If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Sequencing of a 0.65 kb region in the intron 40 of the vWF gene demonstrated a complex variability. Five STRs named Pol K, F, P1, P2-a and P2-b and an indel polymorphisms (I) are present. We established a routine analysing method to puzzle out the Pol K/F/I/P haplotypes which does not require a sequencing procedure. To recognise the combined polymorphisms as haplotypes, we performed short and middle range PCRs in combination with Nde I and BsmA I restriction tests. Comparison of the amplicon and restriction fragment length reveals the most likely haplotypes of each person involved a kinship test. Furthermore, a SNP allele specific PCR was employed. Additional information can be achieved by typing Pol P2-a and P2-b. Establishing of intron 40 vWF haplotypes using the methods described here can greatly support the resolution of complex kinship cases. This statement is illustrated by demonstration of a family study.
Family studies and prenatal diagnosis in severe von Willebrand disease by polymerase chain reaction amplification of a variable number tandem repeat region of the von Willebrand factor gene.
]. One of this STRs is the well known CODIS marker. Recently, we described the complex variability examined by sequencing analysis of a 0.65 kb region of the intron 40. We demonstrated five STRs which we named Pol K, F, P1, P2-a and P2-b (Fig. 1). Furthermore, two indel polymorphisms and five SNPs were found and could be arranged into a system of three haplotypes (a–c). SNP haplotype c is associated with Pol K allele 14 and shows an atypical repeat structure [
PCR (25 μl): 0.1–1 ng DNA, 200 μM each dNTP, 1.5 mM MgCl2, 0.5 μM of each primer, 1 U Taq polymerase (Applied Biosystems); 95 °C, 3 min soak; 94 °C, 30 s; 50/58 °C, 1 min (PTC-200 cycler; MJ Research, USA).
The two-side-labelled long amplicons (A) involving all polymorphisms from K to P were restricted for at least 4 h at with BsmA I to separate the Pol K alleles from the summarized Pol F, I and P alleles, and with Nde I (NEB, Ipswich, USA) to separate the summarized Pol K, F and I alleles from the Pol P alleles, respectively. The resulting PCR products were resolved and detected by capillary electrophoresis in the denaturing polymers POP4 (PerkinElmer) in the ABI 310 sequencer (PerkinElmer) following standard protocols. Amplicon sizing was supported using the Internal Lane Standard 600 (Promega). Calibration of the allelic ladder was done using sequenced samples [
]. Haplotype reconstruction was done by comparing the long amplicons (summarized Pol K, F, I and P alleles) with the restricted fragments and the short amplicons.
3. Results and discussion
Table 2 demonstrates the procedure to puzzle out the vWF Pol K/F/I/P haplotype. The haplotypes of each person can be determined by comparing the length of all amplicons and all restriction fragments. In addition, SNP-allele-specific PCR resulted in amplicons type C hosting Pol F, I and P. This corresponds to the HEX labelled BsmA I restriction fragments produced by cleaving of amplicon A. Table 3 shows additional polymorphic information for this family with P subtyping. Typical haplotypes were known from the sequencing study carried out before [
Estimation of vWF haplotypes including the Pol K, F, Indel, P and SNP type makes available a high number of low frequency intron 40 haplotypes, which provide an enormous potential for kinship analysis. If the vWF Pol K alleles are known from routine casework or multiplexes, three further PCR reactions and one to two restriction analyses have to be done. Pol P subtyping can increase the information, but correct haplotype reconstruction is not possible in all cases.
4. Conclusions
We established a routine analysing method to puzzle out the Pol K/F/I/P haplotypes, which does not require a sequencing procedure. To recognise the combined polymorphisms as haplotypes we performed short and middle range PCRs in combination with Nde I and BsmA I restriction tests. Comparison of the amplicon and restriction fragment length reveals the most likely haplotypes of each person involved a kinship test. Furthermore, a SNP type specific PCR was employed. Additional information can be achieved by typing Pol P2-a and P2-b. Establishing of intron 40 vWF haplotypes using the methods described here can greatly support the resolution of complex kinship cases.
Conflict of interest
None.
References
Mancuso D.J.
Tuley E.A.
Westfield L.A.
Worrall N.K.
Shelton-Inloes B.B.
Sorace J.M.
Alevy Y.G.
Sadler J.E.
Structure of the gene for human von Willebrand factor.
Family studies and prenatal diagnosis in severe von Willebrand disease by polymerase chain reaction amplification of a variable number tandem repeat region of the von Willebrand factor gene.
00258-8&id=gr1.jpg){kind=link}