- Email: [email protected]
Duchenne muscular dystrophy caused by a complex rearrangement between intron 43 of the DMD gene and chromosome 4
Available online at www.sciencedirect.com
Neuromuscular Disorders 21 (2011) 178–182 www.elsevier.com/locate/nmd
Case report
Duchenne muscular dystrophy caused by a complex rearrangement between intron 43 of the DMD gene and chromosome 4 Berivan Baskin a,b,⇑, William T. Gibson c, Peter N. Ray a,b,d a
Division of Molecular Genetics, Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, Canada b The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, Canada c Department of Medical Genetics, Child and Family Research Institute, The University of British Columbia, Vancouver, Canada d Department of Molecular Genetics, The University of Toronto, Toronto, Canada Received 30 September 2010; accepted 15 November 2010
Abstract Deletions/duplications of exons in the DMD gene cause about 70% of all cases of Duchenne muscular dystrophy (DMD). Most remaining mutations are point mutations or small insertion–deletions located mainly in the coding but also in deep intronic regions of the DMD gene. We describe a novel complex rearrangement in a patient affected with DMD that was undetectable using standard molecular diagnostic methods. Analysis of the proband’s mRNA from a muscle biopsy revealed the insertion of an 80 bp cryptic exon from chromosome 4 between exons 43 and 44 of the dystrophin gene. Array comparative genomic hybridization and breakpoint junction sequence analysis indicated this cryptic exon originated from a complex genomic 90 kb insertion of non-coding chromosome 4 into intron 43 of the dystrophin gene. This rearrangement was also detectable in the patient’s mother. The genomic characterization of this novel complex mutation was essential for accurate carrier and genetic counseling of this family and emphasizes the need for comprehensive molecular diagnosis of patients with clinical signs of DMD and clear pathological changes. Ó 2010 Elsevier B.V. All rights reserved. Keywords: DMD; Duchenne muscular dystrophy; Rearrangement; mRNA; Insertion
1. Introduction Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene, the largest gene in the genome measuring 2.3 Mb although only 11 kb (0.6%) of the gene is coding. Approximately 65% of mutations causing disease are deletions of one or more exons and 5% are due to duplications [1]. These types of mutations are routinely detected using dosage assays such as multiplex ligation probe amplification (MLPA), which can detect 100% of deletion/duplication in all 79 exons in the DMD gene. Most
⇑ Corresponding author at: Division of Molecular Genetics, Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, 555 University Avenue, Toronto, Canada M5G 1X8. Fax: +1 416 813 7732. E-mail address: [email protected] (B. Baskin).
0960-8966/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2010.11.008
remaining mutations causing DMD are point mutations, including nonsense, missense, splice site mutations and small indels in the coding exons and exon/intron boundaries. The majority of point mutations are detected using direct sequence analysis of all 79 exons and exon/intron boundaries of the DMD gene. Thus these two of methodologies, dosage and direct sequence analysis, detect 98% of mutations DMD causing mutations when used on genomic DNA [2]. However, several reports have described mutations in the DMD gene embedded in deep intronic regions, causing exon skipping or intron retention [3,4]. Deep intronic mutations can be identified using mRNA analysis, but this requires muscle tissue from the patients and so is not routinely done. However comprehensive analysis of the DMD gene in patients with clear clinical signs of DMD should include not only dosage and point mutation analysis on genomic DNA but also mRNA analysis of a muscle
B. Baskin et al. / Neuromuscular Disorders 21 (2011) 178–182
biopsy to increase the yield of pathogenic sequence alterations in the dystrophin gene [1]. Complex mutations such as inversions and rearrangements within the DMD gene and rearrangements involving the dystrophin gene and neighboring genes or chromosomes are rare but have been described in a few cases [5,6]. We describe the characterization of a novel complex rearrangement of the dystrophin gene in a boy affected with DMD. Routine analysis on genomic DNA did not reveal any mutation in the DMD gene. RT-PCR analysis revealed a cryptic exon of 80 bp inserted between exon 43 and 44 originating from chromosome 4. Further analysis using array CGH and breakpoint junction sequence analysis revealed a complex rearrangement and insertion of 90 kb of chromosome 4 into intron 43 by non-homologous end joining. This genomic rearrangement was also detectable in the patient’s mother but not in the maternal aunt who was 6 weeks pregnant. The characterization of the mutation at the genomic level was essential not only for the confirmation of diagnosis but also for carrier detection and genetic counseling in relatives of the proband. 2. Results 2.1. Mutation analysis The patient was diagnosed with DMD at 5 years of age on the basis of clinical presentation, elevated serum creatine kinase (>23,000 U/L), and negative dystrophin staining on immunohistochemistry. As of his most recent
179
follow-up, he remains ambulatory (10 years, 9 months of age) but frequently uses a motorized chair. Routine diagnostic testing on genomic DNA from peripheral leukocytes from the patient did not reveal deletions/duplications of any of the 79 exons in the dystrophin gene. Direct sequencing of the genomic DNA did not identify a mutation in the coding regions or at the invariant nucleotides at the donor/ acceptor splice sites. Since clinical and pathology data was consistent with a DMD phenotype we performed mRNA analysis from a muscle biopsy on the patient. Reverse transcriptase PCR was performed using primers to amplify the entire dystrophin gene in 30 overlapping fragments, which were visualized by agarose gel electrophoresis. Two of the amplicons showed abnormal size, and when separated by capillary electrophoresis were 80 bp larger than expected with peak sizes of 473 and 585 bp, respectively (Fig. 1A). Direct sequence analysis of the two aberrant size fragments revealed an 80 bp insertion of a sequence perfectly spliced between exons 43 and 44 (Fig. 1B). BLAST analysis on the 80 bp sequence against the human genome (NCBI Build 36.2) was performed and revealed 100% alignment with sequence from chromosome 4 (183,199,811–183,199, 890). This region on chromosome 4 does not code for any apparent protein. We predicted that there was a larger region of chromosome 4 inserted in the genomic DNA in intron 43 of the DMD gene and that the 80 bp sequence was spliced in as a cryptic exon between exons 43 and 44 in the dystrophin transcript.
Fig. 1. Analysis of dystrophin mRNA prepared from muscle biopsy. (A) The dystrophin transcript was amplified and separated by capillary electrophoreses. Two fragments encompassing exons 42–44 and 43–44, respectively were larger than expected. In the normal control, peaks of the expected sizes 393 bp (top left) and 505 bp (top right) were detected, whereas in the patient sample both fragments were 80 bp larger with peak sizes of 473 and 585 bp, respectively. (B) Sequence analysis of the abnormal sized fragments showed an 80 bp insertion between exons 43 and 44. The insert aligned 100% with a non-coding region of chromosome 4 (183199811–183199890).
180
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2.2. Array CGH and breakpoint junction analysis To determine how much of chromosome 4 was inserted into intron 43 of the dystrophin gene we analyzed the patient with a Nimblegen CGH chromosome 4 tiling array. The array CGH revealed an insertion of approximately 80– 90 kb of chromosome 4 (Fig. 2A). We then established the breakpoint in the DMD gene by performing long range PCR using overlapping primers covering the entire 70465 bp of intron 43. The approximate location of the breakpoint was determined by the inability to amplify a segment of intron 43. Using long range PCR we then successfully amplified the DMD intron 43/chromosome 4 break point junctions (Fig. 2B). Junction sequence analysis revealed that the breakpoint in intron 43 of the DMD gene had a five base pair deletion and showed no homology to any repeat elements. The 50 breakpoint junction of the chromosome 4 insertion demonstrated simple, (TA)n, repeating elements and four base pairs of microhomology with the DMD intron 43 junction sequence. The 30 junction of the insertion showed no repeat elements and no homology to the DMD intron 43 junction. By sequence analysis we determined that the inserted chromosome 4 segment revealed more complexity, consisting of two segments derived from chromosome 4; one large (86976 bp) and one small (2596 bp). The sequence of the smaller segment was not contiguous with the 80 kb chromosome 4 shown on array CGH but originated from a region 10 kb upstream of the larger fragment on the chromosome 4 map. This smaller fragment was not detected by array
CGH because there were no probes located in this region on the array. The 30 end of the larger segment and the 50 end of the smaller segment both showed simple (GGAA)n repeating elements. 2.3. Diagnostic testing of family members Once all the break point junctions were determined in the patient, we were able to assess the carrier status in female relatives. Two forward PCR primers; one located upstream of the intron 43 junction and the other located upstream of the 30 junction of chromosome 4 and a reverse primer located downstream of the intron 43 junction were used (Fig. 3A). Amplification from the normal chromosome X generated a 345 bp fragment while the mutant chromosome X produced a 1607 bp fragment (Fig. 3B). We were able to determine that the mother of the index case (CK level 248 U/L, Ref. range <140) was a carrier of the insertion and that the maternal aunt who was 6 weeks pregnant was not a carrier of the mutation. 3. Discussion The most common dystrophin mutations are deletions or duplications of one or more exons in the DMD gene. These types of rearrangements account for approximately 70% of all dystrophin mutations. Other types of rearrangements such as inversions and insertions are much less common [5]. Rearrangements involving the dystrophin gene with neighboring loci leading to insertion of entire or
Fig. 2. Identification of the breakpoint junction sequence. (A) Array-CGH profile of chromosome 4 of the patient. Genomic DNA from the patient was hybridized to a Nimblegen oligo tiling array to determine the size of the chromosome 4 insertion in intron 43 of the DMD gene. Arrows indicate the approximate breakpoints for the insert (B). Long-range PCR was used to identify the break-point junctions of the insertion, which consisted of two segments, one large (87098 bp) and one small (2596 bp) region of chromosome 4; (1a) indicates the co-ordinate of the proximal break point in intron 43 of DMD (1b) the 50 breakpoint junction of the large chromosome 4 segment (2a) the 30 breakpoint junction of the large chromosome 4 segment (2b) the 50 breakpoint junction of the small segment (3a) the 30 breakpoint junction of the small chromosome 4 segment and (3b) the distal break point junction in intron 43 of DMD.
B. Baskin et al. / Neuromuscular Disorders 21 (2011) 178–182
181
Fig. 3. Determination of carrier status of female relatives of proband. Breakpoint junction PCR was used to determine carrier status in proband’s mother and maternal aunt. Primers A and B generate a 345 bp fragment from the normal chromosome X, and primers C and B generate a 1607 bp amplicon from the mutant chromosome X. Only the 1607 bp fragment was detected in the proband as expected. Both fragments were amplified in the mother indicating that she is a carrier and only the 345 bp was detected in the maternal aunt indicating that she is a non-carrier.
partial genes into the DMD gene have been described but are rare [6]. Zhang et al. [7] reported the insertion of IL1RAPL1 into DMD and insertions of transposable element, L1, into the dystrophin gene have also been reported [8,9]. The patient described here presented with dystrophinopathy and was found to be negative for mutations on routine diagnostic testing. Since the patient’s clinical presentation and pathology findings were consistent with DMD, we performed mRNA analysis on a muscle biopsy and detected an insertion of a cryptic exon between exons 43 and 44 originating from chromosome 4. Detailed analysis of genomic DNA revealed a 90 kb insertion of two segments of non-contiguous chromosome 4 sequence into intron 43 of the DMD gene. The primary mechanism causing rearrangements in the dystrophin gene has been proposed to be DNA doublestrand breaks (DBS) followed by non-homologous end joining (NHEJ) [6,10–12], which requires no homology or only microhomology at break point junctions. However
Oshima et al. [6] suggested that a case with an inversion/ deletion rearrangement of the DMD gene was caused by a replication based mechanism rather than NHEJ since breakpoint junction sequence revealed repetitive sequences that could have formed a stem-loop structure causing genomic instability. Other authors, reviewed in Zhang et al. have suggested that complex human chromosomal rearrangements can be caused by a replication based mechanism without the need for extensive homology or stem loop formation [13]. The complex rearrangement in the case described in this report consisted of two non-contiguous chromosome 4 segments; a larger 87 kb fragment and a smaller 2.6 kb fragment with the origin of the smaller segment 10 kb upstream of the larger segment on the chromosome 4 map. The break points of the 2.6 kb segment were located in non-repetitive regions whereas the 87 kb segment breakpoints were both located in simple repeat elements, the 50 end in (TA)n repeats and the 30 end in (GGAA)n repeats. The two segments showed GAA microhomology at their junction. The DMD gene
182
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breakpoint in intron 43 had a five base pair deletion and showed no homology to any repeat elements. The 50 breakpoint junction of the chromosome 4 insertion and the DMD gene showed four base pairs of microhomology, whereas the 30 junction of the insertion showed no microhomology with the DMD intron 43 junction. We conclude that this rearrangement of chromosome 4 with the DMD gene in this patient could be the result of either NHEJ or a replication based mechanism. Based on the characterization of the breakpoint junctions in the patient we were able to offer carrier testing to female relatives. The mother of the index case was determined to be a carrier which was consistent with the elevated creatine kinase levels detected in her. We subsequently received a blood sample on the maternal aunt who was then 6 weeks pregnant and could establish that she did not carry this rearrangement. Analysis of dystrophin mRNA does not only detect deep embedded intronic mutations in the DMD gene leading to the incorporation of cryptic exons and intron retention in the dystrophin transcript but also complex DMD rearrangements that cause exon skipping and insertion of exons from other genes [3,5,7]. This case emphasizes the importance of performing mRNA analysis in DMD patients where no disease causing mutations are found by routine analysis so that accurate genetic counseling and prenatal diagnosis can be offered to the family. Acknowledgements W.T.G. is supported by a Clinician Scientist Salary Award from both CIHR and the CFRI. We gratefully acknowledge Chieko Chijiwa for her administrative assistance during the work presented here. References [1] Aartsma-Rus A, Van Deutekom JC, Fokkema IF, Van Ommen GJ, Den Dunnen JT. Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
cases that confirm the reading-frame rule. Muscle Nerve 2006;34:135–44. Stockley TL, Akber S, Bulgin N, Ray PN. Strategy for comprehensive molecular testing for Duchenne and Becker muscular dystrophies. Genet Test 2006;10:229–43. Zhang Z, Habara Y, Nishiyama A, Oyazato Y, Yagi M, Takeshima Y, et al.. Identification of seven novel cryptic exons embedded in the dystrophin gene and characterization of 14 cryptic dystrophin exons. J Hum Genet 2007;52:607–17. Beroud C, Carrie A, Beldjord C, Deburgrave N, Llense S, Carelle N, et al.. Dystrophinopathy caused by mid-intronic substitutions activating cryptic exons in the DMD gene. Neuromuscul Disord 2004;14:10–8. Madden HR, Fletcher S, Davis MR, Wilton SD. Characterization of a complex Duchenne muscular dystrophy-causing dystrophin gene inversion and restoration of the reading frame by induced exon skipping. Hum Mutat 2009;30:22–8. Oshima J, Magner DB, Lee JA, Breman AM, Schmitt ES, White LD, et al.. Regional genomic instability predisposes to complex dystrophin gene rearrangements. Hum Genet 2009;126:411–23. Zhang Z, Yagi M, Okizuka Y, Awano H, Takeshima Y, Matsuo M. Insertion of the IL1RAPL1 gene into the duplication junction of the dystrophin gene. J Hum Genet 2009;54:466–73. Musova Z, Hedvicakova P, Mohrmann M, Tesarova M, Krepelova A, Zeman J, et al.. A novel insertion of a rearranged L1 element in exon 44 of the dystrophin gene: further evidence for possible bias in retroposon integration. Biochem Biophys Res Commun 2006;347:145–9. Narita N, Nishio H, Kitoh Y, Ishikawa Y, Ishikawa Y, Minami R, et al.. Insertion of a 50 truncated L1 element into the 30 end of exon 44 of the dystrophin gene resulted in skipping of the exon during splicing in a case of Duchenne muscular dystrophy. J Clin Invest 1993;91:1862–7. Toffolatti L, Cardazzo B, Nobile C, Danieli GA, Gualandi F, Muntoni F, et al.. Investigating the mechanism of chromosomal deletion: characterization of 39 deletion breakpoints in introns 47 and 48 of the human dystrophin gene. Genomics 2002;80:523–30. del Gaudio D, Yang Y, Boggs BA, Schmitt ES, Lee JA, Sahoo T, et al.. Molecular diagnosis of Duchenne/Becker muscular dystrophy: enhanced detection of dystrophin gene rearrangements by oligonucleotide array-comparative genomic hybridization. Hum Mutat 2008;29:1100–7. Hegde MR, Chin EL, Mulle JG, Okou DT, Warren ST, Zwick ME. Microarray-based mutation detection in the dystrophin gene. Hum Mutat 2008;29:1091–9. Zhang F, Carvalho CM, Lupski JR. Complex human chromosomal and genomic rearrangements. Trends Genet 2009;25:298–307.
Neuromuscular Disorders 21 (2011) 178–182 www.elsevier.com/locate/nmd
Case report
Duchenne muscular dystrophy caused by a complex rearrangement between intron 43 of the DMD gene and chromosome 4 Berivan Baskin a,b,⇑, William T. Gibson c, Peter N. Ray a,b,d a
Division of Molecular Genetics, Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, Canada b The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, Canada c Department of Medical Genetics, Child and Family Research Institute, The University of British Columbia, Vancouver, Canada d Department of Molecular Genetics, The University of Toronto, Toronto, Canada Received 30 September 2010; accepted 15 November 2010
Abstract Deletions/duplications of exons in the DMD gene cause about 70% of all cases of Duchenne muscular dystrophy (DMD). Most remaining mutations are point mutations or small insertion–deletions located mainly in the coding but also in deep intronic regions of the DMD gene. We describe a novel complex rearrangement in a patient affected with DMD that was undetectable using standard molecular diagnostic methods. Analysis of the proband’s mRNA from a muscle biopsy revealed the insertion of an 80 bp cryptic exon from chromosome 4 between exons 43 and 44 of the dystrophin gene. Array comparative genomic hybridization and breakpoint junction sequence analysis indicated this cryptic exon originated from a complex genomic 90 kb insertion of non-coding chromosome 4 into intron 43 of the dystrophin gene. This rearrangement was also detectable in the patient’s mother. The genomic characterization of this novel complex mutation was essential for accurate carrier and genetic counseling of this family and emphasizes the need for comprehensive molecular diagnosis of patients with clinical signs of DMD and clear pathological changes. Ó 2010 Elsevier B.V. All rights reserved. Keywords: DMD; Duchenne muscular dystrophy; Rearrangement; mRNA; Insertion
1. Introduction Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene, the largest gene in the genome measuring 2.3 Mb although only 11 kb (0.6%) of the gene is coding. Approximately 65% of mutations causing disease are deletions of one or more exons and 5% are due to duplications [1]. These types of mutations are routinely detected using dosage assays such as multiplex ligation probe amplification (MLPA), which can detect 100% of deletion/duplication in all 79 exons in the DMD gene. Most
⇑ Corresponding author at: Division of Molecular Genetics, Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, 555 University Avenue, Toronto, Canada M5G 1X8. Fax: +1 416 813 7732. E-mail address: [email protected] (B. Baskin).
0960-8966/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2010.11.008
remaining mutations causing DMD are point mutations, including nonsense, missense, splice site mutations and small indels in the coding exons and exon/intron boundaries. The majority of point mutations are detected using direct sequence analysis of all 79 exons and exon/intron boundaries of the DMD gene. Thus these two of methodologies, dosage and direct sequence analysis, detect 98% of mutations DMD causing mutations when used on genomic DNA [2]. However, several reports have described mutations in the DMD gene embedded in deep intronic regions, causing exon skipping or intron retention [3,4]. Deep intronic mutations can be identified using mRNA analysis, but this requires muscle tissue from the patients and so is not routinely done. However comprehensive analysis of the DMD gene in patients with clear clinical signs of DMD should include not only dosage and point mutation analysis on genomic DNA but also mRNA analysis of a muscle
B. Baskin et al. / Neuromuscular Disorders 21 (2011) 178–182
biopsy to increase the yield of pathogenic sequence alterations in the dystrophin gene [1]. Complex mutations such as inversions and rearrangements within the DMD gene and rearrangements involving the dystrophin gene and neighboring genes or chromosomes are rare but have been described in a few cases [5,6]. We describe the characterization of a novel complex rearrangement of the dystrophin gene in a boy affected with DMD. Routine analysis on genomic DNA did not reveal any mutation in the DMD gene. RT-PCR analysis revealed a cryptic exon of 80 bp inserted between exon 43 and 44 originating from chromosome 4. Further analysis using array CGH and breakpoint junction sequence analysis revealed a complex rearrangement and insertion of 90 kb of chromosome 4 into intron 43 by non-homologous end joining. This genomic rearrangement was also detectable in the patient’s mother but not in the maternal aunt who was 6 weeks pregnant. The characterization of the mutation at the genomic level was essential not only for the confirmation of diagnosis but also for carrier detection and genetic counseling in relatives of the proband. 2. Results 2.1. Mutation analysis The patient was diagnosed with DMD at 5 years of age on the basis of clinical presentation, elevated serum creatine kinase (>23,000 U/L), and negative dystrophin staining on immunohistochemistry. As of his most recent
179
follow-up, he remains ambulatory (10 years, 9 months of age) but frequently uses a motorized chair. Routine diagnostic testing on genomic DNA from peripheral leukocytes from the patient did not reveal deletions/duplications of any of the 79 exons in the dystrophin gene. Direct sequencing of the genomic DNA did not identify a mutation in the coding regions or at the invariant nucleotides at the donor/ acceptor splice sites. Since clinical and pathology data was consistent with a DMD phenotype we performed mRNA analysis from a muscle biopsy on the patient. Reverse transcriptase PCR was performed using primers to amplify the entire dystrophin gene in 30 overlapping fragments, which were visualized by agarose gel electrophoresis. Two of the amplicons showed abnormal size, and when separated by capillary electrophoresis were 80 bp larger than expected with peak sizes of 473 and 585 bp, respectively (Fig. 1A). Direct sequence analysis of the two aberrant size fragments revealed an 80 bp insertion of a sequence perfectly spliced between exons 43 and 44 (Fig. 1B). BLAST analysis on the 80 bp sequence against the human genome (NCBI Build 36.2) was performed and revealed 100% alignment with sequence from chromosome 4 (183,199,811–183,199, 890). This region on chromosome 4 does not code for any apparent protein. We predicted that there was a larger region of chromosome 4 inserted in the genomic DNA in intron 43 of the DMD gene and that the 80 bp sequence was spliced in as a cryptic exon between exons 43 and 44 in the dystrophin transcript.
Fig. 1. Analysis of dystrophin mRNA prepared from muscle biopsy. (A) The dystrophin transcript was amplified and separated by capillary electrophoreses. Two fragments encompassing exons 42–44 and 43–44, respectively were larger than expected. In the normal control, peaks of the expected sizes 393 bp (top left) and 505 bp (top right) were detected, whereas in the patient sample both fragments were 80 bp larger with peak sizes of 473 and 585 bp, respectively. (B) Sequence analysis of the abnormal sized fragments showed an 80 bp insertion between exons 43 and 44. The insert aligned 100% with a non-coding region of chromosome 4 (183199811–183199890).
180
B. Baskin et al. / Neuromuscular Disorders 21 (2011) 178–182
2.2. Array CGH and breakpoint junction analysis To determine how much of chromosome 4 was inserted into intron 43 of the dystrophin gene we analyzed the patient with a Nimblegen CGH chromosome 4 tiling array. The array CGH revealed an insertion of approximately 80– 90 kb of chromosome 4 (Fig. 2A). We then established the breakpoint in the DMD gene by performing long range PCR using overlapping primers covering the entire 70465 bp of intron 43. The approximate location of the breakpoint was determined by the inability to amplify a segment of intron 43. Using long range PCR we then successfully amplified the DMD intron 43/chromosome 4 break point junctions (Fig. 2B). Junction sequence analysis revealed that the breakpoint in intron 43 of the DMD gene had a five base pair deletion and showed no homology to any repeat elements. The 50 breakpoint junction of the chromosome 4 insertion demonstrated simple, (TA)n, repeating elements and four base pairs of microhomology with the DMD intron 43 junction sequence. The 30 junction of the insertion showed no repeat elements and no homology to the DMD intron 43 junction. By sequence analysis we determined that the inserted chromosome 4 segment revealed more complexity, consisting of two segments derived from chromosome 4; one large (86976 bp) and one small (2596 bp). The sequence of the smaller segment was not contiguous with the 80 kb chromosome 4 shown on array CGH but originated from a region 10 kb upstream of the larger fragment on the chromosome 4 map. This smaller fragment was not detected by array
CGH because there were no probes located in this region on the array. The 30 end of the larger segment and the 50 end of the smaller segment both showed simple (GGAA)n repeating elements. 2.3. Diagnostic testing of family members Once all the break point junctions were determined in the patient, we were able to assess the carrier status in female relatives. Two forward PCR primers; one located upstream of the intron 43 junction and the other located upstream of the 30 junction of chromosome 4 and a reverse primer located downstream of the intron 43 junction were used (Fig. 3A). Amplification from the normal chromosome X generated a 345 bp fragment while the mutant chromosome X produced a 1607 bp fragment (Fig. 3B). We were able to determine that the mother of the index case (CK level 248 U/L, Ref. range <140) was a carrier of the insertion and that the maternal aunt who was 6 weeks pregnant was not a carrier of the mutation. 3. Discussion The most common dystrophin mutations are deletions or duplications of one or more exons in the DMD gene. These types of rearrangements account for approximately 70% of all dystrophin mutations. Other types of rearrangements such as inversions and insertions are much less common [5]. Rearrangements involving the dystrophin gene with neighboring loci leading to insertion of entire or
Fig. 2. Identification of the breakpoint junction sequence. (A) Array-CGH profile of chromosome 4 of the patient. Genomic DNA from the patient was hybridized to a Nimblegen oligo tiling array to determine the size of the chromosome 4 insertion in intron 43 of the DMD gene. Arrows indicate the approximate breakpoints for the insert (B). Long-range PCR was used to identify the break-point junctions of the insertion, which consisted of two segments, one large (87098 bp) and one small (2596 bp) region of chromosome 4; (1a) indicates the co-ordinate of the proximal break point in intron 43 of DMD (1b) the 50 breakpoint junction of the large chromosome 4 segment (2a) the 30 breakpoint junction of the large chromosome 4 segment (2b) the 50 breakpoint junction of the small segment (3a) the 30 breakpoint junction of the small chromosome 4 segment and (3b) the distal break point junction in intron 43 of DMD.
B. Baskin et al. / Neuromuscular Disorders 21 (2011) 178–182
181
Fig. 3. Determination of carrier status of female relatives of proband. Breakpoint junction PCR was used to determine carrier status in proband’s mother and maternal aunt. Primers A and B generate a 345 bp fragment from the normal chromosome X, and primers C and B generate a 1607 bp amplicon from the mutant chromosome X. Only the 1607 bp fragment was detected in the proband as expected. Both fragments were amplified in the mother indicating that she is a carrier and only the 345 bp was detected in the maternal aunt indicating that she is a non-carrier.
partial genes into the DMD gene have been described but are rare [6]. Zhang et al. [7] reported the insertion of IL1RAPL1 into DMD and insertions of transposable element, L1, into the dystrophin gene have also been reported [8,9]. The patient described here presented with dystrophinopathy and was found to be negative for mutations on routine diagnostic testing. Since the patient’s clinical presentation and pathology findings were consistent with DMD, we performed mRNA analysis on a muscle biopsy and detected an insertion of a cryptic exon between exons 43 and 44 originating from chromosome 4. Detailed analysis of genomic DNA revealed a 90 kb insertion of two segments of non-contiguous chromosome 4 sequence into intron 43 of the DMD gene. The primary mechanism causing rearrangements in the dystrophin gene has been proposed to be DNA doublestrand breaks (DBS) followed by non-homologous end joining (NHEJ) [6,10–12], which requires no homology or only microhomology at break point junctions. However
Oshima et al. [6] suggested that a case with an inversion/ deletion rearrangement of the DMD gene was caused by a replication based mechanism rather than NHEJ since breakpoint junction sequence revealed repetitive sequences that could have formed a stem-loop structure causing genomic instability. Other authors, reviewed in Zhang et al. have suggested that complex human chromosomal rearrangements can be caused by a replication based mechanism without the need for extensive homology or stem loop formation [13]. The complex rearrangement in the case described in this report consisted of two non-contiguous chromosome 4 segments; a larger 87 kb fragment and a smaller 2.6 kb fragment with the origin of the smaller segment 10 kb upstream of the larger segment on the chromosome 4 map. The break points of the 2.6 kb segment were located in non-repetitive regions whereas the 87 kb segment breakpoints were both located in simple repeat elements, the 50 end in (TA)n repeats and the 30 end in (GGAA)n repeats. The two segments showed GAA microhomology at their junction. The DMD gene
182
B. Baskin et al. / Neuromuscular Disorders 21 (2011) 178–182
breakpoint in intron 43 had a five base pair deletion and showed no homology to any repeat elements. The 50 breakpoint junction of the chromosome 4 insertion and the DMD gene showed four base pairs of microhomology, whereas the 30 junction of the insertion showed no microhomology with the DMD intron 43 junction. We conclude that this rearrangement of chromosome 4 with the DMD gene in this patient could be the result of either NHEJ or a replication based mechanism. Based on the characterization of the breakpoint junctions in the patient we were able to offer carrier testing to female relatives. The mother of the index case was determined to be a carrier which was consistent with the elevated creatine kinase levels detected in her. We subsequently received a blood sample on the maternal aunt who was then 6 weeks pregnant and could establish that she did not carry this rearrangement. Analysis of dystrophin mRNA does not only detect deep embedded intronic mutations in the DMD gene leading to the incorporation of cryptic exons and intron retention in the dystrophin transcript but also complex DMD rearrangements that cause exon skipping and insertion of exons from other genes [3,5,7]. This case emphasizes the importance of performing mRNA analysis in DMD patients where no disease causing mutations are found by routine analysis so that accurate genetic counseling and prenatal diagnosis can be offered to the family. Acknowledgements W.T.G. is supported by a Clinician Scientist Salary Award from both CIHR and the CFRI. We gratefully acknowledge Chieko Chijiwa for her administrative assistance during the work presented here. References [1] Aartsma-Rus A, Van Deutekom JC, Fokkema IF, Van Ommen GJ, Den Dunnen JT. Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
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