Figure 1-(a). DNA amplification by PCR. Lane 1 and 3, using primer MTHFR-1/MTHFR-2 while lane 2 and 4, using primer MTHFR-2/MTHFR-3; Lane 1 and 2: annealing temperature were 60°C, lane 3 and 4 with annealing temperature at 62°C. b) PCR products of 13 DNA samples showed high specific of 198 bp bands. L: 100 bp ladder. Lane 1-9 are blood samples 3708-3716. Lane 10-13 are: NM, JP800, JP804, and JP 806 respectively.
Figure 2-Sequencing results. Antisense sequence of part of MTHFR gene contains codon 677. JP-800 (T/T): homozygous mutation at codon 677. JP-804 (C/T): heterozygotes of at codon 677. JP-806 (C/C): wild type sample. The blood samples sequencing results were summarized in Table 1.
Figure 3-Aligned sequences of all samples to a previously published sequence (NM_005957) for MTHFR gene from Genbank. Y: stands for C or T.
Two pairs of primers MTHFR-1/MTHFR-2 and MTHFR-2/MTHFR-3 were used to amplify of 246bp and 198 bp fragments of MTHFR gene respectively. Primers MTHFR-2/MTHFR-3 gave a clear 198 bp fragment and primers MTHFR-1/MTHFR-2 gave a diffuse band 246 (Fig. 1a). The annealing temperature of 62°C showed more DNA products than 60°C reaction. So the above parameters were used for all of the following PCR reaction. The PCR products of all 13 samples gave the same bands around 200 bp, and showed high specification. (Fig.1b)
As the 3’ end of primer MTHFR-3 is only 2 bp away from nucleotide 677, the primer MTHFR-2 was used in sequencing to obtain the sequence of MTHFR cDNA. The sequencing results (Fig. 2) clearly show the presence of mutation 677G A (antisense) of MTHFR gene in positive control sample with homozygous A/A at position 677 (NM, JP-800). The heterozygous control sample showed A/G bands at nt 677 (JP-804), while the wild type control (JP-806) as expected showed G/G at nt 677. Thus the method and primer used in this project are appropriate for identification of the nt 677 mutation of MTHFR gene. All 9 PCR products amplified from blood samples were sequenced following the above sequencing method and the nt 677 genotype obtained are in Table 1.
In order to confirm the above sequencing results were the sequence of human MTHFR gene but not other genes, the sequences were compared with previously published MTHFR cDNA in GenBank(NM_005957). Due to reverse primer MTHFR-2 was employed on sequencing, so all the sequencing results were complimented and reversed on Mac Vector before aligned with the published result (Fig.3). JP-804 and 3715 showed difference from NM_005957 from codon 794 (underlined). It can be conclude that exon 4 stops at nt 793 while intron 4 starts from nt 794 as T.
Due to 677C T mutation, antisense MTHFR DNA had 5’ GACTC 3’ sequence from codon 674 to 678 instead of 5’GCCTC 3’, it created a new Hinf I cutting site (G¯ANTC). So Hinf I digestion products separating on agarose gel showed 23-bp and 175-bp 2 bands for homozygous mutation, 23-bp, 175-bp and 198-bp 3 bands for heterozygous and one 198 bp bands for wild type sample. All the results from restriction enzyme digestion match the sequencing data on MTHFR genotyping analysis. (Data not shown).
Meanwhile, the readable sequence of wild type control (JP-806) 100% match the human MTHFR gene from nt 659 to nt 777. While the heterozygotes control (JP-804) and positive patient control (JP-800 and NM) have one point difference at codon 677. So it can be confirmed that the primers used, the PCR and sequencing methods did give the sequence of part of exon 4 in MTHFR gene. The sequences of all the nine random population sample were also compared with GenBank data and all of them are 100% match the MTHFR gene from previous published results (except those samples which had the mutation C→T at nt 677 as listed in Table.1).
Two pairs of primers (MTHFR-1/MTHFR-2, and MTHFR-3/MTHFR-2) from published papers [1,2] were used in this project to amplify the 246 bp or 198 bp fragments of MTHFR gene, which contain codon 677. The combination of primers MTHFR-3 and MTHFR-2 gave a more detail band on PCR than those containing the primers as MTHFR-1/MTHFR-2. This may be due to the fact that the MTHFR-1 has a high G/C content--67% and thus may produce non-specific bands, while MTHFR-3 has a lower G/C content (56%). For all the PCR reaction in this project I used primers MTHFR-3/MTHFR-2 to produce 198 bp fragments of MTHFR gene.
The primer MTHFR-3 is only 2-bp away from codon 677, while the primer MTHFR-2 is 143-bp from codon 677. So MTHFR-2 was used to sequence the DNA.
Comparing with the previous data from GenBank, all of the 13 DNA samples sequenced using the above methods gave a sequence with100% match the human MTHFR gene transcript except with regard to the polymorphism in codon 677. Homozygotes T/T, heterozygotes C/T and wild type (C/C) controls all showed results as expected on sequencing gel. It confirmed that the above methods could be employed as a precise method to identify the point mutation at codon 677 of MTHFR gene.
Blood sample #3710, #3713 and # 3714 have genotypes T/T, #3708, #3709, # 3711 and # 3715 have genotypes C/T, while #3712and #3716 have genotypes C/C. The rate of 677C→T among the 9 Ashkenazi Jewish alleles from this study is 55.6% and obviously higher than the reports from other populations, which is from 9-25%[20, 23, 24]. But it is close to another report on the rate of 677C→T alleles among the Ashkenazi Jewish population (300 alleles tested), which was 47.7% . The frequency of mutant homozygotes is 33.3%, which is a little bit higher than the other report on Ashkenazi Jewish population . The small sample size limits the interpretability of the frequency of this mutation.
Acknowledgement. I would like to thank the unending patience of Rocco Coli, Sabrina Volpi and Ira Daly, without whose support this project could not have been completed. I thank Dr. Berish Y. Rubin for generously supplying blood samples, and his support on this project. I also thank Drs. Richard Finnell, Kneji Nakai and Huiping Zhu generously provided me control DNA samples and Dr. Ling Qiu on helpful discussion. I finally want to acknowledge the assistance from Dr. Sylvia Anderson and Amy Kozak.
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