Methods
Total genomic DNA was extracted from peripheral venous blood of the female patient and her
family members with the TIANamp Genomic DNA Kit (TIANGEN, China) as per the manufacturer’s
instructions. Polymerase Chain Reaction was used to screen for changes in all exons of ENG, ACVRL1
and SMAD4. The primers used for amplification were designed and synthesized by Genewiz, Inc
(Table1-3). Amplification was performed as per the PCR reaction system. PCR conditions included a
hot start step at 95℃for 5min, followed by 9 cycles of 45s at 95℃, 45s at 61℃and 30s at 72℃, and
followed by 34 cycles of 45s at 95℃, 45s at 55℃and 30s at 72℃, with a final extension at 72℃for 5min.
PCR products were purified using PCR Purification Kit (GENERAY, China) and sequenced with the
ABI 3730xl (ABI, USA).
Mutations were analyzed with Chromas 2.22 software (Technelysium Pty. Ltd., Tewantin,
Queensland, Australia). Conversation of amino acids among multiple species were analyzed with
Clustal Omega online (http://www.ebi.ac.uk/Tools/msa/clustalo/). All the reference sequences come
from NCBI gene data.
Table 1 The primers used for amplification of ENG gene
Exons
1
Primer sequences
F
TCCCTGTGTCCACTTCTCCT
R
2
F
3
F
4
F
5
F
6
F
7
F
8
F
9
F
11
F
12
F
13
F
14
F
15
F
385
CAATCCCTCAGAGGCTTCAC
ATCACCTTTGGTGCCTTCCT
R
439
CTAGGCTGCTATGGCTCTGG
CCTAGCAACCATGGCTCAAT
R
500
CAGACTGCCAGGCCACAT
CAGGAAACATTTCAGCAGCA
R
386
GTCAGTGTCCCTGAGCAATG
GGGGTGGGAACTCTTAATTCT
R
690
GAGAGACCAAGAGCGTCACC
GGCAACTCCACAGGGCCATG
R
448
CTTGCAGAGGGACGTGACTT
CTGGGTTGTGGTCAGTCCTT
R
496
GCTGAGCTGAAGGACAAATCA
AGAGCCTGAGAATCGCTTGA
R
447
GAGCACAGTCGCTTTCTCCT
CTCCCATTGTTCCCATGTGC
R
634
CTCAGGATGTGCGCCTCCTTGTG
GAGCACAGTCGCTTTCTCCT
R
480
GCTTGTGGCATGTGAACTGT
ATGGCTCCTCCACCAAATATCAGA
R
468
CATCAACCTGACTCCCACCT
GGCTGATCTGACTGCTAGGG
R
472
TCAGCTCTTCCCACCTGAGT
TGGAAGCATCCAAATCATCA
R
351
GGCGTGAGCTCAGGATACA
TCCCTAGGAGACGTTTGGAA
R
The length of amplification
694
GGCTGGCCTGAATCTGTTT
Table 2 The primers used for amplification of ACVRL gene
Exons
2
Primer sequences
F
GCAGGAGGGAGCCACGGCCA
R
3
F
4
F
5
F
6
F
7
F
397
AAGAGGTTGATGCTGCAGGT
CCAACCTCTGACTCCAGCTC
R
336
ACCGCCTGTGATTCCAGTA
TAAGGGTCTGGGGTTCTGTG
R
440
AATCTCTGGGTGACCCCTCT
AGAGGGGTCACCCAGAGATT
R
488
CCAGAGCATGAGAGGAAAGG
CCTTTCCTCTCATGCTCTGG
R
216
AGGCAGAGCTGATCTGGGCC
TGAGGGAAGGATGACTGAGG
R
The length of amplification
AGACAACTCGTGGGGTCTTG
607
8
F
GGTTTCTGGCCCTTGGATAG
R
9
F
10
F
GGCCCTTGGATAGAGGGTAG
R
452
TTTCACAGGGAAGGTGAAGG
CTCGCTCTCCTTTCCAATGCTCT
R
357
GCCCTAACCAGGACACTCAG
339
GGGCTATTGAATCACTTTAGGCTTC
Table 3 The primers used for amplification of SMAD4 gene
Exons
2
Primer sequences
F
CCAGAGCAATTTCATCTTTTCC
R
3
F
4
F
5
F
6
F
7
F
8
F
9
F
10
F
11
F
12
F
380
TCAAAAATGTCATCATCCCAGT
TTCTTGGCACTTTAGCAGAGAA
R
479
TTCCTTCCACCCAGATTTCA
TCATGTGAGAGGTATAATGAAACTGA
R
492
CCGACAATTAAGATGGAGTGC
TTCATACTACATGCTCCTGACACA
R
378
TGAGAAGTGACCCCATAATTCC
CCAGTTGTTTTGGGTGCATT
R
376
TGAAACAAAATCACAGGATGAA
GCACTTGGCAGATAGCACTG
R
488
TGGTGGTGAGGCAAATTAGG
AGGACAGCAGCAGAATGGAT
R
492
TTTAATGTTTACTTACTTGGAGTTTCC
AAAATGGAATAATTTTGAGCTTCC
R
345
CTGCCGCTCACACAAACTAA
TGACTTTTGCTGGTAAAGTAGTATGC
R
497
CAATGTTCTAAAGGGGCAAAA
TTTTGCCCCTTTAGAACATTG
R
575
TCTGAAGAGATGGAGCACAAA
TGAGTTGGTAGGATTGTGAGGA
R
The length of amplification
CTAGGAGCAAGGCAGCAAAC
497