(Dys)function Follows Form: Nucleic Acid Structure, Repeat Expansion, and Disease Pathology in FMR1 Disorders

Fragile X-related disorders (FXDs), also known as FMR1 disorders, are examples of repeat expansion diseases (REDs), clinical conditions that arise from an increase in the number of repeats in a disease-specific microsatellite. In the case of FXDs, the repeat unit is CGG/CCG and the repeat tract is located in the 5′ UTR of the X-linked FMR1 gene. Expansion can result in neurodegeneration, ovarian dysfunction, or intellectual disability depending on the number of repeats in the expanded allele. A growing body of evidence suggests that the mutational mechanisms responsible for many REDs share several common features. It is also increasingly apparent that in some of these diseases the pathologic consequences of expansion may arise in similar ways. It has long been known that many of the disease-associated repeats form unusual DNA and RNA structures. This review will focus on what is known about these structures, the proteins with which they interact, and how they may be related to the causative mutation and disease pathology in the FMR1 disorders.

DNAzyme Cleavage of CAG Repeat RNA in Polyglutamine Diseases

CAG repeat expansion is the genetic cause of nine incurable polyglutamine (polyQ) diseases with neurodegenerative features. Silencing repeat RNA holds great therapeutic value. Here, we developed a repeat-based RNA-cleaving DNAzyme that catalyzes the destruction of expanded CAG repeat RNA of six polyQ diseases with high potency. DNAzyme preferentially cleaved the expanded allele in spinocerebellar ataxia type 1 (SCA1) cells. While cleavage was non-allele-specific for spinocerebellar ataxia type 3 (SCA3) cells, treatment of DNAzyme leads to improved cell viability without affecting mitochondrial metabolism or p62-dependent aggresome formation. DNAzyme appears to be stable in mouse brain for at least 1 month, and an intermediate dosage of DNAzyme in a SCA3 mouse model leads to a significant reduction of high molecular weight ATXN3 proteins. Our data suggest that DNAzyme is an effective RNA silencing molecule for potential treatment of multiple polyQ diseases.

Characterization of C9orf72 haplotypes to evaluate the effects of normal and pathological variations on its expression and splicing

Expansion of the hexanucleotide repeat (HR) in the first intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in Caucasians. All C9orf72-ALS/FTD patients share a common risk (R) haplotype. To study C9orf72 expression and splicing from the mutant R allele compared to the complementary normal allele in ALS/FTD patients, we initially created a detailed molecular map of the single nucleotide polymorphism (SNP) signature and the HR length of the various C9orf72 haplotypes in Caucasians. We leveraged this map to determine the allelic origin of transcripts per patient, and decipher the effects of pathological and normal HR lengths on C9orf72 expression and splicing. In C9orf72 ALS patients’ cells, the HR expanded allele, compared to non-R allele, was associated with decreased levels of a downstream initiated transcript variant and increased levels of transcripts initiated upstream of the HR. HR expanded R alleles correlated with high levels of unspliced intron 1 and activation of cryptic donor splice sites along intron 1. Retention of intron 1 was associated with sequential intron 2 retention. The SNP signature of C9orf72 haplotypes described here enables allele-specific analysis of transcriptional products and may pave the way to allele-specific therapeutic strategies.

Prevalence of RFC1-Mediated Spinocerebellar Ataxia in a United States Ataxia Cohort

ABSTRACTObjectiveRepeat expansions in RFC1 and DAB1 have recently been identified as causing cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) and spinocerebellar ataxia 37 (SCA37), respectively. We evaluated the prevalence of these repeat-expansions in an undiagnosed ataxia cohort from the United States.MethodsA cohort of 596 patients with undiagnosed familial or sporadic cerebellar ataxia were evaluated at a tertiary referral ataxia center and excluded for common genetic causes of cerebellar ataxia. Patients were then screened for the presence of pathogenic repeat expansions in RFC1 (AAGGG) and DAB1 (ATTTC) using fluorescent repeat primed polymerase chain reaction (RP-PCR). Two additional undiagnosed ataxia cohorts from different centers, totaling 96 and 13 patients respectively, were subsequently screened for RFC1 resulting in a combined 705 subjects tested.ResultsIn the initial cohort, 42 samples were identified with one expanded allele in the RFC1 gene (7.0%), and 9 had two expanded alleles (1.5%). For the additional cohorts, we found 12 heterozygous samples (12.5%) and 7 biallelic samples (7.3%) in the larger cohort, and 1 heterozygous sample (7.7%) and 3 biallelic samples (23%) in the second. In total, 19 patients were identified with biallelic repeat expansions in RFC1 (2.7%). Of these 19 patients, 6 (32%) had a clinical diagnosis of CANVAS, 10 had cerebellar ataxia with neuropathy (53%), and 3 had spinocerebellar ataxia (16%). No patients were identified with expansions in the DAB1 gene.ConclusionIn a large undiagnosed ataxia cohort from the United States, biallelic pathogenic repeat expansion in RFC1 was observed in 2.7%. Testing should be strongly considered in ataxia patients, especially those with CANVAS or neuropathy.

Epigenetics of Friedreich’s Disease: Methylation of the (GAA)n-Repeats Region in FXN Gene

Background: Friedreich’s disease (FD) is the most common hereditary ataxia. It is associated, most frequently, with homozygous GAA repeats expansion in intron 1 of the FXN gene. Methylation of the FXN gene can play an important role in the pathogenesis of FD. Aims: to study methylation pattern in CpG sites flanking GAA-expansion in intron 1 of the FXN gene in patients with FD and their heterozygous relatives as well as its relationship with clinical features. Materials and methods: We studied DNA samples from patients with FD (n=18), their relatives carrying heterozygous GAA expansion (n=12), and control group (n=15). Pattern of methylation was studied by direct sequencing of DNA regions after bisulphide processing. Results: We analyzed 18 CpG sites in the UP-GAA region of the gene (before GAA-repeats) and 12 CpG sites in the DOWN-GAA region (after GAA-repeats). In the UP-region, the mean methylation level of CpG sites in FD patients was higher compared to controls (n=15) (р0.05), while in the DOWN-region there was a decrease of mean methylation level in FD compared to controls (р0.05). Analysis of methylation level in different CpG sites in the UP-GAA region revealed hypermethylation for 15 of 18 CpG-sites as compared to controls (р0.05). The most significant differences in methylation level in the UP-GAA region were seen for CpG sites 50−54, 57 and 58. In contrast, in the DOWN-GAA region almost all CpG sites were fully methylated in the control group, while in FD patients methylation was significantly lower (р0.05). We revealed positive correlation of mean methylation level and more expanded allele length for the UP-GAA region in FD (r=0.63; p=0.03), and no correlations for the DOWN-GAA region. In heterozygous carriers we observed an analogous positive correlations in the UP-GAA region for CpG site 50 (r=0.77; p=0.04), while in the DOWN-GAA region there was inverse correlation of methylation with GAA repeat number in the expanded allele (r=-0.83, p=0.02). Negative correlation was found between the hypermethylation of some CpG-sites in the UP-GAA region and age of the disease onset (p0.05). Conclusion: We revealed hypermethylation in the UP-GAA region and hypomethylation in the DOWN-GAA region in patients with FD compared to controls and correlations of methylation level with the GAA expansion length and age of disease onset.

Expanded alleles of the FMR1 gene are related to unexplained recurrent miscarriages

Up to 50% of recurrent miscarriage cases in women occur without an underlying etiology. In the current prospective case–control study, we determined the impact of CGG trinucleotide expansions of the fragile-X mental retardation 1 (FMR1) gene in 49 women with unexplained recurrent miscarriages. Case group consisted of women with two or more unexplained consecutive miscarriages. Blood samples were obtained and checked for the presence of expanded alleles of the FMR1 gene using PCR. Patients harboring the expanded allele, with a threshold set to 40 repeats, were further evaluated by sequencing. The number of abortions each woman had, was not associated with her respective CGG repeat number (P=0.255). The repeat sizes of CGG expansion in the FMR1 gene were significantly different in the two population groups (P=0.027). All the positive cases involved intermediate zone carriers. Hence, the CGG expanded allele of the FMR1 gene might be associated with unexplained multiple miscarriages; whether such an association is coincidental or causal can be confirmed by future studies using a larger patient cohort.

Predictors of Survival in a Huntington's Disease Population from Southern Italy

Background:The primary aim of the present study was to determine the survival rates and identify predictors of disease duration in a cohort of Huntington's disease (HD) patients from Southern Italy.Methods:All medical records of HD patients followed between 1977 and 2008 at the Department of Neurological Sciences of Federico II University in Naples were retrospectively reviewed and 135 patients were enrolled in the analysis. At the time of data collection, 41 patients were deceased (19 males and 22 females) with a mean ± SD age at death of 56.6 ± 14.9 years (range 18-83).Results:The median survival time was 20 years (95% CI: 18.3-21.7). Cox regression analysis showed that the number of CAG in the expanded allele (HR 1.09 for 1 point triplet increase, p=0.002) and age of onset (HR 1.05 for 1 point year increase, p=0.022) were independent and significant predictors of lower survival rates.Conclusions:We believe that these findings are important for a better understanding of the natural history of the disease and may be relevant in designing future therapeutic trials.