Methods To Identify Mutations In Factor Viii Gene Biology Essay


Various methods are available for mutation detection in F VIII patients, The most advanced and cost effective method is High resolution melting analysis (HRM).

HRM analysis is performed on amplicons after polymerise chain reaction (PCR) on the region of interest of DNA which are first amplified and using a special binding dye (Intercalating dyes) on the double stranded to fluoresce. The amplicon DNA from region of interest is heated from 500C to 950C and denature to separate the double stranded DNA allowing the fluorescence to gradually fade away. The results is plotted to show fluorescence versus tempretaure of the amplicon on Melting curves. The difference in the curves detect mutation on HA patients.

2. Describe the different types of mutation seen in the Factor VIII gene causing Haemophilia A (<750 words)[16 marks]

Haemophilia A also known as classical haemophilia (FVIII deficiency) is an X-linked recessive disorder and is the most common type of haemophilia associated with excessive bleeding. Factor VIII deficiency is mostly an inherited disorder causing dysfunction where one of the proteins needed to form blood clots is missing or reduced owing to heterogeneous mutations in F8 gene. HA is a pan ethnic disease that affects all races equally and has no predilection with approximately 1 in 5000 in males affected. FVIII gene is located on the Xq28 area with the size of 186 kb of the X chromosome. Made up of 26 exons and 25 introns and contains 2332 amino acids on maturation. Most family of approximately 30% do not have family history of HA but condition is due to spontaneous gene mutation. Carriers of severe haemophilia A account to 60% of the hemophilia population and and are due to bleeding sustain after injuries. They are also susceptible to spontaneous bleeding mostly in musles and joints area.

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Haemophilia is present in both males and females but can only be passed through to the next generation through mothers by special gene carrier. A father with non haemophilia carrier and mother with haemophilia result in male child will have 50:50 chance of developing the disorder. A female child of the same parents will also have 50:50 of Haemophilia carrier. However if the father is affected and mother does not carry the haemophilia gene, a female child will be a carrier which is known as obligate, while a male child be clear of the disease gene. This will not be possible to pass on the disease to his future generation.

Haemophilia are categorised in three different levels and are: Mild, moderate and severe.

Mild haemophilia between 6 - 49% patients develop bleeding due to injury sustain during accident or surgery in the hospitals. The mild haemophilia symptoms are diagnosed when unusual bleeding persists during this surgery, accident or just a minor tooth extraction by dentist. The incident usually occurs in adulthood patients.

Moderate haemophilia occurs in patient of about 15% in population. These are injuries resulting in bleeding and occasionally can be bleeding without any reasons known as spontaneous bleeding.

Severe haemophilia occurs in population of around 60% and are subject to injury sustain during accidents. More often, frequent spontaneous bleeding re occur in muscle joints without apparent resons.

There are different types of mutations in the F8 gene which includes; Point mutation, inversions, deletion, and insertions.

Point mutation: This account for nearly 90% mutation gene defects in patients which comprises of nonsense, missesnse and splice site.

Nonsence: Alter specific amino acid codon and replace it with translation stop codon. This distruptions blocks the synthesis of haemophilia protein creating a severe bleeding and poor clotting factors.

Missense mutations: The point mutation changes codon in replacement for different amino acids depending on location and phenotypes. The results give rise to polypeptides chain of a new protein. This substitution of amino acids tends to be less severe to disease if it's not directly associated to structural importance region. The severity however may be detrimental if the regions affected are significant to the protein functionality.

mRNA splice site mutations: mRNA splice site codon that are distorted or where a novel one are created due to mutation. The exon may also skip the original site which may results in frame shift.

Inversions: Factor VIII gene is located at the very end of the megabase (mb) of the X chromosome. The most frequents type of mutation found in severe HA is inversions that occur owing to homologous recombination linking mispaired copies of gene A positioned inside intron 22 of the gene with the size of around 500 kb telomeric. This account for almost half of severe HA cases. Identification of intron 22 with carrier analysis indicated that inversion mutation occurs mostly in males and this are subdivided into distal inversions and proximal inversions.

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Deletions: These mutations are related to complete gene deletion in protein sequence or partial deletion within the gene to multiple base pairs. This type of mutation has the higher tendency to destroy genetic functions. Severe hemophilia diseases are mostly associated with this type of mutation.

Insertions: These types of mutation are very rare which can affect the gene functionality. It occurs when a small pieces of extra gene is added to the nucleotides.

3. Describe the different types of mutation seen in the Factor IX gene causing Haemophilia B (<500 words) [3 marks]


Haemophilia B (HB) also known as FIX is sometimes called Christmas disease. Named after Stephen Christmas, who was the first patient diagnosed with the disease and is located on X chromosome on band xq27. 1-q27.2, with 34 kb and composition of 8 exons and 7 sequences. The FIX gene is significantly smaller in comparison with FVII gene. At maturity stage, the protein gene composed of 415 amino acids. Haemophilia B is also an X-linked recessively hereditary disease caused by heterogeneous mutations in factor IX gene. HB is the results of deficiency of clotting in FIX relatively similar to HA and is second most common haemophilia types that affects approximately 1 in 30,000 males and is a hybrid across ethnic groups evenly. FIX inhibitor can lead to disruption coagulation cascade which results in spontaneous hemorrhage due to trauma. Knee, elbow, CNS, muscles and cardiovascular system are the site of haemorrhage where the trauma can occur.

The carrier is normally rampant in females with FIX level as low 1% during puberty when the rise is significantly increase to as much as 60% due to the effects of testosterone at maturity stage.

Females normaly has two X chromosomes, therefore one defective X chromosome is not enough to cause severe HB disease in gene defects. On the other hand males with one X and Y chromosome and not so lucky making them more susceptible to severe gene mutation of HB. Females HB carrier can easily pass on the disease to their offspring, while male carrier are unable to transfer the mutation to his son but may pass it to his grandson. However male carrier can easily transfer the mutation to his daughter.

Spontaneous mutation can occur in FIX gene similar to FVIII gene. Insertion, point mutations, deletion and inversion are types of mutation associated with factor IX genes, but the most common gene mutation in HB are the point mutations and deletions. Therefore, large portions of defective gene in deletion are rarely causes oF HB unlike HA.

Some changes that occur in the missence nucleotide evolutionarily preserved donor splice and acceptor splice sequences while others generate cryptic splice intersection. This ultimately disrupts initial FIX RNA transcription. Mostly all of the missense mutation occur in the anmino acids coding region and generally affect the conserved amino acids. Nonsence mutation are types of point mutation that produces unbalanced truncated protein and frameshift mutation causing defective translation of coagulation in FIX.


Hemophilia B is heterogeneous mutation disease in clinical severity and can be critical at molecular level. Earlier discovery of coagulation FIX was classified as single base mutation at a specific nucleotide region and was called Leyden-specific region. Some other mutations inside the FIX promoter gene are far away from qualitative changes but lies within nucleotides where the protein binds in transcriptions. The disruption causes the DNA interactions during promoter thus compromising transcription results. Some of the promoter interferes with the normal gene production creating gene abnormality defects throughout life expectancy. Other abnormality with transcription reduction of coagulation FIX in HB is known as Leyden. These are characterized by deficiency of FIX at birth through childhood to adulthood.

4. Describe and explain the techniques used for carrier status detection in Haemophilia A and B (<1000 words) [20 marks]

Hemophilias A and B are X-linked recessive inherited bleeding disorders caused by heterogeneous mutations or absence of normal gene coding of factor VIII or IX genes. Detection of carriers is an important aspect of haemophilia care in other to effect control of the disease. Most carriers of haemophilia are regarded as asymptomatic. Gene mutation significantly can be stressful to the affected family and are tools to enable genetics analysis, but identification of mutation are of a great interest to scientist in the field of research.

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In the recent past carrier detection were performed on standard analysis and clotting assays. Results obtained were unreliable due to lyonisation. Current recombinant DNA analysis with sophisticated technology has improved carrier detections and makes the procedure extremely reliable for scientist in inherited diseases. In comparing various methods used in detection of haemophilia carrier, it showed the likelihood of misdiagnosis when pedigree analysis is only used and certainty in diagnosis with gene probing.

Carriers are usually related to genetic transformation from family history and affected women are classified as obligatory. Women carriers are affected due to their father infected with haemophilia or related male from mother's side. These women who are haemophilia positive are known as asymptomatic. Anxiety of their offspring being affected by haemophilia is one reason why suspected carrier seeks advice. The test carried out may identify patients as; Obligatory carrier, possible carrier or not a carrier.

Due to high heterogeneity of molecular defects in haemophilia, the first step in diagnosis is through genetic trace from family pedigree. This can be achieved by linkage analysis method using restriction fragment length polymorphisms (RFLP) or short tandem repeats (STRs) to detect defective allele in the families affected linked to F8 or F9. If more than one haemophiliac case already exist in the family, then its classified as familial. If its just one case or new case in the family then its known as isolated.

Direct or indirect genetic analyses in F8 or F9 are possible in diagnosing carrier detection with haemophilia. Preferred choices in most cases appropriate are direct strategy detection of mutation by pre-screening techniques. These can be followed by cloning to amplify F8 or F9 genes and allow full diagnosis of affected carrier using DNA methods.

Direct detection of intron 22 of F8 gene can be use to explain half of severe haemophilia A patients, while detection of 1- 5% of haemophilia A cases are in intron 1 inversion. A range of other pathological disorder which includes insertion, point mutations and deletions have been confirmed in both hemophilia A and B characterized by widespread heterogeneity and relatively high level of mutations.

Indirect method

Is the analysis of DNA polymorphisms related to F8 or F9 genes using markers in the introns or nearer the genes. The information derived is useful to families using extragenic and intragenic market of F8 gene. Certain advantages that can affect the use of extragenic markers are misdiagnosis due to crossover of recombinant.

The disease detected in the intron 22 inversion can be severe, moderate or mild cases due to infrequent mutation e.g. deletion, insertion and point mutation. The mutation are detected using DNA sequencing methods. Various types of mutation screening are available i.e. conformation sensitive gel electrophoresis (CSGE), denaturing gradient gel electrophoresis (DGGE), single strand conformation polymorphism (SSCP) and hetero-duplex study which are carried out before DNA sequencing. This first step shows an aberrant migration profile in a particular segment. The sequencing of the segments allows the scientist to understand the characteristics of the mutation.

Direct Methods

Direct method enables the gene defect in female carrier of haemophilia to be determined irrespective of disease in the family. The size of F8 and F9 gene, i.e 186 kb and 33 kb respectively with its genomic complexity has made the study more complicated. Different point mutation analysis techniques in the amplification of DNA now available has made the techniques more easily detected if mutation are present in patient. The results are sequence and linked to female family member to determined carrier status.

Each one individual inherits its DNA from parents which are further replicated with the next offspring. RFLP uses sequence of DNA with restriction site at both ends with a special target in between. This target sequence binds to a probe to form complementary base pairs. The probe having been tagged with a special restriction enzyme allows detection of target sequence and cleaves the DNA from host organisms on binding during analysis. The RFLP methods shows various bands when southern blot are incorporated. DNA molecules consists of four smaller nucleotides namely adenine (A), guanine (G), cytosine (C), and thymine (T). These nucleotides identify individual gene mutation in the DNA regions. Varieties of gene mutation identify includes; Insertion mutation where extra nucleotides are been added to DNA region, Deletions mutation is due to removal of nucleotides and Point mutation due to replacement of nucleotides by different one. Other DNA in mammals contains repetition of the same nucleotides.

RFLP is a molecular biological procedure used in comparing DNA from two different samples prior to polymerase chain reaction (PCR) to target DNA and replicate millions of strand copies similar to its parent. Restriction enzyme is then combined to the amplified DNA to cleave at a specific location. The results are then separated by length through agarose gel electrophoresis. Electrical current transmitted through the gel assist the fragment of DNA to move according to their sizes and length. The results show shorter fragments travelling farther from its origin through the gel. Each of this fragments length are known as allele distinguished to individual genetic traits.

The DNA fragments are labelled using radioactive probe. The gel mixture of the molecules is then x-ray to change colour due to presence of radioactivity. Various samples can be analyse using this methods to show the location of the DNA fragments of intro 22 inversion in the film as patterns. This pattern can be compared for differences to show mutation types causing haemophilia disease.

5. Describe the techniques used for prenatal diagnosis of Haemophilia (<650 words) [6 marks]

Prenatal diagnosis is normally an option requested when termination of pregnancy are considered due to foetus affected by haemophilia having been identified. It can also assist mothers to prepare for planning delivery. The need for the test may be due to family history abnormality in genetic mutation or the parents may have had a child with the gene defect.

The process of prenatal diagnosis constitutes importance of managing haemophilia disease. Recent technology in molecular biology has contributed to safe effective early diagnosis in pregnancy. There are various methods that have been developed in diagnosis of prenatal defects in fetuses to assist mothers suspected at risk of having abnormal mutation which includes; Amniocentesis, Chorionic villus sampling, Fetoscopy and Ultrasound scanning. The most preferred methods however is chorionic villus sampling (CVS).

Chorionic villus sampling (CVS): Procedure is usually caried out at approximately 12 weeks gestation period to determine foetal sex type by performing chromosomal analysis extracted by CVS method. DNA obtained from foetal can be analyzed for haemophilia disease causing mutation. On fertilisation by sperm, cell mass are formed and located in the inner cells is the fetus while placenta are located outside the cell mass.

Catheter is inserted through the abdominal wall and ultrasound scanning used first to confirm length of the pregnancy and locate the placenta. Patient can view the baby's progress from monitor. The ultrasound is continuously used to guide the instrument and extract chorionic villi fragment. This are tiny similar to finger protruding on the placenta. The cells from the placenta contain genetic information that can be analyzed for deformities mutations, metabolic defects and certain genetic defects from the chromosomal make up and also reveal the sex of the baby.

Amniocentesis: Process used to diagnose prenatal foetal defects using a sample of amniotic fluid from the pregnancy that surround the baby in the womb from 15 weeks onward.

The procedure entails surgeon using an ultrasound scan over the mother's tummy and visualise image of the womb which can be seen on a clear TV monitor. This clearly shows the baby's progress and placenta position, thus guided the surgeon of the best place to extract the amniotic fluid with no risk of damaging the placenta and baby's health.

The section of the body to insert a needle may be numbed with anaesthetic using injection into the tummy. This prevents the mothers from experiencing discomfort during the procedure. A long thin needle is then inserted through the abdominal wall into the sac containing the amniotic fluid that surround the baby. The syringe removes a small amount of amniotic fluid sample which are sent to laboratory for analysis. The sample may contain certain chromosomal defects, metabolic disease, and mutation causing haemophilia. Results may take up to four weeks.

Fetoscopy: is a procedure which involves observation of baby in the uterus around 18 weeks with the aid of thin flexible object called fetoscope. A small inscision in made in the stomach wall and the fetoscope inserted inside the uterus with aid of ultrasound which guide the surgeon to the specific place of choice and minimise risk of harm to the baby.

There are two different methods of fetoscopy and includes external and endoscopic.

External fetoscopy: similar to stethoscope and fitted with headpiece are used externally on the mother's abdomen to listen and monitor foetal heart tones of the baby.

Endoscopic fetoscopy: This is the method mostly associated with diagnosis of haemophilia and involves insertion of fibre optic endoscope inside the uterus while both baby and mother are heavily sedated either through abdomen (transabdominally) or through cervix (transcervically) with insertion of needle through the tube. Tissues or blood foetal samples are then taken for analysis. This surgical procedure prevents birth defects in prenatal pregnancy before they manifest to serious disease.

Ultrasound scanning: Usually carried out around 20 to 22 weeks gestation period and entails uses of utrasound high frequency waves which impossible to be heard by normal human hear to scan the foetus. The foetal are visual on the TV monitor and allow skeletal and other mutation to be seen. Measurements of the scan can be analyse for mutation and abnormalities.