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Functional analysis of conserved non-coding regions around the short stature hox gene (shox) in whole zebrafish embryos.

ABSTRACT: Mutations in the SHOX gene are responsible for Leri-Weill Dyschondrosteosis, a disorder characterised by mesomelic limb shortening. Recent investigations into regulatory elements surrounding SHOX have shown that deletions of conserved non-coding elements (CNEs) downstream of the SHOX gene produce a phenotype indistinguishable from Leri-Weill Dyschondrosteosis. As this gene is not found in rodents, we used zebrafish as a model to characterise the expression pattern of the shox gene across the whole embryo and characterise the enhancer domains of different CNEs associated with this gene.Expression of the shox gene in zebrafish was identified using in situ hybridization, with embryos showing expression in the blood, putative heart, hatching gland, brain pharyngeal arch, olfactory epithelium, and fin bud apical ectodermal ridge. By identifying sequences showing 65% identity over at least 40 nucleotides between Fugu, human, dog and opossum we uncovered 35 CNEs around the shox gene. These CNEs were compared with CNEs previously discovered by Sabherwal et al., resulting in the identification of smaller more deeply conserved sub-sequence. Sabherwal et al.'s CNEs were assayed for regulatory function in whole zebrafish embryos resulting in the identification of additional tissues under the regulatory control of these CNEs.Our results using whole zebrafish embryos have provided a more comprehensive picture of the expression pattern of the shox gene, and a better understanding of its regulation via deeply conserved noncoding elements. In particular, we identify additional tissues under the regulatory control of previously identified SHOX CNEs. We also demonstrate the importance of these CNEs in evolution by identifying duplicated shox CNEs and more deeply conserved sub-sequences within already identified CNEs.

SUBMITTER: Kenyon EJ

PROVIDER: S-EPMC3123344 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Functional analysis of conserved non-coding regions around the short stature hox gene (shox) in whole zebrafish embryos.

Kenyon Emma J EJ McEwen Gayle K GK Callaway Heather H Elgar Greg G

PloS one 20110624 6

<h4>Background</h4>Mutations in the SHOX gene are responsible for Leri-Weill Dyschondrosteosis, a disorder characterised by mesomelic limb shortening. Recent investigations into regulatory elements surrounding SHOX have shown that deletions of conserved non-coding elements (CNEs) downstream of the SHOX gene produce a phenotype indistinguishable from Leri-Weill Dyschondrosteosis. As this gene is not found in rodents, we used zebrafish as a model to characterise the expression pattern of the shox ...[more]

PMID: 21731768

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Project description:BackgroundSHOX mutations have previously been described as causes of Léri-Weill dyschondrosteosis (LWD), Langer mesomelic dysplasia (LMD), and idiopathic short stature. The loss of X chromosome-Turner syndrome or mosaic 45,X/46,XX or 46,XY-also leads to the heterozygous loss of SHOX in patients with short stature only or with features similar to LWD. The aim of this study was to assess the efficacy of the targeted screening for SHOX variants, which involved different methods in the laboratory analysis of short stature. We determined the significance and positive predictive value of short stature for the detection of SHOX variants.MethodsTargeted screening for variants in SHOX involving MLPA, sequencing, karyotyping and FISH was performed in the short stature cohort (N = 174) and control cohort (N = 91). The significance of short stature and particular characteristics for the detection of SHOX variants was determined by Fisher's exact test, and the probability of SHOX mutation occurrence was calculated using a forward/stepwise logistic regression model.ResultsIn total, 27 and 15 variants influencing SHOX were detected in the short stature and control cohorts, respectively (p > 0.01). Sex chromosome aberrations and pathogenic CNV resulting in diagnosis were detected in eight (4.6%) and five (2.9%) patients of the short stature group and three (3.3%) and one (1.1%) individuals of the control group. VUS variants were discovered in 14 (8.0%) and 11 (12.1%) individuals of the short stature and control groups, respectively. MLPA demonstrated the detection rate of 13.22%, and it can be used as a frontline method for detection of aberrations involving SHOX. However, only mosaicism of monosomy X with a higher frequency of monosomic cells could be reliably discovered by this method. Karyotyping and FISH can compensate for this limitation; their detection rates in short stature group were 3.55% and 13.46% (N = 52), respectively. FISH proved to be more effective than karyotyping in the study as it could reveal cryptic mosaics in some cases where karyotyping initially failed to detect such a clone. We suggest adding FISH on different tissue than peripheral blood to verify sex-chromosome constitution, especially in cases with karyotypes: 45,X; mosaic 45,X/46,XX or 46,XY; 46,Xidic(Y) detected from blood; in children, where mosaic 45,X was detected prenatally but was not confirmed from peripheral blood. The correlation of short stature with the occurrence of SHOX mutations was insignificant and short stature demonstrates a low positive predictive value-15.5% as unique indicator for SHOX mutations. The typical skeletal signs of LWD, including Madelung deformity and disproportionate growth, positively correlate with the findings of pathogenic SHOX variants (p < 0.01) by Fisher's exact test but not with the findings of VUS variants in SHOX which are more prevalent in the individuals with idiopathic short stature or in the individuals with normal height.

Dataset Information

Functional analysis of conserved non-coding regions around the short stature hox gene (shox) in whole zebrafish embryos.

Publications

Functional analysis of conserved non-coding regions around the short stature hox gene (shox) in whole zebrafish embryos.

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