Mutations in KDSR Cause Recessive Progressive Symmetric Erythrokeratoderma

Abstract

The discovery of new genetic determinants of inherited skin disorders has been instrumental to the understanding of epidermal function, differentiation, and renewal. Here, we show that mutations in KDSR (3-ketodihydrosphingosine reductase), encoding an enzyme in the ceramide synthesis pathway, lead to a previously undescribed recessive Mendelian disorder in the progressive symmetric erythrokeratoderma spectrum. This disorder is characterized by severe lesions of thick scaly skin on the face and genitals and thickened, red, and scaly skin on the hands and feet. Although exome sequencing revealed several of the KDSR mutations, we employed genome sequencing to discover a pathogenic 346 kb inversion in multiple probands, and cDNA sequencing and a splicing assay established that two mutations, including a recurrent silent third base change, cause exon skipping. Immunohistochemistry and yeast complementation studies demonstrated that the mutations cause defects in KDSR function. Systemic isotretinoin therapy has achieved nearly complete resolution in the two probands in whom it has been applied, consistent with the effects of retinoic acid on alternative pathways for ceramide generation.

Keywords: KDSR; TSC10; ceramide; erythrokeratoderma; genome sequencing; ichthyosis; inversion; isotretinoin; skin; splicing.

Figure 1

KDSR Mutations in PSEK Subjects Include Single-Nucleotide Changes That Affect Splicing and a 346 kb Inversion That Abolishes Expression (A) Reverse transcription and amplification of a portion of KDSR RNA spanning exons 6–10 from a wild-type control sample produced a band of the expected size (543 bp, right lane), whereas RNA from subject 429, a subject heterozygous for a substitution at the last base of exon 9 (c.879G>A), also produced a smaller band (441 bp, left lane) in which exon 9 had been skipped (r.778_879del [p.Gln260_Gln293del]). Corresponding Sanger sequences are shown in Figure S1C. (B) Reverse transcription and amplification of a portion of KDSR RNA generated by a wild-type KDSR construct expressed in HEK cells produced a single product including exons 3–5 (450 bp, middle lane), whereas RNA from a KDSR construct with the exon 4 splice acceptor mutation found in subject 101 (c.256−2A>C) produced a smaller band (384 bp, left lane) in which exon 4 had been skipped (r.256_321del [p.Val86_Gln107del]). Corresponding Sanger sequences are shown in Figure S1D. (C) When the same experiment shown in (A) was performed with RNA from subject 1107, who, like subject 429, is heterozygous for the KDSR c.879G>A mutation (causing skipping of exon 9), all amplification products lacked exon 9, demonstrating that his other KDSR allele harbors a mutation that either also affects exon 9 splicing or ablates expression of KDSR entirely. (D) Genome sequencing of DNA from subject 1107 revealed a 346 kb inversion on chromosome 18 (g.63,361,789_63,707,612inv), which flips the genomic sequence between intron 2 of KDSR and intron 1 of SERPINB11, as shown. KDSR is in purple, and SERPINB11 is in green; there are six additional genes between these (not shown). Amplification of genomic DNA across the boundaries of the inversion with the red or blue primer set (arrows) should produce a product only when the inversion is present. (E) Amplification of genomic DNA with primers shown in blue in (D) produced the expected PCR product (533 bp) with DNA from subject 1107 and his mother and with DNA from subject 438 and her father, confirming that both probands are compound heterozygous for this inversion and another deleterious KDSR mutation (one inherited from each parent). The same result was obtained with the primers shown in red in (D) (665 bp, not shown). Corresponding Sanger sequences for both primer sets are shown in Figures S1E and S1F.

Figure 2

Clinical and Histologic PSEK Features Due to KDSR Mutations The face, palms, soles, and genitals are the most severely affected areas in all subjects. (A) Image of the face of subject 101 shows well-demarcated pink-red plaques, with overlying thick yellow-white scale, which are prominent on the cheeks, central forehead, and neck. There are focal, denuded, red areas that showed no evidence of bacterial superinfection but could be the result of over-grooming. (B) Subject 1107 has similar focal plaques with thick scale on the nose, cheeks, and chin. (C and D) Images of the palm of subject 1107 (C) and sole of subject 101 (D) show erythematous palmoplantar hyperkeratosis with peeling scale. (E) Histology of affected skin from the buttock of subject 1107 is remarkable for epidermal thickening (acanthosis), sparse to absent keratohyalin granules in the granular layer, and retention of nuclei in the stratum corneum (arrow). Epidermal layers are labeled as follows: B, basal layer; S, stratum spinosum; G, granular layer; SC, stratum corneum.

Figure 3

KDSR Encodes 3-Ketodihydrosphingosine Reductase, an Enzyme in the Ceramide Synthesis Pathway (A) The three major metabolic pathways capable of producing ceramides (the de novo, sphingomyelinase, and salvage pathways) are shown in purple, aqua, and brown, respectively. KDSR (in red) is critical to the creation of ceramides via de novo synthesis but is not intrinsic to the other two pathways. (B) The domain structure of KDSR and the locations of mutations in PSEK subjects are shown. The TyrXXXLys, Asn, and Ser residues that form the canonical catalytic triad are in orange, the putative ThrGlyXXXGlyxGly NAD binding site and Rossman fold are in light and dark green, respectively, and the putative transmembrane domains are in blue. Putative homodimerization and homotetramerization domains are indicated with boxes. Residues that are substituted or deleted as a result of mutations in PSEK subjects are in red.

Figure 4

Filaggrin Distribution Is Expanded in Affected PSEK Tissue Tissue from a normal donor and affected tissue from subject 1107 was immunostained with primary antibodies to KDSR, KRT14, KRT10, and FLG (Santa Cruz Biotechnology sc-366781, sc-53253, sc-53252, and sc-25897, respectively) and corresponding Cy2 (green) or Cy3 (red) secondary antibodies. DAPI was used as a nuclear counterstain (blue). (A and E) Positive immunostaining for KDSR (green) was observed in all layers of the epidermis in normal (A) and affected (E) tissue. (B and F) Positive immunostaining for KRT14 (red) was limited to the basal layer of the epidermis in normal (B) and affected (F) tissue. (C and G) Positive immunostaining for KRT10 (red), a marker of epidermal differentiation, was limited to the suprabasal epidermis in normal (C) and affected (G) tissue. Normal tissue showed typical autofluorescence of the stratum corneum. (D and H) Positive immunostaining for FLG (green), a marker of the granular layer of the epidermis, was tightly restricted in normal tissue (D, arrow) but expanded within affected tissue (H, asterisk).

Figure 5

Mutant KDSR Does Not Complement Depletion of Endogenous Yeast TSC10 A yeast strain with galactose-inducible and glucose-repressible expression of the KDSR ortholog TSC10 was created,, , and depletion of endogenous TSC10 was verified (Figure S4). Ten-fold serial dilutions of yeast expressing plasmid-borne mutant forms of KDSR in the presence or absence of endogenous yeast TSC10 showed severe growth defects in the absence of endogenous TSC10, whereas both yeast TSC10 and wild-type KDSR fully complemented the loss of endogenous yeast TSC10. The empty vector served as a negative control.