Hum Genet (1991) 86:604-606
9 Springer-Verlag1991
Rett syndrome: exclusion mapping following the hypothesis of germinal mosaicism for new X-linked mutations N . A r c h i d i a c o n o 1, M . L e r o n e 1, M. R o c c h i 1, M. A n v r e t 2, I". O z c e l i k 3, U . Francke 3, and G . R o m e o 1
ILaboratorio di Genetica Motecolare, Istituto G. Gaslini, Largo G. Gaslini, 5, 1-16148 Genoa, Italy 2Department of Clinical Genetics, Karolinska Hospital, Stockholm, Sweden 3Howard Hughes Medical Institute and Department of Genetics, Beckman Center for Molecular and Genetic Medicine, Stanford University Medical Center, Stanford, California, USA Received August 8, 1990 / Revised November 12, 1990
S u m m a r y . The hypothesis of germinal mosaicism in the unaffected mother of two half-sisters affected with Rett syndrome is postulated to explain the unusual recurrence of this genetic disorder affecting only females (1/10000); it might be caused by new X-linked mutations with lethality in male fetuses. The analysis of 34 X-linked restriction fragment length polymorphisms (RFLPs) in these two affected females and in their unaffected mother and half-brother, together with the reconstruction of phase for 15 informative RFLPs in somatic cell hybrids retaining a single X chromosome from each female, has made it possible to exclude some regions of the X chromosome as possible sites of the mutation(s) causing Rett syndrome.
Introduction
Rett syndrome (RTS) is an infantile neurological disorder characterized by psychomotor deterioration and autistic behaviour with presentation after the first year of life. It affects only females, with an incidence of about 1/10000 liveborn females (Hagberg et al. 1983; Hanefeld 1985; Comings 1986). The strongest evidence that favours the genetic origin of RTS comes from the complete concordance in all the known sets of monozygotic twins compared with the discordance among dizygotic twins (Tariverdian et al. 1987; unpublished data kindly provided by the International Rett Syndrome Association). The hypothesis that RTS is caused by X-linked dominant new mutation(s) that would be lethal in male fetuses is therefore generally accepted. According to this hypothesis, new mutations would occur randomly in gametes of RTS parents and a distorted sex ratio among sibs of affected girls is not expected. Indeed, no significant difference in the sex ratio was found when tested in sibships of patients with RTS (Killian 1986), or in previous generations (Wahlstrom and Anvret 1986). Offprint requests to: G. Romeo
The same model of X-linked new mutations with lethality in male fetuses has been invoked for the rarer Aicardi syndrome for which no familial cases have been reported and which has been mapped in Xp22 on the basis of X/autosomal translocations observed in affected females (see review by Davies et al. 1987). The occasional occurrence of new mutations has also been reported for incontinentia pigmenti (IP), which is usually transmitted as an X-linked dominant disorder with lethality in male fetuses (Lenz 1961; and reviews by Mandel et al. 1988, 1989). In order to explain the exceptional recurrence of RTS in a set of half-sisters, we postulated a germinal mosaicism for an X-linked new mutation in their mother and proposed a strategy to map the RTS mutation(s) in such rare predigrees (Romeo et al. 1986). Briefly, this approach consists of the study of X-linked haplotypes based on the identification of the phase of the restriction fragment length polymorphism (RFLP) alleles in the mother and her affected daughters. Since these female individuals were the only members of such a pedigree available for study, the phase of the polymorphic alleles was identified using somatic cell hybrids that retained one human X chromosome. The regions of the maternally inherited X chromosome that show discordance of haplotypes in the two half-sisters have been excluded as possible locations of the RTS mutation(s).
Materials and m e t h o d s
Samples of peripheral blood were drawn in heparin, and lymphoblastoid cell lines were established from the two affected half-sisters, their unaffected mother, and half-brother. DNA from established lymphoblastoid lines and from somatic cell hybrids was extracted according to published procedures (Kunkel et al. 1977), digested with resriction enzymes, electrophoresed and blotted as previously described (Roncuzzi et al. 1985). Probes revealing RFLPs of the X chromosome were radioactively labelled by nick translation (Amersham, Buckinghamshire, kit ~=5500) or oligolabelling (Multiprimer DNA labelling system, Amersham, Buckinghamshire, kit RPN.1601Y). Somatic cell hybrids were prepared
605
Fig. 1. Reconstruction of haplotypes for RFLPs informative in the mother (I-2) and inherited by the two affected half-sisters (II-1 and II-2) and by their healthy brother (II-3). The dotted or black boxes represent the segments from maternal X chromosomes, the hatched (II-1) or grey (II-2) boxes represent the X chromosomes from different fathers. The alleles are indicated according to Kidd et al. (1989). The hybrids used to establish phase are indicated above each corresponding haplotype of I-2, II-1 and II-2
according to a published procedure (Brahe and Serra 1981) by fusing the YH21 hamster cell line with lymphoblastoid cells from propositae and their mother. The hybrid clones were isolated and cytogenetically examined, using Q-banding, for the presence of human chromosomes. Three clones, one from each half sister and one from the mother, were selected because they had the lowest modal chromosome number and the highest mitotic index without any apparent rearrangement of the single X chromosome that they retained after HAT (hypoxanthine, aminopterin, thymidine) selection. When informative, the RFLP analysis always confirmed the presence of a single human X chromosome in these hybrids, which carried one of the 2 alleles present in the genomic DNA from the same female. Linkage analysis was performed using the programs MLINK of LINKAGE V5.03 (Lathrop and Lalouel 1984). The assumptions made to use this program were those of X linkage and of disease present in the mother, since the option of germinal mosaicism was not possible.
Results A total of 34 R F L P s mapping on the X c h r o m o s o m e were analysed in genomic D N A from the four individuals available for the study and in D N A f r o m hybrids of the females (the fathers were not available for study). Figure 1 shows the haplotypes for 15 informative R F L P s reconstructed in the m o t h e r and in her two daughters according to the data from somatic cell hybrids. The hybrids are labelled as follow: H y 9 7 A derived f r o m I-2, Hy81F1 from II-1 (carrying the maternal X chromosome), H y 8 2 L from II-2 (carrying the paternal X c h r o m o s o m e ) . As a further test with respect to the origin of the single X c h r o m o s o m e retained in the hybrids derived from the two affected half sisters, we also considered those RFLPs for which I-2 (mother) was homozygous and II-1 or II-2 were heterozygous. The presence in the hybrid of the maternal or paternal alleles identifies the only X chrom o s o m e retained in the hybrid as being of maternal or paternal origin. This type of analysis (based on homozygosity in the m o t h e r and heterozygosity in a daughter) is informative for 8 RFLPs in II-1 and for 2 RFLPs in II-2. In addition to confirming the maternal origin of the X c h r o m o s o m e in hybrid Hy81F1 and the paternal origin of the X c h r o m o s o m e in hybrid Hy82L, this analysis is in
agreement with the cytogenetic and molecular data that indicate that no r e a r r a n g e m e n t of these X c h r o m o s o m e s has occurred in vitro. According to the strategy described above, the 15 R F L P s for which the m o t h e r is heterozygous were used for the identification of the regions of the maternal X c h r o m o s o m e shared by the two half-sisters (concordant region). In addition, the analysis of the phase in the m o t h e r I-2 allows the identification of the recombination events and of the discordant regions in the X chromosomes inherited by her three children (Fig. 1). These regions can be identified in Xp as those immediately surrounding the loci defined by probes pS232 (DXS278) and pXG-16 (DXS92) and that identified by p B a m X 7 around the centromere, and the region of D X Y S 1 on Xq. A two point linkage analysis between the above markers and the RTS gene yields, in each case, a lod score ___- 2 . 0 8 for 0 = 0.002. For all the other concordant markers, positive but not significant lod scores were obtained ( m a x i m u m lod score = 0.6 for 0 = 0.00).
Discussion The order of polymorphic loci on the X c h r o m o s o m e reported in Fig. 1 derives f r o m information obtained from recent reports (Mandel et al. 1989). As already pointed out ( R o m e o et al. 1986), the peculiarity of this pedigree makes it highly probable that the mutation causing RTS in the two half sisters is of maternal origin. According to the strategy described in the Introduction, our results lead to the exclusion, as possible regions of localization of the RTS gene(s), of those portions of the X chromosome not shared by the two half sisters. These regions can be identified as those surrounding the loci DXS278, DXS92, D X S Z 1 and D X Y S 1 . A quantitative estimate of the exclusion of the RTS gene in the vicinity of each of these loci yields lod scores _< -- - 2 . 0 8 for 0 -- 0.002. It is of interest, however, that where the two affected half sisters are concordant, their half brother shows alleles that are discordant with theirs for a total of 11 RFLPs. With regard to the possible mapping of the RTS
606 m u t a t i o n ( s ) o n t h e X c h r o m o s o m e , two d i f f e r e n t X - a u tosomal translocations involving Xp have been recently d e s c r i b e d in R T S p a t i e n t s ( Z o g h b i et al. 1990; J o u r n e l et al. 1990). T h e c o r r e s p o n d i n g X c h r o m o s o m e b r e a k p o i n t s o c c u r in t h e s e cases in d i f f e r e n t r e g i o n s of X p i d e n t i f i e d as Xp22.11 a n d Xp11.22, r e s p e c t i v e l y ( Z o g h b i et al. 1990; J o u r n e l et al. 1990). G e n e t i c analysis c a r r i e d o u t in the present study does not exclude either localization. Addit i o n a l i n f o r m a t i o n c o m e s f r o m t h e analysis of t h e h y b r i d l a b e l l e d as H Y 8 1 F 1 ( f r o m p r o p o s i t a II-1). T h e X chrom o s o m e , w h i c h is r e t a i n e d in this h y b r i d a n d which is o f m a t e r n a l origin, d i d n o t s h o w a n y d e l e t i o n . T h e r e f o r e , a m i c r o d e l e t i o n of m a t e r n a l origin as a cause of R T S seems u n l i k e l y for all t h e e x a m i n e d loci. T h e v a r i a t i o n o f t h e clinical c o u r s e o b s e r v e d in s o m e p e d i g r e e s with r e c u r r e n c e o f e i t h e r R T S o r I P w o u l d b e in a g r e e m e n t w i t h u n b a l a n c e d L y o n i z a t i o n . Such varieg a t i o n has b e e n o b s e r v e d in two full sisters with R T S ( H a n e f e l d et al. 1986) a n d in t h e f a m i l y with t h e t(X;22) (p11.22;p11) b a l a n c e d t r a n s l o c a t i o n ( J o u r n e l et al. 1990). In a similar way, studies of 5 heterozygous females, from 3 k i n d r e d s s e g r e g a t i n g IP, i n d i c a t e t h a t the cells e x p r e s sing m u t a t i o n s t h a t a r e l e t h a l in m a l e fetuses m a y b e s e l e c t e d a g a i n s t in c a r r i e r f e m a l e s , w h o m i g h t s h o w a p h e n o t y p e less d e l e t e r i o u s t h a n t h a t e x p e c t e d o n the basis o f r a n d o m i n a c t i v a t i o n ( M i g e o n et al. 1989). T h e a p p r o a c h o f e x c l u s i o n m a p p i n g u s e d in this w o r k can o v e r c o m e t h e i n c o n s i s t e n c i e s r e v e a l e d b y t h e two t r a n s l o c a t i o n s r e c e n t l y r e p o r t e d in R e t t p a t i e n t s ( Z o g h b i et al. 1990; J o u r n e l et al. 1990).
Acknowledgements. This work was supported by grants from the Consiglio Nazionale delle Ricerche (Rome) with special reference to the Progetto Finalizzato Ingegneria Genetica and from Ministero della Pubbliea Istruzione, Rome, Italy. The help of Dr.M. Devoto for linkage analysis and the technical assistance from I. Giambarrasi, S. Castagnola and M. Bertorello are acknowledged. T.O. is an Associate and U.F. an Investigator of the Howard Hughes Medical Institute.
References Brahe C, Serra A (1981) A simple method for fusing human lymphocytes with rodent cell in monolayer by polyethylene glycol. Somat Cell Genet 7:109-115 Comings DE (1986) The genetics of Rett syndrome: the consequences of a disorder where every case is a new mutation. Am J Med Genet [Suppl 1] : 383-388 Davies KE, Mandel JL, Wiessebach J, Fellous M (1987) Report of the committee on the genetic constitution of X and Y chromosomes. Cytogenet Cell Genet 46:1-4
Hagberg B, Aicardi J, Dias K, Ramos O (1983) A progressive syndrome of autism, dementia, ataxia, and loss of purposeful hand use in girls: Rett syndrome. Report of 35 cases. Ann Neurol 14 : 471-479 Hanefeld F (1985) The clinicla pattern of Rett syndrome. Brain Dev 7 : 320-325 Hanefeld F, Hanefeld U, Wilichowskie, Schmidtke J (1986) Rettsyndrome - search for genetic markers. Am J Med Genet 24 : 377-382 Journel H, Melki J, Turleau C, Munnich A, Grouchy J de (1990) Rett phenotype with X/autosome translocation: possible mapping of the gene to the short arm. Am J Med Genet 35 : 142147 Kidd KK, Boweock J, Schmidtke J, Track RK, Ricciuti F, Hutchings G, Bale A, et al (1989) Report of the DNA committee and catalogs of cloned and mapped genes and DNA polymorphisms. (10th International Workshop on Human Gene Mapping) Cytogenet Cell Genet 51 : 622-947 Killian W (1986) On the genetics of Rett syndrome: analysis of family and pedigree data. Am J Med Genet [Suppl 1] : 369-376 Kunkel LM, Smith KD, Boyer SH, Borgaonkar DS, Wachtel SS, Miller OJ, Breg WR, et al (1977) Analysis of human Y-chromosome specific reiterated DNA in chromosome variants. Proc Natl Acad Sci USA 74 : 1245-1249 Lathrop GM, Lalouel JM (1984) Easy calculation of lod scores and genetic risks on small computers. Am J Hum Genet 36:460465 Lenz W (1961) Zur Genetik der Incontinentia pigmenti. Ann Paediatr (Basel) 196 : 149-165 Mandel JL, Willard HF, Nussbaum RL, Davies KE, Romeo G (1988) Report of the committee on the genetic constitution of the X chromosome. Cytogenet Cell Genet 49:107-110 Mandel JL, Willard HF, Nussbaum RL, Romeo G, Puck JM, Davies KE (1989) Report of the committee on the genetic constitution of the X chromosome. (10th International Workshop on Human Gene Mapping) Cytogenet Cell Genet 51 : 384-437 Migeon BR, Axelman J, Jan de Beur S, Valle D, Mitchell GA, Rosenbaum KN (1989) Selection against lethal alleles in females heterozygous for incontinentia pigmenti. Am J Hum Genet 44:100-106 Romeo G, Archidiacono N, Ferlini A, Rocchi M (1986) Rett syndrome: lack of association with fragile site Xp22 and strategy for genetic mapping of X-linked new mutations. Am J Med Genet [Suppl 1] : 355-359 Roncuzzi L, Fadda S, Mochi M, Prosperi L, Sangiorgi S, Santamaria R, Sbarra D, et al (1985) Mapping of X-linked Becker muscular dystrophy through crossovers identified by DNA polymorphisms and by haplotype characterization in somatic cell hybrids. Am J Hum Genet 37: 407-417 Tariverdian G, Kantner G, Vogel F (1987) A monozygotic twin pair with Rett syndrome. Hum Genet 75 : 88-90 Wahlstrom J, Anvret M (1986) Chromosome finding in the Rett syndrome and a test of two-step mutation theory. Am J Med Genet [Suppl 1] : 361-368 Zoghbi HY, Ledbetter DH, Schultz R, Percy AK, Glaze DG (1990) A de novo X;3 translocation in Rett syndrome. Am J Med Genet 35 : 148-151
9 Springer-Verlag1991
Rett syndrome: exclusion mapping following the hypothesis of germinal mosaicism for new X-linked mutations N . A r c h i d i a c o n o 1, M . L e r o n e 1, M. R o c c h i 1, M. A n v r e t 2, I". O z c e l i k 3, U . Francke 3, and G . R o m e o 1
ILaboratorio di Genetica Motecolare, Istituto G. Gaslini, Largo G. Gaslini, 5, 1-16148 Genoa, Italy 2Department of Clinical Genetics, Karolinska Hospital, Stockholm, Sweden 3Howard Hughes Medical Institute and Department of Genetics, Beckman Center for Molecular and Genetic Medicine, Stanford University Medical Center, Stanford, California, USA Received August 8, 1990 / Revised November 12, 1990
S u m m a r y . The hypothesis of germinal mosaicism in the unaffected mother of two half-sisters affected with Rett syndrome is postulated to explain the unusual recurrence of this genetic disorder affecting only females (1/10000); it might be caused by new X-linked mutations with lethality in male fetuses. The analysis of 34 X-linked restriction fragment length polymorphisms (RFLPs) in these two affected females and in their unaffected mother and half-brother, together with the reconstruction of phase for 15 informative RFLPs in somatic cell hybrids retaining a single X chromosome from each female, has made it possible to exclude some regions of the X chromosome as possible sites of the mutation(s) causing Rett syndrome.
Introduction
Rett syndrome (RTS) is an infantile neurological disorder characterized by psychomotor deterioration and autistic behaviour with presentation after the first year of life. It affects only females, with an incidence of about 1/10000 liveborn females (Hagberg et al. 1983; Hanefeld 1985; Comings 1986). The strongest evidence that favours the genetic origin of RTS comes from the complete concordance in all the known sets of monozygotic twins compared with the discordance among dizygotic twins (Tariverdian et al. 1987; unpublished data kindly provided by the International Rett Syndrome Association). The hypothesis that RTS is caused by X-linked dominant new mutation(s) that would be lethal in male fetuses is therefore generally accepted. According to this hypothesis, new mutations would occur randomly in gametes of RTS parents and a distorted sex ratio among sibs of affected girls is not expected. Indeed, no significant difference in the sex ratio was found when tested in sibships of patients with RTS (Killian 1986), or in previous generations (Wahlstrom and Anvret 1986). Offprint requests to: G. Romeo
The same model of X-linked new mutations with lethality in male fetuses has been invoked for the rarer Aicardi syndrome for which no familial cases have been reported and which has been mapped in Xp22 on the basis of X/autosomal translocations observed in affected females (see review by Davies et al. 1987). The occasional occurrence of new mutations has also been reported for incontinentia pigmenti (IP), which is usually transmitted as an X-linked dominant disorder with lethality in male fetuses (Lenz 1961; and reviews by Mandel et al. 1988, 1989). In order to explain the exceptional recurrence of RTS in a set of half-sisters, we postulated a germinal mosaicism for an X-linked new mutation in their mother and proposed a strategy to map the RTS mutation(s) in such rare predigrees (Romeo et al. 1986). Briefly, this approach consists of the study of X-linked haplotypes based on the identification of the phase of the restriction fragment length polymorphism (RFLP) alleles in the mother and her affected daughters. Since these female individuals were the only members of such a pedigree available for study, the phase of the polymorphic alleles was identified using somatic cell hybrids that retained one human X chromosome. The regions of the maternally inherited X chromosome that show discordance of haplotypes in the two half-sisters have been excluded as possible locations of the RTS mutation(s).
Materials and m e t h o d s
Samples of peripheral blood were drawn in heparin, and lymphoblastoid cell lines were established from the two affected half-sisters, their unaffected mother, and half-brother. DNA from established lymphoblastoid lines and from somatic cell hybrids was extracted according to published procedures (Kunkel et al. 1977), digested with resriction enzymes, electrophoresed and blotted as previously described (Roncuzzi et al. 1985). Probes revealing RFLPs of the X chromosome were radioactively labelled by nick translation (Amersham, Buckinghamshire, kit ~=5500) or oligolabelling (Multiprimer DNA labelling system, Amersham, Buckinghamshire, kit RPN.1601Y). Somatic cell hybrids were prepared
605
Fig. 1. Reconstruction of haplotypes for RFLPs informative in the mother (I-2) and inherited by the two affected half-sisters (II-1 and II-2) and by their healthy brother (II-3). The dotted or black boxes represent the segments from maternal X chromosomes, the hatched (II-1) or grey (II-2) boxes represent the X chromosomes from different fathers. The alleles are indicated according to Kidd et al. (1989). The hybrids used to establish phase are indicated above each corresponding haplotype of I-2, II-1 and II-2
according to a published procedure (Brahe and Serra 1981) by fusing the YH21 hamster cell line with lymphoblastoid cells from propositae and their mother. The hybrid clones were isolated and cytogenetically examined, using Q-banding, for the presence of human chromosomes. Three clones, one from each half sister and one from the mother, were selected because they had the lowest modal chromosome number and the highest mitotic index without any apparent rearrangement of the single X chromosome that they retained after HAT (hypoxanthine, aminopterin, thymidine) selection. When informative, the RFLP analysis always confirmed the presence of a single human X chromosome in these hybrids, which carried one of the 2 alleles present in the genomic DNA from the same female. Linkage analysis was performed using the programs MLINK of LINKAGE V5.03 (Lathrop and Lalouel 1984). The assumptions made to use this program were those of X linkage and of disease present in the mother, since the option of germinal mosaicism was not possible.
Results A total of 34 R F L P s mapping on the X c h r o m o s o m e were analysed in genomic D N A from the four individuals available for the study and in D N A f r o m hybrids of the females (the fathers were not available for study). Figure 1 shows the haplotypes for 15 informative R F L P s reconstructed in the m o t h e r and in her two daughters according to the data from somatic cell hybrids. The hybrids are labelled as follow: H y 9 7 A derived f r o m I-2, Hy81F1 from II-1 (carrying the maternal X chromosome), H y 8 2 L from II-2 (carrying the paternal X c h r o m o s o m e ) . As a further test with respect to the origin of the single X c h r o m o s o m e retained in the hybrids derived from the two affected half sisters, we also considered those RFLPs for which I-2 (mother) was homozygous and II-1 or II-2 were heterozygous. The presence in the hybrid of the maternal or paternal alleles identifies the only X chrom o s o m e retained in the hybrid as being of maternal or paternal origin. This type of analysis (based on homozygosity in the m o t h e r and heterozygosity in a daughter) is informative for 8 RFLPs in II-1 and for 2 RFLPs in II-2. In addition to confirming the maternal origin of the X c h r o m o s o m e in hybrid Hy81F1 and the paternal origin of the X c h r o m o s o m e in hybrid Hy82L, this analysis is in
agreement with the cytogenetic and molecular data that indicate that no r e a r r a n g e m e n t of these X c h r o m o s o m e s has occurred in vitro. According to the strategy described above, the 15 R F L P s for which the m o t h e r is heterozygous were used for the identification of the regions of the maternal X c h r o m o s o m e shared by the two half-sisters (concordant region). In addition, the analysis of the phase in the m o t h e r I-2 allows the identification of the recombination events and of the discordant regions in the X chromosomes inherited by her three children (Fig. 1). These regions can be identified in Xp as those immediately surrounding the loci defined by probes pS232 (DXS278) and pXG-16 (DXS92) and that identified by p B a m X 7 around the centromere, and the region of D X Y S 1 on Xq. A two point linkage analysis between the above markers and the RTS gene yields, in each case, a lod score ___- 2 . 0 8 for 0 = 0.002. For all the other concordant markers, positive but not significant lod scores were obtained ( m a x i m u m lod score = 0.6 for 0 = 0.00).
Discussion The order of polymorphic loci on the X c h r o m o s o m e reported in Fig. 1 derives f r o m information obtained from recent reports (Mandel et al. 1989). As already pointed out ( R o m e o et al. 1986), the peculiarity of this pedigree makes it highly probable that the mutation causing RTS in the two half sisters is of maternal origin. According to the strategy described in the Introduction, our results lead to the exclusion, as possible regions of localization of the RTS gene(s), of those portions of the X chromosome not shared by the two half sisters. These regions can be identified as those surrounding the loci DXS278, DXS92, D X S Z 1 and D X Y S 1 . A quantitative estimate of the exclusion of the RTS gene in the vicinity of each of these loci yields lod scores _< -- - 2 . 0 8 for 0 -- 0.002. It is of interest, however, that where the two affected half sisters are concordant, their half brother shows alleles that are discordant with theirs for a total of 11 RFLPs. With regard to the possible mapping of the RTS
606 m u t a t i o n ( s ) o n t h e X c h r o m o s o m e , two d i f f e r e n t X - a u tosomal translocations involving Xp have been recently d e s c r i b e d in R T S p a t i e n t s ( Z o g h b i et al. 1990; J o u r n e l et al. 1990). T h e c o r r e s p o n d i n g X c h r o m o s o m e b r e a k p o i n t s o c c u r in t h e s e cases in d i f f e r e n t r e g i o n s of X p i d e n t i f i e d as Xp22.11 a n d Xp11.22, r e s p e c t i v e l y ( Z o g h b i et al. 1990; J o u r n e l et al. 1990). G e n e t i c analysis c a r r i e d o u t in the present study does not exclude either localization. Addit i o n a l i n f o r m a t i o n c o m e s f r o m t h e analysis of t h e h y b r i d l a b e l l e d as H Y 8 1 F 1 ( f r o m p r o p o s i t a II-1). T h e X chrom o s o m e , w h i c h is r e t a i n e d in this h y b r i d a n d which is o f m a t e r n a l origin, d i d n o t s h o w a n y d e l e t i o n . T h e r e f o r e , a m i c r o d e l e t i o n of m a t e r n a l origin as a cause of R T S seems u n l i k e l y for all t h e e x a m i n e d loci. T h e v a r i a t i o n o f t h e clinical c o u r s e o b s e r v e d in s o m e p e d i g r e e s with r e c u r r e n c e o f e i t h e r R T S o r I P w o u l d b e in a g r e e m e n t w i t h u n b a l a n c e d L y o n i z a t i o n . Such varieg a t i o n has b e e n o b s e r v e d in two full sisters with R T S ( H a n e f e l d et al. 1986) a n d in t h e f a m i l y with t h e t(X;22) (p11.22;p11) b a l a n c e d t r a n s l o c a t i o n ( J o u r n e l et al. 1990). In a similar way, studies of 5 heterozygous females, from 3 k i n d r e d s s e g r e g a t i n g IP, i n d i c a t e t h a t the cells e x p r e s sing m u t a t i o n s t h a t a r e l e t h a l in m a l e fetuses m a y b e s e l e c t e d a g a i n s t in c a r r i e r f e m a l e s , w h o m i g h t s h o w a p h e n o t y p e less d e l e t e r i o u s t h a n t h a t e x p e c t e d o n the basis o f r a n d o m i n a c t i v a t i o n ( M i g e o n et al. 1989). T h e a p p r o a c h o f e x c l u s i o n m a p p i n g u s e d in this w o r k can o v e r c o m e t h e i n c o n s i s t e n c i e s r e v e a l e d b y t h e two t r a n s l o c a t i o n s r e c e n t l y r e p o r t e d in R e t t p a t i e n t s ( Z o g h b i et al. 1990; J o u r n e l et al. 1990).
Acknowledgements. This work was supported by grants from the Consiglio Nazionale delle Ricerche (Rome) with special reference to the Progetto Finalizzato Ingegneria Genetica and from Ministero della Pubbliea Istruzione, Rome, Italy. The help of Dr.M. Devoto for linkage analysis and the technical assistance from I. Giambarrasi, S. Castagnola and M. Bertorello are acknowledged. T.O. is an Associate and U.F. an Investigator of the Howard Hughes Medical Institute.
References Brahe C, Serra A (1981) A simple method for fusing human lymphocytes with rodent cell in monolayer by polyethylene glycol. Somat Cell Genet 7:109-115 Comings DE (1986) The genetics of Rett syndrome: the consequences of a disorder where every case is a new mutation. Am J Med Genet [Suppl 1] : 383-388 Davies KE, Mandel JL, Wiessebach J, Fellous M (1987) Report of the committee on the genetic constitution of X and Y chromosomes. Cytogenet Cell Genet 46:1-4
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