Genetic association and characterization of FSTL5 in isolated clubfoot

Anas M Khanshour, Yared Kidane, Julia Kozlitina, Reuel Cornelia, Alexandra Rafipay, Vanessa De Mello, Mitchell Weston, Nandina Paria, Aysha Khalid, Jacqueline T Hecht, Matthew B Dobbs, B. Stephens Richards, Neil Vargesson, F Kent Hamra, Megan Wilson, Carol Wise, Christina A Gurnett, Jonathan J Rios* (Corresponding Author)

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)
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Talipes equinovarus (clubfoot, TEV) is a congenital rotational foot deformity
occurring in 1 per 1,000 births with increased prevalence in males compared to females. The genetic etiology of isolated clubfoot (iTEV) remains unclear. Using a genome-wide association study, we identified a locus within FSTL5, encoding follistatin-like 5, significantly associated with iTEV. FSTL5 is an uncharacterized gene whose potential role in embryonic and post-natal development were previously unstudied. Utilizing multiple model systems, we found that Fstl5 was expressed during later stages of embryonic hindlimb development, and, in mice, expression was restricted to the condensing cartilage anlage destined to form the limb skeleton. In the post-natal growth plate, Fstl5 was specifically expressed in pre-hypertrophic chondrocytes. As Fstl5 knock out rats displayed no gross malformations, we engineered a conditional transgenic mouse
line (Fstl5LSL 54 ) to over-express Fstl5 in skeletal osteochondroprogenitors. We observed that hindlimbs were slightly shorter and that bone mineral density was reduced in adult male, but not female, Prrx1-cre;Fstl5LSL 56 mice compared to control. No overt clubfootlike deformity was observed in Prrx1-cre;Fstl5LSL 57 mice, suggesting FSTL5 may function in other cell types to contribute to iTEV pathogenesis. Interrogating published mouse embryonic single-cell expression data showed Fstl5 was expressed in cell lineage sub60 clusters whose transcriptomes were associated with neural system development. Moreover, our results suggest lineage-specific expression of the Fstl genes correlates with their divergent roles as modulators of TGF-ß and BMP signaling. Results from this study associate FSTL5 with iTEV and suggest an potential sexually dimorphic role for Fstl5 in vivo.
Original languageEnglish
Pages (from-to)3717-3728
Number of pages12
JournalHuman Molecular Genetics
Issue number22
Early online date26 Oct 2020
Publication statusPublished - 15 Nov 2020

Bibliographical note

The Atherosclerosis Risk in Communities Study is carried out as a collaborative
study supported by National Heart, Lung, and Blood Institute contracts (HHSN268201100005C, HHSN268201100006C, HHSN268201100007C,
HHSN268201100008C, HHSN268201100009C, HHSN268201100010C,
HHSN268201100011C, and HHSN268201100012C). The authors thank the staff and participants of the ARIC study for their important contributions. Funding for GENEVA was provided by National Human Genome Research Institute grant U01HG004402 (E.Boerwinkle). We thank H. Hobbs and J. Cohen for contributing control samples for replication genotyping, Nadav Ahituv for sharing RNA-seq data for both bat and mouse embryonic limb buds, Tommy Hyatt for designing the custom genotyping assay, and members of the UT Southwestern Transgenic Core facility, including John Ritter, Mylinh Nguyen, and Robert Hammer. Publicly available mouse embryonic expression analysis results were provided online at (24). The authors acknowledge the contributions and support of the Center for Excellence in Clubfoot Research at Scottish Rite for Children, including Shawne Faulks and Kristhen Atala. Fstl5 mutant rats were produced by the NIH Mutant Rat Resource at UT Southwestern Medical Center (R24RR03232601, R24OD011108, R01HD036022, and (5R01HD053889). This study was supported by funding from the Scottish Rite for Children Research Fund (J.J.R.), Shriners Hospital for Children (J.T.H), and the National Institutes of Health award R01HD043342 (J.T.H.).


  • genome-wide association study
  • clubfoot
  • FSTL5
  • imputation


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