Genomic imprinting is an important biological phenomenon defined by allelic-specific epigenetic modifications, brought about by the parent-of-origin specific gene expression. In other words, the expression of the imprinted genes depends on whether they are inherited maternally or paternally [1
]. Genomic imprinting is tissue-specific, phase-specific and probably race-specific in some cases [3
]. The IGF2 gene encodes for insulin-like growth factor II, which is structurally homologous to insulin and displays growth promoting and metabolic effects on various cell types. IGF2
is the first gene discovered to be imprinted and expressed exclusively from the paternal allele in human. It is also the first imprinted gene found to display loss of imprinting (LOI) or aberrant imprinting in human diseases. Evidently, LOI or reactivation of the normally silent maternal allele of IGF2
is associated with an increase of IGF2
expression that may subsequently play an important role in human diseases [4
]. Research shows that embryos were less likely to survive when both the paternal and the maternal alleles of IGF2
were not expressed. In another case, if the silent maternal allele was activated while the normally expressed paternal allele remained silent, abortion seemed unavoidable as result of the trophoblast cells’ failure to grow [5
]. Research also exhibits that about 25% Beckwith-Wiedemann syndrome (BWS) were related to LOI of IGF2
] while IGF2
LOI were also associated with various tumors [1
]. This study focuses mainly on investigating the imprinting status of IGF2
in cord blood lymphocytes from Han Chinese newborns, to figure out the epigenetic features this ethnic group and evaluate the possible associations between IGF2
LOI and infant phenotypes as well as other related factors.
2. Materials and Methods
2.1 Collection of cord blood samples and clinical data
A total of 4 ml of cord blood samples were collected from 923 Han Chinese term singleton newborns at Nanjing Drum Tower Hospital, affiliated with Nanjing University Medical School, in China from August 2005 to March 2006. This study was approved by the Scientific Research Ethics Committee of Drum Tower Hospital and informed consents were obtained from all participants.
Clinical data were obtained from involved parents and newborns, which included parents family history, heights of mothers and their weight gain during pregnancy, obstetrical history, levels of hemoglobin, hematokrit, and other biochemical profiles in second and third-trimester including albumin, total protein, fasting plasma glucose, triglyceride, cholesterol, L-cholesterol, H-cholesterol, apoprotein A1, apoprotein B in serum. Complications of pregnancy and childbirth events were also noted. In addition, a physical examination was performed for all newborns and their birth weights, heights, and head, chest, and abdominal circumferences, and shoulders radical lines were recorded. Moreover, blood glucose levels in cord blood and heels 2 hours after birth, and bilirubin percutaneously were measured. The neonatal screening program of congenital hypothyrosis, using dissociation-enhanced lanthanide fluroimmunoassay (DELFIA) and phenylketonuria through the fluorescent assay of all normal neonates, were then carried out. Each newborn was followed up for 30 days after birth; after the 30-day period, ultrasound scanning was performed to check the infants’ livers and kidneys.
2.2 Nucleic acid preparation
Nucleated cells from cord blood and peripheral blood samples were separated using gradient centrifugation with Ficoll-Hypaque. DNA was extracted with the proteinase K/phenol chloroform protocol as described previously [7
], while RNA was extracted with Trizol (invitrogen), following the manufacturer’s instruction.
2.3 Screening for informative samples
The IGF2 exon 9 ApaI polymorphism site was detected using PCR. The primer sequences are as follows: forward primer: 5‘-CTTGGACTTTGAGTCAAATTGG-3’; reverse primer: 5‘-GGTCGTGCCAA TTACATTTCA-3’. DNA was denatured at 94 ºC for 3 min, amplified by 36 cycles of 94 ºC for 1 min, 55 ºC for 40 sec, and 72 ºC for 40 sec. PCR products were digested for at least 4 hours with endonuclease ApaI at 30 ºC (10 unites of enzyme for 8 μl PCR product and 1.5 μl buffer in a final volume of 15 μl); ran through a 2% agarose gel and visualized with ethidium bromide. The A (not digested by ApaI) and B alleles (digested by ApaI) are 292 bp and 229 bp, respectively. Samples of heterozygous A/B at the ApaI polymorphism were selected to synthesize cDNA.
2.4 RNA reverse transcription and IGF2 imprinting assay
In order to avoid any possible gDNA contamination, the RNA was treated with RNase-free DNase I (Invitrogen) and RNase inhibitor (Promega) for 25 min at 25 ºC, followed by heating for 15 min at 65 ºC, to inactivate the DNase I, and then the treated RNA was extracted with Trizol once. cDNA was synthesized by SuperScriptIII (invitrogen) with 1 μl
of random hexamer, 1 μl
of 10 mmol/L dNTP, 4 μl
of 5 X buffer, 2 μl
of DTT in a total volume of 15 μl
at 25 ºC for 25 min, and at 42 ºC for 50 min. Reverse transcriptase was inactivated through heating for 10 min at 70 ºC. cDNA was amplified by PCR using the primers for IGF2
, under the same conditions used for gDNA with the only exception of increasing the amount of cDNA to two μl
. A total of 12.5 μl
of RT-PCR product was over digested with ApaI
for at least 4 hours at 30 ºC, under the same conditions as above, ran on a 2% agarose gel, and visualized with ethidium bromide using Labwork 4.5 software (UVP). Loss of imprinting (LOI) was assigned when the ratio of the more-abundant to less-abundant allele was less than 3:1 [7
To ensure the absence of gDNA contamination, all RT-PCR analysis were performed in duplicate, that is, in both the presence and absence of reverse transcriptase.
2.5 Statistical analysis
The various groups were compared using the χ2 test. Differences of means of continuous parametric data were analyzed using the t test. All statistical data were analyzed by means of SPSS version 13.0. All p values are two-sided; a p value smaller than 0.05 indicated significant difference.
, a growth factor indispensable during fetal development, plays an important role in the regulation of placental nutrient supply and subsequently, the modulation of fetal growth [8
]. Many researches have shown an association between LOI of IGF2
and BWS, a congenital overgrowth, malformation, and embryonic tumor predisposition syndrome [10
]. LOI of IGF2
might be an early stage event in some tumors and can be found in over 20 sorts of malignancy [1
]. LOI of IGF2
in peripheral blood lymphocytes (PBL) occurs in about 10% of normal people [12
]. This particular population might be at high risk for developing cancers and diseases. In response to the growing speculation, suggested by researchers, that LOI of IGF2
might be a potential heritable biomarker for neoplasm predisposition [12
], we felt the need to survey IGF2
imprinting status in various kinds of ethnicity and developmental stages. In some small sample studies, IGF2
showed biallelic expression in the cord blood of 20% – 30% infants, which is higher than that of adults [4
]. However, owing to the limited number of samples investigated and a lack of clinical background on related individuals, these studies were unable to identify the traits of newborns with IGF2
LOI in their cord blood. Under strict quality control, we set up a large bank of cord blood samples for both the experimental conditions and clinical data collections. In addition, donations of blood from some parents allowed us to survey the possible hereditary nature of abnormal imprinting. By examining a relatively large number of specimens for the first time, we found that ~20% of Han Chinese newborns showed LOI of IGF2
in cord blood nucleated cells, suggesting that it may represent an epigenetic characteristic for this ethnicity, which has the largest population in the world.
LOI has been linked to many human diseases, the mechanism remains unclear. Theoretically, the total amount of mRNA encoding IGF2
increases one-fold when a silenced maternal allele is expressed. Experimentally, many quantitative research also demonstrated that, on average, the mean level of IGF2
expression increases one-fold [15
] and LOI in local tissue can induce tumor growth by promoting cell proliferation. It was reported that a high expression of IGF2,
induced by LOI, is characteristic of undifferentiated, immature and hyperplasia cells, which promotes the proliferation of not only cancer but also normal cells [16
]. Moreover, epidemiological data consistently prove the existence of a positive relationship between increased body size and colorectal malignancy as well as the IGF2 peptide level in serum, and BMI tends to increase in patients with colorectal cancer [18
Our study indicated no noticeable increase in the mean BW of newborns with LOI of IGF2
. However, it did show the incidence of significant LOI decreases in low BW groups. These results highlighted the notion that epigenetic regulation does indeed have some effects on BW modulation. It is noteworthy to mention that while there were no significant statistical differences in the abdominal perimeters between the LOI group and the normal imprinting group, it tended to increase in the LOI group. Neonates in these two groups also showed a rapid decrease in glycemia after birth. Hypoglycemia could be seen in 30% – 50% of BWS without any symptoms, and could be naturally recovered within the first 3 days, for which hyperinsulinism may be accounted [20
Environmental exposures such as tobacco smoking, alcohol and nutrient ingestion including calcium, folic acid, selenium and fat did not show any correlations to LOI of IGF2
]; we showed that many maternal environmental factors from second and third trimester mothers, including concentration of albumin and lipid in serum, pre-pregnancy BMI, and weight gain during pregnancy, also did not appear to have an impact on imprinting. The latest mouse study demonstrated that maternal nutrient deficiency during development could induce LOI, thus confirming the idea that environmental factors can give rise to the epigenetic alterations [22
]. The fact that the biallelic expression of IGF2
originates from the loss of maternal imprinting suggest that abnormal maternal factors might be the primary cause of LOI [23
This study showed that LOI of IGF2
tended to increase with mother’s age although no significant differences were observed. On the other hand, newborns with LOI also increased significantly with fathers over 35 years old, suggesting possible paternal influences on aberrant imprinting; father’s age may alter offspring imprinting through contributions of abnormal sperms including spermatozoal RNA as well as the epigenetic alterations as previously described [24
Research relating to LOI inheritance in humans is very lacking. Although we have observed a weak association between parental and newborn LOI, it is still too early to draw any conclusions. Additional investigations based on a larger specimen size are needed for further confirmation. In our studies, many newborns with LOI of IGF2 did not indicate significant phenotypes. Thus, we cannot exclude the possibility that epigenetic abnormality induced phenotypes may occur gradually throughout life. One way to account for this problem is to continue to follow these infants to observe any phenotype changes in future. In addition, the relationship between IGF2 imprinting status in lymphocytes and IGF2 peptide concentration in serum remains to be investigated.