Table 1.
Primers sequences used for pyrosequencing analysis.
Table 2.
Primer sequences used for real time PCR analysis.
Table 3.
Clinical characteristics, maternal and perinatal outcomes among cases (HbSS and HbSC) and controls.
Fig 1.
Gross evaluation of one selected placenta from each of the three groups of patients considered: HbSS, HbSC and HbAA (without SCD).
Fetal (A) and maternal (B) sides of the placenta from a HbSS patient with a term cesarean delivery (37 week gestation) with multiple hospital admissions for worsening anemia and previous exchange transfusion at 28 weeks. Fetal (C) and maternal (D) sides of the placenta from a HbSC patient, also delivered by cesarean at term, due to maternal request (37 weeks) with programmed blood transfusions during third trimester and no severe complications. Fetal (E) and maternal (F) sides of the placenta from a patient without SCD, delivered at 39 weeks, by Cesarean section due to 2 previous cesareans. In the HbSS and HbSC placentas it is possible to observe increased subchorionic fibrin deposition and calcifications (non-specific alterations). For methylation analysis, villous tissue was sampled.
Fig 2.
Heatmap generated from the 396 DMPs obtained in the HbSS group compared to the control group.
The rows represent each DMP and the columns each patient in the HbSS group and controls (CTL). The colors represent the methylation levels; the more red the more methylated and the more blue the less methylated. From these 396 DMPs, a total of 68 DMPs were in common with those obtained in the HbSC group.
Fig 3.
Heatmap generated from the 581 DMPs obtained in the HbSC group compared to the control group.
The rows represent each DMP and the columns each patient in the HbSC group and controls (CTL). The colors represent the methylation levels; the more red the more methylated and the more blue the less methylated. From these 581 DMPs, a total of 68 DMPs were in common with those obtained in the HbSS group.
Fig 4.
(A) The distribution of hyper DMPs and hypo DMPs according to their distance from the promoter. TSS1500, 200 to 1500 base pairs upstream of the transcription start site (TSS); TSS200, 200 base pairs upstream of the TSS; 5′UTR, 5′ untranslated region; 1st Exon; 3′UTR, 3′ untranslated region. (B) The distribution of hyper DMPs and hypo DMPs in different genomic region types. Island, a CpG site located within a CpG island; Shore, a CpG site located < 2 kilobases from a CpG island (N_: located at North; S_: located at South); Shelf, a CpG site located > 2 kilobases from a CpG island; Open sea, a CpG site not in an island or annotated gene. Data of DMPs obtained from the comparison between HbSS vs Control groups. *: group of DMPs (hyper or hypomethylated) statistically more frequent in a specific region (p<0.05; chi-square distribution test).
Fig 5.
(A) The distribution of hyper DMPs and hypo DMPs according to their distance from the promoter. TSS1500, 200 to 1500 base pairs upstream of the transcription start site (TSS); TSS200, 200 base pairs upstream of the TSS; 5′UTR, 5′ untranslated region; 1st Exon; 3′UTR, 3′ untranslated region. (B) The distribution of hyper DMPs and hypo DMPs in different genomic region types. Island, a CpG site located within a CpG island; Shore, a CpG site located < 2 kilobases from a CpG island (N_: located at North; S_: located at South); Shelf, a CpG site located > 2 kilobases from a CpG island; Open sea, a CpG site not in an island or annotated gene. Data of DMPs obtained from the comparison between HbSC vs Control groups. *: group of DMPs (hyper or hypomethylated) statistically more frequent in a specific region (p<0.05; chi-square distribution test).
Table 4.
The GO terms for differentially methylated genes between cases (HbSS and HbSC) and controls groups.
GALR2, PTGFR, ADCY4.
Fig 6.
Methylation data from pyrosequencing analysis in the HbSS and HbSC groups compared with the control group (CON).
A: CpGs sites analyzed in the HbSS group. i cg03949391-PTGFR; ii cg3989617-GPR56; iii cg0727418-GALR2 and iv cg23179456-ADCY4. B: CpGs sites analyzed in the HbSC group. i cg24847829-SPOCK1; ii cg24676244-THSD7A and iii cg23179456-ADCY. *p<0.05, **p<0.01 (Student’s unpaired t test).
Table 5.
Correlation analyses for array and pyrosequencing methylation data.
Fig 7.
Expression levels of genes in the HbSS and HbSC groups compared with the control group (CON).
A: Genes assessed in the HbSS group. i PTGFR; ii GPR56; iii GALR2 and iv ADCY4. B: Genes evaluated in the HbSC group. i SPOCK1; ii THSD7A and iii ADCY4. *p<0.05, **p<0.01, (a) Mann-Whitney U test, (b) Student’s unpaired t test.
Fig 8.
Correlation analyses between methylation and expression data performed in genes from case and control groups.
A: Analysis in the HbSS group. i PTGFR; ii GPR56; iii GALR2 and iv ADCY4. B: Analysis in the HbSC group. i SPOCK1; ii THSD7A and iii ADCY4. (a) Spearman method. p-values <0.05 are indicated in bold.