Single cell analysis of CAG repeat in brains of dentatorubral-pallidoluysian atrophy (DRPLA)

doi:10.1016/S0022-510X(01)00596-2

Journal of the Neurological Sciences

Volume 190, Issues 1–2, 15 September 2001, Pages 87-93

https://doi.org/10.1016/S0022-510X(01)00596-2 Get rights and content

Abstract

Somatic mosaicism of an expanded repeat is present in tissues of patients with triplet repeat diseases. Of the spinocerebellar ataxias associated with triplet repeat expansion, the most prominent heterogeneity of the expanded repeat is seen in dentatorubral-pallidoluysian atrophy (DRPLA). The common feature of this somatic mosaicism is the difference in the repeat numbers found in the cerebellum as compared to other tissues. The expanded allele in the cerebellum shows a smaller degree of expansion. We previously showed by microdissection analysis that the expanded allele in the granular layer in DRPLA cerebellum has less expansion than expanded alleles in the molecular layer and white matter. Whether this feature of lesser expansion in granule cells is common to other types of neurons is yet to be clarified. We used a newly developed excimer laser microdissection system to analyze somatic mosaicism in the brains of two patients, one with early- and another with late-onset DRPLA, and used single cell PCR to observe the cell-to-cell differences in repeat numbers. In the late onset patient, repeat expansion was more prominent in Purkinje cells than in granule cells, but less than that in the glial cells. In the early onset patient, repeat expansion in Purkinje cells was greater than in granule cells but did not differ from that in glial cells. These findings suggest that there is a difference in repeat expansion among neuronal subgroups and that the number of cell division cycles is not the only determinant of somatic mosaicism.

Introduction

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder characterized by a combination of the clinical features of epilepsy, myoclonus, choreoathetoid movement, cerebellar ataxia, and dementia [1], [2]. DRPLA is caused by the expansion of the CAG trinucleotide repeat in the DRPLA gene located on chromosome 12p [3], [4].

Tissue-to-tissue variation in triplet repeat expansion was initially reported in blood cells and muscles of patients with myotonic dystrophy. Somatic mosaicism of the triplet repeat expansion in the central nervous system also has been reported in cases of childhood-onset Huntington disease, DRPLA, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, Machado-Joseph disease, and spinal and bulbar muscular atrophy [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. The common feature of the somatic mosaicism in these diseases is that the expansion is least in the cerebellum. Using microdissection analysis of patients with DRPLA, we previously showed that the expanded allele of the cerebellar granular layer is less expanded than in the molecular layer and white matter [11]. Whether this lesser degree of expansion of the triplet repeat is specific to cerebellar granule cells or is a general feature in all neuron types is yet to be clarified.

The brain consists of a heterogeneous variety of cells. To analyze gene expression in neurons of patients with neurodegenerative disorders, the specific types of neurons must be isolated from postmortem brain tissues. We recently developed an excimer laser microdissection system that provides high resolution and minimal thermal damage. We here report its use to analyze CAG repeats in single neurons from DRPLA brains.

Section snippets

Brain samples

Somatic mosaicism was investigated in two autopsied DRPLA brains, stored at −80 °C, which had been used in a previous study [11]. In one case, there was early disease onset and the myoclonus epilepsy phenotype (case 2 in our previous report), and in the other late disease onset (case 8 in our previous report) and the cerebellar ataxia phenotype. The PCR product bands in the latter brain tissue had a wider distribution range in the tissues, except for the cerebellum, than in the former.

Single cell isolation by the excimer laser microdissection system

The

Isolation of single neurons and glial cells

Single cells were dissected individually by the laser microdissection system. Fig. 1 shows the microdissection process of a Purkinje cell.

Genomic PCR

Expanded alleles of 25 granule, 33 Purkinje, and 44 glial cells in the white matter were analyzed in the early onset brain. For the late onset brain, 60 granule, 67 Purkinje, and 57 glial cells in the cerebellar white matter were analyzed. Genomic PCR of individual neurons is shown in Fig. 2. The multiple bands of PCR products of single cells were artifacts

Discussion

Somatic mosaicism of an expanded allele has been noted in triplet repeat diseases. The feature common to this somatic mosaicism is that the lowest degree of expansion is found in the cerebellum. Analysis of microscopically dissected cerebellar tissues showed that the mosaicism in cerebellum is identical to that in the cerebellar granular layer [11].

The smallest expansion pattern has been associated with the anatomical features of the cerebellum and the cerebellar granular layer, in which most

Acknowledgements

This work was supported by grants-in-aid from CREST, Japan Science and Technology.

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Somatic expansions arise in a synchronous manner, in which most mutant alleles in a cell population expand together, gaining similar incremental number of repeats over time. Such stepwise expansions occur in the cells of patients with HD, DM1, and FXS (Goold et al., 2019; Kim et al., 2020; Lokanga et al., 2013; Peterlin et al., 1996; Yang et al., 2003) and in the brains of individuals with HD, SCA1, SCA3, SCA7, spinal and bulbar muscular atrophy (SBMA), and FXS (Hashida et al., 2001; Lokanga et al., 2013; Maia et al., 2017; Martins et al., 2006; Mouro Pinto et al., 2020; Santos et al., 2014; Shelbourne et al., 2007; Telenius et al., 1994; Trang et al., 2015; Watanabe et al., 2000; Yang et al., 2003). In HD mice, expansion rates are ∼3.5 CAG units/month per brain cell (Lee et al., 2010, 2011b; Nakamori et al., 2020).
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Single cell analysis of CAG repeat in brains of dentatorubral-pallidoluysian atrophy (DRPLA)

Abstract

Introduction

Section snippets

Brain samples

Single cell isolation by the excimer laser microdissection system

Isolation of single neurons and glial cells

Genomic PCR

Discussion

Acknowledgements

J. Neurol. Sci.

Trends Genet.

Am. J. Hum. Genet.

Cell

Am. J. Hum. Genet.

Neuron

Genomics

Brain Res. Mol. Brain Res.

Neuroscience

Familial myoclonus epilepsy and choreoathetosis: hereditary dentatorubral-pallidoluysian atrophy

Neurology

Dentatorubral-pallidoluysian atrophy or Naito-Oyanagi disease

Neurogenetics

Unstable expansion of CAG repeat in hereditary dentatorubral-pallidoluysian atrophy (DRPLA)

Nat. Genet.

Dentatorubral and pallidoluysian atrophy expansion of an unstable CAG trinucleotide on chromosome 12p

Nat. Genet.

Somatic and gonadal mosaicism of the Huntington disease gene CAG repeat in brain and sperm

Nat. Genet.

Somatic mosaicism of CAG repeat in dentatorubral-pallidoluysian atrophy (DRPLA)

Hum. Mol. Genet.

Molecular cloning of a full-length cDNA for dentatorubral-pallidoluysian atrophy and regional expressions of the expanded alleles in the CNS

Am. J. Hum. Genet.

Gametic and somatic tissue-specific heterogeneity of the expanded SCA1 CAG repeat in spinocerebellar ataxia type 1

Nat. Genet.

Somatic mosaicism of expanded CAG repeats in brains of patients with dentatorubral-pallidoluysian atrophy: cellular population-dependent dynamics of mitotic instability

Am. J. Hum. Genet.

Brain regional differences in the expansion of a CAG repeat in the spinocerebellar ataxias: dentatorubral-pallidoluysian atrophy, Machado-Joseph disease, and spinocerebellar ataxia type 1

Ann. Neurol.

CAG repeat analyses in frozen and formalin-fixed tissues following primer extension preamplification for evaluation of mitotic instability of expanded SCA1 alleles

Hum. Genet.

Somatic mosaicism of the CAG repeat expansion in spinocerebellar ataxia type 3/Machado-Joseph disease

Hum. Mutat.

Somatic mosaicism of the expanded CAG trinucleotide repeat in mRNAs for the responsible gene of Machado-Joseph disease (MJD), dentatorubral-pallidoluysian atrophy (DRPLA), and spinal and bulbar muscular atrophy (SBMA)

Neurochem. Res.

Mosaicism of unstable CAG repeats in the brain of spinocerebellar ataxia type 2

J. Neurol.