Ataxia-telangiectsia:
the clinical phenotype and epidemiology

The
cerebral degenerative disorder Ataxia-telangiectsia (AT) is an autosomal
recessive disease primarily characterised by cerebellar degeneration which
often begins in infancy, becoming progressively worse by the age of ten at
which point most patients are reliant on a wheelchair (Rothblum-Oviatt et al.,
2016, Perlman et al., 2003). AT is not strictly confined to neurodegeneration
and other associated phenotypes of the disease include immunodeficiency, growth
retardation, hypersensitivity to ionising radiation and thymic dysplasia
(Rothblum-Oviatt et al., 2016, Perkins et al., 2002, Liyanage et al., 2000).
Individuals with AT are also predisposed to an increased likelihood of
malignancy, especially that of lymphoid origin (Rothblum-Oviatt et al., 2016,
Perkins et al., 2002). Patients diagnosed with AT have a 38% increased likelihood
of developing leukaemia and lymphomas during their lifetime  (Liyanage et al., 2000). In fitting with this
magnitude of clinical phenotypes, AT is often referred to as a syndrome of
genomic instability, chromosomal instability and of DNA damage response
(Rothblum-Oviatt et al., 2016). Individuals of all races and ethnicities are
affected by AT with approximately 2% of the population being carriers of the
mutated allele (Rothblum-Oviatt et al., 2016, Su and Swift, 2000). Patients
diagnosed with AT will, on average, only live to 25 years of age (Crawford et
al., 2006). This is mostly as a result of complications associated with AT as
opposed to the consequence of neurodegeneration. The most common cause of death
amongst patients are malignancy, infection and pulmonary failure (Crawford et
al., 2006, Perlman et al., 2003).

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Ataxia-telangiectsia
mutated (ATM)

The
gene responsible for the pleiotropic phenotype of AT is ataxia-telangiectsia
mutated (ATM) which in the majority of AT patients is inactivated as a result
of bialliec mutation (Taylor et al., 2014, Rotman and Shiloh, 1999). The ATM gene
which is located on chromosome 11q22-23 is 150 kb in length and encodes a
serine-threonine kinase belonging to the phosphatidylinositol-3 kinase (PI3K)
family (Gilad et al., 1996, Camacho et al., 2002). The entire length of the ATM
gene covering 62 coding exons, including proximal, central and distal regions
are equally susceptible to mutation (Perlman et al., 2003, Rothblum-Oviatt et
al., 2016). The mutation spectrum which exists within AT is also incredibly
diverse. The majority of mutations that occur are truncating, these are
nonsense mutations which occur due to frameshifts, insertions or deletions and
often generate premature termination codons in the message (Rothblum-Oviatt et
al., 2016, Perlman et al., 2003, Scott et al., 2001). Over 85% of mutations
that occur within AT patients are truncating, resulting in the production of
highly unstable ATM protein fragments which are unable to be detected via
western blotting (Perlman et al., 2003, Rothblum-Oviatt et al., 2016). Other
types of mutation which are known to occur in AT patients, albeit to a lesser
extent, are leaky splice-site mutations and missense mutations which alter the
overall amino acid sequence and (Scott 2001 et al., Rothblum-Oviatt et al.,
2016). These forms of in-frame mutations allow for a small amount of
functioning ATM protein to be produced, which can be detected by western
blotting. The multiple genotypes to exist within AT patients presents varying
phenotypes amongst patients; AT can therefore be categorised as classical or
mild forms, depending on the mutation which occurs (Rothblum-Oviatt et al.,
2016). The two types have differing impacts on the severity of the disease and
the life expectancy of the patient, however, both are based on one underlying
premise which is the loss of function of the ATM kinase (Taylor et al., 2014).

The ATM protein
kinase

ATM
is a 370-kDa protein kinase which belongs to a conserved family of proteins
containing motifs typical to those of the lipid kinase PI3K (Kim et al., 1999,
Shiloh, 2003). This PI3K-like protein kinase (PIIK) is predominately located
within the nucleus of dividing cells, with a small fraction of cytoplasmic
vesicles in certain cell types also containing ATM (Kim et al., 1999).
Structurally, the PI3K domain occupies only 10% of the ATM protein (Shiloh et
al., 2013). Several other structural domains also exist within ATM, including
FAT (conserved sequence in FRAP, ATM and TRRAP) and FATC (FAT-C-terminal)
located in the extreme C-terminus of ATM which play a key role within
regulating kinase activity of the protein (Bhatti et al., 2011). Important
domains located within the N-terminus include the NLS (nuclear localisation
sequence) and SUBS (N-terminal substrate binding site) (Shiloh et al., 2013,
Bhatti et al., 2011). It is the signature PI3K domain which groups ATM into the
PIIK family, other members of which include ATR (ATM and RAD3-related),
DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and mTOR (mammalian
TOR) (Shiloh 2013). The active members of the PIIK family all have an
involvement in responding to different cellular stresses, mediated via phosphorylation
of downstream targets in the corresponding pathways (Shiloh, 2003). Those which
are major players in genotoxic stresses, such as double stranded DNA breaks
(DSBs), include ATM, DNA-PKcs and ATR (Shiloh and Ziv, 2013). These kinases are
notable for their preferential phosphorylation of serine or threonine, followed
by glutamine residues located on their respective target substrates (Shiloh and
Ziv, 2013).

DNA damage
response via ATM

The
DNA damage response (DDR) refers to a highly effective mechanism evolved by
cells overtime to ameliorate any genotoxic stresses which may occur (Marechal
and Zou, 2013). The underlying premise of this process is the ability to both
detect and communicate problems within the DNA and to either activate repair mechanisms,
arrest cell cycle progression or remove cells with unrepairable genomes
(Marechal and Zou, 2013). Alterations to DNA can arise due to a number of
different factors, those of which include spontaneous chemical changes in DNA
constituents and DNA-damaging agents which inflict damage on the DNA (Shiloh,
2003). It is the endogenous and exogenous DNA-damaging agents which pose the
greatest threat to genome stability, inducing a plethora of DNA lesions
(Shiloh, 2003). Well-known clastogens include anti-cancer chemotherapies such
as cisplatin or bleomycin, ionizing radiation which leads to extensive base
damage and reactive chemicals (Mehta and Haber, 2014, Shiloh, 2003). These can
produce a multitude of damage including intra- and interstrand cross-links, base
lesions, single- and double-strand breaks (Mehta and Haber, 2014). The repair
of damaged DNA is essential in maintaining genomic integrity,  if not repaired then the survival of the cell
is threatened and the risk of developing mutations leading to malignancy is
increased (Abraham, 2001).                                                                                                                                                                                                                         
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             

Activation of
ATM and phosphorylation of its targets

A
key mediator of DNA damage response, in particular that of DSBs, is ATM. This
PIIK interacts with activator proteins and nucleic acid/chromatin complexes to
regulate multiple downstream substrates initiating the repair of DSBs (Bhatti et
al., 2011). To exert any kind of signal transduction in response to cellular
stresses, ATM firstly needs to be activated. The PI3K kinase not only undergoes
a spatial relocation from the nucleus to sites of DNA damage but also a catalytic
activation (Shiloh and Ziv, 2013). Both the relocation and full activation of
ATM involves the interaction with a highly conserved protein complex known as
MRN which is composed of MRE11, RAD50 and NBS1 and the histone
acetyltransferase Tip60 HAT (Bhatti et al., 1991, Sun et al., 2005). MRN is one
of the first complexes recruited to DNA DSBs, in an ATM independent manner
(Bhatti et al. 1991). Via the MRE11 and RAD50 subunits, MRN is able to
recognise and directly bind to double-stranded DNA ends producing a MRN-DNA
complex (Marechal and Zou, 2013). This complex is vital for the rapid
localisation and activation of ATM to DNA break sites, all occurring prior to
the autophosphorylation of ATM (Bhatti et al., 1991).

In
healthy cells, ATM molecules lay dormant existing only in their inactive form
involving a dimerized configuration whereby the kinase domain is blocked by the
FAT domain (Shiloh, 2003). Rapidly after DNA damage is detected via MRN the ATM
dimer is autophosphorylated at a key S1981 residue, leading to the
monomerisation of the protein (Bhatti et al., 2011). This process occurs
secondary to the recruitment of unphosphorylated ATM to sites of damage and
leads to the crucial transphosphorylation of multiple ATM molecules within just
minutes after DSBs are detected (Bhatti et al., 1991, Shiloh, 2003). Upon
activation, ATM is able to phosphorylate a diverse repertoire of downstream
signalling targets including Brca1, Chk2 and p53 which are involved in DNA
damage response (Marechal and Zou, 2013). The role of these signalling
molecules is to further regulate and maintain cellular homeostasis mediated via
cell cycle checkpoints, DNA repair and apoptosis (Sun et al., 2005). Cells
deficient in ATM are sensitive to genotoxic agents and are therefore impaired
in the signal transduction pathways which mediate responses to cellular stress
(Rotman and Shiloh, 1999).

ATM response:
managing cell-cycle checkpoints

The
repertoire of downstream signalling molecules which are activated by ATM is
incredibly diverse. Most are involved in interlinking signalling cascades,
downstream of ATM, which using a combined effect are able to maintain cellular
survival in times of genomic stress. Modulating this process via multiple
pathways is the tumour suppressor gene p53, which, post DNA damage is able to
regulate cell-cycle checkpoints (Rotman and Shiloh, 1999). Creating pauses
during times of physiological stress and delaying the progression through the
cell cycle allows time for repair of DNA DSBs before entering the replication
phase (Rotman and Shiloh, 1999, Derheimer and Kastan, 2010). Both p53 and its
interacting proteins MDM2 and CHK2 are modulated by ATM, successfully halting
the G1/S checkpoint. Phosphorylation on the Ser15 residue of p53 activates and
stabilises it; increasing the overall transcriptional activity of the protein
(Shiloh, 2003). This results in the transcriptional induction of p21- an
inhibitor of the cyclin-dependent kinase/cyclinE which controls entry into
S-phase of the cell cycle (Shiloh, 2003). ATM also acts via the checkpoint
kinase CHK2; phosphorylation of this protein allows for the direct inhibition
of MDM2 thus interfering with the inhibitory p53-MDM2 interaction (Shiloh,
2003). Whilst p53 plays a central role in modulating the G/S checkpoint other
proteins are required for different stages of the cell cycle. Breast cancer
associated-1 (Brca1) is phosphorylated by ATM at multiple sites, all of which
elicit different effects on cell cycle progression. Of particular importance is
phosphorylation on the Ser1423 which mediates G2/M arrest (Derheimer and
Kastan, 2010).

ATM: DNA repair
mechanisms

Two
mechanistic approaches are currently put in place within mammalian cells in
order to repair DNA DSBs. The first is a high-fidelity approach which utilises
homologous recombination (HR) between sister chromatids (Shiloh, 2003).
Homologous recombination is mediated by the family of RAD51 associated
proteins, compiled of numerous RAD51 paralogues including RAD52, RAD54
helicase, MRE11 nuclease, XRS2 and BRCA2 (Jackson, 2002, Shiloh, 2003). The
RAD50/MRE11 complex initiates the first stage of HR; processing of DNA ends
produces single 3′ tails which in subsequent steps are used as substrates for
homologous pairing (Tauchi et al., 2002). RAD52 then binds to the
single-stranded ends of DNA enabling the formation of a nucleoprotein filament
on the exposed ends (Shiloh, 2003). This nucleoprotein filament is required for
the interaction with undamaged DNA. Identification of a homologous region leads
to the catalysis of strand exchange events via RAD51, here the undamaged DNA
duplexes are invaded by the damaged DNA, displacing one strand and forming a
D-loop (Jackson, 2002). The use of a homologous template restricts HR to the
G2/S phase of the cell cycle, when a template is available (Davis and Chen,
2013). Extension of the 3′ terminus of damaged DNA is initiated and carried out
by a DNA polymerase which utilises information from the undamaged homologue and
then ends are then ligated by DNA ligase 1 (Jackson, 2002). ATM is thought to
be involved in multiple processes within this pathway. Bakr et al., suggest a
role for ATM in the phosphorylation and activation of the nuclease MRE11, the
recruitment of the RAD50 associated proteins and the phosphorylation or RAD51
via c-Abl (Bakr et al., 2015). It was also demonstrated that ATM-deficient
cells displayed the accumulation of essential proteins such as RAD51, which
were clearly not being phosphorylated and thus activated by ATM (Bakr et al.,
2015).

Non-homologous
end joining

Non-homologous
end joining (NHEJ) is the more predominantly used repair method that produces
local microdeletions in the DNA to seal breaks in a rapid yet effective manner
(Shiloh, 2003). NHEJ of DNA DSBs
is mediated through the direct re-ligation of a broken DNA segment (Davis and
Chen, 2013). Unlike homologous recombination, this process does not require any
kind of homologous sister template and is therefore not restricted to a
particular stage of the cell cycle (Davis and Chen, 2013). Central to the
process of NHEJ is the heterodimeric protein Ku, comprised of two subunits,
Ku70 and Ku80 (Jackson, 2002). This heterodimer binds to DSBs in a sequence
independent manner forming an open ring structure on the exposed ends of DNA (Shiloh,
2003, Jackson, 2002). The DNA-Ku complex then mediates subsequent steps in NHEJ
by initiating the recruitment of DNA-PKs to form a complete holoenzyme
(Jackson, 2002). DNA-PKs are involved in the recruitment and phosphorylation of
further target substrates, with preference for the sequence consensus
serine/threonine, glutamine (Jackson, 2002). Multiple factors are recruited by
this complex, including Apraxtin and PNK like factor (APLF), replication factor
A2 and x-ray cross complementing protein 4 (XRCC4), a ligase IV heterodimer
which seals the breaks (Davis and Chen, 2013, Shiloh, 2003).

 

V(D)J recombination

There
are very few programmed genomic alterations which occur within vertebrates. One
of which takes place within developing B-lymphocytes and T-lymphocytes in a
process known as V(D)J recombination; the driver of antigenic variation amongst
immunoglobulins (Igs) and T-cell receptors (TCR) (Jackson and Bartek, 2009).
Both the TCR and Igs are comprised of variable and constant regions which are
involved in specifying antigen binding and formulating the various Ig classes,
respectively (Jackson and Bartek, 2009). Exons which encode the antigen-binding
regions are composed of three gene segments, V, D and J. It is the amplitude of
re-arrangement possibilities exhibited by these segments that creates great
diversity amongst Igs and TCRs (Jackson and Bartek, 2009). The process of V(D)J
is initiated by recombination signal (RS) sequences, composed of palindromic
heptamers and AT rich nonamers separated by either 12 or 23 base pair spacers
(Bassing et al., 2002). These conserved heptamer-spacer-nonamer sequences are
used to flank regions of the gene that are then recognised by the recombination
activating genes, RAG1 and RAG2 (Bassing et al., 2002, Alt et al., 1992).  RAG1 and RAG2 mediate the induction of blunt
double-stranded breaks between the V, D and J segments of the gene. The coding
ends of the DNA are subsequently modified whilst the flanked regions are
joined, resulting in either the inversion or deletion of the intervening
material (Bassing et al., 2002).  The
re-ligation stage of V(D)J is executed using the process of non-homologous end
joining and the subsequent proteins which are central to this process (Bassing et
al., 2002).

ATM deficient
cells and increased likelihood of malignancy

As
discussed previously, patients diagnosed with the neurodegenerative condition
AT present an increased likelihood of developing malignancy within their
lifetime, demonstrating approximately a 50-fold to 150-fold increase in risk
(Olsen et al, 2001). Likewise, this increased cancer risk can also be seen
within the general population as a result of somatic mutation and
next-generation sequencing recently revealing a total of 167 distinct mutations
spanning the entire ATM gene (Choi et al., 2016). Mutations in ATM often occur
due to a loss of heterogeneity at cytobands 11q-22-23 which can be seen in a
multitude of different cancer types (Khanna, 2000). Those of lymphoid origin
are particularly susceptible and the aberration of ATM has been demonstrated in
B-cell non-Hodgkin lymphoma, mantle cell lymphoma and B-cell chronic
lymphocytic leukaemia (CLL) (Takagi, 2017).

The
most common form of mutations known to occur in haematological malignancies are
chromosomal translocations (Nussenzweig, 2010). Translocations occur as a
result of two separate double stranded breaks joining together on either the
same or different chromosomes (Tepsurporn et al. 2014). V(D)J recombination
initiates multiple DSBs which consequently increases the potential substrates
for chromosomal translocations (Tepsurporn et al., 2014). Recombination is usually
monitored by ATM to ensure the process of NHEJ occurs providing re-ligation of
segments in a reasonable time frame and thus maintaining genomic integrity
(Shiloh, 2003, Tepsurporn et al., 2014). In cells deficient of ATM these breaks
are not efficiently repaired and thus the risk of mutation is rapidly increased,
the event of one or more unrepaired double strand breaks can then increase the chance
of chromosomal translocations (Jackson, 2002).

c-Myc amplification

A
common chromosomal translocation seen within multiple malignancies is that of
c-myc. Myc (c-Myc) is a proto-oncogene that is frequently upregulated in
multiple types of cancers. The basic helix-loop-helix leucine zipper (bHLHZip)
protein functions as a transcription factor which binds to DNA in a sequence
specific manner after dimerization with another protein, known as Max (Campaner
and Amati, 2012). This transcription factor is necessary for many processes
including cell growth and survival, mediated through regulation of the cell
cycle, for metabolism, differentiation and apoptosis (Campaner and Amati,
2012). 

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