ANXA5 and VEGFA Gene Variants in Women with Early Pregnancy Losses from North Macedonia

Figure 1.

Single base extension primers data

Variant	Nucleotide change	PCR fragment size (bp)	SNaPshot primer orientation	SNaPshot result (N/M)	SNaPshot fragment size-N (bp)	SNaPshot fragment size-M (bp)
rs112782763 (c.-229G>A)	G/A	551	Forward	G/A	27.1	30.3
rs28717001 (c.-210A>C)	A/C		Reverse	T/G	21.9	28.1
rs28651243 (c.-184T>C)	T/C		Reverse	A/G	34.1	32
rs113588187 (c.-135G>A)	G/A		Reverse	C/T	36.1	37.1
rs1570360 (c.1154G>A)	G/A	450	Forward	G/A	37.5	38.5
rs3025039 (c.*237C>T)	C/T	427	Reverse	G/A	41.5	42.5

The general results of the variants in the ANXA5 gene in the group of women with EPLs and Control group

Haplotype	Women with EPLs (n=190)	%	p-value	Controls (n=190)	%
N/N	135	71.05	0.0009	161	84.73
N/M1	15	7.89	0.09	8	4.21
M1/M1	0	0.00	/	1	0.52
N/M2	36	18.95	0.0006	14	7.37
M2/M2	4	2.11	0.18	1	0.52
M1/M2	0	0.00	/	5	7.90
Total	190	100.00		190	100.00

Nucleotide sequences of PCR primers

Primer ID	Sequence (5′-3′)	Length (bp)
ANXA5-F	CAGCTACCGGGACAGCTC	18
ANXA5-R	CTCCAAAACCCCGAGCCC	18
VEGFA-F_IGU	TTCCTAGCAAAGAGGGAACG	20
VEGFA-R_IGU	GCTGACCGGTCCACCTAAC	19
VEGFA-F1_3’utr	ACACCATCACCATCGACAGA	20
VEGFA-R1_3’utr	GTCAGGATCTGAGTGGGAACA	21
rs112782763_ SShot	CCCCCCGCGGCCGGCCTGCGGTTG	24
rs28717001_ SShot	CCCCCCCTGCCCGGCTTGGCCCG	23
rs28651243_ SShot	CCCCCCCCCCCCCCCCGGAAACGCCAGCGGCCCC	34
rs113588187_ SShot	CCCCCCCGCCGAGATGCAGACGCTGAAGGATC	32
rs1570360_Sshot	CCCCCCCCCCCCCCCCGCCCGAGCCGCGTGTGGA	34
rs3025039_Sshot	CCCCCCCCCCCCCCTGGCGAATCCAATTCCAAGAGGGACC	40

The genotypic frequency of the examined VEGFA variants in the group of women with EPLs and Control groups_

c.-1154G>A	Women with EPLs (n=190)	%	p-value	Controls (n=190)	%
GG	92	48.42	0.47	99	52.11
GA	76	40.00	0.53	70	36.84
AA	22	11.58	0.87	21	11.05
Total	190	100.00		190	100.00
c.*237C>T
CC	137	72.10	0.64	141	74.21
CT	50	26.32	0.64	46	24.21
TT	3	1.58	1	3	1.58
Total	190	100.00		190	100.00