Deletions, duplications and novel restriction fragment length polymorphsm in Duchenne and Becker m us d a r dystrophies Lau YL, Srivastava G, Wong V, Liu YT, Ho FCS, Yeung CY. Deletions, duplications and novel restriction fragment length polymorphism in Duchenne and Becker muscular dystrophies. Clin Genet 1992: 41: 252-258. To determine the mutations of Southern Chinese with Duchenne and Becker muscular dystrophies (DMD, BMD), we analysed 28 DMD and BMD patients in 24 unrelated families for intragenic deletions and duplications by using cDNA probes covering the entire 14 kb of the dystrophin gene. Deletions were detected in nine unrelated patients (seven patients by probe 8 and two by probe 2b-3). Gene duplications were detected by probe 1-2a in two patients with the duplication bands confirmed in both Hind 111 and Bgl I1 digests and by densitometry. A third patient was found to have a junction fragment with Bgl I1 and a duplication band with Hind 111 by probe 5b-7. Therefore 50% of the 24 unrelated families were found to have either deletions or duplications. A previously undescribed restriction fragment length polymorphism (RFLP) was found in one fiimily with probe 5b-7 in Bgl I1 digests which was found to segregate with the disease phenotype. This new RFLP was not detected in over 70 unrelated X chromosomes we have examined so far, and appeared to be “private” for this family. The presence of this new restriction site may or may not be the mutation responsible for the disease phenotype.
Duchenne and Becker muscular dystrophies (DMD, BMD) are alleleic X-linked recessive disorders, affecting about 1 in 3300 liveborn males (Emery 1986). The gene has been located at Xp21 by identification of unusual patients with cytologically visible deletions of Xp21 (Francke et al. 1985) or balanced X;autosome translocation (Boyd & Buckle 1986). Subsequent linkage studies using randomly cloned DNA probes from this region confirmed the localization of the gene to Xp21 (Davies et al. 1983, Kingston et al. 1984). Genomic probes (PERT 87 and XJ) were soon obtained by enrichment for sequences from within the deleted region of a male patient (Kunkel et al. 1985) and by cloning of the translocation junction from a female t(X;21) translocation patient (Ray et al. 1985). About 5%10% of the patients had gene deletions detected by these genomic probes (Monaco et al. 1985, Kunkel et al. 1986). Expressed sequences were identified in these regions and used to clone the complementary DNA (cDNA) (Koenig et al. 1987, Cross et al. 1987, Burghes et al. 1987). The entire ICkilobase (kb) transcript was found to be encoded for by more than 70 exons spread over 2500 k b of genomic DNA (Koenig et al. 1987, 252
1. L lap, G. Srlvastrvi’, V. Won!, 1. T. Llu’, F. C. S. Ha’ and C. Y. Young Departments of Paediatrics and ’Pathology, University ot Hong Kong
-
Key words: Becker muscular dystrophy Duchenne muscular dystrophy gene mutathns restriction fragment length polymorphism
-
-
Dr. %Lung Lau, Department ot Paediatrics, Queen Mary Hospital, Poktuhm Road, University of Hong Kong, Hong Kong Received 16 July, revised 25 November, accepted for pubiication 4 December 1991
Van Ommen et al. 1986).These cDNA probes were found to be able to detect deletions or duplications of one or more exons in over 50% of patients (Lindl6f et al. 1989, Gillard et al. 1989, Koenig et al. 1989). The aim of the present study was to analyse the DMD and BMD mutations in Southern Chinese, as this has not been done before, although a study on DMD mutations in Northern Chinese was published recently (Zeng et al. 1991). We analysed 28 DMD and BMD Chinese patients in 24 unrelated families for intragenic deletions and duplications by using the cDNA probes described by Koenig et al. (1987). Deletions were detected in nine patients and duplications in three. A patient was found to have a novel RFLP which was shown to be linked with the disease within the family and has not been detected in any other individuals examined so far.
Patients, mitorials and mithods Patients All of the patients have been followed for several years in the Neurology Clinic of the University Department of Paediatrics and have been diag-
Mutations in Duchenne muscular dystrophy nosed as DMD or BMD on the basis of grossly raised serum creatine kinase activity, pseudohypertrophy of the calf muscles, myopathic electromyographic findings, consistent muscle biopsy changes and progression of muscle weakness. Patients were classified as DMD if they became wheelchairbound before the age of 12 years and as BMD if still ambulant at age 16 years. There were 18 unrelated DMD patients, 3 BMD patients and 3 not yet classified. Southern blot analysis
High molecular weight DNA was extracted from 5-10 ml of whole blood anticoagulated in EDTA using standard proteinase K procedure. DNA (20 pg) was digested with the restriction endonucleases Hind 111 and Bgl 11. The samples were then subjected to electrophoresis in 0.8% agarose gels. After denaturation and neutralization, DNA was transferred to Nylon membranes (Gene Screen Plus, NEN, Du Point, Boston, MA) and hybridization was carried out at 42°C for 40 h in 50% formamide, 1 M sodium chloride, 1% SDS, IP/i dextran sulphate, 0.2% polyvinylpyrrolidone, 0.20/0 Ficoll, 0.2% bovine serum albumin, 50 mM Tris-HCI (pH 7 3 , 0.1%) sodium pyrophosphate, denatured salmon sperm DNA (200 pg/ml) and the denatured ["PI-oligolabelled DNA probe (0.5-5 ng/ml). The final wash was 0.2 x SSC ( I x SSC; 150 mM sodium chloride and 15 mM sodium citrate), 1% SDS, 0.1% sodium pyrophosphate at 65°C for 40 min. Autoradiography was performed using Kodak films (X-OMAT, XAR 5) at -70°C for 2-7 days. The membranes were stripped in 0.1 x SSC, 1% SDS, 0.1 m M EDTA at 95°C for 40 min and autoradiographed to check for residual radioactivity before reprobing. As hybridization probes, we used eight contiguous segments, labelled 1-2a, 2b-3, &5a, 5b-7, 8, 9, 10 and 11-14 that cover the entire 14-kb dystrophin cDNA. The cDNA probes were obtained from the American Type Culture Collection (ATCC). The cloned segments were released from the vectors with appropriate restriction endonucleases. For cDNA 9-14, EcoRI insert was further cleaved by BamHI to give 9, 10 and 11-14 probes. Inserts were purified from agarose gels using Gene Clean Kit I (Bresatec, Australia). The oligolabelling of the isolated inserts was done according to the supplier's conditions (Bresatec, Australia). Transmission densitometry scanning
The autoradiographic film was scanned using a transmission densitometer (Helena Laboratory
Cliniscan 2), according to the method described by Hu et al. (1988, 1989, 1990). for the three patients with duplications. Briefly, the area under each peak represents the intensity of each hybridization band. The peak area divided by the sum of the peak area of all bands in that lane will give the relative peak value in percentage. Since the bands with double (gene duplication) intensity increase the total peak area and alter the relative peak value, a normalization factor is obtained by calculating the ratio of the mean peak values of a constant band showing single intensity in a normal male control and similar intensity in the duplication case. All the relative peak values from the duplication case were multiplied by this factor. Because the hybridization band intensity (relative peak value) is estimated by the ratio of the intensity of each band to that of all bands in the same lane, the results are not affected by the variation of the DNA load. Results Deletions
Deletions involving portions of the gene were found in nine unrelated patients. Seven were detected by probe 8 and two by probe 2b-3. All these deletions were confirmed by using two restriction enzymes Hind 111 and Bgl TI, showing absence of the corresponding exon-containing restriction fragments. The deletion frequency was thus 37.5%. The extent of the deletions is shown in Table 1. Exon number 45 of 0.5 kb was difficult to demonstrate, and the border of deletion at this point was not absolutely certain. The disease was classified as DMD in six of the deletion families, and three patients were too young to be classified. Duplications
Three unrelated patients were found to have duplications of portions of the gene (Table 1). Two were DMD patients and one was a BMD patient. Two restriction enzymes Hind I11 and Bgl I1 were used to avoid uncertainty due either to small size of some exon-containing fragments or to co-migrating fragments, and to confirm any duplication results obtained. Two duplication cases were detected by probe 1-2a, and the duplications were confirmed in both Hind 111 and Bgl I1 digests (Fig. la, b) 7.5 kb and 4.6 kb fragments detected by probe 1-2a in Hind I11 digests correspond to 6.5 kb and 1.4 kb fragments in Bgl I1 digests, respectively. The other duplication case was detected by probe 5b-7. The duplication of specific bands was verified by using a transmission densitometer in Patients a and b (Table 2). Densitometry scanning was not feasible in Patient c because of close proximity between the 253
.
cn a
N
~
DMD (004; c)
~
BMD (023; b)
~~
DMD (019)
DMD (043)
DMD (029)
I
I
I
3.2 3.25 4.2 8.5 3.1 8 4.6 7.5
(c)
Chssificatim
DMD (012)
2b-3
4-5a
5b-7
10.5
4 6.6 2.7 6 1.7 12 3 7.3 12
20
5.2
4.7 11 18.0 1.8 0.4 1.3 1.5 6.1
-J
6.2
49 50 51 52
8
4.2 11 4.1 0.5 1.5 10 1.2 3.9 1.6 3.7 3.1 7
3 4 5 6 7 8+9 10+11 12 13 14+1516 17 18 19 20 21 22+25 26+27 28 29 30+33 34 35 36 37 38+39 40+41 42 43 44 45 46 47 48
1
(b)
2
1-2a
clilvcal
(a)
Table 1. Deletrw and duplications in DMD and BMD patients relative to the exon containq Hind 111 fragments detected with cDNA probes
Mutations in Duchenne muscular dystrophy F P M
FPM
F P M
FPM
F P M
)k-
.* -
)zll -+tr \.
**
I
e
HhdIII
Bgl I4
&
b
a
C
Hindlll
Hind Ill
Bgl I I
Fig. 1. Southern blot analysis of the three duplication patients (P), normal males (M) and normal females (F). Duplicated bands in patients are indicated by arrows. (a) Probe I-2a showing duplication o f 7.5 and 4.6 kb fragments with Hind 111 and 6.5 k b fragment with Bgl I 1 in Patient a. (b) Probe I-2a showing duplication of 4.6 kb fragment with Hind 111 and 1.4 kb fragment with Bgl I1 in Patient b. (c) Probe 5b-7 showing duplication of 6.2 k b fragment with Hind Ill and junction fragment (JF) with Bgl I 1 in Patient c.
duplicated band and a single-intensity band with Hind 111 digests (Fig. Ic). However, an altered-size restriction fragment (JF) was detected with Bgl TI digests (Fig. lc). This fragment probably contains the duplication junction, since it was detected in the presence of all the normal size fragments. It is unlikely that this novel fragment represents a polymorphic allele or results from other types of DNA rearrangements such as deletion or insertion (Den Dunnen et al. 1989). The duplication frequency was thus 12.5%.
Table 2. Hybndization intensity in duplication cases Relative peak value'
(a)
0)
*
Hybridization band (probe 1-2a)
Male control
Patient
Ratio Pafientcontrd
Patient a 7.5 kb (Hind Ill) 4.6 kb (Hind Ill) 6.5 kb (Bgl II)
12.2 14.3 44.8
22.45 33.70 84.00
1.84 2.36 1.80
Patient b 4.6 kb (Hind Ill) 1.4 kb (Bgi II)
13.7 8.2
31.40 14.40
2.29 1.76
Relatie peak value of each band represents the percentage of the peak area ot that band divided by the sum of the peak area of all the bands in that lane. The relative peak value of the duplication case was normalized as described in Methods.
Therefore, 50"h of our 24 unrelated families had either deletions or duplications as detected by Southern blotting using cDNA probes.
Novel RFLP
It was found that the restriction fragment pattern of a DMD patient with probe 5b-7 and Bgl I1 was unique (Fig. 2), with absence of the 21 kb fragment (band a) and acquisition of two smaller fragments (bands b and c). The 2 smaller exon-containing restriction fragments (12.7 kb and 8.3 kb) add up to 21 kb. His family was analysed (Fig. 2) and his mother, two aunts and sister also had the two smaller bands with the 21 kb band as well. None of his healthy maternal uncles had the two smaller fragments. The most likely explanation for this observation is a previously unreported RFLP, which seems to exist in this family only. We have examined over 70 unrelated X chromosomes and did not find this RFLP outside this family. This RFLP and another previously reported RFLP (Darras & Francke 1988) (bands d ore), as detected by probe 5b-7 with Bgl 11, were shown to link with the disease phenotype in this family (Fig. 2), implying the proband's two aunts and sister are carriers. 255
Lau et al.
v= FC YC -3
~b
Duchenne and Becker muscular dystrophies (DMD, BMD) are alleleic X-linked recessive disorders, affecting about 1 in 3300 liveborn males (Emery 1986). The gene has been located at Xp21 by identification of unusual patients with cytologically visible deletions of Xp21 (Francke et al. 1985) or balanced X;autosome translocation (Boyd & Buckle 1986). Subsequent linkage studies using randomly cloned DNA probes from this region confirmed the localization of the gene to Xp21 (Davies et al. 1983, Kingston et al. 1984). Genomic probes (PERT 87 and XJ) were soon obtained by enrichment for sequences from within the deleted region of a male patient (Kunkel et al. 1985) and by cloning of the translocation junction from a female t(X;21) translocation patient (Ray et al. 1985). About 5%10% of the patients had gene deletions detected by these genomic probes (Monaco et al. 1985, Kunkel et al. 1986). Expressed sequences were identified in these regions and used to clone the complementary DNA (cDNA) (Koenig et al. 1987, Cross et al. 1987, Burghes et al. 1987). The entire ICkilobase (kb) transcript was found to be encoded for by more than 70 exons spread over 2500 k b of genomic DNA (Koenig et al. 1987, 252
1. L lap, G. Srlvastrvi’, V. Won!, 1. T. Llu’, F. C. S. Ha’ and C. Y. Young Departments of Paediatrics and ’Pathology, University ot Hong Kong
-
Key words: Becker muscular dystrophy Duchenne muscular dystrophy gene mutathns restriction fragment length polymorphism
-
-
Dr. %Lung Lau, Department ot Paediatrics, Queen Mary Hospital, Poktuhm Road, University of Hong Kong, Hong Kong Received 16 July, revised 25 November, accepted for pubiication 4 December 1991
Van Ommen et al. 1986).These cDNA probes were found to be able to detect deletions or duplications of one or more exons in over 50% of patients (Lindl6f et al. 1989, Gillard et al. 1989, Koenig et al. 1989). The aim of the present study was to analyse the DMD and BMD mutations in Southern Chinese, as this has not been done before, although a study on DMD mutations in Northern Chinese was published recently (Zeng et al. 1991). We analysed 28 DMD and BMD Chinese patients in 24 unrelated families for intragenic deletions and duplications by using the cDNA probes described by Koenig et al. (1987). Deletions were detected in nine patients and duplications in three. A patient was found to have a novel RFLP which was shown to be linked with the disease within the family and has not been detected in any other individuals examined so far.
Patients, mitorials and mithods Patients All of the patients have been followed for several years in the Neurology Clinic of the University Department of Paediatrics and have been diag-
Mutations in Duchenne muscular dystrophy nosed as DMD or BMD on the basis of grossly raised serum creatine kinase activity, pseudohypertrophy of the calf muscles, myopathic electromyographic findings, consistent muscle biopsy changes and progression of muscle weakness. Patients were classified as DMD if they became wheelchairbound before the age of 12 years and as BMD if still ambulant at age 16 years. There were 18 unrelated DMD patients, 3 BMD patients and 3 not yet classified. Southern blot analysis
High molecular weight DNA was extracted from 5-10 ml of whole blood anticoagulated in EDTA using standard proteinase K procedure. DNA (20 pg) was digested with the restriction endonucleases Hind 111 and Bgl 11. The samples were then subjected to electrophoresis in 0.8% agarose gels. After denaturation and neutralization, DNA was transferred to Nylon membranes (Gene Screen Plus, NEN, Du Point, Boston, MA) and hybridization was carried out at 42°C for 40 h in 50% formamide, 1 M sodium chloride, 1% SDS, IP/i dextran sulphate, 0.2% polyvinylpyrrolidone, 0.20/0 Ficoll, 0.2% bovine serum albumin, 50 mM Tris-HCI (pH 7 3 , 0.1%) sodium pyrophosphate, denatured salmon sperm DNA (200 pg/ml) and the denatured ["PI-oligolabelled DNA probe (0.5-5 ng/ml). The final wash was 0.2 x SSC ( I x SSC; 150 mM sodium chloride and 15 mM sodium citrate), 1% SDS, 0.1% sodium pyrophosphate at 65°C for 40 min. Autoradiography was performed using Kodak films (X-OMAT, XAR 5) at -70°C for 2-7 days. The membranes were stripped in 0.1 x SSC, 1% SDS, 0.1 m M EDTA at 95°C for 40 min and autoradiographed to check for residual radioactivity before reprobing. As hybridization probes, we used eight contiguous segments, labelled 1-2a, 2b-3, &5a, 5b-7, 8, 9, 10 and 11-14 that cover the entire 14-kb dystrophin cDNA. The cDNA probes were obtained from the American Type Culture Collection (ATCC). The cloned segments were released from the vectors with appropriate restriction endonucleases. For cDNA 9-14, EcoRI insert was further cleaved by BamHI to give 9, 10 and 11-14 probes. Inserts were purified from agarose gels using Gene Clean Kit I (Bresatec, Australia). The oligolabelling of the isolated inserts was done according to the supplier's conditions (Bresatec, Australia). Transmission densitometry scanning
The autoradiographic film was scanned using a transmission densitometer (Helena Laboratory
Cliniscan 2), according to the method described by Hu et al. (1988, 1989, 1990). for the three patients with duplications. Briefly, the area under each peak represents the intensity of each hybridization band. The peak area divided by the sum of the peak area of all bands in that lane will give the relative peak value in percentage. Since the bands with double (gene duplication) intensity increase the total peak area and alter the relative peak value, a normalization factor is obtained by calculating the ratio of the mean peak values of a constant band showing single intensity in a normal male control and similar intensity in the duplication case. All the relative peak values from the duplication case were multiplied by this factor. Because the hybridization band intensity (relative peak value) is estimated by the ratio of the intensity of each band to that of all bands in the same lane, the results are not affected by the variation of the DNA load. Results Deletions
Deletions involving portions of the gene were found in nine unrelated patients. Seven were detected by probe 8 and two by probe 2b-3. All these deletions were confirmed by using two restriction enzymes Hind 111 and Bgl TI, showing absence of the corresponding exon-containing restriction fragments. The deletion frequency was thus 37.5%. The extent of the deletions is shown in Table 1. Exon number 45 of 0.5 kb was difficult to demonstrate, and the border of deletion at this point was not absolutely certain. The disease was classified as DMD in six of the deletion families, and three patients were too young to be classified. Duplications
Three unrelated patients were found to have duplications of portions of the gene (Table 1). Two were DMD patients and one was a BMD patient. Two restriction enzymes Hind I11 and Bgl I1 were used to avoid uncertainty due either to small size of some exon-containing fragments or to co-migrating fragments, and to confirm any duplication results obtained. Two duplication cases were detected by probe 1-2a, and the duplications were confirmed in both Hind 111 and Bgl I1 digests (Fig. la, b) 7.5 kb and 4.6 kb fragments detected by probe 1-2a in Hind I11 digests correspond to 6.5 kb and 1.4 kb fragments in Bgl I1 digests, respectively. The other duplication case was detected by probe 5b-7. The duplication of specific bands was verified by using a transmission densitometer in Patients a and b (Table 2). Densitometry scanning was not feasible in Patient c because of close proximity between the 253
.
cn a
N
~
DMD (004; c)
~
BMD (023; b)
~~
DMD (019)
DMD (043)
DMD (029)
I
I
I
3.2 3.25 4.2 8.5 3.1 8 4.6 7.5
(c)
Chssificatim
DMD (012)
2b-3
4-5a
5b-7
10.5
4 6.6 2.7 6 1.7 12 3 7.3 12
20
5.2
4.7 11 18.0 1.8 0.4 1.3 1.5 6.1
-J
6.2
49 50 51 52
8
4.2 11 4.1 0.5 1.5 10 1.2 3.9 1.6 3.7 3.1 7
3 4 5 6 7 8+9 10+11 12 13 14+1516 17 18 19 20 21 22+25 26+27 28 29 30+33 34 35 36 37 38+39 40+41 42 43 44 45 46 47 48
1
(b)
2
1-2a
clilvcal
(a)
Table 1. Deletrw and duplications in DMD and BMD patients relative to the exon containq Hind 111 fragments detected with cDNA probes
Mutations in Duchenne muscular dystrophy F P M
FPM
F P M
FPM
F P M
)k-
.* -
)zll -+tr \.
**
I
e
HhdIII
Bgl I4
&
b
a
C
Hindlll
Hind Ill
Bgl I I
Fig. 1. Southern blot analysis of the three duplication patients (P), normal males (M) and normal females (F). Duplicated bands in patients are indicated by arrows. (a) Probe I-2a showing duplication o f 7.5 and 4.6 kb fragments with Hind 111 and 6.5 k b fragment with Bgl I 1 in Patient a. (b) Probe I-2a showing duplication of 4.6 kb fragment with Hind 111 and 1.4 kb fragment with Bgl I1 in Patient b. (c) Probe 5b-7 showing duplication of 6.2 k b fragment with Hind Ill and junction fragment (JF) with Bgl I 1 in Patient c.
duplicated band and a single-intensity band with Hind 111 digests (Fig. Ic). However, an altered-size restriction fragment (JF) was detected with Bgl TI digests (Fig. lc). This fragment probably contains the duplication junction, since it was detected in the presence of all the normal size fragments. It is unlikely that this novel fragment represents a polymorphic allele or results from other types of DNA rearrangements such as deletion or insertion (Den Dunnen et al. 1989). The duplication frequency was thus 12.5%.
Table 2. Hybndization intensity in duplication cases Relative peak value'
(a)
0)
*
Hybridization band (probe 1-2a)
Male control
Patient
Ratio Pafientcontrd
Patient a 7.5 kb (Hind Ill) 4.6 kb (Hind Ill) 6.5 kb (Bgl II)
12.2 14.3 44.8
22.45 33.70 84.00
1.84 2.36 1.80
Patient b 4.6 kb (Hind Ill) 1.4 kb (Bgi II)
13.7 8.2
31.40 14.40
2.29 1.76
Relatie peak value of each band represents the percentage of the peak area ot that band divided by the sum of the peak area of all the bands in that lane. The relative peak value of the duplication case was normalized as described in Methods.
Therefore, 50"h of our 24 unrelated families had either deletions or duplications as detected by Southern blotting using cDNA probes.
Novel RFLP
It was found that the restriction fragment pattern of a DMD patient with probe 5b-7 and Bgl I1 was unique (Fig. 2), with absence of the 21 kb fragment (band a) and acquisition of two smaller fragments (bands b and c). The 2 smaller exon-containing restriction fragments (12.7 kb and 8.3 kb) add up to 21 kb. His family was analysed (Fig. 2) and his mother, two aunts and sister also had the two smaller bands with the 21 kb band as well. None of his healthy maternal uncles had the two smaller fragments. The most likely explanation for this observation is a previously unreported RFLP, which seems to exist in this family only. We have examined over 70 unrelated X chromosomes and did not find this RFLP outside this family. This RFLP and another previously reported RFLP (Darras & Francke 1988) (bands d ore), as detected by probe 5b-7 with Bgl 11, were shown to link with the disease phenotype in this family (Fig. 2), implying the proband's two aunts and sister are carriers. 255
Lau et al.
v= FC YC -3
~b
