AUTHOR=Bradshaw Gabrielle , Lualhati Robbie R. , Albury Cassie L. , Maksemous Neven , Roos-Araujo Deidre , Smith Robert A. , Benton Miles C. , Eccles David A. , Lea Rod A. , Sutherland Heidi G. , Haupt Larisa M. , Griffiths Lyn R. 

TITLE=Exome Sequencing Diagnoses X-Linked Moesin-Associated Immunodeficiency in a Primary Immunodeficiency Case

JOURNAL=Frontiers in Immunology

VOLUME=Volume 9 - 2018

YEAR=2018

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2018.00420

DOI=10.3389/fimmu.2018.00420

ISSN=1664-3224

ABSTRACT=Background

We investigated the molecular aetiology of a young male proband with confirmed immunodeficiency of unknown cause, presenting with recurrent bacterial and Varicella zoster viral infections in childhood and persistent lymphopaenia into early adulthood. 

Aim

To identify causative functional genetic variants related to an undiagnosed primary immunodeficiency.

Method

Whole genome microarray copy number variant (CNV) analysis was performed on the proband followed by whole exome sequencing (WES) and trio analysis of the proband and family members. A >4kbp deletion identified by repeated CNV analysis of exome sequencing data along with three damaging missense single nucleotide variants were validated by Sanger sequencing in all family members. Confirmation of the causative role of the candidate gene was performed by qPCR and Western Blot analyses on the proband, family members and a healthy control. 

Results

CNV identified our previously reported IL25 amplification in the proband, however the variant was not validated to be a candidate gene for immunodeficiency. WES trio analysis, data filtering and in-silico prediction identified a novel, damaging (SIFT: 0; Polyphen 1; Grantham score: 101) and disease-causing (MutationTaster) single base mutation in the X chromosome (c.511C>T p.Arg171Trp) MSN gene not identified in the UCSC Genome Browser database. The mutation was validated by Sanger sequencing, confirming the proband was hemizygous X-linked recessive (-/T) at this locus and inherited the affected T allele from his non-symptomatic carrier mother (C/T), with other family members (father, sister) confirmed to be wild type (C/C). Western Blot analysis demonstrated an absence of moesin protein in lymphocytes derived from the proband, compared with normal expression in lymphocytes derived from the healthy control, father and mother. qPCR identified significantly lower MSN mRNA transcript expression in the proband compared to an age- and sex-matched healthy control subject in whole blood (P = 0.02), and lymphocytes (P = 0.01). These results confirmed moesin deficiency in the proband, directly causative of his immunodeficient phenotype.

Conclusion

These findings confirm X-linked moesin-associated immunodeficiency in a proband previously undiagnosed up to 24 years of age. This study also highlights the utility of WES for the diagnosis of rare or novel forms of primary immunodeficiency disease.