Imprinting disorders are associated with mutations and epimutations affecting imprinted genes, that is those whose expression is restricted by parent of origin. Their diagnosis is challenging for two reasons: firstly, their clinical features, particularly prenatal and postnatal growth disturbance, are heterogeneous and partially overlapping; secondly, their underlying molecular defects include mutation, epimutation, copy number variation, and chromosomal errors, and can be further complicated by somatic mosaicism and multi-locus methylation defects. It is currently unclear to what extent the observed phenotypic heterogeneity reflects the underlying molecular pathophysiology; in particular, the molecular and clinical diversity of multilocus methylation defects remains uncertain. To address these issues we performed comprehensive methylation analysis of imprinted genes in a research cohort of 285 patients with clinical features of imprinting disorders, with or without a positive molecular diagnosis. 20 of 91 patients (22%) with diagnosed epimutations had methylation defects of additional imprinted loci, and the frequency of developmental delay and congenital anomalies was higher among these patients than those with isolated epimutations, indicating that hypomethylation of multiple imprinted loci is associated with increased diversity of clinical presentation. Among 194 patients with clinical features of an imprinting disorder but no molecular diagnosis, we found 15 (8%) with methylation anomalies, including missed and unexpected molecular diagnoses. These observations broaden the phenotypic and epigenetic definitions of imprinting disorders, and show the importance of comprehensive molecular testing for patient diagnosis and management.

BackgroundThe rare autosomal genetic disorder, Spondylo-meta-epiphyseal dysplasia with short limbs and abnormal calcifications (SMED-SL), is reported to be caused by missense or splice site mutations in the human discoidin domain receptor 2 (DDR2) gene. Previously our group has established that trafficking defects and loss of ligand binding are the underlying cellular mechanisms of several SMED-SL causing mutations. Here we report the clinical characteristics of two siblings of consanguineous marriage with suspected SMED-SL and identification of a novel disease-causing mutation in the DDR2 gene.MethodsClinical evaluation and radiography were performed to evaluate the patients. All the coding exons and splice sites of the DDR2 gene were sequenced by Sanger sequencing. Subcellular localization of the mutated DDR2 protein was determined by confocal microscopy, deglycosylation assay and Western blotting. DDR2 activity was measured by collagen activation and Western analysis.ResultsIn addition to the typical features of SMED-SL, one of the patients has an eye phenotype including visual impairment due to optic atrophy. DNA sequencing revealed a novel homozygous dinucleotide deletion mutation (c.2468_2469delCT) on exon 18 of the DDR2 gene in both patients. The mutation resulted in a frameshift leading to an amino acid change at position S823 and a predicted premature termination of translation (p.S823Cfs*2). Subcellular localization of the mutant protein was analyzed in mammalian cell lines, and it was found to be largely retained in the endoplasmic reticulum (ER), which was further supported by its N-glycosylation profile. In keeping with its cellular mis-localization, the mutant protein was found to be deficient in collagen-induced receptor activation, suggesting protein trafficking defects as the major cellular mechanism underlying the loss of DDR2 function in our patients.ConclusionsOur results indicate that the novel mutation results in defective trafficking of the DDR2 protein leading to loss of function and disease. This confirms our previous findings that DDR2 missense mutations occurring at the kinase domain result in retention of the mutant protein in the ER.

IEM was a major cause of NIHF in this cohort. The AARS2 variant accounts for a significant number of cases with NIHF in this cohort of Omani patients.

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