Molecular deletion patterns in Turkish Duchenne and Becker muscular dystrophy patients

Molecular deletion patterns in Turkish Duchenne and Becker muscular dystrophy patients
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  ~t~ LS VI R Brain & Development 1996; 18:91-94 Original article Molecular deletion patterns in Turkish Duchenne and ecker muscular dystrophy patients a v b* .... a a Pervin Dinner , Haluk Topaloglu , ~ukruye Ayter , Meral Ozgii~ , • • Haydar Ah Ta~dermr , Yavuz Renda h a Detmrtment ~?/.Medical Biology. Faculty Of Medicine. University ~ Hacettepe. 06 I00 Anhtra. Ttcrke3 h Department ~/" Child Neurology. Faculty Of Medicine. Unit'er.sity t?/Hacettepe. 06 I00 Ankara. Turkey Received 24 July 1995; accepted 3 October 1995 The dystrophin gene deletion patterns of Duchenne/Becker muscular dystrophy were investigated in 57 DMD, 7 BMD and 1 DMD-BMD intermediate muscular dystrophy patients. Deletions, analyzed by multiplex amplification of selected exons, were observed in 58% (38 cases) of the patients. It was found that exon 48 was the most frequently affected, while exon 44 was the least frequently affected. The number of deleted exons was variable, but single exon deletions were more frequent (41%) than larger deletions in our population and the great majority of deletions began distal to exon 44. The application of PCR to deletion analysis in D/BMD was found to be very useful in delineating the extent of the deletion in most of the cases (82%). It was seen that the frequency of deletion breakpoints in distal part of the dystrophin gene (exons 42-52) was detected in 64% of our cases. In our group, the frequency of deletion breakpoints in the same area of the dystrophin gene was between that of the French and the Finnish patients. The distribution of deletion breakpoints within the dystrophin gene of the Turkish population seems to have some differences from other populations. Deletion breakpoints were found to be clustered mainly in three separate regions covering introns 44, 45 and 50 within the central region of the dystrophin gene. Intron 44 was mostly 5' breakpoints but it was found not to be involved as 3' breakpoints. The correlation between phenotype and type of deletion agreed with the reading frame theory except for one DMD case. Kevword.~: l)uchenne muscular dystrophy; Becker muscular dystrophy; Deletion; Dystrophin 1. INTRODUCTION Duchenne and Becker muscular dystrophies (D/BMD) are allelic and X linked recessive neuromuscular disorders that result from mutations in the dystrophin gene. Some 65% of DMD patients exhibit deletions, which can be found by cDNA hy- bridization [1-3] or, more recently, by PCR analysis [4-7]. Deletions are mainly clustered in a region near the center of the dystrophin gene and to a lesser extent near its 5' end [3,8]. The size of the deletion is not directly correlated with the severity of disease [9]. It has been shown that some exon deletions cause DMD whilc others cause BMD, because deletions causing DMD shift thc translational reading frame, resulting in a severely • Corresponding author. Fax: (90) (312) 3106262. 0387-7604/96/ 15.00 ,~ 1996 Elsevier Science B.V. All rights reserved SSDI 0387.7604(95)00128-X truncated protein gene product, whereas in BMD the translational reading frame may be maintained in phase, resulting in an abnormal, but partially functional protein product [10]. However. the reading frame hypothesis is not without exceptions [9,1 I]. Specifically, about one-third of patients with a deletion in the 5' end of the gene have a phenotype that is not predictable on the basis of the effect of the deletion on the reading frame. Excep- tions to the reading frame hypothesis have been explained at the molecular level due to the use of alternative initiator methionine codons (ATG) or alternative splicing [12]. In the present study, we analyzed 65 D/BMD patients fbr intragenic deletions using multiplex amplification of 15 exons and we observed the most and the least frequently affected exons and the number of deleted exons. We mapped the breakpoint regions of the deletions, discussed the distribution of deletion breakpoints within introns with differences between DMD and BMD deletions and examined the affect of the deletions on the  92 P. Dinqer et al. / Brain Development 1996; 18:91-94 translational reading flame of dystrophin mRNA using additional PCR reactions. 2 METHODS 2 1 Patients . . .2 50 T ~ ll;' ;=;;;llqi *r~.::~l,** ~,]q. : ~'IIIT~; ll,:, '~ lli=li,l¢fftqlli;,~; fk;,,r~ ~ , ,,;. ~,et, .~j O 45 ~', ,~,o],,: ;',. , ,::,,= 2,T,,i, T~, ~:,,HlJi','$ 2~j 17 ~ i= i Fifty-seven DMD, 7 BMD, and 1 DMD-BMD intermediate muscular dystrophy patients who were of Turkish srcin were enrolled in this study. All patients were diagnosed in the Pediatric Neurology Department, Children's Hospital, Hacettepe Univer- sity in the htst 2 years. Diagnosis was based on clinical symp- toms, high serum creatine kinase levels and histopathological and dystrophin analysis of muscle biopsies. The age at which patients lost ambulation was the main clinical parameter used to differen- tiate DMD from intermediate and BMD phenotypes. For this purpose, children who lost amulation before the age of 13 were classified as DMD, those who were still ambulant at the age of 16 were classified as BMD, and those losing ambulation between the ages of 13 and 16 years were classified as intermediate. (One of the patients with exon 13-19 deletions was classified as intermediate.) Children who were still too young to be assigned to a category were classified on the basis of family history (if there was any) or a combination of clinical and biochemical parameters and dystrophin immunocytochemistry of muscle biop- sies. 2 2 DNAstudies DNA was extracted from leukocytes either by extraction with phenol-chloroform [13] or by salting out the proteins [14]. PCR deletion analysis of the dystrophin gene was carried out with the primer sets of Chamberlain et al. [4,5] and Gibbs et al. [6] using conditions as described. Fifteen exons were coamplified in two multiplex PCR reactions (9 exon PCR reaction: exons 4, 8, 12, 17, 19, 44, 45, 48, 51; and 6 exon PCR reaction: Pm+ I, cxons 13. 43, 47, 50, 52). These primer, sets were sometimes supplemented with additional primers according to the 5' and 3' localization of the dystrophin exons originally described by Abbs et al. [71 Amplified DNA fragments were separated by electrophoresis through 3f+ agarose (NuSieve, FMC Bioproducts, Rockland, USA) + I agarose or 1.5 agarose gels. Deletions were diag- nosed when one of the bands present in the amplified control DNA was absent from the patient DNA. The localization of deletion breakpoints was detected by PCR methods, which amplified exons adjacent to the deleted exons. To detect the localization of breakpoints to particular introns, additional exon primers (exons 42, 46, 49, 53) were used. For simplicity, it was assumed that each breakpoint occurred within an intron. 3. RESULTS Wc analyzed a total of 65 unrelated D/BMD patients for intragenic deletions of the dystrophin gene. The frequency and distribution of dystrophin gene deletions was assessed by multi- plcx amplification of selected exons. The overall detection rate for deletions was 58 . In DMD patients, we observed a deletion 0 2 4 6 8 10 12 Number of Patients Fig. I. Numbers of patients with deletions in each exon of the dystrophin gene. frequency of 60 . For BMD, this proportion was 43 , although the sample size (n = 7) for this group is too small for statistical purposes. The majority of the deletions (26/38) were found to be localized within the central region of the gene (exons 43-52), the remaining deletions (12/38) were mapped to the proximal hot spot (exons 4-19). Exon 48 was the most frequently affected (12 out of 38 deletion patients) and of all exons showing a deletion, exon 44 was the least frequently deleted. The great majority of deletions began distal to exon 44 (Fig. I). Since 68 of the deletions were seen in the central region of the gene, exons 42, 46, 49 and 53 were amplified to detect the deletion sizes in our patients. Deletion sizes were shown in 22 of 26 deletions (82 ) which were localized in the distal part of the gene using PeR technology. In 4 patients, the deletions covered exon 53, so we could not observe the localization of endpoints, because we did not use primers distal to exon 53. Having precisely localized the deletion endpoints in 22 pa- tients, we evaluated the localization of 5' and 3' breakpoints. In addition to 5 patients whose only 5' or 3' breakpoint regions were known, a total of 26 starting breakpoint regions and 23 ending breakpoint regions were analyzed (Fig. 2). In total. 76 break- points (38 deletion cases) were assigned to different introns, 49 (64 ) in the distal part of the gene (introns 42-52). Nineteen of 20 DMD patients had deletions leading to a frameshift of the dystrophin transcripts. Both of the mutations described for BMD patients were in frame deletions. This result showed the correlation between clinical phenotype and genotype. The number of deleted exons was variable in 22 patients. Among 22 patients with deletions. 9 (41 ) had single exon deletions, 4 (18 ) had two exon deletions. 6 (27 ) had three and six exon 8UU-I ~i ~ ~ :- I i 1 j .......... .... = 3, bre.k°o~o,s 41 42 43 44 45 46 47 48 49 50 51 52 53 Intron NO Fig. 2. Distribution of 5' and 3' deletion breakpoints within introns along the dystrophin gene. Data include 49 deletion breakpoints in 24 DMD and 2 BMD patients.  P. Dinqer et al./ Brain & Derelopment 1996; 18:91-94 93 093 = o.8J • ~ 0,7 l ~ '~ '~ 0 6 = : )4 ¢ - "~ o 4 ~1~ II"-l~l:~ ll-- o o.3 ;t i: : ....... --d:?LJH i ..... : ;;_ ~;~ lk Countries, Fig. 3. Frequency of breakpoints occuring in introns 41-60 of the dystrophin gone in different countries. Data from different countries except Turkey were obtained from the study of Danieli et al. [20]. deletions, and 3 (14%) had four, five and eight exon deletions. 33% of 'single exon deletions' had "cxon 51' deletion. 4 DISCUSSION Various studies have demonstrated that a high percentage of patients (65%) with D/BMD have deletions within the dys- trophin gene. Deletions are shown by direct methods. Detection of deletion as a genetic fault in DMD families provides the opportunity for prenatal diagnosis with a theoretical 100ok accu- racy. We detected deletions in 58% of our patients with D/BMD by amplifying 15 exons of the dystrophin gene. Deletions were more frequent (26/38) in the central region of the dystrophin genc than in the 5' terminal region of the dystrophin genc (12/38). The frequencies and the distribution of deletions in the dystrophin gcne for DMD were similar between patients from different countrics [ 15-19]. In different populations, there is a variation in the frequencies of breakpoints in introns 41-60. In our study, we did not use primers extending beyond exon 53, and still compared our results with groups who have done exon screening between 41 and 60. However, this does not affect our results, because deletion break- points after cxon 53 are extremely rare (Fig. 3) (data obtained from the stud~ of Danieli et al. [20]). It was observed that our data are between those of France and Finland. There were some differences in polarity of deletion breaks for each intron between our population and other populations [20]. Intron 44 was involved mostly as a starting breakpoint region whereas intron 50 and intron 52 appeared to be involved as the ending breakpoint region (Fig. 2). 27¢7~ of 5' breakpoints were localized in intron 44 and 31% of them were localized in intron 45 and 50 in DMD patients. The 5' hreakpoints of 2 BMD patients were localized in introns 44 and 47. 35% of 3' breakpoints were localized in introns 50 and 52 in DMD patients. The 3' breakpoints of 2 BMD patients were localized in introns 48 and 49. Intron 44 was found not to bc involved as a 3' breakpoint and there was no 5' breakpoint in intron 43 in our population, whereas they appear to be involved as the 5' or 3' breakpoints in some cases in European countries studied [20]. As we mentioned betbre, the great majority of deletions in our population began distal to exon 44. The polarity of deletion breaks for intron 50 was different from that of European countries studied [20]. Intron 50 appeared tO he equally involved as the starting or ending breakpoint in our population (Fig. 2). We could predict the effect of deletions on the translational open reading frame (ORF) of the dystrophin rnRNA from the published sequences of the entire eDNA and of the intron/exon boundaries of main parts of the dyslrophin gene [8]. As predicted by Monaco ct al. [10], many studies [8,1 I] have demonstrated that the effect of deletions on the translational reading frame of dystrophin mRNA may account for the phenotypic differences between DMD and BMD rather than the size and k~cation of deletions. The detection of clinical phenotype especially will be very important in the cases which are not known DMD or BMD Our data, with one exception, agree with most of the studies [15,19]. One patient, who had an in frame deletion removing exons 48-49, showed a typical DMD phenotype. As a result, in 95~ of cases the predicted effect on the translational reading frame was concordant with clinical severity. The number of deleted exons was distributed heterogeneously in 22 patients whose deletion starting and ending points were known. 4lea of 22 patients had single exon deletions. 33'7; of single exon deletions had exon 51' deletion. Thc~,e results suggest that screening of all exons in lhe central region of Ihe gene would increase the frequency with which deletions are detected. ACKNOWI,EDGEMENTS ]'his stud'.' was supported by the Turkish Govcrment Planning Agency and the Turkish Child Neurology Associa- tion. Some deletion primers were gift,; fiom S. Abbs. Division of Medical and Molecular Genetics, Pcdialric Research Unit. (;uy's Hospital, l,ondon and from HGMP Resource Center. Clinical Research Centre. Cambridge, UK. We would like to thank Dr. Francesco Munloni fi~r crilical reattmg of our paper. REFERENCES I. Dan'as BT, Francke U. Normal human genomic restriction fragment patterns and polymorphisms revealed by hybridization with the entire dystrophin eDNA. Am ] Hunt Genet 1988: 43: 612-9. 2. Koenig M. Hoffinan EP, Berlelson C.J. Monaco AP. Feener C. Kunkel I,M Complete cloning of the Duchenne muscular dystrophy (DMD) eDNA and preliminary genomic organization of the I)MI) gene in normal and affected individuals. ('ell 1987: 50:509 17 3. Den Dunnen JT. Grootschohen PM, Bakker E. el al. Topograph',, ol the DMD gene: FIGE and eDNA analysis oJ 194 cases reveals 115 deletions and 13 duplications. Am ] H,m (h,net 1989: 45:835-3.7 4, Chamberlain .IS. Gibbs RA. Ranier JE. Nguyen PN. Ca~,key ("'F. Deletion screening of the DMD locus via muhiplex DNA amplifica- lion. Nucleic Acids Res 1988; 16: I 1141--56. 5. Chamberlain .JS. Gibbs RA. Ranier JE. Caskey CT. Muhiplex PCR for the diagnosis of DMD. In: Innis MA. Gelt:and DH. Sninski J. White T, eds. PCR protocols: ~ guide m method,~ and apidication~ San l)iego: Academic Press, 1990: 272-81. 6. Gibbs RA. Chamberlain JS. Caskcy CT. F'nnciples and applications for DNA amplification. In: Erlich HA. cd. PCR teclmoh(¢y. Nov.. York: Stockton Press. 1989:171-91 7. Abbs S, Yau S, Clark S. Malhew CG. Bobrow B. A convenient multiplex PCR system for the detection of dystmphin gene deletions: a comparative analysis with eDNA hybridization shows mistyping by both methods. J Med Genet 1991: 28: 304-I I. 8 Koenig M. Monaco AP, Kunkel LM. The complete sequence of dystrophin predicts a rod shaped cymskclctal protein. ('ell 1988: 53: 219-28  94 P. Dinqer et al./ Brain Deeehqmlent 1996; 18: 91.-94 9. Kocnig M, Beggs AH, Moyer M, et al. The molecular ba.~is for I)uchcmle versus Becket muscular dystrophy: Correlation of severity with type of deletions. Am J Hum Genet 1989: 45: 498-506. I0. Monaco AP, Bcrtclson C J, Liechti-Gallati S, Moscr H, Kunkel LM. An explanation for the phenotyping differences between patients bearing partial deletions of the Duchenne muscular dystrophy locus. (;emmtics 1988; 2: 90-5. II, Gillard EF, Chamberlain JS, Murphy EG, et al. Molecular and phenotypic analysis of patients with deletions with the deletion rich region of the Duchennc muscular dystrophy (1)MD) gene. Ant J Hum (;enet 1989: 45: 507-20. 12. Muntoni F, Gobbi P, Sewry C, ct al. Deletions in the 5 region of dystrophin and resulting phenotypes. J Med Genet 1994; 31: 843-7. 13. Maniatis T, Fritsch EF, Sambrook J. Molecular cloning: A htbora- tol'v mamml. 2nd cdn. New York: Cold Spring Harbor Laboratory, 1989. 14. Miller lVl. Dykes DD. Polesky HF. A simple salting out procedure for extracting DNA from hunlan nucleated cells. Nucleic Acids Res 1988: 16: 1215. 15. Claustres M, Tuffery S, Chevron MP, et al. Molecular deletion patterns in families from Southern France with Duchcnne/Bccker muscular dystrophies. Hunt Genet 1991; 8g: 179-84. 16. Florentin L. Mavrau A, Kekou K, Metaxou C. Deletion patterns of l)uchenne and Bccker muscular dystrophies in Greece. J Med Genet 1995: 32: 48-51. 17. hnoto N, Arinami T, Hamano K, et al. Topographic pattern of the rearrangement of the dystrophin gene in Japanese Duchennc muscu- lax dystrophy. Hunt Genet 1993; 92: 533-6. 18. Seydc SN, Slomski R, Rininsland F, Ellermcyer U, Kwiatkowska J, Reiss J. Molecular genetic analysis of 67 patients with Duchenne/Bcckcr muscular dystrophy. Hum Genet 1992: 90: 65-70. 19. Simard LR, Gingras F, Delvoye N. Vanesse M, Melanffon SB, lzabuda D. Deletions in the dystrophin gene: analysis of Duchennc and Becker muscular dystrophy patients in Quebec. Hum Genet 1992: 89: 419-24, 20. Danieli GA, Mioni F, Miillcr CR. Vitiello L. Mostacciuolo ML, Grimm T, Patterns of deletions of lhc dystrophin gene in different European populations, tlunt Genet 1993: 91: 342-6.
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