Homozygous COQ7 mutation: a new cause of potentially treatable distal hereditary motor neuropathy.

Distal hereditary motor neuropathy represents a group of motor inherited neuropathies leading to distal weakness. We report a family of two brothers and a sister affected by distal hereditary motor neuropathy in whom a homozygous variant c.3G>T (p.1Met?) was identified in the COQ7 gene. This gene encodes a protein required for coenzyme Q10 biosynthesis, a component of the respiratory chain in mitochondria. Mutations of COQ7 were previously associated with severe multi-organ disorders characterized by early childhood onset and developmental delay. Using patient blood samples and fibroblasts derived from a skin biopsy, we investigated the pathogenicity of the variant of unknown significance c.3G>T (p.1Met?) in the COQ7 gene and the effect of coenzyme Q10 supplementation in vitro. We showed that this variation leads to a severe decrease in COQ7 protein levels in the patient's fibroblasts, resulting in a decrease in coenzyme Q10 production and in the accumulation of 6-demethoxycoenzyme Q10, the COQ7 substrate. Interestingly, such accumulation was also found in the patient's plasma. Normal coenzyme Q10 and 6-demethoxycoenzyme Q10 levels were restored in vitro by using the coenzyme Q10 precursor 2,4-dihydroxybenzoic acid, thus bypassing the COQ7 requirement. Coenzyme Q10 biosynthesis deficiency is known to impair the mitochondrial respiratory chain. Seahorse experiments showed that the patient's cells mainly rely on glycolysis to maintain sufficient ATP production. Consistently, the replacement of glucose by galactose in the culture medium of these cells reduced their proliferation rate. Interestingly, normal proliferation was restored by coenzyme Q10 supplementation of the culture medium, suggesting a therapeutic avenue for these patients. Altogether, we have identified the first example of recessive distal hereditary motor neuropathy caused by a homozygous variation in the COQ7 gene, which should thus be included in the gene panels used to diagnose peripheral inherited neuropathies. Furthermore, 6-demethoxycoenzyme Q10 accumulation in the blood can be used to confirm the pathogenic nature of the mutation. Finally, supplementation with coenzyme Q10 or derivatives should be considered to prevent the progression of COQ7-related peripheral inherited neuropathy in diagnosed patients.

Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions.

Microsatellites are short tandem repeats, ubiquitous in all eukaryotes and represent ~2% of the human genome. Among them, trinucleotide repeats are responsible for more than two dozen neurological and developmental disorders. Targeting microsatellites with dedicated DNA endonucleases could become a viable option for patients affected with dramatic neurodegenerative disorders. Here, we used the Streptococcus pyogenes Cas9 to induce a double-strand break within the expanded CTG repeat involved in myotonic dystrophy type 1, integrated in a yeast chromosome. Repair of this double-strand break generated unexpected large chromosomal deletions around the repeat tract. These deletions depended on RAD50, RAD52, DNL4 and SAE2, and both non-homologous end-joining and single-strand annealing pathways were involved. Resection and repair of the double-strand break (DSB) were totally abolished in a rad50Δ strain, whereas they were impaired in a sae2Δ mutant, only on the DSB end containing most of the repeat tract. This observation demonstrates that Sae2 plays significant different roles in resecting a DSB end containing a repeated and structured sequence as compared to a non-repeated DSB end. In addition, we also discovered that gene conversion was less efficient when the DSB could be repaired using a homologous template, suggesting that the trinucleotide repeat may interfere with gene conversion too. Altogether, these data show that SpCas9 may not be the best choice when inducing a double-strand break at or near a microsatellite, especially in mammalian genomes that contain many more dispersed repeated elements than the yeast genome.

Trinucleotide repeat instability during double-strand break repair: from mechanisms to gene therapy.

Trinucleotide repeats are a particular class of microsatellites whose large expansions are responsible for at least two dozen human neurological and developmental disorders. Slippage of the two complementary DNA strands during replication, homologous recombination or DNA repair is generally accepted as a mechanism leading to repeat length changes, creating expansions and contractions of the repeat tract. The present review focuses on recent developments on double-strand break repair involving trinucleotide repeat tracts. Experimental evidences in model organisms show that gene conversion and break-induced replication may lead to large repeat tract expansions, while frequent contractions occur either by single-strand annealing between repeat ends or by gene conversion, triggering near-complete contraction of the repeat tract. In the second part of this review, different therapeutic approaches using highly specific single- or double-strand endonucleases targeted to trinucleotide repeat loci are compared. Relative efficacies and specificities of these nucleases will be discussed, as well as their potential strengths and weaknesses for possible future gene therapy of these dramatic disorders.

Kallikrein-related peptidase 7 (KLK7) is a proliferative factor that is aberrantly expressed in human colon cancer.

Emerging evidence indicates that serine proteases of the tissue kallikrein-related peptidases family (KLK) are implicated in tumorigenesis. We recently reported the ectopic expression of KLK4 and KLK14 in colonic cancers and their signaling to control cell proliferation. Human tissue kallikrein-related peptidase 7 (KLK7) is often dysregulated in many cancers; however, its role in colon tumorigenesis has not yet been established. In the present study, we analyzed expression of KLK7 in 15 colon cancer cell lines and in 38 human colonic tumors. In many human colon cancer cells, KLK7 mRNA was observed, which leads to KLK7 protein expression and secretion. Furthermore, KLK7 was detected in human colon adenocarcinomas, but it was absent in normal epithelia. KLK7 overexpression in HT29 colon cancer cells upon stable transfection with a KLK7 expression plasmid resulted in increased cell proliferation. Moreover, subcutaneous inoculation of transfected cells into nude mice led to increased tumor growth that was associated with increased tumor cell proliferation as reflected by a positive Ki-67 staining. Our results demonstrate the aberrant expression of KLK7 in colon cancer cells and tissues and its involvement in cell proliferation in vitro and in vivo. Thus, KLK7 may represent a potential therapeutic target for human colon tumorigenesis.

A multiple animal and cellular models approach to study frataxin deficiency in Friedreich Ataxia.

Friedreich's ataxia (FA) is one of the most frequent inherited recessive ataxias characterized by a progressive sensory and spinocerebellar ataxia. The main causative mutation is a GAA repeat expansion in the first intron of the frataxin (FXN) gene which leads to a transcriptional silencing of the gene resulting in a deficit in FXN protein. The nature of the mutation (an unstable GAA expansion), as well as the multi-systemic nature of the disease (with neural and non-neural sites affected) make the generation of models for Friedreich's ataxia quite challenging. Over the years, several cellular and animal models for FA have been developed. These models are all complementary and possess their own strengths to investigate different aspects of the disease, such as the epigenetics of the locus or the pathophysiology of the disease, as well as being used to developed novel therapeutic approaches. This review will explore the recent advancements in the different mammalian models developed for FA.

TALEN-Induced Double-Strand Break Repair of CTG Trinucleotide Repeats.

Trinucleotide repeat expansions involving CTG/CAG triplets are responsible for several neurodegenerative disorders, including myotonic dystrophy and Huntington's disease. Because expansions trigger the disease, contracting repeat length could be a possible approach to gene therapy for these disorders. Here, we show that a TALEN-induced double-strand break was very efficient at contracting expanded CTG repeats in yeast. We show that RAD51, POL32, and DNL4 are dispensable for double-strand break repair within CTG repeats, the only required genes being RAD50, SAE2, and RAD52. Resection was totally abolished in the absence of RAD50 on both sides of the break, whereas it was reduced in a sae2Δ mutant on the side of the break containing the longest repeat tract, suggesting that secondary structures at double-strand break ends must be removed by the Mre11-Rad50 complex and Sae2. Following the TALEN double-strand break, single-strand annealing occurred between both sides of the repeat tract, leading to repeat contraction.

A fast, sensitive and cost-effective method for nucleic acid detection using non-radioactive probes.

Nucleic acid detection and quantification using a labeled DNA probe is a very common molecular biology procedure. Here, we describe a new method, based on commonly used laboratory solutions, for nucleic acid hybridization and detection with digoxigenin-labeled DNA probes. The protocol described is faster, more sensitive and much cheaper than a standard protocol using commercial solutions. Comparison with a classical radioactive detection method shows that the latter exhibits less background and shows a greater linear response. Hence, the proposed protocol may be routinely performed for qualitative detection of nucleic acid, but when precise signal quantitation needs to be obtained, radioactive probe hybridization associated to phosphorimaging technology is more reliable.

Replication stalling and heteroduplex formation within CAG/CTG trinucleotide repeats by mismatch repair.

Trinucleotide repeat expansions are responsible for at least two dozen neurological disorders. Mechanisms leading to these large expansions of repeated DNA are still poorly understood. It was proposed that transient stalling of the replication fork by the repeat tract might trigger slippage of the newly-synthesized strand over its template, leading to expansions or contractions of the triplet repeat. However, such mechanism was never formally proven. Here we show that replication fork pausing and CAG/CTG trinucleotide repeat instability are not linked, stable and unstable repeats exhibiting the same propensity to stall replication forks when integrated in a yeast natural chromosome. We found that replication fork stalling was dependent on the integrity of the mismatch-repair system, especially the Msh2p-Msh6p complex, suggesting that direct interaction of MMR proteins with secondary structures formed by trinucleotide repeats in vivo, triggers replication fork pauses. We also show by chromatin immunoprecipitation that Msh2p is enriched at trinucleotide repeat tracts, in both stable and unstable orientations, this enrichment being dependent on MSH3 and MSH6. Finally, we show that overexpressing MSH2 favors the formation of heteroduplex regions, leading to an increase in contractions and expansions of CAG/CTG repeat tracts during replication, these heteroduplexes being dependent on both MSH3 and MSH6. These heteroduplex regions were not detected when a mutant msh2-E768A gene in which the ATPase domain was mutated was overexpressed. Our results unravel two new roles for mismatch-repair proteins: stabilization of heteroduplex regions and transient blocking of replication forks passing through such repeats. Both roles may involve direct interactions between MMR proteins and secondary structures formed by trinucleotide repeat tracts, although indirect interactions may not be formally excluded.

Highly specific contractions of a single CAG/CTG trinucleotide repeat by TALEN in yeast.

Trinucleotide repeat expansions are responsible for more than two dozens severe neurological disorders in humans. A double-strand break between two short CAG/CTG trinucleotide repeats was formerly shown to induce a high frequency of repeat contractions in yeast. Here, using a dedicated TALEN, we show that induction of a double-strand break into a CAG/CTG trinucleotide repeat in heterozygous yeast diploid cells results in gene conversion of the repeat tract with near 100% efficacy, deleting the repeat tract. Induction of the same TALEN in homozygous yeast diploids leads to contractions of both repeats to a final length of 3-13 triplets, with 100% efficacy in cells that survived the double-strand breaks. Whole-genome sequencing of surviving yeast cells shows that the TALEN does not increase mutation rate. No other CAG/CTG repeat of the yeast genome showed any length alteration or mutation. No large genomic rearrangement such as aneuploidy, segmental duplication or translocation was detected. It is the first demonstration that induction of a TALEN in an eukaryotic cell leads to shortening of trinucleotide repeat tracts to lengths below pathological thresholds in humans, with 100% efficacy and very high specificity.