Novel Pathogenetic Variants in PTHLH and TRPS1 Genes Causing Syndromic Brachydactyly

Abstract

Skeletal disorders, including both isolated and syndromic brachydactyly type E, derive from genetic defects affecting the fine tuning of the network of pathways involved in skeletogenesis and growth-plate development. Alterations of different genes of this network may result in overlapping phenotypes, as exemplified by disorders due to the impairment of the parathyroid hormone/parathyroid hormone-related protein pathway, and obtaining a correct diagnosis is sometimes challenging without a genetic confirmation. Five patients with Albright's hereditary osteodystrophy (AHO)-like skeletal malformations without a clear clinical diagnosis were analyzed by whole-exome sequencing (WES) and novel potentially pathogenic variants in parathyroid hormone like hormone (PTHLH) (BDE with short stature [BDE2]) and TRPS1 (tricho-rhino-phalangeal syndrome [TRPS]) were discovered. The pathogenic impact of these variants was confirmed by in vitro functional studies. This study expands the spectrum of genetic defects associated with BDE2 and TRPS and demonstrates the pathogenicity of TRPS1 missense variants located outside both the nuclear localization signal and the GATA ((A/T)GATA(A/G)-binding zinc-containing domain) and Ikaros-like binding domains. Unfortunately, we could not find distinctive phenotypic features that might have led to an earlier clinical diagnosis, further highlighting the high degree of overlap among skeletal syndromes associated with brachydactyly and AHO-like features, and the need for a close interdisciplinary workout in these rare patients. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Keywords: CELL/TISSUE SIGNALING - ENDOCRINE PATHWAYS; DISEASES AND DISORDERS OF/RELATED TO BONE; DISORDERS OF CALCIUM/PHOSPHATE METABOLISM; GENETIC RESEARCH; ISORDERS OF CALCIUM/PHOSPHATE METABOLISM; PARATHYROID-RELATED DISORDERS; PTH/VIT D/FGF23.

Fig. 1

(A,B) Electropherograms of the confirmatory direct sequencing of genetic variants discovered by WES. (C) Schematic representation of the PTHLH isoform NM_198965.1, its proteolytic processing pattern into bioactive peptides and known mutations mapping in coding regions. Prohormone: 177 amino acids. Bioactive peptides: mature N‐terminal (having PTH‐like and growth regulatory activities) (amino acids 37–70), mid‐region (regulating calcium transport and cell proliferation and containing the nuclear localization signal, NLS) (amino acids 103–130) and C‐terminal secretory (modulating osteoclast activity) (amino acids 143–146) peptides. (D) Schematic representation of the TRPS1 isoform NM_14112.2, protein domains and known mutations in coding regions. Protein: 1294 amino acids. Protein domains of the TRPS1 transcription factor predicted by SMART: at the N‐terminal, region involved in the nuclear translocation, 7 zinc finger motifs (amino acids 235–260/346–371/447–472/536–567/627–650/679–702/705–728); one DNA‐binding zinc finger GATA‐type motif (amino acids 903–953), surrounded by the putative NLSs; at the C‐terminal, region involved in the homodimer formation, and 2 zinc finger IKAROS‐like (amino acids 1228–1250/1256–1280). Known coding mutations are reported in the upper part (mutations found in our patients reported in bold; @ highlights recurrent mutations). NLS = nuclear localization signal; SMART = simple modular architecture research tool; WES = whole‐exome sequencing.

Fig. 2

Gene expression analysis in transfected the HepG2 cell line and nuclear and cytoplasmatic localization of TRPS1 GFP‐tagged wt and mutated proteins. (A) By immunocytochemistry the cellular localization of TRPS1 GFP‐tagged (green) transcription factors after 48 hours transfection of pCMV6‐AC‐GFP TRPS1 plasmids in HepG2. Nuclei were counterstained by DAPI (blue). The graph summarizes the quantification of TRPS GFP‐tagged by ImageJ software. Scale bars = 50 μm. Values obtained by the TRPS1 localization analysis of mutants were statistically significant (p < 0.001, Student’s t test). (B) Gene expression analysis of TRPS1 target genes, SOX9 and STAT3 in HepG2 pCMV6‐AC‐GFP TRPS1 transfected. The y‐axis reports the binary logarithm (ln₂) RQ in mRNA expression versus mock, used as calibrator reference. Statistical analysis: ordinary one‐way ANOVA. SOX9: F = 95.75, p <0.0001; multiple comparisons p values: wt = <0.0001, c.47G>A = 0.1434, c.203A>C = 0.8592, c.343C>T* = 0.0032, c.422C>T* = 0.0002, c.2894G>A = <0.001. STAT3: F = 804.5, p = <0.0001; multiple comparisons p values: wt = <0.0001, c.47G>A = >0.9999, c.203A>C = 0.0008, c.343C>T* = <0.0001, c.422C>T* = 0.0009, c.2894G>A = 0.9956. RQ = relative fold expression change.

Fig. 3

RUNX2 gene and protein expression analysis in the L88/5 cell line by immunocytochemistry. (A) RUNX2 expression by rhodamine immunostaining (red) in L88/5 (36 hours after pCMV6‐AC‐GFP TRPS1 plasmids transfection) and nuclear counterstaining by DAPI (blue). The graph resumes the staining quantification by ImageJ tool. Scale bars = 50 μm. RUNX2 immunocytochemistry values in mutants were statistically significant, but the one for the c.343C>T variant (p < 0.001, Student's t test). (B) Gene expression analysis of RUNX2 in wild‐type and pCMV6‐AC‐GFP TRPS1 transfected L88/5 cells. The y‐axis reports the binary logarithm (ln₂) RQ in mRNA expression versus mock, used as calibrator reference. Statistical analysis: ordinary one‐way ANOVA. RUNX2: F = 220.9, p = <0.0001; multiple comparisons p values: wt = <0.0001, c.47G>A = 0.0368, c.203A>C = 0.0846, c.343C>T* = <0.0001, c.422C>T* = 0.0011, c.2894G>A = <0.0001. RQ = relative fold expression change.