Whole Exome Sequencing in Inborn Errors of Immunity

Use the Power but Mind the Limits

Giorgia Bucciol; Erika Van Nieuwenhove; Leen Moens; Yuval Itan; Isabelle Meyts


Curr Opin Allergy Clin Immunol. 2017;17(6):421-430. 

In This Article

Validation of the Functional Impact of a Variant

No prediction tool has the power to prove the pathogenicity of a variant. This is why, upon discovery of a potential new disease-causing gene or variant, it is fundamental to experimentally ascertain the causal connection between the variant and the clinical and immunological phenotype, to prevent ascribing to a false-positive result a disease-causing effect.[7] A multidisciplinary team meeting involving the clinician (scientist), genetic counsellor, bioinformatician, and clinical and nonclinical researcher is optimal for efficient prioritizing of variants that need further validation. Especially for single-patient genetic studies, solid evidence to show that the genetic variant is responsible for the destruction, impairment of function or alteration of expression or function of the protein product is necessary.[4] First, the effect of the variant on RNA or protein expression (for instance, by Western Blot or flow cytometry) must be investigated. In the case of unaltered protein expression, altered function can lead to disease. Functional studies are therefore a crucial step. For instance, in the study of mutations in genes affecting the type I interferon pathway, the in-vitro assay of viral load in cell lines relevant to the phenotype can be pursued. In case of a de novo mutation, different cell types should be tested to exclude a somatic mutation. Finally, for LOF mutations the cellular phenotype should be rescued by transduction with the wild-type allele, whereas for GOF mutations rescue should be obtained by knockdown/knockout or correction of the mutant allele.[4] The functional confirmation of causality represents the most difficult and time-consuming part in the characterization of new disease-associated genes or mutations, but remains essential for the scientific validation of the findings and hence also for the diagnostic value of a reported variant. Reports of novel 'mutations', even if in genes known to cause inborn errors of immunity, should be interpreted with caution if the validation step is missing.