Добірка наукової літератури з теми "Polyalanine tract mutations"

ABSTRACT The Schizosaccharomyces pombe DNA repair generhp51 + encodes a RecA-like protein with the DNA-dependent ATPase activity required for homologous recombination. The level of the rhp51 + transcript is increased by a variety of DNA-damaging agents. Its promoter has twocis-acting DNA damage-responsive elements (DREs) responsible for DNA damage inducibility. Here we report identification of Rdp1, which regulates rhp51 + expression through the DRE of rhp51 +. The protein contains a zinc finger and a polyalanine tract similar to ones previously implicated in DNA binding and transactivation or repression, respectively. In vitro footprinting and competitive binding assays indicate that the core consensus sequences (NGG/TTG/A) of DRE are crucial for the binding of Rdp1. Mutations of both DRE1 and DRE2 affected the damage-induced expression ofrhp51 +, indicating that both DREs are required for transcriptional activation. In addition, mutations in the DREs significantly reduced survival rates after exposure to DNA-damaging agents, demonstrating that the damage response ofrhp51 + enhances the cellular repair capacity. Surprisingly, haploid cells containing a complete rdp1deletion could not be recovered, indicating thatrdp1 + is essential for cell viability and implying the existence of other target genes. Furthermore, the DNA damage-dependent expression of rhp51 + was significantly reduced in checkpoint mutants, raising the possibility that Rdp1 may mediate damage checkpoint-dependent transcription ofrhp51 +.

Eighteen severe human diseases have so far been associated with trinucleotide repeat expansions coding for either polyalanine (encoded by a GCN repeat tract) or polyglutamine (encoded by a CAG repeat tract). Among them, oculopharyngeal muscular dystrophy (OPMD), spinocerebellar ataxia type-3 (SCA3), and Huntington’s disease (HD) are late-onset autosomal-dominant disorders characterized by the presence of intranuclear inclusions (INIs). We have previously identified the OPMD causative mutation as a small expansion (from 2 in normal to 7 in disease) of a GCG repeat tract in the PABPN1 gene. In addition, -1 ribosomal frameshifting has been reported to occur in expanded CAG repeat tracts in the ATXN3 (SCA3) and HTT (HD) genes, resulting in the translation of a hybrid CAG/GCA repeat tract and the production of a polyalanine-containing peptide. Previous studies on OPMD suggest that polyalanine-induced toxicity is very sensitive to the dosage and length of the alanine stretch. Here we report the characterization of a polyclonal antibody that selectively recognizes pathological expansions of polyalanine in PABPN1. Furthermore, our antibody also detects the presence of alanine proteins in INIs of SCA3 and HD patient samples.

Background Congenital central hypoventilation syndrome (CCHS), which is caused by PHOX2B with phenotypic variations, has a point of controversy: CCHS is putatively involved in autopsy cases of sudden unexpected infant death (SUID) including sudden infant death syndrome. Objective The relation of CCHS to SUID cases was investigated by extensive genotyping of PHOX2B. Methods We analyzed 93 DNA samples of less than one-year-old SUID cases that were autopsied in our department. Unrelated adult volunteers (n = 942) were used as the control. Results No polyalanine tract expansion was detected in the SUID cases. The allelic frequencies of repeat contractions and SNP (rs28647582) in intron 2 were not significantly different from that in those control group. Further extensive sequencing revealed a non-polyalanine repeat mutation (NPARM) of c.905A>C in a sudden death case of a one-month-old male infant. This missense mutation (p.Asn302Thr), registered as rs779068107, was annotated to ‘Affected status is unknown’, but it might be associated with the sudden death. Conclusion NPARM was more plausibly related to sudden unexpected death than expansions because of severe clinical complications. This finding indicates possible CCHS involvement in forensic autopsy cases without ante-mortem diagnosis.

Eight diseases, exemplified by Huntington's disease and spinocerebellar ataxia type 1, are caused by CAG-repeat expansion mutations. The CAG repeats are translated into expanded polyglutamine tracts, which are associated with deleterious novel functions. While these diseases are characterized by intraneuronal aggregate formation, it is unclear whether the aggregates cause disease. We have addressed this debate by generating intracellular aggregates with green fluorescent protein (GFP) fused to 19-37 alanines. No aggregates were seen in cells expressing native GFP or GFP fused to seven alanines. Aggregate-containing cells expressing GFP fused to 19-37 polyalanines show high rates of nuclear fragmentation compared with cells expressing the same constructs without aggregates, or cells expressing GFP fused to seven alanines. This suggests an association between aggregate formation and cell death.

The aristaless homeobox gene, ARX, belongs to a large family of homeodomain transcription factors with essential roles in forebrain, pancreas, muscle tissues and testes development in human and mouse. Mutation of ARX in humans results in mental retardation with or without ambiguous genitalia. We used comparative analyses to examine the evolutionary conservation of the mammalian ARX gene. We characterised ARX in a marsupial, the tammar wallaby, to determine if this gene is highly conserved in the homeodomain, aristaless domain, octapeptide motif and polyalanine tracts of all mammals. We further investigated the mRNA distribution in the developing head of tammar with in situ hybridisation, and found that it is expressed in forebrain and olfactory bulb as expected. Besides these regions, very strong expression was detected in the epithelium of the tongue and nasal pits. In the gonads, there is very strong staining in the interstitial cells and some of the germ cells in the developing ovary; strong staining was also seen in the cytoplasm of Sertoli cells and some of the germ cells, weak staining was also detected in the interstitium of the testis, possibly within the vessel endothelial cells and interstitial fibroblast-like cells. In addition, we investigated mRNA distribution in adult testes based on a very strong signal observed with northern blotting. Interestingly, mRNA expression was restricted to the round spermatids, and was not seen before or after this stage. In order to confirm this new role for ARX in the adult testis, we further investigated mRNA distribution of Arx in adult mouse testis, and found the same expression pattern, which implies a conserved function for ARX in spermatogenesis and may explain why humans with ARX mutations are infertile. This is the first report that ARX gene is involved in spermatogenesis in addition to its conserved roles in early mammalian development.

Intellectual disability (ID) is a highly prevalent disorder that affects 1-3% of the population, with ~ 100 causative genes mapped to the X-chromosome. The Aristaless-related homeobox gene (ARX) is an important transcription factor with critical roles in development, particularly in the developing brain. Variants are not well tolerated within ARX, with missense mutations resulting in phenotypes that always involve ID with frequent comorbidities of epilepsy, infantile spasms, hand dystonia, autism or dysarthria. Historically, it was thought that only males were affected by mutations in ARX due to their single X-chromosome. In this thesis, we describe a family with multiple affected individuals, including two females with a novel insertion mutation within ARX. We furthermore review the reported phenotype of females with mutations in ARX and highlight the importance of screening ARX in both male and female patients with ID and seizures. The majority of patients with ARX mutations are affected by expansion mutations in polyalanine tract 1 and 2 within the protein, giving rise to ID with or without epilepsy and movement disorders of varying severity. Mice modelling the two most frequent polyalanine expansion mutations (Arx(GCG)7 referred to as PA1 and Arx432-455dup24 referred to as PA2) recapitulate many of the clinical features seen in humans (Kitamura et al. 2009, Jackson et al. Submitted 2017). To dissect the molecular basis of different polyalanine expansions in vivo, we used 12.5 dpc brain samples from PA1 and PA2 mice to analyse disruptions in gene expression in the developing forebrain to capture the primary disruption leading to the developmental phenotypes caused by these mutations. A greater number of genes deregulated in the more severe PA1 mice was shown, with the majority of genes also perturbed in the milder PA2 mice, but failed to reach significance compared to WT at this early stage of development. We saw a significant overlap with a number of known direct targets of ARX (5%) and genes implicated in ID, epilepsy and autism (12%). From my analysis, I predict a core pathway of transcription regulators as potential drivers of the ID and infantile spasms in patients with ARX polyalanine expansion mutations. Next, I investigated the mechanisms driving the partial loss of function. My data indicates this reduced function does not occur through disruptions of binding to DNA or protein interactors in relation to the region of ARX spanning both polyalanine tract 1 and 2. However, in this thesis, I demonstrate a marked reduction in polyalanine mutant protein may be the contributing factor to disease manifestation. Transcription activity assays indicated ARX responds in a dose-depend manner, and greater reduction in protein leads to an increased severity of the disease. Investigations into the molecular mechanism contributing to this reduction in protein level show no significant change in protein stability (in vitro). Instead, initial studies indicate inefficiency of translation resulting in reduced protein abundance. From my data and other previous studies, I discuss the likelihood of a multiple hit model contributing to the partial loss of function and leading to the variability of clinical presentations.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2017