mice and controls. While patients show reduced levels of blood BCL2 transcripts, this measurement displays low accuracy to discriminate patients from matched controls. However, increased levels of blood BAX transcripts and decreased BCL2/BAX ratio are associated with earlier onset of disease, indicating a possible association with MJD pathogenesis. Post-mortem MJD brains show increased BCL2/BAX transcript ratio in the dentate cerebellar nucleus (DCN) and increased BCL2/BAX insoluble protein ratio in the DCN and pons, suggesting that in these regions, severely affected by degeneration in MJD, cells show signs of apoptosis resistance. Interestingly, a follow-up study of 18 patients further shows that blood BCL2 and TP53 transcript levels increase over time in MJD patients. Furthermore, while the similar levels of blood BCL2, BAX, and TP53 transcripts observed in preclinical subjects and controls is mimicked by pre-symptomatic MJD mice, the expression profile of these genes in patient brains is partially replicated by symptomatic MJD mice. Globally, our findings indicate that there is tissue-specific vulnerability to apoptosis in MJD subjects and that this tissue-dependent behavior is partially replicated in a MJD mouse model.

Blood transcriptome sequencing identifies biomarkers able to track disease stages in spinocerebellar ataxia type 3.

Transcriptional dysregulation has been described in spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD), an autosomal dominant ataxia caused by a polyglutamine expansion in the ataxin-3 protein. As ataxin-3 is ubiquitously expressed, transcriptional alterations in blood may reflect early changes that start before clinical onset and might serve as peripheral biomarkers in clinical and research settings. Our goal was to describe enriched pathways and report dysregulated genes, which can track disease onset, severity or progression in carriers of the ATXN3 mutation (pre-ataxic subjects and patients). Global dysregulation patterns were identified by RNA sequencing of blood samples from 40 carriers of ATXN3 mutation and 20 controls and further compared with transcriptomic data from post-mortem cerebellum samples of MJD patients and controls. Ten genes-ABCA1, CEP72, PTGDS, SAFB2, SFSWAP, CCDC88C, SH2B1, LTBP4, MEG3 and TSPOAP1-whose expression in blood was altered in the pre-ataxic stage and simultaneously, correlated with ataxia severity in the overt disease stage, were analysed by quantitative real-time PCR in blood samples from an independent set of 170 SCA3/MJD subjects and 57 controls. Pathway enrichment analysis indicated the Gαi signalling and the oestrogen receptor signalling to be similarly affected in blood and cerebellum. SAFB2, SFSWAP and LTBP4 were consistently dysregulated in pre-ataxic subjects compared to controls, displaying a combined discriminatory ability of 79%. In patients, ataxia severity was associated with higher levels of MEG3 and TSPOAP1. We propose expression levels of SAFB2, SFSWAP and LTBP4 as well as MEG3 and TSPOAP1 as stratification markers of SCA3/MJD progression, deserving further validation in longitudinal studies and in independent cohorts.

The Homogeneous Azorean Machado-Joseph Disease Cohort: Characterization and Contributions to Advances in Research.

Machado-Joseph disease (MJD)/spinocerebellar ataxia type 3 (SCA3) is the most common autosomal dominant ataxia worldwide. MJD is characterized by late-onset progressive cerebellar ataxia associated with variable clinical findings, including pyramidal signs and a dystonic-rigid extrapyramidal syndrome. In the Portuguese archipelago of the Azores, the worldwide population cluster for this disorder (prevalence of 39 in 100,000 inhabitants), a cohort of MJD mutation carriers belonging to extensively studied pedigrees has been followed since the late 1990s. Studies of the homogeneous Azorean MJD cohort have been contributing crucial information to the natural history of this disease as well as allowing the identification of novel molecular biomarkers. Moreover, as interventional studies for this globally rare and yet untreatable disease are emerging, this cohort should be even more important for the recruitment of trial participants. In this paper, we profile the Azorean cohort of MJD carriers, constituted at baseline by 20 pre-ataxic carriers and 52 patients, which currently integrates the European spinocerebellar ataxia type 3/Machado-Joseph disease Initiative (ESMI), a large European longitudinal MJD cohort. Moreover, we summarize the main studies based on this cohort and highlight the contributions made to advances in MJD research. Knowledge of the profile of the Azorean MJD cohort is not only important in the context of emergent interventional trials but is also pertinent for the implementation of adequate interventional measures, constituting relevant information for Lay Associations and providing data to guide healthcare decision makers.

Early onset obesity due to a mutation in the human leptin receptor gene.

Summary: Leptin is secreted by adipocytes in response to fat storage and binds to its receptor (LEPR), which is ubiquitously expressed throughout the body. Leptin regulates energy expenditure and is anorexigenic. In this study, we describe the clinical and hormonal findings of three siblings with a personal history of rapid weight gain during the first months of life. They had delayed puberty, high levels of FSH (15.6 ± 3.7 mUI/mL; reference: 1.5-12.4) and LH (12.3 ± 2.2 mUI/mL; reference: 1.7-8.6), normal oestradiol and total testosterone and successful fertility. None of the patients had dyslipidemia, diabetes or thyroid disease. Next-generation sequencing identified a pathogenic homozygous variant c.2357T>C, p.(Leu786Pro) in LEPR. Their parents and children were heterozygous for this mutation. We compared clinical and biochemical findings of homozygous carriers with first-degree heterozygous family members and ten randomly selected patients with adult-onset morbid obesity. Homozygous carriers of the mutation had significantly higher BMI (32.2 ± 1.7 kg/m2 vs 44.5 ± 7.1 kg/m2, P = 0.023) and increased serum levels of leptin (26.3 ± 9.3 ng/mL vs 80 ± 36.4 ng/mL, P = 0.028) than their heterozygous relatives. Compared with the ten patients with adult-onset morbid obesity, serum levels of leptin were not significantly higher in homozygous carriers (53.8 ± 24.1 ng/mL vs 80 ± 36.4 ng/mL, P = 0.149), and thus serum levels of leptin were not a useful discriminative marker of LEPR mutations. We described a rare three-generation family with monogenic obesity due to a mutation in LEPR. Patients with early onset obesity should be considered for genetic screening, as the identification of mutations may allow personalized treatment options (e.g. MC4R-agonists) and targeted successful weight loss. Learning points: The early diagnosis of monogenic forms of obesity can be of great interest since new treatments for these conditions are becoming available. Since BMI and leptin levels in patients with leptin receptor mutations are not significantly different from those found in randomly selected morbid obese patients, a careful medical history is mandatory to suspect this condition. Loss of leptin receptor function has been associated with infertility. However, our patients were able to conceive, emphasizing the need for genetic counselling in affected patients with this condition.

Novel Machado-Joseph disease-modifying genes and pathways identified by whole-exome sequencing.

Machado-Joseph disease (MJD/SCA3) is a neurodegenerative polyglutamine disorder exhibiting a wide spectrum of phenotypes. The abnormal size of the (CAG)n at ATXN3 explains ~55% of the age at onset variance, suggesting the involvement of other factors, namely genetic modifiers, whose identification remains limited. Our aim was to find novel genetic modifiers, analyse their epistatic effects and identify disease-modifying pathways contributing to MJD variable expressivity. We performed whole-exome sequencing in a discovery sample of four age at onset concordant and four discordant first-degree relative pairs of Azorean patients, to identify candidate variants which genotypes differed for each discordant pair but were shared in each concordant pair. Variants identified by this approach were then tested in an independent multi-origin cohort of 282 MJD patients. Whole-exome sequencing identified 233 candidate variants, from which 82 variants in 53 genes were prioritized for downstream analysis. Eighteen disease-modifying pathways were identified; two of the most enriched pathways were relevant for the nervous system, namely the neuregulin signaling and the agrin interactions at neuromuscular junction. Variants at PARD3, NFKB1, CHD5, ACTG1, CFAP57, DLGAP2, ITGB1, DIDO1 and CERS4 modulate age at onset in MJD, with those identified in CFAP57, ACTG1 and DIDO1 showing consistent effects across cohorts of different geographical origins. Network analyses of the nine novel MJD modifiers highlighted several important molecular interactions, including genes/proteins previously related with MJD pathogenesis, namely between ACTG1/APOE and VCP/ITGB1. We describe novel pathways, modifiers, and their interaction partners, providing a broad molecular portrait of age at onset modulation to be further exploited as new disease-modifying targets for MJD and related diseases.

Promoter Variation and Expression Levels of Inflammatory Genes IL1A, IL1B, IL6 and TNF in Blood of Spinocerebellar Ataxia Type 3 (SCA3) Patients.

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.

Triplet Repeat Primed PCR (TP-PCR) in Molecular Diagnostic Testing for Spinocerebellar Ataxia Type 3 (SCA3).

INTRODUCTION AND OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: The present TP-PCR protocol developed for SCA3 should constitute a reliable complementary technique to overcome the limitations of standard PCR.

Novel candidate blood-based transcriptional biomarkers of Machado-Joseph disease.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Nystagmus as an early ocular alteration in Machado-Joseph disease (MJD/SCA3).

BACKGROUND: Machado-Joseph disease (MJD), also named spinocerebellar ataxia type 3 (SCA3) is the most common autosomal dominant ataxia worldwide. Although nystagmus is one of the most frequently reported ocular alterations in MJD patients its behaviour during the course of the disease, namely in its early stages, has only recently started to be investigated. The main goal of this work was to characterize the frequency of nystagmus in symptomatic and presymptomatic carriers of the MJD mutation, and investigate its usefulness as an early indicator of the disease. METHODS: We conducted an observational study of Azorean MJD family members, comprising a total of 158 subjects which underwent neurological evaluation. Sixty eight were clinically and molecularly diagnosed with MJD, 48 were confirmed asymptomatic carriers and 42 were confirmed non-carriers of the MJD mutation. The frequency of nystagmus was calculated for the 3 groups. RESULTS: Nystagmus was present in 88% of the MJD patients. Seventeen percent of the at-risk subjects with a carrier result in the molecular test and none of the 42 individuals who received a non-carrier test result displayed nystagmus (p < 0.006). Although not reaching statistical significance, symptomatic subjects showing nystagmus had a tendency for a higher length of the CAG tract in the expanded allele, when compared to individuals who did not have nystagmus. CONCLUSIONS: The frequency of nystagmus in asymptomatic carriers and its absence in non-carriers of the mutation, suggests that nystagmus may appear before gait disturbance and can thus be considered an early sign of MJD.

Nonoptical massive parallel DNA sequencing of BRCA1 and BRCA2 genes in a diagnostic setting.

The introduction of the benchtop massive parallel sequencers made it possible for the majority of clinical diagnostic laboratories to gain access to this fast evolving technology. In this study, using the Ion Torrent Personal Genome Machine, we present a strategy for the molecular diagnosis of hereditary breast and ovarian cancer and respective analytical validation. The methodology relies on a multiplex PCR amplification of the BRCA1 and BRCA2 genes combined with a variant prioritization pipeline, designed to minimize the number of false-positive calls without the introduction of false-negative results. A training set of samples was used to optimize the entire process, and a second set was used to validate and independently evaluate the performance of the workflow. Performing the study in a blind manner relative to the variants in the samples and using conventional Sanger sequencing as standard, the workflow resulted in a strategy with a maximum analytical sensitivity ≥98.6% with a confidence of 95% and a specificity of 96.9%. Importantly, no true variant was missed. This study presents a comprehensive massive parallel sequencing-Sanger sequencing based strategy, which results in a high analytical sensitivity assay that provides a time- and cost-effective strategy for the identification of mutations in the BRCA1 and BRCA2 genes.

Psychological well-being and family satisfaction levels five years after being confirmed as a carrier of the Machado-Joseph disease mutation.

The present study on long-term outcome of presymptomatic testing for Machado-Joseph disease (MJD) aimed to evaluate the psychological well-being and the familial satisfaction of subjects that 5 years prior received an unfavorable result in the predictive testing (PT). The study included 47 testees of Azorean origin (23 from the island of Flores and 24 from S. Miguel) that completed the fourth evaluation session of the MJD protocol, and undertook a neurological examination at the moment of participation in the study. Nearly 50% of testees were symptomatic at the time of the study. Psychological well-being of the 47 participants was evaluated using the Psychological General Well-Being Index (PGWB). The family satisfaction scale by adjectives was applied to obtain information on family dynamics. The average PGWB score of the total participants was of 73.3, a value indicative of psychological well-being. Nearly half of the testees presented scores indicating psychological well-being, whereas scores indicating moderate (28.9%) or severe (23.7%) stress were found in the remaining. The average score in the PGWB scale was lower in symptomatic than in asymptomatic subjects; moreover, the distinct distribution of the well-being categories seen in the two groups shows an impact of the appearance of first symptoms on the psychological state. Motives for undertaking the test, provided 5 years prior, failed to show an impact in well-being. The average score for familial satisfaction was of 134, a value compatible with high familial satisfaction, which represented the most frequent category (59.6%). Results demonstrate that well-being and family satisfaction need to be monitored in confirmed carriers of the MJD mutation. The inclusion of acceptance studies, after PT, as well as the development of acceptance training actions, should be of major importance to anticipate the possibility of psychological damage.

Sequence analysis of 5' regulatory regions of the Machado-Joseph disease gene (ATXN3).

Machado-Joseph disease (MJD) is a late-onset autosomal dominant neurodegenerative disorder, which is caused by a coding (CAG)(n) expansion in the ATXN3 gene (14q32.1). The number of CAG repeats in the expanded alleles accounts only for 50 to 75 % of onset variance, the remaining variation being dependent on other factors. Differential allelic expression of ATXN3 could contribute to the explanation of different ages at onset in patients displaying similar CAG repeat sizes. Variation in 5' regulatory regions of the ATXN3 gene may have the potential to influence expression levels and, ultimately, modulate the MJD phenotype. The main goal of this work was to analyze the extent of sequence variation upstream of the ATXN3 start codon. A fragment containing the core promoter and the 5' untranslated region (UTR) was sequenced and analyzed in 186 patients and 59 controls (490 chromosomes). In the core promoter, no polymorphisms were observed. In the 5' UTR, only one SNP (rs3814834) was found, but no improvements on the explanation of onset variance were observed, when adding its allelic state in a linear model. Accordingly, in silico analysis predicted that this SNP lays in a nonconserved position for CMYB binding. Therefore, no functional effect could be predicted for this variant.

The APOE ε2 allele increases the risk of earlier age at onset in Machado-Joseph disease.

OBJECTIVE: To investigate a modulating effect of the apolipoprotein E (APOE) polymorphism on age at onset of Machado-Joseph disease (MJD). DESIGN: We collected blood samples from 192 patients with MJD and typed the APOE polymorphism. Patients The 192 patients with MJD included 59 from the Azores, 73 from mainland Portugal, and 60 from Brazil. SETTING: Academic research center. RESULTS: Cases with the ε2/ε3 genotype had an earlier onset compared with those with the ε3/ε3 or the ε3/ε4 genotype. In this series of patients, the presence of an APOE ε2 allele implies a decrease of nearly 5 years in the age at onset. When combining several other predictors in a general linear model, namely, the presence/absence of the APOE ε2 allele, with the size of the (CAG)(n) in expanded alleles, the model was significantly improved and the explanation of onset variance was raised from 59.8% to 66.5%. Furthermore, the presence of the ε2 allele was associated with an onset before age 39 years (odds ratio, 5.00; 95% CI, 1.18-21.14). CONCLUSION: The polymorphism at the APOE gene plays a role as a genetic modifier of MJD phenotype.

Parkinsonian phenotype in Machado-Joseph disease (MJD/SCA3): a two-case report.

BACKGROUND: Machado-Joseph disease (MJD), or spinocerebellar ataxia type 3 (SCA3), is an autosomal dominant neurodegenerative disorder of late onset, which is caused by a CAG repeat expansion in the coding region of the ATXN3 gene. This disease presents clinical heterogeneity, which cannot be completely explained by the size of the repeat tract. MJD presents extrapyramidal motor signs, namely parkinsonism, more frequently than the other subtypes of autosomal dominant cerebellar ataxias. Although parkinsonism seems to segregate within MJD families, only a few MJD patients develop parkinsonian features and, therefore, the clinical and genetic aspects of these rare presentations remain poorly investigated. The main goal of this work was to describe two MJD patients displaying the parkinsonian triad (tremor, bradykinesia and rigidity), namely on what concerns genetic variation in Parkinson's disease (PD) associated loci (PARK2, LRRK2, PINK1, DJ-1, SNCA, MAPT, APOE, and mtDNA tRNA(Gln) T4336C). CASE PRESENTATION: Patient 1 is a 40 year-old female (onset at 30 years of age), initially with a pure parkinsonian phenotype (similar to the phenotype previously reported for her mother). Patient 2 is a 38 year-old male (onset at 33 years of age), presenting an ataxic phenotype with parkinsonian features (not seen either in other affected siblings or in his father). Both patients presented an expanded ATXN3 allele with 72 CAG repeats. No PD mutations were found in the analyzed loci. However, allelic variants previously associated with PD were observed in DJ-1 and APOE genes, for both patients. CONCLUSIONS: The present report adds clinical and genetic information on this particular and rare MJD presentation, and raises the hypothesis that DJ-1 and APOE polymorphisms may confer susceptibility to the parkinsonian phenotype in MJD.

Dyskeratosis congenita--two siblings with a new missense mutation in the DKC1 gene.

Dyskeratosis congenital is reported in two siblings. They presented with the classic triad of mucocutaneous features: leukoplakia of the tongue, dystrophic nails, and a widespread reticulate pigmentation on the neck and upper chest. A genetic analysis was performed and a new missense mutation S356P, hemizygous, was identified in the DKC1 gene in both patients. Acitretin was started at a low-dose in both patients, resulting in clinical improvement and important, positive psychosocial effects.

Mutation of the MIF type II receptor in two brothers.

The Müllerian inhibiting factor (MIF) is responsible for regression of Müllerian ducts during male sexual differentiation. Mutations in MIF or its type II receptor lead to persistence of the uterus and Fallopian tubes in male children--i.e., persistent Müllerian duct syndrome (PMDS). Both are rare autosomal recessive disorders. We report a 7-month-old male infant who underwent inguinal herniorrhaphy. Remnants of vas deferens and gonads with macroscopic characteristics of ovaries, along with Fallopian tubes and a rudimentary uterus, were found. Karyotype confirmed male sex. Molecular genetics revealed the most frequent MIF type II receptor gene mutation--27 bp deletion. Investigation of the older brother presenting bilateral cryptorchidism at 7 years of age led to similar clinical findings and the same mutation. We report here an MIF type II receptor mutation in two brothers, with the particularity that the surgical findings in the younger son initiated the diagnostic process in both children.

Increased transcript diversity: novel splicing variants of Machado-Joseph disease gene (ATXN3).

Machado-Joseph disease (MJD) is a late-onset neurodegenerative disorder that presents clinical heterogeneity not completely explained by its causative mutation. MJD is caused by an expansion of a CAG tract at exon 10 of the ATXN3 gene (14q32.1), which encodes for ataxin-3. The main goal of this study was to analyze the occurrence of alternative splicing at the ATXN3 gene, by sequencing a total of 415 cDNAs clones (from 20 MJD patients and 14 controls). Two novel exons are described for the ATXN3 gene. Fifty-six alternative splicing variants, generated by four types of splicing events, were observed. From those variants, 50 were not previously described, and 26 were only found in MJD patients samples. Most of the variants (85.7%) present frameshift, which leads to the appearance of premature stop codons. Thirty-seven of the observed variants constitute good targets to nonsense-mediated decay, the remaining are likely to be translated into at least 20 different isoforms. The presence of ataxin-3 domains was assessed, and consequences of domain disruption are discussed. The present study demonstrates high variability in the ATXN3 gene transcripts, providing a basis for further investigation on the contribution of alternative splicing to the MJD pathogenic process, as well as to the larger group of the polyglutamine disorders.

The (CAG)n tract of Machado-Joseph Disease gene (ATXN3): a comparison between DNA and mRNA in patients and controls.

Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder of late onset (occurring at a mean age of 40.2 years). The clinical manifestation of MJD is dependent on the presence of an expansion of the (CAG)(n) motif within exon 10 of the ATXN3 gene, located at 14q32.1. The variance in onset of MJD is only partially correlated (approximately 50-80%) with the extension of the CAG tract in genomic DNA (gDNA). The main aim of this work was to determine whether there are discrepancies in the size of the (CAG)(n) tract between gDNA and mRNA, and to establish whether there is a better association between age at onset and repeat size at the mRNA level. We typed gDNA and cDNA samples for the (CAG)(n) tract totalizing 108 wild-type and 52 expanded ATXN3 alleles. In wild-type alleles no differences were found between gDNA and cDNA. In expanded alleles, the CAG repeat size in gDNA was not always directly transcribed into the mRNA; on average there were differences of +1 repeat at the cDNA level. The slight discrepancies obtained were insufficient to cause significant differences in the distribution of the expanded alleles, and therefore no improvement in onset variance explanation was obtained with mRNA.

Mutations in the MECP2 gene are not a major cause of Rett syndrome-like or related neurodevelopmental phenotype in male patients.

Rett syndrome is a genetic neurodevelopmental disorder that affects mainly girls, but mutations in the causative MECP2 gene have also been identified in boys with classic Rett syndrome and Rett syndrome-like phenotypes. We have studied a group of 28 boys with a neurodevelopmental disorder, 13 of which with a Rett syndrome-like phenotype; the patients had diverse clinical presentations that included perturbations of the autistic spectrum, microcephaly, mental retardation, manual stereotypies, and epilepsy. We analyzed the complete coding region of the MECP2 gene, including the detection of large rearrangements, and we did not detect any pathogenic mutations in the MECP2 gene in these patients, in whom the genetic basis of disease remained unidentified. Thus, additional genes should be screened in this group of patients.