CHAPTER 1

INTRODUCTION

Leukaemia is a heterogenous disease characterized by dysregulation in proliferation

of blood precursor cells of myeloid or lymphoid origin. It is common malignant cancer
affecting children with peak of age 2 to 5 years old. Cases of haematologicalmalignancies
were reported to have increased with approximately one third of cases in children were
reported annually (Conter et al, 2004 and Kwan et al, 2009). Major morphologic subtypes of
leukaemia are acute lymphoblastic leukaemia (ALL) with B-cell precursor phenotype and
acute myeloid leukaemia (AML) characterized by chromosomal abnormalities which have
been shown to originate in utero (Lightfoot and Roman, 2004).

Chromosomal translocations are the most common structural chromosome changes

observed in childhood leukaemia which induced by breakage in double-strand DNA and arise
in haematopoietic stem cells. However, chromosomal translocations alone are not sufficient
to cause leukaemogenesis in the offspring therefore required additional postnatal events
(Greaves and Wiemels, 2003). Smith et al (2005) reported that translocations has been
present in most childhood leukaemias before birth and indoor pesticide exposure during
pregnancy caused an increased cancer risk in the offspring highlighted the influence of
environmental factors in the disease development. Commented [NA1]:

Several studies concluded that childhood leukaemia may arise before birth suggesting
that maternal and perinatal exposures such as chemical or infectious agents during pregnancy
may increase risk of childhood cancer (Greaves and Wiemels, 2003; McHale and Smith,
2004 and Ma et al, 2002). The exact causes of childhood leukaemia remain largely unknown
however several environmental exposures have been proposed to be causal in the
development of childhood leukaemia including radiation, maternal smoking, maternal alcohol
consumption and maternal nutrient status all being suggested to influence cancer risk in the
offspring (Belson et al, 2007; Davis et al, 2004; Pufulete et al, 2005).
 Figure 1.1 Metabolic pathways involving folate and vitamin B12 (taken from Fenech et al,
2001). Commented [NA2]:

Deficiency in folate (erythrocyte folate<140 ng/ml or plasma folate<3 ng/ml) and

vitamin B12 have been significantly associated with cancer development are well
documented (Blount et al, 1997 and Hercberg et al, 1992). More recently, maternal dietary Commented [NA3]:

folate intake during pregnancy has been suggested to influence the risk of leukaemia
development in children. Study by Bailey et al (2012) reported a possible protective effects of Commented [NA4]: Folate intake during pregnancy has been
found to be associated with ALL
folate in maternal with higher folate and vitamin B12 intake during pregnancy and
–Thompson et al, Lancet (2001) 8;358(9297):1935-40
association with decreased risk of ALL in the offspring. In addition, Thompson et al (2001) –Amigou et al, Cancer Causes Control (2012) 23(8):1265-77
–Bailey et al, Nutr Cancer (2012) 64(7):1122-30
also presented similar finding whereby folate supplement administered in mothers during
pregnancy reduces acute lymphoblastic leukaemia risk in the offspring. Furthermore, it was
found an inverse association between maternal folic acid supplementation before and during
pregnancy with risk of ALL in the child (Amigou et al, 2012). Commented [NA5]:

Folate is a water-soluble vitamin that can be found mainly in fortified cereals, citrus
fruits and juices, asparagus, baked beans, lentils and others (Tapiero et al, 2001). It is
important in maintaining DNA stability and provides one-carbon methyl groups for the one-
carbon metabolic pathway which is involved in nucleic acid synthesis and cell division,
regulation of gene expression DNA synthesis and repair mechanisms as well as in the
methylation pathway. Folate is important for providing 5-MTHF for the methylation of
homocysteine to methionine which is then converted to S-adenosylmethionine (SAM), the
universal methyl donor essential for the methylation of biomolecules including DNA (Figure
1.1) (Bailey et al, 2012 and Langie et al, 2013). Variation of dietary intake of this methyl-
group source and others involved in this pathway including vitamin B12 (Figure 1.1) could
therefore potentially affect patterns of DNA methylation and therefore influence cancer risk
susceptibility via altered gene expression (Davis et al, 2004).

DNA methylation is one of an epigenetic series mechanism which can affect the
expression of genes without altering the genomic sequence. It can be defined as addition of a
methyl group to cytosine bases at the carbon 5’ position in CpG dinucleotide site (Davis et al,
2004). CpG island characterized by cluster of methylated CpG sites and associated with
transcriptional repression (Lilycrop et al, 2005). Commented [NA6]:

DNA methylation patterns are established during development in utero and early life
and can influences specific gene expression via transcriptional regulation (McKay et al,
2011). In general, it is assume that methylated promoter region leads to gene silencing while
unmethylated promoter region is associated with gene expression (Figure 1.2). Commented [NA7]:

Figure 1.2 DNA methylation and gene expression

(http://missinglink.ucsf.edu/lm/genes_and_genomes/methylation.html).
 Reports from animal studies have suggested that maternal nutritional status during
pregnancy is highly associated with altered DNA methylation and gene expression patterns in
the offspring which coincide with phenotypic variations in the offspring (McKay et al, 2011;
Burdge et al, 2007; Lilycrop et al, 2008 and Dolinoy et al, 2006).

In one of the first examples of this, Cooney et al (2002) showed that the offspring of
female mice that were fed with methyl-supplemented diet influenced the changes in the
molecular marks including DNA methylation which consequently influenced phenotypic
changes in the pups whereby they present difference in colour coat. This suggested that in Commented [NA8]:

utero exposure to maternal nutritional status could influence epigenetic markings of the genes
in the offspring leading to a clear phenotypic consequence (Davis et al, 2004). Whilst this
example described a dietary change involving multiple methyl-donors, changes in folate
intake alone during pregnancy have been observed to alter DNA methylation in the offspring.

Feeding pregnant rats with protein-restricted (PR) diet causes an increased

glucocorticoid receptor (GR) and PPARα expression in the liver of the offspring due to
hypomethylation of their specific promoter regions which decreased DNA methyltransferase-
1 expression and increased rate of transcription in histone modifications (Lilycorp et al,
2007). The results were reversed whereby there were increased in GR and PPARα expression
when folic acid content of the PR diet was increased.

Furthermore, depletion in maternal folate status in mice during pregnancy causes a

reduction in genome-wide methylation and methylation of p53 gene in the small intestine of
the adult offspring (McKay et al, 2011a, McKay et al, 2011b), further demonstrating that
epigenetic patterns of the offspring are highly susceptible towards environmental exposures
during development and that changes in epigenetic marks may persist to adult life in response
to changes maternal folate status in rodent models. Commented [I9]:

Evidence is also emerging from molecular epidemiological studies to suggest that

maternal nutrient intake during pregnancy is associated with altered DNA methylation of the
offspring. Mothers who consumed folic acid supplements had children with increased
methylation in the IGF2 DMR compared to those born to mothers who did not consumed
supplements (Steegers-Theunissen et al, 2009). Study by van Rooij et al (2003) demonstrated
that low maternal serum B12 was associated with increased risk of orofacial cleft
development in the offspring. McKay et al (2012) reported the reduction in global DNA
methylation was found in maternal with higher vitamin B12 intake during pregnancy. Commented [NA10]:

These observations suggested that maternal folate status during pregnancy and
potentially other vitamins involved in one carbon metabolism including vitamin B12 is
significantly associated with epigenetic patterns in the offspring therefore this may
potentially modulate risk of childhood leukaemia as maternal folate status could alter DNA
methylation patterns (potential mechanism in the causal pathway to cancers). Therefore it is
highly plausible that DNA methylation might be one mechanism by which maternal folate
status during pregnancy is associated with development of childhood leukaemia.

In order to investigate this potential aetiological mechanism, we have previously

identified genes which were aberrantly methylated in response to low maternal folate intake
during pregnancy in a mouse model. Furthermore, genes found to be aberrantly methylated in
childhood leukaemia from previous studies have been identified and were compared with the
‘folate-responsive’ gene list to give a final set of candidate genes in which to investigate the Commented [NA11]:

hypothesis that low maternal folate status may potentially influence childhood leukaemia risk
via changes in DNA methylation. Folate and vitamin B12 status in the mothers during
pregnancy were previously measured as well as for the infants therefore correlate these data
in association with changes in DNA methylation. Commented [NA12]:

1.1 Hypothesise
  Primary: Maternal folate status during pregnancy contributes in the development of
childhood leukaemia through alteration in DNA methylation of disease-associated
genes.
 Secondary: Infant folate status may be associated with DNA methylation in the
offspring
 Secondary: Maternal and infant B12 status influences the degree of DNA methylation
in the infant.

1.2 Objectives

 To validate the methylation assay of target disease-associated genes.

 To compare methylation pattern of normal patient’s samples with acute lymphoblastic
leukaemia’s samples in order to confirm hypermethylation in ALL and at the specific
loci investigated
 To investigate DNA methylation of disease associated genes in offspring in relation to
maternal folate status during pregnancy.