Am. J. Hum. Genet. 54:575-585, 1994 Gonosomal Mosaicism in Myotonic Dystrophy Patients: Involvement of Mitotic Events in (CTG)n Repeat Variation and Selection against Extreme Expansion in Sperm Gert Jansen,* Patrick Willems,* Marga Coerwinkel,* Willy Nillesen,t Hubert Smeets,t Lieve Vits,* Chris Howeler,1 Han Brunnert and Be Wieringa* Department of Cell Biology and Histology and tDepartment of Human Genetics, Faculty of Medical Sciences, University of Nijmegen, Nijmegen; $Department of Medical Genetics, University of Antwerp, Antwerp; and IDepartment of Neurology, Academic Hospital Maastricht, University of Maastricht, Maastricht Summary Myotonic dystrophy (DM) is caused by abnormal expansion of a polymorphic (CTG). repeat, located in the DM protein kinase gene. We determined the (CTG). repeat lengths in a broad range of tissue DNAs from patients with mild, classical, or congenital manifestation of DM. Differences in the repeat length were seen in somatic tissues from single DM individuals and twins. Repeats appeared to expand to a similar extent in tissues originating from the same embryonal origin. In most male patients carrying intermediate- or small-sized expansions in blood, the repeat lengths covered a markedly wider range in sperm. In contrast, male patients with large allele expansions in blood (>700 CTGs) had similar or smaller repeats in sperm, when detectable. Sperm alleles with >1,000 CTGs were not seen. We conclude that DM patients can be considered gonosomal mosaics, i.e., combined somatic and germ-line tissue mosaics. Most remarkably, we observed multiple cases where the length distributions of intermediate- or small-sized alleles in fathers' sperm were significantly different from that in their offspring's blood. Our combined findings indicate that intergenerational length changes in the unstable CTG repeat are most likely to occur during early embryonic mitotic divisions in both somatic and germ-line tissue formation. Both the initial CTG length, the overall number of cell divisions involved in tissue formation, and perhaps a specific selection process in spermatogenesis may influence the dynamics of this process. A model explaining mitotic instability and sex-dependent segregation phenomena in DM manifestation is discussed. Introduction 1991), myotonic dystrophy (DM) (Brook et al. 1992; Fu Dynamic mutation in simple-sequence motifs (Richards et al. 1992; Mahadevan et al. 1992), Huntington disease and Sutherland 1992) is a new principle in human mo- (HD) (Huntington's Disease Collaborative Research lecular genetics. It has now been demonstrated that Group 1993), spinocerabellar ataxia type 1 (Orr et al. simple-sequence-motif instability is causally involved in 1993), and fraxE (Knight et al. 1993). Though quite spinal and bulbar muscular atrophy (or Kennedy dis- different disorders, fragile X syndrome, DM, and HD ease; La Spada et al. 1991), fragile X syndrome (Fu et al. may have several mutational/mechanistic aspects in 1991; Kremer et al. 1991; Verkerk et al. 1991; Yu et al. common. All three diseases show atypical segregation patterns, referred to as the "Sherman paradox" in frag- ile X syndrome (Sherman et al. 1985) and as "anticipa- Received August 9, 1993; accepted for publication December 3, tion" in DM (H6weler et al. 1989) and HD (Ridley et al. 1993. 1988). In each of these disorders, founder chromo- Address for correspondence and reprints: Be Wieringa, Depart- somes are implicated (Conneally et al. 1989; Richards et ment of Cell Biology and Histology Faculty of Medical Sciences, al. 1992; Imbert et al. 1993), repeat length and disease University of Nijmegen, P.O. Box 9101, 6500 HB, Nijmegen, The severity are correlated (Yu et al. 1992; Harley et al. Netherlands. © 1994 by The American Society of Human Genetics. All rights reserved. 1993; Huntington's Disease Collaborative Research 0002-9297/94/5404-0001$02.OO Group 1993), and there is an almost-identical threshold 575 576 Jansen et al. length for the trinucleotide motifs (approximately 35- To assess the occurrence of mitotic repeat-length varia- 40 trinucleotides) above which instability occurs (Brun- tions within DM patients, we analyzed a broad range of ner et al. 1992; Macpherson et al. 1992; Huntington's tissues from both single patients and twins with mild, Disease Collaborative Research Group 1993). Although average, and severe disease manifestation. To address this suggests that similar molecular mechanisms under- further the mechanism and developmental timing of the lie the unstable behavior of the mutations, in DM fami- CTG repeat instability, we also studied repeat lengths in lies the enormous fluctuations in repeat length and germ-line tissues and compared CTG length in father's clinical severity are most evident and easy to study. The sperm with that in his child's blood. Our findings show high prevalence of the disorder, the parental influences that intergenerational CTG-repeat-length changes oc- on disease severity (Harper 1989), the availability of cur mainly during early embryonic mitotic divisions, large families, and tissue biopsy samples render DM a resulting in both somatic and germ-line tissue mosaicism. particularly interesting model in which to study dy- namic mutations. Material and Methods The causative mutation in DM, an expanding (CTG)n repeat, is located in the 3' UTR of a gene encoding a Patient Tissues putative protein kinase, known as "DM-PK" (Brook et Blood samples were collected from a large panel of al. 1992; Fu et al. 1992; Jansen et al. 1992b; Mahadevan DM patients, investigated by the diagnostic DNA ser- et al. 1992). The gene is expressed predominantly in vices at the University of Nijmegen; the Department of smooth, skeletal, and heart muscle and at low levels in Medical Genetics, University of Antwerp; and the De- brain and endocrine tissues (Jansen et al. 1992b). This partment of Neurology, University of Maastricht. pattern of expression parallels the tissues affected by Sperm samples were obtained from selected patients, the DM phenotype. In normal individuals, the (CTG). by the diagnostic DNA services at the University of repeat may vary in length between 3 and 37 repeat units Nijmegen and the Department of Medical Genetics of (Brunner et al. 1992; H. Smeets, H. Brunner, and W. the University of Antwerp. DNA was isolated accord- Nillesen, personal communication) with length distri- ing to standard procedures (Miller et al. 1988). butions clustered at 5, 11-14, and 18-22 CTGs in the Tissue samples obtained at autopsy from one female normal population (Brook et al. 1992; Brunner et al. DM patient (patient F, who died at age 66 years and 1992; Fu et al. 1992; Mahadevan et al. 1992; Imbert et who presented classical DM features including myo- al. 1993). When tested within individual pedigrees, tonia, cataract, apathetic behavior, and progressive di- CTG repeats in the 3-37-trinucleotide size range segre- gestive and respiratory problems, with onset at age 42 gate stably in a Mendelian fashion. This suggests that years) were taken within 4 h of death. Samples of skele- the mutation rate for "normal" alleles has an upper tal muscle (quadriceps), heart muscle, ovary, uterus, limit similar to that estimated for many other microsat- skin, liver, kidney, brain (frontal cortex and thalamus), ellites typed in human and mouse Ueffreys et al. 1988), lung, adrenal gland, thymus, pancreas, mesenteric typically between 10-2 and 10'-. Expanded repeats are lymph node, and spleen of this patient were snap frozen much more unstable, and there is a positive correlation in liquid nitrogen and stored at -80'C. Similarly, tis- between the length of the repeat and its instability sues from a congenital case (patient L), two congenital (Hunter et al. 1992; Shelbourne et al. 1992; Brunner et twins (28-wk-old prematures who died 2 wk after birth al. 1993a; Lavedan et al. 1993b). The length of the re- from respiratory insufficiency), and an affected embryo peat ranges from 42 to approximately 150 in nonmani- (P) aborted in week 12 were prepared and stored at festing or mildly affected patients to several thousand -80°C for DNA analysis. DNA was isolated using CTG units in more severely affected individuals or con- standard procedures (Maniatis et al. 1988). Clinical genital cases of DM (Hunter et al. 1992; Brunner et al. diagnosis of patients was as based on the criteria estab- 1993a; Harley et al. 1993). lished by the Myotonic Dystrophy Working Group While studying CTG repeat lengths in tissue speci- (Griggs et al. 1989). mens from various DM individuals, we observed that CTG repeats up to approximately 100 repeat units were Validation and Optimalization of the (CTG)n Length detected as defined, though often faint, bands. Larger Analyses: Southern Blotting versus PCR Analysis repeat alleles appeared as a diffuse smear because of For the examination of genomic DNAs from individ- mitotic instability (Brunner et al. 1992; Hunter et al. ual tissue samples Southern blot analysis and PCR typ- 1992; Mahadevan et al. 1992; Shelbourne et al. 1992). ing were used. Southern blot analysis is the only suit- Gonosomal Mosaicism in Myotonic Dystrophy Patients S77 able method for the analysis of long and heterogeneous EDTA, 50 mM NaCl) were equilibrated for 30 min in CTG expansions in tissues from severely affected classi- 30 mM NaOH, 1 mM EDTA; and PCR samples were cal DM patients and cases of congenital DM. For each adjusted to 10 mM NaOH, 0.2 mM EDTA, 0.6% Fi- sample, 10 ,ug of genomic DNA was digested with coll-400, 0.01% bromophenol blue dye, before loading HindIII (Bethesda Research Laboratories), electro- on the gel. Alkaline gels were electrophoresed for 24 h phoresed on 0.7% agarose gels, and transferred onto at 20 V in 30 mM NaOH, 1 mM EDTA, and blotted nylon membranes (Hybond N+; Amersham). Southern and probed as described for the standard assay above. blots were probed with the 1.8-kbp HindIII/BamHI By using the denaturing electrophoresis where heterolo- fragment of probe pGB2.6 (Aslanidis et al.