Preliminary investigation into the genetic etiology of short stature in children through whole exon sequencing of the core family

Abstract A comprehensive survey was carried out to investigate the genetic etiology of short stature in children by whole exon sequencing of a core family cohort to find and study mutations in multiple genes to assess their potential correlations to low height in children. The study included 56 pediatric patients from the Department of Pediatrics at the Zhangzhou Affiliated Hospital of Fujian Medical University. The participants met strict inclusion criteria, including age, Han Chinese ethnicity, low height standard deviation score, and the absence of known causes for short stature. Core pedigrees were identified using exome sequencing. After sequencing, variations were categorized and interpreted according to a variety of factors, including inheritance, location, type, and disease-causing gene databases. Variants were verified by Sanger sequencing. Most of the 97 gene mutations were missense. ACAN, PHEX, and COL2A1 were the most common gene mutations. Copy number variations were identified, particularly associated with the PHEX gene. Protein functional studies revealed that the mutations had a considerable influence on disease-promoting damage. The chromosomal locations with the highest enrichment of these genes were chr12, chr5, and chr2. In conclusion, the study revealed numerous genetic changes that may substantially impact physiological processes and disease. These findings establish the basis for further investigations into their diagnostic and therapeutic capabilities.


Introduction
Short stature, defined as a height below the third percentile for a child's age and gender, is a prevalent problem in pediatric medicine [1,2].It affects about 3% of children and can have serious consequences for their physical and psychological health [3].While some cases of short stature are attributed to environmental factors, such as malnutrition or chronic illness, a substantial proportion is believed to have a genetic basis [4][5][6].
In the broader domains of personalized medicine and genetics, it is critical to comprehend the genetic variations and their prospective effects on biological systems and human health.Genetic variations are of paramount importance in ascertaining an individual's medication response, disease susceptibility, and overall health.The ability to recognize and analyze these variations yields vital knowledge regarding the genetic predispositions of an individual, thus facilitating personalized strategies for healthcare and treatment.In the context of genetics, the study of genetic variations is essential for comprehending the complexity of human genetic diversity.Millions of genetic variants comprise the human genome, and each of them has the potential to affect a variety of physiological characteristics, including height, disease susceptibility, and drug metabolism.An increased comprehension of the genetic basis of human diversity and susceptibility to various health conditions results from the investigation of these variations.Furthermore, in the field of personalized medicine, the analysis of genetic variations holds great promise for tailoring medical treatments and interventions to individual patients.Healthcare providers can enhance the efficacy and precision of medical care by providing personalized suggestions for disease prevention, diagnosis, and treatment through the identification of specific genetic variants associated with certain conditions.
Hence, it is imperative to comprehend the genetic basis of short stature to facilitate accurate diagnosis, prognosis, and potentially targeted therapeutic interventions [7,8].Over the years, numerous genes have been implicated in the regulation of skeletal growth and development [9].For example, genetic short stature can result from defects in the growth hormone-insulin-like growth factor axis (GHRH-GH-IGF-1) [10,11].This pathway includes the production and release of growth hormone-releasing hormone (GHRH) from the hypothalamus, which stimulates the secretion of growth hormone (GH) from the pituitary gland [3,12].GH subsequently stimulates insulin-like growth factor 1 (IGF-1) production in the liver and other tissues, thus promoting overall and skeletal development [13,14].
Additionally, certain short-statured individuals may be affected by growth hormone insensitivity (GHI), which is also referred to as Laron syndrome [20].Gene mutations including those in GHR, STAT5B, PTPN11, IKBKB, IGF1, IGFALS, and insulin-like growth factor 1 receptor gene (IGF1R) can lead to GHI.Some patients who were previously diagnosed with idiopathic short stature were subsequently discovered to have GHI [18,19].However, there is still a group of children that are suspected to have problems in the GHRH-GH-IGF-1 axis but do not show any known pathogenic mutations when screened for the above-mentioned genes [2,3].This observation implies the existence of unidentified genes that play a role in this endocrine axis.Additionally, many of the functionally relevant genes have not been thoroughly investigated for pathogenic mutations.Hence, to clarify the cause of short stature in this population, it is imperative to conduct additional research and extensive investigations into this endocrine axis.
The primary aim of this study was to conduct a comprehensive investigation into the genetic factors contributing to short stature in children, in particular, to find and analyze genetic mutations through whole exon sequencing to ascertain whether they are associated with low height in pediatric patients; to address the knowledge gap regarding the genetic causes of short stature to pave the way for future developments in the field of diagnosis and treatment; to source a core family cohort; to perform whole exon sequencing; to classify and interpret variations; and to validate results using Sanger sequencing.The study was driven by the hypothesis that short stature is substantially influenced by genetic mutations and that whole exon sequencing can provide important insights into the genetic basis of this condition.

Case source
This is a prospective observational study.The majority of patients included in this study were from the Department of Pediatrics at the Zhangzhou Affiliated Hospital of FuJian Medical University.These patients were referred by experienced clinicians who specialize in diagnosing and treating short stature.These clinicians did thorough clinical examinations, made initial diagnoses, gave treatment assistance, and conducted follow-ups.
Informed consent: Informed consent has been obtained from all individuals included in this study.

Ethical approval:
The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance with the tenets of the Helsinki Declaration, and has been approved by the authors' institutional review board or equivalent committee.

Inclusion and exclusion criteria
The criteria for inclusion were as follows: (1) minimum age requirement of 18 years; (2) Han Chinese ethnicity; (3) height standard deviation score for sex and age (HtSDS) not exceeding −2.5 (as per the standardized growth curve for Chinese children and adolescents, 2009); (4) comprehensive medical records, excluding patients with a clearly defined cause for short stature; (5) informed consent obtained from the patients and/or their guardians; and (6) collection of complete familial blood samples, including at least the patient and both parents' EDTA anticoagulated blood samples.

Patient recall
The procedures for patient recall were as follows: (1) obtaining informed permission forms; (2) collecting venous blood samples from enrolled patients and preserving them at −20°C following the extraction of genomic DNA using DNA extraction kits; and (3) collecting pre-and post-treatment clinical data for the enrolled patients and creating a clinical database using EXCEL software for archival and management purposes.

Whole exon sequencing technology to detect the families of enrolled children
Exome sequencing of the core pedigrees was performed using the Agilent SureSelectXT Library Prep Kit ILM reagent set for DNA library preparation, along with the Human All Exon V5 probes developed by Agilent.The specific procedures followed the standard SureSelect exome capture protocol, including genomic DNA fragmentation, end repair, adapter ligation, PCR amplification, probe hybridization to target regions, and enrichment using magnetic beads.Sequencing was carried out on Illumina HiSeq2500 or Next500 platforms, with a sequencing length of 2 × 125 bp.Data processing involved the following steps: (1) after sequencing, raw data in FASTQ format were obtained from the sequencing platform.Using command-line tools in a Linux work environment, the data was processed as follows: (i) the BWA software (Burrows-Wheeler Alignment tool) compared the raw sequencing data with the human reference genome (hg19) to obtain BAM-formatted files, which could be visualized using the IGV software.(ii) The Picard software was used to remove duplicate reads and evaluate data quality.(iii) Single-nucleotide variations and insertions/deletions (Indels) were identified using the GATK software (Genome Analysis Toolkit), which produced files in VCF format.The wANNOVAR software (http://wannovar.usc.edu/) was then utilized to annotate the variants from various perspectives, facilitating subsequent filtering.Variant filtering mainly relied on mutation frequency, as well as the location and type of the variants.Databases such as the 1000 Genome Project, ESP6500, and ExAC (Exome Aggregation Consortium) were used as references for mutation frequency.
The analysis and interpretation of the results involved the following steps: rare variants that passed the frequency and type filtering criteria were evaluated based on whether they were inherited from parents and whether they were documented in OMIM (http://www.omim.org/)and HGMD (http://www.hgmd.org/)as disease-causing genes.Clinical presentations and inheritance patterns documented in previous reports, along with predictive analysis of the variants using tools such as PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2), SIFT (http://sift.jcvi.org/),and Mutation Taster (http://www.mutationtaster.org/),were considered in a comprehensive assessment of the pathogenicity of the variants.The ACMG classification criteria for variants were consulted, and considering the experience from previous studies, rare variants that matched the clinical presentations and inheritance patterns were considered pathogenic if they fell into the following categories: previously reported pathogenic mutations and new mutations in known disease-causing genes (including nonsense, frameshift, start codon, stop codon, and splice-site mutations).Further investigation of novel missense variants, which have been identified as disease-causing genes and have been verified to be associated with height variation using GWAS, but have not been reported to have pathogenic mutations, would have scientific significance.
Primers were designed utilizing Primer Premier 5.0 software to validate the results.Sanger sequencing was then conducted to confirm the pathogenic or potentially pathogenic variants and determine their parental origin.If no pathogenic variants were found through exome sequencing, further genetic chip testing would be conducted.
Short stature genes  3

General information
This study included a total of 56 patients, consisting of 39 males (69.64%) and 17 females (30.36%).The mean age of the participants at the clinic presentation was 6.12 ± 3.14 years.For male and female patients, the mean genetic target heights were 165.98 ± 3.67 and 156.57± 4.36 cm, respectively.Regarding the HtSDS, our population presented a mean score of −3.29 ± 0.91, indicating an overall reduced stature compared to the standard population.Meanwhile, the mean peak GH value was found to be 8.63 ± 4.04.

Possible pathogenic gene mutation
A total of 97 different gene mutations were discovered among the 56 subjects included in the study.The top three most frequent gene mutations were ACAN, PHEX, and COL2A1.Of these potential pathogenic genes, missense mutation was the most common mutation type, followed by deletion mutation, intragenic mutation, splicing mutation, nonsense mutation, frameshift mutation, and other mutations (Figure 1).

Copy number variations (CNV) of possible pathogenic genes
CNV is a type of genetic variation that involves alterations in the number of copies of a specific segment of DNA.Normally, a particular DNA segment exists in two copies to correspond with the common diploid human body.However, in the case of genotype copy number variation, this number could range from one to three or even higher.CNV can vary in size, ranging from 1 kilobase (kb) to several megabases (Mb).Among these potential pathogenic genes, both non-CNV and deletion-CNV were identified.It is worth noting that deletion-CNV was most closely associated with PHEX (Figure 2 and Table 1).

Prediction of protein functional damage caused by these possible pathogenic gene mutations
To assess the protein functional impact of these potential pathogenic gene mutations, analyses were performed utilizing MutationTaster, SIFT, and PolyPhen-2.The results of the MutationTaster analysis revealed a substantial increase in the disease-causing protein functional damage.On the other hand, SIFT analysis revealed a predominant enrichment of tolerated protein functional damage.In addition, PolyPhen-2 analysis revealed a significant prevalence of functional damage to benign proteins (Figure 3).

The chromosomal location of possible pathogenic genes
Figure 4 displays the chromosomal locations that exhibited the highest enrichment of potential pathogenic genes, with chr12, chr5, and chr2 being the top three.Furthermore, Table 2 provides detailed information on the nucleotide alterations and corresponding amino acid changes of these potential pathogenic genes.

Discussion
The comprehensive genomic survey presented in this study identified a diverse range of genetic variations in pediatric patients with short stature.The study revealed a total of 97 gene mutations, with the most common mutations found in genes like ACAN, PHEX, and COL2A1.These variations included missense, deletion, splicing, and nonsense mutations, along with copy number variations, potentially impacting protein function and physiological processes.These genetic alterations have the potential to significantly influence biological systems and human health, underscoring the importance of understanding the genetic basis of short stature for clinical management and therapeutic strategies.The rationale for the study stems from the significant impact of genetic variations on human health, particularly in the context of conditions such as short stature, diabetes, and cancer.It is vital to comprehend the particular genetic variations selected for examination and their significance to human health to clarify their possible consequences and develop personalized healthcare strategies.Due to their capacity to affect vital cellular processes and contribute to susceptibilities to disease, the genetic variations selected for investigation, including missense mutations, nonsense mutations, and silent mutations are of the utmost importance to human health.These variations were selected based on their known associations with regulatory pathways governing skeletal growth, insulin signaling, and cellular proliferation, all of which have direct implications for physiological well-being and disease states.Genetic variations and short stature: Short stature is a prevalent issue in the field of pediatric medicine, with a significant number of cases thought to be caused by genetic factors.The genes that control the growth and development of the skeleton, specifically those involved in the GHRH-GH-IGF-1 pathway, have a significant impact on an individual's stature.Mutations in these genes, including missense mutations, can directly impact skeletal growth and developmental pathways, making them pertinent targets for investigation in the context of short stature.Numerous missense mutations were identified in genes including HUWE1 (NM_031407), IGF1R (NM_000875), and IGF2 (NM_000612.6), among others.Missense mutations result in the change of a single nucleotide, leading to a different amino acid in the protein sequence.The resultant proteins may experience substantial structural and functional changes due to these substitutions, which may affect the biological processes that they regulate.For example, HUWE1 (HECT, UBA, and WWE domain containing 1) is a gene known to be involved in protein ubiquitination, a process critical for regulating diverse cellular functions.Mutations in this gene have been associated with X-linked mental retardation and may contribute to oncogenesis in various tissues, but there is no direct correlation provided between HUWE1 missense mutations and short stature.Mutations in the IGF1R gene, which encodes the insulin-like growth factor 1 receptor, could potentially disrupt insulin signaling and glucose metabolism, contributing to diseases like diabetes or cancer.Some of the identified missense mutations could affect genes involved in insulin signaling pathways, such as the IGF1R [25].Missense mutations can cause a functional disruption of IGF1R, which can result in altered insulin signaling.This can have an impact on glucose metabolism and insulin sensitivity.These alterations may potentially contribute to the  onset of insulin resistance, which is a defining characteristic of type 2 diabetes.Besides, missense mutations in genes related to glucose metabolism, such as IGF2, may perturb normal glucose homeostasis [26].Similarly, mutations in IGF2, an important growth factor in human development and growth [27,28], may cause abnormal growth and development, and potentially contribute to the onset of diseases such as Beckwith-Wiedemann syndrome or cancer.sVariations in the function of IGF2 may have an impact on the utilization and regulation of glucose, which may have implications for the risk of developing diabetes.Missense mutations in genes involved in cell signaling pathways, such as IGF1R, can impact cellular processes related to proliferation and survival.Dysregulated IGF1R signaling due to missense mutations can contribute to uncontrolled cell growth and reduced apoptosis, which are characteristic features of cancer development and progression.Most notably, the ZNF764 (XM_001300919) and IFITM5 (NM_001025295) genes have been identified to contain nonsense mutations.The introduction of premature stop codons into the coding sequence by these mutations almost certainly results in a truncated protein product.Depending on the location of the truncation, the protein may completely lose its functional capability or gain novel, inappropriate functions, a phenomenon known as gain-of-function mutation [29,30].Some of these mutations have the potential to cause severe damage, manifesting as disease states or aberrant cellular behavior.When examining the nonsense mutations that have been identified in ZNF764 and IFITM5, it is critical to consider the possible mechanisms by which truncated protein products could acquire inappropriate and novel functions.Additionally, it is crucial to consider the implications of these mechanisms on cellular behavior and disease states.Novel functions of truncated proteins may manifest as aberrant interactions, altered subcellular localization, or dominant-negative interference.Additionally, these mutations can lead to dysregulated signaling pathways, disruption of protein complexes, and altered gene expression.
Notably, silent mutations have been detected in our dataset as well, specifically in the KMT2C and POLR1A genes.Silent mutations, also known as synonymous mutations, are DNA sequence changes that do not result in an alteration of the encoded amino acid within the corresponding protein [31].Traditionally, silent mutations were regarded as benign conditions that did not affect the function of proteins.On the other hand, recent studies have revealed that these mutations have the potential to impact protein synthesis and overall cellular function by causing disruptions in splicing regulation, gene expression, and mRNA stability [32].Silent mutations can influence gene expression levels by altering codon usage, affecting the rate of protein translation, and potentially impacting overall protein abundance.Furthermore, these mutations have the potential to affect the structure and stability of mRNA, which may affect the efficacy of mRNA degradation and processing.Furthermore, it has been discovered that it exerts an impact on alternative splicing, thereby influencing the synthesis of distinct protein isoforms from a single gene.An investigation into the inherited blood disorder beta-thalassemia demonstrates how silent mutations can affect splicing efficacy, ultimately resulting in the onset of the disease [33].These silent mutations affect the splicing of pre-mRNA, resulting in aberrant splicing patterns and the production of abnormal hemoglobin, contributing to the pathogenesis of beta-thalassemia.Further research is needed to determine the exact effects of silent mutations in the POLR1A and KMT2C genes, however, when evaluating their significance to disease processes, it is crucial to take into account the possible regulatory functions of these mutations in gene expression and protein synthesis.The present investigation identified particularly noteworthy mutations in the genes PHEX (NM_000444), ZC4H2 (chrX), and KMT2C.These mutations led to a condition known as 'amino acid deficiency'.Mutations of PHEX and ZC4H2, it likely indicate missing segments in the amino acid sequence due to nucleotide deletions [34].Amino acid deficiencies arising from nucleotide deletions can have a direct impact on the functionality of the encoded proteins in the PHEX and ZC4H2 contexts, which are associated with X-linked hypophosphatemia and Wieacker-Wolff syndrome, respectively.This may disrupt critical biological pathways, leading to the characteristic signs and symptoms associated with these genetic disorders.Further investigation is required to definitively identify what is meant by 'amino acid deficiency' and the implication it has on protein function and overall physiology.
Furthermore, synonymous mutations or mutations resulting in an apparent "no amino acid change" were observed, suggesting that these genetic changes do not cause a modification to the sequence of the encoded protein.It has been widely accepted that these mutations remained "silent" and had no discernible impact [35].However, we now know, that synonymous mutations can influence gene expression levels, alter mRNA stability and structure, or affect splicing regulation.Hence, these 'innocuous' mutations might have been overlooked and require thorough investigation.
In earlier research, Chen and colleagues [10] identified 24 potentially harmful or detrimental variants of collagen genes in patients exhibiting skeletal abnormalities and short stature; COL2A1 mutations were the most prevalent, accounting for around 57.7% of each case.Additionally, they identified prevalent mutations associated with skeletal development, encapsulating FGFR3, COMP, NPR2, ACAN, and FBN1.These results have a few similarities to this study, however, this study further added to their study by finding a series of new possible pathogenic gene mutations and presenting the gene feature of Chinese patients with short stature.
The identified genetic alterations discovered in this study represent novel and significant findings with potential implications for both diagnostic and therapeutic applications in a clinical setting.The diversity of genetic variations, including missense, deletion, splicing, and nonsense mutations, presents a wealth of potential targets for further exploration in the context of personalized medicine for individuals with short stature.Particularly in individuals with growth-related disorders, the existence of missense mutations in genes including IGF1R, IGF2, and HUWE1 holds potential for the advancement of targeted therapies.The identified mutations may have clinical significance across a range of growth-related disorders, as they have been linked to growth failure, developmental disorders, and Xlinked mental retardation.Additionally, the presence of silent mutations and their impact on gene expression, mRNA stability, and splicing regulation suggests the potential for personalized treatment approaches tailored to the specific genetic profile of individuals with short stature.Utilizing the identified genetic alterations as diagnostic markers could enable more precise and individualized diagnostics, leading to earlier detection and intervention for individuals with underlying genetic causes of short stature.Furthermore, the potential therapeutic relevance of the identified genetic alterations extends to the development of novel treatment modalities, including gene therapy and targeted pharmacological interventions.By understanding the specific genetic variations contributing to short stature in individual patients, tailored therapeutic strategies could be developed to address specific molecular defects, potentially leading to more effective treatments and improved clinical outcomes.The implications of these findings extend beyond short stature, as the genetic variations identified in this study may also have relevance to a broader range of growth and developmental disorders.The discovery could greatly benefit our understanding of short stature and the development of personalized treatment methods for many different genetic disorders that impair growth and development by revealing the genetic basis of these conditions.Therefore, additional studies are needed to confirm the functional effects of the found genetic variations, particularly in the context of short stature and related growth and developmental disorders.Experimental validation is crucial to elucidate the specific consequences of the identified mutations on protein function, cellular processes, and overall physiological outcomes.
Despite the comprehensive nature of the genomic survey conducted in this study, several inherent limitations need to be addressed.First, a significant constraint is the absence of functional validation for the identified mutations.Although various genetic variations, such as missense, deletion, splicing, and nonsense mutations, were effectively identified in the study, their experimental validation did not establish their functional impact on protein activity or cellular function.This limitation hinders the ability to definitively assess how these mutations may alter biological processes and contribute to disease susceptibility or pathogenesis.Additionally, the study did not consider the interplay with the epigenetic landscape.Epigenetic factors, such as DNA methylation and histone modifications, can modulate gene expression independently of changes in the DNA sequence.The potential regulatory effects on protein function and gene expression were not taken into consideration due to the lack of analysis concerning epigenetic modifications.Considering the significant role of epigenetics in regulating gene expression and cellular behavior, this oversight limits the comprehensive understanding of the genetic contributions to short stature.Furthermore, the study did not address the potential interactions between multiple mutations within the same individual.It is increasingly recognized that the cumulative effects of multiple mutations may have synergistic or antagonistic impacts on protein function and cellular pathways.Understanding the potential interactions between different mutations is vital for unraveling the complex genetic architecture underlying short stature.Therefore, the lack of consideration for potential epistatic interactions between mutations represents a notable limitation in the interpretation of the results.These limitations collectively impact the interpretation of the results by highlighting the need for caution when drawing direct associations between the identified mutations and the observed phenotypes.Without functional validation, insights into the specific consequences of the mutations on protein function and cellular processes remain speculative.Additionally, the absence of considerations regarding the epigenetic landscape and potential interactions between multiple mutations may result in an incomplete representation of the genetic contributions to short stature, potentially leading to oversimplified or erroneous conclusions regarding the genetic etiology of the condition.

Conclusion
In summary, our investigation reveals an extensive range of genetic variations that could have various and significant implications for short stature.The results of this study pave the way for future research to explore the functional implications of these genetic variations and evaluate their potential as therapeutic targets or diagnostic indicators.However, the incorporation of these genetic alterations into predictive or therapeutic models necessitates careful consideration, considering the possible modification of their effects by other genetic or epigenetic factors, environmental influences, or biological randomness, which still requires further comprehension.

Figure 1 :
Figure 1: Possible pathogenic gene mutation and its variation classification.

Figure 2 :
Figure 2: Copy number variations of possible pathogenic genes.

Figure 3 :
Figure 3: Prediction of protein functional damage caused by these possible pathogenic gene mutations.

Figure 4 :
Figure 4: The chromosomal location of possible pathogenic genes.

Table 1 :
Possible pathogenic gene mutation and its variation type, related disease, and source of variation

Table 2 :
The nucleotide alteration and amino acid change of possible pathogenic genes