Blut

Blut (1990) 60:31-36

© Springer-Verlag 1990

Original article

Diagnosis of haemophilia B using the polymerase chain reaction J. Reiss, U. Neufeldt, K. Wieland, and B. Zoll Institut fiir Humangenetik, Gosslerstrasse 12d, D-3400 G6ttingen, Federal Republic of Germany Received April 6, 1989/Accepted July 20, 1989

Summary. The p o l y m e r a s e chain reaction (PCR) was used to a m p l i f y specific D N A sequences within the factor IX gene o f h a e m o p h i l i a B patients and their relatives. Three o f the amplified fragments contain p o l y m o r p h i c sites, which can be used as markers in segregation analyses. These restriction f r a g m e n t length p o l y m o r p h i s m s (RFLPs) were until recently detected by Southern blotting after digestion with the restriction enzymes Taq I, D d e I and X m n 1. All three R F L P ' s are located in introns o f the factor I X gene and together are informative in a p p r o x i m a t e l y 70°70 o f all cases. Each o f the polym o r p h i s m s was successfully used in carrier detection studies after amplification of the relevant fragments. This m e t h o d is also suitable for rapid antenatal diagnosis. Additionally we were able to amplify all eight exons of the factor IX gene including the splice junctions and a p a r t of the 5'-region. Large deletions or insertions can be detected without further analysis. Several possibilities for the rapid detection o f point m u t a t i o n s after D N A amplification have been described recently. The complete amplification o f all functional parts o f the Factor I X gene in c o m b i n a t i o n with these new techniques should enable us to detect the m a j o r i t y o f m u t a tions leading to h a e m o p h i l i a B. Key words: H a e m o p h i l i a B - Diagnosis - R F L P s - PCR - DNA Amplification

Introduction H a e m o p h i l i a B (Christmas disease) is an X-linked recessive disorder, affecting a p p r o x i m a t e l y 1 in 30,000 males, which is caused by defects in the factor IX gene. C o a g u l a t i o n studies used to identify Offprinl requests to." J. Reiss

carriers lack accuracy, and for antenatal diagnosis such tests can be used no earlier t h a n the second trimester [26]. The characterization o f restriction f r a g m e n t length p o l y m o r p h i s m s ( R F L P s ) within the factor I X gene has permitted indirect gene diagnosis o f h a e m o p h i l i a B [3,4, 11,14,27]. For routine diagnosis, segregation analysis using intragenie R F L P s has been the m e t h o d o f choice. Since the Factor I X gene covers a b o u t 33.5 kb [30], screening for all possible m u t a t i o n s resulting in h a e m o philia B is therefore hardly feasible [12]. Until recently, genotyping for these R F L P s was carried out by Southern blot analysis [25], a laborious technique using radioactive materials. A rapid alternative, which does not require use o f radioactive substances, is specific D N A a m p l i f i c a t i o n using the p o l y m e r a s e chain reaction (PCR) [22], which has already been applied to the diagnosis o f h a e m o p h i l i a A [15]. F r o m the published sequence o f the factor I X gene [30] we derived primer sequences for the amplification o f three polym o r p h i c regions within this gene as well as each o f its 8 exons. Using these primers we have d e m o n s t r a ted the application o f P C R to the clinical diagnosis o f h a e m o p h i l i a B.

Materials and methods DNA was isolated from 10-20 ml peripheral blood using repeated phenol/chloroform extraction. Chorionic villi were digested with proteinase K and extracted with phenol/chloroform, Restriction enzymes were used according to the manufacturer's recommendation (New England Biolabs, USA). Southern blot analysis was done according to standard protocols [17]. DNA amplification was essentially performed as described by Kogan et al. [15] but modified as follows. The total reaction volume was 50/A containing 500 ng genomic DNA. A paraffin layer was used to prevent evaporation and centrifugation steps therefore could be omitted. Each amplification cycle consisted of 90 s elongation at 64°C, 30 s denaturation at 90°C and 40 s annealing at room temperature. Amplification samples were processed with a programmable robot a r m ( P & P F!ektronik,

J. Reiss et al.: Diagnosis of haemophilia B

32 Niirnberg, FRG). Forty to sixty cycles were performed adding 1.25 units Taq DNA Polymerase [6] (New England Biolabs, USA) before rounds 1 and 31. One fifth of the amplified sample was analysed on 1.2% agarose or 12% polyacrylamide gels. In

case of restriction enzyme treatment, the DNA was digested directly after a dilution of 1 : 10. Oligonucleotides were synthesised automatically by the phosphoramidite method and purified by HPLC, detritylation and repeated co-evaporation with distilled water.

Results The sequences of the oligonucleotides used as primers for RFLP analysis and exon amplification are listed in Tables 1 and 2. The primers were usually 24 bases long and of an intermediate G/C-content. They all initiated the amplification of one single sequence; the product size varied between 150 bp and 702 bp. RFLP segregation analysis, an indirect diagnostic approach, was feasible within one day

compared to nearly a week necessary for Southern blot analysis. Figure 1 illustrates an example of a family study for each of the three intragenic polymorphisms. In one case, a consultand could be successfully excluded as a carrier (a). In another case, a prenatal diagnosis with a high degree of certainty could be offered (b). In the third case, the consultand could again be excluded as a carrier, although the segregation pattern could not be fully resolved (c). Further details are given in the legend to Fig. 1. The Taq I and Xmn I RFLPs are defined by the presence or absence of the indicated restriction site. To control the digestion of the amplified fragments, D N A of a woman who was homozygous for the presence of the restriction site, was simultaneously amplified and digested using the same reaction mixture (buffer, enzyme and further ingredients). The Dde I RFLP is of the insertion/deletion type, where

Table 1. Oligonucleotide sequences for RFLP studies and the corresponding PCR products Primer

Sequence

Fragment size

Enzyme

Digestion products

Location

JR 17 JR18

5'-GTC TTA GAA CCT AAT GAA AGT TTG 5'-AGG CAA GAA GGG TAA TGG GGA GGG

150 bp

Xmn l

100+50 bp

Intron 3

JR 19 JR20

5'-CTG GAG TAT GAC TGG CCA ATT ATC 5'-GTC GGA A A A AGA AAT AAT CTA GGC

200 bp

Taq I

125 + 75 bp

Intron 4

JR29 JR30

5'-GGG ACC ACT GTC GTA TAA TGT GGT 5'-CAC TCC TGA ACT CTG GAG GAT AGA

381/331 bp

--

a

Intron 1

a

No digestion necessary for allele detection

Table 2. Oligonucleotide sequences for exon amplification Primer

Sequence

Fragment size

Exon

JR41 JR42

5'-TGT GCT GCC ACA GTA AAT GTA GCC 5'-CGT GCT GGC TGT TAG ACT CTT CAA

511 bp

a

JR39 JR40

5'-GGC TCC TAT GCC CTA AAG AGA AAT 5'-GGT TGG ACT GAT CTT TCT GAG TCC

635 bp

b + c

JR3 JR4

5'-AGG ACC GGG CAT TCT AAG CAG TTT A 5'-CAG TTT CAA CTT GTT TCA GAG G G A A

234 bp

d

JR37 JR38

5'-CCC ATA CAT GAG TCA GTA GTT CCA 5'-GGA CAC AGA AAG AAT TCA GGT TGT

426 bp

e

JR35 JR36

5'-TGA CAA GGA TGG GCC TCA ATC TCA 5'-TCT TGC CAG CTG AGC TCC AGT TTT

344 bp

f

JR33 JR34

5'-GCC TAT TCC TGT 5'-TAT GAC CCT TCT

AAC CAG CAC ACA GCC TTT AGC CCA

327 bp

g

JR31 JR32

5'-GTC AGT GGT CCC AAG TAG TCA CTT 5'-AAG TGA TTA GTT AGT GAG AGG CCC

702 bp

h*

* Only the translated part of exon h is amplified

J. Reiss et al.: Diagnosis of haemophilia B

a

I

~_@

33

b

c

IT ~ 0

C

NI ]~1

nl ]]]I]~2

]~3 ]]2

M

]]I ]]2

C

]-[3 I1

[2

~I

2

M

[1

]]2

.~I

12

Fig. 1. Pedigrees of haemophilia B families (above) and the results of RFLP analysis after amplification, digestion and electrophoresis, a Segregation analysis using the Taq I RFLP. The consultand was individual IV/I. Absence of allele 2 (fragments of 125 bp and 75 bp) as observed in the affected male I11, 3 excluded her as a carrier of the familial haemophilia B. 12% polyacrylamide gel. b Segregation analysis using theXmn i RFLP. The consultand was II/1. Her mother is homozygous for allele 1 (fragment of 150 bp). The pedigree therefore is not fully informative. Detection of the grandpaternal allele 2 (fragments of 100 bp and 50 bp) in prenatal diagnosis would exclude a male fetus as being affected. The small 50 bp fragment stains very poorly with ethidium bromide and is frequently not visible. 12% polyacrylamide gel. c Analysis o[ a sporadic case with the Dde I polymorphism. The consultand was ll/1. The affected male Ill/1 carries allele 2 (fragment of 331 bp). If lhis is the grandpaternal allele, a new mutation excludes I1/1 as carrier. If the affcctcd male carries a grandmaternal allele, then segregation analysis excludes the carrier status for II/1 as well. 1.2% agarosc gel. M - molecular weight standards (KB-I.adder, gethesda Research Laboratories, USA), C = controls with alleles 2-2

o I

bc Ii

~o-m

~b+cm

5" M

d • ~d~

e I ~e~

f I

gh i'm ~f~

~gm

3' ~h~

Fig. 2. Amplification of exons and splicing sequences of the factor 1X genc with DNA from a male healthy control. Tile genomic organization is given on top, the picture shows the amplified sequence (double tests). The fragment size does not necessarily correspond to the length of the exons. The neighbouring exons b and c are amplified as one fragment. The transcribed portion of exon h is not amplified completely, but the amplification product includes all translated sequences. 1.2% agarose gel. M - - molecular weight standard (KB-Ladder, BRI.)

j. Reiss et al.: Diagnosis of haemophilia B

34 a 50 bp long D N A fragment is either present or absent [30]. The corresponding amplification products differ in size and the alleles can be recognized without further enzyme digestion. Placing the oligonucleotide primers at suitable positions, the different alleles could be easily distinguished. In contrast to the former Southern blot analysis, allele sizes can be directed - by choosing the appropriate primer target sequences - to fit the applied resolution procedure. If, in a particular family, the intragenic polymorphisms are not informative for diagnosis, the detailed analysis of the mutation in question - direct D N A analysis - is clearly desirable. The eight exons of the factor IX gene can be individually amplified for this purpose. We used D N A from a healthy male control to amplify all exons with the flanking splice junction sequences and a certain region 5' of the gene. Figure 2 shows the amplified exon regions of the control. All fragments could be

amplified in the same P C R cycle. To further simplify this analysis, we combined the primers for exons a + d, exons e + h and exons f + (b + c) to amplify the regions in one reaction. The two products of each combination can be easily distinguished by their fragment length, although it was found necessary to anneal the primers for exons e and h in an ice-water-bath. Deletions should be recognized by the absence of amplification products or, in case of very small deletions, by shorter fragments. Figure 3 shows such a deletion detected by P C R analysis and its confirmation by Southern blotting. This deletion is almost certainly the disease-causing mutation. Carrier analysis by gene dosage is difficult to perform with Southern blot analysis and, at least in standard laboratories, impossible by PCR. Prenatal diagnosis, however, can be offered to the patient's mother or other female relatives at risk in subsequent pregnancies. Direct comparison of the fetal D N A with that of the patient should either confirm or exclude the presence of the mutation in the fetal genome. Discussion

Fig. 3. a Deletion detection in a haemophilia B patient by the total absence of the PCR product representing exon g and its flanking sequences. 1.2°70agarose gel. C = control, M = molecular weight standard (KB-Ladder, BRL), P = patient, b Confirmation of the deletion described in a by Southern blotting using Taq I digested genomic DNA and a 32p-labelled cDNA probe [1]. The cDNA probe also detects the Taq I RFLP close to exon d. The two alleles are represented by two different bands in a heterozygouswoman (Cc). The patient (P) carries the large allele, which is clearly demonstrated in comparison to a hemizygous, healthy male (C), who carries the same allele (4th band from top). The three exons f, g and h are missing, represented by 2nd, 3rd and 5th band from top in the controls

The polymerase chain reaction technique has many applications in both medical investigations and molecular biology. The ability to repeatedly investigate a D N A fragment of known standard sequence is of major diagnostic value. Inherited diseases such as the haemophilias, which are heterogeneous in nature and exhibit a wide spectrum of possible disease-causing mutations, are routinely diagnosed using R F L P markers. The R F L P alleles can be determined very quickly and conveniently with the use of primer-directed D N A amplification. Much less starting material and the avoidance of radioactive probes are further advantages compared to the classical hybridization procedure. An estimate for the reliability of the P C R technique has been presented [161. The three intragenic RFLPs described above which are used in the diagnosis of haemophilia B are located within 6 kb of each other on the Xchromosome. Due to linkage disequilibrium, joint analysis of all three polymorphic sites would yield 66°70 heterozygosity [7, 27]. Other intragenic factor IX RFLPs are known, but they are unlikely to add significant information in an individual case [10, 28]. Recently, a potentially useful RFLP, 3' to the factor IX gene, has been described, which, probably due to a methylation effect can be detected using P C R but not by Southern blot analysis [29]. The recruitment of extragenic polymorphisms

J. Reiss et al.: Diagnosis of haemophilia B

[9, 19,20] to the PCR technique would require sequence data that are not yet available. Furthermore, the conclusions drawn would still be probabilistic due to the uncertainties of recombination. Since risk figures of approximately 10% are commonly found with extragenic R F L P analysis, genetic counselling of a specific family is not supported satisfactorily by this approach. In uninformative families the diagnostic method of choice must be the amplification of all functional parts of a given gene from the affected person's DNA. If a deletion is not readily recognized, the exons, splice junction sequences and 5'-regions, likely to control expression, can rapidly be examined by direct sequencing. The rapid localization of single base-pair changes should be possible either through the altered melting behaviour [2] or by chemical mismatch analysis [18]. Examples of the characterization of point mutations by amplification of DNA from haemophilia B patients have already been described [5, 8, 13, 21,24]. Unequivocal identification of the diseasecausing mutation can sometimes be difficult because several deviations from the known wild-type sequence may be observed. This is however irrelevant for diagnostic purposes, since for carrier analysis, only a comparison with the affected patient is required. The patient's sequence can be identified in other family members by individual patterns that need not necessarily result from the disease-causing mutation(s). In our experience with haemophilia A, PCR is also suitable for antenatal diagnosis using chorionic villi (data not shown). DNA preparation time included, such a diagnosis can be completed within 24 h, if RFLPs are informative. This procedure can now also be used for cases of haemophilia B. The final characterization of the actual disease-causing mutation by molecular investigations such as DNA sequencing, should not only increase our understanding of the mutational processes underlying inherited disease but also provide insight into the relationship between structure and functions at the protein level. Acknowledgements. This work was supported by the Bundesministerium far Forschung und Technologie (SVG 07065320). We thank G.G. Brownlee for making the cDNA probe available, H. P. Geithe for synthesizing the oligonucleotides for us, D. Immke for preparing the manuscript, M. Krawczak and D.N. Cooper for helpful comments.

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J. Reiss et al.: Diagnosis of haemophilia B 25. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503--517 26. Thompson AR (1986) Structure, function and molecular defects of factor IX. Blood 67: 565-572 27. Winship PR, Anson DS, Rizza CR, Brownlee GG (1984) Carrier detection in haemophilia B using two further intragenic restriction fragment length polymorphisms. Nucl Acids Res 12:8861-8872 28. Winship PR, Brownlee GG (1986) Diagnosis of haemophilia B carriers using intragenic oligonucleotide probes. Lancet lI: 218-219 29. Winship PR, Rees DJG, Alkan M (1989) Detection of polymorphisms at cytosine phosphoguanadine dinucleotides and diagnosis of haemophilia B carriers. Lancet I: 631 -634 30. Yoshitake S, Schach BG, Foster DC, Davie EW, Kurachi K (1985) Nucleotide sequence of the gene for human factor IX (antihemophilic factor B). Biochemistry 24:3736-3750