Karyotyping in Genetic Counseling Programme Essay Example

Karyotyping in Genetic Counseling Programme Essay Example Karyotyping in Genetic Counseling Programme Paper Karyotyping in Genetic Counseling Programme Paper 1997). In another, clients were asked to rate counselling sessions in terms of clarity, depth of discussion and their willingness to raise issues; the ratings were examined for evidence of influence from the sex of the counsellor (Zare, 1984). However, both studies acknowledged the limitations of their approach, stressing the importance of relating such findings to qualitative analysis of the actual counselling sessions. Reported satisfaction is a questionable measure of process, since it does not necessarily relate to what actually occurred during the session. As Clarke et al. highlight, while research has focused on outcome, effectiveness is also fundamentally related to process. In their view, while outcome measures are valid in a research context, they are useless in practice, a position echoed by other commentators who argue that outcome measures used alone are both inappropriate and methodologically inadequate (Kessler, 1992). 3. Structural and Numerical Abnormalities There are two types of aberrations that karyotyping could be identified in the foetus – these are numerical and structural. Numerical aberrations depict loss or gain of chromosomes which might be one or more. The title aneuploidy has been given to such kinds of aberrations. The term trisomy expresses the occurrence of a single extra chromosome and the term polyploidy shows the occurrence of two or more chromosomes (Mosby, 2005). Structural chromosome rearrangements are considered to be the other main sort of aberration; this is an outcome of partition and reunification in a different configuration of chromosome. These aberrations also contain translocations, which includes the exchange of genetic materials among chromosomes. One of the most famous aberrations identified during the prenatal period are aneuploidies such as trisomy 21 (Down’s syndrome), trisomy 18 (Edward syndrome), trisomy 13 (Patau syndrome), and X and Y sex chromosome aneuploidies (Divane, 1994). 95% of live-born chromosomal aberrations occur as a result of them (Whiteman, 1991). Chronologically occurrences of various chromosome aberrations are very difficult in composition because certain aberrations have such negative side effect that the survival chances of the foetus are significantly reduced. Statistics and facts that are gathered on the occurrence of different aberrations on certain occasions must have to be present in relation to the number of births (which are before or after birth), occasionally in comparison to the numbers of amniotic samples examined, or at other times in terms of pregnancies. . Table1. Chromosome abnormalities commonly detected their frequency and consequences (Grimshaw, 2003). Chromosome number, shape, and size form the karyotype. In spite of the fact that every karyotype is varied for each organism, every cell in every organism has the same karyotype. Chromosomal abnormalities can be separated into two categories: numeric and structural (Figure 1) (Todd, 2000). Changes in chromosome numbers result in the addition (trisomy or triploidy) or loss (monosomy or aneuploidy) of a chromosome. Between and within chromosomes structural changes could appear. Regions between chromosomes can be traded (translocation) or donated from one to the other (insertion). In the same chromosome, regions can be lost (deletion), duplicated (amplification), or reversed (inversion). It is a challenge for medical professionals to correctly identify these structural alterations and following that counsel the patients (Todd, 2000). Figure:1 Diagrammatic representation of chromosomes and their structural alteration: A) Chromosomes 3 and 8 stained with giemsa (G-banding) at a resolution of the 400 band level. Each chromosome has a short (p) and long (q) arm that is separated by a centromere at one end and a telomere at the other. Chromosomes are described as metacentric (chromosome 3) or acrocentric (chromosome 8), depending on the position of the centromere. Bands and sub-bands are numbered from the centromere outward. B) Structural aberrations involving 2 chromosomes. Illustrated is a fragment from chromosomes 3 (yellow) and 8 (blue) undergoing translocation [t (3;8)(p21-pter;p21-pter)] and insertion [ins (3;8)(q21. 2-21. 3;q13. 3-22)]. C) Structural alterations involving a single chromosome illustrated (using chromosome 3) include amplification [dup(3)(p21-pter)], inversion [inv(3)(p21-pter)], and deletion [del(3)(p21-pter)] (Todd, 2000). Figure 2: The human karyotype and aberrations. A) The normal human karyotype consists of 46 chromosomes (23 pairs). Autosomes are chromosome pairs 1-22. The sex chromosomes consist of a pairing of the X and Y chromosomes (XX _ female; XY _ male). B) Each chromosome is composed of two chromatids. At the end of each chromatid is the telomere. The centromere (â€Å"clear zone†) is found in a centric or paracentric position. C) Numeric aberrations of the human karyotype appear in many diseases and syndromes. Most commonly, the change is an addition (trisomy) of loss (aneuploidy) of a chromosome. Down’s syndrome is an important example. D) Structural alterations are another important chromosomal aberration. Many types of structural alterations exist in human disease and syndromes. The Philadelphia chromosome, found in chronic myelogenous leukemia, is an important example and a major prognostic factor (Todd, 2000). 4. Methods Used in Genetic Counselling Programme 4. 1 Amniocentesis: Amniocentesis is one of several diagnostic tests that are carried out for mothers undergoing Genetic counselling. It detects the chromosome disorders that can occur in the unborn child. In this process, a sample of the fluid from the amnion is removed and then tested for disorders like Down’s syndrome, anaemia etc. This test is carried out during the 15th week of pregnancy. Amniotic fluid is used for different tests in the laboratory like karyotyping etc. However, amniocentesis increases the risk of miscarriage and therefore, this test is recommended only for women who have a high risk of chromosome abnormality. 4. 2 Conventional cytogenetics: For prenatal diagnosis the methods that are mainly used in genetic counselling are classified into two categories: Conventional Genetics and Molecular Cytogenetics (Bui, 2002). 4. 2. 1 Karyotyping In 1969 there was an expansion of karyotyping techniques for banding chromosomes, which allowed the detection of more subtle structural chromosome abnormalities. A karyotype is the exact organization (matching and alignment) of the chromosome complement of a cell. In a karyotype, chromosomes are arranged and numbered by size, from the largest to the smallest. Karyotype is the normal classification, which illustrates the normal or abnormal, constitutional or acquired chromosomal complement of an individual, tissue or cell line. To determine the numerical chromosomal abnormalities or structural rearrangements –mainly translocations- the conventional cytogenetic techniques should be used (Catalina, 2007). When full karyotype analysis is performed all the samples should be cultured enough so dividing cells are present. Then the cells are harvested, and the metaphase chromosomes are spread onto a microscope slide. The chromosomes are banded by enzyme digestion and then are analyzed by a cytogenetic expert. Advantages: Until now the gold standard for genetic tests is the conventional cytogenetic study, since it is the best one currently available for assessing the whole karyotype at one time. Moreover, it is inexpensive and detects abnormalities bigger than 3Mb in size (Catalina, 2007). Disadvantages: Only dividing cells can be assessed, there is a need for metaphase stage cells. No frozen tissue can be used. Moreover, it is a time-consuming method and due to the lack of automation in sample processing, the time needed to analyze and generate the final report is almost two weeks. Due to the difficulties of analyzing and interpreting the data, an experienced cytogenetic specialist is required (Catalina, 2007). 4. 2. 2 Molecular Cytogenetics Even though, Karyotyping remains the gold standard of chromosome analysis and still is the most frequently used genetic method in prenatal diagnosis, development of fluorescence in situ hybridization (FISH) technologies (Bui, 2002) is the most significant step in cytogenetics during the past 20 years. Moreover, over the past 30 years modifications in cytogenetic techniques have provided an opportunity to increase sensitive detection of chromosome abnormalities. The invention of FISH techniques has provided the most prominent advances in the fields of research and diagnosis. A complete dissection of complex chromosome rearrangements can be achieved by the new multicolour karyotyping techniques and also provides the prospect of identifying new recurring chromosome rearrangements. Comparatively interphase fluorescence in situ hybridization and genomic hybridization both hindered the use of metaphase chromosomes altogether and have allowed the genetic analysis of previously problematic and unidentifiable targets. New advances in comparative genomic hybridization to DNA microarrays help in achieving high resolution and automated screening for chromosomal imbalances. Rather than replacing conventional cytogenetics, these new techniques have extended the range of cytogenetic analyses when they are applied in a complementary fashion (Kearney, 2001). 4. 2. 2. 1 FISH Background: The most common practice that has been carried out in molecular cytogenetics is known as FISH. In 1988 it was first introduced in USA based clinical cytogenetics, and speedy progress has subsequently been witnessed in this field. In 1991 the first test was made in the UK. This method acquires chromosome-based probes accompanied by fluorescent labels which are attached to them; now these probes can be found in commercial kit form. Microscope systems are required for detection, which are available as basic fluorescence microscopes to advanced image analysis systems known as microscope and camera devices of cooled charged-coupled type. This test involves more effort and resources than presently practising FISH, which is a harder labour test than the existing karyotyping technique (Grimshaw, 2003). Prenatal diagnosis and FISH Test: Fluorescent In-Situ Hybridization studies have been conducted earlier on uncultured amniocytes with the acquisition of single chromosome-specific probes (for example for chromosome 21). However, these probes (centromeric repetitive or alphoid) showed vivid evidence of cross-hybridization between certain chromosomes (e. g. the two probes interacted for chromosomes 13 and 21). This finding paved the roads to the development of different types of probes (cosmid contig and YAC probes). It was successfully demonstrated that the usage of the cosmid prodes to identify Down’s syndrome (trisomy 21) in 1994, in a study of 500 uncultured amniotic fluid samples. After a couple of years, the UK introduced the use of YAC probes so to ensure speedy first reports on samples (Lowther, 1996). Although, evidence emerged which predicted that a mixture of five FISH probes could be utilised as a combined multicolour FISH hybridization experiment, when testing the five chromosomes most commonly linked with chromosomal abnormalities (21, 18, 13, X, Y). Further into that these 5-probe FISH test kits were manufactured and made available commercially (Grimshaw, 2003). Figure 3. Diagram of FISH procedures (Carpenter, 2001). A number of techniques, such as multicolour FISH (MFISH) and SKY FISH, have been developed from the FISH-based karyotyping of chromosomes. Fluorescent dyes used spectral karyotyping methods that jot together particular chromosomes regions. By utilizing a chain of specific probes each with changing quantity of dyes, unique spectral characteristics were found in different pairs of chromosomes (Catalina, 2007). There is a wide range of FISH techniques for both diagnostic and research applications. Since commercial availability of probes is increasing most clinical laboratories now use FISH as an addition to cytogenetic diagnosis. Metaphase FISH with specific gene probes provides an accurate assessment of rearrangements with a defined diagnostic or prognostic value, and interphase FISH provides the possibility of analysis on samples that would otherwise fail. One of the most significant advances has been in the development of multicolour FISH technologies which has culminated in FISH-based karyotyping methods. Metaphase CGH provides a global screening approach allowing the analysis of samples previously intractable to cytogenetic analysis. More recently, the development of CGH to DNA previously intractable to cytogenetic analysis. More recently, the development of CGH to DNA (Kearney, 2001). Advantages: FISH is a very rapid method, the results are ready within 24-48 hours. Also it is a sensitive and cost-effective and identifies both numerical and structural chromosomal abnormalities in interphase and metaphase nuclei, and permits rapid sex determination (Catalina, 2007). Limitations: However, FISH has some limitations such as cross-hybridization of non-specific fluorescence signals, non-specific background, and suboptimal signal strength. Though, small deletions, duplications and inversions cannot be identified by painting prodes (Catalina, 2007). 4. 2. 2. 2 Multicolour Whole-chromosome Painting (M-FISH AND SKY) The most prominent characteristic of FISH is its ability to simultaneously identify several targets by using variant colours (multicolour FISH). As early as 1989, as many as three targets could be visualized at the same time (Nederlof, 1989). By the early 1990s 7 ±12 different probes in different colours could be simultaneous detected (Dauwerse, 1992), (Ried, 1992). However, it was not until 1996 that developments in probe labelling and digital imaging systems allowed the visualization of the entire chromosome complement in 24 different colours (Schrock, 1996), (Speicher, 1996). The two techniques, M-FISH and SKY, both utilize DOP-PCR amplification of flow-sorted chromosomes and a ‘combinatorial’ labelling approach. The principle behind this for both M-FISH and SKY is the generation of more colours than there are fluorochromes available, by labelling with 1:1 mixtures of fluorochromes. The theoretical number of targets which can be discriminated in this way is 2n=1, where n represents the number of fluorochromes available. Using only five fluorochromes, this allows painting of the whole chromosome in twenty-four coloured complement (see Figure 5). Figure 5. M-FISH colour karyotype of a bone marrow metaphase from an AML patient. G-banding identified a balanced t(1;3)(p32;p13), and this was confirmed by M-FISH (arrows). However, M-FISH also identified a cryptic der(6)t(6;22) not visible by G-banding (arrow). Two copies of the der(6) are present in this cell (Kearney, 2001). The imaging system which is used to discriminate fluorochrome combinations is the only difference between SKY and M-FISH. M-FISH is acquiring different fluorochrome pictures for each of the five fluorochromes using specifically selected narrow band pass filter sets (Eils, 1998),(Speicher, 1996). SKY on the other hand uses a single exposure of the image and a grouping of cooled charge coupled device (CCD) imaging and Fourier transform spectrometry to analyze spectrum of the fluorochrome combinations (Schrock, 1996). Both of these methods use dedicated software to transfer the unique labelling combination for each chromosome into a pseudocolour. It is important to mention that both of them have already demonstrated hidden chromosome rearrangements in complex karyotypes such as in tumour cell lines and in haematological malignancies (Speicher, 1996), (Veldman, 1997). Disadvantages: As with other whole-chromosome painting methods, both M-FISH and SKY are not capable to detect small intrachromosomal rearrangements (inversions, deletions, duplications). Both techniques can not detect mosaic cells. In particular, the limit of resolution for telomeric rearrangements is 2 ±2. 5 Mb (Brown,2000), (Uhrig,1999). Additionally, to overcome these limitations complementary FISH approaches are required. In addition to this, latest reports state that although M-FISH and SKY have proved to be extremely useful in prenatal, postnatal, and cancer cytogenetics, these technologies have innate limitations that, in certain cases, could result in chromosomal misclassification. Most multicolour karyotyping errors have a similar mechanistic origin. Structural rearrangements, which compare non-homologous chromosome material, often come up in overlapping fluorescence at the interface of the translocated segments; called occasionally as â€Å"flaring† (Lu, 2000). This effect can obscure or alter the fluorescence pattern of adjacent chromatin, which could lead to misinterpretation (Lee, 2001). 4. 2. 2. 3 Comparative genomic hybridization (CGH) CGH is a technique that presents an overview of the whole genome and allows the detection of DNA copy number changes. It is a powerful option instead of chromosome banding and FISH. This method can detect a genome screening of chromosomal differencies without previous information about genomic regions which could be a potential target. CGH is a substitute method which reveals unbalanced chromosomal changes that may happen in hESCs lines during lengthy-span cultures, especially in cases where it seems difficult to obtain high quality metaphases (Catalina, 2007). Advantages and Limitations of CGH: The obvious edge of the CGH technique is that it requires only the genomic DNA; moreover, CGH does not require prior knowledge of the genomic region of interest. CGH can also identify copy number changes, increases and losses of regions of chromosome. Though, CGH is able to identify a number of quantitative genetic alterations including duplication or deletion of single chromosome bands. The CGH analysis also indicates the presence of genetic abnormalities that are not detected by other cytogenetic or molecular approaches. The turgidity of this technique in detecting low copy number gains or losses is in between 10-20Mb, therefore the detection limit of amplification is 2Mb. However, CGH has several limitations such as inability to detect chromosomal balanced translocation, inversions, and intragenic rearrangements (Catalina, 2007). Figure 7: Comparison of cytogenetic techniques for identifying chromosomal abnormalities (Speicher, 2005) Although the advances of the techniques utilized in genetic counselling are major, all of them come with their own share of advantages and disadvantages. The same is summarized in Table 2. Quantitative fluorescent polymerase chain reaction (QF-PCR) QF-PCR combines the benefits of relative and competitive RT-PCR. It is accurate, specific, high throughput and relatively easy to execute. Real time PCR automates the lengthy relative RT-PCR process by quantitating reaction products for each sample in every cycle. RT-PCR systems detect and quantify the fluorescent reporter. The signal of this reporter increases in direct proportion to the amount of PCR product in the reaction. The reporter is a double-strand DNA which is bound to a specific dye (SYBR Green) and upon excitation emits light. If the dye is included in PCR reaction as PCR product accumulates the fluorescence increase. An alternative technique for quantifying PCR products is TaqMan, which depends on fluorescent resonance energy transfer (FRET) of hybridization probes for quantitation. The probe hybridizes to an internal region of a PCR product. After irradiation the excited reporter dye transfers energy to the nearby quenching dye, which results in a non-fluorescent substrate. The advantages of this method are that it is inexpensive, simple to use, and sensitive The future of prenatal diagnosis: Full karyotype or molecular cytogenetics tests? The introduction of rapid molecular testing of all prenatal samples has brought up the question of the need for full karyotype analysis of all samples. When ultrasonography shows chromosomal abnormality, and there is no aneuploidy can be identified by frequent testing, full karyotyping is definitely required. However, when women have been identified by serum screening and/or maternal age as being at increased risk of Down’s syndrome they undergo persistent testing. â€Å"Double testing† of these women in a public-funded health service could be considered as unjustifiable and there are also down points for the parents. The time between the results of the rapid test and the full karyotyping could cause needless anxiety. Additionally most parents do not realize the significance of the full karyotyping; because they only worry about Down’s syndrome and not for the possibility of other abnormalities. Full karyotype analysis could identify abnormalities of unidentified importance, likewise the presence of very small â€Å"marker† chromosomes, clearly balanced chromosome rearrangements, or regions of variability, which could be hereditary. These outcomes could frequently create counselling difficulties, and cause problems for the parents in how to deduce and choose between anxiety and pregnancy termination during an ongoing pregnancy. When a chromosome rearrangement is found in one of the parents, full karyotyping is needed to test for abnormalities arising as a result of the rearrangement. However, recent advances in the policy of pre-implantation genetic diagnosis for rearrangement carriers (Scrivn, 1998) have permited rapid prenatal testing for chromosome imbalance using sub telomere probes (Pettenati, 2002). The UK National Screening Committee (UKNSC) suggested in 2004 that there is no need for karyotyping when screening for Down’s syndrome and instead prenatal diagnosis with FISH (fluorescence in-situ hybridisation) or PCR as rapid diagnostic tests as should be offered. Furthermore, UKNSC also suggested that the two previous tests should only be included for trisomies 13, 18, and 21. Before introducing the radical step of rapid testing alone for pregnancies at risk of Down’s syndrome, it is important to set up the significance and predicted effect of such a change in policy. Full karyotype results of prenatal samples from these referral categories can be audited to determine how many clinically significant chromosome abnormalities are likely to be undetected if rapid testing alone had been carried out. Recent studies which are investigativing karyotype abnormalities in prenatal samples referred for raised maternal age (RMA) or increased Down’s syndrome risk identified by serum screening will be reviewed in the following paragraph: In 1,130 prenatal samples, which were all referred for RMA or elevated risk of Down’s syndrome, an important clinical abnormality in chromosome 8 was identified (deletion of the short arm), which would not be identified by rapid testing (Thein et al). This is responsible for 0. 08% of the sample group. Thilaganathan et al. reported 3,203 amniotic fluid samples, were referred for a number of reasons. Rapid testing here did not detect all clinically significant abnormalities, which were detected with ultrasound. On another study, Ryall et al.reported 2,737 prenatal samples from pregnancies referred as serum screen +ve and an abnormality in chromosome 2 and 6 were detected. In the largest cohort study with 20,923 referrals around 30 important abnormalities were identified which includes four small marker chromosomes (Lewin et al. ). Among them, three pregnancies had trisomy 8, three trisomy 9, and three trisomy 16; and were all non-mosaic and therefore non-viable. Additionally thirteen cases of structurally abnormal chromosomes were identified, and complex abnormalities were found in four pregnancies. Rapid testing would detected as far as 99. 2% of clinical significant abnormalities, when pregnencies are reffered to RMA or serum +ve. In these studies, 196 balanced rearrangements or other good prognosis anomalies were found, which would have required parental karyotyping. This would lead to anxiety and in some cases termination of pregnancy. 5. Summary The future seems very exciting since the new developments in genetic information will present great challenges for genetic counselling. Medical doctors are currently facing problems in understanding and retaining genetic information outside their own scientific area. In some cases it is still not clear when test should be offered, since if a family is affected legal action could be used to question why an available test was not offered. Eventually, more diagnostic techniques would help to identify and treat more effectively, but not without false positives. Though, queries about which disorders to diagnose and when, (e. g. premarital, pre-conceptional, foetal, childhood, adulthood) will continue. It is important to mention that molecular cytogenetic increase the progress of prenatal diagnosis used in genetic counselling programmes to reveal chromosomal abnormality. The molecular cytogenetic techniques provide speed, accurate, ease and reliable diagnosis although there are some limitations associated with these techniques. However, if combination of the conventional techniques and molecular ones wil