High frequency of pathogenic ACAN variants including an intragenic deletion in selected individuals with short stature

Abstract

Context: Defining the underlying etiology of idiopathic short stature (ISS) improves the overall management of an individual.

Objective: To assess the frequency of pathogenic ACAN variants in selected individuals.

Design: The single-center cohort study was conducted at a tertiary university children's hospital. From 51 unrelated patients with ISS, the 16 probands aged between 3 and 18 years (12 females) with advanced bone age and/or autosomal dominant inheritance pattern of short stature were selected for the study. Fifteen family members of ACAN-positive probands were included. Exome sequencing was performed in all probands, and additional copy number variation (CNV) detection was applied in selected probands with a distinct ACAN-associated phenotype.

Results: Systematic phenotyping of the study cohort yielded 37.5% (6/16) ACAN-positive probands, with all novel pathogenic variants, including a 6.082 kb large intragenic deletion, detected by array comparative genomic hybridization (array CGH) and exome data analysis. All variants were co-segregated with short stature phenotype, except in one family member with the intragenic deletion who had an unexpected growth pattern within the normal range (-0.5 SDS). One patient presented with otosclerosis, a sign not previously associated with aggrecanopathy.

Conclusions: ACAN pathogenic variants presented a common cause of familial ISS. The selection criteria used in our study were suggested for a personalized approach to genetic testing of the ACAN gene in clinical practice. Our results expanded the number of pathogenic ACAN variants, including the first intragenic deletion, and suggested CNV evaluation in patients with typical clinical features of aggrecanopathy as reasonable. Intra-familial phenotypic variability in growth patterns should be considered.

Figure 1

Structure of the aggrecan protein (RefSeq NP_037359.3) (6, 14) and ACAN gene (RefSeq NM_013227.3). Positions of the current pathogenic variants (bottom) with respective predicted changes in the amino acid sequence are shown. Blue boxes on the genomic DNA (gDNA) denote the coding regions (exons 1–18), drawn approximately to the related protein. Light blue boxes represent untranslated regions. G1, globular domain 1; G2, globular domain 2; G3, globular domain 3; IGD, interglobular domain; KS, keratan sulfate attachment region; CS1, chondroitin sulfate attachment region 1; CS2, chondroitin sulfate attachment region 2; EGF1, 2, epidermal growth factor-like domain 1, 2; CLD, C-type lectin domain; CRP, complement regulatory like domain; **large deletion encompassing exons 3–6 (NG_012794.1: g. 39409_45491del; NP_037359.3: p.His25_Thr350del)

Figure 2

Three methods detecting heterozygous deletion in the ACAN gene. (A) The result of the NGS CNVkit detection algorithm indicating a possible intragenic deletion in the ACAN gene. (B) The result of array CGH confirming the deletion of exons 3–5 in the ACAN gene (arr (GRCh 37) 15q26.1 (89381207_89386488)x1). (C) LR-PCR with the NGS sequence analysis determining exact nucleotide positions of the deletion (NG_012794.1: g. 39409_45491del), encompassing exons 3–6 of the ACAN gene. The red line indicates the deletion, and green color marks indicate deletion coordinates. For comparison, control cases without deletion are shown. CES, clinical exome sequencing (TruSight One); P1, proband no. 1; P1F, father of proband no 1.

Figure 3

Pedigrees and growth charts. (A) Pedigrees of six unrelated families with ACAN pathogenic mutations. M, allele with mutation; Wt, wild type allele. (B) Growth charts of family members with a heterozygous multi-exon deletion in the ACAN gene. Red points indicate the growth of P1S without short stature in comparison to her father (P1F; black points) with early growth cessation and sister (P1; green points) with short stature and profound bone age advancement. The left end of each horizontal arrow represents the proband’s height at chronological age and the right end represents the bone age. Vertical arrow shows the start of growth hormone (GH) treatment. Cross signs mark mother’s (above) and father’s (below) final height. P1, proband no. 1, P1S – sister of proband no. 1, P1F, father of proband no. 1.