- Basics of Genetics and Genomic InvestigationsPravin D Potdar
- Carrier Screening and Genetic Risk PredictionSunita Bijarnia-Mahay, Kanika Singh
- Pedigree Charting and Inheritance: Essential for Genetic CounselingDhanlaxmi Shetty
- Genetic Counseling: Principles and PracticeUsha Dave
- Psychosocial and Behavioral Aspects of Genetic CounselingGayatri Iyer, A Swarna Kumari, Qurratulain Hasan
- Credentials of a Genetic Counselor: Qualification, Skills, and Experience
HISTORY OF GENETICS AND MOLECULAR MEDICINE
Gregor Mendel was an Austrian scientist in the 1800, who was a founder of modern genetics for his laws of inheritance (Fig. 1). Unfortunately, Mendel could not get recognition for his important work in genetics during his lifetime. However, it was only once his papers were rediscovered in the early 20th century by scientists who realized his findings applied to explain many observed patterns of inheritance. Mendelian inheritance patterns follow Mendelian law of segregation, independent assortment and dominance. Understanding the concepts of human genetics, the role of genes, behavior, and the environmental factors are important for applying genetic and genomic technologies advances for improving diagnosis of various diseases.
Since 1901, several Nobel Laureate scientists were involved in innovation in technologies developed in the field of Genetics and Molecular Medicine. In 1910, Albrecht Kossel was awarded the first Nobel Prize for his chemical descriptions of deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). The whole world learned about the nitrogen bases that comprise DNA and RNA are Adenine, Thymine, Guanine, Cytosine, and Uracil often represented by A, T, G, C, and U, respectively. In 1933, Thomas H Morgan, was awarded the Nobel Prize for his work that gave very important information about heritability which passed to the next generation. These foundational discoveries in the nascent field of genetics are named after Dr Morgan. In 1958, George Wells Beadle, Edward Lawrie Tatum, and Joshua Lederberg, were given the Nobel Prize for their research findings showing that DNA sequences contain information required to make proteins. In 1959, Ochoa and Kornberg described very important findings on how new copies of DNA and RNA are made. In 1963, Francis Crick, James Watson, and Maurice Wilkins were the most well-known Nobel laureates who were able to determine the double-stranded structure of DNA. After this work, Robert W Holley, Har Gobind Khorana, and Marshall W Nirenberg, 41968 were awarded the Nobel Prize for their discoveries describing how information in the DNA sequence is coded and further shown that DNA sequences are segmented into discrete 3-base units, known as codons. Paul Burg, Walter Gilbert, and Frederick Sanger, 1980 awarded the Nobel Prize for their role in developing scientific methods that allow us to determine the sequence of DNA. In 1983, Barbara McClintock, was awarded the Nobel Prize for her work describing the ability of DNA to move between locations within the genome. Thereafter Kary B Mullis and Michael Smith, awarded Nobel prize for their work in establishing scientific methods that allow us to study particular regions of the DNA. Mullis developed a technique known as PCR. This method is used to make numerous copies of a specific region of DNA. Roger Kornberg, 2006, won the Nobel prize for his work in describing and imaging proteins responsible for reading DNA. In 2009, Elizabeth Blackburn, Carol Greider, and Jack Szostak, 2009 have won the Nobel Prize for their work describing the structure and maintenance of telomeres, regions of DNA located at the ends of chromosomes. This chapter on basic molecular genetics will provide fundamental information about basic concepts in genetics and molecular biology as well as the innovative technology involved in diagnosis and therapies of various diseases. It will also emphasize the role of genetic counseling in understanding hereditary factors involved in various genetic disorders and how it can be prevented by genetic counseling.
HUMAN CELLS AND ITS COMPONENTS
The human body consists of 11 important organ systems which work together to maintain the functioning of the human body. All these organs are made of bunches of cells. A cell's cytoplasm is enclosed in cell membrane, which contains many biomolecules such as proteins and nucleic acids. Cells are visible under a light microscope whereas under electron microscopy gives a much higher resolution showing greatly detailed cell structure (Fig. 2).
5Electron microscopic studies have shown that there are several types of organelles suspended into the cytoplasm of cells. It includes nucleus, mitochondria, Golgi apparatus, endoplasmic reticulum and lysosomes. The cytosol is having the gelatinous fluid with various organelles suspended in it. Each of these organelles has a specific function.
Cell Nucleus
Cell nucleus is a membrane-bound large organelle that is found in human cells. It has double membrane nuclear envelope that encloses the entire organelle and isolates its contents from the cellular cytoplasm and the nuclear matrix. The cell nucleus contains multiple long linear DNA molecules in a complex with a large variety of proteins, called histones, to form chromosomes. Various genes are structured within these chromosomes which promote cell functions and therefore nucleus is also called the powerhouse of the cell.
Deoxyribonucleic Acid
Deoxyribonucleic acid (DNA) is a molecule that carries genetic information of all living cells. The DNA consists of two strands wind around one another to form a double helix structure as shown in Figure 3. Each strand is made of alternating sugar (deoxyribose) and phosphate groups and one of four bases—adenine (A), cytosine (C), guanine (G), and thymine (T) is attached to each sugar molecule. The double helix structure of DNA molecules was first discovered by Crick and Watson in 1953 for which they got the Nobel Prize in Medicine in year 1963. The most common methods of DNA 6extraction are phenol/chloroform extraction methods which isolate DNAs from proteins and lipids. After the DNA is extracted from the sample, it can be analyzed, by restriction fragment length polymorphism (RFLP) analysis or qualitative and quantitative analysis by polymerase chain reaction (PCR) to diagnose any disease.
Ribonucleic Acid
Ribonucleic acid (RNA) is a single stranded polymeric molecule that plays very important roles in coding, decoding, regulation and expression of genes. Each nucleotide of RNA molecules contains a ribose sugar with four bases such as adenine (A), cytosine (C), guanine (G), or uracil (U). The adenine and guanine are purines, and cytosine and uracil are pyrimidines (Fig. 3). These bases form hydrogen bonds between cytosine and guanine, between adenine and uracil and between guanine and uracil. There are major three types of RNA which are involved in regulation of gene expression and protein synthesis include—messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA). In protein synthesis, tRNA delivers amino acids to the ribosome, where rRNA then links amino acids together to form coded proteins. In the transcription process information in a strand of DNA is copied into a new molecule, mRNA by the enzyme called RNA polymerase. This mRNA carries information from DNA to the ribosome at the sites of protein synthesis. This process is called “translation”. The most prominent examples of noncoding RNAs which are involved in the process of translation are tRNA and rRNA. RNA is further characterized into small RNA and long RNA. Large RNAs, mainly include lncRNA and mRNA whereas, small RNAs include rRNA, tRNA, microRNA (miRNA) and small interfering RNA (siRNA).
Proteins
Proteins are macromolecules having one or more long chains of amino acid residues. Proteins perform various functions within cells. They provide structure to cells, help in transporting molecules from one location to another, catalyzing metabolic reactions, involved in DNA replication and also respond to various stimuli. Proteins differ from one another primarily in their sequence of amino acids present and by the nucleotide sequence of their genes. The linear chain of amino acid residues in protein is called a polypeptide whereas protein containing less than 20–30 residues are commonly called peptides or oligopeptides. In all proteins, amino acid residues are bonded together by peptide bonds. The protein lifespan is measured in terms of its half-life which range from minutes to years. In mammalian cells it is around 1–2 days. It is now estimated that in the human body there are almost 80,000–400,000 proteins. Proteins can be purified by using a variety of techniques such as ultracentrifugation, precipitation, electrophoresis, and chromatography. The proteins have primary, secondary, tertiary, and quaternary protein structure and these structures are very much useful in understanding the nature and function of each level of protein. Protein can be studied by various technologies such as immunohistochemistry, site-directed mutagenesis, X-ray crystallography, nuclear magnetic resonance and mass spectrometry. Insulin is the first protein sequenced by Frederick Sanger in 1949 for which he was awarded Noble prize in Medicine in 1958. In protein, genetic code is having a three-nucleotide set called codons and each three-nucleotide combination designates an amino acid. Proteins can be informally divided into three main classes such as globular proteins, 7fibrous proteins and membrane proteins. All globular proteins are enzymes. Fibrous proteins are the major component of connective tissue, or keratin and membrane proteins often serve as receptors.
Chromosomes
Three German scientists, Dr Matthias Schleiden, Rudolf Virchow and Theodor Schwann were the first scientists who published the structures of chromosomes which are presently familiar to us. Thereafter in 1923, Dr Theophilus Painter counted 24 pairs of chromosomes, i.e., 48 chromosomes under the microscope. However, his error was corrected only in 1956 when Dr Joe Hin Tjio, an Indonesian cytogeneticist, who determined its true number of chromosomes to 46. In general chromosomes are defined as an organized package of DNA found in the nucleus of the cell. In humans there are 23 pairs of chromosomes in which 22 pairs of numbered chromosomes are called autosomes, and one pair of chromosome called sex chromosomes and designate as X and Y chromosome. It is shown that each parent contributes one chromosome to each pair so that offspring get half of their chromosomes from their mother and half from their father.
Chromosomes are made up of a long DNA molecule having genetic material of respective organisms. Chromosomes have its packaging proteins called histones which bind DNA molecules and condense it to maintain its integrity (Fig. 4). The histone protein provides structural support to a chromosome and gives a more compact and complex three-dimensional structure which plays a significant role in transcriptional regulation.
During cell division, mitosis is referred to as a specialized process which separates the duplicated genetic material carried in the nucleus. In metaphase, chromosomes are duplicated and this phase is called S phase. These duplicated copies of chromosomes are joined by a centromere, resulting in an X-shaped structure. During this process, chromosome segregation occurs to form pairs of homologous chromosomes which are separated from each other and migrate to opposite poles of the nucleus. These joined copies are now called sister chromatids. During metaphase the X-shaped structure chromosomes are formed which are highly condensed and thus easiest to distinguish for chromosomal analysis. These metaphase chromosomes aligned in the center of the cell in their condensed form are normally visible under a light microscope which can be photographs, counted and easily karyotype for genetic study.
Meiosis is a special type of cell division of germ cells which produce the gametes. In meiosis, the homologous chromosomes duplicate exchange genetic information during the first division, called meiosis I. Then it divides again in meiosis II by 8splitting up sister chromatids to form haploid gametes. These two haploid gametes are fused together again during fertilization to form a diploid cell with a complete set of paired chromosomes.
Chromosomal and Mitochondrial Inheritance
Mitochondria is one of the important organelles found in cells and is often called the powerhouse of the cell. Mitochondrial DNA (MtDNA) is a special type of DNA present in human mitochondria which is circular in size. Mitochondrial DNA contains 37 genes. The main characteristic of MtDNA is that this DNA is maternally inherited. Males and females inherit a copy of MtDNA from their mother which is passed entirely unchanged through the maternal line and cannot pass to their offspring through males MtDNA. Offspring can only inherit a copy of MtDNA from their mother only and not from father. This mode of inheritance is called mitochondrial inheritance. There are mitochondrial DNA testing services available which can help to determine maternal lineage.
Mitochondrial Diseases
Mitochondria are widely spread in the human body and control incredibly diverse functions. Therefore, the diseases of the mitochondria are also very much diverse. The severity of a mitochondrial disease in a child depends on the percentage of abnormal mutations in mitochondria in the cell that formed him or her. If abnormalities are present in the mother's mitochondria, it will be inherited by her offspring but if the father has this abnormality, he will not pass this defect to his children as males do not pass on their MtDNA to their offspring. The ineffective MtDNA functioning can lead to the cell malfunctioning or cellular death altogether. The brain, heart, liver, skeletal muscles, kidney and endocrine and respiratory systems organs are also affected by MtDNA diseases.
They most commonly cause of neuromuscular diseases called mitochondrial myopathies that have typical symptoms of muscular weakness, loss of tone and restricted movement as well as sensory loss and loss of motor control which include Leigh syndrome, Leber's hereditary optic neuropathy, Wolff-Parkinson-White syndrome: Diabetes and Deafness and other diseases include abnormalities of the muscles in the gastrointestinal tract, limbs, heart, lungs, etc. Another subcategory is mitochondrial myopathies include Kearns-Sayre syndrome (KSS), mitochondrial depletion syndrome (MDS), mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS). Mitochondrial diseases are intensive and cause mild to severe organ dysfunction resulting in a poor quality of life, often leading to death.
Karyotype and Abnormalities
Karyotyping is the technology which analyses the pairs of all chromosomes of the cell to provide the snapshot of an individual's chromosomes. Karyotypes are prepared by using standard staining protocol which will reveal the characteristic structural features of each chromosome. Karyotyping can detect changes in chromosome number associated with polyploidy or aneuploidy of the cells. It also detects more specific structural changes, such as chromosomal deletions, duplications, translocations and inversions occurring in the cells to some avoidable conditions. Thus, karyotyping is becoming an important diagnostic test which gives information about specific birth defects, 9genetic disorders, and even development of cancers in humans.
Detection of chromosomal abnormalities by doing karyotype: Now days G-banded karyotyping is routinely used to diagnose a wide range of chromosomal abnormalities in the individuals. Aneuploidy, which is often caused by the absence or addition of a chromosome, is simply detected by karyotype analysis. Similarly, translocations can be very well confirmed by doing karyotype. Researchers often are interested in identifying candidate genes on specific chromosomes by doing karyotype. This work was greatly facilitated by the completion of the Human Genome Project in 2003 by Dr Francis Collins. Several researchers are now able to implement various molecular cytogenetic techniques to achieve even higher resolution of genomic changes including fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) that can potentially identify abnormalities in chromosomes at individual genes level. Presently, molecular cytogenetics is a dynamic discipline which can implement innovative technologies for diagnosis and therapies for various genetic disorders. In humans, 20% of conceptions have chromosomal defects. Many times it is found that the irregularities of chromosomes are as a result of a failure of meiosis in the production of sperm and ova. There are two major categories of chromosomal abnormalities. One is irregular number of chromosomes and second structural modification in a chromosome. Both these abnormalities usually result from nondisjunction errors during meiosis process.
Irregular number of chromosomes: The major error that shows up in karyotypes of human cells is a variation in the number of chromosomes from the normal 46. Normally human cells have 46 chromosomes (23 pairs) where both mother and father equally contribute 23 numbers of chromosomes each resulting in 23 homologous pairs in their child. However, in disease conditions it can be complete multiples of sets, e.g., 23 + 23 + 23 which is called polyploidy. Whereas there can be an addition or loss of chromosomes within a set, e.g., 23 + 22 or 23 + 24 is called aneuploidy. When there is one or few chromosome abnormalities found in chromosome number it is called a monosomy. But when one or many more chromosomes are abnormal the condition is called a trisomy because one homologous pair has three chromosomes instead of two.
Structural modification of a chromosome: The structural modification of a chromosome is a potentially devastating kind of error that occurs in the human chromosome. This usually occurs when there is a breakage and loss of a portion of a chromatid arm or a reunion of the arm of chromosome at a different site of another chromosome. This is also called a translocation of specific chromosomes to other numbered chromosomes such as 9:22 translocation in chronic myeloid leukemia. In most of these cases, the actual cause of these breakages is not yet known. However, experimentally breakage can be created with radiation, with some chemicals, and with viruses.
Mosaicism: Chromosomal abnormalities do not exist in every human cells. However sometimes there is a mosaic pattern shown by some cells and tissues which are carrying these abnormalities. It is thought that mosaicism usually results from mutations that occur during mitosis at an early stage of embryonic development. It is now possible that we can link specific medical syndromes to the particular chromosomal errors. 10The examples of these mosaicism are Mosaic Down syndrome and Mosaic Klinefelter syndrome.
Complete and incomplete dominance: Locus is a specific position of genes on the chromosome. Each locus has an allelic form and complete set of alleles in an individual is its genotype. The visible effect of these alleles on the structure or function of that individual is called its phenotype. Complete dominance is called when only one allele in the genotype is seen in the phenotype. Take an example of brown eyes, it is a characteristic which exhibits complete dominance. Someone who has a copy of the gene for brown eyes will always have brown eyes. Incomplete dominance is a mixture of the alleles in the genotype which are seen in the phenotype. Best example for incomplete dominance is a Tay-Sachs disease. When one parent has straight hair and the other have curly hair and if the born child has wavy hair, this dominance is an example of incomplete dominance. Codominance is a relationship between two alleles of a gene where no allele is recessive and thus the phenotypes of both alleles are expressed. The ABO blood group is a best example of codominance. In ABO group, A and B alleles are codominant with each other. When the person has both A and B, he will have type AB blood group. In codominance, it does not matter whether the alleles in the homologous chromosomes are dominant or recessive.
GENETIC DEFECTS
A genetic disorder is caused by one or more abnormalities in the human genome. It can be caused by a mutation in a single gene, in multiple genes and by the chromosomal abnormality. The mutation responsible to cause disease can occur spontaneously before embryonic development or it can be inherited from their parents who are carriers of a faulty gene is called autosomal recessive inheritance. Whereas, when this has been inherited from the parent who is already suffering from this disorder is called autosomal dominant inheritance. Some disorders are caused by a mutation on the X chromosome. There are more than 6,000 known genetic disorders in which around 600 diseases can be treatable. Around 1 out of 50 people are suffering from known single-gene mutation and around 1 out of 263 people are affected by chromosomal abnormalities and around 65% of people are suffering due to congenital genetic mutations. Cancers are also one of the genetic disorders which are caused by genetic mutations in which some are hereditary whereas others are caused by some other factors.
X-inactivation
X-inactivation of chromosomes is defined when one of the copies of the X chromosome is inactivated in female mammals. The inactive X chromosome is silenced by it and is packaged into a transcriptionally inactive structure called heterochromatin. In 1961, Mary Lyon, a geneticist, first figured out that in females who have two copies of the X chromosome, one copy of each gene is turned off permanently in one chromosome or another. So that females, who have two copies of the X chromosome, and males, who have one copy of the X chromosome, can both operate fairly normally. So this process of turning off one copy of one gene or another on the X chromosome is called lyonization of X-inactivation. This is so-called X-linked disease. The X- linked disease in the female, inherits one copy of abnormal gene and one copy of normal gene. The abnormal gene is 11always turned off and the normal gene is always allowed to stay on. Some of the examples of disease include females with hemophilia B, myotubular myopathy, X-linked hemolytic anemia, X-linked thrombocytopenia. RETT syndrome is a neurodevelopmental disorder caused by mutations in the X-linked MeCP2 gene; it is characterized by autism, dementia and ataxia. Becker muscular dystrophy is also one of the X-inactivation diseases.
Barr Bodies
Barr bodies are important for regulation of X-linked gene products which are being transcribed. In a normal female with the genotype 46 XX have 1 Barr body. Whereas, XX females have one Barr body per cell and XXX females have 2 Barr bodies per cell. In XXY Klinefelter syndrome, males have one Barr body per cell. No Barr bodies are observed in XY male. No Barr body is found in Turner's syndrome because they only have one X. So Turner's syndrome patient, has 45 chromosomes and one sex chromosome, thus has no Barr bodies and is therefore it is called as X-chromatin negative. Turner syndrome, is a condition where one of the X sex chromosome is missing. This disease is only found in females. Turner syndrome can cause a variety of developmental problems including short height, failure of the ovaries to develop and heart defects.
Mutations
Mutation is a change in DNA sequence, either due to error in DNA replication process or due to exposure to environmental factors such as UV light and cigarette smoke. Base substitutions, deletions and insertions are the major three types of DNA mutations observed in the cells. Mutations may or may not produce discernible changes in the characteristics or phenotype of an organism. Mutations play an important role in both normal as well abnormal biological processes including evolution, cancer, and the development of the immune system. Various diseases are identified according to their mutational pattern such as point mutation is caused in various cancers, sickle cell anemia, beta-thalassemia, cystic fibrosis, etc. Chromosomal mutation in caused in leukemia and lymphomas and many other genetic disorders and copy number variation with gene expression in breast cancer. Several neurological disorders are diagnosed with their extended repeat sequences such as Huntington disease, spinocerebellar ataxia (SCA), Fragile X syndrome, etc.
Expression and Penetrance
Expressivity is defined as the degree of phenotype expressed by the individuals having a particular genotype. Expressivity is related to the intensity of a given phenotype and it differs from penetrance. Penetrance is referred to as the proportion of individuals with a particular genotype but actually expresses the phenotype. The example of this disease is that the multiple people with the same disease can have the same genotype but one may express more severe symptoms, while another carrier may appear normal. These differences in expression can be influenced by epigenetic factors, environmental conditions and various genes modifiers. The cis-regulatory elements, an epigenetic factor, can also cause variability in expression. Marfan syndrome, Van der Woude syndrome, and neurofibromatosis are three common syndromes that involve phenotypic variability due to expressivity. Mutations in the FBN1 gene on chromosome 15 which encodes fibrillin-1 is responsible for the 12cause of Marfan syndrome. This syndrome affects connective tissue in the body and also cardiovascular disease. Another example is neurofibromatosis (NF1) also known as Von Recklinghausen disease. It is a genetic disorder that is caused by a mutation in the neurofibromin gene NF1 on chromosome 17. A loss of function mutation in the tumor suppressor gene can cause tumors on the nerves called neurofibromas.
Penetrance is defined as the proportion of measure that of individuals in a population who carry a specific gene and express the related phenotype. Just take an example of an autosomal dominant disorder which has 95% penetrance, that means 95% of those people with the mutation will develop that particular disease, whereas 5% will not develop this disease by this mutation. It is said that in autosomal dominant inherited disorder there is a complete penetration (100%) when clinical symptoms are present in all individuals causing this mutation. Their 100% penetrance is neurofibromatosis type 1. Highly penetrant alleles, and highly heritable symptoms can be easily demonstrated. Clinicians and geneticist can easily notice the alleles which are highly penetrant and show symptoms which are highly heritable.
Genome Imprinting: Epigenetics
Genomic imprinting is a phenomenon where genes can be expressed in a parent-of-origin-specific manner. These include Prader-Willi and Angelman syndromes, Silver-Russell syndrome, Beckwith-Wiedemann syndrome, Albright hereditary osteodystrophy, uniparental disomy and male infertility. So far there are 260 imprinted genes in mice and 228 imprinted genes in humans are reported. There are maternal imprinting and parental imprinting. Maternal imprinting in which the allele of a particular gene inherited from the mother, which is transcriptionally silent and the paternally inherited allele is active whereas in paternal imprinting paternally-inherited allele is silenced and the maternally-inherited allele is active.
GENETIC DIAGNOSTIC TESTS
Karyotyping
Chromosomal karyotyping is one of the diagnostic tests, which evaluates the structure and number of chromosomes present in cell type to access the abnormality present if any. Karyotype tests can be used for finding out birth defects or genetic disorders such as cancer. Suppose you have had trouble getting pregnant or have had several miscarriages, the doctor may want to check whether you or your partner have any chromosomal problem. Similarly this test can find out if you have a disorder that you could pass down to your child or have any genetic issue. Karyotyping can also find the cause of certain physical or developmental problems of your baby or young child facing in their life span.
Karyotyping can be done by using following materials.
Chorionic villus sampling (CVS): Small sample of your baby's cells (between 10 and 13 weeks of gestation) from the chorionic villi is sent to the laboratory for karyotyping analysis. Results of this study will diagnose whether your baby is normal or has any chromosomal abnormality such as Down syndrome with trisomy 13 or trisomy 18, or other genetic disorders. This testing is only to be recommended by your doctor because it may cause some risk to the baby.
Amniocentesis: For amniocentesis, doctors get samples of your baby's cells by taking a small amount of amniotic fluid that 13surrounds your baby in the womb between 15 and 20 weeks of gestation, with a long needle through your abdomen. They send the cells to a laboratory for karyotyping analysis. Test results will give a report for any abnormality present in his or her chromosomes karyotyped.
Bone marrow aspiration, biopsy or whole blood: Bone marrow, biopsy or whole blood sample is taken for karyotyping to detect cancer or a blood disorder. They usually take it from your hip bone with a special needle sometimes under local anesthesia.
Karyotyping Protocol for Peripheral Blood Lymphocyte Culture
Collect 2 mL of fresh whole blood in the heparin tube under strict aseptic conditions. Prepare growth medium containing 7 mL of RPMI 1640 medium with Pen/Strep antibiotics with L-glutamine and 100 µL of phytohemagglutinin. Mix well and add 0.7 mL of whole blood in a 15 mL culture tube. The culture is mixed well by inverting gently several times and incubated at 37°C in the CO2 incubator for 70–72 hours. The tubes are placed at the angle of 45 degrees, so that the culture can grow efficiently. Harvesting this culture after 72 hours by addition of 100 µL of 0.2% colcemid. Again incubate it for 2 hours at the same condition. The culture tube is then centrifuged at 3,500 rpm for 8–10 minutes at room temperature. The supernatant is discarded and the pellet is mixed with 8 mL of hypertonic solution. Repeat the centrifugation and the obtained pellet is resuspended in 2 mL of chilled fixative containing 3 part methanol and 1 part glacial acetic acid. Repeat the fixative-centrifugation step until clear-white pellet appears. Dissolve the pellet in the 2 mL fixative and store the tube at 4°C until further study.
Preparation of Slide and Giemsa Staining
Prepared Grease free slides by washing with proper detergents and kept it in the freeze till further use. While making the slide, drop down 2–3 drops of the culture solution from around 2 feet height on the chilled slide. Heat fix the slide by heating it gently on the heater at 37°C for a few minutes and then stain it with Giemsa stain for 5–10 minutes. After staining, wash the slide with the running tap water and dry it and observe under an inverted microscope having 100X lens. Take 10 good spread metaphases photographs from each slide for interpreting the results (Figs. 5A and B).
Figs. 5A and B: (A) G-banding karyotype of a normal human cell; (B) Fluorescence in situ hybridization (FISH) to localization of BCR-ABL signal in chronic myeloid leukemia (CML).
Fluorescence in situ Hybridization Technology
Fluorescence in situ hybridization technology is used for detecting specific genes of interest on specific chromosomes. The technique is mainly based on the principle of exposing chromosomes to a small DNA sequence called a probe that has attached a fluorescent molecule to it for visualization. Several FISH probes are available in the cytogenetic laboratory to diagnose many types of chromosomal abnormalities in patients. For FISH testing, we can use cells or patient's tissue, which is especially colored by using a specific probe which is attached to specific parts of certain chromosomes in order to visualize and count them under a fluorescent microscope for detecting presence of abnormal genes (Fig. 5B). The conventional karyotyping is limited to the detection of rearrangements involving more than 5 Mb of DNA. The resolution of the FISH technique is about 100 kb–1 Mb in size. FISH technology is very much useful for understanding the presence of HER2 gene expression for targeted therapy of breast cancer. FISH testing usually gives positive or negative reports. Positive means your breast cancer cells make too much HER2 and your doctor should treat you with drugs that target that protein and negative means the protein is not involved in the growth of your tumor.
There are presently several applications of FISH technology for diagnosis of various cancer and many genetic disorders. Some of these disorders are myelogenous leukemia, acute lymphoblastic leukemia, 22q13 deletion syndrome, chronic Cri-du-chat, velocardiofacial syndrome, and Down syndrome. The analysis of chromosomes 21, X, and Y by FISH is enough to identify oligozoospermic individuals at risk in infertility cases.
Chromosomal Microarray Analysis
Chromosomal microarray analysis (CMA) technology is an innovative and sensitive genetic testing to detect specific chromosomal abnormalities which cannot be detected by routine karyotyping method. This chromosomal microarray technology is mainly the recommended for the first-line genetic test for any developmental delay (DD) disorder or intellectual disability (ID) or autism spectrum disorders (ASD). However, CMA does not identify fragile X syndrome (FXS), which is also an intellectual disability disorder. In prenatal diagnosis with normal karyotype, CMA is able to diagnose a clinically significant chromosomal aberration in almost 1% of structurally normal pregnancies. There are two CMA techniques used: (1) comparative genomic hybridization (CGH) and (2) single nucleotide polymorphisms (SNP).
Comparative Genomic Hybridization
Comparative genomic hybridization (CGH) based arrays mainly compare a patient's DNA with normal control DNA to identify areas that are either over- or under-expressed in the patient sample. In the CGH technology patient's and control DNA samples are cut into fragments then labeled with different fluorescent colors usually green and red. The probes are mixed together in equal proportions and placed on a glass slide array having multiple probes from representative sequences across the human genome. These DNA templates are hybridized in a competitive manner to complementary sequences of DNA probes on the array slide. In postnatal studies, most of the laboratories which are performing CGH will report clinically significant in the range of 50–100 Kb whereas, in prenatal studies it may vary according to the indication for testing.15
Single Nucleotide Polymorphisms Microarray Analysis
Single nucleotide polymorphisms microarray analysis (SOMA) uses high-density oligonucleotide-based arrays in which target probes are chosen from DNA locations which vary from individuals by a single base pair mutation. In the SOMA technology, only a patient's fetal DNA is labeled and hybridized to the SNP array. The fluorescence probe intensities of patient samples are compared with intensities of normal controls, which give exact copies of abnormal genes on specific chromosomes. Most SNP arrays used in a clinical setting contain both SNP probes and copy number probes. The density of these probes on these hybrid arrays is as high as 2.7 million probes. Uniparental disomy (UPD), mosaicism, zygosity, maternal cell contamination, parent of origin and consanguinity are very well evaluated by SOMA. Lastly, triploidy which is not detected by CGH, can be identified by using SOMA technology.
Inborn Errors of Metabolism and Mass Spectroscopy
Inborn errors of metabolism (IEM) are also called congenital or inherited metabolic disorders. In 1908, British physician Archibald Garrod described the term inborn errors of metabolism. Traditionally the inherited metabolic diseases are classified as disorders of carbohydrate metabolism such as G6PD deficiency, amino acid metabolism such as phenylketonuria, organic acid metabolism such as 2-hydroxyglutaric acidurias or lysosomal storage diseases such as Gaucher's disease. Several congenital metabolic diseases are detectable by newborn screening tests by using mass spectrometry (MS). There is a revolution in gas chromatography–mass spectrometry (GCMS) based technology with an integrated analytics system, which has now made it possible to test newborn errors for more than 100 genetic metabolic disorders.
Metabolomics is a new approach to the diagnosis of IEM, when the clinical presentations of diseases are non-specific. Metabolomic analysis is mainly focused on the complete set of all small molecule metabolites present in biological specimens. It has stated that there are more than 700 different metabolites linked to IEM and more than 400 endogenous metabolites are identified by MS-based metabolomics technology. Besides, diagnosis, metabolomics can lead to the discovery of new IEM, novel biomarkers, and a better overall understanding of IEM. It is most important to develop advanced bioinformatics solutions, computer methodologies and software to convert the huge amount of data generated by this approach into effective clinically actionable tools that can aid in decision-making. The field of IEM continues to grow as innovative technologies such as NGS. The comprehensive metabolomic profiling strategies will provide deeper insight into mechanisms of disease and phenotypic differences between individuals with the same disorder. Although the number of IEM is daunting, a systematic and logical approach to test selection in a patient with a clinical presentation of a metabolic defect, regardless of age, can lead to a high degree of diagnostic success.
Molecular Genetic Methods
Since successful implementation of the human genome project in 2003, several new innovative technologies have been developed which reshape human genomic approaches for better diagnosis and therapies of various diseases. Nucleic acid–based testing is 16becoming a crucial diagnostic tool not only for the diagnosis of inherited genetic disease but also used in diagnosis and therapies of a wide variety of neoplastic and infectious diseases. The molecular testing can help clinicians to manage appropriate therapy by identifying specific therapeutic targets of several newly tailored drugs which can reduce cytotoxicity and drug resistance. Molecular diagnostics offers a great tool for assessing disease prognosis and therapy response and detecting minimal residual disease in cancer and infectious diseases patients. Now most of the laboratories routinely carry our testing which is based on DNA or RNA analysis for precise medicine. This part of the chapter provides a brief review of some principles and applications of molecular diagnostic techniques such as polymerase chain reaction (PCR), real-time PCR, DNA sequencing, microarray technology and next generation sequencing (NGS) beside fluorescent in situ hybridization (FISH) and chromosomal microarray analysis (CMA) described before in this chapter.
Polymerase Chain Reaction
Nowadays PCR is the most important molecular technology used in a molecular pathology laboratory. In PCR a pair of complementary sequences of oligonucleotide primers from the flanking location of interest gene is chosen to make desired primers together with unique heat-resistant polymerases DNA. Polymerase enzymes for multifraction of targeted DNA copies of chimeric gene can be obtained. Each PCR cycle involves three basic steps: (1) denaturing, (2) annealing, and (3) polymerization. During denaturing, double stranded DNA is separated by heating at 90–95°C, while during annealing, oligonucleotide primers are bound to their complementary bases on the single-stranded DNA. This step requires a much cooler temperature, i.e., 55°C. In the polymerization process, the polymerase enzyme, Taq polymerase, reads the template strand and matches it with the appropriate nucleotides to give new strands of DNA. This process is repeated 30–40 times where each cycle doubles the amount of the targeted genetic material. At the end of the PCR, millions of identical copies of the original specific DNA are formed which give a single band when run on electrophoretic gel (Fig. 6A).
Reverse Transcriptase-PCR
Polymerase chain reaction can also be used to amplify an RNA template, the procedure is termed as a reverse transcriptase PCR or RT-PCR. The RNA sequence is first converted to a double-stranded nucleic acid sequence (cDNA) by using a reverse transcriptase enzyme. The cDNA sequence can then be amplified by using the same PCR conditions described earlier. RT-PCR technology is used for detection of RNA expression of various viruses, such as HIV and hepatitis C virus, COVID-19, etc., beside many cancer genes. Since RNA is not as stable as DNA, fresh samples are generally required for RNA analysis. DNA samples can be stored for a longer time than RNA samples.
Real-time PCR
The recent development in “Real-time” PCR technology also called q-PCR has great advantages over traditional PCR. qPCR technology allows us to analyze specific genes in quantitative manners which is also called a copy number of this gene. The q-PCR instrument measures the amount of fluorescence emitted from a dye intercalated in the double-helix DNA product and the amount of fluorescence is proportional 17to the number of copies of the amplification target (Fig. 6B). q-PCR therefore offers a great rapid quantitative advantage over conventional PCR. q-PCR is very much useful for assessment of minimal residual disease following novel targeted therapy for chronic myeloid leukemia (CML) or many other cancers. It is also useful in knowing the exact copy number of RNAs or DNAs viruses such as HBV, HIV, HCV and COVID 19 viruses in human body fluids.
DNA Sequencing
DNA sequencers play a very important role in basic biological research such as medical diagnosis, biotechnology, forensic biology and virology. DNA sequencing is a process which determines the nucleic acid sequences, i.e., adenine, guanine, cytosine, and thymine. The rapid speed of sequencing with modern DNA sequencing technology helped in completing DNA sequencing during the human genome project. The first DNA sequencers were obtained in the early 1970s. Development of fluorescence-based sequencing methods with a DNA sequencer, Sanger's sequencing became a method of choice to analyze human samples more precisely and accurately. Therefore DNA sequencing is called Gold Standard Technology in Medical Sciences. For DNA sequencing, genomic or cDNA was taken in 18a PCR tube with specific primers for the gene of interest along with 4 nucleotides (dATP, dTTP, dGTP, and dCTP) and DNA polymerase as an enzyme. The dye-labeled, chain-terminating dideoxy nucleotides are also added in this mixture. The mixture is then kept in a PCR machine at specific cycling condition as per the manufacturer instructions. The resultant product is then run on an automated DNA sequencer. The results obtained will be analyzed by using Bioinformatics tools (Fig. 6C).
Microarray Analysis Techniques
Microarray analysis technique allows researchers to investigate the expression state of a large number of genes at one time. Microarray is a chip-based technology. We can get DNA RNA, and protein microarrays. However, a major drawback in microarray technology is we get large quantities of data which is difficult to analyze without the help of computer programmers and Bioinformatics. In microarray analysis samples on gene chips undergo various processes which then produce a large amount of data. Affymetrix and Agilent technology are the major manufacturers of microarray instruments and they both provide data analysis software alongside with their microarray products (Fig. 6D).
Next Generation Sequencing
Next generation sequencing is innovative DNA sequencing technology which has revolutionized genomic research in recent years due to its speed and specificity. Now using NGS sequencing an entire human genome would have been completed within a single day. Present situation, though NGS technology has superseded conventional Sanger sequencing, however, it has not been used in routine clinical practice because of the high cost of these instruments and maintenance. There are a number of different NGS platforms using different sequencing technologies Bioinformatics software tools are used for analysis the data generated by NGS sequencers. NGS can be used to sequence the human whole genome or specific gene of interest or small numbers of individual genes.
Potential use of NGS in clinical practice: There are various applications of NGS in biomedical research to improve patient care. NGS is a most sensitive technology which allows detection of mosaic mutations. NGS plays a very important role in the field 19of microbiology to characterize various pathogens more efficiently and accurately than present conventional methods such as morphology, staining methods. NGS has several applications in the field of oncology for diagnosis and therapy of cancers. NGS is also useful in some of the diagnosis of various infectious diseases and neurological and genetic disorders. There are various NGS technology platforms developed by various companies such as Illumina (Solexa) sequencing, Roche 454 sequencing and Ion Torrent, Proton/PGM sequencing.
SIGNIFICANCE OF GENETIC KNOWLEDGE IN GENETIC COUNSELING
Recent completion of the human genome project, genetic testing will increasingly become available for a greater number of medical conditions such as cancers, cardiovascular disease, diabetes, infectious diseases and neurological and genetic disorders. The definition of genetic services focuses on genetic testing and genetic counseling. Genetic counseling is a communication process which deals with the human heredity problems associated with the risk of an occurrence of a genetic disorder in the family. This process involves a trained genetic expert who can discuss the hereditary disease problem of their family with one of the members of that family to explain the advantages and disadvantages of genetic counseling. It is important that Genetic counselor should meet one of the responsible family members of the heredity family to give all details about medical facts of that particular disease, possibility of diagnosis of that disease for pre or postnatal care and how this disease can manage any available therapy. Besides this Genetic counselor should inform his family member about any possibility of risk of recurrence of this disease to his specialized relatives. Further, Genetic counselor should make it understand the family member about the alternatives for dealing with the risk of occurrence of this disease as per their family goals, and their ethical and religious standards. All Genetic counselors have their own moral responsibility to make the best possible adjustment to his or her client disorder in such a way that the affected family member will get relief from his or her services for the same.
Recently, Biesecker and Peters (2001) have presented a working definition of genetic counseling to the affected family which mainly emphasizes a possible therapeutic solution between provider and clients.
Genetic counseling is nothing but the dynamic psychoeducational process extended on genetic information to the clients who have family history of that particular genetic disorder. They should help these clients to know and personalize technical and probabilistic genetic information. They have a pleasant duty to try their best to promote self-determination and to enhance clients’ ability to adapt to this situation, so that there will be a meaningful way to minimize psychological distress of this affected family.
Just to take an example of Tay-Sachs syndrome. It is a degenerative, neurological disorder in small children in which a child can die within the age of 5 years. In this situation, the parents are mutation carriers for this disease and want to have children in near future. As they are aware that there is a 1 in 4 probability of having an affected child. After approaching Genetic counselor in accordance with the above noted problem, Genetic counselor would focus on helping the parents to understand the condition, how it is inherited, and how much is the risk of 20having an affected child. Genetic counselor will give an option to them to do prenatal testing to know whether the child have any such abnormality and help them to cope with the outcome of their decision mean if the test is negative they can carry forward pregnancy but if test is positive they need to take a decision to abort this fetus at desire time as per the advice of their doctor. Genetic counselors play an important role in handling this situation more carefully so that both parents agree for this decision.
The major goals specified for genetic testing and counseling are as follows: (1) to educate and inform the clients about the genetic condition, (2) to provide support and help them cope, (i.e., psychological and social support to families, referral to appropriate support services), and (3) to facilitate the informed decision-making. The effectiveness and success of these genetic services will depend on the extent to which these goals are attained. However, the outcome of criteria for genetic services is problematic. The most commonly examined criteria of existing studies have focused on the knowledge acquisition and risk comprehension, psychological distress, patient satisfaction, and reproductive decision making. Diseases which can be detected through genetic testing and genetic counseling are breast and ovarian cancer, celiac disease, age-related macular degeneration (AMD), bipolar disorder, obesity, Parkinson's disease, Huntington disease and psoriasis.
CONSIDERING GENETIC COUNSELING AND TESTING FOR BREAST CANCER FAMILY (CASE STUDY)
Breast cancer is one of dreadful cancers in the world. It is proved that 15% of breast cancers are hereditary, it means that if mother is having breast cancer, the daughter also will be susceptible to breast cancer in near future. It is now well-established that the age of development of this cancer in related family members is decreasing day by day. So it happens that a daughter gets breast cancer earlier than her mother. My laboratory at Jaslok Hospital and Research Centre, Mumbai has done extensive work on breast cancer incidence in Indian population. We have established the most important diagnostic BRCA1 and BRCA2 mutational testing in my laboratory. We have scanned more than 45 breast cancer affected families for studying these mutations. Each family has at least 11–22 members in which at 2–5 members are affected with breast or other cancers. We have labeled these families as high risk family (more than 5 members are affected) or low risk family (1–2 members are affected).
We have personally called breast cancer patients and their relatives for discussion in our office. We have first of all cleared them that this research work may benefit the patients relative daughter, son, sister, etc., but not to the breast cancer patient because this testing or study may not be useful for patient therapy or cure of breast cancer. This study may prevent the incidence of breast cancer in younger generations or immediate relatives by evaluation of BRCA1 and BRCA2 mutational testing. During this study, we have collected all information about the incidence of breast or other cancer in their family. It was also observed that if both parents have family history of breast or other cancer, even then the next generation has a greater risk of development of breast cancer or other cancer. We have done complete sequencing of whole BRCA1 and BRCA2 genes in all these patients. Our study found 21out the specific founder BRCA2 mutation in the family member of the breast cancer patient who may be susceptible for breast cancer in near future. Those who have got this founder mutation, we have advised them to keep on watch on any early symptoms of breast cancer so that they can be advised for possible preventive therapy.
SUGGESTED READING
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