Detecting Disease-Causing Mutations Lecture Notes PDF

Republic of Iraq Ministry of Higher Education Stage:S2 and Scientific Research Module: MGD Wasit Universty College of medicine :Lecture Title Detecting disease causing mutations Lecturer Name: Dr –Dhamyaa K....

Republic of Iraq Ministry of Higher Education Stage:S2 and Scientific Research Module: MGD Wasit Universty College of medicine :Lecture Title Detecting disease causing mutations Lecturer Name: Dr –Dhamyaa K. Kadhim  Intended learning outcomes At the end of this lecture you should be able to: Recognise the fundamental importance of PCR in the diagnosis of genetic disease. (LO 11.8) Provide an overview of the different genetic tests available for the detection of mutations in genes. (LO 11.9) Show an appreciation of the ethical issues associated with genetic testing. (LO 11.10) Detection of Mutations ❖ Describe the application of molecular genetics techniques to analyse and detect genetic mutations that cause disease. ❖ Most human mutations are single base substitutions that cause single amino acid substitutions. ❖ Common mutations have been characterised for many genes of clinical interest precise alterations at the DNA.  Detection of Mutations Most DNA-based clinical diagnoses rely on the PCR amplification: After PCR amplification, detecting the mutation could follow from: For instance ✔ Information regarding a loss or gain of a restriction enzyme site in the PCR product (e.g. loss of an MstII site in the Sickle Cell mutation). ✔ Information regarding the size of the PCR product (e.g. detection of the 3bp deletion in the ΔF508 CF mutation). ✔ Presence or absence of a PCR product (e.g. using allele-specific PCR). ✔ DNA sequence of the PCR product.  Detection of Mutations Sickle Cell Anaemia Gene: HBB (hemoglobin, beta) Location: 11p15.5 Gene size: 1,600 bp, 3 exons Mutation: 1 bp substitution Sickle Cell Anaemia patients carry single base pair mutation (A>T) in the 7th codon at exon 1 of their β-globin gene. The sickle cell mutation results in just one amino acid change (Glu>Val) in the human β-haemoglobin protein. SCA: 5'ATG GTG CAT CTG ACT CCT GTG GAG AAG TCT….3‘ Healthy: 5'ATG GTG CAT CTG ACT CCT GAG GAG AAG TCT.3' HBB protein: N-met val his leu thr pro glu glu lys ser….-C  Detection of Mutations Sickle Cell Anaemia: There are several alternative methods that could be used to check for SCA mutation ✔ PCR amplification followed by DNA sequencing ✔ PCR amplification followed by digestion with MstII restriction enzyme:  Detection of Mutations Sickle Cell Anaemia ✔ PCR amplification followed by hybridisation to an allele-specific oligonucleotide: For known disease-causing mutations, the allele-specific probes can be design for detection of this mutation. This allele-specific oligonucleotide (ASO) can distinguish between the two different alleles in a hybridisation experiment.  Detection of Mutations Sickle Cell Anaemia ✔ PCR amplification, check for absence/presence of product (e.g. using allele-specific PCR): For known disease-causing mutations, allele-specific PCR is designed for discriminate between templates which differ at a specific single base. Two different allele-specific primers are designed that differ at 3‘ terminus at which a base corresponds with the base found in wild-allele or mutant-allele  Detection of Mutations Sickle Cell Anaemia ✔ PCR amplification, check for absence/presence of product (e.g. using allele-specific PCR): One common primer is used in conjunction with two different allele-specific primers: Two PCR amplification achieved, one having common primer with one allele-specific primer and other PCR amplification having common primer with other allele-specific primer. A PCR product will only be amplified if the allele-specific primer is perfectly matched with the template DNA at its 3’end  Detection of Mutations Many others mutation found, many variant result in...  Detection of Mutations Cystic Fibrosis (CF) Gene: CFTR (cystic fibrosis transmembrane conductance regulator) The CFTR gene encodes to CFTR protein containing 1480 amino acids which acts as chloride channel across cell membrane. Location: 7q31.2 Gene size: 189,000 bp, 27 exons Mutation: More than 1000 mutations (point and frameshift) are identified in CFTR gene, approximately 70% of the mutations in CF patients correspond to a specific deletion of 3 base pairs (CTT) at exon 11, which results in the loss of a phenylalanine residue (ΔF508) in the CFTR protein. This causes misfolding of the newly synthesized mutant CFTR ion channel so that does not integrate into the cell membrane, but remains in the cytoplasm (where it is degraded): CF: 5‘-…ATC ATT GGT GTT TCC TAT GAT GAA TAT….-3' Healthy: 5‘..ATC ATC TTT GGT GTT TCC TATGATGAATAT..-3' CFTR protein: N-…ile ile phe gly val ser tyr asp glu tyr…-C  Detection of Mutations Cystic Fibrosis (CF) ✔ PCR amplification, check the size of product by gel electrophoresis: Information regarding the size of the PCR product (e.g. detection of the 3bp deletion in the ΔF508 CF mutation).  Southern blotting ❑ Not all mutations can be easily detected using PCR-based methods. ❑ Southern blotting allows investigation of an individual gene in a background of all other genes. It is the technique of choice when there is a need to analyses larger segments of DNA within and around a gene. ❑ for instance: It used to investigate (partial) gene inversions (e.g. haemophilia A), the gross organisation of the gene needs to be investigated. Southern blotting is also used to analyse triplet repeat disorders, such as Huntington’s disease, Mytonic dystrophy and Fragile X syndrome. Southern blotting Haemophilia A : XLR Haemophilia A is caused by mutations in the F8 gene at Xq28 which encodes clotting factor VIII of the blood clotting cascade. Several different mutations are known. The common point mutations are well characterised and be screened easily. About 40% of all severe cases involve a gene inversion involving 500 kb of DNA. Southern blotting Haemophilia A a) F8 gene has 26 exons spanning 187 kb of DNA. b) Red box indicates a sequence in intron 22, of which two copies also found 360 kb and 435 kb away from the start of gene( The arrows show the orientation). c) During meiosis, the DNA may loop around to allow two copies of repeated sequences to pair in the same orientation. d) Recombination between paired sequences occurred, which results in chromosomal inversion of about 500kb (included inversion of partial F8 gene) that disrupt F8 gene ( non-functional gene) Southern blotting Haemophilia A All exons of the gene are intact, so PCR-based methods reveal no mutations. Southern blot can be used to check the size of DNA fragment of F8 gene. If the inversion is present, the fragment is an abnormal size. Southern blotting Myotonic Dystrophy (MD): AD MD is the most common heritable neuromuscular disorder. Patient suffers from progressive weakness and myotonia MD is caused by a triplet repeat expansion of CTG (dynamic mutation) in the 3' UTR of the myotonin gene at 19q13.3. The myotonin gene is responsible for synthesis of myotonic dystrophy protein kinase and at present it is not known that how this protein kinase causing clinical features of MD. The normal number of CTG repeats is 5-37. Affected individuals have CTG repeats from 50 to many thousand (full mutation). The triplet repeats expansion from 38-49 known as pre-mutation without clinical features. Southern blotting Myotonic Dystrophy (MD): Triplet repeat expansion is unstable and may increase when transmitted from parents to child, resulting in significant worsening across generations either causing the disease to appear at an earlier or be of worse severity, this phenomena is called anticipation Southern blot can be used to detect this triple repeat expansion Southern blotting Myotonic Dystrophy (MD): Myotonic dystrophy - expanded repeat not in the exon. Expansion may affect export of the mRNA from nucleus.  Triplet repeat disorders Normal # of Disease Disease Repeat copies # of copies Fragile X syndrome CGG or 6-50 200-2000 CCG Freidreich ataxia GAA 6-29 200-900 Haw River syndrome CAG 7-25 49-75 Huntington disease CAG 10-34 40-121 Jacobsen syndrome CGG 11 100-1000 Myotonic dystrophy type 1 CTG 5-37 50-1000 Myotonic dystrophy type 2 CCTG < 10 > 100 Spinal and bulbar muscular CAG 14-32 40-55 atrophy Spinocerebellar ataxia CAG 4-44 40-130  Array Comparative Genomic Hybridisation (Array CGH) ▪ Array CGH is used to scan the entire genome for any copy number change (deletion or duplication) involving sequences of a few kb or longer: Ex, This technique is used to screen for sub-microscopic chromosomal deletions for which the location cannot be deduced from the patient’s phenotype. Unlike FISH, it does not require prior knowledge of chromosomal location of any variants ▪ Array CGH can not detect the balanced chromosomal rearrangements or inversion THANK YOU