BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
47, 195-197 (1992)
BRIEF COMMUNICATION Detection of Molecular Deletions in the Chinese DMD Patients Using Two Amplified Dystrophin Sequences This Brief Communication reports the detection of molecular deletions in Chinese DMD patients using two new amplified dystrophin DNAs involving the regions of exon 49 and 50. The results show that over 50% of the DMD deletions can be rapidly detected by PCR amplification of these two dystrophin sequences. 0 1992 Academic Press, Inc.
Duchenne muscular dystrophy (DMD) is probably the most common and severe X-linked disease, with an incidence of 1 in 3000 male births in China. Our previous study has shown that nearly 60% of the Chinese patients with DMD have partial deletions within the dystrophin gene when detected using a panel of cDNA probes, and the deletions are concentrated in the region of probe 8 (1). Thus, it is also possible to detect Chinese DMD deletions using polymerase chain reaction (PCR) amplification. We recently used multiplex PCR of nine sets of amplified dystrophin DNAs described by Chamberlain et al. (2) to detect the Chinese DMD deletions. However, exon 49 and 50 which represent the other two main regions of Chinese DMD deletions are not involved in the nine amplified dystrophin DNAs. Accordingly, we designed and applied two additional amplified sequences involving the exon 49 and 50 regions, respectively, to detect Chinese patients with DMD. We herein report the research results. EXPERIMENTAL
PROCEDURES
Genomic DNA was prepared from the peripheral leucocytes of 74 Chinese male patients with DMD. DNA (0.1 pg) was subjected to multiplex PCR of dystrophin DNA fragments using Taq DNA polymerase in a Perkin-Elmer Cetus DNA thermal cycler for 30 rounds. The amplification primers corresponding to exons 49 and 50 are 5’-CCAGGCAGAAATI’GATCGC-3’ and 5’-GACCACGTCAATGGCAAATGT3’;5’-CACCAAATGGATTAAGATGT-3’ and 5’-TAGCTAGAGCCAAAGAGAATGGG-3’, which amplify 297 bp (SI) and 226 bp (S2) segments of dystrophin DNA, respectively. A small aliquot of the PCR products was then electrophorescd in 3% Nu Sieve agarose gel. The gels were visually examined for the presence or absence of the specific amplified fragments by staining with ethidium bromide. 195 0885-4505192 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
196
BRIEF COMMUNICATION
:xon
Probe
45 48 19 17 51 8 12 49 44 50 4
Primer
7 8 3 3
e f ii h
a
a g 4
lb 2 8 7
d sa i
a la
FIG. 1. Detection of dystrophin DNA deletions by multiplex PCR amplification using 3% NuSieve agarose gel electrophoresis in TBE buffer at 40 mA for 6 h.
RESULTS AND DISCUSSION The results showed that 38 patients exhibited a deletion of at least one amplified dystrophin DNA fragment, accounting for 51.3% of the total number of patients examined when all 11 amplified dystrophin DNAs were used (Fig. 1). However, as shown in Table 1, the deletions are concentrated in F, Sl, S2 DNA fragments which involved exons 48, 49, and 50; but the highest deletion frequencies focus
Deletion Distributions
Exon regions (i)” 4 W 8 (P)” 12 (b)” 17 (Cy 19 (d)” 44 (ey 45 (f)” 48 Sib 49 s26 50 (h)” 51
TABLE 1 and Frequencies Detected with Multiplex
Length of amplification @PI 196 360 331 416 456 268 547 506 297 226 388
’ According to Chamberlain et al. (1989). b Designed by the authors.
Deletions detected (“ro) l/74 4174 9/74 10/74 4/74 4/74 10/74 17174 18/74 20/74 10/74
(1.4) (5.4) (12.2) (13.5) (5.4) (5.4) (13.5) (23.0) (24.3) (27.2) (13.5)
PCR Frequency in all deletions (%I l/38 4/38 9138 lo/38 4/38 4/38 lo/38 17/38 18/38 20/38 lo/38
(2.6) (10.5) (23.7) (26.3) (10.5) (10.5) (26.3) (44.7) (47.4) (52.6) (26.3)
BRIEF COMMUNICATION
197
on the S2 and Sl regions. Our data also showed that over 50% of the molecular deletions in the Chinese patients with DMD are capable of detection by PCR amplification of these two dystrophin sequences, Sl and S2. If we chose either Sl or S2 combined with amplified fragment b or g, 70% of the dystrophin DNA deletions will be diagnosed. Together with the nine amplified fragments described by Chamberlain et al. (2), it is anticipated that over 90% of the Chinese DMD deletions can be rapidly screened via multiplex PCR amplification. ACKNOWLEDGMENT This work was supported by China National Research Grant 863. We thank Dr. J. S. Chamberlain, the University of Michigan Medical School, for providing the human dystrophin sequence data.
REFERENCES 1. Zeng YT, Chen MJ, Ren ZR, Qiu XK, Huang SZ. Analysis of RPLPs and DNA deletions in the Chinese Duchenne muscular dystrophy. J Med Genet 28:167-170, 1991. 2. Chamberlain JS, Gibbs RA, Ranier JE, Caskey CT. Multiplex PCR for the diagnosis of Duchenne muscular dystrophy. In PCR: Protocals and Applications-A Laboratory Manual (Innis M, Gelfand D, Sninski J, White T, Eds.). San Diego: Academic Press, 1989, pp. 272-281.
ZENG YI-TAO CHEN MEI-JUE REN ZHAO-RUI HUANG YING QIU XIAO-KUN HUANG SHU-ZHEN Shanghai Institute of Medical Shanghai Children’s Hospital Shanghai 200040, China Received October 17, 1991
Genetics
MEDICINE
AND
METABOLIC
BIOLOGY
47, 195-197 (1992)
BRIEF COMMUNICATION Detection of Molecular Deletions in the Chinese DMD Patients Using Two Amplified Dystrophin Sequences This Brief Communication reports the detection of molecular deletions in Chinese DMD patients using two new amplified dystrophin DNAs involving the regions of exon 49 and 50. The results show that over 50% of the DMD deletions can be rapidly detected by PCR amplification of these two dystrophin sequences. 0 1992 Academic Press, Inc.
Duchenne muscular dystrophy (DMD) is probably the most common and severe X-linked disease, with an incidence of 1 in 3000 male births in China. Our previous study has shown that nearly 60% of the Chinese patients with DMD have partial deletions within the dystrophin gene when detected using a panel of cDNA probes, and the deletions are concentrated in the region of probe 8 (1). Thus, it is also possible to detect Chinese DMD deletions using polymerase chain reaction (PCR) amplification. We recently used multiplex PCR of nine sets of amplified dystrophin DNAs described by Chamberlain et al. (2) to detect the Chinese DMD deletions. However, exon 49 and 50 which represent the other two main regions of Chinese DMD deletions are not involved in the nine amplified dystrophin DNAs. Accordingly, we designed and applied two additional amplified sequences involving the exon 49 and 50 regions, respectively, to detect Chinese patients with DMD. We herein report the research results. EXPERIMENTAL
PROCEDURES
Genomic DNA was prepared from the peripheral leucocytes of 74 Chinese male patients with DMD. DNA (0.1 pg) was subjected to multiplex PCR of dystrophin DNA fragments using Taq DNA polymerase in a Perkin-Elmer Cetus DNA thermal cycler for 30 rounds. The amplification primers corresponding to exons 49 and 50 are 5’-CCAGGCAGAAATI’GATCGC-3’ and 5’-GACCACGTCAATGGCAAATGT3’;5’-CACCAAATGGATTAAGATGT-3’ and 5’-TAGCTAGAGCCAAAGAGAATGGG-3’, which amplify 297 bp (SI) and 226 bp (S2) segments of dystrophin DNA, respectively. A small aliquot of the PCR products was then electrophorescd in 3% Nu Sieve agarose gel. The gels were visually examined for the presence or absence of the specific amplified fragments by staining with ethidium bromide. 195 0885-4505192 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
196
BRIEF COMMUNICATION
:xon
Probe
45 48 19 17 51 8 12 49 44 50 4
Primer
7 8 3 3
e f ii h
a
a g 4
lb 2 8 7
d sa i
a la
FIG. 1. Detection of dystrophin DNA deletions by multiplex PCR amplification using 3% NuSieve agarose gel electrophoresis in TBE buffer at 40 mA for 6 h.
RESULTS AND DISCUSSION The results showed that 38 patients exhibited a deletion of at least one amplified dystrophin DNA fragment, accounting for 51.3% of the total number of patients examined when all 11 amplified dystrophin DNAs were used (Fig. 1). However, as shown in Table 1, the deletions are concentrated in F, Sl, S2 DNA fragments which involved exons 48, 49, and 50; but the highest deletion frequencies focus
Deletion Distributions
Exon regions (i)” 4 W 8 (P)” 12 (b)” 17 (Cy 19 (d)” 44 (ey 45 (f)” 48 Sib 49 s26 50 (h)” 51
TABLE 1 and Frequencies Detected with Multiplex
Length of amplification @PI 196 360 331 416 456 268 547 506 297 226 388
’ According to Chamberlain et al. (1989). b Designed by the authors.
Deletions detected (“ro) l/74 4174 9/74 10/74 4/74 4/74 10/74 17174 18/74 20/74 10/74
(1.4) (5.4) (12.2) (13.5) (5.4) (5.4) (13.5) (23.0) (24.3) (27.2) (13.5)
PCR Frequency in all deletions (%I l/38 4/38 9138 lo/38 4/38 4/38 lo/38 17/38 18/38 20/38 lo/38
(2.6) (10.5) (23.7) (26.3) (10.5) (10.5) (26.3) (44.7) (47.4) (52.6) (26.3)
BRIEF COMMUNICATION
197
on the S2 and Sl regions. Our data also showed that over 50% of the molecular deletions in the Chinese patients with DMD are capable of detection by PCR amplification of these two dystrophin sequences, Sl and S2. If we chose either Sl or S2 combined with amplified fragment b or g, 70% of the dystrophin DNA deletions will be diagnosed. Together with the nine amplified fragments described by Chamberlain et al. (2), it is anticipated that over 90% of the Chinese DMD deletions can be rapidly screened via multiplex PCR amplification. ACKNOWLEDGMENT This work was supported by China National Research Grant 863. We thank Dr. J. S. Chamberlain, the University of Michigan Medical School, for providing the human dystrophin sequence data.
REFERENCES 1. Zeng YT, Chen MJ, Ren ZR, Qiu XK, Huang SZ. Analysis of RPLPs and DNA deletions in the Chinese Duchenne muscular dystrophy. J Med Genet 28:167-170, 1991. 2. Chamberlain JS, Gibbs RA, Ranier JE, Caskey CT. Multiplex PCR for the diagnosis of Duchenne muscular dystrophy. In PCR: Protocals and Applications-A Laboratory Manual (Innis M, Gelfand D, Sninski J, White T, Eds.). San Diego: Academic Press, 1989, pp. 272-281.
ZENG YI-TAO CHEN MEI-JUE REN ZHAO-RUI HUANG YING QIU XIAO-KUN HUANG SHU-ZHEN Shanghai Institute of Medical Shanghai Children’s Hospital Shanghai 200040, China Received October 17, 1991
Genetics
