Trafficking defects and loss of ligand binding are the underlying causes of all reported DDR2 missense mutations found in SMED-SL patients

Spondylo-meta-epiphyseal dysplasia (SMED) with short limbs and abnormal calcifications (SMED-SL) is a rare, autosomal recessive human growth disorder, characterized by disproportionate short stature, short limbs, short broad fingers, abnormal metaphyses and epiphyses, platyspondyly and premature calcifications. Recently, three missense mutations and one splice-site mutation in the DDR2 gene were identified as causative genetic defects for SMED-SL, but the underlying cellular and biochemical mechanisms were not explored. Here we report a novel DDR2 missense mutation, c.337G>A (p.E113K), that causes SMED-SL in two siblings in the United Arab Emirates. Another DDR2 missense mutation, c.2254C>T (p.R752C), matching one of the previously reported SMED-SL mutations, was found in a second affected family. DDR2 is a plasma membrane receptor tyrosine kinase that functions as a collagen receptor. We expressed DDR2 constructs with the identified point mutations in human cell lines and evaluated their localization and functional properties. We found that all SMED-SL missense mutants were defective in collagen-induced receptor activation and that the three previously reported mutants (p.T713I, p.I726R and p.R752C) were retained in the endoplasmic reticulum. The novel mutant (p.E113K), in contrast, trafficked normally, like wild-type DDR2, but failed to bind collagen. This finding is in agreement with our recent structural data identifying Glu113 as an important amino acid in the DDR2 ligand-binding site. Our data thus demonstrate that SMED-SL can result from at least two different loss-of-function mechanisms: namely defects in DDR2 targeting to the plasma membrane or the loss of its ligand-binding activity

Molecular characterization of the Portuguese patients with defects in GlcNAc-phosphotransferase: a key enzyme in the M6-P dependent lysosomal trafficking

Introduction: GlcNAc-phosphotransferase is one of the enzymes responsible for the formation of M6P residues and plays a key role in lysosomal trafficking, since most soluble acid hydrolases reach these organelles through the M6P pathway. It is composed of six subunits (α2β2γ2), products of two genes recently cloned: GNPTAB (mutated in mucolipidosis II/IIIA patients) and GNPTG (mutated in MLIIIC patients). Methods: Using both gDNA and cDNA extracted from patient’s fibroblasts, we performed a molecular study of both genes in 13 MLII/III patients (10Portuguese, 1Finnish, 1Spanish of Arab origin and 1Indian). Expression studies were performed by quantitative real-time PCR. Results: We identified 11 different mutations, 8 of them novel: 6 in the GNPTAB gene (c.121delG;c.440delC;c.2249_50insA;W81L;I403T and E667) and 2 in the GNPTG gene (c.610-1G.T and c.639delT). Interestingly, although the MLII-causing mutations have been mostly found to be private or rare, there is one (c.3503_3504delTC) that shows a broad distribution having been detected among different populations. This same mutation was also the most frequent one in our patients. Such distribution pattern prompted us to perform a haplotypic study. We analysed 37 patients (23Italians, 8Arab Muslims, 1Turkish and 5Portuguese) for 3 intragenic polymorphisms and 2 microsatellite markers flanking the GNPTAB gene, identifying a common haplotype. Regarding the mRNA expression studies, real-time results suggest the existence of feedback regulation mechanisms between α/β and the γ subunits. Discussion/Conclusion: This work enabled the establishment of a strong genotype-phenotype correlation, which is of crucial importance to an improved genetic counselling for ML families. The sharing of an ancestral haplotype by patients carrying the deletion implies a common origin of this mutation, while the higher level of diversity observed at the most distant locus indicates that it is a relatively ancient one. The developed strategies constitute valuable tools that allow carrier detection and prenatal- molecular diagnostics of these diseases

Mutations in DDR2 Gene Cause SMED with Short Limbs and Abnormal Calcifications

The spondylo-meta-epiphyseal dysplasia [SMED] short limb-hand type [SMED-SL] is a rare autosomal-recessive disease, first reported by Borochowitz et al. in 1993.1 Since then, 14 affected patients have been reported.2–5 We diagnosed 6 patients from 5 different consanguineous Arab Muslim families from the Jerusalem area with SMED-SL. Additionally, we studied two patients from Algerian and Pakistani ancestry and the parents of the first Jewish patients reported.1 Using a homozygosity mapping strategy, we located a candidate region on chromosome 1q23 spanning 2.4 Mb. The position of the Discoidin Domain Receptor 2 (DDR2) gene within the candidate region and the similarity of the ddr2 knockout mouse to the SMED patients' phenotype prompted us to study this gene6. We identified three missense mutations c.2254 C > T [R752C], c. 2177 T > G [I726R], c.2138C > T [T713I] and one splice site mutation [IVS17+1g > a] in the conserved sequence encoding the tyrosine kinase domain of the DDR2 gene. The results of this study will permit an accurate early prenatal diagnosis and carrier screening for families at risk

Mucolipidosis II is caused by mutations in GNPTA encoding the alpha/beta GlcNAc-1-phosphotransferase

Tiede S, Storch S, Lübke T, et al. Mucolipidosis II is caused by mutations in GNPTA encoding the alpha/beta GlcNAc-1-phosphotransferase. Nature Medicine. 2006;11(10):1109-1112

Development of Genomic DNA Reference Materials for Genetic Testing of Disorders Common in People of Ashkenazi Jewish Descent

Many recessive genetic disorders are found at a higher incidence in people of Ashkenazi Jewish (AJ) descent than in the general population. The American College of Medical Genetics and the American College of Obstetricians and Gynecologists have recommended that individuals of AJ descent undergo carrier screening for Tay Sachs disease, Canavan disease, familial dysautonomia, mucolipidosis IV, Niemann-Pick disease type A, Fanconi anemia type C, Bloom syndrome, and Gaucher disease. Although these recommendations have led to increased test volumes and number of laboratories offering AJ screening, well-characterized genomic reference materials are not publicly available. The Centers for Disease Control and Prevention-based Genetic Testing Reference Materials Coordination Program, in collaboration with members of the genetic testing community and Coriell Cell Repositories, have developed a panel of characterized genomic reference materials for AJ genetic testing. DNA from 31 cell lines, representing many of the common alleles for Tay Sachs disease, Canavan disease, familial dysautonomia, mucolipidosis IV, Niemann-Pick disease type A, Fanconi anemia type C, Bloom syndrome, Gaucher disease, and glycogen storage disease, was prepared by the Repository and tested in six clinical laboratories using three different PCR-based assay platforms. A total of 33 disease alleles was assayed and 25 different alleles were identified. These characterized materials are publicly available from Coriell and may be used for quality control, proficiency testing, test development, and research