Human Molecular Genetics, 1993, Vol. 2, No. 8 1309-1310

A mutation in the 3' untranslated regionof the factor IX gene in four families withhemophilia BErica Vielhaber, David P.Jacobson, Rhett P.Ketterling, Jing-Zhong Liu and Steve S.Sommer*Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, MN 55905, USA

Received April 29, 1993; Revised and Accepted June 4, 1993

Hemophilia B is an X-linked coagulopathy caused by defects inthe factor IX gene. We have examined the putative promoterregion, the coding sequence, and splice junctions in 320consecutive families with hemophilia B. Mutations have beenfound in all but 18 of these families (94%). In the search forthe undefined mutations in these 18 cases, the region adjacentto the polyadenylation site was sequenced. In four families(HB95, HB150, HB263, and HB318), an A to G transition wasdetected at base pair 32,528 [numbering system from (1)], 208bp 5' of the polyadenylation signal. This transition creates apotential splice donor site in the 3' untranslated region of thefactor IX gene (Figure 1).

The affected individuals have clinically severe diseaseassociated with factor IX coagulants of less than 3 %, and factorIX antigens of less than 1 % (except for HB318 for whom dataare not available) (Table 1). Haplotype analyses suggest that themutation occurred independently at least three times (Table 1).

A PASA assay (PCR amplification of specific alleles; alsoknown as allele specific amplification) (2, 3) was utilized for therapid detection of this mutation (Figure 2). The assay revealedthat the mother of HB263 (HB263M) did not have the mutationin DNA isolated from her blood. The absence of the mutationat bp 32,528 was confirmed by direct sequencing of a sampleof DNA extracted from a second tube of her blood. Furthermore,HB263M confirmed that she was the biological mother of HB263.Additional haplotype analyses reveal that HB263, HB263M, andthe father of HB263M possess a rare haplotype present in only1% of the Caucasian population (Table 1). These data indicatethat the mutation in HB263 originated in the germline of hismother.

To confirm that the transition at bp 32,528 was absent in thenormal population, the PASA assay was used to screen 723unrelated chromosomes from individuals of predominantlyNorthern European extraction (data not shown). The transitionat bp 32,528 was not detected on any of these chromosomes.

In conclusion, the aggregate data indicate that the A to Gtransition at bp 32,528 causes severe hemophilia B, possibly byactivating a cryptic splice site that bypasses the polyadenylationsignal thereby destablizing the mRNA or perhaps altering thesplicing of the previous intron. To our knowledge, this is thefirst report of a mutation in the 3' untranslated region of the factorIX gene [see compendium of 806 mutations (4)]. In our sampleof 320 patients, mutations at bp 32,528 occurred independentlyat least three times, constituting 1.3% of the 227 independent

mutations. Thus, this site may be a minor hotspot of mutation,since no other transitions or transversions at non-CpGdinucleotides have independently occurred more than two timesin our sample.

ACKNOWLEDGEMENTS

We thank Mary Johnson for excellent secretarial help. The work was supportedby RO1 HL39762.

REFERENCES

1. Yoshitake,S., Schach.B.G., Foster.D.C, Davie.E.W. and Kurachi.K. (1985)Biochemistry 24, 3736-3750.

2. Sommer.S.S., Cassady,J.D., Sobell.J.L. and Bottema,C.D.K. (1989) MayoClinic Proc. 64, 1361-1372.

3. Sommer.S.S., Groszbach,A.R. and Bottema.C.D.K. (1992) BioTechniques12, 82-87.

4. Giannelli,F., Green.P.M., High.K.A., Sommer.S., Poon,M.-C., Ludwig.M.,Schwaab.R., Reitsma,P.H., Goossens,M., Yoshioka.A. and Brownlee.G.G.(1993) Nucleic Acids Res. 21, in press.

5. Eyster.M.E., LewisJ.H., Shapiro,S.S., Gill,F., Kajani.M., Prager,D.,Djerassi.I., Rice.S., Lusch,C. and Keller,A. (1980) Am. J. Hematol. 9,277-286.

6. Parquet-Gemez,A., Mazurier,C, Amiral.J. and MartinoliJ.L. (1984) Throm.Res. 35, 703-712.

7. Sarkar,G., Paynton.C. and Sommer.S.S. (1991) Nucleic Acids Res. 19,631-636.

8. Dutton.C, Bottema.C.D.K. and Sommer.S.S. (1993) Hum. Mutation, inpress.

9. Mount,S.M. (1982) Nucleic Acids Res. 10, 459-472.10. Sarkar.G. and Sommer.S.S. (1989) Science 244, 331-334.

5 ' . . . nGCCTAGACCAGAGGACATAAGTATCATG... 196 bp...MrMACTGGTGTTt

32.528

5' SPLICE JUNCTIONCONSENSUS SEQUENCE' CAG/GT*AGT

Figure 1. Sequence of the region flanking the A to G mutation (underlined) atposition 32,528 in the 3' untranslated region of the factor IX gene. The newlycreated potential splice donor site is in large bold type and the 6 bp polyadenylationsignal is in large italicized type. The splice donor consensus sequence from (9)is shown for comparison.

* To whom correspondence should be addressed

at University of T

oronto Library on N

ovember 19, 2014

http://hmg.oxfordjournals.org/

Dow

nloaded from

1310 Human Molecular Genetics, 1993, Vol. 2, No. 8

Table 1. Patient information

Patient no.

95150263318

ClinicalSeverity1

severeseveresevereN/A5

Factor DCAntigen2

< 1 %< 1 %< 1%N/A

Ethnicity

BohemianN/A (Caucasian)Irish/GermanChinese

Haplotype

Taql

_-

_

Subset3

Mai

ThrTteAlaThr

HhalHaplotype4

Frequency

1%

'Criteria of Eyster et al. (5).2Measured with a kit from Diagnostica Stago (6).3The informative polymorphisms are shown; In addition, five other polymorphisms were examined. The polymorphisms and the alleles connnon to all patients areas follows: BamHI ( - ) , intron 1 RY(i) (AB) (7), Xmnl ( - ) , AIu4a(T) (8), 3' RY(i) (II) (7)."•The frequency of the haplotype composed of a total of eight polymorphisms analyzed in Caucasians (HB95, HB150, HB263) or Asians (HB318) (Vielhaber etal., manuscript in preparation).5Not available.

Figure 2. PASA assays for the normal and mutant alleles at bp 32,528. Lanes1 —4: PCR amplification of the normal allele with oligonucleotides I and n. Lane1 = HB95; Lane 2 HB263; Lane 3 = noncarrier mother of HB263; Lane 4= unaffected individual. Lanes 5 —8: PCR amplification of the mutant allele witholigonucleotides I and m. Lane 5 = HB95; Lane 6 = HB263; Lane 7 = noncarriermother of HB263; Lane 8 = unaffected individual. Lanes S = HaeUl digestionof </>X174 DNA; Lane N = No DNA control. Oligonucleotides for PASA Assay,the allele specific base in oligonucleotides n and III is underlined [see (10) forinformative oligonucleotide nomenclature]: I = F9-(T7-23) E8 (32125)-41D =TAA TAC GAC TCA CTA TAG GGA GAC TTT CAG AGA GTT AAG TT;H = F9-E8 (32544)-17U(TN) = GGA GAC ATG ATA CTT AT; m =F9-E8(32544)-17U (CN) = GGA GAC ATG ATA CTT AC

at University of T

oronto Library on N

ovember 19, 2014

http://hmg.oxfordjournals.org/

Dow

nloaded from