2018-2020 : MitoMatcher, a genetic-phenotypic database for the diagnosis of mitochondrial diseases

The advent of high throughput sequencing (NGS) techniques has revolutionized the genetic diagnosis of mitochondrial diseases with an increase in the amount of data generated but also in their complexity, particularly with the identification of many variants of unknown significance (VSI) in nuclear and mitochondrial genomes (mtDNA). The objective of the MitoDiag network, 11 French hospital laboratories specializing in the diagnosis of mitochondrial diseases and supported by the Filnemus sector, was therefore to create a French clinical-biological database, MitoMatcher, which collects for each patient the set of variants mtDNA, clinical and demographic data to facilitate exchanges between the various network laboratories and NGS data analysis.
The MitoMatcher database was designed around 3 modules: a clinical database, a genetic database of mtDNA variants and a query interface. The clinical database is provided by PhenoTIPS, specialized in the collection and analysis of phenotypic information from patients with genetic diseases and using the terminology HPO. The genetic database contains, for each patient, all the information contained in the VCF file as well as metadata (nature of the sample, type of analysis). For the query and information sharing interface between the labs, CafeVariome was set up in collaboration with Anthony Brookes coordinating the European project GEN2PHEN.
As a first step, the MitoMatcher project focused on the analysis of the NGS data of the mtDNA. After developing the structure of the database of genetic variants, it was tested by integrating the data of nearly 800 patients from a previous study conducted by the laboratories of the network. Interoperability between clinical information from PHENOTIPS and the genetic database was made possible by the integration of CafeVariome into MitoMatcher. The interface has been developed to allow information sharing between laboratories and the extraction of new knowledge through the "elastic search" technology.
Mitomatcher is a French clinical-biological database that simplifies the sharing of genetic and phenotypic information between laboratories and facilitates the interpretation of mtDNA VSIs. The deployment of the tool within the network is planned for the year 2020. In a second time, its use is envisaged for more complex queries (co-occurrence of variants, influence of mitochondrial haplogroups ...). Finally, in the medium term, the tool can be extended to variants of nuclear genes involved in mitochondrial diseases, and the Mitomatcher architecture used for other pathologies.
MitoMatcher has got a poster award at the congress "10ème Assises de Génétique humaine et médicale" by the Association BioinfoDiag.

2017 : Workshop Meetochondrie

The Association Network "MeetOchondrie" organized from 8 to 10 March 2017, in Angers, a workshop entitled "La Génétique mitochondriale dans tous ses états".

This workshop was aimed at people wishing to learn and acquire basic skills in molecular biology and molecular anthropology for the observation and analysis of population genetics including pathology. Participants were given the opportunity to perform basic molecular biology techniques (mtDNA extraction, PCR, library preparations and NGS sequencing), which allowed them to determine their own mitochondrial haplogroup and initiate bioinformatic analysis of mitochondrial DNA sequences.


2014 : Workshop Meetochondrie

The Association Network "MeetOchondrie" organized from 25 to 27 June 2014, in Angers, a workshop entitled "La Génétique mitochondriale dans tous ses états".

This workshop was aimed at people wishing to learn and acquire basic skills in molecular biology and molecular anthropology for the observation and analysis of population genetics including pathology. Participants were given the opportunity to perform basic molecular biology techniques (mtDNA extraction, PCR, library preparations and NGS sequencing), which allowed them to determine their own mitochondrial haplogroup and initiate bioinformatic analysis of mitochondrial DNA sequences.


2008-2012 : STIC implementation and medico-economic evaluation of the exhaustive mtDNA analysis by Surveyor and Mitochips techniques

This work resulted in a publication.

Mitochondrial DNA (mtDNA) diseases are rare disorders whose prevalence is estimated around 1 in 5000. Patients are usually tested only for deletions and for common mutations of mtDNA which account for 5-40% of cases, depending on the study. However, the prevalence of rare mtDNA mutations is not known.
We analysed the whole mtDNA in a cohort of 743 patients suspected of manifesting a mitochondrial disease, after excluding deletions and common mutations. Both heteroplasmic and homoplasmic variants were identified using two complementary strategies (Surveyor and MitoChip). Multiple correspondence analyses followed by hierarchical ascendant cluster process were used to explore relationships between clinical spectrum, age at onset and localisation of mutations.
7.4% of deleterious mutations and 22.4% of novel putative mutations were identified. Pathogenic heteroplasmic mutations were more frequent than homoplasmic mutations (4.6% vs 2.8%). Patients carrying deleterious mutations showed symptoms before 16 years of age in 67% of cases. Early onset disease (< 1 year) was significantly associated with mutations in protein coding genes (mainly in complex I) while late onset disorders (>16 years) were associated with mutations in tRNA genes. MTND5 and MTND6 genes were identified as 'hotspots' of mutations, with Leigh syndrome accounting for the large majority of associated phenotypes.
Rare mitochondrial DNA mutations probably account for more than 7.4% of patients with respiratory chain deficiency. This study shows that a comprehensive analysis of mtDNA is essential, and should include young children, for an accurate diagnosis that is now accessible with the development of next generation sequencing technology.

Bannwarth S, Procaccio V, Lebre AS, Jardel C, Chaussenot A, Hoarau C, Maoulida H, Charrier N, Gai X, Xie HM, Ferre M, Fragaki K, Hardy G, Mousson de Camaret B, Marlin S, Dhaenens CM, Slama A, Rocher C, Paul Bonnefont J, Rotig A, Aoutil N, Gilleron M, Desquiret-Dumas V, Reynier P, Ceresuela J, Jonard L, Devos A, Espil-Taris C, Martinez D, Gaignard P, Le Quan Sang KH, Amati-Bonneau P, Falk MJ, Florentz C, Chabrol B, Durand-Zaleski I, and Paquis-Flucklinger V. (2013) Prevalence of rare mitochondrial DNA mutations in mitochondrial disorders. J Med Genet 50: 704-714. PubMed

2004-2007 : Evaluation of Mitochips Technology (Affymetrix) for complete resequencing of mtDNA: 133 patients were tested in collaboration with PartnerChip (support GIS-MR).

This work resulted in a publication.

Mitochondrial DNA (mtDNA) mutations have been implicated in non-syndromic hearing loss either as primary or as predisposing factors. As only a part of the mitochondrial genome is usually explored in deafness, its prevalence is probably under-estimated. Among 1350 families with non-syndromic sensorineural hearing loss collected through a French collaborative network, we selected 29 large families with a clear maternal lineage and screened them for known mtDNA mutations in 12S rRNA, tRNASer(UCN) and tRNALeu(UUR) genes. When no mutation could be identified, a whole mitochondrial genome screening was performed, using a microarray resequencing chip: the MitoChip version 2.0 developed by Affymetrix Inc. Known mtDNA mutations was found in nine of the 29 families, which are described in the article: five with A1555G, two with the T7511C, one with 7472insC and one with A3243G mutation. In the remaining 20 families, the resequencing Mitochip detected 258 mitochondrial homoplasmic variants and 107 potentially heteroplasmic variants. Controls were made by direct sequencing on selected fragments and showed a high sensibility of the MitoChip but a low specificity, especially for heteroplasmic variations. An original analysis on the basis of species conservation, frequency and phylogenetic investigation was performed to select the more probably pathogenic variants. The entire genome analysis allowed us to identify five additional families with a putatively pathogenic mitochondrial variant: T669C, C1537T, G8078A, G12236A and G15077A. These results indicate that the new MitoChip platform is a rapid and valuable tool for identification of new mtDNA mutations in deafness.

Lévêque M, Marlin S, Jonard L, Procaccio V, Reynier P, Amati-Bonneau P, Baulande S, Pierron D, Lacombe D, Duriez F, Francannet C, Mom T, Journel H, Catros H, Drouin-Garraud V, Obstoy MF, Dollfus H, Eliot MM, Faivre L, Duvillard C, Couderc R, Garabedian EN, Petit C, Feldmann D, Denoyelle F. Whole mitochondrial genome screening in maternally inherited non-syndromic hearing impairment using a microarray resequencing mitochondrial DNA chip. Eur J Hum Genet 2007;15:1145-55. PubMed

2000-2009 : Inter-laboratory coordination work on enzymatic assays of the respiratory chain.

This work resulted in consensual protocols and an article of recommendations.

Diversity of respiratory chain spectrophotometric assays may lead to difficult comparison of results between centers. The French network of mitochondrial diseases diagnostic centers undertook comparison of the results obtained with different protocols in the French diagnostic centers. The diversity of protocols was shown to have striking consequences, which prompted the network to undertake standardization and optimization of the protocols with respect to clinical diagnosis, i.e. high velocity while maintaining linear kinetics relative to time and enzyme concentration. Assays were set up on animal tissues and verified on control human muscle and fibroblasts. Influence of homogenization buffer and narrow range of optimal concentration of phosphate, substrate and tissue were shown. Experimental data and proposed protocols have been posted on a free access website. Their subsequent use in several diagnostic centers has improved consistency for all assays.

Medja F, Allouche S, Frachon P, Jardel C, Malgat M, Mousson de Camaret B, Slama A, Lunardi J, Mazat JP, Lombès A. Development and implementation of standardized respiratory chain spectrophotometric assays for clinical diagnosis. Mitochondrion 2009;9:331-9. PubMed

2006-2008 : Role of mitochondrial haplogoups in MELAS.

This work resulted in a publication.

The A3243G mutation in the tRNALeu gene (UUR), is one of the most common pathogenic mitochondrial DNA (mtDNA) mutations in France, and is associated with highly variable and heterogeneous disease phenotypes. To define the relationships between the A3243G mutation and mtDNA backgrounds, we determined the haplogroup affiliation of 142 unrelated French patients - diagnosed as carriers of the A3243G mutation - by control-region sequencing and RFLP survey of their mtDNAs.
The analysis revealed 111 different haplotypes encompassing all European haplogroups, indicating that the 3243 site might be a mutational hot spot. However, contrary to previous findings, we observed a statistically significant underepresentation of the A3243G mutation on haplogroup J in patients (p = 0.01, OR = 0.26, C.I. 95%: 0.08-0.83), suggesting that might be due to a strong negative selection at the embryo or germ line stages.
Thus, our study supports the existence of mutational hotspot on mtDNA and a "haplogroup J paradox," a haplogroup that may increase the expression of mtDNA pathogenic mutations, but also be beneficial in certain environmental contexts.

Pierron D, Rocher C, Amati-Bonneau P, Reynier P, Martin-Négrier ML, Allouche S, Batandier C, Mousson de Camaret B, Godinot C, Rotig A, Feldmann D, Bellanne-Chantelot C, Arveiler B, Pennarun E, Rossignol R, Crouzet M, Murail P, Thoraval D, Letellier T. New evidence of a mitochondrial genetic background paradox: impact of the J haplogroup on the A3243G mutation. BMC Med Genet. 2008 May 7;9:41. PubMed

2004-2006 : Epidemiology of mitochondrial diseases.

Reynier P; Réseau "Maladies Mitochondriales". [Epidemiology of mitochondrial cytopathies: contribution from the French mitochondrial diseases network]. Ann Biol Clin (Paris) 2006; 64:584-6. French. PubMed