origin of the supernumerary x chromosome in a patient with fragile x and klinefelter syndrome

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American Journal of Medical Genetics 38440-444 (1991) Origin of the Supernumerary X Chromosome in a Patient With Fragile X and Klinefelter Syndrome Kenneth G. Kupke, A. Lee Soreng, and Ulrich Muller Division of Genetics and Mental Retardation Center (K.G.K., U.M.) and Joint Program in Neonatology (K.G.K.), Children$ Hospital and Department of Pediatrics, Harvard Medical School (K.G.K., U.M.); Division of Diagnostic Molecular Biology, Pathology Department, Brigham and Women’s Hospital (A .L.S.), Boston, Massachusetts We report on a 10-year-old patient with the fragile X [fra(X)] syndrome and a 47,XXY karyotype. He had Martin-Bell syndrome, in- cluding typical craniofacial findings and mental retardation. The fra(X) was detected on both X chromosomes of the patient in 8% of the metaphases examined. DNA analysis using X chromosome sequences from the peri- centromeric region and from distal Xq sug gests that the patient is homozygous at the fra(X) locus due to maternal nondisjunction during meiosis 11. KEY WORDS Klinefelter syndrome, nondis- junction, meiosis, RFLP anal- ysis INTRODUCTION The concurrence of the fragile X [fra(X)Iand Klinefel- ter syndromes has been previously reported [Wilmot et al., 1980; F’roster-Iskenius et al., 1983; OBrien et al., 1982; Filippi et al., 1983; Fryns et al., 1983, 1984; Schnur et al., 1986; Pueschel et al., 19871. One report documented the coexistence of the Xq27 fragile site in 2 of 32 Klinefelter patients [Filippi et al., 19881.Recently, another investigation reported the occurrence of Klinefelter syndrome in 3 of 465 fra(X) patients for a frequency of 1:155 [Fryns and Van den Berghe, 19883. The high rate of Klinefelter syndrome among fra(X) patients significantly exceeds the 1:1,000 incidence of 47,XXY in the general population [Nielsen and Sillesen, 19751.Thus, these findings raise the possibility that the fra(X) mutation may predispose the X chromosome to nondisjunction [Watson et al., 19881. Despite the possible biologic relationship between the fra(X) mutation and meiotic nondisjunction [Filippi et al., 19881, few studies have investigated the origin and Received for publication November 22, 1989; revision received February 12, 1990. a2 Address reprint requests to Dr. Kenneth G. Kupke, Division of Genetics, Children’s Hospital, 300 Longwood Avenue, Boston MA 02115. stage of meiotic nondisjunction of the supernumerary X chromosome in these patients. In one such study, a fam- ily with a 47,fra(X)(q27)XY male was investigated by RFLP analysis and paternal nondisjunction was demon- strated [Schnur et al., 19861. Another case was similarly evaluated, and maternal meiosis I nondisjunction was shown [Filippi et al., 1983; Purrello et al., 19871. In addition, 2 patients have been described in which the fragile site was cytogenetically observed in both X chro- mosomes within the same cell [Fryns et al., 1984; Pueschel et al., 19871. These latter findings suggest homozygosity at the fra(X) locus due to maternal non- disjunction. However, molecular studies of parental ori- gin were not performed in either case. In the current study, we report the clinical, cytogene- tic, and molecular aspects of another patient with both Klinefelter and Martin-Bell syndrome. CLINICAL REPORT The propositus, now a 10-year-old boy, was the 3,300 g product of the second full-term pregnancy of a 27-year- old woman. The pregnancy was normal apart from the occurrence of streptococcal pharyngitis during the sec- ond month of gestation, which was treated with erythromycin. Labor and delivery were normal. During infancy, the patient was noted to be hypotonic; gross and fine motor and expressive language skills were mildly delayed in early childhood. At age 3 years, he developed a seizure disorder for which he was treated with anticon- vulsants. One year later, he underwent a genetic evalua- tion due to a family history of mental retardation in the mother’s family. His examination showed his weight, height, and head circumference at the 25th, loth, 50th centiles, respectively. He had prominent ears with hypo- plastic antihelices, a left ear tag, a single right trans- verse palmar crease, mild tibia1 torsion, mild hypotonia, and moderate developmental delay. Results of plasma and urinary amino acid studies were normal. A routine karyotype showed 47,XXY. Subsequently, a fra(X) study of 120 peripheral blood lymphocytes cultured in medium 199 showed 7% of cells to be positive for fra(X1. Subsequent cytogenetic analyses of other relatives showed the presence of the fragile site at Xq27.3 in chromosomes from the patient’s unaffected sister and in 5 mentally retarded males, including 2 first cousins, 2 0 1991 Wiley-Liss, Inc.

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American Journal of Medical Genetics 38440-444 (1991)

Origin of the Supernumerary X Chromosome in a Patient With Fragile X and Klinefelter Syndrome

Kenneth G. Kupke, A. Lee Soreng, and Ulrich Muller Division of Genetics and Mental Retardation Center (K.G.K., U.M.) and Joint Program in Neonatology (K.G.K.), Children$ Hospital and Department of Pediatrics, Harvard Medical School (K.G.K., U.M.); Division of Diagnostic Molecular Biology, Pathology Department, Brigham and Women’s Hospital (A .L.S.), Boston, Massachusetts

We report on a 10-year-old patient with the fragile X [fra(X)] syndrome and a 47,XXY karyotype. He had Martin-Bell syndrome, in- cluding typical craniofacial findings and mental retardation. The fra(X) was detected on both X chromosomes of the patient in 8% of the metaphases examined. DNA analysis using X chromosome sequences from the peri- centromeric region and from distal Xq sug gests that the patient is homozygous at the fra(X) locus due to maternal nondisjunction during meiosis 11.

KEY WORDS Klinefelter syndrome, nondis- junction, meiosis, RFLP anal- ysis

INTRODUCTION The concurrence of the fragile X [fra(X)I and Klinefel-

ter syndromes has been previously reported [Wilmot et al., 1980; F’roster-Iskenius et al., 1983; OBrien et al., 1982; Filippi et al., 1983; Fryns et al., 1983, 1984; Schnur et al., 1986; Pueschel et al., 19871. One report documented the coexistence of the Xq27 fragile site in 2 of 32 Klinefelter patients [Filippi et al., 19881. Recently, another investigation reported the occurrence of Klinefelter syndrome in 3 of 465 fra(X) patients for a frequency of 1:155 [Fryns and Van den Berghe, 19883. The high rate of Klinefelter syndrome among fra(X) patients significantly exceeds the 1:1,000 incidence of 47,XXY in the general population [Nielsen and Sillesen, 19751. Thus, these findings raise the possibility that the fra(X) mutation may predispose the X chromosome to nondisjunction [Watson et al., 19881.

Despite the possible biologic relationship between the fra(X) mutation and meiotic nondisjunction [Filippi et al., 19881, few studies have investigated the origin and

Received for publication November 22, 1989; revision received February 12, 1990. a2 Address reprint requests to Dr. Kenneth G. Kupke, Division of Genetics, Children’s Hospital, 300 Longwood Avenue, Boston MA 02115.

stage of meiotic nondisjunction of the supernumerary X chromosome in these patients. In one such study, a fam- ily with a 47,fra(X)(q27)XY male was investigated by RFLP analysis and paternal nondisjunction was demon- strated [Schnur et al., 19861. Another case was similarly evaluated, and maternal meiosis I nondisjunction was shown [Filippi et al., 1983; Purrello et al., 19871. In addition, 2 patients have been described in which the fragile site was cytogenetically observed in both X chro- mosomes within the same cell [Fryns et al., 1984; Pueschel et al., 19871. These latter findings suggest homozygosity at the fra(X) locus due to maternal non- disjunction. However, molecular studies of parental ori- gin were not performed in either case.

In the current study, we report the clinical, cytogene- tic, and molecular aspects of another patient with both Klinefelter and Martin-Bell syndrome.

CLINICAL REPORT The propositus, now a 10-year-old boy, was the 3,300 g

product of the second full-term pregnancy of a 27-year- old woman. The pregnancy was normal apart from the occurrence of streptococcal pharyngitis during the sec- ond month of gestation, which was treated with erythromycin. Labor and delivery were normal. During infancy, the patient was noted to be hypotonic; gross and fine motor and expressive language skills were mildly delayed in early childhood. At age 3 years, he developed a seizure disorder for which he was treated with anticon- vulsants. One year later, he underwent a genetic evalua- tion due to a family history of mental retardation in the mother’s family. His examination showed his weight, height, and head circumference at the 25th, loth, 50th centiles, respectively. He had prominent ears with hypo- plastic antihelices, a left ear tag, a single right trans- verse palmar crease, mild tibia1 torsion, mild hypotonia, and moderate developmental delay. Results of plasma and urinary amino acid studies were normal. A routine karyotype showed 47,XXY. Subsequently, a fra(X) study of 120 peripheral blood lymphocytes cultured in medium 199 showed 7% of cells to be positive for fra(X1.

Subsequent cytogenetic analyses of other relatives showed the presence of the fragile site a t Xq27.3 in chromosomes from the patient’s unaffected sister and in 5 mentally retarded males, including 2 first cousins, 2

0 1991 Wiley-Liss, Inc.

Fragile X and Klinefelter Syndrome 441

ously described [Aldridge et al., 19841. The DNA was digested with the following restriction endonucleases according to the manufacturer's recommendations: MspI, TaqI, BclI, BglI, BglII, PstI, EcoRI, and XmnI. Gel electrophoresis with 2 kg per lane, Southern transfer to nylon membranes (Hybond N, Amersham), and hybrid- ization with 32-P-radiolabelled DNA probes were per- formed as previously described [Muller et al., 19861. After hybridization with X chromosomal DNA probes, the same filters were hybridized with the autosomal probe 16-32 in order to confirm equal DNA loading in each lane. Laser densitometry was performed on each informative Southern blot as previously described [Kupke and Muller, 19891. The relative DNA content of each lane was calculated from the intensity of hybrid- ization with p16-32. The amount of DNA in the father's lane was arbitrarily assigned a value of 1.00, and the amount of the mother's and patient's DNA was calcu- lated relative to the father's. Signal doses were obtained by calculating the motherchild ratio of densitometric integrated areas for informative alleles.

RESULTS/DISCUSSION This patient has manifestations consistent with

fra(X) syndrome, namely, his craniofacial anomalies, mental retardation, seizure disorder, and characteristic speech pattern. His current prepubescent age precludes a fair assessment of the contribution of Klinefelter syn- drome to his phenotype. However, previous case reports indicate that these patients do not develop the macro- orchidism typical of fra(X) syndrome [Froster-Iskenius et al., 1983; Fryns et al., 1984; Fryns and Van den Berghe, 1988; Mavrou et al., 19881. Rather, they usually display hypogonadism, eunuchoid secondary sexual characteristics, and occasionally gynecomastia.

Cytogenetic analysis of the propositus shows a 47,fra(X)(q27), fra(X)(q27), Y karyotype. The fra(X) was expressed in 54% (54/100) of metaphases with 8% (8/100) of the metaphases expressing the fragile site on both X chromosomes (Fig. 2). These karyotypic findings are consistent with homozygosity of the fra(X) locus. Cytogenetic analysis of maternal cells showed a 46,XX normal female karyotype with no apparent expression of the fra(X) site.

DNA from the relatives was also hybridized with DNA probes derived from the distal X long arm (see Materials and Methods). Of these probes, DX13 (DXS15) which was previously assigned to Xq28 [Davies

male second cousins, and a great-uncle (Fig. 1). These males reportedly all have the Martin-Bell syndrome. The paternal grandfather was reportedly of normal in- telligence.

At age 10 years, the patient's weight and height were at the 50th and 25th centiles, respectively, and the up- per to lower segment ratio was 0.95. He had a long face with a broad forehead, large protuberant ears, a normal hair pattern, and a prominent jaw. He demonstrated some mild joint laxity in his fingers and elbows but no clinodactyly. The genitalia were prepubescent with a stretched penile length of 5 cm and small testes (4 ml). He was friendly, but hyperactive with perseverative speech, and intellectually, he was moderately delayed. No additional neurologic abnormalities were noted. He was enrolled in special education classes and his seizure disorder was relatively well controlled with anticonvul- sants.

MATERIALS AND METHODS Cytogenetic Analysis

Fra(X) karyotype analysis was performed on PHA- stimulated T-lymphocytes cultured in medium 199 [Sutherland, 19771 supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 50 U/ml penicillin, and 50 kg/ml streptomycin. Heparinized whole blood was col- lected from the propositus and his mother and 0.5 ml were cultured in 10 ml of medium 199 for 96 hours, including one hour in 0.075 pg/ml colcemid. Slides were GTG-banded [Seabright, 19711.

Hybridization Probes A total of 13 X chromosomal DNA probes were uti-

lized to study DNA from close relatives. Six probes rec- ognize pericentromeric loci: DXS14 (p58-11, MAO-A (A2R/D7) [Ozelius et al., 19881, DXS255 (M27p), DXSl (~€9, DXS159 (cpX73), and PGKl (pHPGK-7e) [Kidd et al., 19891. Seven other probes recognize loci linked to the fra(X) at Xq27.3: DXS15 [Davies et al., 19851, DXS52 (St 14-1) [Oberle et al., 19871, DXS105 (CX55.71, DXS98 (4D-8), F8C (p1.8 and p114.12), and DXS51 (p52A) [Kidd et al., 19891. An autosomal probe 16-32, assigned to 16p [Harris et al., 19871, was applied as an internal standard for equal DNA loading.

DNA Analysis Genomic DNA was extracted from peripheral blood

leukocytes from the patient and his parents as previ-

I

T - O

Fig. 1. Pedigree of family. denotes fra(X) syndrome; N, normal intelligence; D, developmental delay but fra(X) unconfirmed.

442 Kupke et al.

Fig. 2. GTG-banded metaphase chromosomes of the patient expressing the fragile site on both X chromosomes (long arrows). The Y chromosome is marked by a short arrow.

et al., 19851, was informative. The DXS15 locus has been shown to be linked to the fra(X) locus with a recombina- tion frequency of 0.15 estimated by linkage [Brown et al., 19881. As shown in Figure 3A, DX13 hybridizes with a 2.8 kb BglII restriction fragment in the father, with a 2.8 kb and a 5.4 kb fragment in the mother, and a 5.4 kb fragment in the child. Accounting for the relative amount of DNA loaded in each lane, the motherchild ratio of hybridization intensity was found to be 1.0:2.47, indicating the presence of 2 copies of the 5.4 kb allele in the patient. This double dosage demonstrates homo- zygosity at this locus in the patient, which likely indi- cates homozygosity at the fra(X) locus. These findings thus confirm the “homozygous” cytogenetic findings of fra(X) site expression within the same cell.

The parental origin of the supernumerary X chromo- some in the patient was determined by the hybridization of polymorphic X chromosomal DNA sequences. One pericentromeric DNA probe, A2R/D7 (MAO-A), was in- formative in the nuclear family. The probe A2R/D7 rec- ognizes a 12.0 kb MspI restriction fragment in the fa- ther, fragments of 12.0 kb and 4.0 kb in the mother, and a 4.0 kb fragment in the child (Fig. 3B). Taking into account the relative DNA loading for each lane, the motherchild ratio of the hybridization intensity of the 4.0 kb fragment was calculated to be 1.00:2.05. Two copies of the 4.0 kb allele are evidently present in the

patient. Given that the MAO-A locus detected by A2R/ D7 is in close proximity to the centromere [Ozelius et al., 19881, its recombination frequency relative to the cen- tromere must be small. Therefore, the finding of double dosage of one maternal allele most likely reflects mater- nal nondisjunction during or after meiosis 11. The lack of chromosomal mosaicism in the patient makes a post- zygotic error less likely. X chromosomal meiosis I1 non- disjunction was also found to result in homozygosity of other X-linked disease loci such as the mutated an- drogen receptor gene in a case of 47,XXYltesticular fem- inization [Muller et al., 19901.

Previous parental origin studies of the X chromosome in populations of Klinefelter syndrome patients have shown no clear-cut predominance of paternal or mater- nal origin. In one study using Xg blood group, GGPD, and color blindness as markers, 58% of the supernumer- ary X chromosomes were of maternal and 42% were of paternal origin [Carothers and Filippi, 19883. In an- other investigation of 35 47,XXY patients, RFLP anal- ysis using X chromosomal DNA probes showed the ori- gin of the supernumerary X to be due to errors of paternal meiosis I in 53%, maternal meiosis I in 34% and maternal meiosis I1 in 9% of the cases [Jacobs et al., 19881.

The present case is the third reported patient with 47,XXY/fra(X) in whom the parental origin of the extra

Fragile X and Klinefelter Syndrome 443

Fig. 3. Differential hybridization of probes DX13 (A) and A2RiD7 (B) to BglI-A) and BIMspI-cleaved DNA from father (lane 11, mother (lane 21, and 47,fra(X)(q27),fra(X)(q27),Y patient (lane 3). Size stan- dards are given in kb. Hybridization results using p16-32 obtained with the same blot are shown below each lane to indicate the relative amount of DNA loading. The 5.4 kb BglII fragment detected by DX13 in A is in double dosage in the patient, taking into account the slight underloading of the child’s lane as compared to the mother’s (for further details, see text). In B the mother is heterozygous for 12.0 and 4.0 kb MspI fragments detected by A2RiD7. The 4.0 kb allele is present in double dosage in the child as compared to the mother.

X chromosome was determined. Thus, it is not yet clear if the distribution of parental origin and meiotic nondis- junction is different from that in populations of Klinefel- ter patients without the fra(X1 syndrome. If there is truly an increased predisposition to meiotic errors among fratX) heterozygotes, one would expect a predom- inance of cases due to maternal nondisjunction. Further studies will show whether nondisjunction preferentially occurs during meiosis I1 as in the present case.

ACKNOWLEDGMENTS We thank Drs. X. Breakefield, S . Orkin, I. W. Craig, L.

M. Kunkel, G. Bruns, and the ATCC for DNA probes. K. G. K. was the recipient of an NIH postdoctoral training grant in genetics. This work was supported by NIH grant HD24381.

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