X-inactivation in girls with Rett svndrome J
Kormann-Bortolotto MH, Woods CG, Green SH,Webb T. X-inactivation in girls with Rett syndrome. Clin Genet 1992: 42: 296301.
M. H. Kormann-Borteletto', C. 6. Woods', S. H. Greenf and 1. Webb'
Cytogenetic studies have been carried out on a series of nine girls with Rett syndrome, six of their mothers and nine normal female controls. No abnormality of the Xchromosome has been observed in any subject. Xinactivation studies using various methods of detecting the timing of individual band replication were performed. The overall pattern seen was essentially the same in all subjects, but in the patients with Rett syndrome there may be an alteration in the timing of the X-inactivation process in the region Xpl1.3 or 4-rXp21.
Departments of 'Clinical Genetics and *Paediatrics, University of Biiingham, Birmingham, and 'Department of Medical Genetics, Churchl Hospnal. Oxford, U.K.
Key words: Rett syndrome
- X-inactivation
Maria Helena Kormann-Boltdotto, Department of Clinical Genetics, Birmingham Maternity Hospital, Edgbaston. Birmingham B15 2TG, UK Received 3 March, revbed version received 3 August, accepted for publication 10 August 1992
Rett syndrome (RS) is a neurological disorder causing severe mental retardation which appears to be limited to the female sex. Its prevalence has been estimated at between 1:IOOOO and 1:lSOOO female births (Hagberg 1985, Engerstrom 1990). Although by far the majority of cases are sporadic (> 99%), there are a few reports of familial recurrence (Hanefeld et al. 1986, Haenggeli et al. 1990, Buhler et al. 1990). Twins have also been reported, to date monozygotic pairs being concordant while dizygotic twins are discordant for the disorder (Tariverdian 1990). The aetiology of RS is still not defined. Genetic heterogeneity is possible and several different causative mechanisms have been proposed. The most likely explanation would be an X-linked dominant mutation with lethality in the male and reproductive lethality in the female (Comings 1986, Killian 1986). However, this does not explain the rare familial cases, and it seems that there is no distortion of the expected 1:1 sex ratio in Rett sibships. The familial cases could be explained by gonadal mosaicism (Killian 1986) or extremes of X-inactivation in obligate carriers. Other possible mechanisms include allelic and nonallelic metabolic interference (Buhler et a]. 1990), mitochondria1 DNA mutation (Eeg-Olofsson et al. 1990) and a disturbance in the X-inactivation process (Riccardi 1986). Any disruption of the late replicating X-chromosome process could disturb the genetic inactivation of one or more genes on this chromosome. Riccardi (1986) observed some X-chromosome homologue discrepancies in BrdU 296
pulse labelled cells from one Rett syndrome patient. Martinho et a]. (1990) proposed a variant pattern of late-replicating X-chromsome in Rett patients. Non-random X-chromosome inactivation was observed by Zoghbi et al. (1990a) in peripheral tissues from RS patients. With the aim of searching for a possible structural aberration on the X-chromosome and to study the X-chromosome late-replication patterns in Rett syndrome, a series of patients, their mothers and controls have been studied utilizing different cytogenetic procedures. Materials and methods Subjects
A total of nine Rett syndrome patients, six Rett mothers and nine unrelated healthy female controls were studied. The Rett group was composed of seven sporadic cases and one pair of monozygotic twins. The patients fulfilled the diagnostic criteria and clinical course described by Trevathen & Naidu (1988). Due to the ages of the study patients, we were confident that none had Angelman syndrome, which can be confused with RS in younger girls. Ages ranged from 9 to 31 years. Cytogenetic study
Peripheral lymphocytes were cultured for 72 h according to standard techniques. Different procedures were used as follows:
X-inactivation in Rett syndrome 1) To search for chromosomal abnormalities elongated chromosomes at the 850 band level were obtained from synchronised cultures. After 48 h culture time cells were blocked with an excess of thymidine (300 mg/l) and released after a further 24 h with deoxycytidine (lod4 hd), for 4 or 5 h. Harvesting and slide making were by routine methods and the slides were GTG banded. 2) To study the sequence of appearance of the earliest replicating bands on the: late X-chromosome, cells from 7 RS patients, 4 RS mothers and 9 controls were cultured with the addition of 5BrdU M). Replicates were exposed for either the last 8, 6 or 4 h of culture time, harvested by routine methods and the slides R lbanded according to Benn & Perle (1986). The presence of all darkstaining R-bands on the late replicating X-chromosome was noted for each mitosis studied. A dynamic picture of the sequence of appearance of the first bands to replicate was built up by observing their presence or absence in a series of mitoses. Where possible, 50 cells were studied from each subject. 3) Cultures from 3 RS patients and their mothers M) and were also treated with both 5-BrdU M) for the last 6 and 2 h of 5-azacytidine culture, respectively. The slides were R-banded as above. Where possible, at least 40 cells were analysed per individual. 4) To investigate the sequence of appearance of the latest replicating bands on thie late-X, tritiated thymidine ['HI dTTP at a final concentration of 5 p U m l was added for the last 4 a.nd 3 h of culture. Slides from 6 RS girls, 4 RS mothers and 4 controls were first G-banded, 30-50 cells were drawn and the positions of the two X chromosomes noted. After destaining the slides.,they were dipped into Ilford Nuclear Research emu.lsion K2 and kept in the dark at 4°C for 10 to 14 days before being developed and restained with L,eishman. The Xchromosomes were relocated in each cell from the original drawings and the late and early homologues were identified from the d.ensity of radioactivity in the film layer above each cell. Where identifiable bands were obtained in the late replicating Xchrornosome, their positions were noted. 5 ) Cells from 7 RS patients, 5 EIS mothers and 2 normal sisters of RS patients were cultured and 5azacytidine (lo-' M) was added for 2 h before harvest, to investigate the conderisation level of the Xchromosomes (Haaf et al. 1988). Results 1. Investigation of chromosomal abnomnalities
The analysis of elongated, G-banded chromosomes at approximately the 850 band level did not show
any chromosomal abnormality in the patients' cells, except in one case where the affected girl and her mother presented an inv (9) (pl lql2) which was assumed to be coincidental to the presence of the syndrome. 2. Identification of early replicating bands on the inactive Xchromosome by A-banding
With the use of 5-BrdU in three different culture times, it was possible to obtain large numbers of cells with the late X-chromosome in different stages of replication. A late replication pattern was delineated for each individual late X according to the frequency with which each positive ' R-band (earliest bands to replicate) appeared. The three groups, RS girls, RS mothers and control females, showed the same basic replication pattern with Xp22 as the first band to replicate (firstly Xp22.3 and then Xp22.1), followed by Xp11.4, Xq13, Xq26, Xpll.2, Xq28, Xq24, Xq22, Xql 1 and Xp21 (Fig. la and 2a). Variations of this main pattern were sometimes observed between individuals from the same group and even between cells from the same individual. The frequency with which the bands were observed in the three different groups is shown in Table 1. Chi-square tests were performed to compare the three groups using the frequency with which each band was present. The groups differed for two bands: Xp2 I (Fig. 2b) (x2 = 10.04; 2 df; P
Kormann-Bortolotto MH, Woods CG, Green SH,Webb T. X-inactivation in girls with Rett syndrome. Clin Genet 1992: 42: 296301.
M. H. Kormann-Borteletto', C. 6. Woods', S. H. Greenf and 1. Webb'
Cytogenetic studies have been carried out on a series of nine girls with Rett syndrome, six of their mothers and nine normal female controls. No abnormality of the Xchromosome has been observed in any subject. Xinactivation studies using various methods of detecting the timing of individual band replication were performed. The overall pattern seen was essentially the same in all subjects, but in the patients with Rett syndrome there may be an alteration in the timing of the X-inactivation process in the region Xpl1.3 or 4-rXp21.
Departments of 'Clinical Genetics and *Paediatrics, University of Biiingham, Birmingham, and 'Department of Medical Genetics, Churchl Hospnal. Oxford, U.K.
Key words: Rett syndrome
- X-inactivation
Maria Helena Kormann-Boltdotto, Department of Clinical Genetics, Birmingham Maternity Hospital, Edgbaston. Birmingham B15 2TG, UK Received 3 March, revbed version received 3 August, accepted for publication 10 August 1992
Rett syndrome (RS) is a neurological disorder causing severe mental retardation which appears to be limited to the female sex. Its prevalence has been estimated at between 1:IOOOO and 1:lSOOO female births (Hagberg 1985, Engerstrom 1990). Although by far the majority of cases are sporadic (> 99%), there are a few reports of familial recurrence (Hanefeld et al. 1986, Haenggeli et al. 1990, Buhler et al. 1990). Twins have also been reported, to date monozygotic pairs being concordant while dizygotic twins are discordant for the disorder (Tariverdian 1990). The aetiology of RS is still not defined. Genetic heterogeneity is possible and several different causative mechanisms have been proposed. The most likely explanation would be an X-linked dominant mutation with lethality in the male and reproductive lethality in the female (Comings 1986, Killian 1986). However, this does not explain the rare familial cases, and it seems that there is no distortion of the expected 1:1 sex ratio in Rett sibships. The familial cases could be explained by gonadal mosaicism (Killian 1986) or extremes of X-inactivation in obligate carriers. Other possible mechanisms include allelic and nonallelic metabolic interference (Buhler et a]. 1990), mitochondria1 DNA mutation (Eeg-Olofsson et al. 1990) and a disturbance in the X-inactivation process (Riccardi 1986). Any disruption of the late replicating X-chromosome process could disturb the genetic inactivation of one or more genes on this chromosome. Riccardi (1986) observed some X-chromosome homologue discrepancies in BrdU 296
pulse labelled cells from one Rett syndrome patient. Martinho et a]. (1990) proposed a variant pattern of late-replicating X-chromsome in Rett patients. Non-random X-chromosome inactivation was observed by Zoghbi et al. (1990a) in peripheral tissues from RS patients. With the aim of searching for a possible structural aberration on the X-chromosome and to study the X-chromosome late-replication patterns in Rett syndrome, a series of patients, their mothers and controls have been studied utilizing different cytogenetic procedures. Materials and methods Subjects
A total of nine Rett syndrome patients, six Rett mothers and nine unrelated healthy female controls were studied. The Rett group was composed of seven sporadic cases and one pair of monozygotic twins. The patients fulfilled the diagnostic criteria and clinical course described by Trevathen & Naidu (1988). Due to the ages of the study patients, we were confident that none had Angelman syndrome, which can be confused with RS in younger girls. Ages ranged from 9 to 31 years. Cytogenetic study
Peripheral lymphocytes were cultured for 72 h according to standard techniques. Different procedures were used as follows:
X-inactivation in Rett syndrome 1) To search for chromosomal abnormalities elongated chromosomes at the 850 band level were obtained from synchronised cultures. After 48 h culture time cells were blocked with an excess of thymidine (300 mg/l) and released after a further 24 h with deoxycytidine (lod4 hd), for 4 or 5 h. Harvesting and slide making were by routine methods and the slides were GTG banded. 2) To study the sequence of appearance of the earliest replicating bands on the: late X-chromosome, cells from 7 RS patients, 4 RS mothers and 9 controls were cultured with the addition of 5BrdU M). Replicates were exposed for either the last 8, 6 or 4 h of culture time, harvested by routine methods and the slides R lbanded according to Benn & Perle (1986). The presence of all darkstaining R-bands on the late replicating X-chromosome was noted for each mitosis studied. A dynamic picture of the sequence of appearance of the first bands to replicate was built up by observing their presence or absence in a series of mitoses. Where possible, 50 cells were studied from each subject. 3) Cultures from 3 RS patients and their mothers M) and were also treated with both 5-BrdU M) for the last 6 and 2 h of 5-azacytidine culture, respectively. The slides were R-banded as above. Where possible, at least 40 cells were analysed per individual. 4) To investigate the sequence of appearance of the latest replicating bands on thie late-X, tritiated thymidine ['HI dTTP at a final concentration of 5 p U m l was added for the last 4 a.nd 3 h of culture. Slides from 6 RS girls, 4 RS mothers and 4 controls were first G-banded, 30-50 cells were drawn and the positions of the two X chromosomes noted. After destaining the slides.,they were dipped into Ilford Nuclear Research emu.lsion K2 and kept in the dark at 4°C for 10 to 14 days before being developed and restained with L,eishman. The Xchromosomes were relocated in each cell from the original drawings and the late and early homologues were identified from the d.ensity of radioactivity in the film layer above each cell. Where identifiable bands were obtained in the late replicating Xchrornosome, their positions were noted. 5 ) Cells from 7 RS patients, 5 EIS mothers and 2 normal sisters of RS patients were cultured and 5azacytidine (lo-' M) was added for 2 h before harvest, to investigate the conderisation level of the Xchromosomes (Haaf et al. 1988). Results 1. Investigation of chromosomal abnomnalities
The analysis of elongated, G-banded chromosomes at approximately the 850 band level did not show
any chromosomal abnormality in the patients' cells, except in one case where the affected girl and her mother presented an inv (9) (pl lql2) which was assumed to be coincidental to the presence of the syndrome. 2. Identification of early replicating bands on the inactive Xchromosome by A-banding
With the use of 5-BrdU in three different culture times, it was possible to obtain large numbers of cells with the late X-chromosome in different stages of replication. A late replication pattern was delineated for each individual late X according to the frequency with which each positive ' R-band (earliest bands to replicate) appeared. The three groups, RS girls, RS mothers and control females, showed the same basic replication pattern with Xp22 as the first band to replicate (firstly Xp22.3 and then Xp22.1), followed by Xp11.4, Xq13, Xq26, Xpll.2, Xq28, Xq24, Xq22, Xql 1 and Xp21 (Fig. la and 2a). Variations of this main pattern were sometimes observed between individuals from the same group and even between cells from the same individual. The frequency with which the bands were observed in the three different groups is shown in Table 1. Chi-square tests were performed to compare the three groups using the frequency with which each band was present. The groups differed for two bands: Xp2 I (Fig. 2b) (x2 = 10.04; 2 df; P
