cells Review Applying Bioinformatic Platforms, In Vitro, and In Vivo Functional Assays in the Characterization of Genetic Variants in the GH/IGF Pathway Affecting Growth and Development Sabina Domené, Paula A. Scaglia, Mariana L. Gutiérrez and Horacio M. Domené * Centro de Investigaciones Endocrinológicas ‘Dr César Bergadá’, (CEDIE) CONICET, FEI, División de Endocrinología, Hospital de Niños Ricardo Gutiérrez), Buenos Aires C1425EFD, Argentina; [email protected] (S.D.); [email protected] (P.A.S.); [email protected] (M.L.G.) * Correspondence: [email protected]; Tel.: +54-11-4963-5931; Fax: +54-11-4963-5930 Abstract: Heritability accounts for over 80% of adult human height, indicating that genetic variabil- ity is the main determinant of stature. The rapid technological development of Next-Generation Sequencing (NGS), particularly Whole Exome Sequencing (WES), has resulted in the characterization of several genetic conditions affecting growth and development. The greatest challenge of NGS remains the high number of candidate variants identified. In silico bioinformatic tools represent the first approach for classifying these variants. However, solving the complicated problem of variant interpretation requires the use of experimental approaches such as in vitro and, when needed, in vivo functional assays. In this review, we will discuss a rational approach to apply to the gene variants identified in children with growth and developmental defects including: (i) bioinformatic tools; (ii) in silico modeling tools; (iii) in vitro functional assays; and (iv) the development of in vivo models. Citation: Domené, S.; Scaglia, P.A.; While bioinformatic tools are useful for a preliminary selection of potentially pathogenic variants, Gutiérrez, M.L.; Domené, H.M. in vitro—and sometimes also in vivo—functional assays are further required to unequivocally de- Applying Bioinformatic Platforms, In termine the pathogenicity of a novel genetic variant. This long, time-consuming, and expensive Vitro, and In Vivo Functional Assays process is the only scientifically proven method to determine causality between a genetic variant and in the Characterization of Genetic a human genetic disease. Variants in the GH/IGF Pathway Affecting Growth and Development. Cells 2021, 10, 2063. https:// Keywords: bioinformatics; functional assays; growth and development; GH-IGF axis; short stature; doi.org/10.3390/cells10082063 next-generation sequencing Academic Editor: Haim Werner Received: 10 July 2021 1. Introduction Accepted: 9 August 2021 Heritability accounts for more than 80% of adult human height [1], indicating that Published: 12 August 2021 genetic variability is the main determinant of stature. Over 700 common variants (single nucleotide polymorphisms, SNPs) across more than 400 loci have been independently Publisher’s Note: MDPI stays neutral associated with height [2]. with regard to jurisdictional claims in The GH/IGF axis plays an important role in pre and postnatal growth. In 1981, published maps and institutional affil- Phillips III and their colleagues described the first molecular characterization in this axis: iations. the complete GH1 gene deletion, resulting in familial isolated complete GH deficiency [3]. Since then, molecular defects in more than 48 different genes have been described all along the GH/IGF axis [4,5]. These defects result in alteration of GH synthesis and secretion, impairment on GH action, in IGF1 synthesis, transport and action, and in Copyright: © 2021 by the authors. IGF2 synthesis. Most of these molecular defects were discovered by the candidate gene Licensee MDPI, Basel, Switzerland. approach using clinical data and biochemical profiles to select the more likely candidate This article is an open access article gene(s) to be studied. In recent years, the candidate gene approach to the molecular distributed under the terms and diagnosis of genetic diseases has been replaced with copy number variation (CNV) analysis conditions of the Creative Commons based on chromosomal microarrays, sequencing of a genetic panel (including several Attribution (CC BY) license (https:// candidate genes), whole-exome sequencing (WES), and whole-genome sequencing (WGS) creativecommons.org/licenses/by/ technologies (Figure1). The diagnostic effectiveness to detect a genetic defect as etiology 4.0/). Cells 2021, 10, 2063. https://doi.org/10.3390/cells10082063 https://www.mdpi.com/journal/cells Cells 2021, 10, x FOR PEER REVIEW 2 of 25 Cells 2021, 10, 2063 2 of 24 genes), whole-exome sequencing (WES), and whole-genome sequencing (WGS) technol- ogies (Figure 1). The diagnostic effectiveness to detect a genetic defect as etiology of short statureof short using stature these using technologies these technologies is about is 2 about5–40% 25–40% [6]. Since [6]. 2012 Since, with 2012, the with extended the extended use of theseuse of techniques these techniques,, novel genetic novel geneticclinical clinicalconditions conditions have been have elucidated been elucidated in short patients in short wherepatients clinical where and clinical biochemical and biochemical data did datanot suggest did not an suggest obvious an candidate obvious candidate gene. In addi- gene. tion,In addition, this novel this approach novel approach has also has detected also detected molecular molecular defects defects affecting affecting growth growth in genes in beyondgenes beyond the GH/IGF the GH/IGF axis. axis. Figure 1. Systematic approach to identify potentialpotential pathogenic genetic variants. Flowchart for a systematic approachapproach to select and characterize potential pathogenic genetic variants in children with growth and development disorders. The select and characterize potential pathogenic genetic variants in children with growth and development disorders. The selection of patient(s) can be directed to individual patients, a trio including the patient and his/her parents, multiple selection of patient(s) can be directed to individual patients, a trio including the patient and his/her parents, multiple families presenting several affected index cases, or a cohort of patients sharing a similar phenotype. Next-Generation familiesSequencing presenting (NGS) can several be performed affected indexusing cases,gene panels, or a cohort Whole of Exome patients Sequencing sharing a (WES), similar or phenotype. Whole Genome Next-Generation Sequencing Sequencing(WGS). For variant(s) (NGS) can prioritization, be performed population, using gene disease, panels, Wholeand sequence Exome Sequencingdatabases are (WES), used. orSystematic Whole Genome literature Sequencing review is (WGS).also required. For variant(s) Variants prioritization, are then classified population, according disease, to ACMG and sequence criteria as databases pathogenic, are likely used. pathogenic, Systematic literatureof uncertain review sig- isnificance, also required. likely benign, Variants or are benign then. classifiedIn silico functional according assessment to ACMG criteria is based as upon pathogenic, several likelycomputational pathogenic, predictive of uncertain pro- significance,grams. Finally, likely to unequivocally benign, or benign. determine In silico the pathogenicity functional assessment of a novel isgenetic based variant, upon several further computationalin vitro functional predictive assays programs.and sometimes Finally, in tovivo unequivocally animal models determine are required. the pathogenicity of a novel genetic variant, further in vitro functional assays and sometimes in vivo animal models are required. One of the greatest challenges of WES and WGS remains the high number of candi- date variantsOne of the identified. greatest Most challenges of these of are WES novel and or WGS ultra- remainsrare variants the high that numberneed to be of clas- can- sifieddidate according variants identified.to their pathogenicity. 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