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Framshift deletion mechanisms in Egyptian Duchenne and Becker muscular dystrophy families. Mol

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Framshift deletion mechanisms in Egyptian Duchenne and Becker muscular dystrophy families. Mol
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   Mol. Cells , Vol. 18, No. 2, pp. 141-149 Frameshift Deletion Mechanisms in Egyptian Duchenne and Becker Muscular Dystrophy Families Elhawary Nasser A * , Rabah Mohamad Shawky 1 , and Nemat Hashem  Medical Genetics Center, Ain Shams University, Cairo, Egypt; 1  Department of Pediatrics and Genetics, Faculty of Medicine, AinShams University, Cairo, Egypt. (Received March 25, 2004; Accepted July 16, 2004) Partial gene deletion is the major type of mutation leading to Duchenne muscular dystrophy (DMD) and its mild allelic form, Becker muscular dystrophy (BMD). Amplification of the genomic DNAs of 152 unrelated dystrophin patients using multiple primers detected 78 (51.3%) probands with deletion mutations. We predicted the translational reading frame for all the deletions in Egyptian dystrophin males. The frameshift rule was confirmed positively ranging for 50 to 67% of the cases depending on the type of dis-ease. We discuss ways of accounting for some excep-tions from the frameshift hypothesis in the central and proximal regions. These explanations may help in de-veloping procedures for reducing the severity of dys-trophin phenotypes to restore the correct frame by disrupting the translational fidelity. Great efforts have been put into the development of effective ‘gene cor-rection’ procedures via  such intrinsic mechanisms.   In addition, we mapped regional difference in deletion mutation frequencies within the  DMD  gene locus be-tween the different Egyptian governorates.   There were no double deletions in the Egyptian dystrophin males.   Keywords: Dystrophin Gene; Egyptian Population; Frameshift Deletions; Multiplex PCR. Introduction The Duchenne and Becker muscular dystrophies (DMD/ BMD) are X-linked muscle diseases (1:3500 newborn males) typically caused by frame-shifting (DMD) or non-frame-shifting (BMD) mutations in the dystrophin-coding  DMD  gene (Hoffman et al. , 1987; 1988; Koenig et al. , * To whom correspondence should be addressed. Tel: 20-2-531-1741; Fax: 20-2-258-5577 E-mail: nasgenet@hotmail.com 1989; Monaco et al. , 1988). DMD patients suffer from lethal muscle degeneration (Behrman, 2000). The  DMD  gene spans over 3 Mb of genomic DNA and encodes a 14-kb mRNA transcript, made up of 79 exons, that translates into the 427 kDa cytoskeletal protein, dystrophin (Roberts, 1995). Dystrophin is an essential component of the dys-trophin-glycoprotein complex that maintains the mem-brane integrity of muscle fibers (Ervasti and Campbell, 1991; Koenig et al. , 1988; Yoshida et al. , 1990). A hypothesis known as the reading-frame hypothesis proposes that deletions that alter the reading frame of dystrophin mRNA produce no functional dystrophin and cause severe DMD, while in-frame deletions may produce partly-functional internally deleted dystrophin leading to the milder Becker disease (Monaco et al. , 1988). Al-though exceptions have been observed (Malhotra et al. , 1988; Thanh et al. , 1995; Winnard et al. , 1995), the read-ing frame rule has been confirmed in > 90% of cases. Intragenic deletions and duplications together account for over two thirds of mutations leading to DMD and BMD (den Dunnen et al. , 1989; Koenig et al. , 1989). Most can be detected by multiplex PCR (Beggs et al. , 1990; Chamberlain et al. , 1988) and are clustered in 2 high-frequency deletion regions (HFDRs), one in the 5 ′  (centromeric) portion of the gene, the other in the 3 ′  half of the gene (Baumbach et al. , 1989; Kim et al  ., 2002; Koenig et al. , 1989). A small proportion ranging from 0 to 6% of the mutations within the dystrophin gene involves duplications (Hu    et al. , 1990; Mendell   et al. , 2001). More than 200 dystrophin point mutations are known (http:// www.dmd.nl/). Previous studies of dystrophin deletion mutations in Egyptian males have not dealt with the frameshifting hypothesis (Abdel-Fattah et al. , 2003; Effat et al. , 2000). In the present paper, we present an analysis of excep-tions to the frameshift rule and their implications for dys-trophin in males. A plausible explanation of such excep-tions suggests how one might reduce disease severity via   MoleculesandCells © KSMCB 2004  142 Frameshift Deletion Mechanisms in BMD/DMD exon skipping (van Deutekom et al. , 2001) and restore the correct frame by interfering with translational fidelity (Wagner et al. , 2001). Based on this study, we can ac-count for many cases of at-risk carriers scanned with de-naturing high performance liquid chromatography (DHPLC) (in preparation). Materials and Methods Patients  We categorized patients as DMD if they were wheel-chair-bound at/before the age of 12 y, and as BMD if they were still ambulant at age 16. Patients were classified as intermediate (B/DMD) if they became wheelchair bound between the ages of 12 and 16 y (Dubowitz, 1990). Patients labeled as not deter-mined “ND” were too young to permit a definitive diagnosis, and are grouped separately (Table 1). Dystrophin patients were selected from more than 300 Egyptian dystrophin families (Hashem, 1982) registered in the database-records of the Medi-cal Genetics Center, AinShams University (ASMGC) (Abdel-Fattah et al. , 2003; Elsayed, 1998). Our sample contained 152 proband males. The age of onset of the B/DMD patients ranged from 1.5 to 21.5 y. Available dystrophin probands were sub- jected to muscle-strength testing, which included hand-held dynamometry and quantitative muscle testing (QMT) for nine muscle groups on one side of the body (unpublished data). DNA analyses   DNA samples were purified directly from pe-ripheral blood lymphocytes (DNA Isolation from Blood-Spin column, Qiagen), blood spotted on Guthrie cards (McCabe et al. , 1987) or buccal mucosa (Richards et al. , 1993). A 5 µ l volume of buccal cells typically sufficed for PCR amplification.   Multiplex PCR    Oligonucleotide primer sets were self-synthesized using a DNA synthesizer (ABI 392 Model). Routine multiplex PCR amplification was performed using primer sets flanking exons 4, 8, 12, 17, 19, 44, 45, 48, and 51 (Chamberlain et al. , 1990). We did not add DMSO as this resulted in a lower PCR  yield (Abdel-Fattah et al. , 2003). PCR amplicons were separated on 3% NuSieve agarose (BMA) (Fig. 1). Results For the sake of accuracy, we focused only on those pro-bands for whom specific clinical information was avail-able. The great majority of genetic defects in the  DMD  gene, the longest known so far, are large deletions involv-ing either major or minor hot spots. However, the  DMD  gene of 152 unrelated Egyptian dystrophin patients was amplified and we detected deletions in 78 of them (51.3%). The distribution of the dystrophin families in Egyptian governorates was 30%, 51%, and 19% in the Cairo zone, Lower and Upper Egypt, respectively. The much lower dystrophin deletion frequency in Upper Fig. 1.  Multiplex PCR of the human dystrophin gene electro-phoresed on a 3% NuSieve gel-ethidium dye. 1C; represented ‘normal control male’, MW is Φ 174/  Hae III size marker, cases 111, 112, and 115 showed missing bands due to exon deletions, and cases 110, 113, 116, 117 gave no deletions.   Egypt may be due to the stringent rules and habits among Upper Egyptians aimed at maintaining purity of srcin and prohibiting consanguinity with foreigners. Social life in Lower Egypt tends to be much more civilized, coopera-tive and cosmopolitan. The median onset age was 5, while that of presenting age was 12. Furthermore, a higher fre-quency of deletions was observed in familial cases (55%) than in sporadic ones (Table 1). Deletion analyses   We detected 18 deletions (23%) in the proximal HFDRs located within the region of the gene encoding the first 19 exons; six (33%) were deletions of individual exons, while the remaining 12 (67%) had dele-tions of exon clusters. We detected no single exon dele-tion confined to exon 17. We also observed that the low-est frequency of deletions (1.28%) affected exons 8, 12, 19, as single, and exons 4-12, 8-17, 12-44, 17-19 as gross exon deletions (Table 2). Sixty deletions (77%) were de-tected within the central hotspot consistent with the litera-ture (Baumbach et al. , 1989). 61.7%, of single deletion involving exon 51 were found in the central region of the  DMD  gene (Table 2).  Frameshift and in-frame mutations We predicted the translational reading frame of 78 deletions using the data-base in the Leiden Muscular Dystrophy website. Deletion of a DNA segment consisting of one or more exons and ending with a non-integral number of triplet codons should cause a shift in the reading frame. On the other hand, deletion of exon(s) containing an integral number of triplet codons should maintain the reading frame (Beggs et al. , 1991; Malhotra et al. , 1988). We predicted a shift of the reading frame due to exon(s) deletion in 25   DMDs, 7 intermediates, 1 BMD, and 11 “ND” cases. On the other hand, in-frame deletions were predicted in 14 DMDs, 7   Elhawary Nasser A et al  . 143 Table 1. Clinical data on 78 deletions in the hotspots of the dystrophin gene in 152 Egyptian patients. Age (years) Family  No. a   Phenotypes Age b  (years) Onset Mild c  Mod. d  Severe e  WCB f   F.H. g  98 BMD 16 05 05 -   145 BMD 26 18 18 22 -   175 h  BMD 27.5 21.5 21.5 22.5 + 4 DMD 12 09 09 10.5 11 + 11 i  DMD (D) 16 03 03 06 08 + 22NS DMD 15 05 05 09 +   30NS DMD 10.5 05 06 07 08 09.5 - 43  j  DMD 17 05 05 07 10 11 + 47 DMD 11 06 11 11 + 50 k  DMD 12 01.5 04 08.5 + 79 DMD 08 03 06 08 09 -   92 DMD 07 05 05 07 09 + 115 DMD 12 04 08 10 -   119 DMD 14 02 02 09 11 + 124 DMD 14.5 05 06 07 + 128 DMD 12 06 06 07 -   133 DMD 15 08 08 09 10 -   135 DMD 14 06 08.5 10 + 136 DMD 10 05 07 09 -   150 DMD 11.5 04 06 10 11 -   153 DMD 16 02.5 03.5 08 11 -   155 l  DMD 10.5 06 06 08 + 160 m  DMD 07 06 06 9.5 + 187 DMD 11 07 08 09 + 188 DMD 12 05 06 08 09 -   189 DMD 12 10 10 11 -   191 DMD 13 04 04 06 07 -   192 DMD 11 04 04 06 08 + 197 DMD 11.5 07 07 08 10 -   198 DMD 08 05 06 07 11 + 201 DMD 23 08 09 10 11 + 205 DMD 15 05 05 08 10 -   215 DMD 12 07 09 10 + 216 DMD 09 07 07 10.5 + 218 DMD 11 03 09 11 11.5 - 219 DMD 19 02 08.5 11 - 220 DMD 17 05 05 07 09 + 241 DMD 05 02 03 04 05 + 75NS DMD 12 02 05 06 11 + 89NS DMD 30 05.5 05.5 07 08 - 94NS DMD 18 05 07 09 11 + 98NS DMD 12 04 07 08 09 + 8 B/DMD 20 06 08 10 13 -   22 B/DMD 11 04 08 11 12 -   55NS B/DMD 16 08 10 12 + 84NS B/DMD 12 03 10 12 14 + 100 B/DMD 11 09 09 12 + 111 B/DMD 14 09 13 14 + 166 B/DMD 19 08 08 12 15 + 202 B/DMD 13 06 06 08 10 12 -   207 B/DMD 16 06 06 08 12 + 208 B/DMD 15 10 10 12 14 + 210 B/DMD 15 08 08 10 13 15 -   212 B/DMD 15 10 10 12 15 + 223 B/DMD 23 10 10 12 13 14 + 227 B/DMD 30 04 25 + (Continued)  144 Frameshift Deletion Mechanisms in BMD/DMD Age (years) Family  No. a   Phenotypes Age b  (years) Onset Mild c  Mod. d  Severe e  WCB f   F.H. g  2 ND 7 03 03 05 + 13NS ND 5 05 04.5 + 19NS ND 8 01.5 06 08 + 25NS ND 7 04 07 + 58NS ND 8.5 05.5 07 + 61NS ND 8 05 07 + 66NS ND 14 10 10 14 +   109NS ND 10 06 08 + 144 ND 9.5 03 03 05 + 149 ND 7 03 6.5 -   156 ND 7 05 05 -   159 ND 8 05 05 06 08 -   176 ND 9 04 04 06 08 -   211 ND 7.5 03 03 05 -   222 ND 10 05 08 10 - 225 ND 13 04 08 13 - 230 ND 6 01.5 06 - 234 ND 8 07 08 - 235 ND 8 06 08 10 - 240 ND 10 03.5 05 10 - 242 ND 7 04 07 07.5 08.5 - 243 ND 11 04 04 12 - a   These numbers only refer to one affected proband. b The age of initial examination. c Mild = fatigue and/or any detectable weakness including clumsiness, falling, abnormal gait, toe walking, and slow running in the absence of positive Gower’s signs. d  Moderate = positive Gower’s sign, difficulty with stairs, and/or waddling gait. e Severe = inability to rise without assistance and/or walking only with effort and/or severe wasting of muscles. f   WCB = wheelchair bound. g Family history (F.H.) considered (+) if there are more than one affected individuals in the family, and (-) if the affected male was a sporadic case. h A distant relative has delayed motor mental milestones. I The patient died at age 16 y of cardiomyopathy, and his DMD brother (WCB at 9 y) died at age 15 y of dilated cardiomyopathy.  j The patient had 3 DMD brothers died of dilated cardiomyopathy (not screened) at ages of 20, 21, and 22 y. k  Patient #50 has 2 affected DMD brothers (not screened); one moderate DMD phenotype presenting with a low IQ = 80% (onset 3 y, wad-dling gait and febrile convulsions, while the other, 4.5 years old, had delayed mental and motor milestones (IQ = 80%) and febrile convul-sions (onset 1.5 y). l An affected brother died at age of 23 y of cardiomyopathy (onset at 3.5 years with abnormal gait, weakness of lower limbs and pseudomus-cular hypertrophy) (not screened). m The proband has a 3-year-old brother, not diagnosed as a DMD patient until now, but affected with retinoblastoma and right eye replaced by an artificial one. intermediates, 2 BMDs, and 11 “ND” cases and we pre-dicted the deleted codons as well as the resulting protein sizes in these cases. Genotype-phenotype analyses Based on our molecular data and the available information on the patients’ pheno-types, 78 dystrophin   patients agreed to some extent with the reading frame model; namely 2/3 (67%) of the BMDs, 25/39 (64%) of the DMDs, and 7/14 (50%) of the B/DMDs, on the assumption that B/DMD patients make truncated dystrophin proteins (Table 3). 22 cases could not be explained by the frameshift rule: 1 BMD (#145), 14 DMDs (e.g. #75NS, #92, #98NS, #119) and 7   B/DMDs (e.g. #8, #84NS, #166). Discussion Most  DMD  gene therapy strategies are based on ‘gene addition’ by viral or non-viral delivery of dystrophin se-quences to muscle tissue. However, the alternative gene therapy approach aimed at  gene correction  instead of gene addition is gaining increasing attention (Bartlett et al. , 2000; Rando et al. , 2000). Two recent studies of in-version/deletion mechanisms at the  DMD  gene locus ex-amined loss of multiple exons together with inversion or     Elhawary Nasser A et al  . 145 Table 2.  Exon deletion frequencies in 78 Egyptian dystrophin patients. Exon(s) deleted No. of deletionsDeletion frequency (%)4 3 3.85 8 1 1.28 12 1 1.28 19 1 1.28 4-12 1 1.28 4-17 2 2.56 4-19 3 3.85 > 4 to end 2 2.56 8-17 1 1.28 12-44 1 1.28 17-19 1 1.28 44 4 5.13 45 10 12.82 48 6 7.69 51 17 21.80 45-48 4 5.13 45-51 11 14.10 48-51 8 10.26 insertion of fragments to improve prediction of out-of-frame shifting resulting in mild BMD phenotypes (Cagliani et al. , 2004; Wheway et al. , 2003). Our study is aimed essentially at developing gene correction procedures to restore the correct translational reading frame. We have accounted for some exceptions to the frameshift rule in both the proximal and central HFDRs of the dystrophin gene. Our approach was based on examining the exon-intron borders of the two exons that flank the deletion, and clas-sifying them into one of three types depending on the po-sition of their ends with respect to coding triplets. A dele-tion that juxtaposes two exons with two similar-type bor-ders maintains the reading frame, while those with differ-ent-type borders alter the reading frame, leading to early termination of translation (Fig. 2). This interpretation suggests an approach to treatment aimed at inducing exon skipping to reduce the severity of mutant phenotypes and restore the reading frame by disrupting translational fidel-ity. The most notable exception in our series was one BMD mutant (#145) that had an apparent frameshift deletion of exon 51 in the dystrophin. A similar case was previously observed in a BMD male (Baumbach et al. , 1989; Gillard et al. , 1989). In patient (#145) the frame would be re-stored if alteration of a normal splice-site consensus re-sulted in failure to include either exon that flanks the dele- tion. The reading frame could be restored by including in-tron sequences in the message, by activating a cryptic splice site or by generating a novel splice site within the A B C Fig. 2.  Diagramatic representation of the mechanisms of the out- and in-frame deletions in the central HFDRs of the dystro-phin gene. A) and B) represent +1 and +2 translational frameshifts, respectively, and C) shows how further deletion may correct the translational reading frame resulting from the first deletion. flanking introns. This approach has been followed in an interesting antisense-based system to induce skipping and reduce the severity of the phenotypes (Ginjaar et al.,  2000; van Deutekom et al. , 2001). Also, gentamicin, an aminoglycosides antibiotics, has been used to disrupt translational fidelity and to allow the incorporation of amino acids at stop codons (Barton-Davis et al. , 1999; Wagner et al. , 2001). 76.9% of the deletions examined were confined to the central region of dystrophin distal to exon 44. When the deletion was restricted to exon 45, the reading frame was distorted, resulting in a DMD phenotype. Deletion of exon 51, on the other hand, resulted in a frame-shift muta-tion leading to a severe DMD phenotype. Deletion of one or more additional exons (45-48, 48-51, and 45-51) in this region resulted in a BMD phenotype, suggesting that fur-ther deletions correct the translational reading frame re-sulting from the first deletion. Splicing deletions of exons 45-48 (e.g. #133 and #175), 45-51 (#136, #166, and #187) and 48-51 (e.g. #8, #92, #119, and #128) are examples of in-frame mutations (Table 3). The severe phenotype in patient WCB (#119), and other cases (e.g. #159, #176, #207) in which exon 48 was missing, involve an apparent in-frame deletion of the gene that might also be explained by altered splicing. The translational reading frame in those patients might be dis-rupted by inclusion of a cryptic splice site or by the gen-eration of a novel splice site in flanking introns. Both ex-
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