Genetic testing

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Genetic_testing

 

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 congenital hypothyroidism (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.
• Genealogical DNA test - used to determine ancestry or ethnic heritage for genetic genealogy.
• 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.

In popular culture

Some possible future ethical problems of genetic testing were considered in the science fiction film Gattaca, the novel Next, and the science fiction anime series "Gundam Seed". Also, some films which include the topic of genetic testing include The Island, Halloween: The Curse of Michael Myers, and the Resident Evil series.

Ethics

Pediatric genetic testing

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.

Chromosome abnormality

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Chromosome_abnormality

 

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.
• Duplications: A portion of the chromosome is duplicated, resulting in extra genetic material. Known human disorders include Charcot-Marie-Tooth disease type 1A, which may be caused by duplication of the gene encoding peripheral myelin protein 22 (PMP22) on chromosome 17.
• Translocations: A portion of one chromosome is transferred to another chromosome. There are two main types of translocations:
  • Reciprocal translocation: Segments from two different chromosomes have been exchanged.
  • 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.

• For the prenatal diagnosis of a foetus, amniocentesis, chorionic villus sampling or circulating foetal cells would be collected and analysed in order to detect possible chromosomal abnormalities.
• For the preimplantational diagnosis of an embryo, a blastocyst biopsy would be performed.
• For a lymphoma or leukemia screening the technique used would be a bone marrow biopsy.

Karyotype

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Karyotype

 

Karyotyping is the process by which cytogeneticists take photographs of chromosomes in order to determine the chromosome complement of an individual, including the number of chromosomes and any abnormalities. The term is also used for the complete set of chromosomes in a species or in an individual organism and for a test that detects this complement or measures the number.

Karyotypes describe the chromosome count of an organism and what these chromosomes look like under a light microscope. Attention is paid to their length, the position of the centromeres, banding pattern, any differences between the sex chromosomes, and any other physical characteristics. The preparation and study of karyotypes is part of cytogenetics.

Karyogram of human male using Giemsa staining

The study of whole sets of chromosomes is sometimes known as karyology. The chromosomes are depicted (by rearranging a photomicrograph) in a standard format known as a karyogram or idiogram: in pairs, ordered by size and position of centromere for chromosomes of the same size. 

The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). Thus, in humans 2n = 46. 

So, in normal diploid organisms, autosomal chromosomes are present in two copies. There may, or may not, be sex chromosomes. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies. 

The study of karyotypes is important for cell biology and genetics, and the results may be used in evolutionary biology (karyosystematics) and medicine. Karyotypes can be used for many purposes; such as to study chromosomal aberrations, cellular function, taxonomic relationships, and to gather information about past evolutionary events.

History of karyotype studies

Chromosomes were first observed in plant cells by Carl Wilhelm von Nägeli in 1842. Their behavior in animal (salamander) cells was described by Walther Flemming, the discoverer of mitosis, in 1882. The name was coined by another German anatomist, Heinrich von Waldeyer in 1888. It is New Latin from Ancient Greek κάρυον karyon, "kernel", "seed", or "nucleus", and τύπος typos, "general form")

The next stage took place after the development of genetics in the early 20th century, when it was appreciated that chromosomes (that can be observed by karyotype) were the carrier of genes. Lev Delaunay in 1922 seems to have been the first person to define the karyotype as the phenotypic appearance of the somatic chromosomes, in contrast to their genic contents. The subsequent history of the concept can be followed in the works of C. D. Darlington and Michael JD White.

Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912, Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia, concluding an XX/XO sex determination mechanism. Painter in 1922 was not certain whether the diploid of humans was 46 or 48, at first favoring 46, but revised his opinion from 46 to 48, and he correctly insisted on humans having an XX/XY system. Considering the techniques of the time, these results were remarkable. 

Fusion of ancestral chromosomes left distinctive remnants of telomeres, and a vestigial centromere

 

In textbooks, the number of human chromosomes remained at 48 for over thirty years. New techniques were needed to correct this error. Joe Hin Tjio working in Albert Levan's lab was responsible for finding the approach:

1. Using cells in tissue culture
2. Pretreating cells in a hypotonic solution, which swells them and spreads the chromosomes
3. Arresting mitosis in metaphase by a solution of colchicine
4. Squashing the preparation on the slide forcing the chromosomes into a single plane
5. Cutting up a photomicrograph and arranging the result into an indisputable karyogram.

The work took place in 1955, and was published in 1956. The karyotype of humans includes only 46 chromosomes. The great apes have 48 chromosomes. Human chromosome 2 is now known to be a result of an end-to-end fusion of two ancestral ape chromosomes.

Observations on karyotypes

Staining

The study of karyotypes is made possible by staining. Usually, a suitable dye, such as Giemsa, is applied after cells have been arrested during cell division by a solution of colchicine usually in metaphase or prometaphase when most condensed. In order for the Giemsa stain to adhere correctly, all chromosomal proteins must be digested and removed. For humans, white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. Sometimes observations may be made on non-dividing (interphase) cells. The sex of an unborn fetus can be determined by observation of interphase cells (see amniotic centesis and Barr body).

Observations

Six different characteristics of karyotypes are usually observed and compared:

1. Differences in absolute sizes of chromosomes. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family. For example, the legumes Lotus tenuis and Vicia faba each have six pairs of chromosomes, yet V. faba chromosomes are many times larger. These differences probably reflect different amounts of DNA duplication.
2. Differences in the position of centromeres. These differences probably came about through translocations.
3. Differences in relative size of chromosomes. These differences probably arose from segmental interchange of unequal lengths.
4. Differences in basic number of chromosomes. These differences could have resulted from successive unequal translocations which removed all the essential genetic material from a chromosome, permitting its loss without penalty to the organism (the dislocation hypothesis) or through fusion. Humans have one pair fewer chromosomes than the great apes. Human chromosome 2 appears to have resulted from the fusion of two ancestral chromosomes, and many of the genes of those two original chromosomes have been translocated to other chromosomes.
5. Differences in number and position of satellites. Satellites are small bodies attached to a chromosome by a thin thread.
6. Differences in degree and distribution of heterochromatic regions. Heterochromatin stains darker than euchromatin. Heterochromatin is packed tighter. Heterochromatin consists mainly of genetically inactive and repetitive DNA sequences as well as containing a larger amount of Adenine-Thymine pairs. Euchromatin is usually under active transcription and stains much lighter as it has less affinity for the giemsa stain. Euchromatin regions contain larger amounts of Guanine-Cytosine pairs. The staining technique using giemsa staining is called G banding and therefore produces the typical "G-Bands".

A full account of a karyotype may therefore include the number, type, shape and banding of the chromosomes, as well as other cytogenetic information.

Variation is often found:

1. between the sexes,
2. between the germ-line and soma (between gametes and the rest of the body),
3. between members of a population (chromosome polymorphism),
4. in geographic specialization, and
5. in mosaics or otherwise abnormal individuals.

Human karyotype

human karyotype (male)

 

The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes (allosomes). Normal karyotypes for females contain two X chromosomes and are denoted 46,XX; males have both an X and a Y chromosome denoted 46,XY. Any variation from the standard karyotype may lead to developmental abnormalities.

Diversity and evolution of karyotypes

Although the replication and transcription of DNA is highly standardized in eukaryotes, the same cannot be said for their karyotypes, which are highly variable. There is variation between species in chromosome number, and in detailed organization, despite their construction from the same macromolecules. This variation provides the basis for a range of studies in evolutionary cytology. 

In some cases there is even significant variation within species. In a review, Godfrey and Masters conclude:

In our view, it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed ... But, used in conjunction with other phylogenetic data, karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species, which were previously inexplicable.

Although much is known about karyotypes at the descriptive level, and it is clear that changes in karyotype organization has had effects on the evolutionary course of many species, it is quite unclear what the general significance might be.

We have a very poor understanding of the causes of karyotype evolution, despite many careful investigations ... the general significance of karyotype evolution is obscure.

— Maynard Smith

Changes during development

Instead of the usual gene repression, some organisms go in for large-scale elimination of heterochromatin, or other kinds of visible adjustment to the karyotype.

• Chromosome elimination. In some species, as in many sciarid flies, entire chromosomes are eliminated during development.
• Chromatin diminution (founding father: Theodor Boveri). In this process, found in some copepods and roundworms such as Ascaris suum, portions of the chromosomes are cast away in particular cells. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost. In A. suum, all the somatic cell precursors undergo chromatin diminution.
• X-inactivation. The inactivation of one X chromosome takes place during the early development of mammals. In placental mammals, the inactivation is random as between the two Xs; thus the mammalian female is a mosaic in respect of her X chromosomes. In marsupials it is always the paternal X which is inactivated. In human females some 15% of somatic cells escape inactivation, and the number of genes affected on the inactivated X chromosome varies between cells: in fibroblast cells up about 25% of genes on the Barr body escape inactivation.

Number of chromosomes in a set

A spectacular example of variability between closely related species is the muntjac, which was investigated by Kurt Benirschke and his colleague Doris Wurster. The diploid number of the Chinese muntjac, Muntiacus reevesi, was found to be 46, all telocentric. When they looked at the karyotype of the closely related Indian muntjac, Muntiacus muntjak, they were astonished to find it had female = 6, male = 7 chromosomes.

They simply could not believe what they saw ... They kept quiet for two or three years because they thought something was wrong with their tissue culture ... But when they obtained a couple more specimens they confirmed [their findings].

— Hsu p. 73-4

The number of chromosomes in the karyotype between (relatively) unrelated species is hugely variable. The low record is held by the nematode Parascaris univalens, where the haploid n = 1; and an ant: Myrmecia pilosula. The high record would be somewhere amongst the ferns, with the adder's tongue fern Ophioglossum ahead with an average of 1262 chromosomes. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at 372 chromosomes. The existence of supernumerary or B chromosomes means that chromosome number can vary even within one interbreeding population; and aneuploids are another example, though in this case they would not be regarded as normal members of the population.

Fundamental number

The fundamental number, FN, of a karyotype is the number of visible major chromosomal arms per set of chromosomes. Thus, FN ≤ 2 x 2n, the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. Humans have FN = 82, due to the presence of five acrocentric chromosome pairs: 13, 14, 15, 21, and 22 (the human Y chromosome is also acrocentric). The fundamental autosomal number or autosomal fundamental number, FNa or AN, of a karyotype is the number of visible major chromosomal arms per set of autosomes (non-sex-linked chromosomes).

Ploidy

Ploidy is the number of complete sets of chromosomes in a cell.

• Polyploidy, where there are more than two sets of homologous chromosomes in the cells, occurs mainly in plants. It has been of major significance in plant evolution according to Stebbins. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30–35%, but in grasses the average is much higher, about 70%. Polyploidy in lower plants (ferns, horsetails and psilotales) is also common, and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants.Polyploidy in animals is much less common, but it has been significant in some groups.
  Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid.
• Haplo-diploidy, where one sex is diploid, and the other haploid. It is a common arrangement in the Hymenoptera, and in some other groups.
• Endopolyploidy occurs when in adult differentiated tissues the cells have ceased to divide by mitosis, but the nuclei contain more than the original somatic number of chromosomes. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication, the daughter chromosomes separating from each other inside an intact nuclear membrane.
  In many instances, endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). The cells do not always contain exact multiples (powers of two), which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate.
  This process (especially studied in insects and some higher plants such as maize) may be a developmental strategy for increasing the productivity of tissues which are highly active in biosynthesis.
  The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to humans; it is diverse and complex, and serves differentiation and morphogenesis in many ways.
• See palaeopolyploidy for the investigation of ancient karyotype duplications.

Aneuploidy

Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost. Abnormalities in chromosome number usually cause a defect in development. Down syndrome and Turner syndrome are examples of this.

Aneuploidy may also occur within a group of closely related species. Classic examples in plants are the genus Crepis, where the gametic (= haploid) numbers form the series x = 3, 4, 5, 6, and 7; and Crocus, where every number from x = 3 to x = 15 is represented by at least one species. Evidence of various kinds shows that trends of evolution have gone in different directions in different groups. In primates, the great apes have 24x2 chromosomes whereas humans have 23x2. Human chromosome 2 was formed by a merger of ancestral chromosomes, reducing the number.

Chromosomal polymorphism

Some species are polymorphic for different chromosome structural forms. The structural variation may be associated with different numbers of chromosomes in different individuals, which occurs in the ladybird beetle Chilocorus stigma, some mantids of the genus Ameles, the European shrew Sorex araneus. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast, that the two chromosome morphs are adapted to different habitats.

Species trees

The detailed study of chromosome banding in insects with polytene chromosomes can reveal relationships between closely related species: the classic example is the study of chromosome banding in Hawaiian drosophilids by Hampton L. Carson. 

In about 6,500 sq mi (17,000 km²), the Hawaiian Islands have the most diverse collection of drosophilid flies in the world, living from rainforests to subalpine meadows. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera, Drosophila and Scaptomyza, in the family Drosophilidae.

The polytene banding of the 'picture wing' group, the best-studied group of Hawaiian drosophilids, enabled Carson to work out the evolutionary tree long before genome analysis was practicable. In a sense, gene arrangements are visible in the banding patterns of each chromosome. Chromosome rearrangements, especially inversions, make it possible to see which species are closely related.

The results are clear. The inversions, when plotted in tree form (and independent of all other information), show a clear "flow" of species from older to newer islands. There are also cases of colonization back to older islands, and skipping of islands, but these are much less frequent. Using K-Ar dating, the present islands date from 0.4 million years ago (mya) (Mauna Kea) to 10mya (Necker). The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll, which can be dated to 30 mya. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer, at least into the Cretaceous. Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain.

All of the native Drosophila and Scaptomyza species in Hawaiʻi have apparently descended from a single ancestral species that colonized the islands, probably 20 million years ago. The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches. Although it would be possible for a single gravid female to colonise an island, it is more likely to have been a group from the same species.

There are other animals and plants on the Hawaiian archipelago which have undergone similar, if less spectacular, adaptive radiations.

Chromosome banding

Chromosomes display a banded pattern when treated with some stains. Bands are alternating light and dark stripes that appear along the lengths of chromosomes. Unique banding patterns are used to identify chromosomes and to diagnose chromosomal aberrations, including chromosome breakage, loss, duplication, translocation or inverted segments. A range of different chromosome treatments produce a range of banding patterns: G-bands, R-bands, C-bands, Q-bands, T-bands and NOR-bands.

Depiction of karyotypes

Types of banding

Cytogenetics employs several techniques to visualize different aspects of chromosomes:

• G-banding is obtained with Giemsa stain following digestion of chromosomes with trypsin. It yields a series of lightly and darkly stained bands — the dark regions tend to be heterochromatic, late-replicating and AT rich. The light regions tend to be euchromatic, early-replicating and GC rich. This method will normally produce 300–400 bands in a normal, human genome.
• R-banding is the reverse of G-banding (the R stands for "reverse"). The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions).
• C-banding: Giemsa binds to constitutive heterochromatin, so it stains centromeres.The name is derived from centromeric or constitutive heterochromatin. The preparations undergo alkaline denaturation prior to staining leading to an almost complete depurination of the DNA. After washing the probe the remaining DNA is renatured again and stained with Giemsa solution consisting of methylene azure, methylene violet, methylene blue, and eosin. Heterochromatin binds a lot of the dye, while the rest of the chromosomes absorb only little of it. The C-bonding proved to be especially well-suited for the characterization of plant chromosomes.
• Q-banding is a fluorescent pattern obtained using quinacrine for staining. The pattern of bands is very similar to that seen in G-banding.They can be recognized by a yellow fluorescence of differing intensity. Most part of the stained DNA is heterochromatin. Quinacrin (atebrin) binds both regions rich in AT and in GC, but only the AT-quinacrin-complex fluoresces. Since regions rich in AT are more common in heterochromatin than in euchromatin, these regions are labelled preferentially. The different intensities of the single bands mirror the different contents of AT. Other fluorochromes like DAPI or Hoechst 33258 lead also to characteristic, reproducible patterns. Each of them produces its specific pattern. In other words: the properties of the bonds and the specificity of the fluorochromes are not exclusively based on their affinity to regions rich in AT. Rather, the distribution of AT and the association of AT with other molecules like histones, for example, influences the binding properties of the fluorochromes.
• T-banding: visualize telomeres.
• Silver staining: Silver nitrate stains the nucleolar organization region-associated protein. This yields a dark region where the silver is deposited, denoting the activity of rRNA genes within the NOR.

Classic karyotype cytogenetics

Karyogram from a human female lymphocyte probed for the Alu sequence using FISH.

 

In the "classic" (depicted) karyotype, a dye, often Giemsa (G-banding), less frequently mepacrine (quinacrine), is used to stain bands on the chromosomes. Giemsa is specific for the phosphate groups of DNA. Quinacrine binds to the adenine-thymine-rich regions. Each chromosome has a characteristic banding pattern that helps to identify them; both chromosomes in a pair will have the same banding pattern.

Karyotypes are arranged with the short arm of the chromosome on top, and the long arm on the bottom. Some karyotypes call the short and long arms p and q, respectively. In addition, the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. For example, Cri du chat syndrome involves a deletion on the short arm of chromosome 5. It is written as 46,XX,5p-. The critical region for this syndrome is deletion of p15.2 (the locus on the chromosome), which is written as 46,XX,del(5)(p15.2).

Multicolor FISH (mFISH) and spectral karyotype (SKY technique)

Spectral karyogram of a human female

 

Multicolor FISH and the older spectral karyotyping are molecular cytogenetic techniques used to simultaneously visualize all the pairs of chromosomes in an organism in different colors. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. Because there are a limited number of spectrally distinct fluorophores, a combinatorial labeling method is used to generate many different colors. Fluorophore combinations are captured and analyzed by a fluorescence microscope using up to 7 narrow-banded fluorescence filters or, in the case of spectral karyotyping, by using an interferometer attached to a fluorescence microscope. In the case of an mFISH image, every combination of fluorochromes from the resulting original images is replaced by a pseudo color in a dedicated image analysis software. Thus, chromosomes or chromosome sections can be visualized and identified, allowing for the analysis of chromosomal rearrangements. In the case of spectral karyotyping, image processing software assigns a pseudo color to each spectrally different combination, allowing the visualization of the individually colored chromosomes.

Spectral human karyotype

Multicolor FISH is used to identify structural chromosome aberrations in cancer cells and other disease conditions when Giemsa banding or other techniques are not accurate enough.

Digital karyotyping

Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. This method is also known as virtual karyotyping.

Chromosome abnormalities

Chromosome abnormalities can be numerical, as in the presence of extra or missing chromosomes, or structural, as in derivative chromosome, translocations, inversions, large-scale deletions or duplications. Numerical abnormalities, also known as aneuploidy, often occur as a result of nondisjunction during meiosis in the formation of a gamete; trisomies, in which three copies of a chromosome are present instead of the usual two, are common numerical abnormalities. Structural abnormalities often arise from errors in homologous recombination. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body, or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. 

Chromosomal abnormalities that lead to disease in humans include

• Turner syndrome results from a single X chromosome (45,X or 45,X0).
• Klinefelter syndrome, the most common male chromosomal disease, otherwise known as 47,XXY, is caused by an extra X chromosome.
• Edwards syndrome is caused by trisomy (three copies) of chromosome 18.
• Down syndrome, a common chromosomal disease, is caused by trisomy of chromosome 21.
• Patau syndrome is caused by trisomy of chromosome 13.
• Trisomy 9, believed to be the 4th most common trisomy, has many long lived affected individuals but only in a form other than a full trisomy, such as trisomy 9p syndrome or mosaic trisomy 9. They often function quite well, but tend to have trouble with speech.
• Also documented are trisomy 8 and trisomy 16, although they generally do not survive to birth.

Some disorders arise from loss of just a piece of one chromosome, including

• Cri du chat (cry of the cat), from a truncated short arm on chromosome 5. The name comes from the babies' distinctive cry, caused by abnormal formation of the larynx.
• 1p36 Deletion syndrome, from the loss of part of the short arm of chromosome 1.
• Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing; a deletion of the maternal genes, example of imprinting disorder.
• Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing; a deletion of the paternal genes, example of imprinting disorder.

• Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual; one well-documented example is the Philadelphia chromosome, a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia.