Whole exome sequencing in three families segregating a pediatric case of sarcoidosis

Abstract

Background: Sarcoidosis (OMIM 181000) is a multi-systemic granulomatous disorder of unknown origin. Despite multiple genome-wide association (GWAS) studies, no major pathogenic pathways have been identified to date. To find out relevant sarcoidosis predisposing genes, we searched for de novo and recessive mutations in 3 young probands with sarcoidosis and their healthy parents using a whole-exome sequencing (WES) methodology.

Methods: From the SARCFAM project based on a national network collecting familial cases of sarcoidosis, we selected three families (trios) in which a child, despite healthy parents, develop the disease before age 15 yr. Each trio was genotyped by WES (Illumina HiSEQ 2500) and we selected the gene variants segregating as 1) new mutations only occurring in affected children and 2) as recessive traits transmitted from each parents. The identified coding variants were compared between the three families. Allelic frequencies and in silico functional results were analyzed using ExAC, SIFT and Polyphenv2 databases. The clinical and genetic studies were registered by the ClinicalTrials.gov - Protocol Registration and Results System (PRS) ( https://ichgcp.net/clinical-trials-registry/NCT02829853" title="See in ClinicalTrials.gov">NCT02829853 and has been approved by the ethical committee (CPP LYON SUD EST - 2 - REF IRB 00009118 - September 21, 2016).

Results: We identified 37 genes sharing coding variants occurring either as recessive mutations in at least 2 trios or de novo mutations in one of the three affected children. The genes were classified according to their potential roles in immunity related pathways: 9 to autophagy and intracellular trafficking, 6 to G-proteins regulation, 4 to T-cell activation, 4 to cell cycle and immune synapse, 2 to innate immunity. Ten of the 37 genes were studied in a bibliographic way to evaluate the functional link with sarcoidosis.

Conclusions: Whole exome analysis of case-parent trios is useful for the identification of genes predisposing to complex genetic diseases as sarcoidosis. Our data identified 37 genes that could be putatively linked to a pediatric form of sarcoidosis in three trios. Our in-depth focus on 10 of these 37 genes may suggest that the formation of the characteristic lesion in sarcoidosis, granuloma, results from combined deficits in autophagy and intracellular trafficking (ex: Sec16A, AP5B1 and RREB1), G-proteins regulation (ex: OBSCN, CTTND2 and DNAH11), T-cell activation (ex: IDO2, IGSF3), mitosis and/or immune synapse (ex: SPICE1 and KNL1). The significance of these findings needs to be confirmed by functional tests on selected gene variants.

Keywords: Candidate genes; Sarcoidosis; Whole-exome sequencing (WES).

Conflict of interest statement

Ethics approval and consent to participate

This project has been approved by the ethical committee (CPP LYON SUD EST – 2 – REF IRB 00009118 – September 21, 2016), according to the Laws Nos. 94–548 of 1 July 1994, Nos. 94–653 and 94–654 of 29 July 1994, taking into account the amendments further introduced by the laws n ° 2004–800 of 6 August 2004 and n ° 2011–814 of 7 July 2011, both relating to bioethics. This approval included an informed consent form and a detailed information sheet for patients. According to the article R. 1131–4 of the French Public Health Code, for the participants who are under 18 yr., the consent has been given by both mother and father during clinical and genetic counseling. In the three cases, there were the natural parents which have the parental authority. In addition, the consent of the minor is systematically sought if he is able to express his will and participate in the decision. The clinical and genetic studies were registered by the Consent for publication

The SARCFAM protocol was presented to the ethical committee (CPP LYON SUD EST – 2 – REF IRB 00009118) dated September 21, 2016 and includes consent for genetic studies and an information document on the objectives of genetic analysis and the scientific SARCFAM project. This document is an informed consent for the publication of scientific and clinical data subject to a complete anonymity of the information. Our study does not present any photographic data. This informed consent is valid for adult patients and for children (< 18 yr) after signature of both legal parents.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1

Flowchart of WES (Whole Exome Sequencing) analysis: T1, T2, T3 design the three nuclear families respectively, p (paternal) for the father, m (maternal) for the maternal and c for the affected child. The number in dark boxes indicate the total number of variants identified respectively in T1, T2 and T3 and inherited in affected children as (a) homozygous and (b) compound heterozygous variants. As mentioned in the text, we selected only those variants which were observed in at least two trios and putatively pathogenic as suggested by SIFT and/or POLYPHENv2 software

Fig. 2

Parallel visualization of two gene variants identified in T1 by Integrative Genomics Viewer (IGV) (Broad Institute©) and Sanger Sequencing. a + b Confirmation of the de novo c.1912 T > G (c.1912A > C on reverse strand by WES) variant in the SPICE1 gene inherited by T1c (a + b), either on IGV (a) and Sanger sequence (b). c + d Confirmation of the homozygous c.2806A > G variant in the KNL1 (CASC5) gene inherited by T1c, either on IGV (c) and Sanger sequence (d). The IGV profiles show the absence of c.1912 T > G (SPICE1) in both T1p and T1 m parents (a) and the status of heterozygous carriers of T1p and T1 m for the c.2806A > G (KNL1) variant (c)