GJC2 promoter mutations causing Pelizaeus–Merzbacher-like disease

doi:10.1016/j.ymgme.2013.12.001

Molecular Genetics and Metabolism

Volume 111, Issue 3, March 2014, Pages 393-398

https://doi.org/10.1016/j.ymgme.2013.12.001 Get rights and content

Highlights

•
A homozygous mutation in the GJC2 regulatory region was found in two cases of PMLD.
•
c.170A > G is the second promoter mutation found in the SOX10 binding site of GJC2.
•
Reduced transcription is shown by both mutations through a luciferase reporter assay.
•
PMLD-causing mutations may result in profound down-regulation of the GJC2 promoter.
•
We provide genetic confirmation of pathogenicity by promoter mutations in PMLD.

Abstract

Objective

Pelizaeus–Merzbacher-like disease is a rare hypomyelinating leukodystrophy caused by autosomal recessive mutations in GJC2, encoding a gap junction protein essential for production of a mature myelin sheath. A previously identified GJC2 mutation (c.-167G > A) in the promoter region is hypothesized to disrupt a putative SOX10 binding site; however, the lack of additional mutations in this region and contradictory functional data have limited the interpretation of this variant.

Methods

We describe two independent Pelizaeus–Merzbacher-like disease families with a novel promoter region mutation and updated in vitro functional assays.

Results

A novel GJC2 mutation (c.-170G > A) in the promoter region was identified in Pelizaeus–Merzbacher-like disease patients. In vitro functional assays using human GJC2 promoter constructs demonstrated that this mutation and the previously described c.-167G > A mutation similarly diminished the transcriptional activity driven by SOX10 and the binding affinity for SOX10.

Interpretation

These findings support the role of GJC2 promoter mutations in Pelizaeus–Merzbacher-like disease. GJC2 promoter region mutation screening should be included in the evaluation of patients with unexplained hypomyelinating leukodystrophies.

Introduction

Hypomyelinating leukodystrophies are a rare cause of disease of the central nervous system (CNS) characterized by abnormal myelin formation [1]. The prototype condition for hypomyelinating leukodystrophies is Pelizaeus–Merzbacher disease (PMD) (OMIM 312080), an X-linked condition [2] that is due to a mutation in the proteolipid protein 1 gene (PLP1) (OMIM 300401). Pelizaeus–Merzbacher-like disease (PMLD) (OMIM 608804) is a clinically similar disease without detectable abnormalities within the PLP1 gene. PMLD is instead an autosomal recessive hypomyelinating leukodystrophy that was shown to be caused by mutations in the gap junction protein gamma-2 gene (GJC2) (OMIM 608803) that encodes the connexin 47 protein (Cx47), a connexin family member and gap junction protein important in astrocytes and oligodendrocytes [3], [4]. Mutation of GJC2 does not allow Cx47 to reach the membrane, resulting in loss of function [5]. Additionally, the GJC2 promoter region contains SOX10 transcriptional factor binding sites, which allow for SOX10 to play a role in myelin formation [5].

More than twenty different coding mutations have so far been identified in the GJC2 coding region [2], [4], [6], [7], [8], [9], [10], [11]. An additional mutation, c.-167A > G, was identified in the putative promoter region in individuals with the phenotype of PMLD [3], [5], [12], [13]. This promoter mutation was first identified in the homozygous state, has now been reported in 15 individuals from 5 families [3], [5], [12], [13], and has additionally been found in two patients [12] in the heterozygous state with another previously published mutation [7] within the coding sequence of GJC2. There is evidence suggesting that some c.-167A > G cases arose from a single founder [3], [13] and this mutation is thought to account for nearly a third of GJC2-PMLD phenotypes [13]. GJC2 mutations account overall for only 10% of unsolved cases of hypomyelination, suggesting that mutations in GJC2 and its promoter region at the SOX10 binding site are a rare cause of this phenotype [6]. Mutation c.-167A > G was demonstrated to result in decreased SOX10 dependent transcription of the luciferase reporter gene in constructs containing mouse Gjc2 promoter region [5]. However, previous studies using the human GJC2 regulatory region did not show that the c.-167A > G mutation disrupts the SOX10-dependent transcription [12].

Here we present two PMLD-affected individuals with a novel homozygous mutation (c.-170A > G) in the SOX10 binding site within the GJC2 regulatory region. We also demonstrate that both the c.-167A > G and c.-170A > G mutations reduce transcription of human GJC2 using a new luciferase reporter assay. Together, these studies further our understanding of the underlying causes of PMLD and the role of SOX10 in regulation of GJC2 in CNS myelin formation.

Section snippets

Clinical material collection and evaluation

Individual 1 and his unaffected brother were identified prospectively, as part of evaluation of individuals with unsolved leukodystrophies in the IRB-approved Myelin Disorders Bioregistry Project at Children's National Medical Center. Individual 2 and her unaffected family members were enrolled in an IRB-approved research protocol at Nemours Alfred I. duPont Hospital for Children. Affected individuals were examined by author AV (1) and authors JSS and KM (2). Informed consent was obtained. PMLD

Clinical data

Case histories are detailed in supplemental data (Supplement 2) and in Table 1. Both affected individuals presented in the newborn period with congenital nystagmus, and had significant motor delays in early childhood. Neither individual gained independent walking, but both walked supported for some time. Receptive language appeared relatively spared, as with cognition, but expressive language was severely limited by dysarthria. Both individuals had a combination of pyramidal and extrapyramidal

Discussion

In this report, we identified a new c.-170 A > G mutation in the GJC2 promoter region in two independent families with PMLD. The mutation is located in the putative SOX10 binding site, which lies within a highly conserved segment and is predicted to play an important role in GJC2 transcription. Previously, another promoter mutation, c.-167A > G, was identified in multiple families. Although the c.-167A > G mutation was shown to be deleterious using well-studied mouse promoter constructs, another

Authorship and contributions

Leo Gotoh, Ken Inoue, Grace Hobson and Adeline Vanderver took primary responsibility for writing and revising the paper. Leo Gotoh, Yu-ichi Goto and Ken Inoue performed experiments on promoter region function. Guy Helman, Sara Mora, Kiran Maski, Janet S. Soul, Miriam Bloom, Sarah Evans and Adeline Vanderver examined, counseled and provided patient specific information, Ljubica Caldovic and Grace M. Hobson provided critical review of sequencing results and in silico analyses.

Conflict of interest

Sara Mora and Dr. Grace Hobson report that they provide some services for A. I. duPont Molecular Diagnostics Laboratory. They could not determine whether a third party paid for diagnostic testing of the patients in the work under consideration. In any case, the patients were recruited into the study after the diagnostic testing was done (conception and planning of the study was done after the diagnostic testing), but the paper mentions the diagnostic test results. Dr. Hobson also reports grants

Acknowledgments

This work was funded in part by the Pelizaeus Merzbacher Disease Foundation (to AV and Gr Ho), grants from the Health and Labour Sciences Research Grants, Research on Rare and Intractable Diseases (H24-Nanchitou-Ippan-072, to K.I.), and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (KAKENHI: 21390103 and 23659531, to K.I.). Gr Ho is supported by NIH R01NS058978. Gu He is funded by the Delman family fund. Individual material

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¹: Dr Inoue and Leo Gotoh share the role of first author in this publication.

²: Dr Hobson and Dr Vanderver share the role of senior author in this publication.

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GJC2 promoter mutations causing Pelizaeus–Merzbacher-like disease

Highlights

Abstract

Objective

Methods

Results

Interpretation

Introduction

Section snippets

Clinical material collection and evaluation

Clinical data

Discussion

Authorship and contributions

Conflict of interest

Acknowledgments

Am. J. Hum. Genet.

Brain Dev.

Mol. Genet. Metab.

J. Mol. Biol.

Genomics

Genotype-phenotype correlation in inherited brain myelination defects due to proteolipid protein gene mutations. Clinical European Network on Brain Dysmyelinating Disease

Eur. J. Hum. Genet.

Three or more copies of the proteolipid protein gene PLP1 cause severe Pelizaeus-Merzbacher disease

Brain

Molecular confirmation of founder mutation c.-167A > G in Tunisian patients with PMLD disease

Gene

Disrupted SOX10 regulation of GJC2 transcription causes Pelizaeus-Merzbacher-like disease

Ann. Neurol.

Pelizaeus-Merzbacher like disease in a family with variable phenotype and a novel splicing gjc2 mutation

J. Child