7 Med Genet 1992; 29: 774-779 774
The correlation of age of onset with CTG trinucleotide repeat amplification in myotonic dystrophy Alasdair Hunter, Catherine Tsilfidis, Gabrielle Mettler, Pierre Jacob, Mani Mahadevan, Linda Surh, Robert Korneluk
Abstract The gene for myotonic dystrophy (DM) has recently been isolated and amplification of an unstable CTG trinucleotide repeat, located within the DM gene, has been identified in virtually all patients studied to date. A high proportion of DM families who are studied show a progressively earlier age of onset with succeeding generations and, in the few pedigrees reported so far, an increasing degree of amplification of the CTG repeat has been noted to parallel this trend. It has been implicit in several of the original reports on the nature of the changes in the DM gene that knowledge of CTG amplification status at the DM locus of a person will provide useful information concerning prognosis. However, no studies of genotype-phenotype correlation have been reported and there are no specific data on which to base such counsel. In this paper we report the correlation between the degree of CTG amplification and age of onset in 109 DM gene carriers from 17 families. Included are parent-child and sib-sib comparisons which provide a framework in which to incorporate DNA diagnostic studies when counselling subjects and families at risk for DM. (J Med Genet 1992;29:774-9)
Division of Genetics, Children's Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, Ontario KlH 8L1, Canada. A G W Hunter C Tsilfidis G Mettler P Jacob M Mahadevan L Surh R G Korneluk Correspondence
to
Dr Hunter. Received 8 June 1992. Revised version accepted 30 June 1992.
increase with successive generations and the clinical severity/age of onset to correlate with the degree of expansion of the allele. However, no substantive clinical data have been provided to support this contention and authors have tended to simplify the definition of severity into the three broad categories described by Harper8 (congenital; late adolescence and early adulthood onset; and middle to old age with cataracts and minimal if any muscle weakness). The age of onset and severity of DM are a continuum. Marked variation between generations is characteristic, and while correlation between sibs is relatively high (r = 066),9 significant differences may be seen between sibs. Differences in allele size between sibs have been reported.3 Any means of providing prognosis specific to an individual person is therefore of paramount importance. The studies published to date suggest but do not prove a correlation between allelic expansion and severity. The specific characteristics of this relationship need to be established before changes in practice in genetic counselling for DM. This is especially so with respect to prenatal diagnosis. Our regional genetic service for Eastern Ontario and Western Quebec has a large French Canadian population and DM is the most common single gene disorder seen for genetic counselling. We have therefore reassessed the signs and symptoms of affected members of a subset of our DM families to explore the relationship of a subject's gene expansion status to disease prognosis. Myotonic dystrophy (DM) is the most prelevant Although detailed clinical data were obtained form of muscular dystrophy and is estimated for all patients in this report, the analysis is to affect 1 in 8000 persons worldwide. Further- simplified to an analysis by age of onset, which more, it is present in up to 1/475 inhabitants of correlates well with ultimate disease severity.8 the Saguenay-Lac St Jean region of Quebec,l and maintains a high rate in the general French Canadian population. Recently variable expansion of an unstable DNA fragment Materials and methods at a polymorphic site on chromosome 19q13.3 DNA ANALYSIS has been reported in patients with DM.'2 The Genomic DNA was extracted from either periunstable site has been identified as a CTG pheral blood samples or lymphoblast cultures trinucleotide repeat found within the 3' using modifications of the methods of Madisen untranslated region of a gene showing homo- et aP or Miller et al.'0 The DNA (3 to 5 pg) was logy to the serine-threonine specific family of digested with EcoRI (New England Biolabs), protein kinases.i The repeat is highly poly- electrophoresed on 0-8% agarose gels, Southmorphic in the general population where it ern transferred onto nylon membrane occurs with a range of between five and 38 (HybondTm,Amersham), and probed overnight with repeats. Almost all affected subjects have a radiolabelled 2-2 kb BamHI/EcoRI subclone shown significant amplification of the repeat of probe pGB2.6.7 This genomic probe detects the CTG trinucleotide variable length polyover that seen in the general population, although two of 98 families in one survey morphism associated with DM. Membranes showed no expansion7 and merit further study. were washed in 0-2 x saline sodium citrate The number of repeats has been reported to (SSC), 041% sodium dodecyl sulphate (SDS)
775
The correlation of age of onset with CTG trinucleotide repeat amplification in myotonic dystrophy
at 60°C and exposed to Kodak XAR film for one to four days at - 80°C. CTG trinucleotide repeat length was classified using the following scale: EQ, no visible expansion on Southern blot but proven to be a DM gene carrier by linkage analysis; El, expansion of 0< 1-5 kilobase pairs (kb); E2, expansion of 15< 30 kb; E3, expansion of 3*04-5 kb. Broad classification categories were used owing to the high degree of somatic heterogeneity associated with the CTG repeat, whose complex pattern does not readily lend itself to specific analysis and categorisation.
SDS at 45°C and exposed to Kodak XAR film for 1-0 to 2-5 hours at -800C. FAMILIES
Fifteen families who had been genotyped for the DM mutation, lived close by, and were of sufficient size were chosen from the over 120 kindreds whose DNA had been referred to our laboratory. Two additional small families presented during the six week study period and were included. The clinical investigators were aware who were DM positive by linkage analysis but they were blinded to the degree of allele expansion until after the study. A total of 109 DNA proven positive DM PCR BASED ANALYSIS subjects was evaluated using a standard history Polymerase chain reaction (PCR) amplifica- and examination protocol. Most families were tion of the CTG repeat provides a more accur- seen as a group, usually at one member's house ate assessment of its size. It detects expansion but occasionally in the genetic clinic. This not visible on Southern blots of EQ subjects turned out to have a significant advantage in and was used to rank the size of expansion terms of corroborating data and examining between family members who had the same EQ family photographs. In one family, telephone to E2 class. Genomic DNA (1 gig) was PCR interviews, medical records, and photographs amplified with primers 406 and 4097 using a were used for some members. All physical standard protocol of 30 cycles at 94°C for one examinations were carried out by one or two minute, 60GC for one minute, and 72°C for 1-5 clinicians (AH, PJ); interviews were carried minutes. Amplified DNA was electrophoresed out by AH or GM. Ophthalmological assesson 1% agarose gels, vacuum blotted onto ny- ments were carried out using a portable slit lon membrane (HybondTmersham), and probed lamp (AH), and in case of doubt patients were with a labelled (CTG),o oligonucleotide. referred to a genetic-ophthalmologist for asMembranes were washed in 6 x SSC, 0-1% sessment. Persons were categorised by age of onset as follows: congenital, postnatal and < 5 years, 5 Relationship of allele size to age of onset to
The correlation of age of onset with CTG trinucleotide repeat amplification in myotonic dystrophy Alasdair Hunter, Catherine Tsilfidis, Gabrielle Mettler, Pierre Jacob, Mani Mahadevan, Linda Surh, Robert Korneluk
Abstract The gene for myotonic dystrophy (DM) has recently been isolated and amplification of an unstable CTG trinucleotide repeat, located within the DM gene, has been identified in virtually all patients studied to date. A high proportion of DM families who are studied show a progressively earlier age of onset with succeeding generations and, in the few pedigrees reported so far, an increasing degree of amplification of the CTG repeat has been noted to parallel this trend. It has been implicit in several of the original reports on the nature of the changes in the DM gene that knowledge of CTG amplification status at the DM locus of a person will provide useful information concerning prognosis. However, no studies of genotype-phenotype correlation have been reported and there are no specific data on which to base such counsel. In this paper we report the correlation between the degree of CTG amplification and age of onset in 109 DM gene carriers from 17 families. Included are parent-child and sib-sib comparisons which provide a framework in which to incorporate DNA diagnostic studies when counselling subjects and families at risk for DM. (J Med Genet 1992;29:774-9)
Division of Genetics, Children's Hospital of Eastern Ontario, 401 Smyth Road, Ottawa, Ontario KlH 8L1, Canada. A G W Hunter C Tsilfidis G Mettler P Jacob M Mahadevan L Surh R G Korneluk Correspondence
to
Dr Hunter. Received 8 June 1992. Revised version accepted 30 June 1992.
increase with successive generations and the clinical severity/age of onset to correlate with the degree of expansion of the allele. However, no substantive clinical data have been provided to support this contention and authors have tended to simplify the definition of severity into the three broad categories described by Harper8 (congenital; late adolescence and early adulthood onset; and middle to old age with cataracts and minimal if any muscle weakness). The age of onset and severity of DM are a continuum. Marked variation between generations is characteristic, and while correlation between sibs is relatively high (r = 066),9 significant differences may be seen between sibs. Differences in allele size between sibs have been reported.3 Any means of providing prognosis specific to an individual person is therefore of paramount importance. The studies published to date suggest but do not prove a correlation between allelic expansion and severity. The specific characteristics of this relationship need to be established before changes in practice in genetic counselling for DM. This is especially so with respect to prenatal diagnosis. Our regional genetic service for Eastern Ontario and Western Quebec has a large French Canadian population and DM is the most common single gene disorder seen for genetic counselling. We have therefore reassessed the signs and symptoms of affected members of a subset of our DM families to explore the relationship of a subject's gene expansion status to disease prognosis. Myotonic dystrophy (DM) is the most prelevant Although detailed clinical data were obtained form of muscular dystrophy and is estimated for all patients in this report, the analysis is to affect 1 in 8000 persons worldwide. Further- simplified to an analysis by age of onset, which more, it is present in up to 1/475 inhabitants of correlates well with ultimate disease severity.8 the Saguenay-Lac St Jean region of Quebec,l and maintains a high rate in the general French Canadian population. Recently variable expansion of an unstable DNA fragment Materials and methods at a polymorphic site on chromosome 19q13.3 DNA ANALYSIS has been reported in patients with DM.'2 The Genomic DNA was extracted from either periunstable site has been identified as a CTG pheral blood samples or lymphoblast cultures trinucleotide repeat found within the 3' using modifications of the methods of Madisen untranslated region of a gene showing homo- et aP or Miller et al.'0 The DNA (3 to 5 pg) was logy to the serine-threonine specific family of digested with EcoRI (New England Biolabs), protein kinases.i The repeat is highly poly- electrophoresed on 0-8% agarose gels, Southmorphic in the general population where it ern transferred onto nylon membrane occurs with a range of between five and 38 (HybondTm,Amersham), and probed overnight with repeats. Almost all affected subjects have a radiolabelled 2-2 kb BamHI/EcoRI subclone shown significant amplification of the repeat of probe pGB2.6.7 This genomic probe detects the CTG trinucleotide variable length polyover that seen in the general population, although two of 98 families in one survey morphism associated with DM. Membranes showed no expansion7 and merit further study. were washed in 0-2 x saline sodium citrate The number of repeats has been reported to (SSC), 041% sodium dodecyl sulphate (SDS)
775
The correlation of age of onset with CTG trinucleotide repeat amplification in myotonic dystrophy
at 60°C and exposed to Kodak XAR film for one to four days at - 80°C. CTG trinucleotide repeat length was classified using the following scale: EQ, no visible expansion on Southern blot but proven to be a DM gene carrier by linkage analysis; El, expansion of 0< 1-5 kilobase pairs (kb); E2, expansion of 15< 30 kb; E3, expansion of 3*04-5 kb. Broad classification categories were used owing to the high degree of somatic heterogeneity associated with the CTG repeat, whose complex pattern does not readily lend itself to specific analysis and categorisation.
SDS at 45°C and exposed to Kodak XAR film for 1-0 to 2-5 hours at -800C. FAMILIES
Fifteen families who had been genotyped for the DM mutation, lived close by, and were of sufficient size were chosen from the over 120 kindreds whose DNA had been referred to our laboratory. Two additional small families presented during the six week study period and were included. The clinical investigators were aware who were DM positive by linkage analysis but they were blinded to the degree of allele expansion until after the study. A total of 109 DNA proven positive DM PCR BASED ANALYSIS subjects was evaluated using a standard history Polymerase chain reaction (PCR) amplifica- and examination protocol. Most families were tion of the CTG repeat provides a more accur- seen as a group, usually at one member's house ate assessment of its size. It detects expansion but occasionally in the genetic clinic. This not visible on Southern blots of EQ subjects turned out to have a significant advantage in and was used to rank the size of expansion terms of corroborating data and examining between family members who had the same EQ family photographs. In one family, telephone to E2 class. Genomic DNA (1 gig) was PCR interviews, medical records, and photographs amplified with primers 406 and 4097 using a were used for some members. All physical standard protocol of 30 cycles at 94°C for one examinations were carried out by one or two minute, 60GC for one minute, and 72°C for 1-5 clinicians (AH, PJ); interviews were carried minutes. Amplified DNA was electrophoresed out by AH or GM. Ophthalmological assesson 1% agarose gels, vacuum blotted onto ny- ments were carried out using a portable slit lon membrane (HybondTmersham), and probed lamp (AH), and in case of doubt patients were with a labelled (CTG),o oligonucleotide. referred to a genetic-ophthalmologist for asMembranes were washed in 6 x SSC, 0-1% sessment. Persons were categorised by age of onset as follows: congenital, postnatal and < 5 years, 5 Relationship of allele size to age of onset to
