Duchenne Muscular Dystrophy in a Female Patient with a Karyotype of 46,X,I(X)(Q10)

Tohoku J. Exp. Med., 2010, 222, 149-153Karyotype Analysis of a Female Patient with DMD 149

Duchenne Muscular Dystrophy in a Female Patient with a Karyotype of 46,X,i(X)(q10)

Zhanhui Ou,1 Shaoying Li,1 Qing Li,1 Xiaolin Chen,1 Weiqiang Liu1 and Xiaofang Sun1

1Institute of Gynecology and Obstetrics, The Third Affiliated Hospital of Guangzhou Medical College, Duobao Road, Guangzhou, China

Duchenne muscular dystrophy (DMD) is a severe recessive X-linked form of muscular dystrophy caused by mutations in the dystrophin gene and it affects males predominantly. Here we report a 4-year-old girl with DMD from a healthy family, in which her parents and sister have no DMD genotype. A PCR-based method of multiple ligation-dependent probe amplification (MLPA) analysis showed the deletion of exons 46 and 47 in the dystrophin gene, which led to loss of dystrophin function. No obvious phenotype of Turner syndrome was observed in this patient and cytogenetic analysis revealed that her karyotype is 46,X,i(X)(q10). In conclusion, we describe the first female patient with DMD who carries a de novo mutation of the dystrophin gene in one chromosome and isochromosome Xq, i(Xq), in another chromosome.

Duchenne muscular dystrophy (DMD) is a severe an uneventful pregnancy. At birth, her growth parameters recessive X-linked form of muscular dystrophy which is were normal. Her motor development was delayed: she characterized by rapid progression of muscle degeneration, could sit at 10 months and walk at 15 months, but fell down eventually leading to loss of ambulation and death. It easily. She shows abnormal gait, never runs and jumps, has affects approximately 1/3,500 male births (Mehler 2000). difficulty in standing up as well as rising from the floor, she The disorder is caused by mutations in the dystrophin gene also had calf hypertrophy, muscle weakness and Gowers’ located on Xp21.2. Dystrophin gene encodes a protein of sign, but without any neurological deficits or mental abnor- the membrane cytoskeleton in skeletal muscle (Hoffman et mality. Blood examination revealed consistently elevated al. 1987). Among DMD probands affected by the patho- serum levels of creatine kinase, 9,487 U/L (normal: 15-200 genic mutations, 60% have deletions and 5-10% have dupli- U/L), creatinine kinase isoenzyme MB, 409 U/L (normal: cations of the exons (Den Dunnen et al. 1989). The remain- 0-25 U/L), aspartate aminotransferase, 259 U/L (normal: ing about 30% patients are caused by mutations at the 0-40 U/L), and alanine aminotransferase, 568 U/L (normal: nucleotide level (Den Dunnen et al. 1989; Roberts et al. 0-40 U/L). Neither her parents nor her younger sister has 1992). A recent study revealed that in about two thirds of the same phenotype. Patient’s muscle biopsy is not avail- patients, mothers are carriers and the remaining one third able. Her height was 98 cm (-1SD), and weight was 20 kg, patients are due to de novo mutations in dystrophin gene without any other clinical signs of Turner syndrome at pres- (Alcántara et al. 2001). ent. Multiplex Ligation-dependent Probe Amplification Clinical Findings (MLPA) is a method to establish the copy number of up to The study protocol has been reviewed and approved by 45 nucleic acid sequences in one single reaction (also called the Research Ethics Committee of The Third Affiliated multiplex PCR reaction). MLPA reactions result in a very Hospital of Guangzhou Medical College. Informed consent reproducible gel pattern with fragments ranging from 130 to was obtained from all participants. 490 bp. Comparison of this gel pattern to that obtained with The 4-year-old propositus is the first pregnancy, was a control sample indicates which sequences show an aber- born to a 25-year-old man and his 24-year-old wife. Both rant copy number. By the MLPA (SALSA MLPA KIT parents are healthy and nonconsanguineous with an unre- P034/P035 DMD/Becker, MRC Holland, Amsterdam, markable family history. She was referred to our institute Netherlands) analysis, we detected the deletion of exons 46 for muscle weakness. The proposita was born at term after and 47 in the proposita but not in her parents or her younger

Received August 10, 2010; revision accepted for publication September 22, 2010. doi: 10.1620/tjem.222.149 Correspondence: Xiaofang Sun, Institute of Gynecology and Obstetrics, The Third Affiliated Hospital of Guangzhou Medical College, Duobao Road, Guangzhou, China. e-mail: [email protected]

149 150 Z. Ou et al.

Fig. 1. MLPA analysis of the family members. The arrows in the Set 1 of the patient indicate the deletion of exons 46 and 47, and arrowheads indicate the existence of exons 45 and 48. The number below the peak represented the correspond- ing exon. c: abbreviation for control. sister (Fig. 1), indicating that the patient’s deletion in the mutations are out of frame transcription, which may be sus- dystrophin gene was caused by de novo event. Further PCR ceptible to nonsense-mediated decay (NMD) process and analysis confirmed the specific deletion of these exons in prevent the translation. Therefore, these mutations are caus- the patient but not in her parents and sister (data not shown). ative for the phenotype of DMD. The results of test for the Based on website http://www.dmd.nl, we found that the short tandem repeat (STR) loci in the dystrophin gene and Karyotype Analysis of a Female Patient with DMD 151

Fig. 2. Pedigree of this family. A, Dystrophin gene linkage analysis in the family, it is obvious that the patient has the identical STR with her mother in these six loci. The primer sequences of these markers were published in Leiden Muscular Dystrophy Pages (http://www.dmd.nl). B, The three loci in the factor VIII gene of Xq28 analysis (Lalloz et al. 1991, 1994; Fang et al. 2006), showing that the patient has the same haplotype as her father. Square= male, Circle =female, fully shaded = affected. The number given beside each marker denoted the size of the STR marks products, “-” repre- sent missing the locus.

Xq implied that the mutations in X chromosome in the Instead, i(Xq) formation in human most likely results from patient originated from her mother (Fig. 2A, B). sister chromatid breakage and reunion in proximal Xp Cytogenetic analysis revealed the karyotype in the proposita (Callen et al. 1987; James et al. 1997). In addition, it was is 46,X,i(X)(q10) (Fig. 3). In other words, this patient is a reported that the increasing parental age was not associated female hemizygous for X linked genes located on the X with isochromosome formation, no matter maternally or chromosome short arm, as she has only one Xp chromo- paternally derived (James et al. 1997). In our case, the some, while her parents and younger sister have normal patient was born when her father and mother were in their karyotype. age of 25 and 24, respectively, demonstrating that isochromosome formation also occurs in the young couples’ child. Discussion Interestingly, this index case is free from the obvious It has long been proposed that i(Xq) formation is phenotype of Turner syndrome. It is possible that the girl is caused by centromere misdivision. However, several stud- too young to manifest the symptoms of Turner syndrome, as ies demonstrated that centromere misdivision is not a com- a previous study concluded that the median age of female mon but a rare mechanism of i(Xq) formation in humans. with i(Xq) to be diagnosed is 14.2 years (Stochholm et al. 152 Z. Ou et al.

Fig. 3. Karyotype analysis of the patient. The abnormal X chromosome has two long arms compared with the normal one (indicated by arrow).

2006) . The only abnormal phenotype we observed is short et al. 1986), the karyotype of our patient is unique. We stature because of haploinsufficiency for SHOX gene, which present the first female patient with DMD who carries an is located in X-chromosome pseudoautosomal region on the atypical karyotype of Turner syndrome and harbors a de distal Xp. It is important to note that this region is identical novo mutation in dystrophin gene in the remaining X chro- on X- and Y-chromosomes and does not undergo X-inacti mosome. vation (Blaschke and Rappold 2000). To our knowledge, several different genetic abnormali- References ties have been documented for female DMD and the milder Alcántara, M.A., Garcia-Cavazos, R., Hernandez, U.E., allelic form Becker muscular Dystrophy (BMD): (1) an González-del, Angel. A., Carnevale, A. & Orozco, L. (2001) X-autosome reciprocal translocation and a preferential inac- Carrier detection and prenatal molecular diagnosis in a Duch- enne muscular dystrophy family without any affected relative tivation of the normal X-chromosome (Verellen-Dumoulin available. Ann. Genet., 44, 149-153. et al. 1984); (2) in a classical 45, X0 karyotype of Turner Azofeifa, J., Voit, T., Hubner, C. & Cremer, M. (1995) X-chromo- syndrome, simultaneously, the only X-chromosome with a some methylation in manifesting and healthy carriers of dys- dystrophin mutation (Chelly et al. 1986); (3) skewed X trophinopathies: concordance of activation ratios among first degree female relatives and skewed inactivation as cause of the inactivation in the normal X-chromosome of the female affected phenotypes. Hum. Genet., 96, 167-176. DMD mutation carriers. (Azofeifa et al. 1995); (4) unipa- Blaschke, R.J. & Rappold, G.A. (2000) SHOX: growth, Leri-Weill rental disomy of female with DMD mutation in both and Turner syndromes. Trends Endocrinol. Metab., 11, 227- X-chromosome (Quan et al. 1997); (5) co-occurrence of 230. Callen, DF., Mulley, J.C., Baker, E.G. & Sutherland, G.R. (1987) mutations in both dystrophin and androgen-receptor genes Determining the origin of human X isochromosomes by use of in the patient (Katayama et al. 2006); and (6) girl with DNA sequence polymorphisms and detection of an apparent homozygous dystrophin mutation caused by consanguinity, i(Xq) with Xp sequences. Hum. Genet., 77, 236-240. whose parents have the same mutations in the DMD gene Chelly, J., Marlhens, F., Le Marec, B., Jeanpierre, M., Lambert, M., (Fujii et al. 2009). While our patient is similar to the case Hamard, G., Dutrillaux, B. & Kaplan, J.C. (1986) De novo DNA microdeletion in a girl with Turner syndrome and Duch- of Turner syndrome with the dystrophin gene mutation in enne muscular dystrophy. Hum. Genet., 74, 193-196. the remaining X chromosome as originally reported (Chelly Den Dunnen, J.T., Grootscholten, P.M., Bakker, E., Blonden, L.A., Karyotype Analysis of a Female Patient with DMD 153

Ginjaar, H.B., Wapenaar, M.C., van Paassen, H.M., van able dinucleotide repeat within the factor VIII gene. Lancet, Broeckhoven, C., Pearson, P.L. & van Ommen, G.J. (1989) 338, 207-211. Topography of the Duchenne muscular dystrophy (DMD) Lalloz, M.R., Schwaab, R., McVey, J.H., Michaelides, K. & gene: FIGE and cDNA analysis of 194 cases reveals 115 dele- Tuddenham, E.G. (1994) Haemophilia A diagnosis by simul- tions and 13 duplications. Am. J. Hum. Genet., 45, 835-847. taneous analysis of two variable dinucleotide tandem repeats Fang, Y., Wang, X.F., Dai, J. & Wang, H.L. (2006) A rapid multi- within the factor VIII gene. Br. J. Haematol., 86, 804-809. fluorescent polymerase chain reaction for genetic counselling Mehler, M.F. (2000) Brain dystrophin, neurogenetics and mental in Chinese haemophilia A families. Haemophilia, 12, 62-67. retardation. Brain Res. Brain Res. Rev., 32, 277-307. Fujii, K., Minami, N., Hayashi, Y., Nishino, I., Nonaka, I., Tanabe, Quan, F., Janas, J., Toth-Fejel, S., Johnson, D.B., Wolford, J.K. & Y., Takanashi, J. & Kohno, Y. (2009) Homozygous female Popovich, B.W. (1997) Uniparental disomy of the entire X Becker muscular dystrophy. Am. J. Med. Genet. A, 149A, chromosome in a female with Duchenne muscular dystrophy. 1052-1055. Am. J. Hum. Genet., 60, 160-165. Hoffman, E.P., Brown, R.H. Jr. & Kunkel, L.M. (1987) Dystro- Roberts, R.G., Bobrow, M. & Bentley, D.R. (1992) Point muta- phin: the protein product of the Duchenne muscular dystrophy tions in the dystrophin gene. Proc. Natl. Acad. Sci. USA, 89, locus. Cell, 51, 919-928. 2331-2335. James, R.S., Dalton, P., Gustashaw, K., Wolff, D.J., Willard, H.F., Stochholm, K., Juul, S., Juel, K., Naeraa, R.W. & Gravholt, C.H. Mitchell, C. & Jacobs, P.A. (1997) Molecular characterization (2006) Prevalence, incidence, diagnostic delay, and mortality of isochromosomes of Xq. Ann. Hum. Genet., 61, 485-490. in Turner syndrome. J. Clin. Endocrinol. Metab., 91, 3897- Katayama, Y., Tran, V.K., Hoan, N.T., Zhang, Z., Goji, K., Yagi, M., 3902. Takeshima, Y., Saiki, K., Nhan, N.T. & Matsuo, M. (2006) Verellen-Dumoulin, C., Freund, M., De Meyer, R., Laterre, C., Co-occurrence of mutations in both dystrophin- and androgen- Frederic, J., Thompson, M.W., Markovic, V.D. & Worton, R.G. receptor genes is a novel cause of female Duchenne muscular (1984) Expression of an X-linked muscular dystrophy in a dystrophy. Hum. Genet., 19, 516-519. female due to translocation involving Xp21 and non-random Lalloz, M.R., McVey, J.H., Pattinson, J.K. & Tuddenham, E.G. inactivation of the normal X chromosome. Hum. Genet., 67, (1991) Haemophilia A diagnosis by analysis of a hypervari- 115-119.