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The enigma of the fragile X chromosome
r iews
TIG -- Atrd1198,5
The enigma of the fragile X
The fragile X chromosome is now known to be associated with the commonest inherited form of mental handicap in man. This is rather remarkable Grant R. Sutherland for a condition which was first reported in 1969 and At the beginning of this decade thefragile X chromosome was recogni~d as being assonot recognized as affectciated with the commonest inhen"tedform of mental handicap in man. De~'te intensive ing more than a single study, fragile X remains a mystery; cytogeneticstudy of it is difficult, the exactnature of family until the midits ass~'iation with mental retardation L~unknou,n, its genetics are bizarre and its mole1970sk cular nature speculatit~. Many people working in the area originally thought that the fragile X was associated with a form Unifying concepts of mental retardation without any distinguishing Two unifying concepts have been proposed to clinical features and which followed classical X-linked explain the phenotypic features. Turners suggested inheritance, that is only males were affected and that the features can be accounted for in terms of females were normal but carried the condition2. All generalized overgrowth - increased birth weight, these early thoughts and assumptions are now known large ears, head and testes, prominent jaws etc. Optiz 4 to be either partially or totally incorrect. Solving the problems associated with the cytogenetics, genetics Table 1. Feature~ of males uqth .fragile X-linked mental and clinical role of the fragile X has become a major retardation challenge to those working in medical genetics and molecular biology. Birthweight Normal but usuallygreater than sibs, mean at about 70th percentile. The c l i n i c a l p i c t u r e Height Mostly between 50th and 97th In considering the clinical status of fragile X indipercentiles in infancyand childhood; viduals a distinction between affected and unaffected mostly below the 50th percentile in individuals and the sexes needs to be made. Unadulthood. affected individuals have no abnormal clinical Head circumference Slightly increased in childhood, features. usuallyabove the 50th percentile in adulthood. Affected males Forehead Prominent, especiallyin older These males were originally thought to be phenochildren and adults. typically normal until it was noted that many of them J a w s Prominent, especiallyin adults. had large testes (macroorchidism). Since this clinical Ears Prominent and mildlydysmorphic. feature is not consistent (about 80% of affected mares Long ears. have it~) and occurs in at least two other forms of Genitalia Macroorchidism usuallyseen in X-linked mental retardation, there was controversy adults, occasionallyin children. about its association with fragile X for some time. Penis usually normal length. Scrotal Once clinical attention was diverted from this fascinskin sometimes thickened. Possibly ating sign, a range of other clinical features were increased incidenceof hypospadias, noted (see Table 1)1'2. It should be noted that not all cryptorchidism, and other affected males have all or even many of these signs. genitourinaryproblems. Some males have a remarkably normal phenotype Connectivetissue Hyperextensibilityof joints, while others have a very florid expression of it (Fig. 1). particularly fingers. Fine velvety The degree of mental handicap ranges from very skin with striae. High arched or cleft palate. Mitral valve proplapse and slight to profound. Most ma/es are moderately to dilatationof ascending aorta. severely handicapped. Behaviour problems, Torticollisand kyphoscoliosis.Flat especially before adolescence, are also a feature. feet. Inguinalhernia. Many males have a marked attention-deficitdisorder; Epilepsy. Hyper-reflexiaof lower others are labelled as autistic or psychotic4. Indeed the Other features limbs. Gynaecomastia. behaviour problems often cause parents more diffiBehaviour Stereotyped with odd mannerisms, culty in coping than the mental handicap. autistic, hyperactive,excessive shyness, mild self mutilation(biting). A f f ected f ema les The intellectual impairment in females is much less Speech Litany speech, perseveration, than in males. About 30% of female carriers are echolalia, better language form than content. affected and are mostly borderline or mildly retarded, although severely retarded fragile X females have Cognitivedefects Better verbal than spatial abilities, been reported re. Most of the mildly affected females deficit in digit span, deficit in verbal abstractions, no specific verbal have been reported to be phenotypically normal but versus performance difference, show many of the features of the males, these features possible specific left-hemisphere becoming more evident as the severity of the retardadefect. tion becomes more marked z.
chromosome
© 1985,EBevierScience ~ubli.,~he~B V, A~n~erdam 0168-9525t851502.00
T I G - April 1985
revieww
Fig. 1. Left:fragile X male aged 15 years with few facial features of the syndrome; Centre"40-yearold male showing particularly florid facial features of the syndrome, his ears have been pinned and cosmetically improved by surgery; Top right.' 70-year-old male with few features of the ~ndrome,' Bottom right,"marked macroorchidism in another fragile X male, te.sticularvolume is comparedwith a 25 rnl orchidometer bead. Reproduced from Sutherland and Hecht2 by permission of Ordord University Press.
suggested a generalized connective tissue dysplasia as the underlying defect. It remains to be shown that affected fragile X individuals have an abnormal or absent gene product. Folate metabolism has been extensively studied and no abnormality found 4. Endocrine studies have revealed no specific abnormality. Treatment Lejeune 6 claimed that megadoses of folic acid and other folates greatly help behavioural problems in this condition. Results from other studies have been equivocal* '7 and as there is no rational basis for this treatment it cannot be advocated2. Lejeunes stated that trimethoprim, an antifolate antibiotic, exacerbates the condition; it may be best avoided if suitable alternative antibiotics are available until this matter has been resolved.
Cytogenetics Examples of fragile X are shown in Fig. 2. Details of the methods for tissue culture and cytogenetics to detect fragile X have recently been reviewed z.3,s.The fragile X (along with the other fragile sites) is not seen in all metaphases examined and the reasons for this are unknown. Presumably each cell has the DNA sequence required for expression, yet this only occurs in a proportion of cells which can range from less than 1% up to about 80°70,. In males, the proportion expressing in any individual remains fairly constant over time and appears to be a familial characteristic9. Most retarded males express fragile X in 10-30% of their cellsa. The very few normal carrier (nonpenetrant) fragile X males studied cytogenetically do not express the fragile site.
The situation for females is differenP. Most retarded females express the fragile X. The majority of adult female carriers of normal intelligence do not express the fragile X, and those who do usually express it in <5% of their cells. In the author's experience, young (<20 years) fragile X females express the fragile site, regardless of intelligence. This experience is not, however, universal 1and there is a need for longitudinal studies on young fragile X females of normal intelligence to determine whether the frequency of expression of the fragile X decreases with time. The fragile site can be expressed on both active and inactive X chromosomes*. There is some evidence that there is a higher proportion of active fragile-X-expressing chromosomes in retarded fragile X females than in similar, intellectually normal females and that the mental impairment of female carriers is a result of differential X inactivation. However, this matter is by no means settled. The proportion of affected female carriers is much higher than for other X-linked recessive disorders where differential X inactivation is considered to be responsible for this phenomenon. Another cytogenetic problem with fragile X is its detection in cell types other than phytohemagglutinin (PHA)-stimulated lymphocytes in short-term tissue culture. Fragile sites are not expressed in EpsteinBarr virus-transformed lymphoblastoid cell lines grown under conditions of folate depletion, but can be induced in them by 5-fluorodeoxyuridine (FUdR) s. Skin fibroblast and amniotic fluid cell strains also present problems of fragile site detection. Several methods inducing fragile sites in these cell types have been published; these mainly involve induction with methotrexate or FUdR. Most of these methods work
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TIG -- April 1985
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Fig. 2. Thefragile X chromosomeas seen in metaphasechromosomepreparationsfrom lymphocytecultures (exceptfor g): (a-OExpression of thefragile site on chromosomesat different stages of compaction; (d) despirilization of the chromosome material distal to the fragile site; (ed)double-sateUitedappearanceequivalent to a tn'radialformation indicative of truefragility at thefragile site,"(g) expression of thefragile site in skin fibroblast metaphase; (h-j) appearanceof thefragile X on G-banding; (k) early replicationof normal X (small arrow)in a single metaphase;and iT)late replicationoffragile X (broadarrow)by BrdU-labelling~qth late replicationof normal X (small arrow)from a single metaphase. Reproduced from Sutherland"1by permissionof Academic Press. on some patients some of the time but none are completely satisfactory for diagnostic use, making prenatal diagnosis of the fragile X uncertain2. Do different cell types have differences in nucleotide metabolism or are differences in cell replication kinetics responsible for the greater difficulties in inducing fragile sites in cell types other than PHAstimulated lymphocytes? The fragile X chromosome has been introduced into rodent/human hybrid cells and can be induced to express itself in these, even in the absence of all the human autosomes ~°. Standard cytogenetic methods have contributed much to knowledge of fragile sites but have limitations in the detection of some fragile X males, many fragile X females and intellectually normal carriers of both sexes. Attempts to overcome this now involve the use of restriction fragment length polymorphisms (RFLPs) and genetic linkage.
Genetics The fragile X was originally thought to be a cytogenetic marker of a recessive gene for mental retardation. The nature of the association between the fragile site and the mental retardation was unknown (and remains so, especially since the fragile X 'syndrome' can exist without the fragile X) but the gene was considered to follow classical X-linked inheritance. It soon became apparent that there were too many affected females in fragile X families for the gene to be
considered truly recessive. The gradual realization that fragile X could be transmitted by males who were both intellectually and cytogenetically normal finally demonstrated that fragile X is unique among genetic phenomena in man L'L~2. Systematic studies of the genetics of fragile X H.12 have revealed the following: 1. The segregation ratio (proportion of affected males) is 0.4, not the expected 0.5 for an X-linked recessive gene. 2. No males are the result of new mutation occurring in the egg of their mother; that is, the mothers of all fragile X males are fragile X carriers. 3. The segregation ratio for sons of affected fragile X females is 0.5. 4. Affected females receive the fragile X from their mothers, not their fathers. 5. The fragile X daughters of normal carrier males are unaffected. 6. The fragile X mothers of normal transmitting males are normal themselves and have a low risk of having affected fragile X children. These findings suggest that some form of activation or 'switching on' of the mutation leading to mental retardation with the fragile X is required and that this probably occurs in the egg. It is not known whether, once activated, this mutation can be switched off. The solution to these problems awaits detailed linkage studies with DNA probes closely linked to the
review
TIG--Apri11985
fragile X and the characterization of the DNA which results in a fragile site.
25
Molecular biology
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The fragile X chromosome was first described by Lubs 1:~who documented it in a family with X-linked mental retardation. Confirmationthat this findingwas more than isolated curiosity took a long time. This was probably because, at that time, many cytogenetics laboratories where changing their methodologies to use newly developed culture media. These yielded higher quality chromosome preparation than TC199, which was almost universally used for blood lymphocyte culture in the earlier stages of human cytogenetics. The fragile X (along with other rare fragile sites on human chromosomes) was originally found to be only expressed when lymphocytes were cultured in TC199 and not in several other commercially available culture media3. Hence this general change to improved technology was probably largely responsible for inhibiting progress in the study of the fragile X. Subsequently, it has been shown that the fragile X, along with the other folate-sensitive fragile sites2, is seen when lymphocytes are cultured in media free of folic acid and thymidine and can be induced by inhibitors of folate metabolism such as methotrexate and inhibitors of thymidylate synthetase such as FUdR or FCdR 2'8. This led to a general view that the essential requirement for expression of the fragile X was a deficiency of thymidylate during DNA synthesis. Recently, it has been shown that an excess of thymidine, but not its analog, bromodeoxyuridine (BrdU), will induce the folate-sensitive fragile sites and that the critical factor involved in their expression is the dCTP/dTTP ratio during DNA synthesisf4. The fragile sites are only expressed when this ratio is very high or very low. It was hypothesized that the fragile site could result from the amplification of a DNA sequence which was an alternating repeating polypurineJpolypyrimidine structure such as (AGAGAG)nv (TCTCTC)n2 (Ref. 14). Such a structure would have single-strand gaps when replicating if either dTTP or dCTP was limiting but not in the presence of BrdU which can pair with A or G. Such under-replicated DNA would be difficult to adequately package for mitosis. A fragile site is a region of DNA which is not properly packaged for mitosis. Apart from this hypothesis, which is currently untested, there is no knowledge of what a fragile site is at the DNA level or of how the tissue culture factors which lead to fragile site expression relate to the cytogenetic observation of a gap or break in the chromosome structure.
Linkage relationships A map of the distal end of the X chromosome is shown in Fig. 3. The fragile site is linked to the DNA probes F9, DX13, 52A and 114IS. Whilst there is ample opportunity for recombination between the fragile X and these probes, and not every family will be informative for linkage, the application of all these probes to any one family should help in many cases. Combined with cytogenetic results, this should allow carrier detection and possibly prenatal diagnosis. Probes which are more closely linked to the fragile X than
TIG-5
(Lesch-Nyllan syndrome)
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9 (Haemophlha B) site (fragile :xl linked mental retarda|lon) ~14, Factor 8 (Haemophlha A) Adrenoleukodyslrophy Ihs keloids, crypforchtdism, renal dysDlasta syndrome
Fig. 3. Provisional map of the distal end of the X chromosome. those currently available are urgently needed for these diagnostic purposes.
Population studies It has been estimated that the incidence of affected fragile X males and females is about the same, and is of the order of one in 2500 live births n. The only reported study of randomly selected neonates for the fragile X failed to detect it in the 3458 subjects studied f~. Studies of retarded populations gave different results, depending upon the type of population1~'~. Fragile X individuals usually live in the community. The incidence of this condition amongst institutionalized retarded males is 1-2%. Among children at special schools for the retarded, the incidence is probably 3-5%. Perspective The fragile X is one of a group of 15 fragile sites known as the folate-sensitive fragile sites2'~7,and the folate-sensitive fragile sites are only one group of fragile sites on human chromosomes 17. Much that has been learned about the fragile X comes from a study of the folate-sensitive fragile sites. Some of the more extravagant statements made about fragile X have neglected to see it as possibly a special case (and the only special aspect of it may be that it is on the X chromosome) of a more general phenomenon.
Acknowledgement Studies in the author's laboratory have been supported by the National Health and Medical Research Council of Australia and the Adelaide Children's Hospital Research Trust.
References Mainly review articles and recently published articles which are not covered in the reviews are cited. Opitz and Sutherland4 contains a bibliographyof all articles pertaining to the fragile X entered in MEDLINE up to midOctober 1983. 1 Turner, G. and Jacohs, P. A. (1984) Marker (X) linked mental retardation. Adv. Hgm. C,,enet. 13, 83-112 2 Sutherland, G. R. and Hecht, F. (1985) Fragile Sites on Human Chromosomes, Oxford University Press, New York 3 Sutherland, G. R. (1983) The fragile X chromosome. Int. Rev. CytoL 81,107-143
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4 0 p i t z , J. and Sutherland, G. R. (1984) Conference Report. International Workshop on the Fragile X and X-linked mental retardation. Am. f Med. Genet. 17, 5-94 5 Turner,G. (1982) FragileX-linkedmentalretardation. In Human Genetics (Bonne-Tamir, B., ed.), pp. 311-314, Alan Liss 6 Lejeune, J., Legrand, N., Lafourcade, J., Rethor6, M. O., Raoul, O. and Maunoury, C. (1982) Fragilit~du chromosome X et effets de la trim6thoprime. Ann. G~,u~t. 25, 149-151 7 Hagermann, R. J. and McBogg, P. M. (1983) The Fra~le X S)~Irome, Spectra Publishing Co. Inc., Dillon, Colorado 8 Glover, T. W. (1983) The fragile X chromosome: factors influencing its expression in vqtro. In C)'togenetics of the Mammalian X Chromosome, Part B. (Sandberg, A. A., ed.), pp. 415-430, Alan Liss 9 Soudek, D., Partington, M. W. and Lawson, J. S. (1984) The fragile X syndrome. I. Familialvariation in the proportion of lymphocytes with the fragile sites in males. Am. J. Med. Genet. 17, 241-252 10 Warren, S. T. and Davidson, R. L. (1984) Expression of fragile X chromosomein human-rodent somatic cell hybrids. Somat. Cell Molec. Genet. 10, 409-413 11 Sherman, S. L., Morton, N. E., Jacobs, P. A. and Turner, G. (1984) The marker (X) syndrome: a cytogenetic and genetic analysis. Ann. Hum. Genet. 48,
April 1985
21-37 12 Sherman, S. L., Jacobs, P. A., Morton, N. E., FrosterIskenius, U., Howard-Peebles, P. N., Nielsen, K. B., Partington, M. W., Sutherland, G. R., Turner, G. and Watson, M. Further segregation analysis of the fragile X syndrome with special reference to transmitting males. Hum. Genet., in press 13 Lubs, H. A. (1969) A marker X chromosome. Am. f Hum. Genet. 21,231--244 14 Sutherland, G. R., Baker, E. and Fratini, A. Excess thymidine induces folate sensitive fragile sites. Science, submitted 15 Drayna, D., Davies, K., Hartley, D., Mandel, J.-L., Camerino, G., Williamson, R. and White, R. (1984) Genetic mapping of the human X chromosome by using restriction fragment length polymorphisms. Proc. Natl Acad. Sci. USA 81, 2836-2839 16 Sutherland, G. R. (1985) Heritable fragile sites on human chromosomes. XII. Population cytogenetics. Ann. Hum. Genet. 49, 153-160 17 Sutherland, G. R., Parslow, M. I. and Baker, E. New classes of fragile sites induced by 5-azacytidine and BrdU. Hum. Genet., in press G. R. Sutherland is at the Department of H istopathology, Cytogenetics Unit, The Adelaide Children "s Hospital Inc., North Adelaide, South Australia 5006.
The regulation of the bithorax complex
One of the key features of the anatomy of insects, and indeed of many animals, is segmental organization. The body is built up of a series of homologous units, each of which exhibits its own The importance of the differential expression of the bithorax complex (BX-O in directing the diversification of segments in Drosophila is now well established. What unique specializations. remains to be discovered is how this differential expression is achieved. Identification of How these segments other genes essential for the expression of the complex is one route to a detailed arise and diversify is of understanding of its regulation. great interest to developmental biologists. A combined genetic and developmental analysis of the fruit- primordia. The best candidates for selector genes in fly, Drosophila, has contributed significantly to our Drosophila occur in two clusters on the third chromounderstanding of this problem. Cell-lineage studies some. Genetic analysis has shown that genes of one have revealed that segments are composed of two cluster, the Antennapedia complex (ANT-C) 5, are lineage units, termed compartments 1. The lineage required primarily for the development of the anterior restrictions which establish compartments and hence segments of the fly, whilst those of the other cluster, segments, occur very early in development, at or the bithorax complex (BX-C)s, control the around the cellular blastoderm stage, when the development of the posterior segments. Recent analyses of these two gene complexes at the embryo consists simply of a hollow sphere formed by a monolayer of undifferentiated cells. From this point molecular level have provided compelling support for onwards, the cells of each segment follow their own the selector gene hypothesis. First, using the technique of in situ hybridization it has been found genetically programmed developmental pathway2. Two sets of genes are involved in the establishment that transcripts deriving from the ANT-C7and BX-C8 and diversification of segments. The segmentation (also M. Akam and A. Martinez-Afias, personal genes3 control the subdivision of the embryo into the communication) do indeed accumulate in a segmentcorrect number of segmental units, but without specific manner from the blastoderm stage onwards. Second, structural analyses of the genes of the two specifying the developmental pathway which each will follow. This decision depends upon the activity of complexes have revealed a region of highly conserved the members of the second set termed selector genes4. sequence homology, termed the homoeoboxa, which Garcia-Bellido 4 proposed that selector genes, by is shared by genes of both the ANT-C and BX-C. This controlling the expression of different sets of finding considerably strengthens the notion that the 'realisator genes', are essentially responsible for genes of the two complexes have similar functions: selecting between different pathways of develop- more specifically, though the precise role of the ment. Accordingly, the diversification of segments homoeobox is at present unclear, the likelihood that it would be accomplished by arranging for different encodes a DNA-binding domain '° is directly in line selector genes to be active in different segmental with the proposal that selector genes regulate the
Philip Ingham
":" 1985. Elsevier .'-;clencePublishers B,V. A m s t e ~ a m O168 952.5/85/$0"2 00
TIG -- Atrd1198,5
The enigma of the fragile X
The fragile X chromosome is now known to be associated with the commonest inherited form of mental handicap in man. This is rather remarkable Grant R. Sutherland for a condition which was first reported in 1969 and At the beginning of this decade thefragile X chromosome was recogni~d as being assonot recognized as affectciated with the commonest inhen"tedform of mental handicap in man. De~'te intensive ing more than a single study, fragile X remains a mystery; cytogeneticstudy of it is difficult, the exactnature of family until the midits ass~'iation with mental retardation L~unknou,n, its genetics are bizarre and its mole1970sk cular nature speculatit~. Many people working in the area originally thought that the fragile X was associated with a form Unifying concepts of mental retardation without any distinguishing Two unifying concepts have been proposed to clinical features and which followed classical X-linked explain the phenotypic features. Turners suggested inheritance, that is only males were affected and that the features can be accounted for in terms of females were normal but carried the condition2. All generalized overgrowth - increased birth weight, these early thoughts and assumptions are now known large ears, head and testes, prominent jaws etc. Optiz 4 to be either partially or totally incorrect. Solving the problems associated with the cytogenetics, genetics Table 1. Feature~ of males uqth .fragile X-linked mental and clinical role of the fragile X has become a major retardation challenge to those working in medical genetics and molecular biology. Birthweight Normal but usuallygreater than sibs, mean at about 70th percentile. The c l i n i c a l p i c t u r e Height Mostly between 50th and 97th In considering the clinical status of fragile X indipercentiles in infancyand childhood; viduals a distinction between affected and unaffected mostly below the 50th percentile in individuals and the sexes needs to be made. Unadulthood. affected individuals have no abnormal clinical Head circumference Slightly increased in childhood, features. usuallyabove the 50th percentile in adulthood. Affected males Forehead Prominent, especiallyin older These males were originally thought to be phenochildren and adults. typically normal until it was noted that many of them J a w s Prominent, especiallyin adults. had large testes (macroorchidism). Since this clinical Ears Prominent and mildlydysmorphic. feature is not consistent (about 80% of affected mares Long ears. have it~) and occurs in at least two other forms of Genitalia Macroorchidism usuallyseen in X-linked mental retardation, there was controversy adults, occasionallyin children. about its association with fragile X for some time. Penis usually normal length. Scrotal Once clinical attention was diverted from this fascinskin sometimes thickened. Possibly ating sign, a range of other clinical features were increased incidenceof hypospadias, noted (see Table 1)1'2. It should be noted that not all cryptorchidism, and other affected males have all or even many of these signs. genitourinaryproblems. Some males have a remarkably normal phenotype Connectivetissue Hyperextensibilityof joints, while others have a very florid expression of it (Fig. 1). particularly fingers. Fine velvety The degree of mental handicap ranges from very skin with striae. High arched or cleft palate. Mitral valve proplapse and slight to profound. Most ma/es are moderately to dilatationof ascending aorta. severely handicapped. Behaviour problems, Torticollisand kyphoscoliosis.Flat especially before adolescence, are also a feature. feet. Inguinalhernia. Many males have a marked attention-deficitdisorder; Epilepsy. Hyper-reflexiaof lower others are labelled as autistic or psychotic4. Indeed the Other features limbs. Gynaecomastia. behaviour problems often cause parents more diffiBehaviour Stereotyped with odd mannerisms, culty in coping than the mental handicap. autistic, hyperactive,excessive shyness, mild self mutilation(biting). A f f ected f ema les The intellectual impairment in females is much less Speech Litany speech, perseveration, than in males. About 30% of female carriers are echolalia, better language form than content. affected and are mostly borderline or mildly retarded, although severely retarded fragile X females have Cognitivedefects Better verbal than spatial abilities, been reported re. Most of the mildly affected females deficit in digit span, deficit in verbal abstractions, no specific verbal have been reported to be phenotypically normal but versus performance difference, show many of the features of the males, these features possible specific left-hemisphere becoming more evident as the severity of the retardadefect. tion becomes more marked z.
chromosome
© 1985,EBevierScience ~ubli.,~he~B V, A~n~erdam 0168-9525t851502.00
T I G - April 1985
revieww
Fig. 1. Left:fragile X male aged 15 years with few facial features of the syndrome; Centre"40-yearold male showing particularly florid facial features of the syndrome, his ears have been pinned and cosmetically improved by surgery; Top right.' 70-year-old male with few features of the ~ndrome,' Bottom right,"marked macroorchidism in another fragile X male, te.sticularvolume is comparedwith a 25 rnl orchidometer bead. Reproduced from Sutherland and Hecht2 by permission of Ordord University Press.
suggested a generalized connective tissue dysplasia as the underlying defect. It remains to be shown that affected fragile X individuals have an abnormal or absent gene product. Folate metabolism has been extensively studied and no abnormality found 4. Endocrine studies have revealed no specific abnormality. Treatment Lejeune 6 claimed that megadoses of folic acid and other folates greatly help behavioural problems in this condition. Results from other studies have been equivocal* '7 and as there is no rational basis for this treatment it cannot be advocated2. Lejeunes stated that trimethoprim, an antifolate antibiotic, exacerbates the condition; it may be best avoided if suitable alternative antibiotics are available until this matter has been resolved.
Cytogenetics Examples of fragile X are shown in Fig. 2. Details of the methods for tissue culture and cytogenetics to detect fragile X have recently been reviewed z.3,s.The fragile X (along with the other fragile sites) is not seen in all metaphases examined and the reasons for this are unknown. Presumably each cell has the DNA sequence required for expression, yet this only occurs in a proportion of cells which can range from less than 1% up to about 80°70,. In males, the proportion expressing in any individual remains fairly constant over time and appears to be a familial characteristic9. Most retarded males express fragile X in 10-30% of their cellsa. The very few normal carrier (nonpenetrant) fragile X males studied cytogenetically do not express the fragile site.
The situation for females is differenP. Most retarded females express the fragile X. The majority of adult female carriers of normal intelligence do not express the fragile X, and those who do usually express it in <5% of their cells. In the author's experience, young (<20 years) fragile X females express the fragile site, regardless of intelligence. This experience is not, however, universal 1and there is a need for longitudinal studies on young fragile X females of normal intelligence to determine whether the frequency of expression of the fragile X decreases with time. The fragile site can be expressed on both active and inactive X chromosomes*. There is some evidence that there is a higher proportion of active fragile-X-expressing chromosomes in retarded fragile X females than in similar, intellectually normal females and that the mental impairment of female carriers is a result of differential X inactivation. However, this matter is by no means settled. The proportion of affected female carriers is much higher than for other X-linked recessive disorders where differential X inactivation is considered to be responsible for this phenomenon. Another cytogenetic problem with fragile X is its detection in cell types other than phytohemagglutinin (PHA)-stimulated lymphocytes in short-term tissue culture. Fragile sites are not expressed in EpsteinBarr virus-transformed lymphoblastoid cell lines grown under conditions of folate depletion, but can be induced in them by 5-fluorodeoxyuridine (FUdR) s. Skin fibroblast and amniotic fluid cell strains also present problems of fragile site detection. Several methods inducing fragile sites in these cell types have been published; these mainly involve induction with methotrexate or FUdR. Most of these methods work
iews
o
TIG -- April 1985
"
|
fo'
.
d
q Pg...
l
k"
/
nh
I
Fig. 2. Thefragile X chromosomeas seen in metaphasechromosomepreparationsfrom lymphocytecultures (exceptfor g): (a-OExpression of thefragile site on chromosomesat different stages of compaction; (d) despirilization of the chromosome material distal to the fragile site; (ed)double-sateUitedappearanceequivalent to a tn'radialformation indicative of truefragility at thefragile site,"(g) expression of thefragile site in skin fibroblast metaphase; (h-j) appearanceof thefragile X on G-banding; (k) early replicationof normal X (small arrow)in a single metaphase;and iT)late replicationoffragile X (broadarrow)by BrdU-labelling~qth late replicationof normal X (small arrow)from a single metaphase. Reproduced from Sutherland"1by permissionof Academic Press. on some patients some of the time but none are completely satisfactory for diagnostic use, making prenatal diagnosis of the fragile X uncertain2. Do different cell types have differences in nucleotide metabolism or are differences in cell replication kinetics responsible for the greater difficulties in inducing fragile sites in cell types other than PHAstimulated lymphocytes? The fragile X chromosome has been introduced into rodent/human hybrid cells and can be induced to express itself in these, even in the absence of all the human autosomes ~°. Standard cytogenetic methods have contributed much to knowledge of fragile sites but have limitations in the detection of some fragile X males, many fragile X females and intellectually normal carriers of both sexes. Attempts to overcome this now involve the use of restriction fragment length polymorphisms (RFLPs) and genetic linkage.
Genetics The fragile X was originally thought to be a cytogenetic marker of a recessive gene for mental retardation. The nature of the association between the fragile site and the mental retardation was unknown (and remains so, especially since the fragile X 'syndrome' can exist without the fragile X) but the gene was considered to follow classical X-linked inheritance. It soon became apparent that there were too many affected females in fragile X families for the gene to be
considered truly recessive. The gradual realization that fragile X could be transmitted by males who were both intellectually and cytogenetically normal finally demonstrated that fragile X is unique among genetic phenomena in man L'L~2. Systematic studies of the genetics of fragile X H.12 have revealed the following: 1. The segregation ratio (proportion of affected males) is 0.4, not the expected 0.5 for an X-linked recessive gene. 2. No males are the result of new mutation occurring in the egg of their mother; that is, the mothers of all fragile X males are fragile X carriers. 3. The segregation ratio for sons of affected fragile X females is 0.5. 4. Affected females receive the fragile X from their mothers, not their fathers. 5. The fragile X daughters of normal carrier males are unaffected. 6. The fragile X mothers of normal transmitting males are normal themselves and have a low risk of having affected fragile X children. These findings suggest that some form of activation or 'switching on' of the mutation leading to mental retardation with the fragile X is required and that this probably occurs in the egg. It is not known whether, once activated, this mutation can be switched off. The solution to these problems awaits detailed linkage studies with DNA probes closely linked to the
review
TIG--Apri11985
fragile X and the characterization of the DNA which results in a fragile site.
25
Molecular biology
26
The fragile X chromosome was first described by Lubs 1:~who documented it in a family with X-linked mental retardation. Confirmationthat this findingwas more than isolated curiosity took a long time. This was probably because, at that time, many cytogenetics laboratories where changing their methodologies to use newly developed culture media. These yielded higher quality chromosome preparation than TC199, which was almost universally used for blood lymphocyte culture in the earlier stages of human cytogenetics. The fragile X (along with other rare fragile sites on human chromosomes) was originally found to be only expressed when lymphocytes were cultured in TC199 and not in several other commercially available culture media3. Hence this general change to improved technology was probably largely responsible for inhibiting progress in the study of the fragile X. Subsequently, it has been shown that the fragile X, along with the other folate-sensitive fragile sites2, is seen when lymphocytes are cultured in media free of folic acid and thymidine and can be induced by inhibitors of folate metabolism such as methotrexate and inhibitors of thymidylate synthetase such as FUdR or FCdR 2'8. This led to a general view that the essential requirement for expression of the fragile X was a deficiency of thymidylate during DNA synthesis. Recently, it has been shown that an excess of thymidine, but not its analog, bromodeoxyuridine (BrdU), will induce the folate-sensitive fragile sites and that the critical factor involved in their expression is the dCTP/dTTP ratio during DNA synthesisf4. The fragile sites are only expressed when this ratio is very high or very low. It was hypothesized that the fragile site could result from the amplification of a DNA sequence which was an alternating repeating polypurineJpolypyrimidine structure such as (AGAGAG)nv (TCTCTC)n2 (Ref. 14). Such a structure would have single-strand gaps when replicating if either dTTP or dCTP was limiting but not in the presence of BrdU which can pair with A or G. Such under-replicated DNA would be difficult to adequately package for mitosis. A fragile site is a region of DNA which is not properly packaged for mitosis. Apart from this hypothesis, which is currently untested, there is no knowledge of what a fragile site is at the DNA level or of how the tissue culture factors which lead to fragile site expression relate to the cytogenetic observation of a gap or break in the chromosome structure.
Linkage relationships A map of the distal end of the X chromosome is shown in Fig. 3. The fragile site is linked to the DNA probes F9, DX13, 52A and 114IS. Whilst there is ample opportunity for recombination between the fragile X and these probes, and not every family will be informative for linkage, the application of all these probes to any one family should help in many cases. Combined with cytogenetic results, this should allow carrier detection and possibly prenatal diagnosis. Probes which are more closely linked to the fragile X than
TIG-5
(Lesch-Nyllan syndrome)
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9 (Haemophlha B) site (fragile :xl linked mental retarda|lon) ~14, Factor 8 (Haemophlha A) Adrenoleukodyslrophy Ihs keloids, crypforchtdism, renal dysDlasta syndrome
Fig. 3. Provisional map of the distal end of the X chromosome. those currently available are urgently needed for these diagnostic purposes.
Population studies It has been estimated that the incidence of affected fragile X males and females is about the same, and is of the order of one in 2500 live births n. The only reported study of randomly selected neonates for the fragile X failed to detect it in the 3458 subjects studied f~. Studies of retarded populations gave different results, depending upon the type of population1~'~. Fragile X individuals usually live in the community. The incidence of this condition amongst institutionalized retarded males is 1-2%. Among children at special schools for the retarded, the incidence is probably 3-5%. Perspective The fragile X is one of a group of 15 fragile sites known as the folate-sensitive fragile sites2'~7,and the folate-sensitive fragile sites are only one group of fragile sites on human chromosomes 17. Much that has been learned about the fragile X comes from a study of the folate-sensitive fragile sites. Some of the more extravagant statements made about fragile X have neglected to see it as possibly a special case (and the only special aspect of it may be that it is on the X chromosome) of a more general phenomenon.
Acknowledgement Studies in the author's laboratory have been supported by the National Health and Medical Research Council of Australia and the Adelaide Children's Hospital Research Trust.
References Mainly review articles and recently published articles which are not covered in the reviews are cited. Opitz and Sutherland4 contains a bibliographyof all articles pertaining to the fragile X entered in MEDLINE up to midOctober 1983. 1 Turner, G. and Jacohs, P. A. (1984) Marker (X) linked mental retardation. Adv. Hgm. C,,enet. 13, 83-112 2 Sutherland, G. R. and Hecht, F. (1985) Fragile Sites on Human Chromosomes, Oxford University Press, New York 3 Sutherland, G. R. (1983) The fragile X chromosome. Int. Rev. CytoL 81,107-143
r iews
TIG-
4 0 p i t z , J. and Sutherland, G. R. (1984) Conference Report. International Workshop on the Fragile X and X-linked mental retardation. Am. f Med. Genet. 17, 5-94 5 Turner,G. (1982) FragileX-linkedmentalretardation. In Human Genetics (Bonne-Tamir, B., ed.), pp. 311-314, Alan Liss 6 Lejeune, J., Legrand, N., Lafourcade, J., Rethor6, M. O., Raoul, O. and Maunoury, C. (1982) Fragilit~du chromosome X et effets de la trim6thoprime. Ann. G~,u~t. 25, 149-151 7 Hagermann, R. J. and McBogg, P. M. (1983) The Fra~le X S)~Irome, Spectra Publishing Co. Inc., Dillon, Colorado 8 Glover, T. W. (1983) The fragile X chromosome: factors influencing its expression in vqtro. In C)'togenetics of the Mammalian X Chromosome, Part B. (Sandberg, A. A., ed.), pp. 415-430, Alan Liss 9 Soudek, D., Partington, M. W. and Lawson, J. S. (1984) The fragile X syndrome. I. Familialvariation in the proportion of lymphocytes with the fragile sites in males. Am. J. Med. Genet. 17, 241-252 10 Warren, S. T. and Davidson, R. L. (1984) Expression of fragile X chromosomein human-rodent somatic cell hybrids. Somat. Cell Molec. Genet. 10, 409-413 11 Sherman, S. L., Morton, N. E., Jacobs, P. A. and Turner, G. (1984) The marker (X) syndrome: a cytogenetic and genetic analysis. Ann. Hum. Genet. 48,
April 1985
21-37 12 Sherman, S. L., Jacobs, P. A., Morton, N. E., FrosterIskenius, U., Howard-Peebles, P. N., Nielsen, K. B., Partington, M. W., Sutherland, G. R., Turner, G. and Watson, M. Further segregation analysis of the fragile X syndrome with special reference to transmitting males. Hum. Genet., in press 13 Lubs, H. A. (1969) A marker X chromosome. Am. f Hum. Genet. 21,231--244 14 Sutherland, G. R., Baker, E. and Fratini, A. Excess thymidine induces folate sensitive fragile sites. Science, submitted 15 Drayna, D., Davies, K., Hartley, D., Mandel, J.-L., Camerino, G., Williamson, R. and White, R. (1984) Genetic mapping of the human X chromosome by using restriction fragment length polymorphisms. Proc. Natl Acad. Sci. USA 81, 2836-2839 16 Sutherland, G. R. (1985) Heritable fragile sites on human chromosomes. XII. Population cytogenetics. Ann. Hum. Genet. 49, 153-160 17 Sutherland, G. R., Parslow, M. I. and Baker, E. New classes of fragile sites induced by 5-azacytidine and BrdU. Hum. Genet., in press G. R. Sutherland is at the Department of H istopathology, Cytogenetics Unit, The Adelaide Children "s Hospital Inc., North Adelaide, South Australia 5006.
The regulation of the bithorax complex
One of the key features of the anatomy of insects, and indeed of many animals, is segmental organization. The body is built up of a series of homologous units, each of which exhibits its own The importance of the differential expression of the bithorax complex (BX-O in directing the diversification of segments in Drosophila is now well established. What unique specializations. remains to be discovered is how this differential expression is achieved. Identification of How these segments other genes essential for the expression of the complex is one route to a detailed arise and diversify is of understanding of its regulation. great interest to developmental biologists. A combined genetic and developmental analysis of the fruit- primordia. The best candidates for selector genes in fly, Drosophila, has contributed significantly to our Drosophila occur in two clusters on the third chromounderstanding of this problem. Cell-lineage studies some. Genetic analysis has shown that genes of one have revealed that segments are composed of two cluster, the Antennapedia complex (ANT-C) 5, are lineage units, termed compartments 1. The lineage required primarily for the development of the anterior restrictions which establish compartments and hence segments of the fly, whilst those of the other cluster, segments, occur very early in development, at or the bithorax complex (BX-C)s, control the around the cellular blastoderm stage, when the development of the posterior segments. Recent analyses of these two gene complexes at the embryo consists simply of a hollow sphere formed by a monolayer of undifferentiated cells. From this point molecular level have provided compelling support for onwards, the cells of each segment follow their own the selector gene hypothesis. First, using the technique of in situ hybridization it has been found genetically programmed developmental pathway2. Two sets of genes are involved in the establishment that transcripts deriving from the ANT-C7and BX-C8 and diversification of segments. The segmentation (also M. Akam and A. Martinez-Afias, personal genes3 control the subdivision of the embryo into the communication) do indeed accumulate in a segmentcorrect number of segmental units, but without specific manner from the blastoderm stage onwards. Second, structural analyses of the genes of the two specifying the developmental pathway which each will follow. This decision depends upon the activity of complexes have revealed a region of highly conserved the members of the second set termed selector genes4. sequence homology, termed the homoeoboxa, which Garcia-Bellido 4 proposed that selector genes, by is shared by genes of both the ANT-C and BX-C. This controlling the expression of different sets of finding considerably strengthens the notion that the 'realisator genes', are essentially responsible for genes of the two complexes have similar functions: selecting between different pathways of develop- more specifically, though the precise role of the ment. Accordingly, the diversification of segments homoeobox is at present unclear, the likelihood that it would be accomplished by arranging for different encodes a DNA-binding domain '° is directly in line selector genes to be active in different segmental with the proposal that selector genes regulate the
Philip Ingham
":" 1985. Elsevier .'-;clencePublishers B,V. A m s t e ~ a m O168 952.5/85/$0"2 00