WITHDRAWN: SHOX variations in Idiopathic Short Stature in North India and its overall prevalence in Asia

Abstract

Background

Short stature homeobox (SHOX) haploinsufficiency underlies idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). The worldwide prevalence of SHOX variations in ISS varies from 2.5–15%. This study aims to assess the implication of SHOX variation in ISS in North India and its overall prevalence in Asian population.

Methods

SHOX gene analysis carried out by Multiplex ligation dependent probe amplification (MLPA) followed by Sanger sequencing in 54 patients with variable phenotypes. Meta-analysis was done to evaluate its prevalence which comprises 11 previous studies including present, carried out on a total of 979 participants.

Results

SHOX gene analysis resulted in 12.9% of positivity (7.4% deletions and 5.5% duplications). SHOX association was seen significantly related to gender, with predominance in female sex (P-value: 0.047). Short arms and forearms were the only significantly associated trait seen in 51.85% of children. We have found a rare association of multiple exostosis with LWD in a child with deletion. The prevalence of SHOX variation came as 15.2% in Asians. No significant difference was found in geographical region-specific analysis.

Conclusion

This meta-analysis compiles findings from the last decade and provides an updated picture of the prevalence of SHOX variations in Asians, emphasizing their potential as therapeutic targets in ISS patients.

Introduction

Idiopathic short stature (ISS) is defined as height of a child which is more than 2 Standard deviations (>2 SD) below the respective mean height in a given age, sex, and population without features suggestive of endocrine, chromosomal, nutritional or any systemic abnormality and having a normal stimulated growth hormone (GH) levels (Cohen et al., 2007). ISS affects around 2 % of the children. It is a genetically heterogeneous condition. With the advancing techniques, a battery of genes is known to be involved in causing longitudinal growth in human beings, but despite this fact, genetic causes constitute only a small proportion of cases of short stature (Rosenfeld et al., 2004). One of the genes which is highly implicated in this process is the short-stature homeobox-containing gene (SHOX), which is responsible for encoding a pseudoautosomal homeobox-containing osteogenic gene; PHOG which is known for causing short stature in Turner syndrome (Rao et al., 2001). The importance of this gene is known from the fact that it is one of the most common genetic causes leading to short stature in children with either isolated or familial short stature.

The location of SHOX gene is characteristic and is responsible for the variable phenotype its variation produces. Being located over the tip of the short arms of both sex chromosomes X and Y inside the telomeric part of pseudo-autosomal region 1 (PAR1), which is named due to its presence over both these chromosomes. The location also is pivotal as genes in this area escape the process of lyonization. It is seen that SHOX gene functions in a dose-dependent manner i.e., loss-of-function variation which involves only a part of the gene i.e., involving one of the SHOX allele, technically known as haploinsufficiency, it can lead to a wide variety of phenotype manifesting as the deficiency of the gene and resulting in short stature of varying degrees. From the knowledge that exists regarding the gene, SHOX deletions are the most common type of variations encountered worldwide, known to account for 80% of all variations. These deletions can be of varying sizes and can involve either the gene itself or regions above and below it, which contains a regulatory enhancer region. Other variations which have also gained prominence are the nonsense and missense sequence alterations seen commonly within the gene. Most common variations are often seen in exons 3 and 4 which are known to encode the functional part of this pivotal homeodomain (Schneider et al., 2005).

In different studies across the world, estimates of the presence of SHOX gene variation leading to this idiopathic type of short stature ranges from 2.5% to 15% (Huber et al., 2006; Jorge et al., 2007; Hirschfeldova et al., 2012; Binder et al., 2011). The prevalence of ISS in India due to SHOX variations is not known. In the last decades, many studies have been done on the association between different gene variations and ISS. The SHOX variations are one of the most common cause of ISS. The prevalence of this variation varies in between 2-15% according to the ethnicity and selection of patients. Some studies from Asian populations have reported important frequencies in comparison to the worldwide populations. In this study, we have selected 54 individuals with ISS (height less than 2SD of population reference range height, with no identified cause). These 54 selected individuals had their detailed anthropometric evaluation, dysmorphism evaluation and skeletal survey by radiographs. Multiplex ligation dependent probe amplification (MLPA) and Sanger sequencing of exon 2 and 6 were done to identify SHOX deletions and sequence variations, respectively. Additionally, we aimed to estimate and evaluate the potential relationship between SHOX variation and ISS in Asian populations.

Materials And Methods

Subjects: This was a prospective study conducted at the Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, from July 2020 to March 2022, with approval from the Ethics Committee of the institute (Approval no.: NK/6656/MS/315). A detailed physical examination of children with idiopathic short stature was done to look for dysmorphic features “micrognathia, high-arched palate, short forearm, cubitus valgus, short hands, characteristic Madelung deformity, dislocation of ulna at the elbow, short lower leg, bowing of tibia, genu valgus, short feet, scoliosis, muscular hypertrophy. The study children were measured for the following anthropometric parameters using standardized techniques and instruments, in the Growth Laboratory/ Clinic: Height (cm), Weight (kg), Sitting Height (cm), Arm Span (cm). Body Mass Index (kg/m²), Arm span to height ratio as well as upper segment to lower segment ratio was also calculated. A written informed consent was obtained from each patient and their parents or legal guardians, in case of minors after explaining the purpose and type of all procedures used.

Consent to participate- Written informed consent was taken from the parents.

Rappold scoring: Rappold scoring was done for the better delineation of the clinical expression in our cohort (Rappold et al., 2007).

Molecular analysis: Genomic DNA was extracted from peripheral blood by QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany). SHOX analysis was done using SALSA P018-G2 kit (MRC-Holland, Netherlands – Resnova, Italy) as per the manufacturer’s instructions. The entire coding region of the SHOX gene (exon 2–exon 6a/6b) and splice junctions were sequenced by Sanger sequencing using automatic sequencer ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). Primer sequences will be available upon request.

Meta-analysis: Meta-analysis of prevalence was done using MedCalc software. This study followed the PRISMA guidelines (Liberati et al., 2009). To identify the publications interested in studying the association of SHOX gene variations in ISS in Asian, we have done the thorough literature mining through various databases including PubMed, Scopus and Web of Science. The search string was a combination of the following keywords: “Idiopathic Short Stature”, “SHOX”, “deletions/duplications/variations”, “copy number variations”. All the studies published in last 10 years (2012-2022) have been selected for this study. The selected studies met the following eligibility criteria:

- The region: Asian.

- The period: last 10 years.

- The language: English.

- Peer reviewed publication.

Included studies were original research articles written in English, related to SHOX prevalence, variations and/or incidence along with the number of individuals. The authors independently assess the risk of bias for the included studies, using Newcastle-Ottawa Scale (NOS) tool for quality assessment (Wells et al., 2015).

Statistical analysis: The strength of the association between SHOX variation and ISS in Asian population was evaluated by calculating the odds ratio (OR) using the Mantel-Haenszel statistic method. Fixed/random-effect model was applied along with a corresponding 95% confidence interval (CI). Fixed-effects models were used to assess the pooled prevalence of genes for results with low heterogeneity (I2 ≤50%). Otherwise, random-effects models were applied for the analyses. MedCalc software, (Version 20.111) was used for all data synthesis and statistical analyses. Chi² and I² statistics were used for calculating heterogeneity across individual studies and subgroups. Population bias was assessed by Funnel-plot analysis. Additionally, subgroup analysis was done according to the ethnicity/regions of the population.

Results

Demographic characteristics: Among the 54 enrolled participants, 5 (9.25%) were in the age group 1-5 years, 20 (37%) were in the age group of 5-10 years, 26 patients (48%) lied in 10-15 years age group and 3 patients (5.5%) were above 15 years of age. The median (IQR) age of children in this study was 11years [8years,13years]. The study included a total of 26 females (48.1%) and 28 males (51.9%) (Table 1). 

Physical characteristics: Phenotypic characteristics of enrolled patients are given in Table 2. Short upper limbs (arms and forearms) was the most consistent feature (found 28 of 54 subjects) and was common than short lower limbs (24 of 54 subjects). Short upper limbs were a significant finding in the variation positive subjects (Table 4B). One subject had multiple exostosis around both knee joints (Fig 1a, b). She was found to have SHOX deletion. 

Anthropometric data: Mean, SD and Range for the measured anthropometric parameters among the study cohort (n=54) is shown in Supplementary Table 1. Most of the anthropometric parameters as analyzed using SPSS software represented a moderately skewed distribution (almost towards normal distribution for practical purposes) with a skewness index ranging between +1 to -1. While, detailed physical growth and pubertal characteristics of study subjects who were positive for SHOX gene (n=7) are depicted in Table 3. Interestingly, four out of these seven cases had initially presented in the endocrinology OPD of our Department and were advised Growth Hormone therapy. However, due to financial constraints or loss to follow-up, growth hormone could not be initiated in these children except one (case 3) in whom a marked increase in height gain was noticed (from -6.4 SDS at 8 years to -3.29 SDS at 13 years).

SHOX gene testing: SHOX del/dup in the study was found in 12.9% cases in the study (7 out of 54 children) using MLPA, wherein heterozygous deletion of exons was seen in 4 children (7.4%), and duplications in SHOX gene was noticed in 3 children (5.5%). Among positive patients, 14-year female child had heterozygous deletion of exon 4. Heterozygous deletion of exon 5 of SHOX gene was seen in Case 2. Thirteen-year male child showed heterozygous deletion of exon 5 and 6 and 4^th case showed heterozygous deletion of exon 4. Exon 4 deletion of SHOX gene was found in 3 patients out of 7 positive patients. Rest of 3 patients showed duplications; one had complete duplication of SHOX gene, while another showed duplication of exons 1, 2, 3 including upstream region. One patient was found to carry duplication downstream region of SHOX gene. MLPA results are given in Supplementary Fig 1.

Clinical characteristics of positive patients: Clinical parameters of seven positive patients, their Rappold scoring (Supplementary Table 2) and phenotypic features are given in Table 3 and 4 respectively.

Correlation of gender with SHOX gene variation: Among a total of 54 cases, SHOX gene variation was present in 3.5 % boys and 23.07% of girls enrolled. This was significant relation and as was found from Fischer’s exact T- test, there is a significant relationship (p=0.047) between SHOX gene variations and female sex (Table 4A).

Correlation of phenotypic parameters with SHOX gene variations: From among all the traits which were noted among the enrolled patients, short arms and forearms were found to be statistically significant and related to those with ISS having SHOX gene variations, whereas rest of the phenotypic characters were not found to be linked with variation. The phenotypic characters and their respective association are summarized in (Table-4B). Cubitus valgus and madelung deformity, which are characteristic features of LWD, were observed in 1 out of 7 (14%) variation positive subjects.   

Correlation with Anthropometric data: No significant association was found in height, Mid-parental height weight, BMI, US:LS ratio, Arm span to linear height ratio (data not shown).

Correlation with radiological parameters: Radiologic survey was done using X-ray of wrist, arms and forearm, legs, thoracolumbar spine and chest X-ray. However, no significant association was established between abnormal radiological findings and SHOX gene variation.

Meta-analysis in Asian population: A total of 852 studies were retrieved from different databases and screening criteria. After the removal of duplicates (692 studies), 160 studies were considered potentially eligible for evaluation, but 149 did not meet all the inclusion criteria, leaving 11 studies (Fig 2). This meta-analysis comprises 11 previous studies including present study, carried out on a total of 979 participants.

Study Characteristics: Detailed characteristics of the studies are provided in Table 5 with NOS scoring. Studies were conducted on 6 different countries of Asia which were further subcategorised into South, West and Eastern part. Most of the studies used MLPA as a major method, followed by Sanger sequencing, chromosomal microarray (CMA) and fluorescent in situ hybridisation (FISH) analysis. 

Prevalence of SHOX variation: Variations in SHOX gene were identified in 83 patients from total 979 patients. Using random effect model, the mean prevalence of SHOX variations was 14.3% (95%CI, 5.037-2.699, p <0.0001, I2 = 95.10%) (Fig 3a, Table 6A). The funnel plot was asymmetric, suggesting the possibility of publication bias Fig 3b.

SHOX variations in South, West and East Asia: Further prevalence and association were calculation on the basis of different regions of Asia. It has been divided into South Asia (134), West Asia (226) and East Asia (619). No significant differences were observed in these rates in different regions of the Asia. The mean frequency of SHOX variations in South Asia was 10.4%, with this rate being higher than the rates in other regions, including East Asia (8.4%), and West Asia (7.5%) (Table 6B, Fig 4).

Discussion

SHOX gene deficiency is one of the single gene disorders of the bone resulting in highly variable osteodysplasia and hence affecting the overall height in children. However, as the knowledge of the gene and allelic alterations in the gene have evolved over time, it is now well known that isolated deficiency of either the SHOX gene or its modifiers up and down the Par region are responsible in a spectrum of disorders ranging from simple idiopathic short stature to more severe spectrum of disorders like “Leri–Weill dyschondrosteosis (LWD, MIM 127300) and Langer Mesomelic dysplasia (LMD, MIM 249700)”. In present study, SHOX gene variation was found in 12.9% cases in the study (7 out of 54 children) using MLPA and Sanger sequencing, wherein heterozygous deletion of exons was seen in 4 children (7.4%), and duplications in SHOX gene was noticed in 3 children (5.5%). 

The skeletal findings in Leri–Weill dyschondrosteosis are characteristic, however, in one of our subjects, multiple exostosis was observed around the knee joints. It’s rare and was observed in some members in a multigenerational non-consanguineous North African family, reported by Al Kaissi A et al. 2016.

In studies conducted earlier, and as mentioned in SHOX database, this value ranges from 2-15% among children with Idiopathic short stature (Huber et al., 2006; Jorge et al., 2007; Hirschfeldova et al., 2012; Binder et al., 2011). In study by Hirschfeldova et al, MLPA analysis detected a total of 13.7% of SHOX gene anomalies including 4 deletions, 1 duplication with an “ambiguous effect” and 2 SHOX gene point variations (Hirschfeldova et al., 2012). In the same study single isolated enhancer duplication was also observed in LWD group. In our study, 3 duplications (5.5%) are observed, with no enhancer effect noticed, and was found in children with short stature. In present study, SHOX gene association was seen significantly to be related to gender, with predominance in female sex (P value: 0.047), and had no significant age correlation. Short arms and forearms were the only trait seen in 51.85% of the children and was found to be significantly related to SHOX gene variation in the study (P value: 0.024) and the rest of phenotypic traits were not found to be significantly associated. Seven children who had SHOX gene mutated were thoroughly studied for the phenotypic- genotypic correlation.

For the better delineation of the clinical expression in our cohort, the Rappold scoring was done for the investigated patients. Interestingly, all patients had varied scores with a median value of 3 (range 0-10). Short forearm was the most observed feature (62.5%), while cubitus valgus was observed in 2 patients. All the reported BMI were less than 50th percentile. Unexpectedly, arm span/height ratio was uncommon (28.5%), whereas bowing of forearm and tibia was present in only 1 patient. Sitting height/height ratio (>55.5%), dislocation of ulna at the elbow and muscular hypertrophy were absent in all the subject (Table 1).

In different studies conducted in different populations, the prevalence of SHOX variations in ISS children represent a wide array. The study conducted by Suppia et al., 2003 showed around 7% patients with ISS having SHOX gene deletion, while Musebeck et al., 2001 conducted a study over 35 patients of ISS, none of whom had the gene deletion.

This meta-analysis aimed to investigate the association between the SHOX variation and ISS in Asia. Data was included from 11 selected studies, which involved 979 ISS cases. Overall prevalence of SHOX variation in Asia was found to be 14.3%. These results indicate the implication and the association of these variations with ISS in Asia. For comparison, the participants were divided into South, West and East Asian sub-groups. In South Asia, the results indicated the presence of SHOX variations in 10.4% of the patients with ISS.

Among South Asians, Kumar et al., 2020 reported pathogenic heterozygous variants in 4 children (6.5%). Variations included exon 5 duplication, splice site point variant c.278-1G>C in exon 3, partial deletion and complete deletion. Singh et al., 2018 showed SHOX haploinsufficiency in two patients, while one patient had mosaic gain in SHOX. 

In West Asian population, Dalil et al., 2016 found one patient (2.6%) with SHOX variation. Contrarily, Bakir et al., 2018 has not found any variation in the exonic sequences and exon/intron boundaries of the SHOX gene. Alharthi et al., 2017 found only one variation in exon 4 of SHOX gene while rest of patients had polymorphisms in exons 1, 2, 4, and 6. Gursoy et al., 2020 showed three different point variations and one whole SHOX gene deletion 15 patients from four different families. Overall prevalence in West Asia came around 7.5%. 

In Eastern Asia, Fukami et al., 2015 reported six rare CNVs in PAR1 in 245 patients. Lee et al., 2021 have confirmed SHOX deficiency in 23 patients from 15 unrelated families. In a study by Tung et al., 2018 SHOX gene intragenic deletions were found in five patients, one deletion in the regulatory region, and a missense variation at exon 5. Prevalence of variation in SHOX gene varied in between different geographical regions of Asia, being highest in South Asia. While Shima et al., 2016 stated SHOX abnormalities in 3.8% of ISS and 50% of LWD cases. These results indicate the difference in the prevalence of the SHOX variations according to the selection criteria, used methods, sample size, and population.  

The meta-analysis is a compilation of findings from the last decade and an updated picture of overall prevalence of SHOX variations in Asians, underscoring its potential as a main target in ISS patients. Further investigations of higher quality, large cohort size with functional validation are warranted to validate this association.

Declarations

Acknowledgements: The authors wish to thank the families of all the children with ISS who participated in the study.

Conflict of interest- Authors have no relevant financial or non-financial interests to disclose.

Funding: This work was supported by the Institute’s special research grant for DM/MD thesis.

Data availability: All data generated or analysed during this study are included in this published article.

Author contributions: All listed authors have made substantial, contributions and have approved the submitted version. All authors have read and agreed to the published version of the manuscript. Conceptualization: PS, Data curation: AT, CB, Patient recruitment: DD, KM, SP, AK, IP, Experimentation: AK, AT, SS, Analysis: PS, AT, HK, Literature search and quality scoring: CB, AT, PS, writing original draft: PS, AT, Writing-review and editing: HK, AK, IP, DD, KM.

Ethical approval- The study protocol was approved by Thesis Committee and Ethics Committee of the Postgraduate Institute of Medical Education and Research, Chandigarh, India. 

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Tables

Table 1: Demographic details of enrolled patients

Table 2: Physical characteristics of the recruited patients

Table 3: Clinical parameters of positive patients

Table 4A: Correlation of gender with SHOX gene mutation

Table 4B: Association of phenotypic features with SHOX mutation

Table 5: Characteristics of studies included for analysis

Table 6A: Prevalence of SHOX mutation in Asian population along with test for heterogeneity and Publication bias

Table 6B: Prevalence of SHOX mutation in South, West and East Asian population