Identification of biallelic EXTL3 mutations in a novel type of spondylo-epi-metaphyseal dysplasia

Abstract

Spondylo-epi-metaphyseal dysplasia (SEMD) is a group of inherited skeletal diseases characterized by the anomalies in spine, epiphyses and metaphyses. SEMD is highly heterogeneous and >20 distinct entities have been identified. Here we describe a novel type of SEMD in two unrelated Turkish patients who presented with severe platyspondyly, kyphoscoliosis, pelvic distortion, constriction of the proximal femora and brachydactyly. Although these phenotypes overlap considerably with some known SEMDs, they had a novel causal gene, exostosin-like glycosyltransferase 3 (EXTL3), that encodes a glycosyltransferase involved in the synthesis of heparin and heparan sulfate. The EXTL3 mutation identified in the patients was a homozygous missense mutation (c.953C>T) that caused a substitution in a highly conserved amino acid (p.P318L). The enzyme activity of the mutant EXTL3 protein was significantly decreased compared to the wild-type protein. Both patients had spinal cord compression at the cranio-vertebral junction and multiple liver cysts since early infancy. One of the patients showed severe immunodeficiency, which is considered non-fortuitous association. Our findings would help define a novel type of SEMD caused by EXTL3 mutations.

Figure 1

Patient 1. (a, b) Clinical pictures at age 5 months. Note frontal bossing, prominent eye, depressed nasal bridge, micrognathia and severe brachydactyly. (c–f) Radiographs at age 5 months. Note broad thorax, severe platyspondyly with kyphosis at the thoracolumbar junction, broad ilia with short greater notches, broad ischia and pubes, broad metaphyses of the long tubular bones, and severe brachydactyly. (g) Spinal magnetic resonance imaging at age 8 months prior to surgery showing spinal cord compression at the cranio-vertebral junction. A full color version of this figure is available at the Journal of Human Genetics journal online.

Figure 2

Patient 2. (a, b) Clinical pictures at age 8 months showing skeletal disorders. (c) Spinal magnetic resonance imaging at age 5 months showing spinal cord compression at the cranio-vertebral junction. (d–h) Radiographs of the skeleton at age 9 months (d, e) and age 17 months (f–h). The skeletal changes are identical with those of patient 1; however, brachydactyly is more severe and metaphyseal deformities are more conspicuous. A full color version of this figure is available at the Journal of Human Genetics journal online.

Figure 3

EXTL3 mutation. (a) Electropherograms of Sanger sequence of family 1. A homozygous mutation (c.953C>T; p.P318L) is found in the patients (II-1); the parents (I-1 and I-2) are heterozygous for the mutation. (b) Western blotting of the conditioned medium from the cells transfected with the wild-type (WT) and mutant (P318L) EXTL3 showing comparable amounts of the recombinant EXTL3 proteins (arrow). (c) Glycosyltransferase assay of the purified recombinant EXTL3s using UDP-[³H]GlcNAc as the donor substrate and heparin sulfate (HS) as the acceptor substrate. GlcNAc transferase activity was determined by incorporation of [³H]GlcNAc into the non-reducing end of GlcUA residue in HS. Mock: without expression vector. GlcNAc transferase activity of the mutant was significantly reduced. Values were mean±s.e. (n=3). *P<0.005 versus WT was calculated using the Student’s t-test. A full color version of this figure is available at the Journal of Human Genetics journal online.