Thierry Frébourg

Le Professeur Thierry Frébourg est décédé brutalement le 13 mars 2021 à l’âge de 60 ans. Ses collègues et amis généticiens de la Filière FILNEMUS lui rendent hommage, lui qui a tant fait pour les maladies rares

Implementation of a Reliable Next-Generation Sequencing Strategy for Molecular Diagnosis of Dystrophinopathies

International audienceDiagnosis of dystrophinopathies needs to combine several techniques for detecting copy number variations (CNVs; two-thirds of mutations) and single nucleotide variations (SNVs). We participated in the design of an amplicon-based PCR kit (Multiplicom) for sequencing with a GS-Junior instrument (Roche) and later with a MiSeq instrument (Illumina). We compared two different software programs, MiSeq Reporter (Illumina) and SeqNext (JSI Medical Systems) for data analyses. Testing of six patient DNA samples carrying 72 SNVs in the DMD gene showed an experimental sensitivity of 91.7% with MiSeq Reporter, 98.6% with SeqNext, and >99.9% with both, demonstrating the need to use two different software programs. Analytical specificity was >98%. Fifty-eight additional patient DNAs were analyzed, and 25 deleterious mutations were identified, without false-negative results. We also tested the possibility for our protocol to identify CNVs. We performed additional next-generation sequencing experiments on 50 DNAs and identified 28 CNVs, all confirmed by multiple ligation probe amplification. Statistical analyses on amplicons without CNV (n = 3797), amplicons with heterozygous deletions (n = 51) or duplications (n = 191), and with hemizygous duplications (n = 63) showed a sensitivity and specificity of >99.9%. We implemented a strategy to simultaneously detect SNVs and CNVs in the DMD gene with one comprehensive technique, allowing considerable reduction of time and cost burden for diagnosis of dystrophinopathies

Iron metabolism in mice with partial frataxin deficiency.

Friedreich ataxia (FRDA), the most common autosomal recessive inherited ataxic disorder, is the consequence of deficiency of the mitochondrial protein frataxin, typically caused by homozygous intronic GAA expansions in the corresponding gene. The yeast frataxin homologue (yfh1p) is required for cellular respiration. Yfh1p appears to regulate mitochondrial iron homeostasis and protect from free radical toxicity. Complete loss of frataxin in knockout mice leads to early embryonic lethality, indicating an important role for frataxin during development. Heterozygous littermates with partial frataxin deficiency are apparently healthy and have no obvious phenotype. Here we evaluate iron metabolism and sensitivity to dietary and parenteral iron loading in heterozygote frataxin knockout mice (Fx(+/-)). Iron concentrations in the liver, heart, pancreas and spleen, and cellular iron distribution patterns were compared between wild type and Fx(+/-) mice. Response to parenteral iron challenge was not different between Fx(+/-) mice and wild type littermates, while sporadic iron deposits were observed in the hearts of dietary iron-loaded Fx(+/-) mice. Finally, we evaluated the effect of partial frataxin deficiency on susceptibility to cardiac damage in the mouse model of hereditary hemochromatosis (HH), the Hfe knockout mice. HH, an iron overload disease, is one of the most frequent genetic diseases in populations of European origin. By breeding Hfe(-/-) with Fx(+/-) mice, we obtained compound mutant mice lacking both Hfe and one frataxin allele. Sparse iron deposits in areas of mild to moderate cardiac fibrosis were found in the majority of these mice. However, they did not develop any neurological symptoms. Our studies indicate an association between frataxin deficiency, iron deposits and cardiac fibrosis, but no obvious association between iron accumulation and neurodegeneration similar to FRDA could be detected in our model. In addition, these results suggest that frataxin mutations may have a modifier role in HH, that predisposes to cardiomyopathy.Journal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.info:eu-repo/semantics/publishe

Use of SNP array analysis to identify a novel TRIM32 mutation in limb-girdle muscular dystrophy type 2H.

International audienceMolecular diagnosis of monogenic diseases with high genetic heterogeneity is usually challenging. In the case of limb-girdle muscular dystrophy, multiplex Western blot analysis is a very useful initial step, but that often fails to identify the primarily affected protein. We report how homozygosity analysis using a genome-wide SNP array allowed us to solve the diagnostic enigma in a patient with a moderate form of LGMD, born from consanguineous parents. The genome-wide scan performed on the patient's DNA revealed several regions of homozygosity, that were compared to the location of known LGMD genes. One such region indeed contained the TRIM32 gene. This gene was previously found mutated in families with limb-girdle muscular dystrophy type 2H (LGMD2H), a mild autosomal recessive myopathy described in Hutterite populations and in 4 patients with a diagnosis of sarcotubular myopathy. A single missense mutation was found in all these patients, located in a conserved domain of the C-terminal part of the protein. Another missense mutation affecting the N-terminal part of TRIM32, observed in a single consanguineous Bedouin family, was reported to cause the phenotypically unrelated and genetically heterogeneous Bardet-Biedl syndrome, defining the BBS11 locus. Sequencing of TRIM32 in our patient revealed a distal frameshift mutation, c.1753_1766dup14 (p.Ile590Leu fsX38). Together with two recently reported mutations, this novel mutation confirms that integrity of the C-terminal domain of TRIM32 is necessary for muscle maintenance

Frataxin knockin mouse.

Friedreich ataxia is the consequence of frataxin deficiency, most often caused by a GAA repeat expansion in intron 1 of the corresponding gene. Frataxin is a mitochondrial protein involved in iron homeostasis. As an attempt to generate a mouse model of the disease, we introduced a (GAA)(230) repeat within the mouse frataxin gene by homologous recombination. GAA repeat knockin mice were crossed with frataxin knockout mice to obtain double heterozygous mice expressing 25-36% of wild-type frataxin levels. These mice were viable and did not develop anomalies of motor coordination, iron metabolism or response to iron loading. Repeats were meiotically and mitotically stable.Journal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.info:eu-repo/semantics/publishe