Hum Genet (1991) 87 : 353-360
9 Springer-Verlag1991
Genotype-phenotype correlation and germline mosaicism in DMD/BMD patients with deletions of the dystrophin gene A. E. Covone, M. Lerone, and G. Romeo Laboratorio di Genetica Molecolare,Istituto G. Gaslini, Largo G. Gaslini, 5, 1-16148 Genova, Italy Received August 29, 1990 / Revised January 11, 1991
Summary. The molecular analysis of 127 DMD/BMD patients showed that 73 of them (57%) had deletions in the dystrophin gene. Two different methods were used in this study: (a) hybridization of HindIII-digested genomic DNA with nine cDNA probes corresponding to the entire 14 kb eDNA of the DMD gene; and (b) simultaneous amplification of nine exons of the DMD gene (multiplex DNA amplification) by the polymerase chain reaction (PCR). When the deletion breakpoints of the intragenic deletions were analyzed with regard to their phenotypic consequences, nine patients were found to represent exceptions to the reading-frame hypothesis. Information regarding mental development was also available for 61 of the 73 deleted patients and for 34 of the 54 non-deleted ones. The proportion of mentally retarded patients was found to be similar in the two groups (deleted, 15%; non-deleted, 18%). Finally, in one family, a junction fragment present in the patient was not found in the peripheral blood DNA of the mother but was present in the sister, thus indicating germline mosaicism in the mother.
Introduction Duchenne and Becker muscular dystrophies (DMD and BMD) are allelic, X-linked, neuromuscular disorders mapping at Xp21. The incidence of DMD is 1/3 500 male births (Brooke et al. 1983; Emery 1987; Rowland 1988) while BMD is approximately 8-10 times less frequent. These two forms of myopathy have similar symptoms but can be discriminated on the basis of their clinical course. DMD is characterized by progressive muscular weakness with the majority of patients being confined to a wheelchair before the age of 12 (Emery 1987); few patients survive much beyond the age of 20. BMD is characterized by a slower rate of progression; most BMD patients can usually walk until the age of 16 and some of them even until the age of 40-50 or beyond (Walton et al. 1981). Offprint requests to." A. E. Covone
The DMD/BMD gene is the largest human gene isolated to date (Monaco and Kunkel 1988), spanning more than 2300 kb of genomic DNA, and is composed of at least 75 exons that encode a 14 kb transcript (Chamberlain et al. 1990), which is translated into a 427 kDa protein named dystrophin (Hoffman et al. 1987; Koenig et al. 1988). DMD or BMD results from mutations in this gene. Mutations that cause a total distruption of the protein product are responsible for the more severe phenotype (DMD), while mutations causing the production of a semi-functional protein are responsible for the milder form (BMD). It has been observed that about 60% of Duchenne and Becker muscular dystrophies are due to deletions in the dystrophin gene which, owing to the enormous size of the locus, are for the vast majority partial and intragenic, removing one or more exons. (Koenig et al. 1987, 1989; Darras et al. 1988b; Wapenaar et al. 1988; Baumbach et al. 1989; Lindl6f et al. 1989). Accurate prenatal diagnosis and carrier detection based on deletion analysis is possible by Southern blotting using eDNA or genomic probes and by PCR (Darras et al. 1988a; Cole et al. 1988; Speer et al. 1989; Chamberlain et al. 1988, 1989, 1990). Our study on 127 affected males represents a deletion mapping of the DMD gene. Southern and PCR analysis are the techniques involved in this study. We used the 14 kb cDNA for Southern analysis and nine sets of primers for PCR analysis. Nine exons, located in deletionprone regions, were simultaneously amplified. The detection rate of deletions, using the latter technique, was equal to 83% of the deletions detected by cDNA analysis. The strategy we used for a rapid identification of deletions was based on amplification of genomic DNA as a first step of the analysis and, if no deletion was observed, on analysis through Southern blotting using eDNA clones. In eight DMD and one BMD patients (12% of the deleted patients) the deletion pattern does not follow the reading-frame hypothesis postulated by Monaco et al. (1988). According to this model, the deletions which create a shift of the reading frame lead to the more severe form of muscular dystrophy (DMD). If the open
354 T a b l e 1. C l i n i c a l d e s c r i p t i o n of the 73 D M D / B M D p a t i e n t s b e a r i n g a d e l e t i o n in t he d y s t r o p h i n ge ne . A n e m p t y s pa c e i n d i c a t e s t h a t inform a t i o n is n o t a v a i l a b l e . P a t i e n t 50 is i n c l u d e d b e c a u s e he is an e x c e p t i o n to t he r e a d i n g f r a m e h y p o t h e s i s P a t i e n t no.
A g e at d i a g n o s i s
MR"
A g e (ye a rs ) A t confinem e n t to w h e e l c h a i rb
F/I d
Exons involvede
At time of s t u d y c
Cornm en ts f
BMD
5
18years
-
-
F
45 > 47
BMD
10
7years
-
-
11
F
46 > 48
+
BMD
10
3years
-
-
7
F
46 > 48
+
BMD
11
-
53
63
F
48 > 49
BMD
12
5years
-
-
15
F
45 > 48
BMD
19
14 y e a r s
-
-
BMD
22
12years
-
-
BMD
22
9years
-
BMD
24
4years
-
DMD
I
45 > 48
51
F
45 > 47
-
45
F
45 > 47
-
12
I
45 > 47
5
F
06 > 07
DMD
10
I
46 > 47
DMD
18
7years
-
-
11
I
DMD
28
8 years
-
12
16
I
DMD
31
6years
+
-
11
I
DMD
33
5 years
-
9
11
I
45
DMD
34
4years
-
9
d14
I
22 > 25
DMD
36
I
52
DMD
40
5years
-
8
d14
F
48 > 50
DMD
42
2 years
-
-
9
F
03 > 07
F
46 > 48
4years
-
10
14
I
(01) + 02
DMD
43
DMD
44
02 > 07 (48) 45 > 52
DMD
45
I
45 > 50
DMD
47
9 years
-
13
20
I
50
DMD
49
5years
-
14
24
F
44 > 52
DMD
50
DMD
51
5years
-
-
13
I
DMD
52
5years
+
14
DMD
54
4years
-
9
03 + 04
19
+
45 > 52
I
18 > 25
I
28 > 43
DMD
56
3 years
-
-
12
I
48 > 54
DMD
57
2years
-
-
6
I
03 > 35
DMD
58
F
49 > 52
DMD
60
-
-
6
I
51 > 53
*
DMD
62
18months
+
-
5
I
46 > 55
*
DMD
64
5years
+
-
9
I
47 > 52
*
DMD
65
F
45 > 52
DMD
68
DMD
71
4years
5years
-
-
9
I
05 > 36
F
(08)~
33 18
DMD
72
3 years
-
15
d20
I
08 >
DMD
73
6 years
+
10
18
F
45
DMD
74
5 years
-
-
11
I
51
DMD
75
4years
-
8
17
F
45 > 50
DMD
76
9years
-
10
20
I
48 ~ 52
DMD
77
7years
-
-
10
I
46 + 47
DMD
79
DMD
80
4years
+
-
DMD
82
3years
+
-
DMD
83
DMD
84
DMD
86
DMD
I
47 )
10
I
03 > 07
10
F
45 > 52
*
+*
52
F
49 ~ 52
I
40 ~ 42
10years
+
-
13
I
16
89
5 years
-
11
21
I
50
DMD
90
4years
-
10
18
F
46 + 47
DMD
91
8years
-
-
14
F
45 4- 46
+
355
Table 1 (continued) Patient no.
DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD
91 92 93 95 96 100 101 106 107 108 111 112 115 119 120 121 123 124 127 129 130 133 134 136
Age at diagnosis
5years 6 years 6years 9months 4years 3 years 6years 3 years 6years 4years 6 years 3 years 6years 2years 2 years 6years 5 years 18months 2 years 18months 9 months 9years 1 year 4years
MR a
+ + -
F/I d
Age (years) A t confinement to wheelchair b
A t time of study c
11 12 7 13 9 11 12 9 10 11 12 -
12 9 20 6 d23 14 14 19 19 14 17 5 12 3 3 7 20 5 4 3 1 18 11 14
F I I I I I I I F I F I I I F I I I I I F I F I
Exons involved e
45 46 45 42 44 44 48 43 03 48 45 (04) 48 45 46 46 49 46 (01) -47 52 47 43 47
Comments f
+ > > + > > > + > >
46 48 52 43 51 51 50 44 15 50
+ *
> > > + > > > > + > > > >
13 52 50 47 52 53 51 60 48 ? 51 51 50
*
*
+ +
* * + * * +* +
a Mental retardation: + , present; - , absent. According to the Stanford-Binet intelligence test, patients 80, 84 and 124 have a high degree of M R , patients 64, 73, 82, and 107, a moderate de~ree, and patients 31 and 52, a low degree A dash indicates that the patient can still walk c Figures preceded by 'd' indicate age at death for patients already deceased
a Familial/isolated Key: > more than two exons deleted; +, two deleted; 0 , breakpoints not yet clearly determined; ?, second breakpoints not yet identified f Key: + , deletion does not follow reading frame hypothesis; * patients too young to be classed definitely under B M D or D M D but in whom the clinical progression suggests D M D
r e a d i n g f l a m e ( O R F ) is m a i n t a i n e d b e y o n d t h e d e l e t i o n , the milder form of the disease (BMD) occurs. In the present study we also report a DMD family in which a deletion associated with a junction fragment was transmitted to more than one offspring by a woman who s h o w e d n o e v i d e n c e f o r m u t a t i o n i n h e r s o m a t i c cells. This indicates that the deletion must have occurred during mitosis in early germline proliferation, leading to germline mosaicism. This phenomenon has important implications for the counselling of DMD families. In a recent study on 28 families with a new DMD/BMD mut a t i o n B a k k e r e t al. ( 1 9 8 9 ) s u g g e s t t h e u s e o f a r e c u r r e n c e r i s k o f a p p r o x i m a t e l y 7 % ( 1 4 % if t h e h a p l o t y p e a s s o c i a t e d w i t h t h e m u t a t i o n is k n o w n ) i n c o u n s e l l i n g o f sisters and mothers of a sporadic DMD/BMD patient.
B M D (Brooke et al. 1983), such as evaluation of creatinine kinase (CK) levels and myopathic changes detectable on muscle biopsy. We classified our patients with regard to the clinical follow-up of the disease into: (a) D M D patients who became confined to wheelchairs between the ages of 7 and 15 years and (b) B M D who were still ambulatory without support at age 15 (Hoffman et al. 1988). Nevertheless, some of our patients were too young to be classified as D M D or B M D (Table 1). All samples were analyzed by Southern analysis using the six c D N A clones of the D M D gene (Koenig et al. 1987). High molecular weight genomic D N A was extracted from 20 ml of peripheral blood according to standard laboratory procedures. The D N A samples were digested with two restriction enzymes (HindIII, PstI) to increase the possibility of detecting RFLPs or junction fragments. Electrophoresis, Southern blotting and hybridization were carried out using established procedures (Church and Gilbert 1984). The c D N A probes used for hybridization were nine contiguous segments of the dystrophin c D N A as reported by Koenig et al. (1987): 1-2a, 2 b - 3 , 4 - 5 a , 5 b - 6 , 7-8, 9a, 9-10, 11, 12-14. The cloned c D N A fragments were released from the vectors by digestion with E c o R I or EcoRI-HindIII. Probes 5 b 6 and 7 were obtained by EcoRI + HincII digestion of clone 5 b - 7 . Probes 9-10, 11 and 12-14 were obtained by EcoRI + HinclI digestion of clone 9-14. Probe 9a corresponds to the first 3 0 0 b p of probe 9-10 (EcoRI-PstI fragment). This is a subclone of the 9-10
Materials and methods The 127 D M D / B M D patients analyzed in our laboratory were diagnosed using standard clinical diagnostic criteria for D M D and
356
clone and was used in two separate hybridizations to distinguish the two HindIII fragments of I kb revealed by the original clone. In this way, smaller cDNA fragments were used as probes for separate hybridizations of genomic DNA thus resolving comigrating fragments. The hybridization probes were separated in low melting point agarose gels (LMP Agarose, IBI) and then radiolabeled (Feinberg and Vogelstein 1983, 1984). The PCR analysis was carried out using primer kits, made available by Drs. J. S. Chamberlain and T. Caskey, containing nine sets of primers for the multiplex amplification, and following established reaction conditions (Chamberlain et al. 1988, 1989).
c D N A probes (see Materials and methods). Of the 39 PCR non-deleted samples, 5 were subsequently found deleted in those regions which do not amplify in the multiplex reaction. The extension of the 73 deletions encompassing the D M D gene is shown in Fig. 1. In five D M D patients, c D N A analysis revealed a deletion associated with a junction fragment. In these cases D N A samples from the patients and the other family members were digested with the following restriction enzymes: HindlII, PstI, EcoRI, TaqI and BglII in order to discriminate between a pattern given by a junction fragment (which should be present with most of the restriction enzymes) and a rare D N A variant. In one of these families ( D M D 34) D N A samples from the proband, his father, mother and sister were analyzed. The proband showed a deletion of the 20 kb HindlII band (exons 22 to 25) and an additional HindlII band of about 14 kb when hybridized with the 4 - 5 a c D N A probe (data not shown). In this patient, deleted and junction fragments were also observed with PstI (Fig. 2, lane 4) and EcoRI, TaqI, BgllI digests (data not shown). While the sister of this patient (Fig. 2, lane 3) shows the same additional band as the proband, neither parent has such a band. Therefore, the mutation which gives rise to the junction fragment must be present in the germline of the mother. This junction fragment was generated by deletion of one or
Results Using the c D N A probes and HindIII-digested genomic D N A , we detected 73 deletions in 127 D M D / B M D patients (57%). In most cases the PstI digests were also analyzed to confirm the results obtained with HindIII digestion. A subset of 6 4 D M D / B M D patients was analyzed using P C R as a first screening for the detection of deletions. The 25 deletions thus found (39%) were subsequently fully characterized by c D N A hybridization. The genomic D N A samples which did not show any deletion by P C R analysis were subjected to further screening, initially with 4 - 5 a , 5 b - 6 , 9a and 9 - 1 0 c D N A probes and then, if no deletion was found, with the remaining b
a
c
d
e
2.4
2.55 6.7 12 2.8
lO
5b-6
4-5a
2b-3
I
l-2a I
60 58+59 57 56 55 54 53 52 51 50 49 48 47 46 45 44 43 42 40+41 38+39 37 36 35 34 30>33 29 28 26+27 22>25 21 20 19 18 17 16 14+15 13 12 i0+ii 8+9 7 6 5 4 3 2 1
3.5 6.0 8.8 1.0 2.3 8.3 7.8+1.0 7.0 3.1 3.7 1.6 1.2+3.9 I0 1.5 0.5 4.1 ii 4.2 6.2 6.1 1.5 1.3 0.4 1.8 18 Ii 4.7 5.2 20 12 7.3 3.0 12 1.7 6.0 2.7 6.6 4.0 10.5 7.5 4.6 8.0 3.1 8.5 4.2 3.25 3.2
3 3
3 2 3 1 3 1 3 3 3
2
3
3
2
l
3 2 3 2 3 3 3 3 3 3 3
1 3 1 3 2 3 3 3 3 2 3 1 2 3 3 3 3 1
I*
Fig. 1. Extent of the deletions in the dystrophin gene observed in 73 DMD/BMD patients by cDNA and PCR analysis: cDNA probes used for Southern analysis, numbered 1-10 from the 5' end to the 3' end of the 14 kb dystrophin cDNA. Probes 11 and 12-14 are not shown because we did not observe any deletion in that region of the gene (a); exon numbers (b); exon-containing HindlII genomic fragments (sizes in kb) (c); exon border type according to Koenig et al. (1989) (d); and extent of the deletions observed in 73 DMD/BMD patients (e). Thick bars denote BMD patients; thin bars denote DMD patients. Squares (m) indicate the deletions which are exceptions to the reading-frame hypothesis. Asterisks (*) indicate deletions that are apparently exceptions to the reading-frame hypothesis but are associated with a junction fragment. Thin bars with free ends indicate that the breakpoint/s of the deletions concerned are not yet clear
357 patient was digested with PstI (Fig. 3, lane 3), TaqI and B g l l I (data not shown). Since this junction fragment was
Fig. 2. The restriction fragment pattern of PstI in family DMD 34 following hybridization with the cDNA fragment 4-5a. In this family the affected boy (lane 4) shows a deletion and an anomalous size band (arrow). A band of the same size is observed in the PstI pattern of his sister (lane 3) but is absent from the blood DNA of his mother (lane 1). In lane 2 the PstI pattern of the father of the patient is shown
Fig. 3. The restriction fragment pattern of HindIII and PstI of family DMD 52 following hybridization with the cDNA fragment 45a: lanes 3 and 4 contain the PstI and HindIII digests, respectively, of the affected boy. This deletion involves the 20kb, 12kb and 7.3 kb bands and is associated with a junction fragment of approximately 6 kb in the HindIII pattern (lower arrow, lane 4). A deletion and a junction fragment of approximately 23 kb (upper arrow, lane 3) are also observed in the PstI pattern of the affected boy. In lanes 5 and 6 the DNA from the mother of the patient shows normal Psd and HindIII patterns. The same result is evident for the PstI and HindIII digests of the father shown in lanes i and 2
m o r e exons (exons 22-25) corresponding to the 2 0 k b H i n d l I I band. In another family, D M D 52, D N A samples from the patient and his parents were analyzed. This patient shows a deletion of the 20 kb, 12 kb and 7.3 kb H i n d l I I bands, and the presence of an additional band of about 6 kb, when hybridized with the 4 - 5 a c D N A p r o b e (Fig. 3, lane 4). F u r t h e r m o r e , this patient showed a deletion of the 3.0 kb and 12 kb bands when hybridized with probe 2 b - 3 . The deletion and the presence of an extra band, of about 23 kb, were also observed when the D N A of this
not present in the D N A of the parents, this indicates that the deletion observed in the affected male is a new mutation. In this patient, the deletion involves exons 18-21 and probably m o r e than one exon of the 22-25 group. E v e n in this case, the junction fragment was generated by deletion of some of the exons of the 22-25 group. T h r e e other D M D patients ( D M D 57, D M D 87, D M D 134) showed a deletion associated with a junction fragm e n t (data not shown). Finally, prenatal diagnosis was p e r f o r m e d on 20 male fetuses at risk for D M D / B M D (caused by a deletion previously identified) using c D N A analysis or P C R amplification. Deletions were found in 6 out of the 12 fetuses analyzed with c D N A probes, and the other 6 were normal. P C R analysis was used for 8 prenatal diagnoses, and 3 fetuses were found to bear a deletion, 5 being diagnosed as normal.
Discussion In the present study, screening of deletions was carried out by c D N A analysis and multiplex P C R amplification. Using the former approach, 57% of the patients were found to be deleted, which is comparable to the detection rates reported by other laboratories (Forrest et al. 1988; Koenig et al. 1989; den D u n n e n et al. 1989). T h e P C R analysis, used as a first approach in 64 patients, led to the identification of 25 deletions (39%). This multiplex P C R allows simultaneous amplification of nine exons, particularly of those which have the highest frequency of deletion breakpoints. In our study it was possible to identify 83% of the deletions that can be revealed by c D N A analysis, which is in good a g r e e m e n t with the 90% reported by Chamberlain et al. (1989). The extension of the deletions identified by P C R was confirmed using c D N A analysis, which was also used to further investigate those patients who did not show any deletion by the P C R method. Five of these patients showed deletions in the gene on further c D N A analysis. T h e region corresponding to probes 11 and 12-14 was not deleted in any of our patients. Out of the 73 deletions, 53 (71%) encompassed the central high frequency deletion region ( H F D R ) of the gene, located approximately 1200kb from the p r o m o t e r , and 13 (17.5%) encompassed the proximal H F D R located approximately 500 kb from the 5' end of the gene. As reported in other studies, m o r e than 60% of all the intragenic breakpoints are located in this limited n u m b e r of exons. On the other hand, the location of the second breakpoint of these deletions is widely variable. The possibility of identifying the molecular basis of the phenotypic differences in D M D / B M D patients has been investigated by m a n y authors. A first study, by Monaco et al. (1988), suggested an association between the effect that an intragenic deletion would have on the translational reading f r a m e of the m R N A and the clinical severity of the disease. Although no apparent correlation was observed between the location and size of de-
358 letions with the clinical severity of the disease, more recent studies on the predicted structure of the dystrophin molecule show that in-frame deletions involving the Nterminal "actin binding" domain of the protein (including up to 1400 aminoacids or exons 1 to 34) usually lead to the milder form of muscular dystrophy (BMD). Furthermore, in-frame deletions involving exons 31-44 are predicted to lead to asymptomatic individuals (Koenig et al. 1989). This hypothesis is based on the observation that no BMD deletion starting in this very large intron (160-180 kb) between exons 44 and 45 and extending towards exon 31 have yet been found. On the contrary, the deletion breakpoints occurring in this intron are highly frequent in D M D patients bearing out-of-frame deletions extending between exon 44 and exon 52 (Koenig et al. 1989; den D u n n e n et al. 1989; Gilgenkrantz et al. 1989). Another very large intron (110 kb) is located between exons 7 and 8 and is also very frequently involved in deletion breakpoints. Furthermore, deletions involving the cystein-rich domain and the C-terminal domain (exons 54-58) lead to the production of a non-functional or unstable protein and, therefore, to a D M D phenotype. This region is thought to be essential for the correct function of the dystrophin molecule because it is highly conserved during evolution (Lemaire et al. 1988). Interestingly, deletions in these regions are extremely rare. Exceptions to the reading-frame hypothesis (Malhotra et al. 1988; Koenig et al. 1989; Baumbach et al. 1989; Gillard et al. 1989) were present in 9 out of the 73 patients with deletions (12%) analyzed in this study (Fig. 1, Table 1). Eight D M D patients were found to bear inframe deletions. In four D M D patients (patient 106 with a deletion of exons 43 and 44; patient 107 with a deletion of exons 3-15; patient 123 with a deletion spanning from exon 49 to 53, and patient 133 with a deletion of exons 47-51), despite the maintained reading frame following the deletion event, the clinical course of the disease seems to be that of a typical D M D case. An explanation for this could be that the exon/s involved in these deletions might be critical for the function of the protein. Two brothers (DMD 91) bearing an in-frame deletion of exons 45 and 46 show a slightly milder form of the disease; at 12 and 14 years of age they can still walk with the aid of calipers. Therefore, despite an early onset, the clinical course in these patients seems to be less severe than that in a typical DMD case. In the young DMD patients, 68 (deletions of exons 5-36) and 129 (deletion of exons 47 and 48), the deletions do not cause a frame shift in the transcript. In these patients the future clinical progression of the disease might be less severe than in a typical D M D case. A deletion of exons 46-48, causing a shift of the reading frame, occurred in two BMD brothers (BMD 10) whose clinical features do not indicate increased severity of the disease. These two B M D patients are still too young (7 and 11 years of age) to show a correlation between the type of deletion and the severity of the disease. Unfortunately, no accurate clinical data are available for the D M D patient (50) whose deletion of exons 3 and 4 does not cause a shift of the reading frame, and he, therefore, is another exception to the reading-frame rule. Two of these exceptions, namely D M D patients 68 and 91, have
already been reported in a previous paper (Koenig et al. 1989), while the remaining seven are not similar to any of those described in previous studies (Malhotra et al. 1988; Baumbach et al. 1989; Gillard et al. 1989). Malhotra and collaborators (1988) were the first to observe that 6 patients with BMD and 5 with severe BMD, out of 29 total D M D / B M D patients, bore a deletion of exons 3-7 that created a shift in the O R F if exon 2 was spliced to exon 8 in the mRNA. These authors hypothesized three possible mechanisms to explain these exceptions to the reading-frame hypothesis: (1) differential splicing (Feener et al. 1989) which could mask the frame-shift mutations creating an m R N A with an in-frame deletion thereby resulting in the production of a partially functional protein (e.g., splicing of exon 2 to exon 9 or of exon 1 to exon 8); (2) reinitiation of protein synthesis at the next potential initiation A T G codon which is present in-frame in exon 8; (3) reinitiation of transcription from a second promoter downstream from the deletion in the very large intron ( l l 0 k b ) between exons 7 and 8. In this way a partially functional protein could be generated in these patients, leading to a mild form of the disease. In three of our patients (DMD 57, DMD 86 and DMD 134) the deletions do not seem to follow the readingframe hypothesis, but on the other hand, a junction fragment is present in each case. These patients cannot be considered as exceptions because it is not possible to identify the deletion borders by this analysis. In family D M D 34 the detection of a junction fragment in association with the deletion observed in the D M D patient was very useful for the identification of carriers. The presence of this junction fragment in the sister of the patient leads to the conclusion that she is a carrier of the same deletion as observed in the patient. Furthermore, since their mother did not show the junction fragment in her blood cells, whereas the same deletion occurred in both siblings, we conclude that the mutation must have occurred in a germline cell clone of the mother, as already previously hypothesized for other pedigrees (Romeo et al. 1986; Bakker et al. 1987; Darras et al. 1987, 1988b; Hall 1988). Mental retardation occurred in 9 (15%) of the 61 patients with deletions for whom this type of information was available (see Table 1). The two D M D patients (31 and 82) have the same type of deletion, comprising exons 45-52. Three additional D M D patients showing mental retardation have overlapping deletions: patient 62 shows a deletion of exons 46-55; patient 124 bears a deletion involving exons 46-51; and patient 64 has a deletion of exons 47-52. Interestingly, all these five D M D patients with mental retardation lack exons 47-51 located in the central H F D R of the gene. Two other D M D patients showing mental retardation have a deletion in this region: patient 73 shows a deletion of exon 45 only, while the deletion in patient 84 involves exons 40-42. The remaining two mentally retarded DMD patients show overlapping deletions involving the proximal H F D R of the dystrophin gene and, in particular, patient 80 has a deletion of exons 3-7, while patient 107 has an in-frame deletion spanning exons 3-5. Clinical information about mental retardation was available in 34 out of the 54 non-
359 d e l e t e d p a t i e n t s , a n d 6 ( 1 8 % ) o f t h e s e w e r e m e n t a l l y retarded. The proportion of patients showing mental retardation is a l m o s t the s a m e in t h e two g r o u p s (with a n d witho u t d e l e t i o n s ) . H o w e v e r , in five o f t h e n i n e d e l e t e d p a tients in w h o m m e n t a l r e t a r d a t i o n o c c u r r e d t h e d e l e t i o n i n v o l v e d e x o n s c o n t a i n e d in p r o b e 8. S i m i l a r d a t a w e r e p r e s e n t e d b y Lindl/Sf et al. (1989), w h o f o u n d t h a t six out of seven mentally retarded DMD patients bore overl a p p i n g d e l e t i o n s in p r o b e 8, all o f t h e m i n v o l v i n g e x o n 50. T h e s e a u t h o r s suggest t h a t this r e g i o n m i g h t b e imp o r t a n t in n o r m a l m e n t a l d e v e l o p m e n t in m a l e s . N e v e r theless, m o r e r e c e n t findings r e g a r d i n g i n v o l v e m e n t o f a s e c o n d p r o m o t e r r e g i o n in t h e p r o d u c t i o n o f t h e dyst r o p h i n p r e s e n t in b r a i n ( b r a i n p r o m o t e r ) , l o c a t e d 5' to t h e m u s c u l a r p r o m o t e r , i n d i c a t e t h a t this r e g i o n m i g h t b e r e s p o n s i b l e f o r m e n t a l r e t a r d a t i o n (F. M. B o y c e , A . H . B e g g s , C. F e h n e r , a n d L. M. K u n k e l , in p r e p a r a t i o n ) . F u r t h e r studies o n g e n o t y p e - p h e n o t y p e c o r r e l a t i o n s in D M D p a t i e n t s s h o w i n g M R m i g h t h e l p in clarifying this point.
Acknowledgements. We thank Drs. M. Koenig and A.P. Monaco for helpful discussions and critical reading of the manuscript and Messrs. F. Caroli, I. Giambarrasi and M. Bertorello and Ms. A. Cereseto for technical assistance. We also thank the many physicians who helped in the collection of blood samples and clinical information. This work was carried out with funds from the Progetto Finalizzato Ingegneria Genetica of the Italian National Research Council (CNR) and from the Italian Union against Muscular Dystrophy (U.I.L.D.M.).
References Bakker E, Van Broeckhoven C, Bonten EJ, Van de Vooren MJ, Veenema H, Van Hul W, VanOmmen GJB, Vandenberghe A, Pearson PL (1987) Germline mosaieism and Duchenne muscular dystrophy mutations. Nature 329 : 554-558 Bakker E, Veenema H, Den Dunnen JT, Van Broeckhoven C, Grootscholten PM, Bonten E J, Van Ommen GJB, Pearson PL (1989) Germinal mosaicism increases the recurrence risk for 'new' Duchenne muscular dystrophy mutations. J Med Genet 26 : 553-559 Baumbach LL, Chamberlain JS, Ward PA, Farwell NJ, Caskey CT (1989) Molecular and clinical correlations of deletions leading to Duchenne and Becket muscular dystrophies. Neurology 39 : 465-474 Brooke MH, Fenichel GM, Griggs RC, Mendell JR, Moxley R, Miller JP, Province MA, and the CIDD Group (1983) Clinical investigations in Duchenne muscular dystrophy. II. Determination of the "power" of therapeutic trials based on the natural history. Muscle Nerve 6:91-103 Chamberlain JS, Caskey CT (1990) Duchenne muscular dystrophy. Curr Neurol 10 : 65-103 Chamberlain JS, Gibbs RA, Ranier JE, Nguyen PN, Caskey CT (1988) Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res 16 : 11141-11156 Chamberlain JS, Gibbs RA, Ranier JE, Caskey CT (1989) Multiplex PCR for diagnosis of Duchenne muscular dystrophy. In: Innis M, Gelfand D, Sninski J, White T (eds) PCR: protocols and applications - a laboratory manual. Academic Press, New York, pp 272-281 Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci USA 81 : 1991-1995
Cole CG, Coyne A, Hart KA, Sheridan R, Walker A, Johnson L, Hodgson S, Bobrow M (1988) Prenatal testing for Duehenne and Becket muscular dystrophy. Lancet I : 262-265 Darras BT, Francke U (1987) A partial deletion of the muscular dystrophy gene transmitted twice by an unaffected male. Nature 329 : 556-558 Darras BT, Koenig M, Kunkel LM, Francke U (1988a) Direct method for prenatal diagnosis and carrier detection in Duchenne/ Becket muscular dystrophy using the entire dystrophin eDNA. Am J Med Genet 29 : 713-726 Darras BT, Blattner P, Harper JF, Spiro AJ, Alter S, Franeke U (1988b) Intragenic deletions in 21 Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) families studied with the dystrophin eDNA: location of breakpoints on HindIII and BglII exon-containing fragment maps, meiotic and mitotic origin of the mutations. Am J Hum Genet 43 : 620-629 Den Dunnen JT, Grootseholten PM, Bakker E, Blonden LAJ, Ginjaar HB, Wapenaar MC, Van Paassen HMB, Van Broeckhoven C, Pearson PL, Van Ommen GJB (1989) Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am J Hum Genet 45 : 835-847 Emery A E H (1987) Duchenne muscular dystrophy. (Oxford Monographs on Medical Genetics, no 15). Oxford University Press, Oxford, pp 71-91 Feener CA, Koenig M, Kunkel LM (1989) Alternative splicing of human dystrophin mRNA generates isoforms at the carboxy terminus. Nature 338:509-511 Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13 Feinberg AP, Vogelstein B (1984) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Addendum. Anal Biochem 137 9267-268 Forrest SM, Cross GS, Flint T, Speer A, Robson KJH, Davies K (1988) Further studies of gene deletions that cause Duchenne and Becker muscular dystrophies. Genomics 2:109-114 Gilgenkrantz H, Chelly J, Lambert M, Recan D, Barbot JC, Van Ommen GJB, Kaplan JC (1989) Analysis of molecular deletions with eDNA probes in patients with Duehenne and Becket muscular dystrophies. Genomics 5 : 574-580 Gillard EF, Chamberlain JS, Murphy EG, Duff CL, Smith B, Burghes AHM, Thompson MW, Sutherland J, Oss I, Bodrug SE, Klamut HJ, Ray PN, Worton RG (1989 Molecular and phenotypic analysis of patients with deletions within the deletion-rich region of the Duchenne muscular dystrophy (DMD) gene. Am J Hum Genet 45 : 507-520 Hall JG (1988) Review and hypotheses: somatic mosaicism: observations related to clinical genetics. Am J Hum Genet 43 : 355363 Hoffman EP, Brown RH Jr, Kunkel LM (1987) Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51 : 919-928 Hoffman EP, Fishbeck KH, Brown RH, Johnson M, Medori R, Loike JD, Harris JB, Waterston R, Brooke M, Specht L, Kupsky W, Chamberlain J, Caskey CT, Shapiro F, Kunkel LM (1988) Dystrophin quality and quantity determines the clinical severity of Duchenne Becker muscular dystrophies. N Engl J Med 318 : 1363-1368 Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener C, Kunkel LM (1987) Complete cloning of the Duchenne muscular dystrophy (DMD) eDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 50 : 509-517 Koenig M, Monaco AP, Kunkel LM (1988) The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell 53 : 219-228 Koenig M, Beggs AH, Moyer M, Scherpf S, Heindrich K, Bettecken T, Meng G, Muller CR, Lindli3f M, K~i~iriainen H, Chapelle A de la, Kiuru A, Savontaus M-L, Gilgenkrantz H, Recan D, Chelly J, Kaplan J-C, Covone AE, Archidiacono N,
360 Romeo G, Liechti-Gallati S, Schneider V, Braga S, Moser H, Darras BT, Murphy P, Francke U, Chen D, Morgan G, Denton M, Greenberg CR, Wrogemann K, Blonden LAJ, Van Paassen HMB, Van Ommen GJB, Kunkel LM (1989) The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am J Hum Genet 45 : 498-506 Lemaire C, Heilig R, Mandel J-L (1988) The chicken dystrophin cDNA: striking conservation of the C-terminal coding and 3' untranslated regions between man and chicken. EMBO J 7: 4157-4162 Lindl6f M, Kiuru A, K~ifiri~iinenH, Kalimo H, Lang H, Pihko H, Rapola J, Somer H, Somer M, Savontaus M-L, Capelle A de la (1989) Gene deletions in X-linked muscular dystrophy. Am J Hum Genet 44: 496-503 Malhotra SB, Hart KA, Klamut HJ, Thomas NST, Bodrug SE, Burghes AHM, Bobrow M, Harper PS, Thompson MW, Ray PN, Worton RG (1988) Frame-shift deletions in patients with Duchenne and Becker muscular dystrophy. Science 242:755759 Monaco AP, Kunkel LM (1988) Cloning of the Duchenne/Becker muscular dystrophy locus. Adv Hum Genet 17 : 61-98 Monaco AP, Bertelson CJ, Liechti-Gallati S, Moser H, Kunkel LM (1988) An explanation for the phenotypic differences be-
tween patients bearing partial deletions of the DMD locus. Genomics 2 : 90-95 Romeo G, Roncuzzi L, Ferlini A, Pirozzi A, Nobile C (1986) New mutations in Duchenne and Becker muscular dystrophies (abstract). Am J Hum Genet 39 [Suppl] : 99 Rowland LP (1988) Clinical concepts of Duchenne muscular dystrophy. The impact of molecular genetics. Brain 111:479-495 Speer A, Spiegler AWJ, Hanke R, Grade K, Giertler U, Schieck J, Forrest S, Davies KE, Neumann R, Bollmann R, Bommer C, Sommer D, Coutelle C (1989) Possibilities and limitation of prenatal diagnosis and carrier determination for Duchenne and Becker muscular dystrophy using cDNA probes. J Med Genet 26 : 1-5 Walton JN, Gardner-Medwin D (1981) In: Walton J (ed) Disorders of voluntary muscle. Churchill Livingstone, Edinburgh, pp 481-524 Wapenaar MC, Kievits T, Hart KA, Abbs S, Blonden LAJ, Den Dunnen JT, Grootscholten PM, Bakker E, Verellen-Dumoulin C, Bobrow M, Van Ommen GJB, Pearson PL (1988) A deletion hot spot in the Duchenne muscular dystrophy gene. Genomics 2 : 101-108
9 Springer-Verlag1991
Genotype-phenotype correlation and germline mosaicism in DMD/BMD patients with deletions of the dystrophin gene A. E. Covone, M. Lerone, and G. Romeo Laboratorio di Genetica Molecolare,Istituto G. Gaslini, Largo G. Gaslini, 5, 1-16148 Genova, Italy Received August 29, 1990 / Revised January 11, 1991
Summary. The molecular analysis of 127 DMD/BMD patients showed that 73 of them (57%) had deletions in the dystrophin gene. Two different methods were used in this study: (a) hybridization of HindIII-digested genomic DNA with nine cDNA probes corresponding to the entire 14 kb eDNA of the DMD gene; and (b) simultaneous amplification of nine exons of the DMD gene (multiplex DNA amplification) by the polymerase chain reaction (PCR). When the deletion breakpoints of the intragenic deletions were analyzed with regard to their phenotypic consequences, nine patients were found to represent exceptions to the reading-frame hypothesis. Information regarding mental development was also available for 61 of the 73 deleted patients and for 34 of the 54 non-deleted ones. The proportion of mentally retarded patients was found to be similar in the two groups (deleted, 15%; non-deleted, 18%). Finally, in one family, a junction fragment present in the patient was not found in the peripheral blood DNA of the mother but was present in the sister, thus indicating germline mosaicism in the mother.
Introduction Duchenne and Becker muscular dystrophies (DMD and BMD) are allelic, X-linked, neuromuscular disorders mapping at Xp21. The incidence of DMD is 1/3 500 male births (Brooke et al. 1983; Emery 1987; Rowland 1988) while BMD is approximately 8-10 times less frequent. These two forms of myopathy have similar symptoms but can be discriminated on the basis of their clinical course. DMD is characterized by progressive muscular weakness with the majority of patients being confined to a wheelchair before the age of 12 (Emery 1987); few patients survive much beyond the age of 20. BMD is characterized by a slower rate of progression; most BMD patients can usually walk until the age of 16 and some of them even until the age of 40-50 or beyond (Walton et al. 1981). Offprint requests to." A. E. Covone
The DMD/BMD gene is the largest human gene isolated to date (Monaco and Kunkel 1988), spanning more than 2300 kb of genomic DNA, and is composed of at least 75 exons that encode a 14 kb transcript (Chamberlain et al. 1990), which is translated into a 427 kDa protein named dystrophin (Hoffman et al. 1987; Koenig et al. 1988). DMD or BMD results from mutations in this gene. Mutations that cause a total distruption of the protein product are responsible for the more severe phenotype (DMD), while mutations causing the production of a semi-functional protein are responsible for the milder form (BMD). It has been observed that about 60% of Duchenne and Becker muscular dystrophies are due to deletions in the dystrophin gene which, owing to the enormous size of the locus, are for the vast majority partial and intragenic, removing one or more exons. (Koenig et al. 1987, 1989; Darras et al. 1988b; Wapenaar et al. 1988; Baumbach et al. 1989; Lindl6f et al. 1989). Accurate prenatal diagnosis and carrier detection based on deletion analysis is possible by Southern blotting using eDNA or genomic probes and by PCR (Darras et al. 1988a; Cole et al. 1988; Speer et al. 1989; Chamberlain et al. 1988, 1989, 1990). Our study on 127 affected males represents a deletion mapping of the DMD gene. Southern and PCR analysis are the techniques involved in this study. We used the 14 kb cDNA for Southern analysis and nine sets of primers for PCR analysis. Nine exons, located in deletionprone regions, were simultaneously amplified. The detection rate of deletions, using the latter technique, was equal to 83% of the deletions detected by cDNA analysis. The strategy we used for a rapid identification of deletions was based on amplification of genomic DNA as a first step of the analysis and, if no deletion was observed, on analysis through Southern blotting using eDNA clones. In eight DMD and one BMD patients (12% of the deleted patients) the deletion pattern does not follow the reading-frame hypothesis postulated by Monaco et al. (1988). According to this model, the deletions which create a shift of the reading frame lead to the more severe form of muscular dystrophy (DMD). If the open
354 T a b l e 1. C l i n i c a l d e s c r i p t i o n of the 73 D M D / B M D p a t i e n t s b e a r i n g a d e l e t i o n in t he d y s t r o p h i n ge ne . A n e m p t y s pa c e i n d i c a t e s t h a t inform a t i o n is n o t a v a i l a b l e . P a t i e n t 50 is i n c l u d e d b e c a u s e he is an e x c e p t i o n to t he r e a d i n g f r a m e h y p o t h e s i s P a t i e n t no.
A g e at d i a g n o s i s
MR"
A g e (ye a rs ) A t confinem e n t to w h e e l c h a i rb
F/I d
Exons involvede
At time of s t u d y c
Cornm en ts f
BMD
5
18years
-
-
F
45 > 47
BMD
10
7years
-
-
11
F
46 > 48
+
BMD
10
3years
-
-
7
F
46 > 48
+
BMD
11
-
53
63
F
48 > 49
BMD
12
5years
-
-
15
F
45 > 48
BMD
19
14 y e a r s
-
-
BMD
22
12years
-
-
BMD
22
9years
-
BMD
24
4years
-
DMD
I
45 > 48
51
F
45 > 47
-
45
F
45 > 47
-
12
I
45 > 47
5
F
06 > 07
DMD
10
I
46 > 47
DMD
18
7years
-
-
11
I
DMD
28
8 years
-
12
16
I
DMD
31
6years
+
-
11
I
DMD
33
5 years
-
9
11
I
45
DMD
34
4years
-
9
d14
I
22 > 25
DMD
36
I
52
DMD
40
5years
-
8
d14
F
48 > 50
DMD
42
2 years
-
-
9
F
03 > 07
F
46 > 48
4years
-
10
14
I
(01) + 02
DMD
43
DMD
44
02 > 07 (48) 45 > 52
DMD
45
I
45 > 50
DMD
47
9 years
-
13
20
I
50
DMD
49
5years
-
14
24
F
44 > 52
DMD
50
DMD
51
5years
-
-
13
I
DMD
52
5years
+
14
DMD
54
4years
-
9
03 + 04
19
+
45 > 52
I
18 > 25
I
28 > 43
DMD
56
3 years
-
-
12
I
48 > 54
DMD
57
2years
-
-
6
I
03 > 35
DMD
58
F
49 > 52
DMD
60
-
-
6
I
51 > 53
*
DMD
62
18months
+
-
5
I
46 > 55
*
DMD
64
5years
+
-
9
I
47 > 52
*
DMD
65
F
45 > 52
DMD
68
DMD
71
4years
5years
-
-
9
I
05 > 36
F
(08)~
33 18
DMD
72
3 years
-
15
d20
I
08 >
DMD
73
6 years
+
10
18
F
45
DMD
74
5 years
-
-
11
I
51
DMD
75
4years
-
8
17
F
45 > 50
DMD
76
9years
-
10
20
I
48 ~ 52
DMD
77
7years
-
-
10
I
46 + 47
DMD
79
DMD
80
4years
+
-
DMD
82
3years
+
-
DMD
83
DMD
84
DMD
86
DMD
I
47 )
10
I
03 > 07
10
F
45 > 52
*
+*
52
F
49 ~ 52
I
40 ~ 42
10years
+
-
13
I
16
89
5 years
-
11
21
I
50
DMD
90
4years
-
10
18
F
46 + 47
DMD
91
8years
-
-
14
F
45 4- 46
+
355
Table 1 (continued) Patient no.
DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD DMD
91 92 93 95 96 100 101 106 107 108 111 112 115 119 120 121 123 124 127 129 130 133 134 136
Age at diagnosis
5years 6 years 6years 9months 4years 3 years 6years 3 years 6years 4years 6 years 3 years 6years 2years 2 years 6years 5 years 18months 2 years 18months 9 months 9years 1 year 4years
MR a
+ + -
F/I d
Age (years) A t confinement to wheelchair b
A t time of study c
11 12 7 13 9 11 12 9 10 11 12 -
12 9 20 6 d23 14 14 19 19 14 17 5 12 3 3 7 20 5 4 3 1 18 11 14
F I I I I I I I F I F I I I F I I I I I F I F I
Exons involved e
45 46 45 42 44 44 48 43 03 48 45 (04) 48 45 46 46 49 46 (01) -47 52 47 43 47
Comments f
+ > > + > > > + > >
46 48 52 43 51 51 50 44 15 50
+ *
> > > + > > > > + > > > >
13 52 50 47 52 53 51 60 48 ? 51 51 50
*
*
+ +
* * + * * +* +
a Mental retardation: + , present; - , absent. According to the Stanford-Binet intelligence test, patients 80, 84 and 124 have a high degree of M R , patients 64, 73, 82, and 107, a moderate de~ree, and patients 31 and 52, a low degree A dash indicates that the patient can still walk c Figures preceded by 'd' indicate age at death for patients already deceased
a Familial/isolated Key: > more than two exons deleted; +, two deleted; 0 , breakpoints not yet clearly determined; ?, second breakpoints not yet identified f Key: + , deletion does not follow reading frame hypothesis; * patients too young to be classed definitely under B M D or D M D but in whom the clinical progression suggests D M D
r e a d i n g f l a m e ( O R F ) is m a i n t a i n e d b e y o n d t h e d e l e t i o n , the milder form of the disease (BMD) occurs. In the present study we also report a DMD family in which a deletion associated with a junction fragment was transmitted to more than one offspring by a woman who s h o w e d n o e v i d e n c e f o r m u t a t i o n i n h e r s o m a t i c cells. This indicates that the deletion must have occurred during mitosis in early germline proliferation, leading to germline mosaicism. This phenomenon has important implications for the counselling of DMD families. In a recent study on 28 families with a new DMD/BMD mut a t i o n B a k k e r e t al. ( 1 9 8 9 ) s u g g e s t t h e u s e o f a r e c u r r e n c e r i s k o f a p p r o x i m a t e l y 7 % ( 1 4 % if t h e h a p l o t y p e a s s o c i a t e d w i t h t h e m u t a t i o n is k n o w n ) i n c o u n s e l l i n g o f sisters and mothers of a sporadic DMD/BMD patient.
B M D (Brooke et al. 1983), such as evaluation of creatinine kinase (CK) levels and myopathic changes detectable on muscle biopsy. We classified our patients with regard to the clinical follow-up of the disease into: (a) D M D patients who became confined to wheelchairs between the ages of 7 and 15 years and (b) B M D who were still ambulatory without support at age 15 (Hoffman et al. 1988). Nevertheless, some of our patients were too young to be classified as D M D or B M D (Table 1). All samples were analyzed by Southern analysis using the six c D N A clones of the D M D gene (Koenig et al. 1987). High molecular weight genomic D N A was extracted from 20 ml of peripheral blood according to standard laboratory procedures. The D N A samples were digested with two restriction enzymes (HindIII, PstI) to increase the possibility of detecting RFLPs or junction fragments. Electrophoresis, Southern blotting and hybridization were carried out using established procedures (Church and Gilbert 1984). The c D N A probes used for hybridization were nine contiguous segments of the dystrophin c D N A as reported by Koenig et al. (1987): 1-2a, 2 b - 3 , 4 - 5 a , 5 b - 6 , 7-8, 9a, 9-10, 11, 12-14. The cloned c D N A fragments were released from the vectors by digestion with E c o R I or EcoRI-HindIII. Probes 5 b 6 and 7 were obtained by EcoRI + HincII digestion of clone 5 b - 7 . Probes 9-10, 11 and 12-14 were obtained by EcoRI + HinclI digestion of clone 9-14. Probe 9a corresponds to the first 3 0 0 b p of probe 9-10 (EcoRI-PstI fragment). This is a subclone of the 9-10
Materials and methods The 127 D M D / B M D patients analyzed in our laboratory were diagnosed using standard clinical diagnostic criteria for D M D and
356
clone and was used in two separate hybridizations to distinguish the two HindIII fragments of I kb revealed by the original clone. In this way, smaller cDNA fragments were used as probes for separate hybridizations of genomic DNA thus resolving comigrating fragments. The hybridization probes were separated in low melting point agarose gels (LMP Agarose, IBI) and then radiolabeled (Feinberg and Vogelstein 1983, 1984). The PCR analysis was carried out using primer kits, made available by Drs. J. S. Chamberlain and T. Caskey, containing nine sets of primers for the multiplex amplification, and following established reaction conditions (Chamberlain et al. 1988, 1989).
c D N A probes (see Materials and methods). Of the 39 PCR non-deleted samples, 5 were subsequently found deleted in those regions which do not amplify in the multiplex reaction. The extension of the 73 deletions encompassing the D M D gene is shown in Fig. 1. In five D M D patients, c D N A analysis revealed a deletion associated with a junction fragment. In these cases D N A samples from the patients and the other family members were digested with the following restriction enzymes: HindlII, PstI, EcoRI, TaqI and BglII in order to discriminate between a pattern given by a junction fragment (which should be present with most of the restriction enzymes) and a rare D N A variant. In one of these families ( D M D 34) D N A samples from the proband, his father, mother and sister were analyzed. The proband showed a deletion of the 20 kb HindlII band (exons 22 to 25) and an additional HindlII band of about 14 kb when hybridized with the 4 - 5 a c D N A probe (data not shown). In this patient, deleted and junction fragments were also observed with PstI (Fig. 2, lane 4) and EcoRI, TaqI, BgllI digests (data not shown). While the sister of this patient (Fig. 2, lane 3) shows the same additional band as the proband, neither parent has such a band. Therefore, the mutation which gives rise to the junction fragment must be present in the germline of the mother. This junction fragment was generated by deletion of one or
Results Using the c D N A probes and HindIII-digested genomic D N A , we detected 73 deletions in 127 D M D / B M D patients (57%). In most cases the PstI digests were also analyzed to confirm the results obtained with HindIII digestion. A subset of 6 4 D M D / B M D patients was analyzed using P C R as a first screening for the detection of deletions. The 25 deletions thus found (39%) were subsequently fully characterized by c D N A hybridization. The genomic D N A samples which did not show any deletion by P C R analysis were subjected to further screening, initially with 4 - 5 a , 5 b - 6 , 9a and 9 - 1 0 c D N A probes and then, if no deletion was found, with the remaining b
a
c
d
e
2.4
2.55 6.7 12 2.8
lO
5b-6
4-5a
2b-3
I
l-2a I
60 58+59 57 56 55 54 53 52 51 50 49 48 47 46 45 44 43 42 40+41 38+39 37 36 35 34 30>33 29 28 26+27 22>25 21 20 19 18 17 16 14+15 13 12 i0+ii 8+9 7 6 5 4 3 2 1
3.5 6.0 8.8 1.0 2.3 8.3 7.8+1.0 7.0 3.1 3.7 1.6 1.2+3.9 I0 1.5 0.5 4.1 ii 4.2 6.2 6.1 1.5 1.3 0.4 1.8 18 Ii 4.7 5.2 20 12 7.3 3.0 12 1.7 6.0 2.7 6.6 4.0 10.5 7.5 4.6 8.0 3.1 8.5 4.2 3.25 3.2
3 3
3 2 3 1 3 1 3 3 3
2
3
3
2
l
3 2 3 2 3 3 3 3 3 3 3
1 3 1 3 2 3 3 3 3 2 3 1 2 3 3 3 3 1
I*
Fig. 1. Extent of the deletions in the dystrophin gene observed in 73 DMD/BMD patients by cDNA and PCR analysis: cDNA probes used for Southern analysis, numbered 1-10 from the 5' end to the 3' end of the 14 kb dystrophin cDNA. Probes 11 and 12-14 are not shown because we did not observe any deletion in that region of the gene (a); exon numbers (b); exon-containing HindlII genomic fragments (sizes in kb) (c); exon border type according to Koenig et al. (1989) (d); and extent of the deletions observed in 73 DMD/BMD patients (e). Thick bars denote BMD patients; thin bars denote DMD patients. Squares (m) indicate the deletions which are exceptions to the reading-frame hypothesis. Asterisks (*) indicate deletions that are apparently exceptions to the reading-frame hypothesis but are associated with a junction fragment. Thin bars with free ends indicate that the breakpoint/s of the deletions concerned are not yet clear
357 patient was digested with PstI (Fig. 3, lane 3), TaqI and B g l l I (data not shown). Since this junction fragment was
Fig. 2. The restriction fragment pattern of PstI in family DMD 34 following hybridization with the cDNA fragment 4-5a. In this family the affected boy (lane 4) shows a deletion and an anomalous size band (arrow). A band of the same size is observed in the PstI pattern of his sister (lane 3) but is absent from the blood DNA of his mother (lane 1). In lane 2 the PstI pattern of the father of the patient is shown
Fig. 3. The restriction fragment pattern of HindIII and PstI of family DMD 52 following hybridization with the cDNA fragment 45a: lanes 3 and 4 contain the PstI and HindIII digests, respectively, of the affected boy. This deletion involves the 20kb, 12kb and 7.3 kb bands and is associated with a junction fragment of approximately 6 kb in the HindIII pattern (lower arrow, lane 4). A deletion and a junction fragment of approximately 23 kb (upper arrow, lane 3) are also observed in the PstI pattern of the affected boy. In lanes 5 and 6 the DNA from the mother of the patient shows normal Psd and HindIII patterns. The same result is evident for the PstI and HindIII digests of the father shown in lanes i and 2
m o r e exons (exons 22-25) corresponding to the 2 0 k b H i n d l I I band. In another family, D M D 52, D N A samples from the patient and his parents were analyzed. This patient shows a deletion of the 20 kb, 12 kb and 7.3 kb H i n d l I I bands, and the presence of an additional band of about 6 kb, when hybridized with the 4 - 5 a c D N A p r o b e (Fig. 3, lane 4). F u r t h e r m o r e , this patient showed a deletion of the 3.0 kb and 12 kb bands when hybridized with probe 2 b - 3 . The deletion and the presence of an extra band, of about 23 kb, were also observed when the D N A of this
not present in the D N A of the parents, this indicates that the deletion observed in the affected male is a new mutation. In this patient, the deletion involves exons 18-21 and probably m o r e than one exon of the 22-25 group. E v e n in this case, the junction fragment was generated by deletion of some of the exons of the 22-25 group. T h r e e other D M D patients ( D M D 57, D M D 87, D M D 134) showed a deletion associated with a junction fragm e n t (data not shown). Finally, prenatal diagnosis was p e r f o r m e d on 20 male fetuses at risk for D M D / B M D (caused by a deletion previously identified) using c D N A analysis or P C R amplification. Deletions were found in 6 out of the 12 fetuses analyzed with c D N A probes, and the other 6 were normal. P C R analysis was used for 8 prenatal diagnoses, and 3 fetuses were found to bear a deletion, 5 being diagnosed as normal.
Discussion In the present study, screening of deletions was carried out by c D N A analysis and multiplex P C R amplification. Using the former approach, 57% of the patients were found to be deleted, which is comparable to the detection rates reported by other laboratories (Forrest et al. 1988; Koenig et al. 1989; den D u n n e n et al. 1989). T h e P C R analysis, used as a first approach in 64 patients, led to the identification of 25 deletions (39%). This multiplex P C R allows simultaneous amplification of nine exons, particularly of those which have the highest frequency of deletion breakpoints. In our study it was possible to identify 83% of the deletions that can be revealed by c D N A analysis, which is in good a g r e e m e n t with the 90% reported by Chamberlain et al. (1989). The extension of the deletions identified by P C R was confirmed using c D N A analysis, which was also used to further investigate those patients who did not show any deletion by the P C R method. Five of these patients showed deletions in the gene on further c D N A analysis. T h e region corresponding to probes 11 and 12-14 was not deleted in any of our patients. Out of the 73 deletions, 53 (71%) encompassed the central high frequency deletion region ( H F D R ) of the gene, located approximately 1200kb from the p r o m o t e r , and 13 (17.5%) encompassed the proximal H F D R located approximately 500 kb from the 5' end of the gene. As reported in other studies, m o r e than 60% of all the intragenic breakpoints are located in this limited n u m b e r of exons. On the other hand, the location of the second breakpoint of these deletions is widely variable. The possibility of identifying the molecular basis of the phenotypic differences in D M D / B M D patients has been investigated by m a n y authors. A first study, by Monaco et al. (1988), suggested an association between the effect that an intragenic deletion would have on the translational reading f r a m e of the m R N A and the clinical severity of the disease. Although no apparent correlation was observed between the location and size of de-
358 letions with the clinical severity of the disease, more recent studies on the predicted structure of the dystrophin molecule show that in-frame deletions involving the Nterminal "actin binding" domain of the protein (including up to 1400 aminoacids or exons 1 to 34) usually lead to the milder form of muscular dystrophy (BMD). Furthermore, in-frame deletions involving exons 31-44 are predicted to lead to asymptomatic individuals (Koenig et al. 1989). This hypothesis is based on the observation that no BMD deletion starting in this very large intron (160-180 kb) between exons 44 and 45 and extending towards exon 31 have yet been found. On the contrary, the deletion breakpoints occurring in this intron are highly frequent in D M D patients bearing out-of-frame deletions extending between exon 44 and exon 52 (Koenig et al. 1989; den D u n n e n et al. 1989; Gilgenkrantz et al. 1989). Another very large intron (110 kb) is located between exons 7 and 8 and is also very frequently involved in deletion breakpoints. Furthermore, deletions involving the cystein-rich domain and the C-terminal domain (exons 54-58) lead to the production of a non-functional or unstable protein and, therefore, to a D M D phenotype. This region is thought to be essential for the correct function of the dystrophin molecule because it is highly conserved during evolution (Lemaire et al. 1988). Interestingly, deletions in these regions are extremely rare. Exceptions to the reading-frame hypothesis (Malhotra et al. 1988; Koenig et al. 1989; Baumbach et al. 1989; Gillard et al. 1989) were present in 9 out of the 73 patients with deletions (12%) analyzed in this study (Fig. 1, Table 1). Eight D M D patients were found to bear inframe deletions. In four D M D patients (patient 106 with a deletion of exons 43 and 44; patient 107 with a deletion of exons 3-15; patient 123 with a deletion spanning from exon 49 to 53, and patient 133 with a deletion of exons 47-51), despite the maintained reading frame following the deletion event, the clinical course of the disease seems to be that of a typical D M D case. An explanation for this could be that the exon/s involved in these deletions might be critical for the function of the protein. Two brothers (DMD 91) bearing an in-frame deletion of exons 45 and 46 show a slightly milder form of the disease; at 12 and 14 years of age they can still walk with the aid of calipers. Therefore, despite an early onset, the clinical course in these patients seems to be less severe than that in a typical DMD case. In the young DMD patients, 68 (deletions of exons 5-36) and 129 (deletion of exons 47 and 48), the deletions do not cause a frame shift in the transcript. In these patients the future clinical progression of the disease might be less severe than in a typical D M D case. A deletion of exons 46-48, causing a shift of the reading frame, occurred in two BMD brothers (BMD 10) whose clinical features do not indicate increased severity of the disease. These two B M D patients are still too young (7 and 11 years of age) to show a correlation between the type of deletion and the severity of the disease. Unfortunately, no accurate clinical data are available for the D M D patient (50) whose deletion of exons 3 and 4 does not cause a shift of the reading frame, and he, therefore, is another exception to the reading-frame rule. Two of these exceptions, namely D M D patients 68 and 91, have
already been reported in a previous paper (Koenig et al. 1989), while the remaining seven are not similar to any of those described in previous studies (Malhotra et al. 1988; Baumbach et al. 1989; Gillard et al. 1989). Malhotra and collaborators (1988) were the first to observe that 6 patients with BMD and 5 with severe BMD, out of 29 total D M D / B M D patients, bore a deletion of exons 3-7 that created a shift in the O R F if exon 2 was spliced to exon 8 in the mRNA. These authors hypothesized three possible mechanisms to explain these exceptions to the reading-frame hypothesis: (1) differential splicing (Feener et al. 1989) which could mask the frame-shift mutations creating an m R N A with an in-frame deletion thereby resulting in the production of a partially functional protein (e.g., splicing of exon 2 to exon 9 or of exon 1 to exon 8); (2) reinitiation of protein synthesis at the next potential initiation A T G codon which is present in-frame in exon 8; (3) reinitiation of transcription from a second promoter downstream from the deletion in the very large intron ( l l 0 k b ) between exons 7 and 8. In this way a partially functional protein could be generated in these patients, leading to a mild form of the disease. In three of our patients (DMD 57, DMD 86 and DMD 134) the deletions do not seem to follow the readingframe hypothesis, but on the other hand, a junction fragment is present in each case. These patients cannot be considered as exceptions because it is not possible to identify the deletion borders by this analysis. In family D M D 34 the detection of a junction fragment in association with the deletion observed in the D M D patient was very useful for the identification of carriers. The presence of this junction fragment in the sister of the patient leads to the conclusion that she is a carrier of the same deletion as observed in the patient. Furthermore, since their mother did not show the junction fragment in her blood cells, whereas the same deletion occurred in both siblings, we conclude that the mutation must have occurred in a germline cell clone of the mother, as already previously hypothesized for other pedigrees (Romeo et al. 1986; Bakker et al. 1987; Darras et al. 1987, 1988b; Hall 1988). Mental retardation occurred in 9 (15%) of the 61 patients with deletions for whom this type of information was available (see Table 1). The two D M D patients (31 and 82) have the same type of deletion, comprising exons 45-52. Three additional D M D patients showing mental retardation have overlapping deletions: patient 62 shows a deletion of exons 46-55; patient 124 bears a deletion involving exons 46-51; and patient 64 has a deletion of exons 47-52. Interestingly, all these five D M D patients with mental retardation lack exons 47-51 located in the central H F D R of the gene. Two other D M D patients showing mental retardation have a deletion in this region: patient 73 shows a deletion of exon 45 only, while the deletion in patient 84 involves exons 40-42. The remaining two mentally retarded DMD patients show overlapping deletions involving the proximal H F D R of the dystrophin gene and, in particular, patient 80 has a deletion of exons 3-7, while patient 107 has an in-frame deletion spanning exons 3-5. Clinical information about mental retardation was available in 34 out of the 54 non-
359 d e l e t e d p a t i e n t s , a n d 6 ( 1 8 % ) o f t h e s e w e r e m e n t a l l y retarded. The proportion of patients showing mental retardation is a l m o s t the s a m e in t h e two g r o u p s (with a n d witho u t d e l e t i o n s ) . H o w e v e r , in five o f t h e n i n e d e l e t e d p a tients in w h o m m e n t a l r e t a r d a t i o n o c c u r r e d t h e d e l e t i o n i n v o l v e d e x o n s c o n t a i n e d in p r o b e 8. S i m i l a r d a t a w e r e p r e s e n t e d b y Lindl/Sf et al. (1989), w h o f o u n d t h a t six out of seven mentally retarded DMD patients bore overl a p p i n g d e l e t i o n s in p r o b e 8, all o f t h e m i n v o l v i n g e x o n 50. T h e s e a u t h o r s suggest t h a t this r e g i o n m i g h t b e imp o r t a n t in n o r m a l m e n t a l d e v e l o p m e n t in m a l e s . N e v e r theless, m o r e r e c e n t findings r e g a r d i n g i n v o l v e m e n t o f a s e c o n d p r o m o t e r r e g i o n in t h e p r o d u c t i o n o f t h e dyst r o p h i n p r e s e n t in b r a i n ( b r a i n p r o m o t e r ) , l o c a t e d 5' to t h e m u s c u l a r p r o m o t e r , i n d i c a t e t h a t this r e g i o n m i g h t b e r e s p o n s i b l e f o r m e n t a l r e t a r d a t i o n (F. M. B o y c e , A . H . B e g g s , C. F e h n e r , a n d L. M. K u n k e l , in p r e p a r a t i o n ) . F u r t h e r studies o n g e n o t y p e - p h e n o t y p e c o r r e l a t i o n s in D M D p a t i e n t s s h o w i n g M R m i g h t h e l p in clarifying this point.
Acknowledgements. We thank Drs. M. Koenig and A.P. Monaco for helpful discussions and critical reading of the manuscript and Messrs. F. Caroli, I. Giambarrasi and M. Bertorello and Ms. A. Cereseto for technical assistance. We also thank the many physicians who helped in the collection of blood samples and clinical information. This work was carried out with funds from the Progetto Finalizzato Ingegneria Genetica of the Italian National Research Council (CNR) and from the Italian Union against Muscular Dystrophy (U.I.L.D.M.).
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