457
killer (CD 16, CD56) cells were seen. This observation suggests that from the heterogeneous cell populations included in LAK-cells, T -lymphocytes but not natural killer cells can migrate to tumour sites in melanoma patients. Tumour recognition and traffic are probably restricted to specific T lymphocytes, as is also suggested by Swift and colleagues’ data. However, the peripheral blood seems to contain a considerable number of these T cells, which can be used for tumour imaging after an interleukin-2-induced short-time activation. Out of the 14 patients who received radiolabelled LAK-cells, 5 were treated in a clinical trial with dacarbazine and interleukin-2.3 Of 3 scintigraphically-positive patients, 2 responded to the treatment, whereas the 2 negative patients did not respond. Although these preliminary data should be interpreted with
caution, we postulate that metastases respond to immunotherapies that use interleukin-2 possibly through a T lymphocyte-tumour cell HLA-DR-dependent interaction.3 LAK-cell scintigraphy might be a fast and economic method to analyse individual properties and might have prognostic relevance for immunotherapies that use interleukin-2.
axial
lipomas characteristic of multiple symmetric lipomatosis (MSL) 4s Morphological and biochemical evidence suggests that familial and sporadic MSL is associated with mitochondrial dysfunction. An additional patient in our series of MSL, who did not have myoclonus epilepsy, also had the tRNAlys mutation. Moreover, the index case of the US family in whom the MERRF mutation was originally described’ now also has lipomas (J. M. Shoffner, personal communication). It thus seems that MSL, at least in some cases, is a manifestation of this mutation. Although the clinical features of the mitochondrial encephalopathies are diverse, there are recognisable clinical patterns, for which genotypic specificity is emerging. The clinician should now ask for analysis of mitochondrial DNA from blood or muscle to search for the tRNAlys mutation in cases of possible MERRF, MSL, or Leigh’s syndrome. However, all three of these clinical phenotypes are probably also caused by other yet to be discovered molecular lesions. Department of Neurology, Hospital,
Austin
Melbourne, Victoria 3084, Australia
Department of Dermatology, University Hospital Zurich, CH-8091 Zurich, Switzerland
R. DUMMER
Department of Nuclear Medicine, University of Würzburg, Wurzburg, Germany
E. SCHÄFER C. EILLES W. BÖRNER
Department of Dermatology, University Hospital Zurich
G. BURG
Montreal
Neurological Institute,
Montreal, Quebec, Canada
S. F. BERKOVIC E. A. SHOUBRIDGE F. ANDERMANN E. ANDERMANN S. CARPENTER G. KARPATI
1. Schoffner
1. Schafer E, Dummer
R, Eilles C, Martin M, Börner W, Burg G. Imaging pattern of radiolabeled lymphokine-activated killer cells in malignant melanoma patients. Eur
J Nucl Med 1991; 18: 106-10. 2. Dummer R, Becker
JC, Kahlhammer U, et al. Combined chemo- and immunotherapy using dacarbazine and continuous infusion of interleukin 2 in metastatic malignant melanoma patients. Eur J Dermatol (in press). 3. Cohen PJ, Lotze MT, Roberts JR, Rosenberg SA, Jaffe ES. The immunopathology of sequential tumor biopsies in patients treated with interleukin-2. Correlation of response with T-cell infiltration and HLA-DR expression. Am J Patlol 1987; 129: 208-16.
JM, Lott MT, Lezza AMS, Seibel P, Ballinger SW, Wallace DC. Myoclonic epilepsy and ragged-red fiber disease (MERRF) is associated with a mitochondrial DNA tRNAlys mutation. Cell 1990; 61: 931-37. 2. Zeviani M, Amati P, Bresolin N, et al. Rapid detection of the A→G (8344) mutation of mtDNA in Italian families with myoclonus-epilepsy and ragged red fibres (MERRF). Am J Hum Genet 1991; 48: 203-11. 3. Marseille Consensus Group. Classification of progressive myoclonus epilepsies and related disorders. Ann Neurol 1990; 28: 113-16. 4. Berkovic SF, Carpenter S, Evans A, et al. Myoclonus epilepsy and ragged-red fibers (MERRF) 1: a clinical, pathological, biochemical, magnetic resonance spectroscopic and positron emission tomographic study. Brain 1989; 112: 1231-60. 5. Berkovic SF, Andermann F, Shoubridge EA, et al. Mitochondrial dysfunction in multiple symmetric lipomatosis. Ann Neurol 1991; 29: 566-69.
Severity of cystic fibrosis Clinical spectrum of mitochondrial DNA mutation at base pair 8344 SIR,-Dr Hammans and colleagues (June 1, p 1311) identify a mutation at base pair 8344 of the mitochondrial genome in blood samples of 5 of 7 patients with the syndrome ofmyoclonic epilepsy and ragged red fibres (MERRF), confirming previous studies with muscle mitochondrial DNA.l,2 This mutation, which is in the mitochondrial gene for lysine transfer RNA (tRNAlys), was also found in 1 patient with myoclonus and ataxia who lacked ragged red fibres, but was absent in many other syndromes associated with mitochondrial disease. We describe further phenotypes associated with this mutation. The clinical spectrum of MERRF is broad relative to the other causes of progressive myoclonus epilepsy from which it should be distinguished.3We have reported 13 patients with MERRF, including one family with 6 affected members.4 The 2 most severely affected (subjects 5 and 6) had the pathological features of Leigh’s syndrome, in addition to the system degeneration characteristic of MERRF.4 We have now identified the tRNAlys mutation in this family. This confirms Hammans and colleagues’ observation of Leigh’s syndrome lesions at necropsy in 1 of their MERRF patients with this mutation. The neuropathological lesions characterising Leigh’s syndrome are strikingly different from those in uncomplicated cases of MERRF, yet they now seem to be consequences of the same mitochondrial DNA mutation in some families. On the basis of positron emission tomographic data,4 we have suggested that such clinicopathological diversity can be explained by the peculiarities of mitochondrial inheritance, resulting in a spectrum of severity of cerebral metabolic deficits. In the more severe cases, sudden clinical and metabolic deterioration can happen with additional stresses, such as fever, resulting in the pathological picture of Leigh’s syndrome. The proband of our MERRF family also had large disfiguring
SIR,-Dr Johansen and colleagues (March 16, p 631) report that just over three-quarters of patients with cystic fibrosis are homozygous for the AF508 mutation. The rest have other mutations, many of which have been recognised, raising the possibility that the clinical expression of the disease is determined by the mutation that is present. We report a 45-year-old man in whom the disease has been unusually mild and whose genotype has been determined. The patient was an only child born in 1946. At 5 years old he had steatorrhoea and respiratory infections, and fibrocystic disease of the pancreas was diagnosed. He was treated with pancreatic enzyme supplements and made such good progress that by age 15 the diagnosis was thought to have been incorrect and the enzyme supplements were stopped. In his early twenties steatorrhoea recurred and a jejunal biopsy showed an abnormality consistent with coeliac disease. Failure of his symptoms to resolve completely on a gluten-free diet was attributed to non-compliance. At the age of 27 intestinal obstruction developed and at laparotomy the terminal 06 metres of ileum proved to be occluded by inspissated faecal-like material. At about this time he was investigated for a persistent productive cough and bronchography showed bilateral bronchiectasis. By age 38, when he moved to Devon, he was beginning to be troubled by breathlessness on exertion. Since then he has had planned admissions for physiotherapy about twice a year in addition to postural drainage at home, but he has never required admission for an acute infective exacerbation. Chest radiography showed extensive basal emphysema with upper zone fibrosis. His lung function has declined over the past 7 years (FEV 925 ml and FVC 1929 ml in 1984; and 550 ml and 1350 ml, respectively, in 1991), and Pseudomwnas aeruginosa has frequently been cultured from his sputum. His sweat sodium concentration was 124 mmol/1 in 1984. He has maintained a steady weight during this time (51 kg, height
458
175 cm), but lately complained of passing 3-4 loose motions per day with steatorrhoea. These symptoms completely resolved when pancreatic enzymes were re-started. He worked successfully as a musician until breathlessness forced him to stop at 40. He is a non-smoker. The patient always believed that he had cystic tibrosis, even though this was not consistently the opinion of his doctors. On DNA analysis he proved heterozygous for the AF508 mutation and for the G551D mutation in exon 11. Weconclude that this genotype. is compatible with mild expression of the disease. South Devon Healthcare,
Torbay Hospital, Lawes Bridge, Torquay TQ2 7AA, UK
J. ROBINSON G. A. RICHARDSON
A.
Prenatal 1.
diagnosis of two CF families by simultaneous PCR amplification of IVS8CA microsatellite and the exon 10 region
Johansen HK, Nir M, Hoiby N, Koch C, Schwartz M. Seventy of cystic fibrosis in patients homozygous and heterozygous for &Dgr;F508 mutation. Lancet 1991; 337:
of CFTR that contains the AF508 mutation.
631-34
diagnosis of cystic fibrosis by multiplex PCR of mutation and
Prenatal
microsatellite alleles SIR,-Genetic analysis of cystic fibrosis (CF) has been considerably improved since the isolation of the CF gene and the identification of the major CF mutation (AF508 deletion),l which is seen in about 70% of North American and North European subjects, but in only 50% among those living in the Mediterranean region.2 Although more than 80 CF mutations have been identified (CF Genetic Analysis Consortium), most are very uncommon in the general patient population.3,4 Therefore, DNA markers closely linked to the disease locus are still being used, alone or in combination with mutation analysis, for between 40% (northern Europe and North America) and 75% (southern Europe) of CF families.5 We have identified a microsatellite polymorphism in intron 8 of the CFTR gene (IVS8CA)6 and developed PCR conditions for multiplex DNA amplification of microsatellite alleles and the AF508 mutation. The IVS8CA microsatellite detects up to 10 alleles and has a heterozygosity of 80%. The study of the IVS8CA microsatellite in several hundred families, which include 664 meioses, has not revealed new mutant alleles, and shows that it can safely be applied to prenatal diagnosis and carrier detection of CF.66 The table shows the distribution of IVS8CA microsatellite alleles in AF508, non-AF508, and normal chromosomes. Two common alleles are associated with AF508 chromosomes, 23 (52%) and 17 (40%), whereas these alleles account for only 7-6% and 11 % of normal chromosomes, respectively. For non-AF508 chromosomes three common alleles were found, 23 (31%), 17 (10%), and 16 (50%). Allele 16 is the most common (76%) in normal chromosomes. The figure shows two prenatal diagnosis analyses done in a 50% informative and in a non-informative AF508 mutation CF family, which were fully informative for the IVS8CA marker. The simultaneous analysis of the microsatellite and the AF508 mutation allows prenatal diagnosis in a single tube reaction experiment per sample. Since the AF508 mutation accounts for only 50% of the CF ALLELE DISTRIBUTION* OF &Dgr;F508, NON-&Dgr;F508, AND NORMAL CHROMOSOMES FOR IVS8CA MICROSATELLITE IN CFTR GENE
Sequences for the amplification of exon 10 are E1 OD2 (5’-TTCACTTCTAATGATGATTAT) and E10R1 (5’-GTTGGCATGCTTTGATGACGTTTC), and for IVS8CA are NUR-1 (5’-TCTATCTCATGTTAATGCTG) and NUR-2 (5’-GTTTCTAGAGGACATGATC) PCR was done with 500 ng DNA, 200 µmol of each dNTP, 10 pmol of primer NUR-1,1 6 pmol each of end-labelled primers NUR-1 and EX1 OR in total volume of 25 wl. Annealing was at 50.C, and elongation was for 1 min 30 pmol each of primers NUR-1 and EX10R were end-labelled, with 20 wCi of [&ggr;-32P]ATP (3000 Ci/mmol) (1 Ci=3.7x1010Bq) and 5 units of polynucleotide kinase 2 µl of amplification products were mixed with 2 µl of formamide stop solution, loaded mto 6% polyacrylamide gel, and electrophoresed at 1500 V for 3 h Dried gel was exposed against radiographic film for 20-45 min at room temperature M, mother; F, father; CF, cystic fibrosis; CV, chorionic villi. A23, A21, A17, A16, and A15= number of repeats for CA/GT alleles.
chromosomes in the Mediterranean region, and other described mutations in the gene are very uncommon, only 25% of families are fully informative for prenatal diagnosis and 50% are halfinformative with mutation analysis only. When microsatellite and AF508 analysis are combined, families became more than 95% informative. Since some linkage disequilibrium exists between microsatellite alleles and non-AF508 CF chromosomeshaplotype data generated with microsatellite alleles can also be used to improve genetic counselling in couples with less than a 1 -in-4 risk, and in CF couples with a deceased CF child who are negative for the &Dgr;F508 mutation.’ Although several restriction fragment length polymorphisms (RFLPs) have been identified in the CFTR gene, they are not very informative, mainly because they show strong allelic association with other allelic systems in the CF locus, and because they are two-allele polymorphisms.1 The large heterogeneity found in the non-AF508 mutant allele pool suggests that microsatellite and haplotype analysis may be the method of choice for genetic testing in several circumstances. This is especially true in most Mediterranean countries, where only one-third of the estimated 75 000 CF families are expected to be fully informative for mutational
analysis.’"
Molecular Genetics Department, Cancer Research Institute, Hospital Duran i Reynals, 08907 Barcelona, Catalonia, Spain
1. Kerem
X. ESTIVILL N. MORRAL T. CASALS V. NUNES
B-S, Rommens JM, Buchanan JA, et al. Identification of the cystic fibrosis genetic analysis Science 1989; 245: 1073-80.
gene:
Cysuc Fibrosis Genetic Analysis Consortium. Worldwide survey of the &Dgr;F508 mutation-report from the Cystic Fibrosis Genetic Analysis Consortium. AmJ
2. The
Hum Genet 1991; 47: 354-59
Cutting GR, Kash LM, Rosenstein BJ, et al. A cluster of cystic fibrosis mutations in the first nucleotide-binding fold of the cystic fibrosis conductance regulator protein Nature 1990; 346: 366-39. 4. Dean M, White MB, Amos J, et al. Multiple mutations in highly conserved residues are found in mildly affected cystic fibrosis patients. Cell 1990; 61: 863-70. 5. Novelli G, Sangiuolo F, Dallapiccola B, et al. &Dgr;F508 gene deletion and prenatal diagnosis of cystic fibrosis in Italian and Spanish families. Prenat Diagn 1990; 10: 3.
*Alleles indicate the number of CA/GT repeats
413-14. 6. Morral N, Nunes V, Casals T, Estivill X. CA/GT microsatellite alleles within the cystic fibrosis transmembrane regulator (CFTR) gene are not generated by unequal crossingover. Genomics 1991; 10: 692-98. 7. Casals T, Nunes V, Lázaro C, et al. Mutation and linkage disequilibrium analysis in genetic counselling of Spanish cystic fibrosis families. J Med Genet (in press)
killer (CD 16, CD56) cells were seen. This observation suggests that from the heterogeneous cell populations included in LAK-cells, T -lymphocytes but not natural killer cells can migrate to tumour sites in melanoma patients. Tumour recognition and traffic are probably restricted to specific T lymphocytes, as is also suggested by Swift and colleagues’ data. However, the peripheral blood seems to contain a considerable number of these T cells, which can be used for tumour imaging after an interleukin-2-induced short-time activation. Out of the 14 patients who received radiolabelled LAK-cells, 5 were treated in a clinical trial with dacarbazine and interleukin-2.3 Of 3 scintigraphically-positive patients, 2 responded to the treatment, whereas the 2 negative patients did not respond. Although these preliminary data should be interpreted with
caution, we postulate that metastases respond to immunotherapies that use interleukin-2 possibly through a T lymphocyte-tumour cell HLA-DR-dependent interaction.3 LAK-cell scintigraphy might be a fast and economic method to analyse individual properties and might have prognostic relevance for immunotherapies that use interleukin-2.
axial
lipomas characteristic of multiple symmetric lipomatosis (MSL) 4s Morphological and biochemical evidence suggests that familial and sporadic MSL is associated with mitochondrial dysfunction. An additional patient in our series of MSL, who did not have myoclonus epilepsy, also had the tRNAlys mutation. Moreover, the index case of the US family in whom the MERRF mutation was originally described’ now also has lipomas (J. M. Shoffner, personal communication). It thus seems that MSL, at least in some cases, is a manifestation of this mutation. Although the clinical features of the mitochondrial encephalopathies are diverse, there are recognisable clinical patterns, for which genotypic specificity is emerging. The clinician should now ask for analysis of mitochondrial DNA from blood or muscle to search for the tRNAlys mutation in cases of possible MERRF, MSL, or Leigh’s syndrome. However, all three of these clinical phenotypes are probably also caused by other yet to be discovered molecular lesions. Department of Neurology, Hospital,
Austin
Melbourne, Victoria 3084, Australia
Department of Dermatology, University Hospital Zurich, CH-8091 Zurich, Switzerland
R. DUMMER
Department of Nuclear Medicine, University of Würzburg, Wurzburg, Germany
E. SCHÄFER C. EILLES W. BÖRNER
Department of Dermatology, University Hospital Zurich
G. BURG
Montreal
Neurological Institute,
Montreal, Quebec, Canada
S. F. BERKOVIC E. A. SHOUBRIDGE F. ANDERMANN E. ANDERMANN S. CARPENTER G. KARPATI
1. Schoffner
1. Schafer E, Dummer
R, Eilles C, Martin M, Börner W, Burg G. Imaging pattern of radiolabeled lymphokine-activated killer cells in malignant melanoma patients. Eur
J Nucl Med 1991; 18: 106-10. 2. Dummer R, Becker
JC, Kahlhammer U, et al. Combined chemo- and immunotherapy using dacarbazine and continuous infusion of interleukin 2 in metastatic malignant melanoma patients. Eur J Dermatol (in press). 3. Cohen PJ, Lotze MT, Roberts JR, Rosenberg SA, Jaffe ES. The immunopathology of sequential tumor biopsies in patients treated with interleukin-2. Correlation of response with T-cell infiltration and HLA-DR expression. Am J Patlol 1987; 129: 208-16.
JM, Lott MT, Lezza AMS, Seibel P, Ballinger SW, Wallace DC. Myoclonic epilepsy and ragged-red fiber disease (MERRF) is associated with a mitochondrial DNA tRNAlys mutation. Cell 1990; 61: 931-37. 2. Zeviani M, Amati P, Bresolin N, et al. Rapid detection of the A→G (8344) mutation of mtDNA in Italian families with myoclonus-epilepsy and ragged red fibres (MERRF). Am J Hum Genet 1991; 48: 203-11. 3. Marseille Consensus Group. Classification of progressive myoclonus epilepsies and related disorders. Ann Neurol 1990; 28: 113-16. 4. Berkovic SF, Carpenter S, Evans A, et al. Myoclonus epilepsy and ragged-red fibers (MERRF) 1: a clinical, pathological, biochemical, magnetic resonance spectroscopic and positron emission tomographic study. Brain 1989; 112: 1231-60. 5. Berkovic SF, Andermann F, Shoubridge EA, et al. Mitochondrial dysfunction in multiple symmetric lipomatosis. Ann Neurol 1991; 29: 566-69.
Severity of cystic fibrosis Clinical spectrum of mitochondrial DNA mutation at base pair 8344 SIR,-Dr Hammans and colleagues (June 1, p 1311) identify a mutation at base pair 8344 of the mitochondrial genome in blood samples of 5 of 7 patients with the syndrome ofmyoclonic epilepsy and ragged red fibres (MERRF), confirming previous studies with muscle mitochondrial DNA.l,2 This mutation, which is in the mitochondrial gene for lysine transfer RNA (tRNAlys), was also found in 1 patient with myoclonus and ataxia who lacked ragged red fibres, but was absent in many other syndromes associated with mitochondrial disease. We describe further phenotypes associated with this mutation. The clinical spectrum of MERRF is broad relative to the other causes of progressive myoclonus epilepsy from which it should be distinguished.3We have reported 13 patients with MERRF, including one family with 6 affected members.4 The 2 most severely affected (subjects 5 and 6) had the pathological features of Leigh’s syndrome, in addition to the system degeneration characteristic of MERRF.4 We have now identified the tRNAlys mutation in this family. This confirms Hammans and colleagues’ observation of Leigh’s syndrome lesions at necropsy in 1 of their MERRF patients with this mutation. The neuropathological lesions characterising Leigh’s syndrome are strikingly different from those in uncomplicated cases of MERRF, yet they now seem to be consequences of the same mitochondrial DNA mutation in some families. On the basis of positron emission tomographic data,4 we have suggested that such clinicopathological diversity can be explained by the peculiarities of mitochondrial inheritance, resulting in a spectrum of severity of cerebral metabolic deficits. In the more severe cases, sudden clinical and metabolic deterioration can happen with additional stresses, such as fever, resulting in the pathological picture of Leigh’s syndrome. The proband of our MERRF family also had large disfiguring
SIR,-Dr Johansen and colleagues (March 16, p 631) report that just over three-quarters of patients with cystic fibrosis are homozygous for the AF508 mutation. The rest have other mutations, many of which have been recognised, raising the possibility that the clinical expression of the disease is determined by the mutation that is present. We report a 45-year-old man in whom the disease has been unusually mild and whose genotype has been determined. The patient was an only child born in 1946. At 5 years old he had steatorrhoea and respiratory infections, and fibrocystic disease of the pancreas was diagnosed. He was treated with pancreatic enzyme supplements and made such good progress that by age 15 the diagnosis was thought to have been incorrect and the enzyme supplements were stopped. In his early twenties steatorrhoea recurred and a jejunal biopsy showed an abnormality consistent with coeliac disease. Failure of his symptoms to resolve completely on a gluten-free diet was attributed to non-compliance. At the age of 27 intestinal obstruction developed and at laparotomy the terminal 06 metres of ileum proved to be occluded by inspissated faecal-like material. At about this time he was investigated for a persistent productive cough and bronchography showed bilateral bronchiectasis. By age 38, when he moved to Devon, he was beginning to be troubled by breathlessness on exertion. Since then he has had planned admissions for physiotherapy about twice a year in addition to postural drainage at home, but he has never required admission for an acute infective exacerbation. Chest radiography showed extensive basal emphysema with upper zone fibrosis. His lung function has declined over the past 7 years (FEV 925 ml and FVC 1929 ml in 1984; and 550 ml and 1350 ml, respectively, in 1991), and Pseudomwnas aeruginosa has frequently been cultured from his sputum. His sweat sodium concentration was 124 mmol/1 in 1984. He has maintained a steady weight during this time (51 kg, height
458
175 cm), but lately complained of passing 3-4 loose motions per day with steatorrhoea. These symptoms completely resolved when pancreatic enzymes were re-started. He worked successfully as a musician until breathlessness forced him to stop at 40. He is a non-smoker. The patient always believed that he had cystic tibrosis, even though this was not consistently the opinion of his doctors. On DNA analysis he proved heterozygous for the AF508 mutation and for the G551D mutation in exon 11. Weconclude that this genotype. is compatible with mild expression of the disease. South Devon Healthcare,
Torbay Hospital, Lawes Bridge, Torquay TQ2 7AA, UK
J. ROBINSON G. A. RICHARDSON
A.
Prenatal 1.
diagnosis of two CF families by simultaneous PCR amplification of IVS8CA microsatellite and the exon 10 region
Johansen HK, Nir M, Hoiby N, Koch C, Schwartz M. Seventy of cystic fibrosis in patients homozygous and heterozygous for &Dgr;F508 mutation. Lancet 1991; 337:
of CFTR that contains the AF508 mutation.
631-34
diagnosis of cystic fibrosis by multiplex PCR of mutation and
Prenatal
microsatellite alleles SIR,-Genetic analysis of cystic fibrosis (CF) has been considerably improved since the isolation of the CF gene and the identification of the major CF mutation (AF508 deletion),l which is seen in about 70% of North American and North European subjects, but in only 50% among those living in the Mediterranean region.2 Although more than 80 CF mutations have been identified (CF Genetic Analysis Consortium), most are very uncommon in the general patient population.3,4 Therefore, DNA markers closely linked to the disease locus are still being used, alone or in combination with mutation analysis, for between 40% (northern Europe and North America) and 75% (southern Europe) of CF families.5 We have identified a microsatellite polymorphism in intron 8 of the CFTR gene (IVS8CA)6 and developed PCR conditions for multiplex DNA amplification of microsatellite alleles and the AF508 mutation. The IVS8CA microsatellite detects up to 10 alleles and has a heterozygosity of 80%. The study of the IVS8CA microsatellite in several hundred families, which include 664 meioses, has not revealed new mutant alleles, and shows that it can safely be applied to prenatal diagnosis and carrier detection of CF.66 The table shows the distribution of IVS8CA microsatellite alleles in AF508, non-AF508, and normal chromosomes. Two common alleles are associated with AF508 chromosomes, 23 (52%) and 17 (40%), whereas these alleles account for only 7-6% and 11 % of normal chromosomes, respectively. For non-AF508 chromosomes three common alleles were found, 23 (31%), 17 (10%), and 16 (50%). Allele 16 is the most common (76%) in normal chromosomes. The figure shows two prenatal diagnosis analyses done in a 50% informative and in a non-informative AF508 mutation CF family, which were fully informative for the IVS8CA marker. The simultaneous analysis of the microsatellite and the AF508 mutation allows prenatal diagnosis in a single tube reaction experiment per sample. Since the AF508 mutation accounts for only 50% of the CF ALLELE DISTRIBUTION* OF &Dgr;F508, NON-&Dgr;F508, AND NORMAL CHROMOSOMES FOR IVS8CA MICROSATELLITE IN CFTR GENE
Sequences for the amplification of exon 10 are E1 OD2 (5’-TTCACTTCTAATGATGATTAT) and E10R1 (5’-GTTGGCATGCTTTGATGACGTTTC), and for IVS8CA are NUR-1 (5’-TCTATCTCATGTTAATGCTG) and NUR-2 (5’-GTTTCTAGAGGACATGATC) PCR was done with 500 ng DNA, 200 µmol of each dNTP, 10 pmol of primer NUR-1,1 6 pmol each of end-labelled primers NUR-1 and EX1 OR in total volume of 25 wl. Annealing was at 50.C, and elongation was for 1 min 30 pmol each of primers NUR-1 and EX10R were end-labelled, with 20 wCi of [&ggr;-32P]ATP (3000 Ci/mmol) (1 Ci=3.7x1010Bq) and 5 units of polynucleotide kinase 2 µl of amplification products were mixed with 2 µl of formamide stop solution, loaded mto 6% polyacrylamide gel, and electrophoresed at 1500 V for 3 h Dried gel was exposed against radiographic film for 20-45 min at room temperature M, mother; F, father; CF, cystic fibrosis; CV, chorionic villi. A23, A21, A17, A16, and A15= number of repeats for CA/GT alleles.
chromosomes in the Mediterranean region, and other described mutations in the gene are very uncommon, only 25% of families are fully informative for prenatal diagnosis and 50% are halfinformative with mutation analysis only. When microsatellite and AF508 analysis are combined, families became more than 95% informative. Since some linkage disequilibrium exists between microsatellite alleles and non-AF508 CF chromosomeshaplotype data generated with microsatellite alleles can also be used to improve genetic counselling in couples with less than a 1 -in-4 risk, and in CF couples with a deceased CF child who are negative for the &Dgr;F508 mutation.’ Although several restriction fragment length polymorphisms (RFLPs) have been identified in the CFTR gene, they are not very informative, mainly because they show strong allelic association with other allelic systems in the CF locus, and because they are two-allele polymorphisms.1 The large heterogeneity found in the non-AF508 mutant allele pool suggests that microsatellite and haplotype analysis may be the method of choice for genetic testing in several circumstances. This is especially true in most Mediterranean countries, where only one-third of the estimated 75 000 CF families are expected to be fully informative for mutational
analysis.’"
Molecular Genetics Department, Cancer Research Institute, Hospital Duran i Reynals, 08907 Barcelona, Catalonia, Spain
1. Kerem
X. ESTIVILL N. MORRAL T. CASALS V. NUNES
B-S, Rommens JM, Buchanan JA, et al. Identification of the cystic fibrosis genetic analysis Science 1989; 245: 1073-80.
gene:
Cysuc Fibrosis Genetic Analysis Consortium. Worldwide survey of the &Dgr;F508 mutation-report from the Cystic Fibrosis Genetic Analysis Consortium. AmJ
2. The
Hum Genet 1991; 47: 354-59
Cutting GR, Kash LM, Rosenstein BJ, et al. A cluster of cystic fibrosis mutations in the first nucleotide-binding fold of the cystic fibrosis conductance regulator protein Nature 1990; 346: 366-39. 4. Dean M, White MB, Amos J, et al. Multiple mutations in highly conserved residues are found in mildly affected cystic fibrosis patients. Cell 1990; 61: 863-70. 5. Novelli G, Sangiuolo F, Dallapiccola B, et al. &Dgr;F508 gene deletion and prenatal diagnosis of cystic fibrosis in Italian and Spanish families. Prenat Diagn 1990; 10: 3.
*Alleles indicate the number of CA/GT repeats
413-14. 6. Morral N, Nunes V, Casals T, Estivill X. CA/GT microsatellite alleles within the cystic fibrosis transmembrane regulator (CFTR) gene are not generated by unequal crossingover. Genomics 1991; 10: 692-98. 7. Casals T, Nunes V, Lázaro C, et al. Mutation and linkage disequilibrium analysis in genetic counselling of Spanish cystic fibrosis families. J Med Genet (in press)
