Association between single nucleotide polymorphisms and viral load in congenital cytomegalovirus infection

Human cytomegalovirus (HCMV) causes the most common mother-to-a-child intrauterine infection. Congenital cytomegalovirus (cCMV) infection concerns 1% of newborns, which results in about 40,000 infected newborns in the United States each year.1 cCMV is diagnosed when HCMV is detected in newborn urine until 21st day of life by qualitative real-time polymerase chain reaction (PCR).2 Viral load (VL) in whole blood (viremia) and urine (viruria) is usually measured at diagnosis and during antiviral treatment in cCMV infection. Infants with cCMV infection have significantly higher viruria than infants with postnatal CMV (pCMV) infection, acquired through the birth canal or breast milk.2, 3 Moreover, women who transmit HCMV to neonate during prenatal period have higher HCMV DNA levels in urine, blood and amniotic fluid.4, 5 It has been suggested that higher VL could be a marker of the increased risk of significant neurological sequelae in cCMV infection.6 However, the real predictive value of VL for short- and long-term outcomes in cCMV infection is still unclear. There are limited data on factors that affect the level of viremia and viruria in cCMV-infected newborn infants. Monocytes, cytokines and pattern recognition receptors (PRRs) seem to be the first line of host immune defense against HCMV.7 Single nucleotide polymorphisms (SNPs) in genes encoding cytokines and cytokine receptors might participate in the complex interplay between the host immune system and HCMV and determine VL.8, 9, 10, 11, 12, 13, 14

This study aimed to evaluate association between SNPs in genes encoding cytokines and cytokine receptors (IL1B rs16944, IL12B rs3212227, IL28B rs12979860, CCL2 rs1024611, DC-SIGN rs735240, TLR2 rs5743708,TLR4 rs4986791, TLR9 rs352140) and viremia and viruria in newborn infants with cCMV infection.

This prospective study investigated associations between eight SNPs in genes encoding cytokines and cytokine receptors (IL1B rs16944, IL12B rs3212227, IL28B rs12979860, CCL2 rs1024611, DC-SIGN rs735240, TLR2 rs5743708, TLR4 rs4986791 and TLR9 rs352140) and VL in blood and urine in newborn infants with cCMV infection.

We found the novel associations between IL12B polymorphism and VL in neonates with cCMV infection. We observed that heterozygous T/G genotype of IL12B rs3212227 polymorphism was associated with higher viruria than T/T genotype in cCMV-infected neonates. The strength of these results was not supported by alleles analysis. Neither T nor G allele of IL12B rs3212227 promoted higher viruria separately. We assume that probably only heterozygous T/G status (both alleles altogether) uphold increased HCMV DNA concentration in urine. Interestingly, T allele of IL12B rs3212227 was associated with higher viremia in symptomaticneonates. However, neither T/T nor T/G genotype was connected with higher VL in blood as could be expected.

Popescu et al. proved that impaired CMV-specific T helper (Th) cellular immunity is highly dependent on IL-12.25 IL-12 is an inflammatory cytokine that initiates T-cell diferentiation towards Th1 and inhibits Th2 lymphocyte activity.26 So far, SNPs of IL-12 were found to be associated with other viral infections, such as hepatitis B virus (HBV), hepatitis C virus (HCV) and also with HCMV infection among adult patients.27, 28, 29 Seegers et al. link IL12 rs3212227 SNP polymorphism with increased IL-12 production.30 We only might speculate that newborn infants carrying heterozygous T/G genotype of IL12B rs3212227 produce higher amount of IL-12. Presumably, IL-12 could promote higher urinary HCMV excretion to eliminate the virus. Our study might support a hypothesis on the protective role of IL12 polymorphism in a host defense against HCMV infection. However, we could not explain contradictory data of T allele and T-linked genotypes in relation to viremia. It should be underlined that the number of newborns in each subgroup was relatively small, despite the fact, it was one of the largest population of cCMV-infected neonates that was genetically studied so far. Certainly, further investigations are warranted to confirm the association between SNP of IL12B rs3212227 and higher viruria and verify the role of T allele in HCMV DNA concentration in blood.

There is still no consensus whether viruria is a result of kidney virus filtering during viremia or HCMV origins from infected renal cells. HCMV kidney tropism was widely documented, mostly among renal allograft recipients.31 In relation to cCMV infection, the highest amount of HCMV was observed in pancreas, liver, kidney and brain of the fetus, which suggested a disseminated HCMV infection.32 On the other hand, cCMV-infected infants tend to excrete virus in the urine for even 3–5 years,33 even if HCMV is not detected in the blood. Our results may support previous findings showing that viruria might be less likely to be secondary to HCMV DNA concentration in blood than to renal cells infection.

Symptomatic infants with cCMV infection excrete higher amount of HCMV DNA in urine than asymptomatic infants.6 We also observed significantly higher viruria and viremia in symptomatic infants. It should be underlined that higher VL can be linked to an increased risk of unfavourable sequelae of cCMV infection.34, 35 Smiljkovic et al. showed that infant would have <75% probability of being symptomatic above 18,770 copies/mL, with a threshold of 100,000 copies/ mL approaching a 100% probability.36 On the other hand, according to Marsico et al., majority of newborns on antiviral therapy without viral suppression still had improved hearing.37 The predictive role of the VL and unfavourable sequelae in infants with cCMV infection is still questionable and needs further investigations.6, 38

There are limited data on the associations between SNPs and VL in cCMV infection. Most of them concerned SNPs of TLRs. Paradowska et al. documented that heterozygous variant of TLR9-1486T<C (rs187084) was associated with statistically higher HCMV DNA concentration in blood in HCMV-infected infants.18 Studzińska et al. reported higher viremia and viruria and increased risk of symptomatic HCMV infection in infants with heterozygous genotype of TLR3 rs3775291. Higher HCMV viremia was also noted in heterozygous genotype of TLR7 rs5741880.39 However, above-mentioned reports included both children with cCMV and pCMV infection. As far as cCMV infection was concerned, no associations were found between TLR2 2258 G<A SNP (rs5743708) and HCMV VL in neonates, but G/A variants in TLR2 2258 and TLR9 2848 C<T (rs352140) SNPs were correlated with higher VLs in fetal amniotic fluid and maternal urine samples.19 In our study, none of three examined TLR SNPs (TLR2 rs5743708, TLR4 rs4986791, TLR9 rs352140) were associated with higher viremia or viruria. We also found no association between CCL2 and DC-SIGN SNPs and VL in blood and urine.

We compared allele frequencies of our study population to the reference population from the Reference SNP Report.40 As we expected, our study population allele frequencies were very similar to the frequencies in the reference European population (the difference level did not exceed 5 percentage points) (Table S1 in Supplementary Materials).

Some limitations of our study should be acknowledged. We are aware that there are some confounding factors which may affect the results, but the current study was not designed to address these issues. First, the genetic background and the serological status of the mother are relevant for the risk of primary infection. Undoubtedly, the type of maternal infection (primary or non-primary) and the time of fetal infection may impact the genetic study results. Moreover, the diversity of HCMV strains may afect the results of the study. Although the current study population is larger than in our previous report,11 the sample size might be still too small for genetic associations. In addition, no correction for multiple testing was applied, and so type I error cannot be completely ruled out.

The important strength of our study is its prospective design. Furthermore, we included homogeneous population consisted of Caucasian central European newborn infants with cCMV infection, what let us eliminate potential race dependent genetic factors. We focused on congenital infection only because we thought that diferent genetic mechanisms might determine congenital and postnatal HCMV infection. What is more, five of eight selected SNPs were analyzed for the first time in the context of cCMV infection.15 To the best of our knowledge, this is the first and the largest study that analyzed the association between the eight SNPs and VL, and the first study that suggests the novel association between IL-12B polymorphism and VL in cCMV infection. However, future studies are necessary to replicate these findings in the larger study population.

To summarise, we found the novel associations between IL-12B polymorphism and VL in cCMV-infected newborns. Our results confirmed that symptomatic newborn infants had higher viremia and viruria than asymptomatic newborn infants. Indisputably, the role of SNPs in the pathogenesis of cCMV requires further investigations, which may change prognosis for newborn infants with cCMV in the future.

SNPs in genes encoding cytokines and cytokine receptors might determine the level of VL in cCMV infection.

Association between eight SNPs of IL1B, IL12B, IL28B, CCL2, DC-SIGN, TLR2 and TLR4, TLR9 and VL in cCMV infection was studied.

T/G genotype of IL12B rs3212227 was associated with higher viruria than T/T genotype in cCMV infection.

T allele of IL12B rs3212227 was associated with higher viremia in comparison to G allele in symptomatic cCMV infection. Symptomatic newborns had significantly higher viremia and viruria.

No other associations between SNPs and VL were reported in newborns with cCMV-infection.

SNPs analysis might be suitable for better identification and diagnosis of newborns (or even fetuses) with cCMV infection.