651
could be used for administration of other pressurised aerosols.7 The only side-effect observed was oral candidiasis, found in 3 children on active treatment and 3 on placebo. Budesonide has a favourable ratio between topical and systemic effects,8,9 and metabolism is increased in younger patients.1o Nevertheless, a systemic effect from the proposed treatment cannot be excluded, and individually increased susceptibility may also be a risk; thus, treatment with budesonide should be reserved for children with moderate to severe symptoms. Topical corticosteroids could be administered as a nebulised suspension, which may be an effective treatment for recurrent wheezing in children below 3 years of age11,12 and avoids the use of freon. However, the nebuliser and air-compressor are costly and require both electricity and maintenance, and there is a risk of contamination.13,14 The procedure may also be timeconsuming and uncomfortable or frightening, and the child’s face may be exposed to an aerosol of a potent topical glucocorticosteroid for long periods, with the risk of a steroid skin rash. Our findings indicate that budesonide aerosol delivered through a spacer and facemask with a 1-way valve gives significant symptom relief in young children with recurrent wheezing who were already treated with inhaled &bgr;2 agonist, and that the need for additional treatment with oral prednisolone was reduced. Further work is needed to quantify the amount of active agent delivered to the airways through this device, and to determine the lowest dose of budesonide that achieves a satisfactory clinical response, but this delivery system may be simpler, and have less risk of side-effects, than use of nebulised corticosteroids.
REFERENCES 1. Wilson NM. Wheezing bronchitis revisited. Arch Dis Child 1989; 64: 1194-99. 2. Lee DA, Winslow NR, Speight ANP, Hey EN. Prevalence and spectrum of asthma in childhood. Br Med J 1983; 286: 1256-68. 3. Bisgaard H, Ohlsson S. PEP-spacer: an adaption for administration of MDI to infants. Allergy 1989; 44: 363-64.
4. Polgar G, Promadhat V. Pulmonary function testing in children: techniques and standards. London: WB Saunders, 1971. 5. Bisgaard H, Dalgaard P, Nyboe J. Risk factors for wheezing during infancy. Acta Paediatr Scand 1987; 76: 719-26. 6. Gleeson JGA, Price JF. Controlled trial of budesonide given by the Nebuhaler in preschool children with asthma. Br Med J 1988; 297: 163-66. 7.
8.
O’Callaghan C, Milner AD, Swarbrick A. Spacer device with face mask attachment for giving bronchodilators to infants with asthma. Br Med J 1989; 298: 160-61. Bisgaard H, Damkjaer Nielsen M, Andersen B, et al. Adrenal function in children
with
bronchial asthma treated with
beclomethasone
dipropionate or budesonide. J Allergy Clin Immunol 1988; 81: 1088-95. 9. Bisgaard H, Pedersen S, Damkjaer M, Østerballe O. Adrenal function in asthmatic children treated with inhaled budesonide. Acta Paediatr Scand (in press). 10. Pedersen S, Steffensen G, Ekman I, Tønnesson M, Borgå O. Pharmacokinetics of budesonide in children with asthma. Eur J Clin Pharmacol 1987; 31: 579-82. 11. Carlsen KH, Leegaard J, Larsen S, Ørstavik I. Nebulized beclomethasone dipropionate in recurrent obstructive episodes after acute bronchiolitis. Arch Dis Child 1988; 63: 1428-33. 12. Pedersen S, Ramsgaard-Hansen O, Nikander K. Budesonide suspension for nebulization in children with asthma. Eur Respir J 1989; 2 (suppl 8): abstr 82. 13. Pierce AK, Sanford JP, Thomas GD, Leonard JS. Long-term evaluation of decontamination of inhalation therapy equipment and the occurrence of necrotizing pneumonia. N Engl J Med 1970; 282: 528-31. 14. Barnes KL, Clifford R, Holgate ST, Murphy D, Comber P, Bell E. Bacterial contamination of home nebulizers. Br Med J 1987; 295: 812-14.
Location of facioscapulohumeral muscular dystrophy gene on chromosome 4
dominant disorder autosomal faciomuscular scapulohumeral dystrophy (FSHD) is the last of the major progressive muscular dystrophies in which the gene had not been located. In linkage analysis on ten Dutch families with this disorder a lod score of 6·34 at a recombination fraction of 0·13 was obtained with the microsatellite marker Mfd 22 (D4S171). This maps the FSHD gene to chromsome 4. Only one family was uninformative for this marker. We found no evidence of genetic
The
heterogeneity. Introduction
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder with presenting symptoms of facial or shoulder girdle weakness usually at the end of the first or in the second decade. The disease is progressive in most patients, with successive involvement of the abdominal, foot-extensor, upper-arm, and pelvic-girdle muscles.1,2 The rate of progression is variable between and within families. Approximately 10% of gene carriers will become wheelchair dependent, usually at an older age.3
One-third of all gene carriers have no complaints but can be identified by physical examination.2 The muscle disorder is clinically homogeneous with an almost complete, but age-dependent penetrance.2,4 The prevalence is thought to be 1 in 20 000.2High-frequency hearing loss in some patients,s a retinal vasculopathy observed in 56 out of 75 affected sibs,6and the association of FSHD and polyposis coli in two families7 have contributed to discussion about the possible genetic heterogeneity of FSHD. Several linkage studies yielded negative results.8,9 We have been participating in an international consortium which has published the exclusion of almost 90% of the genome.10 We have also excluded the region around the gene for familial adenomatous polyposis on 5q21-q2211 and the regions around the spinal muscular atrophy locus on
ADDRESSES. Department of Neurology, Leiden University, Leiden, Netherlands (C. Wijmenga, MSc, O. F. Brouwer, MD, P. Moerer, G. W. Padberg, MD); Department of Human Genetics, Leiden University (C. Wijmenga, R. R. Frants, PhD, P. Moerer); and Marshfield Medical Research Foundation, Marshfield, Wisconsin, USA (J L. Weber, PhD). Correspondence to Dr G. W. Padberg, Department of Neurology, University Hospital Leiden, PO Box 9600, 2300RC Leiden, Netherlands.
652
TWO-POINT LOD SCORES FOR LINKAGE BETWEEN D4S171 AND FSHD
Lod scores calculated with the MLINK program of the LINKAGE package V5.3," assuming equal recombination fractions in males and females, 0,95 penetrance, and a gene frequency of 0 00005 One non- informative family with two affected sibs is not shown. The maximum lod score is calculated by the LINK program
Segregation of Mfd 22 marker (locus D4S171) in a small part of family 1. Individual genotypes indicated below pedigree. Size of DNA fragments (bp) indicated on left-hand side.
5q11.2-q13.312 and around the dystrophin-like gene on the long arm of chromsome 613 (unpublished). Patients and methods had been obtained we studied ten Dutch 69 affected and 58 nonaffected sibs and 25 spouses. All sibs had a physical examination and were included only if their clinical condition could be stated with confidence, which implied that most of them were older than 20 years. In each kindred at least 1 patient had a muscle biopsy and electromyography to establish the diagnosis of FSHD.1 All sibs underwent audiometry to look for the hearing loss associated with this condition, and in every kindred at least the proband was examined by fluorescein angiography of retinal vessels, which strongly suggested that retinal vasculopathy and FSHD are associated conditions in these kindreds, as in the families reported by Fitzsimons et al.6 Genomic DNA was isolated from freshly collected blood." Standard polymerase chain reactions were carried out in a 15III volume containing 30 ng genomic DNA template, 30 ng of each oligodeoxynucleotide primer,15 200 pmol each dGTP, dCTP and dTTP, 2-5 umol dATP, and 0-75 µCi &agr;32P-dATP at 800 Ci/mmol, plus 50 mmol/1 KCI, 10 mmol/1 "Tris" Cl (pH 8-3), 1-5 mmol/1 MgCl2, 0-01% gelatine, and 045 unit Taq polymerase (Perkin Elmer Cetus). Samples were overlaid with mineral oil and were processed through 27 temperature cycles of 1 min at 94°C (denaturation), 2 min at 55°C (annealing), and 1 min at 72°C (elongation). The last elongation step was lengthened with 6 min. Samples of the amplified DNA were mixed with 2 volumes of formamide sample buffer and subjected to electrophoresis on standard denaturing polyacrylamide DNA sequencing gels. 16 After fixing and drying, the gels were exposed for 1 day. Fragment size standards were dideoxy sequencing ladders of M13mp10.
After informed
consent
multigeneration families consisting of
from 143 to 163 bp (figure). The heterozygosity of the marker is 0-75 (PIC value 0-67). The results of the two-point linkage analysis between the FSHD and D4S171 loci are presented in the table. Only one of the ten families was uninformative for this marker. The maximum lod score is 6-34 at a recombination fraction of 0-13 (1 lod unit confidence interval: 0-07-0-24). We therefore conclude that the genetic defect causing FSHD is located on chromosome 4. Microsatellite markers are very useful in the screening phase of linkage studies: the technique is much faster than conventional Southern blotting, necessitates only 30 ng of genomic DNA, and allows multiple markers to be analysed in a single experiment. The high information content is of great value, especially for small families. The microsateltites are evenly distributed all over the genome, rendering them highly useful in linkage studies, as in this study, which is the first time that a human disease gene has been located with the use of this type of marker. The positive linkage results obtained in all individual kindreds argue against genetic heterogeneity of FSHD. It is unclear if the muscle disease, the hearing loss, and the retinal vasculopathy are all caused by a defect in the same gene or whether these conditions are caused by different, but linked, genes. The mapping of the FSHD gene will permit the isolation of additional markers and eventually the gene itself, leading to improvements in diagnosis and in insight into the defect that causes this disease. This study would not have been possible without the support of the patients, their families, and their organisation, the Vereniging Spierziekten Nederland. We thank the Prinses Beatrix Fonds and the Association Française contre les Myopathies for financial support, and Dr A. W. Eriksson, Dr G. J. B. van Ommen, Mr Y. Poortman, Dr L. A. Sandkuyl, and Dr A. R. Wintzen for their valuable comments.
Results and Discussion
Having obtained negative lod scores with forty-nine conventional polymorphic DNA markers plus the published excluded regions1o we turned to a new class of highly informative polymorphisms-the so-called microsatellite markers,14 most of which have a heterozygosity frequency of more than 50%. We analysed sixty microsatellite loci for linkage to FSHD. Linkage was demonstrated between the putative FSHD locus and marker Mfd 22 (locus D4S171), mapped to chromosome ç4.15 This marker shows ten different alleles ranging in size
REFERENCES 1. Munsat TL.
Facioscapulohymeral dystrophy
and the
scapuloperoneal
syndrome. In: Engel AG, Banker BQ, eds. Myology. New York: McGraw Hill, 1986: 1251-66. 2. Padberg GW. Facioscapulohumeral disease. Thesis, Leiden University, 1982. 3. Lunt PW. A workshop on facioscapulohumeral (Landouzy-Dejerine) disease. J Med Genet 1989; 26: 535-37. 4. Lunt PW, Compston DA, Harper PS. Estimation of age dependent penetrance in facioscapulohumeral muscular dystrophy by minimizing ascertainment bias. J Med Genet 1989; 26: 755-60. 5. Voit T, Lammprecht A, Lenard HG, Goebel HH. Hearing loss in facioscapulohumeral dystrophy. Eur J Pediatr 1980; 145: 280-85.
653
6. Fitzsimons RB, Gurwin EB, Bird AC. Retinal vascular abnormalities in facioscapulohumeral muscular dystrophy. Brain 1987; 110: 631-48. 7. Blake DM, Brown R, Gilliam TC, Warburton D, Rowland LP. The second family with facioscapulohumeral muscular dystrophy and familial polyposis coli. Neurology 1989; 39: (S1)404. 8. Padberg GW, Eriksson AW, Volkers WS, et al. Linkage studies in autosomal dominant facioscapulohumeral muscular dystrophy. J Neurol Sci 1984; 65: 261-68. 9. Padberg GW, Klasen EC, Volkers WS, de Lange GG, Wintzen AR. Linkage studies in facioscapulohumeral muscular dystrophy. Muscle Nerve 1988; 11: 833-35. 10. Sarfarazi M, Upadhyaya M, Padberg G, et al. An exclusion map for facioscapulohumeral (Landouzy-Déjérine) disease. J Med Genet 1989; 26: 481-84. 11.Wijmenga C, Frants RR, Brouwer OF, et al. Facioscapulohumeral muscular dystrophy gene in Dutch families is not linked to markers for familial adenomatous polyposis on the long arm of chromosome 5. J Neurol Sci 1990; 95: 225-29.
12. 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; 334: 540-41. 13. Love DR, Hill DF, Dickson G, Spurr NK, et al. An autosomal transcript in skeletal muscle with homology to dystrophy. Nature 1989; 339: 55-58. 14. Weber JL, May PE. Abundant class of human DNA polymporhisms which can be typed using the polymerase chain reaction. Am J Hum Genet 1989; 44: 388-96. 15. Weber
JL, May PE. Dinucleotide repeat polymorphism at the D4S171 locus. Nucleic Acids Res 1990; 18: 2202. 16. Biggin MD, Gibson TJ, Hong GF. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci (USA) 17.
1983; 80: 3963-65. Lathrop GM, Lalouel JM, Julien C, Ott J. Strategies for multilocus linkage anlaysis in human. Proc Natl Acad Sci (USA) 1984; 81: 3443-46.
Absence of close linkage between maternal genes for susceptibility to pre-eclampsia/eclampsia and HLA DR&bgr;
To test the
possibility that maternally expressed susceptibility genes for pre-eclampsia/eclampsia are closely linked to the HLA region on chromosome 6 of the human genome, members of pedigrees with multiple cases of these disorders were typed for HLA DR&bgr; restriction
ten
fragment length polymorphisms by means of Taql digests. The data were analysed by the LIPED program to calculate lod scores, by several programs to detect potential heterogeneity of recombination fraction between pedigrees, and by the affected-sibling and the affected-pedigreemember methods. The results exclude close linkage. If the putative susceptibility genes lie on chromosome 6 they must lie at least 5 centiMorgans, and probably more, from the HLA DR&bgr; loci. No indication of linkage at higher recombination fractions was found. The main
maternally expressed genes affecting susceptibility to pre-eclampsia are not in the HLA region. Lancet 1990; 336: 653-57.
Introduction
Despite a century of investigation, the aetiology of preeclampsia and eclampsia remains unknown. Studies of the frequency of the disorder in blood relatives of index cases show that susceptibility is highly heritable.l-6 The power of conventional genetic analysis to reveal the exact mode of inheritance is limited by the facts that the phenotype is confmed to the one sex and that it occurs only in pregnancy, so it is possible that susceptibility is determined by the maternal genotype, by the fetal genotype, by an interaction between the two, or by some combination of these. Most of the simple pedigree data point to a major role for the
maternal genotype,3-5 although some details are more easily interpreted in terms of an effect of the fetal genotype. 6,7 On the other hand, the various kinds of association between susceptibility and HLA types decisively implicate the fetal genotype. The reported differences from control groups include: greater homozygosity at the HLA B locus in women affected by pre-eclampsia and in their spouses;8 greater antigen sharing at the A and B loci9,10 and greater sharing of HLA DR47,11 between affected women and their spouses; association of HLA DR4 with risk of recurrence of non-proteinuric pregnancy hypertension,"z increased heterozygosity at the B locus in eclamptic women,"and a higher frequency of HLA B35 in babies born to eclamptic women. 14 Since the discoveries of an association between HLA and susceptibility to pre-eclampsia,8,9 the possibility that there are susceptibility genes linked to the HLA locus has been raised several times.7.11,12,15 Kilpatrick and colleagues,7 have
lately proposed a very specific hypothesis-that preeclampsia occurs when both mother and fetus are homozygous for the one HLA-linked recessive gene. We have examined this hypothesis by testing for linkage between maternally expressed genes for susceptibility and HLA DR&bgr;. Patients and methods Ten multi-case pedigrees were ascertained, each through a single index case, by interviewing patients who presented at the Monash ADDRESSES: School of Biological Sciences, Macquarie University, New South Wales (A. N. Wilton, PhD, Prof D. W. Cooper, PhD, P. Marshall, BSc) and Monash Perinatal Unit, Department of Obstetrics and Gynaecology, Monash University, Victoria, Australia (S. P. Brennecke, FRACOG, S, M. Bishop, RN). Correspondence to Prof D. W. Cooper, School of Biological Sciences, Macquarie University, NSW 2109, Australia.
could be used for administration of other pressurised aerosols.7 The only side-effect observed was oral candidiasis, found in 3 children on active treatment and 3 on placebo. Budesonide has a favourable ratio between topical and systemic effects,8,9 and metabolism is increased in younger patients.1o Nevertheless, a systemic effect from the proposed treatment cannot be excluded, and individually increased susceptibility may also be a risk; thus, treatment with budesonide should be reserved for children with moderate to severe symptoms. Topical corticosteroids could be administered as a nebulised suspension, which may be an effective treatment for recurrent wheezing in children below 3 years of age11,12 and avoids the use of freon. However, the nebuliser and air-compressor are costly and require both electricity and maintenance, and there is a risk of contamination.13,14 The procedure may also be timeconsuming and uncomfortable or frightening, and the child’s face may be exposed to an aerosol of a potent topical glucocorticosteroid for long periods, with the risk of a steroid skin rash. Our findings indicate that budesonide aerosol delivered through a spacer and facemask with a 1-way valve gives significant symptom relief in young children with recurrent wheezing who were already treated with inhaled &bgr;2 agonist, and that the need for additional treatment with oral prednisolone was reduced. Further work is needed to quantify the amount of active agent delivered to the airways through this device, and to determine the lowest dose of budesonide that achieves a satisfactory clinical response, but this delivery system may be simpler, and have less risk of side-effects, than use of nebulised corticosteroids.
REFERENCES 1. Wilson NM. Wheezing bronchitis revisited. Arch Dis Child 1989; 64: 1194-99. 2. Lee DA, Winslow NR, Speight ANP, Hey EN. Prevalence and spectrum of asthma in childhood. Br Med J 1983; 286: 1256-68. 3. Bisgaard H, Ohlsson S. PEP-spacer: an adaption for administration of MDI to infants. Allergy 1989; 44: 363-64.
4. Polgar G, Promadhat V. Pulmonary function testing in children: techniques and standards. London: WB Saunders, 1971. 5. Bisgaard H, Dalgaard P, Nyboe J. Risk factors for wheezing during infancy. Acta Paediatr Scand 1987; 76: 719-26. 6. Gleeson JGA, Price JF. Controlled trial of budesonide given by the Nebuhaler in preschool children with asthma. Br Med J 1988; 297: 163-66. 7.
8.
O’Callaghan C, Milner AD, Swarbrick A. Spacer device with face mask attachment for giving bronchodilators to infants with asthma. Br Med J 1989; 298: 160-61. Bisgaard H, Damkjaer Nielsen M, Andersen B, et al. Adrenal function in children
with
bronchial asthma treated with
beclomethasone
dipropionate or budesonide. J Allergy Clin Immunol 1988; 81: 1088-95. 9. Bisgaard H, Pedersen S, Damkjaer M, Østerballe O. Adrenal function in asthmatic children treated with inhaled budesonide. Acta Paediatr Scand (in press). 10. Pedersen S, Steffensen G, Ekman I, Tønnesson M, Borgå O. Pharmacokinetics of budesonide in children with asthma. Eur J Clin Pharmacol 1987; 31: 579-82. 11. Carlsen KH, Leegaard J, Larsen S, Ørstavik I. Nebulized beclomethasone dipropionate in recurrent obstructive episodes after acute bronchiolitis. Arch Dis Child 1988; 63: 1428-33. 12. Pedersen S, Ramsgaard-Hansen O, Nikander K. Budesonide suspension for nebulization in children with asthma. Eur Respir J 1989; 2 (suppl 8): abstr 82. 13. Pierce AK, Sanford JP, Thomas GD, Leonard JS. Long-term evaluation of decontamination of inhalation therapy equipment and the occurrence of necrotizing pneumonia. N Engl J Med 1970; 282: 528-31. 14. Barnes KL, Clifford R, Holgate ST, Murphy D, Comber P, Bell E. Bacterial contamination of home nebulizers. Br Med J 1987; 295: 812-14.
Location of facioscapulohumeral muscular dystrophy gene on chromosome 4
dominant disorder autosomal faciomuscular scapulohumeral dystrophy (FSHD) is the last of the major progressive muscular dystrophies in which the gene had not been located. In linkage analysis on ten Dutch families with this disorder a lod score of 6·34 at a recombination fraction of 0·13 was obtained with the microsatellite marker Mfd 22 (D4S171). This maps the FSHD gene to chromsome 4. Only one family was uninformative for this marker. We found no evidence of genetic
The
heterogeneity. Introduction
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder with presenting symptoms of facial or shoulder girdle weakness usually at the end of the first or in the second decade. The disease is progressive in most patients, with successive involvement of the abdominal, foot-extensor, upper-arm, and pelvic-girdle muscles.1,2 The rate of progression is variable between and within families. Approximately 10% of gene carriers will become wheelchair dependent, usually at an older age.3
One-third of all gene carriers have no complaints but can be identified by physical examination.2 The muscle disorder is clinically homogeneous with an almost complete, but age-dependent penetrance.2,4 The prevalence is thought to be 1 in 20 000.2High-frequency hearing loss in some patients,s a retinal vasculopathy observed in 56 out of 75 affected sibs,6and the association of FSHD and polyposis coli in two families7 have contributed to discussion about the possible genetic heterogeneity of FSHD. Several linkage studies yielded negative results.8,9 We have been participating in an international consortium which has published the exclusion of almost 90% of the genome.10 We have also excluded the region around the gene for familial adenomatous polyposis on 5q21-q2211 and the regions around the spinal muscular atrophy locus on
ADDRESSES. Department of Neurology, Leiden University, Leiden, Netherlands (C. Wijmenga, MSc, O. F. Brouwer, MD, P. Moerer, G. W. Padberg, MD); Department of Human Genetics, Leiden University (C. Wijmenga, R. R. Frants, PhD, P. Moerer); and Marshfield Medical Research Foundation, Marshfield, Wisconsin, USA (J L. Weber, PhD). Correspondence to Dr G. W. Padberg, Department of Neurology, University Hospital Leiden, PO Box 9600, 2300RC Leiden, Netherlands.
652
TWO-POINT LOD SCORES FOR LINKAGE BETWEEN D4S171 AND FSHD
Lod scores calculated with the MLINK program of the LINKAGE package V5.3," assuming equal recombination fractions in males and females, 0,95 penetrance, and a gene frequency of 0 00005 One non- informative family with two affected sibs is not shown. The maximum lod score is calculated by the LINK program
Segregation of Mfd 22 marker (locus D4S171) in a small part of family 1. Individual genotypes indicated below pedigree. Size of DNA fragments (bp) indicated on left-hand side.
5q11.2-q13.312 and around the dystrophin-like gene on the long arm of chromsome 613 (unpublished). Patients and methods had been obtained we studied ten Dutch 69 affected and 58 nonaffected sibs and 25 spouses. All sibs had a physical examination and were included only if their clinical condition could be stated with confidence, which implied that most of them were older than 20 years. In each kindred at least 1 patient had a muscle biopsy and electromyography to establish the diagnosis of FSHD.1 All sibs underwent audiometry to look for the hearing loss associated with this condition, and in every kindred at least the proband was examined by fluorescein angiography of retinal vessels, which strongly suggested that retinal vasculopathy and FSHD are associated conditions in these kindreds, as in the families reported by Fitzsimons et al.6 Genomic DNA was isolated from freshly collected blood." Standard polymerase chain reactions were carried out in a 15III volume containing 30 ng genomic DNA template, 30 ng of each oligodeoxynucleotide primer,15 200 pmol each dGTP, dCTP and dTTP, 2-5 umol dATP, and 0-75 µCi &agr;32P-dATP at 800 Ci/mmol, plus 50 mmol/1 KCI, 10 mmol/1 "Tris" Cl (pH 8-3), 1-5 mmol/1 MgCl2, 0-01% gelatine, and 045 unit Taq polymerase (Perkin Elmer Cetus). Samples were overlaid with mineral oil and were processed through 27 temperature cycles of 1 min at 94°C (denaturation), 2 min at 55°C (annealing), and 1 min at 72°C (elongation). The last elongation step was lengthened with 6 min. Samples of the amplified DNA were mixed with 2 volumes of formamide sample buffer and subjected to electrophoresis on standard denaturing polyacrylamide DNA sequencing gels. 16 After fixing and drying, the gels were exposed for 1 day. Fragment size standards were dideoxy sequencing ladders of M13mp10.
After informed
consent
multigeneration families consisting of
from 143 to 163 bp (figure). The heterozygosity of the marker is 0-75 (PIC value 0-67). The results of the two-point linkage analysis between the FSHD and D4S171 loci are presented in the table. Only one of the ten families was uninformative for this marker. The maximum lod score is 6-34 at a recombination fraction of 0-13 (1 lod unit confidence interval: 0-07-0-24). We therefore conclude that the genetic defect causing FSHD is located on chromosome 4. Microsatellite markers are very useful in the screening phase of linkage studies: the technique is much faster than conventional Southern blotting, necessitates only 30 ng of genomic DNA, and allows multiple markers to be analysed in a single experiment. The high information content is of great value, especially for small families. The microsateltites are evenly distributed all over the genome, rendering them highly useful in linkage studies, as in this study, which is the first time that a human disease gene has been located with the use of this type of marker. The positive linkage results obtained in all individual kindreds argue against genetic heterogeneity of FSHD. It is unclear if the muscle disease, the hearing loss, and the retinal vasculopathy are all caused by a defect in the same gene or whether these conditions are caused by different, but linked, genes. The mapping of the FSHD gene will permit the isolation of additional markers and eventually the gene itself, leading to improvements in diagnosis and in insight into the defect that causes this disease. This study would not have been possible without the support of the patients, their families, and their organisation, the Vereniging Spierziekten Nederland. We thank the Prinses Beatrix Fonds and the Association Française contre les Myopathies for financial support, and Dr A. W. Eriksson, Dr G. J. B. van Ommen, Mr Y. Poortman, Dr L. A. Sandkuyl, and Dr A. R. Wintzen for their valuable comments.
Results and Discussion
Having obtained negative lod scores with forty-nine conventional polymorphic DNA markers plus the published excluded regions1o we turned to a new class of highly informative polymorphisms-the so-called microsatellite markers,14 most of which have a heterozygosity frequency of more than 50%. We analysed sixty microsatellite loci for linkage to FSHD. Linkage was demonstrated between the putative FSHD locus and marker Mfd 22 (locus D4S171), mapped to chromosome ç4.15 This marker shows ten different alleles ranging in size
REFERENCES 1. Munsat TL.
Facioscapulohymeral dystrophy
and the
scapuloperoneal
syndrome. In: Engel AG, Banker BQ, eds. Myology. New York: McGraw Hill, 1986: 1251-66. 2. Padberg GW. Facioscapulohumeral disease. Thesis, Leiden University, 1982. 3. Lunt PW. A workshop on facioscapulohumeral (Landouzy-Dejerine) disease. J Med Genet 1989; 26: 535-37. 4. Lunt PW, Compston DA, Harper PS. Estimation of age dependent penetrance in facioscapulohumeral muscular dystrophy by minimizing ascertainment bias. J Med Genet 1989; 26: 755-60. 5. Voit T, Lammprecht A, Lenard HG, Goebel HH. Hearing loss in facioscapulohumeral dystrophy. Eur J Pediatr 1980; 145: 280-85.
653
6. Fitzsimons RB, Gurwin EB, Bird AC. Retinal vascular abnormalities in facioscapulohumeral muscular dystrophy. Brain 1987; 110: 631-48. 7. Blake DM, Brown R, Gilliam TC, Warburton D, Rowland LP. The second family with facioscapulohumeral muscular dystrophy and familial polyposis coli. Neurology 1989; 39: (S1)404. 8. Padberg GW, Eriksson AW, Volkers WS, et al. Linkage studies in autosomal dominant facioscapulohumeral muscular dystrophy. J Neurol Sci 1984; 65: 261-68. 9. Padberg GW, Klasen EC, Volkers WS, de Lange GG, Wintzen AR. Linkage studies in facioscapulohumeral muscular dystrophy. Muscle Nerve 1988; 11: 833-35. 10. Sarfarazi M, Upadhyaya M, Padberg G, et al. An exclusion map for facioscapulohumeral (Landouzy-Déjérine) disease. J Med Genet 1989; 26: 481-84. 11.Wijmenga C, Frants RR, Brouwer OF, et al. Facioscapulohumeral muscular dystrophy gene in Dutch families is not linked to markers for familial adenomatous polyposis on the long arm of chromosome 5. J Neurol Sci 1990; 95: 225-29.
12. 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; 334: 540-41. 13. Love DR, Hill DF, Dickson G, Spurr NK, et al. An autosomal transcript in skeletal muscle with homology to dystrophy. Nature 1989; 339: 55-58. 14. Weber JL, May PE. Abundant class of human DNA polymporhisms which can be typed using the polymerase chain reaction. Am J Hum Genet 1989; 44: 388-96. 15. Weber
JL, May PE. Dinucleotide repeat polymorphism at the D4S171 locus. Nucleic Acids Res 1990; 18: 2202. 16. Biggin MD, Gibson TJ, Hong GF. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci (USA) 17.
1983; 80: 3963-65. Lathrop GM, Lalouel JM, Julien C, Ott J. Strategies for multilocus linkage anlaysis in human. Proc Natl Acad Sci (USA) 1984; 81: 3443-46.
Absence of close linkage between maternal genes for susceptibility to pre-eclampsia/eclampsia and HLA DR&bgr;
To test the
possibility that maternally expressed susceptibility genes for pre-eclampsia/eclampsia are closely linked to the HLA region on chromosome 6 of the human genome, members of pedigrees with multiple cases of these disorders were typed for HLA DR&bgr; restriction
ten
fragment length polymorphisms by means of Taql digests. The data were analysed by the LIPED program to calculate lod scores, by several programs to detect potential heterogeneity of recombination fraction between pedigrees, and by the affected-sibling and the affected-pedigreemember methods. The results exclude close linkage. If the putative susceptibility genes lie on chromosome 6 they must lie at least 5 centiMorgans, and probably more, from the HLA DR&bgr; loci. No indication of linkage at higher recombination fractions was found. The main
maternally expressed genes affecting susceptibility to pre-eclampsia are not in the HLA region. Lancet 1990; 336: 653-57.
Introduction
Despite a century of investigation, the aetiology of preeclampsia and eclampsia remains unknown. Studies of the frequency of the disorder in blood relatives of index cases show that susceptibility is highly heritable.l-6 The power of conventional genetic analysis to reveal the exact mode of inheritance is limited by the facts that the phenotype is confmed to the one sex and that it occurs only in pregnancy, so it is possible that susceptibility is determined by the maternal genotype, by the fetal genotype, by an interaction between the two, or by some combination of these. Most of the simple pedigree data point to a major role for the
maternal genotype,3-5 although some details are more easily interpreted in terms of an effect of the fetal genotype. 6,7 On the other hand, the various kinds of association between susceptibility and HLA types decisively implicate the fetal genotype. The reported differences from control groups include: greater homozygosity at the HLA B locus in women affected by pre-eclampsia and in their spouses;8 greater antigen sharing at the A and B loci9,10 and greater sharing of HLA DR47,11 between affected women and their spouses; association of HLA DR4 with risk of recurrence of non-proteinuric pregnancy hypertension,"z increased heterozygosity at the B locus in eclamptic women,"and a higher frequency of HLA B35 in babies born to eclamptic women. 14 Since the discoveries of an association between HLA and susceptibility to pre-eclampsia,8,9 the possibility that there are susceptibility genes linked to the HLA locus has been raised several times.7.11,12,15 Kilpatrick and colleagues,7 have
lately proposed a very specific hypothesis-that preeclampsia occurs when both mother and fetus are homozygous for the one HLA-linked recessive gene. We have examined this hypothesis by testing for linkage between maternally expressed genes for susceptibility and HLA DR&bgr;. Patients and methods Ten multi-case pedigrees were ascertained, each through a single index case, by interviewing patients who presented at the Monash ADDRESSES: School of Biological Sciences, Macquarie University, New South Wales (A. N. Wilton, PhD, Prof D. W. Cooper, PhD, P. Marshall, BSc) and Monash Perinatal Unit, Department of Obstetrics and Gynaecology, Monash University, Victoria, Australia (S. P. Brennecke, FRACOG, S, M. Bishop, RN). Correspondence to Prof D. W. Cooper, School of Biological Sciences, Macquarie University, NSW 2109, Australia.
