A fusion gene is a hybrid gene formed from two previously independent genes. It can occur as a result of translocation, interstitial deletion, or chromosomal inversion. Fusion genes have been found to be prevalent in all main types of human neoplasia. The identification of these fusion genes play a prominent role in being a diagnostic and prognostic marker.
A schematic showing the ways a fusion gene can occur at a chromosomal level.
History
The first fusion gene
was described in cancer cells in the early 1980s. The finding was based
on the discovery in 1960 by Peter Nowell and David Hungerford in
Philadelphia of a small abnormal marker chromosome in patients with chronic myeloid leukemia—the first consistent chromosome abnormality detected in a human malignancy, later designated the Philadelphia chromosome. In 1973, Janet Rowley in Chicago showed that the Philadelphia chromosome had originated through a translocation between chromosomes 9 and 22,
and not through a simple deletion of chromosome 22 as was previously
thought. Several investigators in the early 1980s showed that the
Philadelphia chromosome translocation
led to the formation of a new BCR/ABL1 fusion gene, composed of the 3'
part of the ABL1 gene in the breakpoint on chromosome 9 and the 5' part
of a gene called BCR in the breakpoint in chromosome 22. In 1985 it was
clearly established that the fusion gene on chromosome 22 produced an
abnormal chimeric BCR/ABL1 protein with the capacity to induce chronic myeloid leukemia.
Oncogenes
It has been known for 30 years that the corresponding gene fusion plays an important role in tumorgenesis.
Fusion genes can contribute to tumor formation because fusion genes
can produce much more active abnormal protein than non-fusion genes.
Often, fusion genes are oncogenes that cause cancer; these include BCR-ABL, TEL-AML1 (ALL with t(12 ; 21)), AML1-ETO (M2 AML with t(8 ; 21)), and TMPRSS2-ERG with an interstitial deletion on chromosome 21, often occurring in prostate cancer.
In the case of TMPRSS2-ERG, by disrupting androgen receptor (AR)
signaling and inhibiting AR expression by oncogenic ETS transcription
factor, the fusion product regulates the prostate cancer.
Most fusion genes are found from hematological cancers, sarcomas, and prostate cancer.
BCAM-AKT2 is a fusion gene that is specific and unique to high-grade serous ovarian cancer.
Oncogenic fusion genes may lead to a gene product with a new or
different function from the two fusion partners. Alternatively, a
proto-oncogene is fused to a strong promoter, and thereby the oncogenic function is set to function by an upregulation caused by the strong promoter of the upstream fusion partner. The latter is common in lymphomas, where oncogenes are juxtaposed to the promoters of the immunoglobulin genes. Oncogenic fusion transcripts may also be caused by trans-splicing or read-through events.
Since chromosomal translocations play such a significant role in
neoplasia, a specialized database of chromosomal aberrations and gene
fusions in cancer has been created. This database is called Mitelman
Database of Chromosome Aberrations and Gene Fusions in Cancer.
Diagnostics
Presence
of certain chromosomal aberrations and their resulting fusion genes is
commonly used within cancer diagnostics in order to set a precise
diagnosis. Chromosome banding analysis, fluorescence in situ hybridization (FISH), and reverse transcription polymerase chain reaction
(RT-PCR) are common methods employed at diagnostic laboratories. These
methods all have their distinct shortcomings due to the very complex
nature of cancer genomes. Recent developments such as high-throughput sequencing and custom DNA microarrays bear promise of introduction of more efficient methods.
Evolution
Gene
fusion plays a key role in the evolution of gene architecture. We can
observe its effect if gene fusion occurs in coding sequences. Duplication, sequence divergence, and recombination are the major contributors at work in gene evolution.
These events can probably produce new genes from already existing
parts. When gene fusion happens in non-coding sequence region, it can
lead to the misregulation of the expression of a gene now under the
control of the cis-regulatory
sequence of another gene. If it happens in coding sequences, gene
fusion cause the assembly of a new gene, then it allows the appearance
of new functions by adding peptide modules into multi domain protein.
The detecting methods to inventory gene fusion events on a large
biological scale can provide insights about the multi modular
architecture of proteins.
Detection
In
recent years, next generation sequencing technology has already become
available to screen known and novel gene fusion events on a genome wide
scale. However, the precondition for large scale detection is a
paired-end sequencing of the cell's transcriptome.
The direction of fusion gene detection is mainly towards data analysis
and visualization. Some researchers already developed a new tool called
Transcriptome Viewer (TViewer) to directly visualize detected gene
fusions on the transcript level.
Research applications
Biologists may also deliberately create fusion genes for research purposes. The fusion of reporter genes
to the regulatory elements of genes of interest allows researches to
study gene expression. Reporter gene fusions can be used to measure
activity levels of gene regulators, identify the regulatory sites of
genes (including the signals required), identify various genes that are
regulated in response to the same stimulus, and artificially control the
expression of desired genes in particular cells. For example, by creating a fusion gene of a protein of interest and green fluorescent protein, the protein of interest may be observed in cells or tissue using fluorescence microscopy. The protein synthesized when a fusion gene is expressed is called a fusion protein.
Chromosomal reciprocal translocation of the 4th and 20th chromosome.
In genetics, chromosome translocation is a phenomenon that results in unusual rearrangement of chromosomes. This includes balanced and unbalanced translocation, with two main types: reciprocal-, and Robertsonian translocation. Reciprocal translocation is a chromosome abnormality caused by exchange of parts between non-homologous chromosomes.
Two detached fragments of two different chromosomes are switched.
Robertsonian translocation occurs when two non-homologous chromosomes
get attached, meaning that given two healthy pairs of chromosomes, one
of each pair "sticks" together.
A gene fusion may be created when the translocation joins two otherwise-separated genes. It is detected on cytogenetics or a karyotype of affected cells. Translocations can be balanced (in an even exchange of material with no genetic information extra or missing, and ideally full functionality) or unbalanced (where the exchange of chromosome material is unequal resulting in extra or missing genes).
Reciprocal translocations
Reciprocal
translocations are usually an exchange of material between
non-homologous chromosomes. Estimates of incidence range from about 1 in
500 to 1 in 625 human newborns. Such translocations are usually harmless and may be found through prenatal diagnosis. However, carriers of balanced reciprocal translocations have increased risks of creating gametes with unbalanced chromosome translocations, leading to Infertility, miscarriages or children with abnormalities. Genetic counseling and genetic testing
are often offered to families that may carry a translocation. Most
balanced translocation carriers are healthy and do not have any
symptoms.
It is important to distinguish between chromosomal translocations occurring in gametogenesis, due to errors in meiosis, and translocations that occur in cellular division of somatic cells, due to errors in mitosis.
The former results in a chromosomal abnormality featured in all cells
of the offspring, as in translocation carriers. Somatic translocations,
on the other hand, result in abnormalities featured only in the affected
cell line, as in chronic myelogenous leukemia with the Philadelphia chromosome translocation.
Nonreciprocal translocation
Nonreciprocal translocation involves the one-way transfer of genes from one chromosome to another nonhomologous chromosome.
Robertsonian translocations
Robertsonian translocation is a type of translocation caused by breaks at or near the centromeres of two acrocentric chromosomes. The reciprocal exchange of parts gives rise to one large metacentric
chromosome and one extremely small chromosome that may be lost from the
organism with little effect because it contains few genes. The
resulting karyotype in humans leaves only 45 chromosomes, since two chromosomes have fused together.
This has no direct effect on the phenotype, since the only genes on
the short arms of acrocentrics are common to all of them and are present
in variable copy number (nucleolar organiser genes).
Robertsonian translocations have been seen involving all
combinations of acrocentric chromosomes. The most common translocation
in humans involves chromosomes 13 and 14 and is seen in about 0.97 / 1000 newborns.
Carriers of Robertsonian translocations are not associated with any
phenotypic abnormalities, but there is a risk of unbalanced gametes that
lead to miscarriages or abnormal offspring. For example, carriers of
Robertsonian translocations involving chromosome 21 have a higher risk of having a child with Down syndrome. This is known as a 'translocation Downs'. This is due to a mis-segregation (nondisjunction)
during gametogenesis. The mother has a higher (10%) risk of
transmission than the father (1%). Robertsonian translocations involving
chromosome 14 also carry a slight risk of uniparental disomy 14 due to trisomy rescue.
Infertility: One of the would-be parents carries a balanced translocation, where the parent is asymptomatic but conceived fetuses are not viable.
Down syndrome is caused in a minority (5% or less) of cases by a Robertsonian translocation of the chromosome 21 long arm onto the long arm of chromosome 14.
Chromosomal translocations between the sex chromosomes can also result in a number of genetic conditions, such as
XX male syndrome: caused by a translocation of the SRY gene from the Y to the X chromosome
The International System for Human Cytogenetic Nomenclature (ISCN) is used to denote a translocation between chromosomes. The designation t(A;B)(p1;q2) is used to denote a translocation between chromosome
A and chromosome B. The information in the second set of parentheses,
when given, gives the precise location within the chromosome for
chromosomes A and B respectively—with p indicating the short arm of the chromosome, q
indicating the long arm, and the numbers after p or q refers to
regions, bands and subbands seen when staining the chromosome with a staining dye. See also the definition of a genetic locus.
The translocation is the mechanism that can cause a gene to move from one linkage group to another.
Examples
For an explanation of the symbols and abbreviations used in these examples, see Cytogenetic notation.
In 1938, Karl Sax, at the Harvard University Biological Laboratories, published a paper entitled "Chromosome Aberrations Induced by X-rays", which demonstrated that radiation could induce major genetic
changes by affecting chromosomal translocations. The paper is thought
to mark the beginning of the field of radiation cytology, and led him to
be called "the father of radiation cytology".
Hematopoietic stem cells (HSCs) are the stem cells that give rise to other blood cells. This process is called haematopoiesis. This process occurs in the red bone marrow, in the core of most bones. In embryonic development, the red bone marrow is derived from the layer of the embryo called the mesoderm.
Haematopoiesis
is the process by which all mature blood cells are produced. It must
balance enormous production needs (the average person produces more than
500 billion blood cells every day) with the need to regulate the number
of each blood cell type in the circulation. In vertebrates, the vast
majority of hematopoiesis occurs in the bone marrow and is derived from a
limited number of hematopoietic stem cells that are multipotent and
capable of extensive self-renewal.
Stem and progenitor cells can be taken from the pelvis, at the iliac crest, using a needle and syringe.
The cells can be removed as liquid (to perform a smear to look at the
cell morphology) or they can be removed via a core biopsy (to maintain
the architecture or relationship of the cells to each other and to the
bone).
Subtypes
A colony-forming unit is a subtype of HSC. (This sense of the term is different from colony-forming units of microbes, which is a cell counting unit.) There are various kinds of HSC colony-forming units:
The above CFUs are based on the lineage. Another CFU, the colony-forming unit–spleen (CFU-S), was the basis of an in vivo
clonal colony formation, which depends on the ability of infused bone
marrow cells to give rise to clones of maturing hematopoietic cells in
the spleens of irradiated mice after 8 to 12 days. It was used
extensively in early studies, but is now considered to measure more
mature progenitor or transit-amplifying cells rather than stem cells.
Isolating stem cells
Since hematopoietic stem cells cannot be isolated as a pure population, it is not possible to identify them in a microscope. Hematopoietic stem cells can be identified or isolated by the use of flow cytometry where the combination of several different cell surface markers (particularly CD34)
are used to separate the rare Hematopoietic stem cells from the
surrounding blood cells. Hematopoietic stem cells lack expression of
mature blood cell markers and are thus called Lin-. Lack of expression
of lineage markers is used in combination with detection of several
positive cell-surface markers to isolate hematopoietic stem cells. In
addition, hematopoietic stem cells are characterised by their small size
and low staining with vital dyes such as rhodamine 123 (rhodamine lo) or Hoechst 33342 (side population).
Function
Diagram of cells that arise from Hematopoetic stem cells during the process of hematopoiesis.
Haematopoiesis
Hematopoietic
stem cells are essential to haematopoiesis, the formation of the cells
within blood. Hematopoietic stem cells can replenish all blood cell
types (i.e., are multipotent)
and self-renew. A small number of Hematopoietic stem cells can expand
to generate a very large number of daughter Hematopoietic stem cells.
This phenomenon is used in bone marrow transplantation,
when a small number of Hematopoietic stem cells reconstitute the
hematopoietic system. This process indicates that, subsequent to bone
marrow transplantation, symmetrical cell divisions into two daughter
Hematopoietic stem cells must occur.
Stem cell self-renewal is thought to occur in the stem cell niche in the bone marrow, and it is reasonable to assume that key signals present in this niche will be important in self-renewal.
There is much interest in the environmental and molecular requirements
for HSC self-renewal, as understanding the ability of HSC to replenish
themselves will eventually allow the generation of expanded populations
of HSC in vitro that can be used therapeutically.
Quiescence
Hematopoietic stem cells, like all adult stem cells, mostly exist in a state of quiescence,
or reversible growth arrest. The altered metabolism of quiescent HCSs
helps the cells survive for extended periods of time in the hypoxic bone
marrow environment.
When provoked by cell death or damage, Hematopoietic stem cells exit
quiescence and begin actively dividing again. The transition from
dormancy to propagation and back is regulated by the MEK/ERK pathway and PI3K/AKT/mTOR pathway.
Dysregulation of these transitions can lead to stem cell exhaustion, or
the gradual loss of active Hematopoietic stem cells in the blood
system.
Mobility
Hematopoietic stem cells have a higher potential than other immature blood cells to pass the bone marrow barrier, and, thus, may travel in the blood from the bone marrow in one bone to another bone. If they settle in the thymus, they may develop into T cells. In the case of fetuses and other extramedullary hematopoiesis, Hematopoietic stem cells may also settle in the liver or spleen and develop.
This enables Hematopoietic stem cells to be harvested directly from the blood.
DNA damage with aging
DNA strand breaks accumulate in long term Hematopoietic stem cells during aging. This accumulation is associated with a broad attenuation of DNA repair and response pathways that depends on HSC quiescence. Non-homologous end joining
(NHEJ) is a pathway that repairs double-strand breaks in DNA. NHEJ is
referred to as "non-homologous" because the break ends are directly
ligated without the need for a homologous template. The NHEJ pathway
depends on several proteins including ligase 4, DNA polymerase mu and NHEJ factor 1 (NHEJ1, also known as Cernunnos or XLF).
DNA ligase 4 (Lig4) has a highly specific role in the repair of
double-strand breaks by NHEJ. Lig4 deficiency in the mouse causes a
progressive loss of Hematopoietic stem cells during aging. Deficiency of lig4 in pluripotent stem cells results in accumulation of DNA double-strand breaks and enhanced apoptosis.
In polymerase mu mutant mice, hematopoietic cell development is
defective in several peripheral and bone marrow cell populations with
about a 40% decrease in bone marrow cell number that includes several
hematopoietic lineages.
Expansion potential of hematopoietic progenitor cells is also reduced.
These characteristics correlate with reduced ability to repair
double-strand breaks in hematopoietic tissue.
Deficiency of NHEJ factor 1 in mice leads to premature aging of
hematopoietic stem cells as indicated by several lines of evidence
including evidence that long-term repopulation is defective and worsens
over time.
Using a human induced pluripotent stem cell model of NHEJ1 deficiency,
it was shown that NHEJ1 has an important role in promoting survival of
the primitive hematopoietic progenitors.
These NHEJ1 deficient cells possess a weak NHEJ1-mediated repair
capacity that is apparently incapable of coping with DNA damages induced
by physiological stress, normal metabolism, and ionizing radiation.
The sensitivity of haematopoietic stem cells to Lig4, DNA
polymerase mu and NHEJ1 deficiency suggests that NHEJ is a key
determinant of the ability of stem cells to maintain themselves against
physiological stress over time. Rossi et al.
found that endogenous DNA damage accumulates with age even in wild type
Hematopoietic stem cells, and suggested that DNA damage accrual may be
an important physiological mechanism of stem cell aging.
Clinical significance
Transplant
Hematopoietic stem cell transplantation (HSCT) is the transplantation of multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. It may be autologous (the patient's own stem cells are used), allogeneic (the stem cells come from a donor) or syngeneic (from an identical twin).
It is most often performed for patients with certain cancers of the blood or bone marrow, such as multiple myeloma or leukemia.
In these cases, the recipient's immune system is usually destroyed with
radiation or chemotherapy before the transplantation. Infection and graft-versus-host disease are major complications of allogeneic HSCT.
In order to harvest stem cells from the circulating peripheral blood, blood donors are injected with a cytokine,
such as granulocyte-colony stimulating factor (G-CSF), that induces
cells to leave the bone marrow and circulate in the blood vessels.
In mammalian embryology, the first definitive Hematopoietic stem cells are detected in the AGM (aorta-gonad-mesonephros), and then massively expanded in the fetal liver prior to colonising the bone marrow before birth.
Hematopoietic stem cell transplantation remains a dangerous
procedure with many possible complications; it is reserved for patients
with life-threatening diseases. As survival following the procedure has
increased, its use has expanded beyond cancer to autoimmune diseases and hereditary skeletal dysplasias; notably malignant infantile osteopetrosis and mucopolysaccharidosis.
Research
Behavior in culture
A cobblestone area-forming cell (CAFC)assay is a cell culture-based empirical assay. When plated onto a confluent culture of stromal feeder layer,
a fraction of Hematopoietic stem cells creep between the gaps (even
though the stromal cells are touching each other) and eventually settle
between the stromal cells and the substratum (here the dish surface) or
trapped in the cellular processes between the stromal cells. Emperipolesis is the in vivo phenomenon in which one cell is completely engulfed into another (e.g. thymocytes into thymic nurse cells); on the other hand, when in vitro, lymphoid lineage cells creep beneath nurse-like cells, the process is called pseudoemperipolesis. This similar phenomenon is more commonly known in the HSC field by the cell culture terminology cobble stone area-forming cells (CAFC), which means areas or clusters of cells look dull cobblestone-like
under phase contrast microscopy, compared to the other Hematopoietic
stem cells, which are refractile. This happens because the cells that
are floating loosely on top of the stromal cells are spherical and thus
refractile. However, the cells that creep beneath the stromal cells are
flattened and, thus, not refractile. The mechanism of
pseudoemperipolesis is only recently coming to light. It may be mediated
by interaction through CXCR4 (CD184) the receptor for CXC Chemokines (e.g., SDF1) and α4β1integrins.
Repopulation kinetics
Hematopoietic
stem cells (HSC) cannot be easily observed directly, and, therefore,
their behaviors need to be inferred indirectly. Clonal studies are
likely the closest technique for single cell in vivo studies of HSC.
Here, sophisticated experimental and statistical methods are used to
ascertain that, with a high probability, a single HSC is contained in a
transplant administered to a lethally irradiated host. The clonal
expansion of this stem cell can then be observed over time by monitoring
the percent donor-type cells in blood as the host is reconstituted. The
resulting time series is defined as the repopulation kinetic of the
HSC.
The reconstitution kinetics are very heterogeneous. However, using symbolic dynamics, one can show that they fall into a limited number of classes. To prove this, several hundred experimental repopulation kinetics from clonal Thy-1lo SCA-1+ lin− c-kit+
HSC were translated into symbolic sequences by assigning the symbols
"+", "-", "~" whenever two successive measurements of the percent
donor-type cells have a positive, negative, or unchanged slope,
respectively. By using the Hamming distance,
the repopulation patterns were subjected to cluster analysis yielding
16 distinct groups of kinetics. To finish the empirical proof, the Laplace add-one approach
was used to determine that the probability of finding kinetics not
contained in these 16 groups is very small. By corollary, this result
shows that the hematopoietic stem cell compartment is also heterogeneous
by dynamical criteria.
It was originally believed that all Hematopoietic stem cells were
alike in their self-renewal and differentiation abilities. This view
was first challenged by the 2002 discovery by the Muller-Sieburg group
in San Diego, who illustrated that different stem cells can show
distinct repopulation patterns that are epigenetically predetermined
intrinsic properties of clonal Thy-1lo Sca-1+ lin−c-kit+ HSC. The results of these clonal studies led to the notion of lineage bias. Using the ratio
of lymphoid (L) to myeloid (M) cells in blood as a quantitative marker,
the stem cell compartment can be split into three categories of HSC. Balanced (Bala) Hematopoietic stem cells
repopulate peripheral white blood cells in the same ratio of myeloid to
lymphoid cells as seen in unmanipulated mice (on average about 15%
myeloid and 85% lymphoid cells, or 3 ≤ ρ ≤ 10). Myeloid-biased (My-bi) Hematopoietic stem cells give rise to very few lymphocytes resulting in ratios 0 < ρ < 3, while lymphoid-biased (Ly-bi) Hematopoietic stem cells generate
very few myeloid cells, which results in lymphoid-to-myeloid ratios of ρ
> 10. All three types are normal types of HSC, and they do not
represent stages of differentiation. Rather, these are three classes of
HSC, each with an epigenetically fixed differentiation program. These
studies also showed that lineage bias is not stochastically regulated or
dependent on differences in environmental influence. My-bi HSC
self-renew longer than balanced or Ly-bi HSC. The myeloid bias results
from reduced responsiveness to the lymphopoetin interleukin 7 (IL-7).
Subsequently, other groups confirmed and highlighted the original findings.
For example, the Eaves group confirmed in 2007 that repopulation
kinetics, long-term self-renewal capacity, and My-bi and Ly-bi are
stably inherited intrinsic HSC properties. In 2010, the Goodell group provided additional insights about the molecular basis of lineage bias in side population (SP) SCA-1+ lin− c-kit+ HSC. As previously shown for IL-7 signaling, it was found that a member of the transforming growth factor family (TGF-beta) induces and inhibits the proliferation of My-bi and Ly-bi HSC, respectively.
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