Polytene chromosomes in a Chironomus salivary gland cell
Polytene chromosome
Polytene chromosomes are large chromosomes which have thousands of DNA strands. They provide a high level of function in certain tissues such as salivary glands.
They are produced when repeated rounds of DNA replication without cell division forms a giant chromosome. Thus polytene chromosomes form when multiple rounds of replication produce many sister chromatidswhich stay fused together.
Polytene chromosomes, at interphase,
are seen to have distinct thick and thin banding patterns. These
patterns were originally used to help map chromosomes, identify small chromosome mutations, and in taxonomic identification. They are now used to study the function of genes in transcription.
Function
In
addition to increasing the volume of the cells' nuclei and causing cell
expansion, polytene cells may also have a metabolic advantage as
multiple copies of genes permits a high level of gene expression. In Drosophila melanogaster, for example, the chromosomes of the larval salivary glands undergo many rounds of endoreduplication to produce large quantities of adhesive mucoprotein (“glue”) before pupation. Another example within the fly itself is the tandem duplication of various polytene bands located near the centromere of the X chromosome which results in the Bar phenotype of kidney-shaped eyes.
The interbands are involved in the interaction with the active chromatin proteins, nucleosome remodeling, and origin recognition complexes. Their primary functions are: to act as binding sites for RNA pol II, to initiate replication and, to start nucleosome remodeling of short fragments of DNA.
Structure
In
insects, polytene chromosomes are commonly found in the salivary glands;
they are also referred to as "salivary gland chromosomes". The large
size of the chromosome is due to the presence of many longitudinal
strands called chromonemata;
hence the name polytene (many stranded). They are about 0.5 mm in
length and 20 μm in diameter. The chromosomal strands are formed after
repeated division of the chromosome in the absence of cytoplasmic
division. This type of division is called endomitosis.
The polytene chromosome contains two types of bands, dark bands and
interbands. The dark bands are darkly stained and the inter bands are
lightly stained with nuclear stains. The dark bands contain more DNA and
less RNA. The interbands contain more RNA and less DNA. The amount of
DNA in interbands ranges from 0.8 - 25%.
The bands of polytene chromosomes become enlarged at certain
times to form swellings called puffs. The formation of puffs is called
puffing. In the regions of puffs, the chromonemata uncoil and open out
to form many loops. The puffing is caused by the uncoiling of individual
chromomeres in a band. The puffs indicate the site of active genes
where mRNA synthesis takes place.
The chromonemata of puffs give out a series of many loops laterally. As
these loops appear as rings, they are called Balbiani rings after the
name of the researcher who discovered them. They are formed of DNA, RNA
and a few proteins. As they are the site of transcription, transcription
mechanisms such as RNA polymerase and ribonucleoproteins are present.
In protozoans, there is no transcription, since the puff consists only of DNA.
History
Polytene chromosomes were originally observed in the larval salivary glands of Chironomus midges by Édouard-Gérard Balbiani in 1881.
Balbiani described the chromosomal puffs among the tangled thread
inside the nucleus, and named it "permanent spireme". In 1890, he
observed similar spireme in a ciliated protozoan Loxophyllum meleagris. The existence of such spireme in Drosophila melanogaster
was reported by Bulgarian geneticist Dontcho Kostoff in 1930. Kostoff
predicted that the discs (bands) which he observed were "the actual
packets in which inherited characters are passed from generation to
generation."
The hereditary nature of these structures was not confirmed until they were studied in Drosophila melanogaster in the early 1930s by German biologists Emil Heitz and Hans Bauer. In 1930, Heitz studied different species of Drosophila (D. melanogaster, D. simulans, D. hydei, and D. virilis)
and found that all their interphase chromatins in certain cells were
swollen and messy. In 1932, he collaborated with Karl Heinrich Bauer
with whom he discovered that the tangled chromosomes having distinct
bands are unique to the cells of the salivary glands, midgut, Malphigian
tubules, and brain of the flies Bibio hurtulunus and Drosophila funebris. The two papers were published in the early 1933. Unaware of these papers, an American geneticist Theophilus Shickel Painter reported in December 1933 the existence of giant chromosome in D. melanogaster (followed by a series of papers the following year). Learning of this, Heitz accused Painter of deliberately ignoring their original publication to claim priority of discovery. In 1935, Henry J. Muller and A.A. Prokofyeva established that the individual band or part of a band corresponds with a gene in Drosophila. The same year, P.C. Koller hesitantly introduced the name "polytene" to describe the giant chromosome, writing:
It seems that we can regard these
chromosomes as corresponding with paired pachytene chromosomes at
meiosis in which the intercalary parts between chromomeres have been
stretched and separated into smaller units, and in which, instead of two
threads lying side by side, we have 16 or even more. Hence they are
"polytene" rather than pachytene; I do not, however, propose to use this
term; I shall refer to them as "multiple threads."
Occurrence
Polytene chromosomes are present in secretory tissues of dipteran insects such as the Malpighian tubules of Sciara and also in protists, plants, mammals, or in cells from other insects. Some of the largest polytene chromosomes described thus far occur in larval salivary gland cells of the chironomid genus Axarus.
In plants, they are found in only a few species, and are restricted to ovary and immature seed tissues such as in Phaseolus coccineus and P. vulgaris (Nagl, 1981), and the anther tapetum of Vigna unguiculata and of some Phaseolus species.
Polytene chromosomes are also used to identify the species of
chironomid larvae that are notoriously difficult to identify. Each
morphologically distinct group of larvae consists of a number of
morphologically identical (sibling) species that can only be identified
by rearing adult males or by cytogenetic analysis of the polytene
chromosomes of the larvae. Karyotypes are used to confirm the presence
of specific species and to study genetic diversity in species with a
wide range of genetic variation.
The amount of non-coding DNA varies greatly among species. Often,
only a small percentage of the genome is responsible for coding
proteins, but an increasing percentage is being shown to have regulatory
functions. When there is much non-coding DNA, a large proportion
appears to have no biological function, as predicted in the 1960s. Since
that time, this non-functional portion has controversially been called
"junk DNA".
The international Encyclopedia of DNA Elements (ENCODE) project uncovered, by direct biochemical approaches, that at least 80% of human genomic DNA has biochemical activity. Though this was not necessarily unexpected due to previous decades of research discovering many functional non-coding regions, some scientists criticized the conclusion for conflating biochemical activity with biological function. Estimates for the biologically functional fraction of the human genome based on comparative genomics range between 8 and 15%. However, others have argued against relying solely on estimates from comparative genomics due to its limited scope. Non-coding DNA has been found to be involved in epigenetic activity and complex networks of genetic interactions and is being explored in evolutionary developmental biology.
The amount of total genomic DNA varies widely between organisms, and
the proportion of coding and non-coding DNA within these genomes varies
greatly as well. For example, it was originally suggested that over 98%
of the human genome does not encode protein sequences, including most sequences within introns and most intergenic DNA, while 20% of a typical prokaryote genome is non-coding.
In eukaryotes, genome size, and by extension the amount of non-coding DNA, is not correlated to organism complexity, an observation known as the C-value enigma. For example, the genome of the unicellular Polychaos dubium (formerly known as Amoeba dubia) has been reported to contain more than 200 times the amount of DNA in humans. The pufferfishTakifugu rubripes
genome is only about one eighth the size of the human genome, yet seems
to have a comparable number of genes; approximately 90% of the Takifugu genome is non-coding DNA.
Therefore, most of the difference in genome size is not due to
variation in amount of coding DNA, rather, it is due to a difference in
the amount of non-coding DNA.
In 2013, a new "record" for the most efficient eukaryotic genome was discovered with Utricularia gibba, a bladderwort
plant that has only 3% non-coding DNA and 97% of coding DNA. Parts of
the non-coding DNA were being deleted by the plant and this suggested
that non-coding DNA may not be as critical for plants, even though
non-coding DNA is useful for humans.
Other studies on plants have discovered crucial functions in portions
of non-coding DNA that were previously thought to be negligible and have
added a new layer to the understanding of gene regulation.
Promoters facilitate the transcription of a particular gene and are typically upstream of the coding region. Enhancer sequences may also exert very distant effects on the transcription levels of genes.
Introns
Simple illustration of an unspliced mRNA precursor, with two introns and three exons (top). After the introns have been removed via splicing, the mature mRNA sequence is ready for translation (bottom).
Studies of group I introns from Tetrahymenaprotozoans
indicate that some introns appear to be selfish genetic elements,
neutral to the host because they remove themselves from flanking exons during RNA processing and do not produce an expression bias between alleles with and without the intron. Some introns appear to have significant biological function, possibly through ribozyme functionality that may regulate tRNA and rRNA
activity as well as protein-coding gene expression, evident in hosts
that have become dependent on such introns over long periods of time;
for example, the trnL-intron is found in all green plants and appears to have been vertically inherited for several billions of years, including more than a billion years within chloroplasts and an additional 2–3 billion years prior in the cyanobacterial ancestors of chloroplasts.
Pseudogenes
Pseudogenes are DNA sequences, related to known genes, that have lost their protein-coding ability or are otherwise no longer expressed
in the cell. Pseudogenes arise from retrotransposition or genomic
duplication of functional genes, and become "genomic fossils" that are
nonfunctional due to mutations that prevent the transcription of the gene, such as within the gene promoter region, or fatally alter the translation of the gene, such as premature stop codons or frameshifts.
Pseudogenes resulting from the retrotransposition of an RNA
intermediate are known as processed pseudogenes; pseudogenes that arise
from the genomic remains of duplicated genes or residues of inactivated genes are nonprocessed pseudogenes. Transpositions of once functional mitochondrial genes from the cytoplasm to the nucleus, also known as NUMTs, also qualify as one type of common pseudogene. Numts occur in many eukaryotic taxa.
While Dollo's Law
suggests that the loss of function in pseudogenes is likely permanent,
silenced genes may actually retain function for several million years
and can be "reactivated" into protein-coding sequences and a substantial number of pseudogenes are actively transcribed.
Because pseudogenes are presumed to change without evolutionary
constraint, they can serve as a useful model of the type and frequencies
of various spontaneous genetic mutations.
Over 8% of the human genome is made up of (mostly decayed)
endogenous retrovirus sequences, as part of the over 42% fraction that
is recognizably derived of retrotransposons, while another 3% can be
identified to be the remains of DNA transposons.
Much of the remaining half of the genome that is currently without an
explained origin is expected to have found its origin in transposable
elements that were active so long ago (> 200 million years) that
random mutations have rendered them unrecognizable. Genome size variation in at least two kinds of plants is mostly the result of retrotransposon sequences.
Telomeres
Telomeres are regions of repetitive DNA at the end of a chromosome, which provide protection from chromosomal deterioration during DNA replication.
Recent studies have shown that telomeres function to aid in its own
stability. Telomeric repeat-containing RNA (TERRA) are transcripts
derived from telomeres. TERRA has been shown to maintain telomerase
activity and lengthen the ends of chromosomes.
Junk DNA
The term "junk DNA" became popular in the 1960s. According to T. Ryan Gregory,
the nature of junk DNA was first discussed explicitly in 1972 by a
genomic biologist, David Comings, who applied the term to all non-coding
DNA. The term was formalized that same year by Susumu Ohno, who noted that the mutational load
from deleterious mutations placed an upper limit on the number of
functional loci that could be expected given a typical mutation rate.
Ohno hypothesized that mammal genomes could not have more than 30,000
loci under selection before the "cost" from the mutational load would
cause an inescapable decline in fitness, and eventually extinction. This
prediction remains robust, with the human genome containing
approximately 20,000 genes. Another source for Ohno's theory was the
observation that even closely related species can have widely
(orders-of-magnitude) different genome sizes, which had been dubbed the C-value paradox in 1971.
Though the fruitfulness of the term "junk DNA" has been questioned on the grounds that it provokes a strong a priori
assumption of total non-functionality and though some have recommended
using more neutral terminology such as "non-coding DNA" instead; "junk DNA" remains a label for the portions of a genome sequence for which no discernible function has been identified and that through comparative genomics analysis appear under no functional constraint suggesting that the sequence itself has provided no adaptive advantage. Since the late 70s it has become apparent that the majority of non-coding DNA in large genomes finds its origin in the selfish amplification of transposable elements, of which W. Ford Doolittle and Carmen Sapienza in 1980 wrote in the journal Nature:
"When a given DNA, or class of DNAs, of unproven phenotypic function
can be shown to have evolved a strategy (such as transposition) which
ensures its genomic survival, then no other explanation for its
existence is necessary."
The amount of junk DNA can be expected to depend on the rate of
amplification of these elements and the rate at which non-functional DNA
is lost. In the same issue of Nature, Leslie Orgel and Francis Crick wrote that junk DNA has "little specificity and conveys little or no selective advantage to the organism". The term occurs mainly in popular science and in a colloquial
way in scientific publications, and it has been suggested that its
connotations may have delayed interest in the biological functions of
non-coding DNA.
Several lines of evidence indicate that some "junk DNA" sequences are
likely to have unidentified functional activity and that the process of exaptation of fragments of originally selfish or non-functional DNA has been commonplace throughout evolution.
ENCODE Project
In 2012, the ENCODE project, a research program supported by the National Human Genome Research Institute, reported that 76% of the human genome's non-coding DNA sequences were transcribed and that nearly half of the genome was in some way accessible to genetic regulatory proteins such as transcription factors.
However, the suggestion by ENCODE that over 80% of the human genome is
biochemically functional has been criticized by other scientists,
who argue that neither accessibility of segments of the genome to
transcription factors nor their transcription guarantees that those
segments have biochemical function and that their transcription is selectively advantageous.
Furthermore, the much lower estimates of functionality prior to ENCODE
were based on genomic conservation estimates across mammalian lineages.
In response to such views, other scientists argue that the wide spread
transcription and splicing that is observed in the human genome directly
by biochemical testing is a more accurate indicator of genetic function
than genomic conservation because conservation estimates are relative
due to incredible variations in genome sizes of even closely related
species, it is partially tautological, and these estimates are not based
on direct testing for functionality on the genome.
Conservation estimates may be used to provide clues to identify
possible functional elements in the genome, but it does not limit or cap
the total amount of functional elements that could possibly exist in
the genome since elements that do things at the molecular level can be
missed by comparative genomics. Furthermore, much of the apparent junk DNA is involved in epigenetic regulation and appears to be necessary for the development of complex organisms.
In a 2014 paper, ENCODE researchers tried to address "the question of
whether nonconserved but biochemically active regions are truly
functional". They noted that in the literature, functional parts of the
genome have been identified differently in previous studies depending on
the approaches used. There have been three general approaches used to
identify functional parts of the human genome: genetic approaches (which
rely on changes in phenotype), evolutionary approaches (which rely on
conservation) and biochemical approaches (which rely on biochemical
testing and was used by ENCODE). All three have limitations: genetic
approaches may miss functional elements that do not manifest physically
on the organism, evolutionary approaches have difficulties using
accurate multispecies sequence alignments since genomes of even closely
related species vary considerably, and with biochemical approaches,
though having high reproducibility, the biochemical signatures do not
always automatically signify a function.
They noted that 70% of the transcription coverage was less than 1
transcript per cell. They noted that this "larger proportion of genome
with reproducible but low biochemical signal strength and less
evolutionary conservation is challenging to parse between specific
functions and biological noise". Furthermore, assay resolution often is
much broader than the underlying functional sites so some of the
reproducibly "biochemically active but selectively neutral" sequences
are unlikely to serve critical functions, especially those with
lower-level biochemical signal. To this they added, "However, we also
acknowledge substantial limitations in our current detection of
constraint, given that some human-specific functions are essential but
not conserved and that disease-relevant regions need not be selectively
constrained to be functional." On the other hand, they argued that the
12–15% fraction of human DNA under functional constraint, as estimated
by a variety of extrapolative evolutionary methods, may still be an
underestimate. They concluded that in contrast to evolutionary and
genetic evidence, biochemical data offer clues about both the molecular
function served by underlying DNA elements and the cell types in which
they act. Ultimately genetic, evolutionary, and biochemical approaches
can all be used in a complementary way to identify regions that may be
functional in human biology and disease.
Some critics have argued that functionality can only be assessed in reference to an appropriate null hypothesis.
In this case, the null hypothesis would be that these parts of the
genome are non-functional and have properties, be it on the basis of
conservation or biochemical activity, that would be expected of such
regions based on our general understanding of molecular evolution and biochemistry.
According to these critics, until a region in question has been shown
to have additional features, beyond what is expected of the null
hypothesis, it should provisionally be labelled as non-functional.
Evidence of functionality
Many non-coding DNA sequences must have some important biological function. This is indicated by comparative genomics studies that report highly conserved regions of non-coding DNA, sometimes on time-scales of hundreds of millions of years. This implies that these non-coding regions are under strong evolutionary pressure and positive selection. For example, in the genomes of humans and mice, which diverged from a common ancestor
65–75 million years ago, protein-coding DNA sequences account for only
about 20% of conserved DNA, with the remaining 80% of conserved DNA
represented in non-coding regions. Linkage mapping
often identifies chromosomal regions associated with a disease with no
evidence of functional coding variants of genes within the region,
suggesting that disease-causing genetic variants lie in the non-coding
DNA. The significance of non-coding DNA mutations in cancer was explored in April 2013.
Non-coding genetic polymorphisms play a role in infectious disease susceptibility, such as hepatitis C.[49] Moreover, non-coding genetic polymorphisms contribute to susceptibility to Ewing sarcoma, an aggressive pediatric bone cancer.
Some specific sequences of non-coding DNA may be features essential to chromosome structure, centromere function and recognition of homologous chromosomes during meiosis.
According to a comparative study of over 300 prokaryotic and over 30 eukaryotic genomes,
eukaryotes appear to require a minimum amount of non-coding DNA. The
amount can be predicted using a growth model for regulatory genetic
networks, implying that it is required for regulatory purposes. In
humans the predicted minimum is about 5% of the total genome.
Over 10% of 32 mammalian genomes may function through the formation of specific RNA secondary structures. The study used comparative genomics to identify compensatory DNA mutations that maintain RNA base-pairings, a distinctive feature of RNA
molecules. Over 80% of the genomic regions presenting evolutionary
evidence of RNA structure conservation do not present strong DNA
sequence conservation.
Non-coding DNA separates genes from each other with long gaps, so mutation in one gene or part of a chromosome, for example deletion or insertion, does not have a frameshift effect
on the whole chromosome. When genome complexity is relatively high,
like in the case of human genome, not only between different genes, but
also inside many genes, there are gaps of introns
to protect the entire coding segment and minimise the changes caused by
mutation. Non-coding DNA may perhaps serve to decrease the probability
of gene disruption during chromosomal crossover.
Regulating gene expression
Some non-coding DNA sequences determine the expression levels of
various genes, both those that are transcribed to proteins and those
that themselves are involved in gene regulation.
Transcription factors
Some non-coding DNA sequences determine where transcription factors attach.
A transcription factor is a protein that binds to specific non-coding
DNA sequences, thereby controlling the flow (or transcription) of
genetic information from DNA to mRNA.
Operators
An operator is a segment of DNA to which a repressor
binds. A repressor is a DNA-binding protein that regulates the
expression of one or more genes by binding to the operator and blocking
the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes. This blocking of expression is called repression.
Enhancers
An enhancer is a short region of DNA that can be bound with proteins (trans-acting factors), much like a set of transcription factors, to enhance transcription levels of genes in a gene cluster.
Silencers
A silencer is a region of DNA that inactivates gene expression when
bound by a regulatory protein. It functions in a very similar way as
enhancers, only differing in the inactivation of genes.
Promoters
A promoter is a region of DNA that facilitates transcription of a
particular gene when a transcription factor binds to it. Promoters are
typically located near the genes they regulate and upstream of them.
Insulators
A genetic insulator is a boundary element that plays two distinct
roles in gene expression, either as an enhancer-blocking code, or rarely
as a barrier against condensed chromatin. An insulator in a DNA
sequence is comparable to a linguistic word divider such as a comma in a sentence, because the insulator indicates where an enhanced or repressed sequence ends.
Pseudogene sequences appear to accumulate mutations more rapidly than coding sequences due to a loss of selective pressure.
This allows for the creation of mutant alleles that incorporate new
functions that may be favored by natural selection; thus, pseudogenes
can serve as raw material for evolution and can be considered "protogenes".
A study published in 2019 shows that new genes (termed de novo gene birth) can be fashioned from non-coding regions. Some studies suggest at least one-tenth of genes could be made in this way.
Long range correlations
A
statistical distinction between coding and non-coding DNA sequences has
been found. It has been observed that nucleotides in non-coding DNA
sequences display long range power law correlations while coding
sequences do not.
The current standard for forensic
DNA testing relies on an analysis of the chromosomes located within the
nucleus of all human cells. 'The DNA material in chromosomes is composed
of "coding" and "non-coding" regions. The coding regions are known as
genes and contain the information necessary for a cell to make proteins.
. . . Non-protein coding regions . . . are not related directly to
making proteins, [and] have been referred to as "junk" DNA.' The
adjective "junk" may mislead the lay person, for in fact this is the DNA
region used with near certainty to identify a person.
Genetic testing, also known as DNA testing, is used to identify changes in DNA sequence or chromosome structure. Genetic testing can also include measuring the results of genetic changes, such as RNA analysis as an output of gene expression, or through biochemical analysis to measure specific protein output.
In a medical setting, genetic testing can be used to diagnose or rule
out suspected genetic disorders, predict risks for specific conditions,
or gain information that can be used to customize medical treatments
based on an individual's genetic makeup.
Genetic testing can also be used to determine biological relatives,
such as a child's parentage (genetic mother and father) through DNA paternity testing, or be used to broadly predict an individual's ancestry.
Genetic testing of plants and animals can be used for similar reasons
as in humans (e.g. to assess relatedness/ancestry or predict/diagnose
genetic disorders), to gain information used for selective breeding, or for efforts to boost genetic diversity in endangered populations.
The variety of genetic tests has expanded throughout the years.
Early forms of genetic testing which began in the 1950s involved
counting the number of chromosomes per cell. Deviations from the
expected number of chromosomes (46 in humans) could lead to a diagnosis
of certain genetic conditions such as trisomy 21 (Down syndrome) or monosomy X (Turner syndrome). In the 1970s, a method to stain specific regions of chromosomes, called chromosome banding, was developed that allowed more detailed analysis of chromosome structure and diagnosis of genetic disorders that involved large structural rearrangements. In addition to analyzing whole chromosomes (cytogenetics), genetic testing has expanded to include the fields of molecular genetics and genomics which can identify changes at the level of individual genes, parts of genes, or even single nucleotide "letters" of DNA sequence. According to the National Institutes of Health, there are tests available for more than 2,000 genetic conditions, and one study estimated that as of 2017 there were more than 75,000 genetic tests on the market.
Types
Genetic testing is "the analysis of chromosoms (DNA), proteins, and certain metabolites in order to detect heritable disease-related genotypes, mutations, phenotypes, or karyotypes for clinical purposes." It can provide information about a person's genes and chromosomes throughout life.
There are a number of types of testing available, including:
Cell-free fetal DNA
(cffDNA) testing - a non-invasive (for the fetus) test. It is performed
on a sample of venous blood from the mother, and can provide
information about the fetus early in pregnancy. As of 2015 it is the most sensitive and specific screening test for Down syndrome.
Newborn screening
- used just after birth to identify genetic disorders that can be
treated early in life. A blood sample is collected with a heel prick
from the newborn 24–48 hours after birth and sent to the lab for
analysis. In the United States, newborn screening procedure varies state
by state, but all states by law test for at least 21 disorders. If
abnormal results are obtained, it does not necessarily mean the child
has the disorder. Diagnostic tests must follow the initial screening to
confirm the disease.
The routine testing of infants for certain disorders is the most
widespread use of genetic testing—millions of babies are tested each
year in the United States. All states currently test infants for phenylketonuria (a genetic disorder that causes mental illness if left untreated) and congenitalhypothyroidism (a disorder of the thyroid
gland). People with PKU do not have an enzyme needed to process the
amino acid phenylalanine, which is responsible for normal growth in
children and normal protein use throughout their lifetime. If there is a
buildup of too much phenylalanine, brain tissue can be damaged, causing
developmental delay. Newborn screening can detect the presence of PKU,
allowing children to be placed on special diets to avoid the effects of
the disorder.
Diagnostic testing
- used to diagnose or rule out a specific genetic or chromosomal
condition. In many cases, genetic testing is used to confirm a diagnosis
when a particular condition is suspected based on physical mutations
and symptoms. Diagnostic testing can be performed at any time during a
person's life, but is not available for all genes or all genetic
conditions. The results of a diagnostic test can influence a person's
choices about health care and the management of the disease. For
example, people with a family history of polycystic kidney disease (PKD)
who experience pain or tenderness in their abdomen, blood in their
urine, frequent urination, pain in the sides, a urinary tract infection
or kidney stones may decide to have their genes tested and the result
could confirm the diagnosis of PKD.
Carrier testing
- used to identify people who carry one copy of a gene mutation that,
when present in two copies, causes a genetic disorder. This type of
testing is offered to individuals who have a family history of a genetic
disorder and to people in ethnic groups with an increased risk of
specific genetic conditions. If both parents are tested, the test can
provide information about a couple's risk of having a child with a
genetic condition like cystic fibrosis.
Preimplantation genetic diagnosis - performed on human embryos prior to the implantation as part of an in vitro fertilization
procedure. Pre-implantation testing is used when individuals try to
conceive a child through in vitro fertilization. Eggs from the woman and
sperm from the man are removed and fertilized outside the body to
create multiple embryos. The embryos are individually screened for
abnormalities, and the ones without abnormalities are implanted in the
uterus.
Prenatal diagnosis - used to detect changes in a fetus's
genes or chromosomes before birth. This type of testing is offered to
couples with an increased risk of having a baby with a genetic or
chromosomal disorder. In some cases, prenatal testing can lessen a
couple's uncertainty or help them decide whether to abort the pregnancy. It cannot identify all possible inherited disorders and birth defects,
however. One method of performing a prenatal genetic test involves an
amniocentesis, which removes a sample of fluid from the mother's
amniotic sac 15 to 20 or more weeks into pregnancy. The fluid is then
tested for chromosomal abnormalities such as Down syndrome (Trisomy 21)
and Trisomy 18, which can result in neonatal or fetal death. Test
results can be retrieved within 7–14 days after the test is done. This
method is 99.4% accurate at detecting and diagnosing fetal chromosome
abnormalities. Although there is a risk of miscarriage associated with
an amniocentesis, the miscarriage rate is only 1/400. Another method of
prenatal testing is Chorionic Villus Sampling (CVS). Chorionic villi are
projections from the placenta that carry the same genetic makeup as the
baby. During this method of prenatal testing, a sample of chorionic
villi is removed from the placenta to be tested. This test is performed
10–13 weeks into pregnancy and results are ready 7–14 days after the
test was done. Another test using blood taken from the fetal umbilical cord is percutaneous umbilical cord blood sampling.
Predictive and presymptomatic testing
- used to detect gene mutations associated with disorders that appear
after birth, often later in life. These tests can be helpful to people
who have a family member with a genetic disorder, but who have no
features of the disorder themselves at the time of testing. Predictive
testing can identify mutations that increase a person's chances of
developing disorders with a genetic basis, such as certain types of cancer. For example, an individual with a mutation in BRCA1 has a 65% cumulative risk of breast cancer.
Hereditary breast cancer along with ovarian cancer syndrome are caused
by gene alterations in the genes BRCA1 and BRCA2. Major cancer types
related to mutations in these genes are female breast cancer, ovarian,
prostate, pancreatic, and male breast cancer.
Li-Fraumeni syndrome is caused by a gene alteration on the gene TP53.
Cancer types associated with a mutation on this gene include breast
cancer, soft tissue sarcoma, osteosarcoma (bone cancer), leukemia and
brain tumors. In the Cowden syndrome there is a mutation on the PTEN
gene, causing potential breast, thyroid or endometrial cancer. Presymptomatic testing can determine whether a person will develop a genetic disorder, such as hemochromatosis
(an iron overload disorder), before any signs or symptoms appear. The
results of predictive and presymptomatic testing can provide information
about a person's risk of developing a specific disorder, help with
making decisions about medical care and provide a better prognosis.
Pharmacogenomics
- determines the influence of genetic variation on drug response. When a
person has a disease or health condition, pharmacogenomics can examine
an individual's genetic makeup to determine what medicine and what
dosage would be the safest and most beneficial to the patient. In the
human population, there are approximately 11 million single nucleotide
polymorphisms (SNPs) in people's genomes, making them the most common
variations in the human genome. SNPs reveal information about an
individual's response to certain drugs. This type of genetic testing can
be used for cancer patients undergoing chemotherapy.
A sample of the cancer tissue can be sent in for genetic analysis by a
specialized lab. After analysis, information retrieved can identify
mutations in the tumor which can be used to determine the best treatment
option.
Non-diagnostic testing includes:
Forensic testing - uses DNA sequences to identify an individual
for legal purposes. Unlike the tests described above, forensic testing
is not used to detect gene mutations associated with disease. This type
of testing can identify crime or catastrophe victims, rule out or
implicate a crime suspect, or establish biological relationships between
people (for example, paternity).
Paternity testing
- uses special DNA markers to identify the same or similar inheritance
patterns between related individuals. Based on the fact that we all
inherit half of our DNA from the father, and half from the mother, DNA
scientists test individuals to find the match of DNA sequences at some
highly differential markers to draw the conclusion of relatedness.
Research testing - includes finding unknown genes, learning how
genes work and advancing understanding of genetic conditions. The
results of testing done as part of a research study are usually not
available to patients or their healthcare providers.
Medical procedure
Genetic
testing is often done as part of a genetic consultation and as of
mid-2008 there were more than 1,200 clinically applicable genetic tests
available.
Once a person decides to proceed with genetic testing, a medical
geneticist, genetic counselor, primary care doctor, or specialist can
order the test after obtaining informed consent.
Genetic tests are performed on a sample of blood, hair, skin, amniotic fluid (the fluid that surrounds a fetus during pregnancy), or other tissue. For example, a medical procedure called a buccal smear
uses a small brush or cotton swab to collect a sample of cells from the
inside surface of the cheek. Alternatively, a small amount of saline mouthwash
may be swished in the mouth to collect the cells. The sample is sent to
a laboratory where technicians look for specific changes in
chromosomes, DNA, or proteins, depending on the suspected disorders,
often using DNA sequencing. The laboratory reports the test results in writing to a person's doctor or genetic counselor.
Routine newborn screening tests are done on a small blood sample obtained by pricking the baby's heel with a lancet.
Risks and limitations
The
physical risks associated with most genetic tests are very small,
particularly for those tests that require only a blood sample or buccal
smear (a procedure that samples cells from the inside surface of the
cheek). The procedures used for prenatal testing carry a small but
non-negligible risk of losing the pregnancy (miscarriage) because they
require a sample of amniotic fluid or tissue from around the fetus.
Many of the risks associated with genetic testing involve the
emotional, social, or financial consequences of the test results. People
may feel angry, depressed, anxious, or guilty about their results. The
potential negative impact of genetic testing has led to an increasing
recognition of a "right not to know".
In some cases, genetic testing creates tension within a family because
the results can reveal information about other family members in
addition to the person who is tested. The possibility of genetic discrimination
in employment or insurance is also a concern. Some individuals avoid
genetic testing out of fear it will affect their ability to purchase
insurance or find a job.
Health insurers do not currently require applicants for coverage to
undergo genetic testing, and when insurers encounter genetic
information, it is subject to the same confidentiality protections as
any other sensitive health information. In the United States, the use of genetic information is governed by the Genetic Information Nondiscrimination Act (GINA) (see discussion below in the section on government regulation).
Genetic testing can provide only limited information about an
inherited condition. The test often can't determine if a person will
show symptoms of a disorder, how severe the symptoms will be, or whether
the disorder will progress over time. Another major limitation is the
lack of treatment strategies for many genetic disorders once they are
diagnosed.
Another limitation to genetic testing for a hereditary linked
cancer, is the variants of unknown clinical significance. Because the
human genome has over 22,000 genes, there are 3.5 million variants in
the average person's genome. These variants of unknown clinical
significance means there is a change in the DNA sequence, however the
increase for cancer is unclear because it is unknown if the change
affects the gene's function.
A genetics professional can explain in detail the benefits,
risks, and limitations of a particular test. It is important that any
person who is considering genetic testing understand and weigh these
factors before making a decision.
Other risks include incidental findings—a discovery of some possible problem found while looking for something else.
In 2013 the American College of Medical Genetics and Genomics (ACMG)
that certain genes always be included any time a genomic sequencing was
done, and that labs should report the results.
Direct-to-consumer genetic testing
Direct-to-consumer
(DTC) genetic testing (also called at-home genetic testing) is a type
of genetic test that is accessible directly to the consumer without
having to go through a health care professional. Usually, to obtain a
genetic test, health care professionals such as physicians, nurse
practitioners, or genetic counselors
acquire their patient's permission and then order the desired test,
which may or may not be covered by health insurance. DTC genetic tests,
however, allow consumers to bypass this process and purchase DNA tests
themselves. DTC genetic testing can entail primarily
genealogical/ancestry-related information, health and trait-related
information, or both.
There is a variety of DTC tests, ranging from tests for breast cancer alleles to mutations linked to cystic fibrosis. Possible benefits of DTC testing are the accessibility of tests to consumers, promotion of proactive healthcare, and the privacy of genetic information.
Possible additional risks of DTC testing are the lack of governmental
regulation, the potential misinterpretation of genetic information,
issues related to testing minors, privacy of data, and downstream expenses for the public health care system. In the United States, most DTC genetic test kits are not reviewed by the Food and Drug Administration (FDA), with the exception of a few tests offered by the company 23andMe.
As of 2019, the tests that have received marketing authorization by the
FDA include 23andMe's genetic health risk reports for select variants
of BRCA1/BRCA2, pharmacogenetic
reports that test for selected variants associated with metabolism of
certain pharmaceutical compounds, a carrier screening test for Bloom syndrome, and genetic health risk reports for a handful of other medical conditions, such as celiac disease and late-onset Alzheimer's.
Controversy
DTC
genetic testing has been controversial due to outspoken opposition
within the medical community. Critics of DTC testing argue against the
risks involved, the unregulated advertising and marketing claims, and the overall lack of governmental oversight.
DTC testing involves many of the same risks associated with any
genetic test. One of the more obvious and dangerous of these is the
possibility of misreading of test results. Without professional
guidance, consumers can potentially misinterpret genetic information,
causing them to be deluded about their personal health.
Some advertising for DTC genetic testing has been criticized as
conveying an exaggerated and inaccurate message about the connection
between genetic information and disease risk, utilizing emotions as a
selling factor. An advertisement for a BRCA-predictive genetic test for breast cancer stated: “There is no stronger antidote for fear than information.”
Apart from rare diseases that are directly caused by specific,
single-gene mutation, diseases "have complicated, multiple genetic links
that interact strongly with personal environment, lifestyle, and
behavior."
Ancestry.com, a company providing DTC DNA tests for genealogy purposes, has reportedly allowed the warrantless search of their database by police investigating a murder.
The warrantless search led to a search warrant to force the gathering
of a DNA sample from a New Orleans filmmaker; however he turned out not
to be a match for the suspected killer.
Government regulation in the United States
With regard to genetic testing and information in general, legislation in the United States called the Genetic Information Nondiscrimination Act
prohibits group health plans and health insurers from denying coverage
to a healthy individual or charging that person higher premiums based
solely on a genetic predisposition
to developing a disease in the future. The legislation also bars
employers from using individuals’ genetic information when making hiring, firing, job placement, or promotion decisions. The legislation, the first of its kind in the United States, was passed by the United States Senate on April 24, 2008, on a vote of 95-0, and was signed into law by President George W. Bush on May 21, 2008. It went into effect on November 21, 2009.
In June 2013 the US Supreme Court issued two rulings on human
genetics. The Court struck down patents on human genes, opening up
competition in the field of genetic testing. The Supreme Court also ruled that police were allowed to collect DNA from people arrested for serious offenses.
The American Academy of Pediatrics (AAP) and the American College of Medical Genetics (ACMG) have provided new guidelines for the ethical issue of pediatrics genetic testing and screening of children in the United States.
Their guidelines state that performing pediatric genetic testing
should be in the best interest of the child. In hypothetical situations
for adults getting genetically tested 84-98% expressing interest in
getting genetically tested for cancer predisposition.
Though only half who are at risk of would get tested. AAP and ACMG
recommend holding off on genetic testing for late-onset conditions until
adulthood. Unless diagnosing genetic disorders during childhood and
start early intervention can reduce morbidity or mortality. They also
state that with parents or guardians permission testing for asymptomatic
children who are at risk of childhood onset conditions are ideal
reasons for pediatrics genetic testing. Testing for pharmacogenetics and newborn screening is found to be acceptable by AAP and ACMG guidelines.
Histocompatibility
testing guideline states that it's permissible for children of all ages
to have tissue compatibility testing for immediate family members but
only after the psychosocial,
emotional and physical implications has been explored. With a donor
advocate or similar mechanism should be in place to protect the minors
from coercion and to safeguard the interest of said minor.
Both AAP and ACMG discourage the use of direct-to-consumer
and home kit genetic because of the accuracy, interpretation and
oversight of test content.
Guidelines also state that if parents or guardians should be encouraged
to inform their child of the results from the genetic test if the minor
is of appropriate age. If minor is of mature appropriate age and request
results, the request should be honored. Though for ethical and legal
reasons health care providers should be cautions in providing minors
with predictive genetic testing without the involvement of parents or
guardians. Within the guidelines AAP and ACMG state that health care provider
have an obligation to inform parents or guardians on the implication of
test results. To encourage patients and families to share information
and even offer help in explain results to extend family or refer them to
genetic counseling.
AAP and ACMG state any type of predictive genetic testing for all types
is best offer with genetic counseling being offer by Clinical genetics, genetic counselors or health care providers.
Israel
Israel
uses DNA testing to determine if people are eligible for immigration.
The policy where "many Jews from the Former Soviet Union (‘FSU’) are
asked to provide DNA confirmation of their Jewish heritage in the form
of paternity tests in order to immigrate as Jews and become citizens
under Israel's Law of Return" has generated controversy.
Costs
The cost of
genetic testing can range from under $100 to more than $2,000. This
depends on the complexity of the test. The cost will increase if more
than one test is necessary or if multiple family members are getting
tested to obtain additional results. Costs can vary by state and some
states cover part of the total cost.
From the date that a sample is taken, results may take weeks to
months, depending upon the complexity and extent of the tests being
performed. Results for prenatal testing are usually available more
quickly because time is an important consideration in making decisions
about a pregnancy. Prior to the testing, the doctor or genetic counselor
who is requesting a particular test can provide specific information
about the cost and time frame associated with that test.
The three major single-chromosome mutations: deletion (1), duplication (2) and inversion (3).
The two major two-chromosome mutations: insertion (1) and Translocation (2)
A chromosomal disorder, anomaly, aberration, or mutation is a missing, extra, or irregular portion of chromosomal DNA. It can be from a typical number of chromosomes or a structural abnormality in one or more chromosomes. Chromosome mutation was formerly used in a strict sense to mean a change in a chromosomal segment, involving more than one gene. The term "karyotype" refers to the full set of chromosomes from an individual; this can be compared to a "normal" karyotype for the species via genetic testing.
A chromosome anomaly may be detected or confirmed in this manner.
Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. There are many types of chromosome anomalies. They can be organized into two basic groups, numerical and structural anomalies.
Numerical disorders
This is called aneuploidy
(an abnormal number of chromosomes), and occurs when an individual
either is missing a chromosome from a pair (monosomy) or has more than
two chromosomes of a pair (trisomy, tetrasomy, etc.).
An example of trisomy in humans is Down syndrome,
which is a developmental disorder caused by an extra copy of chromosome
21; the disorder is therefore also called trisomy 21. Having an extra
copy of this chromosome means that individuals have three copies of each
of its genes instead of two, making it difficult for cells to properly
control how much protein is made. Producing too much or too little
protein can have serious consequences. Genes on chromosome 21 that
specifically contribute to the various symptoms of Down syndrome are now
being identified. The frequency of Trisomy 21 has been determined to be
a function of advanced maternal age.
An example of monosomy is Turner syndrome, where the individual is born with only one sex chromosome, an X.
Sperm aneuploidy
Exposure of males to certain lifestyle, environmental and/or occupational hazards may increase the risk of aneuploid spermatozoa. In particular, risk of aneuploidy is increased by tobacco smoking, and occupational exposure to benzene, insecticides, and perfluorinated compounds. Increased aneuploidy is often associated with increased DNA damage in spermatozoa.
Structural abnormalities
When the chromosome's structure is altered, this can take several forms:
Deletions: A portion of the chromosome is missing or deleted. Known disorders in humans include Wolf-Hirschhorn syndrome, which is caused by partial deletion of the short arm of chromosome 4; and Jacobsen syndrome, also called the terminal 11q deletion disorder.
Robertsonian translocation:
An entire chromosome has attached to another at the centromere - in
humans these only occur with chromosomes 13, 14, 15, 21, and 22.
Inversions: A portion of the chromosome has broken off, turned upside down, and reattached, therefore the genetic material is inverted.
Insertions: A portion of one chromosome has been deleted from its normal place and inserted into another chromosome.
Rings:
A portion of a chromosome has broken off and formed a circle or ring.
This can happen with or without loss of genetic material.
Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere.
Chromosome instability syndromes
are a group of disorders characterized by chromosomal instability and
breakage. They often lead to an increased tendency to develop certain
types of malignancies.
Inheritance
Most
chromosome abnormalities occur as an accident in the egg cell or sperm,
and therefore the anomaly is present in every cell of the body. Some
anomalies, however, can happen after conception, resulting in Mosaicism (where some cells have the anomaly and some do not). Chromosome anomalies can be inherited from a parent or be "de novo".
This is why chromosome studies are often performed on parents when a
child is found to have an anomaly. If the parents do not possess the
abnormality it was not initially inherited; however it may be transmitted to subsequent generations.
Acquired chromosome abnormalities
Most cancers, if not all, could cause chromosome abnormalities,
with either the formation of hybrid genes and fusion proteins,
deregulation of genes and overexpression of proteins, or loss of tumor
suppressor genes.
Furthermore, certain consistent chromosomal abnormalities can turn
normal cells into a leukemic cell such as the translocation of a gene,
resulting in its inappropriate expression.
DNA damage during spermatogenesis
During the mitotic and meiotic cell divisions of mammalian gametogenesis, DNA repair is effective at removing DNA damages. However, in spermatogenesis the ability to repair DNA damages decreases substantially in the latter part of the process as haploid spermatids undergo major nuclear chromatin remodeling into highly compacted sperm nuclei. As reviewed by Marchetti et al., the last few weeks of sperm development before fertilization
are highly susceptible to the accumulation of sperm DNA damage. Such
sperm DNA damage can be transmitted unrepaired into the egg where it is
subject to removal by the maternal repair machinery. However, errors in
maternal DNA repair of sperm DNA damage can result in zygotes with chromosomal structural aberrations.
Melphalan is a bifunctional alkylating agent frequently used in chemotherapy.
Meiotic inter-strand DNA damages caused by melphalan can escape
paternal repair and cause chromosomal aberrations in the zygote by
maternal misrepair. Thus both pre- and post-fertilization DNA repair appear to be important in avoiding chromosome abnormalities and assuring the genome integrity of the conceptus.
Detection
Depending on the information one wants to obtain, different techniques and samples are needed.
