ACAN Repeat Number Polymorphism in Patients with Idiopathic Short Stature Open Access

Idiopathic short stature (ISS) is non-syndromic growth failure without endocrine abnormalities, skeletal dysplasia, or other chronic disorders [1]. ISS usually refers to postnatal growth failure [2], although some known ISS cohorts included individuals born small-for-gestational age (SGA) who exhibited no catch-up growth after birth (SGA-SS) [3, 4]. SGA-SS and non-SGA ISS are multifactorial conditions caused by genetic and environmental factors [1, 4]. SGA-SS is known to arise from monogenic mutations, pathogenic copy number variations, and imprinting disorders; however, these abnormalities jointly explain less than half of the cases [5, 6]. Likewise, although mutations in more than 15 genes, including ACAN and SHOX, have been implicated in non-SGA ISS [1, 3, 7], these abnormalities account for only a small percentage of the cases [8, 9]. These findings suggest that unrecognized factors play an important role in the etiology of SGA-SS and non-SGA ISS.

Aggrecan is a major component of the extracellular matrix of growth plate cartilage [10]. An aggrecan molecule consists of a core protein encoded by ACAN and chondroitin sulfate chains [11]. Aggrecan molecules are indispensable for cartilage development. Monoallelic loss-of-function variants of ACAN lead to SGA-SS or non-SGA ISS, while biallelic ACAN mutations result in spondyloepimetaphyseal dysplasia [9, 12, 13]. ACAN exon 12 contains a variable number of tandem repeats (VNTR) of 57 nucleotides (19 amino acids) [14, 15]. This VNTR regulates the function of aggrecan molecules, because each repeat provides attachment sites for chondroitin sulfate chains that mediate water retention [15, 16]. Short repeats of ACAN VNTR are known to increase the risk of intervertebral disk degeneration [17]. Moreover, Mukamel et al. [18] linked this VNTR to height variation in the general population. The authors reported that the repeat numbers of ACAN VNTR were almost linearly correlated with the height of 415,280 individuals of European ancestry.

To date, however, no studies have examined the possible association between ACAN VNTR and ISS. Usually, a part of ACAN exon 12 is excluded from mutation analyses for patients with ISS, because this region cannot be analyzed by standard short-read next-generation sequencing (NGS) [19, 20]. Here, we developed two hypotheses: (i) an extremely short repeat in ACAN VNTR can cause ISS as a Mendelian disorder, or (ii) relatively short repeats increase the risk of ISS through multifactorial mechanisms. To test these hypotheses, we analyzed the repeat numbers of 128 Japanese patients with ISS. We also examined the numbers in 100 ethnicity-matched control individuals, because Mukamel et al. [18] reported that people of African and European ancestries showed different distribution patterns of repeat numbers.

The patient group comprised 128 Japanese individuals (75 males and 53 females, aged between 9 months and 25 years) clinically diagnosed with ISS. Of the 128 patients, 63 (35 males and 28 females) satisfied the standard diagnostic criteria of SGA (birth weight and/or length of ≤−2.0 standard deviation score [SDS]) [4, 21] and were classified into the SGA-SS group, while the remaining 65 (40 males and 25 females) were categorized into the non-SGA ISS group.

These patients were selected from 240 unrelated Japanese individuals who underwent clinical evaluations between 2013 and 2024 because of their short stature. The 128 patients satisfied the following criteria: (i) height ≤−2.0 SDS of sex- and age-matched Japanese reference data; (ii) no growth hormone deficiency or thyroid hormone abnormalities; (iii) no other chronic disorder or environmental exposure that may affect growth; and (iv) no signs of skeletal dysplasia or congenital malformation syndrome. In addition, we excluded patients who carried pathogenic variants in major causative genes for ISS. To this end, we performed short-read NGS using a custom-made panel targeting 11 genes (ACAN, FGFR3, GH1, GHR, GHRHR, IGF1, IGF1R, IGFALS, NPR2, SHOX, and STAT5B) (Kazusa DNA Research Institute, Chiba, Japan). Moreover, we conducted methylation-specific multiplex ligation-dependent probe amplification or pyrosequencing for patients with SGA-SS and excluded those with imprinting disorders [4, 5].

The control DNA samples were purchased from the DNA Bank of the Japanese Collection of Research Bioresources (https://cellbank.nibiohn.go.jp/english) [22, 23]. These samples were obtained from healthy adult volunteers from various regions of Japan. Reportedly, these samples reflect the genomic data of the Japanese general population [23]. We utilized DNA samples from 50 males and 50 females.

Genomic DNA was extracted from peripheral leukocytes. The genomic region in ACAN exon 12 encompassing the VNTR was PCR-amplified. Primers used in this study were described previously [14]. PCR products were run on agarose gels and visualized with SYBR green. The number of repeats in each sample was assessed based on the size of the PCR products (online suppl. Fig. 1; for all online suppl. material, see https://doi.org/10.1159/000545736). Agarose gel electrophoresis was performed in duplicates.

To increase the accuracy of this experiment, we conducted long-read NGS on 12 samples with different-sized PCR products and used these samples as size markers. Four additional samples were also subjected to long-read NGS to confirm the results of gel electrophoresis. Libraries were prepared using the Native Barcoding Kit 24 V14 (Oxford Nanopore Technologies [ONT], Oxford, UK), and long-read NGS was performed using PromethION 2 Solo (ONT). The sequence reads were base-called using the MinKNOW software integrated with the Dorado Basecaller Server (ONT). The reads were mapped using minimap2 (biocrates life sciences, Innsbruck, Austria) to the human reference genome (GRCh38) that contains 27 repeats in ACAN VNTR. The results were visualized using Integrative Genomics Viewer (https://igv.org/).

The distribution of the repeat numbers of all alleles of the patient group was compared to that of the control group. We also analyzed the repeat numbers of the shorter and longer alleles in each individual, as well as the average numbers of the two alleles. The normal distribution of the number of repeats was confirmed visually. A linear mixed model was used to analyze the statistical difference in the number of all alleles (two variables from each participant) between the two groups, while Welch’s t test was used to analyze the remaining items (single variable from each individual). p values of less than 0.05 were considered significant. We also examined whether some patients carried a very small number of repeats (less than 13 repeats [17]).

Next, the distribution of the repeat numbers of the SGA-SS and non-SGA ISS groups was analyzed separately. Furthermore, we examined the correlation between the repeat numbers and height SDSs using a Pearson correlation coefficient, for patients whose recent height data were available.

The repeat numbers of the patient and control groups ranged from 20 to 31 and from 21 to 31, respectively, and showed a normal distribution (Table 1; Fig. 1a–c). The repeat numbers of the patient group were comparable to those of the control group, and none of the patients had a very small number of repeats. The repeat numbers of the shorter and longer alleles in each individual, as well as the average number of the two alleles, were similar between the two groups. The height SDSs obtained from 106 patients did not correlate with the repeat numbers (Pearson correlation coefficient 0.112, p = 0.253) (Fig. 1d).

Next, we analyzed the results of the SGA-SS and non-SGA ISS groups separately (Table 1; Fig. 1c, 2). Both groups had similar repeat numbers to those of the control group. Furthermore, there were no significant differences in the repeat numbers of the shorter and longer alleles or the average repeat numbers of the two alleles between the SGA-SS or non-SGA ISS groups, and the control group.

This is the first study that examined ACAN VNTR in patients with ISS. We initially hypothesized that small repeat numbers of this VNTR are involved in the development of ISS, because the functional importance of this VNTR in human skeletal growth has been suggested by several studies [14, 18]. However, the results of the present study do not support the hypothesis. Indeed, the smallest repeat number of the 128 patients was comparable to that of the control individuals (19 vs. 21), indicating that reduced repeat numbers of ACAN VNTR are unlikely to be a common cause of autosomal dominant ISS. In this context, Mukamel et al. [18] documented that, in their cohort, the difference between the maximum and minimum repeat numbers (44 and 13, respectively) yielded a height difference of only ∼3.2 cm [18]. Therefore, the size reduction of ACAN VNTR appears to be insufficient to cause ISS as a Mendelian disorder, although extremely small repeat numbers of the VNTR (less than 5, for example) may be hidden in some cases with ISS.

More importantly, we found no difference in the repeat number distribution between the patient and control groups. Both groups showed a normal distribution similar to that observed in European people [18]. Furthermore, the repeat numbers of the SGA-SS and non-SGA ISS groups were comparable to those of the control group. At the start of this study, we expected that reduced repeat numbers of ACAN VNTR would predominantly increase the risk of non-SGA ISS, because previous studies have shown that patients with heterozygous ACAN mutations typically manifest normal or low-normal body length at birth and severe growth failure thereafter [9, 24]. However, we found no association between the repeat numbers and non-SGA ISS or the entire ISS. In addition, the height SDSs of the patients did not correlate with the repeat numbers. These results imply that ACAN VNTR plays no or only minor roles in the etiology of ISS.

The major limitation of this study is the small number of patients and the lack of height data of the control individuals. Indeed, the negative results of this study may reflect the presence of individuals with ISS in the control group. Further studies are needed to validate our results.

The results of this study indicate that ACAN VNTR is a non-contributing factor in the vast majority of ISS cases. The repeat numbers of our Japanese patients and control individuals were similar to those of the European general population. Given the small number of study subjects, our results await further validation.

We would like to thank Drs. M. Adachi, H. Aoyagi, S. Dateki, Y. Dowa, M. Goto, Y. Goto, T. Hamajima, J. Hamada, Y. Hasegawa, M. Honda, R. Horikawa, M. Itoh, M. Izawa, W. Jyo, K. Matsui, M. Mitani-Konno, S. Mizuno, Y. Miyoshi, T. Miyoshi, T. Nagata, Y. Naiki, Y. Nakamura, C. Numakura, M. Obata, A. Ochi, N. Ohno, N. Sasaki, T. Shibazaki, T. Shono, S. Soneda, T. Tanaka, H. Tateishi, T. Urakami, H. Yagi, and S. Yatsuga for providing clinical data and samples of the patients.

This study was approved by the Institutional Review Board Committee at the National Center for Child Health and Development (project #519). Written informed consent was obtained from the participants and/or their parents.

M.F. was a member of the Editorial Board of this journal at the time of submission. The other authors declare no conflicts of interest to declare.

This study was supported by grants from the National Center for Child Health and Development (2023B-4), the Canon Foundation, the Japan Endocrine Society, and the Takeda Science Foundation. The funders had no role in the design, data collection, data analysis, and reporting of this study.

S.N., Y.K., and M.F. designed the study, interpreted the results, and wrote the manuscript; K.P. performed statistical analysis; T.K. and T.M. performed clinical analyses; A.I. supervised the study. All authors approved the final manuscript.

The data that support the findings of this study are not publicly available due to their containing information that could compromise the privacy of research participants but are available from the corresponding author upon reasonable request.