chipping (of genes)

The genetic chip is an innovative technology for detailed testing to detect tiny deficiencies or excesses in the genome and greatly improves the ability to diagnose

Idit Maya and Mordechai Shochat Galileo

Classical genetics is divided into two main branches: cytogenetics, the "cellular" genetics, which refers to all genetic information in its basic packaging - the chromosomes; And molecular genetics, the "code" genetics, in which the nucleic acid sequence of DNA, which is the "letters" that encode the basic unit of information, the gene, is decoded.

In each of the nucleated cells of our body there are 23 pairs of chromosomes (46 in total), of which 22 pairs are identical for males and females, and one pair of sex chromosomes: two X chromosomes for females and one X and one Y chromosome for males. The DNA helix, the sequence of letters that constitutes the genetic information that is coded for building proteins, is folded and packed inside the chromosomes in an orderly and precise manner, enabling the functioning of each of our body's cells. You can compare all the genetic information in each of our body cells to a large encyclopedia with 46 volumes containing many chapters and many entries. Each such volume is a chromosome and inside it a very large number of letters of information.

In this article we will focus on cytogenetics and the different methods to diagnose deficiencies or excesses of different "volumes" or different "chapters" therein. We will mention the old and well-known methods on the one hand, and on the other hand we will focus on a modern cytogenetic method called the genetic chip.

The karyotype method: counting and staining

In the classic method, called "karyotype" and known in genetics for about 50 years, you look at a cell that is in the stages of division and in which you can see the chromosomes under a light microscope. With this method, the chromosomes are counted and by means of a special staining, called Giemsa staining, we also look at their internal structure, which is seen as a sort of black and white blur. With this method, the amount of chromosomes and their structure are checked, for example in white blood cells taken in a regular blood test, or from fetal cells extracted by amniotic fluid puncture or placental cyst testing (CVS). Both of these tests are invasive tests: in the placental cyst test, the area of ​​the placenta is reached through the vagina and a cell sample is taken from there. In amniotic fluid acupuncture, the membranous sac (amniotic sac) in which the fetus swims (and by way of the abdominal wall and the wall of the mother's uterus) is punctured in order to draw a sample of the amniotic fluid. From these samples, the fetal cells are filtered, grown in culture in the laboratory and tested using the karyotype method using the light microscope in search of cells that are in the stages of division (mitosis), since in these cells the chromosomes can be seen. It should be remembered that this is a procedure that is not without risks and it is known that following it there is a risk of miscarriage that is 1:300-1:500 in the amniotic fluid test and around 1:100 in the placental cyst test.

A karyotype test makes it possible to diagnose genetic syndromes resulting from a lack or excess of whole chromosomes, or of large segments within the chromosomes (of at least several million nucleotides). An example of a syndrome diagnosed by this method is Down syndrome, trisomy 21 (a trio of chromosomes number 21 instead of a pair of chromosomes). It is possible to diagnose Down's syndrome and other chromosomal problems both after birth and by examining placental cysts or amniotic fluid during pregnancy. It is also possible to perform a karyotype test on cancer cells originating from the bone marrow or various organs, in search of structural or quantitative changes in chromosomes that indicate different clinical characteristics of the tumors. For example, in cancer that originates in the bone marrow called "Chronic Myelocytic Leukemia" (CML), a structural change (translocation - copying a chromosome segment to another location) is common in the cancer cells, and in this case - a connection between chromosome 9 and chromosome 22. This structural change causes the growth of Controlled bone marrow cells and the appearance of the malignant tumor. According to these and other changes observed in the tumor cells, we can sometimes characterize the type of tumor and sometimes choose the most appropriate chemotherapy treatment.

The genetic chip: detection of extensive changes

Genetic chips are a state-of-the-art technology that allows checking many areas (hundreds of thousands of sites) in the subject's genetic material at once. There are different types of genetic chips; Of those that test changes of single bases (SNP: Single Nucleotide Polymorphism), that is, a change from one "letter" to another (between one nucleotide to another) within the genetic code, and of those that test differences of tiny segments between the patient sample and a control sample. The genetic chip is called in the scientific language CMA (Chromosomal Micro-arrayAnalysis) or by its other name Array Comparative Genomic Hybridization, and for short Array CGH. This is a new cytogenetic method, which allows the detection of smaller structural or quantitative changes than the classic karyotype test. With this method, all the chromosomes are separated into several thousand tiny segments and compared to control segments. If we find a lack or excess of such a tiny segment, then we can find out the content of the genes in it and thus know if it is possible to associate this laboratory finding with the set of symptoms in the patient. This test has already been conducted on thousands of healthy people, and thus we can know which of these quantitative changes (deficiency or excess) are common in the population and present in healthy individuals, and which changes are rare and can cause vulnerabilities such as mental retardation or birth defects. A finding that is present in one out of a hundred healthy individuals (that is, about 1% of a certain population) is usually treated as a common finding and a finding that is present at a lower frequency as rare.

Using the genetic chip method, it is possible to detect hundreds of well-known genetic syndromes, such as Palate-Cardiofacial Syndrome, also known as Velo-Cardiofacial Syndrome or D-George Syndrome (resulting from a deficiency of a small segment on chromosome 22), or Williams Syndrome (resulting from a deficiency of A tiny segment on chromosome 7; and see: "cracks in", Galileo 43, January-February 2001). Compared to the classical karyotype method, the resolution of this test is up to a hundred times higher, and it is possible to detect tiny deficiencies or excesses of several tens of thousands or hundreds of thousands of nucleotides. Moreover, with the old method it is impossible to diagnose syndromes resulting from the deficiency of such a small segment. On the other hand, balanced structural changes (such as balanced translocations) will not be detected by the genetic chip method, because there is no lack of a certain genetic sequence, but only a change in its position on the surface of the genome.

"Fishing" tiny chromosome damage

Another cytogenetic technology known as FISH (Fluorescent in Situ Hybridization) has existed for many years. In this method, fluorescent detectors are used to search in a targeted manner for a deficiency or an excess of a tiny segment at a specific location on the chromosome. For example, in cleft palate syndrome (D-George syndrome) a specific detector is used for the long arm of chromosome 22, in segment number 11, which is known to be missing in patients with this syndrome. Such a FISH test is done because of suspicion of the existence of the syndrome, due to clinical characteristics in the child or fetus such as heart defects, cleft palate and lip or immune deficiency. Compared to this test, the great advantage of the genetic chip test lies in the fact that it detects hundreds of such syndromes at the same time, without the need for a targeted detector, and in fact without a specific clinical suspicion. The chip test can also be performed on patients for whom we do not know the clinical diagnosis, such as patients suffering from mental retardation without accompanying clinical characteristics (such as congenital damage to the kidneys or heart) or patients with autism. It is also possible to use this method as part of a prenatal diagnosis through the examination of placental secretions or amniotic fluid even without any findings in the fetus.

There are various chips from competing companies that use this technology, for example from Blugnome and Signature Genomics. They are all based on the same principle, but divide all the genetic material under test into a different number of DNA segments. The resolution in this test will be greater if the DNA segments are smaller and more numerous. When we come to look for an explanation of a clinical picture in patients in whom genetic syndromes are suspected, we will use a chip with a higher resolution. On the other hand, in the examination of fetuses (if from a sample of the fetal cells extracted by acupuncture of the amniotic fluid and if appropriate taken from the placenta) chips with a less detailed resolution tend to be used to reduce the chance of having to deal with an answer whose clinical significance is unknown at this stage. The more such tests are carried out in Israel and in the world, on individuals with mental retardation as well as on healthy individuals in different populations of origin, the more information will be accumulated and then we will be able to know the meaning of tiny deficiencies or excesses, of a few tens of thousands or hundreds of thousands of nucleotides (such deficiencies and excesses are common) on the subject's health. In the future, if this technology becomes widely used, then we can also use the high-resolution chips in embryo tests.

Analysis of test results

There are three options for the results of this test, and these are:

A. Normal result: no deficiency or excess of a tiny chromosomal segment is found, or a deficiency or excess of a tiny segment is found, which is recognized as a "normal variation" in healthy people, so no medical problem is expected.

B. Incorrect result: We will detail below what are the options that this result brings up.

third. A result whose clinical significance is not entirely clear: a lack or excess of a tiny chromosomal segment was found, which means that it is apparently normal, but there may be a small doubt that cannot be proven at this stage due to a paucity of information in the medical literature.

If the result is not normal, that is - an excess or deficiency of a tiny chromosomal segment is found, which is clearly of medical significance - proof of a certain clinical indication has been found. In this case there are several options:

A. A lack or excess of a tiny chromosomal segment was found which means a severe genetic syndrome and/or mental retardation or severe autism. It is possible to confirm the presence of the deficiency or excess in most cases using the specific FISH test. This is a finding of clear clinical significance, both if the sample is from a patient and if it is from fetal cells. If it is a sample of fetal cells, you can debate with the couple whether to terminate the pregnancy or continue it. The presence of the genetic change in the chip test in a patient or fetus makes it possible to test his parents for carriers of this change, in order to know how to define it: is it a new occurrence (de novo) or does it already exist in one of the parents? This step is carried out through a targeted search for the lack or The excess in the parents in the FISH test. According to the parents' carriers, it is possible to give precise genetic counseling regarding the chance of the syndrome recurring in the future pregnancies of the parents and other family members, and even offer them diagnostic tests to detect the deficiency or excess.

B. A lack or excess of a chromosomal segment was found, which probably means a severe genetic syndrome, and the certainty about it is high (over 30%) but not absolute. There are several criteria that we take into account when we come to analyze the clinical significance of a lack or excess of such a chromosomal segment. The first and most important criterion is information about previous cases in the medical literature: is it found among patients with a certain clinical picture, and then there is a tendency to interpret the genetic change as pathological and clinically significant, or is it found in the healthy population and then there is a tendency to interpret the genetic change as unimportant? The reason there is no absolute certainty in these cases is that regarding clinical cases with certain tiny changes we still do not have sufficient information. In these situations, we analyze the data according to the size of the missing or excess genetic segment and the gene content in it. If the segment is very large, and contains many genes that are known to be important in the development and function of vital organs and pathways, or there are known Mendelian genetic syndromes caused by a point mutation in the sequence of these genes, then we assume that their complete lack will cause some clinical picture. The fourth characteristic that is taken into account in the analysis of the results of the genetic chip is the presence of the lack or excess in the parents. Although many cases of variation in the clinical expression have been described, when the parent with the deficiency only suffers from learning disabilities, and his offspring may cause severe cognitive impairment to the point of mental retardation, but still the presence of the deficiency or excess in one of the healthy parents will guide us to analyze the chip results as normal.

It is important to understand that in the face of an abnormal answer of this type if it is a sample of fetal cells, it is possible to debate with the couple whether to terminate the pregnancy or continue it. Even in the face of such a result, according to the results of the FISH test for the parents, we can assess the risk of recurrence in their future pregnancies.

third. A lack or excess of a tiny chromosomal segment was found, the clinical meaning of which is recognized health changes that can be treated, for example a tendency to diabetes in old age. Often these health changes exist among several family members, and the finding simply explains the existence of these phenomena or features in them. If it is detected in the fetus, it is not required to terminate the pregnancy.

Incorrect results

What is the chance of finding an abnormal result in a subject's blood sample using the genetic chip method? It is important to understand that the chance of finding an abnormal result, one that indicates one of the genetic syndromes that can be detected with this technology, depends on the source of the sample that was sent for genetic chip testing.

If it is a blood sample from a patient (child or adult) with developmental delay or mental retardation, in an isolated form (called non-syndromic mental retardation), or in combination with other physical findings (called syndromic mental retardation), then the chance of finding a positive finding ranges from -10 20% It is important to understand that the patient's blood sample is sent for this test in most cases after a karyotype test was performed for him and it was found to be normal; So a positive finding on the genetic chip is a great advance in terms of diagnostic ability (even though we still do not have an explanation for a significant portion of the cases), a diagnostic ability that was not available to us just a few years ago. If it is a blood sample from a patient (child or adult) with autism, the chance according to different series ranges from 5-10%. That is, in over 90% of autism cases, especially in the milder cases, we will not be able to find a change in the genetic chip test.

What is the chance of finding an abnormal result in a fetal cell sample on the genetic chip? The chance of finding a lack or excess of a tiny genetic segment in the genetic chip decreases considerably when it comes to fetal cell sampling (from benign fluid or placental cyst acupuncture) compared to the chance in patients with mental retardation. This chance is affected by the degree of suspicion and the risk factors that existed in the pregnancy before the test was performed, and what was the reason for performing the invasive test to collect the fetal cells in the first place. In the genetic chip method, DNA is extracted from the cell culture or from a direct sample of the amniotic fluid.

If the amniotic fluid acupuncture was performed due to a single abnormal finding in the ultrasound (such as multiple fingers, abnormal nuchal translucency, heart defect or structural deformity of the kidneys or skeleton), the chance of an abnormal finding in the genetic chip test is about 1:40, after found that the karyotype is normal. The greater the degree of severity of the ultrasound findings, the greater the chance of finding a lack or excess of a tiny chromosomal segment and can reach up to 10% (when it comes to multiple defects in major systems such as the heart, limbs, kidneys and brain).

If the acupuncture was performed due to a family history - a brother or a child with mental retardation or autism - the chance of an abnormal finding in the genetic chip is around 1:50-1:100. If the acupuncture was performed due to a mother's age over 35 years (the main reason for amniotic fluid tests in Israel) or because an increased risk of Down's syndrome is found in the routine pregnancy follow-up tests, such as biochemical screening in the first and second trimesters of pregnancy, then the chance of an abnormal finding on the chip is around 1:50. It is important to understand that even in pregnancies where the acupuncture was performed without any medical reason, but due to the parents' will only, there is still a chance of an abnormal finding in the genetic chip, but with a lower probability, around 1:100-1:200.

When is it recommended to do a genetic chip test when it is known that the classic karyotype is not normal? As mentioned, a karyotype test detects deficiencies, excesses or large structural changes in chromosomes. In most cases, the answer of this test is unequivocal: a correct or incorrect answer, such as in the case of chromosome trisomies, such as trisomy 21 which causes Down syndrome, or other trisomies: trisomy of chromosome 13 (Pato syndrome), trisomy 18 (Edwards syndrome) , the trisomy of the sex chromosome X. There are situations in which doubt remains regarding the clinical significance of the finding in the karyotype, such as in cases of an excess of an isolated chromosomal segment, whose gene content is unclear (a finding called a "marker" in the professional language), or in cases of the exchange of segments of genetic material between two Chromosomes (a finding called translocation). In these cases, the genetic chip test can add essential information - if there is a lack or excess of genetic information in the patient or the fetus. A finding of an abnormal genetic chip can explain a clinical picture in a patient, or confirm the suspicion that the clinical picture observed in the fetus is the result of a chromosomal change and lack or excess of essential genes. On the other hand, a normal genetic chip answer increases the confidence that the genetic load of the patient or the fetus is balanced, and that most likely the chromosomal finding observed in the karyotype test is clinically insignificant.

The genetic chip test: advantages and disadvantages

As you remember, this is a test whose resolution is 100 times higher than the classic karyotype test, so the chip test detects deficiencies or excesses that are too small to be detected by the old test. Second, it is inclusive and diagnoses changes in all of our genetic information, even without clinical suspicion of a specific syndrome, unlike the spot FISH tests. In addition to this, this method is also able to diagnose changes that exist only in some of our body's cells, in a condition called "mosaic", even when it is only 7% of the body's cells, a finding that was not detected in the karyotype test. Also, this is a relatively quick test that is obtained after ten days to two weeks. A quick answer is especially important when the test is performed on a sample of fetal cells when they want an answer as early as possible in the pregnancy to allow the parents to decide on the options for terminating it.

It is recommended to perform a chip test in every patient suffering from mental retardation or who has a suspicion of a genetic syndrome for which no explanation can be found through a karyotype test or other routine tests. It is possible to consider performing the test in patients with autism or in those who have been diagnosed with several congenital defects (such as a congenital heart defect). The indications for performing the genetic chip test are less unambiguous and agreed upon when it comes to fetal cells. There is a consensus that if structural defects in the various organs are detected by ultrasound during pregnancy, it is recommended to perform it: but regarding the other pregnancies, in which benign fluid acupuncture is performed for other reasons such as high maternal age or without any medical reason, it is not clear at this stage whether it is worth performing the test and what the cost ratio is - Benefit from its performance. The decision of the Association of Genetic Doctors in Israel at this stage is to inform every woman who undergoes invasive testing during pregnancy about the possibility of performing a genetic chip test on the fetal cell sample, and to allow her the choice of whether to perform it. It must be remembered that when using any new technology there may be errors in decoding the answers; Results that are currently decoded as normal may in the future turn out to indicate problems and vice versa: changes that are currently decoded as abnormal may in the future turn out to be benign.

One of the notable disadvantages of this method, and one of the obstacles on the way to accumulating information in this field, is the high cost of the test. This test is currently performed in some of the genetic institutes in Israel as well as in some private clinical laboratories in the world and the minimum cost is around NIS 4,000. The financing is from the private pocket of the patient and his family, and many of the patients whose arm does not achieve this are therefore denied the test.

Summary

Genetic counseling is a complex procedure that is important to many families. Deciphering a genetic chip test after an examination by a qualified person can make a significant contribution to the decisions of the family and future parents. The genetic chip test does not yet replace the standard karyotype test performed on a blood sample or benign fluid. Despite the reservations, the genetic chip test is an important test, with which much experience has already been gained, and it increases the resolution and increases the percentages of detection and diagnosis. Most likely, in the near future, when knowledge and experience increase, this test will be part of the routine tests performed on patients and fetal cell samples.

Dr. Eidit Maya She graduated from the medical school of the Hebrew University in Jerusalem with an MD degree. Specialist in internal medicine and currently specializes in a super-specialty in medical genetics at the Raphael Recanati Genetic Institute, Beilinson Hospital, Rabin Medical Center in Petach-Tikva.

Professor Mordechai (Motti) Shohat He graduated from the Tel Aviv University School of Medicine with an MD degree. Specialist in pediatrics and medical genetics and director of the Raphael Recanati Genetics Institute at Beilinson Hospital, Rabin Medical Center in Petach Tikva. Professor of Medical Genetics at Tel Aviv University.