American Journal of Medical Genetics 42:281-287 (1992)

Use of Dystrophin Genomic and cDNA Probes for Solving Difficulties in Carrier Detection and Prenatal Diagnosis of Duchenne Muscular Dystrophy Ruth Shomrat, Nava Driks, Cyril Legum, and Yosef Shiloh The Genetic Institute, Tel Aviv Medical Center (R.S., N J ) . , C.L.) and Department of Human Genetics, Sackler School of Medicine, Tel Aviv University (C.L., Y.S.), Israel Duchenne muscular dystrophy (DMD) results from mutations in the X-linked gene coding for the muscular protein dystrophin. The isolation of genomic and cDNA probes for this gene has greatly facilitated the detection of DMD carriers, which previously relied mainly on measurements of serum creatine kinase (CK), and has enabled prenatal diagnosis of this disease. However, the relatively large size of the gene and the high frequency of recombination and mutation events within the dystrophin locus continue to pose difficulties in the genetic counselling and prenatal diagnosis of DMD, and render the conclusions of molecular analysis less clear cut. This communication presents examples of two such difficulties: the distinction between sporadicand inherited cases in families with a single patient and normal CK levels in all females, and the distinction between mutant and normal dystrophin alleles in families in which the patients have died. The combined use of genomic and cDNA probes allows one to make these distinctions. An additional complicating factor, gonadal mosaicism, is demonstrated.

KEY WORDS: DMD, CK, genetic counselling INTRODUCTION Duchenne muscular dystrophy (DMD)is the most severe and common of muscular dystrophies, affecting 1in 3,500 males [Harper, 19891. The disease is caused by defects in the X-linked gene coding for the muscle pro-

Received for publication November 6, 1990; revision received May 3, 1991. Address reprint requests to Ruth Shomrat, The Genetic Institute, Ichilov Hospital, 6 Weizmann Street, Tel Aviv, Israel.

0 1992 Wiley-Liss, Inc.

tein dystrophin [Monaco and Kunkel, 1987; Love and Davies, 1989; Worton and Thompson, 19881.About 10% of dystrophin mutations result in the less severe Becker muscular dystrophy (BMD).As the inheritance of DMD and BMD is X-linked, it is of particular importance to be able to detect carrier status among female relatives of the patients. Until recent years, prediction of carrier status and recurrence risks in DMD relied on Bayesian calculations, which were based on pedigree data and measurements of serum creatine kinase (CK) levels. Since only two thirds of obligate carriers show elevated CK levels [Emery, 19651, this analysis was useful only in certain families. Another difficulty in genetic counselling of DMD and BMD families stems from the relatively high mutation rate of the dystrophin gene which results in an exceptionally high frequency of de novo mutations [Harper, 19891. The molecular cloning of genomic fragments from the DMD locus has enabled carrier detection and prenatal diagnosis of DMD and BMD by linkage analysis based on restriction fragment length polymorphisms (RFLPs) [Bakker et al., 1986; Lindolf et al., 1986;Hodgson et al., 19871.However, this approach is not as straightforward in DMD as in many other genetic diseases, because of the large size of the gene which necessitates the use of several intragenic and flanking probes in order to rule out recombination [Cole et al., 1988; Kelly et al., 1990; Williams et al., 19861. An important advance in the molecular diagnosis of DMD was made when full length cDNA of the dystrophin gene became available [Burghes et al., 1987; Koenig et al., 19873.The cDNA probes detect intragenic deletions andlor rearrangements in 65% of the patients, greatly reducing the number of diagnostic dilemmas [Gillard et al., 1989; Hodgson et al., 1989; Speer et al., 1989; Ward et al., 19891. We present here several examples of the combined use of dystrophin genomic and cDNA probes which enabled us to solve two typical diagnostic difficulties in DMD families with single cases and normal CK levels in all females, and families who seek genetic counselling or prenatal diagnosis when the affected memberb) are no longer alive.

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MATERIALS AND METHODS The Genetic Institute of the Tel Aviv Medical Center is the referral center for molecular diagnosis of DMD and BMD for the entire state of Israel. Followingclinical examination of the patients and pedigree construction, serum CK levels were measured using the BoehrringerMannheim laboratory kits. Isolation of genomic DNA from peripheral blood, Southern blotting, and hybridization were performed according to previously published methods [Sambrook et al., 1989; Shiloh et al., 19863. A battery of genomic and cDNA probes (Table I) was used and RFLP haplotypes were constructed on the basis of the RFLPs for which each family was informative. RESULTS Families With Single DMD Patients and Normal CK Levels in All Females In family A (Fig. 1)carrier testing was requested by the proband's four sisters, who had normal CK levels. Bayesian calculations, taking into account the normal CK levels of the sisters, indicated that their carrier risk was 41%.Haplotype segregation among the brothers (Fig. 1)did not support the possibility that the patient represented a new mutation, since all 5 healthy brothers had the maternal haplotype M1, while the patient had the M2 haplotype. However, the carrier risk of 11-6,II-7,and 11-8, who inherited the M1 haplotype, now dropped to that of the general population (approximately 0.03%), while that of 11-10,who had the M2 haplotype, was now identical to the mother's risk, 66%. Southern blotting analysis carried out at a later stage with the dystrophin full cDNA changed the situation once again. A junction fragment was detected in the proband by probe 8, using several restriction enzymes (Fig. 2), but was not present in the DNA of any other relative. We concluded that the proband was, after all, a result of a new mutation and the carrier risk of 11-10, now also dropped to that of the normal female population. In family B (Fig. 1)Bayesian analysis based on CK levels gave the mother and the proband's sister carrier

TABLE I. Molecular Probes and Polymorphisms Used in This Study Probe RFLP Alleles Location D-2

Pvu I1

6.616

99-6 J-Bir p87-30 p87-15

XJ2.3 XJ1.2 XJ1.l 754

Pst I BamH I Bgl I1 Taq I Xmn I BamH I Msp I Xmn I Taq I Bcl I Taq I Pst I

22/13 2115 3018 3.3i3.1 2.811.6 +- 1.2 917 + 2 4i1.7 8.717.5 7.816.4 211.6 3.813.1 1219

754-11

Bgl I1

6.315.6

lrrnlly R -

u-

I

p87-30 p87-1

2l 121 cpk 56

----

II

M2

64

I

; . Z : q

87 c*

1

12cpkXb

q ¶ 4 f 23+71 l 6 7.5 8 7 12 9 0

II

Fig. 1. Segregation of dystrophin haplotypes in three families with DMD. The box at the top right of each family shows the probes corresponding to each RFLP and their order. (cpk, creaiine phosphokinase).

Distal

p87-1

Intragenic

Proximal

risks of 41% and 20%, respectively. RFLP segregation showed that 11-1 inherited from her mother the same haplotype as her affected brother, thus increasing her carrier risk to 41%; cDNA probe analysis again changed the situation: the probes 2b-3 and 4-5a uncovered a deletion in the patient's DNA, while probe 2b-3 also detected a junction fragment (Fig. 3). The junction fragment, detected by using 3 additional restriction enzymes (not shown), was present also in the DNA of the proband's sister but not in the mother's DNA. We con-

DMD Diagnosis

I

2

m

II

1

9 10 5

Kb

1Y

283

m ,

5 10 9

Kb -12

15.89.2

-

-7

6.2+

- 5.6 -5.2

4.6-

e3.8 3.2-

Xmn I

Pst I

Fig. 2. Southern blotting analysis of Family A with the eDNA probe 8. A junction fragment is detected in the DNA of the proband.

cluded that the mother represented a case of gonadal mosaicism and that the sister was definitely a carrier. In Family C (Fig. 1) the risk for being a carrier by Bayesian analysis incorporating CK levels was 41% for the mother and 20% for each daughter. Haplotype analysis showed that the affected and the healthy brothers inherited the same dystrophin haplotype from their mother, indicating that the patient, 11-4,probably represented a new mutation. This was further substantiated when the cDNA probe 8 detected a junction fragment in the patient’s DNA (Fig. 41, which was not present in the unaffected brother or in the mother’s DNA. The carrier risk for the two sisters was determined to be similar to that of the general population.

Families With No Living Patients In families D-I (Figs. 5, 6) no rearrangements (indicated by junction bands) were observed using any of the genomic and cDNA probes. Carrier risks were initially determined using Bayesian calculations following CK measurements and then compared to those obtained after adding the linkage data. In family D, the consultant, 11-1, and her mother had normal CK levels. DNA analysis showed that 11-1had inherited the same hap-

lotype as her unaffected brother, 11-2. This lowered her carrier risk from a value of 20 to about 7%. This figure takes into account the possibility of maternal gonadal mosaicism when the proband’s haplotype is unknown [Bakker et al., 19891. Families E and F (Fig. 5) represent a situation where neither living patients nor healthy brothers are available, but the females with high CK levels, who are probable carriers may enable the distinction between mutant and normal haplotypes. The a priori carrier risk of the consultands 11-2in Family E and 111-2in Family F was 50%. Following haplotype analysis the carrier risk for 11-2 in Family E dropped to about 5%, which is the probability for recombination between the mutation and probe PERT 87-15, the only probe for which the mother was informative. The risk for 111-2 in Family F now became less than 5%, since haplotype analysis did not indicate any recombination within the DMD locus proximal to J-Bir. Following genetic counselling, prenatal diagnosis of DMD in family F was requested by a carrier, 11-2,on the basis of her CK and dystrophin haplotypes (Fig. 5). Ultrasound examination showed a twin pregnancy and cytogenetic analysis of chorionic villus samples of each fetus indicated that both were male. DNA analysis

Shomrat et al.

284

P2

1 1

Kb

2

II

3 4

ia 4

3

Kb

- 11

+21

- 7.4 4.4

-

3.0

-

2.3

-5.4

--3.84.2

-3.5

“+-a

+2.7 -2.6

-2

0.9

-

-0.9

0.5-

^**

Toq I

-1.0

-/-c

Taq I

Bgl I I

Fig. 3. Southern blotting analysis of Family B With the CDNA probes 2b-3 and4-5a. Adeletion and ajunction fragment are detected in the patient’s DNA. The junction fragment is observed also in the patient’s sister, but not in their mother.

Fig. 4. Southern blotting analysis of Family C with cDNA probe 8. The probe detects a junction fragmentin the patient’s DNA,

showed that one had the mutant (Ml) dystrophin haplotype, and the other had the normal (M2) haplotype. The parents elected to terminate the pregnancy immediately. In family G (Fig. 6) prenatal diagnosis of DMD was requested by the sister of three deceased brothers, 11-1. CK levels and haplotype analysis led to the identification of the mutant haplotype, M1. Although the male fetus was shown to have the M1 haplotype, for religious reasons the parents elected to continue the pregnancy. The baby was born a t term and the CK level at age 1year was 2,500 IU/ml. In family H (Fig. 6) prenatal diagnosis was requested by the sister of the deceased proband, 11-3. High serum CK and the presence of haplotype M2 indicated that she was a carrier. Unfortunately, the first pregnancy of a male fetus who had the normal (P) haplotype terminated spontaneously. The male fetus in the second preg-

nancy was found t o have the mutant (M2) haplotype and the parents elected to terminate this pregnancy. Examination of DNA from a chorionic villus sample from the male fetus in the third pregnancy showed a recombinant haplotype that represented a crossing-over between D-2 and J-Bir and contained the portion of the normal haplotype (P)proximal to D-2. Based on the recombination frequency between D-2 and J-Bir, the risk of that fetus being affected was estimated below 20%. The parents elected t o continue the pregnancy. The baby was born a t term and found to have serum CK level of 181 IUiml at the age of 6 months. Further CK testing was refused by the parents. At 18 months of age the child appears to be healthy. Finally, family I (Fig. 7) represents a family with no living patients in whom several recombination events in different individuals precluded reliable carrier risk estimation.

DMD Diagnosis Family D

285

FamilyG

I

I

q Y

M2

7.8

6.4

124

I

I I XJ 2.3

;"I,

cuk 33

7.8

12

5.6

M

M.

L Fig. 6. Segregation of dystrophin haplotypes in two DMD families in which prenatal diagnosis of the disease was requested. (Topsa, termination of pregnancy, spontaneous abortion; Topel, termination of pregnancy, elective abortion.)

Fig. 5. Segregation of dystrophin haplotypes in three DMD families in which the patients are deceased.

DISCUSSION This report presents several counselling and diagnostic difficulties resulting from the unusual size and relatively high mutation rate of the dystrophin gene. However, the combined use of genomic and cDNA probes for linkage and structural analyses can reduce the number of unresolved problems to a minimum [Darras et al., 1988a,b;LeRoy et al., 1988; Speer et al., 1989; Sugino et al., 1989; Ward et al., 19891. The high frequency of rearrangements in the dystrophin gene not only con-

tributes to the frequency of new mutations but may also simplify their identification by Southern blotting. The appearance of rearranged bands particularly facilitates carrier detection [Darras et al., 1988a,b; Gillard et al., 1989;Lindolf et al., 19891.Several labs have had success in detecting heterozygous deletions using dosage blotting [Mao and Cremer, 19891, but this method is not easily reproduced and is subject to experimental error because it is based on the detection of a 50%reduction in signal intensity on Southern blots. The proportion of rearrangements detected in heterozygotes can be increased further by pulsed-field gel electrophoresis [Chen et al., 1988; Den Dunnen et al., 19871, but this method is not generally available in diagnostic laboratories. It should be borne in mind that some 30%of DMD patients represent point mutations which at this point are not amenable to direct identification [Beggs and Kunkel, 19901. Family B represents a problem recently recognized in surprising frequency in DMD families, namely, gonadal mosaicism [Bakker et al., 1987; 1989; Darras et al., 1987; Lanman et al., 1987; Wood et al., 1988; Speer et

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286 Family I

the Israel Society for Muscular Diseases (Ilmesh) and the families for their kind cooperation and support.

I M 22

21

30 3 1

7+2 17 87

PI

P

0 4

7.5 12

M

1+2 12

111

h 8.7

Fig. 7. Segregation of dystrophin haplotypes in a family with no living patients.

al., 19891. It has been estimated that up to 14% of “sporadic” cases of DMD actually represent gonadal mosaicism [Bakker et al., 19891,raising the possibility of this phenomenon as being important for DMD counselling in families with single patients. Family I is an example of a diagnostic dilemma encountered in families with no living patients, no rearrangements, and several crossover events in key members of the family or in a fetus [LeRoy et al., 19881. In this family, identification of the grandpaternal X-chromosome in the fetuses of the consultands 11-1 and 11-4 would exclude the at risk X-chromosome. Certain cases still pose counselling problems. Families with no living patients, no unaffected males, and no females with elevated CK levels on the one hand, and families with no genomic rearrangements and multiple recombination events on the other hand. These difficulties preclude a clear definition of mutant and normal haplotypes. Counselling and diagnostic information revert in such cases to probability calculations, and a major component in the family’s decision becomes their subjective risk perception [Shiloh and Saxe 19891. Immunohistochemical staining of muscle sections with dystrophin antibodies in putative carrier females [Arahata et al., 1989; Bieber et al., 19891,while not always clear cut, may in the future answer the question of carrier detection in such families.

ACKNOWLEDGMENTS We are grateful to Dr. L. Kunkel for providing - us with probes, to the genomic probes and length Dr. GJB. van Ommen for probes J-66HI and p-20, and to

REFERENCES Arahata K, Hoffman EP, Kunkel LM, Ishiura S, Tsukahara T, Ishihara T, Sunohara N, Nonaka I, Ozawa E, Sugita H (1989): Dystrophin diagnosis: Comparison of dystrophin abnormalities by immunofluorescent and immunoloblot analysis. Proc Natl Acad Sci USA 86:7154-7158. Bakker E, Bonten EJ, de Lange LF, Veenema H, Majoor-Krakauer D, Hofker MH, van Ommen, GJB, Pearson PL (1986): DNA probe analysis for carrier detection and prenatal diagnosis of Duchenne muscular dystrophy: A standard diagnostic procedure. J Med Genet 23:573-580. Bakker E, van Broeckhoven C, Bonten EJ, van de Vooren MJ, Veenema H, van Hul W, van Ommen GJB, Vandenberger A, Pearson PL (1987): Germline mosaicism and Duchenne muscular dystrophy mutations. Nature 329554-556. Bakker E, Veenema H, den Dunnen JT, van Broeckhoven C, Grootscholten PM, Bonten EJ, van Ommen GJB, Pearson, PL (1989):Germinal mosaicism increases the recurrence risk for ‘new’ Duchenne muscular dystrophy mutations. J Med Genet 26553559. Beggs AH, Kunkel LM (1990):Improved diagnosis of Duchenne/Becker muscular dystrophy. J Clin Invest 85613-619. Bieber FR, Hoffman EP, Amos J A (19891: Dystrophin analysis in Duchenne muscular dystrophy: use in fetal diagnosis and in genetic counseling. Am J Hum Genet 45362-367. Burghes AHM, Logan C, Hu X, Belfall B, Worton, RG,Ray, PN (1987)A cDNA clone from the Duchenne/Becker muscular dystrophy gene. Nature 328:434-437. Chen JD, Denton M, Morgan G, Pearn JH, Mackinlay AG (1988): The use of field-inversion gel electrophoresis for deletion detection in Duchenne muscular dystrophy. Am J Hum Genet 42:777-780. Cole C, Coyne A, Hart KA, Sheridan R, Walker A, Johnson L, Hodgson S, Bobrow M (1988): Prenatal testing for Duchenne and Becker muscular dystrophy. Lancet I:262-265. Darras BT, Blattner P, Harper JF, Spiro AJ, Alter S, Francke U (1988a): Intragenic deletions in 21 Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) families studied with the dystrophin cDNA: location of breakpoints on HindIII and BgIII exon-containing fragment maps, meiotic and mitotic origin of the mutations. Am J Hum Genet 43:620-629. Darras BT, Koenig M, Kunkel LM, Francke U (1988b): Direct method for prenatal diagnosis and carrier detection in Duchenne/Becker muscular dystrophy using the entire dystrophin cDNA. Am J Med Genet 29:713-726. Darras BT, Francke U (1987):Apartial deletion ofthe muscular dystrophy gene transmitted twice by a n unaffected male. Nature 329556-558. Den Dunnen, JT, Bakker E, Klein Breteler EG, Pearson PL, van Ommen GJB (1987):Direct detection ofmore than 50%ofthe Duchenne muscular dystrophy mutations by field inversion gels. Nature 329:640-642. Emery AEH (1965) Carrier detection in sex-linked muscular dystrophy. J Genet Hum 14:318-329. 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 45507520. Harper, PS (1989) The muscular dystrophies. In Scriver CR, Beaudet, AL, Sly WS, Valle, D (eds): “The Metabolic Basis of Inherited Disease.’’ New York: McGraw-Hill co., 6th Edition, pp 2869-2902. Hodgson S, Hart K, Abbs S, Heckmatt J, Rodillo E, Bobrow M, Dubowitz V (1989): Correlation of clinical and deletion data in Duchenne and Becker muscular dystrophy. J Med Genet 26:682-693. Hodeson S. Walker A. Cole C. Hart K. Johnson L. Heckmatt J . Dubuowitz V, Bobrow M (1987j: The application of linkage analysis to genetic counselling in families with Duchenne or Becker muscular dystrophy. J Med Genet 24:152-159.

DMD Diagnosis Kelly ED, Graham CA, Nevin NC (1990):Carrier estimations in Duchenne muscular dystrophy families in Northern Ireland using RFLP analysis. J Med Genet 27:lOl-104. Koenig M, Hoffman EP, Bertelson CJ, Monaco AP,Feener C, Kunkel LM (1987):Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 59:509-517. Lanman JT, Pericak-Vance MA, Bartlett RJ, Chen JC, Yamaoka L, Koh J, Speer MC, Hung W-Y, Roses AD (1987): Familial inheritance of a DXS164 deletion mutation from a heterozygous female. Am J Hum Genet 41:138-144. LeRoy BS, Uhrhammer NA, Steere KJ, Boehm CD, King RA, Rich SS, Williams PP, Smith SA, de Martinville B (1988):Identification of carriers of Duchenne muscular dystrophy: Value of molecular analysis. Am J Med Genet 31:709-721. Lindolf M, Kaariainen H, Davies KE, de la Chapelle A (1986):Carrier detection and prenatal diagnosis in X linked muscular dystrophy using restriction fragment length polymorphisms. J Med Genet 23:560-572. LindolfM, Kiuru A, KaariainenH, Kalimo H, Lang H, Pihko H, Rapola J , Somer H, Somer M, Savontaus M-L, de la Chapelle A (1989):Gene deletions in X-linked muscular dystrophy. Am J Hum Genet 44~496-503. Love DR, Davies KE (1989):Duchenne muscular dystrophy: The gene and the protein. Mol Biol Med 67-17. Mao Y, Cremer M (1989) Detection of Duchenne muscular dystrophy carriers by dosage analysis using the DMD cDNA clone 8. Hum Genet 81:193-195. Monaco AP,Kunkel LM (1987): A giant locus for the Duchenne and Becker muscular dystrophy gene. lkends Genet 3:33-37. Sambrook EF, Fritsch Y, Maniatis T (1989): Molecular Cloning: A

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Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press. Shiloh, S, Saxe, L (1989) Perception of recurrence risks by genetic counselees. Psychology and Health 3:45-61. Shiloh Y, Korf B, Kohl NE, Sakai K, Brodeur GM, Harris P, Kanda N, Seeger RC, Alt FW, Latt SA (1986):Amplification and rearrangement of DNA sequences from the chromosomal region 21324 in human neuroblastomas. Cancer Res 46:5297-5301. Speer A, Spiegler AW, Hanke R, Grade K, Giertler U, SchieckJ, Forrest S, Davies KE, Neuman 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:l-5. Sugino S, Fujishita S, Kamimura N, Matsumoto T, Wapenaar MC, Deng HX, Shibuya N, Miike T, Naiikawa N (1989): Moleculargenetic study of Duchenne and Becker muscular dystrophies: Deletion analysis of 45 Japanese patients and segregation analyses in their families with RFLPs based on the data from normal Japanese families. Am J Med Genet 34:555-561. Ward, PA, Hejtmancik J F , Witkowski JA, Baumbach LL, Gunnel1 S, Speer J , Hawley P, Tantravahi U, Caskey CT (1989): Prenatal diagnosis of Duchenne muscular dystrophy: Prospective linkage analysis and retrospective dystrophin cDNA analysis. Am J Hum Genet 44:270-281. Williams H, Sarfarazi M, Brown C, Thomas N, Harper PS (1986):The use of flanking markers in prediction for Duchenne muscular dystrophy. Arch Dis Child 61218-222. Wood S, McGillivary BC (1988): Germinal mosaicism in Duchenne muscular dystrophy. Hum Genet 78:282-284. Worton RG, Thompson MW (1988): Genetics of Duchenne muscular dystrophy. Annu Rev Genet 22601-629.

Use of dystrophin genomic and cDNA probes for solving difficulties in carrier detection and prenatal diagnosis of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) results from mutations in the X-linked gene coding for the muscular protein dystrophin. The isolation of genomic and...
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