Three novel ANO5 missense mutations in Caucasian and Chinese families and sporadic cases with gnathodiaphyseal dysplasia

Lingling Jin,Michael T Collins,Ying Hu,Ernst J Reichenberger,Albert S Woo,Keith Blackwell,Yi Liu,Fanyue Sun

doi:10.1038/srep40935

Abstract

Gnathodiaphyseal dysplasia (GDD; MIM#166260) is an autosomal dominant syndrome with characteristic cemento-osseous lesions of jawbones, bone fragility, and diaphyseal sclerosis of tubular bones. To date, only five mutations in the proposed calcium-activated chloride channel ANO5/TMEM16E gene have been identified. In this study, we describe two families and two singular patients with three new mutations. One Caucasian family with seven affected members exhibited frequent bone fractures and florid osseous dysplasia (p.Cys356Tyr), while one Chinese family with two affected members suffered from cementoma and purulent osteomyelitis (p.Cys360Tyr). In addition, two different novel mutations (p.Gly518Glu and p.Arg215Gly) were identified in sporadic patients without family history. In vitro studies overexpressing GDD mutations (p.Cys356Tyr and p.Cys360Tyr) showed significantly reduced ANO5 protein. It appears that all GDD mutations known so far locate in an extracellular domain following the first transmembrane domain or in the 4th putative transmembrane domain. Both wild-type and mutant ANO5 protein localize to the endoplasmic reticulum. After Ano5 gene knock-down with shRNA in MC3T3-E1 osteoblast precursors we saw elevated expression of osteoblast-related genes such as Col1a1, osteocalcin, osterix and Runx2 as well as increased mineral nodule formation in differentiating cells. Our data suggest that ANO5 plays a role in osteoblast differentiation.

Highlights

Hallmarks of gnathodiaphyseal dysplasia are the development of cemento-osseous lesions of the mandible and increased bone fragility
The mandibular mass of the proband from Family 1 developed around age 12 years and her fractures began to occur around the age of 7
The proband’s mother developed a lesion that was initially described as “ossifying fibroma”, but should be better termed as florid osseous dysplasia, at age 21 years and her brother was first diagnosed at age 12 years

Summary

Introduction

The gene responsible for GDD (GDD1, TMEM16E, ANOCTAMIN5, ANO5; MIM#608662) is a member of the TMEM16 gene family of calcium-activated chloride channels[12]. ANO5 encodes for a 913 amino-acid protein and belongs to a large family of transmembrane proteins which share a common predicted eight-transmembrane topology with N-and C-terminal cytoplasmic tails. We report two families of Caucasian and Chinese origin with autosomal dominant GDD caused by a p.Cys356Tyr mutation in ANO5 and a novel p.Cys360Tyr mutation, respectively as well as two novel heterozygous missense mutations (p.Gly518Glu and p.Arg215Gly) in two unrelated patients without family history. We describe the clinical features of the probands in detail as well as stimulatory effects on osteoblastogenesis by knocking down ANO5 in a pre-osteoblastic cell line and study the effects of p.Cys356Tyr and p.Cys360Tyr ANO5 mutations on protein expression

Methods

Results

Conclusion