Saturday 9 May
No 8802
1992
ORIGINAL ARTICLES
Unstable DNA sequence in
A variable DNA sequence has been detected in
patients with myotonic dystrophy.
We set out to
determine whether identification of this specific molecular defect would improve clinical management of patients and families with myotonic
dystrophy. 127 affected patients who were studied had an expanded DNA fragment not seen in 73 normal controls. The increase in length of the fragment correlated broadly with disease severity, and we noted expansion of the sequence in successive generations of the same family. Progressive expansion of the affected gene provides a molecular explanation for an apparently earlier onset in successive generations (anticipation) in myotonic dystrophy and supports the role of an unstable repeat sequence as the basis of the defect. The specificity of this finding will assist in accurate diagnosis of myotonic dystrophy and genetic counselling of affected families.
myotonic dystrophy
Although myotonic dystrophy follows autosomal dominant inheritance, it shows several unusual genetic features. Extreme variability of clinical features may occur even between different affected members of the same family, there may be anticipation2(an apparently earlier onset in successive generations), and a severe congenital form of the disease may occur-but only in the offspring of affected women.44 No primary protein abnormality had been identified in muscle or other tissues but molecular techniques have now localised the gene to a small region of chromosome 19q. We and colleagues have recently reported a variable and apparently specific DNA change that seems to be directly related to the fundamental abnormality in myotonic dystrophy, and which involves the myotonic dystrophy gene itself.9 What are the clinical implications of these fmdings, and how might they be applied to diagnosis and genetic prediction in patients and relatives?
Patients and methods 127 subjects with myotonic dystrophy, from an extensive series in our department over 20 years, were examined by at least one of us and allocated to one of three broad clinical groups: congenital (with neuromuscular problems, usually severe, already manifest at seen
Introduction
Myotonic dystrophy, the commonest muscular dystrophy of adult life, is one of the most variable inherited disorders known in clinical medicine. Features may appear at almost any age and its severity ranges from a profound generalised muscle weakness with mental retardation to the occurrence of cataract with little obvious neuromuscular disease. Myotonia is the consistent sign that distinguishes the condition from all other muscular dystrophies.’
ADDRESSES Institute of Medical Genetics, University of Wales College of Medicine, Heath Park, Cardiff CF4 6EJ, UK (H G Harley, PhD, S. A. Rundle, MSc, W. Reardon, MD, J. Myring, BSc, S Crow, BSc, Prof P. S. Harper, MD, D. J Shaw, PhD) and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA (J. D. Brook, PhD) Correspondence to Dr Helen Harley
1126
RELATION OF CLINICAL STATUS TO MOLECULAR ABNORMALITY IN MYOTONIC DYSTROPHY
birth); classic (typical neuromuscular features with myotonia and dystrophic changes, with onset usually in adolescence or early adult life); or minimal (slight weakness and wasting, onset in later life, commonly presenting because of cataract). A fourth group with early childhood onset and similar clinical features to the congenital group, but without clearly documented onset at birth, was kept separate to enable comparisons with previous studies. DNA from venous blood of each patient and from 73 unrelated normal controls was digested with EcoRI, separated by agarose gel electrophoresis for 24 h at 1 -4 V/cm, transferred by Southern blot onto a nylon membrane, and hybridised with a radiolabelled probe
(D19S95/pBBO 7) which identifies a two-allele polymorphism in normal individuals (with alleles of 9 and 10 kb). Each sample was scored for the presence of one or both of the normal alleles. One of these normal alleles together with a novel band of variable size was observed in all samples from patients with myotonic dystrophy: when present, the size of the variable allele was estimated to the nearest 500 bp and its appearance noted (eg, whether sharply defined or as a smear, indicating a range of variable allele sizes in one individual); the lowest limit of a smear was used to classify these individuals by size of insert.
Lane
myotonic
Fig 2-Anticipation and correlation of severity of dystrophy with size of abnormal DNA band in one family.
All samples show constant band (C) of 16 kb. Lanes 1 and 5 are normal spouses; lane 2 (grandmother) has fragment expanded by about 05 kb, lane 3 (mother) shows 25 kb expansion; and lane 4 (congenitally affected child) shows a 4 kb expansion.
(see table). The amount of additional DNA caused by expansion of the unstable sequence varies from 100 bp to 6-0 kb, although patients with smears have shown expansions of up to 8 kb. The approximate correlation of band size with disease type is shown in fig 1: congenitally affected patients showed the largest bands while minimally affected individuals have bands only slightly larger than the normal 10 kb. 2 shows correlation of phenotype with band size one family. A grandmother (lane 2) was mildly affected with cataracts removed at 48 years of age but a diagnosis of myotonic dystrophy was only made after the ’1. birth of a granddaughter (lane 4) who had congenital myotonic dystrophy, neonatal hypotonia, respiratory distress, facial diplegia, and mental handicap. Lane 3 shows the child’s mother, who had moderately severe myotonia and muscle weakness with onset in the mid-teens. Thus, increased severity and earlier onset is matched by an increased band size-which provides a biological explanation for the phenomenon of anticipation. Fig 3 shows findings in a similar family in which the increase in severity of disease and of band size mainly occurs between the affected mother (lane 4), with symptoms of myotonia since the age of 20 years and wasting of facial, jaw, and
Fig
within
Results DNA typings were obtained for 73 normal individuals and 127 patients with myotonic dystrophy, of whom 7 were minimally affected, 87 had classic adult onset, 9 had onset in early childhood, and 24 had congenital disease. The variable band was detected in all patients but in none of the controls MINIMAL
CLASSIC ADULT
Lane and somatic DNA
Fig 3-Anticipation myotonic dystrophy. Band size
Fig 1-Distribution of size of expansion of unstable sequence by type of myotonic dystrophy.
instability in family with
All samples show constant band (C) of 16 kb Lanes 1 and 5 are normal spouses; lane 3 shows smeared band of mildly affected grandfather; lane 4 shows 3 kb expansion in his daughter; and lane 2 shows 5 kb expansion in severely affected grandson.
1127
stemomastoid muscles, and her son (lane 2), who had prenatal onset with polyhydramnios, severe hypotonia from birth with delayed but improving motor development, and
striking weakness of jaw and facial muscles. The mildly affected grandfather (lane 3), with cataracts, ptosis, and mild facial weakness only, shows a smear of DNA rather than a discrete band-indicative of somatic instability of the variable band.
Discussion How might DNA analysis for an unstable sequence assist in the clinical management of patients and families affected by myotonic dystrophy? The extreme clinical variability of myotonic dystrophy leads to problems in diagnosis and in prediction for family members at risk for the disorder. Use of linked DNA markers is subject to error because of recombination, and such tests depend on the availability of several family members for their interpretation. Demonstration of a specific molecular defect will allow accurate diagnosis and genetic prediction in many families or individuals for whom previous techniques have been unreliable or impossible. The apparent relation of the size of expansion of the unstable sequence to disease severity will be especially helpful for prenatal diagnosis, in which a parental decision about termination of pregnancy may depend on the likely degree of disability. A retrospective analysis of samples taken from a family with 2 sisters with moderate symptoms of myotonic dsytrophy who requested chorionic villus sampling in pregnancies illustrates this potential benefit in prenatal testing. With the closely linked marker D19S63 (less than 1% recombination proximal to myotonic dystrophy) 2 pregnancies were shown to be at high and 2 at low risk of myotonic dystrophy, the high-risk pregnancies were terminated at the parents’ request. Analysis of residual DNA available for 3 pregnancies confirmed the previous prediction in each case, with an expanded band clearly seen in the pregnancies predicted as high risk but not in the pregnancy predicted to be normal, which ended in the birth of a clinically normal child. The variable bands in the abnormal pregnancies were much larger (2-5 and 3-5 kb) than that in the moderately affected mother (1 -0 kb), which suggests that had these pregnancies continued to term the children would have been severely affected. The technique could also be applied to differential diagnosis of apparent congenital myotonic dystrophy. In one family whom we have followed, a male infant was initially thought to have myotonic dystrophy on the basis of generalised hypotonia, severe feeding problems, an older brother who had died aged 10 weeks with an undiagnosed neuromuscular problem, and a finding of apparent mild myotonia on examination of his 32-year-old mother. However, after muscle biopsy a revised diagnosis of congenital fibre-type disproportion was made. Reassessment at 11 years showed facial and jaw weakness with clear percussion and grip myotonia, and myotonia in the mother was confirmed. Use of DNA analysis revealed an expansion of 3-4 kb, with a smaller expansion (0-5 kb) in the mother: availability of this specific molecular test at the initial assessment would have prevented subsequent misdiagnosis and an inappropriate assessment of prognosis and genetic risks, which was not corrected for 10 years. Similarly, the technique could be used to exclude myotonic dystrophy in siblings of congenitally affected individuals. In one family the first and third born daughters were severely affected with congenital myotonic dystrophy and their
mother was moderately affected with myotonia and general muscle weakness from 25 years of age. The second daughter had had recurrent otitis media and also complained of muscle pains on exercise. The parents were convinced that she was affected, despite normal clinical findings and electromyography at another hospital. In view of continued parental concern and the possible need for an ear operation under general anaesthetic, as well as to assess long-term prognosis, conventional linkage analysis was done at 7 years of age and the closely linked marker D19S63 showed a low risk. Subsequent analysis for the variable sequence confirmed her unaffected status, with no expanded fragment, whereas expansions were found in the affected mother and both affected sisters. Finally, identification of the variable sequence might help in accurate diagnosis of individuals with mild or minimal symptoms and signs suggestive of myotonic dystrophy. When cataract is the only abnormality in an individual thought to have transmitted the myotonic dystrophy gene the diagnosis may often be uncertain. For example, in one family a 65-year-old man had a son and grandson with classic myotonic dystrophy. He himself had no neuromuscular symptoms or abnormal neurological signs, and no abnormalities on electromyography; cataracts had been removed in his 50s. His 63-year-old wife had no
neurological,
electromyographic,
or
ophthalmic
abnormalities. DNA analysis of the grandfather showed an expansion of015 kb, and a similar abnormality was seen in a sample preserved from his dead mother who had also had cataracts.
Recognition of an unstable DNA sequence may also help explain some of the puzzling clinical and genetic features of myotonic dystrophy. The progressive expansion of the sequence in successive generations (as seen in figs 2 and 3) provides molecular explanation for the phenomenon of anticipation, first recognised in myotonic dystrophy 70 years ago,10 but whose validity has been debated by geneticists ever since.2,3 This finding is not only helpful for genetic counselling but may help to explain the mechanisms underlying expression of the disease, although it remains unclear why the severe congenital form of muscular dystrophy has exclusively maternal transmission because in our series expansion in band size is seen in the offspring of to
affected men and women alike. These findings in myotonic dystrophy show similarities the intergenerational variation seen in fragile X syndrome, in which transmission of an unstable DNA sequence has also been demonstrated. On the basis of these findings Sutherland et all’ pointed out the possible relevance of unstable DNA to anticipation, incomplete penetrance, expression, and other genetic variability, and predicted that a comparable mechanism might operate in other disorders including myotonic dystrophy. The myotonic dystrophy gene has now been cloned and its sequence determined. The unstable sequence is due to a triplet repeat (CTG)n located in the 3’ untranslated region of a gene whose predicted product is a member of the protein kinase family.9,12 The expansion in fragment length seen in myotonic dystrophy patients is caused by an increase in the copy number (n) of the CTG repeat: in normal individuals n is between 5 and 27, but in affected individuals n ranges from about 50 to 2000 or more. Most apparently unrelated cases of myotonic dystrophy from a wide area of Europe have probably arisen from a single ancestral mutation. 13 The restriction fragment length polymorphism identified by the gene probe that detects the unstable sequence shows to
1128
complete linkage disequilibrium with myotonic dystrophy,6 such that all individuals with myotonic dystrophy have the same allele. This finding suggests that the original event (probably an increase from an allele with n 5-27 to one with n :., 50) may have resulted in minimal or no clinical disturbance, but that an adverse clinical outcome arose from enlargement of this allele in subsequent generations because of its instability. How this change produces the observed =
clinical effects on muscle and other symptoms is still to be determined, but the fmding of hereditary unstable DNA in patients with myotonic dystrophy helps to explain anticipation and variable manifestations among patients with this condition.
We thank Mr Lodewijk Sandkinjl and Dr lain Fenton for help with data and Dr Gertrude Kohn, Dr Dian Donnai, and Prof Victor Dubowitz for referring families. Our work is supported by the Muscular Dystrophy Group of Great Britain, Muscular Dystrophy Association and Piton Foundation (USA), the Wellcome Trust, and the Medical Research Council.
analysis,
REFERENCES 1.
Harper PS. Myotonic dystrophy. Philadelphia: Saunders, 1989.
Insulin resistance and
Cigarette smoking is associated with increases in plasma triglycerides and decreases in plasma high density-lipoprotein-cholesterol concentration. These changes not only increase risk of coronary heart disease but also are secondary to resistance to insulin-stimulated glucose uptake or hyperinsulinaemia. To see whether there is a relation between cigarette smoking and insulin-mediated glucose uptake we measured plasma lipid and
lipoprotein concentrations, plasma glucose and insulin response to an oral glucose challenge, and insulin-mediated glucose uptake in 40 matched healthy volunteers (20 non-smokers, 20 smokers). Smokers had significantly higher mean (SEM) very-low-density-lipoprotein triglycerides (0·66 [0·10] vs 0·39 [0·03] mmol/l, p
No 8802
1992
ORIGINAL ARTICLES
Unstable DNA sequence in
A variable DNA sequence has been detected in
patients with myotonic dystrophy.
We set out to
determine whether identification of this specific molecular defect would improve clinical management of patients and families with myotonic
dystrophy. 127 affected patients who were studied had an expanded DNA fragment not seen in 73 normal controls. The increase in length of the fragment correlated broadly with disease severity, and we noted expansion of the sequence in successive generations of the same family. Progressive expansion of the affected gene provides a molecular explanation for an apparently earlier onset in successive generations (anticipation) in myotonic dystrophy and supports the role of an unstable repeat sequence as the basis of the defect. The specificity of this finding will assist in accurate diagnosis of myotonic dystrophy and genetic counselling of affected families.
myotonic dystrophy
Although myotonic dystrophy follows autosomal dominant inheritance, it shows several unusual genetic features. Extreme variability of clinical features may occur even between different affected members of the same family, there may be anticipation2(an apparently earlier onset in successive generations), and a severe congenital form of the disease may occur-but only in the offspring of affected women.44 No primary protein abnormality had been identified in muscle or other tissues but molecular techniques have now localised the gene to a small region of chromosome 19q. We and colleagues have recently reported a variable and apparently specific DNA change that seems to be directly related to the fundamental abnormality in myotonic dystrophy, and which involves the myotonic dystrophy gene itself.9 What are the clinical implications of these fmdings, and how might they be applied to diagnosis and genetic prediction in patients and relatives?
Patients and methods 127 subjects with myotonic dystrophy, from an extensive series in our department over 20 years, were examined by at least one of us and allocated to one of three broad clinical groups: congenital (with neuromuscular problems, usually severe, already manifest at seen
Introduction
Myotonic dystrophy, the commonest muscular dystrophy of adult life, is one of the most variable inherited disorders known in clinical medicine. Features may appear at almost any age and its severity ranges from a profound generalised muscle weakness with mental retardation to the occurrence of cataract with little obvious neuromuscular disease. Myotonia is the consistent sign that distinguishes the condition from all other muscular dystrophies.’
ADDRESSES Institute of Medical Genetics, University of Wales College of Medicine, Heath Park, Cardiff CF4 6EJ, UK (H G Harley, PhD, S. A. Rundle, MSc, W. Reardon, MD, J. Myring, BSc, S Crow, BSc, Prof P. S. Harper, MD, D. J Shaw, PhD) and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA (J. D. Brook, PhD) Correspondence to Dr Helen Harley
1126
RELATION OF CLINICAL STATUS TO MOLECULAR ABNORMALITY IN MYOTONIC DYSTROPHY
birth); classic (typical neuromuscular features with myotonia and dystrophic changes, with onset usually in adolescence or early adult life); or minimal (slight weakness and wasting, onset in later life, commonly presenting because of cataract). A fourth group with early childhood onset and similar clinical features to the congenital group, but without clearly documented onset at birth, was kept separate to enable comparisons with previous studies. DNA from venous blood of each patient and from 73 unrelated normal controls was digested with EcoRI, separated by agarose gel electrophoresis for 24 h at 1 -4 V/cm, transferred by Southern blot onto a nylon membrane, and hybridised with a radiolabelled probe
(D19S95/pBBO 7) which identifies a two-allele polymorphism in normal individuals (with alleles of 9 and 10 kb). Each sample was scored for the presence of one or both of the normal alleles. One of these normal alleles together with a novel band of variable size was observed in all samples from patients with myotonic dystrophy: when present, the size of the variable allele was estimated to the nearest 500 bp and its appearance noted (eg, whether sharply defined or as a smear, indicating a range of variable allele sizes in one individual); the lowest limit of a smear was used to classify these individuals by size of insert.
Lane
myotonic
Fig 2-Anticipation and correlation of severity of dystrophy with size of abnormal DNA band in one family.
All samples show constant band (C) of 16 kb. Lanes 1 and 5 are normal spouses; lane 2 (grandmother) has fragment expanded by about 05 kb, lane 3 (mother) shows 25 kb expansion; and lane 4 (congenitally affected child) shows a 4 kb expansion.
(see table). The amount of additional DNA caused by expansion of the unstable sequence varies from 100 bp to 6-0 kb, although patients with smears have shown expansions of up to 8 kb. The approximate correlation of band size with disease type is shown in fig 1: congenitally affected patients showed the largest bands while minimally affected individuals have bands only slightly larger than the normal 10 kb. 2 shows correlation of phenotype with band size one family. A grandmother (lane 2) was mildly affected with cataracts removed at 48 years of age but a diagnosis of myotonic dystrophy was only made after the ’1. birth of a granddaughter (lane 4) who had congenital myotonic dystrophy, neonatal hypotonia, respiratory distress, facial diplegia, and mental handicap. Lane 3 shows the child’s mother, who had moderately severe myotonia and muscle weakness with onset in the mid-teens. Thus, increased severity and earlier onset is matched by an increased band size-which provides a biological explanation for the phenomenon of anticipation. Fig 3 shows findings in a similar family in which the increase in severity of disease and of band size mainly occurs between the affected mother (lane 4), with symptoms of myotonia since the age of 20 years and wasting of facial, jaw, and
Fig
within
Results DNA typings were obtained for 73 normal individuals and 127 patients with myotonic dystrophy, of whom 7 were minimally affected, 87 had classic adult onset, 9 had onset in early childhood, and 24 had congenital disease. The variable band was detected in all patients but in none of the controls MINIMAL
CLASSIC ADULT
Lane and somatic DNA
Fig 3-Anticipation myotonic dystrophy. Band size
Fig 1-Distribution of size of expansion of unstable sequence by type of myotonic dystrophy.
instability in family with
All samples show constant band (C) of 16 kb Lanes 1 and 5 are normal spouses; lane 3 shows smeared band of mildly affected grandfather; lane 4 shows 3 kb expansion in his daughter; and lane 2 shows 5 kb expansion in severely affected grandson.
1127
stemomastoid muscles, and her son (lane 2), who had prenatal onset with polyhydramnios, severe hypotonia from birth with delayed but improving motor development, and
striking weakness of jaw and facial muscles. The mildly affected grandfather (lane 3), with cataracts, ptosis, and mild facial weakness only, shows a smear of DNA rather than a discrete band-indicative of somatic instability of the variable band.
Discussion How might DNA analysis for an unstable sequence assist in the clinical management of patients and families affected by myotonic dystrophy? The extreme clinical variability of myotonic dystrophy leads to problems in diagnosis and in prediction for family members at risk for the disorder. Use of linked DNA markers is subject to error because of recombination, and such tests depend on the availability of several family members for their interpretation. Demonstration of a specific molecular defect will allow accurate diagnosis and genetic prediction in many families or individuals for whom previous techniques have been unreliable or impossible. The apparent relation of the size of expansion of the unstable sequence to disease severity will be especially helpful for prenatal diagnosis, in which a parental decision about termination of pregnancy may depend on the likely degree of disability. A retrospective analysis of samples taken from a family with 2 sisters with moderate symptoms of myotonic dsytrophy who requested chorionic villus sampling in pregnancies illustrates this potential benefit in prenatal testing. With the closely linked marker D19S63 (less than 1% recombination proximal to myotonic dystrophy) 2 pregnancies were shown to be at high and 2 at low risk of myotonic dystrophy, the high-risk pregnancies were terminated at the parents’ request. Analysis of residual DNA available for 3 pregnancies confirmed the previous prediction in each case, with an expanded band clearly seen in the pregnancies predicted as high risk but not in the pregnancy predicted to be normal, which ended in the birth of a clinically normal child. The variable bands in the abnormal pregnancies were much larger (2-5 and 3-5 kb) than that in the moderately affected mother (1 -0 kb), which suggests that had these pregnancies continued to term the children would have been severely affected. The technique could also be applied to differential diagnosis of apparent congenital myotonic dystrophy. In one family whom we have followed, a male infant was initially thought to have myotonic dystrophy on the basis of generalised hypotonia, severe feeding problems, an older brother who had died aged 10 weeks with an undiagnosed neuromuscular problem, and a finding of apparent mild myotonia on examination of his 32-year-old mother. However, after muscle biopsy a revised diagnosis of congenital fibre-type disproportion was made. Reassessment at 11 years showed facial and jaw weakness with clear percussion and grip myotonia, and myotonia in the mother was confirmed. Use of DNA analysis revealed an expansion of 3-4 kb, with a smaller expansion (0-5 kb) in the mother: availability of this specific molecular test at the initial assessment would have prevented subsequent misdiagnosis and an inappropriate assessment of prognosis and genetic risks, which was not corrected for 10 years. Similarly, the technique could be used to exclude myotonic dystrophy in siblings of congenitally affected individuals. In one family the first and third born daughters were severely affected with congenital myotonic dystrophy and their
mother was moderately affected with myotonia and general muscle weakness from 25 years of age. The second daughter had had recurrent otitis media and also complained of muscle pains on exercise. The parents were convinced that she was affected, despite normal clinical findings and electromyography at another hospital. In view of continued parental concern and the possible need for an ear operation under general anaesthetic, as well as to assess long-term prognosis, conventional linkage analysis was done at 7 years of age and the closely linked marker D19S63 showed a low risk. Subsequent analysis for the variable sequence confirmed her unaffected status, with no expanded fragment, whereas expansions were found in the affected mother and both affected sisters. Finally, identification of the variable sequence might help in accurate diagnosis of individuals with mild or minimal symptoms and signs suggestive of myotonic dystrophy. When cataract is the only abnormality in an individual thought to have transmitted the myotonic dystrophy gene the diagnosis may often be uncertain. For example, in one family a 65-year-old man had a son and grandson with classic myotonic dystrophy. He himself had no neuromuscular symptoms or abnormal neurological signs, and no abnormalities on electromyography; cataracts had been removed in his 50s. His 63-year-old wife had no
neurological,
electromyographic,
or
ophthalmic
abnormalities. DNA analysis of the grandfather showed an expansion of015 kb, and a similar abnormality was seen in a sample preserved from his dead mother who had also had cataracts.
Recognition of an unstable DNA sequence may also help explain some of the puzzling clinical and genetic features of myotonic dystrophy. The progressive expansion of the sequence in successive generations (as seen in figs 2 and 3) provides molecular explanation for the phenomenon of anticipation, first recognised in myotonic dystrophy 70 years ago,10 but whose validity has been debated by geneticists ever since.2,3 This finding is not only helpful for genetic counselling but may help to explain the mechanisms underlying expression of the disease, although it remains unclear why the severe congenital form of muscular dystrophy has exclusively maternal transmission because in our series expansion in band size is seen in the offspring of to
affected men and women alike. These findings in myotonic dystrophy show similarities the intergenerational variation seen in fragile X syndrome, in which transmission of an unstable DNA sequence has also been demonstrated. On the basis of these findings Sutherland et all’ pointed out the possible relevance of unstable DNA to anticipation, incomplete penetrance, expression, and other genetic variability, and predicted that a comparable mechanism might operate in other disorders including myotonic dystrophy. The myotonic dystrophy gene has now been cloned and its sequence determined. The unstable sequence is due to a triplet repeat (CTG)n located in the 3’ untranslated region of a gene whose predicted product is a member of the protein kinase family.9,12 The expansion in fragment length seen in myotonic dystrophy patients is caused by an increase in the copy number (n) of the CTG repeat: in normal individuals n is between 5 and 27, but in affected individuals n ranges from about 50 to 2000 or more. Most apparently unrelated cases of myotonic dystrophy from a wide area of Europe have probably arisen from a single ancestral mutation. 13 The restriction fragment length polymorphism identified by the gene probe that detects the unstable sequence shows to
1128
complete linkage disequilibrium with myotonic dystrophy,6 such that all individuals with myotonic dystrophy have the same allele. This finding suggests that the original event (probably an increase from an allele with n 5-27 to one with n :., 50) may have resulted in minimal or no clinical disturbance, but that an adverse clinical outcome arose from enlargement of this allele in subsequent generations because of its instability. How this change produces the observed =
clinical effects on muscle and other symptoms is still to be determined, but the fmding of hereditary unstable DNA in patients with myotonic dystrophy helps to explain anticipation and variable manifestations among patients with this condition.
We thank Mr Lodewijk Sandkinjl and Dr lain Fenton for help with data and Dr Gertrude Kohn, Dr Dian Donnai, and Prof Victor Dubowitz for referring families. Our work is supported by the Muscular Dystrophy Group of Great Britain, Muscular Dystrophy Association and Piton Foundation (USA), the Wellcome Trust, and the Medical Research Council.
analysis,
REFERENCES 1.
Harper PS. Myotonic dystrophy. Philadelphia: Saunders, 1989.
Insulin resistance and
Cigarette smoking is associated with increases in plasma triglycerides and decreases in plasma high density-lipoprotein-cholesterol concentration. These changes not only increase risk of coronary heart disease but also are secondary to resistance to insulin-stimulated glucose uptake or hyperinsulinaemia. To see whether there is a relation between cigarette smoking and insulin-mediated glucose uptake we measured plasma lipid and
lipoprotein concentrations, plasma glucose and insulin response to an oral glucose challenge, and insulin-mediated glucose uptake in 40 matched healthy volunteers (20 non-smokers, 20 smokers). Smokers had significantly higher mean (SEM) very-low-density-lipoprotein triglycerides (0·66 [0·10] vs 0·39 [0·03] mmol/l, p
