Neonatal Sepsis: Role of Genomics in the Diagnosis and Management

INTRODUCTION

Genetic linkage and phenotypic variations in relation with susceptibility, severity and outcome are the current interest in diseases like cancer, autoimmune diseases, infectious diseases, etc. Along with other known risk factors of neonatal sepsis, genomic risk factors also play a major role in host response to infections. Genetic variations in regulatory and coding regions of inflammatory related genes may result in phenotypic changes which reflect in host immune response. Although limited studies are available on neonatal and pediatric sepsis, genetic variations are extensively studied in adult sepsis. It is well-known that the probability of genetic modification through human life is very less and hence the evidence from adult sepsis can be used for diagnosis, management and prognosticate neonates with sepsis on the basis of genetic variations. This chapter briefly discusses the genetic variations associated with sepsis and its clinical implications.

HUMAN GENOME PROJECT

The human genome project was aimed at deciphering the nucleotide sequence that makes up human DNA. The project was initiated in 1989 and accomplished successfully in 2003 providing the complete sequence of approximtately 3 billion base pairs in human genome.1 The database GenBank houses the gene sequences and is maintained by National Center for Biotechnology Information (NCBI). The sequence in the database is available to everyone at free of cost. Human Genome Project leads Genomic research to the next level, assessing the genetic variations in relation with human health which eventually started the $138 million project called HapMap project.2

HAPMAP PROJECT

The primary goal of HapMap (Haplotype Map) project was to identify and stratify genetic variations among different populations of the world. The project was started in 2002, as a collaborative project of six countries, the United States, China, the United Kingdom, Canada, Japan and Nigeria covering the African, Asian and European ancestry. The project was completed in 2009 with dataset of three phases revealing the genetic variations among the studied population.3 These data can be accessed from the public website: http://www.ncbi.nlm.nih.gov/SNP/.

With the success of HapMap project, researchers have initiated another two mega projects: Human Variome Project and 1000 Genome Project. Human Variome Project started officially in 2010 and aims at collecting information regarding the genetic variations that affect human health. This will improve human health management and development of personalized medicine.4 1000 Genome project is an extension of HapMap project with primary objective of sequencing more than a thousand individuals from different population to identify genetic variations with at least 1% frequency in the population. This project was started in 2008 and with the help high throughput technology, next generation sequencing is likely to be completed in 2012.5 The known measures of genetic variations are discussed briefly below.

SINGLE NUCLEOTIDE POLYMORPHISMS

The genes that are involved in the pathophysiology of diseases are called candidate genes. Rather approaching genetic variation by whole genome sequencing, studying genetic variations in candidate genes and their association with disease is cost-effective and may lead to the development of novel prognostic genomic markers.

Single Nucleotide Polymorphisms (SNPs) are found in millions throughout the human genome. A single change in the nucleotide sequence of a gene may bring about a drastic change in gene expression and also in phenotype. In view of genotype, SNPs can be explained as allelic variations, homozygous (wild/mutant) or heterozygous. SNPs in inflammatory related genes are being studied for more than two decades. In particular, genetic variations in TNF-α, TLR4, IL-6, IL-10 are significantly associated with sepsis. Table 19.1 depicts some of the well-studied SNPs in relation with sepsis.

 Table 19.1 SNPs and their association with sepsis

Apart from these well-known genes, genetic variations were also studied in genes like AQP548, NOS249, HMOX150, SOD251, BCL245, etc. It is impossible to screen each newborn for all these genes which showed significant association with sepsis. But screening of two or three specific genes with respect to the appropriate targets like susceptibility, mortality, outcome or type of organism, along with prevailing risk factors can result in remarkable improvement of neonatal sepsis outcome.

Tagging SNPs

Tag SNPs are single nucleotide polymorphisms which are non-randomly associated with SNPs at other loci in the chromosome, possibly causal SNPs. Analysis of Tag SNPs reduces the burden of studying each individual SNPs separately and helps in understanding the interaction between the SNPs. Tag SNPS can be identified using HapMap database by Linkage Disequilibrium analysis (Haploview program) or PHASE (software used to reconstruct haplotype and estimation of recombination rate). Zeng et al found three SNPs, rs7843858, rs11465996 and rs2114169 as tagging SNPs, but only rs11465996 is associated with sepsis susceptibility and MODS in both Chonging and Zhejiang populations.52 In TLR2 gene, out of the three tagging SNPs, rs1898830, rs3804099, and rs7656411, only rs3804099 was significantly associated with sepsis morbidity and MODS.53 Association of tagging SNPs with sepsis was also found in Protein C gene (Chinese Han population)54, DEFB1 gene (Chinese population)55, Fibrinogen-beta-gene (Caucasians)56 and IL-10.57

Haplogroups

Haplogroups define genetic populations with similar haplotypes having the same SNPs expressing similar phenotypic changes. Y-chromosome (Y-DNA) haplogroups and mitochondrial DNA (mtDNA) haplogroups are two types of haplogroups, presently known to researchers. Haplogroups come from a common ancestor and are restricted to specific geographical locations. Genotyping a single SNP in a haplogroup serves as a prognostic marker of the associated disease for the entire population. Researchers have studied haplogroups in relation with sepsis. mtDNA haplogroup R predicts the outcome of septic encephalopathy in Chinese Han population. R haplogroup delivers high probability of neurological recovery when compared to non-R haplogroup.58 Also R haplogroup serves as predictor of sepsis outcome and associated with long-term survival of patients.59 The MHC haplotype, AH 8.1 confers protective effect towards septic shock in COPD (chronic obstructive pulmonary disease) patients in Caucasian population.60 mtDNA haplogroup H predicted outcome associated with 180 days survival in European patients with severe sepsis.61

Tandem Repeats

Tandem repeats are the repetition of two or more nucleotides placed adjacent to each other. When the number of repeating nucleotides are small (2-6 base pairs), it is called microsatellites and more than 10 repeating nucleotides, it is called minisatellites.

Microsatellites

Very few studies are available correlating microsatellites and sepsis. Microsatellites in genes like HMOX151, TNFa & b62, eNOS63, IL-1064 were studied in relation with sepsis. Flores et al found significant association  of CXCL2 -665(AC)n microsatellite with sepsis susceptibility in Spanish population.65

Copy Number Variations

Copy number variations are the presence of abnormal number of copies of DNA sequences. Human genome contains approximately 12% of copy number variations. Abnormal copies may result in increased production of proteins which may cause phenotypic changes. Chen et al.66 found that high copy number of DEFA1/DEFA3 (>8) is associated with severe sepsis. Conversely, no significant association was found in DEFB4/DEFB103 copy numbers (2 to

9) in relation with S.aureus sepsis.67 As there is insufficient evidence of CNVs role in sepsis susceptibility and its outcome, further research is required to use CNVs as a significant screening test for risk stratification in neonatal sepsis.

Epigenomics

Epigenomics is an advanced genomics in which gene expression is modified without change in nucleotide sequence. These modifications can be heritable. It may result from the environmental factors surrounding the host. Epigenomic variations are mainly of three kinds:

1. Histone modification
2. DNA methylation
3. miRNA

The role of epigenetic variations in sepsis was studied prominently in vitro. No human study is available to date. But miRNAs had been studied widely in human sepsis. The findings of the available studies are briefed below.

Histone Modification

Out of the different types of histone modifications, histone acetylation and histone methylation are only clearly known. Modification in histone proteins results in diverse packing of DNA and concurrently its expression. The expression of immune related gene involved in immunosuppression which follows severe sepsis can be modified by Histone acetylation/methylation.

In naive CD4+ T-cells of sepsis mice models, repressive histone methylation was found at the promoter region of IFN-g and GATA-3 transcription factor.68 Histone acetylation is primarily controlled by histone acetylases (Histone Acetyl Transferases-HATs) and histone deacetlyases (HDACs). Li et al.69 revealed that septic shock caused hypoacetylation of nuclear proteins which was reversed by administering HDAC inhibitors (HDACI). The HDACI effectively prevented cell death, reduced inflammation and eventually improved survival of septic shock animal models.69

DNA Methylation

DNA methylation is the process by which a methyl group is added to DNA by the enzymes DNA methyl transferases (DNMTs). This can be repaired by another set of enzymes known as DNA Demethylases. Methylation of DNA occurs mostly in the 5th position of cytosine residues which is followed by a guanine residue, forming the CpG dinucleotides. DNA methylation predominantly results in gene suppression and demethylation is associated with gene activation. Gazzar et al72 showed that silencing of TNF-a expression in vitro during endotoxin tolerance occurred by the combined action of H3K9 methylation and TNF-a promoter CpG methylation. H3 histone is methylated on lysine 9 by histone methyl transferase, G9a. TNF-a promoter CpG methylation is catalyzed by Dnmt 3a/b (DNA methyl transferases) and HP1 (Heterochromatin protein 1).72,73

miRNA

miRNAs (micro RNAs) are non-coding RNAs with 17 to 25 base pairs. More than 2000 miRNAs had been studied in humans. The details of the miRNAs can be accessed from the public website, http://www.mirbase.org/. miRNA binds to the complementary sequences in mRNA and down regulates the translation process. The binding of miRNA to the target mRNA may also result in the decay of target mRNA. miRNAs are regulated by DNA methylation and histone modification processes. The role of miRNAs in the diagnosis of sepsis was extensively studied. In particular, miR-146a and miR-223 were demonstrated to be significant prognostic/diagnostic markers of sepsis.71,72 Some other miRNAs like miR-15071,73, miR-499-5p74, miR-15a and miR-1675 are still under infantry research for their role in sepsis. miRNAs are also capable of distinguishing sepsis from systemic inflammatory response syndrome.

Nutrigenomics

Nutrigenomics is the new research area of genomics revealing the interaction between diet, gene and health. It explains the effects of nutrients on gene expression. As poor feeding is one of the clinical signs of neonatal sepsis,76 the diet intake of newborn obviously reflects in health management and immune response to infections. Although no studies are available in relation with

sepsis, nutritional genomics found to play major role in chronic diseases like type 2 diabetes77, cardiovascular diseases78 and cancer.79 Dietary nucleotides which are found abundant in all animal and vegetable origin, plays vital role in newborn humoral immune response.80,81 The functioning of neonatal immune system was also found to be related with maternal dietary factors.82 Future research will reveal the importance of nutrition in fighting infections, on genome basis.

CLINICAL APPLICATIONS

The knowledge acquired from studies of adult sepsis can be applied for newborns with sepsis. Newborn genetic screening had been initiated in diseases like cystic fibrosis,83 sickle cell disease,84,85 and deafness.86,87 Genetic screening helps to decipher the neonatal susceptibility to infections and predetermine the outcome. Based on the genetic findings, the treatment strategies can be improved for better results.

Sepsis genomics studies revealed the association of genetic variations with type of micro-organism causing sepsis.31,32,38 Hence genetic screening of newborns may help in deciding antibiotic treatment with respect to the predetermined micro-organism. This will result in the reduced antibiotic usage with simultaneously reduced antibiotic resistance.

The lifestyle of parents may be expressed in newborn phenotype, epigenetically. Folic acid, which is prescribed to almost all pregnant women, is a methylating agent.88 Folic acid was found to increase global DNA methylation and decreased inflammation in animal models of Helicobacter associated gastric cancer.89 The consequences of DNA methylation caused by folic acid on newborn health are still hidden. Multivitamins and green vegetables are also involved in DNA methylation process,90 which may be inherited to their offspring. Detailed research in epigenetics may disclose the importance of epigenetic variations in modifying human health.

The influence of diet in newborn health and immune response towards infectious diseases is well known.91 By studying the genomic basis of effects of nutrients on gene expression, newborn health can be improved by nutritional therapy itself, rather than depending on the drugs.

LIMITATIONS OF GENOMIC MARKERS                                             

Genetic variations differ with populations, culture, food habits, etc. It may take few more decades to study each ethnic group of the world in view of genetic variations. Although the HapMap project gives some data, in countries like India, where diversity among population is greater, extensive research has to be done.

Genetic association studies require very large sample size to establish a significant result. The available literatures about genetic associations were done on small sample size which cannot give true picture in an area with large population.92

As discussed above, human genome consists of millions of SNPs and they may be inter-linked (Linkage disequilibrium). Without knowing the exact causal SNP of the disease, screening the tagging SNPs by mistake may result in prognostic/diagnostic errors.93,94

The cost involved in genotyping is high to be implemented for routine newborn screening, particularly in developing countries with dense popu- lation. New cost-effective technologies have to be developed in order to improve the applicability of genetic screening of newborns.

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