American Journal of M e d i c a l Genetics 44830-833 (1992)
Infantile Autism-Fragile X: Molecular Findings Support Genetic Heterogeneity Helena Malmgren, Karl-Henrik Gustavson, J a n Wahlstrom, Ingrid Arpi-Henriksson, Jurgen Bensch, Ulf Pettersson, and Niklas Dahl Department of Medical Genetics, Biomedical Centre, Uppsala University iH.M., U.P., N.D.) and Department of Clinical Genetics, University Hospital, Uppsala (K.-H.G.,N.D.), Department of Clinical Genetics, Ostra Hospital, Gothenburg ( J .W.), Department of Child Psychiatry, County Hospital, Karlstad (Z.A.-H.), and Department of Pediatrics, Orebro Hospital, Orebro (J.B.), Sweden
Twenty-two members of 18 families with autism have been examined for the presence of mutations and abnormal methylation in the FMR-1 region at Xq27.3.All patients fulfilled diagnostic criteria of infantile autism. A characteristic pattern of insertion and methylation were detected after Southern blot analysis in 7 autistic individuals expressing the fragile site at Xq27.3. Normal DNA patterns were observed in 15 autistic boys cytogenetically negative for the fragile site. The results indicate a lack of involvement of the FMR-1 region in infantile autists negative for fragile X expression. o 1992 Wiley-Liss, Inc.
al., 19911. The responsible gene (FMR-1)has been isolated and includes a 200 base-pair (bp) fragment of repeated CGGs which are targets for mutations in the fra(X) syndrome [Kremer et al., 1991; Yu et al., 1991; Verkerk et al., 19911. Cloning of this region yielded a DNA probe, StB12.3, that detects both the abnormal methylation and the mutations in the FMR-1 gene [Oberle et al., 1991;Rousseau et al., 19911.The use of the probe or amplification of the region by the polymerase chain reaction provides the basis for a highly specific and sensitive test of the fra(X) mutation. Individuals with a low degree of expression or even carriers of the premutation can easily be detected [Rousseau et al., 1991;Fu et al., 19911.The use of a double digest with the restriction endonucleases EcoRI and EagI allows mutations to be detected and at the same time methylation KEY WORDS: fragile X syndrome, FMR-1 status to be determined [Malmgren et al., 19923.Normal gene, mutations males exhibit a 2.8 kb fragment, and normal females exhibit a 2.8 kb fragment and a 5.2 kb fragment resulting from the inactive X-chromosome. The premutation INTRODUCTION is detected as fragment(s1 slightly larger (< 500 bp) Infantile autism, originally described by Kanner than 2.8 kb but with no abnormal methylation. Carrier [19431, was first assumed to be a single disease. The females normally have a small insertion which gives concept today is that infantile autism is an hetero- rise t o 2 slightly larger fragments in addition to the geneous disorder [Coleman and Gillberg, 19851.A possi- normal ones. Affected males exhibit a large fragment or ble associationbetween autism and the fragile X [fra(X)] a smear of fragments above 5.2 kb. Affected females syndrome has been studied by several groups and has exhibit a large fragment or a smear as in affected males yielded conflicting results. Coexistence of autism and in addition to the normal fragments. the fra(X)syndrome was first reported in 1982 [Brownet In this study, we have investigated DNA from paal., 1982; Meryash et al., 19821. Subsequent studies tients diagnosed as cases of infantile autism for the have found a variable association [Watson et al., 1984; presence of abnormal methylation and mutations in the Blomquist et al., 1985; Brown et al., 1986; Bregman et FMR-1 region at Xq27.3. The purpose was to examine: al., 19881and one study has failed to detect any signifi- 1) if autistic individuals without fra(X) expression cant association [Einfield et al., 19891. showed an abnormal pattern in the FMR-1 region and, Recent studies showed that a region a t Xq27.3 is ab- 2) if autistic individuals expressing fra(X) differed in normally methylated in persons affected by the fra(X) mutation and methylation patterns as compared to nonsyndrome [Bell et al., 1991;Heitz et al., 1991;Vincent et autistic fra(X) carriers. Received for publication March 4, 1991; revision received June 16, 1992. Address reprint requests t o Dr. Niklas Dahl, Department of Clinical Genetics and Pediatrics, University Hospital, S-751 85 Uppsala, Sweden.
0 1992 Wiley-Liss, Inc.
MATERIALS AND METHODS Patients A total of 22 patients aged 4-47 years (21 males and one girl) had a diagnosis of infantile autism according to DSM-I11 criteria. Seven patients (6 boys, one girl) from 4
Autism and Fragile X families were included in the present study because they were positive for fra(X) expression from previous analyses (Table I). The remaining 15 patients, including 2 brothers, were chosen randomly as being autistic males (Table I). Cytogenetic analysis of the fra(X) site was performed according to standard procedures and at least 100 cells were analyzed. DNA Analysis Blood samples were collected from patients in EDTA vacutainer tubes. Patient leukocyte DNA was extracted and double digested with the restriction enzymes EcoRI and EagI, subjected to gelelectrophoresis, and transferred onto nylon membranes using standard methods. The DNA probe StB12.3 and the control probe F33 [Oberle et al., 1991; Rousseau et al., 19911 were radiolabeled by random priming (Pharmacia oligolabeling kit) and hybridized to the filters at 65°C in a hybridization solution (5 x SSPE, 5 x Denhart's buffer, 0.2% SDS, and 250 mg/ml yeast RNA). The filters were washed to a stringency of 0.1 x SSC and 0.1% SDS at 65°C and exposed to Kodak XAR5 films at - 70°C.
TABLE I. Distribution of Sex, Fra(X) Expression, and Age Among 22 Autistic Individuals Investigated Family 1
No. of patients 1 1 4 1 15
2 3 4 5-18
DISCUSSION In the present study we failed to detect any abnormal patterns in the FMR-1 region among 15 fra(X) negative autistic individuals. Seven autistic patients positive for fra(X) expression showed a typical pattern for individuals affected by the fra(X) syndrome. These results are consistent with the hypothesis that infantile autism is not primarily due to aberrations in the FMR-1 region. The conflicting data about the variable correlation between autism and the fra(X)syndrome can have many different causes. One reason is that different diagnostic criteria have been used for defining autism. Although most fra(X) males are not autistic, the majority have
Sex
% fra(X)
M
24 2 8-20 22 0
M M F M
~
Age (years)
12 21 10-47 20 4-36'
"One patient in this group also had cerebral gigantism.
some autistic features. The peculiar but characteristic pattern of inheritance in the fra(X) syndrome is not observed in families afflicted by autism without fra(X) expression. This speaks against amplification of the CGG-repeat in the FMR-1 gene being the primary defect in autism. However, the possibility of other allelic mutations has not be considered. The results of this study support the hypothesis that autism is a heterogeneous disease [Rapin, 19871. It has been suggested that the autistic features in patients with the fra(X) syndrome are caused by other genes on
RESULTS Cytogenetic Investigation Seven autistic individuals (6 boys, one girl) were earlier diagnosed as positive for fra(X) expression. Fifteen autistic boys, not subject to earlier cytogenetic investigation, were all negative for the fra(X) site. DNA Analyses DNA from the 7 patients with autism expressing the fragile site at Xq27.3 showed a similar pattern as seen in fra(X) patients. A large insertion (> 600 bp) was observed and the adjacent CpG island is methylated (Fig. 1). The large size of the mutation did not allow for a precise estimation of the number of CGC-repeat as described [Fu et al., 19911. The single autistic female investigated showed a pattern identical to that of a woman affected by the fra(X) syndrome with a smear of enlarged and methylated fragments (Fig. 2). Among 15 autistic boys negative for the fragile site Xq27.3, no alterations were detected with the probe StB12.3 (Fig. 2). There were no indications of insertions or deletions within the limit of the resolution (about 100 bp). Methylation patterns appeared normal as detected by EagI/ EcoRI digestion.
831
w 5.2 kb-
2.8 kb-
Fig. 1. Hybridization of the probe StB12.3 to DNA from a family including a son with fra(X) and autistic features. The DNA pattern of the son is similar to that ofmales with the fra(X) syndrome. The mother shows the pattern of a fra(X) carrier female and the sister expresses a full mutation.
832
Malmgren et al.
1
2
3
4
5
6
7
C
C
5.2 kb-
-5.2 kb
-2.8 kb
2.8 kb-
Fig. 2. The probe StB12.3 hybridized to DNA from autistic individuals. One fra(X) positive girl with autism (lane 1)and 6 fra(X) negative males with autism (lanes 2-71, For lane 7, a longer exposure has confirmed that no other bands exist than the 2.8 kb fragment. C, control DNA from normal females.
the X-chromosomeinfluenced by the fra(X) site [Cohen et al., 19911.Another possibility is that a nonfunctional FMR-1 gene may induce secondary nonspecific organic defects affecting the central nervous system. In conclusion, no indications of aberrations in the FMR-1 region in fra(X) negative autism were found in the present study. We suggest that mutations in the FMR-1 gene is not the primary cause of autism in fra(X) negative patients. Autism in fra(X) positive individuals is probably a secondary event caused by unknown mechanisms. Still, the behavioral phenotype in the fra(X) syndrome might have implications for the etiology of autism. The present study included a limited number of autistic patients and a larger collection of samples from autistic individuals will have to be investigated in order to confirm our hypothesis.
ACKNOWLEDGMENTS We thank Prof. J.-L. Mandel for providing the probes used in this study. This work was supported by grants from the Beijer Foundation, the Savstaholm Society,the Swedish Medical Research Council, and the Ehle Foundation.
REFERENCES Bell VM, Hirst MC, Nakahori Y, MacKinnon RN, Roche A, Flint TJ, Jacobs PA, Tommerup N, Tranebjaerg L, Froster-Iskenius U, Kerr B, Tbrner G, Lindenbaum RH, Winter R, Pembrey M, Thibodeau S, Davies KE (1991): Physical mapping across the fragile X: Hypermethylation and clinical expression of the fragile X syndrome. Cell 64861-866. Blomquist H K:son, Bohman M, Edvinsson SO, Gillberg C, Gustavson K-H, Holmgren G, Wahlstrom J (1985): Frequency of the fragile X syndrome in infantile autism. Clin Genet 27:113-117. Bregman JD, Lerkman J F , Ort SI (1988):Fragile X syndrome: Genetic predisposition to psychopathology. J Autism Dev Disord 18:343354. Brown WT, Jenkins EC, Cohen IL, Fisch GS, Wolf-Schein EG, Gross A, Waterhouse L, Fein D, Maison-Brothers A, Ritvo E, Ruttenberg BA, Bentley W, Castells S (1986): Fragile X and autism: A multicenter survey. Am J Med Genet 23:341-352. Brown WT, Jenkins EC, F’riedman E, Brooks J, Wisniewski K, Raguthu S, French J (1982): Autism is associated with the fragile X syndrome. J Autism Dev Disord 12:303-307. Cohen IL, Sudhalter V, Pfadt A, Jenkins EC, Brown WT, Vietze PM (19911: Why are autism and the fragile-X syndrome associated? Conceptual and methodlogical issues. Am J Hum Genet 48: 195-202. Coleman M, Gillberg C (EdsX1985): “The biology of the Autistic Syndromes.” New York F’raeger.
Autism and Fragile X Einfeld S, Molony H, Hall W (1989): Autism is not associated with the fragile X syndrome. Am J Med Genet 34:187-193. Fu Y-H, Kuhl DPA, Pizzuti A, Pieretti M, Sutcliffe JS, Richards S, Verkerk A, Holden J, Fenwick Jr R, Warren ST, Oostra BA, Nelson DL, Caskey CT (1991):Variation of the CGG repeat at the fragile X site results in genetic instability: Resolution of the Sherman paradox. Cell 67:1047-1058. Heitz D, Rousseau F, Devys D, Saccone S, Abderrahim H, Le Paslier D, Cohen D, Vincent A, Toniolo D, Della Valle G, Johnson S, Schlessinger D, Oberle I, Mandel J-L (1991):Isolation of sequences that span the fragile X and identification of a fragile X-related CpG island. Science 251:1236-1239. Kanner L (1943):Autistic disturbances of affective contact. Nerv Child 2:217-250. Kremer EJ, Pritchard M, Lynch M, Yu S, Holman K, Baker E, Warren ST, Schlessinger D, Sutherland GR, Richards RI (1991):Mapping of DNA instability a t the fragile X to a trinucleotide repeat sequence p(CCG)n. Science 252:1711-1714. Malmgren H, SteBn-Bondeson M-L, Gustavbon K-H, Seemanova E, Holmgren G , Over16 I, Mandel J-L, Pettersson u, Dahl N (1992): Methylation and mutation patterns in the fragile X syndrome, Am J Med Genet 43:268-278. Meryash DL, Szymanski LS, Gerald ps (1982):Infantile autism associated with the fragile X syndrome. J AutismDev Disord 12:2g5-301. Oberle I, Rousseau F, Heitz D, Kretz C, Devys D, Hanauer A, Boue J, Bertheas MF, Mandel J-L (1991): Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome. Science 252:1097- 1102.
833
Rapin I (1987): Searching for the cause of autism: A neurological perspective. In Cohen DJ, Donnellan AM (eds): “Handbook of Autism and Pervasive Developmental Disorders.” Silver Spring, MD: Winston, pp 710-717. Rousseau F, Heitz D, Biancalana V, Blumenfeld S, Kretz C, Boue J, Tommerup N, Van Der Hagen C, DeLozier-Blanchet C, Croquette M-F, Gilgenkrantz S,Jalbert P, Voelckel M-A, Oberle I, Mandel J-L (1991):Direct diagnosis by DNA analysis ofthe Fragile X syndrome of mental retardation. N Engl J Med 325:1673-1681. Verkerk AJMH, Pieretti M, Sutcliffe JS, Fu Y-H, Kuhl DPA, Pizzuti A, Reiner 0, Richards S, Victoria MF, Zhang F, Eussen BE, van Ommen G-JB. Blonden LAJ. Rieeens GJ. Chastain JL. Kunst CB. Galhard H, Caskey CT, NeIsG-DL, Oostra BA, Warren ST (1991). Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variations in fragile x syndrome, cell 65:905-914, Vincent A, Heitz D, Petit C, Kertz C, Oberle I, Mandel J-L (1991): Abnormal pattern detected in fragile-X patients by pulsed-field gel electrophoresis. Nature 349524-626. Watson MS, Leckman JF, Annex B, Breg WR, Boles D, Volkmar FR, Cohen DJ (1984):EkagileX in a sunrey of75 autistic males, NEngl J Med 310:1462. yu s,kitchard M, Kremer E, Lynch M, Nancarrow J , Baker E, Holman K, Mulley JC, Warren ST, Schlessinger D, Sutherland GR, Richards RI (1991):Fragile X genotype characterized by an unstable region of DNA, science 252:1179-1181,
Infantile Autism-Fragile X: Molecular Findings Support Genetic Heterogeneity Helena Malmgren, Karl-Henrik Gustavson, J a n Wahlstrom, Ingrid Arpi-Henriksson, Jurgen Bensch, Ulf Pettersson, and Niklas Dahl Department of Medical Genetics, Biomedical Centre, Uppsala University iH.M., U.P., N.D.) and Department of Clinical Genetics, University Hospital, Uppsala (K.-H.G.,N.D.), Department of Clinical Genetics, Ostra Hospital, Gothenburg ( J .W.), Department of Child Psychiatry, County Hospital, Karlstad (Z.A.-H.), and Department of Pediatrics, Orebro Hospital, Orebro (J.B.), Sweden
Twenty-two members of 18 families with autism have been examined for the presence of mutations and abnormal methylation in the FMR-1 region at Xq27.3.All patients fulfilled diagnostic criteria of infantile autism. A characteristic pattern of insertion and methylation were detected after Southern blot analysis in 7 autistic individuals expressing the fragile site at Xq27.3. Normal DNA patterns were observed in 15 autistic boys cytogenetically negative for the fragile site. The results indicate a lack of involvement of the FMR-1 region in infantile autists negative for fragile X expression. o 1992 Wiley-Liss, Inc.
al., 19911. The responsible gene (FMR-1)has been isolated and includes a 200 base-pair (bp) fragment of repeated CGGs which are targets for mutations in the fra(X) syndrome [Kremer et al., 1991; Yu et al., 1991; Verkerk et al., 19911. Cloning of this region yielded a DNA probe, StB12.3, that detects both the abnormal methylation and the mutations in the FMR-1 gene [Oberle et al., 1991;Rousseau et al., 19911.The use of the probe or amplification of the region by the polymerase chain reaction provides the basis for a highly specific and sensitive test of the fra(X) mutation. Individuals with a low degree of expression or even carriers of the premutation can easily be detected [Rousseau et al., 1991;Fu et al., 19911.The use of a double digest with the restriction endonucleases EcoRI and EagI allows mutations to be detected and at the same time methylation KEY WORDS: fragile X syndrome, FMR-1 status to be determined [Malmgren et al., 19923.Normal gene, mutations males exhibit a 2.8 kb fragment, and normal females exhibit a 2.8 kb fragment and a 5.2 kb fragment resulting from the inactive X-chromosome. The premutation INTRODUCTION is detected as fragment(s1 slightly larger (< 500 bp) Infantile autism, originally described by Kanner than 2.8 kb but with no abnormal methylation. Carrier [19431, was first assumed to be a single disease. The females normally have a small insertion which gives concept today is that infantile autism is an hetero- rise t o 2 slightly larger fragments in addition to the geneous disorder [Coleman and Gillberg, 19851.A possi- normal ones. Affected males exhibit a large fragment or ble associationbetween autism and the fragile X [fra(X)] a smear of fragments above 5.2 kb. Affected females syndrome has been studied by several groups and has exhibit a large fragment or a smear as in affected males yielded conflicting results. Coexistence of autism and in addition to the normal fragments. the fra(X)syndrome was first reported in 1982 [Brownet In this study, we have investigated DNA from paal., 1982; Meryash et al., 19821. Subsequent studies tients diagnosed as cases of infantile autism for the have found a variable association [Watson et al., 1984; presence of abnormal methylation and mutations in the Blomquist et al., 1985; Brown et al., 1986; Bregman et FMR-1 region at Xq27.3. The purpose was to examine: al., 19881and one study has failed to detect any signifi- 1) if autistic individuals without fra(X) expression cant association [Einfield et al., 19891. showed an abnormal pattern in the FMR-1 region and, Recent studies showed that a region a t Xq27.3 is ab- 2) if autistic individuals expressing fra(X) differed in normally methylated in persons affected by the fra(X) mutation and methylation patterns as compared to nonsyndrome [Bell et al., 1991;Heitz et al., 1991;Vincent et autistic fra(X) carriers. Received for publication March 4, 1991; revision received June 16, 1992. Address reprint requests t o Dr. Niklas Dahl, Department of Clinical Genetics and Pediatrics, University Hospital, S-751 85 Uppsala, Sweden.
0 1992 Wiley-Liss, Inc.
MATERIALS AND METHODS Patients A total of 22 patients aged 4-47 years (21 males and one girl) had a diagnosis of infantile autism according to DSM-I11 criteria. Seven patients (6 boys, one girl) from 4
Autism and Fragile X families were included in the present study because they were positive for fra(X) expression from previous analyses (Table I). The remaining 15 patients, including 2 brothers, were chosen randomly as being autistic males (Table I). Cytogenetic analysis of the fra(X) site was performed according to standard procedures and at least 100 cells were analyzed. DNA Analysis Blood samples were collected from patients in EDTA vacutainer tubes. Patient leukocyte DNA was extracted and double digested with the restriction enzymes EcoRI and EagI, subjected to gelelectrophoresis, and transferred onto nylon membranes using standard methods. The DNA probe StB12.3 and the control probe F33 [Oberle et al., 1991; Rousseau et al., 19911 were radiolabeled by random priming (Pharmacia oligolabeling kit) and hybridized to the filters at 65°C in a hybridization solution (5 x SSPE, 5 x Denhart's buffer, 0.2% SDS, and 250 mg/ml yeast RNA). The filters were washed to a stringency of 0.1 x SSC and 0.1% SDS at 65°C and exposed to Kodak XAR5 films at - 70°C.
TABLE I. Distribution of Sex, Fra(X) Expression, and Age Among 22 Autistic Individuals Investigated Family 1
No. of patients 1 1 4 1 15
2 3 4 5-18
DISCUSSION In the present study we failed to detect any abnormal patterns in the FMR-1 region among 15 fra(X) negative autistic individuals. Seven autistic patients positive for fra(X) expression showed a typical pattern for individuals affected by the fra(X) syndrome. These results are consistent with the hypothesis that infantile autism is not primarily due to aberrations in the FMR-1 region. The conflicting data about the variable correlation between autism and the fra(X)syndrome can have many different causes. One reason is that different diagnostic criteria have been used for defining autism. Although most fra(X) males are not autistic, the majority have
Sex
% fra(X)
M
24 2 8-20 22 0
M M F M
~
Age (years)
12 21 10-47 20 4-36'
"One patient in this group also had cerebral gigantism.
some autistic features. The peculiar but characteristic pattern of inheritance in the fra(X) syndrome is not observed in families afflicted by autism without fra(X) expression. This speaks against amplification of the CGG-repeat in the FMR-1 gene being the primary defect in autism. However, the possibility of other allelic mutations has not be considered. The results of this study support the hypothesis that autism is a heterogeneous disease [Rapin, 19871. It has been suggested that the autistic features in patients with the fra(X) syndrome are caused by other genes on
RESULTS Cytogenetic Investigation Seven autistic individuals (6 boys, one girl) were earlier diagnosed as positive for fra(X) expression. Fifteen autistic boys, not subject to earlier cytogenetic investigation, were all negative for the fra(X) site. DNA Analyses DNA from the 7 patients with autism expressing the fragile site at Xq27.3 showed a similar pattern as seen in fra(X) patients. A large insertion (> 600 bp) was observed and the adjacent CpG island is methylated (Fig. 1). The large size of the mutation did not allow for a precise estimation of the number of CGC-repeat as described [Fu et al., 19911. The single autistic female investigated showed a pattern identical to that of a woman affected by the fra(X) syndrome with a smear of enlarged and methylated fragments (Fig. 2). Among 15 autistic boys negative for the fragile site Xq27.3, no alterations were detected with the probe StB12.3 (Fig. 2). There were no indications of insertions or deletions within the limit of the resolution (about 100 bp). Methylation patterns appeared normal as detected by EagI/ EcoRI digestion.
831
w 5.2 kb-
2.8 kb-
Fig. 1. Hybridization of the probe StB12.3 to DNA from a family including a son with fra(X) and autistic features. The DNA pattern of the son is similar to that ofmales with the fra(X) syndrome. The mother shows the pattern of a fra(X) carrier female and the sister expresses a full mutation.
832
Malmgren et al.
1
2
3
4
5
6
7
C
C
5.2 kb-
-5.2 kb
-2.8 kb
2.8 kb-
Fig. 2. The probe StB12.3 hybridized to DNA from autistic individuals. One fra(X) positive girl with autism (lane 1)and 6 fra(X) negative males with autism (lanes 2-71, For lane 7, a longer exposure has confirmed that no other bands exist than the 2.8 kb fragment. C, control DNA from normal females.
the X-chromosomeinfluenced by the fra(X) site [Cohen et al., 19911.Another possibility is that a nonfunctional FMR-1 gene may induce secondary nonspecific organic defects affecting the central nervous system. In conclusion, no indications of aberrations in the FMR-1 region in fra(X) negative autism were found in the present study. We suggest that mutations in the FMR-1 gene is not the primary cause of autism in fra(X) negative patients. Autism in fra(X) positive individuals is probably a secondary event caused by unknown mechanisms. Still, the behavioral phenotype in the fra(X) syndrome might have implications for the etiology of autism. The present study included a limited number of autistic patients and a larger collection of samples from autistic individuals will have to be investigated in order to confirm our hypothesis.
ACKNOWLEDGMENTS We thank Prof. J.-L. Mandel for providing the probes used in this study. This work was supported by grants from the Beijer Foundation, the Savstaholm Society,the Swedish Medical Research Council, and the Ehle Foundation.
REFERENCES Bell VM, Hirst MC, Nakahori Y, MacKinnon RN, Roche A, Flint TJ, Jacobs PA, Tommerup N, Tranebjaerg L, Froster-Iskenius U, Kerr B, Tbrner G, Lindenbaum RH, Winter R, Pembrey M, Thibodeau S, Davies KE (1991): Physical mapping across the fragile X: Hypermethylation and clinical expression of the fragile X syndrome. Cell 64861-866. Blomquist H K:son, Bohman M, Edvinsson SO, Gillberg C, Gustavson K-H, Holmgren G, Wahlstrom J (1985): Frequency of the fragile X syndrome in infantile autism. Clin Genet 27:113-117. Bregman JD, Lerkman J F , Ort SI (1988):Fragile X syndrome: Genetic predisposition to psychopathology. J Autism Dev Disord 18:343354. Brown WT, Jenkins EC, Cohen IL, Fisch GS, Wolf-Schein EG, Gross A, Waterhouse L, Fein D, Maison-Brothers A, Ritvo E, Ruttenberg BA, Bentley W, Castells S (1986): Fragile X and autism: A multicenter survey. Am J Med Genet 23:341-352. Brown WT, Jenkins EC, F’riedman E, Brooks J, Wisniewski K, Raguthu S, French J (1982): Autism is associated with the fragile X syndrome. J Autism Dev Disord 12:303-307. Cohen IL, Sudhalter V, Pfadt A, Jenkins EC, Brown WT, Vietze PM (19911: Why are autism and the fragile-X syndrome associated? Conceptual and methodlogical issues. Am J Hum Genet 48: 195-202. Coleman M, Gillberg C (EdsX1985): “The biology of the Autistic Syndromes.” New York F’raeger.
Autism and Fragile X Einfeld S, Molony H, Hall W (1989): Autism is not associated with the fragile X syndrome. Am J Med Genet 34:187-193. Fu Y-H, Kuhl DPA, Pizzuti A, Pieretti M, Sutcliffe JS, Richards S, Verkerk A, Holden J, Fenwick Jr R, Warren ST, Oostra BA, Nelson DL, Caskey CT (1991):Variation of the CGG repeat at the fragile X site results in genetic instability: Resolution of the Sherman paradox. Cell 67:1047-1058. Heitz D, Rousseau F, Devys D, Saccone S, Abderrahim H, Le Paslier D, Cohen D, Vincent A, Toniolo D, Della Valle G, Johnson S, Schlessinger D, Oberle I, Mandel J-L (1991):Isolation of sequences that span the fragile X and identification of a fragile X-related CpG island. Science 251:1236-1239. Kanner L (1943):Autistic disturbances of affective contact. Nerv Child 2:217-250. Kremer EJ, Pritchard M, Lynch M, Yu S, Holman K, Baker E, Warren ST, Schlessinger D, Sutherland GR, Richards RI (1991):Mapping of DNA instability a t the fragile X to a trinucleotide repeat sequence p(CCG)n. Science 252:1711-1714. Malmgren H, SteBn-Bondeson M-L, Gustavbon K-H, Seemanova E, Holmgren G , Over16 I, Mandel J-L, Pettersson u, Dahl N (1992): Methylation and mutation patterns in the fragile X syndrome, Am J Med Genet 43:268-278. Meryash DL, Szymanski LS, Gerald ps (1982):Infantile autism associated with the fragile X syndrome. J AutismDev Disord 12:2g5-301. Oberle I, Rousseau F, Heitz D, Kretz C, Devys D, Hanauer A, Boue J, Bertheas MF, Mandel J-L (1991): Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome. Science 252:1097- 1102.
833
Rapin I (1987): Searching for the cause of autism: A neurological perspective. In Cohen DJ, Donnellan AM (eds): “Handbook of Autism and Pervasive Developmental Disorders.” Silver Spring, MD: Winston, pp 710-717. Rousseau F, Heitz D, Biancalana V, Blumenfeld S, Kretz C, Boue J, Tommerup N, Van Der Hagen C, DeLozier-Blanchet C, Croquette M-F, Gilgenkrantz S,Jalbert P, Voelckel M-A, Oberle I, Mandel J-L (1991):Direct diagnosis by DNA analysis ofthe Fragile X syndrome of mental retardation. N Engl J Med 325:1673-1681. Verkerk AJMH, Pieretti M, Sutcliffe JS, Fu Y-H, Kuhl DPA, Pizzuti A, Reiner 0, Richards S, Victoria MF, Zhang F, Eussen BE, van Ommen G-JB. Blonden LAJ. Rieeens GJ. Chastain JL. Kunst CB. Galhard H, Caskey CT, NeIsG-DL, Oostra BA, Warren ST (1991). Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variations in fragile x syndrome, cell 65:905-914, Vincent A, Heitz D, Petit C, Kertz C, Oberle I, Mandel J-L (1991): Abnormal pattern detected in fragile-X patients by pulsed-field gel electrophoresis. Nature 349524-626. Watson MS, Leckman JF, Annex B, Breg WR, Boles D, Volkmar FR, Cohen DJ (1984):EkagileX in a sunrey of75 autistic males, NEngl J Med 310:1462. yu s,kitchard M, Kremer E, Lynch M, Nancarrow J , Baker E, Holman K, Mulley JC, Warren ST, Schlessinger D, Sutherland GR, Richards RI (1991):Fragile X genotype characterized by an unstable region of DNA, science 252:1179-1181,
