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PRIMA1 mutation: a new cause of nocturnal frontal lobe epilepsy.

Hildebrand MS, Tankard R, Gazina EV, Damiano JA, Lawrence KM, Dahl HH, Regan BM, Shearer AE, Smith RJ, Marini C, Guerrini R, Labate A, Gambardella A, Tinuper P, Lichetta L, Baldassari S, Bisulli F, Pippucci T, Scheffer IE, Reid CA, Petrou S, Bahlo M, Berkovic SF - Ann Clin Transl Neurol (2015)

Bottom Line: No additional PRIMA1 mutations were found in 300 other NFLE cases.PRiMA knockout mice have reduction of AChE and accumulation of acetylcholine at the synapse; our minigene analysis suggests that the c.93+2T>C mutation leads to knockout of PRIMA1.Mutations with gain of function effects in acetylcholine receptor subunits cause autosomal dominant NFLE.

View Article: PubMed Central - PubMed

Affiliation: Epilepsy Research Centre, Department of Medicine, Austin Health, University of Melbogurne Melbourne, Victoria, Australia.

ABSTRACT

Objective: Nocturnal frontal lobe epilepsy (NFLE) can be sporadic or autosomal dominant; some families have nicotinic acetylcholine receptor subunit mutations. We report a novel autosomal recessive phenotype in a single family and identify the causative gene.

Methods: Whole exome sequencing data was used to map the family, thereby narrowing exome search space, and then to identify the mutation.

Results: Linkage analysis using exome sequence data from two affected and two unaffected subjects showed homozygous linkage peaks on chromosomes 7, 8, 13, and 14 with maximum LOD scores between 1.5 and 1.93. Exome variant filtering under these peaks revealed that the affected siblings were homozygous for a novel splice site mutation (c.93+2T>C) in the PRIMA1 gene on chromosome 14. No additional PRIMA1 mutations were found in 300 other NFLE cases. The c.93+2T>C mutation was shown to lead to skipping of the first coding exon of the PRIMA1 mRNA using a minigene system.

Interpretation: PRIMA1 is a transmembrane protein that anchors acetylcholinesterase (AChE), an enzyme hydrolyzing acetycholine, to membrane rafts of neurons. PRiMA knockout mice have reduction of AChE and accumulation of acetylcholine at the synapse; our minigene analysis suggests that the c.93+2T>C mutation leads to knockout of PRIMA1. Mutations with gain of function effects in acetylcholine receptor subunits cause autosomal dominant NFLE. Thus, enhanced cholinergic responses are the likely cause of the severe NFLE and intellectual disability segregating in this family, representing the first recessive case to be reported and the first PRIMA1 mutation implicated in disease.

No MeSH data available.


Related in: MedlinePlus

Minigene analysis reveals exon skipping. (A) RT-PCR results showing skipping of the first coding exon of PRIMA1 due to the c.93+2T>C mutation 48 h following transfection into HEK293T cells. A 100 base pair marker (lane 1) was run. The 124 base pair first exon of PRIMA1 is only expressed from the wild-type minigene construct (lane 2), resulting in a 369 base pair product. Whereas the mutant (lane 3) and empty vector control (lane 4) both lack the first exon producing 245 base pair products. The same result was observed in cells harvested 24 h posttransfection (data not shown). (B) Schematic illustration of exon skipping caused by the c.93+2T>C mutation compared to the wild-type splicing.
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fig03: Minigene analysis reveals exon skipping. (A) RT-PCR results showing skipping of the first coding exon of PRIMA1 due to the c.93+2T>C mutation 48 h following transfection into HEK293T cells. A 100 base pair marker (lane 1) was run. The 124 base pair first exon of PRIMA1 is only expressed from the wild-type minigene construct (lane 2), resulting in a 369 base pair product. Whereas the mutant (lane 3) and empty vector control (lane 4) both lack the first exon producing 245 base pair products. The same result was observed in cells harvested 24 h posttransfection (data not shown). (B) Schematic illustration of exon skipping caused by the c.93+2T>C mutation compared to the wild-type splicing.

Mentions: Since PRIMA1 was not expressed in lymphocytes and we did not have access to other tissues from family members, we examined the effect of the mutation using a minigene assay. PRIMA1 DNA fragments containing exon 2 (124 bp) with flanking intronic sequences (wild-type and c.93+2T>C) were amplified from the genomic DNA of unaffected and affected family members, respectively, and cloned into a splicing-competent minigene vector. HEK293T cells were transfected with the minigenes and the vector, and incubated for 24 or 48 h. Cellular RNA was then extracted and the splicing of minigene-derived RNA was analyzed by RT-PCR using PCR primers binding to the 5′ and 3′ exons of the vector. The results revealed incorporation of exon 2 in RNA produced by the wild-type minigene and skipping of exon 2 in RNA produced by the mutant minigene (Fig.3).


PRIMA1 mutation: a new cause of nocturnal frontal lobe epilepsy.

Hildebrand MS, Tankard R, Gazina EV, Damiano JA, Lawrence KM, Dahl HH, Regan BM, Shearer AE, Smith RJ, Marini C, Guerrini R, Labate A, Gambardella A, Tinuper P, Lichetta L, Baldassari S, Bisulli F, Pippucci T, Scheffer IE, Reid CA, Petrou S, Bahlo M, Berkovic SF - Ann Clin Transl Neurol (2015)

Minigene analysis reveals exon skipping. (A) RT-PCR results showing skipping of the first coding exon of PRIMA1 due to the c.93+2T>C mutation 48 h following transfection into HEK293T cells. A 100 base pair marker (lane 1) was run. The 124 base pair first exon of PRIMA1 is only expressed from the wild-type minigene construct (lane 2), resulting in a 369 base pair product. Whereas the mutant (lane 3) and empty vector control (lane 4) both lack the first exon producing 245 base pair products. The same result was observed in cells harvested 24 h posttransfection (data not shown). (B) Schematic illustration of exon skipping caused by the c.93+2T>C mutation compared to the wild-type splicing.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4554443&req=5

fig03: Minigene analysis reveals exon skipping. (A) RT-PCR results showing skipping of the first coding exon of PRIMA1 due to the c.93+2T>C mutation 48 h following transfection into HEK293T cells. A 100 base pair marker (lane 1) was run. The 124 base pair first exon of PRIMA1 is only expressed from the wild-type minigene construct (lane 2), resulting in a 369 base pair product. Whereas the mutant (lane 3) and empty vector control (lane 4) both lack the first exon producing 245 base pair products. The same result was observed in cells harvested 24 h posttransfection (data not shown). (B) Schematic illustration of exon skipping caused by the c.93+2T>C mutation compared to the wild-type splicing.
Mentions: Since PRIMA1 was not expressed in lymphocytes and we did not have access to other tissues from family members, we examined the effect of the mutation using a minigene assay. PRIMA1 DNA fragments containing exon 2 (124 bp) with flanking intronic sequences (wild-type and c.93+2T>C) were amplified from the genomic DNA of unaffected and affected family members, respectively, and cloned into a splicing-competent minigene vector. HEK293T cells were transfected with the minigenes and the vector, and incubated for 24 or 48 h. Cellular RNA was then extracted and the splicing of minigene-derived RNA was analyzed by RT-PCR using PCR primers binding to the 5′ and 3′ exons of the vector. The results revealed incorporation of exon 2 in RNA produced by the wild-type minigene and skipping of exon 2 in RNA produced by the mutant minigene (Fig.3).

Bottom Line: No additional PRIMA1 mutations were found in 300 other NFLE cases.PRiMA knockout mice have reduction of AChE and accumulation of acetylcholine at the synapse; our minigene analysis suggests that the c.93+2T>C mutation leads to knockout of PRIMA1.Mutations with gain of function effects in acetylcholine receptor subunits cause autosomal dominant NFLE.

View Article: PubMed Central - PubMed

Affiliation: Epilepsy Research Centre, Department of Medicine, Austin Health, University of Melbogurne Melbourne, Victoria, Australia.

ABSTRACT

Objective: Nocturnal frontal lobe epilepsy (NFLE) can be sporadic or autosomal dominant; some families have nicotinic acetylcholine receptor subunit mutations. We report a novel autosomal recessive phenotype in a single family and identify the causative gene.

Methods: Whole exome sequencing data was used to map the family, thereby narrowing exome search space, and then to identify the mutation.

Results: Linkage analysis using exome sequence data from two affected and two unaffected subjects showed homozygous linkage peaks on chromosomes 7, 8, 13, and 14 with maximum LOD scores between 1.5 and 1.93. Exome variant filtering under these peaks revealed that the affected siblings were homozygous for a novel splice site mutation (c.93+2T>C) in the PRIMA1 gene on chromosome 14. No additional PRIMA1 mutations were found in 300 other NFLE cases. The c.93+2T>C mutation was shown to lead to skipping of the first coding exon of the PRIMA1 mRNA using a minigene system.

Interpretation: PRIMA1 is a transmembrane protein that anchors acetylcholinesterase (AChE), an enzyme hydrolyzing acetycholine, to membrane rafts of neurons. PRiMA knockout mice have reduction of AChE and accumulation of acetylcholine at the synapse; our minigene analysis suggests that the c.93+2T>C mutation leads to knockout of PRIMA1. Mutations with gain of function effects in acetylcholine receptor subunits cause autosomal dominant NFLE. Thus, enhanced cholinergic responses are the likely cause of the severe NFLE and intellectual disability segregating in this family, representing the first recessive case to be reported and the first PRIMA1 mutation implicated in disease.

No MeSH data available.


Related in: MedlinePlus