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

SCA10 expansion templates for SMRT sequencing.

<p>(A) PCR amplification of the SCA10 expansion from gDNA extracted from blood lymphocytes (Subjects A and B) or from somatic cell hybrid lines (subject C). Lanes are cropped from non-adjacent lanes of the same gel. The full gel is shown is <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135906#pone.0135906.s001" target="_blank">S1 Fig</a>. Arrows indicate the size of bands that were excised for cloning and sequencing (subject A, the 6.5 kb band; subject B, the 5.9 kb band; subject C, the 4.7 kb band) (B) Purified template from cloned PCR products in Fig 1A for SMRT sequencing. L: 1 kb ladder (New England Biolabs). The full gels are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135906#pone.0135906.s002" target="_blank">S2 Fig</a>.</p

Schematic representations of the repeat expansions.

<p>(A) SCA10 expansion in subject A. (B) SCA10 expansion in subject B. (C) SCA10 expansion in subject C. Rectangles represent sequence motifs, as indicated by the color key, in the 5’ (upper left) to 3’ (lower right) direction. Black rectangles indicate unverified motifs described further in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135906#pone.0135906.g003" target="_blank">Fig 3B</a> and are indicated as follows: A: ATTTCT, ATTTCT; B: ATTTCT, A, ATTTCT, A, ATTCCT, TAC, ATTTCT, A, ATT, ACTTCT, ATTCA, ATTTCT, ATTTCT, T, ACTTTCT, TCTTTCT, ATTT, ATTTCT, ATCT, ATTTCT, ATTTCT, ATTTCT, ATTTCT, ATTTCT, T, ATCC, ATTC, ATTTCC, C, ATTTCC, TTCCC, ATTTCC, CATCC, ATTTCC, C, C, C, C, ATTC, ATTTCC, ATTCC; C: ATCT, ATCT, ATCT, AT, ATCT, T, ATC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, C, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, ATCC, C</p

pJAZZ-OCmin vector.

<p>The telN gene and several non-essential genes from phage N15 (purple boxes) were deleted from the vector pJAZZ-OC. repA, cB: replication protein genes; Cam^r, chloramphenicol resistance; T, terminator. Closed terminal hairpin structures are indicated by black circles.</p

SMRT sequencing results summary.

<p>^§as estimated by gel electrophoresis of cloned expansion fragment excised from plasmid backbone.</p><p>^<b>¶</b><b>As compared with Sanger sequencing.</b></p><p>*Alignment based on reference genome sequence (NC_000022.11; GI: 568815576, Region: 45794523..45796589) using LALIGN.</p><p>^as determined by counting motif blocks in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135906#pone.0135906.g002" target="_blank">Fig 2</a>. Rpts, repeats; nts, nucleotides; bp, base pairs</p><p>SMRT sequencing results summary.</p

Proportion of repeat motifs in SCA10 expansions.

<p>Proportions are calculated as the percentage of nucleotides of each motifs divided by the total number of nucleotides for each expansion. Motifs present in SMRT sequence results that are verified by Sanger sequencing methods (“shotgun”) comprise the majority of motifs seen (A) while some motifs are unverified (B). Green, SMRT sequencing results from subject A; Green hatched, random shotgun sequencing results from subject A; Blue, SMRT sequencing results from subject B; Red, SMRT sequencing result from subject C; Red hatched, random shotgun sequencing results from subject C.</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