Novel subtype of mucopolysaccharidosis caused by arylsulfatase K (ARSK) deficiency

Abstract

Background: Mucopolysaccharidoses (MPS) are monogenic metabolic disorders that significantly affect the skeleton. Eleven enzyme defects in the lysosomal degradation of glycosaminoglycans (GAGs) have been assigned to the known MPS subtypes (I-IX). Arylsulfatase K (ARSK) is a recently characterised lysosomal hydrolase involved in GAG degradation that removes the 2-O-sulfate group from 2-sulfoglucuronate. Knockout of Arsk in mice was consistent with mild storage pathology, but no human phenotype has yet been described.

Methods: In this study, we report four affected individuals of two unrelated consanguineous families with homozygous variants c.250C>T, p.(Arg84Cys) and c.560T>A, p.(Leu187Ter) in ARSK, respectively. Functional consequences of the two ARSK variants were assessed by mutation-specific ARSK constructs derived by site-directed mutagenesis, which were ectopically expressed in HT1080 cells. Urinary GAG excretion was analysed by dimethylene blue and electrophoresis, as well as liquid chromatography/mass spectrometry (LC-MS)/MS analysis.

Results: The phenotypes of the affected individuals include MPS features, such as short stature, coarse facial features and dysostosis multiplex. Reverse phenotyping in two of the four individuals revealed additional cardiac and ophthalmological abnormalities. Mild elevation of dermatan sulfate was detected in the two subjects investigated by LC-MS/MS. Human HT1080 cells expressing the ARSK-Leu187Ter construct exhibited absent protein levels by western blot, and cells with the ARSK-Arg84Cys construct showed markedly reduced enzyme activity in an ARSK-specific enzymatic assay against 2-O-sulfoglucuronate-containing disaccharides as analysed by C18-reversed-phase chromatography followed by MS.

Conclusion: Our work provides a detailed clinical and molecular characterisation of a novel subtype of mucopolysaccharidosis, which we suggest to designate subtype X.

Keywords: genetics; human genetics; orthopedics; pediatrics; phenotype.

Figure 1

Phenotype of skull, thorax and spine. Lateral skull radiographs show mildly thickened calvarias of S1 (A, B) and S2 (C–D). S1 and S2 show an open bite (A, C). Radiographs of chest and spine show broad (oar-shaped) ribs (S1 at 17 years (E, F) and S2 at 14 years (H)), broad vertebral bodies, platyspondyly and irregular endplates (S1 at 17 years (G), S3 at 18 years (J)). S1 shows a bell-shaped thorax (E). Radiographs of lateral spine additionally illustrate anterior beaking and irregular vertebral end plates (J). Lateral spine radiograph of the lumbar area shows platyspondyly with scalloping of the posterior end plates (S1 at 12 years (G)). Anterior inferior beaking of the cervical spine of S3 at 20 years (I). S1–S4, subjects 1–4.

Figure 2

Radiological abnormalities of the extremities. Hand radiographs demonstrate short third, fourth and fifth metacarpals, small carpal bones and small epiphyses of lower ends of radius and ulna (S2 at 14 years (A), S4 at 17 years (B), S3 at 18 years (C)). Posterior-Anterior (PA) projection hand radiograph of S2 at 14 years shows defective ossification of the carpal bones associated with retarded bone age. Apparent metaphysial irregularities and dysplasia along the metacarpophalangeal joints (A). PA right pelvis radiograph of S2 at 14 years (D) shows dysplasia and fragmentation of the capital femoral epiphysis associated with acetabular dysplasia. The shaft of the femur is overtubulated. Radiographs of the femora and tibiae demonstrate vertical striae in the metaphyses (S2 at 14 years (E), S4 at 17 years (F)). Tibia and fibula of S2 at 14 years show overtubulation of the shafts (G). Epi-metaphysial dysplasia of the inferior ends of the tibia and fibula. Metaphysial irregularities are associated with metaphysial striation (G). S1–S4, subjects 1–4.

Figure 3

Overview of the subjects, their phenotype and health problems. Family 1: (A) pedigree, (C–E) S1 at the age of 16 years, (F, G) S2 at the age of 14 years. Family 2: (B) pedigree. S1–S2 show mild coarse facial features, midface retrusion, full lips (C, F), short-trunk short stature (D, F, G), (relative) macrocephaly (D, F, G), short neck (E, G) and genua valga (F). Hands are shaped normally (H, I). Onset of symptoms in S1 and S2 is depicted in the timeline, age in years (J). MPS, mucopolysaccharidoses; S1–S4, subjects 1–4; WT, wild type. M1=mutation in family 1; M2=mutation in family 2.

Figure 4

Investigation of the functional consequences of the ARSK variants. ARSK-WT but not ARSK-Arg84Cys desulfates synthetic 2-sulfoglucuronate-N-acetylglucosamine (G2A0) disaccharides (A, B). (A) G2A0 treated with cell lysates expressing ARSK-WT resulted in a minor peak at 26.5 mL representing the 2-O-sulfated educt (m/z 670.15) and a major peak at 28 mL retention volume representing the desulfated product (m/z 590.19), indicating the loss of a sulfate group (highlighted in yellow). Analysis with C18-reversed-phase chromatography. (B, C) Incubation of AMAC-labelled G2A0 disaccharide with ARSK-Arg84Cys cell lysates or with cell lysates of untransfected cells resulted in a main AMAC-peak at 26 mL (m/z 670.15). The G0A0-mediated fluorescence signal remained the minor peak in both samples. Of note, this minor peak in untransfected as well as in ARSK-Arg84Cys transfected cells results most likely from the activity of the endogenous ARSK of the HT1080 cells. The ubiquitous peak in the right of the chromatogram (>30 mL of retention volume) was not analysed in more detail as it was also present in unreacted samples. Western blot analysis (D): Comparable expression levels for ARSK-WT and ARSK-Arg84Cys but lack of ARSK-Leu187Ter in HT1080 cell lysates. This indicates comparable stability of ARSK-WT and ARSK-Arg84Cys and probable nonsense mediated mRNA-decay as a consequence of the Leu187Ter variant. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. WT, wild type.