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. 2019 Jul;56(7):434-443.
doi: 10.1136/jmedgenet-2018-105893. Epub 2019 Apr 24.

Functional analysis of novel desert hedgehog gene variants improves the clinical interpretation of genomic data and provides a more accurate diagnosis for patients with 46,XY differences of sex development

Katie Ayers  1   2 , Jocelyn van den Bergen  1 , Gorjana Robevska  1 , Nurin Listyasari  3 , Jamal Raza  4 , Irum Atta  4 , Stefan Riedl  5   6 , Karen Rothacker  7 , Catherine Choong  7   8 , Sultana M H Faradz  9 , Andrew Sinclair  1   2
Affiliations

Functional analysis of novel desert hedgehog gene variants improves the clinical interpretation of genomic data and provides a more accurate diagnosis for patients with 46,XY differences of sex development

Katie Ayers et al. J Med Genet. 2019 Jul.

Abstract

Background: Desert hedgehog (DHH) gene variants are known to cause 46,XY differences/disorders of sex development (DSD). We have identified six patients with 46,XY DSD with seven novel DHH gene variants. Many of these variants were classified as variants of uncertain significance due to their heterozygosity or associated milder phenotype. To assess variant pathogenicity and to refine the spectrum of DSDs associated with this gene, we have carried out the first reported functional testing of DHH gene variant activity.

Methods: A cell co-culture method was used to assess DHH variant induction of Hedgehog signalling in cultured Leydig cells. Protein expression and subcellular localisation were also assessed for DHH variants using western blot and immunofluorescence.

Results: Our co-culture method provided a robust read-out of DHH gene variant activity, which correlated closely with patient phenotype severity. While biallelic DHH variants from patients with gonadal dysgenesis showed significant loss of activity, variants found as heterozygous in patients with milder phenotypes had no loss of activity when tested with a wild type allele. Taking these functional results into account improved clinical interpretation.

Conclusion: Our findings suggest heterozygous DHH gene variants are unlikely to cause DSD, reaffirming that DHH is an autosomal recessive cause of 46,XY gonadal dysgenesis. Functional characterisation of novel DHH variants improves variant interpretation, leading to greater confidence in patient reporting and clinical management.

Keywords: DHH; desert hedgehog; disorders of sex development; gonadal dysgenesis.

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Conflict of interest statement

Competing interests: None declared.

Figures

Figure 1
Figure 1
Previously reported and novel variants in desert hedgehog (DHH). A schematic figure showing the protein position of both previously reported variants in DHH (upperside), and the new ones reported in this study (underside). Publication references are in bold superscript after each variant. Yellow circles show missense variants, blue are in-frame deletions and red are deletions causing frameshifts. ** depicts those that were homozygous and * those that were heterozygous, and ? where the zygosity is unknown. Compound heterozygous variants are depicted by a dotted line joining the two variants.
Figure 2
Figure 2
Variants in desert hedgehog (DHH) do not affect protein localisation. Immunofluorescence staining for HEK293t cells transfected with the different DHH variants, using a DHH antibody (green) and co-stained with DAPI (4′,6-diamidino-2-phenylindole) (blue). (A). Wild type DHH is expressed throughout the cytoplasm and at the plasma membrane. This staining is also seen for the four previously published pathogenic DHH variants (B–E). (F–L) Staining for the new DHH variants reported here. None of the variants appeared to significantly affect protein levels or localisation in this assay, yet the p.Arg164Pro variant had reduced overall number of cells with staining (see online supplementary figure S6).
Figure 3
Figure 3
New desert hedgehog (DHH) gene variants do not significantly affect protein expression. Western blot analysis of protein from overexpression of DHH variants in HEK293t cells, using an anti-FLAG antibody. Tubulin is used as a control. Only the previously reported p.Leu363Cysfs*4, which causes a frameshift, caused a loss of DHH protein, seen as a smaller fragment due to the premature stop codon introduced. Staining with an anti-DHH antibody showed similar results (online supplementary figure S3 and data not shown).
Figure 4
Figure 4
A co-culture assay for functional analysis of desert hedgehog (DHH) variant activity. (A) A schematic representation of the co-culture assay, where human embyronic kidney/HEK293t cells are transfected to express DHH (wild type [WT] or variant), and are co-cultured directly with mouse Leydig (Tm3) cells. DHH secreted by the HEK cells directly activates the Hedgehog signalling pathway in the adjacent Tm3 cells, assessed by qPCR analysis of pathway targets Gli1 and Ptch1. (B) Immunofluorescence staining of the co-culture, showing the HEK cells expressing DHH (red) growing in close proximity with the Tm3 cells (stained with Carboxyfluorescein succinimidyl ester (CSFE) green). DAPI stains nuclei (blue). (C) mGli1 expression in the co-culture assayed by qPCR, for four previously reported DHH variants. (D) mPtch1 expression in the co-culture assayed by qPCR, for four previously reported DHH variants. (E) mGli1 expression in the co-culture assayed by qPCR, for novel DHH variants reported here, either alone or in combination (where found in compound heterozygous or heterozygous format). (F) mGli1 expression in the co-culture assayed by qPCR, for novel DHH variants reported here, either alone or in combination. For all qPCRs the mouse Tbp gene is used as a housekeeper. Expression is relative to Tbp and the control (empty plasmid). Three biological replicates were performed and each qPCR was performed with technical triplicates. P values are calculated using one-way ANOVA. *p>0.05, **p>0.01 ***p>0.001. Activity is calculated as a percentage of WT DHH relative to the negative control.
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