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Differences in intracellular localisation of ANKH mutants that relate to mechanisms of calcium pyrophosphate deposition disease and craniometaphyseal dysplasia.

ABSTRACT: ANKH mutations are associated with calcium pyrophosphate deposition disease and craniometaphyseal dysplasia. This study investigated the effects of these ANKH mutants on cellular localisation and associated biochemistry. We generated four ANKH overexpression-plasmids containing either calcium pyrophosphate deposition disease or craniometaphyseal dysplasia linked mutations: P5L, E490del and S375del, G389R. They were transfected into CH-8 articular chondrocytes and HEK293 cells. The ANKH mutants dynamic differential localisations were imaged and we investigated the interactions with the autophagy marker LC3. Extracellular inorganic pyrophosphate, mineralization, ENPP1 activity expression of ENPP1, TNAP and PIT-1 were measured. P5L delayed cell membrane localisation but once recruited into the membrane it increased extracellular inorganic pyrophosphate, mineralization, and ENPP1 activity. E490del remained mostly cytoplasmic, forming punctate co-localisations with LC3, increased mineralization, ENPP1 and ENPP1 activity with an initial but unsustained increase in TNAP and PIT-1. S375del trended to decrease extracellular inorganic pyrophosphate, increase mineralization. G389R delayed cell membrane localisation, trended to decrease extracellular inorganic pyrophosphate, increased mineralization and co-localised with LC3. Our results demonstrate a link between pathological localisation of ANKH mutants with different degrees in mineralization. Furthermore, mutant ANKH functions are related to synthesis of defective proteins, inorganic pyrophosphate transport, ENPP1 activity and expression of ENPP1, TNAP and PIT-1.

SUBMITTER: Vijen S

PROVIDER: S-EPMC7198517 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Differences in intracellular localisation of ANKH mutants that relate to mechanisms of calcium pyrophosphate deposition disease and craniometaphyseal dysplasia.

Vijen Sunny S Hawes Chris C Runions John J Russell R Graham G RGG Wordsworth B Paul BP Carr Andrew J AJ Pink Ryan C RC Zhang Yun Y

Scientific reports 20200504 1

ANKH mutations are associated with calcium pyrophosphate deposition disease and craniometaphyseal dysplasia. This study investigated the effects of these ANKH mutants on cellular localisation and associated biochemistry. We generated four ANKH overexpression-plasmids containing either calcium pyrophosphate deposition disease or craniometaphyseal dysplasia linked mutations: P5L, E490del and S375del, G389R. They were transfected into CH-8 articular chondrocytes and HEK293 cells. The ANKH mutants d ...[more]

PMID: 32366894

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Project description:BackgroundCraniometaphyseal dysplasia (CMD) is a rare genetic disorder. Autosomal dominant CMD (AD-CMD) is caused by mutations in the ANKH gene. Affected individuals typically have distinctive facial features including progressive thickening of the craniofacial bones. Treatment for AD-CMD primarily consists of surgical intervention to release compression of the cranial nerves and the brain stem/spinal cord. To alleviate progression of the clinical course and improve the quality of life in children waiting to undergo the necessary surgery, we investigated clinical changes in a diagnosed patient with AD-CMD over three years.Case summaryA 17-mo-old boy presented with progressive nasal obstruction, snoring and hearing loss symptoms. Physical examination showed enlargement of the head circumference and clinical features such as wide nasal bridge, paranasal bossing, widely spaced eyes with an increased bizygomatic width, and a prominent mandible. The patient underwent otolaryngological examination, endoscopy, hearing test, laboratory examination of phosphorus and bone metabolism, cranial and femoral computed tomography, X-ray and next-generation sequencing. The patient was diagnosed with AD-CMD due to p.Phe377 deletion (c.1129_1131del) on exon 9 of the ANKH gene. After adherence to a prescribed low-calcium diet, the boy's alkaline phosphatase (ALP) levels continuously decreased to within the normal range. However, after 14 mo of dietary intervention, his parents altered his diet to an intermittent low-calcium diet to include milk and eggs. The patient's ALP was slightly higher than normal after the dietary change but remained close to the normal range. His serum osteocalcin changed to within normal levels after dietary regulation for 33 mo. His serum combined beta C-terminal telopeptide of type I collagen also continuously decreased after the nutritional intervention, although still slightly higher than normal levels. Despite fluctuating blood test results, the boy's nasal symptoms were markedly relieved and steadily improved after dietary intervention. No significant changes were found in the craniofacial bones by cranial radiography. Close monitoring of clinical features is still ongoing. Calcitriol treatment is currently under consideration and a surgical procedure is planned as necessary in the future.ConclusionWe herein report the first Chinese case of AD-CMD with heterozygous mutation of p.Phe377 deletion (c.1129_1131del) on the ANKH gene. Biochemical alterations were significantly improved after dietary intervention indicating that a low-calcium diet may be applied in pediatric AD-CMD patients with ANKH mutations to help alleviate phenotypic manifestations and improve the quality of life before surgical intervention. Further large scale studies are needed to replicate these findings and to establish the appropriate timing for nutritional and surgical interventions.

Dataset Information

Differences in intracellular localisation of ANKH mutants that relate to mechanisms of calcium pyrophosphate deposition disease and craniometaphyseal dysplasia.

Publications

Differences in intracellular localisation of ANKH mutants that relate to mechanisms of calcium pyrophosphate deposition disease and craniometaphyseal dysplasia.

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