A Genetic Approach in the Evaluation of Short Stature

Short stature is considered a condition in which the height is 2 standard deviations below the mean height of a given age, sex, and population group. Human height is a polygenic and heterogeneous characteristic, and its heritability is reported to be approximately 80%. More than 600 variants associated with human growth were detected in the genome-wide association studies. Rare and common variants concurrently affect human height. The rare variations that play a role in human height determination and have a strong impact on protein functions lead to monogenic short stature phenotypes, which are a highly heterogeneous group. With rapidly developing technologies in the last decade, molecular genetic tests have begun to be used widely in clinical genetics, and thus, the genetic etiology of several rare diseases has been elucidated. Identifying the genetic etiology underlying idiopathic short stature which represents phenotypically heterogeneous group of diseases ranging from isolated short stature to severe and syndromic short stature has promoted the understanding of the genetic regulation of growth plate and longitudinal bone growth. In cases of short stature, definite molecular diagnosis based on genetic evaluation enables the patient and family to receive genetic counseling on the natural course of the disease, prognosis, genetic basis, and recurrence risk. The determination of the genetic etiology in growth disorders is essential for the development of novel targeted therapies and crucial in the development of mutation-specific treatments in the future.


Introduction
Short stature (SS) is considered a condition in which the height is 2 standard deviations (SD) below the mean height of a given age, sex, and population group. 1 Short stature is one of the most common causes of admission to pediatric endocrinology clinics.In addition to the constitutional delay of growth and puberty and familial short stature (FSS) known as "normal growth variants, " chronic diseases, hormonal diseases, and genetic causes play a role in the etiology of this condition. 1Idiopathic short stature (ISS) is a term used for children with SS without any systemic, endocrine, nutritional, or chromosomal abnormality.Idiopathic short stature comprises a wide range of patient group with phenotypic and genotypic heterogeneity, and most of the short children are followed up with this diagnosis. 1A multidisciplinary approach is required for the diagnosis of ISS, and clarification of the molecular diagnosis can be a guide in terms of follow-up and treatment. 1owth and height growth in children is a multifactorial condition characterized by both genetic and environmental factors. 2 The prevalence of pathological SS in different populations ranges from 1.3% to 19.8%. 3 In a study conducted on school children in South India, the prevalence of SS was found to be 2.86%. 4Although the frequency of SS among children and adolescents in Shanghai was 3.26%, it was 0.7% in a study evaluating 79 495 children in Utah. 5,6In a study conducted on school children in the United Kingdom, the frequency of SS was 1.3%. 7[10] Human height is a polygenic and heterogeneous characteristic, and its heritability is reported to be approximately 80%. 113][14] Rare and common variants concurrently affect human height.Height differences within the normal range are related to common variants (multiple polymorphisms), and these variants are known to be involved in growth plate functions. 15,16The rare variations that play a role in human height determination have a strong impact on protein functions and lead to monogenic SS phenotypes, which are a highly heterogeneous group. 14th rapidly developing technologies in the last decade, molecular genetic tests have begun to be used widely in clinical genetics, and thus, the genetic etiology of several rare diseases has been elucidated. 17In diseases with high genetic heterogeneity such as ISS, microarray, methylation studies, and next-generation sequencing (NGS) technologies allow the determination of etiology in some cases.In a study on pediatric patients with isolated growth hormone deficiency and ISS, NGS was shown to be beneficial in determining the genetic etiology. 18In recent years, the evaluation of SS cases along with clinical findings and genetic analysis results has provided a better understanding of the clinical variability and genetic heterogeneity of SS syndromes. 19In cases of SS, definite molecular diagnosis based on genetic evaluation enables the patient and family to receive genetic counseling on the natural course of the disease, prognosis, genetic basis, and recurrence risk.In this study, we aimed to summarize the molecular mechanisms underlying the genetic causes of SS cases and to discuss the genetic approach algorithm to these cases.

Molecular Genetic Mechanisms of Short Stature
Defects in Hormonal Signaling Pathway (Growth Hormone/Insulin-Like Growth Factor 1 System) Mutation in genes involved in the GH/IGF1 signaling pathway causes growth retardation and SS.Isolated GH deficiency is observed owing to defects in growth hormone 1 (GH1), growth hormone-releasing hormone receptor genes, and transcription factors (HESX1, SOX2, SOX3, LHX3, LHX4, PTX1, PTX2, OTX2, PROP1, and POUF1) involved in pituitary gland development in this pathway. 20Defects in GH receptor (GHR) and signal transducer and activator of transcription 5b (STAT5B) gene result in the development of GH resistance. 21,22In addition to SS, immune dysregulation is observed in STAT5B defects. 23[26] Defects in Paracrine Signaling Paracrine factors are effective in the proliferation and differentiation of chondrocytes in the growth plate. 27Paracrine signaling pathways include fibroblast growth factor (FGF)-FGF receptor signaling, parathyroid hormone-related protein and Indian hedgehog signaling, bone morphogenetic protein signaling, WNT signaling pathway, C-type natriuretic peptide signaling, and insulin-like growth factor 2 signaling.2][33] IHH mutations lead to brachydactyly type A1 and acrocapitofemoral dysplasia phenotypes. 346][37] Biallelic inactivating mutations in the natriuretic peptide receptor 2 (NPR2) gene cause acromesomelic dysplasia Maroteaux

Main Points
• In short stature, the determination of genetic epidemiology plays an important role in the followup and treatment of other system pathologies.
• Molecular genetic studies guide the identification of rare short-stature variants.• Molecular diagnosis in the short stature allows the family to receive genetic counseling.type, and monoallelic mutations lead to the ISS phenotype. 38Paternal point mutations in IGF2 and the loss of methylation at the imprinting control region (ICR1) site on chromosome 11p15.5[40] Defects in Cartilage Extracellular Matrix The extracellular matrix synthesized by chondrocytes comprising collagen, proteoglycan, and non-collagen proteins is vital for the structure and functions of the growth plate. 41[48][49][50] Defects in Constitutive Cellular Processes Because the genes involved in constitutive cellular processes are crucial not only in the growth plate but also in all cells, findings such as microcephaly, skeletal dysplasia, and facial dysmorphism are observed in addition to SS and growth retardation in these cases.Defects in constitutive cellular processes can be divided into 3 groups according to its molecular mechanism.

Transcription Factors
Mutations in genes encoding transcription factors lead to various syndromic SS phenotypes.SOX9 mutations cause campomelic dysplasia. 51iallelic mutations of SS homeobox-containing gene (SHOX) lead to Langer mesomelic dysplasia, which is a severe skeletal dysplasia, whereas monoallelic mutations lead to Léri-Weill dyschondrosteosis phenotype, a milder skeletal dysplasia. 52[55][56] DNA Repair DNA repair defects cause severe SS, microcephaly, photosensitivity, leukemia, and syndromic SS that predispose to other types of cancer.[64] Intracellular Signaling Intracellular defects cause an extremely heterogeneous group of diseases that affect different signaling pathways, of which the most commonly known is the rat sarcoma (RAS) -mito gen-a ctiva ted protein kinase pathway. 65Diseases that occur as a result of mutations in the genes involved in this signaling pathway are known as RASopathies. 66The most common RASopathy is Noonan syndrome, most commonly caused by mutations in PTPN11 (50%). 67[70]

Diagnostic Approach
The first step in the diagnostic examination of SS comprises obtaining family and patient history; physical examination; evaluation of growth velocity status and bone age; and a set of laboratory examinations.In pediatric endocrinology clinics, patients are evaluated in terms of FSS and constitutional delay in growth and puberty which are the variants of normal as well as hormonal dysfunctions and chronic diseases affecting growth as the first step of clinical examination.After hormonal examinations, targeted single gene analyses can be planned in cases with suspected anomalies in the GH/ IGF1 signaling pathway.Careful evaluation of body proportion is important in the diagnosis of skeletal dysplasia, and in cases suspected of skeletal dysplasia, the entire skeleton should be evaluated with a bone survey. 71Skeletal dysplasia findings are detected in 22% of ISS cases, and this rate increases to 33% in the presence of an affected parent. 71In the evaluation of bone age, delayed or advanced bone age status can be detected in different clinical entities. 1,72,735][76][77] Therefore, the presence of radiographic findings (radiolucency, pyramidalization, and triangularization) on the wrist radiograph suggests that SHOX deficiency should be carefully evaluated. 78In addition, it should be noted that radiographic findings become evident in the late childhood period.In cases where SS is accompanied by dysmorphic findings, genetic consultation should be considered.Even if the typical stigmata of Turner syndrome are not found in girls with SS, karyotype should be requested. 79merican College of Medical Genetics and Genomics practice resource": The algorithm suggested in the genetic approach to short stature cases according to the "Genetic evaluation of short stature" guideline is summarized in Figure 1.80

Genetic Testing Strategies
Using the genetic analyses mentioned in the previous section at appropriate time and order specific to patient will increase the rate of diagnosis.Genetic analyses used in the evaluation of SS cases can be performed through a wide range of conventional karyotyping, methylation analysis, microarray analysis, single-gene sequencing, NGS panel analysis, and exome sequencing.
It should be noted that each technique has its advantages and limitations.

Chromosome Analysis
The first step in the evaluation of a child with SS is karyotyping.It is recommended for all female cases with SS suspected of having Turner syndrome or even without typical stigmata.Fluorescence in situ hybridization (FISH) analysis is used for diagnosis when micro delet ion/m icrod uplic ation syndromes that cannot be detected by karyotyping are suspected.Since 80%-90% of SHOX deficiency cases are associated with deletions, FISH analysis should be performed for the SHOX gene as the first step.In cases where deletion is not detected (10%-20% of the affected individuals), sequence analysis of the SHOX gene should be performed as the second step. 86It should be noted that FISH analysis may not be sufficient in some cases depending on the location and size of the deletion, and chromosomal microarray/multiplex ligation-dependent probe amplification analyses should be planned in these cases. 86ryotyping and FISH analyses are used in the analysis of recurrent micro delet ion/m icrod uplic ation syndromes accompanied by SS.In addition to SS, microcephaly, facial dysmorphism, developmental delay, and congenital malformations can be seen in these syndromes.

Single Gene Testing
In ISS cases, targeted single-gene analyses should be selected if a specific single-gene defect is suspected based on the findings of clinical, laboratory, and radiographic examinations.Analyses of genes such as GHR, IGF1, IGF1R, IGFALS, and PAPPA2 can be planned after clinical and laboratory evaluation of individuals with suspected GH/ IGF-I axis defect.In cases where SHOX deficiency is considered, the test algorithm specified in the previous section should be applied.Although the clinical severity varies, sequence analysis for ACAN should be planned to investigate biallelic mutations in cases with spondyloepimetaphyseal dysplasia and severe SS. 72 Mild signs such as skeletal dysplasia, SS, midface hypoplasia, and advanced bone age suggest monoallelic mutations in the ACAN gene. 72Heterozygous mutations in the ACAN gene were detected in 1.4% of ISS cases. 87Genetic analysis should be performed for the point mutation in FGFR3 at c.1138G>A (p.Gly380Arg), which is known to be responsible for 99% of the cases, in patients with suspected achondroplasia.In cases with hypochondroplasia, point mutation analysis of FGFR3 at c.1620C>A/G (p.Asn540Lys) noted in 70%-80% of cases should be performed.If mutation cannot be detected, whole gene sequencing of FGFR3 should be requested.The genetic evaluation for suspected thanatophoric dysplasia type II and thanatophoric dysplasia type I include analysis of the point mutation in FGFR3 gene at p.Lys650Glu which is known to cause 99% of the cases and at p.Arg248Cys & p.Tyr373Cys which are known to cause 90% of cases, respectively.If mutation cannot be detected, whole gene sequencing of FGFR3 should be requested.Although the prevalence varies in different studies, heterozygous loss-offunction mutations in the NPR2 gene have been detected in ISS cases and it has been shown that biallelic mutations cause acromesomelic dysplasia (Maroteaux type). 88

Multi-Gene Testing Approach
[91] Genome-Wide Testing Approach Comparative genomic hybridization (Array-CGH) or SNP arrays platforms, which assess the whole genome for copy number variations (CNVs), and whole-exome sequencing (WES) analyses for the whole protein-encoding genome are comprehensive genetic tests that can be used in ISS cases.Comparative genomic hybridization (Array-CGH) or SNP arrays platforms are recommended to be applied before WES in ISS cases because they are cheaper and easily accessible. 80][94][95] Analysis of all previously identified SS genes is possible through WES analysis.In addition, WES analysis is a genetic tool that enables the discovery of novel genes and signaling pathways associated with SS and growth retardation.Molecular diagnosis is easier in the presence of pathogenic or likely pathogenic variant compatible with the patient' s clinic; however, several variants of uncertain significance (VUS) are observed during WES analysis.During the interpretation of these VUS variants, all clinical, laboratory, and radiographic data of the patient and segregation analysis studies are used.
It should be noted that rare genetic syndromes that occur with epigenetic mechanisms may be responsible for the etiology of ISS cases. 96ussell-Silver syndrome is characterized by prenatal and postnatal growth retardation, feeding difficulties, recurrent hypoglycemia, body asymmetry, prominent forehead, and relative macrocephaly at birth. 97Molecular etiology can be detected in 60% of the cases, and the most common loss of methylation (30%-60%) on chromosome 11p15, followed by maternal uniparental disomy for chromosome 7 (5%-10%), is observed. 97In cases with suspected RSS, the first step in genetic evaluation should be methylation analysis for the chromosome 11p15 region and maternal UPD analysis for chromosome 7. 97

Figure 1 .
Figure 1.Algorithm for the genetic evaluation of short stature (this algorithm is proposed by American College of Medical Genetics and Genomics practice resource: Genetic evaluation of short stature).
The approach to cases of SS first comprises physical examination, family history, measurement of body proportion, definition of facial dysmorphic findings, analysis of laboratory and radiological findings, and evaluation of bone age.In the presence of specific prediagnosis, genetic evaluation methods should be planned in accordance with the genetic test algorithm recommended in the previous sections.Clarification of genetic etiology is helpful to obtain individualized medical management of patients, determine the prognosis, provide appropriate genetic counseling, and avoid unnecessary tests.The determination of the genetic etiology in growth disorders is essential for the development of novel targeted therapies and crucial in the development of mutation-specific treatments in the future.Author Contributions: Concept -A.T., A.Ç.; Design -A.S.D., A.Ç.; Supervision -A.Ç.; Materials -A.T., A.S.D.; Data Collection and/or Processing -A.T., A.S.D.; Analysis and/or Interpretation -A.T., A.Ç.; Literature Review -A.T.; Writing Manuscript -A.T.; Critical Review -A.Ç.Declaration of Interests: All authors have no conflict of interest regarding this paper.Funding: This research received no grant from any funding agency in the public, commercial, or not-forprofit sectors.94.Hauer NN, Popp B, Schoeller E, et al.Clinical relevance of systematic phenotyping and exome sequencing in patients with short stature.Genet Med.2018;20(6):630-638.[CrossRef] 95.Huang Z, Sun Y, Fan Y, et al.Genetic evaluation of 114 Chinese short stature children in the next generation era: a single center study.Cell Physiol Biochem.2018;49(1):295-305.[CrossRef] 96.Piedrahita JA.The role of imprinted genes in fetal growth abnormalities.Birth Defects Res A Clin Mol Teratol.2011;91(8):682-692.[CrossRef] 97.Wakeling EL, Brioude F, Lokulo-Sodipe O, et al.Diagnosis and management of Silver-Russell syndrome: first international consensus statement.Nat Rev Endocrinol.2017;13(2):105-124.[CrossRef] 98. Stessman HA, Turner TN, Eichler EE.Molecular subtyping and improved treatment of neurodevelopmental disease.Genome Med.2016;8(1):22.[CrossRef] Peer-review: Externally peer-reviewed.