gb-2007-8-3-r32 GBJ Research <p>Alternative ion channel splicing in mesial temporal lobe epilepsy and Alzheimer's disease</p> Heinzen L Erin e.heinzen@duke.edu Yoon Woohyun yoon0001@mc.duke.edu Weale E Michael mweale@duke.edu Sen Arjune a.sen@ion.ucl.ac.uk Wood W Nicholas n.wood@ion.ucl.ac.uk Burke R James james.burke@duke.edu Welsh-Bohmer A Kathleen kwe@duke.edu Hulette M Christine hulet001@mc.duke.edu Sisodiya M Sanjay s.sisodiya@ion.ucl.ac.uk Goldstein B David d.goldstein@duke.edu

Institute for Genome Sciences and Policy, Center for Population Genomics and Pharmacogenetics, Duke University, Durham, NC 27710, USA

Department of Clinical and Experimental Epilepsy, Institute of Neurology, Queen Square, London WC1N 3BG, UK

Department of Molecular Neuroscience, Institute of Neurology, Queen Square, London WC1N 3BG, UK

Joseph and Kathleen Bryan Alzheimer's Disease Research Center, Duke University, Durham, NC 27710, USA

 Genome Biology 1465-6906 2007 8 3 R32 http://genomebiology.com/2007/8/3/R32 17343748 10.1186/gb-2007-8-3-r32 6 11 2006 16 2 2007 7 3 2007 7 3 2007 2007 Heinzen et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Alternative splicing in neuropathology

A novel microarray technology that permits the screening of alternative splice variants identifies disease-associated alternative splicing patterns in ion channel genes of patients with mesial temporal lobe epilepsy and Alzheimers disease.

Abstract

Background

Alternative gene transcript splicing permits a single gene to produce multiple proteins with varied functions. Bioinformatic investigations have identified numerous splice variants, but whether these transcripts are translated to functional proteins and the physiological significance of these alternative proteins are largely unknown. Through direct identification of splice variants associated with disease states, we can begin to address these questions and to elucidate their roles in disease predisposition and pathophysiology. This work specifically sought to identify disease-associated alternative splicing patterns in ion channel genes by comprehensively screening affected brain tissue collected from patients with mesial temporal lobe epilepsy and Alzheimer's disease. New technology permitting the screening of alternative splice variants in microarray format was employed. Real time quantitative PCR was used to verify observed splice variant patterns.

Results

This work shows for the first time that two common neurological conditions are associated with extensive changes in gene splicing, with 25% and 12% of the genes considered having significant changes in splicing patterns associated with mesial temporal lobe epilepsy and Alzheimer's disease, respectively. Furthermore, these changes were found to exhibit unique and consistent patterns within the disease groups.

Conclusion

This work has identified a set of disease-associated, alternatively spliced gene products that represent high priorities for detailed functional investigations into how these changes impact the pathophysiology of mesial temporal lobe epilepsy and Alzheimer's disease.

 Neurobiology Genome studies

Background

The complexity of the genome lies not only in the many genes comprising it, but also in the many levels of processing that influence the proteins that are produced and their abundance. One key site of regulation is the splicing of precursor RNAs to their associated mRNA transcripts. This process alone allows a single gene to have multiple different mRNA transcripts, producing proteins that may differ substantially from one another, even to the extent of having opposing effects 1. Overall, however, little is known about the functional differences amongst the alternative proteins produced from the same gene. Because the functional characterization of proteins can be laborious, it would be useful to be able to prioritize alternative transcripts more likely to have biological significance. One direction for prioritization is on the basis of association with human disease.

Alternative splicing of key genes generates alternative proteins that contribute to several prominent human diseases, for example, the spinal motor neuron protein in spinal muscular atrophy 2, cardiac troponin T, insulin receptor, myotubularin-related 1, and other proteins in myotonic dystrophies 345, and the tau protein in frontotemporal dementia and Alzheimer's disease 34 (other examples are reviewed extensively in 5). Furthermore, alternative splicing of a sodium channel gene, SCN1A, has also recently been associated with altered response to antiepileptic medications 6. There are potentially many more undetected examples of splicing alterations associated with disease pathophysiology and drug response variation in humans.

Studies of alternative splicing have usually been restricted to a single gene or small gene family. To date, there are only a few reports of splice variation screens in human disease and none has been reported for any central nervous system disease. Recently, new technology has become available that allows for the comprehensive investigation of alternative splicing through the use of splice variant microarrays. This technology uses probes in a microarray format and screens for unique exon-exon junctions specific to a particular splicing event 78910. Here we applied this systematic approach to assess the relationship between alternative splicing and two common and important neurological conditions, with the aim of identifying alternative splicing patterns of potential relevance to human disease.

Mesial temporal lobe epilepsy (mTLE) and Alzheimer's disease (AD) are highly complex neurological diseases characterized by aberrant neuronal excitation and neurodegeneration. While the pathological processes differ substantially, both diseases exhibit pathophysiology linked to ion channel activity. Seizure activity characteristic of epilepsy is the result of a dysregulation of inhibitory and excitatory neuronal signaling largely controlled by ion channel activity 11. Likewise, abnormal ion channel function also has been associated extensively with AD. AD-related neurodegeneration is believed to be, in part, caused by the overactivation of N-methyl-D-aspartate receptor activation and subsequent increases in intracellular calcium, oxidative stress, and neurodegeneration 12. Other ion channels, including glutamate receptors, nicotinic cholinergic receptors, and calcium and potassium channels, also have been implicated in AD pathophysiology 131415161718. Little information exists regarding the impact of splicing variation of ion channel genes on mTLE and AD. Our work sought to comprehensively evaluate ion channel splice variation in these two neurological diseases using a microarray format (ExonHit Therapeutics). We evaluated 1,665 known and potential splice events across 287 ion channel genes in human brain tissue samples collected from patients with AD and mTLE. In addition to identifying disease-associated splicing variation, a secondary aim of this work was to assess the reliability of the array-based identification of splicing changes through the use of real time PCR (rtPCR) to validate associations detected using the high-throughput platform.

Results

Mesial temporal lobe epilepsy

Following our initial screen of 1,665 possible alternative splicing events, a total of 221 splicing changes were identified as statistically significantly changed in mTLE samples, with p < 0.05, when comparing splice variant ratio (SVR) values calculated using equation 1 (see Materials and methods). Selected statistically significant events representing a range of p values were chosen for rtPCR confirmation. Of 13 splice array-identified alternative splicing events with an associated p value of less than 0.05, 9 were verified using rtPCR in a larger sample size. Evaluation of discrete groups of p value ranges revealed increased success rates with lower p values (0.02 < p < 0.05, 0% success (two events evaluated); 0.01 < p < 0.02, 70% success (seven events evaluated); p < 0.01, 100% success (3 events evaluated)). In order to report changes most likely to be real, only events that had a p value in splice array analyses of less than 0.02 are reported (Table 1). With this additional filter, a total of 126 alternative splicing events were observed in brain tissue collected from mTLE patients. As additional confirmation, four events not identified as changed in the splice array screen (p > 0.05) were confirmed not to be changed using rtPCR. The compiled list of rtPCR confirmed mTLE-associated alternative splicing events are included in Table 2.

<p>Table 1</p>

List of ion channel genes exhibiting alternative splicing in patients with mTLE

Genes by gene class

GenBank accession number (reference transcript*)

GenBank accession number (variant transcript*)

Alternative splicing event: change of mRNA transcript composition in epilepsy†

Alternatively spliced region

Splice array p value

Calcium channels

CACNA1A

NM_023035

U79668

ES: 26% ↓ variant/reference ratio

EXON 48

0.002

NM_023035

CQ723237

ASA: 24% ↓ reference/total ratio

EXON 23

0.003

NM_023035

BE972738

NE: 36% ↓ variant/total ratio

3' downstream

<0.001

CACNA1C

NM_000719

CQ722696

ASD: 94% ↓ variant/reference ratio

EXON 2

<0.001

NM_000719

CQ722691

ASD: 23% ↓ variant/reference ratio

EXON 24

0.017

CACNA1H

NM_021098

CQ730788

ASA: 25% ↓ variant/reference ratio

EXON 34

<0.001

CACNA1I

NM_001003406

AX068892

NE: 36% ↓ variant/reference ratio

INTRON 1

0.012

NM_001003406

AX068892

NE: 23% ↓ variant/reference ratio

INTRONS 20/21

0.009

CACNA2D1

NM_000722

BG211441

ES: 28% ↑ reference/total ratio

EXON 8

0.003

CACNB1

NM_000723

NM_199247

NE: 28% ↑ variant/reference ratio

INTRON 6

<0.001

NM_000723

BP316738

NE: 28% ↑ variant/reference ratio

INTRON 6

0.002

CACNB2

NM_201596

AF465485

ES: 20% ↓ variant/reference ratio

EXON 7

0.020

CACNB3

NM_000725

AK122911

NEs: 28% ↓ variant/total ratio

5' upstream

<0.001

CACNB4

NM_000726

AY054985

NE: 31% ↑ variant/reference ratio

INTRON 2

0.012

CACNG4

NM_014405

AW134993

PIED: 45% ↑ reference/total ratio

EXON 4

0.009

NM_014405

AI675178

PIED: 41% ↓ variant/reference ratio

EXON 4

0.003

Chloride channels

CLCN2

NM_004366

BC021578

ES: 38% ↓ reference/total ratio

EXON 13

<0.001

CLCN3

NM_001829

CQ732096

ASA: 22% ↓ variant/reference ratio

EXON 2

0.004

NM_001829

CQ736554

NE: 32% ↑ reference/total ratio

INTRON 12

0.012

CLCN6

NM_001286

BC050457

ASA: 16% ↓ variant/reference ratio

EXON 20

0.016

CLCN7

NM_001287

AK096963

IR: 36% ↓ variant/reference ratio

INTRON 18

<0.001

NM_001287

BQ920088

ASA: 21% ↓ variant/reference ratio

EXON 12

0.004

CLIC5

NM_016929

BC039380

ASA: 20% ↓ reference/total ratio

EXON 5

0.013

Sodium channels

SCN2A2

NM_021007

BC029489

ES: 23% ↑ reference/total ratio

EXON 1

0.006

SCN9A

NM_002977

BG108767

ASD: 26% ↓ variant/reference ratio

EXON 16

0.003

SCNN1A

NM_001038

BF033087

PIED: 21% ↓ variant/reference ratio

EXON 13

<0.001

SCNN1D

NM_002978

AX230571

NE: 52% ↑ variant/total ratio

INTRON 2

0.012

NM_002978

AK127357

NE: 77% ↑ variant/total ratio

5' upstream

0.002

Potassium channels

KCNAB2

NM_003636

BG720519

ES: 35% ↑ variant/reference ratio

EXON 1

0.014

NM_003636

BM823724

NEs: 19% ↓ variant/total ratio

5' upstream

0.015

NM_003636

CA495339

IR: 24% ↓ reference/total ratio

INTRON 11

0.020

KCNC3

NM_004977

AK127492

ASD: 27% ↓ variant/reference ratio

EXON 5

0.020

KCNH5

NM_172376

NM_139318

NE: 24% ↑ variant/total ratio

3' downstream

<0.001

KCNJ1

NM_153767

NM_000220

NE: 27% ↓ variant/reference ratio

INTRON 3

<0.001

KCNJ6

NM_002240

CQ738104

NEs: 24% ↓ variant/total ratio

INTRON 2

0.017

KCNJ15

NM_170736

NM_002243

NE: 108% ↑ variant/total ratio

5' upstream

<0.001

NM_170736

NM_170737

NE: 2.5x ↑ reference/total ratio

INTRON 2

<0.001

NM_170736

CQ732921

NEs: 42% ↓ variant/reference ratio

INTRON 2

0.005

NM_170736

BM544058

NE: 98% ↑ variant/total ratio

5' upstream

<0.001

NM_170736

BF105170

NE: 78% ↑ variant/total ratio

5' upstream

0.003

NM_170736

BI518753

NE: 2.5x ↑ reference/total ratio

INTRON 2

<0.001

KCNK1

NM_002245

AV733795

PIED: 34% ↓ variant/reference ratio

EXON 2

0.010

NM_002245

BG699040

NE: 45% ↓ variant/total ratio

INTRON 1

0.009

NM_002245

BF212472

NEs: 23% ↓ variant/total ratio

INTRON 1

0.017

NM_002245

BU661246

NEs: 48% ↓ variant/total ratio

INTRON 1

<0.001

KCNK2

NM_014217

BU956092

ES: 62% ↑ reference/total ratio

EXON 4

0.008

KCNK4

NM_016611

NM_033310

NE: 102% ↑ variant/total ratio

5' upstream

0.005

KCNK12

NM_022055

AX302031

NEs: 20% ↓ variant/total ratio

INTRON 1

0.014

KCNMA1

NM_002247

CQ870200

NE: 24% ↓ variant/reference ratio

EXON 20

0.006

NM_002247

CQ870204

PIED: 32% ↓ variant/reference ratio

3' downstream

0.020

NM_002247

BG185868

NE: 28% ↓ variant/reference ratio

EXON 9

0.014

KCNN2

NM_021614

NM_170775

NE: 26% ↓ variant/total ratio

INTRON 3

0.002

KCNQ2

NM_004518

AY358189

NE: 28% ↓ variant/reference ratio

INTRON 7

0.018

NM_004518

BG772772

NEs: 23% ↓ variant/reference ratio

INTRON 7

0.018

HCN3

NM_020897

CQ715247

EsS: 15% ↓ variant/reference ratio

EXONS 3-4

0.012

SLICK

NM_198503

CQ728754

EsS: 28% ↓ reference/total ratio

EXONS 22-25

0.019

NM_198503

CQ728754

ES: 57% ↑ variant/reference ratio

EXON 15

0.003

GABA receptors

GABRA2

NM_000807

CD014113

ES: 26% ↑ reference/total ratio

EXON 8

<0.001

NM_000807

CD014116

EsS: 29% ↑ reference/total ratio

EXONS 4-9

<0.001

NM_000807

CD014112

EsS: 35% ↑ reference/total ratio

EXON 4

<0.001

NM_000807

CD014107

EsS: 31% ↑ reference/total ratio

EXONS 7/8

<0.001

NM_000807

CD014110

EsS: 36% ↑ reference/total ratio

EXONS 6-8

<0.001

NM_000807

CD014109

EsS: 40% ↑ reference/total ratio

EXONS 6/7

<0.001

GABRA3

NM_000808

AX897950

ES: 17% ↓ reference/total ratio

EXON 7

0.007

GABRG2

NM_198904

NM_198903

ASA: 22% ↑ reference/total ratio

EXON 4

0.005

GABRR1

NM_002042

CB959800

NEs: 17% ↓ variant/total ratio

5' upstream

0.007

Ionotropic glutamate receptors

GRIA1

NM_000827

A46050

NE: 34% ↓ variant/reference ratio

INTRON 14

0.012

NM_000827

M64752

NE: 39% ↓ variant/reference ratio

INTRON 14

0.008

GRIA2

NM_000826

BC010574

NE: 44% ↓ variant/reference ratio

INTRON 13

0.007

NM_000826

AX147452

NE: 61% ↓ variant/reference ratio

INTRON 13/EXON14

0.005

NM_000826

AV748963

NE: 44% ↓ variant/reference ratio

EXON 14

0.015

GRIA3

NM_007325

NM_181894

NEs: 19% ↑ variant/reference ratio

INTRON 2

0.006

GRIK2

NM_021956

CQ715784

ASD: 65% ↓ reference/total ratio

EXON 10

<0.001

GRIK4

NM_014619

CQ869986

NEs: 29% ↓ variant/total ratio

5' upstream

0.006

NM_014619

CQ715345

NE: 31% ↓ variant/total ratio

INTRON 4

0.015

NM_014619

CQ715345

ES: 23% ↑ reference/total

EXON 7

0.005

GRIK5

NM_002088

CQ719647

ASD: 24% ↓ reference/total ratio

EXON 16

0.008

NM_002088

AX665460

ASD: 23% ↓ variant/reference ratio

EXON 18

0.004

NM_002088

CN420154

EsS: 27% ↓ variant/reference

EXONS 12-14

0.020

FXYD domain ion transport regulators

FXYD1

NM_021902

BQ181273

IR: 51% ↓ variant/reference ratio

INTRON 7

<0.001

FXYD6

NM_022003

AX430335

NE: 31% ↓ variant/total ratio

5' upstream

<0.001

NM_022003

AX892598

NE: 35% ↓ variant/total ratio

5' upstream

<0.001

NM_022003

BC018652

NEs: 38% ↓ variant/reference ratio

EXON 1/INTRON 1

<0.001

NM_022003

AL832811

NE: 33% ↓ variant/reference ratio

INTRON 1

<0.001

NM_022003

AW270073

ES: 34% ↓ variant/reference ratio

EXON 9

<0.001

NM_022003

BI823239

NE: 48% ↓ variant/reference ratio

INTRON 1

<0.001

NM_022003

BF527041

NE: 36% ↓ variant/reference ratio

INTRON 1

0.002

NM_022003

BP372334

NE: 45% ↓ variant/reference ratio

INTRON 1

<0.001

NM_022003

BE263758

NE: 37% ↓ variant/reference ratio

EXON 2

<0.001

NM_022003

BX488702

ASA: 50% ↓ variant/reference ratio

INTRON 2

<0.001

NM_022003

BI598749

NE: 40% ↓ variant/reference ratio

INTRON 1

0.006

FXYD7

NM_022006

CQ722304

ES: 21% ↓ variant/reference ratio

EXON 4

0.007

Transient receptor potential cation channels

TRPA1

NM_007332

BF570694

ASD: 37% ↓ reference/total ratio

EXON 25

<0.001

TRPC4

NM_016179

AF421362

ES: 24% ↓ reference/total ratio

EXON 3

0.004

TRPM1

NM_002420

AX480882

ASD:43% ↓ variant/reference ratio

EXON 17

<0.001

TRPM7

NM_017672

CQ728707

EsS: 35% ↑ variant/reference ratio

EXONS 7-11

<0.001

TRPV1

NM_080704

NM_080705

NE: 31% ↑ variant/total ratio

INTRON 1

0.007

NM_080704

AX686983

ES: 35% ↑ variant/reference ratio

EXON 8

0.005

TRPV2

NM_016113

BQ645005

ASD: 25% ↓ variant/reference ratio

EXON 15

0.005

Glycine receptors

GLRB

NM_000824

CD013911

ES: 31% ↑ variant/reference ratio

EXON 9

0.004

Inositol triphosphate receptors

ITPR1

NM_002223

CQ727353

EsS: 22% ↑ variant/reference ratio

EXONS 52-54

0.020

Purinergic receptors

P2RX1

NM_002558

BM469621

NE: 57% ↑ variant/total ratio

INTRON 3

<0.001

NM_002558

BP372168

ASD: 17% ↑ reference/total ratio

EXON 1

0.002

P2RXL1

NM_005446

BC064805

ASD: 24% ↑ variant/reference ratio

EXON 1

0.013

Cyclic nucleotide gated channels