Computational protocol: Identification of a Novel Homozygous Nonsense Mutation Confirms the Implication of GNAT1 in Rod Cone Dystrophy

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Protocol publication

[…] Blood samples of the index (CIC01293, II.1) and his parents (CIC01294, I.1 and CIC06690, I.2) were collected for genetic research and genomic DNA was extracted as previously reported [] (). Research procedures adhered to the tenets of the Declaration of Helsinki and were approved by the local Ethics Committee (CPP, Ile de France V). Prior to genetic testing, written informed consent, which had been previously approved by the CPP, was obtained from each study participant. Targeted next generation sequencing (NGS) and whole exome sequencing (WES) were performed in collaboration with a company (IntegraGen, Evry, France) [,]. A panel of 123 genes known to be associated with retinal dystrophies was used for targeted NGS as previously described []. Subsequently, WES was performed in the trio (proband and parents): exons of DNA samples were captured and investigated as shown before with in-solution enrichment methodology (SureSelect Clinical Research Exome, Agilent, Massy, France) and NGS (Illumina HISEQ, Illumina, San Diego, CA, USA) []. For all subjects, overall WES coverage of the captured regions was 96% and 90.67% for 10x and 25x depth of coverage respectively resulting in a mean sequencing depth of 82x per base (– Tables). Image analysis and base calling were performed with Real Time Analysis software (Illumina) []. Genetic variation annotations were realized by an in-house pipeline (IntegraGen), and results were provided per sample or family in tabulated text files. Stringent filtering criteria were used to select most likely pathogenic variant(s): only nonsense, missense, splice site variants or small deletions or insertions (InDels) with a minor allelic frequency ≤0.005 in Exome Variants Server (EVS,, HapMap (, 1000Genomes ( and Exome Aggregation Consortium (ExAC, were considered to be putative disease-causing. Variant pathogenicity was predicted with bioinformatic tools: Polymorphism Phenotyping v2 (PolyPhen2,, Sorting Intolerant From Tolerant (SIFT,, MutationTaster ( and amino acid conservation across species was studied with UCSC Genome Browser (; Human GRCh37/hg19 Assembly).The GNAT1 variant selected after WES was validated in the index case and the unaffected parents as following: 2 ng of Genomic DNA was amplified by polymerase chain reaction using oligonucleotides (human GNAT1 reference sequence NM_144499.2, Forward: 5’-GAGCCCAGAGAGCAGGTG-3' and Reverse: 5’-GGAGCTGGACGGGGCTG-3') (Sigma, Saint Quentin Fallavier, France) at a concentration of 10 μM each with 1.5 mM MgCl2 (Solis BioDyne, Tartu, Estonie), 3x S solution (Solis BioDyne), 1x B2 buffer (Solis BioDyne), 0.004 U of a polymerase (Hot Fire Polymerase, Solis BioDyne) and 0.2 mM dNTPs (Solis BioDyne) with an annealing temperature of 60°C and as previously described []. The novel GNAT1 nonsense variant identified in this study has been deposited in dbSNP database ( prior to publication.A cohort of additional 384 probands with RCD was studied by targeted NGS, including GNAT1 as previously described with an updated panel covering 195 different genes implicated in RCDs [,]. [...] To identify GNAT1 proteins previously crystallized, BLAST searches (protein Basic Local Alignment Search Tool, BLAST, were performed against the Protein Data Bank (PDB, using the GNAT1 protein sequence (human GNAT1 reference sequence NP_653082.1) as a query. The sequences (Bovine NP_851365.1 GNAT1, human, wild-type and the two RCD mutant forms from NP_653082.1, and the crystallized bovine GNAT1 (1TND, PDB ID)) were then aligned using Clustal Omega ( The three GNAT1 3D-structures, the full-length and the two truncated mutants of NP_653082.1, were predicted (Protein Homology/analogY Recognition Engine V2.0, Phyre2,, []; Iterative Threading ASSEmbly Refinement, I-TASSER,, []; TM-Align,, []). The PyMOL Molecular Graphics System, Version 1.7.x Schrödinger, LLC, was used to model GNAT1 interactions with GDP (PDB code 1GOT), GTP (PDB code 1TND), and with RHO (PDB code 4A4M). […]

Pipeline specifications

Software tools BLASTN, Clustal Omega, Phyre, I-TASSER, TM-align, PyMOL
Organisms Homo sapiens