A proper genetic diagnosis leads to implementation of the appropriate treatment for SS. SHOX is one of the main genes involved in human growth and is an important candidate gene used to test SS in children. The majority of SHOX defects are due to complete deletions (70–80%) or partial deletion (2–6%) of SHOX and/or its regulatory enhancer region. A total of 20–25% of SHOX defects is due to mutations [6]. SHOX enhancers are conserved non-coding DNA elements (CNEs). There are three CNEs enhancers upstream of SHOX (CNE-5, CNE-3, and CNE-2), and there are eight CNE enhancers downstream of SHOX, four of which have transcriptional activity (CNE4, CNE5, ECR1 (CNE7), and ECS4 (CNE9)). Recently, ZED, the fifth enhancer, has been reported as a more likely candidate than CNE9 as a critical limb enhancer in the analyzed downstream region [13, 14].
SHOX haploinsufficiency results in syndromic and non-syndromic SS. SHOX haploinsufficiency is primarily due to submicroscopic CNVs in PAR1, encompassing SHOX exons and/or the cis-acting enhancers [2]. However, only one case out of the 50 studied (2%) showed a large de novo SHOX/PAR1 deletion, including complete SHOX and its regulatory regions. This deletion is associated with normal birth weight and low growth hormone levels.
In contrast, duplications inside or outside the SHOX area were detected in 18% of the children studied. All of these children had normal birth weights and growth hormone levels. SHOX duplications have been described in individuals with SS. Studying LWD and ISS patients identified that duplications in SHOX and its associated elements are less frequent than deletions of SHOX. SHOX-associated elements are conserved non-coding elements (CNE) adjacent to the 3′ end (CNE-3,4,5,7,8,9) and 5′ end (CNE-2,3,5) of the gene [4, 7]. Previous studies have investigated the frequency and distribution of duplications within SHOX and its associated elements between population samples and merged groups of ISS/LWD patients [7]. This group proposed that partial SHOX duplications and small duplications encompassing the CNE-9 enhancer could be highly penetrant alleles associated with ISS and LWD development.
While intragenic SHOX duplications lead to SS, whole gene duplications are associated with more heterogeneous phenotypes [4, 13]. Upstream duplication of CNE-5 and CNE-3 and downstream duplication of ECS4/CNE-9 have been described in ISS [4, 15, 16]. Here, we report three heterozygous duplications (6%) in the SHOX area. These include one duplication at exon one of SHOX, and two cases with duplications at exon 5, and one case with a duplication at exon 6, both of which interfere with the SHOXa transcript.
Moreover, we report a heterozygous duplication (2%) at hg19; chrX:1,029,698 (probe L24245) (SHOX down area). While studying a cohort of 1200 patients, previous studies have reported a recurrent 325 kb duplication in the SHOX area (SALSA MLPA probemix P018-G1, probes L20176-L24245) in seven families presenting with SS [13]. Four families presented with LWD in the affected family members, and three families presented with familial short stature. The findings in this study suggest that duplications of upstream and downstream long-range cis-regulatory DNA elements can result in a SHOX-related phenotype. Controversially, Hirschfeldova and Solc described this duplication area only as a polymorphism, as they detected it in their population sample (0.40%) and in only one case from their LWD/ISS samples (0.28%) [7].
The precise mechanisms of the pathogenic effects of microduplications are not yet understood. These duplications most likely exert a negative effect on SHOX transcription, which leads to the reduction of its expression, ultimately resulting in SHOX haploinsufficiency [13]. Partial SHOX duplications appear to have a more deleterious effect than complete SHOX duplications, due to the disruption of the gene sequence [4, 13].
Small duplications at the CNE-9 enhancer are associated with SS. This duplication could decrease effective communication between the CNE-9 enhancer and the SHOX promoter, negatively effecting SHOX expression [7]. However, no duplication of the CNE-9 enhancer was detected in the group of children that we studied.
Five children out of the 50 children studied (10%) had copy number gain outside of the SHOX area. Four of these children had an exon 8 duplication in the ASMT. Acetyl serotonin methyltransferase (ASMT) is the last enzyme in the melatonin biosynthesis pathway and is located in the PAR1 region of the sex chromosomes outside the SHOX area. Melatonin is a synchronizer of many physiological processes and abnormal melatonin signaling is associated with a broad range of diseases. There is data that suggests that there is a relationship between melatonin and growth hormone (GH) secretion, with reciprocal negative feedback regulation [17, 18]. Hirschfeldova et al. reported a recurrent duplication of the ASMT probe (SALSA MLPA probemix P018-G1, probe L00712) with a frequency of 2.3% in their cohort of 352 LWD/ISS patients [3]. However, Hirschfeldova and Solc detected this duplication at a frequency of 2.0% in their population of 250 individuals [3]. These frequencies are comparable to frequencies obtained by Cai et al., where the duplication of ASMT was reported to be associated with autism spectrum disorders [19]. None of the individuals from the Hirschfeldova et al. and Hirschfeldova and Solc studies that carry the ASMT duplication suffer from autism spectrum disorders [3, 7]. Similarly, none of the four cases in the present study that carry the ASMT duplication suffer from autism spectrum disorders. All these findings suggest the possible involvement of the ASMT in SS. Further studies with a larger sample size are needed to develop a better understanding of the role of the ASMT in SS.
The fifth child with a copy number gain outside of the SHOX area had CRLF2, (exon 2 hg19; chrX:1327689) and CSF2RA (exon 4 hg19; chrX:1401596) duplications. Mitka et al. reported an ISS patient with a duplication encompassing the Xp22.33-PAR1 regulatory region and CRLF2 and CSF2RA, which comprises 1.8% of the cases in this study [20].
Our study demonstrated that there was significantly higher expression of serum miR-140 in children with SS as compared to age and sex-matched healthy subjects (p < 0.05). Miyaki et al. identified that miR-140 null mice exhibit long bone growth retardation, which suggests reduced chondrocyte proliferation, although the structure of the articular joint cartilage appeared grossly normal [11]. Loss of miR-140 in mice leads to growth defects of the endochondral bones, resulting in dwarfism and craniofacial deformities [21]. Integrated study of miRNA expression and mRNA expression during chondrogenesis highlights the importance of miR-140, particularly the −5p strand, and identified positive regulation of the Wnt signaling pathway by miR-140-5p. Wnt signaling has a complex role in chondrogenesis and skeletal development [22]. The significantly high serum miR-140 expression levels observed in children with SS in the present study suggest that serum miRNA is associated with the pathogenesis of SS.