American Journal of Medical Genetics 35314-318 (1990)
Cytogenetic and Molecular Study of the Angelman Syndrome Kiyoshi Imaizumi, Fumio Takada, Yoshikazu Kuroki, Kenji Naritomi, Junichi Hamabe, and Norio Niikawa Division of Medical Genetics, Kanagawa Children’s Medical Center, Kanagawa (K.I.,F.T., Y.K.); Department of Pediatrics, Ryukyu University School of Medicine, Okinawa (K.N.);Department of Human Gen.etics, Nagasaki University School of Medicine, Nagasaki (J.H., N.N.), Japan. Six patients, including two sibs, with Angelman syndrome (AS; three females and three males, aged 11to 18 years) were studied cytogenetically. Molecular analysis was also performed. Using high-resolution banding technique, we detected a microdeletion in the proximal region of chromosome 15q in four cases. The deleted segment was heterogenous between these patients, and the common deleted region appeared to be 15q11.2. Four patients with deleted 15q were all sporadic cases, whereas in the sib cases we could not detect a visible deletion in the long arm of chromosome 15. However, there was no clinical difference between sporadic cases and sib cases. Densitometric analysis of autoradiographic bands of Southern hybridization using two DNA segments, pML34 and pTD3-21,as probes demonstrated that two patients had only one copy for each of the probes. In the remaining four patients, including the sibs, two copies of each sequence were retained. The probes used here detect a molecular deletion in most Prader-Willi syndrome patients. Thus the segment causing AS is localized adjacent to the critical segment of Prader-Willi syndrome. There seemed to be heterogeneity for the molecular deletion within AS individuals.
KEY WORDS: deletion 15q, molecular deletion, Prader-Willi syndrome INTRODUCTION In 1965, Angelman described the disorder of severe mental retardation, absent speech, seizure, easily provoked paroxysms of laughter, and a puppet-like jerky Fkceived for publication January 5,1989; revision received July 5, 1989. Address reprint requests to Kiyoshi Imaizumi, Kanagawa Children’s Medical Center, Mutsugawa 2-138-4, Minamiku Yokohama, Japan.
0 1990 Wiley-Liss, Inc.
gait. It was coined “the happy-puppet syndrome,” which is generally known as Angelman syndrome (AS).Approximately 80 patients with AS have been reported, and most cases are sporadic [Angelman, 1965; Bower and Jeavons, 1967; Berg and Pakula, 1972; Bergreen, 1972; Kibel and Burness, 1983; Massey and Roy, 1973; Mayo et al., 1973; Moor and Jeavons, 1973; Elian, 1975; Batenburg-Plenter, 1976; Godfrey, 1976; Hala and Cagnon, 1976; Pelc et al., 1976; Williams and Frias, 1982; Bjerre e t al., 1984; Dooly et al., 1984; Eber et al., 1986; Pascul e t al., 1986; Kaplan et al., 1987; Magenis et al., 1987; Dorries et al., 1988; Pembrey et al., 1989; Donlon, 1988; Knoll et al., 1989; Williams e t al., 1989a,bl. Four reports of affected sibs and a n affected monozygotic twin pair have been described CKuroki et al., 1980; Hersh et al., 1981; Pashayan et al., 1982; Fisher et al., 1987; Willems et al., 19871. In most cases, chromosomes were reported to be normal except for two cases, one with a pericentric inversion of chromosome 3 with no evident chromosome loss [Williams and Frias, 19821 and the other with a 21p - chromosome [Pashayan et al., 19821. Recently, with the development of high-resolution chromosome banding techniques, Kaplan et al. [1987] described three patients with a n interstitial deletion of chromosome 15qll-q12, which had been detected in more than 50% of the F’rader-Willi syndrome (PWS) patients. One was a girl with AS, one was a boy with Williams syndrome, and one was a girl with an unknown diagnosis. Magenis et al. [1987J also reported three unrelated girls with AS having a deletion of the proximal long arm of chromosome 15,bands q l l - q l 3 in all cells. To investigate the relationship between AS and the 15q interstitial deletion, we performed cytogenetic analyses on six Japanese patients with typical AS by high-resolution banding techniques. In addition, we investigated molecular deletion analysis with Southern blot hybridization using two clonal DNA segments specific for the 15qll-q12 region as probes that could detect a molecular deletion in most PWS patients.
MATERIALS AND METHOD Patients We studied six patients (three females and three males; Fig. 1) aged 11 to 18 years. They had clinical
Angelman Syndrome manifestations of AS such as severe mental retardation (IQs 20-401, absent speech, seizure disorder, easily provoked paroxysms of laughter, and puppet-like jerky gait. Four patients (Nos. 1, 2, 3, and 6) were sporadic cases, while two patients (4 and 5) were sibs. The clinical manifestations of six patients are shown in Table I. Secondary sexual characteristics were appropriate for chronologic age.
Fig. 7. Facial photographs ofpatients 1(A) at age 8 years, 2 (B) at 16 years, 3 (C) at 9 years, 4 (D) at 6 years, 5 (E) at 4 years, and 6 (F) at 11 years. Patients 4 and 5 are sibs.
Cytogenetic Analysis High-resolution chromosome preparations [Yunis, 19761were obtained from peripheral blood lymphocytes. The chromosomes were stained by the GTG-banding method [Seabright, 19711.Particular attention was paid to chromosome 15.
Southern Hybridization The probes used in the present study were pTD3-21, pML34, and pPAl. Two probes, pTD3-21 and pML34 (kindly provided by Dr. S.A. Latt, Boston), are 2.2 kb and 6.4 kb Hind I11 subclones of A-phage libraries, respectively, from a flow-sorted inverted duplication chromosome 15 and were mapped to the region 15qll-q12 [DonIon et al., 19861. A 0.67 kb human prealbumin cDNA clone, pPAl [Mita et al., 19841, assigned to 18qll-q11.2 [Jinno et al., 19861, was used as a n internal standard for a comparative analysis of hybridization density. Genomic DNA of each patient extracted by the standard method from their peripheral blood leukocytes was digested by a n endonuclease Hind 111. Southern blots of each DNA sample on nylon membranes were hybridized with the triple probes, which are described elsewhere [Kamei et al., 19881. The gene doses ofboth pTD3-21 and pML34 in each patient were determined by measuring the hybridization densities of the two probes and comparing them with that for pPA1. Densitometric Analysis The gene doses of both pTD3-21 and pML34 in each individual were determined by taking the average of ratios of their hybridization densities to the pPA1 gene obtained from densitometric analysis of several separate hybridizations.
TABLE I. Clinical Manifestations in Six Patients With Angelman Syndrome and Those of Reported Cases Patients" Clinical manifestations 1 2 3 4 5 6 Reported cases (%) 15 16 15 18 17 11 Age Sex F M M F F M MIF = 20132 General Severe mental retardation + + t + + + 52/52 (100) Paroxysms of laughter + + t + + + 45152 (87) Absent speech + + + + t t 48/48 (100) Craniofacial + + + + + 36/50 (72) Microcephaly 36/45 (78) i t + + + ? Brachycephaly 16/26 (62) - + Occipital groove 25/28 (89) + + + + + Macrostomia 41/49 (84) + t + Tongue protrusion 44149 (90) + + + + + + Mandibular prognathism Neurological 41/42 (98) + + + + + + Stiff gait 46/51 (90) + + + + + + Seizure disorder 38/51 (76) + + + + + + Hyper-reflexia Abnormal EEG I& "Patients 4 and 5 are sibs.
315
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Fig. 2. Chromosome 15 pairs of patients by GTG-banding. A: From patients 1, 2, and 3. The deleted chromosomes arc represented on the right side of each pair, and breakpoints are denoted by arrows. B:From patients 4 and 5. No visible deletion was found.
RESULTS Cytogenetic Analysis An interstitial microdeletion of the proximal long arm of one chromosome 15 was detected in most cells of four patients (1, 2, 3, and 6; Fig. 2 ) . In patient 1 eight cells could be scored, and six of them appeared to have a n interstitial deletion of chromosome 15. bands qll-q12. In the remaining cells, the presence of a chromosome deletion was uncertain. In the same way, seven of eight cells in patient 2 and five of six cells in patient 3 appeared to have a deletion of 15q11.2-ql2. An R-banding technique was used in the analysis of patient 6; we could detect clearly a n interstitial deletion in chromosome 15, band q11.2, in five of six cells (Fig. 3).Patients who were proved to have the chromosome deletion were all sporadic cases. In patients 4 and 5, no microscopic deletion could be detected in the proximal long arm of chromosome 15 in spite of a n extremely careful search (Fig. 2). No mosaicism was observed in any cases. Gene-Dose Studies Probes pTD3-21 and pML34 hybridized to a 6.4 kb and a 2.2. kb Hind I11fragment. The results of the gene-dose studies on each individuals are shown in Table I1 and Figure 4.In two patients with AS and one patient with PWS who have a chromosome deletion in 15q11.2-ql2, the hybridization densities of both probes to the pPAl gene density showed a single copy. The rest, including the two sib cases and one normal control, showed two copies. DISCUSSION As the clinical manifestation of AS showed minimal variability, the d i a p o s i s of AS was not as confusing as
U
RBGder[151 Fig. 3. Chromosome 15 pairs ofpatient 6 hy R-banding. Right-sided chromosomes are those with l5q11.2 deletion.
that of PWS. In the AS patients, four sporadic cases, except the sib pair, had a n interstitial deletion of 15q. The deleted region was identical in three patients (15q11.2-ql2); one patient had a microdeletion of l5q11.2 alone. The smallest region compatible with AS was supposed to be 15q11.2. In 1987, Kaplan et al. described three patients with heterogenous clinical manifestation who had a n interstitial deletion of 15q11.2. One ofthem was a typical As patient. In addition, cases with cytogenetic deletion of the long arm of chromosome 15 in AS have been reported [Magenis et al., 1987; Donlon, 1988; Pembrey et al.,
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TABLE 11. Gene Dose of the Genomic Sequence Corresponding to pML34 and pTD3-21 in AS and PWS patients." Copy No. of fragment
Corrected density ratio Patient
CIA 1.2
0.9 0.9 2.1 2.4 1.9 1.9 2.0
0.9 0.9 1.9 2.4 1.9 1.9 2.0
PWS AS 1
BIA 1.2
2 3
4 5 6 Control
B
C
1
1
DNA deletion
1
1 1 2 2 2 2 2
DNA deletion DNA deletion No DNA deletion No DNA deletion No DNA deletion No DNA deletion No DNA deletion
1 2 2 2 2 2
Interpretation
*A. 4.6 kb fragment for the prealbumin gene as a n inner standard; B, 6.4 kh fragment for pML34; C, 2.2 kb fragment for pTD3-21. Corrected ratios are calculatcd by dividing B/A or CIA ratios each by the average ratio among normal cuntrols.
cytogenetic deletions and in two sibs without deletions, we could not detect the identical molecular deletions. Four patients with AS having a cytogenetic deletion of 6.4 15qll-13 were analyzed using five probes [Knoll et al., -PMW 19891, and all were demonstrated to have deletions of every five cloned DNA segments. Donlon I19881 re*PA1 4. 6 ported that five of six patients with PWS and two of three with AS, all having a 15q11.2 deletion, were shown to have deletions of five of nine probes. In two reports, DNA segments demonstrating molecular deletions were identical in patients with AS and in patients with PWS. The compound recessive disorder was suggested [Donlon, 19881, and the clinical heterogeneity 2.2 -pTD3-21 between PWS and AS patients would be based on a function of different combinations of abnormal recessive alleles a t separate but closely mapping loci. Fourteen 2 2 2 1 1 1 2 2 patients with AS had been analyzed molecularly, and nine patients were proved t o have molecular deletions. Fig. 4. Autoradiograms of patients with the use of pML34 and pTD3-21 as probes and pPA1 as a n inncr control probe. Patient number The rate of detection of deletion in AS was less frequent is shown above the column, and copy number of genes (genomic sethan that in PWS patients. These results suggest that quence corresponding to pML34 and pTD3-21) is shown at the hottom. the critical region of PWS was separated from the region of AS and that the probes used here might be situated between two critical regions. Furthermore, these probes might lie closer to the region of PWS than t o that of AS. In contrast to the paternal transmission of the deleted 1989; Williams et a]., 19891; and the detectable rate of cytogenetic deletion in AS was about 60%.These reports chromosome 15 observed in the majority of PWS pasupport the involvement ofthe critical region of 15ql1.2. tients, the maternal inheritance of the deleted chromoIn cytogenetic examinations of patients with AS some 15 was demonstrated in five cases with AS: Four [Pembrey et al., 19891, six patients had a chromosome were detected cytogenetically [Cooke et al., 19881 and deletion of 15q12, while one sib pair appeared not to one molecularly LWilliams et al., 19881. In addition, five have a chromosome deletion. This suggests that the cases with AS were detected to be maternal in origin inheritance in AS might be a combination of 15qll-13 [Knoll et a]., 1988; Williams, 19891; however, one paterchromosome deletion and of autosomal recessive inheri- nal origin of the deleted chromosome was demonstrated tance. This hypothesis seems to be reasonable, because in a patient with AS [Donlon, 19881. The difference in the empiric recurrence risk of AS was estimated t o be parental origin of deletions involving AS and PWS 1-2% based on study of 48 cases [Willems et al., 19871. seems to be important to clarify the cause of AS. ParenThe molecular analyses of our six patients with AS tal imprinting is hypothesized to be a reason for the were performed using two probes, pTD3-21 and pML34. difference in clinical expression between AS and PWS which had been reported to detect molecular deletion in patients [Knoll et al., 19891. Reported cases, however, the majority of PWS patients [Kamei et al., 19891. The were too small to detect the mode of inheritance of AS. molecular deletion was confirmed in only two cases with Further molecular and cytogenetic studies are needed to visible 15q deletion; however, in another two cases with elucidate the mechanism of AS. Pwsl
kb
2
3
4
5
6
C
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REFERENCES Angelman H 11965):“Puppet” children: A report on three cases. Dev Med Neurol 7:681-688. Batenburg-Plenter AM (1976): En Zeldzaam(?) “vrolijk” oligofreen syndrome. Ned Tjidschr Geneeskd 120:1528-1530. Berg JM, Pakula Z (1972): Angelman’s(“happy puppet”) syndrome. Am J Dis Child 123172-74. Bergreen S (1972): “Happy puppet” syndrome. Ugeskr Laeger 134:1174. Bjerre I, Fagher B, Ryding E, Rosen I (1984): The Angelman or “happy puppet” syndrome. Clinical and electroencephalographic features and cerebral blood flow. Acta Paediatr Scand 73:398-402. Bower BD, Jeavons PM (1967): The “happy puppet” syndrome. Arch Dis Child 42:298-302. Donlon TA (1988):Similar molecular deletions of chromosome 15q11.2 are encountered in both the Prader-Willi and Angelman syndromes. Hum Genet 80:322-328. Donlon TA, Lalande M, Wyman A, Bruns G, Latt SA (1986):Isolation of molecular probes associated with the chromosome 15 instability in the F’rader-Willi syndrome. R o c Natl Acad Sci USA 83:4408-4412 (correction 83:6964). Dooly JM, Berg JM, Pakula Z, MacGregor DL (1981):The puppet-like syndrome of Angelman. Am J Dis Child 135:621-624. Dorries A, Spohr HL, Kunze J (1988): Angelman (“happy puppet”) syndrome-Seven new cases documented by cerebral computed tomography: Review of the literature. Eur J Pediatr 148:270-273. Eber SW, Joost S, Gabriel M (1986):Angelman syndrome. Monatsschr Kinderheilkd 134:158-160. Elian M (1975): Fourteen happy puppets. Clin Pediatr 14:902-908. Fisher JA, Burn J, Alexander FW, Gardner-Medwin D (1987): Angelman (happy puppet) syndrome in a girl and her brother. J Med Genet 24:294-298. Godfrey M (1976): Happy puppet syndrome. Nurs Mirror 143:45-46. Hala F, Chagnon J (1976): Le syndrome de la “Marionnette Joyeuse” Union Md Can 105:1077-1083. Hersh JH, Bloom AS, Zimmerman AW, Dinno ND, Breenstein RM, Weisskopf B, Reese AH (1981): Behavioral correlates in the happy puppet syndrome: A characteristic profile’?Dev Med Child Neruol 23:792-800. Kamei T, Hamabe J , Matsumoto T, Abe K, Harada N, Ishikiriyama S, Hasegawa T, Miymaki K, Mizuno S, Narahara K, Yukizane S, Niikawa N (1988): A molecular deletion study with Southern hybridization on typical Prader-Willi syndrome (PWS) patients with various chromosome abnormalities involving 15q11-12 and on an atypical PWS patient with apparently normal karyotype. Jpn Hum Genet 33:477-486. Kaplan LC, Wharton R, Elias E, Mamdel F, Donlon T, Latt S (1987): Clinical heterogeneity associated with deletions in the long arm of
chromosome 15: Report of 3 new cases and their possible genetic significance. Am J Med Genet 28:45-53. Kibel MA, Burncss FR 11973):The “happy puppet” syndrome. Centr Afr J Med 19:91-93. Knoll JHM, Nicholls RD, Magenis RE, Graham J M Jr, Lalande M, Latt SA (1989):Angelman and Yrader-Willi syndrome share a common chromosome 15 deletion but differ in parental origin of the deletion. Am J Med Genet 32:285-290. Kuroki Y, Matsui I, Yamamoto Y, Ieshima A (1980): The “happy puppet” syndrome in two siblings. Hum Genet 56:227-229. Magenis RE, Brown MG, Lacy DA, Budden S, LaFranchi S (1987):Is A4ngelmansyndrome an alternate result of de1(15)!qllq13)? Am J Med Genet 28329-838. Massey JY, Roy FH 11973):Ocular manifestations of the happy puppet syndrome. J Pediatr Ophthalmol 10:282-284. Mayo 0, Nelson MM, Townsend HRA (1973):Three more “happy puppets.” Dev Med Child Neurol 15:63-74. Moor JR, Jeavons PM (1973): The “happy puppet” syndrome: Two new cases and a review of five previous cases. Neuropaedatrie 4:172-179. Pascul Pascul SI, CamposTullot R, Escudero B, Garcia-Simon R (1986): Sindrome (happy puppet) o de Angelman. %vision a proposito de tres casos. An Esp Pediatr 24:311-316. Pashayan JM, Singer W, Bove C, Eisenberg E, Seto B (1982): The Angelman syndrome in two brothers. Am J Med Genet 13:295-298. Pelc S,Levy J, Point G (1976):“Happy puppet” syndrome ou syndrome du “pantin hilare.” Helv Paediatr Acta 31:183-188. Pembrey M, Fennel1 SJ, Van Den Berghe J, Fitchett M, Summers D, Butler L, Clarke C, Griffiths M, Thompson E, Super M, Barailser M (1989): The association of Angelman’s syndrome with deletions within 15qll-13. J Med Genet 26:73-77. Rolando S, Schenone R, Shappapieta M (1983):La sindrome di Angelman. Minerva Pediatr 35:727-731. Seabright M (1971): A rapid banding technique for human chromosomes. Lancet 2:271-272. Willems P J , Dijkstra I, Oebele F, Smit PG (1987): Recurrence risk in the Angelman (“happy puppet”) syndrome. Am J Med Genet 27:773-780. Williams CA, F’rias J L (1982): The Angelman (“happy puppet”) syndrome. Am J Med Genet 11:453-460. Williams CA, Gray BA, Hendrickson JE, Stone JW, Cantu ES 11989a): Incidence of 15q deletions in the Angelman syndrome: A survey of 12 affected persons. Am J Med Genet 32:339-345. Williams CA, Hendrickson J E , Cantu ES, Donlon TA (1989bJ:Angelman syndrome in a daughter with de1(15)(qllq13iassociated with brachycephaly, hearing loss, enlarged foramen magnum, and ataxia in the mother. Am J Med Genet 32:333-338. Yunis JJ (1976): High resolution of human chromosomes. Science 19:1268- 1270.
Cytogenetic and Molecular Study of the Angelman Syndrome Kiyoshi Imaizumi, Fumio Takada, Yoshikazu Kuroki, Kenji Naritomi, Junichi Hamabe, and Norio Niikawa Division of Medical Genetics, Kanagawa Children’s Medical Center, Kanagawa (K.I.,F.T., Y.K.); Department of Pediatrics, Ryukyu University School of Medicine, Okinawa (K.N.);Department of Human Gen.etics, Nagasaki University School of Medicine, Nagasaki (J.H., N.N.), Japan. Six patients, including two sibs, with Angelman syndrome (AS; three females and three males, aged 11to 18 years) were studied cytogenetically. Molecular analysis was also performed. Using high-resolution banding technique, we detected a microdeletion in the proximal region of chromosome 15q in four cases. The deleted segment was heterogenous between these patients, and the common deleted region appeared to be 15q11.2. Four patients with deleted 15q were all sporadic cases, whereas in the sib cases we could not detect a visible deletion in the long arm of chromosome 15. However, there was no clinical difference between sporadic cases and sib cases. Densitometric analysis of autoradiographic bands of Southern hybridization using two DNA segments, pML34 and pTD3-21,as probes demonstrated that two patients had only one copy for each of the probes. In the remaining four patients, including the sibs, two copies of each sequence were retained. The probes used here detect a molecular deletion in most Prader-Willi syndrome patients. Thus the segment causing AS is localized adjacent to the critical segment of Prader-Willi syndrome. There seemed to be heterogeneity for the molecular deletion within AS individuals.
KEY WORDS: deletion 15q, molecular deletion, Prader-Willi syndrome INTRODUCTION In 1965, Angelman described the disorder of severe mental retardation, absent speech, seizure, easily provoked paroxysms of laughter, and a puppet-like jerky Fkceived for publication January 5,1989; revision received July 5, 1989. Address reprint requests to Kiyoshi Imaizumi, Kanagawa Children’s Medical Center, Mutsugawa 2-138-4, Minamiku Yokohama, Japan.
0 1990 Wiley-Liss, Inc.
gait. It was coined “the happy-puppet syndrome,” which is generally known as Angelman syndrome (AS).Approximately 80 patients with AS have been reported, and most cases are sporadic [Angelman, 1965; Bower and Jeavons, 1967; Berg and Pakula, 1972; Bergreen, 1972; Kibel and Burness, 1983; Massey and Roy, 1973; Mayo et al., 1973; Moor and Jeavons, 1973; Elian, 1975; Batenburg-Plenter, 1976; Godfrey, 1976; Hala and Cagnon, 1976; Pelc et al., 1976; Williams and Frias, 1982; Bjerre e t al., 1984; Dooly et al., 1984; Eber et al., 1986; Pascul e t al., 1986; Kaplan et al., 1987; Magenis et al., 1987; Dorries et al., 1988; Pembrey et al., 1989; Donlon, 1988; Knoll et al., 1989; Williams e t al., 1989a,bl. Four reports of affected sibs and a n affected monozygotic twin pair have been described CKuroki et al., 1980; Hersh et al., 1981; Pashayan et al., 1982; Fisher et al., 1987; Willems et al., 19871. In most cases, chromosomes were reported to be normal except for two cases, one with a pericentric inversion of chromosome 3 with no evident chromosome loss [Williams and Frias, 19821 and the other with a 21p - chromosome [Pashayan et al., 19821. Recently, with the development of high-resolution chromosome banding techniques, Kaplan et al. [1987] described three patients with a n interstitial deletion of chromosome 15qll-q12, which had been detected in more than 50% of the F’rader-Willi syndrome (PWS) patients. One was a girl with AS, one was a boy with Williams syndrome, and one was a girl with an unknown diagnosis. Magenis et al. [1987J also reported three unrelated girls with AS having a deletion of the proximal long arm of chromosome 15,bands q l l - q l 3 in all cells. To investigate the relationship between AS and the 15q interstitial deletion, we performed cytogenetic analyses on six Japanese patients with typical AS by high-resolution banding techniques. In addition, we investigated molecular deletion analysis with Southern blot hybridization using two clonal DNA segments specific for the 15qll-q12 region as probes that could detect a molecular deletion in most PWS patients.
MATERIALS AND METHOD Patients We studied six patients (three females and three males; Fig. 1) aged 11 to 18 years. They had clinical
Angelman Syndrome manifestations of AS such as severe mental retardation (IQs 20-401, absent speech, seizure disorder, easily provoked paroxysms of laughter, and puppet-like jerky gait. Four patients (Nos. 1, 2, 3, and 6) were sporadic cases, while two patients (4 and 5) were sibs. The clinical manifestations of six patients are shown in Table I. Secondary sexual characteristics were appropriate for chronologic age.
Fig. 7. Facial photographs ofpatients 1(A) at age 8 years, 2 (B) at 16 years, 3 (C) at 9 years, 4 (D) at 6 years, 5 (E) at 4 years, and 6 (F) at 11 years. Patients 4 and 5 are sibs.
Cytogenetic Analysis High-resolution chromosome preparations [Yunis, 19761were obtained from peripheral blood lymphocytes. The chromosomes were stained by the GTG-banding method [Seabright, 19711.Particular attention was paid to chromosome 15.
Southern Hybridization The probes used in the present study were pTD3-21, pML34, and pPAl. Two probes, pTD3-21 and pML34 (kindly provided by Dr. S.A. Latt, Boston), are 2.2 kb and 6.4 kb Hind I11 subclones of A-phage libraries, respectively, from a flow-sorted inverted duplication chromosome 15 and were mapped to the region 15qll-q12 [DonIon et al., 19861. A 0.67 kb human prealbumin cDNA clone, pPAl [Mita et al., 19841, assigned to 18qll-q11.2 [Jinno et al., 19861, was used as a n internal standard for a comparative analysis of hybridization density. Genomic DNA of each patient extracted by the standard method from their peripheral blood leukocytes was digested by a n endonuclease Hind 111. Southern blots of each DNA sample on nylon membranes were hybridized with the triple probes, which are described elsewhere [Kamei et al., 19881. The gene doses ofboth pTD3-21 and pML34 in each patient were determined by measuring the hybridization densities of the two probes and comparing them with that for pPA1. Densitometric Analysis The gene doses of both pTD3-21 and pML34 in each individual were determined by taking the average of ratios of their hybridization densities to the pPA1 gene obtained from densitometric analysis of several separate hybridizations.
TABLE I. Clinical Manifestations in Six Patients With Angelman Syndrome and Those of Reported Cases Patients" Clinical manifestations 1 2 3 4 5 6 Reported cases (%) 15 16 15 18 17 11 Age Sex F M M F F M MIF = 20132 General Severe mental retardation + + t + + + 52/52 (100) Paroxysms of laughter + + t + + + 45152 (87) Absent speech + + + + t t 48/48 (100) Craniofacial + + + + + 36/50 (72) Microcephaly 36/45 (78) i t + + + ? Brachycephaly 16/26 (62) - + Occipital groove 25/28 (89) + + + + + Macrostomia 41/49 (84) + t + Tongue protrusion 44149 (90) + + + + + + Mandibular prognathism Neurological 41/42 (98) + + + + + + Stiff gait 46/51 (90) + + + + + + Seizure disorder 38/51 (76) + + + + + + Hyper-reflexia Abnormal EEG I& "Patients 4 and 5 are sibs.
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Fig. 2. Chromosome 15 pairs of patients by GTG-banding. A: From patients 1, 2, and 3. The deleted chromosomes arc represented on the right side of each pair, and breakpoints are denoted by arrows. B:From patients 4 and 5. No visible deletion was found.
RESULTS Cytogenetic Analysis An interstitial microdeletion of the proximal long arm of one chromosome 15 was detected in most cells of four patients (1, 2, 3, and 6; Fig. 2 ) . In patient 1 eight cells could be scored, and six of them appeared to have a n interstitial deletion of chromosome 15. bands qll-q12. In the remaining cells, the presence of a chromosome deletion was uncertain. In the same way, seven of eight cells in patient 2 and five of six cells in patient 3 appeared to have a deletion of 15q11.2-ql2. An R-banding technique was used in the analysis of patient 6; we could detect clearly a n interstitial deletion in chromosome 15, band q11.2, in five of six cells (Fig. 3).Patients who were proved to have the chromosome deletion were all sporadic cases. In patients 4 and 5, no microscopic deletion could be detected in the proximal long arm of chromosome 15 in spite of a n extremely careful search (Fig. 2). No mosaicism was observed in any cases. Gene-Dose Studies Probes pTD3-21 and pML34 hybridized to a 6.4 kb and a 2.2. kb Hind I11fragment. The results of the gene-dose studies on each individuals are shown in Table I1 and Figure 4.In two patients with AS and one patient with PWS who have a chromosome deletion in 15q11.2-ql2, the hybridization densities of both probes to the pPAl gene density showed a single copy. The rest, including the two sib cases and one normal control, showed two copies. DISCUSSION As the clinical manifestation of AS showed minimal variability, the d i a p o s i s of AS was not as confusing as
U
RBGder[151 Fig. 3. Chromosome 15 pairs ofpatient 6 hy R-banding. Right-sided chromosomes are those with l5q11.2 deletion.
that of PWS. In the AS patients, four sporadic cases, except the sib pair, had a n interstitial deletion of 15q. The deleted region was identical in three patients (15q11.2-ql2); one patient had a microdeletion of l5q11.2 alone. The smallest region compatible with AS was supposed to be 15q11.2. In 1987, Kaplan et al. described three patients with heterogenous clinical manifestation who had a n interstitial deletion of 15q11.2. One ofthem was a typical As patient. In addition, cases with cytogenetic deletion of the long arm of chromosome 15 in AS have been reported [Magenis et al., 1987; Donlon, 1988; Pembrey et al.,
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TABLE 11. Gene Dose of the Genomic Sequence Corresponding to pML34 and pTD3-21 in AS and PWS patients." Copy No. of fragment
Corrected density ratio Patient
CIA 1.2
0.9 0.9 2.1 2.4 1.9 1.9 2.0
0.9 0.9 1.9 2.4 1.9 1.9 2.0
PWS AS 1
BIA 1.2
2 3
4 5 6 Control
B
C
1
1
DNA deletion
1
1 1 2 2 2 2 2
DNA deletion DNA deletion No DNA deletion No DNA deletion No DNA deletion No DNA deletion No DNA deletion
1 2 2 2 2 2
Interpretation
*A. 4.6 kb fragment for the prealbumin gene as a n inner standard; B, 6.4 kh fragment for pML34; C, 2.2 kb fragment for pTD3-21. Corrected ratios are calculatcd by dividing B/A or CIA ratios each by the average ratio among normal cuntrols.
cytogenetic deletions and in two sibs without deletions, we could not detect the identical molecular deletions. Four patients with AS having a cytogenetic deletion of 6.4 15qll-13 were analyzed using five probes [Knoll et al., -PMW 19891, and all were demonstrated to have deletions of every five cloned DNA segments. Donlon I19881 re*PA1 4. 6 ported that five of six patients with PWS and two of three with AS, all having a 15q11.2 deletion, were shown to have deletions of five of nine probes. In two reports, DNA segments demonstrating molecular deletions were identical in patients with AS and in patients with PWS. The compound recessive disorder was suggested [Donlon, 19881, and the clinical heterogeneity 2.2 -pTD3-21 between PWS and AS patients would be based on a function of different combinations of abnormal recessive alleles a t separate but closely mapping loci. Fourteen 2 2 2 1 1 1 2 2 patients with AS had been analyzed molecularly, and nine patients were proved t o have molecular deletions. Fig. 4. Autoradiograms of patients with the use of pML34 and pTD3-21 as probes and pPA1 as a n inncr control probe. Patient number The rate of detection of deletion in AS was less frequent is shown above the column, and copy number of genes (genomic sethan that in PWS patients. These results suggest that quence corresponding to pML34 and pTD3-21) is shown at the hottom. the critical region of PWS was separated from the region of AS and that the probes used here might be situated between two critical regions. Furthermore, these probes might lie closer to the region of PWS than t o that of AS. In contrast to the paternal transmission of the deleted 1989; Williams et a]., 19891; and the detectable rate of cytogenetic deletion in AS was about 60%.These reports chromosome 15 observed in the majority of PWS pasupport the involvement ofthe critical region of 15ql1.2. tients, the maternal inheritance of the deleted chromoIn cytogenetic examinations of patients with AS some 15 was demonstrated in five cases with AS: Four [Pembrey et al., 19891, six patients had a chromosome were detected cytogenetically [Cooke et al., 19881 and deletion of 15q12, while one sib pair appeared not to one molecularly LWilliams et al., 19881. In addition, five have a chromosome deletion. This suggests that the cases with AS were detected to be maternal in origin inheritance in AS might be a combination of 15qll-13 [Knoll et a]., 1988; Williams, 19891; however, one paterchromosome deletion and of autosomal recessive inheri- nal origin of the deleted chromosome was demonstrated tance. This hypothesis seems to be reasonable, because in a patient with AS [Donlon, 19881. The difference in the empiric recurrence risk of AS was estimated t o be parental origin of deletions involving AS and PWS 1-2% based on study of 48 cases [Willems et al., 19871. seems to be important to clarify the cause of AS. ParenThe molecular analyses of our six patients with AS tal imprinting is hypothesized to be a reason for the were performed using two probes, pTD3-21 and pML34. difference in clinical expression between AS and PWS which had been reported to detect molecular deletion in patients [Knoll et al., 19891. Reported cases, however, the majority of PWS patients [Kamei et al., 19891. The were too small to detect the mode of inheritance of AS. molecular deletion was confirmed in only two cases with Further molecular and cytogenetic studies are needed to visible 15q deletion; however, in another two cases with elucidate the mechanism of AS. Pwsl
kb
2
3
4
5
6
C
318
Imaizumi et al.
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