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Characterization of C9orf72 haplotypes to evaluate the effects of normal and pathological variations on its expression and splicing


Authors: Israel Ben-Dor aff001;  Crystal Pacut aff002;  Yuval Nevo aff003;  Eva L. Feldman aff002;  Benjamin E. Reubinoff aff001
Authors place of work: The Hadassah Human Embryonic Stem Cell Research Center, The Goldyne Savad Institute of Gene Therapy, Hadassah Medical Center, Jerusalem, Israel aff001;  Department of Neurology, University of Michigan Medical School, Ann Arbor, MI, United States of America aff002;  Computation Center, Hebrew University–Hadassah Medical School, Jerusalem, Israel aff003
Published in the journal: Characterization of C9orf72 haplotypes to evaluate the effects of normal and pathological variations on its expression and splicing. PLoS Genet 17(3): e1009445. doi:10.1371/journal.pgen.1009445
Category: Research Article
doi: https://doi.org/10.1371/journal.pgen.1009445

Summary

Expansion of the hexanucleotide repeat (HR) in the first intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in Caucasians. All C9orf72-ALS/FTD patients share a common risk (R) haplotype. To study C9orf72 expression and splicing from the mutant R allele compared to the complementary normal allele in ALS/FTD patients, we initially created a detailed molecular map of the single nucleotide polymorphism (SNP) signature and the HR length of the various C9orf72 haplotypes in Caucasians. We leveraged this map to determine the allelic origin of transcripts per patient, and decipher the effects of pathological and normal HR lengths on C9orf72 expression and splicing. In C9orf72 ALS patients’ cells, the HR expanded allele, compared to non-R allele, was associated with decreased levels of a downstream initiated transcript variant and increased levels of transcripts initiated upstream of the HR. HR expanded R alleles correlated with high levels of unspliced intron 1 and activation of cryptic donor splice sites along intron 1. Retention of intron 1 was associated with sequential intron 2 retention. The SNP signature of C9orf72 haplotypes described here enables allele-specific analysis of transcriptional products and may pave the way to allele-specific therapeutic strategies.

Keywords:

Alleles – Fibroblasts – Gene mapping – Haplotypes – Heterozygosity – Introns – Polymerase chain reaction – Single nucleotide polymorphisms

Introduction

Amyotrophic lateral sclerosis (ALS [OMIM: 612069]) and frontotemporal dementia (FTD [OMIM: 600274]) are two fatal neurodegenerative diseases. ALS is characterized by motor neuron (MN) degeneration in the brain and spinal cord, which causes progressive muscle wasting and paralysis. FTD is characterized by neuronal degeneration in the frontal and temporal brain lobes, leading to deterioration in language, behavior control, and emotional management. About 10% of ALS and 30–50% of FTD cases have a family history of the disease, typically inherited in an autosomal dominant fashion [1,2].

GGGGCC hexanucleotide repeat (HR) expansion in chromosome 9 open reading frame 72 (C9orf72) gene is the most common genetic cause of ALS and FTD in Caucasians [3,4]. It accounts for around 39% of familial and 7% of sporadic ALS cases and 25% of familial and 6% of sporadic FTD cases in the Caucasians [5]. The HR is located between two alternative 5’ non-coding exons, 1a and 1b (S1 Fig). In approximately half of all alleles, the HR is repeated twice, and in over 98% of the alleles its length is less than 17 repeats [3,6,7]. In diseased alleles, HR length is typically hundreds or thousands of repeats, while intermediate lengths of repeats (disease threshold of ~30) are relatively rare [3,4,8]. In the nucleus, HR expansion is bidirectionally transcribed to sense and antisense transcripts that sequester crucial RNA binding proteins [9]. In the cytoplasm, these transcripts undergo repeat-associated non-ATG (RAN) translation, resulting in the production and accumulation of pathogenic dipeptide repeat proteins [10]. C9orf72 expression from the mutant gene is variable and depends on its methylation status [1114]. Hypermethylation occurs in 36% and 17% of C9orf72-ALS and FTD patients, respectively [15], but not in normal and intermediate repeat lengths [14,16].

Toxic mRNA and dipeptide buildup concurrent with reduced levels of normal C9orf72 protein [17,18] interferes with basic cellular functions, such as splicing, translation, nucleocytoplasmic transport, autophagy, and organelle structure and function, eventually leading to neurodegeneration [19,20].

The C9orf72 gene contains two alternative promoters, three alternative first exons, and two alternative last exons in the three annotated transcript variants: V1 (NM_145005), V2 (NM_018325), and V3 (NM_001256054). V1 and V3 share a common start site, and their first exon ends at two alternative splice sites upstream to the HR. Hence, after V1 and V3 are transcribed, the HR is removed by post-transcriptional splicing. V2, the major C9orf72 variant that normally accounts for 85–95% of C9orf72 transcripts [13,21], starts downstream of the HR, which is therefore not present in transcribed V2 (S1 Fig). The majority of C9orf72 transcripts carry 11 exons that encode a 481-amino acid protein, while a fraction of the transcripts carry 5 exons with an alternative polyA site at an extended exon 5, which encodes a 222-amino acid protein (S1 Fig).

It has been demonstrated that all C9orf72 ALS and FTD (C9-ALS/FTD) patients share a common haplotype that is also abundant in healthy individuals. This risk (R) haplotype was initially characterized by a single nucleotide polymorphic site (SNP) rs3849942G>A, located 3.2 kb downstream to the C9orf72 gene 3’ terminus [3,22]. The risk haplotype was further characterized by 20 SNPs in a 120 kb block that spans C9orf72 with two additional genes [23,24], while more detailed maps demonstrated 44 and 82 SNP signatures along 110 kb [25,26]. Altogether, these maps include 19 SNPs within the 27.3 kb transcribed region of C9orf72 (underlined in Table 1), some are R haplotype specific SNPs.

Tab. 1. Detailed genetic map of the major C9orf72 haplotypes.
Detailed genetic map of the major <i>C9orf72</i> haplotypes.

Here, we established a map of over 100 SNPs and indels along the transcribed region of C9orf72 and identified the SNP signature of the R haplotype, as well as the other major haplotypes in Caucasians. This enables identification of the allelic origin of C9orf72 transcripts in patient and control cells. We show that pathological expansion of the HR is associated with lower V2 levels, while the V1 and V3 levels are comparable with normal R alleles. We also show that pathological HR expansion lowers C9orf72 first- and second-intron splicing efficiency and increases aberrant splicing at first-intron cryptic donor splice sites. We also determined the repeat length that characterizes each haplotype and show that increased HR length in the normal range is associated with higher C9orf72 expression. Deciphering the genetic profile of C9orf72 haplotypes and characterizing deleterious transcripts from the mutant gene are invaluable for developing disease allele-specific DNA or RNA targeting therapeutics.

Results

Characterization of C9orf72 haplotypes

We sought to create a detailed molecular map for the C9orf72 haplotypes to enable biallelic expression analysis and study the effects of HR length on its expression and splicing. Since most of the C9-ALS patients are Caucasians, we focused our efforts on Caucasian Americans. We first analyzed 12 C9-ALS patients for the surrogate marker rs3849942-A (S1 Table, #26). Eleven of these C9-ALS patients were heterozygous (A/G), while one patient (D-184) was homozygous (A/A). This patient carried an R haplotype with a pathological HR expansion (hereafter called Rd; d = disease) and an R haplotype with a normal HR length (hereafter called Rh; h = healthy). By directly sequencing PCR products along the C9orf72 locus in this patient (S1 Table, #1–26), we determined the SNP composition of the R haplotype, while the full SNP signature was accomplished by deep sequencing data (Tables 1 and S2).

We further confirmed the R haplotype and characterized the complementary non-R haplotype in the 11 heterozygous (A/G) C9-ALS patients by the same methodology. Six of the patients (D-007, D-540, D-836, D-338, D-431, D-478) shared a common haplotype that we named "K", carrying the previously described SNP rs10757668 G to A substitution in the 5’ untranslated region (UTR) of exon 2 [3]. Four of the patients (D-312, D-805, D-733, D-850) shared a common haplotype that we named "F" and one patient (D-883) carried a rare haplotype that we named "N". Similar analysis of 42 control subjects confirmed these haplotypes and revealed three additional haplotypes that we named P, J, and Q (S3 Table). The full characterization of these haplotypes was accomplished by deep sequencing data (Tables 1 and S2).

The distribution of the normal haplotypes among our 54 Caucasian American subjects was 28.4% for Rh, 22.1% for F, 20% for K, 16.8% for P, and 7.4%, 3.2%, and 2.1% for the haplotypes J, N, and Q, respectively (Fig 1A, left chart). Only one allele was identified with a "hybrid" haplotype, in which F was replaced by K towards the 3’ end of the gene (H-157 in S3 Table). Each haplotype, except Q, had three or more haplotype-specific SNPs (marked in italics in Tables 1 and S2). According to 1000 Genomes data, the frequency of these SNPs in Europeans is similar along the entire C9orf72 locus and particularly between exons 1 and 7 (Fig 1B). Based on these data, we predicted haplotype frequency among Europeans (Fig 1A, right chart), which was comparable to the haplotype distribution in our sample of Caucasian Americans (Fig 1A, left chart). Sequencing analysis of two PCR-amplified products in intron 1 (503 bp) and intron 6 (666 bp), which together carry nine polymorphic sites (S1 Table, #9, 21), suffices to distinguish between these seven haplotypes (Table 1). Notably, we identified an additional haplotype that we named "Z" from available deep sequencing data. We verified its sequence in several Asian samples from the 1000 Genomes Project, which we identified as homozygous for this haplotype [e.g., NA21124 (Gujarati) and HG00473 (Han Chinese)]. The Z haplotype contains three unique polymorphic sites: rs76412392T>A, rs117867610C>T and rs2305045A>C (Tables 1 and S2), and is rare in Europeans (~0.5%), and thus was not represented in our sample. Distinguishing between Q and Z haplotypes requires PCR analysis in addition to the two described above.

Fig. 1. C9orf72 haplotype frequency and HR lengths.
<i>C9orf72</i> haplotype frequency and HR lengths.
(A) C9orf72 haplotype frequency. The left chart represents the distribution in our sample of 54 Caucasian Americans (95 normal non-mutated alleles) described in S3 Table. The right chart represents the distribution in Europeans, based on haplotype-specific SNP frequency according to the 1000 Genomes data (viewed in Ensembl, http://www.ensembl.org/Homo_sapiens/Search/Results?q=; facet_feature_type=; site=ensembl;facet_species=Human;page=1). The Q haplotype does not harbor haplotype-specific SNPs; therefore, its frequency was calculated from the frequency of SNPs that are shared by Q and an additional haplotype with a known frequency. (B) Frequency of haplotype-specific SNPs along the transcribed region of C9orf72 gene and its 5’ and 3’ flanking regions among Europeans (S2 Table). SNPs are represented by dots, and their position in the graph indicates their position on chromosome 9 and in the C9orf72 gene (x-axis), and their frequency within the European population (y-axis). The SNPs of each haplotype are connected by a line with the same color per haplotype from (A). (C) Histogram of HR length distribution across different haplotypes in 101 non-mutated alleles (S3 Table). Each haplotype is represented by its specific color.

Altogether, we created a detailed molecular map for eight haplotypes at the C9orf72 locus that enables identification of the allelic origin of transcripts that carry heterozygous SNP sites.

The HR length may play a role in C9orf72 expression. We therefore completed our molecular map by analyzing the HR length. Previous studies have shown that HR length in normal R haplotype (Rh) is usually longer than non-R haplotypes (rs3849942 A/A versus G/G) [3,6,7,25]. Yet, the specific SNP signature and identity of the various non-R haplotypes was not known in these studies, so their HR length could not be determined. We therefore analyzed the HR length in Caucasian American subjects for whom we had identified their haplotype, as described above (Fig 1A, left chart), and in additional cell lines by PCR using primers flanking the HR [3]. Matching between the haplotype and HR length was initially determined in the 12 C9-ALS patients carrying a single normal allele copy (S2B and S2C Fig). As expected, we easily determined the length of the normal allele in each patient, while the expanded allele was detected in only one patient (D-478) as sawtooth peaks of 20–35 repeats with a maximal peak at 27 repeats (S2C Fig). For simplicity, we hereafter refer to this length as "intermediate", and to larger alleles that were undetectable by PCR as "large expansions". We next determined the HR length in 15 Caucasian subjects with homozygous haplotype (S2A Fig) and/or with equal HR length in both alleles, which were represented by a single peak. In another 30 subjects in whom HR length could not be absolutely linked to a specific haplotype, the linkage between the haplotype and HR length was in line and complementary to the data from the 27 informative subjects (S3 Table). We found that K, F, and Q haplotypes harbored 2 HR units, whereas N and P contained 4 and 5 HR units, respectively. The J haplotype predominantly had 6 HR units (7 out of 9 tested alleles) and Rh most frequently harbored 8 units (9 out of 26 tested alleles). A variable number of repeats were found in the other tested Rh alleles (Fig 1C and S3 Table). Because of HR length variation in J and R haplotypes, we hereafter specify HR length for these haplotypes (e.g., R haplotype with 12 units is designated R12).

Altogether, we established a molecular map of the haplotypes and their HR length in Caucasians.

Association between HR length and C9orf72 gene expression

Reduced levels of normal C9orf72 transcripts and proteins from the mutated allele can contribute to neural dysfunction in C9-ALS/FTD disease [2729]. Hence, the expression level from the complementary non-mutated allele may be important for the overall C9orf72 protein level per cell and thus modify the disease phenotype. We therefore used our genetic map to search for possible associations between HR length and C9orf72 expression from normal alleles.

It should be noted that a previous analysis of postmortem cerebellum samples from corticobasal degeneration patients revealed a positive correlation between V3 expression and the length of the longest HR allele, in a range of 2 to 28 repeats [30]. Yet this analysis measured the combined contribution of the two alleles and not the relative contribution of each allele to V3 expression.

To determine the relative allelic contribution to RNA transcripts, we used our SNP haplotype mapping for peak height analysis of heterozygous SNP sites in Sanger sequencing chromatograms of RT-PCR products. For accurate analysis, the peak height ratio of the two alternative nucleotides was corrected according to the ratio in which the two alleles contribute equally [3133]. We verified the reliability and the validity of the method to analyze the relative contribution of two alleles (S3 Fig).

We analyzed skin fibroblasts from 12 individuals carrying K and/or P alleles who are therefore heterozygous for SNP rs10757668 in exon 2 and/or rs17769294 in exon 5 (Fig 2A and S2 Table). The second allele in these heterozygous individuals included all haplotypes except Q, and HR lengths of 2, 4, 5, 6, 7, 8, 10, 12, 16, and ~30 repeats. By using a forward primer in exon 1a (nt 59–80) that is common to both V1 and V3 and a reverse primer in exon 2 or 5 (Fig 2A, blue arrows, and S1 Table, #27, 28), we determined the relative allelic expression of these upstream promoter transcripts. Remarkably, in all fibroblast cultures, the allele with the longer HR expressed more combined V1 and V3 than its complementary allele with the shorter HR. Moreover, a linear correlation was observed between the HR-length ratio (e.g., in a cell with 10 and 2 repeats, the ratio is 5) and the expression ratio between the two alleles (Fig 2B, R2 = 0.91, p = 1.8e-06). We further evaluated the relation between HR length and V1 and V3 expression in RhF fibroblasts, by amplifying V1 and V3 transcripts that also carry an extended exon 5 with rs774359 G/A heterozygous site (Fig 2A, orange arrows, and S1 Table, #29). Rh alleles (that contained 8–12 repeats) expression was dramatically higher than the complementary F alleles (2 repeats), supporting a positive effect of HR length on V1 and V3 expression (n = 4). We next evaluated the relation between HR length and V2 expression in fibroblasts by using the same methodology and heterozygous SNP sites (Fig 2A and S1 Table, #30–32). The HR length did not correlate significantly with V2 expression (Fig 2C, R2 = 0.22, p = 0.07).

Fig. 2. Association between HR length and C9orf72 gene expression.
Association between HR length and <i>C9orf72</i> gene expression.
(A) Schematic illustration of C9orf72 variants analysis. Black and green blocks represent translated and untranslated exonal regions, respectively. RT-PCR primers (arrows) and SNPs used for analyses are indicated. (B) Scatterplot showing significant positive correlation between V1+V3 expression level and HR length in normal and intermediate fibroblasts. The x-axis represents the HR length ratio between the longer to the shorter allele. The y-axis represents the expression ratio between the longer and the shorter allele. The line shows the best-fit regression line for the normal alleles. The intermediate R30K2 was not included in the equation due to its uncertain HR length. The inserts show the C/T heterozygous SNP rs10757668 (asterisk) within the RT-PCR amplified products of F2K2 (HR ratio of 1) and R30K2 (HR ratio of ~15). Blue peaks of the C nucleotide represent the F and the R30 alleles, and red peaks of the T nucleotide represent the K allele. (C) Scatterplot showing non-significant correlation between V2 expression level and HR length in non-mutated fibroblasts. The x- and y-axes are as in (B). (D) The overall allelic C9orf72 expression from Rh alleles that carry 7–16 repeats compared to complementary F, K and P alleles, which carry 2–5 repeats. The allelic ratio determined by the analysis of heterozygous SNPs along exons 2, 5 and 11 (S1 Table, #12, 24 and 38). Data presented as mean ± SEM. (E, F) Boxplots showing the relative expression from Rh allele with 7–16 repeat units (blue box) and from Rd allele with expanded HR (pink box), relative to the complementary shorter alleles. The R30 allele is depicted in black circle between the Rh and the Rd boxes. (E) V1+V3, (F) V2. Boxplots show median and quartiles and whiskers are 1.5 times the interquartile range. The orange horizontal line delineates a ratio of 1 and represents the non-R allele expression level. ns: non-significant.

To evaluate if the increased HR length is associated with an increase in overall allelic C9orf72 expression, we compared C9orf72 expression between Rh alleles with 7–16 repeats and their complementary K, F and P alleles, which carry 2–5 repeats. We observed a significant 1.25-fold increase in the overall allelic Rh levels compared to the non-R alleles (Fig 2D, p = 0.00016).

Next, we determined the relation between large HR expansions and C9orf72 expression in C9-ALS fibroblasts (RdK and RdF; n = 5 and n = 3, respectively). For V1 and V3, Rd allele expression was significantly higher than the K and F alleles in 7 out of 8 samples (Fig 2E), but did not significantly differ from the Rh allele (Fig 2E). For V2, Rd allele expression was significantly lower than the complementary K or F allele (Fig 2F; Rd values lower than 1, p = 0.0001). Moreover, the ratio between Rd alleles with large expansions and their complementary alleles was significantly lower than between the Rh alleles with 7–16 repeats and their complementary allele (Fig 2F, p = 0.0003). Notably, for the intermediate R30 allele, V2 expression was higher than its complementary K allele (Fig 2C and 2F, black dot). Thus, the effect of intermediate length of 30 repeats on C9orf72 expression differs from large expansions, as previously reported [14].

Association between HR length and introns 1 and 2 levels

There is uncertainty about the effect of C9orf72 HR expansion on intron 1 levels [12,3436]. We therefore performed RNA-seq to compare the RNA levels of C9orf72 introns in control and C9-ALS cells. We previously generated iPSC lines from control and C9-ALS fibroblasts [37], which express C9orf72 transcripts about three times more than skin fibroblasts (S4 Table), and are therefore more suitable for RNA-seq analysis (see Materials and Methods section for the methodology of analysis of relative RNA levels of introns).

In C9-ALS iPSCs, the mean normalized values for introns 1 and 2 were 3.1 and 2.7 times higher than control iPSCs, respectively (S4 Table and Fig 3A, p = 6e-06 and 0.0002, respectively), while no significant accumulation was observed in other introns (Fig 3A). Similar results were obtained in RNA-seq analysis of skin fibroblasts (S4 Table).

Fig. 3. Association between HR length and introns 1 and 2 levels.
Association between HR length and introns 1 and 2 levels.
(A) The relative frequency of RNA-seq reads aligned to C9orf72 introns in control (white) and C9-ALS (blue) iPSC lines. Intron reads from each iPSC line were normalized to those of exons 2–5. Data represented as fold-change versus control iPSCs. (B) RNA-seq reads of C9-ALS iPSCs that overlap with heterozygous SNP sites are identifiable from their allelic origin from either the normal (dashed columns) or Rd (solid columns) allele. Identifiable reads within intron 1 are depicted in red, and within other C9orf72 regions (without intron 2) in green. (C) Allelic ratio in introns 1 and 2 of C9-ALS fibroblasts determined by Sanger sequencing of RT-PCR amplicons, which was normalized to the ratio in C9orf72 exons. (D) The results in (C) displayed separately for the RdK fibroblasts (n = 4) and for other C9-ALS fibroblasts (RdF and RdN; n = 4). Data presented as mean ± SEM. ns: non-significant.

Our genetic map allows us to identify the allelic origin of reads that overlap heterozygous SNP sites (S4 Fig). Intron 1 carries multiple SNP sites and is therefore appropriate for such an analysis. In C9-ALS iPSCs, a mean of 138 versus 13 identifiable intron 1 reads were derived from the Rd and the normal allele, respectively, while for the rest of the gene, a mean of 96 reads derived from the Rd allele compared with 51 from the normal allele (Fig 3B; n = 3). These results show that in intron 1 of our C9-ALS iPSCs, there is over a 10-fold increase in the number of Rd reads versus its complementary allele. They also suggest that 1.9-fold of this increase is attributable to higher Rd expression and 5.6-fold is probably attributable to inefficient Rd splicing relative to the complementary allele.

We next analyzed the relative contribution of the Rd allele to intron 1 in C9-ALS fibroblasts by using Sanger sequencing chromatograms of heterozygous SNP sites (see Materials and Methods). The Rd allele contribution was 4.5-fold higher for intron 1 and 2.5-fold higher for intron 2 (Fig 3C; n = 8) but did not significantly differ in other introns that we examined (introns 3, 4, and 6). Since the analyses included amplicons that spanned intron 1-exon 2 and intron 2-exon 3 boundaries (S1 Table, #11 and 14), the relative increase in the Rd allele might be attributed to a decrease in its splicing efficiency rather than to an accumulation of excised introns or lariat intermediates.

We next examined the results according to the identity of the complementary allele. We found that for RdK cells, the ratio between the Rd to K allele was 2.8-fold for intron 1 (p = 0.004) and non-significant 1.5-fold higher for intron 2. In cells that did not carry the K allele (RdF and RdN cells), the Rd allele was 6.2-fold higher for intron 1 (p = 8e-08) and 3.4-fold higher for intron 2 (p = 0.0003). Thus, the Rd to K ratio was significantly lower than the Rd to F and N ratios (Fig 3D, p = 0.0002 for intron 1, p = 0.006 for intron 2) indicating that intron 1 and 2 levels in K allele transcripts were higher than in other normal alleles.

To verify this observation, we analyzed normal fibroblast cultures that are heterozygous to the K allele. Remarkably, the K allele contribution to introns 1 and 2 was on average two times higher than the complementary allele (S5A Fig). The increased contribution of K allele was not observed for other introns that we analyzed (S6 Fig).

Interestingly, intron 1 levels of K allele were 2.6 times higher than complementary alleles with 2–7 repeats, but only 1.4 times higher than alleles with 10–30 repeats indicating higher intron 1 levels in moderate and intermediate repeat lengths (S5A Fig). Moreover, analysis of fibroblasts from multiple donors with RK haplotypes supported a correlation between the HR length and intron 1 levels (S7 Fig). To verify the effect of moderate HR lengths, we further analyzed introns 1 and 2 in R12F and R12P fibroblast cultures. Intron 1 RNA levels were 1.6-fold higher in the R12 versus complementary F2 and P5 alleles (S5B Fig). Altogether, these results demonstrate that increased HR length is associated with higher intron 1 levels in normal and pathological ranges.

HR expansion activates cryptic donor splice sites within intron 1

It was previously shown that C9orf72 transcripts can use alternative donor splice sites in intron 1, downstream to exon 1b [35]. We therefore searched for alternative donor splice sites along intron 1 in both control and C9-ALS fibroblasts, by performing RT-PCR with several alternative forward primers along intron 1 and a reverse primer in exon 2 (S1 Table, #33–37). We identified four alternative splice donor sites in fibroblasts located 665, 910, 2295, and 2308 bp downstream to the V2 donor splice site at chr9: 27572766, 27572520, 27571136, and 27571123 (GRCh38/hg38), respectively (Fig 4A). These RT-PCR products were easily detected in C9-ALS cells but barely detectable in control cells. We therefore defined these splice sites as cryptic sites and named them C1, C2, C3, and C4, respectively (Fig 4A). C1 and C2 were analyzed separately, while C3 and C4 were analyzed together due to their proximity (13 bp) from each other. The relative allelic usage of these cryptic splice sites was determined according to the peak height ratio in heterozygous SNP sites (S8 Fig, asterisk).

Fig. 4. HR expansion activates cryptic donor splice sites in intron 1.
HR expansion activates cryptic donor splice sites in intron 1.
(A) Schematic presentation for the location and sequence of intron 1 cryptic donor splice sites C1-C4. (B) Boxplots showing relative allelic usage of cryptic splice-sites (C1-C4) in fibroblasts. K alleles (relative to F, N, P, J, and Rh alleles) are in green, Rh alleles with 7–16 repeats (relative to K, F, and P alleles) are in blue, the R30 allele (relative to K allele) is in grey, and the Rd alleles (relative to K, F, and N alleles) are in pink. The orange horizontal line delineates a ratio of 1 and represents the expression level of the complementary allele. (C) The expression of normal, unspliced, and cryptically spliced C9orf72 transcripts in control and C9-ALS iPSCs, based on the RNA-seq results. Data are presented as mean ± SEM. ns: non-significant.

In control fibroblasts, if cryptically spliced products were detectable, we frequently did not observe substantial differences between the two alleles (S8A and S8B Fig). Yet, a comparison between fibroblasts that carry heterozygous K and/or Rh allele revealed a possible trend for higher cryptic transcript level in the Rh versus K allele (Fig 4B, green and blue boxes). A similar non-conclusive trend was observed for D-478, who carried ~30 HR repeats (Fig 4B, grey box). Thus, it is possible that moderate and intermediate repeat lengths exert a minor effect on C1-C4 levels. In contrast, in C9-ALS fibroblasts that carry large HR expansions, most of C1-C4 transcripts were derived from the Rd allele (Figs 4B(pink box) and S8C-E). Moreover, the ratio between R allele and its complementary non-R allele was significantly higher for the Rd allele than for the Rh allele (Fig 4B, p = 0.0001).

To improve the RNA-seq mapping algorithm’s ability to identify reads originating from cryptic splicing events, we added C1-C4 to the Ensembl gene annotation file. In the three control iPSCs, we identified a single read containing a cryptic splice junction. In contrast, in the three C9-ALS iPSCs, we identified 24 reads containing a cryptic splice junction, and all but one joined to the 5’-end of exon 2 (Figs 4C and S9). Notably, eight of these reads that contained heterozygous SNP sites were all identified as Rd allele products.

To evaluate the portion of unspliced and cryptically spliced transcripts from total C9orf72 transcripts in normal and C9-ALS iPSCs, we counted reads that overlapped the 5’ boundary of exon 2 and contained either the terminus of authentic or cryptic exons as well as unspliced intron transcripts (S9 Fig). Based on this analysis, we estimate that in the control non-mutated iPSCs, about 91% of C9orf72 transcripts are normal while 8% are unspliced, and less than 1% are cryptically spliced. In contrast, in the C9-ALS iPSCs, only 57% of C9orf72 transcripts are normal, whereas 31% and 12% are unspliced and cryptically spliced, respectively (Fig 4C).

Discussion

To allow biallelic analysis of C9orf72 gene transcripts, we generated a detailed genetic variation map of the C9orf72 locus. We fully described the R haplotype and six other haplotypes (F, K, P, N, J and Q) that are common in Caucasians. In addition, we described the Z haplotype (Tables 1 and S2), which is common in China (>20%) and in India (>10%) but not among Europeans (~0.5%). Interestingly, among Asian C9-ALS patients, published genotypic analysis showed that neither allele matched the R haplotype. Thus, it was deduced that the diseased allele among Asian C9-ALS patients originated from a founder other than the one bearing the R haplotype [3840]. However, these studies did not characterize the diseased Asian allele or identified its unique SNPs. Based on the limited published genotyping results of Asian patients [3840], combined with our haplotype molecular map, it appears that the Z haplotype may be the Asian risk haplotype. However, to confirm this, additional studies are needed.

Our molecular mapping included the analysis of HR length in all haplotypes except haplotype Z. The HR length was constant in haplotypes harboring up to 5 repeat units, but not in haplotype J, which typically harbors 6 repeats. The highest level of HR length diversity was observed in haplotype R, which most frequently harbors 8 repeats (Fig 1C). These results are aligned with the reported correlation between tandem repeat length and the rate of change in length in subsequent generations [41,42]. Notably, the shortest documented HR length that changed in the subsequent generation was observed in a father and his daughter who had 11 and 12 repeats, respectively [6], whereas there are tremendous differences in pathological HR length between patient’s cells and tissues [6,4345].

We used our genetic map as a platform for biallelic expression analysis of cells from a single donor, instead of a more conventional comparison between cells from different donors with distinct genetic and environmental backgrounds, possibly with differing RNA or cDNA quality. Since the analysis is direct between the two alleles, it easily identifies unique allelic expression features, which are not mitigated but rather emphasized by the second conventional allele.

By analyzing the peak height ratio of two alternative nucleotides, we determined the relative allelic expression of C9orf72 transcripts in fibroblasts. We showed that the expression of the upstream promoter transcripts, V1 and V3, increases with HR length in the range of 2-~30 repeats (Fig 2B). Our results are in accordance with RT-qPCR results observed in the cerebellum of corticobasal degeneration patients [30]. The levels of V1 and V3 expression from complementary non-mutated alleles may be relevant to the disease pathogenesis. Elevated V1 and V3 expression from non-mutated alleles is associated with an increase in overall allelic C9orf72 expression (Fig 2D). Since a reduction in overall C9orf72 expression from the Rd allele can contribute to the C9-ALS/FTD disease [2729], a complementary non-mutated allele with increased C9orf72 expression might be beneficial to patients, when the Rd allele is strongly repressed. On the other hand, the increased upstream promoter activity of the non-mutated allele, which is correlated with higher HR length within the normal range, is associated with production of pre-spliced RNAs with a moderate HR length. This may aggravate the effects of the pathologically expanded HR RNAs.

Our study on Rd allele transcription revealed some common features among C9-ALS patients. Rd allele expression of V1 and V3 was higher than the complementary non-R allele in most samples, yet its expression levels was not higher than Rh allele. V2 expression from Rd alleles that carry a large but not intermediate expansion, was significantly lower than the complementary alleles, including the Rh allele, consistent with previous studies [14,21,43]. Given the reduced V2 levels from Rd compared to Rh alleles, the similar V1 and V3 levels, combined with the high levels of cryptic and unspliced intron 1 transcripts from the Rd allele (Fig 4C), it appears, as previously noted [13,21], that HR expansion in C9-ALS patients is associated with a marked shift in Rd allele promoter utilization from the downstream harmless promoter to the upstream and potentially harmful promoter.

We further evaluated the effects of pathological HR expansion on intron 1 splicing. Since our research strategy was based on comparative biallelic analysis, we studied intron 1 splicing in normal haplotypes. We observed retention of intron 1 in the K haplotype, which potentially could result from SNP rs10757668G>A in exon 2, just 18 bp downstream of intron 1 (S10 Fig) [46,47]. Therefore, we stratified normal alleles into K and non-K alleles for accurate evaluation of intron 1-retention levels of the Rd allele, when performing allelic evaluation (Fig 3D).

We showed in C9-ALS patients that there are higher unspliced intron 1 levels and activation of cryptic donor splice sites. These changes might be due to a repressive effect of the HR RNA on splicing factors at adjacent donor splice sites, resulting in HR containing transcripts that are either unspliced or cryptically spliced at sites that are more distal. Alternatively, these changes may reflect an adverse impact of the HR chromatin on the adjacent C9orf72 V2 promoter that resulted in activation of more downstream transcription start sites that produce either unspliced or cryptically spliced HR-less transcripts. Previous experiments showed that antisense oligonucleotides (ASO) designed to bind downstream of the HR efficiently reduce the number of HR-containing nuclear foci [21,48,49], supporting the first explanation.

We further demonstrated that in both Rd and K alleles, the increase in intron 1 levels is associated with an increase in intron 2 levels. This finding is not surprising, since mutation in a splicing site of one intron may affect the nearby intron [50]. Based on the number of RNA-seq reads in C9-ALS iPSCs (S4 Table) and the length of introns 1 and 2, we estimate that about 40% of the Rd allele transcripts that contain an unspliced intron 1 also carry an unspliced intron 2.

Notably, our results regarding C9orf72 transcripts and cryptic splicing were observed in fibroblasts and iPSCs, and it will be important to evaluate the generalizability of the results in MNs and, more preferably, MNs derived from iPSCs of ALS patients.

ASO [21,48,49,51] and CRISPR manipulations [52] have great therapeutic potential for C9-ALS/FTD. However, such therapies may also affect C9orf72 transcripts that originate from the normal allele. Reducing C9orf72 protein levels increases inflammatory conditions [53,54] and increases toxicity of dipeptide repeat proteins [20,28]. ASO designed against the HR sequence may confer a preferential targeting against the Rd allele [51]. However, since GGGGCC repeats are found in multiple genes [55], an ASO that targets the pathogenic repeat may reduce the expression of multiple genes. Therefore, ASO exploiting heterozygous SNP sites for specifically targeting the Rd allele and minimize interference to normal allele transcripts might be the best strategy, as previously demonstrated in Huntington disease [5658]. Based on our results, introns 1 and 2 might be good candidates for allelic specific targeting, while the 5’ region of intron 1, which is also retained in cryptically spliced transcripts, might be the best target region. Notably, since the repertoire of the heterozygous SNP sites in patients depends on the identity of the complementary allele, the preferred target site might differ between patients.

Our present molecular map of the C9orf72 gene (Tables 1 and S2) spans a relatively short genomic region of about 30 kb that exhibits a relatively low recombination rate of ~0.35 cM/Mb (centiMorgans per megabase). Since recombination events along this region are rare, most of the Caucasian alleles are of "pure" haplotype (S3 Table) and the allelic origin of heterozygous SNPs can be easily determined. Our map is straightforward, user friendly, and enables the study of specific effects of Rd and other alleles, design of allelic-specific ASOs, and evaluation of the impact of drugs and ASOs on allelic expression of beneficial transcripts such as V2, and harmful transcripts such as unspliced and cryptic transcripts.

In a wider perspective, deciphering the major haplotypes for other genes, and the formation of a straightforward and accessible molecular map, may improve a researcher’s ability to determine effects of genetic variations and to identify imprinted genes. Most importantly, this may enable specific targeting of toxic alleles in protein misfolding disorders.

In summary, we generated a detailed genetic map of the major C9orf72 haplotypes and applied polymorphic sites to determine the effects of HR length and SNP variation on C9orf72 transcript expression and splicing in normal and C9-ALS cells. The characterization of C9orf72 genetic variations may pave the way to allele-specific therapeutic strategies.

Materials and methods

Ethics statement

The ALS patients and the healthy control participants signed an informed consent that included genetic analysis of acquired biospecimens. The study was approved by the University of Michigan Medical School Institutional Review Board (Protocol # HUM00028826). Established lines HEK293, HES-1 (a human embryonic stem cell line; NIH code ES01), OL (a kind gift from Michel Revel) and FOR002 (both are foreskin lines) were used to correlate the HR length with specific haplotypes.

Fibroblast isolation

Human skin fibroblast cultures were established from punch biopsies from Caucasian control and ALS patients from the University of Michigan ALS Clinic. Biopsies were placed in fibroblast growth media (GM) [high-glucose DMEM supplemented with 10% fetal calf serum, 1× Glutamax-1 and 1× MEM NEAA (Gibco/Thermo Fisher Scientific, Waltham, MA)]. Tissues were washed three times and then cut into small pieces that were resuspended in 0.5 ml GM and plated in a minimal amount of GM in two T25 flasks in a humidified atmosphere with 5% CO2 at 37°C. At day 3 and day 6, one ml of GM was added to each flask. Keratinocytes migrated out of the tissue pieces at day 7, and fibroblasts migrated out at day 10 or later. At ~day 21, the cells in both T25 flasks were trypsinized with 0.25% trypsin-EDTA (Gibco) and split 1:5 into 100 mm dishes (Falcon or Corning). After 7–10 days, the fibroblasts reached ~85% confluency and were trypsinized to make 9 working stock vials in freezing medium [GM + 10% DMSO (Sigma-Aldrich, St Louis, MO)], which were stored in liquid nitrogen in collaboration with the Michigan ALS Consortium at the University of Michigan.

Cell culture

Skin fibroblasts were cultured in high-glucose DMEM (Invitrogen/Thermo Fisher Scientific) supplemented with 15% fetal calf serum (Biological Industries, Beit Haemek, Israel), 2 mM l-glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin (all from Invitrogen). iPSC lines were maintained on mitomycin-C (MMC, 10 μg/ml; Sigma-Aldrich)-treated human foreskin fibroblasts in gelatin-coated 6-well plates (3 × 105 feeders/well; Nunc, Roskilde, Denmark) cultured in hESC medium, which consisted of Knockout DMEM supplemented with 16% KnockOut SR, 2 mM l-glutamine, 1% nonessential amino acids, 0.1 mM β-mercaptoethanol, 50 U/ml penicillin, 50 μg/ml streptomycin (all from Invitrogen), and 5 ng/ml bFGF (PeproTech, Rocky Hill, NJ) in a 5% O2 incubator. iPSCs were passaged weekly by mechanical dissection or dissociation with 1 mg/ml collagenase IV (Gibco).

Genomic DNA extraction

Skin fibroblasts were washed with PBS and dissociated with 0.04% trypsin–0.17 mM EDTA (Invitrogen). Following centrifugation, cells were resuspended to an estimated cell concentration of 106 cells/ml in PBS. Five microliters of the resuspended cells were added to 15 μl pre-chilled lysis buffer (final concentration: 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.1% Triton X-100, 0.1% Tween 20) with 0.2 μl proteinase K (Roche) in 0.2 ml Eppendorf tube. Cells were then incubated at 56°C for 2 h followed by 96°C for 25 min and 15°C for 15 min. For iPSC analysis, half of an iPSC colony was washed with PBS, collected in 5 μl PBS, and treated as described for fibroblasts.

RNA isolation and cDNA preparation

Total RNA was isolated from fibroblasts (passages 4–7) and iPSCs (passages 6–10) using a PerfectPure RNA Cultured Cell Kit (5Prime, Gaithersburg, MD) following the manufacturer’s protocol, including on-column DNase treatment. For RT-PCR, 2 μg of total RNA was reverse transcribed using the High-Capacity cDNA RT kit (Applied Biosystems/Thermo Fisher Scientific) with the provided random hexamer primers. Reaction mixtures were prepared with and without RT to confirm there was no detectable genomic DNA contamination in the samples.

PCR analysis

PCR amplifications were performed with 2× PCRBIO HS Taq Mix (PCR Biosystems, United Kingdom), KAPA HiFi HotStart ReadyMix (2×, KAPA Biosystems/Roche), and Phusion Hot Start High-Fidelity DNA polymerase (Finnzymes/Thermo Fisher Scientific), using the primers listed in S1 Table. PCR products were excised from agarose gels and extracted using Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI). Amplicons were Sanger sequenced using BigDye Terminator v1.1 Cycle Sequencing Kit and 3730xl DNA Analyzer (Applied Biosystems, CA). Sequence Scanner Software 2 (Applied Biosystems, CA) was used for sequence data analyses. To calculate the allelic expression ratio, the peak height ratio of the two alternative nucleotides within a cDNA amplified product was normalized to the peak height ratio measured in genomic DNA PCR samples, which represents a 1:1 allelic ratio. At the sites with allele-specific background, the allelic background noise was subtracted from the measured peak height, while samples with high background noise were not included in the analyses. All observations were validated by alternative SNP sites and primer sets. To evaluate the retention levels of introns, the expression ratios in heterozygous SNPs along introns were normalized to the ratio in heterozygous SNPs along exons 2, 5, and 11 (S1 Table, #12, 24, and 38). To avoid misinterpreting the RT-PCR results due to co-localization of intron sense sequences and antisense transcripts derived from the opposite strand [35], intron 1 analyses was performed at least 0.5 kb downstream to the HR (S1 Table, #4–11), where the level of antisense transcripts is very low (S11 Fig).

Fluorescent fragment length analysis of PCR amplified fragments containing the HR was performed as previously described ([59]; laboratory B method) using the KAPA2G Robust PCR Kit and buffer A (KAPA Biosystems). The sequences of the corresponding primers are listed in S1 Table, #3.

RNA sequencing

RNA integrity was confirmed on the 2200 TapeStation (Agilent Technologies, Santa Clara, CA) and quantified with Qubit RNA HS Assay Kit (Invitrogen). Only RNA with a RNA integrity number (RIN) ≥ 9.4 was used for RNA-seq. To retain immature transcripts and introns in the analysis, the poly(A) mRNA enrichment step was omitted. Instead, rRNA was depleted from 1 μg of total RNA using the Ribo-Zero rRNA Removal Kit (Epicentre Biotechnologies/Illumina, Madison, WI). Barcoded cDNA libraries were constructed using the RNA Library Prep Kit v2 (Illumina, San Diego, CA) or the KAPA Stranded mRNA-Seq Kit (KAPA Biosystems) according to the manufacturer protocol and sequenced using NextSeq 500 System, High-Output mode 75 cycles kit (Illumina).

Raw reads were quality-trimmed at both ends with a quality threshold of 32, then adapter sequences were removed with Cutadapt (version 1.9.1; https://cutadapt.readthedocs.io/en/v1.9.1/index.html), only retaining reads at least 15 nt long. Low quality reads were then filtered using the FASTQ Quality Filter program of the FASTX-toolkit (version 0.0.13; http://hannonlab.cshl.edu/fastx_toolkit/download.html), with parameters–q20 –p90. Processed reads were mapped to the GRCh38 human reference genome using TopHat (v2.0.14; https://ccb.jhu.edu/software/tophat/index.shtml). Mapping used gene annotations from Ensembl (release 78), together with the C1-C4 splice sites information to identify reads that originated from these genomic locations as C9orf72 splicing variants. The relative abundance of abnormal transcripts to total C9orf72 transcripts was determined. Reads that overlap the 5’ boundary of exon 2 were counted and identified as an authentic variant, a cryptically spliced transcript, or an unspliced transcript. To overcome bias against the C9orf72 5’ end [21], the average coverage per base along exons 2–5 was taken as an exon 2 boundary, and the portion of authentic transcripts (V1, V2, and V3 altogether) from the total transcripts was evaluated by subtracting the unspliced and cryptically spliced transcripts from the average value. To evaluate the retention levels of C9orf72 introns, intronic read counts were normalized with the read counts along exons 2–5, which are shared by all C9orf72 transcripts (S1 Fig). The RNA-seq data has been deposited at the GEO database, accession number GSE162943 (http://www.ncbi.nlm.nih.gov/geo).

Statistical analysis

Data are expressed as the mean ± SEM. Statistical comparisons of means were performed by a two-tailed unpaired Student’s t-test. The value of p≤0.05 was considered significant. Boxplots were produced using BoxPlotR web-tool (http://shiny.chemgrid.org/boxplotr/) and represent median values (black line), interquartile ranges (colored regions), and Tukey whiskers (define values within 1.5 times the interquartile range from the upper and lower quartile). P-values corresponding to the regression coefficients were computed using MATLAB (MathWorks, Natick, MA).

Supporting information

S1 Fig [pdf]
Schematic illustration of variants V1, V2, and V3.

S2 Fig [a]
Fluorescence fragment length analyses of PCR fragments containing the HR.

S3 Fig [a]
Sanger sequencing can determine the relative expression of two alleles.

S4 Fig [pdf]
RNA-seq reads that overlap with heterozygous SNP sites are identifiable for their allelic origin.

S5 Fig [a]
K allele and increased normal HR length are associated with increased intron 1 and 2 levels.

S6 Fig [pdf]
K allele is associated with increased introns 1 and 2 levels.

S7 Fig [red]
HR length is associated with introns 1 and 2 levels in RK cells.

S8 Fig [e]
Determination of the allelic contribution to cryptic splice products by Sanger sequencing.

S9 Fig [a]
Identification and quantification of unspliced and cryptically spliced transcripts by using RNA-seq data.

S10 Fig [a]
Possible mechanisms of intron 1 retention in the K haplotype.

S11 Fig [as]
Distribution of sense and antisense reads along the 5’ region of .

S1 Table [docx]
PCR primer pairs used for SNP analysis in this study.

S2 Table [docx]
Detailed genetic map of the locus.

S3 Table [docx]
Haplotype and HR length results of the research participants.

S4 Table [docx]
RNA-seq reads of the gene in iPSCs and fibroblasts from control and C9-ALS patients.

S1 Data [xlsx]
Numerical data that underlines graphs.


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