Leri-Weill Dyschondrosteosis Caused by a Leaky Homozygous SHOX Splice-Site Variant

Abstract

SHOX deficiency is a common genetic cause of short stature of variable degree. SHOX haploinsufficiency causes Leri-Weill dyschondrosteosis (LWD) as well as nonspecific short stature. SHOX haploinsufficiency is known to result from heterozygous loss-of-function variants with pseudo-autosomal dominant inheritance, while biallelic SHOX loss-of-function variants cause the more severe skeletal dysplasia, Langer mesomelic dyschondrosteosis (LMD). Here we report for the first time the pseudo-autosomal recessive inheritance of LWD in two siblings caused by a novel homozygous non-canonical, leaky splice-site variant in intron 3 of SHOX: c.544+5G>C. Transcript analyses in patient-derived fibroblasts showed homozygous patients to produce approximately equal amounts of normally spliced mRNA and mRNA with the abnormal retention of intron 3 and containing a premature stop codon (p.Val183Glyfs*31). The aberrant transcript was shown to undergo nonsense-mediated mRNA decay, and thus resulting in SHOX haploinsufficiency in the homozygous patient. Six healthy relatives who are of normal height are heterozygous for this variant and fibroblasts from a heterozygote for the c.544+5G>C variant produced wild-type transcript amounts comparable to healthy control. The unique situation reported here highlights the fact that the dosage of SHOX determines the clinical phenotype rather than the Mendelian inheritance pattern of SHOX variants. This study extends the molecular and inheritance spectrum of SHOX deficiency disorder and highlights the importance of functional testing of SHOX variants of unknown significance in order to allow appropriate counseling and precision medicine for each family individual.

Keywords: SHOX; gene dosage; haploinsufficiency; leaky splice-site; precision medicine; pseudo-autosomal inheritance; short stature; skeletal disorder.

Figure 1

Clinical features in two siblings with biallelic SHOX variants. (A,B): Madelung deformity and mild mesomelia but absence of LMD characteristics on skeletal radiographs in P1 and P2. (C) LWD phenotype with Madelung deformity, mild mesomelic shortening of extremities, genu varum in P1 at age 12 years. (D,E): Genotype-based blotting of body measurements and ratios of 15 individuals from the pedigree show homozygotes having short stature with mesomelic shortening of the upper and lower extremities. Heterozygotes have normal H but predominantly cluster in the lower normal range for H and length of extremities when compared to wild-types. Genotype groups are defined as follows: wild-type (WT/WT) vs. heterozygotes (WT/M), vs. homozygotes without GHT (M/M), vs. homozygotes with GHT (M/M with GHT).

Figure 2

Pedigree, SHOX c.544+5G>C variant detection and consequences on the transcript level. (A) Simplified pedigree and segregation of the SHOX variant. (B) Sequence chromatograms to demonstrate the novel SHOX variant (arrow). (C) Genotype-based transcript analysis in cDNA shows that the homozygous SHOX c.544+5G>C variant leads to production of both wild-type and an aberrant transcript, which retains intron 3 ((C) upper panel). The abnormal transcript undergoes NMD as demonstrated by RT-PCRs in cDNA obtained from fibroblasts with (+) and without (−) NMD inhibition with puromycin. M/M, M/WT, WT/WT represent homozygous mutant, heterozygous mutant and wild-type genotypes, M denotes a molecular size marker, lane 7 No template control. In M/M, a ratio of aberrant SHOX transcript vs. wild-type transcript of 1.54 in (+) puromycin-treated patient cells (+), and of 0.36 in non-treated (-) cells was found, assessed by allele-specific qPCRs; the relative ratio of aberrant to wild-type SHOX transcript is shown (means and standard deviations of four technical replicates) ((C) lower panel)). Sanger sequencing reveals wild-type splicing of all SHOX exons of the RT-PCR product common to patient, parent and control (blue arrow) and reveals retention of intron 3 in the aberrant fragment seen in the patient only (yellow arrow) (D).

Figure 3

Scheme of human phenotypes in relation to SHOX genotypes and SHOX expression. (A) SHOX is located within the pseudo-autosomal region 1 (PAR1). Genes within PAR1 and PAR2 on the tips of both sex chromosomes are inherited such as autosomal genes. (A–C) Scheme of reported SHOX genotypes (A) in relation to SHOX expression (B) and human phenotypes (C). There is a SHOX-dosage-phenotype correlation with SHOX haploinsufficiency resulting in LWD/SS spectrum with pseudo-autosomal dominant (PAD) inheritance and complete loss of SHOX resulting in the severe LMD phenotype (B,C). In contrast SHOX-geno-phenotype, correlation is variable as biallelic SHOX variants can cause both, the LWD phenotype with pseudo-autosomal recessive inheritance (PAR) in case of biallelic hypomorphic SHOX variants inherited from healthy parents and the severe LMD phenotype in case of biallelic null variants inherited from clinically LWD-affected parents. Individuals without a SHOX disease phenotype might be carriers for pseudo-autosomal recessive LWD. In the presented model, the remaining SHOX transcript is assumed to represent the level of SHOX protein.