Progress in the treatment of Friedreich ataxia

Friedreich ataxia (FRDA) is a progressive neurological disorder affecting approximately 1 in 29,000 individuals of European descent. At present, there is no approved pharmacological treatment for this condition however research into treatment of FRDA has advanced considerably over the last two decades since the genetic cause was identified. Current proposed treatment strategies include decreasing oxidative stress, increasing cellular frataxin, improving mitochondrial function as well as modulating frataxin controlled metabolic pathways. Genetic and cell based therapies also hold great promise. Finally, physical therapies are being explored as a means of maximising function in those affected by FRDA

Pharmacological screening using an FXN-EGFP cellular genomic reporter assay for the therapy of Friedreich ataxia

Copyright @ 2013 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Friedreich ataxia (FRDA) is an autosomal recessive disorder characterized by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein frataxin. There is a correlation between expansion length, the amount of residual frataxin and the severity of disease. As the coding sequence is unaltered, pharmacological up-regulation of FXN expression may restore frataxin to therapeutic levels. To facilitate screening of compounds that modulate FXN expression in a physiologically relevant manner, we established a cellular genomic reporter assay consisting of a stable human cell line containing an FXN-EGFP fusion construct, in which the EGFP gene is fused in-frame with the entire normal human FXN gene present on a BAC clone. The cell line was used to establish a fluorometric cellular assay for use in high throughput screening (HTS) procedures. A small chemical library containing FDA-approved compounds and natural extracts was screened and analyzed. Compound hits identified by HTS were further evaluated by flow cytometry in the cellular genomic reporter assay. The effects on FXN mRNA and frataxin protein levels were measured in lymphoblast and fibroblast cell lines derived from individuals with FRDA and in a humanized GAA repeat expansion mouse model of FRDA. Compounds that were established to increase FXN gene expression and frataxin levels included several anti-cancer agents, the iron-chelator deferiprone and the phytoalexin resveratrol.Muscular Dystrophy Association (USA), the National Health and Medical Research Council (Australia), the Friedreich’s Ataxia Research Alliance (USA), the Brockhoff Foundation (Australia), the Friedreich Ataxia Research Association (Australasia), Seek A Miracle (USA) and the Victorian Government’s Operational Infrastructure Support Program

The structure of the tetrasialoganglioside from human brain

Autosomal dominant retinal vasculopathy with cerebral leukodystrophy is a microvascular endotheliopathy with middle- age onset. In nine families, we identified heterozygous C- terminal frameshift mutations in TREX1, which encodes a 3'-5' exonuclease. These truncated proteins retain exonuclease activity but lose normal perinuclear localization. These data have implications for the maintenance of vascular integrity in the degenerative cerebral microangiopathies leading to stroke and dementias

Bioinformatics-Based Identification of Expanded Repeats: A Non-reference Intronic Pentamer Expansion in RFC1 Causes CANVAS

Genomic technologies such as next-generation sequencing (NGS) are revolutionizing molecular diagnostics and clinical medicine. However, these approaches have proven inefficient at identifying pathogenic repeat expansions. Here, we apply a collection of bioinformatics tools that can be utilized to identify either known or novel expanded repeat sequences in NGS data. We performed genetic studies of a cohort of 35 individuals from 22 families with a clinical diagnosis of cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS). Analysis of whole-genome sequence (WGS) data with five independent algorithms identified a recessively inherited intronic repeat expansion [(AAGGG)exp] in the gene encoding Replication Factor C1 (RFC1). This motif, not reported in the reference sequence, localized to an Alu element and replaced the reference (AAAAG)11 short tandem repeat. Genetic analyses confirmed the pathogenic expansion in 18 of 22 CANVAS-affected families and identified a core ancestral haplotype, estimated to have arisen in Europe more than twenty-five thousand years ago. WGS of the four RFC1-negative CANVAS-affected families identified plausible variants in three, with genomic re-diagnosis of SCA3, spastic ataxia of the Charlevoix-Saguenay type, and SCA45. This study identified the genetic basis of CANVAS and demonstrated that these improved bioinformatics tools increase the diagnostic utility of WGS to determine the genetic basis of a heterogeneous group of clinically overlapping neurogenetic disorders

Iron-overload-related disease in HFE hereditary hemochromatosis

Background: Most persons who are homozygous for C282Y, the HFE allele most commonly asssociated with hereditary hemochromatosis, have elevated levels of serum ferritin and transferrin saturation. Diseases related to iron overload develop in some C282Y homozygotes, but the extent of the risk is controversial. Methods: We assessed HFE mutations in 31,192 persons of northern European descent between the ages of 40 and 69 years who participated in the Melbourne Collaborative Cohort Study and were followed for an average of 12 years. In a random sample of 1438 subjects stratified according to HFE genotype, including all 203 C282Y homozygotes (of whom 108 were women and 95 were men), we obtained clinical and biochemical data, including two sets of iron measurements performed 12 years apart. Disease related to iron overload was defined as documented iron overload and one or more of the following conditions: cirrhosis, liver fibrosis, hepatocellular carcinoma, elevated aminotransferase levels, physician-diagnosed symptomatic hemochromatosis, and arthropathy of the second and third metacarpophalangeal joints. Results: The proportion of C282Y homozygotes with documented iron-overload-related disease was 28.4% (95% confidence interval [CI], 18.8 to 40.2) for men and 1.2% (95% CI, 0.03 to 6.5) for women. Only one non-C282Y homozygote (a compound heterozygote) had documented iron-overload-related disease. Male C282Y homozygotes with a serum ferritin level of 1000 μg per liter or more were more likely to report fatigue, use of arthritis medicine, and a history of liver disease than were men who had the wild-type gene. Conclusions: In persons who are homozygous for the C282Y mutation, iron-overload-related disease developed in a substantial proportion of men but in a small proportion of women

Mutations in DCC cause isolated agenesis of the corpus callosum with incomplete penetrance

Brain malformations involving the corpus callosum are common in children with developmental disabilities. We identified DCC mutations in four families and five sporadic individuals with isolated agenesis of the corpus callosum (ACC) without intellectual disability. DCC mutations result in variable dominant phenotypes with decreased penetrance, including mirror movements and ACC associated with a favorable developmental prognosis. Possible phenotypic modifiers include the type and location of mutation and the sex of the individual

Development and validation of a targeted gene sequencing panel for application to disparate cancers

Next generation sequencing has revolutionised genomic studies of cancer, having facilitated the development of precision oncology treatments based on a tumour’s molecular profile. We aimed to develop a targeted gene sequencing panel for application to disparate cancer types with particular focus on tumours of the head and neck, plus test for utility in liquid biopsy. The final panel designed through Roche/Nimblegen combined 451 cancer-associated genes (2.01 Mb target region). 136 patient DNA samples were collected for performance and application testing. Panel sensitivity and precision were measured using well-characterised DNA controls (n = 47), and specificity by Sanger sequencing of the Aryl Hydrocarbon Receptor Interacting Protein (AIP) gene in 89 patients. Assessment of liquid biopsy application employed a pool of synthetic circulating tumour DNA (ctDNA). Library preparation and sequencing were conducted on Illumina-based platforms prior to analysis with our accredited (ISO15189) bioinformatics pipeline. We achieved a mean coverage of 395x, with sensitivity and specificity of >99% and precision of >97%. Liquid biopsy revealed detection to 1.25% variant allele frequency. Application to head and neck tumours/cancers resulted in detection of mutations aligned to published databases. In conclusion, we have developed an analytically-validated panel for application to cancers of disparate types with utility in liquid biopsy

An original phylogenetic approach identified mitochondrial haplogroup T1a1 as inversely associated with breast cancer risk in BRCA2 mutation carriers

Introduction: Individuals carrying pathogenic mutations in the BRCA1 and BRCA2 genes have a high lifetime risk of breast cancer. BRCA1 and BRCA2 are involved in DNA double-strand break repair, DNA alterations that can be caused by exposure to reactive oxygen species, a main source of which are mitochondria. Mitochondrial genome variations affect electron transport chain efficiency and reactive oxygen species production. Individuals with different mitochondrial haplogroups differ in their metabolism and sensitivity to oxidative stress. Variability in mitochondrial genetic background can alter reactive oxygen species production, leading to cancer risk. In the present study, we tested the hypothesis that mitochondrial haplogroups modify breast cancer risk in BRCA1/2 mutation carriers. Methods: We genotyped 22,214 (11,421 affected, 10,793 unaffected) mutation carriers belonging to the Consortium of Investigators of Modifiers of BRCA1/2 for 129 mitochondrial polymorphisms using the iCOGS array. Haplogroup inference and association detection were performed using a phylogenetic approach. ALTree was applied to explore the reference mitochondrial evolutionary tree and detect subclades enriched in affected or unaffected individuals. Results: We discovered that subclade T1a1 was depleted in affected BRCA2 mutation carriers compared with the rest of clade T (hazard ratio (HR) = 0.55; 95% confidence interval (CI), 0.34 to 0.88; P = 0.01). Compared with the most frequent haplogroup in the general population (that is, H and T clades), the T1a1 haplogroup has a HR of 0.62 (95% CI, 0.40 to 0.95; P = 0.03). We also identified three potential susceptibility loci, including G13708A/rs28359178, which has demonstrated an inverse association with familial breast cancer risk. Conclusions: This study illustrates how original approaches such as the phylogeny-based method we used can empower classical molecular epidemiological studies aimed at identifying association or risk modification effects.Peer reviewe

The FANCM:p.Arg658* truncating variant is associated with risk of triple-negative breast cancer

Abstract: Breast cancer is a common disease partially caused by genetic risk factors. Germline pathogenic variants in DNA repair genes BRCA1, BRCA2, PALB2, ATM, and CHEK2 are associated with breast cancer risk. FANCM, which encodes for a DNA translocase, has been proposed as a breast cancer predisposition gene, with greater effects for the ER-negative and triple-negative breast cancer (TNBC) subtypes. We tested the three recurrent protein-truncating variants FANCM:p.Arg658*, p.Gln1701*, and p.Arg1931* for association with breast cancer risk in 67,112 cases, 53,766 controls, and 26,662 carriers of pathogenic variants of BRCA1 or BRCA2. These three variants were also studied functionally by measuring survival and chromosome fragility in FANCM−/− patient-derived immortalized fibroblasts treated with diepoxybutane or olaparib. We observed that FANCM:p.Arg658* was associated with increased risk of ER-negative disease and TNBC (OR = 2.44, P = 0.034 and OR = 3.79; P = 0.009, respectively). In a country-restricted analysis, we confirmed the associations detected for FANCM:p.Arg658* and found that also FANCM:p.Arg1931* was associated with ER-negative breast cancer risk (OR = 1.96; P = 0.006). The functional results indicated that all three variants were deleterious affecting cell survival and chromosome stability with FANCM:p.Arg658* causing more severe phenotypes. In conclusion, we confirmed that the two rare FANCM deleterious variants p.Arg658* and p.Arg1931* are risk factors for ER-negative and TNBC subtypes. Overall our data suggest that the effect of truncating variants on breast cancer risk may depend on their position in the gene. Cell sensitivity to olaparib exposure, identifies a possible therapeutic option to treat FANCM-associated tumors

Heterozygous ANKRD17 loss-of-function variants cause a syndrome with intellectual disability, speech delay, and dysmorphism

ANKRD17 is an ankyrin repeat-containing protein thought to play a role in cell cycle progression, whose ortholog in Drosophila functions in the Hippo pathway as a co-factor of Yorkie. Here, we delineate a neurodevelopmental disorder caused by de novo heterozygous ANKRD17 variants. The mutational spectrum of this cohort of 34 individuals from 32 families is highly suggestive of haploinsufficiency as the underlying mechanism of disease, with 21 truncating or essential splice site variants, 9 missense variants, 1 in-frame insertion-deletion, and 1 microdeletion (1.16 Mb). Consequently, our data indicate that loss of ANKRD17 is likely the main cause of phenotypes previously associated with large multi-gene chromosomal aberrations of the 4q13.3 region. Protein modeling suggests that most of the missense variants disrupt the stability of the ankyrin repeats through alteration of core structural residues. The major phenotypic characteristic of our cohort is a variable degree of developmental delay/intellectual disability, particularly affecting speech, while additional features include growth failure, feeding difficulties, non-specific MRI abnormalities, epilepsy and/or abnormal EEG, predisposition to recurrent infections (mostly bacterial), ophthalmological abnormalities, gait/balance disturbance, and joint hypermobility. Moreover, many individuals shared similar dysmorphic facial features. Analysis of single-cell RNA-seq data from the developing human telencephalon indicated ANKRD17 expression at multiple stages of neurogenesis, adding further evidence to the assertion that damaging ANKRD17 variants cause a neurodevelopmental disorder.Neurolog