Hum Genet (1988) 78 : 282-284

© Springer-Verlag 1988

Germinal mosaicism in Duchenne muscular dystrophy

Stephen Wood and Barbara C. McGillivray

Department of Medical Genetics, U.B.C., 6174 University Boulevard, Vancouver, B.C. V6T lW5, Canada

Summary. We have identified a Duchenne muscular dys- trophy (DMD) pedigree where the disease is associated with a molecular deletion within the DMD locus. We have examined the meiotic segregation products of the common female ances- tor using marker restriction fragment length polymorphisms (RFLPs) detected by probes that lie within this deletion. These studies show that this female has transmitted three dis- tinct types of X chromosome to her offspring. This observa- tion may be explained by postulating that the mutation arose as a postzygotic deletion within this common ancestor, who was consequently germinally mosaic.

Introduction

Duchenne muscular dystrophy (DMD) is a severe disorder of muscle wasting showing X-linked inheritance. This mutation is a genetic lethal. Recently it has become possible to assess X chromosome segregation using restriction fragment length polymorphic (RFLP) markers that are linked to the DMD gene or that lie within the DMD gene (Bakker et al. 1985; Monaco et al. 1985; Ray et al. 1985). Thus genetic prediction of carrier status and prenatal prediction of disease is possible in informative families where the phase is known, or can be reliably inferred. Genetic prediction for relatives of isolated cases of DMD however, is confounded by the frequent occur- rence of mutation.

Direct detection of the molecular defect is possible in the approximately 8% of patients that exhibit deletions (Kunkel et al. 1986) using conventional electrophoretic methods with currently available probes. Female relatives of deletion pa- tients who are heterozygous for marker sites within the dele- tion would be considered to be non-carriers of the deletion. This is not true for mothers of isolated deletion patients since a de novo mutant gamete may be a member of a clone within a mosaic individual. We report here that the common female ancestor of DMD deletion patients has transmitted to her off- spring both alternative allelic forms of a RFLP marker site that is contained within the deletion.

Materials and methods

Marker typing

Genomic DNA was isolated from white cells, restriction di- gested, and Southern blots prepared. Restriction fragments

Offprint requests to: S. Wood

were separated by electrophoresis on horizontal 0.8% agarose gels and transferred to Nytran. DNA probes were labeled with 32-P using random priming (Feinberg and Vogelstein 1984). Prehybridization for at least 2h at 62°C, in 6 × SSC (sodium chloride/sodium citrate), 5 × Denhardt's, 100ug/ml sheared salmon sperm DNA, 0.1% SDS (sodium dodecyl sul- fate), was followed by overnight hybridization in the same so- lution containing labeled DNA probe. Filters were washed in 0.1 × SSC, 0.1% SDS for 15min at room temperature and twice at 62°C for 30 min. Filters were autoradiographed for 2 days at -70°C using Kodak XRP-1 X-ray film with Dupont Cronex Lightening-Plus intensifying screens. The markers typed are listed in Table 1. The allele designations and D numbers are taken from HGM8 (Willard et al. 1985) or the Human Gene Mapping Library, Yale, where available. Ini- tially individuals II-1 to II-4 were tested for the distal markers DXS43 (pD2), DXS41 (p99-6), and DXS28 (C7); markers within the DMD gene using DXS164 (pERT87) and DXS206 (pXJl.1); and the proximal markers DXS84 (754) and DXS7 (L1.28). Subsequently when additional family members were typed and individual III-3 was found to have a deletion involv- ing DXS164, the flanking DXS28 and DXS206 markers to- gether with DXS7 were typed.

Table 1. Markers typed in this family (the loci are listed according to their order from telomere to centromere)

Locus Probe Enzyme Allele Fragment size kb

DXS43 pD2 PvuII AI 6.0

A2 6.6

DXS41 p99.6 PstI A1 22

A2 13

DXS28 C7 EcoRV A1 7.5

A2 8.0

DXS164 pERT87-15 XmnI F1 1.2;1.6

F2 2.8

DXS164 pERT87-15 TaqI G1 3.1

G2 3.3

DXS206 pXJ1.1 TaqI A1 3.1

A2 3.8

DXS84 754 PstI A1 12

A2 9

DXS7 L1.28 TaqI A1 12

A2 9

Table 2. Sizes of restriction fragments observed in family members

Pedigree pD2 p99.6 C7 pERT87-15 pERT87-15 pXJl.1 754 L1.28 number PvuII PstI EcoRV XmnI TaqI TaqI PstI TaqI

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II-1 6.0;6.6 22;13 7.5 1.2;1.6;2.8 3.1;3.3 3.1 9 12;9

II-2 6.0 22;13 7.5;8.0 2.8 3.1 3.1;3.8 9 12;9

II-3 6.0 22;13 7.5;8.0 2.8 3.1 3.1;3.8 9 12;9

II-4 6.0;6.6 22;13 7.5 1.2;1.6;2.8 3.1;3.3 3.1 9 12;9

II-5 NT NT 7.5;8.0 2.8 3.1 3.1;3.8 NT a 12;9

II-6 NT NT 8.0 2.8 3.1 3.1 ;3.8 NT 12 ;9

II-7 NT NT 7.5 1.2;1.6 3.1 3.1 NT 12

III-1 NT NT 7.5;8.0 1.2;1.6 3.1 3.8 NT 9

III-2 NT NT 7.5;8.0 1.2;1.6 3.1 3.1;3.8 NT 12;9

III-3 NT NT 8.0 No bands No bands 3.8 NT 12

a NT indicates not typed

Results

The sizes of restriction fragments observed are shown in Table 2 while Fig. 1 shows two Southern blots aligned with the typed markers of the pedigree, together with our genotypic interpre- tation of this data. Duchenne muscular dystrophy (DMD) in

I

I I

I I I

ii Y Y A1 A1 F2

A1 A2 A2 A1 A2 F1 F del F2 F1 NT

G1 G2 ]del NT G1 I del AIG1

A2 A2 A1 A2 A1 A2 A2 A1 A2 A1 A2 NT A2 A2 A2 A2 NT FI deL NT

2 2 G1 de[ A2 A2

NT

individual III-3 is associated with a deletion involving the DXS164 locus (Fig. 1). The distal or telomeric extent of the deletion has not been defined, but involves the pERT87-30 se- quences. Proximally the deletion does not extend into DXS206 since normal-sized fragments are detected by pXJ10.1 (data not shown). Individuals III-1 and III-2 both exhibit non- maternal inheritance of the DXS164 marker pERT87-15: XmnI. DNA typing shows the F1, 1.2kb (kilobase pair) + 1.6 kb, allele only in these daughters and the F2, 2.8 kb, allele only in their mothers. Thus these daughters are both carriers, inheriting the deletion chromosome from their mothers. This deletion chromosome is inherited by III-1, III-2, and III-3 to- gether with the A2 alleles at the DXS28 and DXS206 loci. These close flanking markers are likely to maintain coupling with the deletion chromosome, particularly DXS206 which lies within 100 kb of the deletion breakpoint.

Individuals II-1 and II-4 are not carriers of DMD, since they are heterozygous for DXS164 markers, inheriting the haplotype F2G1 from their father and F1G2 from their mother. They also inherit from their mother the A1 alleles at both flanking DXS28 and DXS206 loci. Individuals II-2, II-3, and II-5 inherit the alternate maternal A2 alleles at the DXS28 and DXS206 loci. Whereas both II-2 and II-5 are obli- gate carriers, II-3 has produced three unaffected sons. The pERT87-15 hybridizing fragments from II-3 are surprisingly intense (Fig. 1), inconsistent with the monosomy for this re- gion that is suggested by the flanking DXS28 and DXS206 markers. The autoradiograph of the TaqI digests that were simultaneously probed with pERT87-15 and L1.28 (Fig. 1) was evaluated by densitometric scanning. The pERT87- 15 : L1.28 ratio was 0.97 for II-3, whereas the ratios for the ob-

Fig. 1. Pedigree of family and genotypic interpretation of DNA typing results. II-2, II-5, and II-6 are shown as obligate heterozygotes since they all have had at least one son affected with DMD. The diagram- matic representation of DNA markers indicates (from top to bottom, telomeric to centromeric) typing for DXS43 (pD2), DXS41 (p99-6), DXS28 (C7), DXS164 (pERT87-15:XmnI and pERT87-15:TaqI), DXS206 (pXJl.1), DXS84 (754), and DXS7 (L1.28). N T indicates not typed and del indicates a deletion. Maternally derived markers only are shown for II-1 to II-5 with their inferred shared, paternally derived markers being indicated by their untyped deceased father. The Southern blots show typing for L1.28:TaqI (A1 is 12kb and A2 is 9kb), pERT87-15:TaqI (G1 is 3.1kb and G2 is 3.3kb) and pERT87-15:XmnI (F1 is 1.2kb + 1.6kb and F2 is 2.8kb)

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ligate deletion carriers and their daughters were all below 0.5. Thus the G1 allelic fragment in II-3 is present in two copies. Consequently the common female ancestor I-2 has transmit- ted a chromosome with a DXS164 deletion to II-2, II-5 and I1- 6, whereas II-1 and II-4 have inherited a DXS164 F1G2 hap- lotype and II-3 a DXS164 F2G1 haplotype.

Discussion

These family studies show that four types of X chromosome, as defined by RFLP markers within the DXS164 locus, are shared among the five full sisters II-1 to II-5. We propose that one chromosome type is paternally derived and three types are maternally derived. II-3 is homozygous for the F2G1 hap- lotype, II-1 and II-4 are heterozygous F1G2/F2G1, and II-2 and II-5 are hemizygous deletionlF2G1.

The mother (1-2), who had no siblings or maternal rela- tives affected with DMD, must have transmitted the F2G1 haplotype to II-3 since this daughter is homozygous. The mother also transmitted a null deletion haplotype to II-2, II-5 and II-6 (as it is unlikely that both of her husbands were mosaic for a DMD deletion). We propose that the mother also transmitted a third FIG2 haplotype to II-1 and II-4, being consistent with all five full sisters having the same biological father. We feel that an alternative hypothesis of non-paternity is less tenable. This would require non-paternity for both II-1 and II-4 and either non-paternity for II-3 or a double cross- over in the intervals DXS28-DXS164 and DXS164-DXS206. We find this latter explanation less likely than the hypothesis of transmission of three distinctive DXS164 haplotypes by I-2.

Thus we consider this data consistent with, and indicative of, a de novo postzygotic deletion of the chromosome bearing the A2 alleles at the DXS28 and DXS206 loci in this common maternal ancestor (I-2), who was thus germinally and perhaps somatically mosaic. This chromosome, bearing the A2 alleles at both DXS28 and DXS206, has been evaluated in four meio- tic products and found to be intact only once. This provides an estimate of the fraction of the deletion clone in I-2 as 0.75. Haldane (1935) showed that for an X-linked lethal mutation the fraction of mutant phenotypes that arose de novo was 1/3 (if the mutation rates showed no sex difference). He defined the mutation rate as the probability of finding in any particular gamete an X-chromosome carrying a mutant allele although that chromosome carried a normal allele when the individual was conceived. In calculating the genetic risk for the mother of an isolated case of DMD it is usual to consider the a priori risk of the carrier state as 2/5, meaning the probability that the mother was conceived a carrier, and to ignore the possibility of the mother being mosaic, Presently the frequency of such mosaicism is unknown. This question has recently been ad-

dressed by Edwards (1986) who has introduced the concept of "embryonic mutation rate," where the de novo event occurs sufficiently early in embryogenesis to produce mosaicism.

If the mutation is a deletion, the status of the mother, at least in somatic cells, may be tested. Thus demonstration of heterozygosity for markers within a deletion shows that the deletion arose de novo. This means that the mother was con- ceived as a non-carrier but does not mean that her risk is not increased. The mutation may have occurred during gameto- genesis leading to no increased risk, or during embryogenesis leading to an increased risk that depends upon the size of the mutant clone in the mosaic mother. Thus it seems prudent to provide DNA testing for sisters of isolated DMD deletion pa- tients even when the mother appears not to be a deletion car- rier.

Acknowledgements. We thank Drs. L. M. Kunkel, P. L. Pearson, J.-L. Mandel, and R. G. Worton for gifts of their probes used in this study. This work was supported by the Muscular Dystrophy Association of Canada.

References

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Kunkel LM, et al (1986) Analysis of deletions in DNA from patients with Becker and Duchenne muscular dystrophy. Nature 322:73- 77

Monaco AP, Bertelson CJ, Middlesworth W, Colletfi C-A, Aldridge J, Fischbeck KH, Bartlett R, Pericak-Vance MA, Roses AD, Kunkel LM (1985) Detection of deletions spanning the Duchenne muscular dystrophy locus using a tightly linked DNA segment. Nature 316 : 842-845

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Received July 7, 1987 / Revised September 25, 1987