J. Inher. Metab. Dis. 15 (1992) 161-170 © SSIEM and KluwerAcademicPublishers. Printed in the Netherlands
DNA-based Presymptomatic Diagnosis of Wilson Disease D. GAFFNEYt, J. L. WALKERl, J. G. O'DONNELL 1, G. S. FELL 1, K. F. O'NEILL 3, R. H. R. PARK2, R. I. RUSSELL2 1Institute of Biochemistry, and 2Gastroenterology Unit, Glasgow Royal Infirmary, Castle St, Glasgow G40SF; 3Midlock Medical Centre, 7 Midlock St, Glasgow G51 1SL, Scotland
Summary: Investigation using DNA markers in a family with Wilson disease revealed that an apparently normal child of 10 years of age with non-diagnostic copper biochemistry had the disease. The procedure used linked restriction fragment length polymorphic markers. Demonstration of increased liver copper concentration from a liver biopsy confirmed the diagnosis and the child was started on chelation therapy. Two other asymptomatic siblings were shown, using the same techniques, not to have the disease. Similar analysis was carried out on another family with just one index case.
Wilson disease (McKusick 27790) is a rare autosomal recessive disorder characterized by the toxic accumulation of copper in liver, basal ganglia of the brain, kidney, cornea of the eye (resulting in Kayser-Fleischer rings) and bone and heart tissue. This is caused by a decreased excretion of biliary copper. Affected individuals are usually non-symptomatic as young children although the accumulation of copper has already begun. Patients may present with neurological, hepatic, or hematological disease, usually in adolescence or early adulthood. Because of its familial nature, positive diagnosis of one member of a previously unaffected family means that all siblings are at risk, with a 25% chance of having the disease and a 50% chance of being carriers. If low concentrations of serum copper and serum ceruloplasmin are found in siblings of the affected individual then a liver biopsy to determine the extent of copper accumulation is required. The importance of positive diagnosis is that the disease is treatable with chelating agents (Scheinberg and Sternlieb, 1984). The gene for Wilson disease has been localized on chromosome 13 (Frydman et al., 1985). Although the disease may present in different forms and exists in different ethnic groups, this linkage can always be shown. As yet the gene itself has not been identified, but probes which detect polymorphisms are available from either side of the gene. By studying the inheritance of these polymorphic sites, deductions can be made about the inheritance of mutant or normal alleles for Wilson disease itself. A study by Figus and colleagues (1989) from Italy used restriction fragment length
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polymorphisms (RFLPs) to study inheritance patterns in Sardinian families, where the prevalence of the disease is unusually high. Other polymorphic loci have also been defined from both sides of the gene locus (Bowcock et al., 1988a). This produced a refined map of DNA markers from the region 13q 14-q21. The order of the markers along chromosome 13 and the genetic distance between them in centimorgans (cM) is shown in Figure 1. We used probes from three loci in combination to study two families similar to earlier studies (Farrer et al., 1988; Yuzbasiyan-Gurkan et al., 1988; Houwen et al., 1990). The closest known marker (D13S26) is at a considerable genetic distance from the W N D locus (7 cM) so, per generation, the two loci are linked 93% of the time (Figure 1). However, using markers from both sides of the gene we were able to make predictions of risk for most individuals that were considerably more accurate than this. In one family the analysis successfully diagnosed a presymptomatic affected individual.
P A T I E N T S AND M E T H O D S The individuals are referred to by the generation number followed by an individual number as on the pedigree diagrams (Figures 2 and 3). The relevant biochemical details are presented in Table 1 for all the patients and available healthy individuals. Some of the biochemical data (asterisked) were collected as a direct consequence of the R F L P results and their interpretation. Family 1 are Asians with several consanguinous marriages. The first four known patients (II.1, II.3, III.8 and III.9) had been diagnosed after presentation with predominantly neurological symptoms. The presymptomatic diseased boy, III.11, had been noted at age 5 to have tow serum copper and low ceruloplasmin. The actual Families and individuals:
c h r o m o s o m e 13 segment
~///////////////////. ESD ]
ApaI
RB1 1
]
WND 9
RsaI XbaI KpnI BamHI
I
D13S26 7
loci
]
HphI Bsp1286 EcoRI
genetic distances (cM)
}
Enzymes for RFLP detection
Figure 1 A map of the relevant segment of chromosome 13. The known genetic loci are shown in the correct orientation. The ESD locus is the Esterase D locus, RB1 locus is the retinoblastoma susceptibility gene locus, WND is the uncharacterized locus for the Wilson disease gene (as it has not yet been cloned and identified, no RFLPs are known), and D13S26 is an anonymous segment of DNA which maps to the other side of the WND locus (Keats et al., 1989) J. Inher. Metab. Dis. 15 (1992)
bO
. .
. 720
. .
. .
7.5 Low a . . -
. .
7.0 120 . 630
Tremor 15
Tremor 15 Low 111 . . 827
WD
II1.9
WD
111.8
2% 98% 0%
-
16 274
--c -
C
III.10
99% 1% 0%
12 b 160 b 2.6 b 821 b
10
WD b Presymptomatic
III.11
. -
10 175 0.73
-
C
11.3
. -
.
15 260 0.20 -
-
C
11.4
. ~99% ~ 1%
10.5 200 0.23 -
-
-
Cb
III.1
Family 2
.
111.3
-
4 < 30 3.1 327
10
WD Presymptomatic
. . . . . -
42 75 544
15
WD Liver failure
111.2
"WD indicates disease; C indicates carrier bNot known at the start of this study CDash indicates either the measurement was inappropriate, was not made or could not be traced dLow: this was obtained from the case notes. The value was not quoted ~Laboratory reference ranges: Serum copper, 10-25 pmol/L; Serum ceruloplasmin, 150-300 mg/L; Urinary copper, < 1/xmol/24 h; Liver copper, 15-60 #g/g dry wt. Levels above 250#g/g are considered diagnostic for Wilson's disease.
S e r u m c o p p e r (/tmol/L) Ceruloplasmin (mg/L) Urinary copper (#mol/24h) Liver c o p p e r ( # g / g d r y wt) R F L P studies L i k e l i h o o d of W D Likelihood carrier Likelihood normal
Biochemistry at presentation ~
Age at p r e s e n t a t i o n
11.3
WD WD Hepatic cirrhosis T r e m o r 22 21
II.1
Family 1
Key members of both families, with available clinical and biochemical data summarized
W i l s o n disease s t a t u s a Presentation C
Table 1
tao
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Gaffney et al.
164
4. 3 I
,
ndnd 2 2 W+ 4 4
.
li==O
1
1
2
12 22 WW 44
11 22 -~ ? 44
3
1 2 22 WW 44
[,
°
11 24 W+ 42
2
3
4
5
6
7
8
9
10
11
12
11 22 W? 44
11 22 W? 44
21 22 W? 44
21 22 W? 44
21 22 W? 44
21 22 W? 44
21 22 WW 44
21 22 WW 42 ~
21 24 W+ 42
21 22 WW 44
11 24 W+ 42
Figure 2 Pedigree with chromosome 13 loci for family 1 with Wilson disease. Half-shaded individuals are carriers and fully shaded individuals have the disease. A double line connecting two individuals indicates a consanguineous union. The relevant sections of chromosome 13 are shown vertically below each family member from whom DNA was obtained. The paternal chromosome is the leftmost of the two chromosomes shown for each individual. The topmost allele is at the ESD locus with alleles 1 or 2; the next is the RB1 locus where 2 indicates A5,B2 and 4 indicates A7,B1 (see Table 1). The next allele is w for Wilson disease or + for normal and this has been deduced. Below this is the D13S26 region with allele 2 indicating B1, and allele 4 indicating B2. An arrowhead indicates a known crossover from the previous generation values were not quoted but were not considered low enough to warrant further action despite the family history. The mother (11.4) of the affected children is the first cousin of her affected husband. Since the disease is inherited in an autosomal recessive manner, from the time of the first diagnosis of disease in one of her children it was apparent that she must be a carrier. Family 2 are Caucasians from Southern Scotland. The symptomatic patient (111.2, Figure 3) had an acute and fatal hepatic presentation (O'Donnell et al., 1990).
D N A analysis: DNA was prepared from blood samples collected with potassium EDTA as anticoagulant by the method of Kunkel and colleagues (1977). For R F L P analysis, 5 #g DNA was digested with the appropriate restriction enzyme and analysed by means of a Southern blot (Sambrook et al., 1989). The probes were labelled according to the random priming method of Feinberg and Vogelstein (1984). Probes and enzymes: The Esterase D (ESD) probe, EL22, detected a polymorphic site when the DNA was digested with ApaI (Lee and Lee, 1986). Four probes were available from the RB1 locus: p68RS2.0, which detected a multiallelic variablenumber tandem repeat polymorphism when the DNA was digested with RsaI; J. Inher. Metab. Dis. 15 (1992)
DNA-based Diagnosis of Wilson Disease
165
=@
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4
1 1
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12
+ +.
32 +W
44
14
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12
12
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+ +
24
24
5
4 11 32 ~-W 24
6
7
21 52 +W
11 33 ÷ ! ~
11 32 ÷W
14
24
14
III 1
2
3
2t 22 *W
21 12 WW
44
24
Figure 3 Pedigree with chromosome 13 loci for family 2 with Wilson disease. Shading of the individuals and the chromosomal loci are as indicated for Figure 2. For the RB1 locus (second down) 1 indicates A4,B1,G1; 3 indicates A1,B1,G1; 5 indicates A7,B1,Gt and 2 is the same as for Figure 2, i.e. A5,B2,G2. At the Dt3S26 region (bottom) allele 1 indicates B1,D1; 2 indicates B1,D2; 3 indicates B2,D1 and 4 indicates B2,D2
p88R2.5, which detected an XbaI RFLP; p123M1.8, which detected a BamHI RFLP; and p95HS0.5, which detected a KpnI R F L P (Wiggs et al., 1988). The D13S26 locus was on the other side of the W D N locus and the same probe (pH2-10) detects a HphI, a Bsp1286I and an EcoRI R F L P (Bowcock et aI., 1988b) (see Figure 1). Only informative R F L P analyses have been reported (Tables 2 and 3).
Bayes' theorem
Bayes' theorem is a mathematical method of combining all the information available to determine disease risk for an individual. Each possible set of linked alleles for the individual is examined and its probability of being correct, based on the genotypes for the individuals relations, is determined (Connor and Ferguson-Smith, 1991). The information available to us was known diagnosed cases, known carriers (parents of a patient) and R F L P data from the pedigree. For family 1 this method was used to look at all possible sets of linked alleles from the mother (11.4) and to assign each a probability using the rest of the pedigree data. The risk for III.11 was then calculated by examining what he could have inherited from his mother in each case. For family 2 this rigorous approach became impracticable because the key individuals, II.3 and 11.4, are both heterozygotic for both flanking markers.
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Table 2
Genotypes of family 1"
Locus symbol: ESD Probe: EI_22 Enzyme. Apal 1.3 II.1 I1.2 I1,3 11.4 Ili.2 III.3 III.4 Ili.5 III.6 111.7 111.8 111.9 III.10 III.11 111.12
ND b A1A2 A1A1 A1A2 A1A1 A1A1 A1A1 A1A2 A1A2 A1A2 A1A2 AIA2 A1A2 A1A2 A1A2 A1A1
RB1 p68RS2.0 p88RO.6 Rsal XbaI A5A5 A5A5 A5A5 A5A5 A5A7 A5A5 A5A5 A5A5 A5A5 A5A5 A5A5 A5A5 A5A5 A5A7 A5A5 A5A7
B2B2 B2B2 B2B2 B2B2 B1B2 B2B2 B2B2 B2B2 B2B2 B2B2 B2B2 B2B2 B2B2 B1B2 B2B2 B1B2
Dt3S26 pHff lO Hphl B2B2 B2B2 B2B2 B2B2 B1B2 B2B2 B2B2 B2B2 B2B2 B2B2 B2B2 B2B2 BiB2 BIB2 B2B2 B1B2
aln this family the BamHI RFLP at the RB1 locus and the EcoR1 RFLP at the D13S26 locus were uninformative for all members and therefore are not shown in the table bND, not determined
Table 3
Genotypes of family 2
Locus symbol: ESD Probe: EL22 Enzyme: Apal 1.2 1.4 II.1 11.2 II.3 I1.4 II.5 I1,6 II.7 1II.1 Ill.3
A1A2 AIA1 A1A1 A1A2 A2A2 A1A1 A1A2 A1AI A1A1 A1A2 A1A2
J. Inher. Metab. Dis. 15 (1992)
D13S26 pH2 10
RB1 p68RS2.0 RsaI
p88RO.6 XbaI
p123M1.8 BamHI
Hphl
EcoRl
A5A5 AIA5 A4A5 A4A5 A4A5 A1A5 A5A7 A1A1 A1A5 A5A5 A4A5
B2B2 BIB2 B1B2 B1B2 B1B2 B1B2 B1B2 B1B1 BiB2 B2B2 B1B2
G2G2 G1G2 GIG2 G1G2 G1G2 G1G2 G1G2 G1G1 G1G2 G2G2 G1G2
B2B2 B1B2 B1B2 B1B2 B1B2 B1B2 B1B2 B1B2 B1B2 B2B2 B1B2
D2D2 D1D2 D2D2 D2D2 D2D2 D2D2 D1D2 D2D2 DID2 D2D2 D2D2
DNA-based Diagnosis of Wilson Disease
167
RESULTS
RFLP analysis Genomic DNA for all individuals was prepared and the R F L P analysis was carried out. The data from each locus were collated. We have assumed that within loci no crossovers have occurred. This is reasonable because the largest of these loci, the RB1 region, is about 140kb in size (McGee et aL, 1989), giving an approximate probability of crossover per generation of less than 1/700. Therefore, the set of RFLPs detected in the RB 1 region have been assumed to be inherited as a unit, or haplotype, and are described in Figures 2 and 3 by a simple number. The D13S26 region RFLPs are also described by a number. The informative alleles for the individuals are shown in Table 2 for family 1 and Table 3 for family 2. The KpnI R F L P at the RB1 locus and the Bsp1286I R F L P detected by the D13S26 locus were uninformative (all members were homozygous for the same allele) for all members of either family for whom they were used. The variable number tandem repeat R F L P associated with the RB1 locus has multiple alleles of which four were detected within these two families.
Haplotype deduction Having collated the data for each locus, most probable haplotypes had to be deduced. This was easier for family 1 because some individuals were homozygous. For example, to determine whether the mother II.4 had the arrangement of alleles shown in Figure 2 or some alternative pattern - - e.g. 1,2,w,2 on one chromosome and 1,4,+,4 on the other - - her children are examined. The father II.3, can only have passed on 1,2,w,4 or 2,2,w,4 to any of the children. We can 'subtract' his genetic contribution from each child, to see what each child has inherited from the mother. The genotypes of children III.8, III.10, III.tl, and III.12 give support to the illustrated haplotype. The data from child III.9 would support the alternative possibility, as 1,2,w,2 was inherited from his mother. However, in the illustrated arrangement this could have been inherited from his mother if one crossover is assumed and where this occurred is indicated by an arrowhead. This one crossover event is more likely than the four which would have to be assumed to have occurred for the other children if the mother's arrangement of genes were 1,2,w,2 on one chromosome and 1,4,+,4, on the other. The same sort of logic was used throughout the two pedigrees to determine the linkages. Figures 2 and 3 show the most likely arrangement of these alleles along the relevant section of chromosome 13 for each individual in the kindreds. This arrangement is also the most simple arrangement, that which assumed minimal crossovers per generation. As a double check to confirm this, the number of crossovers expected within a pedigree were predicted. RBI and D13S26 loci are 16 cM apart, so, with ten informative meioses within a family tree, 1.6 crossover events would be predicted, or in real terms one or two. Both family trees had the expected number of detectable
J. Inher. Metab. Dis. 15 (1992)
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Gaffney et al.
crossovers (one for each family, arrowed in the figures) and so the illustrations are likely to represent the true arrangement of alleles on chromosome 13.
Analysis of family 1: Presymptomatie diagnosis The most likely arrangement of alleles derived from Table 2 is presented in Figure 2. This analysis allowed us to address the question whether individuals III.10, III.11 and III.12 are likely to have the disease. At the time the family samples were collected, III.8 and III.9 had both been diagnosed (see Table 1). A priori, with a carrier mother and a diagnosed father, the risk for the individual child to have the disease is 1/2. However, it can be seen from Figure 2 that III.I1 has inherited exactly what his affected elder sister (III.8) has inherited. Application of Bayes' theorem to the pedigree data indicated percentage likelihood of disease as shown in Table 1. On the basis of this, further biochemical measurements were done on III.11 and these are also shown in Table 1. His serum copper and serum ceruloplasmin appeared within the normal range but his urine copper results might have given some cause for concern. However, the evidence of the genetic studies was enough to suggest a liver biopsy. The high liver copper confirmed what the genetics had indicated, and penicillamine therapy was initiated. This confirmation actually lowers the calculated risk for III.10 and III.12 to 1 in 100, as they have both inherited different markers from both sides of the W D N locus compared with III.11 or III.8. Interestingly, had III.9 not been diagnosed by liver biopsy, he would have been impossible to diagnose by genetic tests owing to his inheritance of a chromosome 13 segment including a crossover from his mother. The marriage between II. 1 and II.2 is also consanguinous and surprisingly uninformative for the closely linked RFLPs. Owing to the relationship, II.2 was considered at high risk of being a carrier but she has now had seven normal children, four of whom are over 17 years old; she probably (94%) is not a carrier. All her children are of course carriers, since their father has symptomatic Wilson disease.
Analysis of family 2: Assignment of carrier status The parents of the two affected children, individuals II.3 and II.4, originally described their marriage as being consanguinous, but on closer investigation, including a pedigree going back to 1674, no consanguinity between II.3 and II.4 could be established. It is possible that the relationship between 1.3 and 1.4 (who are first cousins once removed) and several earlier marriages between members of II.3 and II.2's individual families was the reason the couple considered themselves 'related'. Therefore, the Wilson disease mutant alleles may have had an independent origin in both lineages, or even further back in history a common ancestor may have existed. In either situation, it is not a surprise that the R F L P results show the linkage of the mutant allele in this family to two quite distinct genetic backgrounds. The family tree and most likely haplotypes, worked out as described for family 1, are shown in Figure 3. For individuals, the precise risk of being a carrier is more J. lnher. Metab. Dis. 15 (1992)
DNA-based Diagnosis o f Wilson Disease
169
difficult to calculate than for the members of family 1. This is because both the known carriers, the father (II.3) and the mother (II.4) are heterozygous for both flanking markers. Hence, for formal mathematical application of Bayes' theorem, 16 different pairings would have to be considered. It is also difficult to confirm any assumptions about which alleles the mutant Wilson disease gene is linked to, since in this case DNA studies were carried out on one patient only, the only surviving index case. For the allele arrangement shown in Figure 3, the brother (III.1) has inherited one chromosome in common with his affected sister, so, barring a double crossover (probability approximately 1%), he will be a carrier for the condition. For the aunts and uncles (generation II), however, risk of being a carrier cannot easily be confirmed. II.1 and II.2 have apparently inherited a different paternal haplotype compared with that which II.3 has inherited, so this may exclude them from being carriers. But there is at least an 8% chance of a crossover which would change their status to being that of a carrier. Similar arguments apply to the designation of carrier status for II.5-7.
DISCUSSION This study shows the value of DNA markers in the diagnosis of Wilson disease, especially when more than one affected individual in the family is available. The analysis may help when copper biochemistry is equivocal (Farrer et al., 1988). Since the results of genetic analysis are independent of the onset of symptoms, DNA analysis could be used as a first screen for very young children of known carriers of the Wilson disease gene.
ACKNOWLEDGEMENTS The authors thank the following people and grant-giving bodies whose help contributed to the work described. Probes for RB1 were obtained from Dr T. P. Dryja of Harvard Medical School and ESD probe was obtained from Dr W.-H. Lee of the University of California. Help with the genetic analysis was provided by Professor J. Michael Connor and Dr Douglas Wilcox of the Duncan Guthrie Institute of Medical Genetics, Glasgow. Family tree information was collected by Mrs Joan Garrigan from Midlock Medical Centre. Patient information was obtained from Dr Alistair Beattie of the Southern General Hospital, Glasgow. Data collection and secretarial help was provided by Mrs Patricia McCabe of the McGill trust. Financial assistance was obtained from the Scottish Home and Health Department mini-grant number K/MRS/41/10/1/F2 and from the Barbara McGill Trust.
REFERENCES
Bowcock, A. M., Farrer, L. A., Hebert, J. M., Agger, M., Sternlieb, I., Scheinberg, I. H., Buys, C. H. C. M., Scheffer, H., Frydman, M., Chajek-Saul, T., Bonne-Tamir, B. and CavalliSforza, L. L. Eight closelylinked loci place the Wilson disease locus within 13q14-q21. Am. J. Hum. Genet. 43 (1988a) 664-674 J. lnher. Metab. Dis. 15 (1992)
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Bowcock, A. M., Hebert, J. M. and Cavalli-Sforza, L. L. Polymorphisms revealed by random probe H2-10 [D13S26] which maps to chromosome 13q21-q22. Nucleic Acids Res. 16 (1988b) 2745 Connor, J. M. and Ferguson-Smith, M. A. Essential Medical Genetics, Blackwell Scientific Publications, Oxford, 1991, pp. 240-242 Farrer, L. A., Bonne-Tamir, B., Frydman, M., Magazanik, A., Kidd, K. K., Bowcock, A. M. and Cavalli-Sforza, L. L. Predicting genotypes at loci for autosomal recessive disorders using linked genetic markers: application to Wilson's disease. Hum. Genet. 79 (1988) 109-117 Feinberg, A. and Vogelstein, B. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Addendum. Anat. Biochem. 137 (1984) 266-267 Figus, A., Lampis, R., Devoto, M., Ristaldi, M. S., Ideo, A., DeVirgilis, S.~ Nurchi, A. M., Corrias, A., Corda, R., Lai, M. A., Tocco, A., Deplano, A., Solinas, A., Zancan, L., Lee, W. H., Cao, A., Pirastu, M. and Balestrieri, A. Carrier detection and early diagnosis of Wilson's disease by restriction fragment length polymorphism analysis. J. Med. Genet. 26 (1989) 78-82 Frydman, M., Bonne-Tamir, B., Farrer, L. A., Conneally, P. M., Magazanik, A., Ashbel, S. and Goldwitch, Z. Assignment of the gene for Wilson disease to chromosome 13: Linkage to the esterase D locus. Proc. Natl Acad. Sci. USA 82 (I985) 1819-1821 Houwen, R. H. J., Scheffer, H., te Meerman, G. J., van der Vlies, P. and Buys, C. H. C. M. Close linkage of the Wilson's disease locus to D13S12 in the chromosomal region 13q21 and not to ESD in 13q14. Hum. Genet. 85 (1990) 560-562 Keats, B., Ott, J. and Connealty, M. Report of the committee on linkage and gene order. Cytogenet. Cell. Genet. 51 (1989) 459-502 Kunkel, L. M., Smith, K. D., Boyer, S. H., Borgaonkar, D. S., Wachtel, S. S., Miller, O. J., Breg, W. R., Jones, H. W. and Rary, J. M. Analysis of human Y-chromosome-specific reiterated DNA in chromosome variants. Proc. Natt Acad. Sci. USA 74 (1977) 1245-1249 Lee, E. Y. H. P. and Lee, W. H. Molecular cloning of the human esterase D gene, a genetic marker of retinoblastoma. Proc. Natl Acad. Sci. USA 83 (1986) 6337-6341 McGee, T. L., Yandell, D. W. and Dryja, T. P. Structure and partial genomic sequence of the human retinoblastoma susceptibility gene. Gene 80 (1989) 119-128 O'Donnell, J. G., Watson, I. D., Fell, G. S., Allison, M. E. M., Russell, R. I. and Mills, P. R. Wilson's disease presenting as acute fulminant hepatic failure. Scot. Med. J. 35 (1990) 118-119 Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular C l o n i n g - A Laboratory Manual, Cold Spring Harbour Laboratory Press, 1989, pp. 9.31-9.52 Scheinberg, I. H. and Sternlieb, I. Wilson's disease. In Smith, J. R. (ed.) Major Problems in Internal Medicine, vol. XXIII, W. B. Saunders Co., Philadelphia, t984 Wiggs, J., Nordenskjold, M., YandelI, D., Rapaport, J., Grondin, V., Janson, M., Werelius, B., Petersen, R., Craft, A., Riedel, K., Liberfarb, R., Walton, D., Wilson, W. and Dryja, T. D. Prediction of the risk of hereditary retinoblastoma, using DNA polymorphisms within the retinoblastoma gene. N. Engt J. Med. 318 (1988) 151-157 Yuzbasiyan-Gurkan, V., Brewer, G. J., Boerwinkle, E. and Ventra, P. J. Linkage of the Wilson disease gene to chromosome 13 in North America pedigrees. Am. J. Hum. Genet. 42 (1988) 825-829
J. Inher. Metab. Dis. 15 (1992)
DNA-based Presymptomatic Diagnosis of Wilson Disease D. GAFFNEYt, J. L. WALKERl, J. G. O'DONNELL 1, G. S. FELL 1, K. F. O'NEILL 3, R. H. R. PARK2, R. I. RUSSELL2 1Institute of Biochemistry, and 2Gastroenterology Unit, Glasgow Royal Infirmary, Castle St, Glasgow G40SF; 3Midlock Medical Centre, 7 Midlock St, Glasgow G51 1SL, Scotland
Summary: Investigation using DNA markers in a family with Wilson disease revealed that an apparently normal child of 10 years of age with non-diagnostic copper biochemistry had the disease. The procedure used linked restriction fragment length polymorphic markers. Demonstration of increased liver copper concentration from a liver biopsy confirmed the diagnosis and the child was started on chelation therapy. Two other asymptomatic siblings were shown, using the same techniques, not to have the disease. Similar analysis was carried out on another family with just one index case.
Wilson disease (McKusick 27790) is a rare autosomal recessive disorder characterized by the toxic accumulation of copper in liver, basal ganglia of the brain, kidney, cornea of the eye (resulting in Kayser-Fleischer rings) and bone and heart tissue. This is caused by a decreased excretion of biliary copper. Affected individuals are usually non-symptomatic as young children although the accumulation of copper has already begun. Patients may present with neurological, hepatic, or hematological disease, usually in adolescence or early adulthood. Because of its familial nature, positive diagnosis of one member of a previously unaffected family means that all siblings are at risk, with a 25% chance of having the disease and a 50% chance of being carriers. If low concentrations of serum copper and serum ceruloplasmin are found in siblings of the affected individual then a liver biopsy to determine the extent of copper accumulation is required. The importance of positive diagnosis is that the disease is treatable with chelating agents (Scheinberg and Sternlieb, 1984). The gene for Wilson disease has been localized on chromosome 13 (Frydman et al., 1985). Although the disease may present in different forms and exists in different ethnic groups, this linkage can always be shown. As yet the gene itself has not been identified, but probes which detect polymorphisms are available from either side of the gene. By studying the inheritance of these polymorphic sites, deductions can be made about the inheritance of mutant or normal alleles for Wilson disease itself. A study by Figus and colleagues (1989) from Italy used restriction fragment length
MS received 16.8.91 Accepted 7.10.91 161
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Gaffney et al.
polymorphisms (RFLPs) to study inheritance patterns in Sardinian families, where the prevalence of the disease is unusually high. Other polymorphic loci have also been defined from both sides of the gene locus (Bowcock et al., 1988a). This produced a refined map of DNA markers from the region 13q 14-q21. The order of the markers along chromosome 13 and the genetic distance between them in centimorgans (cM) is shown in Figure 1. We used probes from three loci in combination to study two families similar to earlier studies (Farrer et al., 1988; Yuzbasiyan-Gurkan et al., 1988; Houwen et al., 1990). The closest known marker (D13S26) is at a considerable genetic distance from the W N D locus (7 cM) so, per generation, the two loci are linked 93% of the time (Figure 1). However, using markers from both sides of the gene we were able to make predictions of risk for most individuals that were considerably more accurate than this. In one family the analysis successfully diagnosed a presymptomatic affected individual.
P A T I E N T S AND M E T H O D S The individuals are referred to by the generation number followed by an individual number as on the pedigree diagrams (Figures 2 and 3). The relevant biochemical details are presented in Table 1 for all the patients and available healthy individuals. Some of the biochemical data (asterisked) were collected as a direct consequence of the R F L P results and their interpretation. Family 1 are Asians with several consanguinous marriages. The first four known patients (II.1, II.3, III.8 and III.9) had been diagnosed after presentation with predominantly neurological symptoms. The presymptomatic diseased boy, III.11, had been noted at age 5 to have tow serum copper and low ceruloplasmin. The actual Families and individuals:
c h r o m o s o m e 13 segment
~///////////////////. ESD ]
ApaI
RB1 1
]
WND 9
RsaI XbaI KpnI BamHI
I
D13S26 7
loci
]
HphI Bsp1286 EcoRI
genetic distances (cM)
}
Enzymes for RFLP detection
Figure 1 A map of the relevant segment of chromosome 13. The known genetic loci are shown in the correct orientation. The ESD locus is the Esterase D locus, RB1 locus is the retinoblastoma susceptibility gene locus, WND is the uncharacterized locus for the Wilson disease gene (as it has not yet been cloned and identified, no RFLPs are known), and D13S26 is an anonymous segment of DNA which maps to the other side of the WND locus (Keats et al., 1989) J. Inher. Metab. Dis. 15 (1992)
bO
. .
. 720
. .
. .
7.5 Low a . . -
. .
7.0 120 . 630
Tremor 15
Tremor 15 Low 111 . . 827
WD
II1.9
WD
111.8
2% 98% 0%
-
16 274
--c -
C
III.10
99% 1% 0%
12 b 160 b 2.6 b 821 b
10
WD b Presymptomatic
III.11
. -
10 175 0.73
-
C
11.3
. -
.
15 260 0.20 -
-
C
11.4
. ~99% ~ 1%
10.5 200 0.23 -
-
-
Cb
III.1
Family 2
.
111.3
-
4 < 30 3.1 327
10
WD Presymptomatic
. . . . . -
42 75 544
15
WD Liver failure
111.2
"WD indicates disease; C indicates carrier bNot known at the start of this study CDash indicates either the measurement was inappropriate, was not made or could not be traced dLow: this was obtained from the case notes. The value was not quoted ~Laboratory reference ranges: Serum copper, 10-25 pmol/L; Serum ceruloplasmin, 150-300 mg/L; Urinary copper, < 1/xmol/24 h; Liver copper, 15-60 #g/g dry wt. Levels above 250#g/g are considered diagnostic for Wilson's disease.
S e r u m c o p p e r (/tmol/L) Ceruloplasmin (mg/L) Urinary copper (#mol/24h) Liver c o p p e r ( # g / g d r y wt) R F L P studies L i k e l i h o o d of W D Likelihood carrier Likelihood normal
Biochemistry at presentation ~
Age at p r e s e n t a t i o n
11.3
WD WD Hepatic cirrhosis T r e m o r 22 21
II.1
Family 1
Key members of both families, with available clinical and biochemical data summarized
W i l s o n disease s t a t u s a Presentation C
Table 1
tao
e~
e¢
Gaffney et al.
164
4. 3 I
,
ndnd 2 2 W+ 4 4
.
li==O
1
1
2
12 22 WW 44
11 22 -~ ? 44
3
1 2 22 WW 44
[,
°
11 24 W+ 42
2
3
4
5
6
7
8
9
10
11
12
11 22 W? 44
11 22 W? 44
21 22 W? 44
21 22 W? 44
21 22 W? 44
21 22 W? 44
21 22 WW 44
21 22 WW 42 ~
21 24 W+ 42
21 22 WW 44
11 24 W+ 42
Figure 2 Pedigree with chromosome 13 loci for family 1 with Wilson disease. Half-shaded individuals are carriers and fully shaded individuals have the disease. A double line connecting two individuals indicates a consanguineous union. The relevant sections of chromosome 13 are shown vertically below each family member from whom DNA was obtained. The paternal chromosome is the leftmost of the two chromosomes shown for each individual. The topmost allele is at the ESD locus with alleles 1 or 2; the next is the RB1 locus where 2 indicates A5,B2 and 4 indicates A7,B1 (see Table 1). The next allele is w for Wilson disease or + for normal and this has been deduced. Below this is the D13S26 region with allele 2 indicating B1, and allele 4 indicating B2. An arrowhead indicates a known crossover from the previous generation values were not quoted but were not considered low enough to warrant further action despite the family history. The mother (11.4) of the affected children is the first cousin of her affected husband. Since the disease is inherited in an autosomal recessive manner, from the time of the first diagnosis of disease in one of her children it was apparent that she must be a carrier. Family 2 are Caucasians from Southern Scotland. The symptomatic patient (111.2, Figure 3) had an acute and fatal hepatic presentation (O'Donnell et al., 1990).
D N A analysis: DNA was prepared from blood samples collected with potassium EDTA as anticoagulant by the method of Kunkel and colleagues (1977). For R F L P analysis, 5 #g DNA was digested with the appropriate restriction enzyme and analysed by means of a Southern blot (Sambrook et al., 1989). The probes were labelled according to the random priming method of Feinberg and Vogelstein (1984). Probes and enzymes: The Esterase D (ESD) probe, EL22, detected a polymorphic site when the DNA was digested with ApaI (Lee and Lee, 1986). Four probes were available from the RB1 locus: p68RS2.0, which detected a multiallelic variablenumber tandem repeat polymorphism when the DNA was digested with RsaI; J. Inher. Metab. Dis. 15 (1992)
DNA-based Diagnosis of Wilson Disease
165
=@
© 2
4
1 1
12 22
II 1 11
12
+ +.
32 +W
44
14
E-1 2
3 22 12 W+ 24
12
12
+ ÷
+ +
24
24
5
4 11 32 ~-W 24
6
7
21 52 +W
11 33 ÷ ! ~
11 32 ÷W
14
24
14
III 1
2
3
2t 22 *W
21 12 WW
44
24
Figure 3 Pedigree with chromosome 13 loci for family 2 with Wilson disease. Shading of the individuals and the chromosomal loci are as indicated for Figure 2. For the RB1 locus (second down) 1 indicates A4,B1,G1; 3 indicates A1,B1,G1; 5 indicates A7,B1,Gt and 2 is the same as for Figure 2, i.e. A5,B2,G2. At the Dt3S26 region (bottom) allele 1 indicates B1,D1; 2 indicates B1,D2; 3 indicates B2,D1 and 4 indicates B2,D2
p88R2.5, which detected an XbaI RFLP; p123M1.8, which detected a BamHI RFLP; and p95HS0.5, which detected a KpnI R F L P (Wiggs et al., 1988). The D13S26 locus was on the other side of the W D N locus and the same probe (pH2-10) detects a HphI, a Bsp1286I and an EcoRI R F L P (Bowcock et aI., 1988b) (see Figure 1). Only informative R F L P analyses have been reported (Tables 2 and 3).
Bayes' theorem
Bayes' theorem is a mathematical method of combining all the information available to determine disease risk for an individual. Each possible set of linked alleles for the individual is examined and its probability of being correct, based on the genotypes for the individuals relations, is determined (Connor and Ferguson-Smith, 1991). The information available to us was known diagnosed cases, known carriers (parents of a patient) and R F L P data from the pedigree. For family 1 this method was used to look at all possible sets of linked alleles from the mother (11.4) and to assign each a probability using the rest of the pedigree data. The risk for III.11 was then calculated by examining what he could have inherited from his mother in each case. For family 2 this rigorous approach became impracticable because the key individuals, II.3 and 11.4, are both heterozygotic for both flanking markers.
J. Inher. Metab. Dis. 15 (1992)
Gaffney et al.
166
Table 2
Genotypes of family 1"
Locus symbol: ESD Probe: EI_22 Enzyme. Apal 1.3 II.1 I1.2 I1,3 11.4 Ili.2 III.3 III.4 Ili.5 III.6 111.7 111.8 111.9 III.10 III.11 111.12
ND b A1A2 A1A1 A1A2 A1A1 A1A1 A1A1 A1A2 A1A2 A1A2 A1A2 AIA2 A1A2 A1A2 A1A2 A1A1
RB1 p68RS2.0 p88RO.6 Rsal XbaI A5A5 A5A5 A5A5 A5A5 A5A7 A5A5 A5A5 A5A5 A5A5 A5A5 A5A5 A5A5 A5A5 A5A7 A5A5 A5A7
B2B2 B2B2 B2B2 B2B2 B1B2 B2B2 B2B2 B2B2 B2B2 B2B2 B2B2 B2B2 B2B2 B1B2 B2B2 B1B2
Dt3S26 pHff lO Hphl B2B2 B2B2 B2B2 B2B2 B1B2 B2B2 B2B2 B2B2 B2B2 B2B2 B2B2 B2B2 BiB2 BIB2 B2B2 B1B2
aln this family the BamHI RFLP at the RB1 locus and the EcoR1 RFLP at the D13S26 locus were uninformative for all members and therefore are not shown in the table bND, not determined
Table 3
Genotypes of family 2
Locus symbol: ESD Probe: EL22 Enzyme: Apal 1.2 1.4 II.1 11.2 II.3 I1.4 II.5 I1,6 II.7 1II.1 Ill.3
A1A2 AIA1 A1A1 A1A2 A2A2 A1A1 A1A2 A1AI A1A1 A1A2 A1A2
J. Inher. Metab. Dis. 15 (1992)
D13S26 pH2 10
RB1 p68RS2.0 RsaI
p88RO.6 XbaI
p123M1.8 BamHI
Hphl
EcoRl
A5A5 AIA5 A4A5 A4A5 A4A5 A1A5 A5A7 A1A1 A1A5 A5A5 A4A5
B2B2 BIB2 B1B2 B1B2 B1B2 B1B2 B1B2 B1B1 BiB2 B2B2 B1B2
G2G2 G1G2 GIG2 G1G2 G1G2 G1G2 G1G2 G1G1 G1G2 G2G2 G1G2
B2B2 B1B2 B1B2 B1B2 B1B2 B1B2 B1B2 B1B2 B1B2 B2B2 B1B2
D2D2 D1D2 D2D2 D2D2 D2D2 D2D2 D1D2 D2D2 DID2 D2D2 D2D2
DNA-based Diagnosis of Wilson Disease
167
RESULTS
RFLP analysis Genomic DNA for all individuals was prepared and the R F L P analysis was carried out. The data from each locus were collated. We have assumed that within loci no crossovers have occurred. This is reasonable because the largest of these loci, the RB1 region, is about 140kb in size (McGee et aL, 1989), giving an approximate probability of crossover per generation of less than 1/700. Therefore, the set of RFLPs detected in the RB 1 region have been assumed to be inherited as a unit, or haplotype, and are described in Figures 2 and 3 by a simple number. The D13S26 region RFLPs are also described by a number. The informative alleles for the individuals are shown in Table 2 for family 1 and Table 3 for family 2. The KpnI R F L P at the RB1 locus and the Bsp1286I R F L P detected by the D13S26 locus were uninformative (all members were homozygous for the same allele) for all members of either family for whom they were used. The variable number tandem repeat R F L P associated with the RB1 locus has multiple alleles of which four were detected within these two families.
Haplotype deduction Having collated the data for each locus, most probable haplotypes had to be deduced. This was easier for family 1 because some individuals were homozygous. For example, to determine whether the mother II.4 had the arrangement of alleles shown in Figure 2 or some alternative pattern - - e.g. 1,2,w,2 on one chromosome and 1,4,+,4 on the other - - her children are examined. The father II.3, can only have passed on 1,2,w,4 or 2,2,w,4 to any of the children. We can 'subtract' his genetic contribution from each child, to see what each child has inherited from the mother. The genotypes of children III.8, III.10, III.tl, and III.12 give support to the illustrated haplotype. The data from child III.9 would support the alternative possibility, as 1,2,w,2 was inherited from his mother. However, in the illustrated arrangement this could have been inherited from his mother if one crossover is assumed and where this occurred is indicated by an arrowhead. This one crossover event is more likely than the four which would have to be assumed to have occurred for the other children if the mother's arrangement of genes were 1,2,w,2 on one chromosome and 1,4,+,4, on the other. The same sort of logic was used throughout the two pedigrees to determine the linkages. Figures 2 and 3 show the most likely arrangement of these alleles along the relevant section of chromosome 13 for each individual in the kindreds. This arrangement is also the most simple arrangement, that which assumed minimal crossovers per generation. As a double check to confirm this, the number of crossovers expected within a pedigree were predicted. RBI and D13S26 loci are 16 cM apart, so, with ten informative meioses within a family tree, 1.6 crossover events would be predicted, or in real terms one or two. Both family trees had the expected number of detectable
J. Inher. Metab. Dis. 15 (1992)
168
Gaffney et al.
crossovers (one for each family, arrowed in the figures) and so the illustrations are likely to represent the true arrangement of alleles on chromosome 13.
Analysis of family 1: Presymptomatie diagnosis The most likely arrangement of alleles derived from Table 2 is presented in Figure 2. This analysis allowed us to address the question whether individuals III.10, III.11 and III.12 are likely to have the disease. At the time the family samples were collected, III.8 and III.9 had both been diagnosed (see Table 1). A priori, with a carrier mother and a diagnosed father, the risk for the individual child to have the disease is 1/2. However, it can be seen from Figure 2 that III.I1 has inherited exactly what his affected elder sister (III.8) has inherited. Application of Bayes' theorem to the pedigree data indicated percentage likelihood of disease as shown in Table 1. On the basis of this, further biochemical measurements were done on III.11 and these are also shown in Table 1. His serum copper and serum ceruloplasmin appeared within the normal range but his urine copper results might have given some cause for concern. However, the evidence of the genetic studies was enough to suggest a liver biopsy. The high liver copper confirmed what the genetics had indicated, and penicillamine therapy was initiated. This confirmation actually lowers the calculated risk for III.10 and III.12 to 1 in 100, as they have both inherited different markers from both sides of the W D N locus compared with III.11 or III.8. Interestingly, had III.9 not been diagnosed by liver biopsy, he would have been impossible to diagnose by genetic tests owing to his inheritance of a chromosome 13 segment including a crossover from his mother. The marriage between II. 1 and II.2 is also consanguinous and surprisingly uninformative for the closely linked RFLPs. Owing to the relationship, II.2 was considered at high risk of being a carrier but she has now had seven normal children, four of whom are over 17 years old; she probably (94%) is not a carrier. All her children are of course carriers, since their father has symptomatic Wilson disease.
Analysis of family 2: Assignment of carrier status The parents of the two affected children, individuals II.3 and II.4, originally described their marriage as being consanguinous, but on closer investigation, including a pedigree going back to 1674, no consanguinity between II.3 and II.4 could be established. It is possible that the relationship between 1.3 and 1.4 (who are first cousins once removed) and several earlier marriages between members of II.3 and II.2's individual families was the reason the couple considered themselves 'related'. Therefore, the Wilson disease mutant alleles may have had an independent origin in both lineages, or even further back in history a common ancestor may have existed. In either situation, it is not a surprise that the R F L P results show the linkage of the mutant allele in this family to two quite distinct genetic backgrounds. The family tree and most likely haplotypes, worked out as described for family 1, are shown in Figure 3. For individuals, the precise risk of being a carrier is more J. lnher. Metab. Dis. 15 (1992)
DNA-based Diagnosis o f Wilson Disease
169
difficult to calculate than for the members of family 1. This is because both the known carriers, the father (II.3) and the mother (II.4) are heterozygous for both flanking markers. Hence, for formal mathematical application of Bayes' theorem, 16 different pairings would have to be considered. It is also difficult to confirm any assumptions about which alleles the mutant Wilson disease gene is linked to, since in this case DNA studies were carried out on one patient only, the only surviving index case. For the allele arrangement shown in Figure 3, the brother (III.1) has inherited one chromosome in common with his affected sister, so, barring a double crossover (probability approximately 1%), he will be a carrier for the condition. For the aunts and uncles (generation II), however, risk of being a carrier cannot easily be confirmed. II.1 and II.2 have apparently inherited a different paternal haplotype compared with that which II.3 has inherited, so this may exclude them from being carriers. But there is at least an 8% chance of a crossover which would change their status to being that of a carrier. Similar arguments apply to the designation of carrier status for II.5-7.
DISCUSSION This study shows the value of DNA markers in the diagnosis of Wilson disease, especially when more than one affected individual in the family is available. The analysis may help when copper biochemistry is equivocal (Farrer et al., 1988). Since the results of genetic analysis are independent of the onset of symptoms, DNA analysis could be used as a first screen for very young children of known carriers of the Wilson disease gene.
ACKNOWLEDGEMENTS The authors thank the following people and grant-giving bodies whose help contributed to the work described. Probes for RB1 were obtained from Dr T. P. Dryja of Harvard Medical School and ESD probe was obtained from Dr W.-H. Lee of the University of California. Help with the genetic analysis was provided by Professor J. Michael Connor and Dr Douglas Wilcox of the Duncan Guthrie Institute of Medical Genetics, Glasgow. Family tree information was collected by Mrs Joan Garrigan from Midlock Medical Centre. Patient information was obtained from Dr Alistair Beattie of the Southern General Hospital, Glasgow. Data collection and secretarial help was provided by Mrs Patricia McCabe of the McGill trust. Financial assistance was obtained from the Scottish Home and Health Department mini-grant number K/MRS/41/10/1/F2 and from the Barbara McGill Trust.
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Bowcock, A. M., Farrer, L. A., Hebert, J. M., Agger, M., Sternlieb, I., Scheinberg, I. H., Buys, C. H. C. M., Scheffer, H., Frydman, M., Chajek-Saul, T., Bonne-Tamir, B. and CavalliSforza, L. L. Eight closelylinked loci place the Wilson disease locus within 13q14-q21. Am. J. Hum. Genet. 43 (1988a) 664-674 J. lnher. Metab. Dis. 15 (1992)
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Bowcock, A. M., Hebert, J. M. and Cavalli-Sforza, L. L. Polymorphisms revealed by random probe H2-10 [D13S26] which maps to chromosome 13q21-q22. Nucleic Acids Res. 16 (1988b) 2745 Connor, J. M. and Ferguson-Smith, M. A. Essential Medical Genetics, Blackwell Scientific Publications, Oxford, 1991, pp. 240-242 Farrer, L. A., Bonne-Tamir, B., Frydman, M., Magazanik, A., Kidd, K. K., Bowcock, A. M. and Cavalli-Sforza, L. L. Predicting genotypes at loci for autosomal recessive disorders using linked genetic markers: application to Wilson's disease. Hum. Genet. 79 (1988) 109-117 Feinberg, A. and Vogelstein, B. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Addendum. Anat. Biochem. 137 (1984) 266-267 Figus, A., Lampis, R., Devoto, M., Ristaldi, M. S., Ideo, A., DeVirgilis, S.~ Nurchi, A. M., Corrias, A., Corda, R., Lai, M. A., Tocco, A., Deplano, A., Solinas, A., Zancan, L., Lee, W. H., Cao, A., Pirastu, M. and Balestrieri, A. Carrier detection and early diagnosis of Wilson's disease by restriction fragment length polymorphism analysis. J. Med. Genet. 26 (1989) 78-82 Frydman, M., Bonne-Tamir, B., Farrer, L. A., Conneally, P. M., Magazanik, A., Ashbel, S. and Goldwitch, Z. Assignment of the gene for Wilson disease to chromosome 13: Linkage to the esterase D locus. Proc. Natl Acad. Sci. USA 82 (I985) 1819-1821 Houwen, R. H. J., Scheffer, H., te Meerman, G. J., van der Vlies, P. and Buys, C. H. C. M. Close linkage of the Wilson's disease locus to D13S12 in the chromosomal region 13q21 and not to ESD in 13q14. Hum. Genet. 85 (1990) 560-562 Keats, B., Ott, J. and Connealty, M. Report of the committee on linkage and gene order. Cytogenet. Cell. Genet. 51 (1989) 459-502 Kunkel, L. M., Smith, K. D., Boyer, S. H., Borgaonkar, D. S., Wachtel, S. S., Miller, O. J., Breg, W. R., Jones, H. W. and Rary, J. M. Analysis of human Y-chromosome-specific reiterated DNA in chromosome variants. Proc. Natt Acad. Sci. USA 74 (1977) 1245-1249 Lee, E. Y. H. P. and Lee, W. H. Molecular cloning of the human esterase D gene, a genetic marker of retinoblastoma. Proc. Natl Acad. Sci. USA 83 (1986) 6337-6341 McGee, T. L., Yandell, D. W. and Dryja, T. P. Structure and partial genomic sequence of the human retinoblastoma susceptibility gene. Gene 80 (1989) 119-128 O'Donnell, J. G., Watson, I. D., Fell, G. S., Allison, M. E. M., Russell, R. I. and Mills, P. R. Wilson's disease presenting as acute fulminant hepatic failure. Scot. Med. J. 35 (1990) 118-119 Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular C l o n i n g - A Laboratory Manual, Cold Spring Harbour Laboratory Press, 1989, pp. 9.31-9.52 Scheinberg, I. H. and Sternlieb, I. Wilson's disease. In Smith, J. R. (ed.) Major Problems in Internal Medicine, vol. XXIII, W. B. Saunders Co., Philadelphia, t984 Wiggs, J., Nordenskjold, M., YandelI, D., Rapaport, J., Grondin, V., Janson, M., Werelius, B., Petersen, R., Craft, A., Riedel, K., Liberfarb, R., Walton, D., Wilson, W. and Dryja, T. D. Prediction of the risk of hereditary retinoblastoma, using DNA polymorphisms within the retinoblastoma gene. N. Engt J. Med. 318 (1988) 151-157 Yuzbasiyan-Gurkan, V., Brewer, G. J., Boerwinkle, E. and Ventra, P. J. Linkage of the Wilson disease gene to chromosome 13 in North America pedigrees. Am. J. Hum. Genet. 42 (1988) 825-829
J. Inher. Metab. Dis. 15 (1992)
