A Chinese SCA36 pedigree analysis of NOP56 expansion region based on long-read sequencing

Abstract

Introduction: Spinocerebellar ataxias 36 (SCA36) is the neurodegenerative disease caused by the GGCCTG Hexanucleotide repeat expansions in NOP56, which is too long to sequence using short-read sequencing. Single molecule real time (SMRT) sequencing can sequence across disease-causing repeat expansion. We report the first long-read sequencing data across the expansion region in SCA36. Methods: We collected and described the clinical manifestations and imaging features of Han Chinese pedigree with three generations of SCA36. Also, we focused on structural variation analysis for intron 1 of the NOP56 gene by SMRT sequencing in the assembled genome. Results: The main clinical features of this pedigree are late-onset ataxia symptoms, with a presymptomatic presence of affective and sleep disorders. In addition, the results of SMRT sequencing showed the specific repeat expansion region and demonstrated that the region was not composed of single GGCCTG hexanucleotides and there were random interruptions. Discussion: We extended the phenotypic spectrum of SCA36. We applied SMRT sequencing to reveal the correlation between genotype and phenotype of SCA36. Our findings indicated that long-read sequencing is well suited to characterize known repeat expansion.

Keywords: GGCCTG; NOP56 gene; SMRT sequencing; repeat interruptions; spinocerebellar ataxia.

FIGURE 1

Identification of Expanded GGCCTG Repeat within NOP56 in the SCA family pedigree. (A) Genealogical structure of the family with spinocerebellar ataxia. The outco me of RP-PCR about II₅ (B) The conventional PCR of II₅ showed only one peak indicated homozygous or with a significant expansion. (C) II₅ showed a characteristic ladder pattern with a 6-bp periodicity indicated repeat expansion in NOP56. (D) The conventional PCR of outcome II₅ shows the CAG repeat number is 41 times in TBP gene, Sanger sequencing shows the CAG repeat number is 43.

FIGURE 2

The MRI findings of the SCA family. (A–B) The 3D-T1 analysis showed the patient’s (II₅) midbrain and thalamus volumes were normal, but the volumes of gray matter and white matter volumes in the cerebellum had decreased. (C) The patient’s (II₅) DTI analysis showed the FA, DEC, AD and white matter bundles followed by fiber quantification. (D) MRI T1 images are in order II₅,II₇,II₉ and II₁₁, and T1 indicates atrophy of the cerebellum.

FIGURE 3

The classification and statistics of the subreads based on SMRT sequencing. (A) The numbers of classification d (GGCCTG) n-supported subreads. (B) The full_dGGCCTGn subreads of length distribution in three patients. (C) The full_dGGCCTGn subreads of repeat number distribution in three patients. These data suggests the existence of somatic heterogeneity in SCA36 patients.

FIGURE 4

The analysis and illustration of the motifs based on the SMRT sequencing. The motif of all subreads ratio distribution in the region of repeat expansions. The ratio is calculated as in percent of the nucleotides of each motif divided by the total number of nucleotides for each expansion. The Top 10 motif (A), Top 10 5 nt-motif (B), 6 nt-motif (C) and 7 nt-motif (D) of all subreads ratio distributions in the region of repeat expansions.

FIGURE 5

NOP56 repeat expansion sequence schematics based on long-read sequencing with the highest GGCCTG repeat number full-length subreads of three patients. The schematics and expansion size shown are based on the highest GGCCTG number of repeats for the patients in the pedigree as follows: II₅ (A), II₇ (B), II₁₁ (C). The Rectangles represent sequence motifs, as indicated by the color key, in the 5’ (upper left) to 3’ (lower right) direction. Black rectangles indicated the motif is less than 1% of all the full-length subreads.