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. 2010 Oct;11(4):409-15.
doi: 10.1007/s10048-010-0245-6. Epub 2010 Apr 28.

Analysis of an insertion mutation in a cohort of 94 patients with spinocerebellar ataxia type 31 from Nagano, Japan

Haruya Sakai  1 , Kunihiro Yoshida, Yusaku Shimizu, Hiroshi Morita, Shu-ichi Ikeda, Naomichi Matsumoto
Affiliations

Analysis of an insertion mutation in a cohort of 94 patients with spinocerebellar ataxia type 31 from Nagano, Japan

Haruya Sakai et al. Neurogenetics. 2010 Oct.

Abstract

Spinocerebellar ataxia type 31 (SCA31) is a recently defined subtype of autosomal dominant cerebellar ataxia (ADCA) characterized by adult-onset, pure cerebellar ataxia. The C/T substitution in the 5'-untranslated region of the puratrophin-1 gene (PLEKHG4) or a disease-specific haplotype within the 900-kb SCA31 critical region just upstream of PLEKHG4 has been used for the diagnosis of SCA31. Very recently, a disease-specific insertion containing penta-nucleotide (TGGAA)(n) repeats has been found in this critical region in SCA31 patients. SCA31 was highly prevalent in Nagano, Japan, where SCA31 accounts for approximately 42% of ADCA families. We screened the insertion in 94 SCA31 patients from 71 families in Nagano. All patients had a 2.6- to 3.7-kb insertion. The size of the insertion was inversely correlated with the age at onset but not associated with the progression rate after onset. (TAGAA)(n) repeats at the 5'-end of the insertion were variable in number, ranging from 0 (without TAGAA sequence) to 4. The number of (TAGAA)(n) repeats was inversely correlated to the total size of the insertion. The number of (TAGAA)(n) repeats was comparatively uniform within patients from the three endemic foci in Nagano. Only one patient, heterozygous for the C/T substitution in PLEKHG4, had the insertions in both alleles; they were approximately 3.0 and 4.3 kb in size. Sequencing and Southern hybridization using biotin-labeled (TGGAA)(5) probe strongly indicated that the 3.0-kb insertion, but not the 4.3-kb insertion, contained (TGGAA)(n) stretch. We also found that 3 of 405 control individuals (0.7%) had the insertions from 1.0 to 3.5 kb in length. They were negative for the C/T substitution in PLEKHG4, and neither of the insertions contained (TGGAA)(n) stretch at their 5'-end by sequencing. The insertions in normal controls were clearly detected by Southern hybridization using (TAAAA)(5) probe, while they were not labeled with (TGGAA)(5) or (TAGAA)(5) probe. These data indicate that control alleles very rarely have a nonpathogenic large insertion in the SCA31 critical region and that not only the presence of the insertion but also its size is not sufficient evidence for a disease-causing allele. We approve of the view that (TGGAA)(n) repeats in the insertion are indeed related to the pathogenesis of SCA31, but it remains undetermined whether a large insertion lacking (TGGAA)(n) is nonpathogenic.

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Figures

Fig. 1
Fig. 1
Correlation between the insertion size and age at onset (n = 89). Representative PCR screening for the SCA31 insertion (a). Agarose gel electrophoresis of PCR products before and after HaeIII digestion is shown. M GeneRulerTM 1-kb Ladder (Fermentas Life Sciences, Burlington, Canada). The size of the insertion is inversely correlated with the age at onset (b). For 5 of 94 patients, age at onset could not be clearly defined by medical interview
Fig. 2
Fig. 2
Distribution of the insertion size in endemic foci in Nagano. The location of the three endemic foci (Kiso, Ina, and Saku) in Nagano prefecture is indicated (a). The distribution of the size of insertions in the three endemic foci is shown (b). The distribution (vertical bar), the average size (horizontal bar), and the standard deviation of the size of insertion (shaded square) in all the patients (n = 94) are shown in left. ## p < 0.01
Fig. 3
Fig. 3
Sequence of the 5′-end of the insertion. The number of (TAGAA)n repeats (underlined in red) is variable, ranging from 0 to 4
Fig. 4
Fig. 4
Correlation between the sizes of (TAGAA)n repeats preceding (TGGAA)n repeats and the insertion size. ##p < 0.01; #p < 0.05. n.s. not significant
Fig. 5
Fig. 5
PCR amplification for the insertion. The patient (ID 256, lane 5) had insertions on both alleles, instead of lacking a wild-type 1.5-kb band without the insertion. The 4.3-kb band is indicated by the arrow
Fig. 6
Fig. 6
Southern hybridization for the insertion. HaeIII-undigested PCR products were separated in a 1% agarose gel, stained with ethidium bromide (a), and then blotted to a nylon membrane. The membrane was hybridized with biotin-labeled (TAAAA)5 probe (b), (TGGAA)5 probe (c), or (TAGAA)5 probe (d). The insertions in SCA31 patients (lanes 1–3) were clearly detected by (TGGAA)5 or (TAGAA)5 probe (c and d) . In patient ID 254 (lane 3), the 3.0-kb insertion (arrow), but not the 4.3-kb insertion (arrowhead), was clearly labeled with (TGGAA)5 probe (c). In contrast, the 4.3-kb insertion (arrowhead), as well as the insertions in normal controls (lanes 4–6), was more intensively labeled with (TAAAA)5 probe than the insertions in SCA31 patients (b). The 1.5-kb fragments derived from a normal allele were visualized by (TAAAA)5 probe (b) because (TAAAA)n repeats are included in the original genomic sequence. Lanes 1–3 ,SCA31 patients (lane 3, patient ID 254); lanes 4–6, control individuals with the insertion (lane 4: control 1; lane 5: control 2; lane 6: control 3 in Table 1); lanes 7 and 8, control individuals without the insertion
Fig. 7
Fig. 7
Correlation between SARA and age at examination (a) or duration of disease (b) in SCA31 patients. The patients were divided into three groups based on the size of insertion; groups I (insertion size >3,300 bp, closed square), II (3,000–3,300 bp, open circle), and III (<3,000 bp, open triangle). SARA was performed 14 times in 11 patients from group I, 34 times in 29 patients from group II, and 19 times in 17 patients from group III. Broken, solid, and dotted lines indicate linear regression lines for groups I, II, and III, respectively
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