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Technical Briefs |
1 Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli "Federico II" and CEINGE-Biotecnologie Avanzate, I-80131 Napoli, Italy
2 Facoltà di Scienze, Università del Molise, 86100 Isernia, Italy
3 Centro Emofilia e Trombosi, Ospedale S.G. Bosco, 80141 Napoli, Italy
4 Centro di Coordinamento Regionale Emocoagulopatie, Dipartimento di Medicina Clinica e Sperimentale, Università di Napoli "Federico II", Napoli, Italy
aaddress correspondence to this author at: Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli "Federico II", via S. Pansini 5, I-80131 Naples, Italy; fax 39-081-746-3650, e-mail salvator{at}unina.it
Hemophilia B (HB) is an X-linked recessive bleeding disorder caused by mutations that produce factor IX (FIX) deficiency. The incidence of HB is 
1:30 000 live male births. The FIX gene spans 34 kb and contains eight exons. The disease results from a myriad of mutations, and because of the rapid turnover of FIX mutations, there is no common mutation pattern in any ethnic group (1). Carrier and prenatal diagnosis can be made by linkage analysis (1)(2), which is rapid and inexpensive but limited by noninformative families, recombinant events, and the high incidence of germline mutations (1). Denaturing gradient gel electrophoresis (3)(4) and direct gene sequencing (5)(6)(7)(8) have been used for the direct identification of FIX mutations. Denaturing reversed-phase HPLC (D-HPLC), which has been used to scan several disease genes, is more sensitive than other scanning procedures and less expensive than direct sequencing (9)(10)(11)(12)(13). In addition, the post-PCR analysis can be automated.
We developed an original D-HPLC screening procedure for the whole FIX gene and analyzed a cohort of 18 unrelated HB patients from Southern Italy previously typed by direct sequencing. In all patients, diagnosis was confirmed by FIX assay (Table 1
). The study was approved by the institutional ethics committee.
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After obtaining informed consent, we collected blood samples by venipuncture at the time of sampling for routine molecular analyses and extracted the DNA with the "Nucleon" procedure (Amersham). Each DNA sample was amplified by PCR for all FIX exons and for the promoter region and then was analyzed by sequencing and by D-HPLC. FIX exons 2 to 7 were amplified using primers described elsewhere (5). Exon 1 and the upstream region of FIX (i.e., from the nucleotide -482), which includes the promoter, were amplified using novel primers:
Promoter forward: 5'-CAAGCTACAGGCTGGAGACA-3'
Promoter reverse: 5'-TCTCCCTCAATGGGTCTTTG-3'
Exon 1 forward: 5'-TTCAGACTCAAATCAGCCACA-3'
Exon 1 reverse: 5'-AAAAGGCAAGCATACTCAATGT-3'
Exon 8 was amplified in two fragments of 400 bp, each using the following novel primers:
Exon 8 (proximal) forward: 5'-TTGCCAATTAGGTCAGTGGTC-3'
Exon 8 (proximal) reverse: 5'-ATGTGGCTCGGTCAACAAGT-3'
Exon 8 (distal) forward: 5'-TTTGCATTGCTGACAAGGAA-3'
Exon 8 (distal) reverse: 5'-GCCCTGTTAATTTTCAATTCCA-3'
The PCR mixture included, for each amplification, the following reagents in a final volume of 50 µL: 100 ng of DNA, 1x PCR buffer (Applied Biosystems), 250 µM each deoxynucleotide triphosphate (Amersham), 600 nM each of the primers (forward and reverse), 1.5 mM MgCl2, and 1 U of Taq polymerase (Applied Biosystems). The PCR conditions were set up to amplify all of the fragments with the same program. The protocol was as follows: denaturation, 1 cycle at 94 °C for 3 min; amplification, 14 touchdown cycles at 94 °C for 20 s, 61 °C for 40 s (decreasing 0.5 °C/cycle), and 72 °C for 45 s; 25 cycles at 94 °C for 20 s, 54 °C for 40 s, and 72 °C for 45 s; and primer extension, 1 cycle at 72 °C for 7 min.
Sequence analysis was performed using the Sanger protocol (14) with an automated procedure in which the four terminator reactions were marked with fluorescent dideoxynucleotides; the fragments were analyzed with the 3100 genetic analyzer (Applied Biosystems).
We used the WAVE system 3500 (Transgenomic) for D-HPLC analysis. PCR samples from HB patients were mixed with a PCR sample from a healthy individual, denatured at 95 °C for 5 min, and left at room temperature for 45 min, a procedure that allowed heteroduplexes to form if a mutation was present in the sample from the patient. DNA aliquots (5–8 µL) were loaded on a preheated C18 reversed-phase column [DNASep; 4.5 (i.d.) x 50 mm; Transgenomic]. The oven temperature for optimal heteroduplex separation with partial DNA denaturation was deduced from the melting profile of the DNA sequence. Wavemaker 4.1.40 software (Transgenomic) was used to compute melting curves and to establish the temperature for analysis, i.e., the temperature at which 30% of the sequence was denatured. DNA was eluted from the column by a linear acetonitrile gradient in 0.1 mmol/L triethylamine acetate (TEAA) buffer, pH 7.0, (Transgenomic) at a constant flow rate of 0.9 mL/min. The gradient was formed by mixing buffer A (0.1 mmol/L TEAA) and buffer B (0.1 mmol/L TEAA containing 250 mL/L acetonitrile). The analytic gradient lasted 4 min, and buffer B was increased by 2% per min. For each fragment, the initial concentration of buffer B was adjusted to obtain a retention time of 4–5 min (see Table 1
). The column was then cleaned with 100% buffer B for 30 s and equilibrated at the starting conditions for 6 s before the next injection. Elution of DNA was detected by the absorbance at 260 nm. HSM software (Transgenomic) regulated each setting of the Wave system during analysis and stored the data.
Sequence analysis identified a mutation in all 18 DNA samples from our HB patients (Table 1
). All variants were point mutations: 4 were nonsense, and the other 14 were missense mutations. A gene variant (-323T
C) was identified 323 nucleotides upstream from the gene in a patient who also carried the A233T mutation in exon 7 (Table 1
, case 9). These findings confirm the heterogeneity of FIX mutations (1)(15). In fact, unlike other frequent genetic disorders, such as hemophilia A, in which 50% of patients carry an inversion of F8C intron 8 (1), and cystic fibrosis, in which the microdeletion DF508 is present in up to 80% of mutated alleles (16), no predominant or ethnicity-specific mutations were present in the FIX gene in patients with HB.
The D-HPLC method identified all 18 mutations. We first performed D-HPLC using the temperature indicated by the Wavemaker software; we then modified the run temperatures in 1 °C steps. For most fragments, the optimal temperature (shown in Table 1
) exceeded that indicated by the software by 1–4 °C; in fact, an increase in oven temperature caused a decrease of the retention time, thereby allowing better resolution of the heteroduplexes.
Several examples of D-HPLC profiles obtained in patients bearing FIX mutations as compared with the D-HPLC wild-type profile are shown in Fig. 1
. Fig. 1A
shows a DNA sample bearing the T38I mutation in exon 2 compared with the wild-type profile; the mutation appears as a two-peak profile. Fig. 1B
shows a DNA sample bearing the A148T polymorphism (exon 6); in this case, the DNA sample bearing the variant also appears as a two-peak profile. Fig. 1C
shows a DNA sample bearing a double variant of exon 6, i.e., the A148T polymorphism and the R180W mutation. In this case there were three peaks.
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The D-HPLC procedure is reproducible: all 18 samples from HB patients were analyzed twice by two operators who obtained the same results. In addition, our study confirms the high sensitivity of D-HPLC reported in the evaluation of CFTR mutations (9)(10) and several other genes (13). The sensitivity of D-HPLC depends on three factors: (a) Each DNA sample must be run at its optimal temperature. A large spectrum of run temperatures must be used with large DNA fragments and with DNA fragments that have several melting domains with different temperatures (9); several software packages are available to calculate the optimal D-HPLC conditions [see, e.g., the Stanford web site (http://insertion.stanford.edu/melt.html)]. (b) The formation of heteroduplexes in DNA samples bearing mutations must be correct. In the case of X-linked diseases, this requires the equimolar mixing of PCR products between a wild-type DNA sample and the sample to be tested. (c) The type of mutations searched for must be detectable by D-HPLC. D-HPLC is more sensitive in the screening of point mutations, which is the case of most FIX mutations (1). In any case, once the D-HPLC conditions have been defined, a prospective study of a novel population sample should be performed to confirm the detection rate of the D-HPLC procedure.
The D-HPLC scanning procedure described here is fast because all of the DNA fragments can be amplified under the same conditions, the post-PCR phase is automated, and each D-HPLC run requires only 6 min. The protocol for D-HPLC scanning of the entire FIX gene is completed in <5 h. Finally, the D-HPLC method is cost-effective: we calculated that it costs approximately US $25 to scan the whole FIX gene, excluding instrument and personnel costs. Direct sequencing has a 100% sensitivity, and rapid protocols have been set up to analyze all FIX gene fragments under the same PCR conditions (6) or in single multiplex PCR amplifications (5). Direct FIX gene sequencing has been shown to be efficient in various ethnic-geographic groups (5)(6)(7)(8). However, direct sequencing is more expensive than D-HPLC. On the other hand, scanning procedures have a sensitivity of 75–90% (10), and in most studies D-HPLC was more sensitive than other scanning procedures, as recently demonstrated for the factor VIII gene (11) and for several other disease genes (12)(13). Furthermore, denaturing gradient gel electrophoresis and single-strand conformation polymorphism analysis are more difficult to automate (9).
In conclusion, D-HPLC scanning of the FIX gene with the procedure described here is suitable for the routine diagnosis of HB and for carrier diagnosis if the proband is not available. Using this procedure, we have confirmed the heterogeneity of FIX gene mutations in HB patients from Southern Italy.
Acknowledgments
We gratefully acknowledge grants from MIUR (DM 623/96), CNR (T.P. Biotecnologie), Ministero della Salute and Regione Campania (L. 502/92), Miistero dlla Salute (Progetti speciali, D.L.vo 299/99, bando 2001-02) and Regione Campania (Ricerca Sanitaria Finalizzata). We are indebted to Jean Ann Gilder for editing the text.
References
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