The repeat expansion detection method in the analysis of diseases with CAG/CTG repeat expansion: Usefulness and limitations

The repeat expansion detection (RED) method was described to detect expansions of trinucleotide repeats of unknown chromosomal location. We have improved the RED method by the use of 8-mer oligonucleotides and assessed its usefulness in 30 samples from patients with spinocerebellar ataxia type 1 (SCA1), Huntington's disease (HD), and Machado Joseph's disease (MJD), for which the number of CAG/CTG repeats was determined by sequencing. There was a good correlation between the number of repeats detected by sequencing and those identified by RED. However, in 17% of samples, the RED gave additional fragments for ligation products of different size than the CAG/CTG repeat expansion detected in the sample by sequencing. The same was observed in a group of control subjects (n = 78) without known clinical abnormalities in which products of more than 40 repeats were detected in 27% of them, indicating that CAG/CTG repeat expansions are common in the general population. Wether this corresponds to unidentified loci with expansions deserves further investigation. Hum Mutat 10:486–488, 1997. © 1997 Wiley-Liss, Inc.     

Detection of CAG repeats in pre-eclampsia/eclampsia using the repeat expansion detection method

Pre-eclampsia/eclampsia is a serious disorder of human pregnancy that has a worldwide incidence of 2-10% and carries a severe morbidity and mortality risk for both mother and child. Its precise cause remains unknown. However, there is increasing evidence of an underlying complex maternal genetic susceptibility. Its high population incidence in the face of strong negative selection pressure suggests that the gene(s) involved have a selective advantage and/or a high mutation rate. One class of genetic diseases that involve a high mutation rate are the trinucleotide repeat expansion diseases. Thus, the aim of this study was to determine whether there is an association between a trinucleotide (CAG) repeat expansion and pre-eclampsia/eclampsia. We have used the repeat expansion detection (RED) method, which was developed to directly identify clinically significant repeat expansions, to analyse genomic DNA from an Australian and New Zealand population. The maximal CAG repeat length for each individual was recorded and the Mann-Whitney U and Wilcoxon rank sum test for independent samples were used to compare distributions for CAG/CTG repeats between two populations. There were no statistically significant differences between the distribution of CAG repeats in normotensive (n = 59) and severe pre-eclampsia (n = 69) (Mann-Whitney U = 1732; P = 0.14), and normotensive (n = 59) and eclamptic (n = 15) populations (Mann-Whitney U = 417, P = 0.726). Therefore, these RED results do not support a role for a large CAG expansion in pre-eclampsia/eclampsia. However, these data do not preclude the possibility that a small CAG expansion is associated with the disorder nor do they negate the hypothesis that a highly mutable gene contributes to the genetic component of pre-eclampsia/eclampsia.     

Analysis of ERDA1, CTG18.1, and uncloned CAG/CTG repeat sequences in familial Parkinson's disease with anticipation

In several neurodegenerative diseases, anticipation or increase in disease severity in succeeding generations within families correlates with expansions of an intragenic CAG/CTG repeat sequence above the normal range through the generations of a pedigree. Some kindreds of familial Parkinson's disease (PD) exhibit genetic anticipation. We used the repeat expansion detection (RED) method to detect repeat expansions directly in DNA samples from the index cases of 34 different PD families with anticipation. The mean age at onset of the younger probands was 48.8 +/- 10.8 years and the mean intergenerational difference was 19.2 +/- 10 years. The distribution of the RED products greater than 40 repeats was not significantly different between patients and controls with the Mann-Whitney U test (U = 510.5, p = 0.67). The samples were then screened for the two expanded-repeat loci, ERDA1 and CTG18.1. We found that in all cases the repeat expansion detected by the RED method may be accounted for by an expansion at these loci. Our results demonstrate that unstable CAG/CTG expansions corresponding to uncloned or cloned sequences (ERDA1, CTG18.1) are not involved in the etiology of rare familial case of PD with genetic anticipation. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 88:738-741, 1999 Copyright 1999 Wiley-Liss, Inc.     

Anticipation in schizophrenia: No evidence of expanded CAG/CTG repeat sequences in French families and sporadic cases

A decrease in age of onset of schizophrenia through consecutive family generations (anticipation) has been found in several studies. Anticipation is known to result from expansion of CAG repeats in genes that determine several neurodegenerative disorders. In a previous study we analysed 26 unilineal two-generation French pedigrees and found clinical evidence of anticipation. A 10-year mean reduction in age of onset of schizophrenia was found in the second generation compared with the parental generation. The repeat expansion detection method was used to screen for CAG expansion in 21 of the 26 families with evidence of anticipation for the disease and in 59 sporadic schizophrenics and 59 controls. Comparison of the frequency distributions of CAG/CTG repeat size observed in schizophrenics and controls showed no significant difference, even when we considered familial (P = 0.23) and sporadic (P = 0.25) affected individuals separately. These results do not support the association between long CAG repeats and schizophrenia. However, the possibility that expansions with fewer than 40 repeats are involved in schizophrenia cannot be excluded. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 81:342–346, 1998. © 1998 Wiley-Liss, Inc.     

Uncloned expanded CAG/CTG repeat sequences in autosomal dominant cerebellar ataxia (ADCA) detected by the repeat expansion detection (RED) method.

In some neurodegenerative diseases, genetic anticipation correlates with expansions of the CAG/CTG repeat sequence above the normal range through the generations of a pedigree. Among these neurodegenerative diseases are late onset autosomal dominant cerebellar ataxias (ADCA). ADCA are genetically heterogeneous disorders with different cloned genes for spinocerebellar ataxia type 1 (SCA1), type 2 (SCA2), type 3 or Machado-Joseph disease (SCA3/MJD), and type 6 (SCA6). Another related dominant ataxia, dentatorubral-pallidoluysian atrophy (DRPLA), also shows CAG/CTG repeat expansions. Genetic anticipation has been reported for all of them except for the recently cloned SCA6 gene. Other, as yet undetected SCA genes may show the same features. We have used the repeat expansion detection (RED) method to detect repeat expansions directly in DNA samples from ADCA patients not resulting from known genes. Our sample consists of 19 affected index cases, corresponding to 52.8% of our ADCA families without CAG/CTG repeat expansions in the SCA1, SCA2, SCA3/MJD, SCA6, or DRPLA genes. Eighty-nine percent of the index cases had expansions of a CAG/CTG sequence greater than 40 repeats by RED, while these were observed in only 26.9% of 78 healthy subjects from the general population (p < 0.0001). The distribution of RED fragments in controls and ADCA patients also shows significant differences with the Mann-Whitney U test (U = 376.5, p = 0.0007). Moreover, there was a significant inverse correlation between the size of expansion and the age of onset (r = -0.54, p = 0.018). These results show CAG/CTG repeat expansions of over 40 repeats in our sample of ADCA families not resulting from known SCA genes.     

Exclusion of CAG/CTG trinucleotide repeat loci which map to chromosome 4 in bipolar disorder and schizophrenia

The hypothesis that expanded trinucleotide repeats contribute to the pathogenesis of schizophrenia and bipolar disorder has been recently supported by three independent studies which have shown that patients with either disorder tend to have larger CAG/CTG repeat expansion detection products than controls. In an attempt to identify the specific expanded CAG/CTG locus or loci which are associated with schizophrenia and bipolar disorder, we determined the repeat size at CAG/CTG loci mapping to candidate regions for psychosis. In this study we report our findings from eight loci which map to chromosome 4. We conclude that these loci are unlikely candidates for CAG/CTG repeat expansion in schizophrenia and bipolar disorder. Am. J. Med. Genet. 74:204–206, 1997. © 1997 Wiley-Liss, Inc.     

European combined analysis of the CTG18.1 and the ERDA1 CAG/CTG repeats in bipolar disorder

Several groups have reported association between large CAG/CTG repeats in the genome and BP disorder using the Repeat Expansion Detection (RED) method. Molecular interpretation studies demonstrated that around 90% of the large CAG/CTG repeats detected by RED can by explained by repeat size at either the CTG18.1 or ERDA-1 locus. In this study we report the findings on a large European BP case-control sample analysed for these two frequently expanded repeats. The frequency of expanded alleles (>40 repeats) at the CTG18.1 locus was significantly higher in the subgroup of patients with a more severe phenotype BPI and a positive first degree family history than in a group of matched controls (9% vs 5%). No difference in ERDA-1 expansion frequency was seen between BP cases and matched controls. We conclude that the ERDA-1 locus is not related to the BP phenotype while expanded alleles at the CTG18.1 locus cannot be excluded as a vulnerability factor for BP disorder.     

Comparison of the myotonic dystrophy associated CTG repeat in European and Japanese populations.

Gene amplification using polymerase chain reaction (PCR) was carried out on DNA samples from a total of 92 normal subjects and 52 subjects with myotonic dystrophy (DM) from European and Japanese populations, to determine the copy number of the CTG repeat associated with DM for each group. In the two populations, the number of repeats on normal chromosomes only were compared, as CTG copy number on DM chromosomes was difficult to determine by PCR alone. In this study, normal chromosomes were found which had as many as 35 copies of the repeat, which is larger than the normal range reported previously but still does not overlap with the repeat number associated with DM pathology, which is at least 50 copies. Using data from normal chromosomes from unrelated subjects, the frequencies of five, 11, and 13 copies of the CTG repeat were found to be significantly different between the two populations, with five and 11 copies more commonly seen in the European population and 13 copies in the Japanese population. This difference may be the result of natural divergence of the normal chromosomes between the population groups.     

Cis-acting modifiers of expanded CAG/CTG triplet repeat expandability: associations with flanking GC content and proximity to CpG islands.

An increasing number of human genetic disorders are associated with the expansion of trinucleotide repeats. The majority of these diseases are associated with CAG/CTG expansions, including Huntington's disease, myotonic dystrophy and many of the spinocerebellar ataxias. Recently, two new expanded CAG/CTG repeats have been identified that are not associated with a phenotype. Expanded alleles at all of these loci are unstable, with frequent length changes during intergenerational transmission. However, variation in the relative levels of instability, and the size and direction of the length change mutations observed, between the CAG/CTG loci is apparent. We have quantified these differences, taking into account effects of progenitor allele length, by calculating the relative expandability of each repeat. Since the repeat motifs are the same, these differences must be a result of flanking sequence modifiers. We present data that indicate a strong correlation between the relative expandability of these repeats and the flanking GC content. Moreover, we demonstrate that the most expandable loci are all located within CpG islands. These data provide the first insights into the molecular bases of cis -acting flanking sequences modifying the relative mutability of dispersed expanded human triplet repeats.     

The evolutionary history of the CD209 (DC-SIGN) family in humans and non-human primates

The CD209 gene family that encodes C-type lectins in primates includes CD209 (DC-SIGN), CD209L (L-SIGN) and CD209L2. Understanding the evolution of these genes can help understand the duplication events generating this family, the process leading to the repeated neck region and identify protein domains under selective pressure. We compiled sequences from 14 primates representing 40 million years of evolution and from three non-primate mammal species. Phylogenetic analyses used Bayesian inference, and nucleotide substitutional patterns were assessed by codon-based maximum likelihood. Analyses suggest that CD209 genes emerged from a first duplication event in the common ancestor of anthropoids, yielding CD209L2 and an ancestral CD209 gene, which, in turn, duplicated in the common Old World primate ancestor, giving rise to CD209L and CD209. K(A)/K(S) values averaged over the entire tree were 0.43 (CD209), 0.52 (CD209L) and 0.35 (CD209L2), consistent with overall signatures of purifying selection. We also assessed the Toll-like receptor (TLR) gene family, which shares with CD209 genes a common profile of evolutionary constraint. The general feature of purifying selection of CD209 genes, despite an apparent redundancy (gene absence and gene loss), may reflect the need to faithfully recognize a multiplicity of pathogen motifs, commensals and a number of self-antigens.     

Analysis of CAG repeat of the Machado-Joseph gene in human, chimpanzee and monkey populations: a variant nucleotide is associated with the number of CAG repeats

Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder associated with an unstable and expanded CAG repeat. We analyzed this locus from various sources including MJD families, Acadian, African American, Caucasian, Greenland Inuit and Thai populations. The range of the CAG repeat size was 14-40 in the normal alleles while the MJD alleles contained 73-78 repeats in our studies. We found 25 different alleles on normal chromosomes with a heterozygosity of 0.86 in combined populations. The most common alleles were 23 (22.9%) and 14 (25.5%) repeats. We also examined 16 chimpanzees and various Old World monkeys: a pigtail macaque, a mangabey and 12 rhesus macaques. The DNA sequences surrounding the CAG repeat did not vary among species. The range of the number of the CAG repeats is 13-14 in macaques, 16 in mangabey and 14-20 in chimpanzees. Variant CAA or AAG triplets in the CAG repeat tracts were found in all 268 human, 28 monkey and 32 chimpanzee chromosomes. As reported in a previous study [Kawaguchi et al. (1994) Nature Genet. 8, 221-228] the common variant positions were the third (CAA), fourth (AAG) and sixth (CAA) positions. However, we found three human chromosomes containing CAG at the sixth position and the mangabey had AAG at the ninth position. In addition, we found CAG at the fourth position and AAG at the sixth position in all macaque chromosomes. The nucleotide following the CAG repeat tract was usually G in all species studied. However, we sometimes found C at this position in human and chimpanzee chromosomes. Interestingly, this variant C was found in all expanded chromosomes and in 54.5% of chromosomes with 27-40 CAG repeats but it was not found in any chromosomes with less than 20 CAG repeats. We hypothesize that the variant C may be associated with CAG repeat instability.      

The role of the motor system in action understanding and communication: Evidence from human infants and non-human primates

There is growing evidence that activation of the motor system during observation of actions, a phenomenon first observed in non-human primates, underlies action understanding and even communication. This review (a) examines the evidence on motor system activity as an underlying neural correlate of action understanding; (b) reviews the theoretical and empirical work linking action understanding and the development of communication, with a specific focus on the role that gestures play as an intermediary; and (c) discusses the research on and existing opportunities for understanding the link between the motor system and communication in both humans and non-human primates, through the lens of action perception. Bringing together findings and perspectives from developmental social cognition in both humans and non-human primates and applying recent neuroscientific perspectives will help to elucidate the processes underlying the ability to understand and communicate with others.     

Spinocerebellar ataxia 1 (SCA1) in the Japanese: Analysis of CAG trinucleitide repeat expansion and instability of the repeat for paternal transmission

Summary SCA1 is caused by expansion of an unstable CAG triplet repeat in a novel gene located on the short arm of chromosome 6. In 126 Japanese individuals from 12 pedigrees with SCA1, studies were done to determine if they carried this mutant gene. All the affected and presymptomatic individuals, determined by haplotype segregation analyses, carried an abnormally expanded allele with the range of 39–63 repeat units. This repeat size inversely correlated with the age at onset. However, contrary to reported results, size of the repeat did not correlate with gender of the transmitting parent. Therefore, the CAG triplet repeat instability on paternal transmission is not likely to be fundamental to SCA1.     

Autoimmunity in non-human primates: the role of major histocompatibility complex and T cells, and implications for therapy.

Two autoimmune disease models were studied in rhesus monkeys: type II collagen-induced arthritis (CIA) and experimental allergic encephalomyelitis (EAE). Unrelated outbred animals were used in these studies. In both models disease resistant and susceptible individuals could be identified. Susceptibility correlated with in vitro cellular responsiveness to antigen in the CIA model. In both models resistant as well as susceptible individuals developed a humoral response to the inducing antigen. However, there is an indication that IgM antibodies play a crucial role in the induction of CIA. No clear association between major histocompatibility complex (MHC) type and disease incidence was found although a higher frequency of a certain DR type was observed in EAE susceptible monkeys. It is likely that both the antigen binding capacity of the MHC class II molecules and the T-cell repertoire play an important role in determining whether disease will develop or not.     

Ancestral differences in the distribution of the {Delta}2642 glutamic acid polymorphism is associated with varying CAG repeat lengths on normal chromosomes: insights into the genetic evolution of Huntington disease

This study addresses genetic factors associated with normalvariation of the CAG repeat in the Huntington disease (HD) gene.To achieve this, we have studied patterns of variation of threetrinucleotide repeats in the HD gene including the CAG and adjacentCCG repeats as well as a GAG polymorphism at residue 2642 (     

Detection of monocyte/macrophage cell populations in effusions: a comparative study using flow cytometric immunophenotyping and immunocytochemistry

The objective of the present study was to compare the efficiency of immunophenotyping using flow cytometry (FCM) and immunocytochemistry (ICC) in the detection of macrophages in serous effusions. Cytoblock sections from 90 effusions were stained for the monocyte/macrophage marker CD14, using ICC. Fresh-frozen samples of all cases were analyzed for CD14 expression, using FCM. Epithelial, lymphoid, and mesothelial cell populations were identified using antibodies against Ber-EP4, CD45, and N-cadherin, respectively. Results were compared with clinical parameters and morphological diagnosis. Thirty-nine specimens were cytologically diagnosed as malignant, containing tumor cells of nonhematologic origin, whereas 46 were interpreted as benign. Two additional specimens were diagnosed as indeterminate or suspicious for malignancy, and 3 specimens contained lymphoma cells. CD14-positive cells were detected in 85/90 (94%) of effusions using FCM, and in all 90 specimens using ICC. The percentage of CD14-positive cells was highly variable, but in some specimens was as high as 76% using FCM and 85% using ICC. A good association was observed between the two methods in the detection of CD14-positive cells (P < 0.001). The presence of macrophages in effusions showed an association with female gender, using both FCM (P = 0.002) and ICC (P = 0.011), but none with effusion site, patient age, clinical and cytological diagnosis, or presence of Ber-EP4-positive cells (P > 0.05). The presence of Ber-EP4-positive cells showed a strong association with the cytological diagnosis of malignancy (P < 0.001). In conclusion, macrophages are a significant cell population in effusions, of both benign and malignant etiology, due to both their size and their possible confusion with cancer cells. Both FCM and ICC aid in the recognition of these cells, and thus provide an effective tool for the identification of different cell populations in effusions.     

Huntington''s disease in Grampian region: correlation of the CAG repeat number and the age of onset of the disease.

The identification of an unstable trinucleotide repeat as the mutation responsible for Huntington's disease (HD) has given the hope that additional information can be provided about age of onset and mode of action of the mutated gene. We present in this paper results of a clinical and molecular study of 82 patients affected with HD from 46 pedigrees within the Grampian region, Scotland. Our results show a correlation between age of onset and size of the CAG expansion. This study has produced no overlap in mutation size between affected and unaffected alleles. The sex of the parent transmitting the mutated allele and the size of the normal allele have no significant effect on the clinical features of the disease. In the three juvenile cases the affected parent was the father but the number of cases is too small to produce statistical significance. An increase in the CAG repeat size is shown in the transmission of the gene in five cases, accompanied by an earlier age of onset in four; in three of these cases, the affected parent was the father. Eleven sib pairs were studied and there is a negative correlation between the difference in age at onset and the difference in repeat size. Thus there is some evidence of a relationship, but this is not statistically significant because of the small numbers involved. The presence of the same or different normal allele had no effect on age of onset in this small group. We suggest that additional factors, as yet unrecognised, influence the age of onset and clinical presentation of HD.