PMC10070732_01
Pedigree of the Chinese family with short stature. (A) The black circles/squares represent the affected patients diagnosed with short stature diagnosis; the white circles/squares represent the unaffected subjects. The arrow indicates the proband. The question mark indicates subject with unknown genotype but known phenotype. (B) X-ray imaging of the proband's left hand at the age of 4 years 5 months and 5 years 8 months. (C) The height growth curve of the proband during the one-year follow-up. (D) The sequencing results of the ACAN mutations. Sequence chromatograms indicate the heterozygosity for a ACAN frameshift mutation (NM_013227.3:c.7230delT; NP_001356197.1:p.Phe2410Leufs*9) in the proband and his mother. The red arrow indicates the mutation. SDS, standard deviation scores; r-hGH, Recombinant human growth hormone; y, year; m, month; BA, bone age.
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F1
The proband, a 4-year-old boy, came to the Xiangya Hospital Central South University with his parents due to growth retardation (Figure 1A). The BA was found to be 5 years and 6 months, as revealed by the X-ray imaging of the left hand (Figure 1B). Meanwhile, the heights of his grandmother (I-1), maternal uncle (II-1), and maternal aunt (II-4) were 145 cm ( -2.78 SDS), 153 cm ( -3.23 SDS), and 165 cm ( 0.28 SDS), respectively (Figure 1A). During this time, the height of the proband increased to 99.0 cm at 4 years 10 months (-2.17 SDS), 101.3 cm at 5 years 1 month (-1.95 SDS), 104.0 cm at 5 years and 5 months (-1.78 SDS), and 105.5 cm at 5 years and 8 months (-1.76 SDS) (Figure 1C). Meanwhile, his weight increased to 18.4 kg (-0.52 SDS) and the BA was 8 years during his latest visit, at the age of 5 years 8 months (Figure 1B). Briefly, (a) Variants within intronic, intergenic, and untranslated regions (UTRs), as well as synonymous single nucleotide variants (SNVs), were excluded from later analysis; (b) Common variants were excluded with minor allele frequency >=0.01 by the 1000 Genomes database, Genome Aggregation Database (gnomAD), version 2.1.1 and an in-house exome database of Berry Genomics; (c) Variants classified as Pathogenic/Likely Pathogenic variants, as well as variant of unknown significance (VUS) by American College of Medical Genetics (ACMG) classification, could be included; (d) Potential causative variants were then screened by a list of growth-associated genes (Supplementary Table S1); (e) Variants that were not existed in the affected family member (II-2) were excluded; (f) Bioinformatics analysis, including Polyphen-2, MutationTaster, SIFT, and CADD, were also carried out to predict the possible impacts of variants; (g) Accordingly, co-segregation analysis was conducted in the family (Supplementary Figure S1). After data filtering of (a), (b), (c), (d), and (e), only a set of 7 variants in seven genes in the proband were identified (Supplementary Figure S1, Supplementary Tables S2, S3). Phe2410Leufs*9) in exon 16 of ACAN was identified in the proband (Figure 1D).
Overview of all known ACAN mutations. (A) The red font symbolizes the mutation identified in this study. (B) The increasing SDS for each year (DeltaSDS/year) of patients with ACAN missense and small deletion mutations during GH treatment. (C) The increasing SDS for each year (DeltaSDS/year) of patients with ACAN null mutations during GH treatment. G1, globular domain 1; IGD, interglobular domain; G2, globular domain 2; KS, keratin sulfate attachment domain; CS, chondroitin sulfate attachment domain; G3, globular domain 3; SDS, standard deviation scores. **p<0.01.
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F2
After data filtering of (a), (b), (c), (d), and (e), only a set of 7 variants in seven genes in the proband were identified (Supplementary Figure S1, Supplementary Tables S2, S3).