Genome analysis

Team

Scientific manager. Judith Melki

The genomics platform makes it possible to identify new genetic causes of human nervous system diseases. It allows for the implementation of genomic technologies (whole exome sequencing, WES), followed by bioinformatic analysis of the data to select variants, Sanger sequencing to validate them, and analysis of the functional consequences of these variants through RNA and protein analysis, and then through the generation of cellular and/or animal models.

Activities

1 et 2. Selection of patients and whole exome sequencing (WES)

Thanks to the collaboration of clinicians and geneticists, we select families of patients with a specific pathology. We then initiate whole-exome sequencing (WES) of several patients (now performed on high-throughput sequencing platforms).

WES is performed using the Twist Bioscience Human Core Exome (Consensus CDS) kit for library preparation and exome enrichment (Integragen).

3. Bioinformatic analysis to predict variants

Bioinformatic analysis of the sequencing data is performed by our group. The sequences are aligned to the human reference genome using the BWA program. Variants are then selected and annotated with SAMtools and Annovar software.

Variants located in coding regions (including missense and nonsense variants), intron-exon junctions (±10 bp), or in-frame deletions or duplications are selected if their frequency in the general population is less than or equal to 0.005 (using the 1000G, ExAC, TopMed, GnomAD, and ALFA databases). Pathogenicity prediction of missense variants is performed using CADD, PolyPhen-2, or SIFT software, splicing variants with Human Splicing Finder software, and ClinVar for previously reported variants (NCBI).

4. Variant validation though Sanger sequencing of quantitative-PCR

Despite sophisticated detection algorithms, whole-exome sequencing (WES) analysis can lead to false positives. The gold standard method for identifying a new mutation relies on Sanger sequencing, which allows for the validation of a specific locus (with a low error rate of <0.1%). The identified and selected variants are validated by Sanger sequencing after PCR amplification (Eurofins Genomics). The resulting sequences are then compared to reference sequences (BLAST, NCBI). Sanger sequencing is also performed to determine the genotype of family members in order to establish the segregation of variants within each family. When splicing variants or premature stop codons are identified, RNA analysis is performed to validate the deleterious effect of the variants on splicing or transcript stability.

5 and 6. Functional validation through RNA and protein analysis and study of the pathophysiological mechanisms related to the new genes

The identification of a new gene is followed by its functional validation, carried out by generating and characterizing animal models (C. elegans or zebrafish) or cell models.

Access

This equipment is available to external users on request, generally within the framework of scientific collaboration.

Main publications

Our group has identified 11 new genes and mecanisms in the 12 last years.

These genes are ASAH1 (Zhou et al. 2012), CNTNAP1 et ADCY6 (Laquérriere et al. 2014), MAGEL2 (Mejlachowicz et al. 2015), GLDN (Maluenda et al. 2016), LGI4 (Xue et al. 2017), UNC50 (Abiusi et al. 2017), EPHB4 (Vivanti et al. 2018), SCN1A (Jaber et al. 2021), NPR1 (Capri et al. 2023) et DST (Capri et al. 2023).

Zhou J, Tawk M, Tiziano FD, Veillet J, Bayes M, Nolent F, Garcia V, Servidei S, Bertini E, Castro-Giner F, Renda Y, Carpentier S, Andrieu-Abadie N, Gut I, Levade T, Topaloglu H, Melki J. Spinal Muscular Atrophy Associated with Progressive Myoclonic Epilepsy Is Caused by Mutations in ASAH1. Am J Hum Genet. 2012;91:5-14.

Laquérriere A, Maluenda J,Camus A,Fontenas L,Dieterich K, Nolent F, Zhou J, Monnier N, Latour P,Gentil D,Héron D, Desguerres I,Landrieu P, Beneteau C, Delaporte B, Bellesme C, Baumann C, Capri Y, Goldenberg A, Lyonnet S, Bonneau D,Estournet B, Quijano-Roy S, Francannet C, Odent S, Saint-Frison Mh, Sigaudy S, Figarella-Branger D, Gelot A, Mussini Jm, Lacroix C, Drouin-Garraud V, Malinge Mc, Attié-Bitach T, Bessieres B, Bonniere M, Encha-Razavi F, Beaufrère Am, Khung-Savatovsky S, Perez Mj, Vasiljevic A, Mercier S, Roume J, Trestard L, Saugier-Veber P, Cordier Mp, Layet V, Legendre M, Vigouroux-Castera A, Lunardi J, Bayes M, Jouk Ps, Rigonnot L, Granier M, Sternberg D, Warszawski J, Gut I,Gonzales M, Tawk M, Melki J. Mutations in CNTNAP1 and ADCY6 are responsible for severe arthrogryposis multiplex congenital with axoglial defects. Hum Mol Genet. 2014;23:2279-89.

Mejlachowicz D, Nolent F, Maluenda J, Ranjatoelina-Randrianaivo H, Giuliano F, Gut I, Sternberg D, Laquerrière A, Melki J. Truncating Mutations of MAGEL2, a Gene within the Prader-Willi Locus, Are Responsible for Severe Arthrogryposis. Am J Hum Genet. 2015;97:616-620.

Maluenda J, Manso C, Quevarec L, Vivanti A, Marguet F, Gonzales M, Guimiot F, Petit F, Toutain A, Whalen S, Grigorescu R, Coeslier Ad, Gut M, Gut I, Laquerrière A, Devaux J, Melki J. Mutations in GLDN, Encoding Gliomedin, a Critical Component of the Nodes of Ranvier, Are Responsible for Lethal Arthrogryposis. Am J Hum Genet. 2016;99:928-933.

Xue S, Maluenda J, Marguet F, Shboul M, Quevarec L, Bonnard C, Ng Ay, Tohari S, Tan Tt, Kong Mk, Monaghan Kg, Cho Mt, Siskind Ce, Sampson Jb, Rocha Ct, Alkazaleh F, Gonzales M, Rigonnot L, Whalen S, Gut M, Gut I, Bucourt M, Venkatesh B, Laquerrière A, Reversade B, Melki J. Loss-of-Function Mutations in LGI4, a Secreted Ligand Involved in Schwann Cell Myelination, Are Responsible for Arthrogryposis Multiplex Congenita. Am J Hum Genet. 2017;100:659-665.

Abiusi E, D’alessandro M, Dieterich K, Quevarec L, Valfort Ac, Mezin P, Jouk Ps, Gut M, Gut I, Bessereau Jl, Melki J. Biallelic mutation of UNC50, encoding a protein involved in AChR trafficking, is responsible for arthrogryposis. Hum Mol Genet. 2017;26:3989-3994.

Vivanti A, Ozanne A., Grondin C, Saliou G, Quevarec L, Maurey H, Aubourg P, Benachi A, Gut M,Gut I,Martinovic J,Sénat Mv,Tawk M,Melki J. Loss of function mutations in EPHB4 are responsible for vein of Galen aneurysmal malformation. Brain. 2018;141:979-988.

Jaber D, Gitiaux C, Blesson S, Marguet F, Buard D, Varela Salgado M, Kaminska A, Saada J, Fallet-Bianco C, Martinovic J, Laquerriere A, Melki J. De novo mutations of SCN1A are responsible for arthrogryposis broadening the SCN1A-related phenotypes. J Med Genet. 2021;58:737-742.

Laquerriere A, Jaber D, Abiusi E, Maluenda J, Mejlachowicz D, Vivanti A, Dieterich K, Stoeva R, Quevarec L, Nolent F, Biancalana V, Latour P, Sternberg D, Capri Y, Verloes A, Bessieres B, Loeuillet L, Attie-Bitach T, Martinovic J, Blesson S, Petit F, Beneteau C, Whalen S, Marguet F, Bouligand J, Heron D, Viot G, Amiel J, Amram D, Bellesme C, Bucourt M, Faivre L, Jouk Ps, Khung-Savatovsky S, Sigaudy S, Delezoide Al, Goldenberg A, Jacquemont Ml, Lambert L, Layet V, Lyonnet S, Munnich A, Van Maldergem L, Piard J, Guimiot F, Landrieu P, Letard P, Pelluard F, Perrin L, Saint-Frison Mh, Topaloglu H, Trestard L, Vincent-Delorme C, Amthor H, Barnerias C, Benachi A, Bieth E, Boucher E, Cormier-Daire V, Delahaye-Duriez A, Desguerre I, Eymard B, Francannet C, Grotto S, Lacombe D, Laffargue F, Legendre M, Martin-Coignard D, Megarbane A, Mercier S, Nizon M, Rigonnot L, Prieur F, Quelin C, Ranjatoelina-Randrianaivo H, Resta N, Toutain A, Verhelst H, Vincent M, Colin E, Fallet-Bianco C, Granier M, Grigorescu R, Saada J, Gonzales M, Guiochon-Mantel A, Bessereau Jl, Tawk M, Gut I, Gitiaux C, Melki J. Phenotypic spectrum and genomics of undiagnosed arthrogryposis multiplex congenita. J Med Genet. 2022;59:559-567.

Capri Y, Kwon T, Boyer O, Bourmance L, Testa N, Baudouin V, Bonnefoy R, Couderc A, Meziane C, Tournier-Lasserve E, Heidet L, Melki J. Biallelic NPR1 loss of function variants are responsible for neonatal systemic hypertension. J Med Genet. 2023;60:993-998.

Capri Y, Bourmance L, Dupont C, Saint-Frison Mh, Guimiot F, Grotto S, Chitrit Y, Laquerrière A, Melki J. DST variants are responsible for neurogenic arthrogryposis multiplex congenita enlarging the spectrum of type VI hereditary sensory autonomic neuropathy. Clin Genet. 2023;104:587-592.

Names in bold caracters are members of our laboratory