Human Molecular Genetics Chapter 18 - Genetic Testing of Individuals - BIOL311 PDF

Tom Strachan Andrew Read Human Molecular Genetics Fourth Edition Chapter 18 Genetic Testing of Individuals Copyright © Garland Science 1 2011  KEY CONCEPTS Testing can...

Tom Strachan Andrew Read Human Molecular Genetics Fourth Edition Chapter 18 Genetic Testing of Individuals Copyright © Garland Science 1 2011  KEY CONCEPTS Testing can be done with either DNA or RNA. RNA must be obtained from a tissue in which the gene in question is expressed. These samples need much more careful handling than DNA samples. RNA analysis may be more economical for a gene that has many small exons. It can reveal abnormal splicing that may not be apparent from DNA testing. 2  KEY CONCEPTS The first step in testing is almost always to amplify the relevant sequences by using PCR, or RT-PCR if RNA is being analyzed. In scanning a gene for any possible mutation, the normal procedure is to sequence the DNA, exon by exon, or to sequence an RT-PCR product. Many methods exist for testing for a pre-defined sequence change 3  Introduction Genetic tests are unusual among clinical tests. A genetic test is normally performed just once. The result forms a permanent part of a person’s health record. It is especially important to avoid mistakes in diagnostic testing. 4 5  What to test and why? DNA for genetic testing can be obtained from any sample containing nucleated cells, but clinical considerations may dictate the choice of sample. 6  What to test and why? Other sources? Bone marrow biopsy Blood cells Skin fibroblast 7 Other sources? We have earlier seen prenatal diagnosis methods such as chorionic villi sampling and amniocentesis. 8 What to test and why? Sometimes it is more appropriate to test RNA or to perform a functional test. If checking enzyme activity  the sample must come from a tissue in which the gene is expressed and/or the gene product is normally functional. 9 Many different types of sample can be used for genetic testing Genetic testing almost always begins with amplification of the DNA or RNA sample by PCR. PCR is sensitive enough to be used for a wide range of tissue samples. 10 11 Many different types of sample can be used for genetic testing Peripheral blood- the best general source of DNA 12 Many different types of sample can be used for genetic testing Mouthwash or buccal scrape noninvasive; usually enough DNA for several dozen PCR tests, but quality and quantity very variable 13 Many different types of sample can be used for genetic testing Skin fibroblast Skin, muscle, etc. for RNA studies; needs to be a tissue where the gene of interest is expressed; requires a biopsy, which is invasive and often unpleasant 14 Many different types of sample can be used for genetic testing Hair roots, semen, cigarette butts, etc. scene-of-crime samples for forensic analysis 15 Many different types of sample can be used for genetic testing Single cell from a blastocyst for pre-implantation diagnosis; technically very demanding 16 Many different types of sample can be used for genetic testing Chorionic villi for prenatal diagnosis at 9–14 weeks 17 Many different types of sample can be used for genetic testing Amniotic fluid a relatively poor source of fetal DNA compared with chorionic villi, but used for testing at 15– 20 weeks of pregnancy 18 Many different types of sample can be used for genetic testing Fetal DNA in maternal blood a promising alternative to chorionic villi; detectable from 6 weeks; paternal alleles only 19  Many different types of sample can be used for genetic testing Pathological specimens vital resource for genotyping dead people; also tumor, etc., biopsies; fixed paraffin embedded tissue requires special procedures for DNA purification 20 Many different types of sample can be used for genetic testing Guthrie card the cards used for neonatal screening have one or more spots of the baby’s blood, not all of which are normally used for the screening; if cards are archived they are a possible source of DNA from a deceased baby 21  RNA or DNA? If a gene has to be scanned for unknown mutations, testing RNA by reverse transcriptase PCR (RT-PCR) offers several advantages. Yet, analyzing RNA has its disadvantages, too. 22  RNA or DNA? Yet, analyzing RNA has its disadvantages, too. – Samples to be handled carefully (degradation is an issue) 23  RNA or DNA? Yet, analyzing RNA has its disadvantages, too. – Gene of interest may not be active in the tissue obtained.  No RNA to collect. – Low level mRNA’s analysis may be difficult. 24  RNA or DNA? Yet, analyzing RNA has its disadvantages, too. – RT-PCR product from a heterozygous person may show only the normal allele. WHY? ‘Cells have a mechanism, nonsense-mediated decay (NMD), that detects mRNAs containing premature termination codons and degrades them. Thus, the usual result of a nonsense mutation is to prevent any expression of the gene.’ 25  RNA or DNA? Yet, analyzing RNA has its disadvantages, too. – RT-PCR product from a heterozygous person may show only the normal allele. Solution: – Treating cultured cells or whole blood with the translation inhibitor puromycin has been shown to inhibit nonsense-mediated decay, which may allow cDNA sequencing to detect transcripts that include premature termination codons. 26  Scanning a gene for mutations For the great majority of diseases there is extensive allelic heterogeneity. Diagnostic testing therefore usually involves searching for mutations that might be anywhere within or near the relevant gene or genes. 27  A gene is normally scanned for mutations by sequencing Sequencing is the method of choice for mutation scanning: either sequencing each exon in genomic DNA or sequencing an RT- PCR product. 28  A gene is normally scanned for mutations by sequencing Sanger Sequencing (dideoxy or capillary electrophoresis sequencing) Its alternatives (next generation sequencing) – Sequencing volume is to be kept in mind. 29 Gauiter (2007, PhD thesis) Simulation of polymer translocation through small channels: A molecular dynamics study and a new Monte Carlo approach 30 December 2021 31 32 33 34  Differences Between NGS and Sanger Sequencing ‘In principle, the concepts behind Sanger vs. next-generation sequencing (NGS) technologies are similar. In both NGS and Sanger sequencing (also known as dideoxy or capillary electrophoresis sequencing), DNA polymerase adds fluorescent nucleotides one by one onto a growing DNA template strand. Each incorporated nucleotide is identified by its fluorescent tag.’ https://www.illumina.com/science/technology/next-generation-sequencing/ngs-vs-sanger-sequencing.html 35  Differences Between NGS and Sanger Sequencing ‘The critical difference between Sanger sequencing and NGS is sequencing volume. While the Sanger method only sequences a single DNA fragment at a time, NGS is massively parallel, sequencing millions of fragments simultaneously per run. This high-throughput process translates into sequencing hundreds to thousands of genes at one time. NGS also offers greater discovery power to detect novel or rare variants with deep sequencing.’ https://www.illumina.com/science/technology/next-generation-sequencing/ngs-vs-sanger-sequencing.html 36  A gene is normally scanned for mutations by sequencing Sequencing gold standard for mutation detection. – DNA quality needs to be good. – Tumor DNA or archive material DNA can be troublesome 37  PCR products Usually less than 200bp long Standard capillary electrophoresis 500-800bp. One can sequence several exons… When sequencing DNA, introns being very large regions can cause problems 38  One problem: - When sequencing DNA, introns being very large regions can cause problems. - Careful designed multiplexed reactions can be used to overcome this problem: - Needs very careful design of primers and meaningful only for analysis which will be performed many times… 39  Sequencing produces a lot of data. Analysis becomes a bottleneck… 40  Remember that the technology is constantly evolving… https://www.nature.com/articles/s41586-020-2547-7 41  After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist1,2. Here we present a human genome assembly that surpasses the continuity of GRCh382, along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome3, we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes. 42 https://www.nature.com/articles/s41586-020-2547-7 A variety of techniques have been used to scan a gene rapidly for possible mutations Sequencing used to be more expensive. Methods were devised to define regions of interest to overcome this issue. 43 44  Scanning methods based on detecting mismatches or heteroduplexes Many tests use the properties of heteroduplexes to detect differences between two sequences. Most mutations occur in heterozygous form; even with autosomal recessive conditions, affected people born to non-consanguineous parents are often compound heterozygotes, with two different mutations. 45 Scanning methods based on detecting mismatches or heteroduplexes Heteroduplexes can be formed simply by heating the PCR product to denature it, and then cooling it slowly. For homozygous mutations, or X-linked mutations in males, it would be necessary to add some reference wild-type DNA 46 Heteroduplexes often have abnormal mobility on non-denaturing polyacrylamide gels. 47 Heteroduplexes have abnormal denaturing profiles. This is exploited in denaturing high-performance liquid chromatography (dHPLC, A) and denaturing gradient gel electrophoresis (DGGE, B). 48 49 50 Scanning methods based on single-strand conformation analysis The protein truncation test (PTT) is a specific test for frameshifts, or splice site or nonsense mutations that create a premature termination codon. PTT is a demanding technique and not very easy to use. 51 DMD mutation scanning with the protein truncation test (PTT). The samples in lanes 3 and 5 produce truncated, faster-running polypeptides. The position of the termination codon within a sample can be determined from the size of the truncated polypeptide. 52 Microarrays allow a gene to be scanned for almost any mutation in a single operation Custom microarrays can be used to interrogate every position in a gene in one assay. Amplified cDNA, or exons of the gene amplified from genomic DNA, are hybridized to a microarray that contains overlapping oligonucleotides corresponding to every part of the sequence. 53 Principle of mutation detection with Affymetrix oligonucleotide arrays. 54  Extra tests would be needed to check for deletions or other larger-scale changes. The range of mutations to be detected has to be defined in advance and designed into the chip. Main role in mutation detection may be for initial scanning of samples to pick up the common mutations. (the difficult cases to be sorted out by other methods.) High set-up costs of microarrays they would only be used for genes such as the breast cancer genes BRCA1/2, where there is a very large demand for mutation analysis. 55 DNA methylation patterns can be detected by a variety of methods Covered in Chapter 11 as well. May be important in disease diagnosis… 56 DNA methylation patterns can be detected by a variety of methods PCR products are always unmethylated, because they are made from the normal monomers supplied in the reaction mix. None of the methods described so far gives any information on the methylation patterns in a DNA sample. 57 DNA methylation patterns can be detected by a variety of methods Genomewide studies of methylation use chromatin immunoprecipitation with an antibody against methylated DNA (see Chapter 11, p. 353) 58 DNA methylation patterns can be detected by a variety of methods To study the methylation status of an individual sequence, two main methods are used: – Restriction enzyme digestion – Bisulphite modification 59 DNA methylation patterns can be detected by a variety of methods Restriction enzyme digestion: – HpaII cuts only unmethylated CCGG, – MspI cuts any CCGG, whether or not it is methylated. 60 DNA methylation patterns can be detected by a variety of methods Restriction enzyme digestion: – Methylated genomic DNA gives different patterns of restriction fragments with the HpaII and MspI enzymes. – Alternatively, a PCR template containing a CCGG sequence can be digested with either HpaII or MspI before amplification. If the CCGG is methylated, the template will not be cleaved by HpaII, and the PCR will yield a product. 61 MspI cuts any CCGG, whether or not it is methylated. No PCR product as a result If the CCGG is methylated, the template will NOT be cleaved by HpaII. The PCR will yield a product. 62 Unclassified variants are a major problem Sequencing the whole of a candidate gene in a large series of patients will reveal many variants. 63 Unclassified variants are a major problem Deletions, frameshifts, nonsense mutations, and changes to the canonical GT...AG splice sites are highly likely to be pathogenic. But deciding whether or not a novel nucleotide substitution is pathogenic (and hence represents the sought-after mutation) can be very difficult. 64  Unclassified variants are a major problem Suggestions: Check the presence or absence on single nucleotide polymorphism databases: – check dbSNP http://www.ncbi.nlm.nih.gov/projects/SNP to see whether the variant has previously been reported – YET, many rare nonpathogenic variants will not be in the database. 65 YET, many rare nonpathogenic variants will not be in the database. 66  Unclassified variants are a major problem Suggestions: Co-segregation with the disease in the family. – Should always be checked where possible, because failure to co-segregate is powerful evidence that the variant is not pathogenic. – However, co-segregation does not prove pathogenicity. 67  Unclassified variants are a major problem Suggestions: Occurrence of a new variant concurrent with the (sporadic) incidence of the disease. – A de novo change (one not present in either parent) in a candidate gene in a de novo case of a dominant disease is highly suspect. 68  Unclassified variants are a major problem Suggestions: Testing ethnically matched controls. – With limitations 69  Unclassified variants are a major problem Suggestions: Functional Studies – Where possible 70  Unclassified variants are a major problem Suggestions: RNA Studies – help identify effects on splicing. 71  Works on human sequences too… www.fruitfly.org/seq_tools/splice.html 72 http://www.umd.be/searchSpliceSite.html 73  Unclassified variants are a major problem Suggestions: In silico predictions of pathogenic effect – This can be attempted where there are good multiple sequence alignments, especially if there is also information on the three- dimensional structure of the gene product or of relevant domains within it. 74  Unclassified variants are a major problem Suggestions: Species conservation provides some useful pointers. If the putative mutant sequence occurs as the wild type in some other species (or in a paralogous gene in humans), the change is unlikely to be pathogenic. 75 TESTING FOR A SPECIFIED SEQUENCE CHANGE Testing for the presence or absence of a known sequence change is a different and much simpler problem than scanning a gene for the presence of any mutation. Samples can always be genotyped by sequencing. BUT conventional sequencing is not an efficient method if only a single nucleotide position is being checked. 76 Some of the main genotyping methods… 77 TESTING FOR A SPECIFIED SEQUENCE CHANGE Many variants of these and other methods have been developed as kits by biotechnology companies. Typical applications include the following: Diagnosis of diseases with limited allelic heterogeneity (Table 18.5). 78 79 TESTING FOR A SPECIFIED SEQUENCE CHANGE Typical applications include the following: Diagnosis within a family. Mutation scanning methods may be needed to define the family mutation, but once it has been characterized, other family members normally need be tested only for that particular mutation. 80 TESTING FOR A SPECIFIED SEQUENCE CHANGE Typical applications include the following: Testing control samples to see whether a change seen in a patient is actually a low-frequency population polymorphism. SNP genotyping. Although the aim is not to find a pathogenic mutation, the problem is identical: to test a DNA sample for a predefined sequence variant. 81  Testing for the presence or absence of a restriction site When a base substitution mutation creates or abolishes the recognition site of a restriction enzyme, this allows a simple direct PCR test for the mutation. 82  Testing for the presence or absence of a restriction site 1. A suitable length sequence containing the potential mutation is PCR amplified. 2. The PCR product is digested with the relevant restriction enzyme 3. The products of digestion are separated by electrophoresis to see whether or not a cut has occurred. 83 Testing for the presence or absence of a restriction site 84 This is all good. But what if you do not have a good restriction site to work with? 85  Testing for the presence or absence of a restriction site Although hundreds of restriction enzymes are known, they almost all recognize symmetrical palindromic sites, and many point mutations will not happen to affect such sequences. In addition, sites for rare and obscure restriction enzymes are unsuitable for routine diagnostic use because the enzymes are expensive and often of poor quality. 86 What to do? Can we introduce the restriction sites artificially? If so, in which step? 87  Testing for the presence or absence of a restriction site If a mutation does not change a suitable site, sometimes one can be introduced by a form of PCR mutagenesis using carefully designed primers. 88 Introducing an artificial diagnostic restriction site. 89 Introducing an artificial diagnostic restriction site. * * An A>T mutation in the intron 4 splice site of the FACC gene does not create or abolish a restriction site. The PCR primer stops short of this altered base but has a single base mismatch (red G- see *) in a non-critical position that does not prevent it from hybridizing to and amplifying both the normal and mutant sequences. 90 Introducing an artificial diagnostic restriction site. The mismatch in the primer introduces an AGTACT restriction site for ScaI into the PCR product from the normal sequence but not the mutant sequence. 91 Where to find the Restriction Enzyme? Ask Sheikh Google… 92 Introducing an artificial diagnostic restriction site. 93