Human Molecular Genetics Chapter 18

Human Molecular Genetics - Chapter 18: Genetic Testing of Individuals

• Genetic testing is unique among clinical tests, generally performed only once, and forming a permanent part of a patient's health record. Accuracy is critically important to avoid errors.

Key Concepts

• Genetic testing can utilize either DNA or RNA.
• RNA testing requires tissue where the gene of interest is expressed, and necessitates more careful handling than DNA samples.
• RNA analysis may be more cost-effective for genes with many small exons, and can reveal abnormal splicing not apparent in DNA testing.
• Amplifying relevant sequences (using PCR or RT-PCR for RNA) is often the first step in testing.
• Exon-by-exon sequencing of DNA or sequencing RT-PCR products is typically used to scan for mutations in a gene.
• Many methods exist for testing for specific sequence changes.

What to Test and Why

• DNA can be obtained from any nucleated cell sample.
• Clinical considerations (e.g., sample accessibility and quality) may influence sample selection.
• Other sources of DNA include blood cells, skin fibroblasts, bone marrow biopsies, prenatal samples (chorionic villus sampling, amniocentesis), and single cells from blastocysts.
• RNA or a functional test may be preferable in some cases (e.g., enzyme activity tests). Samples from the tissue showing gene expression are needed.

DNA or RNA?

• If scanning for unknown mutations, reverse transcriptase PCR (RT-PCR) offers advantages.
• RNA analysis has disadvantages, including handling sensitivity to degradation; the gene of interest might not be expressed, or might be present at a low level.
• Treating samples with translation inhibitors (e.g., puromycin) can sometimes help detect transcripts with premature termination codons missed through NMD.

Scanning a Gene for Mutations

• Most diseases display significant allelic heterogeneity, requiring scans across or near related genes to identify mutations.
• Sequencing (Sanger or next-generation sequencing) is the common choice of method, often exon-by-exon for genomic DNA, or sequencing RT-PCR products.
• The method used might depend on the size/amount of DNA available

A Gene Scanned by Sequencing

• Sanger sequencing (dideoxy or capillary electrophoresis) remains the gold-standard for mutation scans.
• Its alternative, next-generation sequencing, offers a higher throughput of sequencing, but analysis needs to consider sequencing volume.

Scanning Methods Based on Mismatches or Heteroduplexes

• Heteroduplexes can form when a PCR product is heated and then cooled slowly, resulting in a complex DNA strand mixture.
• Heteroduplex formation reveals sequence discrepancies that indicate a mutant DNA strand.
• These differences are exploited in some tests using non-denaturing polyacrylamide gel electrophoresis, dHPLC, and DGGE.

Scanning methods Based on Single-Strand Conformation Analysis

• The protein truncation test (PTT) is used to detect frameshifts, splice site or nonsense mutations creating premature stop codons.
• This is a method more specific to abnormal protein termination, and not always easy to perform

Microarrays

• Microarrays provide a single-operation method to scan for almost any gene mutation.
• Amplified cDNA or gene exons are hybridized to a microarray containing overlapping oligonucleotides corresponding to every part of the sequence.
• Arrays are typically optimized for specific diseases with limited allelic heterogeneity (e.g., BRCA1/2 or cystic fibrosis).

DNA Methylation Patterns

• DNA methylation patterns can be detected by various methods.
• Restriction enzyme digestion is often utilised to determine methylation patterns.
• Methylated DNA exhibits different patterns compared to non-methylated DNA when digested with specific enzymes.
• Alternatively, PCR is used for amplified DNA that undergoes digestion, allowing the detection of methylated or unmethylated sequences.
• Genome-wide methylation studies employ chromatin immunoprecipitation.

Unclassified Variants

• Characterizing variants can be difficult.
• Consider co-segregation with disease within a family to assess pathogenicity; variants arising de novo (parent-absent) in a candidate disease gene strongly suggest pathogenic mutations.
• Testing ethnically matched controls might also be helpful, though with limitations.
• Performing functional studies (e.g., evaluating RNA splicing effects if at all possible) and in-silico predictions of pathogenic effects help.
• Evaluating species conservation of the variant sequence is a relevant pointer for pathogenicity.

Testing for a Specified Sequence Change

• Directly testing for known alterations is more straightforward than a complete gene scan.
• Genotyping of samples can easily be performed via sequencing, but standard sequencing may not be suitable for single-nucleotide-position screening in all cases.
• PCR-based tests can evaluate well-known sequence changes.
• Restriction enzyme tests use a known restriction site and observe if a specific cut happens or not to identify mutations present in the sample.
• PCR mutagenesis can insert artificial restriction sites, allowing to test for presence of particular alterations not previously characterised.