Inheritance patterns of ATCCT repeat interruptions in spinocerebellar ataxia type 10 (SCA10) expansions

<div><p>Spinocerebellar ataxia type 10 (SCA10), an autosomal dominant cerebellar ataxia disorder, is caused by a non-coding ATTCT microsatellite repeat expansion in the ataxin 10 gene. In a subset of SCA10 families, the 5’-end of the repeat expansion contains a complex sequence of penta- and heptanucleotide interruption motifs which is followed by a pure tract of tandem ATCCT repeats of unknown length at its 3’-end. Intriguingly, expansions that carry these interruption motifs correlate with an epileptic seizure phenotype and are unstable despite the theory that interruptions are expected to stabilize expanded repeats. To examine the apparent contradiction of unstable, interruption-positive SCA10 expansion alleles and to determine whether the instability originates outside of the interrupted region, we sequenced approximately 1 kb of the 5’-end of SCA10 expansions using the ATCCT-PCR product in individuals across multiple generations from four SCA10 families. We found that the greatest instability within this region occurred in paternal transmissions of the allele in stretches of pure ATTCT motifs while the intervening interrupted sequences were stable. Overall, the ATCCT interruption changes by only one to three repeat units and therefore cannot account for the instability across the length of the disease allele. We conclude that the AT-rich interruptions locally stabilize the SCA10 expansion at the 5’-end but do not completely abolish instability across the entire span of the expansion. In addition, analysis of the interruption alleles across these families support a parsimonious single origin of the mutation with a shared distant ancestor.</p></div

A minimum spanning network depicting the hypothesized evolution of the ATCCT repeat interruption alleles.

<p>Filled colored circles and numbers correspond to interruption alleles as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175958#pone.0175958.g001" target="_blank">Fig 1</a>. The open blue circle represents a hypothetical allele suggested to exist based on the network. Each bidirectional arrow represents a single repeat unit change between alleles and each arrow notes specific changes. When multiple repeat changes exist between interruption alleles, the order of the repeat changes is not known, i.e. the order of changes between alleles 2 and 3 is not known. The alleles that appear in each family are contained within a red oval and the family (C, M, N, Z) is noted within. The network does not reflect the variation at the distal variable region, theta.</p

The ATCCT repeat interruption is stable through generations.

<p>(A) Schematic of an average length SCA10 expansion allele demonstrating the relative location of the priming sites for the ATCCT PCR amplification and sequencing primers and the relative size of the ATCCT product. White box, ATTCT repeats; hatched black and white box, interruption motifs; green box, presumed pure tract of tandem ATCCT repeat motifs; black box, flanking non-expansion sequences. (B) Detailed schematic of repeat motifs within the ATCCT product. Allele 5 is depicted. White rectangles, ATTCT repeat; orange, ATTTTCT; blue, ATATTCT; green, ATCCT. (C) Seven interruption alleles were observed based on the number of ATTCT repeats observed within each polymorphic stretch (alpha, beta, gamma, delta, epsilon, zeta and eta). (D) SCA10 family pedigrees, only SCA10-positive individuals are shown. Generations are indicated by roman numerals to the left of each pedigree. Square (males) and circles (females) are color-coded by repeat interruption group. Black, undetermined allele; light blue, allele 1; green, allele 2; red, allele 3; yellow, allele 4; grey, allele 5; dark blue, allele 6; tan, allele 7. Numbers below male/female symbols indicate the SCA10 expansion size (in repeat units) determined via Southern blotting; n.d. indicates that the SCA10 expansion size was not determined due to insufficient DNA quality. Thick blue lines, paternal transmissions examined; thick red lines, maternal transmissions examined.</p

The distribution of SCA10 in the American continents and the proposed dispersal pattern of the mutation.

<p>Possible dispersal patterns of Native American and Amerindian populations as they began entering the Americas ∼15,000 years ago are shown as solid blue lines. Asterisks indicate countries where SCA10 patients have documented ancestral ties.</p

Haplotype analysis of single nucleotide polymorphisms (SNPs) surrounding the SCA10 locus in the Sioux SCA10 patient.

&<p>SNPs used in this study were originally studied in Almeida et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081342#pone.0081342-Almeida1" target="_blank">[14]</a>. †Distance of the SNP is relative to the SCA10 expansion and is expressed in base pairs. Locations upstream and downstream of the SCA10 expansion are denoted by negative and positive values, respectively. *, The common disease haplotype of Mexican and Brazilian families in our SCA10 cohort of 31 families <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081342#pone.0081342-McFarland1" target="_blank">[29]</a>. ?The “SCA10 haplotype” originally described in Almeida et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081342#pone.0081342-Almeida1" target="_blank">[14]</a>. NR, not reported by Ameida et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081342#pone.0081342-Almeida1" target="_blank">[14]</a>, although these SNPs are mentioned by this study. ^$, “C” allele segregates with SCA10 expansion. No additional sequence changes were seen outside of the SNPs reported.</p

Southern blot analysis of the SCA10 ATTCT repeat expansion in our Sioux patient with SCA10.

<p>Lane 1: positive control, 2300 repeats (genomic DNA from SCA10 somatic cell hybrid line (SCH)) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081342#pone.0081342-Wakamiya1" target="_blank">[30]</a>; Lane 2: no DNA control; Lane 3: positive control, 800 repeats (genomic DNA from SCA10 SCH); Lane 4: negative control (genomic DNA from normal control SCH); Lane 5: DNA from Sioux SCA10 patient.</p

Path analysis model used to estimate the effect of smoking duration on SS and the role of potential confounding factors.

<p>The standardized path coefficients, significant odds ratios (OR) and p-values (in bold) are shown next to each path. A. Effect of smoking duration on SS, conditional on socioeconomic status; B. Model used to estimate the effect of smoking duration on SS, conditional on body mass index.</p

Logistic Regression models exploring the effect of covariates of smoking on the risk of classification as Sjögren’s Syndrome.

<p>The only significant predictor was smoking duration.</p

Functional characterizations of <i>OAS1</i> isoforms.

<p>(A) Protein expression of OAS1 isoforms was evaluated in EBV-transformed B cells from SS patients (four independent samples from each genotype group) using anti-OAS1 antibody targeting the shared epitope of all the isoforms. The stimulated cells were treated with universal type I IFN (1500U/ml) for 24hrs. The p44 isoform was not detectable using western-blot due to its low expression. The right panel shows quantified band intensity normalized to the GAPDH in each sample. (B) The transcript levels of each <i>OAS1</i> isoform from the same sets of cells described above were determined using real-time PCR. Consistent with the RNA-seq results, the SS-associated risk allele A of rs10774671 was correlated with decreased levels of p46 and increased expression of the p42, p48, and p44 isoforms (significance levels are shown at the bottom). The transcript levels of all the isoforms significantly increased after IFN stimulation (two-tailed <i>t</i> test); however, only p46 had increased protein production after IFN stimulation. (Significance level: ** <i>P</i><0.01; *** <i>P</i><0.001) (C) Individual isoforms of <i>OAS1</i> tagged with Xpress epitope were cloned and transfected into HEK 293T cells for 48hrs. The p48 and p44 isoforms had impaired protein expression compared to p46 and p42, although their transcript levels were equivalent as determined by real-time PCR (n = 4; normalized to <i>HMBS</i>). (D) The full-length and truncated <i>OAS1</i> p48 and p44 isoforms were cloned into HEK 293T cells. Western-blot indicated the lack of expression of the full-length p48 and p44 isoforms, whereas the truncation of both isoform transcripts (T2 and T4) was able to restore protein expression. (E) The 3' alternatively spliced terminus of different <i>OAS1</i> isoforms were linked to the 3'-end of GFP to observe their influence on GFP protein expression in HEK 293T cells. The 3'-terminus from the p48 and p44 isoforms resulted in decreased expression of GFP.</p

Differentially expressed transcripts between 115 anti-Ro/SSA positive SS cases and 56 controls identified through transcriptome profiling.

<p>(A) We identified 73 genes (represented by 83 probes on the heatmap) differentially expressed between anti-Ro/SSA positive SS cases and healthy controls (absolute FC >2 and <i>q</i><0.05). Among the differentially expressed genes, 57 were type I IFN-regulated genes (black bar on right) and formed an IFN signature where most genes were overexpressed in SS patients (yellow indicates overexpressed genes compared to controls). (B) The 57 differentially expressed type I IFN-regulated genes were re-clustered in anti-Ro/SSA positive SS cases using <i>k</i>-means (<i>k</i> = 3) algorithm and heterogeneity of the IFN signature levels in anti-Ro/SSA positive SS cases was observed.</p