The functional genomics laboratory: functional validation of genetic variants

Abstract

Currently, one of the main challenges in human molecular genetics is the interpretation of rare genetic variants of unknown clinical significance. A conclusive diagnosis is of importance for the patient to obtain certainty about the cause of the disease, for the clinician to be able to provide optimal care to the patient and to predict the disease course, and for the clinical geneticist for genetic counseling of the patient and family members. Conclusive evidence for pathogenicity of genetic variants is therefore crucial. This review gives an introduction to the problem of the interpretation of genetic variants of unknown clinical significance in view of the recent advances in genetic screening, and gives an overview of the possibilities for functional tests that can be performed to answer questions about the function of genes and the functional consequences of genetic variants ("functional genomics") in the field of inborn errors of metabolism (IEM), including several examples of functional genomics studies of mitochondrial disorders and several other IEM.

Fig. 1

From genetic test to functional validation. After whole exome (or genome) sequencing, genetic variants are analyzed by bioinformatics tools and additional genetic tests (e.g., segregation analysis, population studies) are performed. On the basis of these data, variants are classified as not/unlikely pathogenic (class 1/2), of unknown pathogenicity (class 3), or likely/definite pathogenic (class 4/5). For class 3 variants, functional validation studies are a powerful tool to obtain evidence for possible pathogenicity

Fig. 2

Examples of functional genomics approaches. These approaches, or combinations thereof, are frequently used to investigate the pathogenicity of genetic variants of unknown clinical significance. The examples shown are described in a clockwise order starting in the top-left corner. Rescue: introduction by lentiviral transduction of wild type LYRM7 cDNA in fibroblasts from a patient with a defect in LYRM7 results in normalization of mitochondrial Rieske Fe-S protein (Hempel et al. 2017). CRISPR/Cas9: Absence of thymidine hydroxylase (TH) staining in iPSC-derived dopaminergic nerve cells from a patient with a defect in PTPS, and normalization of TH expression after CRISPR/Cas9-mediated correction of the PTPS gene (Ishikawa et al. 2016). Biomarkers: detail of a 1H NMR spectrum of a CSF sample from a NANS patient, showing the presence of alpha and beta forms of N-acetylmannosamine (van Karnebeek et al. 2016). iPSC: Abnormal sarcomere organization in iPSC-cardiomyocytes derived from fibroblasts from a Barth-syndrome patient (BTH) with mutations in the tafazzin gene (TAZ), and normalization of sarcomeres after transfection with TAZ-mRNA (Wang et al. 2014). Micro-organism model: Restoration of growth on a non-fermentable carbon source (YPG) of a mitochondrial malate dehydrogenase-deficient yeast strain (mdh1Δ) by transfection with wild type MDH1, but not with various mdh-mutants (Ait-El-Mkadem et al. 2017). Animal model: A cerebellar defect in CLPB knock-down zebrafish embryos, as seen in Ac-tubulin stained embryos (the cerebellum is indicated by the rectangle in the control animal) (Wortmann et al. 2015, 2015)