Common Clinical Problems in Obstetrics & Gynaecology: A Practical Approach Swaraj Batra, Reva Tripathi, Asmita Muthal-Rathore
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1Obstetrics2

Molecular Biology Techniques and Clinical Applications in Obstetrics and GynaecologyChapter One

Sandhya Kabra*
As medical science progressed and obstetric and neonatal care improved, genetic disease became known as a major contributor to ill health in the community and specifically to perinatal loss and morbidity. This has led to an increased demand in reproductive genetic counselling and modification of medical care provided to individuals by using newer molecular techniques in genetics.
The identification of DNA as the constituent of the nucleus by Miescher (in 1869) was followed by a number of discoveries like the demonstration that DNA carries genetic information (in 1946) followed by elucidation of structure of DNA and discovery of polymerases and restriction endonucleases. These laid the foundations of techniques in molecular biology like sequencing, cloning, gene location techniques, etc.
 
STRUCTURE AND FUNCTION OF DNA
DNA is the ‘central dogma’ of the cell which carries the genetic information and controls cell function and division. It transfers this information to mRNA by a process called transcription, and RNA further transfers this information to a protein product by a process called translation. DNA is made up of a pentose sugar called deoxyribose, a nitrogenous base and a phosphate group. The nitrogenous bases consist of the purines which are adenine and guanine, and the pyrimidines which are thymine and cytosine.
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Genetic Code
The DNA structure is a double helical, made up of four base pairs arranged in groups of three, each group coding for an amino acid known as a codon. Gene is a DNA sequence that encodes for a specific product (RNA or protein). All genes together within an organism is known as a genome. The human genome consists of 50,000-100,000 genes, which is nearly 3 billion base pairs. Size of gene and genome is expressed as base pairs (kilobase-103 bases, megabases-106 bases).
A genome is organised into discrete elements called chromosomes. Genes in a chromosome are arranged in a linear fashion. Number of chromosomes in an organism are constant but the gene number per chromosome varies.
 
Gene Expression
It is the processing of information encoded in genetic elements that results in the production of biochemical products, or capability of a gene to produce specific protein or RNA which plays essential metabolic roles in the cell.
Control of expression is at transcriptional, translational and post-translational levels by adjacent regions of the gene.
 
Gene Mutations and Genetic Disease
Mutation is a stable, random heritable change in DNA, which occurs during nuclear division. In somatic cells they are relevant in aging and cancers while in gametes the impact is on the offspring. They can be focal or involve the whole genome, e.g. triploidy. Typical genes which encode for proteins are composed of promoters, exons, introns and polyadenylation signals are subject to a variety of mutations (10−4 to 10−10). These include deletions, expanding triplet repeats, rearrangements, silent, missense, nonsense, frameshift, etc. e.g. thalassemia, sickle-cell anaemia, X-linked lethal disorders, Duchenne's muscular dystrophy, neurofibromatosis, retinoblastoma, etc. Rate of mutations is increased in Duchenne's muscular dystrophy and neurofibromatosis.
 
COMMON TECHNIQUES FOR ANALYSIS OF HUMAN DNA
 
Hybridization
DNA can be isolated from any tissue with nucleated cells. Analysis of DNA requires the formation of single-stranded (SS) or denatured 5DNA. This can be prepared by heating or by using an alkaline solution. These single-stranded DNA will anneal (bind) to their complementary strand under appropriate conditions of temperature. If this process is carried out using probes it is known as hybridization.
 
Probe
A probe is a single-stranded known nucleic acid sequence which has been labeled radioactively or biotinylated, to detect the unknown target sequences which are complementary to it. They can be DNA, RNA or protein in nature, 20-25 base pairs in length. They can be used to isolate RNA in preparations. These can now be synthesised after computerised nucleic acid sequencing.
Probes used to detect genes are called gene probes, which help to identify the presence of individual genes in a DNA preparation and locate a gene when required.
The main application of a gene probe is in the detection of single gene disorders where one gene plays a dominant role in determining disease, e.g. neurofibromatosis, Marfan's syndrome, familial hypercholesterolemia, osteogenesis imperfecta, myotonic dystrophy, achondroplasia, familial polyposis of colon, Huntington's disease, adult polycystic kidney disease, cystic fibrosis, globin disorders, etc.
 
In Situ Hybridization
It provides information on gene expression by a particular cell or tissue by using gene probes to detect sites of production of RNA in tissues or cells.
Probes used are complementary to mRNA of interest. They are then dipped in photographic emulsion and take up silver colour when they are radiolabelled. Chromosome abnormalities like translocations and other rearrangements are identified by fluorescent in situ hybridization (FISH), where the probe is tagged by a fluorescent dye.
 
Cloning
Isolation of DNA fragments and their insertion into the nucleic acid from another biological source (vector) for manipulation and propagation is known as cloning.6
We take, for example, the sentence (gene) for insulin production in humans and paste it in a bacteria, e.g. E.coli. which divides rapidly and produces billions of copies of themselves and hence the human insulin gene sentence. How do we transfer the gene embodying the instruction for insulin production? One approach to achieve the above is to cut the appropriate gene from human DNA and paste, or splice it into plasmid DNA which is circular and can be used as a vehicle for this editing job (see Fig. 1.1). Our ‘scissors’ are the class of enzymes called the Restriction endonucleases which cut a specific base sequence of the DNA molecule in a very precise way.
zoom view
Fig. 1.1: Transfer and cloning of the insulin gene
7With these scissors used singly or in combinations, the segment of the human DNA molecule that specifies insulin gene production can be isolated. The segment is ‘glued’ into place using an enzyme called ligase. The result is an edited, or recombinant DNA molecule. When this recombinant plasmid DNA is inserted into E.coli, the cell will be able to process the instructions to assemble the amino acids for insulin production. More importantly, the new instructions are passed along to the next generation of E.coli cells in the process known as gene cloning.
The genes that have been cloned are of cystic fibrosis, Marfan's syndrome, myotonic dystrophy, familial X-linked mental retardation, Huntington's disease, BRCA 1, hereditary non-polyposis of colon carcinoma.
Phages like M13 and λ, yeast, etc. have been used as vectors. Cloned genes can be used as diagnostic aids, therapeutically for counselling and have research implications. DNA probes for analytic procedures can be procured by cloning. Cloning is invaluable in not only detecting and amplification of DNA, but also in the safe generation of large amounts of purified products encoded by DNA, like clotting factor VIII, insulin, other hormones, etc.
 
Polymerase Chain Reaction (PCR)
PCR is a highly sensitive technique by which minute quantities of specific DNA or RNA can be enzymatically amplified to have sufficient material for detection. The impetus for development of PCR goes to Mullis and his co-workers at the Cetus corporation, CA, USA.
The fundamental of this technique is that each infectious agent possesses a unique signature ‘sequence’ in its DNA/RNA composition by which it can be identified.
 
Methodology
The first step is to denature the DNA which is double-stranded to single-stranded form (95°-100°C).
Single-stranded, oligonucleotide ‘primers’, flank the DNA sequence of interest (i.e. which is to be amplified). This process is known as annealing. Extension of primers by DNA synthesis using Taq polymerase and further denaturation cycle. 30-50 thermal cycles are adequate.
End products are separated by gel electrophoresis.
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Applications
Detection of infectious agents directly in clinical samples e.g. bacteria like Chlamydia trachomatis, Niesseria gonorrheae, Gardnerella vaginalis, fungi like Candida spp., protozoa, e.g. Trichomonas vaginalis, and viruses like HPV.
In spite of the technique being revolutionary in science due to a very high sensitivity in detecting even minute amounts of nuclear material, a major drawback is false positivity by introduction of contaminating nucleic acid in the reaction mixture.
 
ELISA
Enzyme linked immuno sorbent assays (ELISA) are widely used for serologic diagnosis of infectious diseases. Results are reported not as titre but as to how the results relate to the relative amount of signal generated by the patient's serum in comparison to a known serum. Results are repeated as within selective units with a numerical sequence range (5-20 µ/ml) or as a ratio as compared to the control. (Patient 20 µ/ml, control 10 µ/ml, ratio 2).
Both antigen and antibody can be detected.
The basic test consists of antibodies bound to enzymes which retain the ability to catalyse a reaction yielding a visually coloured product.
 
 
Direct ELISA
It is for antigen detection using a labelled antibody (enzyme tagged) yielding a coloured product on addition of the substance.
 
Indirect ELISA
This is used for antibody detection. Antigen is coated on a solid phase to which the patient's serum containing antibodies is added. This is followed by washing and addition of labelled specific antibody, (IgG or IgM). The formation of a coloured product after the addition of substrate is calculated colorimetrically.
Captive ELISAs are used to detect IgM antibody in the presence of IgG antibodies.
The solid phase is first quoted with animal antibody specific for human IgM. The patient's serum is added. After washing, the antigen of interest can be added either already labelled with enzyme 9or unlabelled. In either case, it binds with the specific IgM antibody, if present. Then substrate can be added directly or specific labelled secondary antibody against the specific antigen followed by substrate.
Its advantages are that we do not need to separate the IgG and IgM antibodies in patient's serum but false positives have been recorded because of IgM rheumatoid factor bound to solid phase.
Used in serology for toxoplasma, rubella, CMV, HSV, etc.
 
Monoclonal Antibodies
Monoclonal antibodies are produced by immortal cell lines, against specific site of the antigen which are highly specific and desired in various diagnostic tests. They are produced by fusing normal antibody forming cells with a tumor cell line. After selection of a single clone by dilution or plating out, the clone can be propagated in spinner culture or in mice to produce high titres of the desired monoclonal antibody.
 
Polyclonal Antibodies
Polyclonal antibodies are the antibodies produced against a particular antigen. The exact nature of antibodies is unknown and variable. They are used in EIA, fluorescent assays, etc.
 
Electrophoresis
Electrophoresis is the process of separation of protein components depending on their charge and molecular weight by passage of electric current on agarose or polyacralamide gel. Southern blot is used for DNA detection and Northern blot for RNA detection. Western blot/immunoblot is used for proteins detection.
These combined fractionation and a detection method to provide a sensitive technique for analysis of the above.
 
Microarray Technology
A rapidly evolving approach to genome-scale studies consists of microarrays or DNA chips.
The approaches consists of thousands of synthetic nucleic acid sequences aligned on thin glass or silicon surfaces.
Flourescently labelled test sample of DNA or RNA is hybridized to the chip, and a computerised scanner detects sequence matches.
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These allow detection of variation in DNA sequences and are used in mutational analysis and genotyping.
 
Chromatography
Chromatography: These are the procedures used to separate and characterise substances based on their zone, ionic charge and their solubility in particular solvents.
 
Principle
Mobile phase contains the sample and carries it across through the stationary phase.
 
Mobile Phase: Liquid/Gas
Stationary phase: Liquid/solid: Stationary phase maintains the conditions necessary for separating various substances (analytes).
 
Gas Liquid Chromatography
High performance liquid chromatography, detects protein, carbohydrate, organic acids, fatty acids, etc.
 
APPLICATIONS OF MOLECULAR TECHNIQUES
 
Diagnostic
 
Infections
Rapid diagnosis of viral infections like CMV, HPV, Rubella and HIV in early pregnancy to avoid abortions, birth defects, mental retardation, etc.
  1. PCR using foetal cells to detect specific sequences of the infecting agent.
  2. Chlamydia trachomatis identification by ELISA earlier and PCR now to diagnose and treat PID resulting from upper genital tract pathogens to avoid long-term complications.
 
Y-DNA Probes
Used to identify chromosomal abnormalities associated with sexual ambiguity along with identification of children with dysgenetic gonadal tumours with a high risk of neoplasia.
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Endocrinology
The genes for human chorionic gonadotrophin (hCG), leutinising hormone (LH), and follicle stimulating hormone (FSH) have been isolated, cloned and sequenced, and recombinant varieties are in production for use therapeutically. This knowledge has helped and is being used in determining the expression of hCG, human placental lactogen (hPL) in developing placental tissues, hydatidiform mole and choriocarcinomas. These may give an insight into molecular mechanisms underlying these tumours and help in their therapeutic strategies.
DNA sequencing, PCR and southern blotting help in the diagnosis of congenital adrenal hyperplasia, androgen insensitivity syndrome and hypogonadotrophic hypogonadism.
 
Prenatal Diagnosis
Molecular technique are used in the prenatal diagnosis of chromosomal, single gene, X-linked and dominant disorders. DNA-based genetic diagnosis by gene tracking (linkage analysis) or gene mutation technique (direct technique). Direct technique is used to detect gene mutation, performed by PCR amplification of desired gene and digestion with appropriate restriction enzymes which will generate products of different sizes. For instance, in sickle cell anaemia substitution of valine by glutamine in B globulin gene abolishing the restriction site of Mst II. Unaffected case gives two bands and affected case gives a single band. Similar diagnosis is made in cystic fibrosis and Huntington's disease.
Linkage analysis is needed when specific mutation site is unknown, but chromosomal location of gene is known and by tracking the gene we indirectly can locate the gene site.
 
Thalassaemia
Thalassaemia is an inherited Mendelian disorder characterised by an impairment of haemoglobin production in which there is partial or complete failure to synthesize alpha or beta type of globin chain. Consequences occur because of low intracellular haemoglobin and excess of other chain. The exact nature of the defect varies and is probable that a number of different faults occur along the pathway which translates the genetic information into a polypeptide chain. The gene itself may be deleted as it is usually in alpha thalassaemia. When the abnormality is heterozygous then synthesis of haemoglobin 12is only mildly affected and little disability occurs. Synthesis is grossly impaired when the patient is homozygous and there is imbalance in polypeptide chain production.
β chains are coded by 2 β genes located on chromosome 11 (one of the pair). Heterozygotes have thalassaemia minor with mild anaemia and little or no clinical disability. Homozygotes have thalassaemia major and are unable to synthesise HbA or produce very little and there is profound anaemia after four months of age. Hundred different mutations, mainly point mutations, are responsible.
 
Clinical Presentation
β thalassaemia major: transfusion required.
β thalassaemia minor: transfusion not required.
β thalassaemia intermedia: Severe anaemia, but transfusion not required.
Alpha thalassaemia Reduction or absence of alpha chains synthesis. There are 2 alpha gene loci on chromosome 16 and therefore normally there are 4 alpha genes.
Newborn haemoglobin is α2γ2 so if α less than γ tetramers Hb Barts is formed.
Excess Beta—Beta tetramers—Hb H
Alpha thalassaemia trait: 2 globin genes deleted.
Hydrops faetalis: All 4 alpha chains deleted and Beta tetramers (Hb Barts) fatal.
 
BRCA-1
Reported in 1990, mapped to chromosome 17q 21 familial autosomal dominant). The gene has recently been cloned, 50 per cent chance of child of carrier inheriting this gene. Life time risk of carcinoma breast is 85 per cent in these cases. The onset of carcinoma at around 35 years of age suggests high degree of suspicion BRCA disease allele and should undergo an early mammographic screening for detection of non-palpable breast carcinoma.
In some families there is high risk of carcinoma ovary.
If P53 tumour suppressor gene mutated then associated cancer.
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BRCA-2
Familial female male carcinoma breast.
Genetic testing is critical in risk estimation in females who have P53 mutation or BRCA-1
 
LYNCH-2
Carcinoma of large bowel associated with carcinoma endometrium and other cancers. Random hypermutability in 2p or 3p chromosome.
 
Therapeutic
Gene therapy is the correction of genetic faults at the level of the gene by replacing defective genes in somatic cells with functional genes. Cloned gene is carried by suitable vector for accurate expression.
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SUGGESTED READING
  1. Betty A Forbes, Daniel F Sahm, Alice S Weissfeld, Immunological methods use dfor organism detection, Bailey and Scott's Diagnostic Microbiology, (10th Ed),Mosby Inc,  St Louis,  Missouri 208–219, 1998.
  1. Betty A. Forbes, Daniel F Sahm, Alice S Weissfeld, Molecular methods for microbial identification and characterization: Bailey and Scott's Diagnostic Microbiology (10th Ed), Mosby Inc,  St Louis,  Mussouri, 188–207, 1998.
  1. E Braunwald, AS Fanci, DL Kasper, et al: Genetics and Disease. Harrison's Principles of Internal Medicine (15th Ed), McGraw-Hill  New York  375–96, 2001.
  1. Elmer W. Konenan, Stephen D Allen, WIlliam M Janda et al: Basic bacteriology, concepts of virulence and technologic advances. Color Atlas and Textbook of Diagnostic Microbiology (5th Ed), Lippincott-Raven,  Philadephia  1–67, 1997.
  1. The impact of molecular biology in obstetrics and Gynaecology, John Studd, Obstetrics and Gynaecology, John Studd. Progress in Obstetrics and Gynaecology, Churchill Livingstone,  New Delhi  11: 219–243, 1994.
  1. Vincent T. Devilor Jr, Samuel Hillman, Steven A. Rosenberg, Cancer of the Breast, Cancer: Principles and Practice of Oncology (5th Ed), Lippincott Raven,  Philadelphia  1541–1616, 1997.