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Abstract Background Myotonic dystrophy type 1 (DM1) is caused by CTG repeat expansions in the DMPK gene and is the most common form of muscular dystrophy. Patients can have long delays from onset to diagnosis, since clinical signs and symptoms are often nonspecific and overlapping with other disorders. Clinical genetic testing by Southern blot or triplet‐primed PCR (TP‐PCR) is technically challenging and cost prohibitive for population surveys. Methods Here, we present a high throughput, low‐cost screening tool for CTG repeat expansions using TP‐PCR followed by high resolution melt curve analysis with saturating concentrations of SYBR GreenER dye. Results We determined that multimodal melt profiles from the TP‐PCR assay are a proxy for amplicon length stoichiometry. In a screen of 10,097 newborn blood spots, melt profile analysis accurately reflected the tri‐modal distribution of common alleles from 5 to 35 CTG repeats, and identified the premutation and full expansion alleles. Conclusion We demonstrate that robust detection of expanded CTG repeats in a single tube can be achieved from samples derived from specimens with minimal template DNA such as dried blood spots (DBS). This technique is readily adaptable to large‐scale testing programs such as population studies and newborn screening programs.

Background: Myotonic dystrophy type 1 (DM1) is caused by CTG repeat expansions in the DMPK gene and is the most common form of muscular dystrophy. Patients can have long delays from onset to diagnosis, since clinical signs and symptoms are often nonspecific and overlapping with other disorders. Clinical genetic testing by Southern blot or triplet‐primed PCR (TP‐PCR) is technically challenging and cost prohibitive for population surveys. Methods: Here, we present a high throughput, low‐cost screening tool for CTG repeat expansions using TP‐PCR followed by high resolution melt curve analysis with saturating concentrations of SYBR GreenER dye. Results: We determined that multimodal melt profiles from the TP‐PCR assay are a proxy for amplicon length stoichiometry. In a screen of 10,097 newborn blood spots, melt profile analysis accurately reflected the tri‐modal distribution of common alleles from 5 to 35 CTG repeats, and identified the premutation and full expansion alleles. Conclusion: We demonstrate that robust detection of expanded CTG repeats in a single tube can be achieved from samples derived from specimens with minimal template DNA such as dried blood spots (DBS). This technique is readily adaptable to large‐scale testing programs such as population studies and newborn screening programs. [ABSTRACT FROM AUTHOR]

Friedreich ataxia (FRDA) is an autosomal recessive degenerative disease primarily affecting the nervous system and the heart. The identification of the FRDA gene (FXN) encoding the mitochondrial protein frataxin allowed to better define the clinical and pathological spectrum of the disease, to investigate pathogenesis and to eventually propose novel treatments for this so far incurable disease. Frataxin has homologs in all eukaryotes and in gram-negative bacteria. Studies in yeast and mouse models and biochemical investigations indicate a role in the assembly of iron-sulfur clusters in the mitochondrion. Frataxin deficiency leads to abnormal mitochondrial iron metabolism, decreased activities of iron-sulfur cluster-containing enzymes, reduced oxidative phosphorylation and possibly increased oxidative stress. Complete absence of frataxin is lethal at an embryonic level in all investigated multicellular organisms, in yeast it leads to loss of mitochondrial function and mitochondrial DNA. Patients with FRDA have a unique mutation that results in low levels of structurally and functionally normal frataxin. The mutation is an unstable hyperexpansion of a GAA triplet-repeat sequence in the first intron of the gene. Pathological lengths of the GAA repeat adopt in vitro a non-B, triple helical DNA structure that inhibits transcription. In the nucleus, chromatin condenses at and around the GAA repeat into a heterochromatic state that is associated with repressed transcription. It is not yet known whether the triple helical structure forms in the nucleus and has a role in triggering chromatin condensation. Rare mutations, always occurring in heterozygosity with the GAA repeat expansion in FRDA patients, alter the coding frataxin sequence and result in non-functional or absent protein. Restoring frataxin transcription is an exciting perspective for treatment. Preliminary results in cell models suggest that some inhibitors of histone deacetylases may have this effect. [ABSTRACT FROM AUTHOR]

The goal of all clinical research is to abolish suffering caused by human disease. This can be achieved by the development of suitable intervention, be it treatment, prevention or cure. If the cellular or molecular pathology underlying a specific disease process is understood, therapeutic intervention may be more rapidly realized. For disease where a fraction of the risk is heritable, genetic analysis can be a key strategy: the identification of a genetic variant and subsequent aberrant protein that causes disease lends insight to pathology and subsequent treatment alternatives. One example of this is Fragile X syndrome, where the discovery of the causative gene enabled dissection of the molecular pathway that is disrupted in affected individuals. In this review, we will describe this path to understanding, from discovery of the gene to the current model of disease. [ABSTRACT FROM AUTHOR]