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A SECOND GENETIC LOCUS FOR AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE
8
A SECOND GENETIC LOCUS FOR
AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE G. ROMEO1 G. COSTA1 L. CATIZONE3 G. G. GERMINO4 D. J. WEATHERALL4
M. DEVOTO1 L. RONCUZZI2 P. ZUCCHELLI3 T. KEITH5 S. T. REEDERS4
Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genoa, Italy;1 Laboratorio di Genetica, Clinica Neurologia, and
Dipartimento di Patologia Sperimentale, Universita degli Studi, 40100 Bologna, Italy;2 Divisione di Nefrologia e Dialisi, Ospedale Malpighi, 40138 Bologna, Italy;3 Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK;4 and Collaborative Research Inc, Bedford, Massachusetts 01730, USA5
Summary
Hitherto,
mutations that lead
to
(centromeric) to alpha-globin on chromosome 16 (Reeders ST, Keith T, Green P, et al, unpublished). Reeders et al5 studied twenty-eight Northern European pedigrees in a search for genetic heterogeneity of linkage in ADPKD; heterogeneity of linkage is observed when
genetic loci cause the same no for a second ADPKD found evidence phenotype. They unlinked to a alpha-globin-in their gene-that is, gene Other from various parts of Europe6,7 population. reports have confirmed the linkage between ADPKD and alphaglobin and have likewise yielded no evidence for genetic heterogeneity. Here we report on a single Italian family from Bologna in which no linkage between ADPKD and alpha-globin or other chromosome 16p markers could be detected. In this family ADPKD must be caused by a mutation in a second ADPKD gene that is not linked to mutations at
two or more
alpha-globin.
autosomal
dominant adult-type polycystic kidney disease have been found to be linked to the alpha-globin genes on the short arm of chromosome 16. In an Italian family, absence of linkage between the disease mutation and alpha-globin indicates that the condition can be caused by mutations in a second gene. The clinical features of the disease in this Italian family are indistinguishable from those found in the "linked" families. The finding that there are two polycystic kidney disease genes means that linkage must be demonstrated independently in each family before predictive tests with DNA probes can be used reliably. Introduction RECOMBINANT DNA technology has previously been used to localise mutations in several autosomal dominant ("adult") polycystic kidney disease (ADPKD) families to chromosome 16. Initially, nine ADPKD families were studied and, in each case, the mutation for ADPKD was found to be closely linked to the alpha-globin cluster at a recombination frequency about 5% . Subsequently, genetic linkage was also observed between ADPKD and phosphoglycolate phosphatase (PGP).2 Both alpha-globin and PGP had previously been localised to the distal third of the short arm of human chromosome 16 (band p1.3), thereby localising the ADPKD mutations in these families to the same region. More recently, the localisation has been refined by the identification of anonymous DNA sequences that recognise restriction fragment length polymorphisms (RFLPs) in this region.34 These cloned DNA probes have been used to map the position of the ADPKD gene between the alpha-globin cluster and several loci proximal
13 Terland D. A double blind multicentre crossover general practice study of glyceryl trinitrate delivered by a transdermal therapeutic system in stable angina pectoris Curr Ther Res 1986, 39: 214-22. 14 Muessen G, Agabata IRE, Mueisen L, et al A multicenter trial of transdermal nitroglycerin m exercise-induced angina. Individual anti-anginal response after repeated administration. Am Heart J 1986; 112: 233-38 15 Reichek N, Priest C, Zimrin D, Chandler T, Sutton SJ Antiaginal effects of nitroglycerin patches Am J Cardiol 1984, 54: 1-7 16. Crean PA, Ribeiro P, Crea F, Davies GT, Ratcliffe D, Maseri A Failure of transdermal nitroglycerin to improve chronic stable angina A randomised placebo controlled double blind crossover trial Am Heart J 1984; 108: 1494-500 17. James MA, Walker PR, Papouchado M, Wilkinson PR Efficacy of transdermal glyceryl trinitrate in the treatment of chronic stable angina pectoris Br Heart J 1985; 53: 631-35. 18 Smyth P, Akhras F, Jackson N. Silke B, Taylor SH. Jackson G Nitrate patches are ineffective in stable angina. Circulation 1986; 74: 134-38.
Methods Clinical Evaluation
Symptom-free at-risk family members were assessed by ultrasonography. The criteria for the diagnosis of ADPKD were based on the studies of Bear et al. Individuals were considered to be affected if they were found to have at least one cyst in each kidney and at least two cysts in one kidney.
DNA
Analysis
DNA was extracted from venous blood and digested with the restriction endonuclease appropriate for the DNA probe used in hybridisation experiments. The digested DNA was transferred to nylon filters by Southern blotting and hybridised with 3zP-labelled probes. The following probes were used. The 3’ hypervariable region of the alpha-globin locus (3’HVR) contains 4 kilobases (kb) of a tandemly repeated sequence 8 kb downstream from the genes coding for the alpha-chains of human haemoglobin.9 The 3’HVR probe hybridised to two allelic fragments in each sample. Each allele was identified by an arbitrary letter, 4-y. The probe p090a is an Xbal-HindII fragment from the cosmid CRI-090 which recognises both EcoRI and Dral polymorphisms. The probe p0133i contains a 27 kb EcoRI-BamHI fragment from the cosmid CRI-0133. This probe recognises a HindIII polymorphism. The probe p0327hb contains a 4-3 kb HindIII-BgIII fragment from the cosmid CRI-0327, which recognises a HindIII polymorphism (Reeders ST, Keith T, Green P, et al, unpublished).
Linkage Analysis Lod scores were calculated by use of the LINKAGE computer programs.1o The lod score is the likelihood that two loci are linked at a specified recombination fraction, divided by the likelihood that the loci
19
20. 21 22.
23
24
are
unlinked, expressed as a logarithm to the base 10. A lod
et al. Hemodynamic effects of intermittent transdermal nitroglycerin in chronic congestive heart failure Am J Cardiol 1987, 59: 895-99 Cowan C, Bourke J, Reid DS, Julian DG Tolerance to glyceryl trinitrate patches Prevention by intermittent dosing Br Med J 1987; 294: 544-45. Parker JOM Nitrate therapy in stable angina pectoris N Engl J Med 1987; 316: 1635-42 Leir CV, Huss P, Magorien RD, Unverferth DV Improved exercise capacity and differing arterial and venous tolerance during chronic isosorbide dinitrate therapy for congestive heart failure. Circulation 1983; 67: 817-22 Franciosa JA, Cohn JN. Sustained hemodynamic effects without tolerance during long-term isosorbide dinitrate treatment in chronic left ventricular failure Am J Cardiol 1980, 45: 640-54. Lee G, Mason DT, DeMaria A. Effects of long-term oral administration of isosorbide dinitrate on the antianginal response to nitroglycerin: Absence of cross-tolerance and self-tolerance shown by exercise testing. Am J Cardiol 1978, 41: 82-87
Sharpe N, Coxon R, Webster M,
9 of 3for the linkage between ADPKD and alpha-globin in a that the odds in favour of linkage in 1. A lod score of - 2-0 effectively excludes are 1000 to families these score
given set of families indicates linkage.
Age-of-onset (detection)
curves
were
incorporated
into the
calculations to allow for variation in the sensitivity of ultrasound imaging with age and for false--negative and false-positive diagnoses. These penetrance assumptions are discussed in the Results section.
Admixture Test of Genetic
Heterogeneity
This test was devised by Smith" to determine whether data from number of families support the hypothesis that there is heterogeneity of genetic linkage. It postulates that families in which the disease is segregating can be divided into two classes- a linked class, in which the disease mutation is linked to the marker locus with a true recombination fraction, 80, and an unlinked class. Likelihoods are calculated for a number of values of alpha, the fraction of families postulated to be in the linked class. The method provides a means of estimating (1) the probability that there are two classes, one linked and one unlinked; (2) the fraction of families that are in the linked class, alpha;. and (3) the posterior probability that a particular family is in the linked class. The admixture test was done with the HOMOG computer a
program.12 Results
Clinical Studies The accompanying figure shows the pedigree chart for the family under study (GAS 17). The clinical features of the disease in this family were typical of "adult" type polycystic kidney disease, and the family was indistinguishable, on clinical grounds, from families studied previously in which the disease was linked to alpha-globin. Several patients had symptoms. Individual 11,1has been receiving haemodialysis therapy since the age of 44 yr. 11,3 had a nephrectomy at the age of 37 yr for an infected cyst. Two family members with renal cysts were also found to have hepatic cysts. 1,1died at the age of 76 with a gastric neoplasm and 1,2 died at the age of 53 with uterine cancer and cardiac failure; no necropsies were performed. Individual 111,7 was found to have a single 2 cm cyst in one kidney at the age of 15 yr. The relevance of a single cyst in this age group is not known and this individual was therefore excluded from the analysis.
Linkage Studies The GAS17 family was studied with the 3’HVR probe, which is closely linked to the alpha-globin cluster, and the three probes, described above, which are also closely linked
the ADPKD locus. The order of markers has been determined (Reeders ST, Keith T, Green P, et al, unpublished): 0133i-090a-0327hb-ADPKD-3’HVR. The sex-averaged recombination fractions between 3’HVR and ADPKD and between ADPKD and 090a are —4% and 5-5%, respectively. Two of the four probes studied, 3’HVR and p090a, were informative for parts of this family. The pedigree and genotypes for these probes are shown in the figure. Although it cannot be determined which of the two parents in generation I had ADPKD, several of their children are affected; it is, therefore, extremely unlikely that each of them represents a de-novo mutation. In linkage calculations, both 1,1 and 1,2 were coded as "ADPKD phenotype unknown". Because samples from 1,1 and 1,2 were not available, and because only two genotypes are present in their offspring, the 3’HVR genotypes of these individuals could not be deduced. However, it is clear that alleles A, B, D and L were inherited from 1,1 and 1,2 by their children. Inspection of the pedigree reveals that, irrespective of which of these four alleles was coinherited with the disease mutation, and irrespective of which of the parents passed on the disease, there must be at least four crossovers in a total of ten meioses. If the disease is segregating with the 3’HVR alleles B or D, then there must have been 4 crossovers, whereas if it is segregating with alleles A or L, there must have been at least 6 crossovers. The 090a locus is informative for the children of 11,1,11,3, 11,5, and 11,8. This locus appears to segregate with 3’HVR with no recombination; therefore, if ADPKD lies between 090a and 3’HVR in the GAS 17 family, at least one double crossover must have occurred between these markers.
to
Heterogeneity Testing Lod scores for linkage between 3’HVR and ADPKD, 090a and ADPKD, and 090a and 3’HVR are given for the GAS 17 family in the table. Lod scores for linkage between 3’HVR and ADPKD were combined with previously published lod scores for twenty-eight families5 to compare the GAS 17 family with families that have previously been studied in an identical manner. According to the admixture test, the probability that there is heterogeneity of genetic linkage within the set of twenty-nine families and that some of the families are not linked to alpha-globin was found to be 0-985. The posterior probability that the GAS 17 family is linked, from the whole data set, was 0-014, and that each of the other families are linked to alpha-globin varied between 0-95 and 1 00. Hence the data support the conclusion that the ADPKD mutation in the GAS17 family maps to a
Family pedigree. Filled symbols represent affected individuals. The hatched symbol indicates that the diagnosis is equivocal. Numbers within symbols give the age at which the subject last had a normal ultrasound examination. A to L are 3’HVR alleles. 1 and 2 are 090a alleles.
10 different genetic locus from the ADPKD mutations in the other twenty-eight families that are closely linked to alpha-globin. The fraction of families in the unlinked group was - 3%. This is not, however, a reliable estimate since sampling of families was not random; further studies of a large number of families will be required to determine its true value. LOD SCORES FOR THE
GAS 17
FAMILY
—————————————————————————————
indistinguishable phenotypes, both of which fit the clinical criteria for autosomal dominant polycystic kidney disease. This finding enables us to define two diseases which have different molecular pathologies. The first of these is closely linked to alpha-globin,l lies proximal to alpha-globin on chromosome 16, and has a locus defined by its position within an array of anonymous polymorphic DNA sequences. This locus has been assigned the name PKDI by the Eighth Human Gene Mapping Workshop.13 The second ADPKD gene, which is mutated in the GAS17 described in this report, is currently defined by the fact that it does not map to the interval between ADPKD and 090a. The whereabouts of this second gene is, as yet, unknown. Another family, principally resident in Colorado, USA, but of Sicilian origin, has also shown no evidence of linkage with alpha-globin (Kimberling W, Gabow P, personal communication). These two families come from different parts of Italy but they raise the possibility that the unlinked ADPKD gene is commoner in populations from this part of Europe. On the other hand, del Senno et aF have found no evidence for heterogeneity in Ferrara, a province bordering Bologna from which the GAS 17 family comes. The discovery of these "unlinked" families calls for extensive further study of ADPKD families from different ethnic groups to establish the distribution and frequency of the unlinked form of the disease. These data have important implications for the use of genetic markers in diagnosis of ADPKD. Until such time as the ADPKD genes have been isolated and direct analysis of mutations is possible, DNA-based diagnosis will depend on genetic linkage. However, since there are two ADPKD genes with different linkage relationships, linkage-based tests cannot be used reliably unless linkage has first been independently established in the family under study. This is turn requires that a large number of family members are available for study; the exact number will depend on their relationship and informativeness. When genetic marker data are used to assist in counselling, the chance that the family is of the unlinked type must clearly be taken into account even though this chance seems small.
family
possible factors that could lead to the false detection of genetic heterogeneity were considered. Firstly, multiple errors in diagnosis of ADPKD in the GAS 17 family might lead to an apparent absence of linkage. This is especially important since the sensitivity of cyst detection varies with age, false negative diagnoses occurring more frequently in the first three decades. This possibility is catered for during lod score calculation by the assumption of incomplete penetrance. True values of penetrance are not available since long-term longitudinal studies with repeat ultrasound diagnosis have not been reported; two published estimates of penetrance were, however, available and these were used in two separate sets of calculations. The values incorporated Three
into the lod scores in the table are those cited in Reeders et all and are based on segregation analysis.8 The rest of the assumption that the probability that subjects carrying an ADPKD mutation will manifest the disease rises from 66% in the second decade to 95% in the fourth and subsequent decades. A second set of penetrance values were based on the excess of observed double recombination between 3’HVR and 090a compared with expected numbers derived from the measured recombination rates between these loci (Reeders ST, Keith T, Green P, et al, unpublished). Essentially, these values are equivalent to false-positive and false-negative diagnosis rates of 2%. When these values were incorporated into a second set of lod score calculations (not shown), the support for the hypothesis of heterogeneity was not found to be significant (p=0’l).However, all the above calculations were based on two-point data alone. The use of data from all three informative loci (ADPKD, 090a, 3’HVR) adds further power to the study since ADPKD is known to map to the interval between 3’HVR and 090a in other families. Multipoint analysis permits calculation of the likelihood that the ADPKD mutation in GAS 17 also maps between 3’HVR and 090a. Likelihoods were calculated for a range of locations of ADPKD for each family. These were then used in a modified version of the admixture test as suggested by Ott.l2 Irrespective of which of the above penetrance assumptions were used, the data support the hypothesis of heterogeneity (p <0’001), with a posterior probability of linkage of less than 0001 for the GAS 17 family. It is therefore unlikely that diagnostic error is responsible for the heterogeneity observed here. A second factor that may contribute to apparent heterogeneity is an error in family structure, most commonly caused by nonpaternity. However, no inconsistent genotypes were observed with any of the probes used. Therefore multiple errors of this type, which would be required to explain the absence of linkage, are very unlikely to have occurred. Thirdly, it is possible that both parents in generation I were carriers of an ADPKD mutation. In view of the frequency of the ADPKD gene in the population (-11000), this is unlikely. Nevertheless, lod scores were recalculated on the assumption that both 1,and 1,2 were affected (data not shown). Again the data were found to support heterogeneity (p < 0-01). ).
Discussion The data reported here provide strong evidence that mutations in two separate genes may lead to
We wish to thank the NIDDK, the Wellcome Trust, and the Polycystc Kidney Research Foundation for generous support. This work was realised in part with funds of the "Progetto finalizzato ingegneria genetica e basi molecolari delle malattie ereditarie", C.N.R. (Rome). Mark Lathrop and Jurg Ott kindly provided computer programs and gave useful advice on heterogeneity testing. Correspondence should be addressed to S. T. R., Section of Nephrology, Department of Internal Medicine, School of Medicine, Yale University 2073-LMP, PO Box 3333, New Haven, CT 06510-8056, USA. REFERENCES
ST, Breuning MH, Davies KE, et al. A highly polymorphic DNA marker linked to adult polycystic kidney disease on chromosome 16 Nature 1985, 317: 542-44 2. Reeders ST, Breuning MH, Corney G, et al Two genetic markers closely linked to adult polycystic kidney disease on chromosome 16. Br Med J 1986, 292: 851-53 3. Reeders ST, Keith TP, Green P, et al Linkage studies and physical mapping of the autosomal dominant polycystic kidney disease mutation Cytogen Cell Genet (in 1. Reeders
press
Breuning MH, Reeders ST, Brunner H, et al. Improved early diagnosis of adult polycy stic kidney disease with flanking DNA markers Lancet 1987; ii 1359-61. 5. Reeders ST, Breuning MH, Ryynanen MA, et al. A study of genetic linkage heterogeneity in adult polycystic kidney disease Hum Genet 1987; 76: 438-51 6 Lazarou LP, Davies F, Sarfarazi M, Coles GA, Harper PS. Adult polycystic kidney disease and linked RFLPs at the &agr; globin locus: a genetic study in the South Wales population J Med Genet 1987; 24: 466-73. 7 del Senno L, Castagnoli A, Zamorani G, et al. Use of a genetic marker for the diagnosis of adult polycystic kidney disease in Northern Italy. Prot Biolog Fluids 1987; 35:
4
63-66. JC, McManamon P, Morgan J Age at clinical onset and at ultrasonographic detection of adult polycystic kidney disease—data for genetic counselling. Am J Med Genet 1984; 18: 45-53
8. Bear
11
PATENT FORAMEN OVALE IN YOUNG STROKE PATIENTS A. M. CHANCELLOR M. W. I. WEBSTER D. L. SWIFT H. J. SMITH N. M. BASS SHARPE D. N. L. GLASGOW G.
Departments of Medicine, Neurology, and Cardiology, Auckland Hospital, Auckland, New Zealand The prevalence of patent foramen ovale in patients presenting with non-haemorrhagic stroke or transient ischaemic attacks under the age of 40 years was determined by contrast echocardiography. Studies were performed at rest and with a Valsalva manoeuvre in 40 stroke patients and in an age and sex matched control group. Right-to-left shunting was found in 20 (50%) of the stroke patients and 6 (15%) of the controls (p <0·001). Paradoxical embolism through a patent foramen ovale may be an under-recognised cause of stroke in young adults.
Summary
Introduction ONLY 3% of cerebral infarcts occur in patients under 40 years of age.1 In almost half of these patients a clear underlying cause is not found despite extensive investigation including two-dimensional echocardiography.2 It has been suggested that paradoxical embolism through a patent foramen ovale may cause some of these strokes in the absence of the usual clinical features of deep-vein thrombosis and pulmonary hypertension.3-5 We determined the prevalence of patent foramen ovale as assessed by contrast echocardiography in a population of young stroke
patients. Patients and Methods
Study Population 66 patients under 40 years of age were referred to the neurology department of Auckland Hospital between January 1980 and December 1987 and were diagnosed as having non-haemorrhagic cerebral infarction or transient ischaemic attacks. Diagnosis required the focal neurological deficit to be of acute onset without other explanation after extensive inpatient investigation including computed tomographic scan and/or cerebral angiography. Patients with complicated migraine but no fixed neurological deficit after 24
(ATL Ultramark 8). After
a two-dimensional study, contrast and with a Valsalva manoeuvre was echocardiography 8 ml of performed. physiological saline and 0-5 ml of air were agitated in a Rotamixer Delux (Hook and Tucker Ltd), macroscopic air was extruded, and the saline was injected rapidly into a 20G cannula in a right antecubital vein. Two or more injections were made during normal respiration and then repeated on at least two further occasions during the strain phase of a Valsalva manoeuvre, performed against a mercury manometer to a standard pressure of 40 mm Hg. During each injection the apical fourchamber view was recorded on videotape. Contrast echocardiographs were judged abnormal if microbubbles appeared in the left atrium or left ventricle within three cardiac cycles of their appearance in the right atrium. All studies were interpreted by two non-blinded observers and the diagnosis was made by consensus. The right-to-left shunt was semiquantitatively assessed by counting the maximum number of bubbles seen in the left side of the heart during frame-by-frame analysis. Grade 1 was defined as 1-5 bubbles, grade 2 as 6-20 bubbles, and grade 3 as over 20 bubbles. The same protocol was used for the stroke and normal control
at rest
subjects. Statistical Methods
significance of the frequency of patent foramen ovale in patients was determined by the log likelihood ratio of goodness of fit for single classification frequency distributions (G-test) .6 The expected frequency used was that of the normal The stroke
control group.
Results The stroke group consisted of 17 males and 23 females with an age range of 21-44 years (mean 31-6 years). 32 patients had a single cerebral infarction, 5 had more than one infarct, and 3 had one or more transient ischaemic attacks. All patients were examined by a cardiologist and in none was there clinical, electrocardiographic, or radiological evidence of an atrial septal defect or pulmonary hypertension. A patent foramen ovale was found in 12/40 (30%) at rest and 20/40 (50%) during a Valsalva manoeuvre. In the normal control group a patent foramen ovale was present in 3/40 (7-5%) at rest and 6/40 (15%) with a Valsalva manoeuvre (fig 1). The stroke patients also showed a greater degree of
hours were excluded. Of these patients 3 had died, 7 were lost to follow up, 11 refused contrast echocardiography, and 1 patient with recurrent strokes had already undergone surgical closure of a patent foramen ovale. In another 4 patients echocardiographic views were unsatisfactory, giving a study population of 40 patients. The mean interval between ictus and follow-up was 36 months. Each patient was then prospectively paired with a normal control subject, matched for sex and age (within 2 years). 1 control subject was excluded for poor image quality and was replaced.
Fig I-Prevalence of patent foramen ovale
Method of Assessment Patient
evaluation
Valsalva
included
clinical
assessment,
electrocardiography, chest radiography, and echocardiography
9. Jarman AP, Nicholls RD, Weatherall DJ, Clegg JB, Higgs DR. Molecular characterisation of a hypervariable region downstream of the human &agr;-globin gene cluster. EMBO J1986; 5: 1857-63. 10 Lathrop GM, Lalouel JM. Easy calculations of lod scores and genetic risks on small computers. Am JHum Genet 1984; 36: 460-65.
at rest
and with
a
manoeuvre.
Stroke patients had significantly control group (* denotes p <0’001).
more
patent foramina than the normal
11. Smith CAB. Testing for heterogeneity of recombination fraction values in human genetics. Ann Hum Genet 1963; 27: 175-82. 12. Ott J. A short guide to linkage analysis. In: Davies KE, ed. Human genetic diseases. A practical approach. Oxford. IRL Press, 1987: 19-32. 13. Cox DR, Gedde-Dahl T. Report of the committee on the genetic constitution of chromosomes 13, 14, 15 and 16. Cytogen Cell Genet 1985; 40: 206-41.
A SECOND GENETIC LOCUS FOR
AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE G. ROMEO1 G. COSTA1 L. CATIZONE3 G. G. GERMINO4 D. J. WEATHERALL4
M. DEVOTO1 L. RONCUZZI2 P. ZUCCHELLI3 T. KEITH5 S. T. REEDERS4
Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genoa, Italy;1 Laboratorio di Genetica, Clinica Neurologia, and
Dipartimento di Patologia Sperimentale, Universita degli Studi, 40100 Bologna, Italy;2 Divisione di Nefrologia e Dialisi, Ospedale Malpighi, 40138 Bologna, Italy;3 Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK;4 and Collaborative Research Inc, Bedford, Massachusetts 01730, USA5
Summary
Hitherto,
mutations that lead
to
(centromeric) to alpha-globin on chromosome 16 (Reeders ST, Keith T, Green P, et al, unpublished). Reeders et al5 studied twenty-eight Northern European pedigrees in a search for genetic heterogeneity of linkage in ADPKD; heterogeneity of linkage is observed when
genetic loci cause the same no for a second ADPKD found evidence phenotype. They unlinked to a alpha-globin-in their gene-that is, gene Other from various parts of Europe6,7 population. reports have confirmed the linkage between ADPKD and alphaglobin and have likewise yielded no evidence for genetic heterogeneity. Here we report on a single Italian family from Bologna in which no linkage between ADPKD and alpha-globin or other chromosome 16p markers could be detected. In this family ADPKD must be caused by a mutation in a second ADPKD gene that is not linked to mutations at
two or more
alpha-globin.
autosomal
dominant adult-type polycystic kidney disease have been found to be linked to the alpha-globin genes on the short arm of chromosome 16. In an Italian family, absence of linkage between the disease mutation and alpha-globin indicates that the condition can be caused by mutations in a second gene. The clinical features of the disease in this Italian family are indistinguishable from those found in the "linked" families. The finding that there are two polycystic kidney disease genes means that linkage must be demonstrated independently in each family before predictive tests with DNA probes can be used reliably. Introduction RECOMBINANT DNA technology has previously been used to localise mutations in several autosomal dominant ("adult") polycystic kidney disease (ADPKD) families to chromosome 16. Initially, nine ADPKD families were studied and, in each case, the mutation for ADPKD was found to be closely linked to the alpha-globin cluster at a recombination frequency about 5% . Subsequently, genetic linkage was also observed between ADPKD and phosphoglycolate phosphatase (PGP).2 Both alpha-globin and PGP had previously been localised to the distal third of the short arm of human chromosome 16 (band p1.3), thereby localising the ADPKD mutations in these families to the same region. More recently, the localisation has been refined by the identification of anonymous DNA sequences that recognise restriction fragment length polymorphisms (RFLPs) in this region.34 These cloned DNA probes have been used to map the position of the ADPKD gene between the alpha-globin cluster and several loci proximal
13 Terland D. A double blind multicentre crossover general practice study of glyceryl trinitrate delivered by a transdermal therapeutic system in stable angina pectoris Curr Ther Res 1986, 39: 214-22. 14 Muessen G, Agabata IRE, Mueisen L, et al A multicenter trial of transdermal nitroglycerin m exercise-induced angina. Individual anti-anginal response after repeated administration. Am Heart J 1986; 112: 233-38 15 Reichek N, Priest C, Zimrin D, Chandler T, Sutton SJ Antiaginal effects of nitroglycerin patches Am J Cardiol 1984, 54: 1-7 16. Crean PA, Ribeiro P, Crea F, Davies GT, Ratcliffe D, Maseri A Failure of transdermal nitroglycerin to improve chronic stable angina A randomised placebo controlled double blind crossover trial Am Heart J 1984; 108: 1494-500 17. James MA, Walker PR, Papouchado M, Wilkinson PR Efficacy of transdermal glyceryl trinitrate in the treatment of chronic stable angina pectoris Br Heart J 1985; 53: 631-35. 18 Smyth P, Akhras F, Jackson N. Silke B, Taylor SH. Jackson G Nitrate patches are ineffective in stable angina. Circulation 1986; 74: 134-38.
Methods Clinical Evaluation
Symptom-free at-risk family members were assessed by ultrasonography. The criteria for the diagnosis of ADPKD were based on the studies of Bear et al. Individuals were considered to be affected if they were found to have at least one cyst in each kidney and at least two cysts in one kidney.
DNA
Analysis
DNA was extracted from venous blood and digested with the restriction endonuclease appropriate for the DNA probe used in hybridisation experiments. The digested DNA was transferred to nylon filters by Southern blotting and hybridised with 3zP-labelled probes. The following probes were used. The 3’ hypervariable region of the alpha-globin locus (3’HVR) contains 4 kilobases (kb) of a tandemly repeated sequence 8 kb downstream from the genes coding for the alpha-chains of human haemoglobin.9 The 3’HVR probe hybridised to two allelic fragments in each sample. Each allele was identified by an arbitrary letter, 4-y. The probe p090a is an Xbal-HindII fragment from the cosmid CRI-090 which recognises both EcoRI and Dral polymorphisms. The probe p0133i contains a 27 kb EcoRI-BamHI fragment from the cosmid CRI-0133. This probe recognises a HindIII polymorphism. The probe p0327hb contains a 4-3 kb HindIII-BgIII fragment from the cosmid CRI-0327, which recognises a HindIII polymorphism (Reeders ST, Keith T, Green P, et al, unpublished).
Linkage Analysis Lod scores were calculated by use of the LINKAGE computer programs.1o The lod score is the likelihood that two loci are linked at a specified recombination fraction, divided by the likelihood that the loci
19
20. 21 22.
23
24
are
unlinked, expressed as a logarithm to the base 10. A lod
et al. Hemodynamic effects of intermittent transdermal nitroglycerin in chronic congestive heart failure Am J Cardiol 1987, 59: 895-99 Cowan C, Bourke J, Reid DS, Julian DG Tolerance to glyceryl trinitrate patches Prevention by intermittent dosing Br Med J 1987; 294: 544-45. Parker JOM Nitrate therapy in stable angina pectoris N Engl J Med 1987; 316: 1635-42 Leir CV, Huss P, Magorien RD, Unverferth DV Improved exercise capacity and differing arterial and venous tolerance during chronic isosorbide dinitrate therapy for congestive heart failure. Circulation 1983; 67: 817-22 Franciosa JA, Cohn JN. Sustained hemodynamic effects without tolerance during long-term isosorbide dinitrate treatment in chronic left ventricular failure Am J Cardiol 1980, 45: 640-54. Lee G, Mason DT, DeMaria A. Effects of long-term oral administration of isosorbide dinitrate on the antianginal response to nitroglycerin: Absence of cross-tolerance and self-tolerance shown by exercise testing. Am J Cardiol 1978, 41: 82-87
Sharpe N, Coxon R, Webster M,
9 of 3for the linkage between ADPKD and alpha-globin in a that the odds in favour of linkage in 1. A lod score of - 2-0 effectively excludes are 1000 to families these score
given set of families indicates linkage.
Age-of-onset (detection)
curves
were
incorporated
into the
calculations to allow for variation in the sensitivity of ultrasound imaging with age and for false--negative and false-positive diagnoses. These penetrance assumptions are discussed in the Results section.
Admixture Test of Genetic
Heterogeneity
This test was devised by Smith" to determine whether data from number of families support the hypothesis that there is heterogeneity of genetic linkage. It postulates that families in which the disease is segregating can be divided into two classes- a linked class, in which the disease mutation is linked to the marker locus with a true recombination fraction, 80, and an unlinked class. Likelihoods are calculated for a number of values of alpha, the fraction of families postulated to be in the linked class. The method provides a means of estimating (1) the probability that there are two classes, one linked and one unlinked; (2) the fraction of families that are in the linked class, alpha;. and (3) the posterior probability that a particular family is in the linked class. The admixture test was done with the HOMOG computer a
program.12 Results
Clinical Studies The accompanying figure shows the pedigree chart for the family under study (GAS 17). The clinical features of the disease in this family were typical of "adult" type polycystic kidney disease, and the family was indistinguishable, on clinical grounds, from families studied previously in which the disease was linked to alpha-globin. Several patients had symptoms. Individual 11,1has been receiving haemodialysis therapy since the age of 44 yr. 11,3 had a nephrectomy at the age of 37 yr for an infected cyst. Two family members with renal cysts were also found to have hepatic cysts. 1,1died at the age of 76 with a gastric neoplasm and 1,2 died at the age of 53 with uterine cancer and cardiac failure; no necropsies were performed. Individual 111,7 was found to have a single 2 cm cyst in one kidney at the age of 15 yr. The relevance of a single cyst in this age group is not known and this individual was therefore excluded from the analysis.
Linkage Studies The GAS17 family was studied with the 3’HVR probe, which is closely linked to the alpha-globin cluster, and the three probes, described above, which are also closely linked
the ADPKD locus. The order of markers has been determined (Reeders ST, Keith T, Green P, et al, unpublished): 0133i-090a-0327hb-ADPKD-3’HVR. The sex-averaged recombination fractions between 3’HVR and ADPKD and between ADPKD and 090a are —4% and 5-5%, respectively. Two of the four probes studied, 3’HVR and p090a, were informative for parts of this family. The pedigree and genotypes for these probes are shown in the figure. Although it cannot be determined which of the two parents in generation I had ADPKD, several of their children are affected; it is, therefore, extremely unlikely that each of them represents a de-novo mutation. In linkage calculations, both 1,1 and 1,2 were coded as "ADPKD phenotype unknown". Because samples from 1,1 and 1,2 were not available, and because only two genotypes are present in their offspring, the 3’HVR genotypes of these individuals could not be deduced. However, it is clear that alleles A, B, D and L were inherited from 1,1 and 1,2 by their children. Inspection of the pedigree reveals that, irrespective of which of these four alleles was coinherited with the disease mutation, and irrespective of which of the parents passed on the disease, there must be at least four crossovers in a total of ten meioses. If the disease is segregating with the 3’HVR alleles B or D, then there must have been 4 crossovers, whereas if it is segregating with alleles A or L, there must have been at least 6 crossovers. The 090a locus is informative for the children of 11,1,11,3, 11,5, and 11,8. This locus appears to segregate with 3’HVR with no recombination; therefore, if ADPKD lies between 090a and 3’HVR in the GAS 17 family, at least one double crossover must have occurred between these markers.
to
Heterogeneity Testing Lod scores for linkage between 3’HVR and ADPKD, 090a and ADPKD, and 090a and 3’HVR are given for the GAS 17 family in the table. Lod scores for linkage between 3’HVR and ADPKD were combined with previously published lod scores for twenty-eight families5 to compare the GAS 17 family with families that have previously been studied in an identical manner. According to the admixture test, the probability that there is heterogeneity of genetic linkage within the set of twenty-nine families and that some of the families are not linked to alpha-globin was found to be 0-985. The posterior probability that the GAS 17 family is linked, from the whole data set, was 0-014, and that each of the other families are linked to alpha-globin varied between 0-95 and 1 00. Hence the data support the conclusion that the ADPKD mutation in the GAS17 family maps to a
Family pedigree. Filled symbols represent affected individuals. The hatched symbol indicates that the diagnosis is equivocal. Numbers within symbols give the age at which the subject last had a normal ultrasound examination. A to L are 3’HVR alleles. 1 and 2 are 090a alleles.
10 different genetic locus from the ADPKD mutations in the other twenty-eight families that are closely linked to alpha-globin. The fraction of families in the unlinked group was - 3%. This is not, however, a reliable estimate since sampling of families was not random; further studies of a large number of families will be required to determine its true value. LOD SCORES FOR THE
GAS 17
FAMILY
—————————————————————————————
indistinguishable phenotypes, both of which fit the clinical criteria for autosomal dominant polycystic kidney disease. This finding enables us to define two diseases which have different molecular pathologies. The first of these is closely linked to alpha-globin,l lies proximal to alpha-globin on chromosome 16, and has a locus defined by its position within an array of anonymous polymorphic DNA sequences. This locus has been assigned the name PKDI by the Eighth Human Gene Mapping Workshop.13 The second ADPKD gene, which is mutated in the GAS17 described in this report, is currently defined by the fact that it does not map to the interval between ADPKD and 090a. The whereabouts of this second gene is, as yet, unknown. Another family, principally resident in Colorado, USA, but of Sicilian origin, has also shown no evidence of linkage with alpha-globin (Kimberling W, Gabow P, personal communication). These two families come from different parts of Italy but they raise the possibility that the unlinked ADPKD gene is commoner in populations from this part of Europe. On the other hand, del Senno et aF have found no evidence for heterogeneity in Ferrara, a province bordering Bologna from which the GAS 17 family comes. The discovery of these "unlinked" families calls for extensive further study of ADPKD families from different ethnic groups to establish the distribution and frequency of the unlinked form of the disease. These data have important implications for the use of genetic markers in diagnosis of ADPKD. Until such time as the ADPKD genes have been isolated and direct analysis of mutations is possible, DNA-based diagnosis will depend on genetic linkage. However, since there are two ADPKD genes with different linkage relationships, linkage-based tests cannot be used reliably unless linkage has first been independently established in the family under study. This is turn requires that a large number of family members are available for study; the exact number will depend on their relationship and informativeness. When genetic marker data are used to assist in counselling, the chance that the family is of the unlinked type must clearly be taken into account even though this chance seems small.
family
possible factors that could lead to the false detection of genetic heterogeneity were considered. Firstly, multiple errors in diagnosis of ADPKD in the GAS 17 family might lead to an apparent absence of linkage. This is especially important since the sensitivity of cyst detection varies with age, false negative diagnoses occurring more frequently in the first three decades. This possibility is catered for during lod score calculation by the assumption of incomplete penetrance. True values of penetrance are not available since long-term longitudinal studies with repeat ultrasound diagnosis have not been reported; two published estimates of penetrance were, however, available and these were used in two separate sets of calculations. The values incorporated Three
into the lod scores in the table are those cited in Reeders et all and are based on segregation analysis.8 The rest of the assumption that the probability that subjects carrying an ADPKD mutation will manifest the disease rises from 66% in the second decade to 95% in the fourth and subsequent decades. A second set of penetrance values were based on the excess of observed double recombination between 3’HVR and 090a compared with expected numbers derived from the measured recombination rates between these loci (Reeders ST, Keith T, Green P, et al, unpublished). Essentially, these values are equivalent to false-positive and false-negative diagnosis rates of 2%. When these values were incorporated into a second set of lod score calculations (not shown), the support for the hypothesis of heterogeneity was not found to be significant (p=0’l).However, all the above calculations were based on two-point data alone. The use of data from all three informative loci (ADPKD, 090a, 3’HVR) adds further power to the study since ADPKD is known to map to the interval between 3’HVR and 090a in other families. Multipoint analysis permits calculation of the likelihood that the ADPKD mutation in GAS 17 also maps between 3’HVR and 090a. Likelihoods were calculated for a range of locations of ADPKD for each family. These were then used in a modified version of the admixture test as suggested by Ott.l2 Irrespective of which of the above penetrance assumptions were used, the data support the hypothesis of heterogeneity (p <0’001), with a posterior probability of linkage of less than 0001 for the GAS 17 family. It is therefore unlikely that diagnostic error is responsible for the heterogeneity observed here. A second factor that may contribute to apparent heterogeneity is an error in family structure, most commonly caused by nonpaternity. However, no inconsistent genotypes were observed with any of the probes used. Therefore multiple errors of this type, which would be required to explain the absence of linkage, are very unlikely to have occurred. Thirdly, it is possible that both parents in generation I were carriers of an ADPKD mutation. In view of the frequency of the ADPKD gene in the population (-11000), this is unlikely. Nevertheless, lod scores were recalculated on the assumption that both 1,and 1,2 were affected (data not shown). Again the data were found to support heterogeneity (p < 0-01). ).
Discussion The data reported here provide strong evidence that mutations in two separate genes may lead to
We wish to thank the NIDDK, the Wellcome Trust, and the Polycystc Kidney Research Foundation for generous support. This work was realised in part with funds of the "Progetto finalizzato ingegneria genetica e basi molecolari delle malattie ereditarie", C.N.R. (Rome). Mark Lathrop and Jurg Ott kindly provided computer programs and gave useful advice on heterogeneity testing. Correspondence should be addressed to S. T. R., Section of Nephrology, Department of Internal Medicine, School of Medicine, Yale University 2073-LMP, PO Box 3333, New Haven, CT 06510-8056, USA. REFERENCES
ST, Breuning MH, Davies KE, et al. A highly polymorphic DNA marker linked to adult polycystic kidney disease on chromosome 16 Nature 1985, 317: 542-44 2. Reeders ST, Breuning MH, Corney G, et al Two genetic markers closely linked to adult polycystic kidney disease on chromosome 16. Br Med J 1986, 292: 851-53 3. Reeders ST, Keith TP, Green P, et al Linkage studies and physical mapping of the autosomal dominant polycystic kidney disease mutation Cytogen Cell Genet (in 1. Reeders
press
Breuning MH, Reeders ST, Brunner H, et al. Improved early diagnosis of adult polycy stic kidney disease with flanking DNA markers Lancet 1987; ii 1359-61. 5. Reeders ST, Breuning MH, Ryynanen MA, et al. A study of genetic linkage heterogeneity in adult polycystic kidney disease Hum Genet 1987; 76: 438-51 6 Lazarou LP, Davies F, Sarfarazi M, Coles GA, Harper PS. Adult polycystic kidney disease and linked RFLPs at the &agr; globin locus: a genetic study in the South Wales population J Med Genet 1987; 24: 466-73. 7 del Senno L, Castagnoli A, Zamorani G, et al. Use of a genetic marker for the diagnosis of adult polycystic kidney disease in Northern Italy. Prot Biolog Fluids 1987; 35:
4
63-66. JC, McManamon P, Morgan J Age at clinical onset and at ultrasonographic detection of adult polycystic kidney disease—data for genetic counselling. Am J Med Genet 1984; 18: 45-53
8. Bear
11
PATENT FORAMEN OVALE IN YOUNG STROKE PATIENTS A. M. CHANCELLOR M. W. I. WEBSTER D. L. SWIFT H. J. SMITH N. M. BASS SHARPE D. N. L. GLASGOW G.
Departments of Medicine, Neurology, and Cardiology, Auckland Hospital, Auckland, New Zealand The prevalence of patent foramen ovale in patients presenting with non-haemorrhagic stroke or transient ischaemic attacks under the age of 40 years was determined by contrast echocardiography. Studies were performed at rest and with a Valsalva manoeuvre in 40 stroke patients and in an age and sex matched control group. Right-to-left shunting was found in 20 (50%) of the stroke patients and 6 (15%) of the controls (p <0·001). Paradoxical embolism through a patent foramen ovale may be an under-recognised cause of stroke in young adults.
Summary
Introduction ONLY 3% of cerebral infarcts occur in patients under 40 years of age.1 In almost half of these patients a clear underlying cause is not found despite extensive investigation including two-dimensional echocardiography.2 It has been suggested that paradoxical embolism through a patent foramen ovale may cause some of these strokes in the absence of the usual clinical features of deep-vein thrombosis and pulmonary hypertension.3-5 We determined the prevalence of patent foramen ovale as assessed by contrast echocardiography in a population of young stroke
patients. Patients and Methods
Study Population 66 patients under 40 years of age were referred to the neurology department of Auckland Hospital between January 1980 and December 1987 and were diagnosed as having non-haemorrhagic cerebral infarction or transient ischaemic attacks. Diagnosis required the focal neurological deficit to be of acute onset without other explanation after extensive inpatient investigation including computed tomographic scan and/or cerebral angiography. Patients with complicated migraine but no fixed neurological deficit after 24
(ATL Ultramark 8). After
a two-dimensional study, contrast and with a Valsalva manoeuvre was echocardiography 8 ml of performed. physiological saline and 0-5 ml of air were agitated in a Rotamixer Delux (Hook and Tucker Ltd), macroscopic air was extruded, and the saline was injected rapidly into a 20G cannula in a right antecubital vein. Two or more injections were made during normal respiration and then repeated on at least two further occasions during the strain phase of a Valsalva manoeuvre, performed against a mercury manometer to a standard pressure of 40 mm Hg. During each injection the apical fourchamber view was recorded on videotape. Contrast echocardiographs were judged abnormal if microbubbles appeared in the left atrium or left ventricle within three cardiac cycles of their appearance in the right atrium. All studies were interpreted by two non-blinded observers and the diagnosis was made by consensus. The right-to-left shunt was semiquantitatively assessed by counting the maximum number of bubbles seen in the left side of the heart during frame-by-frame analysis. Grade 1 was defined as 1-5 bubbles, grade 2 as 6-20 bubbles, and grade 3 as over 20 bubbles. The same protocol was used for the stroke and normal control
at rest
subjects. Statistical Methods
significance of the frequency of patent foramen ovale in patients was determined by the log likelihood ratio of goodness of fit for single classification frequency distributions (G-test) .6 The expected frequency used was that of the normal The stroke
control group.
Results The stroke group consisted of 17 males and 23 females with an age range of 21-44 years (mean 31-6 years). 32 patients had a single cerebral infarction, 5 had more than one infarct, and 3 had one or more transient ischaemic attacks. All patients were examined by a cardiologist and in none was there clinical, electrocardiographic, or radiological evidence of an atrial septal defect or pulmonary hypertension. A patent foramen ovale was found in 12/40 (30%) at rest and 20/40 (50%) during a Valsalva manoeuvre. In the normal control group a patent foramen ovale was present in 3/40 (7-5%) at rest and 6/40 (15%) with a Valsalva manoeuvre (fig 1). The stroke patients also showed a greater degree of
hours were excluded. Of these patients 3 had died, 7 were lost to follow up, 11 refused contrast echocardiography, and 1 patient with recurrent strokes had already undergone surgical closure of a patent foramen ovale. In another 4 patients echocardiographic views were unsatisfactory, giving a study population of 40 patients. The mean interval between ictus and follow-up was 36 months. Each patient was then prospectively paired with a normal control subject, matched for sex and age (within 2 years). 1 control subject was excluded for poor image quality and was replaced.
Fig I-Prevalence of patent foramen ovale
Method of Assessment Patient
evaluation
Valsalva
included
clinical
assessment,
electrocardiography, chest radiography, and echocardiography
9. Jarman AP, Nicholls RD, Weatherall DJ, Clegg JB, Higgs DR. Molecular characterisation of a hypervariable region downstream of the human &agr;-globin gene cluster. EMBO J1986; 5: 1857-63. 10 Lathrop GM, Lalouel JM. Easy calculations of lod scores and genetic risks on small computers. Am JHum Genet 1984; 36: 460-65.
at rest
and with
a
manoeuvre.
Stroke patients had significantly control group (* denotes p <0’001).
more
patent foramina than the normal
11. Smith CAB. Testing for heterogeneity of recombination fraction values in human genetics. Ann Hum Genet 1963; 27: 175-82. 12. Ott J. A short guide to linkage analysis. In: Davies KE, ed. Human genetic diseases. A practical approach. Oxford. IRL Press, 1987: 19-32. 13. Cox DR, Gedde-Dahl T. Report of the committee on the genetic constitution of chromosomes 13, 14, 15 and 16. Cytogen Cell Genet 1985; 40: 206-41.