Hyaluronan–binding protein 2 (HABP2) gene variation in women with recurrent miscarriage

All study subjects were recruited from the departments of Obstetrics and Gynecology at Uppsala University Hospital, Karolinska University Hospital, and Danderyd University Hospital, Sweden. The Ethics Boards of Uppsala University and Karolinska Institutet approved the study and all participants gave informed written consent prior to participation.

Eligible cases were identified in the out-patient registers of the participating clinics (with a diagnosis of recurrent miscarriage between 1989 and 2009, depending on the starting point of the out-patient registers at the different centers). Careful review of the medical records was undertaken to assure a correct diagnosis and exclusion of cases with obvious causes for their recurrent miscarriages. Hence, inclusion criterion was three or more verified consecutive miscarriages in the first or second trimester of pregnancy (5–21 completed weeks of gestation), all women were recruited after their third miscarriage. Women with known risk factors for recurrent miscarriages such as Systemic Lupus Erythematosus (SLE), type I diabetes, severe thrombophilia, and major chromosomal aberrations (in either partner) were excluded. Women with type 2 diabetes, any rheumatoid disorder other than SLE, or any autoimmune disease (such as Crohn’s disease or Celiac disease) and coagulopathies other than severe thrombophilia at the time of the initial assessment were kept in the study. All women in this group (n = 165) had conceived naturally. The characteristics of the participating women are displayed as mean + SD in (Table 1).

Peripheral blood samples were collected in EDTA tubes and stored at − 20 °C until DNA extraction. All samples were extracted after all samples had been collected, and at the same time. Genomic DNA was extracted from whole blood in accordance to the QIAamp® DNA Blood Midi/Maxi Handbook (QIAGEN, Netherlands). Five to ten ml of blood was added to 500 μl of QIAGEN protease, the volume was brought up to 10 ml using PBS. Twelve ml of the buffer AL was added with shaking for at least 1 min to ensure complete lysis, and incubated at 70 °C for 10 min. Then 10 ml of 96% ethanol was added to the samples, and thoroughly mixed in order to ensure efficient DNA binding. The lysate solution was carefully transferred into the QIAamp Maxi column and centrifuged at 3000 rpm for 3 min. The filtrate was discarded and 5 ml of AW1 buffer was added followed by centrifugation at 4000 rpm for 2 min. Then 5 ml of AW2 buffer was added, followed by centrifugation at 4000 rpm for 20 min, to evaporate all traces of AW2 buffer from the column. Prior to elution the QIAamp Maxi column was placed in a clean tube, 600 μl of AE buffer was directly pipetted onto the column’s membrane and incubated at room temperature for 5 min and centrifuged at 4000 rpm for 4 min. This step was repeated twice to obtain a maximum yield of the DNA, and then followed by centrifugation at 4000 rpm for 10 min; aliquots of the DNA samples were prepared and stored at − 20 °C until analyzed.

Three polymorphisms in the HABP2 gene were analyzed: rs1157916 (promoter region), rs2240879 (5”UTR region) and rs7080536 (Marburg I, Gly534Glu in exon 13). Besides the Marburg I polymorphism, which has been shown to influence gene expression, our aim was to study common variation (minor allele frequency > 5%) in the regulatory area of the gene. Selection of SNPs was based on data from literature and from NCBI database. Real-time PCR was performed in accordance to TaqMan® SNP Genotyping Assays manufactures’ instruction kit (Applied Biosystems, USA), using 1 to 20 ng of purified genomic DNA as a template. All samples were analyzed at the same time. The reaction mix was prepared using 2X TaqMan Universal PCR Master Mix 12.5 μl/well, 40X Primer and TaqMan Probe SNP Genotyping Assay diluted 1:4 in TE buffer and 1.25 μl of the solution was added per well, DNase free water 10.25 μl/well, 24 μl of the reaction mix was pipetted into the wells of an optical Micro Amp® 96-well reaction plate purchased from Applied Biosystem, and 1 μl of wet DNA was added, giving a final volume of 25 μl per well. The plate was sealed with a plastic cover, and centrifuged to spin down the contents and eliminate air bubbles. The plate design was performed using Applied Biosystems Step One Software version 2.0, PCR was performed on Step One Plus™ Real-Time PCR System (Applied Biosystems, Foster City, USA). Amplification during 40 cycles with denaturation temperature of 95 °C and annealing temperature of 60 °C was performed. Primers and probes were designed for two of the HABP2 polymorphisms using the Primer Express® Software Version 3.0 for allelic discrimination assays in accordance to the Applied Biosystems instruction manual, and diluted according to the Custom TaqMan® SNP Genotyping Assays Protocol. Two non-template controls with DNase free water were used to detect possible contamination and two of the tested samples were randomly chosen and duplicated to check the experiment accuracy. The output was plotted in a graph where the outcome of allelic discrimination was evaluated.

The genotype and allele frequencies of the HABP2 polymorphisms in women with recurrent miscarriage and fertile controls are shown in (Table 2). All the studied polymorphisms were in Hardy-Weinberg equilibrium, except for the rs7080536 in patient group. No significant difference was observed between the two groups. However, rs1157916A allele showed borderline significance being more frequent among RM patients (P = 0.058). One of the RM patients was homozygous for the Marburg I minor allele (AA). This otherwise healthy patient had a history of five consecutive miscarriages and one live birth before the miscarriages.