Elsevier

Biological Psychiatry

Volume 44, Issue 11, 1 December 1998, Pages 1160-1165
Biological Psychiatry

Original Articles
Analysis of CAG/CTG repeat size in chinese subjects with schizophrenia and bipolar affective disorder using the repeat expansion detection method

https://doi.org/10.1016/S0006-3223(97)00492-7Get rights and content

Abstract

Background: Family studies of schizophrenia and bipolar affective disorder provide evidence for genetic anticipation, which (in common with a number of mendelian disorders), may be caused by triplet repeat expansion. This hypothesis is strengthened by evidence from repeat expansion detection (RED) analysis revealing association between the psychoses and long CAG/CTG trinucleotide repeats.

Methods: We performed RED on Han Chinese subjects with schizophrenia (82), bipolar affective disorder (43), and normal controls (61), using a CTG10 oligonucleotide.

Results: Comparison between cases and controls revealed no significant association between long repeats and affected status. We also found no detectable association with age at onset and repeat length in either bipolar affective disorder or schizophrenia. Overall, the size distribution of CAG/CTG repeats in Chinese subjects was not significantly different from those reported previously for Caucasian subjects.

Conclusions: These findings indicate that CAG/CTG repeat expansion is not likely to be a major etiological factor for psychosis in Chinese populations.

Introduction

Recently the discovery of unstable DNA sequence caused by expansion of trinucleotide repeats has defined a new class of disease-causing mutations (reviewed in Sutherland and Richards 1992). These diseases share common clinical phenomena, which are characteristic of anticipation: an inheritance pattern within a pedigree with an increase in disease severity, a decrease in age at onset, or both, in successive generations. Some early observers noted that the clinical phenomena of anticipation was apparent in the inheritance of psychiatric disorders (Mott 1911), but these observations had been discounted as artifacts of ascertainment procedures (Penrose 1948). Interest has been renewed by the discovery that repeat expansion is a mechanism for anticipation in mendelian diseases, and several recent studies provided evidence for anticipation in families with schizophrenia Asherson et al 1994, Bassett and Honer 1994, Sharma et al 1994 and bipolar affective disorder McInnis et al 1993, O’Neil et al 1993, Nylander et al 1994; however in common with earlier studies, there is likely to be a systematic bias toward earlier age at onset in the proband’s generation Penrose 1991, O’Donovan and Owen 1996, Paterson et al 1996, which may simulate anticipation, principally because the families used were ascertained for linkage analysis. Consequently, although compatible with a multifactorial–polygenic model for the inheritance of the major psychoses (Petronis and Kennedy 1995), the evidence for anticipation remains equivocal (Petronis et al 1994).

The repeat expansion detection method (RED) (Schalling et al 1993) provides a direct way to determine if repeat expansions are associated with human genetic diseases. It uses the principle that long trinuclotide repeats are able to serve as templates to catalyze ligation of oligonucleotide into larger multimers, which therefore can provide information on the size of the largest trinucleotide repeats in the genome without prior knowledge of chromosomal location. Several recent independent studies described significant association between longer CAG/CTG repeats and bipolar affective disorder O’Donovan et al 1995, Lindblad et al 1995 and schizophrenia O’Donovan et al 1995, Morris et al 1995 using RED. The preliminary findings of O’Donovan et al (1995) for schizophrenia and bipolar disorder have been replicated in an independent sample (O’Donovan et al 1996a); however, no correlation was found between CAG expansion and a range of dimensional and categorical variables of the schizophrenia phenotype (Cardno et al 1996). Further evidence indicating a role for CAG/CTG expansion in psychosis comes from Schalling et al (1996), who analyzed 14 families multiply affected with bipolar disorder from northern Sweden. Significant association with longer repeats was reported for either subjects with all affective disorders combined or bipolar affective disorder alone (p = .002).

These positive findings are balanced by studies that did not report an association between schizophrenia and expanded CAG/CTG repeats in individuals (Vincent et al 1996) or families (Petronis et al 1996), although Vincent et al reported an association of borderline significance (p = .05) for one of two methods of analysis. In another family study of schizophrenia using RED, Sirugo et al (1997) demonstrated instability and expansion of a CAT/CTG repeat mapping to chromosome 18q21 in several generations of a Danish kindred, including two affected first cousins with long repeats; however, the expanded repeat did not completely segregate with schizophrenia, since some severely affected individuals showed no expansions.

Analysis of CAG/CTG repeats by RED in normal subjects indicates the existence of significant variation in maximum length both within and between different human populations (Sirugo et al 1997). These findings indicate that CTG/CAG repeats may have relatively low mutation rates, allowing population differences to survive random genetic drift. Thus caution must be applied in case–control studies, since population stratification could occur. There is also evidence that maximum CAG/CTG repeat length decreases with age in healthy blood donor populations but not in the general population when not selected for health, or in psychotic subjects (O’Donovan et al 1996b). This provides indirect evidence of age-related deterioration in health related to long CAG/CTG repeats, since U.K. blood donors are screened for a history of cancer, autoimmune diseases, or long-term medication. This effect may act as a confounding variable in case–control studies that use blood donors as controls.

Additional information on triplet repeats in the human genome comes from fluorescence in situ hybridization (Haaf et al 1996). Additional large ATG, CCT, CTT, and TGG repeats beyond the resolution of RED can be detected by this method, and an extremely long (CTG)306 repeat was detected in a subject with schizophrenia, although the significance of this is not clear. Of the 10 possible trinucleotide repeats, expansions of only three (CTG, CCG, and GAA) have been demonstrated to play a direct role in disease pathology. AAG and CCG repeats are common in the human genome, with lengths of 8 repeats or greater occurring every 1400–2000 kb, as estimated from cosmid library hybridization (Gastier et al 1995). CAG repeats are less common, occurring approximately once every 4400 kb, but are predominantly present in exons Stallings 1994, O’Donovan and Guy 1997, where they are the most common simple sequence repeat. Direct sequencing of triplet repeat containing plasmid clones from the human genome also reveals variation in repeat length distribution, with repeats of more than (CAG)11 occurring in about 20% of clones, with a maximum size of 15 repeats observed, whereas (AAG)11 or greater occurred in almost 40% of clones, with a much broader distribution up to 31 repeats (Gastier et al 1995). Furthermore, in a recent screening study of normal individuals by RED, all subjects analyzed had maximal AAG repeat sizes of 180 bp or more (Hofferbert et al 1997). This difference may be a consequence of the strand-specific deficit of (CAG)n in introns, because of their similarity to the 3′ consensus splice sequence CCAG (Stallings 1994).

To further test the hypothesis of repeat expansion in schizophrenia, and in particular whether expansion can be detected for other ethnic groups, we used the RED method to screen for CAG/CTG repeat expansion in the genomes of Han Chinese normal subjects, and patients with schizophrenia and bipolar disorder from south western China.

Section snippets

Subjects

Eighty-two subjects with schizophrenia (49 male, 33 female), 43 subjects with bipolar affective disorder (26 male, 17 female), and 61 controls (38 male, 23 female) were used in the analysis. All the patients and controls are unrelated Han Chinese. The ages of subjects were 15–43 years (25.60 ± 6.41) for schizophrenia, 16–59 years (30.88 ± 8.53) for bipolar affective disorder, and 20–70 years (26.85 ± 8.72) for controls. Age at onset was estimated by the age of first hospital admission and was

Comparison of CAG repeat distribution in chinese and caucasian subjects

Comparison of the distribution of CAG/CTG repeats between Caucasian (O’Donovan et al 1995) and Chinese control subjects using the Wilcoxon Rank Sum test revealed no significant difference in (CAG)n repeat length as detected by RED between control subjects of Caucasian and Chinese ethnic groups (two-tailed p = .83). Comparisons between ethnic groups were also not significant for bipolar affective disorder (two-tailed p = .19) or schizophrenia (two-tailed p = .057), although for schizophrenia the

Discussion

We have screened a series of Han Chinese cases with schizophrenia or bipolar affective disorder and normal controls to further test the hypothesis that these disorders are associated with CAG/CTG repeat expansion. We found no significant association with the length of CAG/CTG repeats in either disorder. This finding is in contrast with other studies that demonstrated a significant increase in CAG/CTG repeat maximum length in schizophrenia Morris et al 1995, O’Donovan et al 1995, O’Donovan et al

Acknowledgments

Supported by the Psychiatry Research Trust (TL, RMM, DAC), the Wellcome Trust (JZ, PCS), the Stanley Foundation (HPV), the Chinese National Nature and Sciences Foundation, and the Doctoral Foundation of the Ministry of Education of China (TL, XL).

We are grateful to the MRC HGMP Resource Centre, Hinxton Hall, Cambridge for provision of oligonucleotide primers for this study, and to the Psychiatry Research Trust and the Royal Society.

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