Mai S. Sater

Relationship between VEGFA polymorphisms and serum VEGF protein levels and recurrent spontaneous miscarriage

STUDY QUESTION Is recurrent spontaneous miscarriage (RSM) associated with changes in vascular endothelial growth factor (VEGF) serum levels, and with polymorphisms in the VEGFA gene? SUMMARY ANSWER Reduced serum VEGF levels, and VEGFA -460T/C (rs833061), 398G/A (rs833068), -583T/C (rs3025020) variants, were associated with RSM. WHAT IS KNOWN ALREADY Reduced expression of VEGF has been linked with spontaneous miscarriage, likely due to defective fetal and placental angiogenesis. Since VEGF production is in part inherited, VEGFA polymorphisms associated with altered VEGF secretion have been investigated for their association with RSM, often with variable conclusions. STUDY DESIGN, SIZE, DURATION A retrospective case-control study, which was conducted between January 2011 and April 15, 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS Subjects comprised 296 women with RSM (mean age: 31.6 ± 5.4 year), and 305 age-matched (mean age: 31.6 ± 4.9 year) control Arab women, who had attended outpatient obstetrics and gynecology clinics in two teaching hospitals in Bahrain. VEGFA -2578C/A (rs699947), -460T/C (rs833061), -1154G/A (rs15703060), -634G/C (rs2010963), 398G/A (rs833068), 497G/A (rs833070), -583T/C (rs3025020) and 936C/T (rs3025039) genotyping was done by real-time PCR, with defined clusters; VEGF serum levels were measured by ELISA. MAIN RESULTS AND THE ROLE OF CHANCE Higher minor allele frequency (MAF) and genotype distribution of -460T/C [corrected P (Pc) = 0.003], 398G/A (Pc = 0.016) and -583T/C (Pc < 0.001) single nucleotide polymorphisms (SNPs) were seen in RSM cases than control women. Increased RSM risk was seen with homozygous -460T/C and 398G/A SNPs and with heterozygous -583T/C, which had a stronger effect when homozygous. Serum VEGF levels were significantly reduced in RSM cases compared with control women (P = 0.016), and correlated with -460T/C, 398G/A and -583T/C genotypes. Haploview analysis revealed heterogeneity in linkage disequilibrium between VEGFA variants, and two blocks were identified: Block 1 comprising -2578C/A, -460T/C and -1154G/A, while Block 2 contained -634G/C, 398G/A, 497G/A, -583T/C and 936C/T. Both negatively and positively RSM-associated 3-locus (Block 1) and 5-locus (Block 2) VEGFA haplotypes were identified, after controlling for a number of covariates. LIMITATIONS, REASONS FOR CAUTION The study was retrospective and can only demonstrate association and not a cause-effect relationship. Furthermore, it was limited to Bahraini Arabs,thereby necessitating parallel studies on other ethnic groups. WIDER IMPLICATIONS OF THE FINDINGS Reduced VEGF secretion, and specific VEGFA variants may contribute to the pathogenesis of RSM. However, the association of VEGFA SNPs with RSM appears to be independent of their association with altered VEGF serum levels. The differential association of VEGFA variants with RSM is in line with previous findings on the contribution of ethnicity/racial background to genetic association studies.

Contribution of VEGF polymorphisms to variation in VEGF serum levels in a healthy population

OBJECTIVE Vascular endothelial growth factor (VEGF) is a pro-angiogenic factor. Variability in VEGF expression, induced by specific VEGFA variants, are involved in angiogenesis-related disorders. This study examined the genotype distribution and functional role (VEGF expression) of rs699947, rs833061, rs1570360, rs2010963, rs833068, rs833070, rs3025020, and rs3025039 VEGFA variants and their haplotypes in 519 healthy Bahraini individuals of both genders. METHODS AND RESULTS The distribution of the eight VEGFA polymorphisms screened was in Hardy-Weinberg equilibrium. The minor allele frequencies of rs699947 (0.42), rs833061 (0.32), rs1570360 (0.31), rs2010963 (0.33), rs833068 (0.37), rs833070 (0.42), rs3025020 (0.33), and rs3025039 (0.13) were generally compared to those established for Caucasians. Of the variants tested, rs3025020 was associated with increased VEGF serum levels (p=0.019), while rs3025039 was associated with decreased levels (p=0.038). Linkage analysis identified two VEGFA blocks, the first, spanning 16 kb, was not associated with altered VEGF levels, while the second, spanning 3 kb containing rs3025020 and rs3025039, was linked with higher VEGF expression, of which the (-583)T/(+936)T haplotype (p=0.008) was linked with higher VEGF levels compared to the (-583)C/(+936)C (all wild-type) haplotype. CONCLUSION These results support the association of rs30250202 and rs3025039, and specific VEGF haplotypes, with altered VEGF serum levels, although the exact functional mechanisms remain to be elucidated.

Anti-phosphatidylserine, anti-cardiolipin, anti-β2 glycoprotein I and anti-prothrombin antibodies in recurrent miscarriage at 8–12 gestational weeks

OBJECTIVE To investigate the association of antibodies to β2-glycoprotein I (anti-β2GPI), cardiolipin (ACA), phosphatidylserine (anti-PS) and prothrombin (anti-PT) with recurrent spontaneous miscarriage (RSM). STUDY DESIGN Case-control study involving 277 RSM cases and 288 controls: autoantibody levels were measured by ELISA. Differences between cases and controls were analyzed by nonparametric Mann-Whitney test, and logistic regression was used in analyzing the association of autoantibodies with RSM. RESULTS Anti-PS IgG, ACA IgM and IgG, and anti-PT IgM were significantly associated with RSM risk, and differential antibody association was noted according to BMI and primary and secondary RSM. Higher prevalence of elevated anti-PS IgG was seen in cases, with the strongest risk above the 99th percentile. For ACA IgM, 28 cases (10.1%) and 5 controls (1.7%) were positive, with increasing OR for increasing cut-off points, which was significant at antibody titers >99th percentile. For ACA IgG, 101 cases (36.5%) and 13 controls (4.5%) were positive, with graded increase in OR for increasing cut-off points, which was significant at titers >90th percentile (maximal at titers >99th percentile). For anti-PT, 23 cases (12.0%) and 9 controls (6.1%) were positive, with increased OR at titers >90th percentile. Regression analyses confirmed the independent association of anti-PS IgG, ACA IgM and IgG with RSM, and significant RSM risk was associated with high anti-PS IgG (P<0.001) and ACA IgM (P<0.001) titers, and a dose-dependent increase in RSM risk was seen with progressively increased ACA IgG titers. No significant association existed between anti-PT IgM and RSM. CONCLUSION Elevated ACA IgM and IgG, and anti-PS IgG antibodies are positively associated with RSM.

Replication study of common variants in CDKAL1 and CDKN2A/2B genes associated with type 2 diabetes in Lebanese Arab population

We investigated the association of CDKAL1 (rs7754840 and rs7756992) and CDKN2A/2B (rs10811661) variants with T2DM. Higher MAF of rs7754840 and rs7756992 were seen in patients, and both were associated with T2DM under additive, dominant, and recessive models. CDKAL1 rs7754840 and rs7756992, but not CDKN2A/2B rs10811661, are associated with T2DM in Lebanese.

The relation of vascular endothelial growth factor (VEGF) gene polymorphisms on VEGF levels and the risk of vasoocclusive crisis in sickle cell disease

The association of vascular endothelial growth factor (VEGFA) variants and VEGF secretion with sickle cell disease (SCD) vasoocclusive crisis (VOC) was investigated in 210 VOC patients and 114 pain‐free control patients.

High Frequency of Anti-Protein Z IgM and IgG Autoantibodies in Women with Idiopathic Recurrent Spontaneous Miscarriage

Citation Sater MS, Finan RR, Al‐Hammad SA, Mohammed FA, Issa AA, Almawi WY. High frequency of anti‐protein Z IgM and IgG autoantibodies in women with idiopathic recurrent spontaneous miscarriage. Am J Reprod Immunol 2011; 65: 526–531

The prevalence of absence of the palmaris longus muscle in the Bahraini population

Absence of the palmaris longus muscle has been well documented in several populations at a prevalence rate ranging between 2.2 and 63.9% which varies according to race, sex, and side of the body. There is little documentation of the prevalence of absence of this muscle from populations in the Arabian Gulf region. We examined 1,043 subjects, 3–85 years old, from the Kingdom of Bahrain for the presence or absence of the palmaris longus muscle using the conventional test for the presence of this muscle. Statistical analyses investigated the association of muscle absence with sex, hand dominance, and laterality. The palmaris longus muscle was absent in 36.8% of subjects. Bilateral absence (19%) was more common than unilateral absence (17.9%) with preponderance in female subjects. The muscle was absent more often on the left side than the right (P = 0.003). In the right upper limbs the muscle was absent in female subjects more than male subjects (P = 0.031). This study reaffirms that there is population variation in the frequency of absence of the palmaris longus muscle. The tendon of the palmaris longus bifurcated at the wrist in 7.1% of subjects, with male subjects showing this feature more frequently than female subjects in the right hand (P = 0.037) and the left hand (P = 0.030). This has not been reported before. The clinical significance of our findings is discussed. Clin. Anat. 23:956–961, 2010.

Effect of the functional VEGFA −583C/T variant on vascular endothelial growth factor levels and the risk of recurrent spontaneous miscarriage

The association of vascular endothelial growth factor (VEGF) -583C/T variant with recurrent miscarriage (RSM) was investigated in 173 RSM cases and 248 control women. Increased minor allele and genotype frequencies of -583C/T, and reduced serum VEGF concentrations were associated with increased risk of RSM.

Anti-annexin V IgM and IgG autoantibodies and the risk of idiopathic recurrent spontaneous miscarriage

Anti-annexin V antibodies have been identified as risk factors for recurrent spontaneous miscarriage (RSM) in some, but not all previous studies. We investigated the association between anti-annexin IgM and IgG in RSM cases and control women. Blood samples from 244 women with idiopathic RSM, and 283 multi-parous control women were tested for anti-annexin V antibodies by ELISA. A significant elevation in anti-annexin V IgM and IgG was seen in the RSM cases. An increased prevalence of elevated anti-annexin V IgM and to a lesser extent anti-annexin V IgG was seen in RSM patients. Receiver operating characteristic analysis indicated that the area under the curve for anti-annexin V IgM was 0.916, and for anti-annexin V IgG was 0.725. A systematic shift in anti-annexin V IgM and IgG distributions toward higher values occurred in RSM women, which was confirmed by percentile analysis. For each of the anti-annexin V isotypes, the adjusted odds ratio increased as the percentile value increased; the strongest risk was for anti-annexin V IgM, in which the 99th percentile (P99) was associated with a 165-fold higher risk than P50, and for anti-annexin V IgG where P99 was associated with a 38-fold higher risk than P50. In addition, a higher prevalence of elevated anti-annexin V IgM and anti-annexin V IgG was seen in RSM cases than in control women. We conclude that anti-annexin V IgM and IgG antibody positivity are independent risk factors for RSM.

Analysis of interleukin-18 promoter polymorphisms and changes in interleukin-18 serum levels underscores the involvement of interleukin-18 in recurrent spontaneous miscarriage

OBJECTIVE To evaluate the association of interleukin-18 (IL-18) promoter single-nucleotide polymorphisms rs1946519 (-656C/A), rs187238 (-137G/C), rs360718 (-119A/C), and rs360717 (-105G/A) and changes in IL-18 serum levels with recurrent spontaneous miscarriage (RSM). DESIGN Case-control study. SETTING Outpatient obstetrics and gynecology clinics. PATIENT(S) Women with confirmed RSM (n = 282), and 283 age- and ethnically matched controls. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) IL-18 genotyping was accomplished by allelic discrimination assays; serum IL-18 levels were measured by ELISA. RESULT(S) The minor allele frequencies of rs360717 and rs1946519, but not rs360718 or rs187238, were higher in patients with RSM. Significant differences in the distribution of the rs360717 and rs1946519 genotypes were noted between patients and controls, and both rs360717 and rs1946519 IL-18 single-nucleotide polymorphisms showed significant association with RSM under additive, dominant, and recessive models. Lower serum IL-18 levels were seen between patients and controls and were more pronounced in rs360717 and rs1946519 heterozygous and homozygous genotypes. Four-locus (rs1946519/rs187238/rs360718/rs360717) IL-18 haplotype analysis identified that the AGAA (Pc<.001), CGAA (Pc<.001), and ACAG (Pc=.018) haplotypes were associated with a reduction in IL-18 secretion and with increased RSM risk, after adjustments for body mass index, menarche, and gravida. CONCLUSION(S) These results demonstrated that reduced IL-18 levels and rs360717 and rs1946519 IL-18 variants are significantly associated with RSM.