Hum Genet (1990) 86:241-243

9 Springer-Verlag1990

Germinal mosaicism from grand-paternal origin in a family with Duchenne muscular dystrophy Mireille Claustres, Paule Kjellberg, Marie Desgeorges, H~i~ne Bellet, and Jacques Demaille INSERM U 249, CRBM / CNRS UPR 8402 and GREPAM, Institut de Biologie, F-34000 MontpeUier, France Received June 22, 1990

Summary. We have identified a Duchenne muscular dystrophy (DMD) pedigree with an unexpected pattern of inheritance. Using marker restriction fragment length polymorphisms detected by probes that lie within and outside the D M D gene, we could demonstrate that the maternal grandfather has transmitted two distinct types of X chromosomes to his offspring. This original observation may be explained by postulating that the DMD mutation must have occurred during mitosis in early germline proliferation, leading to a germline mosaicism within this male ancestor.

Materials and methods

Introduction

D N A analysis

Duchenne muscular dystrophy (DMD) is a lethal Xlinked recessive disorder that affects approximately 1 in 3300 liveborn males and is associated with a high mutation rate. One-third of D M D patients are believed to have the condition as the result of a "new mutation" present in one or more progeny and absent from the lymphocytes of the mother and the grandparents. However, estimations of the recurrence risk and genetic counselling have been drastically complicated by recent reports of new mutations transmitted more than once by a parent in whom lymphocytic chromosomes were normal. The advent of direct detection of the D M D mutation has allowed the demonstration of several cases of germline mosaicism that can explain these observations (Bakker et al. 1987; W o o d and McGillivray 1988; Bakker et al. 1989). H e r e we report a family with an unusual pattern of transmittance of a D M D mutation, for which the examination of haplotypes using marker restriction fragment length polymorphisms (RFLPs) demonstrated that the grandfather had transmitted more than one X-chromosome to his offspring. Offprint requests to: M. Claustres, Laboratoire de Biochimie Grn& tique, Insfitut de Biologie, Boulevard Henri IV, F-34060 Montpellier, France

eamily During the analysis of DMD pedigrees in Southern France, we encountered the case illustrated in Fig. 1. When the family was referred for DNA genotyping, only one boy (IIIa, age 10) was known to have DMD, and the 89 of new mutation was taken into account to calculate the carrier status of the female family members seen. Systematic determination of the serum levels of creatine phosphokinase (CPK) revealed that a second boy was affected with DMD (III6, age 3; CPK levels: 16233 and 19404 units/1 on two different determinations). All individuals other than the two probands had normal CPK levels.

The preparation and analysis of genomic DNA were carried out using standard methodology for familial genotyping with genornic probes. Screening for deletion of the DMD gene was performed by direct amplification of exons by the polymerase chain reaction (PCR "multiplex"; Chamberlain et al. 1988; Beggs et al. 1990) and by classic Southern blotting with the cDNA probes 1-8 (Koenig et al. 1987) on HindIII and TaqI digests. To exclude mislabelling, new blood samples of the two affected boys, their mother and the grandparents were obtained and the whole RFLP analysis repeated.

Results and discussion On the basis of the pedigree data before D N A analysis, the family shown in Fig. 1 seemed to be a typical familial case of DMD: two cousins being affected, their mothers were obligate carriers and the grandmother a presumptive obligate carrier. No deletion or duplication could be detected in the D N A of the probands by PCR or cDNA/ Southern techniques, so that genetic counselling in this family was based only on the indirect analysis of the segregation of markers. A series of intragenic and flanking D N A probes was used to detect informative RFLPs in the family. Unexpectedly, three extragenic flanking RFLPs demonstrated that the grandmother (I2) could not have con-

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Fig. 1. Pedigree of family and genotypic interpretation of DNAtyping results using genomic probes. Distinct haplotypes are represented by vertical bars. 1 Longest restriction allele, 2 smallest restriction allele. Haplotype analysis demonstrates that the chromosome carrying the DMD mutation in the two probands and their mothers came from the grandfather. Asterisks indicate the extragenic RFLPs that are essential in deriving this conclusion. The box contains the genomic probes, which are listed according to the gene order from the most telomeric (top) to the most centromeric (bottom) locus

tributed to the mutant X chromosome of the two probands. She appeared to be homozygous for one telomefic (99.6) and two centromefic (754,754-11) markers, whereas the two affected cousins presented other alleles, which were inherited from the grandfather (I1), at these loci. In the second generation, the two mothers (II2 and II6) of the affected boys inherited the mutant X chromosome from their father. It can be postulated that the daughter (II4), having transmitted the normal grandpaternal X chromosome to her healthy son, is not a carrier. The problem was to ascertain the risk of being a carrier for the youngest daughter (IIs), who transmitted a grandmaternal chromosome to her two healthy sons. Without the presence e l an intragenic specific lesion that would allow the differentiation of the two grandpaternal

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X chromosomes, it was impossible by linkage analysis alone to know if she had inherited the normal or the D M D chromosome from her father. Consequently, the grandfather must have transmitted a D M D - X chromosome to at least two of his daughters, although he himself does not carry the mutation in his somatic cells. This phenotypically normal parent is carrying two types of X chromosomes, most probably because of a post-zygotic mutation within the dystrophin gene; this may have occurred in a germline cell as a mitotic event, leading to gonadal mosaicism. Identification of germinal mosaicism has fundamental implications, since, as reviewed by HaU (1988), this phenomenon could play a major role in mutagenesis in man in X-linked and autosomal disorders. For the clinicians involved in genetic counselling, germline mosaicism appears to be an important diagnostic pitfall (Boileau and Junien 1989) and must be taken into account not only in sporadic but also in familial cases. Our report illustrates the value of extended RFLPs studies in D M D pedigrees, with polymorphisms tested on many individuals from several generations. Acknowledgements. The authors thank Drs. Kunkel, Worton, Koenig and Monaco for making their probes available, Professor B.Echenne and Dr. P. Sarda for referring the pedigree, and M. Nicolas for typing the manuscript. This work was supported by the Association Frangaise centre les Myopathies (AFM).

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References Bakker E, Van Broeckhoven C, Bonten EJ, Van de Vooren MJ, Veenema H, Van Hul W, Ommen GJB van, Vandenberghe A, Pearson PL (1987) Germline mosa'icism and Duchenne muscular dystrophy mutations. Nature 329 : 554-556 Bakker E, Veenema H, Den Dunnen JT, Van Broeckhoven C, Grootscholten PM, Bonten EJ, Ommen GJB van, Pearson PL (1989) Germinal mosa'icism increases the recurrence risk for "new" Duchenne muscular dystrophy mutations. J Med Genet 26: 553-559 Beggs AH, Koenig M, Boyce F, Kunkel LM (1990) PCR primers for the dystrophin gene that complement existing ones to detect over 98% of DMD/BMD deletions. Am J Hum Genet (in press) Boileau C, Junien C (1989) Misdiagnosed normal fetus owing to undetected germinal mosa'icism for DMD deletion. J Med Genet 26: 790-792

Chamberlain JS, Gibbs RA, Ranier JE, Nguyen PN, Caskey CT (1988) Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA ampfification. Nucleic Acids Res 16:11141-11156 Hall JG (1988) Review and hypotheses: somatic mosa'icism: observations related to clinical genetics. Am J Hum Genet 43: 355363 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 50: 509-517 Wood S, McGillivray BC (1988) Germinal mosa'icism in Duchenne muscular dystrophy. Hum Genet 78: 282-284

Germinal mosaicism from grand-paternal origin in a family with Duchenne muscular dystrophy.

We have identified a Duchenne muscular dystrophy (DMD) pedigree with an unexpected pattern of inheritance. Using marker restriction fragment length po...
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