Studies of polyglutamine repeats and their biology in relation to disease

Polyglutamine repeat expansions of the CAG/CTG type frequently lead to disease characterized by progressive neuronal dysfunction. These diseases typically begins in mid-life and result in severe neurodegeneration. To a certain extent they present with similar features and probably share a common mechanism of pathogenesis. There is an inverse relationship between the size of the repeat and the age at onset, though the contribution of the repeat size to age at onset varies according to the protein context. Intergenerational instability leads to further expansion and an earlier age at onset in succeeding generations, a phenomenon called anticipation. These polyglutamine tracts are encoded at the DNA level by CAG/CTG trinucleotide repeats. The Repeat Expansion Detection (RED) method allows detection of long trinucleotide repeats anywhere in the genome without prior knowledge of their location. In paper I, our aim was to determine if repeats of sequence motifs other than those known at that time might expand. Five new trinucleotide motifs were found in repeat sequences larger than 180 nucleotides (nt) in one or more of the 200 individuals analyzed: ATG/CAT, CCT/AGG, CTT/AAG, TGG/CCA and GTT/AAC. CAG/CTG is the motif that has most frequently been associated with diseases. In paper 11, the RED method was made more sensitive and reliable for screening for CAG/CTG expansions. Multivariate analysis was used to optimize the procedure and to determine the most influential factors in the amplification reaction and to identify interacting factors. It was possible to obtain up to a 5.5 fold increase in the yield of the reaction after optimization of the protocol. In parallel with the optimization procedure, candidate diseases were analyzed by the RED method for the presence of expanded CAG trinucleotide repeats in the genome of the patients. Anticipation appeared to characterize familial spastic paraplegia (FSP) linked to the SPG4 locus (2p21-p24), and an association between expanded CAG/CTG repeats and FSP had been reported. In paper 111, 41 affected individuals from five French and one Polish FSP families linked to SPG4 were tested for expanded CAG/CTG repeats using the RED method, PCR for the ERDA1 and CTG18.1 loci as well as by Western blot for expressed polyglutamines. Our study provided evidence against the hypothesis that a large CAG repeat expansion is the basis of SPG4. This has now been confirmed by the cloning of the gene. In paper IV, eight spinocerebellar ataxia type 7 (SCA7) families with marked anticipation were analyzed by RED for the presence of CAG repeat expansion. A 150-240nt CAG repeat was found to cosegregate with the disease (p<0.000001, n=66). Shortly thereafter, the SCA7 gene was cloned, which opened an avenue for pathophysiological studies of SCAT In paper V, normal ataxin-7 (the SCA-7 gene product) was found to be widely distributed in normal human postmortem brain. The pattern of expression was not limited to regions affected in SCAT In SCA7 brain, the mean proportion of immunolabelled nuclei was significantly higher in regions with neuronal loss (21%) than in regions that were spared (4%) (t=2.1; p<0.05), suggesting that nuclear localization of ataxin-7 might be associated with dysfunction. In paper V1, SCA7 cell culture models were established in HEK293 and SH-SY5Y cells, in order to analyse alterations induced by the mutant ataxin-7 and to compare them with observations in human SCA7 brain. Overexpressed ataxin-7 formed large fibrillar inclusions. It also localized to small electron dense structures that might correspond to nuclear bodies and may possibly be precursors of inclusions. There were indirect signs of ongoing abnormal protein folding, including the recruitment of heat shock proteins and proteasome subunits. Occasionally, transcription factors and polyglutamine containing proteins, including the normal forms of ataxin7 and ataxin-3 were recruited into the inclusions. Our results illustrate for the first time the presence of activated caspase-3 in inclusions in a human polyglutamine disease as well as in the SCA7 cell models. Moreover, caspase-3 expression was up- regulated in cortical neurons, suggesting that it may play a role in the disease process. Finally, on the ultrastructural level, there were signs of autophagy and nuclear indentations, indicative of a major stress response in cells expressing mutant ataxin- 7. The optimised RED technique, should prove useful in identifying additional disorders with trinucleotide repeat expansions as in SCAT The SCA7 cell culture models established in this study share many features of SCA7 brain, that are common to all polyglutamine disorders. However, the observation of activated caspase-3 in inclusions in both cell models and SCA7 brain is particularly interesting. Its role in cell dysfunction and death should be tested in stable models

Proximal Deletion 12q with a New Insight to Growth Retardation

Proximal deletion of the long arm of chromosome 12 is a rare chromosomal abnormality described in about 20 patients. Known deletions span the region from 12q11 to 12q13 and include the genes YAF2, AMIGO2, and NELL2. These are suggested as candidate genes for the key phenotypic features such as growth and psychomotor retardation. Here, we present a case with a 3.1-Mb interstitial deletion at 12q12q13.11. The clinical observations of our patient overlap with the major common findings for published cases. The deletion detected in our patient does not involve the previously suggested candidate genes YAF2 and AMIGO2. We draw a correlation between proximal deletion 12q and ARID2 deficiency by comparing patients carrying gross deletions with a cohort of patients carrying small intragenic ARID2 deletions as well as patients with single nucleotide variants (SNVs) in ARID2. Growth retardation &lt;-2 SD is present in cohorts with both gross and small deletions spanning ARID2. However, ARID2 SNVs do not correlate with severe growth retardation

Autism Spectrum Disorders in the Stockholm Youth Cohort: Design, Prevalence and Validity

Reports of rising prevalence of autism spectrum disorders (ASD), along with their profound personal and societal burden, emphasize the need of methodologically sound studies to explore their causes and consequences. We here present the design of a large intergenerational resource for ASD research, along with population-based prevalence estimates of ASD and their diagnostic validity.The Stockholm Youth Cohort is a record-linkage study comprising all individuals aged 0-17 years, ever resident in Stockholm County in 2001-2007 (N = 589,114). ASD cases (N = 5,100) were identified using a multisource approach, involving registers covering all pathways to ASD diagnosis and care, and categorized according to co-morbid intellectual disability. Prospectively recorded information on potential determinants and consequences of ASD were retrieved from national and regional health and administrative registers. Case ascertainment was validated through case-note review, and cross validation with co-existing cases in a national twin study.The 2007 year prevalence of ASD in all children and young people was 11.5 per 1,000 (95% confidence interval 11.2-11.8), with a co-morbid intellectual disability recorded in 42.6% (41.0-44.2) of cases. We found 96.0% (92.0-98.4) of reviewed case-notes being consistent with a diagnosis of ASD, and confirmed ASD in 85.2% (66.2-95.8) of affected twins.Findings from this contemporary study accords with recently reported prevalence estimates from Western countries at around 1%, based on valid case ascertainment. The Stockholm Youth Cohort, in light of the availability of extensive information from Sweden's registers, constitutes an important resource for ASD research. On-going work, including collection of biological samples, will enrich the study further

Distribution of ataxin-7 in normal human brain and retina

International audienceSpinocerebellar ataxia 7 (SCA7) is a neurodegenerative disease caused by the expansion of a CAG repeat encoding a polyglutamine tract in the protein ataxin-7. We developed antibodies directed against two different parts of the ataxin-7 protein and studied its distribution in brain and peripheral tissue from healthy subjects. Normal ataxin-7 was widely expressed in brain, retina and peripheral tissues, including striated muscle, testis and thyroid gland. In the brain, expression of ataxin-7 was not limited to areas in which neurones degenerate, and the level of expression was not related to the severity of neuronal loss. Immunoreactivity was low in some vulnerable populations of neurones, such as Purkinje cells. In neurones, ataxin-7 was found in the cell bodies and in processes. Nuclear labelling was also observed in some neurones, but was not related to the distribution of intranuclear inclusions observed in an SCA7 patient. In this patient, the proportion of neurones with nuclear labelling was higher, on average, in regions with neuronal loss. Double immunolabelling coupled with confocal microscopy showed that ataxin-7 colocalized with BiP, a marker of the endoplasmic reticulum, but not with markers of mitochondria or the trans-Golgi network

Novel PNKP mutations associated with reduced DNA single-strand break repair and severe microcephaly, seizures, and developmental delay

Background: Microcephaly with early-onset seizures (MCSZ) is a neurodevelopmental disorder caused by pathogenic variants in the DNA strand break repair protein, polynucleotide kinase 3′-phosphatase (PNKP). Methods: We have used whole genome sequencing and Sanger sequencing to identify disease-causing variants, followed by a minigene assay, Western blotting, alkaline comet assay, γH2AX, and ADP-ribose immunofluorescence. Results: Here, we describe a patient with compound heterozygous variants in PNKP, including a missense variant in the DNA phosphatase domain (T323M) and a novel splice acceptor site variant within the DNA kinase domain that we show leads to exon skipping. We show that primary fibroblasts derived from the patient exhibit greatly reduced levels of PNKP protein and reduced rates of DNA single-strand break repair, confirming that the mutated PNKP alleles are dysfunctional. Conclusion: The data presented show that the detected compound heterozygous variants result in reduced levels of PNKP protein, which affect the repair of both oxidative and TOP1-induced single-strand breaks, and most likely causes MCSZ in this patient.</p

Expanded trinucleotide CAG repeats in families with bipolar affective disorder

Clinical anticipation has been reported in bipolar affective disorder (BPAD). The hypothesis that expanded trinucleotide repeats are related to anticipation and transmission pattern in families with bipolar affective disorder is tested in this study. Eighty-seven two-generation pairs of patients recruited from 29 bipolar families were analyzed. The repeat expansion detection method was used to detect CAG repeat expansions between successive generations. Significant changes in age at onset and episode frequency in successive generations were observed. Mean trinucleotide CAG repeat length between parental and offspring generation significantly increased when the phenotype increased in severity, i.e. changed from major depression, single episode or unipolar recurrent depression to BPAD. A parent-of-origin effect was also observed with a significant increase in median length CAG between G1 and G2 with maternal inheritance. This increase was observed notably in female offspring. Our findings indicate for the first time that expansion of CAG repeat length could explain the clinical observation of anticipation in families with BPAD. These results provide further support for expanded trinucleotide repeat sequences as risk factors in major affective disorders.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Mutations in HECW2 are associated with intellectual disability and epilepsy

BACKGROUND: De novo mutations are a frequent cause of disorders related to brain development. We report the results of screening patients diagnosed with both epilepsy and intellectual disability (ID) using exome sequencing to identify known and new causative de novo mutations relevant to these conditions. METHODS: Exome sequencing was performed on 39 patient-parent trios to identify de novo mutations. Clinical significance of de novo mutations in genes was determined using the American College of Medical Genetics and Genomics standard guidelines for interpretation of coding variants. Variants in genes of unknown clinical significance were further analysed in the context of previous trio sequencing efforts in neurodevelopmental disorders. RESULTS: In 39 patient-parent trios we identified 29 de novo mutations in coding sequence. Analysis of de novo and inherited variants yielded a molecular diagnosis in 11 families (28.2%). In combination with previously published exome sequencing results in neurodevelopmental disorders, our analysis implicates HECW2 as a novel candidate gene in ID and epilepsy. CONCLUSIONS: Our results support the use of exome sequencing as a diagnostic approach for ID and epilepsy, and confirm previous results regarding the importance of de novo mutations in this patient group. The results also highlight the utility of network analysis and comparison to previous large-scale studies as strategies to prioritise candidate genes for further studies. This study adds knowledge to the increasingly growing list of causative and candidate genes in ID and epilepsy and highlights HECW2 as a new candidate gene for neurodevelopmental disorders