GENETIC TESTING Volume 12, Number 2, 2008 ª Mary Ann Liebert, Inc. Pp. 221–224 DOI: 10.1089=gte.2007.0081
Identification of Duchenne Muscular Dystrophy Female Carriers by Fluorescence In Situ Hybridization and RT-PCR Ana Claudia Vela´zquez-Wong,1 Ce´sar Herna´ndez-Huerta,1 Areli Ma´rquez-Calixto,1 Fidel Omar Herna´ndez-Aguilar,1 Maricela Rodrı´guez-Cruz,2 Fabio Salamanca-Go´mez,1 and Ramo´n Coral-Va´zquez1
Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder caused by mutations in the dystrophin DMD gene located at Xp21.1 region. Up to 65% of the patients present dystrophin gene deletions. Mothers of DMD patients have a two-thirds chance of carrying a dystrophin mutation. The female carrier will transmit the disease gene to half of her sons and half of her daughters. As the recurrence risk for the disease is extremely high, it is very important to detect carrier status among female relatives of the patients to bring an adequate genetic counseling. In this work, results from two methods to identify female carriers are presented. One method is a multicolor fluorescence in situ hybridization (FISH) assay, and the other is reverse transcriptasepolymerase chain reaction (RT-PCR). We showed that FISH is an efficient, sensitive method that brings confident results to detect DMD female carriers as compared to RT-PCR.
Introduction
D
uchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disease caused by mutations in the dystrophin DMD gene. DMD is a rapid progressive form of muscular dystrophy and affects 1 in 3,500 newborn males, whereas BMD has a slower progressive nature and affects 1 in 30,000 newborn males (Blake et al., 2002; Nowak and Davies, 2004). The dystrophin gene is located at Xp21.1 and spans 79 exons (Coffey et al., 1992; Roberts et al., 1993). Approximately 65% of mutations of the DMD gene are large deletions, and they are not evenly distributed because they are concentrated in ‘‘hot spots,’’ one toward the 50 part of the gene, which encompasses exons 3–7, and another in the central portion of the gene, including exons 43–51 (Koenig et al., 1989; Hoffman and Dressman, 2001). Scientific evidence has demonstrated that mothers of affected patients have two-thirds chance of carrying mutations in DMD gene and that 65% of them are deletions (Koenig et al., 1989). Other studies have shown that some mothers are in fact gonadal mosaics and are presumed to carry a dystrophin deletion in a proportion of their gametes (Bunyan et al., 1994; Wilton et al., 1994). Because the X-linked pattern of inheritance has an extremely high recurrence risk, it is very important to detect DMD female carriers.
Analysis of mRNA using reverse transcriptase-polymerase chain reaction (RT-PCR) enables detection of deletions, duplications, and point mutations in the dystrophin gene and encompasses a larger diagnostic extent (Lukas et al., 2001). However, some groups have used the fluorescence in situ hybridization (FISH) assay to identify DMD female carriers since it provides an alternative approach to current molecular methods for carrier detection (Voskova-Goldman et al., 1997; Ligon et al., 2000; Hermanova et al., 2002). Importantly, this method is very useful to detect deletions since it is directed at exon-specific hot spots of the dystrophin gene, which are responsible for the majority of deletions. The number of fluorescent signals determines the copy number of the region to analyze which results are very effective for detecting major deletions. The aim of this study was to detect DMD female carrier status in women from a family with a positive history of DMD using a FISH assay that utilizes specific DMD exon probes and RT-PCR. Materials and Methods Fluorescence in situ hybridization Five women belonging to a family with a positive history of DMD were selected to be screened for carrier status. For the FISH assay we used specific DMD probes for exon 45
1 Unidad de Investigacio´n Me´dica en Gene´tica Humana, and 2Unidad de Investigacio´n Me´dica en Nutricio´n, Hospital de Pediatrı´a, Centro Me´dico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Mexico City, Mexico.
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222 (Cytocell Technologies, Cambridge, UK). The exon 45– specific probe contained both specific exon 45 DNA and specific intronic sequences surrounding the exon. The DMD probe is accompanied by an X chromosome a-satellite probe to provide an internal hybridization control and effective chromosome identification. The DMD probe was directly labeled with Texas Red fluorophore, and X a-satellite was labeled with FITC fluorophore. The procedure was performed according to product recommendations. Briefly, each woman provided a peripheral blood sample, and culture and harvest of the cells were performed according to standard cytogenetic methods (Moorhead et al., 1960). The slides were prepared with metaphase chromosomal suspension by spotting cell suspension onto cleaned microscope slides. The FISH assay consisted of adding 10 mL of probe to the sample slide, and simultaneous denaturation of sample and probe was carried out by heating slide on a hot plate at 758C for 2 min. After that, slides were placed in an incubator at 378C for 16 h. Then, posthybridization washes were performed in 2 SSC. Finally, DAPI antifade was added to the slide, and the samples were analyzed with an Axio Imager Fluorescence Carl Zeiss microscope equipped with a triple band filter. At least 30 chromosomal metaphases were analyzed per individual. Images were captured using Metasystems Ikaros and Isis Image System. RNA isolation and RT-PCR Total RNA from leukocytes was extracted using TRIzol (InvitrogenTM, Carlsbad, CA) according to manufacturer’s instructions. cDNA synthesis was performed using 2 mg total RNA by reverse transcription with MMLV (InvitrogenTM) and random primers. cDNAs were PCR amplified with specific primers for exon 44, sense 50 -CGATTTGACAGATCTG TTGAG-30 , and exon 46, antisense 50 -CTTGACTTGCTCAAG CTTTTC-30 . PCR reaction mixture consisted of 2.5 mL of RT reaction, 10 pmol of each primer, 200 mM of each dNTP, 1.5 mM of MgCl2, 50 mM KCl, 20 mM Tris-HCl pH 8.4, and two units of Taq polymerase (InvitrogenTM) in a final volume of 25 mL. Amplification conditions were one denaturing step at 958C for 5 min followed by 32 cycles with denaturing of 30 s at 958C; annealing of 30 s at 598C; elongation of 30 s at 728C; and final elongation step of 728C for 7 min. PCR products were electrophoretically separated on 2% agarose gel stained with ethidium bromide. Results FISH and RT-PCR assays were performed in women belonging to a family with history of DMD. Previously, we detected a deletion in exon 45 of the DMD gene by multiplex PCR assay (data not shown). RT-PCR results demonstrated that two of the daughters studied presented two bands, as the mother (Fig. 1: I-2, II-2, and II-3), one band corresponding to the size expected (475 bp) for the normal transcript and the other with the calculated size (295 bp) for the transcript with the deletion in the exon 45. According to this, these women are carriers of the mutation associated to DMD. The woman II-1 is a noncarrier since she only presented the band with normal size as the control (C). However, when the FISH assay was performed, female carrier status was determined only in the mother and in one of the daughters. As expected, the mother chromosomal spreads displayed one red signal
FIG. 1. RT-PCR from a DMD female carrier and three daughters.
for the specific exon 45 probe in only one X chromosome, and two green control centromeric signals, one on each X homolog chromosome, demonstrating that she was a DMD female carrier (Fig. 2: I-2). The same results were seen in one of the daughters (Fig. 2: II-2). Three other daughters showed two red and two green signals defining them as non-DMD female carriers (Fig. 2: II-1, II-3, and II-4). Therefore, a woman identified as a DMD carrier by RT-PCR turned out to be a noncarrier by FISH. Discussion The methods most commonly used for molecular diagnosis of deletions at the DMD locus are RT-PCR and Southern blot. These molecular tools are very useful for detecting DMD gene deletions in males, but for females the detection of deletions can be problematic since women possess two X chromosomes (Ligon et al., 2000). More recently, FISH detecting chromosomal deletions has been introduced to identify specific DMD exon deletions (Voskova-Goldman et al., 1997; Ligon et al., 2000; Hermanova et al., 2002). In this study we compared the RT-PCR and FISH results in a family with a positive history of DMD to identify female carriers. According to our results of RT-PCR, we defined two daughters as carriers (Fig. 1: II-2 and II-3). However, by FISH one of these women (II-3) showed one red and one green fluorescent signal on each X chromosome, defining her as a non-DMD carrier. These results were observed in all of the cells of each non-DMD female carrier, either chromosome metaphases and=or interphase nuclei. The inconsistency in the results could have been caused by a normal alternative splicing mechanism present in the DMD gene of this woman. Several alternative spliced forms in DMD gene can produce different size transcripts that can be analyzed by using the RT-PCR technique (Barbieri et al., 1996; Tuffery et al., 1996). This illegitimate transcription of mRNA can be observed in DMD gene as well as in other known genes. According to this, we may suggest that the possible DMD female carriers can present two transcripts when a deletion is present. However, it is very important to remember that DMD gene may suffer alternative splicing in normal conditions. This may explain why one woman was identified as DMD female carrier by RT-PCR. For these reasons, when we compared results of the two methods used, we found a falsepositive result in one healthy woman by RT-PCR. In conclusion, we demonstrated that FISH has the advantage that the number of fluorescent signals determines the
FISH DETECTION OF DMD FEMALE CARRIERS
FIG. 2. FISH chromosomal spreads from a family with positive DMD history. copy number of the region examined, comparing to RT-PCR. Thus, while confirming the results of other groups, FISH using DMD exon-specific probes represents an effective, highly accurate, direct, qualitative approach for establishing DMD carrier status of females (Ligon et al., 2000; Hermanova et al., 2002). Even when there is no cure for DMD patients, providing an accurate diagnosis for female carriers allows bringing the family to a precise genetic counseling. In this way these women could plan their reproductive lives to avoid recurrence risks. To our knowledge, this is the first study that uses Cytocell exon-specific probes to detect DMD female carriers. Acknowledgments We gratefully acknowledge the family that participated in this study, the physicians, and the personnel who collected the samples. This work was supported by Conacyt 2003-7180024 and the Instituto Mexicano del Seguro Social. References Barbieri AM, Soriani N, Ferlini A, Nichelato A, Carrera P (1996) Seven novel additional small mutations and a new alternative splicing in the human dystrophin gene detected by heteroduplex analysis and restricted T-PCR heteroduplex analysis of illegitimate transcripts. Eur J Hum Genet 4(3):183–187. Blake DJ, Weir A, Newey SE, Davies KE (2002) Function and genetics of dystrophin and dystrophin-related proteins in muscle. Physiol Rev 82(2):291–329. Bunyan DJ, Robinson DO, Collins AL, Cockwell AE, Bullman HM, Whittaker PA (1994) Germline and somatic mosaicism in a female carrier of Duchenne muscular dystrophy. Hum Genet 93(5):541–544.
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Address reprint requests to: Ana Claudia Vela´zquez-Wong, M.D. Av. Cuauhtemoc 330 Col. Doctores C. P. 06725 Mexico City Mexico E-mail:
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This article has been cited by: 1. Qian Wang , Jesse Li-Ling , Changkun Lin , Yingyu Wu , Kailai Sun , Hongwei Ma , Chunlian Jin . 2009. Characteristics of Dystrophin Gene Mutations Among Chinese Patients as Revealed by Multiplex Ligation–Dependent Probe AmplificationCharacteristics of Dystrophin Gene Mutations Among Chinese Patients as Revealed by Multiplex Ligation–Dependent Probe Amplification. Genetic Testing and Molecular Biomarkers 13:1, 23-30. [Abstract] [PDF] [PDF Plus] 2. Qian Wang, Jesse Li-Ling, Changkun Lin, Yingyu Wu, Kailai Sun, Hongwei Ma, Chunlian Jin. 2009. Characteristics of Dystrophin Gene Mutations Among Chinese Patients as Revealed by Multiplex Ligation–Dependent Probe Amplification. Genetic Testing, ahead of print090108090224061. [CrossRef] 3. Yupeng Wu, Gengxin Yin, Keqin Fu, De Wu, Qian Zhai, Huarong Du, Zhongjun Huang, Yuhua Niu. 2009. Gene diagnosis for nine Chinese patients with DMD/BMD by multiplex ligation-dependent probe amplification and prenatal diagnosis for one of them. Journal of Clinical Laboratory Analysis 23:6, 380-386. [CrossRef]
