RESEARCH ARTICLE
Anticipation in Myotonic Dystrophy Type 1 Parents with Small CTG Expansions Annabelle Pratte,1* Claude Pre´vost,2 Jack Puymirat,3 and Jean Mathieu4,5 1
Division of Genetic Counseling, CSSS Chicoutimi, Chicoutimi, Quebec, Canada
2
Neuromuscular Clinic, CSSS Jonquiere, Jonquiere, Quebec, Canada Department of Human Genetics, Centre Hospitalier de l’Universite´ Laval, Quebec, Quebec, Canada 4 Faculty of Medicine and Health Sciences, Sherbrooke University, Sherbrooke, Quebec, Canada 3
5
Neuromuscular Clinic, CSSS Jonquiere, Jonquiere, Quebec, Canada
Manuscript Received: 21 March 2014; Manuscript Accepted: 21 December 2014
Myotonic dystrophy type 1 is the most common form of adult muscular dystrophy and has the world’s highest prevalence in the Saguenay-Lac-St-Jean region, due to a founder effect. This autosomal dominant disorder results from an unstable CTG repeat expansion in DMPK. This region of Canada has had a family screening and predictive testing program for this disorder since 1988. Heterozygotes for small expansions (50–100 CTG repeats) can be asymptomatic or minimally affected. The aim of this study was to assess anticipation for these individuals. At the time of this study, the molecular data of 40 individuals and their 76 affected children were available. We compared 76 parent-child pairs. Most offspring (92.1%) had a larger number of repeats than their parent and the median number of repeats in the offspring was 325 (range, 57–2000). The number of CTG repeats was significantly greater when the mutation was transmitted by a father (median, 425 repeats; range, 70–2000), than when it was transmitted by a mother (median, 200 repeats; range, 57–1400). The majority (65.8%) of children also had a more severe phenotype than their parent but the sex of the parent had no significant influence on the severity of the child’s phenotype. No congenital phenotype was observed. These results confirm that anticipation is present even when the parent is heterozygous for a small CTG expansion. The parental sex has an impact on the size of the repeat in the next generation, larger increases being transmitted by males with a small expansion. Ó 2015 Wiley Periodicals, Inc.
Key words: myotonic dystrophy; DMPK gene; CTG repeat; anticipation; genetic counseling
INTRODUCTION Myotonic dystrophy type 1 (DM1; OMIM #160900), also known as dystrophia myotonica or Steinert disease, is the most common form of adult muscular dystrophy. DM1 has an overall worldwide prevalence of approximately 2.1–14.3 per 100,000 [Harper, 2001]. However, its prevalence in the Saguenay-Lac-St-Jean (SLSJ) region, located in the north eastern part of the province of Quebec, Canada, was reported to be of 158 per 100,000 in 2010
Ó 2015 Wiley Periodicals, Inc.
How to Cite this Article: Pratte A, Pre´vost C, Puymirat J, Mathieu J. 2015. Anticipation in myotonic dystrophy type 1 parents with small CTG expansions. Am J Med Genet Part A 167A:708–714.
[Mathieu and Prevost, 2012]. This prevalence is the highest in the world due to a founder effect. This disorder is inherited in an autosomal dominant pattern and results from an unstable cytosine, thymine, and guanine (CTG) repeat expansion in the 3’ untranslated region of the myotonin kinase gene at 19q13.3 [Aslanidis et al., 1992; Brook et al., 1992; Buxton et al., 1992; Harley et al., 1992]. When transcribed into CUG-containing RNA, mutant transcripts aggregate as nuclear foci that sequester RNA-binding proteins, including members of the muscleblind (MBNL) family, resulting in a spliceopathy of downstream effector genes [Jiang et al., 2004]. The DM1 phenotype is a multisystem condition characterized by myotonia, progressive muscle weakness and wasting, cataracts, endocrine disorder, frontal balding, hypogonadism, diabetes mellitus, and cardiac conduction disorder. The severity of the condition is highly variable. The DM1 phenotype is classified according to the age at onset of symptoms: resent Conflict of interest: All authors have no conflict of interest to declare. Grant sponsor: Neuromuscular Partnership Program of Muscular Dystrophy Canada and Canadian Institutes of Health Research (CIHR); Grant number: MOP49556; Grant sponsor: ECOGENE-21 and CIHR; Grant number: CAR43283. Correspondence to: Annabelle Pratte, M.Sc., (C)CGC, Division of Genetic Counseling, CSSS Chicoutimi, 305 St-Vallier, Chicoutimi (Quebec), Canada, G7H 5H6. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 25 February 2015 DOI 10.1002/ajmg.a.36950
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PRATTE ET AL. at birth), childhood (onset between 1 and 10 years of age), early adult (onset from 11 to 20 years of age), adult (onset between 21 and 40 years of age), and mild (onset after 40 years of age) [Koch et al., 1991]. There is a correlation between the number of CTG repeats and the severity of the disease, and an inverse correlation between the number of repeats and the age at onset of symptoms [Hunter et al., 1992; Harley et al., 1993]. Carriers of a relatively small expansion, i.e., 50 to 100 repeats, can be asymptomatic or minimally affected (mostly cataracts) [Arsenault et al., 2006]. The genetics of DM1 is characterized by anticipation [Harley et al., 1992; Harper et al., 1992; Hunter et al., 1992], which is the occurrence of increasing phenotype severity and decreasing age of onset in subsequent generations. This is explained by the fact that the expanded trinucleotide CTG repeat sequence is markedly unstable and prone to expand in successive generations. Two principal factors have been found to determine the degree of expansion (or reduction) in the transmission of the mutation: (1) the size of the parental repeat and (2) the sex of the transmitting parent. According to Martorell et al. [2007]; without regards to the parent’s expansion size, the mean intergenerational variation is minimal when the disease is transmitted by a male and is very high when transmitted by a female. Other studies found the same greater tendency to expansion in female transmission [Lavedan et al., 1993; Redman et al., 1993; Eguchi and Tsuji, 1997; Kim et al., 2008]. According to Harper and Johnson [2001]; over the entire range of repeat lengths, there is no clear sex difference in expansion between the generations [Tsilfidis et al., 1992; Harley et al., 1993]. Some authors have observed that small expansions can be inherited relatively stably for several generations if transmitted by women, but passage through the male germline usually results in a larger increase into the full disease range [Barcelo´ et al., 1993; Brunner et al., 1993; Ashizawa et al., 1994B; Simmons et al., 1998; Martorell et al., 2001; Abbruzzese et al., 2002]. In brief, different studies have been done to address this question and results are variable. There is still confusion regarding anticipation in DM1 for small expansion heterozygotes and there is a need to investigate further. The aim of this study is to assess anticipation in DM1 families when the transmitting parent is heterozygous for a small expansion (50– 100 CTG repeats).
MATERIALS AND METHODS Study Design and Sample At the time of this study, 801 individuals were recruited into the DM1 family screening and predictive testing program coordinated through the SLSJ Neuromuscular Clinic of the CSSS de Jonquie`re. From this group, 302 individuals were affected by DM1 and 499 individuals were homozygous wild type for the mutation. This program is provided by a multidisciplinary team including a neurologist, a genetic counselor and a specialized nurse [Pre´vost et al., 2004]. Participants are recruited in the program if they met the following criteria: (1) at least 18 years of age and mentally capable; (2) having a confirmed family history of DM1 or showing symptoms of the condition. After obtaining a signed informed consent from each participant, a blood sample is drawn for mutation
709 screening. Their demographic and clinical data are stored on a confidential computer database at the clinic since 1983 [Mathieu et al., 2012]. For the present study, data extracted from the registry included date of birth, gender, size of CTG expansion, DM1 phenotype and number of children. Most affected individuals were followed on a yearly basis, permitting to assess their phenotype’s progression regularly. The DM1 phenotype classification used at the Neuromuscular Clinic is based on the age at onset of symptoms [Koch et al., 1991], as described above. However, after the introduction of DNA analysis in predictive testing for DM1 in 1993, subjects were classified as having the mild form of the disease if they presented at least two of the three following criteria: (1) CTG less than 200, (2) Muscular Impairment Rating Scale score of 1 (no muscular impairment) or 2 (minimal signs), and (3) age at onset of symptoms greater than 40 years [Gagnon et al., 2008; Mathieu et al., 2012]. Muscular impairment was defined using a five-point Muscular Impairment Rating Scale (MIRS) [Mathieu et al., 2001]. Genetic testing for DM1 was performed through linkage analysis from 1988 to 1993 and, since 1993, by direct mutation analysis. The molecular analyses were done at the Centre Hospitalier Universitaire de Que´bec (CHUQ) in Quebec city. Genomic DNA was extracted from peripheral blood samples using QIAamp DNA blood kit (Qiagen Sciences, Germantown, Maryland) according to the manufacturer’s instructions. The DNA (3–5 ug) was digested with EcoRI (new England Biolabs), electrophoresed on 0.8% agarose gels, Southern transferred onto a nylon membrane (Hybond, Amersham) and probed overnight with radiolabeled 2.2 kb BamHI/ EcoRI subclone of probe pGB2.66, as previously described [Furling et al., 2001]. A PCR amplification of the CTG repeat was found to provide an accurate assessment of its size. Genomic DNA (1 ug) was PCR-amplified with primers 406 and 409 using standard protocol [Mahadevan et al., 1992]. The subjects enrolled in the study were assigned a code, which separated all clinical data from personally identifiable information. Our study was based on (1) a pedigree analysis and (2) the analysis of the clinical and genetic testing results of the participants to the DM1 testing program. We explored the number of individuals heterozygous for 50 to 100 CTG repeats, the number of CTG repeats and the DM1 phenotype of their affected offspring.
Statistical Analysis The non parametric Mann-Whitney U test and the Chi-square test were used to compare the number of CTG repeats in the offspring according to the sex of the parent. A Mann-Whitney U test was used to compare the difference in the number of CTG repeats between the child and the transmitting parent. A Chi-Square test was also used to compare the DM1 phenotypes among offsprings according to the parental sex. A P-value less than 0.05 was considered significant. Statistical analyses were performed using the SPSS statistical software package (SPSS 21, 2012).
RESULTS Characteristics of the Sample At the time of the study, molecular data were available for 40 parents heterozygous for alleles from 50 to 100 CTG repeats (20 mothers
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and 20 fathers) and their 76 affected children, including three fetuses. Parent-child pairs were obtained by comparing the parent with each of his affected offspring for which molecular testing was done. Parents having more than one affected child were therefore part of more than one pair. In the following analyses, we compared 76 parent-child pairs. Twenty (20) parents contributed one child each, 10 parents contributed two children, six parents had three children, two parents had four children and two parents had five.
Outcome in Offspring Figure 1 shows the number of CTG repeats in the offspring according to the number of repeats in their parent (38 motherchild pairs and 38 father-child pairs). Most offspring (70/76 or 92.1%) had a larger number of repeats than the parent; the median number of repeats in the offspring was 325 (range, 57–2000). The number of repeats in the offspring was significantly greater when the mutation is transmitted by a father (median, 425 repeats; range,
FIG. 1. Number of CTG repeats for 76 parent-child pairs according to the mutation’s parental origin. Most offspring have a larger number of repeats than the parent. The number of repeats in the offspring is greater when the mutation is transmitted by a father, than when it is transmitted by a mother.
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TABLE I. Number of CTG Repeats of 76 Offspring According to the Parental Sex Parental sex CTG Repeats in child 50–100 101–200 201–999 1000 Total
Male n (%) 3 (7.9) 7 (18.4) 20 (52.6) 8 (21.1) 38 (100)
Female n (%) 13 (34.2) 7 (18.4) 13 (34.2) 5 (13.2) 38 (100)
Total n (%) 16 (21.1) 14 (18.4) 33 (43.4) 13 (17.1) 76 (100)
Chi-square, P ¼ 0.038.
70–2000), than when it was transmitted by a mother (median, 200 repeats; range, 57–1400) (P ¼ 0.027). Table I shows the number of repeats in children according to the parental origin. There was a statistically significant difference between the parental sex, a larger proportion of children of affected males being found in the 201–999 and the 1000 CTG groups (P ¼ 0.038); and, conversely, a larger proportion of children of affected females is found in the 50–100 CTG group. The majority (60.5%) of children had more than 200 repeats, a genotype associated with the classic adult onset of DM1 or a more severe phenotype for those having 1000 repeats. Only 21.1% of all children had between 50 and 100 repeats with an expected mild phenotype as observed in their parent. According to these data, the respective risk for a father and a mother heterozygous for a small CTG expansion to have a child with a significant increase, i.e., 200 repeats and higher, was 73.6% (28/38) and 47.4% (18/38). Expressed as the differential change in the number of CTG repeats between the offspring and the transmitting parent, we observed the same significant difference between the parental sex. The median difference was 344 repeats (range, 0–1920) in paternal transmission and 129 repeats (range, 0–1330) in maternal transmission (P ¼ 0.026) (Fig 2). Table II shows the child’s DM1 phenotype according to the sex of the transmitting parent. There was no significant difference between the two groups (P ¼ 0.43). The majority (48/73, 65.8%) of these children had a more severe phenotype than the parent and only 34.2% of the offspring had a mild phenotype, confirming the anticipation phenomenon associated with this disorder. There were no occurrences of congenital DM1 in this cohort, but four occurrences of childhood onset disease (550, 650, 1300 and 1700 CTG) were identified with no parental sex difference (2M:2F). In the 13 children having 1000 repeats, we observed two childhood, eight early adult, and three adult occurrences. In one family, two sisters and one brother had 13 affected children in total. These children showed a large distribution of CTG numbers ranging from 130 to 1400; accordingly, their phenotypes varied from childhood to adult onsets. We therefore observed great intra-familial variability.
DISCUSSION The incidence of myotonic dystrophy in adults is known to be equal in both sexes [Harper, 2001]. Our study is a good representation of
this concept, since approximately half of the individuals heterozygous for a small CTG expansion were men, and the other half were women. People affected with DM1 have a 50% risk of transmitting their mutation to their offspring. However, in our study, we found that only 39.4% of the children received the mutation from their parent. This could be explained by an incomplete assessment, since 39% of the offspring had not yet been investigated at the time of this study. It is also possible that other occurrences of prenatal diagnosis and termination of an affected pregnancy were not reported to the Neuromuscular Clinic. In the present study, we sought to understand the phenomenon of anticipation in myotonic dystrophy families where the transmitting parent was heterozygous for a small CTG expansion (50–100 CTG repeats). Most transmissions showed an intergenerational increase in the number of CTG repeats; 78.9% (60/76) of the offspring had more than 100 CTG units. When looking at the phenotype, 65.8% of the children had an earlier age at onset than their parent. This demonstrates that anticipation is expected not only when parents show multisystem symptoms and a large CTG expansion, but also when parents are heterozygous for a small expansion and have a mild phenotype. We also found that the expansion in the offspring of small CTG expansion heterozygotes depends on the parental sex. Our results illustrate that the intergenerational increase of CTG repeats is higher in paternal than in maternal transmission when the parent is heterozygous for a small CTG expansion. 34.2% of the small CTG expansions were transmitted stably by mothers, while only 7.9% were transmitted by fathers. There are limitations to our approach. In this study, the majority of parents (65% or 26/40) with a small CTG expansion was assessed because one or more of their children had sufficient symptoms of DM1 to be diagnosed with this condition. Other individuals with a small CTG expansion may not have been identified in the population if their children also had a small expansion and had a mild phenotype. Otherwise, in these kinships, the ascertainment was still incomplete; less affected children have not all been assessed. This bias may result in an overestimation of the risk to have a child with a more severe phenotype for a parent with a small expansion. Some parents have contributed to an large number of parentchild pairs. However, when we looked at the CTG expansion and the phenotype in these children, we observed the same variability as the one seen in the other parent-child pairs of the study. Our results show an apparent discrepancy between the larger CTG expansion increase being paternally transmitted and the non statistical difference in children’s phenotype according to the parental sex. Although there was no statistical difference in the offspring’s phenotype relating to the sex of the transmitting parent, a trend toward more severe phenotypes (childhood and early-adult onset) was observed in 60% of children with a paternal transmission of the disease and in only 42% of offspring when the mother was the transmitting parent. A mild phenotype was observed in 43% of the children with a maternal transmission of the disease but in only 26% with a paternal transmission. The data treatment in categories and the small sample size may explain the statistical discrepancy. Finally, all the individuals in this study did not have molecular testing at the same age. We now know that there is a high degree of somatic instability of DM1 mutations, both within and between
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FIG. 2. Boxplots illustrating the differential change in the number of CTG repeats for the 76 parent-child pairs according to the parental sex. Box extends from the 25th to the 75th centile. The line in the middle of the box is the median. Whiskers extend to largest and smallest observed values within 1.5 box lengths. We observe the same parental sex difference, the greater differential change being transmitted by a father.
TABLE II. DM1 Phenotypes of 73 Offspring According to the Parental Sex (Fetuses not included since phenotype not available) Parental sex Child DM1 phenotype Mild Adult Early adult Childhood Total Chi-square, P ¼ 0.43.
Male n (%) 10 (26.3) 5 (13.2) 21 (55.3) 2 (5.3) 38 (100)
Female n (%) 15 (42.9) 5 (14.3) 13 (37.1) 2 (5.7) 35 (100)
Total n (%) 25 (34.2) 10 (13.7) 34 (46.6) 4 (5.5) 73 (100)
different tissues [Anvret et al., 1993; Ashizawa et al., 1993; Lavedan et al., 1993; Thornton et al., 1994; Martorell et al., 1998]. According to Martorell et al. [1998]; the expansion of the CTG repeat in blood cells of patients with DM1 is continuous throughout life; it differs with the age and between tissues of the individuals. However, a lower level of instability in individuals with less than 200 CTG repeats was demonstrated. Recent literature brings out that somatic instability also modifies age at onset of DM1 and may facilitate the formulation of novel therapies [Morales et al., 2012]. In this study, we confirm that anticipation in DM1 is present even when the parent is heterozygous for a small CTG expansion. The results show that larger increases are transmitted by males rather than females carrying a small expansion. However, no congenital phenotype was observed and there was no parental sex difference in the transmission of childhood DM1. The pheno-
PRATTE ET AL. type in the offspring needs to be interpreted not only in terms of the size of the expansion, but also in terms of the age at onset of symptoms. It is important to discuss prenatal diagnosis and other reproductive options with small DM1 expansion heterozygotes. However, individuals with a small CTG expansion may be asymptomatic and may therefore be unaware of their risk of transmitting the disease to their offspring, in a possible more severe form. Individuals at increased risk of DM1 can only be confident of being unaffected when undergoing DNA analysis. This is particularly important for young adults of reproductive age. We hope that this study will help clinicians and genetic counselors to provide more accurate recurrence risks to families affected by DM1 and to promote intra-familial communication of information to at-risk relatives.
ACKNOWLEDGMENTS This research was supported by the Neuromuscular Partnership Program of Muscular Dystrophy Canada and the Canadian Institutes of Health Research (CIHR) (#MOP49556) and ECOGENE21, a research program in community genetics and genomics supported by the Canada Research Chairs Program and by the CIHR (#CAR43283) and was conducted with the Institutional Review Board (IRB) approval from the CSSS de Chicoutimi.
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Anticipation in Myotonic Dystrophy Type 1 Parents with Small CTG Expansions Annabelle Pratte,1* Claude Pre´vost,2 Jack Puymirat,3 and Jean Mathieu4,5 1
Division of Genetic Counseling, CSSS Chicoutimi, Chicoutimi, Quebec, Canada
2
Neuromuscular Clinic, CSSS Jonquiere, Jonquiere, Quebec, Canada Department of Human Genetics, Centre Hospitalier de l’Universite´ Laval, Quebec, Quebec, Canada 4 Faculty of Medicine and Health Sciences, Sherbrooke University, Sherbrooke, Quebec, Canada 3
5
Neuromuscular Clinic, CSSS Jonquiere, Jonquiere, Quebec, Canada
Manuscript Received: 21 March 2014; Manuscript Accepted: 21 December 2014
Myotonic dystrophy type 1 is the most common form of adult muscular dystrophy and has the world’s highest prevalence in the Saguenay-Lac-St-Jean region, due to a founder effect. This autosomal dominant disorder results from an unstable CTG repeat expansion in DMPK. This region of Canada has had a family screening and predictive testing program for this disorder since 1988. Heterozygotes for small expansions (50–100 CTG repeats) can be asymptomatic or minimally affected. The aim of this study was to assess anticipation for these individuals. At the time of this study, the molecular data of 40 individuals and their 76 affected children were available. We compared 76 parent-child pairs. Most offspring (92.1%) had a larger number of repeats than their parent and the median number of repeats in the offspring was 325 (range, 57–2000). The number of CTG repeats was significantly greater when the mutation was transmitted by a father (median, 425 repeats; range, 70–2000), than when it was transmitted by a mother (median, 200 repeats; range, 57–1400). The majority (65.8%) of children also had a more severe phenotype than their parent but the sex of the parent had no significant influence on the severity of the child’s phenotype. No congenital phenotype was observed. These results confirm that anticipation is present even when the parent is heterozygous for a small CTG expansion. The parental sex has an impact on the size of the repeat in the next generation, larger increases being transmitted by males with a small expansion. Ó 2015 Wiley Periodicals, Inc.
Key words: myotonic dystrophy; DMPK gene; CTG repeat; anticipation; genetic counseling
INTRODUCTION Myotonic dystrophy type 1 (DM1; OMIM #160900), also known as dystrophia myotonica or Steinert disease, is the most common form of adult muscular dystrophy. DM1 has an overall worldwide prevalence of approximately 2.1–14.3 per 100,000 [Harper, 2001]. However, its prevalence in the Saguenay-Lac-St-Jean (SLSJ) region, located in the north eastern part of the province of Quebec, Canada, was reported to be of 158 per 100,000 in 2010
Ó 2015 Wiley Periodicals, Inc.
How to Cite this Article: Pratte A, Pre´vost C, Puymirat J, Mathieu J. 2015. Anticipation in myotonic dystrophy type 1 parents with small CTG expansions. Am J Med Genet Part A 167A:708–714.
[Mathieu and Prevost, 2012]. This prevalence is the highest in the world due to a founder effect. This disorder is inherited in an autosomal dominant pattern and results from an unstable cytosine, thymine, and guanine (CTG) repeat expansion in the 3’ untranslated region of the myotonin kinase gene at 19q13.3 [Aslanidis et al., 1992; Brook et al., 1992; Buxton et al., 1992; Harley et al., 1992]. When transcribed into CUG-containing RNA, mutant transcripts aggregate as nuclear foci that sequester RNA-binding proteins, including members of the muscleblind (MBNL) family, resulting in a spliceopathy of downstream effector genes [Jiang et al., 2004]. The DM1 phenotype is a multisystem condition characterized by myotonia, progressive muscle weakness and wasting, cataracts, endocrine disorder, frontal balding, hypogonadism, diabetes mellitus, and cardiac conduction disorder. The severity of the condition is highly variable. The DM1 phenotype is classified according to the age at onset of symptoms: resent Conflict of interest: All authors have no conflict of interest to declare. Grant sponsor: Neuromuscular Partnership Program of Muscular Dystrophy Canada and Canadian Institutes of Health Research (CIHR); Grant number: MOP49556; Grant sponsor: ECOGENE-21 and CIHR; Grant number: CAR43283. Correspondence to: Annabelle Pratte, M.Sc., (C)CGC, Division of Genetic Counseling, CSSS Chicoutimi, 305 St-Vallier, Chicoutimi (Quebec), Canada, G7H 5H6. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 25 February 2015 DOI 10.1002/ajmg.a.36950
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PRATTE ET AL. at birth), childhood (onset between 1 and 10 years of age), early adult (onset from 11 to 20 years of age), adult (onset between 21 and 40 years of age), and mild (onset after 40 years of age) [Koch et al., 1991]. There is a correlation between the number of CTG repeats and the severity of the disease, and an inverse correlation between the number of repeats and the age at onset of symptoms [Hunter et al., 1992; Harley et al., 1993]. Carriers of a relatively small expansion, i.e., 50 to 100 repeats, can be asymptomatic or minimally affected (mostly cataracts) [Arsenault et al., 2006]. The genetics of DM1 is characterized by anticipation [Harley et al., 1992; Harper et al., 1992; Hunter et al., 1992], which is the occurrence of increasing phenotype severity and decreasing age of onset in subsequent generations. This is explained by the fact that the expanded trinucleotide CTG repeat sequence is markedly unstable and prone to expand in successive generations. Two principal factors have been found to determine the degree of expansion (or reduction) in the transmission of the mutation: (1) the size of the parental repeat and (2) the sex of the transmitting parent. According to Martorell et al. [2007]; without regards to the parent’s expansion size, the mean intergenerational variation is minimal when the disease is transmitted by a male and is very high when transmitted by a female. Other studies found the same greater tendency to expansion in female transmission [Lavedan et al., 1993; Redman et al., 1993; Eguchi and Tsuji, 1997; Kim et al., 2008]. According to Harper and Johnson [2001]; over the entire range of repeat lengths, there is no clear sex difference in expansion between the generations [Tsilfidis et al., 1992; Harley et al., 1993]. Some authors have observed that small expansions can be inherited relatively stably for several generations if transmitted by women, but passage through the male germline usually results in a larger increase into the full disease range [Barcelo´ et al., 1993; Brunner et al., 1993; Ashizawa et al., 1994B; Simmons et al., 1998; Martorell et al., 2001; Abbruzzese et al., 2002]. In brief, different studies have been done to address this question and results are variable. There is still confusion regarding anticipation in DM1 for small expansion heterozygotes and there is a need to investigate further. The aim of this study is to assess anticipation in DM1 families when the transmitting parent is heterozygous for a small expansion (50– 100 CTG repeats).
MATERIALS AND METHODS Study Design and Sample At the time of this study, 801 individuals were recruited into the DM1 family screening and predictive testing program coordinated through the SLSJ Neuromuscular Clinic of the CSSS de Jonquie`re. From this group, 302 individuals were affected by DM1 and 499 individuals were homozygous wild type for the mutation. This program is provided by a multidisciplinary team including a neurologist, a genetic counselor and a specialized nurse [Pre´vost et al., 2004]. Participants are recruited in the program if they met the following criteria: (1) at least 18 years of age and mentally capable; (2) having a confirmed family history of DM1 or showing symptoms of the condition. After obtaining a signed informed consent from each participant, a blood sample is drawn for mutation
709 screening. Their demographic and clinical data are stored on a confidential computer database at the clinic since 1983 [Mathieu et al., 2012]. For the present study, data extracted from the registry included date of birth, gender, size of CTG expansion, DM1 phenotype and number of children. Most affected individuals were followed on a yearly basis, permitting to assess their phenotype’s progression regularly. The DM1 phenotype classification used at the Neuromuscular Clinic is based on the age at onset of symptoms [Koch et al., 1991], as described above. However, after the introduction of DNA analysis in predictive testing for DM1 in 1993, subjects were classified as having the mild form of the disease if they presented at least two of the three following criteria: (1) CTG less than 200, (2) Muscular Impairment Rating Scale score of 1 (no muscular impairment) or 2 (minimal signs), and (3) age at onset of symptoms greater than 40 years [Gagnon et al., 2008; Mathieu et al., 2012]. Muscular impairment was defined using a five-point Muscular Impairment Rating Scale (MIRS) [Mathieu et al., 2001]. Genetic testing for DM1 was performed through linkage analysis from 1988 to 1993 and, since 1993, by direct mutation analysis. The molecular analyses were done at the Centre Hospitalier Universitaire de Que´bec (CHUQ) in Quebec city. Genomic DNA was extracted from peripheral blood samples using QIAamp DNA blood kit (Qiagen Sciences, Germantown, Maryland) according to the manufacturer’s instructions. The DNA (3–5 ug) was digested with EcoRI (new England Biolabs), electrophoresed on 0.8% agarose gels, Southern transferred onto a nylon membrane (Hybond, Amersham) and probed overnight with radiolabeled 2.2 kb BamHI/ EcoRI subclone of probe pGB2.66, as previously described [Furling et al., 2001]. A PCR amplification of the CTG repeat was found to provide an accurate assessment of its size. Genomic DNA (1 ug) was PCR-amplified with primers 406 and 409 using standard protocol [Mahadevan et al., 1992]. The subjects enrolled in the study were assigned a code, which separated all clinical data from personally identifiable information. Our study was based on (1) a pedigree analysis and (2) the analysis of the clinical and genetic testing results of the participants to the DM1 testing program. We explored the number of individuals heterozygous for 50 to 100 CTG repeats, the number of CTG repeats and the DM1 phenotype of their affected offspring.
Statistical Analysis The non parametric Mann-Whitney U test and the Chi-square test were used to compare the number of CTG repeats in the offspring according to the sex of the parent. A Mann-Whitney U test was used to compare the difference in the number of CTG repeats between the child and the transmitting parent. A Chi-Square test was also used to compare the DM1 phenotypes among offsprings according to the parental sex. A P-value less than 0.05 was considered significant. Statistical analyses were performed using the SPSS statistical software package (SPSS 21, 2012).
RESULTS Characteristics of the Sample At the time of the study, molecular data were available for 40 parents heterozygous for alleles from 50 to 100 CTG repeats (20 mothers
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and 20 fathers) and their 76 affected children, including three fetuses. Parent-child pairs were obtained by comparing the parent with each of his affected offspring for which molecular testing was done. Parents having more than one affected child were therefore part of more than one pair. In the following analyses, we compared 76 parent-child pairs. Twenty (20) parents contributed one child each, 10 parents contributed two children, six parents had three children, two parents had four children and two parents had five.
Outcome in Offspring Figure 1 shows the number of CTG repeats in the offspring according to the number of repeats in their parent (38 motherchild pairs and 38 father-child pairs). Most offspring (70/76 or 92.1%) had a larger number of repeats than the parent; the median number of repeats in the offspring was 325 (range, 57–2000). The number of repeats in the offspring was significantly greater when the mutation is transmitted by a father (median, 425 repeats; range,
FIG. 1. Number of CTG repeats for 76 parent-child pairs according to the mutation’s parental origin. Most offspring have a larger number of repeats than the parent. The number of repeats in the offspring is greater when the mutation is transmitted by a father, than when it is transmitted by a mother.
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TABLE I. Number of CTG Repeats of 76 Offspring According to the Parental Sex Parental sex CTG Repeats in child 50–100 101–200 201–999 1000 Total
Male n (%) 3 (7.9) 7 (18.4) 20 (52.6) 8 (21.1) 38 (100)
Female n (%) 13 (34.2) 7 (18.4) 13 (34.2) 5 (13.2) 38 (100)
Total n (%) 16 (21.1) 14 (18.4) 33 (43.4) 13 (17.1) 76 (100)
Chi-square, P ¼ 0.038.
70–2000), than when it was transmitted by a mother (median, 200 repeats; range, 57–1400) (P ¼ 0.027). Table I shows the number of repeats in children according to the parental origin. There was a statistically significant difference between the parental sex, a larger proportion of children of affected males being found in the 201–999 and the 1000 CTG groups (P ¼ 0.038); and, conversely, a larger proportion of children of affected females is found in the 50–100 CTG group. The majority (60.5%) of children had more than 200 repeats, a genotype associated with the classic adult onset of DM1 or a more severe phenotype for those having 1000 repeats. Only 21.1% of all children had between 50 and 100 repeats with an expected mild phenotype as observed in their parent. According to these data, the respective risk for a father and a mother heterozygous for a small CTG expansion to have a child with a significant increase, i.e., 200 repeats and higher, was 73.6% (28/38) and 47.4% (18/38). Expressed as the differential change in the number of CTG repeats between the offspring and the transmitting parent, we observed the same significant difference between the parental sex. The median difference was 344 repeats (range, 0–1920) in paternal transmission and 129 repeats (range, 0–1330) in maternal transmission (P ¼ 0.026) (Fig 2). Table II shows the child’s DM1 phenotype according to the sex of the transmitting parent. There was no significant difference between the two groups (P ¼ 0.43). The majority (48/73, 65.8%) of these children had a more severe phenotype than the parent and only 34.2% of the offspring had a mild phenotype, confirming the anticipation phenomenon associated with this disorder. There were no occurrences of congenital DM1 in this cohort, but four occurrences of childhood onset disease (550, 650, 1300 and 1700 CTG) were identified with no parental sex difference (2M:2F). In the 13 children having 1000 repeats, we observed two childhood, eight early adult, and three adult occurrences. In one family, two sisters and one brother had 13 affected children in total. These children showed a large distribution of CTG numbers ranging from 130 to 1400; accordingly, their phenotypes varied from childhood to adult onsets. We therefore observed great intra-familial variability.
DISCUSSION The incidence of myotonic dystrophy in adults is known to be equal in both sexes [Harper, 2001]. Our study is a good representation of
this concept, since approximately half of the individuals heterozygous for a small CTG expansion were men, and the other half were women. People affected with DM1 have a 50% risk of transmitting their mutation to their offspring. However, in our study, we found that only 39.4% of the children received the mutation from their parent. This could be explained by an incomplete assessment, since 39% of the offspring had not yet been investigated at the time of this study. It is also possible that other occurrences of prenatal diagnosis and termination of an affected pregnancy were not reported to the Neuromuscular Clinic. In the present study, we sought to understand the phenomenon of anticipation in myotonic dystrophy families where the transmitting parent was heterozygous for a small CTG expansion (50–100 CTG repeats). Most transmissions showed an intergenerational increase in the number of CTG repeats; 78.9% (60/76) of the offspring had more than 100 CTG units. When looking at the phenotype, 65.8% of the children had an earlier age at onset than their parent. This demonstrates that anticipation is expected not only when parents show multisystem symptoms and a large CTG expansion, but also when parents are heterozygous for a small expansion and have a mild phenotype. We also found that the expansion in the offspring of small CTG expansion heterozygotes depends on the parental sex. Our results illustrate that the intergenerational increase of CTG repeats is higher in paternal than in maternal transmission when the parent is heterozygous for a small CTG expansion. 34.2% of the small CTG expansions were transmitted stably by mothers, while only 7.9% were transmitted by fathers. There are limitations to our approach. In this study, the majority of parents (65% or 26/40) with a small CTG expansion was assessed because one or more of their children had sufficient symptoms of DM1 to be diagnosed with this condition. Other individuals with a small CTG expansion may not have been identified in the population if their children also had a small expansion and had a mild phenotype. Otherwise, in these kinships, the ascertainment was still incomplete; less affected children have not all been assessed. This bias may result in an overestimation of the risk to have a child with a more severe phenotype for a parent with a small expansion. Some parents have contributed to an large number of parentchild pairs. However, when we looked at the CTG expansion and the phenotype in these children, we observed the same variability as the one seen in the other parent-child pairs of the study. Our results show an apparent discrepancy between the larger CTG expansion increase being paternally transmitted and the non statistical difference in children’s phenotype according to the parental sex. Although there was no statistical difference in the offspring’s phenotype relating to the sex of the transmitting parent, a trend toward more severe phenotypes (childhood and early-adult onset) was observed in 60% of children with a paternal transmission of the disease and in only 42% of offspring when the mother was the transmitting parent. A mild phenotype was observed in 43% of the children with a maternal transmission of the disease but in only 26% with a paternal transmission. The data treatment in categories and the small sample size may explain the statistical discrepancy. Finally, all the individuals in this study did not have molecular testing at the same age. We now know that there is a high degree of somatic instability of DM1 mutations, both within and between
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FIG. 2. Boxplots illustrating the differential change in the number of CTG repeats for the 76 parent-child pairs according to the parental sex. Box extends from the 25th to the 75th centile. The line in the middle of the box is the median. Whiskers extend to largest and smallest observed values within 1.5 box lengths. We observe the same parental sex difference, the greater differential change being transmitted by a father.
TABLE II. DM1 Phenotypes of 73 Offspring According to the Parental Sex (Fetuses not included since phenotype not available) Parental sex Child DM1 phenotype Mild Adult Early adult Childhood Total Chi-square, P ¼ 0.43.
Male n (%) 10 (26.3) 5 (13.2) 21 (55.3) 2 (5.3) 38 (100)
Female n (%) 15 (42.9) 5 (14.3) 13 (37.1) 2 (5.7) 35 (100)
Total n (%) 25 (34.2) 10 (13.7) 34 (46.6) 4 (5.5) 73 (100)
different tissues [Anvret et al., 1993; Ashizawa et al., 1993; Lavedan et al., 1993; Thornton et al., 1994; Martorell et al., 1998]. According to Martorell et al. [1998]; the expansion of the CTG repeat in blood cells of patients with DM1 is continuous throughout life; it differs with the age and between tissues of the individuals. However, a lower level of instability in individuals with less than 200 CTG repeats was demonstrated. Recent literature brings out that somatic instability also modifies age at onset of DM1 and may facilitate the formulation of novel therapies [Morales et al., 2012]. In this study, we confirm that anticipation in DM1 is present even when the parent is heterozygous for a small CTG expansion. The results show that larger increases are transmitted by males rather than females carrying a small expansion. However, no congenital phenotype was observed and there was no parental sex difference in the transmission of childhood DM1. The pheno-
PRATTE ET AL. type in the offspring needs to be interpreted not only in terms of the size of the expansion, but also in terms of the age at onset of symptoms. It is important to discuss prenatal diagnosis and other reproductive options with small DM1 expansion heterozygotes. However, individuals with a small CTG expansion may be asymptomatic and may therefore be unaware of their risk of transmitting the disease to their offspring, in a possible more severe form. Individuals at increased risk of DM1 can only be confident of being unaffected when undergoing DNA analysis. This is particularly important for young adults of reproductive age. We hope that this study will help clinicians and genetic counselors to provide more accurate recurrence risks to families affected by DM1 and to promote intra-familial communication of information to at-risk relatives.
ACKNOWLEDGMENTS This research was supported by the Neuromuscular Partnership Program of Muscular Dystrophy Canada and the Canadian Institutes of Health Research (CIHR) (#MOP49556) and ECOGENE21, a research program in community genetics and genomics supported by the Canada Research Chairs Program and by the CIHR (#CAR43283) and was conducted with the Institutional Review Board (IRB) approval from the CSSS de Chicoutimi.
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