271
Mapping of acute (type I) spinal muscular atrophy to chromosome 5q12-q14
Linkage analysis in twenty-five families with acute (type I) spinal muscular atrophy (SMA) showed that the mutant gene responsible for the disorder is tightly linked to the D5S39 locus. The mutation(s) causing the intermediate (type II) and juvenile chronic (type III) forms of SMA were also mapped to DNA marker D5S39 on chromosome 5 (5q12-q14). Thus, the three forms, which have been differentiated clinically on the basis of age of onset and clinical course, are most probably due to different mutations at a single locus on chromosome 5. Prenatal diagnosis of SMA type I will now be possible. Introduction
Hereditary childhood spinal muscular atrophies (SMA) are characterised by degeneration of anterior horn cells of the spinal cord and make up the second most common autosomal recessive disease after cystic fibrosis.1 The diagnosis of these atrophies is based on progressive proximal symmetrical weakness with muscular atrophy. Pathological and electrophysiological evidence of muscle denervation are also found with, in most patients, normal nerve conduction velocities.2,3 The acute form of Werdnig Hoffman disease (type I SMA) is characterised by severe generalised muscle weakness and hypotonia at birth or in the next 3 months. Death, from respiratory failure, usually occurs within the first 2 years. This disease may be distinguished from the intermediate (type 11)4 and juvenile (type III)5,6 forms of chronic SMA by age of onset and slower progression of clinical course. There is controversy over clinical subgroupings within the different forms. SMA has been thought to be either one disorder with a continuous spectrum of clinical expression, or a collection of different diseases with similar pathological changes. 7-9 The underlying biochemical defect remains unknown. Linkage analysis has been used to identify the site of the gene defect. With this technique best results are observed in families with many living affected members, so we began with studies of type II and type IIISMA and found,10 as had others," that these forms of SMA are mapped to DNA marker D5S39 on chromosome 5q 12-q 14. Here we describe our studies in type I disease.
Patients and methods Patients In October, 1988, approximately 400 letters were sent to relevant medical specialists (paediatricians, neuropaediatricians,
neurologists, intensive-care specialists, and geneticists) in France and to European colleagues directly working in this area, requesting pedigree information and clinical histories of, and blood samples from, families with SMA. SMA families were selected on the following diagnostic criteria: (i) proximal, symmetrical limb and trunk weakness; (ii) muscle atrophy without facial or extraocular involvement; (iii) no spasticity, hyperreflexia, sensory loss, or mental retardation; (iv) electromyographic studies showing denervation and diminished motor action potential amplitude with normal or slow nerve conduction velocities;3 and (v) muscle biopsy (done in 71/139 patients) consistent with denervation with no evidence of storage material or other structural abnormalities.2 Patients with confirmed SMA were placed into one of three subgroups. Type I infants were those with generalised muscle weakness, hypotonia, and areflexia starting in the first 3 months of life; these children were unable to sit unaided and respiratory involvement was usually responsible for death before 2 years of age. Type II children were able to sit but unable to stand or walk unaided, and they lived beyond 4 years. Type III patients had proximal muscle weakness, starting after the age of 2.
Methods
Lymphoblastoid cell-lines were established by Epstein-Barr virus transformation of blood samples obtained from every family member. The cell-lines were expanded and the DNA was isolated from these lines or whole blood and then digested with restriction enzymes and electrophoresed on a 0-8% agarose gel for Southern blotting.l2 The p105-153Ra DNA probe (American Type Culture Collection, no 53306) recognises Mspl and Xbal endonuclease polymorphisms at locus D5S39 and identifies two alleles, as previously described;13 it also recognises a PstI polymorphism (unpublished). The computer program MLINK (version 4-9) of the LINKAGE package14 was used to determine whether the DNA probe cosegregated with the locus for SMA. The lod (logarithm of the odds) score indicates the statistical likelihood that two loci are linked. Lod scores vary as a function of 8, which is the frequency with which two loci recombine during meiosis and can be converted to a genetic distance (defmed in centiMorgans, cM) within the genome. Two loci are 1 cM apart (approximately 106 base pairs of DNA) if they recombine in 1 % of all meioses. A lod score above + 3 indicates that the observed data are 1000-fold more likely to occur at a given distance. For the type I form, the significance level of the lod score value was judged as a X2 distribution with one degree of freedom without correction for the prior probability of linkage between two randomly chosen genetic loci since the hypothesis tested was that type I disease is linked to the same marker as type II and type III.
ADDRESSES: INSERM Unité 12, Hôpital des Enfants Malades, Paris (J. Melki, MD, P Sheth, MD, S. Abdelhak, P. Burlet, M. F. Bachelot, Prof J. Frézal, MD, Prof A. Munnich, MD); Centre d’Etudes des Polymorphismes Humains (Prof M. G. Lathrop, PhD). Correspondence to Prof A. Munnich, INSERM Unité 12, Hôpital des Enfants Malades, 149 rue de Sèvies, 75743 Paris, Cedex 15, France.
272
Results Clinical analysis 95 families with childhood SMA were investigated. Clinical information and blood samples from 57 children with type I SMA
provided by paediatricians (20 cases), neuropaediatricians (19 cases), and intensive care specialists (18 cases). These 57 SMA type I children belonged to fifty-six families. In fourteen of these families there had been an affected child who had died; a blood sample was available for only 1 such child. 24 SMA type I patients had at least 1 non-affected sibling and two type I patients belonged to the same were
family. The mean age of affected children was 6 months at the time blood samples were collected. The mean age at death of the 39 affected children who had died was 7-2 months (range 1-23). For chronic SMA, clinical information and blood samples from thirty-nine families with at least 2 living affected children were obtained (these included the twenty-four families previously investigated by linkage analysis10). Twenty-two families had type II and seventeen type III SMA.
Genetic linkage analysis For the purpose of genetic linkage analysis, the twenty-five type I families with at least 1 affected and 1 non-affected child were studied. The maximum lod score between type I locus and the D5S39 marker for combined sexes is 2 36 at &thgr;=0.00 (&khgr;2= 10.87; p < 0-005). LOD SCORES FOR THE SMA LOCUS WITH D5S39
the chronic forms. Taken together, these results strongly indicate that the three SMA independent forms are allelic disorders. The clinical features of childhood SMA are similar to those of Duchenne and Becker muscular dystrophies, in which a wide range of disease expression has been recognized and accounted for by allelic heterogeneity. 18 One would expect a similar result for childhood SMA: one disease composed of several allelic disorders. Prenatal diagnosis of acute SMA disease will now be possible. DNA analysis with probe D5S39 in SMA families will enable affected children to be distinguished from unaffected children when DNA polymorphic sites identify which of each parent’s chromosome 5 carries the mutant allele (informative meiosis). Using three restriction endonucleases, 87% of the families were informative for the D5S39 marker. This findings should be of help in genetic linked locus
as
counselling. At present, when our data are applied in a fully informative pedigree, the probability of error in prediction due to meiotic recombination would be approximately 6% for the single D5S39 marker. When flanking markers are identified, their use should reduce this error rate. It is important to set up DNA banks for affected individuals, especially those with SMA type I, whose life expectancy is quite short. When blood sample or muscle or other tissue sample is not available from deceased affected children, blood collected on filter paper blotters (Guthrie cards) can be stored at room temperature and may be helpful when genotype determination becomes available by PCRamplification.19 A comprehensive programme of family studies should also be undertaken for families who may wish prenatal diagnosis for subsequent pregnancies.
Addendum *Z is the lod
score at
the maximum likelihood estimate of the recombination
fraction,&thgr; 8 families
were
non-informative
(3 type 11, 5 type 1)
Linkage analysis for type II and type III subgroups (table) confirmed our previous finding of the close proximity of the SMA locus to the D5S39 marker. Two recombinants were found within each chronic subgroup, which gave a combined maximum lod score value of 9-06 at a recombination fraction 8 = 0-04. On the basis of the predivided sample test, applied to type I and the combined type II and III subgroups,15 the hypothesis of heterogeneity was rejected. Therefore, the maximum lod score between SMA locus and D5S39 in our study is now 11-08 at Q = 0-03 (0.01-0.06 1-lod-unit confidence interval).
Discussion Acute SMA is the commonest and most severe form of childhood SMA. The clinical subgrouping of childhood SMA has been difficult. Attempts7-9 at improving these classifications on the basis of clinical findings have not always enabled cases to fit neatly into a single subgroup. Since 1983, the reverse genetic approach has been used successfully in the location and identification of the genes responsible for several inherited disorders such as Duchenne muscular dystrophy16 and cystic fibrosis.17 With this method we found that the acute SMA locus is tightly linked to the D5S39 locus. This finding strongly suggests that the three types of SMA have the same map position. Brzustowicz et all’ have reported a two-point lod score for the acute SMA locus with D5S39 of Z =1-6 at 6 = 0- 10, which suggests that acute SMA maps to the same or closely
A study by Gilliam TC, et al, published after acceptance of our paper, also shows genetic homogeneity between acute and chronic forms of spinal muscular atrophy (Nature 1990;
345: 823-25). French Spinal Muscular Atrophy Investigators-Neuropaediatricians: J. Aicardi, Hopital des Enfants-Malades, Paris; J. P. Carriere, Hopital de Purpan, Toulouse; M. Fardeau, Hopital de la Salpetriere, Paris; D. Fontan, Hopital d’Enfants, Bordeaux; P. Landrieu, Hôpital du Kremlin Bicetre, Kremlin Bicetre; G. Ponsot, Hopital Saint-Vincent-de-Paul, Paris. Paediatricians: T. Billette de Villemeur, Hopital Trousseau, Paris; R. Gilly, Centre Hospitalier Lyon-Sud, Pierre Benite; P. Guibaud, Hopital Debrousse, Lyon; S. Gros, Centre Hospitalier, Soissons; J. L. Hasselman, Hôpital Marie-Madeleine, Forbach; J. C. Lambert, Centre Hospitalier, Nice. Intensive care specialists: A. Barois, H6pital Raymond Poincare, Garches; F. Beaufils, Hopital Robert Debre, Paris; J. Costil, Hopital Trousseau, Paris; G. Huault, Hopital du Kremlin Bicetre, Kremlin-Bicetre; C. Puissant, Hopital Nord, Amiens. Geneticists: A. David, Centre Hospitalier, Nantes; H. Joumel, Hopital Prosper Chubert, Vannes; M. Mathieu, Hopital Nord, Amiens; F. Serville, Hopital d’Enfants, Bordeaux; A. Toutain, Hopital Bretonneau, Tours. We thank the families of patients and the physicians from Spain, Belgium, Portugal, Italy, and France for their constant support; Association Francaise contre les Myopathies and the Ministere de la Recherche et de la Technologie (n°88-C0179) for grants; and Mrs Monique Poussiere for secretarial help.
REFERENCES 1. Pearn J. Classification of spinal muscular atrophies. Lancet 1980; i: 919-22. 2. Engel WK. Selective and non-selective susceptibility of muscle fibre types. Arch Neurol 1970; 22: 97-117. 3. Moosa A, Dubowitz V. Motor nerve conduction velocity in spinal muscular atrophy of childhood. Arch Dis Child 1976; 51: 974-77. 4. Dubowitz V. Infantile muscular atrophy. A progressive study with
273
to a slowly progressive variety. Brain 1964; 87: 707-19. 5. Wohlfart G, Fex J, Eliasson S. Hereditary proximal spinal muscular atrophy-a clinical entity simulating progressive muscular dystrophy. Acta Psychiatrica Neurol 1955; 30: 395-406. 6. Kugelberg E, Welander L. Heredofamilial juvenile muscular atrophy simulating muscular dystrophy. Arch Neurol Psychiatry 1956; 75: 500-09. 7. Emery AEH. The nosology of the spinal muscular atrophies. J Med Genet 1971; 8: 481-95. 8. Hausmanowa-Petrusewicz I, Zaremba J, Borkowska JJ. Chronic proximal muscular atrophy of childhood and adolescence: problem of classification and genetic counselling. J Med Genet 1985; 22: 350-53. 9. Emery AEH, Davie AM, Holloway S, Skinner R. International collaborative study of the spinal muscular atrophies. J Neurol Sci 1976; 30: 375-84. 10. Melki J, Abdelhak S, Sheth P, et al. Gene for chronic proximal spinal muscular atrophies maps to chromosome 5q. Nature 1990; 344:
particular reference
767-68.
11. Brzustowicz LM, Lehner T, Castilla LH, et al. Genetic mapping of chronic childhood-onset spinal muscular atrophy to chromosome 5q11·2-13·3. Nature 1990; 344: 540-41.
12. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975; 98: 503-17. 13. Leppert M, Wasmuth J, Overhauser J, et al. A primary genetic linkage map to chromosome 5. Cytogenet Cell Genet 1987; 46: 649. 14. Lathrop GM, Lalouel JM, Julier C, Ott J. Strategies for multilocus linkage analysis in humans. Proc Natl Acad Sci USA 1984; 81: 3443-46. 15. Morton NE. The detection and estimation of linkage between the genes for elliptocytosis and the Rh blood type. Am J Hum Genet 1956; 8: 80-96. 16. Koenig M, Hoffman EP, Bertelson CJ, et al. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 1987; 50: 509-17. 17. Riordan JM, Rommens JM, Kerem B, et al. Identification of the Cystic Fibrosis gene: cloning and characterization of complementary DNA. Science 1989; 245: 1066-73. 18. Monaco AP, Bertelson CJ, Liechti-Gollati S, et al. An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics 1988; 2: 90-95. 19. Lyonnet S, Caillaud C, Rey F, et al. Guthrie cards for detection of point mutations in phenylketonuria. Lancet 1988; ii: 507.
Preliminary report: hepatic vein doppler in the early diagnosis of acute liver transplant rejection
37 transplanted livers (in thirty patients) were assessed by serial doppler ultrasound examination. 18 of 23 biopsy-proved rejection episodes were associated with abrupt damping of the normally pulsatile blood flow of the hepatic veins. In the other 5 episodes, the waveforms were damped at the outset by perioperative ischaemia. There were no rejection episodes with normal traces. Another cause of damping was cholangitis (5 episodes), but was and this distinguishable clinically There were no of biochemically. episodes rejection with normal hepatic vein traces. Serial doppler examination, in combination with clinical evaluation, may allow earlier diagnosis and treatment of liver
rejection. Introduction
of early graft dysfunction after liver is acute rejection1 and many imaging transplantation methods have been advocated for its diagnosis.2 ’ Since only severe rejection is reliably detected, they are of limited value and liver biopsy remains essential. Doppler ultrasound may be helpful in showing hepatic artery or portal vein occlusion,5,6 but as yet has no place in the diagnosis of rejection.’ In the normal liver, doppler of the hepatic veins reveals a pulsatile velocity profile that mirrors the cyclical changes in right atrial pressure. During the cardiac cycle there are periods of retrograde flow in the hepatic veins corresponding to the a and v wave components of the venous pulse8,9 (fig 1). In chronic liver disease this waveform may be severely damped,10 but there are no published data on the appearances in acute liver disease or soon after transplantation. We report our experiences of hepatic vein
The
commonest cause
doppler examination after liver transplantation, in children over eighteen months.
and young adults,
Subjects and
methods
Because our ultrasound resources are limited, we decided to include only those patients passing through the paediatric unit (about one-third of the total number of transplants done). 37 consecutive liver transplants in thirty patients (aged 1-20 yr) were studied for three weeks after transplantation. In most cases a complete child’s liver was transplanted, but in six a cut-down left lobe from an adult donor was used." Hepatic function was assessed daily by measurement of serum bilirubin, alkaline phosphatase,. and aspartate aminotransferase and clotting times (prothrombin and partial thromboplastin times). In all cases of unexplained deterioration in hepatic function or clinically suspected rejection, biopsy specimens were taken before treatment. Routine ultrasound examination of the liver and doppler examination of the hepatic vessels were performed within 24 h of operation and every 2-3 days thereafter. Additional scans were done if clinically indicated and at the time of biopsy. Examinations were made by one of three experienced operators using an Aloka 650 SSD machine with a 3-5 or 5 MHz curvilinear probe. No special patient preparation was required. The scans were done with the patient supine and breathing quietly (or, in the early postoperative period, during mechanical ventilation). Pulsed doppler recordings were obtained from the hepatic artery, portal vein, and the three main hepatic veins. For the hepatic veins, a 10 mm sample volume was used, about 4-6 cm proximal to the inferior vena cava. The large sample volume ensured that part of the
ADDRESSES: Departments of Radiology (R. A. Coulden, FRCR, P D. Britton, FRCR, F Farman, DCR),Paediatrics (G. NobleJamieson, MRCP), and Pathology (D. G. D. Wright, FRCPath), Addenbrooke’s Hospital, Cambridge CB2 2QQ, UK. Correspondence to Dr R. A. Coulden, Department of Cardiology, Manchester Royal Infirmary, Manchester M13 9WL, UK.
Mapping of acute (type I) spinal muscular atrophy to chromosome 5q12-q14
Linkage analysis in twenty-five families with acute (type I) spinal muscular atrophy (SMA) showed that the mutant gene responsible for the disorder is tightly linked to the D5S39 locus. The mutation(s) causing the intermediate (type II) and juvenile chronic (type III) forms of SMA were also mapped to DNA marker D5S39 on chromosome 5 (5q12-q14). Thus, the three forms, which have been differentiated clinically on the basis of age of onset and clinical course, are most probably due to different mutations at a single locus on chromosome 5. Prenatal diagnosis of SMA type I will now be possible. Introduction
Hereditary childhood spinal muscular atrophies (SMA) are characterised by degeneration of anterior horn cells of the spinal cord and make up the second most common autosomal recessive disease after cystic fibrosis.1 The diagnosis of these atrophies is based on progressive proximal symmetrical weakness with muscular atrophy. Pathological and electrophysiological evidence of muscle denervation are also found with, in most patients, normal nerve conduction velocities.2,3 The acute form of Werdnig Hoffman disease (type I SMA) is characterised by severe generalised muscle weakness and hypotonia at birth or in the next 3 months. Death, from respiratory failure, usually occurs within the first 2 years. This disease may be distinguished from the intermediate (type 11)4 and juvenile (type III)5,6 forms of chronic SMA by age of onset and slower progression of clinical course. There is controversy over clinical subgroupings within the different forms. SMA has been thought to be either one disorder with a continuous spectrum of clinical expression, or a collection of different diseases with similar pathological changes. 7-9 The underlying biochemical defect remains unknown. Linkage analysis has been used to identify the site of the gene defect. With this technique best results are observed in families with many living affected members, so we began with studies of type II and type IIISMA and found,10 as had others," that these forms of SMA are mapped to DNA marker D5S39 on chromosome 5q 12-q 14. Here we describe our studies in type I disease.
Patients and methods Patients In October, 1988, approximately 400 letters were sent to relevant medical specialists (paediatricians, neuropaediatricians,
neurologists, intensive-care specialists, and geneticists) in France and to European colleagues directly working in this area, requesting pedigree information and clinical histories of, and blood samples from, families with SMA. SMA families were selected on the following diagnostic criteria: (i) proximal, symmetrical limb and trunk weakness; (ii) muscle atrophy without facial or extraocular involvement; (iii) no spasticity, hyperreflexia, sensory loss, or mental retardation; (iv) electromyographic studies showing denervation and diminished motor action potential amplitude with normal or slow nerve conduction velocities;3 and (v) muscle biopsy (done in 71/139 patients) consistent with denervation with no evidence of storage material or other structural abnormalities.2 Patients with confirmed SMA were placed into one of three subgroups. Type I infants were those with generalised muscle weakness, hypotonia, and areflexia starting in the first 3 months of life; these children were unable to sit unaided and respiratory involvement was usually responsible for death before 2 years of age. Type II children were able to sit but unable to stand or walk unaided, and they lived beyond 4 years. Type III patients had proximal muscle weakness, starting after the age of 2.
Methods
Lymphoblastoid cell-lines were established by Epstein-Barr virus transformation of blood samples obtained from every family member. The cell-lines were expanded and the DNA was isolated from these lines or whole blood and then digested with restriction enzymes and electrophoresed on a 0-8% agarose gel for Southern blotting.l2 The p105-153Ra DNA probe (American Type Culture Collection, no 53306) recognises Mspl and Xbal endonuclease polymorphisms at locus D5S39 and identifies two alleles, as previously described;13 it also recognises a PstI polymorphism (unpublished). The computer program MLINK (version 4-9) of the LINKAGE package14 was used to determine whether the DNA probe cosegregated with the locus for SMA. The lod (logarithm of the odds) score indicates the statistical likelihood that two loci are linked. Lod scores vary as a function of 8, which is the frequency with which two loci recombine during meiosis and can be converted to a genetic distance (defmed in centiMorgans, cM) within the genome. Two loci are 1 cM apart (approximately 106 base pairs of DNA) if they recombine in 1 % of all meioses. A lod score above + 3 indicates that the observed data are 1000-fold more likely to occur at a given distance. For the type I form, the significance level of the lod score value was judged as a X2 distribution with one degree of freedom without correction for the prior probability of linkage between two randomly chosen genetic loci since the hypothesis tested was that type I disease is linked to the same marker as type II and type III.
ADDRESSES: INSERM Unité 12, Hôpital des Enfants Malades, Paris (J. Melki, MD, P Sheth, MD, S. Abdelhak, P. Burlet, M. F. Bachelot, Prof J. Frézal, MD, Prof A. Munnich, MD); Centre d’Etudes des Polymorphismes Humains (Prof M. G. Lathrop, PhD). Correspondence to Prof A. Munnich, INSERM Unité 12, Hôpital des Enfants Malades, 149 rue de Sèvies, 75743 Paris, Cedex 15, France.
272
Results Clinical analysis 95 families with childhood SMA were investigated. Clinical information and blood samples from 57 children with type I SMA
provided by paediatricians (20 cases), neuropaediatricians (19 cases), and intensive care specialists (18 cases). These 57 SMA type I children belonged to fifty-six families. In fourteen of these families there had been an affected child who had died; a blood sample was available for only 1 such child. 24 SMA type I patients had at least 1 non-affected sibling and two type I patients belonged to the same were
family. The mean age of affected children was 6 months at the time blood samples were collected. The mean age at death of the 39 affected children who had died was 7-2 months (range 1-23). For chronic SMA, clinical information and blood samples from thirty-nine families with at least 2 living affected children were obtained (these included the twenty-four families previously investigated by linkage analysis10). Twenty-two families had type II and seventeen type III SMA.
Genetic linkage analysis For the purpose of genetic linkage analysis, the twenty-five type I families with at least 1 affected and 1 non-affected child were studied. The maximum lod score between type I locus and the D5S39 marker for combined sexes is 2 36 at &thgr;=0.00 (&khgr;2= 10.87; p < 0-005). LOD SCORES FOR THE SMA LOCUS WITH D5S39
the chronic forms. Taken together, these results strongly indicate that the three SMA independent forms are allelic disorders. The clinical features of childhood SMA are similar to those of Duchenne and Becker muscular dystrophies, in which a wide range of disease expression has been recognized and accounted for by allelic heterogeneity. 18 One would expect a similar result for childhood SMA: one disease composed of several allelic disorders. Prenatal diagnosis of acute SMA disease will now be possible. DNA analysis with probe D5S39 in SMA families will enable affected children to be distinguished from unaffected children when DNA polymorphic sites identify which of each parent’s chromosome 5 carries the mutant allele (informative meiosis). Using three restriction endonucleases, 87% of the families were informative for the D5S39 marker. This findings should be of help in genetic linked locus
as
counselling. At present, when our data are applied in a fully informative pedigree, the probability of error in prediction due to meiotic recombination would be approximately 6% for the single D5S39 marker. When flanking markers are identified, their use should reduce this error rate. It is important to set up DNA banks for affected individuals, especially those with SMA type I, whose life expectancy is quite short. When blood sample or muscle or other tissue sample is not available from deceased affected children, blood collected on filter paper blotters (Guthrie cards) can be stored at room temperature and may be helpful when genotype determination becomes available by PCRamplification.19 A comprehensive programme of family studies should also be undertaken for families who may wish prenatal diagnosis for subsequent pregnancies.
Addendum *Z is the lod
score at
the maximum likelihood estimate of the recombination
fraction,&thgr; 8 families
were
non-informative
(3 type 11, 5 type 1)
Linkage analysis for type II and type III subgroups (table) confirmed our previous finding of the close proximity of the SMA locus to the D5S39 marker. Two recombinants were found within each chronic subgroup, which gave a combined maximum lod score value of 9-06 at a recombination fraction 8 = 0-04. On the basis of the predivided sample test, applied to type I and the combined type II and III subgroups,15 the hypothesis of heterogeneity was rejected. Therefore, the maximum lod score between SMA locus and D5S39 in our study is now 11-08 at Q = 0-03 (0.01-0.06 1-lod-unit confidence interval).
Discussion Acute SMA is the commonest and most severe form of childhood SMA. The clinical subgrouping of childhood SMA has been difficult. Attempts7-9 at improving these classifications on the basis of clinical findings have not always enabled cases to fit neatly into a single subgroup. Since 1983, the reverse genetic approach has been used successfully in the location and identification of the genes responsible for several inherited disorders such as Duchenne muscular dystrophy16 and cystic fibrosis.17 With this method we found that the acute SMA locus is tightly linked to the D5S39 locus. This finding strongly suggests that the three types of SMA have the same map position. Brzustowicz et all’ have reported a two-point lod score for the acute SMA locus with D5S39 of Z =1-6 at 6 = 0- 10, which suggests that acute SMA maps to the same or closely
A study by Gilliam TC, et al, published after acceptance of our paper, also shows genetic homogeneity between acute and chronic forms of spinal muscular atrophy (Nature 1990;
345: 823-25). French Spinal Muscular Atrophy Investigators-Neuropaediatricians: J. Aicardi, Hopital des Enfants-Malades, Paris; J. P. Carriere, Hopital de Purpan, Toulouse; M. Fardeau, Hopital de la Salpetriere, Paris; D. Fontan, Hopital d’Enfants, Bordeaux; P. Landrieu, Hôpital du Kremlin Bicetre, Kremlin Bicetre; G. Ponsot, Hopital Saint-Vincent-de-Paul, Paris. Paediatricians: T. Billette de Villemeur, Hopital Trousseau, Paris; R. Gilly, Centre Hospitalier Lyon-Sud, Pierre Benite; P. Guibaud, Hopital Debrousse, Lyon; S. Gros, Centre Hospitalier, Soissons; J. L. Hasselman, Hôpital Marie-Madeleine, Forbach; J. C. Lambert, Centre Hospitalier, Nice. Intensive care specialists: A. Barois, H6pital Raymond Poincare, Garches; F. Beaufils, Hopital Robert Debre, Paris; J. Costil, Hopital Trousseau, Paris; G. Huault, Hopital du Kremlin Bicetre, Kremlin-Bicetre; C. Puissant, Hopital Nord, Amiens. Geneticists: A. David, Centre Hospitalier, Nantes; H. Joumel, Hopital Prosper Chubert, Vannes; M. Mathieu, Hopital Nord, Amiens; F. Serville, Hopital d’Enfants, Bordeaux; A. Toutain, Hopital Bretonneau, Tours. We thank the families of patients and the physicians from Spain, Belgium, Portugal, Italy, and France for their constant support; Association Francaise contre les Myopathies and the Ministere de la Recherche et de la Technologie (n°88-C0179) for grants; and Mrs Monique Poussiere for secretarial help.
REFERENCES 1. Pearn J. Classification of spinal muscular atrophies. Lancet 1980; i: 919-22. 2. Engel WK. Selective and non-selective susceptibility of muscle fibre types. Arch Neurol 1970; 22: 97-117. 3. Moosa A, Dubowitz V. Motor nerve conduction velocity in spinal muscular atrophy of childhood. Arch Dis Child 1976; 51: 974-77. 4. Dubowitz V. Infantile muscular atrophy. A progressive study with
273
to a slowly progressive variety. Brain 1964; 87: 707-19. 5. Wohlfart G, Fex J, Eliasson S. Hereditary proximal spinal muscular atrophy-a clinical entity simulating progressive muscular dystrophy. Acta Psychiatrica Neurol 1955; 30: 395-406. 6. Kugelberg E, Welander L. Heredofamilial juvenile muscular atrophy simulating muscular dystrophy. Arch Neurol Psychiatry 1956; 75: 500-09. 7. Emery AEH. The nosology of the spinal muscular atrophies. J Med Genet 1971; 8: 481-95. 8. Hausmanowa-Petrusewicz I, Zaremba J, Borkowska JJ. Chronic proximal muscular atrophy of childhood and adolescence: problem of classification and genetic counselling. J Med Genet 1985; 22: 350-53. 9. Emery AEH, Davie AM, Holloway S, Skinner R. International collaborative study of the spinal muscular atrophies. J Neurol Sci 1976; 30: 375-84. 10. Melki J, Abdelhak S, Sheth P, et al. Gene for chronic proximal spinal muscular atrophies maps to chromosome 5q. Nature 1990; 344:
particular reference
767-68.
11. Brzustowicz LM, Lehner T, Castilla LH, et al. Genetic mapping of chronic childhood-onset spinal muscular atrophy to chromosome 5q11·2-13·3. Nature 1990; 344: 540-41.
12. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975; 98: 503-17. 13. Leppert M, Wasmuth J, Overhauser J, et al. A primary genetic linkage map to chromosome 5. Cytogenet Cell Genet 1987; 46: 649. 14. Lathrop GM, Lalouel JM, Julier C, Ott J. Strategies for multilocus linkage analysis in humans. Proc Natl Acad Sci USA 1984; 81: 3443-46. 15. Morton NE. The detection and estimation of linkage between the genes for elliptocytosis and the Rh blood type. Am J Hum Genet 1956; 8: 80-96. 16. Koenig M, Hoffman EP, Bertelson CJ, et al. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 1987; 50: 509-17. 17. Riordan JM, Rommens JM, Kerem B, et al. Identification of the Cystic Fibrosis gene: cloning and characterization of complementary DNA. Science 1989; 245: 1066-73. 18. Monaco AP, Bertelson CJ, Liechti-Gollati S, et al. An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics 1988; 2: 90-95. 19. Lyonnet S, Caillaud C, Rey F, et al. Guthrie cards for detection of point mutations in phenylketonuria. Lancet 1988; ii: 507.
Preliminary report: hepatic vein doppler in the early diagnosis of acute liver transplant rejection
37 transplanted livers (in thirty patients) were assessed by serial doppler ultrasound examination. 18 of 23 biopsy-proved rejection episodes were associated with abrupt damping of the normally pulsatile blood flow of the hepatic veins. In the other 5 episodes, the waveforms were damped at the outset by perioperative ischaemia. There were no rejection episodes with normal traces. Another cause of damping was cholangitis (5 episodes), but was and this distinguishable clinically There were no of biochemically. episodes rejection with normal hepatic vein traces. Serial doppler examination, in combination with clinical evaluation, may allow earlier diagnosis and treatment of liver
rejection. Introduction
of early graft dysfunction after liver is acute rejection1 and many imaging transplantation methods have been advocated for its diagnosis.2 ’ Since only severe rejection is reliably detected, they are of limited value and liver biopsy remains essential. Doppler ultrasound may be helpful in showing hepatic artery or portal vein occlusion,5,6 but as yet has no place in the diagnosis of rejection.’ In the normal liver, doppler of the hepatic veins reveals a pulsatile velocity profile that mirrors the cyclical changes in right atrial pressure. During the cardiac cycle there are periods of retrograde flow in the hepatic veins corresponding to the a and v wave components of the venous pulse8,9 (fig 1). In chronic liver disease this waveform may be severely damped,10 but there are no published data on the appearances in acute liver disease or soon after transplantation. We report our experiences of hepatic vein
The
commonest cause
doppler examination after liver transplantation, in children over eighteen months.
and young adults,
Subjects and
methods
Because our ultrasound resources are limited, we decided to include only those patients passing through the paediatric unit (about one-third of the total number of transplants done). 37 consecutive liver transplants in thirty patients (aged 1-20 yr) were studied for three weeks after transplantation. In most cases a complete child’s liver was transplanted, but in six a cut-down left lobe from an adult donor was used." Hepatic function was assessed daily by measurement of serum bilirubin, alkaline phosphatase,. and aspartate aminotransferase and clotting times (prothrombin and partial thromboplastin times). In all cases of unexplained deterioration in hepatic function or clinically suspected rejection, biopsy specimens were taken before treatment. Routine ultrasound examination of the liver and doppler examination of the hepatic vessels were performed within 24 h of operation and every 2-3 days thereafter. Additional scans were done if clinically indicated and at the time of biopsy. Examinations were made by one of three experienced operators using an Aloka 650 SSD machine with a 3-5 or 5 MHz curvilinear probe. No special patient preparation was required. The scans were done with the patient supine and breathing quietly (or, in the early postoperative period, during mechanical ventilation). Pulsed doppler recordings were obtained from the hepatic artery, portal vein, and the three main hepatic veins. For the hepatic veins, a 10 mm sample volume was used, about 4-6 cm proximal to the inferior vena cava. The large sample volume ensured that part of the
ADDRESSES: Departments of Radiology (R. A. Coulden, FRCR, P D. Britton, FRCR, F Farman, DCR),Paediatrics (G. NobleJamieson, MRCP), and Pathology (D. G. D. Wright, FRCPath), Addenbrooke’s Hospital, Cambridge CB2 2QQ, UK. Correspondence to Dr R. A. Coulden, Department of Cardiology, Manchester Royal Infirmary, Manchester M13 9WL, UK.
