American Journal of Medical Genetics 85:476–478 (1999) Prenatal Evaluation of a De Novo X;9 Translocation Baruch Feldman,1 Ralph L. Kramer,1 Salah A.D. Ebrahim,2 Dayna J. Wolff,3 and Mark I. Evans1,2,4* 1 Division of Reproductive Genetics, Department of Obstetrics and Gynecology, Wayne State University, Detroit, Michigan 2 Division of Reproductive Genetics, Department of Pathology, Wayne State University, Detroit, Michigan 3 Center for Human Genetics, Case Western Reserve University, Cleveland, Ohio 4 Division of Reproductive Genetics, Department of Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan A case of X-autosome translocation was diagnosed prenatally [46,X,t(X;9)(p21.3∼ 22.1;q22]. We describe the use of fluorescence in situ hybridization (FISH) to estimate the integrity of the Duchenne muscular dystrophy (DMD) gene. X-inactivation studies were used as well to assess the probability of phenotypic abnormalities associated with functional partial disomy X and monosomy 9. Am. J. Med. Genet. 85:476–478, 1999. © 1999 Wiley-Liss, Inc. KEY WORDS: X;9 translocation; prenatal diagnosis; X inactivation; FISH INTRODUCTION X-autosome translocations have generally been shown to be of parental origin or arise de novo. The phenotypic consequences of the translocation will depend on the break point on the X chromosome and the autosome as well as the pattern of X inactivation. Most of these patients are either phenotypically normal or had gonadal dysgenesis, whereas approximately 9% had multiple anomalies and/or mental retardation [Schmidt and Du Sart, 1992]. When the break point occurs within the proper gene, a “classic” X-linked disorder can be phenotypically expressed in a female [Zatz et al., 1981]. We describe a case of fetal X-autosome translocation in which molecular studies were used in order to estimate the X-inactivation pattern and the integrity of the Duchenne muscular dystrophy (DMD) gene. CASE REPORT A 38-year-old patient was seen at 17 weeks of gestation for both elevated maternal serum alpha- *Correspondence to: Mark I. Evans, M.D., Division of Reproductive Genetics, Department of Obstetrics and Gynecology, Hutzel Hospital/Wayne State University, 4707 St. Antoine Boulevard, Detroit, MI 48201. E-mail: [email protected] Received 17 November 1998; Accepted 23 March 1999 © 1999 Wiley-Liss, Inc. fetoprotein (MSAFP) level and advanced maternal age. The patient’s medical, obstetrical, and family history was unremarkable. Ultrasound examination demonstrated no anomalies other than a 3-mm choroid plexus cyst. Cytogenetic analysis of cultured amniocytes revealed the fetal karyotype to be 46,X,t(X;9)(p21.3∼22.1;q22). The translocation arose de novo because both parents were tested and shown to be karyotypically normal. There was concern, however, that the break point on the X chromosome could involve the DMD gene at Xp21.3. In order to obtain a more precise estimate of fetal risk for DMD, assessment of the specific break point was determined with a specific fluorescence in situ hybridization (FISH) probe (Quint-Essential X-Specific DNA Probe, Oncor, Gaithersburg, MD). This probe hybridizes to the Xp21.2-p21.3 region and specifically identifies sequences containing the DMD locus. FISH analysis showed that the Xp21.2-p21.3 locus was present on the normal and the abnormal X-chromosome. No other chromosomes demonstrated a hybridization signal (Fig. 1). This observation suggests that the DMD region was not disrupted, translocated, or deleted by the rearrangement and significantly reduced the likelihood of the fetus affected with DMD. The patient was counseled that the results of the FISH studies are reassuring, however, normal gene function cannot be determined by this technique. Fetal muscle biopsy for dystrophin studies was offered for confirmation of normal gene function, but the couple declined any further invasive procedures. In order to assess the risk of functional partial disomy X and concomitant monosomy 9, an assay to analyze methylation at the fragile X mental retardation gene (FMR1) was performed, as described by Carrel and Willard . DNA from the patient and appropriate controls were digested with the methylation-sensitive enzyme HpaII. This enzyme cleaves at two restriction sites near the CGG repeat of the unmethylated FMR1 gene on the active X chromosome but does not digest these sites on the inactive (methylated) X chromosome. Digested and undigested DNA samples were amplified by polymerase chain reaction. Samples were separated by gel electrophoresis, and X- Prenatal Evaluation of X;9 Translocation Fig. 1. 477 FISH of metaphase chromosomes probed for the DMD region, Xp21.2-p21.3. Positive hybridization is detected on both X chromosomes. inactivation ratios were determined by visual comparison of the cut and uncut fragments obtained from the radiograph. The results of the X-inactivation study strongly suggest nonrandom inactivation of the normal X chromosome (Fig. 2). This significantly reduced the risk of functional partial disomy X and partial monosomy 9. Based on these results the couple decided to continue the pregnancy. Unfortunately, at 33 weeks of gestation, preterm premature rupture of membranes occurred and the patient was hospitalized. At 34 weeks of gestation, chorioamnionitis was diagnosed, but prior to induction of labor, intrauterine fetal death was confirmed. Labor was induced and the patient delivered a stillborn female infant. There were no apparent fetal anomalies, however, a true knot was noted in the umbilical cord. Histological examination of the placenta confirmed the diagnosis of chorioamnionitis but autopsy did not show any other fetal anomalies. Normal dystrophin gene function was demonstrated by positive immunofluorescence staining for dystrophin [Arahata et al, 1989] in tissue obtained from postnatal fetal muscle biopsy. DISCUSSION Fig. 2. Results of FMR1 methylation analysis. For the fetus with the X;9 translocation (lanes 3 and 4), only allele “2” was amplified following digestion. Controls: normal female with random X inactivation (lanes 1 and 2); female with skewed X inactivation pattern (lanes 5 and 6); normal male was included to control for complete digestion (lanes 7 and 8). u, uncut; c, cut. The phenotypic consequences of X-autosome translocation will depend on the breakpoint on the X chromosome and the autosome as well as the pattern of X inactivation. Duchenne muscular dystrophy (DMD) is an X-linked muscle wasting disorder observed with a frequency of approximately 1 in 3,500 newborn males. DMD has also been described in females in which de novo Xautosome translocations have been shown to be the cause [van Bakel et al., 1995]. Expression of the disease in these females is the result of nonrandom inactivation of the normal X chromosome as well as a breakpoint inside or very close to the DMD gene on the translocated chromosome. 478 Feldman et al. X inactivation is the process by which females achieve dosage compensation by silencing one X chromosome. In chromosomally normal females the process is random. However, most females with one abnormal X chromosome demonstrate nonrandom inactivation. Nonrandom inactivation of the normal X chromosome will lead to phenotypically normal fetus. In 10–15% of structurally balanced X-autosome heterozygotes, however, the mechanism fails and some cells with partial disomy of the X chromosome and partial monosomy of the autosomal translocated segment survive and cause phenotypic abnormality [Schmidt and Du Sart, 1992; Wolff et al., 1998]. Our observations on a case of X;9 translocation diagnosed prenatally illustrate the clinical dilemmas associated with X-autosome translocations. We believe that the use of FISH and X-inactivation studies is an important adjunct to conventional cytogenetic techniques in such cases. The use of molecular studies may determine the break points more precisely. It can also provide a more precise estimate of the phenotypic consequences of X-autosome translocation. Unfortunately in the case presented, the fetus was stillborn. This appeared to have resulted from causes unrelated to the X-autosome translocation. 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