~~
Trends in Molecular Medicine
Fragile X Syndrome: Molecular Analysis Reveals a New Mechanism of Mutation in Human Genetic Diseases
Ann Med Downloaded from informahealthcare.com by University of Auckland on 12/07/14 For personal use only.
Annemarie Poustka
The fragile X syndrome belongs to the most common genetic diseases and has a prevalence of one in every 2000 children. The syndrome is named after the fragile site in q27.3 on the X chromosome. The molecular cloning of the DNA containing the fragile site has resulted in the Identification of a heritable unstable DNA sequence revealing a new mechanism of mutation in human genetic disorders. This DNA sequence significantly facilitates the diagnosis and provides a rapid method for carrier detection and prenatal diagnosis. The unstable element is located within a candidate gene, FMR1. The FMR1 protein is not made In fragile X patients and nothing is known about its function. We will have t o await studies on this protein to be able t o understand the variable phenotype of this disease. Key words: fragile site; molecular cloning; heritable unstable sequence; FMRl gene. (Annals of Medicine 24: 453-456,1992)
Introduction The fragile X mental retardation syndrome is the most frequent inherited form of mental retardation and the second most common cause of mental retardation after Downs syndrome. The mental retardation is of varying severity accompanied by behavioural problems and various dismorphic features, making diagnosis often difficult. The syndrome is characterized cytogenetically by the presence of a fragile site at the position Xq27.3. which can be induced in vitro by growth of cells under conditions reducing the availability of precursors for DNA synthesis (1, 2). The inheritance pattern is unusual in showing partial penetrance in both males and females. Especially striking is the observation of phenotypically normal males, who transmit the mutation to daughters, who generally again show no phenotype, while their grandchildren show normal penetrance of the disorder (3). Due to the recent developments of molecular cloning and mapping techniques, rapid progress has been made in the molecular identification and analysis of genes responsible for human genetic diseases. The identification of the mutation responsible for the fragile X phenotype has been one of the last successes on this route, From the Deutsches Krebsforschungszentrum, Heidelberg, Germany. Address and reprint requests: A. Poustka, Ph.D., Deutsches Krebsforschungszentrum, Im Neuenheimerfeld 506, 6900 Heidelberg, Germany.
progressing from the initial cytogenetic and genetic localization, over the closer identification of the position of the fragile region by somatic cell genetic and physical mapping techniques, to the cloning of the region in YAC and cosmid clones, to the identification of the mutation, and to the isolation of a gene involved in the phenotype of the disease.
Progress from Phenotype to Gene This localization of the fragile X locus, ultimately resulting in the molecular isolation of the gene carrying.the mutation has been based on the progressively closer positioning of a number of different elements (the cytogenetic fragile site, a position of enhanced breakage in induced fragile X chromosomes during chromosome transfer, a CpG island found to be selectively methylated in patients, the position of an unstable DNA fragment and the FMR-1 gene) associated with the locus, all of which finally could be shown (within the resolution limits of each assay) to be located within a very short region.
Genetic and cytogenetic mapping localize the fragile X mutation close to the end of the long arm of the X chromosome The first mapping system applied to localize the region implicated in the disease was cytogenetic analysis. In 1969 a fragile site at Xq27.3 was observed and found to be correlated with the phenotype of the disease (4).
454
Poustka
Later, the mutation could be localized to Xq27-qter, based on family analysis data (5-7). Genetic linkage studies, however, could not resolve the order of markers surrounding the mutation, until a physical map of the Xq27.3-Xqter region was constructed using pulsed field gel electrophoresis (8).
Ann Med Downloaded from informahealthcare.com by University of Auckland on 12/07/14 For personal use only.
Physical Localization of the Fragile X Region Using Cell Hybrids The potential for an exact localization of the mutation was increased significantly, when a somatic cell mapping system became available, based on the observation of enhanced breakage of chromosomes carrying the fragile X mutation (9). The construction of cell lines carrying either the fragment proximal or distal to the fragile X induced breakpoint translocated onto a hamster chromosome (10, 1l ) , has speeded up the work of localizing new probes either proximal or distal to the fragile position, increasing the number of probes available for genetic and physical mapping (12, 13). These translocation cell -hybrids also opened the way to mapping the fragile region with molecular techniques, essential to allow a physical localization of the fragile X locus, and to simplify the further molecular analysis. This has been demonstrated in the use of a number of such cell lines, containing the DNA from fragile site to the telomere (Xq27.3-Xqter), to establish a physical map of the area covering over 12 megabases, localizing the breakpoints of a number of independently derived hybrids to a region of 300 kilobases (8). The close proximity of the breakpoints in different cell lines strengthened the assumption that the breaks in the cell lines coincide with the cytogenetically observed region of fragility.
A Selective Methylation Pattern in Fragile X Patients In this 300 kb area defined by the physical map a CpG island was located and shown to be selectively methylated in fragile X patients, the first molecular assay differentiating between disease carriers and patients showing the fragile X phenotype (14-16). The close proximity of this CpG island to the position of the breakpoints in the cell lines, further strengthened the expectation for the involvement of a very short region in most or all of the phenomena associated with the disease.
Isolation of Clones Extending Across the Region of Fragility Opens the Way for a Detailed Molecular Analysis Although large cosmid contigs flanking the cell hybrid breakpoints have been isolated in cosmid clones (16), the identification of a cosmid containing the CpG island and extending across the breakpoints has proven difficult. Yeast artificial chromosome (YAC) clones, however, have been isolated in a number of laboratories in parallel and have been shown by in situ hybridization to extend across the area of cytogenetic fragility (16-19), and to cover the position of the cell line breakpoints and the region of selective methylation in fragile X patients. This
provided for the first time the DNA of interest in cloned form, but also opened the way for the cloning of this area in cosmid or lambda clones. Based on this clone coverage, in a rapid series of development, essentially all elements associated with the fragile X locus could be localized within a restriction fragment of only 5 kb. This 5 kb fragment containing the breakpoints in the cell lines and the CpG island selectively methylated in patients, could also be shown to contain a sequence, which changed its size (20-23) from the length in normal controls over an increased length in asymptomatic carriers, called the premutation. In fragile X patients the fragment is drastically and heterogeneously increased in size to the full mutation.
The Fragile X Mutation is Caused by the Expansion of a Trinucleotide Repeat Located Within a Conserved Gene, Responsible for the Phenotype of the Mutation By sequence determination, this increase in size could be shown to be due to the expansion of a simple repeat sequence p(CCG),, increasing in size from a range of 6 to 54 in normals, over a range of 54 to over 200 in presymptomatic carriers, to a length of 200 to 1500 copies in fragile X patients (24). This sequence is most likely to be located in the first exon of a gene (FMR-1) which shows high conservation between species and is expressed in all tissues (23). While the mRNA remains normally expressed in symptomless carriers, no mRNA can be detected in cells from patients (25). FMR-1 can therefore be considered the prime candidate for causing many, though possibly not all, of the phenotypes of the fragile X mutation. The case for the primary responsibility of the lack of expression of FMR-1 has however been Strengthened further, due to the identification of patients with fragile X phenotype without showing cytogenetically the expression of a fragile site. In two of these cases this could be shown to be due to a deletion of FMR-1. Since the deletion of FMR-1 can cause the same phenotype as the expansion of the repeat, which leads indirectly to the turnoff of the gene, it is therefore likely, that the fragile X phenotype is predominantly caused by the lack of FMR-1 expression (26, 27).
The Fragile X Mutation is One of Three Recently Identified Mutations Caused by Expansion of Trinucleotide Repeats Though considered unexpected, mutations found to be caused by the expansion of trinucleotide repeat sequences have also been found to underly two other common genetic diseases, X-linked spinal muscular atrophy (28) and myotonic dystrophy (29-31), caused by an increase in the size of a p(CAG), trinucleotide repeat sequence. This has not only provided an insight into a new, but obviously quite common, mechanism of mutation, but has also for the first time given a molecular explanation for the phenomenon of anticipation, an increase in the severity of a disease in following generations.
Fragile X Syndrome
Ann Med Downloaded from informahealthcare.com by University of Auckland on 12/07/14 For personal use only.
Diagnosis of the Fragile X Syndrome The availability of a DNA-based test for fragile X diagnosis and carrier detection has revolutionized the diagnosis of this disease (32, 33). Prenatal diagnosis can be based on the detection of an increased fragment size detected by probes located close to the repeat sequence (pfax3 (21), StB12.5 (22), pE5.1 (23), 0x055 (34) and GL30 (Poustka, unpublished)) and can be used to follow the inheritance of the mutation or premutation in pedigrees (32,33,35). The use of PCR techniques, very successful in the clinical diagnosis of many other mutations, is complicated by the selective amplification of fragments containing shorter repeats in samples with multiple repeat length (21,36) and can therefore be used predominantly for carrier detection, allowing improved discrimination of the shorter repeat length. To exclude possible misdiagnosis caused by the lack of amplification of the large repeats, it will, in many cases, have to be used in conjunction with the standard Southern blot approach. In prenatal diagnosis, determination of the size of the repeat fragment can be used to predict the phenotype of individuals. In general, males with repeat amplifications of more than 700 basepairs are considered to be affected, while amplifications of less than 500 basepairs are an indication of carrier status. Females with amplifications of less than 500 basepairs are considered unlikely to be affected, while longer amplification products indicate a significant probability of mental retardation (24). Since the changed methylation pattern indicative of the phenotypic expression cannot be consistently observed in chorion villi, it cannot be used in the prenatal diagnosis (37).
Has the Primary Fragile X Mutation Really been Found? Analysis of the myotonic dystrophy mutation in particular has opened an interesting new aspect underlying the fragile X mutation. This is due to the unexpected combination of an apparently high rate of new mutations observed in myotonic dystrophy, coupled with the observation of strong linkage disequilibrium. This indicates the presence of a second (or actually first) mutation of a specific chromosome, which by some mechanism causes a high probability of expansion of the trinucleotide repeat sequence, which can either be due to a rare primary expansion of the repeat in the myotonic dystrophy gene carried only on chromosomes of this haplotype, or due to the presence of another gene on this chromosome, which induces the expansion of the repeat. It cannot be excluded, therefore, that in the case of the fragile X mutation, the primary change responsible for the expansion of the repeat sequence observed in the premutation could still remain to be located.
References 1. Sutherland GR. The enigma of the fragile X chromosome. Trends Genet 1985; 4: 108-1 3.
455
2. Nussbaum RJ, Ledbetter DH. Fragile X syndrome: a unique mutation in man. Annu Rev Genet 1986; 20: 109-45. 3. Sherman SL, Morton NE, Jacobs PA, Turner G. The marker (X) syndrome: a cytogenetic and genetic analysis. Ann Hum Genet 1984; 48: 21-37. 4. Lubs HA. A marker X-chromosome. Am J Hum Genet 1969; 21: 234-44. 5. Camerlno G, Matte1 MG, Matt61 JF, Jaye M, Mandel JL. Close linkage of fragile X linked mental retardation syndrome to haemophilia B and transmission through a normal male. Nature 1983; 306: 701-7. 6. Oberlb I,Heillg R, Molsan JP, et al. Genetic analysis of the fragile-X mental retardation syndrome with two polymorphic flanking markers. Proc Natl Acad Sci USA 1986; 83: 1016-20. 7. Mulley J, Turner G, Barn S, Sutherland GR. Linkage between the fragile X and F9, DXS52 (St 14). DXS98 (4D8) and DXSlO5 (cX55.7). Am J Med Genet 1988; 30: 567-80. 8. Poustka A, Dletrlch A, Langensteln G, Tonlolo D, Warren ST, Lehrach H. Physical map of human Xq27-qter localising the region of the fragile X mutation. PNAS, 1990 (in press). 9. Warren ST, Davldson RL. Expression of the fragile X chromosome in human-rodent somatic cell hybrids. Somatic Cell Genet 1984; 19: 409-1 3. 10. Warren ST, Zhang F, Llcarneii GR, Peters JF. The fragile X site in somatic cell hybrids: an approach for molecular cloning of fragile sites. Science 1987; 237: 420-3. 11. Warren ST, Knight SJL, Peters JF, Stayton CL, Consalez GG, Zhang F. Isolation of the human chromosomal band Xq28 within somatic cell hybrids by fragile site breakage. Proc Natl Acad Sci USA 1990; 87: 3856-60. 12. Rousseau F, Vincent A, Rlvella S, et al. Four chromosomal breakpoints and four new probes mark out a 10-cM region encompassing the fragile X locus (FRAXA). Am J Hum Genet 1991; 48: 108-1 6. 13. Suthers GK, Mulley JC, Voelckel MA, et al. Genetic mapping of new DNA probes at Xq27 defines a strategy for DNA studies in the fragile X syndrome. Am J Hum Genet 1991; 48: 460-7. 14. Vincent A, Heltz D, Petit C, Kretz C, Oberle I, Mandel JL. Abnormal pattern detected in fragile-X patients by pulsedfield gel electrophoresis. Nature 1991; 349: 624-6. 15. Bell MV, Hlrst MC, Nakahori V, et al. Physical mapping across the fragile X: hypermethylation and clinical expression of the fragile X syndrome. Cell 1991; 64: 861-6. 16. Dietrlch A, Kloschls P, Monaco AP, et al. Molecular cloning and analysis of the fragile X region in man. Nucleic Acids Res 1991; 19,lO: 2567-72. 17. Heitz D. Rousseau F, Devys D, et al. Isolation of normal sequences that span the fragile site and identification of a CpG island involved in fragile X expression. Science 1991; 251: 1236-9. 18. Kremer EJ, Yu S, Prltchard M, et al. Isolation of a human DNA sequence< which spans the fragile X. Am J Hum Genet 1991; 4: 656-61. 19. Hlrst MC, Nakahorl Y, Roche A, et al. A Yac contig across the fragile X site defines the region of fragility. Nucleic Acids Res 1991; 19.12: 3283-8. 20. Kremer EJ, Prltchard M, Lynch M, et al. Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG),. Science 1991; 252; 1711-1 4. 21. Yu S, Prltchard M, Krerner E, et al. Fragile X genotype characterized by an unstable region of DNA. Science 1991; 252: 1179-81. 22. Oberlb I, Rousseau F, Heltz D, et al. Instability of a 500base pair DNA segment and abnormal methylation in fragile X syndrome. Science 1991; 252: 1097-1 102. 23. Verkerk AJMH, Plerettl M, Sutcllffe JS, et al. Identification of a gene (FMR-1) containing a CGG repeat coincident with
456
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25.
26.
27.
28.
Ann Med Downloaded from informahealthcare.com by University of Auckland on 12/07/14 For personal use only.
29.
30.
Poustka
a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 1991; 65: 905-14. Fu Y-H, Kuhl DPA, P i u u t l A, et al. Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell 1991; 67: 1047-58. Pieretti M, Zhang F, Fu Y-H, et al. Absence of expression of the FMR-1 gene in fragile X syndrome. Cell 1991; 66: 1-20. Wohrle D, Rott H-D, Kotzot D, et al. A microdeletion of less than 250 kb, including the proximal part of the FMR-1 gene and the fragile X site, in a male with the clinical phenotype of fragile X syndrome. Am J Hum Genet 1992 (in press). Gedeon AK, Baker E, Robinson H, et al. Fragile X syndrome without CCG amplification. Nature Genetics 1992 (in press). La Spada AR, Wilson EM, Lubahn DB, Harding AE, Fischbeck KH. Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nafure 1991; 352: 77-9. Harley HG, Brook JD, Rundle SA, et al. Expansion of an unstable DNA region and phenotypic variation in mytotonic dystrophy. Nature 1992; 355: 545-6. Brook D, McCurrach ME, Harley HG, et al. Molecular basis of mytotonic dystrophy: Expansion of a trinucleotide
31.
32.
33. 34.
35.
36.
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(CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. Cell 1992; 68: 799-808. Buxton J, Shelbourne P, Davies J, et al. Detection of an unstable fragment of DNA specific to individuals with myotonic dystrophy. Nature 1992; 355: 547-8. Rousseau F, Heltz D, Biancalana V, et al. Direct diagnosis by DNA analysis of the fragile X syndrome of mental retardation, N €ngl J Med 1991; 24: 1673-81. Knight SJL, Hirst MC, Roche A, et al. Molecular studies of the fragile X syndrome. Am J Med Genet 1992; 43: 217-23. Nakahori Y, Knight SJL, Holland J, et al. Molecular heterogeneity of the fragile X syndrome. Nucl Acids Res 1991; 19: 4355-9. Mandel J-L, Hagerman R, Froster U, et al. Conference Report: Fifth international workshop on the fragile X and X-linked mental retardation. Am J Med Genet 1992; 43: 5-27. Richards RI, Sutherland GR. Fragile X syndrome: the molecular picture comes into focus. Trends Genet 1992; 8: 249-55. Sutherland GR, Gedeon A, Kornman L, et al. Prenatal diagnosis of fragile X syndrome by direct detection of the unstable DNA sequence. N Engl J Med 1991; 325: 1720-2.
Trends in Molecular Medicine
Fragile X Syndrome: Molecular Analysis Reveals a New Mechanism of Mutation in Human Genetic Diseases
Ann Med Downloaded from informahealthcare.com by University of Auckland on 12/07/14 For personal use only.
Annemarie Poustka
The fragile X syndrome belongs to the most common genetic diseases and has a prevalence of one in every 2000 children. The syndrome is named after the fragile site in q27.3 on the X chromosome. The molecular cloning of the DNA containing the fragile site has resulted in the Identification of a heritable unstable DNA sequence revealing a new mechanism of mutation in human genetic disorders. This DNA sequence significantly facilitates the diagnosis and provides a rapid method for carrier detection and prenatal diagnosis. The unstable element is located within a candidate gene, FMR1. The FMR1 protein is not made In fragile X patients and nothing is known about its function. We will have t o await studies on this protein to be able t o understand the variable phenotype of this disease. Key words: fragile site; molecular cloning; heritable unstable sequence; FMRl gene. (Annals of Medicine 24: 453-456,1992)
Introduction The fragile X mental retardation syndrome is the most frequent inherited form of mental retardation and the second most common cause of mental retardation after Downs syndrome. The mental retardation is of varying severity accompanied by behavioural problems and various dismorphic features, making diagnosis often difficult. The syndrome is characterized cytogenetically by the presence of a fragile site at the position Xq27.3. which can be induced in vitro by growth of cells under conditions reducing the availability of precursors for DNA synthesis (1, 2). The inheritance pattern is unusual in showing partial penetrance in both males and females. Especially striking is the observation of phenotypically normal males, who transmit the mutation to daughters, who generally again show no phenotype, while their grandchildren show normal penetrance of the disorder (3). Due to the recent developments of molecular cloning and mapping techniques, rapid progress has been made in the molecular identification and analysis of genes responsible for human genetic diseases. The identification of the mutation responsible for the fragile X phenotype has been one of the last successes on this route, From the Deutsches Krebsforschungszentrum, Heidelberg, Germany. Address and reprint requests: A. Poustka, Ph.D., Deutsches Krebsforschungszentrum, Im Neuenheimerfeld 506, 6900 Heidelberg, Germany.
progressing from the initial cytogenetic and genetic localization, over the closer identification of the position of the fragile region by somatic cell genetic and physical mapping techniques, to the cloning of the region in YAC and cosmid clones, to the identification of the mutation, and to the isolation of a gene involved in the phenotype of the disease.
Progress from Phenotype to Gene This localization of the fragile X locus, ultimately resulting in the molecular isolation of the gene carrying.the mutation has been based on the progressively closer positioning of a number of different elements (the cytogenetic fragile site, a position of enhanced breakage in induced fragile X chromosomes during chromosome transfer, a CpG island found to be selectively methylated in patients, the position of an unstable DNA fragment and the FMR-1 gene) associated with the locus, all of which finally could be shown (within the resolution limits of each assay) to be located within a very short region.
Genetic and cytogenetic mapping localize the fragile X mutation close to the end of the long arm of the X chromosome The first mapping system applied to localize the region implicated in the disease was cytogenetic analysis. In 1969 a fragile site at Xq27.3 was observed and found to be correlated with the phenotype of the disease (4).
454
Poustka
Later, the mutation could be localized to Xq27-qter, based on family analysis data (5-7). Genetic linkage studies, however, could not resolve the order of markers surrounding the mutation, until a physical map of the Xq27.3-Xqter region was constructed using pulsed field gel electrophoresis (8).
Ann Med Downloaded from informahealthcare.com by University of Auckland on 12/07/14 For personal use only.
Physical Localization of the Fragile X Region Using Cell Hybrids The potential for an exact localization of the mutation was increased significantly, when a somatic cell mapping system became available, based on the observation of enhanced breakage of chromosomes carrying the fragile X mutation (9). The construction of cell lines carrying either the fragment proximal or distal to the fragile X induced breakpoint translocated onto a hamster chromosome (10, 1l ) , has speeded up the work of localizing new probes either proximal or distal to the fragile position, increasing the number of probes available for genetic and physical mapping (12, 13). These translocation cell -hybrids also opened the way to mapping the fragile region with molecular techniques, essential to allow a physical localization of the fragile X locus, and to simplify the further molecular analysis. This has been demonstrated in the use of a number of such cell lines, containing the DNA from fragile site to the telomere (Xq27.3-Xqter), to establish a physical map of the area covering over 12 megabases, localizing the breakpoints of a number of independently derived hybrids to a region of 300 kilobases (8). The close proximity of the breakpoints in different cell lines strengthened the assumption that the breaks in the cell lines coincide with the cytogenetically observed region of fragility.
A Selective Methylation Pattern in Fragile X Patients In this 300 kb area defined by the physical map a CpG island was located and shown to be selectively methylated in fragile X patients, the first molecular assay differentiating between disease carriers and patients showing the fragile X phenotype (14-16). The close proximity of this CpG island to the position of the breakpoints in the cell lines, further strengthened the expectation for the involvement of a very short region in most or all of the phenomena associated with the disease.
Isolation of Clones Extending Across the Region of Fragility Opens the Way for a Detailed Molecular Analysis Although large cosmid contigs flanking the cell hybrid breakpoints have been isolated in cosmid clones (16), the identification of a cosmid containing the CpG island and extending across the breakpoints has proven difficult. Yeast artificial chromosome (YAC) clones, however, have been isolated in a number of laboratories in parallel and have been shown by in situ hybridization to extend across the area of cytogenetic fragility (16-19), and to cover the position of the cell line breakpoints and the region of selective methylation in fragile X patients. This
provided for the first time the DNA of interest in cloned form, but also opened the way for the cloning of this area in cosmid or lambda clones. Based on this clone coverage, in a rapid series of development, essentially all elements associated with the fragile X locus could be localized within a restriction fragment of only 5 kb. This 5 kb fragment containing the breakpoints in the cell lines and the CpG island selectively methylated in patients, could also be shown to contain a sequence, which changed its size (20-23) from the length in normal controls over an increased length in asymptomatic carriers, called the premutation. In fragile X patients the fragment is drastically and heterogeneously increased in size to the full mutation.
The Fragile X Mutation is Caused by the Expansion of a Trinucleotide Repeat Located Within a Conserved Gene, Responsible for the Phenotype of the Mutation By sequence determination, this increase in size could be shown to be due to the expansion of a simple repeat sequence p(CCG),, increasing in size from a range of 6 to 54 in normals, over a range of 54 to over 200 in presymptomatic carriers, to a length of 200 to 1500 copies in fragile X patients (24). This sequence is most likely to be located in the first exon of a gene (FMR-1) which shows high conservation between species and is expressed in all tissues (23). While the mRNA remains normally expressed in symptomless carriers, no mRNA can be detected in cells from patients (25). FMR-1 can therefore be considered the prime candidate for causing many, though possibly not all, of the phenotypes of the fragile X mutation. The case for the primary responsibility of the lack of expression of FMR-1 has however been Strengthened further, due to the identification of patients with fragile X phenotype without showing cytogenetically the expression of a fragile site. In two of these cases this could be shown to be due to a deletion of FMR-1. Since the deletion of FMR-1 can cause the same phenotype as the expansion of the repeat, which leads indirectly to the turnoff of the gene, it is therefore likely, that the fragile X phenotype is predominantly caused by the lack of FMR-1 expression (26, 27).
The Fragile X Mutation is One of Three Recently Identified Mutations Caused by Expansion of Trinucleotide Repeats Though considered unexpected, mutations found to be caused by the expansion of trinucleotide repeat sequences have also been found to underly two other common genetic diseases, X-linked spinal muscular atrophy (28) and myotonic dystrophy (29-31), caused by an increase in the size of a p(CAG), trinucleotide repeat sequence. This has not only provided an insight into a new, but obviously quite common, mechanism of mutation, but has also for the first time given a molecular explanation for the phenomenon of anticipation, an increase in the severity of a disease in following generations.
Fragile X Syndrome
Ann Med Downloaded from informahealthcare.com by University of Auckland on 12/07/14 For personal use only.
Diagnosis of the Fragile X Syndrome The availability of a DNA-based test for fragile X diagnosis and carrier detection has revolutionized the diagnosis of this disease (32, 33). Prenatal diagnosis can be based on the detection of an increased fragment size detected by probes located close to the repeat sequence (pfax3 (21), StB12.5 (22), pE5.1 (23), 0x055 (34) and GL30 (Poustka, unpublished)) and can be used to follow the inheritance of the mutation or premutation in pedigrees (32,33,35). The use of PCR techniques, very successful in the clinical diagnosis of many other mutations, is complicated by the selective amplification of fragments containing shorter repeats in samples with multiple repeat length (21,36) and can therefore be used predominantly for carrier detection, allowing improved discrimination of the shorter repeat length. To exclude possible misdiagnosis caused by the lack of amplification of the large repeats, it will, in many cases, have to be used in conjunction with the standard Southern blot approach. In prenatal diagnosis, determination of the size of the repeat fragment can be used to predict the phenotype of individuals. In general, males with repeat amplifications of more than 700 basepairs are considered to be affected, while amplifications of less than 500 basepairs are an indication of carrier status. Females with amplifications of less than 500 basepairs are considered unlikely to be affected, while longer amplification products indicate a significant probability of mental retardation (24). Since the changed methylation pattern indicative of the phenotypic expression cannot be consistently observed in chorion villi, it cannot be used in the prenatal diagnosis (37).
Has the Primary Fragile X Mutation Really been Found? Analysis of the myotonic dystrophy mutation in particular has opened an interesting new aspect underlying the fragile X mutation. This is due to the unexpected combination of an apparently high rate of new mutations observed in myotonic dystrophy, coupled with the observation of strong linkage disequilibrium. This indicates the presence of a second (or actually first) mutation of a specific chromosome, which by some mechanism causes a high probability of expansion of the trinucleotide repeat sequence, which can either be due to a rare primary expansion of the repeat in the myotonic dystrophy gene carried only on chromosomes of this haplotype, or due to the presence of another gene on this chromosome, which induces the expansion of the repeat. It cannot be excluded, therefore, that in the case of the fragile X mutation, the primary change responsible for the expansion of the repeat sequence observed in the premutation could still remain to be located.
References 1. Sutherland GR. The enigma of the fragile X chromosome. Trends Genet 1985; 4: 108-1 3.
455
2. Nussbaum RJ, Ledbetter DH. Fragile X syndrome: a unique mutation in man. Annu Rev Genet 1986; 20: 109-45. 3. Sherman SL, Morton NE, Jacobs PA, Turner G. The marker (X) syndrome: a cytogenetic and genetic analysis. Ann Hum Genet 1984; 48: 21-37. 4. Lubs HA. A marker X-chromosome. Am J Hum Genet 1969; 21: 234-44. 5. Camerlno G, Matte1 MG, Matt61 JF, Jaye M, Mandel JL. Close linkage of fragile X linked mental retardation syndrome to haemophilia B and transmission through a normal male. Nature 1983; 306: 701-7. 6. Oberlb I,Heillg R, Molsan JP, et al. Genetic analysis of the fragile-X mental retardation syndrome with two polymorphic flanking markers. Proc Natl Acad Sci USA 1986; 83: 1016-20. 7. Mulley J, Turner G, Barn S, Sutherland GR. Linkage between the fragile X and F9, DXS52 (St 14). DXS98 (4D8) and DXSlO5 (cX55.7). Am J Med Genet 1988; 30: 567-80. 8. Poustka A, Dletrlch A, Langensteln G, Tonlolo D, Warren ST, Lehrach H. Physical map of human Xq27-qter localising the region of the fragile X mutation. PNAS, 1990 (in press). 9. Warren ST, Davldson RL. Expression of the fragile X chromosome in human-rodent somatic cell hybrids. Somatic Cell Genet 1984; 19: 409-1 3. 10. Warren ST, Zhang F, Llcarneii GR, Peters JF. The fragile X site in somatic cell hybrids: an approach for molecular cloning of fragile sites. Science 1987; 237: 420-3. 11. Warren ST, Knight SJL, Peters JF, Stayton CL, Consalez GG, Zhang F. Isolation of the human chromosomal band Xq28 within somatic cell hybrids by fragile site breakage. Proc Natl Acad Sci USA 1990; 87: 3856-60. 12. Rousseau F, Vincent A, Rlvella S, et al. Four chromosomal breakpoints and four new probes mark out a 10-cM region encompassing the fragile X locus (FRAXA). Am J Hum Genet 1991; 48: 108-1 6. 13. Suthers GK, Mulley JC, Voelckel MA, et al. Genetic mapping of new DNA probes at Xq27 defines a strategy for DNA studies in the fragile X syndrome. Am J Hum Genet 1991; 48: 460-7. 14. Vincent A, Heltz D, Petit C, Kretz C, Oberle I, Mandel JL. Abnormal pattern detected in fragile-X patients by pulsedfield gel electrophoresis. Nature 1991; 349: 624-6. 15. Bell MV, Hlrst MC, Nakahori V, et al. Physical mapping across the fragile X: hypermethylation and clinical expression of the fragile X syndrome. Cell 1991; 64: 861-6. 16. Dietrlch A, Kloschls P, Monaco AP, et al. Molecular cloning and analysis of the fragile X region in man. Nucleic Acids Res 1991; 19,lO: 2567-72. 17. Heitz D. Rousseau F, Devys D, et al. Isolation of normal sequences that span the fragile site and identification of a CpG island involved in fragile X expression. Science 1991; 251: 1236-9. 18. Kremer EJ, Yu S, Prltchard M, et al. Isolation of a human DNA sequence< which spans the fragile X. Am J Hum Genet 1991; 4: 656-61. 19. Hlrst MC, Nakahorl Y, Roche A, et al. A Yac contig across the fragile X site defines the region of fragility. Nucleic Acids Res 1991; 19.12: 3283-8. 20. Kremer EJ, Prltchard M, Lynch M, et al. Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG),. Science 1991; 252; 1711-1 4. 21. Yu S, Prltchard M, Krerner E, et al. Fragile X genotype characterized by an unstable region of DNA. Science 1991; 252: 1179-81. 22. Oberlb I, Rousseau F, Heltz D, et al. Instability of a 500base pair DNA segment and abnormal methylation in fragile X syndrome. Science 1991; 252: 1097-1 102. 23. Verkerk AJMH, Plerettl M, Sutcllffe JS, et al. Identification of a gene (FMR-1) containing a CGG repeat coincident with
456
24.
25.
26.
27.
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Ann Med Downloaded from informahealthcare.com by University of Auckland on 12/07/14 For personal use only.
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