- Email: [email protected]
MUSCULAR DYSTROPHY OR SPINAL MUSCULAR ATROPHY?
960
antibodies, and the time-course and pattern of clinical disease were atypical of TBE. We cannot completely rule out concomitant developing multiple sclerosis and unexplained infarction fortuitously a few days after vaccination. However, we do wonder if the repeated simultaneous TBE and tetanus toxoid vaccination
might have caused an allergic cross-reaction with CNS epitopes, leading to multifocal cerebral vasculitis and infarction-as has been postulated for the rare cerebral infarction that follows immunisation against diphtheria.4 Neurological complications to immunisation with TBE virus antigen or tetanus toxoid alone are rare; simultaneous vaccination could be critical and should therfore be avoided. M. SCHABET H. WIETHOELTER W. GRODD A. VALLBRACHT
Neurological Department, Klinikum Schnarrenberg, University of Tubingen, D-7400 Tubingen, West Germany
J. DICHGANS W. BECKER P. A. BERG
Medical Department,
University of Tubingen 1.
Hopf H-Ch. Guillain-Barré syndrome following tetanus toxoid administration. Akt
2.
Rutledge SL, Snead OC. Neurologic complications of immunizations. J Pediatr 1986;
Neurol 1980; 7: 195-200. 109: 917-24. 3. Scholz E, Wiethoelter H. Postvaccinal
neuropathy after active immunization against early-summer meningo-encephalitis (case report). Dtsch Med Wschr 1987; 112: 544-46.
4.
Ehrengut W. Neural complications following immunization against diphtheria. Dtsch Med Wschr
1986; 111: 939-42.
LATE REFERRAL FOR BILIARY ATRESIA
SIR,-Dr Mieli-Vergani and colleagues (Feb 25, p 421) state that late referral of infants with extrahepatic biliary atresia would be lessened if infants were routinely reviewed at four weeks of age instead of the customary examination at 6 weeks. This change is applauded, but does it go far enough when the whole range of neonatal disorders is considered? To ensure that the various organ systems of the baby have adapted successfully to extra-uterine existence, appropriate care consists of physical examination in the first day and close observation in the maternity hospital, followed by repeat examination one week after discharged This arrangement permits the identification of, for example, persisting jaundice, abnormalities missed in hospital (eg, eye defects and heart murmurs), failure to gain weight, and excessive crying. It also gives the mother an opportunity to discuss feeding or other problems she may be having with the baby. Subsequent follow-up with infant welfare or other services can then be arranged. The six-week well-baby review is a remnant of obsteteric tradition that is not based on sound pathophysiological principles. Hamilton Medical
Group, Hamilton, Victoria, 3300, Australia 1. Douglas HM. Some aspects of neonatal 1985-86; 3: 258-74.
HUGH DOUGLAS
care.
Records Adelaide Children’s
Hosp
UNUSUAL CASE OF HAEMOPHILIA B
SIR,-Haemophilia B is an inherited bleeding disorder, resulting from a deficiency in clotting factor IX. The factor IX gene has been well characterised and in many instances the mutations responsible for the disorder have been definedHere we report an unusual case of haemophilia B and identify a point mutation which, we believe, is responsible for the condition. The patient, born in 1965, exhibited a moderately severe bleeding disorder in early life. He presented October, 1971, having had 7 days of sustained bleeding after biting his tongue. There was no family history of bleeding. His partial thromboplastin time was 53 s (control 29 s), prothrombin time normal, and bleeding time 3 min (tests done at Norfolk and Norwich Hospital). Screening tests indicated factor IX deficiency. This was confirmed in a one-stage factor IX assay (Cambridge Haemophilia Centre), which showed his factor IX clotting level to be 27% of normal. Factor VIII clotting level was normal.
patient had on average six bleeding usually after trauma and rarely with haemarthrosis. He was treated successfully with fresh frozen plasma Between 1971 and 1980 the
episodes
per
year,
until 1973 and with factor IX concentrate from 1973 to 1980. He did attend for treatment after 1980. At first this extraordinary lapse was attributed to adolescent revolt but during an admission for an unrelated problem, in March, 1988, the patient explained that he no longer had prolonged bleeding after trauma and no longer required factor IX. His factor IX level was found to have increased to a level compatible with normal haemostasis (35% of normal in March, 1988, and 33% in November, 1988). This unusual clinical course is not unprecedented. Some time ago a similar condition was described in the Netherlands and named haemophilia B Leyden.2 Several patients from the pedigree were reported. The level of factor IX gradually increased after puberty, apparently in response to increasing testosterone.3 An American patient of Armenian descent and a Greek patient also displaying a Leyden-like phenotype have been described.4 In all three cases, DNA analysis revealed mutations in the factor IX promoter region (ie, the region directly responsible for controlling gene expression). Haemophilia B Leyden is associated with a T—A nucleotide transition at position — 205 (relative to the start of transcription, designated + 16); the American patient has a A—G transition at + 13; and the Greek patient has a single base pair deletion, again at + 13.4 In the light of these results we sought to characterise the mutation in our patient. DNA was purified from our patient’s blood and a region - 257 to + 75 was amplified by the polymerase chain reaction. The DNA fragment was sequenced directly by the dideoxy chain termination method of Sanger with slight modifications.’ An AG transition was found at + 13. The patient’s mother carries an A--* G transition at + 13 together with a normal allele. The patient’s two sisters are both free of this mutation. We propose to call our patient’s defect haemophilia B Norwich. Now that a change at + 13 has been associated with this condition in three presumably unrelated individuals, it seems very likely that this mutation is responsible for the Leyden-like phenotype. The elucidation of how the mutation so drastically alters factor IX expression and how hormonal changes at puberty influence expression must await further analysis of the factor IX promoter. not
Sir William Dunn School of Pathology,
University of Oxford, Oxford OX1 3RE, Department of Haematology, Norfolk and Norwich Hospital, and Oxford Haemophilia Centre, Churchill Hospital, Oxford
P. M. CROSSLEY P. R. WINSHIP A. BLACK C. R. RIZZA G. G. BROWNLEE
1. Brownlee, GG. Haemophilia B: a review of patient defects, diagnosis with gene probes and prospects for gene therapy. Rec Adv Haematol 1988; 5: 251-64 2. Veltkamp JJ, Meiloff J, Remmelts HG, van der Vlerk D, Loeliger EA Another genetic variant of haemophilia B: Haemophilia B Leydon. Scand J Haematol 1970; 7: 82-90. 3. Bnet E, Rogier MB, van Tilburg NH, Veltkamp, JJ. A sex-linked hereditary disorder that improves after puberty N EnglJ Med 1982; 306: 788-90. 4. Reitsma PH, Mandalaki T, Kasper CK, Bertina RM, Bnet E. Two novel point mutations correlate with an altered developmental expression of blood coagulation factor IX (haemophilia B Leyden phenotype). Blood 1989; 73: 743-46 5. Reitsma PH, Bertina RM, Ploos van Amstel JK, Riemens A, Bnet E The putative factor IX gene promoter in haemophilia B Leyden. Blood 1988; 72: 1074-76 6. Anson DS, Choo KH, Rees DJG, et al. The gene satructure of human antihaemophilic factor IX. EMBO J 1984; 3: 1053-60. 7. Winship PR An improved method for directly sequencing PCR amplified material using dimethylsulphoxide Nucl Acids Res 1989; 17: 1266.
MUSCULAR DYSTROPHY OR SPINAL MUSCULAR ATROPHY?
SIR,-Dr Clarke and colleagues (Feb 25, p 443) question the existence of adolescent onset X-linked spinal muscular atrophy (SMA). They applied Xp21 DNA probes in two brothers who were diagnosed as SMA mimicking Becker’s muscular dystrophy a decade ago. Deletion of exons were shown in these two brothers and also in other affected males from their large X-linked pedigree. We have seen a similar family. A 33 year-old man had been diagnosed as having KugelbergWelander disease on the basis of onset at the late adolescent stage,
961
neuropathic electromyographic (EMG) findings, and neuropathic/ myopathic changes on muscle biopsy in 1981. He is married to a maternal first-degree cousin (figure). He has two affected brothers who have slowly progressive muscle weakness which started in adolescence (we have not been able to investigate them yet). This man now presents with moderate proximal muscle weakness. His creatine phosphokinase (CPK) is elevated at 1057 U/1 (normal 22-191), and his latest EMG shows a myopathic pattern. His elder son is aged 10 years and has slight difficulty in climbing stairs and heel walking. His CPK is 3508 V jl and he has a myopathic EMG. A muscle biopsy from gastrocnemius showed mild dystrophic changes: rounding of fibres, focal regeneration with minimal increase in endomysial connective tissue, and no evidence of type grouping. A younger 8-year-old son is so far symptomless (CPK 136 Vjl; myopathic EMG). The mother has no muscle complaints (CPK 136 U/1). Most likely this family has Becker type muscular dystrophy. Our index patient was diagnosed as KugelbergWelander disease years ago. This is not surprising; neuropathic findings may be encountered both clinically and on muscle biopsy in Becker’s muscular dystrophy.l,2
Family pedigree. We agree with Clarke and colleagues’ view that juvenile onset X-linked SMA may be a misnomer. The family we present is another example that casts doubt on its existence. Measurement of dystrophin in Duchenne’s and Becker’s muscular dystrophy,3as well as in carriers,4 has introduced a new era to muscle disorders. Routine dystrophin measurement in muscle tissue may help to explain many of the unestablished issues in this group of diseases. Departments of Paediatric Neurology and Pathology, Hacettepe University Children’s Hospital, Ankara, Turkey
HALUK TOPALOGLU YAVUZ RENDA GULSEV KALE KIVILCIM GUCUYENER
1. Bradley WG, Jones MZ, Mussini JM, Fawcett PRN. Becker-type muscular dystrophy. Muscle Nerve 1978; 1: 111-32. 2. Goebel HH, Prange H, Gullotta F, Kiefer H, Jones MZ. Becker’s X-linked muscular dystrophy: histological, enzyme-histochemical and ultrastructural studies of two cases, originally reported by Becker Acta Neuropathol (Berl) 1979, 46: 69-77 3. Hoffmann EP, Fischbeck KH, Brown RH, et al. Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne’s or Becker’s muscular dystrophy. N Engl J Med 1988; 318: 1363-68. 4. Arahata K, Ishihare T, Kamekura K, et al Mosaic expression of dystrophin in symptomatic carriers of Duchenne’s muscular dystrophy. N Engl J Med 1989; 320: 138-42
Blood DNA was analysed in 16 patients with mitochondrial In 1 patient with a deletion of muscle mt-DNAl PCR was used to amplify a 1-5 kb fragment which bridges the mt-DNA
myopathy.
deletion, defined by oligonucleotide primers complementary to sequences at either end of the segment. We used thirty cycles of PCR followed by an additional thirty using nested primers (to increase sensitivity). This deletion is present in the muscle of about 25% of mitochondrial myopathy patients (I. Holt, personal communication). We then used PCR to screen blood DNA from a previously reported series of patients with mitochondrial myopathy2 and 5 controls in the same way. A product of identical size was demonstrated in a further 3 patients but not in controls. Restriction enzyme analysis had previously failed to detect structural rearrangements in blood mt-DNA from these 3 patients. Although muscle DNA is not available, it is highly likely that deleted mt-DNA is present in muscle and blood. All three had the Keams-Sayre phenotype, which is associated with deletions of mt-DNA.4 In two cases restriction mapping of the product was identical to that for the patient known to have a deletion, and in the third the deletion was probably about 60 bp larger. Finally, when one of the internal primers was replaced, an approximately 600 bp product was generated as predicted in all 4 patients. It remains theoretically possible that the PCR products in the 3 patients in whom no muscle was available represent duplications rather than deletions, but these would have to differ greatly from those previously described. Absolute confirmation that the amplified fragments arise from deleted genomes does, of course, require sequence data. These observations confirm the presence of low levels of abnormal mitochondrial genomes in blood of a substantial proportion of patients. The primers were specific for only one of the several deletions that have been described. It seems likely that most or all of the known deletions of mt-DNA known to be present in up to 40% of patients could be detected in blood using different sets of primers. This new approach may enable non-invasive diagnosis in up to 40 % of symptom-free patients with mitochondrial myopathy, and identification of relatives at risk for transmitting the disease. Department of Paediatrics, University of Oxford, John Radcliffe Hospital,
JOANNA POULTON
Oxford OX3 9DU
R. MARK GARDINER
1. Holt IJ, Harding AE, Morgan-Hughes JA. Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature 1988; 331: 717-19. 2. Poulton J, Deadman ME, Gardiner RM. Duplications of mitochondrial DNA in mitochondrial myopathy. Lancet 1989; i: 236-40. 3. Lestienne P, Ponsot G. Keams-Sayre syndrome with muscle mitochondrial DNA
deletion. Lancet 1988; i: 885. M, Morales CT, Di Mauro
4. Zeviani
MYOPATHY
SiR,—In the past year both deletions1 and duplications2 of mitochondrial DNA (mt-DNA) have been demonstrated in patients with mitochondrial myopathy. In patients with mitochondrial myopathy and mt-DNA duplications (a minority), the mt-DNA has been found in every tissue investigated2 so that non-invasive diagnosis on a single blood sample is possible. In patients with deletions, different tissues show a wide variation in proportions of normal and deleted mt-DNA. Muscle is the only tissue in which restriction mapping has demonstrated deletions.’ Dr Johns and colleagues (Feb 18, p 393) report a significant advance-use of the polymerase chain reaction (PCR) to detect deleted genomes at levels below the sensitivity of restriction mapping. Our observations on 4 cases suggest that non-invasive diagnosis may be possible in a substantial proportion of patients.
et
al. Deletions of mitochondrial DNA 38: 1339-46.
in
PRENATALLY DIAGNOSED 45,X/46,XY AND NORMAL PHENOTYPE on 45,X/46,XY mosaicism have postnatally diagnosed cases with mixed gonadal dysgenesis, ambiguous genitalia, or features of Turner syndrome, but anecdotal reports of prenatally diagnosed cases do not support such a high rate of abnormality (see Hsu’s review1 ). We have studied outcome in prenatally diagnosed 45,X/46,XY mosaicism to assess
SiR,—Most publications
focused
NON-INVASIVE DIAGNOSIS OF MITOCHONDRIAL
S,
Keams-Sayre syndrome. Neurology 1988;
on
accurately the true rate of abnormality. 730 questionnaires seeking information on cytogenetic findings and clinical outcome (specifically general phenotype and external genitalia) in prenatally diagnosed 45,X/46,XY were sent to cytogenetic laboratories world wide. We received 174 replies (25%). 93 true mosaic, prenatally diagnosed cases were detected in over 548 000 amniotic fluid samples, a rate of 1-7 per 10 000; this group included 6 cases with a 47,XYY cell line. Postnatal cytogenetic studies were available in 59 cases: 46 were 45,X/46,XY, 2 were 45,X/46, XY j47 ,xYY, 2 were 46,X/47,XYY, 7 were 46,XY, and 2 were 45,X. The outcome of pregnancy was 39 elective terminations (42%), 3 spontaneous abortions, 1 fetal death, 1 stillbirth, and 45 live births (48%); 4 pregnancies were still in progress.
more
antibodies, and the time-course and pattern of clinical disease were atypical of TBE. We cannot completely rule out concomitant developing multiple sclerosis and unexplained infarction fortuitously a few days after vaccination. However, we do wonder if the repeated simultaneous TBE and tetanus toxoid vaccination
might have caused an allergic cross-reaction with CNS epitopes, leading to multifocal cerebral vasculitis and infarction-as has been postulated for the rare cerebral infarction that follows immunisation against diphtheria.4 Neurological complications to immunisation with TBE virus antigen or tetanus toxoid alone are rare; simultaneous vaccination could be critical and should therfore be avoided. M. SCHABET H. WIETHOELTER W. GRODD A. VALLBRACHT
Neurological Department, Klinikum Schnarrenberg, University of Tubingen, D-7400 Tubingen, West Germany
J. DICHGANS W. BECKER P. A. BERG
Medical Department,
University of Tubingen 1.
Hopf H-Ch. Guillain-Barré syndrome following tetanus toxoid administration. Akt
2.
Rutledge SL, Snead OC. Neurologic complications of immunizations. J Pediatr 1986;
Neurol 1980; 7: 195-200. 109: 917-24. 3. Scholz E, Wiethoelter H. Postvaccinal
neuropathy after active immunization against early-summer meningo-encephalitis (case report). Dtsch Med Wschr 1987; 112: 544-46.
4.
Ehrengut W. Neural complications following immunization against diphtheria. Dtsch Med Wschr
1986; 111: 939-42.
LATE REFERRAL FOR BILIARY ATRESIA
SIR,-Dr Mieli-Vergani and colleagues (Feb 25, p 421) state that late referral of infants with extrahepatic biliary atresia would be lessened if infants were routinely reviewed at four weeks of age instead of the customary examination at 6 weeks. This change is applauded, but does it go far enough when the whole range of neonatal disorders is considered? To ensure that the various organ systems of the baby have adapted successfully to extra-uterine existence, appropriate care consists of physical examination in the first day and close observation in the maternity hospital, followed by repeat examination one week after discharged This arrangement permits the identification of, for example, persisting jaundice, abnormalities missed in hospital (eg, eye defects and heart murmurs), failure to gain weight, and excessive crying. It also gives the mother an opportunity to discuss feeding or other problems she may be having with the baby. Subsequent follow-up with infant welfare or other services can then be arranged. The six-week well-baby review is a remnant of obsteteric tradition that is not based on sound pathophysiological principles. Hamilton Medical
Group, Hamilton, Victoria, 3300, Australia 1. Douglas HM. Some aspects of neonatal 1985-86; 3: 258-74.
HUGH DOUGLAS
care.
Records Adelaide Children’s
Hosp
UNUSUAL CASE OF HAEMOPHILIA B
SIR,-Haemophilia B is an inherited bleeding disorder, resulting from a deficiency in clotting factor IX. The factor IX gene has been well characterised and in many instances the mutations responsible for the disorder have been definedHere we report an unusual case of haemophilia B and identify a point mutation which, we believe, is responsible for the condition. The patient, born in 1965, exhibited a moderately severe bleeding disorder in early life. He presented October, 1971, having had 7 days of sustained bleeding after biting his tongue. There was no family history of bleeding. His partial thromboplastin time was 53 s (control 29 s), prothrombin time normal, and bleeding time 3 min (tests done at Norfolk and Norwich Hospital). Screening tests indicated factor IX deficiency. This was confirmed in a one-stage factor IX assay (Cambridge Haemophilia Centre), which showed his factor IX clotting level to be 27% of normal. Factor VIII clotting level was normal.
patient had on average six bleeding usually after trauma and rarely with haemarthrosis. He was treated successfully with fresh frozen plasma Between 1971 and 1980 the
episodes
per
year,
until 1973 and with factor IX concentrate from 1973 to 1980. He did attend for treatment after 1980. At first this extraordinary lapse was attributed to adolescent revolt but during an admission for an unrelated problem, in March, 1988, the patient explained that he no longer had prolonged bleeding after trauma and no longer required factor IX. His factor IX level was found to have increased to a level compatible with normal haemostasis (35% of normal in March, 1988, and 33% in November, 1988). This unusual clinical course is not unprecedented. Some time ago a similar condition was described in the Netherlands and named haemophilia B Leyden.2 Several patients from the pedigree were reported. The level of factor IX gradually increased after puberty, apparently in response to increasing testosterone.3 An American patient of Armenian descent and a Greek patient also displaying a Leyden-like phenotype have been described.4 In all three cases, DNA analysis revealed mutations in the factor IX promoter region (ie, the region directly responsible for controlling gene expression). Haemophilia B Leyden is associated with a T—A nucleotide transition at position — 205 (relative to the start of transcription, designated + 16); the American patient has a A—G transition at + 13; and the Greek patient has a single base pair deletion, again at + 13.4 In the light of these results we sought to characterise the mutation in our patient. DNA was purified from our patient’s blood and a region - 257 to + 75 was amplified by the polymerase chain reaction. The DNA fragment was sequenced directly by the dideoxy chain termination method of Sanger with slight modifications.’ An AG transition was found at + 13. The patient’s mother carries an A--* G transition at + 13 together with a normal allele. The patient’s two sisters are both free of this mutation. We propose to call our patient’s defect haemophilia B Norwich. Now that a change at + 13 has been associated with this condition in three presumably unrelated individuals, it seems very likely that this mutation is responsible for the Leyden-like phenotype. The elucidation of how the mutation so drastically alters factor IX expression and how hormonal changes at puberty influence expression must await further analysis of the factor IX promoter. not
Sir William Dunn School of Pathology,
University of Oxford, Oxford OX1 3RE, Department of Haematology, Norfolk and Norwich Hospital, and Oxford Haemophilia Centre, Churchill Hospital, Oxford
P. M. CROSSLEY P. R. WINSHIP A. BLACK C. R. RIZZA G. G. BROWNLEE
1. Brownlee, GG. Haemophilia B: a review of patient defects, diagnosis with gene probes and prospects for gene therapy. Rec Adv Haematol 1988; 5: 251-64 2. Veltkamp JJ, Meiloff J, Remmelts HG, van der Vlerk D, Loeliger EA Another genetic variant of haemophilia B: Haemophilia B Leydon. Scand J Haematol 1970; 7: 82-90. 3. Bnet E, Rogier MB, van Tilburg NH, Veltkamp, JJ. A sex-linked hereditary disorder that improves after puberty N EnglJ Med 1982; 306: 788-90. 4. Reitsma PH, Mandalaki T, Kasper CK, Bertina RM, Bnet E. Two novel point mutations correlate with an altered developmental expression of blood coagulation factor IX (haemophilia B Leyden phenotype). Blood 1989; 73: 743-46 5. Reitsma PH, Bertina RM, Ploos van Amstel JK, Riemens A, Bnet E The putative factor IX gene promoter in haemophilia B Leyden. Blood 1988; 72: 1074-76 6. Anson DS, Choo KH, Rees DJG, et al. The gene satructure of human antihaemophilic factor IX. EMBO J 1984; 3: 1053-60. 7. Winship PR An improved method for directly sequencing PCR amplified material using dimethylsulphoxide Nucl Acids Res 1989; 17: 1266.
MUSCULAR DYSTROPHY OR SPINAL MUSCULAR ATROPHY?
SIR,-Dr Clarke and colleagues (Feb 25, p 443) question the existence of adolescent onset X-linked spinal muscular atrophy (SMA). They applied Xp21 DNA probes in two brothers who were diagnosed as SMA mimicking Becker’s muscular dystrophy a decade ago. Deletion of exons were shown in these two brothers and also in other affected males from their large X-linked pedigree. We have seen a similar family. A 33 year-old man had been diagnosed as having KugelbergWelander disease on the basis of onset at the late adolescent stage,
961
neuropathic electromyographic (EMG) findings, and neuropathic/ myopathic changes on muscle biopsy in 1981. He is married to a maternal first-degree cousin (figure). He has two affected brothers who have slowly progressive muscle weakness which started in adolescence (we have not been able to investigate them yet). This man now presents with moderate proximal muscle weakness. His creatine phosphokinase (CPK) is elevated at 1057 U/1 (normal 22-191), and his latest EMG shows a myopathic pattern. His elder son is aged 10 years and has slight difficulty in climbing stairs and heel walking. His CPK is 3508 V jl and he has a myopathic EMG. A muscle biopsy from gastrocnemius showed mild dystrophic changes: rounding of fibres, focal regeneration with minimal increase in endomysial connective tissue, and no evidence of type grouping. A younger 8-year-old son is so far symptomless (CPK 136 Vjl; myopathic EMG). The mother has no muscle complaints (CPK 136 U/1). Most likely this family has Becker type muscular dystrophy. Our index patient was diagnosed as KugelbergWelander disease years ago. This is not surprising; neuropathic findings may be encountered both clinically and on muscle biopsy in Becker’s muscular dystrophy.l,2
Family pedigree. We agree with Clarke and colleagues’ view that juvenile onset X-linked SMA may be a misnomer. The family we present is another example that casts doubt on its existence. Measurement of dystrophin in Duchenne’s and Becker’s muscular dystrophy,3as well as in carriers,4 has introduced a new era to muscle disorders. Routine dystrophin measurement in muscle tissue may help to explain many of the unestablished issues in this group of diseases. Departments of Paediatric Neurology and Pathology, Hacettepe University Children’s Hospital, Ankara, Turkey
HALUK TOPALOGLU YAVUZ RENDA GULSEV KALE KIVILCIM GUCUYENER
1. Bradley WG, Jones MZ, Mussini JM, Fawcett PRN. Becker-type muscular dystrophy. Muscle Nerve 1978; 1: 111-32. 2. Goebel HH, Prange H, Gullotta F, Kiefer H, Jones MZ. Becker’s X-linked muscular dystrophy: histological, enzyme-histochemical and ultrastructural studies of two cases, originally reported by Becker Acta Neuropathol (Berl) 1979, 46: 69-77 3. Hoffmann EP, Fischbeck KH, Brown RH, et al. Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne’s or Becker’s muscular dystrophy. N Engl J Med 1988; 318: 1363-68. 4. Arahata K, Ishihare T, Kamekura K, et al Mosaic expression of dystrophin in symptomatic carriers of Duchenne’s muscular dystrophy. N Engl J Med 1989; 320: 138-42
Blood DNA was analysed in 16 patients with mitochondrial In 1 patient with a deletion of muscle mt-DNAl PCR was used to amplify a 1-5 kb fragment which bridges the mt-DNA
myopathy.
deletion, defined by oligonucleotide primers complementary to sequences at either end of the segment. We used thirty cycles of PCR followed by an additional thirty using nested primers (to increase sensitivity). This deletion is present in the muscle of about 25% of mitochondrial myopathy patients (I. Holt, personal communication). We then used PCR to screen blood DNA from a previously reported series of patients with mitochondrial myopathy2 and 5 controls in the same way. A product of identical size was demonstrated in a further 3 patients but not in controls. Restriction enzyme analysis had previously failed to detect structural rearrangements in blood mt-DNA from these 3 patients. Although muscle DNA is not available, it is highly likely that deleted mt-DNA is present in muscle and blood. All three had the Keams-Sayre phenotype, which is associated with deletions of mt-DNA.4 In two cases restriction mapping of the product was identical to that for the patient known to have a deletion, and in the third the deletion was probably about 60 bp larger. Finally, when one of the internal primers was replaced, an approximately 600 bp product was generated as predicted in all 4 patients. It remains theoretically possible that the PCR products in the 3 patients in whom no muscle was available represent duplications rather than deletions, but these would have to differ greatly from those previously described. Absolute confirmation that the amplified fragments arise from deleted genomes does, of course, require sequence data. These observations confirm the presence of low levels of abnormal mitochondrial genomes in blood of a substantial proportion of patients. The primers were specific for only one of the several deletions that have been described. It seems likely that most or all of the known deletions of mt-DNA known to be present in up to 40% of patients could be detected in blood using different sets of primers. This new approach may enable non-invasive diagnosis in up to 40 % of symptom-free patients with mitochondrial myopathy, and identification of relatives at risk for transmitting the disease. Department of Paediatrics, University of Oxford, John Radcliffe Hospital,
JOANNA POULTON
Oxford OX3 9DU
R. MARK GARDINER
1. Holt IJ, Harding AE, Morgan-Hughes JA. Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature 1988; 331: 717-19. 2. Poulton J, Deadman ME, Gardiner RM. Duplications of mitochondrial DNA in mitochondrial myopathy. Lancet 1989; i: 236-40. 3. Lestienne P, Ponsot G. Keams-Sayre syndrome with muscle mitochondrial DNA
deletion. Lancet 1988; i: 885. M, Morales CT, Di Mauro
4. Zeviani
MYOPATHY
SiR,—In the past year both deletions1 and duplications2 of mitochondrial DNA (mt-DNA) have been demonstrated in patients with mitochondrial myopathy. In patients with mitochondrial myopathy and mt-DNA duplications (a minority), the mt-DNA has been found in every tissue investigated2 so that non-invasive diagnosis on a single blood sample is possible. In patients with deletions, different tissues show a wide variation in proportions of normal and deleted mt-DNA. Muscle is the only tissue in which restriction mapping has demonstrated deletions.’ Dr Johns and colleagues (Feb 18, p 393) report a significant advance-use of the polymerase chain reaction (PCR) to detect deleted genomes at levels below the sensitivity of restriction mapping. Our observations on 4 cases suggest that non-invasive diagnosis may be possible in a substantial proportion of patients.
et
al. Deletions of mitochondrial DNA 38: 1339-46.
in
PRENATALLY DIAGNOSED 45,X/46,XY AND NORMAL PHENOTYPE on 45,X/46,XY mosaicism have postnatally diagnosed cases with mixed gonadal dysgenesis, ambiguous genitalia, or features of Turner syndrome, but anecdotal reports of prenatally diagnosed cases do not support such a high rate of abnormality (see Hsu’s review1 ). We have studied outcome in prenatally diagnosed 45,X/46,XY mosaicism to assess
SiR,—Most publications
focused
NON-INVASIVE DIAGNOSIS OF MITOCHONDRIAL
S,
Keams-Sayre syndrome. Neurology 1988;
on
accurately the true rate of abnormality. 730 questionnaires seeking information on cytogenetic findings and clinical outcome (specifically general phenotype and external genitalia) in prenatally diagnosed 45,X/46,XY were sent to cytogenetic laboratories world wide. We received 174 replies (25%). 93 true mosaic, prenatally diagnosed cases were detected in over 548 000 amniotic fluid samples, a rate of 1-7 per 10 000; this group included 6 cases with a 47,XYY cell line. Postnatal cytogenetic studies were available in 59 cases: 46 were 45,X/46,XY, 2 were 45,X/46, XY j47 ,xYY, 2 were 46,X/47,XYY, 7 were 46,XY, and 2 were 45,X. The outcome of pregnancy was 39 elective terminations (42%), 3 spontaneous abortions, 1 fetal death, 1 stillbirth, and 45 live births (48%); 4 pregnancies were still in progress.
more