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Exome Sequencing Identifies a Missense Variant in EFEMP1 Co-Segregating in a Family with Autosomal Dominant Primary Open-Angle Glaucoma.

Mackay DS, Bennett TM, Shiels A - PLoS ONE (2015)

Bottom Line: Primary open-angle glaucoma (POAG) is a clinically important and genetically heterogeneous cause of progressive vision loss as a result of retinal ganglion cell death.Exome sequencing identified a novel missense variant (c.418C>T, p.Arg140Trp) in exon-5 of the gene coding for epidermal growth factor (EGF) containing fibulin-like extracellular matrix protein 1 (EFEMP1) that co-segregated with disease in the family.The recent finding that a common variant near EFEMP1 was associated with optic nerve-head morphology supports the possibility that the EFEMP1 variant identified in this POAG family may be pathogenic.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, United States of America.

ABSTRACT
Primary open-angle glaucoma (POAG) is a clinically important and genetically heterogeneous cause of progressive vision loss as a result of retinal ganglion cell death. Here we have utilized trio-based, whole-exome sequencing to identify the genetic defect underlying an autosomal dominant form of adult-onset POAG segregating in an African-American family. Exome sequencing identified a novel missense variant (c.418C>T, p.Arg140Trp) in exon-5 of the gene coding for epidermal growth factor (EGF) containing fibulin-like extracellular matrix protein 1 (EFEMP1) that co-segregated with disease in the family. Linkage and haplotype analyses with microsatellite markers indicated that the disease interval overlapped a known POAG locus (GLC1H) on chromosome 2p. The p.Arg140Trp substitution was predicted in silico to have damaging effects on protein function and transient expression studies in cultured cells revealed that the Trp140-mutant protein exhibited increased intracellular accumulation compared with wild-type EFEMP1. In situ hybridization of the mouse eye with oligonucleotide probes detected the highest levels of EFEMP1 transcripts in the ciliary body, cornea, inner nuclear layer of the retina, and the optic nerve head. The recent finding that a common variant near EFEMP1 was associated with optic nerve-head morphology supports the possibility that the EFEMP1 variant identified in this POAG family may be pathogenic.

No MeSH data available.


Related in: MedlinePlus

EFEMP1 variant analysis.(A) Sanger sequence trace of the wild-type allele showing translation of arginine at codon 140 (CGG). ^ indicates cut-site for Hpa II. (B) Sanger sequence trace of the mutant allele showing the heterozygous C-to-T transition (denoted Y by the International Union of Pure and Applied Chemistry [IUPAC]) that is predicted to result in the missense substitution of arginine to tryptophan (TGG). (C) Allele-specific restriction fragment length analysis showing loss of an Hpa II restriction-site (5’-C^CGG) that co-segregated with affected individuals heterozygous for the C>T transition (300 bp). M, molecular mass markers (bp). Question marks indicate unconfirmed disease status. Filled symbols indicate affected status. Shaded symbols indicate disease haplotype.
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pone.0132529.g002: EFEMP1 variant analysis.(A) Sanger sequence trace of the wild-type allele showing translation of arginine at codon 140 (CGG). ^ indicates cut-site for Hpa II. (B) Sanger sequence trace of the mutant allele showing the heterozygous C-to-T transition (denoted Y by the International Union of Pure and Applied Chemistry [IUPAC]) that is predicted to result in the missense substitution of arginine to tryptophan (TGG). (C) Allele-specific restriction fragment length analysis showing loss of an Hpa II restriction-site (5’-C^CGG) that co-segregated with affected individuals heterozygous for the C>T transition (300 bp). M, molecular mass markers (bp). Question marks indicate unconfirmed disease status. Filled symbols indicate affected status. Shaded symbols indicate disease haplotype.

Mentions: The remaining, co-segregating novel exome variant (S2 Table) comprised a heterozygous C>T transition in exon-5 of the gene coding for epidermal growth factor (EGF) containing fibulin-like extracellular matrix protein 1 (EFEMP1), also known as fibulin-3, located on chromosome 2p16 (2:56,144,899). This missense change occurred at nucleotide position 418 from the first base of the translation-start codon in the cDNA sequence for EFEMP1 transcript variants 2 and 3 (c.418C>T), and resulted in the loss of an MspI/HpaII restriction site (Fig 2). Sanger sequencing confirmed the presence of the heterozygous c.418C>T change in both of the index affected males (I:1, II:4) and excluded mutations in other exons or splice-sites of EFEMP1 including a previously identified missense mutation (c.1033C>T, p.Arg345Trp) linked with inherited retinal drusen [11]. Allele-specific PCR amplification and Msp1 restriction-fragment-length genotyping further confirmed that the c.418C>T variant co-segregated with known affected but not unaffected relatives in the first two generations of the pedigree (Fig 2). Similar genotyping of the third generation confirmed the presence of the c.418C>T variant in several individuals of unknown disease status with a known affected parent (Fig 1). We note that this variant was not present in one individual in the third generation (III:1) or in her unaffected mother in the second generation (II:3). These observations suggested that the heterozygous c.418C>T variant in EFEMP1 may be predictive for inheriting POAG in this family.


Exome Sequencing Identifies a Missense Variant in EFEMP1 Co-Segregating in a Family with Autosomal Dominant Primary Open-Angle Glaucoma.

Mackay DS, Bennett TM, Shiels A - PLoS ONE (2015)

EFEMP1 variant analysis.(A) Sanger sequence trace of the wild-type allele showing translation of arginine at codon 140 (CGG). ^ indicates cut-site for Hpa II. (B) Sanger sequence trace of the mutant allele showing the heterozygous C-to-T transition (denoted Y by the International Union of Pure and Applied Chemistry [IUPAC]) that is predicted to result in the missense substitution of arginine to tryptophan (TGG). (C) Allele-specific restriction fragment length analysis showing loss of an Hpa II restriction-site (5’-C^CGG) that co-segregated with affected individuals heterozygous for the C>T transition (300 bp). M, molecular mass markers (bp). Question marks indicate unconfirmed disease status. Filled symbols indicate affected status. Shaded symbols indicate disease haplotype.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4498621&req=5

pone.0132529.g002: EFEMP1 variant analysis.(A) Sanger sequence trace of the wild-type allele showing translation of arginine at codon 140 (CGG). ^ indicates cut-site for Hpa II. (B) Sanger sequence trace of the mutant allele showing the heterozygous C-to-T transition (denoted Y by the International Union of Pure and Applied Chemistry [IUPAC]) that is predicted to result in the missense substitution of arginine to tryptophan (TGG). (C) Allele-specific restriction fragment length analysis showing loss of an Hpa II restriction-site (5’-C^CGG) that co-segregated with affected individuals heterozygous for the C>T transition (300 bp). M, molecular mass markers (bp). Question marks indicate unconfirmed disease status. Filled symbols indicate affected status. Shaded symbols indicate disease haplotype.
Mentions: The remaining, co-segregating novel exome variant (S2 Table) comprised a heterozygous C>T transition in exon-5 of the gene coding for epidermal growth factor (EGF) containing fibulin-like extracellular matrix protein 1 (EFEMP1), also known as fibulin-3, located on chromosome 2p16 (2:56,144,899). This missense change occurred at nucleotide position 418 from the first base of the translation-start codon in the cDNA sequence for EFEMP1 transcript variants 2 and 3 (c.418C>T), and resulted in the loss of an MspI/HpaII restriction site (Fig 2). Sanger sequencing confirmed the presence of the heterozygous c.418C>T change in both of the index affected males (I:1, II:4) and excluded mutations in other exons or splice-sites of EFEMP1 including a previously identified missense mutation (c.1033C>T, p.Arg345Trp) linked with inherited retinal drusen [11]. Allele-specific PCR amplification and Msp1 restriction-fragment-length genotyping further confirmed that the c.418C>T variant co-segregated with known affected but not unaffected relatives in the first two generations of the pedigree (Fig 2). Similar genotyping of the third generation confirmed the presence of the c.418C>T variant in several individuals of unknown disease status with a known affected parent (Fig 1). We note that this variant was not present in one individual in the third generation (III:1) or in her unaffected mother in the second generation (II:3). These observations suggested that the heterozygous c.418C>T variant in EFEMP1 may be predictive for inheriting POAG in this family.

Bottom Line: Primary open-angle glaucoma (POAG) is a clinically important and genetically heterogeneous cause of progressive vision loss as a result of retinal ganglion cell death.Exome sequencing identified a novel missense variant (c.418C>T, p.Arg140Trp) in exon-5 of the gene coding for epidermal growth factor (EGF) containing fibulin-like extracellular matrix protein 1 (EFEMP1) that co-segregated with disease in the family.The recent finding that a common variant near EFEMP1 was associated with optic nerve-head morphology supports the possibility that the EFEMP1 variant identified in this POAG family may be pathogenic.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, United States of America.

ABSTRACT
Primary open-angle glaucoma (POAG) is a clinically important and genetically heterogeneous cause of progressive vision loss as a result of retinal ganglion cell death. Here we have utilized trio-based, whole-exome sequencing to identify the genetic defect underlying an autosomal dominant form of adult-onset POAG segregating in an African-American family. Exome sequencing identified a novel missense variant (c.418C>T, p.Arg140Trp) in exon-5 of the gene coding for epidermal growth factor (EGF) containing fibulin-like extracellular matrix protein 1 (EFEMP1) that co-segregated with disease in the family. Linkage and haplotype analyses with microsatellite markers indicated that the disease interval overlapped a known POAG locus (GLC1H) on chromosome 2p. The p.Arg140Trp substitution was predicted in silico to have damaging effects on protein function and transient expression studies in cultured cells revealed that the Trp140-mutant protein exhibited increased intracellular accumulation compared with wild-type EFEMP1. In situ hybridization of the mouse eye with oligonucleotide probes detected the highest levels of EFEMP1 transcripts in the ciliary body, cornea, inner nuclear layer of the retina, and the optic nerve head. The recent finding that a common variant near EFEMP1 was associated with optic nerve-head morphology supports the possibility that the EFEMP1 variant identified in this POAG family may be pathogenic.

No MeSH data available.


Related in: MedlinePlus