Computational protocol: Atypical Manifestation of LPS-Responsive Beige-Like Anchor Deficiency Syndrome as an Autoimmune Endocrine Disorder without Enteropathy and Immunodeficiency

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Protocol publication

[…] Genetic analysis was performed after informed consent by the parents. Genomic DNA from peripheral blood cells was isolated using the QIAamp® DNA Blood Mini Kit (Qiagen, Courtaboeuf, France) according to manufacturer’s instructions. Whole exome sequencing (WES) was performed on the genomic platform of Institut IMAGINE’s. Agilent SureSelect librairies were prepared from 3 μg of genomic DNA sheared with a Covaris S2 Ultrasonicator. Exons regions were captured using the Agilent Sure Select All Exon 51Mb V5 (AGILENT, Les Ulis, France) and sequenced using a HiSeq2500 next generation sequencer (Illumina) on the Genomic Platform of Institut IMAGINE, Paris. Depth of coverage obtained for each sample was around 100× with >98% of the exome covered at least 15-fold. Paired-end sequences were then mapped on the human genome reference (NCBI build37/hg19 version) using the Burrows-Wheeler Aligner. Downstream processing was carried out with the genome analysis toolkit (GATK), SAMtools, and Picard, each following documented best practices. Variant calls were made with the GATK Unified Genotyper. All variants were annotated using the in-house software (PolyWeb) developed by Paris Descartes University Bioinformatics platform. All the annotation process was based on the 72 version of ENSEMBL database. Analysis of genome variations was made using the PolyWeb software. Variants were compared to the ones already present in US National Center for Biotechnology Information database (10) of SNP, 1000 Genome, and Exome Variant Server databases. The impact on the protein function was predicted using three algorithms: Polyphen 2, SIFT (Sorting Intolerant From Tolerant, J. Craig Venter Institute), and Mutation Taster. To confirm the mutation by Sanger sequencing, genomic DNA was amplified by standard techniques using oligonucleotide primers flanking the exon 46 on the Ensembl transcrit ENST00000357115 of LRBA (forward 5′-TTTCCCTCCCTATTGGCAGC-3′, lower 5′-ACAGCAAGCATCTGAAGGGG-3′) using TaqDNA Polymerase (Life Technologies, Saint-Aubin, France). After purification with the QIAquick PCR Purification kit (Qiagen), PCR fragments were sequenced using the same primers by Eurofins on the Genomic Platform of Université Paris Descartes. […]

Pipeline specifications

Software tools BWA, GATK, SAMtools, Picard, PolyPhen, SIFT
Databases Exome Variant Server
Application WES analysis
Organisms Homo sapiens