American Journal of Medical Genetics 3522-27 (1990)
Fragile X Frequency in a Mentally Retarded Population in Brazil Regina C. Mingroni-Netto, Carla Rosenberg, Angela M. Vianna-Morgante, and Rita de Cassia M. Pavanello Human Genetics Laboratory, Department of Biology, University of Scio Paulo, SP, Brazil Seventy-five male and 50 female students from 2 special schools for mildly, moderately retarded, or borderline individuals were screened clinically and cytogenetically in order to estimate the contribution of fragile X [fra(X)]syndrometo the cause of mental retardation in Brazil. We found 6 males (8%)from 4 families and 2 unrelated females (4%) with fra(X) chromosomes. One male and one female were isolated cases. The estimated frequency of Martin-Bell [fra(X)] syndrome among mentally impaired individuals in Brazil was similar to that previously reported in other countries.
KEY WORDS: Martin-Bellsyndrome, mental retardation survey, X-linked mental retardation
INTRODUCTION Martin-Bell or fragile X [fra (X)] syndrome is a n X-linked form of mental retardation associated with a fragile site at Xq27.3, which is expressed in a variable proportion in cells of carriers of the gene. This fragile site is expressed in culture conditions of low levels of folic acid [Sutherland, 19791. This syndrome is known to be the second most frequent cause of genetic mental retardation, and its prevalence is estimated to be 4.4 x among males and 4.1 x among females [Sherman et al., 19841. In screening populations of mentally retarded males, 0.5% to 10.0% of unselected retarded individuals have the fra(X) chromosome. In similar studies in female populations, frequencies from 0% to 7% have been obtained.
Received for publication September 20,1988; revision received, May 30, 1989. Address reprint requests t o Angela M. Vianna-Morgante, Departamento de Biologia, Universidade de SSio Paulo, Caixa Postal 11461; 05499, Sgo Paulo, Brazil.
0 1990 Wiley-Liss, Inc.
In the present study, we estimated, for the first time, the fra(X) contribution to mental retardation in a Brazilian population. Our work was carried out in 2 special schools for mildly, moderately retarded, or borderline individuals.
MATERIALS AND METHODS Patients The 75 males and 50 females were ascertained in 2 special schools for mildly or moderately retarded and borderline individuals (Centro Ocupacional Avanhandava and Centro de HabilitaGao Profissional Sao Joao Batista) in the city of Sao Paulo, Brazil. All students from Sao Jog0 Batista school, enrolled from 1984 to 1987 (55 males and 42 females), were studied cytogenetically, while a t Avanhandava school (20 males and 8 females) consent could not be obtained in all cases. The age of the patients ranged from 6 to 40 years and most were between 10-20 years; they were included in the cytogenetic study regardless of their family history, clinical findings, and previous diagnosis. Clinical Studies All patients from both schools were evaluated clinically. In most males, length (1) and width (w)ofthe testes were measured with a caliper and the volume (V) was calculated according to the formula V = w21a16 [Hansen and With, 19521. The testicular volumes of patients under 18 were compared to the data for normal males compiled by Laron and Zilka [1969]; in the case of adult males, testicular volumes from 10 to 25 ml were considered normal LSchonfeld, 1943; Prader, 1966; Zachmann et al., 19741.
Cytogenetic Studies Chromosome analyses were performed on peripheral lymphocytes cultured for 72 to 86 hours in TC 199 supplemented with 5% fetal calf serum and 13 pgiml trimethoprim. For patients AV-13 and AV-14 trimethoprim was not added. After routine Giemsa staining, 50-100 metaphases were analyzed for each individual. In one patient with a 47,XX, + 21 karyotype, for technical reasons, fewer than 50 metaphases were studied. Whenever a terminal gap was found in a C-group chromosome, the slide was destained and trypsin-banded.
Fragile X Syndrome in Brazil
RESULTS
23
TABLE 11. Summary of Cytogenetic Results in Males
Table I shows the distribution, according to mental status, of 122 of the patients who had a psychological evaluation. Cytogenetic results are presented in Table I1 for males and in Table I11 for females. Among the 69 males who had their testes measured, 5 (7.2%) had macroorchidism (3 prepubertal, one pubertal, and one adult); 2 of them (one prepubertal and one pubertal) had fra(X) syndrome. Four fra(X) male patients (2 prepubertal, one pubertal, and one adult) did not present with macroorchidism. The 8 individuals with fra(X) chromosome belonged to 6 families: one pair of brothers, one pair of male cousins, and 4 cases (2 males and 2 females) without other affected relatives in the schools (2 of the latter were isolated cases). The pedigrees are shown in Figure 1. The frequency of spontaneous abortions in the pedigrees is underestimated, especially in the older generations. Because family data were obtained chiefly through probands’ parents, they frequently ignored the occurrence of abortions in other than close relatives.
Karwtype 46,XY 46 ,XY.fra(X)(q27) 47,XY, + 21 47,XXY 47,XYY Total
No. of individuals (%) 64(85.3) 6 (8.0) 3 (4.0) 1 (1.3) 1 (1.3) 75
was 2.94 ml, normal for his age. A heart murmur was noted. He never had seizures and his intelligence was evaluated as borderline. Speech was described as presenting mild articulation disabilities. The fra(X) frequency was 18%. His elder brother (IV-16) was mentally retarded and showed a fra(X) frequency of 10%. His mother had 1% fra(X)-positive cells. Although 111-3,111-4,111-6,and 111-8were not studied cytogenetically, they received a clinical diagnosis of fra(X)syndrome in another Genetic Counseling Service. All of them presented with mental retardation and Clinical Findings in J?ra(X) Positive Probands macroorchidism. Family SJ-OZ/SJ-lZ (Fig. 1A). SJ-02 (IV-19) was FamilyAV-13/AV-14(Fig, 1B). AV-13 (IV-18) was the only child of normal, non-consanguineous parents, the first child of normal non-consanguineous parents, born by C-section after a normal pregnancy. He was 12 born by normal delivery. At age 14 years, he presented years old when examined, and the clinical findings in- with dolichocephaly, normal OFC, triangular face, and cluded normal head circumference (OFC), narrow fore- highly arched palate. He had hyperextensibility of head, broad nasal bridge, highly arched palate, mild fingerjoints with passive dislocation of the right thumb. bilateral gynecomastia, long hands and fingers, hyper- The average testicular volume was 16.65 ml, which was extensibility of finger joints with passive dislocation of abnormally increased for his age. He had mild mental thumbs. The average testicular volume was 4.29 ml, retardation. He never had seizures. Cytogenetic normal for his age. Seizures were not reported. He was analysis showed 7% of cells with fra(X). moderately retarded. The frequency of fra(X) was 18%. AV-14 (IV-19) was born by normal delivery, and he His mother (111-15) had a fra(X) frequency of 1%. was clinically examined at age 12 years. He presented SJ-12 (IV-17) was the second child born to normal non- with macrocephaly (>98th centile), highly arched palconsanguineous parents after a n uneventful pregnancy ate, hyperextensibility of finger joints, and transitional and normal delivery. Breathing difficulties occurred in palmar crease on the right hand. The average testicular the first days after birth. At age 10 years, he presented volume of 15.86 ml was increased for age. He was dewith normal OFC, dolichocephaly, narrow forehead with scribed as hyperactive, he was mildly mentally retarded low hairline, large protuberant ears, elongated triangu- and never had seizures. The frequency of fra(X) was lar face with midface hypoplasia, prominent mandible 17%. and highly arched palate. The thorax was slightly asymThe proband’s mother (111-7) showed a fra(X) fremetric with pectus excavatum. The fingers and toes quency of 1%.IV-8 was not studied cytogenetically, but were long with hyperextensibility of joints and passive did receive diagnosis of fra(X) syndrome in another dislocation of thumbs. The average testicular volume Genetic Counseling Service, because of his phenotype. Thus, 11-3 and 111-4 were obligate gene carriers. FamiZy SJ-81 (Fig. 1C). SJ-81 (IV-12) was the eighth and last child of normal non-consanguineous parTABLE 1. Distribution of Mental Impairment Among 122 Patients Mental status Severe mental retardation Moderate mental retardation Mild mental retardation Borderline “Normal” Total
Males (%I
Females (%)
g(12.0)
2 (4.3)
Total (8) 11 (9.0)
16(21.3)
17(36.2)
33(27.1)
29(38.7)
16(34.0)
45(36.9)
16(21.3) 5 (6.7) 75
ll(23.4) 1 (2.1) 47
27(22.1) 6 (4.9) 122
TABLE 111. Summary of Cytogenetic Results in Females KarvotvDe 46,XX 46,XX,fra(X)(q27) 46,XX,fra(lO)(q23) 47;XX; + 21 46,XX,del(l)(q25q31) Total
No. of individuals (96)
45(90.0) 2 (4.0) 1 (2.0) 1 (2.0) 1 (2.0) 50
24
Mingroni-Nettoet al.
ents. He was born by C-section, and psychomotor development was slow. He was examined at age 14 years and had macrocephaly (98th centile), large forehead, triangular face, prominent mandible, large and slightly protuberant ears, convergent strabismus, highly arched palate, pectus excavatum, hyperextensibility of finger joints, and broad halluces. The average testicular volume was 13.81 ml, normal for his age. He was mildly retarded and had no seizure history. Autistic behavior was noted. Speech was rated fast and the voice, low. The frequency of fra(X) was 7%. His sibs (IV-6 and IV-101, referred as mentally retarded, were not available for clinical and cytogenetics evaluation. His normal brother (IV-9) as well as his normal niece (V-1) showed a fra(X) frequency of 1%. Family AV-04 (Fig, 1D). AV-04 (IV-3)was the third and last child of normal non-consanguineous parents. He was born by normal delivery. Psychomotor development was delayed, and he was hyperactive. Examination at age 19 years demonstrated normal OFC, broad nose tip with anteverted nostrils, long and wide neck, pectus excavatum, and widely spaced nipples. The upper limbs, hands, and fingers were elongated with hyperextensibility of joints. The average testicular volume was 24.40 ml, which is within the upper normal range for an adult male. He was moderately retarded and without seizures. Speech was repetitive and difficult to understand. The fra(X) frequency was 13%. Family SJ-61 (Fig.IE). SJ-61(111-5)was the second child of normal non-consanguineous parents, born by normal delivery. She was examined at age 18years, and clinical findings included normal OFC, posteriorly angulated ears, long and narrow face, and prominent mandible. She was mildly retarded. She had seizures a t age 4 years. Cytogenetic study demonstrated one cell with the fra(X) among 100 analyzed. In a repeated culture (without trimethoprim), a second fra(X) positive cell was found among 100 analyzed. In spite of the low frequency of fra(X)-positive cells in this patient, the fact that the fra(X) was detected in cells cultured on 2 separate occasions led us to consider this as a positive case. Family AV-26 (Fig.IF). AV-26 (IV-6)was the third child of normal non-consanguineous parents. When examined at age 17 years she presented with normal OFC, normal-size protuberant ears, slightly triangular face with mild prognathism, highly arched palate, clinodactyly of 5th fingers, and hyperextensibility of finger joints. She was mildly retarded. The frequency of fra(X) was 5%.Her mother (111-5)showed a fra(X) frequency of 2%. Three maternal cousins (111-1, IV-1, IV-7) of the proband were mentally retarded. Only IV-1 was available for clinical and cytogenetic evaluation, showing a fra(X) frequency of 11%. His mother (111-4) had a fra(X) frequency of 1%.
DISCUSSION Screening populations for fra(X) chromosome has yielded heterogeneous results, depending on the selection criteria of patients. Some studies in male populations selected individuals with macroorchidism for cytogenetic analysis [Brown et al., 1981; Venter and
Gericke, 1981;Nielsen et al., 1982; Howard-Peebles and Finley, 1983; Primrose et al., 19861. These investigations showed the highest frequencies of males with fra(X) syndrome. Other studies selected male patients with familial mental retardation [Venter and Gericke, 1981; Venter and Op’t Hof, 1982; Howard-Peebles and Finley, 1983; Fryns and Van den Berghe, 1983;Primrose et al., 19861. In most such studies, the selection criterion was mental retardation of unknown cause or mental retardation not associated with chromosomal aberrations LSutherland, 1982; Blomquist et al., 1982, 1983; Kinnel and Banu, 1983; Kirkilionis et al., 1983; Proops et al., 1983; Linna et al., 1984; Webb et al., 1984; Bundey et al., 1985; Sutherland, 1985;Arinami et al., 1986;Sanfilippo et al., 1986;Turner et al., 1986;Webb et al., 1986a,b; Zhou et al., 1986; Arinami et al., 1987; Kahkonen et al., 1987; Thake et al., 19871. Very few studies included all individuals regardless of the cause of mental retardation [Froster-Iskenius et al., 1983;Jacobs et al., 1986; Kahkonen et al., 19861. Obviously, when an unselected group of patients is studied, the proportion of individuals with fra(X) syndrome is lower than in selected samples; however, in selected samples it is not certain that all affected individuals are ascertained, since some of them do not have a family history of mental retardation and, in the case of males, macroorchidism is not always present. In fact, based on screenings that did not select male patients because of family history (Table IV), we estimated that about 25% of affected males are isolated cases. In the few studies of females, familial data are hardly available, but it is worthy noting that one of our 2 affected females is an isolated case. Taking into account both the method of ascertainment as well as the degree of mental retardation, our sample of males is comparable to that of Jacobs et al., [1986] and of Kahkonen et al. [1987]. We found that 8% of male individuals (or 5.6% of the families ascertained) had the fra(Xj chromosome. Our frequency does not differ significantly from that found by Jacobs et al. [1986] among mildly and moderately retarded, but it is significantly higher than the frequency of 0.7% in the study ofK&hkonen et al. [1986](probabilities of 0.12 and 0.01, respectively, generated by Fisher’s exact test). This difference might be due to the greater proportion of borderline individuals and the absence of the moderately and severely retarded in the latter study, since fra(X) positive males more often have moderate and severe mental retardation [Opitz and Sutherland, 19841. There are few unselected population studies of mentally retarded females. The frequency found in our population does not differ from that found by Turner et al. [1980] and Proops et al. [1983] (probabilities of 1.00 for both cases). On the other hand, our incidence is significantly higher than that found by Jacobs et al. [19861 Fig. 1. Pedigrees of 6 kindreds ascertained through fra(X)-positive individuals. Key: MR; fra(X)-positive;0 (3 Normal; fra(X)-positive; @ MR; chromosomes not studied; @ Normal; fra(X)-negative; 0 Carrier of the gene (inferred from genealogy).
Fragile X Syndrome in Brazil
25
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Mingroni-Netto et al.
26
TABLE IV. Frequency of Isolated and Familial Cases Among Males with Fra(X) Syndrome Ascertained Through Screenings of Mentally Retarded Individuals Authors Carpenter et al., 1982 Nielsen et al., 1982 Jacobs et al., 1983 Kahkonen et al., 1983 Fryns, 1984 Bundey et al., 1985 Arinami et al., 1986 Sanfilippo et al., 1986 Present study Total
Familial cases
Isolated cases
3 1 4 5 29 7 6
1 1 1 1 8 5 3 0 1 21(25.3%)
4 3 62(74.7%)
(probability of 0.034), but this sample shows a preponderance of severe and moderate mental retardation, in contrast t o ours, that contains a majority of mildly retarded individuals, and it is known that affected females are usually mildly retarded. These results, taken as a whole, show that the frequency of the fra(X) syndrome among Brazilians is similar to that found in other countries. However, it must be taken into account that our sample comprises students from private schools, in a developed region of the country, the metropolitan region of Siio Paulo. There are no reliable statistics about the incidence of mental retardation in Brazil, but it is thought to be about 3 times higher than in developed countries because of environmental factors [Krynsky, 19831. For this reason, it is reasonable to expect that studies of samples from poorer non-metropolitan regions of the country demonstrate a smaller contribution of fra(X) syndrome to the cause of mental retardation.
ACKNOWLEDGMENTS We are grateful to Dr. Patricia P. Schipper for clinical assistance and to Dr. Paulo A. Otto for critical reading of the manuscript. We thank Ms. Ligia S. Vieira, Ms. Marlice B.P. Robes, and Miss Eliana M. Juvenal for technical assistance. This work was supported by CNPq, FAPESP, and CAPES. REFERENCES Arinami T, Kondo I, Nakajima S (1986): Frequency of the fragile X syndrome on Japanese mentally retarded males. Hum Genet 73:309-313. Arinami T, Kondo I, Nakajima S, Hamaguchi H (1987): Frequency of fragile X syndrome in institutionalized mentally retarded females in Japan. Hum Genet 76:344-347. Blomquist HK, Gustavson K-H, Holmgren G, Nordenson I, Sweins A (1982): Fragile site X chromosomes and X-linked mental retardation in severely retarded boys in a northern Swedish county: A prevalence study. Clin Genet 21:209-214. Blomquist HK, Gustavson K-H, Holmgren G, Nordenson I, PalssonStrae U (1983): Fragile X syndrome in mildly mentally retarded children in a northern Swedish county: A prevalence study. Clin Genet 24:393-398. Brown WT, Mezzacappa PM, Jenkins EC (1981):Screening for fragile X syndrome by testicular size measurement. Lancet 2: 1055. Bundey S,Webb TP, Thake A, Todd J (1985): A community study of severe mental retardation in the West Midlands and the importance of the fragile X chromosome in its aetiology. J Med Genet 22:258-266.
Carpenter NJ, Leichtman LG, Say B (1982): Fragile X-linked mental retardation: A survey of 65 patients with mental retardation of unknown origin. Am J Dis Child 136:392-398. Froster-Iskenius U, Felsch G, Schirren C, Schwinger E (1983):Screening for fra (X)(q)in a population of mentally retarded males. Hum Genet 63:153-157. Fryns J P (1984):The fragile X syndrome: A study of 83 families. Clin Genet 26:497-528. Fryns JP, Van den Berghe H (1983):X-linked mental retardation and fragile (Xq27) site. Clin Genet 23:203-206. Hansen PF, With TK (1952): Clinical measurements of the testes in boys and men. Acta Med Scand Suppl266:457-465. Howard-Peebles PN, Finley WH (1983):Screening of mentally retarded males for macro-orchidism and the fragile X chromosome. Am J Med Genet 15:631-635. Jacobs PA, Mayer M, Matsuura J , Rhoads F, Yee SC (1983):A cytogenetic study of a population of mentally retarded males with special reference the marker (X) syndrome. Hum Genet 63:139-148. Jacobs PA, Mayer M, Abruzzo MA (1986): Studies of the fragile (X) syndrome in populations of mentally retarded individuals in Hawaii. Am J Med Genet 23:567-572. Kahkonen M, Leisti J, Wilska M, Varonen S (1983): Marker X associated mental retardation: A study of 150 retarded males. Clin Genet 23:397-404. Kahkonen M, Leisti J , Thoden CJ, Autio S (1986): Frequency of rare fragile sites among mentally subnormal schoolchildren. Clin Genet 30:234-238. Kahkonen M, Alitalo T, Airaksinen E, Matilainen R, Launiala K, Autio S, Leisti J (1987):Prevalence ofthe fragile X syndrome in four birth cohorts of children of school age. Hum Genet 77:85-87. Kinnel HG, Banu SP (1983):Institutional prevalence of fragile X syndrome. Lancet 2:1427. Kirkilionis A, Sergovich F, Pozsonyi J (1983):Use of testicular volume as a cytogenetic screening criterion. Am J Hum Genet 35:138A. Krynsky S (1983): “Novos Rumos da Defici6ncia Mental.” Sao Paulo: Ed. Sarvier, p 1. Laron Z, Zilka E (1969):Compensatory hypertrophy of testicle in unilateral cryptorchidism. J Clin Endocrinol Metab 29:1409-1413. Linna S-L, Simila S, Haro E, Herva R (1984): Prevalence of fragile-X chromosome. Lancet 1:220-221. Nielsen KB, Tommerup N, Dyggve HV, Schou C (1982): Macrooorchidism and fragile X in mentally retarded males: Clinical, cytogenetic, and some hormonal investigations in mentally retarded males including two with the fragile site at Xq28, fra(Xj(q28).Hum Genet 61:113-117. Opitz JM, Sutherland GR (1984): Conference report: International workshop on the fragile X and X-linked mental retardation. Am J Med Genet 17:5-94. Prader A (1966):Testicular size: Assessment and clinical importance. Triangle 7:240-243. Primrose DA, El-Matmati R, Boyd E, Gosden C, Newton M (1986): Prevalence of the fragile X syndrome in a n institution for the mentally handicapped. Br J Psychiatry 148:655-657. Proops R, Mayer M, Jacobs PA (1983): A study ofmental retardation in children in the Island of Hawaii. Clin Genet 23:81-96. SanfilippoS, Ragusa RM, Musumeci S, Neri G (1986):Fragile X mental retardation: Prevalence in a group of institutionalized patients in Italy and description of a novel EEG pattern. Am J Med Genet 23:589-595. Schonfeld WA (1943j: Primary and secondary sexual characteristics: Study of their development in males from birth through maturity, with biometric study of penis and testes. Am J Dis Child 655355549, Sherman SL, Morton NE, Jacobs PA, Turner G (1984): The marker (XI syndrome: A cytogenetic and genetic analysis. Ann Hum Genet 48:21-37. Sutherland GR (1979):Heritable fragile sites on human chromosomes I. Factors affecting expression in lymphocyte culture. Am J Hum Genet 31:125-135. Sutherland GR (1982):Heritable fragile sites on human chromosomes VIII. Preliminary population cytogenetic data of the folic-acidsensitive fragile sites. Am J Hum Genet 34:452-458.
Fragile X Syndrome in Brazil Sutherland GR i1985):Heritable fragile sites on human chromosomes XII. Population cytogenetics. Ann Hum Genet 48:153-161. Thake A, Todd J, Webb T, Bundey S (1987):Children with the fragile X chromosome a t schools for the mentally retarded. Dev Med Child Neural 29:711-719. Turner G , Brookwell R, Daniel A, Selikowitz M, Zilibowitz M (1980): Heterozygous expression of X-linked mental retardation and X-chromosome marker fra (X)(q27).N Engl J Med 303:662-664. Turner G, Robinson 13, Laing S, Purvis-Smith S (1986): Preventive screening for the fragile X syndrome. N Engl J Med 315:607-609. Venter PA, Gericke GS (1981): A marker X-chromosome and nonspecific male mental retardation. A South African survey (Abstracts). In 6th International Congress of Human Genet.ics, Proceedings, Jerusalem, 1981, p 48. Venter PA, Op’t Hof J (1982):Cytogenetic abnormalities including the marker X chromosome in patients with severe mental retardation. S Afr Med J 62:947-950.
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Webb T, Thake A, Todd 6 ,Bundey S (1984): Prevalence of fragile-X chromosome. Lancet 1:220. Webb TP, Bundey SE, Thake AI, Todd J (1986a):Population incidence and segregation ratios in the Martin-Bell syndrome. Am J Med Genet 23573-580. Webb TP, Bundey S, Thake A, Todd J (1986b): The frequency of the fragile X chromosome among school children in Coventry. J Med Genet 19:44-48. Zachniann NI, Prader A, Kind HP, Hafliger H, Budliger H (1974): Testicular volume during adolescence: Cross-sectional and longitudinal studies. Helv Paediatr Acta 20:61-72. Zhou XT, Xiao GF, Xu BZ, Zhang WJ (1986):The incidence of fragile X syndrome in two Chinese populations. (Abstracts).In 7th International Congress of Human Genetics, Berlin, 1986, p 218.
Fragile X Frequency in a Mentally Retarded Population in Brazil Regina C. Mingroni-Netto, Carla Rosenberg, Angela M. Vianna-Morgante, and Rita de Cassia M. Pavanello Human Genetics Laboratory, Department of Biology, University of Scio Paulo, SP, Brazil Seventy-five male and 50 female students from 2 special schools for mildly, moderately retarded, or borderline individuals were screened clinically and cytogenetically in order to estimate the contribution of fragile X [fra(X)]syndrometo the cause of mental retardation in Brazil. We found 6 males (8%)from 4 families and 2 unrelated females (4%) with fra(X) chromosomes. One male and one female were isolated cases. The estimated frequency of Martin-Bell [fra(X)] syndrome among mentally impaired individuals in Brazil was similar to that previously reported in other countries.
KEY WORDS: Martin-Bellsyndrome, mental retardation survey, X-linked mental retardation
INTRODUCTION Martin-Bell or fragile X [fra (X)] syndrome is a n X-linked form of mental retardation associated with a fragile site at Xq27.3, which is expressed in a variable proportion in cells of carriers of the gene. This fragile site is expressed in culture conditions of low levels of folic acid [Sutherland, 19791. This syndrome is known to be the second most frequent cause of genetic mental retardation, and its prevalence is estimated to be 4.4 x among males and 4.1 x among females [Sherman et al., 19841. In screening populations of mentally retarded males, 0.5% to 10.0% of unselected retarded individuals have the fra(X) chromosome. In similar studies in female populations, frequencies from 0% to 7% have been obtained.
Received for publication September 20,1988; revision received, May 30, 1989. Address reprint requests t o Angela M. Vianna-Morgante, Departamento de Biologia, Universidade de SSio Paulo, Caixa Postal 11461; 05499, Sgo Paulo, Brazil.
0 1990 Wiley-Liss, Inc.
In the present study, we estimated, for the first time, the fra(X) contribution to mental retardation in a Brazilian population. Our work was carried out in 2 special schools for mildly, moderately retarded, or borderline individuals.
MATERIALS AND METHODS Patients The 75 males and 50 females were ascertained in 2 special schools for mildly or moderately retarded and borderline individuals (Centro Ocupacional Avanhandava and Centro de HabilitaGao Profissional Sao Joao Batista) in the city of Sao Paulo, Brazil. All students from Sao Jog0 Batista school, enrolled from 1984 to 1987 (55 males and 42 females), were studied cytogenetically, while a t Avanhandava school (20 males and 8 females) consent could not be obtained in all cases. The age of the patients ranged from 6 to 40 years and most were between 10-20 years; they were included in the cytogenetic study regardless of their family history, clinical findings, and previous diagnosis. Clinical Studies All patients from both schools were evaluated clinically. In most males, length (1) and width (w)ofthe testes were measured with a caliper and the volume (V) was calculated according to the formula V = w21a16 [Hansen and With, 19521. The testicular volumes of patients under 18 were compared to the data for normal males compiled by Laron and Zilka [1969]; in the case of adult males, testicular volumes from 10 to 25 ml were considered normal LSchonfeld, 1943; Prader, 1966; Zachmann et al., 19741.
Cytogenetic Studies Chromosome analyses were performed on peripheral lymphocytes cultured for 72 to 86 hours in TC 199 supplemented with 5% fetal calf serum and 13 pgiml trimethoprim. For patients AV-13 and AV-14 trimethoprim was not added. After routine Giemsa staining, 50-100 metaphases were analyzed for each individual. In one patient with a 47,XX, + 21 karyotype, for technical reasons, fewer than 50 metaphases were studied. Whenever a terminal gap was found in a C-group chromosome, the slide was destained and trypsin-banded.
Fragile X Syndrome in Brazil
RESULTS
23
TABLE 11. Summary of Cytogenetic Results in Males
Table I shows the distribution, according to mental status, of 122 of the patients who had a psychological evaluation. Cytogenetic results are presented in Table I1 for males and in Table I11 for females. Among the 69 males who had their testes measured, 5 (7.2%) had macroorchidism (3 prepubertal, one pubertal, and one adult); 2 of them (one prepubertal and one pubertal) had fra(X) syndrome. Four fra(X) male patients (2 prepubertal, one pubertal, and one adult) did not present with macroorchidism. The 8 individuals with fra(X) chromosome belonged to 6 families: one pair of brothers, one pair of male cousins, and 4 cases (2 males and 2 females) without other affected relatives in the schools (2 of the latter were isolated cases). The pedigrees are shown in Figure 1. The frequency of spontaneous abortions in the pedigrees is underestimated, especially in the older generations. Because family data were obtained chiefly through probands’ parents, they frequently ignored the occurrence of abortions in other than close relatives.
Karwtype 46,XY 46 ,XY.fra(X)(q27) 47,XY, + 21 47,XXY 47,XYY Total
No. of individuals (%) 64(85.3) 6 (8.0) 3 (4.0) 1 (1.3) 1 (1.3) 75
was 2.94 ml, normal for his age. A heart murmur was noted. He never had seizures and his intelligence was evaluated as borderline. Speech was described as presenting mild articulation disabilities. The fra(X) frequency was 18%. His elder brother (IV-16) was mentally retarded and showed a fra(X) frequency of 10%. His mother had 1% fra(X)-positive cells. Although 111-3,111-4,111-6,and 111-8were not studied cytogenetically, they received a clinical diagnosis of fra(X)syndrome in another Genetic Counseling Service. All of them presented with mental retardation and Clinical Findings in J?ra(X) Positive Probands macroorchidism. Family SJ-OZ/SJ-lZ (Fig. 1A). SJ-02 (IV-19) was FamilyAV-13/AV-14(Fig, 1B). AV-13 (IV-18) was the only child of normal, non-consanguineous parents, the first child of normal non-consanguineous parents, born by C-section after a normal pregnancy. He was 12 born by normal delivery. At age 14 years, he presented years old when examined, and the clinical findings in- with dolichocephaly, normal OFC, triangular face, and cluded normal head circumference (OFC), narrow fore- highly arched palate. He had hyperextensibility of head, broad nasal bridge, highly arched palate, mild fingerjoints with passive dislocation of the right thumb. bilateral gynecomastia, long hands and fingers, hyper- The average testicular volume was 16.65 ml, which was extensibility of finger joints with passive dislocation of abnormally increased for his age. He had mild mental thumbs. The average testicular volume was 4.29 ml, retardation. He never had seizures. Cytogenetic normal for his age. Seizures were not reported. He was analysis showed 7% of cells with fra(X). moderately retarded. The frequency of fra(X) was 18%. AV-14 (IV-19) was born by normal delivery, and he His mother (111-15) had a fra(X) frequency of 1%. was clinically examined at age 12 years. He presented SJ-12 (IV-17) was the second child born to normal non- with macrocephaly (>98th centile), highly arched palconsanguineous parents after a n uneventful pregnancy ate, hyperextensibility of finger joints, and transitional and normal delivery. Breathing difficulties occurred in palmar crease on the right hand. The average testicular the first days after birth. At age 10 years, he presented volume of 15.86 ml was increased for age. He was dewith normal OFC, dolichocephaly, narrow forehead with scribed as hyperactive, he was mildly mentally retarded low hairline, large protuberant ears, elongated triangu- and never had seizures. The frequency of fra(X) was lar face with midface hypoplasia, prominent mandible 17%. and highly arched palate. The thorax was slightly asymThe proband’s mother (111-7) showed a fra(X) fremetric with pectus excavatum. The fingers and toes quency of 1%.IV-8 was not studied cytogenetically, but were long with hyperextensibility of joints and passive did receive diagnosis of fra(X) syndrome in another dislocation of thumbs. The average testicular volume Genetic Counseling Service, because of his phenotype. Thus, 11-3 and 111-4 were obligate gene carriers. FamiZy SJ-81 (Fig. 1C). SJ-81 (IV-12) was the eighth and last child of normal non-consanguineous parTABLE 1. Distribution of Mental Impairment Among 122 Patients Mental status Severe mental retardation Moderate mental retardation Mild mental retardation Borderline “Normal” Total
Males (%I
Females (%)
g(12.0)
2 (4.3)
Total (8) 11 (9.0)
16(21.3)
17(36.2)
33(27.1)
29(38.7)
16(34.0)
45(36.9)
16(21.3) 5 (6.7) 75
ll(23.4) 1 (2.1) 47
27(22.1) 6 (4.9) 122
TABLE 111. Summary of Cytogenetic Results in Females KarvotvDe 46,XX 46,XX,fra(X)(q27) 46,XX,fra(lO)(q23) 47;XX; + 21 46,XX,del(l)(q25q31) Total
No. of individuals (96)
45(90.0) 2 (4.0) 1 (2.0) 1 (2.0) 1 (2.0) 50
24
Mingroni-Nettoet al.
ents. He was born by C-section, and psychomotor development was slow. He was examined at age 14 years and had macrocephaly (98th centile), large forehead, triangular face, prominent mandible, large and slightly protuberant ears, convergent strabismus, highly arched palate, pectus excavatum, hyperextensibility of finger joints, and broad halluces. The average testicular volume was 13.81 ml, normal for his age. He was mildly retarded and had no seizure history. Autistic behavior was noted. Speech was rated fast and the voice, low. The frequency of fra(X) was 7%. His sibs (IV-6 and IV-101, referred as mentally retarded, were not available for clinical and cytogenetics evaluation. His normal brother (IV-9) as well as his normal niece (V-1) showed a fra(X) frequency of 1%. Family AV-04 (Fig, 1D). AV-04 (IV-3)was the third and last child of normal non-consanguineous parents. He was born by normal delivery. Psychomotor development was delayed, and he was hyperactive. Examination at age 19 years demonstrated normal OFC, broad nose tip with anteverted nostrils, long and wide neck, pectus excavatum, and widely spaced nipples. The upper limbs, hands, and fingers were elongated with hyperextensibility of joints. The average testicular volume was 24.40 ml, which is within the upper normal range for an adult male. He was moderately retarded and without seizures. Speech was repetitive and difficult to understand. The fra(X) frequency was 13%. Family SJ-61 (Fig.IE). SJ-61(111-5)was the second child of normal non-consanguineous parents, born by normal delivery. She was examined at age 18years, and clinical findings included normal OFC, posteriorly angulated ears, long and narrow face, and prominent mandible. She was mildly retarded. She had seizures a t age 4 years. Cytogenetic study demonstrated one cell with the fra(X) among 100 analyzed. In a repeated culture (without trimethoprim), a second fra(X) positive cell was found among 100 analyzed. In spite of the low frequency of fra(X)-positive cells in this patient, the fact that the fra(X) was detected in cells cultured on 2 separate occasions led us to consider this as a positive case. Family AV-26 (Fig.IF). AV-26 (IV-6)was the third child of normal non-consanguineous parents. When examined at age 17 years she presented with normal OFC, normal-size protuberant ears, slightly triangular face with mild prognathism, highly arched palate, clinodactyly of 5th fingers, and hyperextensibility of finger joints. She was mildly retarded. The frequency of fra(X) was 5%.Her mother (111-5)showed a fra(X) frequency of 2%. Three maternal cousins (111-1, IV-1, IV-7) of the proband were mentally retarded. Only IV-1 was available for clinical and cytogenetic evaluation, showing a fra(X) frequency of 11%. His mother (111-4) had a fra(X) frequency of 1%.
DISCUSSION Screening populations for fra(X) chromosome has yielded heterogeneous results, depending on the selection criteria of patients. Some studies in male populations selected individuals with macroorchidism for cytogenetic analysis [Brown et al., 1981; Venter and
Gericke, 1981;Nielsen et al., 1982; Howard-Peebles and Finley, 1983; Primrose et al., 19861. These investigations showed the highest frequencies of males with fra(X) syndrome. Other studies selected male patients with familial mental retardation [Venter and Gericke, 1981; Venter and Op’t Hof, 1982; Howard-Peebles and Finley, 1983; Fryns and Van den Berghe, 1983;Primrose et al., 19861. In most such studies, the selection criterion was mental retardation of unknown cause or mental retardation not associated with chromosomal aberrations LSutherland, 1982; Blomquist et al., 1982, 1983; Kinnel and Banu, 1983; Kirkilionis et al., 1983; Proops et al., 1983; Linna et al., 1984; Webb et al., 1984; Bundey et al., 1985; Sutherland, 1985;Arinami et al., 1986;Sanfilippo et al., 1986;Turner et al., 1986;Webb et al., 1986a,b; Zhou et al., 1986; Arinami et al., 1987; Kahkonen et al., 1987; Thake et al., 19871. Very few studies included all individuals regardless of the cause of mental retardation [Froster-Iskenius et al., 1983;Jacobs et al., 1986; Kahkonen et al., 19861. Obviously, when an unselected group of patients is studied, the proportion of individuals with fra(X) syndrome is lower than in selected samples; however, in selected samples it is not certain that all affected individuals are ascertained, since some of them do not have a family history of mental retardation and, in the case of males, macroorchidism is not always present. In fact, based on screenings that did not select male patients because of family history (Table IV), we estimated that about 25% of affected males are isolated cases. In the few studies of females, familial data are hardly available, but it is worthy noting that one of our 2 affected females is an isolated case. Taking into account both the method of ascertainment as well as the degree of mental retardation, our sample of males is comparable to that of Jacobs et al., [1986] and of Kahkonen et al. [1987]. We found that 8% of male individuals (or 5.6% of the families ascertained) had the fra(Xj chromosome. Our frequency does not differ significantly from that found by Jacobs et al. [1986] among mildly and moderately retarded, but it is significantly higher than the frequency of 0.7% in the study ofK&hkonen et al. [1986](probabilities of 0.12 and 0.01, respectively, generated by Fisher’s exact test). This difference might be due to the greater proportion of borderline individuals and the absence of the moderately and severely retarded in the latter study, since fra(X) positive males more often have moderate and severe mental retardation [Opitz and Sutherland, 19841. There are few unselected population studies of mentally retarded females. The frequency found in our population does not differ from that found by Turner et al. [1980] and Proops et al. [1983] (probabilities of 1.00 for both cases). On the other hand, our incidence is significantly higher than that found by Jacobs et al. [19861 Fig. 1. Pedigrees of 6 kindreds ascertained through fra(X)-positive individuals. Key: MR; fra(X)-positive;0 (3 Normal; fra(X)-positive; @ MR; chromosomes not studied; @ Normal; fra(X)-negative; 0 Carrier of the gene (inferred from genealogy).
Fragile X Syndrome in Brazil
25
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Mingroni-Netto et al.
26
TABLE IV. Frequency of Isolated and Familial Cases Among Males with Fra(X) Syndrome Ascertained Through Screenings of Mentally Retarded Individuals Authors Carpenter et al., 1982 Nielsen et al., 1982 Jacobs et al., 1983 Kahkonen et al., 1983 Fryns, 1984 Bundey et al., 1985 Arinami et al., 1986 Sanfilippo et al., 1986 Present study Total
Familial cases
Isolated cases
3 1 4 5 29 7 6
1 1 1 1 8 5 3 0 1 21(25.3%)
4 3 62(74.7%)
(probability of 0.034), but this sample shows a preponderance of severe and moderate mental retardation, in contrast t o ours, that contains a majority of mildly retarded individuals, and it is known that affected females are usually mildly retarded. These results, taken as a whole, show that the frequency of the fra(X) syndrome among Brazilians is similar to that found in other countries. However, it must be taken into account that our sample comprises students from private schools, in a developed region of the country, the metropolitan region of Siio Paulo. There are no reliable statistics about the incidence of mental retardation in Brazil, but it is thought to be about 3 times higher than in developed countries because of environmental factors [Krynsky, 19831. For this reason, it is reasonable to expect that studies of samples from poorer non-metropolitan regions of the country demonstrate a smaller contribution of fra(X) syndrome to the cause of mental retardation.
ACKNOWLEDGMENTS We are grateful to Dr. Patricia P. Schipper for clinical assistance and to Dr. Paulo A. Otto for critical reading of the manuscript. We thank Ms. Ligia S. Vieira, Ms. Marlice B.P. Robes, and Miss Eliana M. Juvenal for technical assistance. This work was supported by CNPq, FAPESP, and CAPES. REFERENCES Arinami T, Kondo I, Nakajima S (1986): Frequency of the fragile X syndrome on Japanese mentally retarded males. Hum Genet 73:309-313. Arinami T, Kondo I, Nakajima S, Hamaguchi H (1987): Frequency of fragile X syndrome in institutionalized mentally retarded females in Japan. Hum Genet 76:344-347. Blomquist HK, Gustavson K-H, Holmgren G, Nordenson I, Sweins A (1982): Fragile site X chromosomes and X-linked mental retardation in severely retarded boys in a northern Swedish county: A prevalence study. Clin Genet 21:209-214. Blomquist HK, Gustavson K-H, Holmgren G, Nordenson I, PalssonStrae U (1983): Fragile X syndrome in mildly mentally retarded children in a northern Swedish county: A prevalence study. Clin Genet 24:393-398. Brown WT, Mezzacappa PM, Jenkins EC (1981):Screening for fragile X syndrome by testicular size measurement. Lancet 2: 1055. Bundey S,Webb TP, Thake A, Todd J (1985): A community study of severe mental retardation in the West Midlands and the importance of the fragile X chromosome in its aetiology. J Med Genet 22:258-266.
Carpenter NJ, Leichtman LG, Say B (1982): Fragile X-linked mental retardation: A survey of 65 patients with mental retardation of unknown origin. Am J Dis Child 136:392-398. Froster-Iskenius U, Felsch G, Schirren C, Schwinger E (1983):Screening for fra (X)(q)in a population of mentally retarded males. Hum Genet 63:153-157. Fryns J P (1984):The fragile X syndrome: A study of 83 families. Clin Genet 26:497-528. Fryns JP, Van den Berghe H (1983):X-linked mental retardation and fragile (Xq27) site. Clin Genet 23:203-206. Hansen PF, With TK (1952): Clinical measurements of the testes in boys and men. Acta Med Scand Suppl266:457-465. Howard-Peebles PN, Finley WH (1983):Screening of mentally retarded males for macro-orchidism and the fragile X chromosome. Am J Med Genet 15:631-635. Jacobs PA, Mayer M, Matsuura J , Rhoads F, Yee SC (1983):A cytogenetic study of a population of mentally retarded males with special reference the marker (X) syndrome. Hum Genet 63:139-148. Jacobs PA, Mayer M, Abruzzo MA (1986): Studies of the fragile (X) syndrome in populations of mentally retarded individuals in Hawaii. Am J Med Genet 23:567-572. Kahkonen M, Leisti J, Wilska M, Varonen S (1983): Marker X associated mental retardation: A study of 150 retarded males. Clin Genet 23:397-404. Kahkonen M, Leisti J , Thoden CJ, Autio S (1986): Frequency of rare fragile sites among mentally subnormal schoolchildren. Clin Genet 30:234-238. Kahkonen M, Alitalo T, Airaksinen E, Matilainen R, Launiala K, Autio S, Leisti J (1987):Prevalence ofthe fragile X syndrome in four birth cohorts of children of school age. Hum Genet 77:85-87. Kinnel HG, Banu SP (1983):Institutional prevalence of fragile X syndrome. Lancet 2:1427. Kirkilionis A, Sergovich F, Pozsonyi J (1983):Use of testicular volume as a cytogenetic screening criterion. Am J Hum Genet 35:138A. Krynsky S (1983): “Novos Rumos da Defici6ncia Mental.” Sao Paulo: Ed. Sarvier, p 1. Laron Z, Zilka E (1969):Compensatory hypertrophy of testicle in unilateral cryptorchidism. J Clin Endocrinol Metab 29:1409-1413. Linna S-L, Simila S, Haro E, Herva R (1984): Prevalence of fragile-X chromosome. Lancet 1:220-221. Nielsen KB, Tommerup N, Dyggve HV, Schou C (1982): Macrooorchidism and fragile X in mentally retarded males: Clinical, cytogenetic, and some hormonal investigations in mentally retarded males including two with the fragile site at Xq28, fra(Xj(q28).Hum Genet 61:113-117. Opitz JM, Sutherland GR (1984): Conference report: International workshop on the fragile X and X-linked mental retardation. Am J Med Genet 17:5-94. Prader A (1966):Testicular size: Assessment and clinical importance. Triangle 7:240-243. Primrose DA, El-Matmati R, Boyd E, Gosden C, Newton M (1986): Prevalence of the fragile X syndrome in a n institution for the mentally handicapped. Br J Psychiatry 148:655-657. Proops R, Mayer M, Jacobs PA (1983): A study ofmental retardation in children in the Island of Hawaii. Clin Genet 23:81-96. SanfilippoS, Ragusa RM, Musumeci S, Neri G (1986):Fragile X mental retardation: Prevalence in a group of institutionalized patients in Italy and description of a novel EEG pattern. Am J Med Genet 23:589-595. Schonfeld WA (1943j: Primary and secondary sexual characteristics: Study of their development in males from birth through maturity, with biometric study of penis and testes. Am J Dis Child 655355549, Sherman SL, Morton NE, Jacobs PA, Turner G (1984): The marker (XI syndrome: A cytogenetic and genetic analysis. Ann Hum Genet 48:21-37. Sutherland GR (1979):Heritable fragile sites on human chromosomes I. Factors affecting expression in lymphocyte culture. Am J Hum Genet 31:125-135. Sutherland GR (1982):Heritable fragile sites on human chromosomes VIII. Preliminary population cytogenetic data of the folic-acidsensitive fragile sites. Am J Hum Genet 34:452-458.
Fragile X Syndrome in Brazil Sutherland GR i1985):Heritable fragile sites on human chromosomes XII. Population cytogenetics. Ann Hum Genet 48:153-161. Thake A, Todd J, Webb T, Bundey S (1987):Children with the fragile X chromosome a t schools for the mentally retarded. Dev Med Child Neural 29:711-719. Turner G , Brookwell R, Daniel A, Selikowitz M, Zilibowitz M (1980): Heterozygous expression of X-linked mental retardation and X-chromosome marker fra (X)(q27).N Engl J Med 303:662-664. Turner G, Robinson 13, Laing S, Purvis-Smith S (1986): Preventive screening for the fragile X syndrome. N Engl J Med 315:607-609. Venter PA, Gericke GS (1981): A marker X-chromosome and nonspecific male mental retardation. A South African survey (Abstracts). In 6th International Congress of Human Genet.ics, Proceedings, Jerusalem, 1981, p 48. Venter PA, Op’t Hof J (1982):Cytogenetic abnormalities including the marker X chromosome in patients with severe mental retardation. S Afr Med J 62:947-950.
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Webb T, Thake A, Todd 6 ,Bundey S (1984): Prevalence of fragile-X chromosome. Lancet 1:220. Webb TP, Bundey SE, Thake AI, Todd J (1986a):Population incidence and segregation ratios in the Martin-Bell syndrome. Am J Med Genet 23573-580. Webb TP, Bundey S, Thake A, Todd J (1986b): The frequency of the fragile X chromosome among school children in Coventry. J Med Genet 19:44-48. Zachniann NI, Prader A, Kind HP, Hafliger H, Budliger H (1974): Testicular volume during adolescence: Cross-sectional and longitudinal studies. Helv Paediatr Acta 20:61-72. Zhou XT, Xiao GF, Xu BZ, Zhang WJ (1986):The incidence of fragile X syndrome in two Chinese populations. (Abstracts).In 7th International Congress of Human Genetics, Berlin, 1986, p 218.
