Detection of a novel frameshift mutation and regions ...

Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, 2013; Early Online: 1–8

Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration Downloaded from informahealthcare.com by University of Western Ontario on 01/05/13 For personal use only.

ORIGINAL ARTICLE

Detection of a novel frameshift mutation and regions with homozygosis within ARHGEF28 gene in familial amyotrophic lateral sclerosis

CRISTIAN A. DROPPELMANN1, JIAN WANG2, DANAE CAMPOS-MELO1, BRIAN KELLER1, KATHRYN VOLKENING1,3, ROBERT A. HEGELE2 & MICHAEL J. STRONG1,3 1Molecular

Brain Research Group, Robarts Research Institute, University of Western Ontario, London, Ontario, Biology Research Group, Robarts Research Institute, London, Ontario, and 3Department of Clinical Neurological Sciences, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada

2Vascular

Abstract Rho guanine nucleotide exchange factor (RGNEF) is a novel NFL mRNA destabilizing factor that forms neuronal cytoplasmic inclusions in spinal motor neurons in both sporadic (SALS) and familial (FALS) ALS patients. Given the observation of genetic mutations in a number of mRNA binding proteins associated with ALS, including TDP-43, FUS/ TLS and mtSOD1, we analysed the ARHGEF28 gene (approx. 316 kb) that encodes for RGNEF in FALS cases to determine if mutations were present. We performed genomic sequencing, copy number variation analysis using TaqMan real-time PCR and spinal motor neuron immunohistochemistry using a novel RGNEF antibody. In this limited sample of FALS cases (n7) we identified a heterozygous mutation that is predicted to generate a premature truncated gene product. We also observed extensive regions of homozygosity in the ARHGEF28 gene in two FALS patients. In conclusion, our findings of genetic alterations in the ARHGEF28 gene in cases of FALS suggest that a more comprehensive genetic analysis would be warranted. Key words: Rho guanine nucleotide exchange factor, ARHGEF28, ALS, mutation, homozygosity, protein aggregates, neurofilament

Introduction Amyotrophic lateral sclerosis (ALS) is a fatal adultonset progressive disorder that is characterized by the degeneration of motor neurons in the brain and spinal cord (1). Most cases are clinically sporadic ALS (SALS), while approximately 5 10% have a family history (familial ALS; FALS). These FALS patients can harbour mutations in a diverse array of loci. Mutations in the Cu/Zn superoxide dismutase gene (SOD1) are one of the most frequent causes of ALS, accounting for approximately 20% of FALS and 1% of SALS cases (2,3). An additional 8 10% of FALS can be accounted for by mutations in the TARDBP and FUS/TLS genes (47), while an undetermined percentage can also be accounted for by mutations in UBQLN2, OPTN, PGRN and PFN1 genes (811). Recently, it has been shown that a

hexanucleotide repeat expansion in the C9orf72 gene could account for approximately 23.5% of FALS cases (12,13). Together, these mutations account for approximately 50% of all FALS cases, leaving an additional 50% with unknown genetic aetiology. A hallmark of the pathology of ALS is the formation of neuronal cytoplasmic inclusions (NCIs) demonstrating a range of immunoreactivities, including ubiquitin (14,15), neurofilament (16,17), peripherin (18), TDP-43 (19,20), FUS/TLS (6,7), SOD-1(21,22) and ubiquilin-2 (8). Degenerating spinal motor neurons are also characterized by selective decreases in the levels of polyadenylated mRNA, NFL mRNA, α-internexin mRNA and peripherin (23,24). Considering that TDP-43, FUS/TLS and mtSOD1 are RNA binding proteins (2530), and given the role of many of the known FALS genes in

Correspondence: M. J. Strong, Room C7–120, University Hospital, LHSC, 339 Windermere Road, London, Ontario N6A 5A5, Canada. Fax: 1 519 663 3609. E-mail: [email protected]. (Received 7 December 2012 ; accepted 9 December 2012 ) ISSN 2167-8421 print/ISSN 2167-9223 online © 2013 Informa Healthcare DOI: 10.3109/21678421.2012.758288


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C. A. Droppelmann et al.

the regulation of RNA metabolism, ALS is increasingly considered to be a disorder of RNA processing or metabolism (31 34). Neurofilament (NF) is composed of a highly conserved family of neuronal intermediate filament (IF) proteins: a 68-kDa form (low molecular weight NF; NFL), a 160-kDa form (middle molecular weight NF; NFM), and a 200-kDa form (high molecular weight NF; NFH), which assemble as heteropolymers to form the functional NF protein. The initial polymerization of NFL subunits is required for normal NF architecture formation (35). Disruption of the normal stoichiometry of IF protein expression, such as that observed with the over-expression of peripherin (36) of the selective loss of NFL (37), results in motor neuron dysfunction and death. These data highlight the importance of NF stoichiometry in motor neurons and the regulation of NFL mRNA stability as a possible cause of NF protein inclusions observed in ALS (38,39). We have previously demonstrated that Rho guanine nucleotide exchange factor (RGNEF) is a novel NFL mRNA destabilizing factor that regulates NFL mRNA stability and the levels of cellular NFL proteins and which forms pathological inclusions within the cell bodies of motor neurons of spinal cord from SALS and FALS patients (40). Taking into account the apparent relationship between RNA binding protein inclusion formation and the presence of mutations in their genes observed in FALS, we analysed the ARHGEF28 gene located on chromosome 5q13.2, that encodes RGNEF, to determine if mutations or genetic alterations exist that could be related to its pathology in ALS. We observed a heterozygous mutation in exon 6 of ARHGEF28 that would generate a gene product with a premature truncation. Moreover, we observed that in some FALS patients, ARHGEF28 shows extensive regions of homozygosity without evidence of copy number variation, suggesting that these runs of homozygosity are not due to a loss of chromosome segments (hemizygosity). This study provides the first evidence of genetic alterations in the ALS related gene ARHGEF28.

Materials and methods

mutations in the coding sequence of SOD1, FUS/ TLS and TARDBP were used in these studies. Four of the familial ALS patients presented C9orf72 expanded repeats (cases ALS-3, ALS-5, ALS-6, and ALS-7). RGNEF genomic sequencing Genomic DNA was isolated from frozen brain tissue obtained from FALS and normal control subjects using PureGene DNA isolation kits (Gentra Systems, QIAGEN, Toronto, Ont., Canada). PCR amplifications were performed using primers covering the entire coding region, intron-exon boundaries, and selected non-coding regions of the ARHGEF28 gene (NG_017198). Primer sequences and annealing temperatures for amplification are shown in Supplementary Table I to be found online at http:// informahealthcare.com/doi/abs/10.3109/21678421. 2012.758288. The table is only available in the online version of the journal. Please find this material with the following direct link to the article: http://informahealthcare.com/doi/abs/10.3109/21678421.2012.758288. PCR amplifications were carried out in a 50-μl mixture containing 32 pmole of each primer, 0.2 mM of each of dATP, dCTP, dGTP and dTTP, 1.5 mM MgCl2, 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 1.5 U of Taq Platinum DNA polymerase (Invitrogen, Life Technologies Inc., Burlingotn, Ont., Canada).Thirty amplification cycles were performed, consisting of denaturation at 94oC, annealing at 60oC, and extension at 72oC for 30 s each, followed by a final extension for 10 min at 72oC, and cooling to 4oC. PCR products were purified with Calf Intestinal Phosphatase/ExoI (New England Biolabs, Ipswich, MA, USA) and sequenced on an ABI 3730 DNA Analyzer (Applied Biosystems, Carlsbad, CA, USA). DNA sequences were analysed using SeqScape v2.6 (Applied Biosystems) using GenBank NG_017198 as the reference sequence. Amplification products of exon 6 from patient ALS-5 were subcloned into pCR2.1-TOPO Vector (Invitrogen). Thirty clones were sequenced to resolve multiple amplification products that were observed on direct sequencing of genomic DNA.

Cases All ALS cases were both clinically and neuropathologically confirmed using the El Escorial criteria (World Federation of Neurology Research Group on Neuromuscular Disease, 1994). Written consent for tissue donation at autopsy was obtained from either the patient (ante mortem) or the spouse (post mortem) in accordance with the London Health Sciences Centre ethics policies. Six control (neuropathologically healthy) cases (two male, four female, age range 5375 years) and seven FALS (five male, two female, age range 5471 years) cases without known

Copy number variation analysis using TaqMan real-time PCR Gene copy number analysis was performed by quantitative real-time PCR on an Applied Biosystems 7900HT instrument using ARHGEF28 gene-specific primers and a TaqMan oligonucleotide probe labelled with 6-FAM on its 5′ end and RNAse P primers and 5′ VIC-labelled TaqMan probe in each reaction as normalization control. Experiments were performed in reactions containing 10 μl TaqMan Universal PCR master mix, no AmpErase UNG (Applied


Genetic alterations in ARHGEF28 gene in ALS Biosystems), 2 μl gene primers and probes and 8 μl (10 ng) of genomic DNA. The experiments were performed in quintuplicate. For gene copy number assignment, absolute quantitation raw data (DCT values) were analysed with Copy Caller software v1.0 (Applied Biosystems) using the maximum likelihood analysis method, assuming 2 as the most frequent gene copy number for RGNEF. As negative controls, no template samples were used. The primers and probes used were: RGNEF forward 5′ CGT GGA CAA CAT GGC TTG CAG 3′; RGNEF reverse 5′ TGC AGG CTG TGA GGC GAT TG 3′; RGNEF TaqMan probe [6-Fam]TGG CTC GTC TGC TGG TGA CGC AGG[TAMRA]; RNAse P forward 5′ CAG ATT TGG ACC TGC GAG CG 3′; RNAse P reverse 5′ GAG CGG CTG TCT CCA CAA GT 3′; RNase P TaqMan probe [Vic]TTC TGA CCT GAA GGC TCT GCG CG[TAMRA]. The target of RGNEF primers is inside exon 6 of ARHGEF28 gene. Immunohistochemistry

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controls was performed. Our analysis showed notable findings in the genomic DNA of three patients (Table I, Supplementary Table II). Supplementary Table II is only available in the online version of the journal. Please find this material with the following direct link to the article: http://informahealthcare. com/doi/abs/10.3109/21678421.2012.758288. In the patient ALS-5 we observed heterozygosity for a novel frameshift mutation due to deletion of a single nucleotide (Figure 1A), which would be predicted to cause either: 1) a frameshift mutation with premature truncation, namely K280M fs40X (Figure 1B); or 2) a splicing mutation at the exon 6/ intron 6 splice junction, namely intron 6, 1 delG (GT TT), which would cause exon 6 skipping (Figure 1C). In either case, this allele would be predicted to encode a severely compromised gene product that at best would generate a very short protein product no more than 18% of the total length of the full RGNEF protein (Figure 1D).

Loss of heterozygosity of RGNEF in FALS patients

Archival paraffin-embedded sections of lumbar spinal cord from control and FALS cases were serially sectioned at 6 μm thickness and DAB-IHC was performed. Tissue sections were deparaffinized using standard protocols and then immunolabelled using a goat anti-RGNEF (40) (now commercially available – MediMabs cat# MM-0193-P) (1:500; optimum pH 7.4) antibody. The antigen-antibody complex was visualized using the Vectastain Elite ABC kit (Vector Laboratories, Burlington, Ont., Canada) and an Olympus BX45 microscope with Image-Pro Plus software (LEEDS Precision Instruments Inc, Minneapolis, MN, USA). Results RGNEF mutation in a case of FALS To evaluate the presence of genetic abnormalities in the ARHGEF28 gene, direct sequencing of RGNEF coding regions from ALS patients and normal

In two patients (ALS-2 and ALS-4) we observed extremely long stretches of homozygosity for known single nucleotide polymorphisms (SNPs) (Figure 2A). To confirm the continuity of homozygosity of these long stretches of genomic DNA, we also sequenced deep into the surrounding introns, focusing on regions reported to be highly polymorphic in the genomic reference sequence, essentially those enriched for known SNPs whose heterozygosity was close to 0.5. Specifically, patient ALS-2 had a homozygous block spanning from exon 1 to 9 (~170 kb) and from exon 14 to 33 (~52 kb). Also, patient ALS-4 had a homozygous block spanning from exons 1 to 24 (~258 kb) (Figure 2A). The absence of any heterozygosity, in both patients ALS-2 and ALS-4, over these long genomic stretches that spanned both intronic and exonic sequences could be the consequence of the presence of homozygosity or hemizygosity, the latter consistent with copy number variation for these regions of the gene. To address

Table I. Summary of genetic and pathological fi ndings in this study. A table showing the FALS cases that have RGNEF genetic alterations, C9orf72 expanded repeats and RGNEF pathology (RGNEF cytoplasmic inclusions).

Case ALS-1 ALS-2 ALS-3 (1) ALS-4 ALS-5 (1) ALS-6 ALS-7(3)

Gender

Age at symptom onset

F M F M M M M

62 52 61 60 51 53 56

Site of symptom onset

Disease duration (months)

RGNEF genetic alteration

RGNEF pathology

C9orf72 expanded repeats

bulbar FTD, prominent psychosis limb bulbar limb FTD, prominent behavioural ALSci

54 26 32 26 86 5 (2) 12

No Homozygosis No Homozygosis Frameshift mutation No No

Yes Yes No Yes Yes Yes Yes

No No Yes No Yes Yes Yes

1Siblings. 2Features of behavioural FTD of 60 months duration before onset of bulbar dysfunction; death within fi ve months of bulbar onset. 3Sister deceased with Pick ’s disease.


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Figure 1. Frameshift mutation in RGNEF in a case of FALS. (A) Sequence profi le showing the mutation and the single nucleotide deletion at the exon-intron 6 boundary found in ALS-5. These mutations are predicted to generate a frameshift mutation with premature truncation, namely K280M fs40X ( B ) or a splicing mutation at the exon 6/intron 6 splice junction, namely intron 6, 1 delG (GT TT) (C ), which would cause exon 6 skipping. ( D) Predicted protein products of frameshift mutation compared with full length RGNEF.

Figure 2. Regions with lack of heterozygosity and absence of copy number variation in the ARHGEF28 gene. A) Schematics of ARHGEF28 showing the regions in which a lack of homozygosis was observed (black lines) in ALS-2 and ALS-4. B) A copy number analysis of the ARHGEF28 predicted an n 2 on all cases used in this study. The cases with a lack of heterozygosity are indicated with solid arrows.

Genetic alterations in ARHGEF28 gene in ALS


this issue, we performed real-time PCR of genomic DNA to detect copy number variation over the segments of RGNEF described above. We observed a copy number of n 2 for patients ALS-2 and ALS-4 (Figure 2B). Consequently, the lack of heterozygosity observed in those patients can be attributed only to the presence of homozygosity in these regions of ARHGEF28. RGNEF pathology in cases with genetic alterations We analysed the localization of RGNEF in spinal cord motor neurons of cases with and without ARHGEF28 genetic alterations to determine whether there was apparent pathologic phenotype. Almost all cases showed cytoplasmic inclusions (Table I), as we have recently reported (40). The presence of RGNEF cytoplasmic inclusions, not detected in control samples (Figure 3A), was observed in the cases with homozygosity (Figure 3B, C), the case with mutation in exon 6 (Figure 3D), a case with absence of any ARHGEF28 genetic alteration, including absence of C9orf72 expanded repeats (Figure 3E) and in the cases with absence of ARHGEF28 genetic alterations but with C9orf72 expanded repeats (ALS-7 case is shown in Figure 3F). Only ALS-3 did not show RGNEF pathology (Table I). It is interesting that ALS-5, which bears the mutation that would lead to decreased protein expression and in which we expected less immunoreactivity of full length RGNEF, shows strong immunoreactive inclusions (Figure 3D). These results suggest that the RGNEF pathology observed in ALS patients is independent of the genetic alterations observed in the ARHGEF28 gene or the presence of expanded repeats in the C9orf72 gene. Discussion In the last several years, the discovery of new proteins and mutations associated with ALS has increased significantly (48). However, the finding of a cause or common mechanism to explain ALS pathology remains still elusive. We previously described the presence of cytoplasmic inclusions in ALS spinal motor neurons of a new NFL destabilizing factor called RGNEF (40). Considering that the pathology in ALS of other RNA binding proteins such as FUS/TLS, TDP-43 and mtSOD1 has been associated with mutations in their genes (5 7,30,41,42), we decided to search for genetic alterations in ARHGEF28 to determine whether this could explain the phenotype observed for its protein product in ALS. Although we examined only a limited number of cases, three of seven FALS cases showed genetic alterations within the ARHGEF28 gene. Two of the cases demonstrated stretches of homozygosity (Table I), a finding that has been associated with

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schizophrenia (43), late-onset Alzheimer’s disease (44), and certain cancers (45,46). Given the complex nature of these diseases, however, controversy has arisen from the suggestion that a cause-and-effect relationship with homozygosity exists (47). Despite this controversy, these examples suggest that recessive variants do contribute to the phenotype of complex diseases, as has been described for schizophrenia (43). Our patients demonstrated an average region of homozygosity of 240 kb, representing almost 80% of the gene, raising the possibility of a longer region of homozygosity in chromosome 5 of FALS patients including the ARHGEF28 gene region that could be related to the ALS pathology. The presence or absence of genetic alterations in the ARHGEF28 gene in the cases studied apparently does not affect the RGNEF pathology observed. This is interesting because a similar phenomenon has been found with TDP-43, where protein aggregates are observed with (41,48) or without (49,50) mutations in the TARDBP gene. This suggests that while the presence of genetic alterations in the ARHGEF28 gene is not a requirement for the presence of RGNEF cytoplasmic inclusions, it may be a contributing factor of cellular toxicity as has been suggested for the presence of mutant TDP-43 in ALS (51). The situation observed for the siblings ALS-3 and ALS-5 (Table I) is intriguing. The first case does not show ARHGEF28 genetic alterations or RGNEF pathology and presents a shorter length of the disease while the second has the heterozygous mutation and presents a longer duration of the disease (Table I). Considering that it has been described that shorter fragments of TDP-43 can contribute to the TDP-43 aggregates formation and the pathogenesis of ALS (52 54), it is possible that the presence of shorter fragments of RGNEF are contributing in some as yet unknown way to the RGNEF pathology in the ALS-5 case. Alternatively, it can be hypothesized that the presence in ALS-3 of another genetic factor, not previously evaluated, affects the severity of the disease. To date, there remains no clear association between the presence of expanded repeats in the C9orf72 gene in ALS and the presence of protein aggregates within spinal motor neurons. The presence of these expanded repeats has been directly associated with nuclear RNA foci of C9orf72 mRNA, and p62 positive/TDP-43 negative neuronal cytoplasmic and intranuclear inclusions in cerebellar and hippocampal neurons of C9orf72-linked FTLD and ALS (12,55). Coupled with our previous observation of the lack of C9orf72 protein aggregates in spinal motor neurons of ALS cases without SOD1 mutations (56), it is possible that the formation of RGNEF cytoplasmic inclusions is independent of the pathological process of C9orf72 expanded repeats (Table I). This study suffers from limitations of an extremely small sample size, and thus large-scale


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Figure 3. RGNEF pathology in FALS cases with genetic alterations in RGNEF. Immunohistochemistry of RGNEF in spinal cord motor neurons of a control case (A), ALS-2 ( B ), ALS-4 (C ) ALS-5 ( D), ALS-1 ( E ) and ALS-7 ( F ) cases. RGNEF cytoplasmic inclusions in these FALS cases are indicated by arrows. The presence or absence of genetic alterations are indicated inside of each picture (Scale bar 10 μ m).

population-based studies will be important to determine the significance of alterations in ARHGEF28 gene expression in ALS. Furthermore, it will be critical to determine whether RGNEF acts synergistically with, or modifies the phenotype of, the C9orf72 expansion. As a minimum, however, our study suggests that RGNEF joins the increasing number of RNA binding proteins that form pathological inclusions in ALS and which are associated with genetic mutations. Acknowledgements The authors wish to thank Rosa Rademakers for the C9orf72 genotyping of ALS patients. This work was supported by the Canadian Institute of Health Research (CIHR) and the ALS Society of Canada.

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Supplementary material available online Supplementary Tables I & II.

51.

52.

53.

54.

55.

56.

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