To find out whether the co-occurrence of SHOX and CYP26C1 variants in severe phenotypes is a unique finding specific only to family 1, we screened CYP26C1 in a cohort of 68 individ- uals with LWD and in a cohort of 140 controls with normal height where SHOX deficiency was excluded. The complete list of the variants identified can be found in Appendix Table 2. Synonymous, intronic and common variants were excluded from further analysis.
We identified two further cases with co-occurrence of SHOX and CYP26C1 variants (Figure 3.4). Available clinical data are listed in Table 3.2.
Family 2 had one affected daughter (II:2) presenting with short stature, mesomelia and Madelung deformity. The sister (II:1) presented with short stature but skeletal dysmorphis
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were not diagnosed. Both parents presented with normal stature and no dysmorphisms. A heterozygous SHOX missense mutation c.394C>G (p.Leu132Val) was found in the unaffected father and the two siblings. This variant was previously shown to alter homodimerization and reduce DNA binding abilities42. Screening of CYP26C1 identified a heterozygous missense
mutation c.1133G>A (p.Arg378His) only in the affected daughter (Figure 3.4a, Family 3). The third case was a girl with short stature, mesomelia and Madelung deformity carrying a de novo heterozygous deletion of SHOX and a missense variant in CYP26C1, c.356A>C (p.Gln119Pro). Both parents and the brother were reported having normal stature. The CYP26C1 variant was inherited by the father. Since the SHOX deletion is de novo, this family cannot demonstrate specific co-segregation although it adds to overall evidence that damaging variants in SHOX and CYP26C1 co-occur in individuals with severe LWD pheno- types (Figure 3.4a, Family 2).
Functional analysis of the SHOX and CYP26C1 variants found in these two cases demon- strated their negative impact on the protein activity (Figure 3.4b and c). We conclude that, in addition to family 1, two out of 68 LWD patients with SHOX deficiency presented with damaging variants in CYP26C1 while no damaging mutations were identified in 140 control individuals with normal height. In all families where clinical information was avail- able, all individuals with damaging mutations in both SHOX and CYP26C1 had the more severe skeletal phenotype.
Screening of the controls lead to the identification of common synonymous and intron variants. The variants found are listed in Appendix Table 3.
Family Patients ID Age Gender SD LA SD Madelung deformity
Family 2
I:1 - M - - No I:2 - F - - No II:1 - F - - No II:2 6 F -3.38 -4.71 YesFamily 3
I:1 51 M +0.1 - No I:2 47 F +0.8 -0.1 No II:1 26 M +0.5 -0.9 No II:2 16 F -2.6 -3.6 YesTable 3.2: Families 2 and 3 clinical data. M, male; F, female; -, data not available; Height SD, height standard deviation; LA SD, lower arm standard deviation.
Finally, in order to test whether CYP26C1 mutations alone could lead to short stature or dysmorphic signs, a group of 256 affected individuals (234 ISS and 22 LWD), where SHOX deficiency was excluded, was screened with the GS Junior System (Roche). The variants found are available in Appendix Table 4. We identified two rare missense variants in two independent ISS individuals, c.148C>T (p.Pro50Ser) and c.356A>C (p.Gln119Pro) predicted as damaging (Figure 3.5a and b). Phenotypic information of the family carrying the variant c.148C>T (p.Pro50Ser) were not available. Functional analysis of this variant did not show any significant effect on protein activity (Figure 3.5c). Variant c.356A>C (p.Gln119Pro) was found in a family with three affected siblings. Based on the inheritance of the trait, we obtained no convincing evidence that this variant alone is causative for short stature, although the mutation segregated with the most affected individuals and showed a significant impact
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on CYP26C1 catabolic activity (Figure 3.5a and c). Altogether, these genetic data do not support the hypothesis that CYP26C1 alone is causative for short stature or skeletal abnormalities.
Figure 3.4:Family pedigree charts of the screened LWD patients and functional analysis of the identified variants. (a) Pedigree of the LWD families carrying variants in both SHOX and CYP26C1. Filled symbol, short stature affected individual; red text, SHOX locus; green text, CYP26C1 locus; +, wild type allele; N.A., DNA not available; *, individual with mesomelia and Madelung deformity. (b) Scheme of SHOX protein (upper panel). Leu132 resides in the homeodomain. Luciferase assay testing SHOX Leu132Val mutation on FGFR3 promoter in U2OS cells (n=4). pcDNA4-TO was used as control. Arg153Leu was published as mutation affecting SHOX protein activity and was therefore used as positive control. Data are shown as means ± SD; RLU, Relative Light Units; *** p-value < 0.001, one-way ANOVA Bonferroni’s multiple comparison test. SH3, Src Homology 3 domain. OAR, Otp Aristaless Rax domain. (c) Scheme of CYP26C1 protein. Gln119 and Arg378 reside within the P450 domain. Cignal-RARE system luciferase assay testing CYP26C1 Gln119Pro and Arg378His mutations in U2OS cells treated with 250 nM RA for 24 hours (n=4). pIRES2-EGFP was used as control. The iron binding residue Cys459 was mutated to Ala as positive control. Data are shown as means ± SD; RLU, Relative Light Units; * p-value =0.0286, two-tailed Mann-Whitney non-paramtric t test. TM, Transmembrane helix.
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Figure 3.5:Family pedigree charts of the screened ISS and LWD individuals and func- tional analysis of the identified mutations.(a) Pedigree of the families studied. Filled symbol, short stature affected individual; slash, divorced; green text, CYP26C1 locus; +, wild type allele; N.A. DNA not available; arrow, index patient. (b) Scheme of CYP26C1 protein. Pro50 and Gln119 reside within the P450 domain. TM, Transmembrane helix. Cys459 represents the Iron binding residue. (c) Cignal-RARE system luciferase assay testing CYP26C1 Pro50Ser and Gl119Pro muta- tions in U2OS cells treated with 250 nM RA for 24 hours (n=4). pIRES2-EGFP was used as control. The iron binding residue Cys459 was mutated to Ala as positive control. Data are shown as means ±SD; RLU, Relative Light Units; * p-value = 0.0286, two-tailed Mann-Whitney non-paramtric t test.