American Journal of Medical Genetics 38:80-84 (1991)
Familial Occurrence of Duchenne Dystrophy Through Paternal Lines in Four Families Mayana Zatz, Maria Rita Passos-Bueno, Debora Rapaport, and Mariz Vainzof Departamento de Biologia, Instituto de Bioci&rzcias,Universidade de Scio Paulo, Scio Paulo, Brazil
In a survey of 454 families with patients affected with Duchenne muscular dystrophy (DMD) we have found 4 genealogies with 2 or more affected patients who were related through paternal lines. In 1 of these families, 2 affected cousins showed different DNA deletions suggesting 2 independent mutations; in the other 2, in which only the propositus could be studied, DNA deletions were also detected in the dystrophin gene. In the last one, with 3 affected patients, no DNA deletions were detected but immunohistochemical study of muscle biopsies showed a negative dystrophin pattern typical of DMD. Although one of these families might have occurred by chance, the probability of finding the other 3 in our sample of families with DMD patients constitutes a rare event. It is suggested that other mechanisms, such as the presence of transposable elements in other sites of the genome, could be responsible in some families, for a greater predisposition for the occurrence of pathogenic deletions, duplications or mutations. KEY WORDS: X-linked, Duchenne dystrophy, nonmaternal inheritance, transposable elements INTRODUCTION Here we report 4 families with familial DMD related through paternal lines. SUBJECTS AND METHODS A total of 454 families with patients affected with DMD was ascertained in our center, between 1969 and 1988. In all of them the diagnosis was established through clinical examination, family history, grossly elevated Received for publication February 13, 1990; revision received April 17, 1990. Address reprint requests to Mayana Zatz, Departamento de Biologia, Instituto de Biocihcias, Universidade de SBo Paulo, Sao Paulo, Brazil.
0 1991 Wiley-Liss, Inc.
serum creatine kinase (CK) and, in most, also, serum pyruvate kinase (PK), typical electromyography and/or muscle biopsy. In 294 families, the propositus was a n isolated case while in 160 there were 2 or more affected patients in the family. In 74, there were 2 or more affected boys in the same sibship, in 82 a typical X-linked inheritance was found, while in 4 families affected patients were related through paternal lines. DNA studies, using the cDNA probes cf27, cf23a, cf56a, and cfl15 (donated by Dr. Kay Davies from the University of Oxford) were done in the patients from the 4 families here reported who were personally examined. The method is described in Forrest et al. [1988]. Dystrophin studies through immunofluorescence was performed in muscle biopsies from 3 of the above patients and in 3 DMD female carriers (family 4) according to the method described in Vainzof et al. [1990].
CLINICAL REPORTS The genealogies of the 4 families are illustrated in Figure 1. The description and laboratory findings from each family are reported below: Family 1 The propositus (V-1) is currently 11years old; he was first seen a t age 7 years. He was confined to a wheelchair a t age 10 and he was recently classified a s Vignos 7 (sits erect in a chair; able to roll wheelchair, eats and drinks normally). His serum CK was 1600 Sigma units (normal until 20 S.U.) and his serum PK was 72.8 pmolimlihr (normal up to 6.0 pmolimllhr). A muscle biopsy and electromyography (EMG) was done in 1985; results were typical of a myopathic process. DNA studies showed a deletion of 2 noncontiguous exons (L and J) detected with probe cf56a. The mother had a low estimated probability of being a DMD carrier (less than 20%). The affected cousin (V-3) is currently 7 % years old and was first seen by us a t 6 years. He has typical DMD and was recently classified a s Vignos 3 (climbs stairs only with the aid of the bannister). His serum CK was 1250 S.U.,serum PK 49 pmolimll h r and EMG findings were also characteristic of a myopathic process. His muscle biopsy showed histopathological alterations typical of a myopathic process.
Duchenne Dystrophy Inherited Through Paternal Lines
Family 1
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Dystrophin assessment, studied through immunohistochemistry, showed a negative pattern, characteristic of DMD [Vainzof et al., 19901. His DNA showed a deletion of 3 exons detected with probe cf23a (exons E, F, and G) and 1 exon (I) detected with probe cf56a, which was different from the one observed in his cousin V-1.
The mother also had an estimated low probability of being a carrier of the DMD gene.
Family 2 The propositus (V-5)was first seen a t age 3. His serum CK was 500 S.U., serum PK 53 pmollmlihr, and his muscle biopsy and EMG findings were also typical of a
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myopathic process. He was confined to a wheelchair a t age 7 and died of respiratory infection a t age 12. His DNA showed a deletion of 5 exons, detected with probes cf23a (exons FG) and cf56a (exons IJK). His mother has increased serum CK and PK and a n estimated risk of 100% of being a carrier of the DMD gene. The affected cousin (IV-5)was not examined by us but according to the family he was confined to a wheelchair a t age 7 and died a t age 18 with a clinical course typical of DMD.
Family 3 The propositus (IV-l),currently 10 years old, was first seen a t age @/la years. His serum CK was 920 S.U., serum PK 57.3 pmol/ml/hr, and he had a n EMG and muscle biopsy typical of a myopathic process. The parents refused another muscle biopsy for dystrophin studies. He was confined to a wheelchair at age 8, and he is showing a rapid progression of the myopathic process (Vignos 7). DNA studies showed a deletion of exons F and G (detected with probe cf23a) and of all the exons detected with probe cf56a (IJKLMN). His mother, maternal aunt, and grandmother had normal serum enzymes suggesting a new mutation in the propositus. His affected cousin (111-391,not examined by us, had a diagnosis of DMD and died at age 14. Family 4 The propositus (IV-2),currently 10 years old, was first seen at age 6%. His serum CK was 1800 S.U., serum PK 98.5 pmol/ml/hr, and his EMG was typical of a myopathic process. He had calf hypertrophy and was classified as Vignos 1. His younger affected brother (IV-4),who was 2 years old when first seen at the preclinical stage, had grossly increased serum CK and PK (1480 S.U. and 165 pmoli mlihr, respectively) and his EMG was also characteristic of a myopathic process. No DNA deletions were detected with probes cfl15, cf56a, cf23a, and cf27. A muscle biopsy performed in the 2 affected brothers showed histopathological alterations typical of a myopathic process and a dystrophin immunostaining pattern characteristic of DMD. The mother (111-4) and the 2 sisters (IV-1 and IV-3) had elevated serum enzymes (6-, 3-, and 20 fold-increase, respectively), indicating that they are carriers of the DMD gene. The 3 of them agreed to a muscle biopsy which showed discrete histopathological abnormalities in the mother and older sister. Dystrophin pattern, studied through immunohistochemistry, was normal in all of them. The affected cousin (111-25)was first seen by us a t age 13. He was confined to a wheelchair a t age 10 and was severely disabled (Vignos 9; unable to sit erect without support or unable to eat or drink without assistance). His serum CK was 307 S.U., serum PK 10.6 kmolihr. His EMG was typical of a myopathic process but he refused to have a muscle biopsy.
DNA studies detected no deletions with probes cfl15, cf56a, cf23a, and cf27. The mother has a low estimated probability of being a carrier of the DMD gene.
DISCUSSION With the exception of one family with 3 affected cases of DMD studied by Miciak et al. [19901, we are not aware of any other publication reporting genealogies with DMD affected patients linked through paternal lines. In the 4 families included in the present study an apparently new mutation occurred either in the propositi or in their mothers. An interesting finding was observed in family 1, in which the 2 affected boys had different DNA deletions suggesting 2 independent mutations. In one of them (family 1, V-1) the observed pattern of deletion suggests a complex rearrangement [Passos-Bueno et al., 19901. Noncontiguous deletions have been reported also in 2 other DMD patients [Read et al., 1988; Liechti-Gallatti e t al., 19891. According to Haldane [1935], if the reproductive fitness ( f , of affected individuals is 0, the mutation rate (u) is 1/3 of the incidence, that is, l/d (1 - f , = l/104;and the proportion of DMD carriers is 4u or 41104. In this case, the probability of having 2 new mutations, as for example in family 1, would be approximately 1/104 x 1/104or 11100,000,000.The population of the State of Sao Paulo is around 31 million; thus, the probability for the occurrence of these 4 families by chance, among 454 genealogies (approximately 1%) with DMD affected patients, ascertained in the State of Sao Paulo is so low that it constitutes a n almost impossible event. On the other hand, the family studied by the group of Dr. Sarah Bundey in Birmingham is still more difficult to explain by chance, since the 3 affected DMD patients, linked through paternal lines, have different mutations [Bundey, personal communication]. The probability for the occurrence of 3 new mutations, by chance is 1/104 x 1110~x 1i104, i.e., 1 in 1,OOO,OOO,OOO,OOO! Although we have no explanation, a t present, for these findings, we offer the following hypotheses for our families:
Autosomal Recessive Inheritance We have suggested in a n earlier report [Zatz et al., 19891 t h a t about 2-4% of patients with a “Duchennelike” phenotype might be inherited through autosomal recessive inheritance. Such patients, with a clinical picture very similar to X-linked DMD, would be dystrophin-positive. However, autosomal recessive inheritance would not explain the occurrence of the 4 families described in the present report because (1)in 3 families (1,2, and 3) a deletion in the dystrophin gene was found in the affected males confirming X-linked inheritance; (2) in family 4, in which no DNA deletion was detected, the muscle biopsy from the 2 affected brothers (IV-2 and IV-4) showed a negative dystrophin pattern characteristic of X-linked DMD. In addition, the mother and the 2 sisters of the patients had grossly elevated serum enzymes; (3) no consanguinity was found among the parents of affected patients in any of the 4 families.
Duchenne Dystrophy Inherited Through Paternal Lines
Presence of Male X- Chromosome Mosaicism or Intronic Nonpathogenic Deletion A partial deletion of the dystrophin gene transmitted by an unaffected male was described by Darras and Francke [19871. A similar finding was also reported in X-linked agammaglobulinemia (XLA) by Hendricks et al. [1989] who demonstrated that the XLA defect had been originated from a n healthy male. Intronic nonpathogenic deletions have already been found in normal relatives of DMD patients [Koh et al., 19871and might have a role in the generation of further lethal deletions [Hart et al., 19891. Such hypothesis could account for our family 2. Individual 1-2could carry a nonpathogenic deletion, which would be transmitted to individuals 11-1and 11-3,generate a pathogenic deletion in females 111-2 and 111-12 (who would be heterozygotes) and consequently the 2 affected males IV-5 and v-5. Germinal and somatic mosaicism could also explain our family 2, caused by a mutational event in individual 1-2. If a deletion had occurred during the last DNA replication of the oocyte that gave rise to individuals 11-1 and 11-3, a half chromatid mutation could have been transmitted that would have produced mosaicism of germinal and somatic tissues. In this case, individual 11-1 would be clinically unaffected (as if he were a female carrier of the abnormal gene) as well as his sister 11-3 but both would transmit the deletion to their daughters 111-2and 111-12and subsequently to the 2 affected males IV-5 and V-5. This hypothesis is not yet proven in humans but was suggested by Darras and Francke [19871 to explain the occurrence of their family in which the DMD gene was transmitted twice by an unaffected male to his carrier daughters. According to Bakker et al. [1989], germline mosaicism in both male and female carriers generates a recurrence risk for transmitters and patients of “new” DMD mutations. However, with the exception of family 2, the hypothesis of nonpathogenic intronic deletion or germinal mosaicism being transmitted by individual 1-2 would not explain our other 3 genealogies because it would require male-to-male transmission.
Presence of Transposable Elements in the Family According to Hoegerman and Rary [1986], mutations for fragile X-positive Martin-Bell syndrome and perhaps also achondroplasia may result from the insertion of transposable elements (TEs). Hypothetically, TEs could insert within exons, within introns in positions which would interfere with intron excision, or within regulatory sequences. They suggest that in the case of fragile X, loss of genetic function could result from the loss of genes distal to the site of insertion following subsequent TE excision without ligation of the resulting discontinuity. These authors suggest that in the case of fragile-X, nonaffected transmitting males would originate from a
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zygote in which the causative TE is integrated into another site in the genome. Hypothetically, the TE could either transpose to the X within the germline of “normal” transmitting males or within the germline of their progeny if the TE was transmitted to the progeny on another chromosome. They speculate also that the cytoplasm might be either relatively permissive or restrictive and that not all normal X chromosomes are equally competent for TE insertion. In the present study, with exception of family 4, in which the unrelated mother 111-4is heterozygote for the DMD gene, the presence of TEs a t another locus within the genome could offer a plausible explanation for families 1, 2, and 3, as hypothetically discussed below: In family 1,affected males V-1 and V-3 would have inherited a TE from their father and a normal X chromosome competent for TE insertion from their mother. In both patients, insertion in the maternal X chromosome would have occurred after fertilization following a subsequent TE excision leading to a DNA deletion in the dystrophin gene. Such a mechanism would also provide a n explanation for our finding of 2 different DNA deletions in individuals V-1 and V-3, as well for the rare complex DNA rearrangement reported in individual v-1. The same hypothesis could account for family 2, with the difference that the TE which might be already present in individuals 1-1or 1-2would have been transmitted by the females 111-2 and 111-12, resulting in the DMD carrier IV-3 and the affected male IV-5. In family 3, individual IV-1 could result from the same mechanism described for affected males V-1 and V-3 of family 1, while in the case of his affected cousin 111-39, the TE insertion and abnormal excision might have also occurred in the germline of his mother 11-12. This hypothesis would not explain our family 4, the only one in which no DNA deletions were detected in the affected members. However, if we assume that both the carrier mother 111-4 and the patient 111-25a r e the result of new mutations, the likelihood of the occurrence of one such family would be 4u x = 112500 x 1110,000 or 1in 25,000,000, which would still be possible, by chance, if we take into account the population of the State of Sao Paul0 . An interesting observation is that insertion of L1 sequences (which are human-specific family of long, interspersed, repetitive elements, dispersed throughout the genome) have been reported in 2 unrelated male patients with hemophilia A in a study of 240 unrelated cases screened for DNA studies [Kazazian et al., 19881. No abnormalities were detected in the factor VIII genes in the parents of both patients, suggesting that in both cases the mutations had arisen de novo. According to these authors, these L1 insertions are the first large nonviral insertions described in man which are not due to expansion of short repeats by unequal crossing-over events. They would represent a mechanism for mutation fundamentally different from those previously described. Because it is not known when these events might occur (whether in sperm or ovum, after fertilization, or during early stages of embryogenesis) the proportion of such insertions that are heritable is unknown.
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Yet, the finding of two L1 insertions among 240 patients with hemophilia A suggests that this mechanism of mutation is not uncommon. Most DMBiBMD patients cases are due to DNA deletions; however, the finding of DNA duplications leading to a DMD or BMD phenotype has also been reported in a proportion ofcases [Monaco et al., 1987; Hu et al., 19881. It would be very interesting to investigate if the presence of TEs could not be responsible for some of these duplications through direct insertion into the dystrophin gene or leading to unequal crossing-over. On the other hand, TEs could also offer a n alternative explanation for the observations of intronic nonpathogenic and pathogenic deletions in normal and affected members belonging to the same families [Koh et al., 1987; Hart et al., 19891. We suggest that the presence of TEs could be responsible in some families for a greater predisposition for the occurrence of DNA deletions (pathogenic or nonpathogenic), duplications, mutations, or complex rearrangements. However, such families would not receive the attention when the patient is a n isolated case or the TEs would be transmitted through maternal lines.
ACKNOWLEDGMENTS The collaboration of the following persons is gratefully acknowledged: Dr. Kay Davies for kindly providing the cDNA probes; Drs. Rita de Cassia M. Pavanello and Dr. Ivo Pavanello-Filho for performing the muscle biopsies; Drs. Celia P. Koiffman, Angela M. Vianna-Morgante, Anita Wajntal, and Paul0 A. Otto for helpful suggestions and criticism; to Marcia Luisa das Neves, Marta Canovas, Martha Alessandra de Oliveira Lima, Helena Beatriz Martins da Costa, and Roberto Schreiber; to Marco Antonio Xavier for drawings and photographs; to the staff from ABDIM for their collaboration with the affected patients. This work was supported with grants from FAPESP, CNPq, FINEP, Secretaria de Ci6ncia e Tecnologia do Estado de S. Paulo, and ABDIM.
REFERENCES Bakker E, Veenema H, den Dunnen JT, Van Broeckhoven Ch, Grootscholten PM, Bonten EJ, Van Ommen GJB, Pearson PL (1989): Prenatal diagnosis of Duchenne muscular dystrophy: A three year experience in a rapidly evolving field. J Inher Metab Dis 12 SUPPI 1:174-190.
Bundey S (1990). Personal communication. Darras BT, Francke U (1987):A partial deletion ofthe muscular dystrophy gene transmitted twice by an unaffected male. Nature 328:556-558. Forrest SM, Cross GS, Flint T, Speer A, Robson KJH, Davies KE (1988): Further studies of gene deletions that cause Duchenne and Becker muscular dystrophies. Genomics 2:109-114. Haldane JBS (1935): The rate of spontaneous mutation of a human gene. J Genet 31:317-326. Hart KA, Abbs S, Bobrow M (1989): Pathogenic and nonpathogenic deletions in two families with Duchenne muscular dystrophy. Am J Med Genet 33:142-145. Hendricks RW, Mensink EJBM, Kraakman MEM, Thompson A, Schuurman RKB (1989):Evidence for male X chromosomal mosaicism in X-linked agammaglobulinemia. Human Genet 83:267-270. Hoegerman SF,Rary J M (1986): Speculation on the role of transposable elements in human genetic disease with particular attention to achondroplasia and the fragile X syndrome. Am J Med Genet 23:685-699. Hu X, Burghes AHM, Ray PN, Thompson MW, Murphy EG, Worton RG (1988):Partial gene duplication in Duchenne and Becker muscular dystrophies. J Med Genet 25:369-376. Kazazian HH, Wong C, Youssoufian H, Scott AF, Phillips DG, Antonarakis SE (1988):Haemophilia A resulting from de novo insertion of L1 sequences represents a novel mechanism for mutation in man. Nature 332164-166. Koh J, Pericak-Vance MA, Yamaoka LH, Hung WY, Worton RG, Lee J E , Kandt RS, Bartkett R J , Speer Mc, Phillips K, Ray PN, Gilbert JR, Siddique T, Roses AD (1987): Inherited deletion at Duchenne dystrophy locus in normal male. Lancet I:1154-1155. Liechti-Gallatti S, Koenig M, Kunkel LM, Frey D, Boltshauser E, Schneider V, Braga S, Moser H (1989): Molecular deletion patterns in Duchenne and Becker type muscular dystrophy. Human Genet 81:343-348. Miciak A, Keen A, Jadayet D, Bundey S (1990):Multiple mutation in an extended DMD pedigree. (Manuscript in preparation). Monaco AP, Bertelson CJ, Colletti-Fruner C, Kunkel LM (1987):Localization and cloning of deletion breakpoints in Xp21 involved in muscular dystrophy. Human Genet 75:221-227. Passos-Bueno MR, Rapaport D, Love D, Flint T, Bortolini ER, Zatz M, Davies K (1990): Screening of deletions in the dystrophin gene with the cDNA probes Cf23a, Cf56a and Cfl15. J Med Genet 27:145-150. Read AP, Mouuntford RC, Forrest SM, Kenwrick SJ,Davies KE, Harris R (1988):Patterns of exon deletions in Duchenne and Becker muscular dystrophy. Human Genet 80:152-156. Vainzof M, Pavanello RCM, Pavanello-Filho I, Passos-Bueno MR, Rapaport D, Chu TH, Zatz M (1990): Dystrophin immunostaining in muscles from patients with different types of muscular dystrophy: A Brazilian study. J Neurol Sci (in press). Zatz M, Passos-Bueno MR, Rapaport D (1989):Estimate of the proportion of Duchenne muscular dystrophy with autosomal inheritance. Am J Med Genet 32:407-411.
Familial Occurrence of Duchenne Dystrophy Through Paternal Lines in Four Families Mayana Zatz, Maria Rita Passos-Bueno, Debora Rapaport, and Mariz Vainzof Departamento de Biologia, Instituto de Bioci&rzcias,Universidade de Scio Paulo, Scio Paulo, Brazil
In a survey of 454 families with patients affected with Duchenne muscular dystrophy (DMD) we have found 4 genealogies with 2 or more affected patients who were related through paternal lines. In 1 of these families, 2 affected cousins showed different DNA deletions suggesting 2 independent mutations; in the other 2, in which only the propositus could be studied, DNA deletions were also detected in the dystrophin gene. In the last one, with 3 affected patients, no DNA deletions were detected but immunohistochemical study of muscle biopsies showed a negative dystrophin pattern typical of DMD. Although one of these families might have occurred by chance, the probability of finding the other 3 in our sample of families with DMD patients constitutes a rare event. It is suggested that other mechanisms, such as the presence of transposable elements in other sites of the genome, could be responsible in some families, for a greater predisposition for the occurrence of pathogenic deletions, duplications or mutations. KEY WORDS: X-linked, Duchenne dystrophy, nonmaternal inheritance, transposable elements INTRODUCTION Here we report 4 families with familial DMD related through paternal lines. SUBJECTS AND METHODS A total of 454 families with patients affected with DMD was ascertained in our center, between 1969 and 1988. In all of them the diagnosis was established through clinical examination, family history, grossly elevated Received for publication February 13, 1990; revision received April 17, 1990. Address reprint requests to Mayana Zatz, Departamento de Biologia, Instituto de Biocihcias, Universidade de SBo Paulo, Sao Paulo, Brazil.
0 1991 Wiley-Liss, Inc.
serum creatine kinase (CK) and, in most, also, serum pyruvate kinase (PK), typical electromyography and/or muscle biopsy. In 294 families, the propositus was a n isolated case while in 160 there were 2 or more affected patients in the family. In 74, there were 2 or more affected boys in the same sibship, in 82 a typical X-linked inheritance was found, while in 4 families affected patients were related through paternal lines. DNA studies, using the cDNA probes cf27, cf23a, cf56a, and cfl15 (donated by Dr. Kay Davies from the University of Oxford) were done in the patients from the 4 families here reported who were personally examined. The method is described in Forrest et al. [1988]. Dystrophin studies through immunofluorescence was performed in muscle biopsies from 3 of the above patients and in 3 DMD female carriers (family 4) according to the method described in Vainzof et al. [1990].
CLINICAL REPORTS The genealogies of the 4 families are illustrated in Figure 1. The description and laboratory findings from each family are reported below: Family 1 The propositus (V-1) is currently 11years old; he was first seen a t age 7 years. He was confined to a wheelchair a t age 10 and he was recently classified a s Vignos 7 (sits erect in a chair; able to roll wheelchair, eats and drinks normally). His serum CK was 1600 Sigma units (normal until 20 S.U.) and his serum PK was 72.8 pmolimlihr (normal up to 6.0 pmolimllhr). A muscle biopsy and electromyography (EMG) was done in 1985; results were typical of a myopathic process. DNA studies showed a deletion of 2 noncontiguous exons (L and J) detected with probe cf56a. The mother had a low estimated probability of being a DMD carrier (less than 20%). The affected cousin (V-3) is currently 7 % years old and was first seen by us a t 6 years. He has typical DMD and was recently classified a s Vignos 3 (climbs stairs only with the aid of the bannister). His serum CK was 1250 S.U.,serum PK 49 pmolimll h r and EMG findings were also characteristic of a myopathic process. His muscle biopsy showed histopathological alterations typical of a myopathic process.
Duchenne Dystrophy Inherited Through Paternal Lines
Family 1
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Dystrophin assessment, studied through immunohistochemistry, showed a negative pattern, characteristic of DMD [Vainzof et al., 19901. His DNA showed a deletion of 3 exons detected with probe cf23a (exons E, F, and G) and 1 exon (I) detected with probe cf56a, which was different from the one observed in his cousin V-1.
The mother also had an estimated low probability of being a carrier of the DMD gene.
Family 2 The propositus (V-5)was first seen a t age 3. His serum CK was 500 S.U., serum PK 53 pmollmlihr, and his muscle biopsy and EMG findings were also typical of a
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myopathic process. He was confined to a wheelchair a t age 7 and died of respiratory infection a t age 12. His DNA showed a deletion of 5 exons, detected with probes cf23a (exons FG) and cf56a (exons IJK). His mother has increased serum CK and PK and a n estimated risk of 100% of being a carrier of the DMD gene. The affected cousin (IV-5)was not examined by us but according to the family he was confined to a wheelchair a t age 7 and died a t age 18 with a clinical course typical of DMD.
Family 3 The propositus (IV-l),currently 10 years old, was first seen a t age @/la years. His serum CK was 920 S.U., serum PK 57.3 pmol/ml/hr, and he had a n EMG and muscle biopsy typical of a myopathic process. The parents refused another muscle biopsy for dystrophin studies. He was confined to a wheelchair at age 8, and he is showing a rapid progression of the myopathic process (Vignos 7). DNA studies showed a deletion of exons F and G (detected with probe cf23a) and of all the exons detected with probe cf56a (IJKLMN). His mother, maternal aunt, and grandmother had normal serum enzymes suggesting a new mutation in the propositus. His affected cousin (111-391,not examined by us, had a diagnosis of DMD and died at age 14. Family 4 The propositus (IV-2),currently 10 years old, was first seen at age 6%. His serum CK was 1800 S.U., serum PK 98.5 pmol/ml/hr, and his EMG was typical of a myopathic process. He had calf hypertrophy and was classified as Vignos 1. His younger affected brother (IV-4),who was 2 years old when first seen at the preclinical stage, had grossly increased serum CK and PK (1480 S.U. and 165 pmoli mlihr, respectively) and his EMG was also characteristic of a myopathic process. No DNA deletions were detected with probes cfl15, cf56a, cf23a, and cf27. A muscle biopsy performed in the 2 affected brothers showed histopathological alterations typical of a myopathic process and a dystrophin immunostaining pattern characteristic of DMD. The mother (111-4) and the 2 sisters (IV-1 and IV-3) had elevated serum enzymes (6-, 3-, and 20 fold-increase, respectively), indicating that they are carriers of the DMD gene. The 3 of them agreed to a muscle biopsy which showed discrete histopathological abnormalities in the mother and older sister. Dystrophin pattern, studied through immunohistochemistry, was normal in all of them. The affected cousin (111-25)was first seen by us a t age 13. He was confined to a wheelchair a t age 10 and was severely disabled (Vignos 9; unable to sit erect without support or unable to eat or drink without assistance). His serum CK was 307 S.U., serum PK 10.6 kmolihr. His EMG was typical of a myopathic process but he refused to have a muscle biopsy.
DNA studies detected no deletions with probes cfl15, cf56a, cf23a, and cf27. The mother has a low estimated probability of being a carrier of the DMD gene.
DISCUSSION With the exception of one family with 3 affected cases of DMD studied by Miciak et al. [19901, we are not aware of any other publication reporting genealogies with DMD affected patients linked through paternal lines. In the 4 families included in the present study an apparently new mutation occurred either in the propositi or in their mothers. An interesting finding was observed in family 1, in which the 2 affected boys had different DNA deletions suggesting 2 independent mutations. In one of them (family 1, V-1) the observed pattern of deletion suggests a complex rearrangement [Passos-Bueno et al., 19901. Noncontiguous deletions have been reported also in 2 other DMD patients [Read et al., 1988; Liechti-Gallatti e t al., 19891. According to Haldane [1935], if the reproductive fitness ( f , of affected individuals is 0, the mutation rate (u) is 1/3 of the incidence, that is, l/d (1 - f , = l/104;and the proportion of DMD carriers is 4u or 41104. In this case, the probability of having 2 new mutations, as for example in family 1, would be approximately 1/104 x 1/104or 11100,000,000.The population of the State of Sao Paulo is around 31 million; thus, the probability for the occurrence of these 4 families by chance, among 454 genealogies (approximately 1%) with DMD affected patients, ascertained in the State of Sao Paulo is so low that it constitutes a n almost impossible event. On the other hand, the family studied by the group of Dr. Sarah Bundey in Birmingham is still more difficult to explain by chance, since the 3 affected DMD patients, linked through paternal lines, have different mutations [Bundey, personal communication]. The probability for the occurrence of 3 new mutations, by chance is 1/104 x 1110~x 1i104, i.e., 1 in 1,OOO,OOO,OOO,OOO! Although we have no explanation, a t present, for these findings, we offer the following hypotheses for our families:
Autosomal Recessive Inheritance We have suggested in a n earlier report [Zatz et al., 19891 t h a t about 2-4% of patients with a “Duchennelike” phenotype might be inherited through autosomal recessive inheritance. Such patients, with a clinical picture very similar to X-linked DMD, would be dystrophin-positive. However, autosomal recessive inheritance would not explain the occurrence of the 4 families described in the present report because (1)in 3 families (1,2, and 3) a deletion in the dystrophin gene was found in the affected males confirming X-linked inheritance; (2) in family 4, in which no DNA deletion was detected, the muscle biopsy from the 2 affected brothers (IV-2 and IV-4) showed a negative dystrophin pattern characteristic of X-linked DMD. In addition, the mother and the 2 sisters of the patients had grossly elevated serum enzymes; (3) no consanguinity was found among the parents of affected patients in any of the 4 families.
Duchenne Dystrophy Inherited Through Paternal Lines
Presence of Male X- Chromosome Mosaicism or Intronic Nonpathogenic Deletion A partial deletion of the dystrophin gene transmitted by an unaffected male was described by Darras and Francke [19871. A similar finding was also reported in X-linked agammaglobulinemia (XLA) by Hendricks et al. [1989] who demonstrated that the XLA defect had been originated from a n healthy male. Intronic nonpathogenic deletions have already been found in normal relatives of DMD patients [Koh et al., 19871and might have a role in the generation of further lethal deletions [Hart et al., 19891. Such hypothesis could account for our family 2. Individual 1-2could carry a nonpathogenic deletion, which would be transmitted to individuals 11-1and 11-3,generate a pathogenic deletion in females 111-2 and 111-12 (who would be heterozygotes) and consequently the 2 affected males IV-5 and v-5. Germinal and somatic mosaicism could also explain our family 2, caused by a mutational event in individual 1-2. If a deletion had occurred during the last DNA replication of the oocyte that gave rise to individuals 11-1 and 11-3, a half chromatid mutation could have been transmitted that would have produced mosaicism of germinal and somatic tissues. In this case, individual 11-1 would be clinically unaffected (as if he were a female carrier of the abnormal gene) as well as his sister 11-3 but both would transmit the deletion to their daughters 111-2and 111-12and subsequently to the 2 affected males IV-5 and V-5. This hypothesis is not yet proven in humans but was suggested by Darras and Francke [19871 to explain the occurrence of their family in which the DMD gene was transmitted twice by an unaffected male to his carrier daughters. According to Bakker et al. [1989], germline mosaicism in both male and female carriers generates a recurrence risk for transmitters and patients of “new” DMD mutations. However, with the exception of family 2, the hypothesis of nonpathogenic intronic deletion or germinal mosaicism being transmitted by individual 1-2 would not explain our other 3 genealogies because it would require male-to-male transmission.
Presence of Transposable Elements in the Family According to Hoegerman and Rary [1986], mutations for fragile X-positive Martin-Bell syndrome and perhaps also achondroplasia may result from the insertion of transposable elements (TEs). Hypothetically, TEs could insert within exons, within introns in positions which would interfere with intron excision, or within regulatory sequences. They suggest that in the case of fragile X, loss of genetic function could result from the loss of genes distal to the site of insertion following subsequent TE excision without ligation of the resulting discontinuity. These authors suggest that in the case of fragile-X, nonaffected transmitting males would originate from a
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zygote in which the causative TE is integrated into another site in the genome. Hypothetically, the TE could either transpose to the X within the germline of “normal” transmitting males or within the germline of their progeny if the TE was transmitted to the progeny on another chromosome. They speculate also that the cytoplasm might be either relatively permissive or restrictive and that not all normal X chromosomes are equally competent for TE insertion. In the present study, with exception of family 4, in which the unrelated mother 111-4is heterozygote for the DMD gene, the presence of TEs a t another locus within the genome could offer a plausible explanation for families 1, 2, and 3, as hypothetically discussed below: In family 1,affected males V-1 and V-3 would have inherited a TE from their father and a normal X chromosome competent for TE insertion from their mother. In both patients, insertion in the maternal X chromosome would have occurred after fertilization following a subsequent TE excision leading to a DNA deletion in the dystrophin gene. Such a mechanism would also provide a n explanation for our finding of 2 different DNA deletions in individuals V-1 and V-3, as well for the rare complex DNA rearrangement reported in individual v-1. The same hypothesis could account for family 2, with the difference that the TE which might be already present in individuals 1-1or 1-2would have been transmitted by the females 111-2 and 111-12, resulting in the DMD carrier IV-3 and the affected male IV-5. In family 3, individual IV-1 could result from the same mechanism described for affected males V-1 and V-3 of family 1, while in the case of his affected cousin 111-39, the TE insertion and abnormal excision might have also occurred in the germline of his mother 11-12. This hypothesis would not explain our family 4, the only one in which no DNA deletions were detected in the affected members. However, if we assume that both the carrier mother 111-4 and the patient 111-25a r e the result of new mutations, the likelihood of the occurrence of one such family would be 4u x = 112500 x 1110,000 or 1in 25,000,000, which would still be possible, by chance, if we take into account the population of the State of Sao Paul0 . An interesting observation is that insertion of L1 sequences (which are human-specific family of long, interspersed, repetitive elements, dispersed throughout the genome) have been reported in 2 unrelated male patients with hemophilia A in a study of 240 unrelated cases screened for DNA studies [Kazazian et al., 19881. No abnormalities were detected in the factor VIII genes in the parents of both patients, suggesting that in both cases the mutations had arisen de novo. According to these authors, these L1 insertions are the first large nonviral insertions described in man which are not due to expansion of short repeats by unequal crossing-over events. They would represent a mechanism for mutation fundamentally different from those previously described. Because it is not known when these events might occur (whether in sperm or ovum, after fertilization, or during early stages of embryogenesis) the proportion of such insertions that are heritable is unknown.
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Yet, the finding of two L1 insertions among 240 patients with hemophilia A suggests that this mechanism of mutation is not uncommon. Most DMBiBMD patients cases are due to DNA deletions; however, the finding of DNA duplications leading to a DMD or BMD phenotype has also been reported in a proportion ofcases [Monaco et al., 1987; Hu et al., 19881. It would be very interesting to investigate if the presence of TEs could not be responsible for some of these duplications through direct insertion into the dystrophin gene or leading to unequal crossing-over. On the other hand, TEs could also offer a n alternative explanation for the observations of intronic nonpathogenic and pathogenic deletions in normal and affected members belonging to the same families [Koh et al., 1987; Hart et al., 19891. We suggest that the presence of TEs could be responsible in some families for a greater predisposition for the occurrence of DNA deletions (pathogenic or nonpathogenic), duplications, mutations, or complex rearrangements. However, such families would not receive the attention when the patient is a n isolated case or the TEs would be transmitted through maternal lines.
ACKNOWLEDGMENTS The collaboration of the following persons is gratefully acknowledged: Dr. Kay Davies for kindly providing the cDNA probes; Drs. Rita de Cassia M. Pavanello and Dr. Ivo Pavanello-Filho for performing the muscle biopsies; Drs. Celia P. Koiffman, Angela M. Vianna-Morgante, Anita Wajntal, and Paul0 A. Otto for helpful suggestions and criticism; to Marcia Luisa das Neves, Marta Canovas, Martha Alessandra de Oliveira Lima, Helena Beatriz Martins da Costa, and Roberto Schreiber; to Marco Antonio Xavier for drawings and photographs; to the staff from ABDIM for their collaboration with the affected patients. This work was supported with grants from FAPESP, CNPq, FINEP, Secretaria de Ci6ncia e Tecnologia do Estado de S. Paulo, and ABDIM.
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