Peter Kannu

Diagnostic yield of genetic testing in epileptic encephalopathy in childhood

Epilepsy is a common neurologic disorder of childhood. To determine the genetic diagnostic yield in epileptic encephalopathy, we performed a retrospective cohort study in a single epilepsy genetics clinic.

Detection of cryptic pathogenic copy number variations and constitutional loss of heterozygosity using high resolution SNP microarray analysis in 117 patients referred for cytogenetic analysis and impact on clinical practice

Background: Microarray genome analysis is realising its promise for improving detection of genetic abnormalities in individuals with mental retardation and congenital abnormality. Copy number variations (CNVs) are now readily detectable using a variety of platforms and a major challenge is the distinction of pathogenic from ubiquitous, benign polymorphic CNVs. The aim of this study was to investigate replacement of time consuming, locus specific testing for specific microdeletion and microduplication syndromes with microarray analysis, which theoretically should detect all known syndromes with CNV aetiologies as well as new ones. Methods: Genome wide copy number analysis was performed on 117 patients using Affymetrix 250K microarrays. Results: 434 CNVs (195 losses and 239 gains) were found, including 18 pathogenic CNVs and 9 identified as “potentially pathogenic”. Almost all pathogenic CNVs were larger than 500 kb, significantly larger than the median size of all CNVs detected. Segmental regions of loss of heterozygosity larger than 5 Mb were found in 5 patients. Conclusions: Genome microarray analysis has improved diagnostic success in this group of patients. Several examples of recently discovered “new syndromes” were found suggesting they are more common than previously suspected and collectively are likely to be a major cause of mental retardation. The findings have several implications for clinical practice. The study revealed the potential to make genetic diagnoses that were not evident in the clinical presentation, with implications for pretest counselling and the consent process. The importance of contributing novel CNVs to high quality databases for genotype–phenotype analysis and review of guidelines for selection of individuals for microarray analysis is emphasised.

Recessive Osteogenesis Imperfecta Caused by Missense Mutations in SPARC

Secreted protein, acidic, cysteine-rich (SPARC) is a glycoprotein that binds to collagen type I and other proteins in the extracellular matrix. Using whole-exome sequencing to identify the molecular defect in two unrelated girls with severe bone fragility and a clinical diagnosis of osteogenesis imperfecta type IV, we identified two homozygous variants in SPARC (GenBank: NM_003118.3; c.497G>A [p.Arg166His] in individual 1; c.787G>A [p.Glu263Lys] in individual 2). Published modeling and site-directed mutagenesis studies had previously shown that the residues substituted by these mutations form an intramolecular salt bridge in SPARC and are essential for the binding of SPARC to collagen type I. The amount of SPARC secreted by skin fibroblasts was reduced in individual 1 but appeared normal in individual 2. The migration of collagen type I alpha chains produced by these fibroblasts was mildly delayed on SDS-PAGE gel, suggesting some overmodification of collagen during triple helical formation. Pulse-chase experiments showed that collagen type I secretion was mildly delayed in skin fibroblasts from both individuals. Analysis of an iliac bone sample from individual 2 showed that trabecular bone was hypermineralized on the material level. In conclusion, these observations show that homozygous mutations in SPARC can give rise to severe bone fragility in humans.

Clinical phenotypes associated with type II collagen mutations

COL2A1 mutations give rise to a spectrum of phenotypes predominantly affecting cartilage and bone from the severe disorders that are perinatally lethal to the milder conditions that are recognised in the post‐natal period and childhood. The milder chondrodysplasias are characterised by disproportionate short stature, eye abnormalities, cleft palate and hearing loss. It remains poorly recognised that there is significant variability in the disease presentation, with early onset short stature conditions and later onset milder phenotypes. Similarly, it is under‐acknowledged that COL2A1 mutations may solely cause joint disease in the absence of the other mentioned phenotypic clues. The underlying hypothesis is that there are novel phenotypes caused by mutations in type II collagen that extend from premature arthritis through to more severe bone dysplasias. The importance of finding a COL2A1 mutation lies in the subsequent ability to accurately assess recurrence risks and offer information regarding disease natural history. Most importantly, it enables at‐risk individuals to be identified for implementation of preventative strategies and early ameliorative management of their condition. Such interventions potentially translate into a reduction in health costs associated with musculoskeletal disease.

The skeletal manifestations of the congenital disorders of glycosylation

The congenital disorders of glycosylation (CDG) are a rapidly expanding disease group with protean presentations. Specific end‐organ involvement leads to significant morbidity and mortality, and the skeletal manifestations are often not appreciated, apart from the common association of osteopaenia with CDG‐Ia. We performed a literature review of all documented skeletal manifestations in reported CDG patients, revealing a diverse range of skeletal phenotypes. We discuss the possible underlying mechanisms of these skeletal manifestations observed in CDG that are important and frequently under‐recognized.

Premature arthritis is a distinct type II collagen phenotype

Mutations in the gene encoding type II collagen (COL2A1) give rise to a spectrum of phenotypes predominantly affecting cartilage and bone. These chondrodysplasias are typically characterized by disproportionately short stature, eye abnormalities, cleft palate, and hearing loss. It is less recognized that mutations in COL2A1 can also present as degenerative joint disease in the absence of any other phenotypic clues. We report 2 Australian families presenting with an isolated arthritis phenotype, segregating as a dominant trait affecting both large and small joints, prior to age 30 years. Sequencing of COL2A1 in the propositi revealed 2 sequence changes resulting in glycine substitutions in the triple-helical domain of type II collagen. We review the increasing evidence implicating COL2A1 mutations in individuals presenting with isolated degenerative joint disease, aiming to alert physicians who assess these patients to this possibility. The importance of finding a COL2A1 mutation in such patients lies in the subsequent ability to accurately assess recurrence risks, offer early (including prenatal) diagnosis, and provide information regarding the natural history of the condition. Most importantly, it enables at-risk individuals to be identified for implementation of preventative strategies (i.e., weight loss, joint-friendly exercise programs) and early ameliorative management of their condition.

TRPV4 related skeletal dysplasias: a phenotypic spectrum highlighted byclinical, radiographic, and molecular studies in 21 new families.

BackgroundThe TRPV4 gene encodes a calcium-permeable ion-channel that is widely expressed, responds to many different stimuli and participates in an extraordinarily wide range of physiologic processes. Autosomal dominant brachyolmia, spondylometaphyseal dysplasia Kozlowski type (SMDK) and metatropic dysplasia (MD) are currently considered three distinct skeletal dysplasias with some shared clinical features, including short stature, platyspondyly, and progressive scoliosis. Recently, TRPV4 mutations have been found in patients diagnosed with these skeletal phenotypes.Methods and ResultsWe critically analysed the clinical and radiographic data on 26 subjects from 21 families, all of whom had a clinical diagnosis of one of the conditions described above: 15 with MD; 9 with SMDK; and 2 with brachyolmia. We sequenced TRPV4 and identified 9 different mutations in 22 patients, 4 previously described, and 5 novel. There were 4 mutation-negative cases: one with MD and one with SMDK, both displaying atypical clinical and radiographic features for these diagnoses; and two with brachyolmia, who had isolated spine changes and no metaphyseal involvement.ConclusionsOur data suggest the TRPV4 skeletal dysplasias represent a continuum of severity with areas of phenotypic overlap, even within the same family. We propose that AD brachyolmia lies at the mildest end of this spectrum and, since all cases described with this diagnosis and TRPV4 mutations display metaphyseal changes, we suggest that it is not a distinct entity but represents the mildest phenotypic expression of SMDK.

Metatropic Dysplasia: Clinical and Radiographic Findings in 11 Patients Demonstrating Long-Term Natural History

We report on the largest long‐term follow‐up study in metatropic dysplasia incorporating data collected over a 37‐year period. Case summaries from 11 patients are presented, ranging from 20 weeks of gestation to age 70 years, characterizing the natural history. All patients were seen through the Victorian Clinical Genetics Service and the Southern Cross Bone Dysplasia Centre. Our data lend little support for the current clinical classification of metatropic dysplasia and highlight a spectrum of severity in this rare condition, which we propose has an autosomal dominant inheritance pattern. Complications such as upper respiratory obstruction secondary to laryngo‐tracheal dysfunction need to be carefully monitored for in infancy because this is a preventable cause of mortality. The progression of a thoracic kyphoscoliosis in this condition is often relentless and resistant to surgical treatment. Other causes of morbidity include cervical instability, hearing loss, and functional impairments resulting from degenerative joint deformity. Intellectual outcome in all surviving cases has been normal. Final adult heights ranged from 107 to 135 cm.

Avascular necrosis of the femoral head due to a novel C propeptide mutation in COL2A1.

Avascular Necrosis of the Femoral Head Due to a Novel C Propeptide Mutation in COL2A1 Peter Kannu,* D.D. O’Rielly, J.C. Hyland, and L. Ala Kokko Medical Genetics, Department of Paediatrics, Queen’s University, Kingston, Canada Department of Pathology and Molecular Medicine, Queen’s University, Kingston, Canada Department of Paediatrics, University of Melbourne, Parkville, Melbourne, Australia Connective Tissue Gene Tests, Allentown, Pennsylvania

Two Novel COL2A1 Mutations Associated with a Legg-Calvé-Perthes Disease-like Presentation

BackgroundAbnormal development and growth of the capital femoral epiphysis and acetabulum are associated with a wide variety of underlying etiologies, one of which is Legg-Calvé-Perthes disease.Case DescriptionWe report the cases of two children who presented with abnormal development of both hips and in whom novel mutations in the COL2A1 gene were found. These cases illustrate the importance of identifying individuals with a type II collagen abnormality, as it informs management, allows investigation for other complications, and provides the opportunity for accurate genetic counseling and consideration of other family members who might be at risk.Literature ReviewThe literature documents numerous private mutations in COL2A1 associated with diverse clinical phenotypes including bilateral hip dysplasia and premature osteoarthritis. Some of these mutations are associated with a joint-specific phenotype but few other skeletal or extraskeletal manifestations. Only careful clinical examination of children presenting with hip anomalies therefore will reveal additional findings that warrant an evaluation by a clinical geneticist. DNA mutation analysis may be useful for making a specific diagnosis and identifying other at-risk family members.Purposes and Clinical RelevanceThe purpose of our report is to alert clinicians to the possibility that children who present with bilateral Perthes-like disease of the hip might have an underlying mutation in the gene encoding type II collagen. It is important to consider this in the differential diagnosis and workup of such children as it has specific prognostic, clinical, genetic counseling, and reproductive sequelae.