OR03-05 Male-Dominant Expression of SHOX, a Key Regulator of Growth: Implications for Sex Differences in Height

Abstract Disclosure: A. Hattori: None. A. Seki: None. Y. Naiki: None. A. Nakamura: None. T. Michigami: None. Y. Katoh-Fukui: None. T. Ogata: None. M. Kagami: None. M. Fukami: None. Introduction: Male-specific growth genes, alongside gonadal sex steroids, have been proposed as contributors to sex differences in height; however, no such gene has been identified. SHOX, a key growth gene located in the pseudoautosomal region on the short arm (PAR1) of the sex chromosomes, influences height in a dose-dependent manner. Conventionally, genes on PAR1, including SHOX, were thought to escape X-chromosome inactivation (XCI), thus being expressed equally in men and women. In 2017, Tukiainen et al. reported that most genes on PAR1 are expressed in a male-dominant manner. Nevertheless, sex differences in SHOX expression remain largely unexplored due to its highly tissue-specific expression. Objective: To explore the role of SHOX in sex differences in height. Methods: We compared male and female cartilage and/or chondrocyte samples using the following methods: (i) microarray analysis for X chromosome gene expression (n = 16), (ii) RT-qPCR for SHOX expression (n = 36), (iii) reduced representation bisulfite sequencing (RRBS) for X chromosome DNA methylation (n = 4), and (iv) pyrosequencing for DNA methylation of SHOX-flanking regions (n = 22). Furthermore, we established fibroblast clones derived from a boy and a girl with polydactyly, which enabled us to compare DNA methylation patterns around SHOX among the active and inactive X chromosomes and the Y chromosome using long-read sequencing. Results: Microarray analysis showed male-dominant expression of PAR1 genes, including SHOX. RT-qPCR confirmed male-dominant SHOX expression. RRBS revealed that the X chromosome exhibited female-dominant DNA methylation, which was pronounced in the X-specific region and mild in PAR1. RRBS also showed that SHOX-flanking regions exhibited sexually dimorphic DNA methylation. Specifically, male-dominant DNA methylation was observed in a region upstream of SHOX and a region spanning intron 5-exon 6a, whereas female-dominant DNA methylation was detected in intron 2. These findings were confirmed by pyrosequencing. In silico analysis suggested that the region with female-dominant DNA methylation in intron 2 functions as an enhancer of SHOX. Long-read sequencing of fibroblast clones revealed hypomethylation of intron 2 in the active X and Y chromosomes and hypermethylation in the inactive X chromosome. Discussion: Male-dominant SHOX expression in cartilage tissue indicates a role of SHOX in sex differences in height. Sexually dimorphic DNA methylation around SHOX implies that XCI represses SHOX expression in women. In particular, allele-specific DNA methylation in intron 2 (hypomethylation of the active X and hypermethylation of the inactive X) suggests that this region functions as an enhancer involved in the repression of SHOX expression due to XCI. Conclusion: Male-dominant SHOX expression, driven by the spreading of XCI to SHOX, likely contributes to sex differences in height. Presentation: Saturday, July 12, 2025