Leukocyte telomere shortening in Huntington's disease

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by an expanded CAG repeat. Though symptom onset commonly occurs at midlife and inversely correlates with the CAG repeat expansion, age at clinical onset and progression rate are variable. In the present study we investigated the relationship between leukocyte telomere length (LTL) and HD development. LTL was measured by real-time PCR in manifest HD patients (HD, n = 62), pre-manifest HD patients (pre-HD, n = 38), and age-matched controls (n = 76). Significant LTL differences were observed between the three groups (p < .0001), with LTL values in the order: HD < pre-HD < controls. The relationship between LTL and age was different in the three groups. An inverse relationship between mean LTL and CAG repeat number was found in the pre-HD (p = .03). The overall data seem to indicate that after age 30 years, LT begins to shorten markedly in pre-HD patients according to CAG number and increasing age, up to the values observed in HD. This very suggestive picture allowed us to hypothesize that in pre-manifest HD, LTL could be a measure of time to clinical HD onset. The possible use of LTL as a reliable biomarker to track HD development and progression was evaluated and discussed

Regulation of the expression of the Kluyveromyces lactis PDC1 gene: carbon source-responsive elements and autoregulation

The yeast Kluyveromyces lactis has a single structural gene coding for pyruvate decarboxylase (KIPDC1). In order to study the regulation of the expression of KIPDC1, we have sequenced (EMBL Accession No. Y15435) its promoter and have fused the promoter to the reporter gene lacZ from E. coli. Transcription analysis in a Klpdc1 delta strain showed that KIPDC1 expression is subject to autoregulation. The PDC1 gene from Saccharomyces cerevisiae was able to complement the Rag- phenotype of the Klpdc1 delta mutant strain and it could also repress transcription of the KIPDC1-lacZ fusion on glucose. A deletion analysis of the promoter region was performed to study carbon source-dependent regulation and revealed that at least two cis-acting regions are necessary for full induction of gene expression on glucose. Other cis-elements mediate repression on ethanol

Dramatically different levels of cacna1a gene expression between pre-weaning wild type and leaner mice

Loss of function mutations of the CACNA1A gene, coding for the α1A subunit of P/Q type voltage-gated calcium channel (Ca(V)2.1), are responsible for Episodic Ataxia type 2 (EA2), an autosomal dominant disorder. A dominant negative effect of the EA2 mutated protein, rather than a haploinsufficiency mechanism, has been hypothesised both for protein-truncating and missense mutations. We analysed the cacna1a mRNA expression in leaner mice carrying a cacna1a mutation leading to a premature stop codon. The results showed a very low mutant mRNA expression compared to the wild type allele. Although the mutant mRNA slightly increases with age, its low level is likely due to degradation by nonsense mediated decay, a quality control mechanism that selectively degrades mRNA harbouring premature stop codons. These data have implications for EA2 in humans, suggesting a haploinsufficiency mechanism at least for some of the CACNA1A mutations leading to a premature stop codon

The 'petite negative' yeast Kluyveromyces lactis has a single gene expressing pyruvate decarboxylase activity

We cloned and sequenced the pyruvate decarboxylase (PDC; EC 4.1.1.1) structural gene KlPDCA in the yeast Kluyveromyces lactis and found it to be allelic to the previously isolated rag6 mutation. The putative amino acid sequence of the KlPdcAp appeared to be highly homologous to those of the yeast Pdc proteins identified so far. The disruption of KIPDCA indicated that it is the only PDC structural gene in K. lactis, as evidenced by the lack of PDC activity and ethanol production in the pdcAdelta strains and by the absence of growth on glucose in the presence of respiratory inhibitors. It was observed that expression of the KlPDCA gene is induced by glucose at the transcriptional level. Transcription of the gene was reduced in the ragl, rag2, rag5 and rag8 mutants, which are defective for the low-affinity glucose permease, phosphoglucose isomerase, hexokinase, and a positive regulator of RAG1 expression, respectively

Leukocyte telomere length as potential biomarker of HD progression: A follow-up study

The identification of biomarkers for neurodegenerative disorders such as Huntington's disease (HD) is crucial for monitoring disease progression and therapeutic trial outcomes, especially in the pre-manifest disease stage (pre-HD). In a previous study, we observed that leukocyte telomere length (LTL) was strongly correlated with the estimated time to clinical onset in pre-HD subjects. To validate this hypothesis, we designed a follow-up study in which we analyzed LTL in 45 pre-HD stage subjects at baseline (T0) and then again after clinical onset at follow-up (T1); the follow-up interval was about 3 years, and the CAG range was 39-51 repeats; 90 peripheral blood mononuclear cell samples (PBMCs) were obtained from the Enroll-HD biorepository. In pre-HD subjects at T0, LTL was significantly reduced by 22% compared to the controls and by 14% from T0 at T1. No relationship was observed between the LTL and CAG numbers in subjects carrying different CAG repeats at T0 and at T1, suggesting that LTL reduction occurs independently of CAG number in pre-HD subjects. ROC curve analysis was used to test the validity of LTL as a potential biomarker of HD progression and showed that LTL measurement is extremely accurate in discriminating pre-HD subjects from the controls and even pre-HD from manifest HD, thus yielding a robust prognostic value in pre-HD subjects

A fine physical map of the CACNA1A gene region on 19p13.1-p13.2 chromosome

The P/Q-type Ca(2+) channel alpha(1A) subunit gene (CACNA1A) was cloned on the short arm of chromosome 19 between the markers D19S221 and D19S179 and found to be responsible for Episodic Ataxia type 2, Familial Hemiplegic Migraine and Spinocerebellar Ataxia type 6. This region was physically mapped by 11 cosmid contigs spanning about 1. 4Mb, corresponding to less than 70% of the whole region. The cosmid contig used to characterize the CACNA1A gene accounted only for the coding region of the gene lacking, therefore, the promoter and possible regulation regions. The present study improves the physical map around and within the CACNA1A by giving a complete cosmid or BAC contig coverage of the D19S221-D19S179 interval. A number of new STSs, whether polymorphic or not, were characterized and physically mapped within this region. Four ESTs were also assigned to cosmids belonging to specific contigs

Dissection of the Carboxyl-Terminal Domain of the Proteasomal Subunit Rpn11 in Maintenance of Mitochondrial Structure and Function

We have previously demonstrated that the C-terminal part of Rpn11, a deubiquitinating enzyme in the lid of the proteasome, is essential for maintaining a correct cell cycle and normal mitochondrial morphology and function. The two roles are apparently unlinked as the mitochondrial role is mapped to the Carboxy-terminus, whereas the catalytic deubiquitinating activity is found within the N-terminal region. The mitochondrial defects are observed in rpn11-m1 (originally termed mpr1-1), a mutation that generates Rpn11 lacking the last 31 amino acids. No mitochondrial phenotypes are recorded for mutations in the MPN/JAMM motif. In the present study, we investigated the participation of the last 31 amino acids of the Rpn11 protein by analysis of intragenic revertants and site-specific mutants. We identified a putative -helix necessary for the maintenance of a correct cell cycle and determined that a very short region at the C-terminus of Rpn11 is essential for the maintenance of tubular mitochondrial morphology. Furthermore, we show that expression of the C-terminal part of Rpn11 is able to complement in trans all of the rpn11-m1 mitochondrial phenotypes. Finally, we investigate the mechanisms by which Rpn11 controls the mitochondrial shape and show that Rpn11 may regulate the mitochondrial fission and tubulation processes

A Novel De Novo Mutation of the TITF1/NKX2-1 Gene Causing Ataxia, Benign Hereditary Chorea, Hypothyroidism and a Pituitary Mass in a UK Family and Review of the Literature.

Benign hereditary chorea (BHC) is a rare autosomal dominant condition characterized by early onset, non-progressive chorea, usually caused by mutations in the thyroid transcription factor-1 gene (TITF1). We describe a novel mutation arising de novo in a proband presenting in infancy with delayed walking and ataxia. She later developed chorea, then hypothyroidism and a large cystic pituitary mass. Her daughter presented in infancy with delayed walking and ataxia and went on to develop non-progressive chorea and a hormonally inactive cystic pituitary mass. Mutational analysis of the whole coding region of the TITF1 gene was undertaken and compared with a population study of 160 control subjects. This showed that both affected subjects have a heterozygous A > T substitution at nucleotide 727 of the TITF1 gene changing lysine to a stop codon at residue 211. Genetic analysis of parents and siblings of the proband confirmed that the mutation arose de novo in the proband. The mutated lysine is an evolutionarily highly conserved amino acid in the protein homoeodomain (HD) where most point mutations associated with BHC are located. The range of mutations in BHC is reviewed with particular emphasis on pituitary abnormalities. Cystic pituitary masses and abnormalities of the sella turcica are reported in just 6.4 % of published cases. This is a new nonsense mutation associated with ataxia, benign chorea and pituitary abnormalities which further extends the phenotype of this condition. Mutational screening of TITF1 is important in cases of sporadic or dominant juvenile-onset ataxia, with mild chorea where no other cause is found, particularly if pituitary abnormalities are seen on imaging

Impaired vasoreactivity in mildly disabled CADASIL patients

Background and purpose CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is a rare genetic disease caused by NOTCH3 gene mutations. A dysfunction in vasoreactivity has been proposed as an early event in the pathogenesis of the disease. The aim of this study was to verify whether endothelium dependent and/or independent function is altered in CADASIL patients with respect to controls. Methods Vasoreactivity was studied by a non-invasive pletismographic method in 49 mildly disabled CADASIL patients (30e65 years, 58% male, Rankin scale #2) and 25 controls. Endothelium dependent vasodilatation was assessed by reactive hyperaemia (flow mediated dilationeperipheral arterial tone (FMD-PAT)) and endothelium independent vasoreactivity by glyceryl trinitrate (GTN) administration (GTN-PAT). Results Patients and controls showed comparable age, gender and cardiovascular risk factor distribution. GTN-PAT values were significantly lower in CADASIL patients (1.54 (1.01 to 2.25)) than in controls (1.89 (1.61 to 2.59); p?0.041). FMD-PAT scores did not differ between patients and controls (1.88 (1.57 to 2.43) vs 2.08 (1.81 to 2.58); p?0.126) but 17 CADASIL patients (35%) had FMDPAT scores below the fifth percentile of controls. FMD-PAT and GTN-PAT values correlated both in controls (r?0.648, p<0.001) and CADASIL patients (r?0.563, p<0.001). By multivariable logistic regression for clinical and laboratory variables, only GTN-PAT (OR 0.39, 95% CI 0.15 to 0.97; p?0.044) was independently associated with FMD-PAT below the fifth percentile in CADASIL patients. Conclusions The impaired vasoreactivity observed in CADASIL patients highlights the fact that both endothelial and smooth muscle functional alterations may already be present in mildly disabled subjects. The improvement in vascular function could be a new target for pharmacological trials in CADASIL patients

Correlation of Inter-Locus Polyglutamine Toxicity with CAG•CTG Triplet Repeat Expandability and Flanking Genomic DNA GC Content

Dynamic expansions of toxic polyglutamine (polyQ)-encoding CAG repeats in ubiquitously expressed, but otherwise unrelated, genes cause a number of late-onset progressive neurodegenerative disorders, including Huntington disease and the spinocerebellar ataxias. As polyQ toxicity in these disorders increases with repeat length, the intergenerational expansion of unstable CAG repeats leads to anticipation, an earlier age-at-onset in successive generations. Crucially, disease associated alleles are also somatically unstable and continue to expand throughout the lifetime of the individual. Interestingly, the inherited polyQ length mediating a specific age-at-onset of symptoms varies markedly between disorders. It is widely assumed that these inter-locus differences in polyQ toxicity are mediated by protein context effects. Previously, we demonstrated that the tendency of expanded CAG•CTG repeats to undergo further intergenerational expansion (their ‘expandability’) also differs between disorders and these effects are strongly correlated with the GC content of the genomic flanking DNA. Here we show that the inter-locus toxicity of the expanded polyQ tracts of these disorders also correlates with both the expandability of the underlying CAG repeat and the GC content of the genomic DNA flanking sequences. Inter-locus polyQ toxicity does not correlate with properties of the mRNA or protein sequences, with polyQ location within the gene or protein, or steady state transcript levels in the brain. These data suggest that the observed inter-locus differences in polyQ toxicity are not mediated solely by protein context effects, but that genomic context is also important, an effect that may be mediated by modifying the rate at which somatic expansion of the DNA delivers proteins to their cytotoxic state