- Email: [email protected]
Duchenne muscular dystrophy in a female with compound heterozygous contiguous exon deletions
Accepted Manuscript Title: Duchenne muscular dystrophy in a female with compound heterozygous contiguous exon deletions Author: Eri Takeshita, Narihiro Minami, Kumiko Minami, Mikiya Suzuki, Takeya Awashima, Akihiko Ishiyama, Hirofumi Komaki, Ichizo Nishino, Masayuki Sasaki PII: DOI: Reference:
S0960-8966(17)30001-9 http://dx.doi.org/doi: 10.1016/j.nmd.2017.03.011 NMD 3365
To appear in:
Neuromuscular Disorders
Received date: Revised date: Accepted date:
3-1-2017 13-3-2017 29-3-2017
Please cite this article as: Eri Takeshita, Narihiro Minami, Kumiko Minami, Mikiya Suzuki, Takeya Awashima, Akihiko Ishiyama, Hirofumi Komaki, Ichizo Nishino, Masayuki Sasaki, Duchenne muscular dystrophy in a female with compound heterozygous contiguous exon deletions, Neuromuscular Disorders (2017), http://dx.doi.org/doi: 10.1016/j.nmd.2017.03.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Case report
Duchenne muscular dystrophy in a female with compound heterozygous contiguous exon deletions
Eri Takeshita1), Narihiro Minami2)3), Kumiko Minami3), Mikiya Suzuki4), Takeya Awashima1), Akihiko Ishiyama1), Hirofumi Komaki1), Ichizo Nishino3)5), Masayuki Sasaki1) 1)
Department of Child Neurology, National Center Hospital, National Center of Neurology
and Psychiatry (NCNP), Tokyo, Japan 2)
Department of Laboratory Medicine, National Center Hospital, National Center of
Neurology and Psychiatry (NCNP), Tokyo, Japan 3)
Department of Genome Medicine Development, Medical Genome Center, National Center of
Neurology and Psychiatry (NCNP), Tokyo, Japan 4)
Department of Neurology, Higashi-Saitama Hospital, National Hospital Organization,
Hasuda, Japan 5)
Department of Neuromuscular Research, National Institute of Neuroscience, National Center of
Neurology and Psychiatry (NCNP), Tokyo, Japan
Page 1 of 12
2
Corresponding author: Eri Takeshita, MD, PhD Department of Child Neurology, National Center Hospital, National Center of Neurology and Psychiatry (NCNP), 4-1-1 Ogawahigashi-cho, Kodaira, Tokyo 187-8551, Japan Tel: +81 42 3412711; Fax: +81 42 3446745 E-mail: [email protected]
Highlights This female patient showed Duchenne muscular dystrophy like symptoms and clinical course. Muscle pathology was consistent with Duchenne muscular dystrophy. Gene analysis by MLPA suggested Becker muscular dystrophy carrier. Messenger RNA analysis showed compound heterozygous contiguous exon deletions. Genomic DNA analysis demonstrated the accurate deletion region on each allele.
Page 2 of 12
3
Abstract Females with Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) mutations rarely exhibit clinical symptoms from childhood, although potential mechanisms for symptoms associated with DMD and BMD in females have been reported. We report the case of a female DMD patient with a clinical course indistinguishable from that of a male DMD patient; the female possessed compound heterozygous contiguous exon deletions in the dystrophin gene. She exhibited Gowers’ signs, calf muscle hypertrophy, and high serum creatinine kinase levels at 2 years. Her muscle pathology showed most of the fibers tested negative for dystrophin immunohistochemical staining. She lost ambulatory ability at 11 years. Now, at over 21 years, she has difficulty rolling over by herself. Multiplex ligation-dependent probe amplification analysis of this gene detected one copy of exons 48–53; she was found to be a BMD carrier with an in-frame deletion. Messenger RNA from her muscle demonstrated out-of-frame deletions of exons 48–50 and 51–53 occurring on separate alleles. Genomic DNA from her lymphocytes demonstrated the accurate deletion region on each allele. To our knowledge, this is the first report on a female patient possessing compound heterozygous contiguous exon deletions in the dystrophin gene, leading to DMD.
Keywords: compound heterozygous; Duchenne muscular dystrophy; dystrophin; exon deletion; female
Page 3 of 12
4
1. Introduction Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the dystrophin gene located at Xp21.2. It affects approximately 1 in 3500 boys and causes progressive muscle weakness. DMD patients generally carry frame-shift dystrophin mutations, whereas a milder form called Becker muscular dystrophy (BMD) is due to in-frame mutations. Deletions of one or more exons account for approximately 60%–70% of mutations in individuals with DMD and BMD and duplications are found in approximately 5%–10%. The remaining 25%–35% of DMD cases and 10%–20% of BMD cases are caused by point mutations or other subtle changes in the dystrophin gene [1]. Most heterozygous carrier females with dystrophin mutations exhibit no clinical symptoms, and it is very rare for females with DMD and BMD mutations to exhibit symptoms from childhood. In this study, we report the case of female DMD patient with a clinical course indistinguishable from that of a male DMD patient; the female possessed compound heterozygous contiguous exon deletions in the dystrophin gene. 2. Case report The proband (III-2) was a 21-year-old female, who was the first child of non-consanguineous parents (Fig. 1A). Her mother (II-4) lost an unborn child (III-1) before the patient was born. In addition, her mother was addicted to alcohol and divorced from her father (II-2). She had a half-brother (III-3); however, it was not clear if he was healthy or not. She was born uneventfully; her body weight was 3490 g and body length was 48 cm at
Page 4 of 12
5
birth. At 1 year, she began walking alone but was likely to fall. At 2 years and 2 months, she was referred to the hospital for a heart murmur. The possible existence of heart disease was excluded; however, she exhibited Gowers’ signs, calf muscle hypertrophy, and high serum creatinine kinase levels (CK 18280 IU/L). A muscle biopsy was performed at 2 years and 4 months and most of the fibers tested negative for dystrophin immunohistochemical staining; thus she was diagnosed with DMD (Fig. 1B). At 9 years, she experienced more difficulty going up and down stairs, and she lost ambulatory ability at 11 years. Now, at over 21 years, she can operate her mobility scooter but has difficulty rolling over by herself, and her condition has progressed to deformity of the trunk due to scoliosis. Multiplex ligation-dependent probe amplification (MLPA) analysis of the dystrophin gene detected one copy of exons 48–53, which suggested that an in-frame exon deletion was present in a BMD carrier (Fig. 2A). However, her clinical course and muscle pathology were inconsistent with a BMD carrier and her condition was more similar to female DMD. Chromosomal study by G-banding determined 46 chromosomes with XX, i.e., a normal karyotype. The patterns of X-chromosome inactivation were assessed based on the methylation status at the androgen receptor locus, where the results showed that she possessed a random (non-skewed) X-chromosome inactivation pattern (38.7: 61.3%). These data excluded some possible known mechanisms for female DMD, which we consider later. Thus, we proceeded with additional gene analysis. Messenger RNA was amplified from her muscle by reverse transcription polymerase chain reaction (PCR) using primers located
Page 5 of 12
6
outside the exons of a deletion region (P1 and P2), where the two fragments amplified were shorter than the wild type (Fig. 2B). These fragments were analyzed by direct sequencing. When the P1 forward primer was used for sequencing, exon 47 was followed by exon 48, which overlapped exon 51. Similarly, sequencing with the P2 reverse primer showed that exon 54 was followed by exon 53, which overlapped exon 50 (Fig. 2C). These results suggested that the deletions of exons 48–50 and exons 51–53 were on separate alleles. Thus, we conducted additional sequencing analysis to confirm these results. Using the P3 forward primer located in exon 49 showed that exon 50 was followed by exon 54. Using the P4 reverse primer located in exon 53, we found that exon 51 was followed by exon 47 (Fig. 2D). Thus, the results of the MLPA analysis demonstrated that the two exon deletions occurred on different alleles (Fig. 2E). Additionally, we analyzed genomic DNA from the patient’s lymphocytes to detect the accurate deletion region on each allele. Genomic DNA was amplified by long PCR using primers located outside the deletion region of exons 48–50 (P5 and P6), and subsequent sequencing using the same primers demonstrated that one allele possessed out-of-frame deletion of exons 48–50 (c.6913-23922_7310-1654del123450) (Fig. 3A, 3B). Additional long PCR using primers located outside the deletion region of exons 51–53 (P7 and P8) and sequencing using the same primers demonstrated that the other allele possessed out-of-frame deletion of exons 51– 53 (c.7310-941_7872+4982del100741) (Fig. 3A, 3B). Therefore, we concluded that the subject possessed compound heterozygous contiguous exon deletions, i.e., deletions of
Page 6 of 12
7
exons 48–50 (out-of-frame; c.6913-23922_7310-1654del123450) and exons 51–53 (out-of-frame; c.7310-941_7872+4982del100741); thus, she was genetically diagnosed with female DMD. 3. Discussion We considered that this case was female DMD based on the clinical course and muscle pathology; however, the initial dystrophin gene analysis by MLPA to search for in-frame deletions suggested that she was a BMD carrier. Some mechanisms for female DMD and BMD have been reported previously: Turner syndrome (45, X) with a dystrophin mutation on the remaining X chromosome [2]; skewed X-chromosome inactivation in the carriers of DMD mutations [3,4]; and X-autosome translocations involving the dystrophin gene and preferential inactivation of the normal X chromosome [5,6]. In this case, these mechanisms were excluded based on a chromosomal study using G-banding and X-chromosome inactivation analysis. Therefore, we proceeded with the additional dystrophin gene analysis, where we concluded that she possessed compound heterozygous contiguous exon deletions. Furthermore, some rare genetic abnormalities have been documented as follows: a homozygous dystrophin mutation derived from maternal uniparental disomy [7]; the co-occurrence of mutations in both the dystrophin and androgen receptor genes in a patient with 47, XY [8]; and female BMD with a homozygous dystrophin mutation caused by consanguinity [9]. To the best of our knowledge, only one BMD-like female has been reported who possessed compound heterozygosity for two
Page 7 of 12
8
dystrophin mutations, i.e., an exon deletion and a point mutation in an intron splice site [10]. Although it is rare as mentioned above, several mechanisms which cause female DMD by genetic mutation has occurred because of the presence of both dystrophin alleles. In this case, we could not obtain samples from the subject’s parents to demonstrate whether her compound heterozygous deletion was de novo or not. Her father was healthy and had no muscle weakness, which suggests that he did not possess a DMD mutation, although germline mosaicism is possible. Her mother was long lost, but she exhibited no obvious muscular weakness as an adult. However, her mother may have been a carrier of the deletions of exons 48–50 or exons 51–53. Alternatively, her mother may have been heterozygous for the deletion of exons 48–53, where the deletions of exons 48–50 and exons 51–53 may have occurred because of interchromosomal recombination, and both deletions could have been inherited by maternal uniparental disomy. To the best of our knowledge, this is the first report of a patient with compound heterozygous contiguous exon deletions in dystrophin, thereby causing female DMD. Compound heterozygous contiguous exon deletions are very rare; however, if the clinical presentation of the patient is similar to male DMD, regardless of whether they are female, we consider that she may possess compound heterozygous mutations in dystrophin even if the results of MLPA analysis indicate that the subject is a carrier of an exon deletion. 4. Acknowledgement This study was supported by an Intramural Research Grant (26-6 and 26-7) for
Page 8 of 12
9
Neurological and Psychiatric Disorders of the NCNP. We thank the patient for her participation in the study.
References [1] Darras BT, Miller DT, Urion DK. Updated on November 23, 2011. Dystrophinopathies. In: GeneReviews at GeneTests Medical Genetics Information Resource (database online). Copyright,
University
of
Washington,
Seattle.
1993-2014.
Available
at
http://www.ncbi.nlm.nih.gov/books/NBK1119/ [2] Chelly J, Marlhens F, Le Marec B, Jeanpierre M, Lambert M, Hamard G, et al. De novo DNA microdeletion in a girl with Turner syndrome and Duchenne muscular dystrophy. Hum Genet 1986;74:193–6. [3] Azofeifa J, Voit T, Hübner C, Cremer M. X-chromosome methylation in manifesting and healthy carriers of dystrophinopathies: concordance of activation ratios among first degree female relatives and skewed inactivation as cause of the affected phenotypes. Hum Genet 1995;96:167–76. [4] Yoshioka M, Yorifuji T, Mituyoshi I. Skewed X inactivation in manifesting carriers of Duchenne muscular dystrophy. Clin Genet 1998;53:102–7. [5] Verellen-Dumoulin C, Freund M, De Meyer R, Laterre C, Frédéric J, Thompson MW, et al. Expression of an X-linked muscular dystrophy in a female due to translocation involving
Page 9 of 12
10
Xp21 and non-random inactivation of the normal X chromosome. Hum Genet 1984;67:115–9. [6] Cantagrel V, Lossi AM, Boulanger S, Depetris D, Mattei MG, Gecz J, et al. Disruption of a new X linked gene highly expressed in brain in a family with two mentally retarded males. J Med Genet 2004;41:736–42. [7] Quan F, Janas J, Toth-Fejel S, Johnson DB, Wolford JK, Popovich BW. Uniparental disomy of the entire X chromosome in a female with Duchenne muscular dystrophy. Am J Hum Genet 1997;60:160–5. [8] Katayama Y, Tran VK, Hoan NT, Zhang Z, Goji K, Yagi M, et al. Co-occurrence of mutations in both dystrophin- and androgen-receptor genes is a novel cause of female Duchenne muscular dystrophy. Hum Genet 2006;119:516–9. [9] Fujii K, Minami N, Hayashi Y, Nishino I, Nonaka I, Tanabe Y, et al. Homozygous female Becker muscular dystrophy. Am J Med Genet A 2009;149:1052–5. [10] Soltanzadeh P, Friez MJ, Dunn D, von Niederhausern A, Gurvich OL, Swoboda KJ, et al. Clinical and genetic characterization of manifesting carriers of DMD mutations. Neuromuscul Disord 2010;20:499–504.
Page 10 of 12
11
Figure 1. A: Family tree of the patient (P: proband). B: Muscle pathology of the patient (at 400-fold magnification). Immunohistochemical staining for dystrophin. Most of the fibers tested negative for dystrophin by immunohistochemical staining in this case. Figure 2. A: Multiplex ligation-dependent probe amplification analysis of the dystrophin gene detected one copy of exons 48–53. B: Messenger RNA was amplified from a muscle sample by reverse transcription polymerase chain reaction (PCR) using the primers P1 and P2 (shown in Fig. 2E). Pt: patient and WT: wild type. Two amplified fragments shorter than the WT were identified. C: Fragments from Fig. 2B analyzed by direct sequencing using the primer pair: P1 and P2. Using the P1 forward primer, exon 47 was followed by exon 48, which overlapped exon 51. Using the P2 reverse primer, exon 54 was followed by exon 53, which overlapped exon 50. D: Fragments from Fig. 2B analyzed by direct sequencing using the primers P3 and P4 (shown in Fig. 2E). Using the P3 forward primer, exon 50 was followed by exon 54. Using the P4 reverse primer, exon 51 was followed by exon 47. E: Diagram showing the pattern of dystrophin gene mutations in the subject. The deletions of exons 48–50 and exons 51–53 occurred on separate alleles. P: primer. Figure 3. A: Location of primers for the long PCR depicted in Fig. 3B. P: primer. B: Genomic DNA was amplified from the patient’s lymphocytes by long PCR using primers P5 and P6 (outside the deletion region of exons 48–50) and primers P7 and P8 (outside the deletion region of exons 51–53). Pt: patient and WT: wild type. Each primer set amplified an
Page 11 of 12
12
appropriately sized fragment from the patient DNA but not from the WT DNA for extreme long size. Sequencing analysis using the same primers demonstrated that one allele possessed out-of-frame deletions of exons 48–50 (c.6913-23922_7310-1654del123450), whereas the other
allele
possessed
out-of-frame
deletions
of
exons
51–53
(c.7310-941_7872+4982del100741).
Page 12 of 12
S0960-8966(17)30001-9 http://dx.doi.org/doi: 10.1016/j.nmd.2017.03.011 NMD 3365
To appear in:
Neuromuscular Disorders
Received date: Revised date: Accepted date:
3-1-2017 13-3-2017 29-3-2017
Please cite this article as: Eri Takeshita, Narihiro Minami, Kumiko Minami, Mikiya Suzuki, Takeya Awashima, Akihiko Ishiyama, Hirofumi Komaki, Ichizo Nishino, Masayuki Sasaki, Duchenne muscular dystrophy in a female with compound heterozygous contiguous exon deletions, Neuromuscular Disorders (2017), http://dx.doi.org/doi: 10.1016/j.nmd.2017.03.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Case report
Duchenne muscular dystrophy in a female with compound heterozygous contiguous exon deletions
Eri Takeshita1), Narihiro Minami2)3), Kumiko Minami3), Mikiya Suzuki4), Takeya Awashima1), Akihiko Ishiyama1), Hirofumi Komaki1), Ichizo Nishino3)5), Masayuki Sasaki1) 1)
Department of Child Neurology, National Center Hospital, National Center of Neurology
and Psychiatry (NCNP), Tokyo, Japan 2)
Department of Laboratory Medicine, National Center Hospital, National Center of
Neurology and Psychiatry (NCNP), Tokyo, Japan 3)
Department of Genome Medicine Development, Medical Genome Center, National Center of
Neurology and Psychiatry (NCNP), Tokyo, Japan 4)
Department of Neurology, Higashi-Saitama Hospital, National Hospital Organization,
Hasuda, Japan 5)
Department of Neuromuscular Research, National Institute of Neuroscience, National Center of
Neurology and Psychiatry (NCNP), Tokyo, Japan
Page 1 of 12
2
Corresponding author: Eri Takeshita, MD, PhD Department of Child Neurology, National Center Hospital, National Center of Neurology and Psychiatry (NCNP), 4-1-1 Ogawahigashi-cho, Kodaira, Tokyo 187-8551, Japan Tel: +81 42 3412711; Fax: +81 42 3446745 E-mail: [email protected]
Highlights This female patient showed Duchenne muscular dystrophy like symptoms and clinical course. Muscle pathology was consistent with Duchenne muscular dystrophy. Gene analysis by MLPA suggested Becker muscular dystrophy carrier. Messenger RNA analysis showed compound heterozygous contiguous exon deletions. Genomic DNA analysis demonstrated the accurate deletion region on each allele.
Page 2 of 12
3
Abstract Females with Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) mutations rarely exhibit clinical symptoms from childhood, although potential mechanisms for symptoms associated with DMD and BMD in females have been reported. We report the case of a female DMD patient with a clinical course indistinguishable from that of a male DMD patient; the female possessed compound heterozygous contiguous exon deletions in the dystrophin gene. She exhibited Gowers’ signs, calf muscle hypertrophy, and high serum creatinine kinase levels at 2 years. Her muscle pathology showed most of the fibers tested negative for dystrophin immunohistochemical staining. She lost ambulatory ability at 11 years. Now, at over 21 years, she has difficulty rolling over by herself. Multiplex ligation-dependent probe amplification analysis of this gene detected one copy of exons 48–53; she was found to be a BMD carrier with an in-frame deletion. Messenger RNA from her muscle demonstrated out-of-frame deletions of exons 48–50 and 51–53 occurring on separate alleles. Genomic DNA from her lymphocytes demonstrated the accurate deletion region on each allele. To our knowledge, this is the first report on a female patient possessing compound heterozygous contiguous exon deletions in the dystrophin gene, leading to DMD.
Keywords: compound heterozygous; Duchenne muscular dystrophy; dystrophin; exon deletion; female
Page 3 of 12
4
1. Introduction Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the dystrophin gene located at Xp21.2. It affects approximately 1 in 3500 boys and causes progressive muscle weakness. DMD patients generally carry frame-shift dystrophin mutations, whereas a milder form called Becker muscular dystrophy (BMD) is due to in-frame mutations. Deletions of one or more exons account for approximately 60%–70% of mutations in individuals with DMD and BMD and duplications are found in approximately 5%–10%. The remaining 25%–35% of DMD cases and 10%–20% of BMD cases are caused by point mutations or other subtle changes in the dystrophin gene [1]. Most heterozygous carrier females with dystrophin mutations exhibit no clinical symptoms, and it is very rare for females with DMD and BMD mutations to exhibit symptoms from childhood. In this study, we report the case of female DMD patient with a clinical course indistinguishable from that of a male DMD patient; the female possessed compound heterozygous contiguous exon deletions in the dystrophin gene. 2. Case report The proband (III-2) was a 21-year-old female, who was the first child of non-consanguineous parents (Fig. 1A). Her mother (II-4) lost an unborn child (III-1) before the patient was born. In addition, her mother was addicted to alcohol and divorced from her father (II-2). She had a half-brother (III-3); however, it was not clear if he was healthy or not. She was born uneventfully; her body weight was 3490 g and body length was 48 cm at
Page 4 of 12
5
birth. At 1 year, she began walking alone but was likely to fall. At 2 years and 2 months, she was referred to the hospital for a heart murmur. The possible existence of heart disease was excluded; however, she exhibited Gowers’ signs, calf muscle hypertrophy, and high serum creatinine kinase levels (CK 18280 IU/L). A muscle biopsy was performed at 2 years and 4 months and most of the fibers tested negative for dystrophin immunohistochemical staining; thus she was diagnosed with DMD (Fig. 1B). At 9 years, she experienced more difficulty going up and down stairs, and she lost ambulatory ability at 11 years. Now, at over 21 years, she can operate her mobility scooter but has difficulty rolling over by herself, and her condition has progressed to deformity of the trunk due to scoliosis. Multiplex ligation-dependent probe amplification (MLPA) analysis of the dystrophin gene detected one copy of exons 48–53, which suggested that an in-frame exon deletion was present in a BMD carrier (Fig. 2A). However, her clinical course and muscle pathology were inconsistent with a BMD carrier and her condition was more similar to female DMD. Chromosomal study by G-banding determined 46 chromosomes with XX, i.e., a normal karyotype. The patterns of X-chromosome inactivation were assessed based on the methylation status at the androgen receptor locus, where the results showed that she possessed a random (non-skewed) X-chromosome inactivation pattern (38.7: 61.3%). These data excluded some possible known mechanisms for female DMD, which we consider later. Thus, we proceeded with additional gene analysis. Messenger RNA was amplified from her muscle by reverse transcription polymerase chain reaction (PCR) using primers located
Page 5 of 12
6
outside the exons of a deletion region (P1 and P2), where the two fragments amplified were shorter than the wild type (Fig. 2B). These fragments were analyzed by direct sequencing. When the P1 forward primer was used for sequencing, exon 47 was followed by exon 48, which overlapped exon 51. Similarly, sequencing with the P2 reverse primer showed that exon 54 was followed by exon 53, which overlapped exon 50 (Fig. 2C). These results suggested that the deletions of exons 48–50 and exons 51–53 were on separate alleles. Thus, we conducted additional sequencing analysis to confirm these results. Using the P3 forward primer located in exon 49 showed that exon 50 was followed by exon 54. Using the P4 reverse primer located in exon 53, we found that exon 51 was followed by exon 47 (Fig. 2D). Thus, the results of the MLPA analysis demonstrated that the two exon deletions occurred on different alleles (Fig. 2E). Additionally, we analyzed genomic DNA from the patient’s lymphocytes to detect the accurate deletion region on each allele. Genomic DNA was amplified by long PCR using primers located outside the deletion region of exons 48–50 (P5 and P6), and subsequent sequencing using the same primers demonstrated that one allele possessed out-of-frame deletion of exons 48–50 (c.6913-23922_7310-1654del123450) (Fig. 3A, 3B). Additional long PCR using primers located outside the deletion region of exons 51–53 (P7 and P8) and sequencing using the same primers demonstrated that the other allele possessed out-of-frame deletion of exons 51– 53 (c.7310-941_7872+4982del100741) (Fig. 3A, 3B). Therefore, we concluded that the subject possessed compound heterozygous contiguous exon deletions, i.e., deletions of
Page 6 of 12
7
exons 48–50 (out-of-frame; c.6913-23922_7310-1654del123450) and exons 51–53 (out-of-frame; c.7310-941_7872+4982del100741); thus, she was genetically diagnosed with female DMD. 3. Discussion We considered that this case was female DMD based on the clinical course and muscle pathology; however, the initial dystrophin gene analysis by MLPA to search for in-frame deletions suggested that she was a BMD carrier. Some mechanisms for female DMD and BMD have been reported previously: Turner syndrome (45, X) with a dystrophin mutation on the remaining X chromosome [2]; skewed X-chromosome inactivation in the carriers of DMD mutations [3,4]; and X-autosome translocations involving the dystrophin gene and preferential inactivation of the normal X chromosome [5,6]. In this case, these mechanisms were excluded based on a chromosomal study using G-banding and X-chromosome inactivation analysis. Therefore, we proceeded with the additional dystrophin gene analysis, where we concluded that she possessed compound heterozygous contiguous exon deletions. Furthermore, some rare genetic abnormalities have been documented as follows: a homozygous dystrophin mutation derived from maternal uniparental disomy [7]; the co-occurrence of mutations in both the dystrophin and androgen receptor genes in a patient with 47, XY [8]; and female BMD with a homozygous dystrophin mutation caused by consanguinity [9]. To the best of our knowledge, only one BMD-like female has been reported who possessed compound heterozygosity for two
Page 7 of 12
8
dystrophin mutations, i.e., an exon deletion and a point mutation in an intron splice site [10]. Although it is rare as mentioned above, several mechanisms which cause female DMD by genetic mutation has occurred because of the presence of both dystrophin alleles. In this case, we could not obtain samples from the subject’s parents to demonstrate whether her compound heterozygous deletion was de novo or not. Her father was healthy and had no muscle weakness, which suggests that he did not possess a DMD mutation, although germline mosaicism is possible. Her mother was long lost, but she exhibited no obvious muscular weakness as an adult. However, her mother may have been a carrier of the deletions of exons 48–50 or exons 51–53. Alternatively, her mother may have been heterozygous for the deletion of exons 48–53, where the deletions of exons 48–50 and exons 51–53 may have occurred because of interchromosomal recombination, and both deletions could have been inherited by maternal uniparental disomy. To the best of our knowledge, this is the first report of a patient with compound heterozygous contiguous exon deletions in dystrophin, thereby causing female DMD. Compound heterozygous contiguous exon deletions are very rare; however, if the clinical presentation of the patient is similar to male DMD, regardless of whether they are female, we consider that she may possess compound heterozygous mutations in dystrophin even if the results of MLPA analysis indicate that the subject is a carrier of an exon deletion. 4. Acknowledgement This study was supported by an Intramural Research Grant (26-6 and 26-7) for
Page 8 of 12
9
Neurological and Psychiatric Disorders of the NCNP. We thank the patient for her participation in the study.
References [1] Darras BT, Miller DT, Urion DK. Updated on November 23, 2011. Dystrophinopathies. In: GeneReviews at GeneTests Medical Genetics Information Resource (database online). Copyright,
University
of
Washington,
Seattle.
1993-2014.
Available
at
http://www.ncbi.nlm.nih.gov/books/NBK1119/ [2] Chelly J, Marlhens F, Le Marec B, Jeanpierre M, Lambert M, Hamard G, et al. De novo DNA microdeletion in a girl with Turner syndrome and Duchenne muscular dystrophy. Hum Genet 1986;74:193–6. [3] Azofeifa J, Voit T, Hübner C, Cremer M. X-chromosome methylation in manifesting and healthy carriers of dystrophinopathies: concordance of activation ratios among first degree female relatives and skewed inactivation as cause of the affected phenotypes. Hum Genet 1995;96:167–76. [4] Yoshioka M, Yorifuji T, Mituyoshi I. Skewed X inactivation in manifesting carriers of Duchenne muscular dystrophy. Clin Genet 1998;53:102–7. [5] Verellen-Dumoulin C, Freund M, De Meyer R, Laterre C, Frédéric J, Thompson MW, et al. Expression of an X-linked muscular dystrophy in a female due to translocation involving
Page 9 of 12
10
Xp21 and non-random inactivation of the normal X chromosome. Hum Genet 1984;67:115–9. [6] Cantagrel V, Lossi AM, Boulanger S, Depetris D, Mattei MG, Gecz J, et al. Disruption of a new X linked gene highly expressed in brain in a family with two mentally retarded males. J Med Genet 2004;41:736–42. [7] Quan F, Janas J, Toth-Fejel S, Johnson DB, Wolford JK, Popovich BW. Uniparental disomy of the entire X chromosome in a female with Duchenne muscular dystrophy. Am J Hum Genet 1997;60:160–5. [8] Katayama Y, Tran VK, Hoan NT, Zhang Z, Goji K, Yagi M, et al. Co-occurrence of mutations in both dystrophin- and androgen-receptor genes is a novel cause of female Duchenne muscular dystrophy. Hum Genet 2006;119:516–9. [9] Fujii K, Minami N, Hayashi Y, Nishino I, Nonaka I, Tanabe Y, et al. Homozygous female Becker muscular dystrophy. Am J Med Genet A 2009;149:1052–5. [10] Soltanzadeh P, Friez MJ, Dunn D, von Niederhausern A, Gurvich OL, Swoboda KJ, et al. Clinical and genetic characterization of manifesting carriers of DMD mutations. Neuromuscul Disord 2010;20:499–504.
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11
Figure 1. A: Family tree of the patient (P: proband). B: Muscle pathology of the patient (at 400-fold magnification). Immunohistochemical staining for dystrophin. Most of the fibers tested negative for dystrophin by immunohistochemical staining in this case. Figure 2. A: Multiplex ligation-dependent probe amplification analysis of the dystrophin gene detected one copy of exons 48–53. B: Messenger RNA was amplified from a muscle sample by reverse transcription polymerase chain reaction (PCR) using the primers P1 and P2 (shown in Fig. 2E). Pt: patient and WT: wild type. Two amplified fragments shorter than the WT were identified. C: Fragments from Fig. 2B analyzed by direct sequencing using the primer pair: P1 and P2. Using the P1 forward primer, exon 47 was followed by exon 48, which overlapped exon 51. Using the P2 reverse primer, exon 54 was followed by exon 53, which overlapped exon 50. D: Fragments from Fig. 2B analyzed by direct sequencing using the primers P3 and P4 (shown in Fig. 2E). Using the P3 forward primer, exon 50 was followed by exon 54. Using the P4 reverse primer, exon 51 was followed by exon 47. E: Diagram showing the pattern of dystrophin gene mutations in the subject. The deletions of exons 48–50 and exons 51–53 occurred on separate alleles. P: primer. Figure 3. A: Location of primers for the long PCR depicted in Fig. 3B. P: primer. B: Genomic DNA was amplified from the patient’s lymphocytes by long PCR using primers P5 and P6 (outside the deletion region of exons 48–50) and primers P7 and P8 (outside the deletion region of exons 51–53). Pt: patient and WT: wild type. Each primer set amplified an
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appropriately sized fragment from the patient DNA but not from the WT DNA for extreme long size. Sequencing analysis using the same primers demonstrated that one allele possessed out-of-frame deletions of exons 48–50 (c.6913-23922_7310-1654del123450), whereas the other
allele
possessed
out-of-frame
deletions
of
exons
51–53
(c.7310-941_7872+4982del100741).
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