Conference report: Third International Workshop on the fragile X and X-linked mental retardation

American Journal of Medical Genetics 3O:l-29 (1988)

CONFERENCE REPORT: THIRD INTERNATIONAL WORKSHOP ON THE FRAGILE X AND X-LINKED MENTAL RETARDATION

Giovanni Neri, John M. Opitz, Margareta Mikkelsen, Patricia A. Jacobs, Kay Davies, Gillian Turner

Institutional affiliations of the chairpersons are given in Appendix I: List of participants

INTRODUCTION

Between September 13 and 16, 1987 the Third International Workshop on the Fragile X and X-linked Mental Retardation (fra (X) and XLMR) was held under the presidency of Professor Giovanni Neri in the pleasant environment of the Oasi Institute (Oasi Maria SS) for the study and rehabilitation of the mentally retarded in Troina, Sicily. Organizers and participants are grateful to the President and Scientific Director of the Insti- tute respectively, Fr. Luigi Ferlauto and Prof. Ercole Sega for their gracious hospitality and to the Institute of Human Genetics, Catholic University, Rome for support of the conference. Financial support for the organization of the workshop was also provided by Associazione Oasi Maria SS, Troina, Minister0 della Pubblica Istruzione and Consiglio Nazionale delle Ricerche, Rome, and finally by Carl Zeiss S.p.A. Milano.

The 3 day program was divided into 7 sessions: Clinical (Opitz); prenatal diagnosis and treatment (Mikkelsen); cytogenetics (Jacobs); gene mapping in non-specific XLMR (Jacobs); molecular (Davies); genetics and epidemiology (Turner); hypotheses (Neri).

The impact of molecular genetics on the field of XLMR was most evident in the area of gene mapping in which enormous progress was recorded since the second, Dunk Island Conference. The genetic nature of the fragile site per se has been relatively resistant to the methods of molecular biology, hence, continues to stimulate the postulation of elegant hypotheses,

specific

Address reprint requests to John M. Opitz, Shodair Hospital, PO Box 5539, Helena, MT, !ISA 59604.

0 1988 Alan R. Liss, Inc.

2 Neri et al.

although at a less dizzying pace than previously. The approaches of Stephen Warren of Emory were considered most promising in "cracking" the fragile site and to offer an explanation for the puzzling recombinational variability around the fragile site studied exhaustively by Ted Brown and his collaborators. Steve Warren's methods may perhaps allow him to be the first to clone the fragile site in one or more pieces and to investigate the theory of "imprinting" proposed by Charles Laird of the Univer- sity of Washington, Seattle, Department of Zoology. The imprinting theory, or Laird model, has received considerable attention and has generated some unease. The feeling of the workers in the field about this model were perhaps best summarized by Robert Nussbaum at another meeting sometime later at which he was heard to groan (half facetiously) that the worry of everyone was not so much "that Charles Laird was crazy, but that he might be right", imprinting representing in this case perhaps only generational differences in DNA methylation and not a major structural gene alteration.

The consensus of the participants was that the conference was a great success and should be repeated. Thus, the fourth International Workshop on the Fragile X and XLMR will be held immediately after the 10th Human Gene Mapping Conference under the Presidency of W. Ted Brown of the New York State Institute for Basic Research in Developmental Disabilities (Staten Island, New York) in the spring/early summer of 1989.

SESSION I: CLINICAL

Chairperson: John M. Opitz

Martin-Bell (MBS) or Fra(X) Syndrome

Anthropometric studies have yielded a number of interesting insights. In a small number of boys born after 1970 Karen Br#ndum-Nielsen found accelerated linear growth and reduced weight early in life becoming normal by 4 years; all except 2 occipito-frontal head circumference (OFC) measurements were above the mean.

Studies done by Loesch et a1 at La Trobe University in Australia involved 30 anthropometric measures in 56 men and 91 women with MBS and 119 normal control persons. These show, in adult life, a decrease of stature, upper limb length and of upper face height with increase in jaw length, chest circumference and waist width in affected men and women.

Merlin Butler and his coworkers from Vanderbilt have used a photoanthropometric method, which enables an objective

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description of facial structures, to study 31 boys with MBS below age 12 years. Two of 14 craniofacial indices were abnormal in MBS boys: palpebral fissure length (increased), and inter-inner canthal distance (decreased).

It is the dermatoglyphic changes and crown size asymmetry which are most suggestive of a pleiotropic effect acting from early embryogenesis of a type suggestive of gonosomal aneuploidy.

In a collaborative study between workers from Catania and Troina of 14 fra(X) boys compared with a control group of 191 normal school boys, Milone et a1 found a lower frequency of ulnar loops, particularly on 2nd and 3rd fingers, with a corresponding increase in whorls; a transverse course of mainline A; and, an increased frequency of abnormal palmar creases. The log-score index of Simpson et a1 identified 71.4 of the patients studied by Milone et al; that of Rodewald et a1 identified 64.2% - the difference being attributed to ethnic variability. Langenbeck and coworkers from G6ttingen used the Rodewald index method prospectively in an attempt to find fra(X) cases in a group of 160 institutionalized mentally retarded males without Down syndrome. An abnormal (negative) score was recorded in 32 men, 14 of whom were fra(X) positive. This frequency of 9 2 2% indicates that most if not all MBS patients were detected by this method in that population. This study confirms the important insight of dermatoglyphic developmental abnormalities in the MBS including increased frequency of radial loops, whorls and arches on fingertips, a pronounced transverse course of palmar ridges, rudimentary mainline C, a lower & ridge count due to increased ridge width, a lower frequency of true patterns on soles, abnormal palmar creases and conspicuous sandal creases. In this patient population the Rodewald index method had a specificity of 86-88%.

Peretz et a1 (Hebrew University and Hadassah Faculty of Dental Medicine) documented a statistically significantly increased asymmetry in crown-diameters in 13 males with MBS; indeed, in the mandible the asymmetry was as high as in Down syndrome.

At the moment it seems impractical to incorporate extensive physical, dental and dermatoglyphic measurements into the clinical evaluation of MBS males, but nothing has obviated the need for accurate clinical identification of MBS males at birth. The "rogue's gallery" of photos of fra(X) infants assembled retrospectively from birth till childhood by Hockey and Crowhurst (West Perth, Australia) suggests that it is possible to pick out fra(X) children on the basis of a photographic and verbally descriptive phenotype. Prospective and blind studies of this type are urgently needed on larger, consecutively ascertained

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groups of affected infants.

One of the most important genetic associations with the fra(X) syndrome identified to date involves an apparently increased predisposition to nondisjunction involving chromosome 21 [Watson et al, this issue] and the X (Klinefelter syndrome) - [Fryns and Van den Berghe; Filippi et al, this issue].

A potentially serious manifestation of the connective tissue dysplasia in the MBS was identified by Waldstein and Hagerman of Denver in an 18-year-old MBS boy who died suddenly with viral pneumonia and myocarditis and was found to have striking hypo- plasia of the aorta with relative coarctation and pronounced abnormalities of elastin distribution and structure at the base of the mitral and tricuspid valves associated with severe depletion of mucopolysaccharides. Antemortem echocardiography had failed to visualize any cardiovascular abnormalities except for mild prolapse of the mitral valve in systole. Thus, prospective echocardiographic studies of MBS males and appropriate sib and non-sib control persons are indicated to deter- mine if this is a common manifestation in the fra(X) syndrome.

While the cardiovascular abnormalities in this boy were at most a contributing factor in his death, the question of a truly increased incidence of sudden infant death in the MBS remains open. This issue was investiqated by Fryns and coworkers (Leuven) who concluded that sudden infant death is "frequently" observed in the progeny of obligate femala carriers. Six such deaths were observed in 68 offspring of 8 mentally subnormal fra(X)-positive mothers, and 23 deaths in a total of 388 male and female offspring born to 86 obligate carriers. The deaths were attributed to a possible CNS defect and underscore the need to fund a carefully controlled, multi-center prospective study of infant mortality and morbidity in the fra(X) syndrome with maximum effort exerted to obtain autopsies when indicated.

One of the potentially most important findings in the MBS obtained by the multidisciplinary studies of this condition at the Greenwood Genetic Center was documented by Phelan and her coworkers. They reviewed cancer occurrence in 2 of their patients and in 2 previously reported individuals with the MBS: 1.) a 57-year-old inan with left testicular seminoma at 45, and a right testicular seminoma at 50 years; 2.) a 16-year-old boy with a mucin-producing adenocarcinoma of the colon at 14 years; 3.) a sperm granuloma (without spermatogenesis) in a 34-year-old man; and 4.) a malignant ganglioglioma in a 17-year-old black boy. These are all unusual tumors and suggest again need fo r a prospective multi-center study of the risk of cancer/tumor development in hemi- and heterozygotes of the MBS.

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Nosology of XLMR

One of the most dramatic developments in the field of XLMR nosology is the discovery of what may be called the Thode- Leonard syndrome which is a complex XLMR syndrome due to a tandem dup(Xq) involving segment q13.1->21.1. The abnormal X replicated late in all metaphases of heterozgous females. This MCA/MR syndrome is so specific that it was possible for Alison Thode to predict the correct chromosome findings in 2 affected male cousins demonstrated at the 6th David Smith Morphogenesis Workshop by Claire Leonard of Salt Lake City and said to have had normal chromosomes in a previous study. Thode's prediction has since been confirmed on high-resolution prometaphase studies which also showed the dup(X)(ql3.1->q21.1) in Leonard's patients.

One of the loveliest of the many successes of Michael Partington's stay with Gillian Turner last year was the delineation of what may be called the Partington syndrome of XLMR with dystonic hand movements and its mapping to the segment from Xp21->Xpter.

Simultaneous studies in Rome [Neri et all, Graz and Salzburg [Behmel et all and Plichigan have led to a much more detailed understanding of what was previously known as the Golabi-Rosen syndrome but is more appropriately called the Simpson-Golabi- Behmel syndrome. Interestingly enough, this complex MCA/MR syndrome may be associated with (nearly) normal IQ in some affected hemizygotes and on the other hand a disorder so severe as to be lethal in almost 50% of hemizygotes in a clinical picture suggesting a thesaurismosis due to a large molecular weight inborn error of metabolism. As pointed out by Bryan Hall, there may be considerable phenotypic overlap between severe cases of the Simpson-Golabi-Behme1 syndrome and the FG syndrome [q.v. Opitz et al, this issue].

The observations of Lisbeth Tranebjaerg and her coworkers (Copenhagen) suggest that there exists an entity of XLMR and psoriasis which should be screened for in dermatology clinics and in large populations of individuals with non-specific forms of XLMR.

As this was going to press, Chudley et a1 submitted a paper on a new MCA/XLMR syndrome that may be called the Chudley-Lowry- Hoar syndrome (and will be published in a subsequent issue of the Journal). This disorder (with suggestive resemblance to the Prader-Willi syndrome) deserves further mapping studies, since so far the gene could be located anywhere on the X except for distal Xp and distal Xq.

The true extent of heterogeneity in XLMR is probably still unsuspected by most workers in the field and promises a most

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fruitful era of collaboration between clinician and geneticist. Until such heterogeneity is completely resolved and all variability is accounted for, the Helena workers want to continue to make their facilities available for a registry of XLMR conditions to aid patient care, nosology, delineation, publication and if appropriate, collaborative investigation.

This conference was again highly successful in increasing awareness of the complexity of the subject of XLMR and the successes possible in the collaborative, international study of phenotype, cytogenetic and genetic mechanisms of XLMR.

SESSION 11: PRENATAL DIAGNOSIS AND TREATMENT.

Chairperson: Margareta Mikkelsen

Four groups reported on their experience with prenatal fra(X) detection.

Jenkins and colleagues presented three new cases: 1 chorionic villus sample and 2 amniotic fluid samples and summarized previously studied cases. They used 2 syst.ems, FudR induction and high thymidine induction. They found FudR better than high thymidine. They experienced their first false-negative fra(X) diagnosis and stressed the importance of using several systems for prenatal fra(X) detection.

The Australasian experience was presented by Purvis-Smith, who stressed that chorionic villi are preferable over amniotic fluid, that a minimum of 200 cells should be studied and that at least two methods should be used and possibly two different laboratories. A l s o , the follow-up of the cases is considered of greatest importance.

The European experience with fra(X) detection in chorionic villi from 15 fetuses was presented by Niels Tomunerup, who had used 3 methods, excess thymidine, thymidilate depletion, and RFLP analysis carried out in Jean Louis Mandel's laboratory. He also reported the experience of prenatal diagnosis for advanced maternal age where a single cell with the fragile site was detected. When the amniotic fluid cell cultures were analyzed with the routine protocol for fragile X detection 10% fragile X positive cells were found and after abortion the fragile X was also found in fetal tissues.

Shapiro and his group reported on their experiences using amniotic fluid, chorionic viili, fetal blood and molecular probes in 85 cases with documented family history. Tommerup found a case with discrepancy between cytogenetic and flanking

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marker studies - the latter indicating presence of the abnormal X which could be induced in any of the cultures, before an& after termination of pregnancy. Tessa Webb reported on fetal blood chromosome studies without encountering any difficulties in confirming the prenatal diagnosis.

It seems apparent that the most rewarding way to go in prenatal diagnosis of the fra(X) is to start with chorionic villus sampling and induction with at least two cytogenetic methods, possibly combined with RFLP analysis. When problems occur one can then proceed to fetal blood sampling around week 18 to 19.

In the treatment session Fisch et a1 reported on their blind study of fra(X) males treated with high doses of folic acid. There was no significant improvement of behavior. The fra(X) disappeared from the cells. Tessa Webb followed the cytogenetic status of an autistic boy treated with folic acid over 2 years. She showed disappearance and reappearance with even higher percentage. The same results were recorded previously by Br&dum-Nielsen and Tommerup [1984] in a study of fra(X) males.

Hagerman et a1 used a cross-over trial of CNS stimulant med- ication in the treatment of fra(X) boys with some response. Such treatment would probably not be acceptable in a number of countries and the results were not convincingly effective. In conclusion, folic acid treatment, discussed so extensively in previous meetings, cannot any longer be regarded as adequate therapy for fragile X patients.

Postscript: After the conference D r . Jenkins submitted the following report: "Five C.V.S. studies have been carried out; 2 of the women were obligate carriers and both had male fetuses. One was negative for fra(X) with confirmative DNA marker studies while the other was positive (Table I in our paper). It was not possible to obtain tissue for confirmation in this case due to the method of termination.

In the remaining 3 studies all fetuses had fra(X) family histories. Of these, 2 were females and one was male. In one of the females 7 of 150 cells were fra(X) positive. Confirma- tion is pending the birth of the baby girl. In summary 2 of 5 C.V.S. samples at risk for fra(X) were positive, one coming from an obligate carrier".

SESSION 111: CYTOGENETICS

Chairperson: Patricia A. Jacobs

The inactivation pattern of the X chromosome in fra(X)- positive females was discussed in two papers. J.P. Fryns

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reported that in his hands there was a large excess of active fra(X) chromosomes irrespective of the phenotype or mental status of the patients. In contrast, Ursula Froster-Iskenius reported a correlation between X inactivation status and mental ability, an excess of active fra(X) chromosomes being associated with mental retardation. The difficulty of carrying out these investigations in mentally normal heterozygotes with their low level of fra(X) positive cells was emphasized, as was the resulting over-representation of the few atypical, mentally normal high-expressing females in these series.

A number of papers discussed factors affecting the expression of the fra(X). These included a report by David and Susan Ledbetter that very high doses of aphidicolin in lymphocyte or lymphoblast cultures dramatically increased the proportion of cells showing a fra(X) in normal control males but lowered the proportion of cells showing the fra(X) in retarded individuals with the fra(X) syndrome. These observations supported the idea that there may be a common fragile site at Xq27 inducible with high levels of aphidicolin. However, it is not clear whether this is identical to the fragile site associated with the Martin-Bell syndrome.

Niels Tommerup presented data showing that the fra(X), but not the common fragile sites, is associated with replication processes that depend on DNA polymerase alpha.

Tom Glover reported on a somatic cell hybrid system that contained a single human chromosome 3 or a chromosome 3 and a fra(X). When the cells were cultured with aphidicolin or FUdR, a very high proportion of cells showed breaks and/or rearrangements at or near the common fragile site at 3p14, a similar, but less marked effect was found for the fragile site at Xq27. This system provides an excellent means of generating fragile site- specific rearrangements, and when a number of such rearrangements involving the fragile (X) site are available, they will provide junction fragments that may be extremely useful in the molecular cloning of the fra(X) gene.

Charles Laird gave a stimulating paper in which he pointed out a number of similarities between the fragile site associated with X-linked mental retardation and intercalary heterochromatic regions of Drosophila chromosomes.

Tessa Webb presented the results of an extensive cytogenetic study of 3 fra(X)-positive fetuses. She cultured a number of different tissues and cell types including lung, testes, kidney, skin and muscle and found a fragile (X) to be present in all.

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and understand non-specific XLMR and its heterogeneity.

SESSION V: MOLECULAR GENETICS OF THE MARTIN BELL SYNDROME

Chairperson: Kay E. Davies

Several markers bridge the fragile site and have been studied extensively [see Heilig et al; Brown et al; Mulley et al; et a1 in this volume for details and references]. These studies show that the fra(X) site is very closely linked to the Martin Bell syndrome locus in the vast majority of families. Therefore, the fragile site is either directly involved in the mutation responsible for the phenotype or lies in a region adjacent to it on the chromosome. Some XLMR without the fragile site is due to mutations on the X chromosome other than at Xq27 [e.g. Suthers et al; Sutherland et al, this issue]. Markers in the Xq26-qter region have been mapped by both physical methods, using somatic cell hybrids and deleted or rearranged X chromosomes, and linkage analysis in affected families by several laboratories. The order of the markers is not unequivocally established although the main body of data supports the following order:

Patterson

Collective data suggest a recombination fraction of 0.15 ( 2 max = 41.05) for DXS52-FRAXA and 0.20 (Z max = 23.42) for the distance F9-FRAXA [Davies et al., 19871. The latter distance is a total figure and does not take into account the apparent genetic heterogeneity observed between families in the interval F9-FRAXA. This was first reported by Brown et al. [1985] and has been supported by combining data from published reports and several centers [Brown et al., 19871. The underlying cause of the recombinational variability remains unknown although there may be a relation between the genetic distance and the degree of expression of the fragile site.

Both DXSlO5 and DXS98 map closer to the fragile site and DXS98 in particular may be valuable for DNA studies for antenatal diagnosis in conjunction with cytogenetic analysis. A maximum lod score of 6.15 at a recombination fraction of 0.10 was found for DXS98-FRAXA in one study [Brown et al., this issue], whereas no recombinants were found in the study of Mulley et al. [this issue]. Genetic heterogeneity between DXS105 and FRAXA is similar to that observed in the interval F9-FRAXA. However, there are insufficient data to investigate heterogeneity for the interval DXS98-FRAXA. In clinical application, it must be emphasized that a double recombination event cannot be ruled out even if DXS52 and DXS98 are used as

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bridging markers. There are also problems in relating the DNA linkage results to the severity of the phenotype in females. The use of DNA markers in the clinical prediction of the phenotype in this disease may well have to await a better understanding of the mutation involved and a method for the direct detection of the defect. For example, if the disease is the result of amplification of sequences at the fragile site which disrupts gene expression as suggested by Ledbetter and Nussbaum at the Dunk Island workshop, then this could be followed in a family by pulsed field gel electrophoresis (PFGE).

PHYSICAL MAPPING STUDIES

The ability to fractionate high molecular weight DNA (50-5000 kb) on agarose gels by PFGE has enabled a prelimiary physical map of the Xq27-Xqter region to be constructed. For example, F9 and MCF.2 have been linked within 270 kb of each other and DXS52, DXS15 and DXS33 have been shown to lie within 470 kb [Nguyen et al., and Patterson et al., this issue]. Interestingly, FBC, which is now thought to lie proximal to DXS52, does not show physical linkage to DXS52. Thus, F8C may be quite close to the mutation and may be a more useful marker than hitherto realized.

Attempts to detect the breakpoints in females with terminal deletions beginning at (or around) the fragile site from the closest marker DXS98 have failed so far. Thus, this locus may lie at some distance from the region of interest [Patterson et al., this issue]. More markers are needed to construct a better physical map and to visualize any chromosome changes associated with the syndrome. Preliminary analyses indicate a high level of GC residues in the region distal to the fragile site. This may be correlated with a large number of expressed sequences within this region of the human X chromosome. So far, no specific alterations in methylation patterns have been observed between normal and affected individuals. This effect should be investigated carefully in the light of the hypothesis of Laird [this issue] which would predict such changes.

One of the most promising approaches to the cloning of the fragile site was presented by Warren et al. [this issue]. They have succeeded in isolating candidate sequences for the breakpoints in the X chromosome at or around the fragile site. Human cell lines expressing the fragile X site were fused with Chinese hamster cells such that some human X chromosomes broken at the fragile site become translocated onto Chinese hamster chromosomes. Libraries were made of the hybrid cell DNA, and sequences hybridizing to both human and Chinese hamster DNA were identified. These sequences should lie at the breakpoints present at the fragile site in the X chromosome. Their detailed characterization should lead to some exciting data on the nature

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bridging markers. There are also problems in relating the DNA linkage results to the severity of the phenotype in females. The use of DNA markers in the clinical prediction of the phenotype in this disease may well have to await a better understanding of the mutation involved and a method for the direct detection of the defect. For example, if the disease is the result of amplification of sequences at the fragile site which disrupts gene expression as suggested by Ledbetter and Nussbaum at the Dunk Island workshop, then this could be followed in a family by pulsed field gel electrophoresis (PFGE).

PHYSICAL MAPPING STUDIES

The ability to fractionate high molecular weight DNA (50-5000 kb) on agarose gels by PFGE has enabled a prelimiary physical map of the Xq27-Xqter region to be constructed. For example, F9 and MCF.2 have been linked within 270 kb of each other and DXS52, DXSl.5 and DXS33 have been shown to lie within 470 kb [Nguyen et al., and Patterson et al., this issue]. Interestingly, F8C, which is now thought to lie proximal to DXS52, does not show physical linkage to DXS52. Thus, F8C may be quite close to the mutation and may be a more useful marker than hitherto realized.

Attempts to detect the breakpoints in females with terminal deletions beginning at (or around) the fragile site from the closest marker DXS98 have failed so far. Thus, this locus may lie at some distance from the region of interest [Patterson et al., this issue]. More markers are needed to construct a better physical map and to visualize any chromosome changes associated with the syndrome. Preliminary analyses indicate a high level of GC residues in the region distal to the fragile site. This may be correlated with a large number of expressed sequences within this region of the human X chromosome. So far, no specific alterations in methylation patterns have been observed between normal and affected individuals. This effect should be investigated carefully in the light of the hypothesis of Laird [this issue] which would predict such changes.

One of the most promising approaches to the cloning of the fragile site was presented by Warren et al. [this issue]. They have succeeded in isolating candidate sequences for the breakpoints in the X chromosome at or around the fragile site. Human cell lines expressing the fragile X site were fused with Chinese hamster cells such that some human X chromosomes broken at the fragile site become translated onto Chinese hamster chromosomes. Libraries were made of the hybrid cell DNA, and sequences hybridizing to both human and Chinese hamster DNA were identified. These sequences should lie at the breakpoints present at the fragile site in the X chromosome. Their detailed characterization should lead to some exciting data on the nature

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of the DNA sequences involved.

It is evident that genetic and physical approaches are now giving some interesting insights into the fragile X syndrome. The next year should be a very challenging and rewarding one for those working in the field.

SUMMARY

The molecular analysis of X-linked mental retardation has progressed rapidly since the last Workshop as more DNA markers have become available and a good genetic map of the X chromosome has been constructed. This has allowed a finer localization of the Martin-Bell syndrome and the characterization of other X-linked mental retardation syndromes. Although none of these studies has yet resulted in the identification of the mutations involved, some interesting experiments are now being carried out in several laboratories using new approaches that may well reveal the underlying defect in these diseases very soon.

SESSION VI: GENETICS AND EPIDEMIOLOGY

Chairperson: Gillian Turner

The session on the epidemiology of the fragile X produced reports on a variety of survey methods. Data were available from Sicily, Finland and Greece and a screening slide comparing all the surveys now reported showed that the prevalence figures for affected males were rouqhly all of the same sort of magni- tude ranging from 1:lOOO to 1:2500. Stephanie Sherman then presented a paper which suggested that there is an excess of dizygotic twinning in the offspring of heterozygous, carrier women. Gillian Turner then discussed the problems encountered in case-finding involving a total population in a defined area. This generated some discussion on the ethics of this sort of work; the importance of informed consent was stressed and the fact that families had a right to this knowledge now that it is medically available.

SESSION VII: HYPOTHESES

Chairperson: Giovanni Neri

Laird proposed a mechanism whereby 2 independent events are required for expression of the syndrome: the fr&(X) mutation, and X chromosome inactivation in pre-oogonial cells. The reactivation process that occurs in preparation for oogenesis is blocked by the presence of the fra(X) mutation in a region

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around fra(Xq27), thus causing mental retardation in males receiving such a partially inactivated X chromosome. In females, the presence and degree of mental retardation would depend on the proportion of partially inactivated X chromosomes that, by chance, escape lyonization. Warren proposed that loss of X-linked genes distal to Xq27 is responsible for mental retardation. Although such loss might be expected to cause mitotic failure, cells with the deletion would remain viable and therefore exert an impact mostly on tissues, such as brain, where there is very little cell proliferation. Mental retardation should be proportional to the number of cells in the brain that carry the Xq27-qter deletion.

Finally, Vogel and Steinbach presented a model according to which the fra(X) site is not the locus of the Martin Bell syndrome. This is postulated to be due to a separate mutation on the X chromosome, whose presence enhances the expression of the common fragile site at Xq27. In turn, the Martin Bell mutation is under the influence of an autosomal gene.

Each of these models explains some of the peculiarities of the fra(X) syndrome and has heuristic value inasmuch as they are testable through experimental design and/or clinical observation. However, none of them really provides substantial elements to the practical question of genetic counseling and recurrence risk assessment, which continue to rest on empirical data.

ACKNOWLEDGMENTS

We are most grateful Drs. Susan 0. Lewin and James F. Reynolds for recording and editorial activities at the meeting, to Mrs. LaVelle M. Spano and Ms. Debra Evans for expert secretarial collaboration, to Ms. Schillaci, Randelli and Mascali for organizational work on the conference and to the Montana Department of Health and Environmental Sciences for support through a grant to the Montana Medical Genetics Program under HB716.

REFERENCES

Brown WT, Gross AC, Chan CB, Jenkins EC (1985): Genetic linkage heterogeneity in the fragile X syndrome. Hum Genet 71:ll-18.

Brown WT, Gross A, Chan C, Jenkins E, Mandel JL, Oberlg I, Arveiler B, Novelli G, Thibodeau S, Hagerman R, Summers K, Turner G, White BN, Mulligan L, Forster-Gibson C, Holden JJA, Zoll B, Krawczak M, Goonewardena P, Gustavson KH, Pettersson U, Holmgren G, Schwartz C, Howard-Peebles PN,

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Murphy P, Breg WR, Veenema H, Carpenter NJ (1987): Multi- locus analysis of the fragile X syndrome. Hum Genet in press.

Davies KE, Mandel J-L Weissenbach J, Fellous M (1987): Report of the Committee on the Genetic Constitution of the X and Y Chromosomes. Cytogenet Cell Genet, in press.


APPENDIX I: LIST OF PARTICIPANTS

Ignazio Barberi Policlinico Universitario Messina, Italy

Universits di Cagliari Cagliari, Italy

Karen Brqdndum-Nielsen John F. Kennedy Institute Glostrup, Denmark

Institute for Basic Research Staten Island, New York, USA

John Radcliffe Hospital Oxford, England, U.K.

Cattedra di Genetica Medica Trieste, Italy

Institute for Basic Research Staten Island, New York, USA

Medizinische Universitzt LGbeck, Federal Republic of Germany

Jean-Pierre Fryns Center for Human Genetics Leuven, Belgium

Universits di Palermo Palermo, Italy

Thomas W. Glover University of Michigan Ann Arbor, Michigan, USA

Ponmani Goonewardena Biomedical Center Uppsala, Sweden

Karl-Henrik Gustavson University Hospital Uppsala, Sweden

Randi J. Hagerman University of Colorado Denver, Colorado

University of Colorado Denver, Colorado

Health Department of W.A. West Perth, W. Australia

Paolo Bergonzi

W. Ted Brown

Kay Davies

Giorgio Filippi

Gene Fisch

Ursula Froster-Iskenius

Liborio Giuf f rz

Paul Hagerman

Athel Hockey

Gsta Holmgren University Hospital Ume;, Sweden

Patricia Howard-Peebles Genetic & IVF Inst. Fairfax, VA, USA

Salisbury General Hospital Salisbury, Wiltshire England

Institute for Basic Research Staten Island, NY USA

Patricia A. Jacobs

Edmund C. Jenkins

Bertrand R. Jordan CIML INSERM - CNRS Marseille, France

Hans K:son Blomquist University Hospital Ume;, Sweden

Marketta Kzhkgnen Oulu University Central Hospital Oulu, Finland

Charles Laird University of Washington Seattle, WA, USA

Ulrich Langenbeck University of Frankfurt Frankfurt, FRG

Baylor College of Medicine Houston, Texas, USA

Baylor College of Medicine Houston, Texas, USA

Jaakko Leisti Oulu University Central Hospital Oulu, Finland

Susan 0. Lewin Shodair Children's Hospital Helena, Montana, USA

David H. Ledbetter

Susan A. Ledbetter

16 Neri et al.

Jean Louis Mandel Universitg Louis Pasteur Strasbourg, France

Maria Enrica Martini Ospedale S. Giovanni Roma, Italy

Teresa Mattina Universit; di Catania Catania, Italy

Athens University Medical School Athens, Greece

Margareta Mikkelsen The John F. Kennedy Institute Glostrup, Denmark

Gabriella Milone Universitg di Catania Catania, Italy

Florindo Mollica Universit; di Catania Catania, Italy

Adelaide Children's Hospital North Adelaide, Australia

Institute Oasi Troina, Italy

Universit; G. D'Annunzio Chieti, Italy

Ann-Marie NordstrGm United Laboratories Ltd. Helsinki, Finland

Shodair Children's Hospital Helena, Montana, USA

Queen's University Kingston, Ontario, Canada

Universit; di Catania Catania, Italy

Institu€e-of Child Health London, England, U.K.

Prince of Wales Hospital Randwick, NSW, Australia

Ariadni Mavrou

John Mulley

Sebastiano A. Musumeci

Giovanni Neri

John M. Opitz

Michael Partington

Lorenzo Pavone

Marcus Pembrey

Stuart Purvis-Smith

James F. Reynolds Shodair Children's Hospi t a1 Helena, Montana, USA

Istituto Oasi Troina, Italy



Tamar Schaap Hadassah University Ho sp i t a 1 Jerusalem, Israel

Angela Schmidt Institut fiir Humangenetik Essen, W. Germany

Charles Schwartz Greenwood Genetics Center Greenwood, SC, USA

Ercole Sega Istituto Oasi Troina, Italy

Lawrence R. Shapiro Westchester County Medical Center Valhalla, New York, USA

Stephanie Sherman Memorial Sloan- Kettering Cancer Cntr. New York, New York, USA

Peter Steinbach Universit'at U l m U l m , Germany

Grant R. Sutherland Adelaide Children's Hosp i t a1 North Adelaide, Au s t r a1 ia

Alison Thode Prince of Wales Children's Hospital Randwick, NSW, Au s t r a1 i a

Corrado Romano

Vincenza Sammito

Salvatore Sanfilippo


Harriet von Koskull Helsinki University Central Hospital Helsinki, Finland

Ostra Sjukhuset GGteborg, Sweden

The Children's Hospital Denver, Colorado, USA

Emory University School of Medicine Atlanta, Georgia, USA

Tessa Webb Birmingham Maternity Hospital Brimingham, UK

Jan Wahls t rgm

Gail Waldstein

Stephen T. Warren

Niels Tommerup John F . Kennedy Institute Glostrup, Denmark

Lisbeth Tranebjaerg John F. Kennedy Institute Glostrup, Denmark

Prince of Wales Children's Hospital Randwick, NSW, Australia


Walther Vogel Un ivers it St u 1m Ulm, Germany

Gillian Turner

Giovanni Ventimiglia

18 Nerietal.

APPENDIX 11: Abstracts

ABSTRACT 1

PHYSICAL AND GENETIC MAPPING IN THE Xq27-Xqter REGION OF THE HUMAN X CHROMOSOME. Davies KE, Patterson MN, Bell MV, Dorkins HR, King AW, Kenwrick SJ, Thibodeau S, Ryynanen M , Schwartz C. University of Oxford, UK; Mayo Clinic, USA; University of Helsinki, Finland; Greenwood Genetic Center, USA.

The main aim of this study is to construct a detailed physical and genetic map around the fragile site at Xq27.3 on the human X chromosome. Initially, we used existing probes which map to this region to make a long range restriction map covering several hundred kb using the technique of pulsed-field gel electrophoresis.

We have demonstrated that the loci DXS15 and DXS52 recognise common restriction fragments with several enzymes including an Sfi I fragment of 60 kb. Thus, the distance between the limits of the DXS15 and DXS52 loci may be as little as 60 kb. Given the approximate genetic distance between DXS52 and DXS15 of 2-5 cM, these observations suggest that genetic recombination in this region may be unusually high.

Genetic distances between markers in this region have been obtained by segregation analyses in the CEPH families and fragile X families. A multipoint linkage map will be presented.

ABSTRACT 2

BrdU AND FRA(X)(q) EXPRESSION IN HEMIZYGOUS AND HETEROZYGOUS PATIENTS WITH THE FRA(X)-FORM OF MENTAL RETARDATION. Froster- Iskenius U , Wilhelm D, Schwinger E. Institut fur Humangenetik, Medizinische Universitat zu LGbeck, FRG.

Variable degrees of phenotype expression are observed among carriers of the fra(X)-form of mental retardation.

It was suggested that inactivation of the X-chromosome with the fragile site might partly explain this phenotype variation. However, studies investigating a correlation between the inactivation pattern of the X-chromosome with the fragile site and the phenotype in heterozygotes gave contradictory results [Fryns et al, Hum Genet 65:400, 1984; Knoll et al, Am J Hum Genet 36:640, 1984; Paul et al, Hum Genet 66:344;1984; Tuckerman et al, J Med Genet 22:85, 19851.

BrdU, which is used to distinguish between the active and inactive X-chromosome reduces fra(X) expression. If this sup- pression affects fra(X) expression preferentially either on the active or the inactive X-chromosome, this might influence the interpretation of genotype-phenotype correlation studies in heterozygotes.

We have studied the effect of BrdU on fra(X) expression in 13 hemizygous males and 7 heterozygous fra(X) carriers.


Lymphocyte cultures were set up in parallel using methotrexate for fra(X) expression. To half of the cultures BrdU was added 5 hours before harvesting. Slides were RBA and GAG banded and a minimum of 100 cells per patient and per culture condition was scored.

In hemizygous males no difference of fra(X) expression was observed between the cultures to which BrdU had been added and those with methotrexate alone. In the heterozygous carriers expression of fra(X) in cultures with BrdU was significantly lower as compared to the cultures without BrdU.

From this observation we conclude that BrdU suppresses fra(X) expression preferentially at the inactive X-chromosome.

Furthermore, a negative correlation between fra(X) expression on the active X-chromosome and phenotype in heterozygous carriers was observed.

ABSTRACT 3

SITE-SPECIFIC CHROMOSOME DELETIONS AND REARRANGEMENTS AT FRAGILE SITES IN SOMATIC CELL HYBRIDS. Glover TW, Stein CK. Department of Pediatrics and Human Genetics, University of Michigan, Ann Arbor, Michigan.

Cytologically, fragile sites usually appear as nonstaining gaps. It has been suggested frequently that fragile sites may be involved in breakage and recombination events; however, it is not clear if DNA or chromosome breakage actually occurs at fragile sites. We have shown previously that fragile sites predispose to intrachromosomal exchange as measured by sister chromatid exchanges (SCEs) [Glover and Stein, Am J Hum Genet 39:A114, 19861. This observation strongly suggested that DNA strand breakage frequently, if not always, occurs at fragile sites during expression. We now report that fragile sites also predispose to deletions and interchromosomal exchanges. Using somatic cell hybrids containing either chromosome 3 or the fragile X and chromosome 3 as the only human chromosome, we have shown that chromosome breakage indeed occurs at fragile sites and leads with a high frequency to deletions and translocations in these cells.

Hybrid cells were cultured for 5 days with 0.4 VM or 0.6 VM aphidicolin or FUdR and scored for breaks or rearrangements in the human chromosomes. Between 22 and 33% of the cells had deletions or rearrangements of chromosome 3 and of these 30 to 50% occurred at or very near 3p14. Rearrangements were mostly translocations to hamster chromosomes. Thus, an average of 13% of cells had a deletion or translocation at 3p14. Initial experiments with the fragile X containing cell line showed a similar but somewhat reduced frequency of exchanges at the fragile X site. In one experiment, 11% of cells showed a translocation to hamster chromosomes at or very near the fragile X. Loss or retention of markers flanking the fragile site was

20 Nerietal.

determined by Southern blot analysis DNA from isolated colonies to further substantiate that the breaks were at the fragile sites.

Our data clearly show that fragile sites predispose to chromosome breakage and recombination in somatic cell hybrids. This system provides a unique means for generating site specific chromosome rearrangements in somatic cell hybrids that could be applied to many other fragile sites in addition to 3p14 and Xq28. Such hybrids have the potential of being very useful in the creation of specific hybrid cell panels for gene mapping studies. Moreover, the generation of hybrids containing junctions of human and hamster DNA at translocation breakpoints suggests a strategy for molecular cloning of junction fragments and DNA sequences at fragile sites. We are currently constructing phage and cosmid libraries to screen for such junction fragments.

ABSTRACT 4

PREVALENCE OF THE FRAGILE X OR MARTIN-BELL SYNDROME. K&k&en M, Alitalo T, Airaksinen E, Matilainer R, Launiala K, Autio S , Leisti J. Department of Clinical Genetics, Oulu University Central Hospital; Rinnekoti Institution for the Mentally Retarded; Department of Pediatrics, Kuopio University Central Hospital, Finland.

We studied retrospectively the prevalence of the fra(X) or Martin-Bell syndrome among 12,882 children (6594 boys and 6288 girls) born during the years 1969-1972 in Kuopio province in eastern central Finland. Mentally retarded children had been ascertained from regular schools (by using school achievement tests) and from registers of mentally retarded individuals. In the present study the fra(X) was found in 6/111 mentally retarded children (5.4%), i.e. in 4/61 boys and in 2/50 girls, respectively. It was not detected in 85 healthy control children.

The corrected prevalence of fragile X syndrome among boys in 4 successive birth cohorts was estimated to be 1 in 1210 or 0.8/1000 and among girls 1 in 2418 or 0.4/1000. The overell prevalence was calculated to be 1 in 1612 or 0.6/1000 children.

ABSTRACT 5

THE DISTRIBUTION OF FRAGILE X CHROMOSOMES IN AMNIOTIC CELLS CULTURED BY THE IN SITU TECHNIC. Kerem B, Goitein R, Schaap T. Department of Human Genetics, Hadassah University Hospital; Department of Genetics, The Hebrew University, Jerusalem, Israel.

The fragile site Xq27.3 can be induced in low proportions of cultured cells from fra(X) patients. This may be due to either one of the following causes: a) the cultured cell population is


heterogeneous, and only one or few cell types will show the fragile site; or b) all the cells have an equal and l o w probability of showing the fragile site.

Amniotic cells cultured on coverslips will form colonies stemming from a single or few cells. We applied this method to amniotic fluid cells from a fra(X) patient, and registered cells with fra(X) chromosomes in the various colonies. The distribution of these cells between and within colonies indicates that the cell population is homogenous, with equal and low probability of showing the fragile site.

ABSTRACT 6

HIGH LEVELS OF FRAGILE X EXPRESSION IN NORMAL MALES INDUCED BY APHIDICOLIN. Ledbetter DH, Ledbetter SA. Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas.

We previously demonstrated fragile X expression in two unrelated normal males with no family history of Martin-Bell syndrome (MBS) and in a male chimpanzee using a somatic cell hybrid system [Nature 324:161,1986]. This suggested that Xq27.3 is a common fragile site present in all normal X chromosomes which is altered (?amplified) to produce the MBS, perhaps by unequal recombination events in female meiosis.

We now present additional evidence that the Xq27 fragile site is common. Four affected and 6 unrelated control males were studied. Lymphocyte or lymphoblast cultures were treated with 2 mM thymidine (Td), 0.2 pJl aphidicolin (APC), or 1.5 VM APC. Fifty to one-hundred cells per condition were analyzed in a blind fashion. With 2 mM Td, affected males showed fragile X expression in 78/300 cells (26%, range = 22-31%) and the 6 control males in 1/350 cells. With 0.2 1JM APC, affected males showed expression in 2/200 cells and normal males in 1/350 cells. With 1.5 APC, affected males showed expression in 37/300 cells (12%, range = 8/14%), significantly lower than Td levels ( x 2 =17.2, df = 1, p<O.OOl) but significantly higher than background breakage (x7 = 140.6, df = 1, p<O.OOl). With 1.5 $4 APC, all 6 control males showed fragile X expression, in a total of 82/450 cells (18%, range = 12-28%). This is significantly above breakage (x2 = 249.6, df = 1, p<O.OOl), and is also higher than that seen in affected males (x2 = 4.25, df = 1, p <0.05). These results support our previous suggestion that Xq27 is a common fragile site, and is consistent with data from our lab and others that fragile expression in normal males or females occurs at a frequency of approximately 1/500 cells under conditions of thymidylate stress. Caution is therefore warranted in the interpretation of low frequency expression in diagnostic situations, and individual laboratories should establish "normal ranges" for fragile X expression.

background

22 Nerietal.

ABSTRACT 7

THE FRAGILE X SYNDROME IN NORTHERN FINLAND: A GENEALOGIC STUDY. Leisti J, Kihkb'nen M, Herva R, Winqvist R, Ukkola L, Heino R, VB'isXnen M-L, Rekild A, Linna S-L. Departments of Clinical Genetics, Pediatrics and Pathology, Oulu University Central Hospital, Oulu, Finland.

We have actively searched for the fragile X syndrome during the past few years among the mentally retarded in the province of Oulu (population 430,000) in Northern Finland. Fourty-eight families with 113 sibships and 109 mentally retarded males have been ascertained and shown to express the fragile site Xq27. Thirty-one mentally retarded and 27 mentally normal females were found to be fra(X) positive, respectively; in addition, 104 females were considered to be obligate carriers on the basis of pedigree information (mothers of affected sons or daughters or transmitting males).

To date we have found 6 additional extensive pedigrees, with at least 41 affected sibships and probably 15 mentally normal, non-expressing transmitting males. In the largest pedigree there seem to be five transmitting male cousins who have 14 mentally normal daughters and several affected grand- or great grandchildren. Church records have proven to be useful both in obtaining more extensive information on the pedigrees and in linking of separately ascertained sibships into the same pedigree.

The inheritance of the fra(X) locus is being followed in the families with the aid of chromosome analysis and RFLP analysis using the probes F9, DXSlO5 and DXS52.

ABSTRACT 8

GENETIC AND PHYSICAL MAPPING OF THE FRAGILE X REGION: A) NEW POLYMORPHIC MARKERS; B) IS THE FRAGILE X SITE A PREFERENTIAL BREAKPOINT IN CHROMOSOME REARRANGEMENTS? Oberle' I, Arveiler B, Woelflin A, Hofker M, Pearson P, Mandel JL. INSERM U 184, LGME du CNRS, Faculte' de Me'decine, Strasbourg, France; Department of Human Genetics, Leiden, The Netherlands.

have characterized new polymorphic DNA markers useful for the genetic mapping of the q26-q28 region of the human X chromosome. They include new informative RFLPs for DXS52 (St14), DXSlO5, DXS115, and DXS152. We have shown that DXSlO5 and DXS152 are contained within a 40 kb region. A linkage analysis was performed in fragile X families and in large normal families from the CEPH, using polymorphic loci located in Xq26-q28. This multipoint linkage study allowed us to establish the order: centromere-DXS100-DXS86-DXSl44-DXS5l-DXSlO2-F9-DXSlO5-F~-(F8, DXSlS,DXS52,DXSllS). DXS105 is 7% recombination closer to the FRAXA locus than F9 and should thus be a better marker for analysis of fragile X families. However, the DXSlO5 locus still

We


appears loosely linked to the FRAXA locus in some families. The multipoint estimation for recombination between DXSlOS and FRAXA is 0.17 in our data. Together with other studies, our results define a cluster of 11 loci located in Xq26-q27 and which map within a 10 to 15 cM region. This contrasts with the absence of markers (other than the fragile X locus) between DXS105 in q27 and the G6PD cluster in q28, which are separated by about 30% recombination.

We have also used these probes and a series of non-polymorphic ones previously assigned to the q26-q28 region to analyse X chromosomes with various types of rearrangements in this region: interstitial or terminal deletions, translocations, dicentric X chromosomes, We observed that probes are distributed in two groups: the probes are either proximal to 7 breakpoints analysed, or distal to all 7, with no probes showing an intermediate distribution. For those probes that have been genetically mapped the localisation as defined by the fragile X locus is exactly parallel to the physical localisation with respect to the 7 breakpoints. These results suggest that the region of the fragile x locus is a region of preferential chromosome rearrangement.

ABSTRACT 9

FURTHER NEUROPATHOLOGICAL OBSERVATIONS IN ADULT FRAGILE X. Rudelli R, Madrid R , Brown WT. New York Institute for Basic Research, Staten Island, NY.

We have previously reported on the dendritic spine abnormalities observed in a 62-year-oldI moderately retarded man with the fra(X) syndrome [Rudelli et al, Acta Neuropathol 67:289, 19851. No other significant neuronal or glial changes were seen in that case, with the exception of hemispherical white matter pallor which was then attributed to postmortem tissue alteration.

Since then we have had the opportunity of examining two additional fra(X) cases of juvenile and adult death. Similar deep white matter lesions have been observed in well-preserved specimens from the latter two cases.

These lesions were characterized by myelin loss, relative axon preservation and a diffuse spongy edematous change. No abnormal cells, inclusions, sudanophilic deposits or mineralization were identified. Arcuate and callosal white matter were preserved in all cases. Myelin loss with axon preservation bears marked resemblance to the lesions of the demyelinating diseases, however, they seem to be related to deep white matter edema. Effects of protracted edema on tissues consist of irreversible glial changes and myelin degeneration with relative sparing of axons. The pathogenesis of the edematous lesion is not clear. Deep white matter edema could arise from different factors including abnormal white matter

24 Nerietal.

metabolism. Current ongoing studies include evaluation of interstitial

white matter mucopolysaccharides as well as of a possible myelin metabolic abnormality.

ABSTRACT 10

ANALYSIS OF SITE-SPECIFIC SISTER CHROMATID EXCHANGES AT THE Xq27 FRAGILE SITE. Tommerup N. The John F. Kennedy Institute, Glostrup, Denmark.

high frequency of site-specific sister chromatid exchanges (SCEs) was found at the fragile site at Xq27. In 3 affected males this frequency varied between 30-41%. Despite treatment with agents which increased (caffeine) or decreased (cyclohexi- mide) the overall number of SCEs per cell, the frequency of site-specific SCEs remained remarkably stable, suggesting a close association between the expression of the fragile site and the presence of site-specific SCEs. Two possible mechanisms could explain such a high frequency of SCEs at a specific chro- mosomal site: 1) a recombinational event affecting (a) specific sequence(s) with likely sister-strand exchange in half of the cases; 2) a large amount of DNA-damage within a cluster of highly repetitive sequences, where each damaged site may lead to a SCE with a relatively low probability.

So far, the observation of site specific SCEs in less than half of the fragile sites may support the latter explanation.

A

ABSTRACT 11

EVIDENCE FOR A DNA POLYMERASE a DIRECTED REPLICATIVE PROCESS INVOLVED IN THE EXPRESSION OF THE FRAGILE SITE AT Xq27. Tommerup N. The John F. Kennedy Institute, Glostrup, Denmark.

The competitive inhibitor of DNA polymerases, cytosine-1-B- D-arabinofuranoside (Ara-C), and the specific inhibitor of DNA polymerase a , aphidicolin (APC), both caused inhibition of the expression of the fragile site at Xq27 fra(X). This inhibition occurred when the fragile site was induced by thymidylate depletion (FUdR), and by deoxycytidine depletion (excess thymidine). These data suggest that 1) replication directed by DNA polymerase a is involved in the expression of the fra(X), and 2) a fundamental difference between the fra(X) and the common fragile sites induced by APC.

The conditions necessary for expression of the fra(X) is known to be associated with severe nucleotide pool imbalance(s), and it is thought that this imbalance interferes with replication of specific DNA sequence(s) within the fragile region. One way to interpret these data is that thymidylate or deoxycytidine depletion leads to DNA-damage, that repair of this damage is directed by DNA polymerase a , and that the cytogenetically


detectable fragile site is a consequence of this repair process.

ABSTRACT 12

UNSUSPECTED PRENATAL DIAGNOSIS OF THE FRAGILE X. Tommerup N, Reintoft I, Reske-Nielsen E l Brgndum-Nielsen K, Mikkelsen M. The John F. Kennedy Institute, Glostrup, Denmark; Department of Pathology, Central Hospital, Esbjerg; Department of Neuropatho- logy, Arhus Kommunehospital, Arhus, Denmark.

So far, prenatal diagnosis of the fragile site at Xq27 has been offered to families with a known risk of Martin-Bell syndrome (MBS). However, the high prevalence and mutation rate of the disease indicate that new cases may come to attention in routine amniocentesis programs.

An example of this is illustrated: A woman without any known risk of MBS was offered amniocentesis due to her age (36 years). One out of 12 routinely analysed cells expressed fragility at Xq27. Further analysis of a total of 100 cells in the routine culture did not show additional cells with the fra(X), but 10% positive cells were found in parallel cultures treated with excess thymidine. The parents chose termination after genetic counselling, and the fra(X) was demonstrated in the fetal tissue. Histological examination of the brain showed an unusual number of proliferation foci in the first cortical layer, whereas all other organs were normally developed.

ABSTRACT 13

FIRST TRIMESTER PRENATAL DIAGNOSIS OF THE FRAGILE SITE AT Xq27. Tommerup N, Sdndergaard F, Tdnnesen T I Arveiler B, Bech B, Bdrresen A, Davies K, Froster-Iskenius U, Gustavson K-HI van der Hagen CB, Heiberg A, Mandel JL, Mikkelsen M I Miny P, Rasmussen K, Schinzel A, Wahlstrijm J. The John F. Kennedy Institute, Glostrup, Denmark; Laboratoire de Ggndtique Moleculaire des Eucaryotes, Strasbourg, France; University Hospital of Copenhagen, Denmark; University of O s l o , Norway; University of Oxford, England; Medical University of Liibeck, W. Germany; Akademiska Sjukhuset, Uppsala, Sweden; Westfxlische Wilhelms- Universitgt, W. Germany; University of Arhus, Denmark; University of Ziirich, Switzerland; University of GEteborg, Sweden.

Following chorionic villus sampling, first trimester prenatal diagnosis of the fra(X) was attempted in 15 pregnancies at risk of Martin-Bell syndrome. Preferably, three diagnostic methods were applied in each case; 1) induction of the fra(X) by thymidylate depletion; 2) induction of the fra(X) with excess thymidine; and, 3) analysis of RFLPs detected by linked, flanking DNA probes.

The fetal sex ratio was 9M:6F. The fra(X) was demonstrated

26 Neri et al.

cytogenetically in long-term cultured chorionic villus cells in three male fetuses. In two of these, informative DNA markers suggested that the fetus carried the mutation, and in one case an apparent cross-over between the flanking probes prevented the use of RFLP analysis. Following termination of these pregnancies (TOP), the fra(X) was demonstrated in all 3 fetuses. However, in another male fetus, an apparent discrepancy between the cytogenetic and the RFLP data was found. The fra(X) could not be induced in any of the cell cultures, but both flanking markers suggested that the fetus carried the abnormal X chromosome. The parents opted for TOP, and the fra(X) could not be induced in several fetal tissues, including blood. In one female fetus, only tetraploid mitoses were observed in the 24 hour short term culture; however, flow-cytometric analysis indicated that most chorionic villus cells were diploid, suggesting the presence of placental mosaicism.

ABSTRACT 14

IDENTICAL EXPRESSION OF THE FRAGILE X BUT DISCORDANT CLINICAL MANIFESTATIONS IN MONOZYGOTIC TWINS WITH MARTIN-BELL SYNDROME. Tommerup N, Tranebjaerg L, T$nnesen T, Kastern W, Hansen H, Dissing J. The John F. Kennedy Institute, Glostrup, Denmark; Hagedorn Research Laboratory, Gentofte; University Institute of Forensic Medicine, Copenhagen.

monozygotic twins with the fragile X provide a powerful tool for testing 1) whether the degree of expression of the fragile X is under genetic control, and 2) the strength of association between the fragile X mutation and the phenotype.

We describe a pair of male monozygotic twins with the fragile X. Monozygosity was determined by chromosome analysis, isozyme- and HLA-typing and DNA-fingerprinting. The expression of the fragile site was identical in both twins (20-25% in blood and 6-7% in skin fibroblasts). However, from early childhood twin A was significantly more retarded than twin B and developed a much more striking phenotype with frontal bossing than twin 13, who was considered normal by the parents. Speech problems indicated that twin B was affected to some degree, but considerably less than his brother.

The identical expression of the fragile site in both twins is on line with suggestions that expression of the fragile X is genetically determined. If so, the observed difference in phenotype may suggest, that the fragile site mutation per se does not automatically lead to a certain degree of retardation, but that additional factor(s) or event(s) play a r61e as well.

Male

ABSTRACT 15

THE PROS AND CONS OF PREVENTIVE SCREENING FOR THE FRAGILE (X).


Turner G, Partington MW, Robinson H, Latham M, Kenny J, Van den Berk M, Laing S , Thode A. Department of Medical Genetics, Prince of Wales Children's Hospital, Sydney, Australia.

The New South Wales State Department of Health has funded preventive "screening" for the fra(X) or Martin-Bell syndrome in the intellectually handicapped in order to identify females at risk in the population. The program will take 4 years to complete and it is now in its second year.

Ninety percent of families gave us permission to examine their offspring for this condition. If an individual is identified as being fra(X) positive, the parents usually go through a period of re-grieving. The sisters at risk have all been pleased to acquire genetic information. Staff of sheltered workshops and special schools have been enthusiastic about the program. One of the major difficulties encountered is that the medical profession is generally unaware of the fra(X) or Martin-Bell syndrome and therefore, they have to be included in the educational process.

Our procedure of screening has changed to case-finding. Originally we excluded those with a definite diagnosis or those with head circumference below the 3rd centile or cerebral palsy and looked at the chromosomes of the rest. We have now switched to case-finding using a 10-point score which includes positive family history (2), personality (2). face-length ( 2 ) , ear-size (2) and body-habitus (2). This has reduced considerably the number of specimens for analysis.

The program generates considerable follow-up work: 1) a number of different clinical diagnoses are made; 2) other chromosome abnormalities are found; and 3) there is a follow-up of the proband's family. This has increased the need for regionalized services for genetic counseling in our state.

By all standards, the cost of the program is small and the benefits great. We conclude that now the onus i s on health authorities everywhere to state why they are not screening for the fra(X).

ABSTRACT 16

CYTOGENETIC FRA(X) EXPRESSION AND LINKAGE STUDIES IN EIGHT FINNISH FRA( X) FAMILIES. von Koskull H, Ngrdstrom A-M. Laboratory of Prenatal Genetics, Departments of Obstetrics and Gynecology, Helsinki University Central Hospital; United Laboratories Ltd., Helsinki, Finland.

Fra(X) expression and RFLP-linkage was studied in 62 members of 8 fra(X) families. Cytogenetic expression was induced in lymphocyte cultures by 1) excess of thymidine, 2) fluorodeoxyuridine (FUdR), and 3) low folic acid (serum) concentration; 60 complete metaphases were scored in each category. Fra(X) expression varied as follows:

28 Nerietal.

Method 1 2 3 ( % ) ( % ) ( % )

Mental retardation XY 10-41 5-38 0-30 or subnormality xx 10-36 10-34 10-32

carriers xx 0-16 0- 19 0- 17 Phenotypically normal

Thus, excess thymidine (300 iJg/ml) can be used for the detection of fra(X) in mentally retarded or subnormal individuals, but carrier detection requires several methods.

Preliminary results of tissue cultures (including amniotic fluid, chorionic villus, testis tissue and skin fibroblasts) indicate that excess of thymidine is the most efficient method to demonstrate fra(X) cytogenetically. The level of fra(X) expression seems to be familial and therefore the correlation between fra(X) expression and the level of linkage was calculated. The WLP-linkage studies were performed using two probes: St 14 (from J.-F. Mandel, Strasbourg) and CXSS.7 (from P. Pearson, Leiden). The correlation between fra(X) expression and genetic linkage is discussed.

ABSTRACT 17

CONSTRUCTION OF A SELECTABLE PLASMID TO TEST FOR DNA SEQUENCE FRAGILITY OR BREAKAGE IN MAMMALIAN CELLS. Warren ST, Ngo EJ. Departments of Biochemistry and Pediatrics, Division of Medical Genetics, Emory University School of Medicine, Atlanta, Georgia.

In order to examine DNA fragments for sequence-specific fragility and/or breakage, we have developed a plasmid vector system into which a test DNA sequence can be cloned and the resulting recombinant introduced into mammalian cells for analysis. The plasmid, pGPTK, was constructed by ligating the 3.5 kb Bam HI fragment from pHSV-106, which contains the entire Herpes thymidine kinase gene, into the Bam HI site of pSV2gpt. The resulting 8.7 kb plasmid now contains two selectable genes which confer HAT (hypoxanthine, aminopterin, thymidine) resistance to mammalian cells deficient in HGPRT and TK activities. A test sequence can be cloned into a Hpa I site which separates the GPT and TK genes. The resulting plasmid is then linearized, such that GPT-test sequence-TK moleculars are formed, and then electroporated into CHO B21 cells (which lack HGPRT and TK activities).

Transformants are selected on HAT media and screened by Southern analysis for single copy integration of the linear plasmid (which is common following electroporation, at low DNA concentrations, of linear molecules). A single copy transformant is used to test for sequence-induced breakage. In the absence of HAT selection, the cell line is exposed to conditions which may influence breakage (i.e. thymidine stress, depending upon the test sequence). Following an expression


time, aliquots of cells are plated into media containing either bromodeoxyuridine, hypoxanthine and azaserine (which selects against TK and for GPT) or thioguanine, thymidine and fluorodeoxyuridine (which selects against TK and for GPT). Control dishes containing either growth media alone or HAT media are also inoculated. The resulting colonies are counted and compared to groups that were not exposed to breakage induction or control cell line which are transfected with the plasmid containing a random but equal sized genomic fragment as the test sequence. The segregation of TK from GPT in the induced cell line would be evidence for breakage and is confirmed by Southern analysis. We are currently testing various sequences which structurally might be thought to confer fragility.

Received for publication October 27, 1987; revision received March 7, 1988.

Conference report: Third International Workshop on the fragile X and X-linked mental retardation

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Transcript of Conference report: Third International Workshop on the fragile X and X-linked mental retardation