Study Notes: Medical Genetics - DNA Mutation Detection and Sequencing Methods

SCH3223 Medical Genetics: Lecture 5 Overview

• Topic: How can we check a patient's DNA for mutations?

• Core Learning Objectives:     * Understand the principles of DNA sequencing and interpret Sanger sequence traces (chromatograms).     * Identify clinical applications of Next-Generation Sequencing (NGS).     * Distinguish between checking for specific changes, scanning genes, sequencing panels, or whole-exome sequencing.     * Explain principles of mutation detection methods (e.g., specific mutations vs. deletions/duplications).

Standardized Mutation Nomenclature

• DNA Sequences ($c.$):     * Notation Symbols:         * > indicates a change.         * $\text{del}$ indicates a deletion.         * $\text{ins}$ indicates an insertion.     * Base Numbering: Usually starts from the $AUG$ codon.     * Intron Numbering: Numbered based on the closest exon.         * Use a $+$ sign if they are closer to the end of one exon (e.g., c.84+6G>A).         * Use a $-$ sign if they are closer to the start of the next exon (e.g., c.85-8T>A).     * Specific DNA Examples:         * c.76A>C: The nucleotide at position $76$ changed from $A$ to $C$ (sometimes written as $A76C$).         * $c.76-78\text{del}$: Nucleotides from position $76$ to $78$ have been deleted.         * c.92+1G>A: The first base after the exon ending at nucleotide $92$ changed from $G$ to $A$.

• Protein Sequences ($p.$):     * Labeling is simplified as there are no introns; only amino acid position and the specific change are required.     * Stop Codons: Indicated by $\text{Ter}$ or an asterisk ($<em>$).      Specific Protein Examples:         * $p.Ala26Val$ (or $p.A26V$): The $26\text{th}$ amino acid changed from Alanine to Valine.         * $p.Cys318\text{Ter}$ (or $p.Cys318X$ or $p.C318*$): The $318\text{th}$ amino acid changed from Cysteine to a stop codon.

Case Studies in Mutation Detection (Review)

• Case 4: The Davies Family (Duchenne Muscular Dystrophy - DMD):     * Method: Multiplex PCR identified a partial deletion of the $Dystrophin$ gene (exons $45$, $47$, and $48$).     * Confirmation: Additional PCR confirmed exons $44$ and $46$ were also missing.     * Clinical Status: Diagnosis of DMD confirmed.

• Case 1: The Ashton Family (Huntington's Disease):     * Method: PCR of Alfred’s DNA showed an expansion of the $CAG$ repeat in exon $1$ of the $HTT$ gene.     * Results: Fragment sizes observed included $19\,bp$, $19\,bp$, $18\,bp$, and $56\,bp$.     * Clinical Status: Diagnosis confirmed.

• Case 12: The Lipton Family (Fragile-X Syndrome):     * Method: Southern Blotting of the $FMR1$ gene detected an $800\,CGG$ repeat expansion in Luke.     * Method 2: Triplet-primed PCR identified Luke’s mother, Linda, as a carrier of the premutation with $38$ and $120$ repeats.

Methodologies for Detecting Specific Sequence Changes

• Rationale for Specific Testing:     * Testing for one specific variant when the disease is always caused by the same change.     * Testing common variations that account for most disease cases.     * Family-specific mutations already identified.     * Checking healthy controls when population data is insufficient.

• Oligonucleotide Ligation Assay (OLA):     * Target: Primarily used to detect specific Single Nucleotide Polymorphisms (SNPs).     * Process:         1. DNA is denatured and the probe is hybridized.         2. Three probes are used: A Common probe, a Wild-type specific probe, and a Disease allele specific probe.         3. DNA ligase repairs breaks between adjacent bases only if the probe matches the allele at the $3^{\prime}$ end.         4. If no match exists, no ligation occurs.     * Detection: Ligation products are identified via size differences (electrophoresis) or color differences (fluorescence).

• Allele-Specific PCR:     1. Uses selective amplification of alleles to detect specific SNPs.     2. Involves one common primer and two allele-specific primers.     3. Mechanism: $Taq$ polymerase cannot extend the primer if there is a mismatch at the $3^{\prime}$ end.     4. Requires two separate PCR reactions run side-by-side to determine genotype (Wild-type, Heterozygote, or Mutant).

• Restriction Enzyme Digestion:     * Certain mutations create or destroy recognition sequences for restriction enzymes.     * Process: PCR amplification followed by digestion with the specific enzyme.     * Detection: Products are visualized on standard electrophoresis gels (e.g., fragments of $113\,bp$, $86\,bp$, and $199\,bp$ to distinguish alleles).

Clinical Application: Beta-Thalassemia (Case 14 - Nicolaides Family)

• Patient Profile: Spiros and Elena, healthy Greek Cypriot couple seeking preconception testing.

• Statistics: $1\text{ in }7$ Greek Cypriots is a carrier of the $\beta\text{-thalassemia}$ gene.

• Disease Profile ($\beta\text{-thalassemia}$):     * Inheritance: Autosomal recessive.     * Genetic Basis: Mutations in the $HBB$ gene ($\beta\text{-globin}$) on $11p15.4$.     * Physiological Effects: Low hemoglobin (oxygen lack), shortage of Red Blood Cells (RBCs), abnormal blood clots.     * Symptoms: Pale skin, fatigue, weakness, failure to thrive, jaundice, enlarged organs, misshapen bones, and life-threatening anemia starting before age $2$.     * Clinical Signs:         * Hypochromia: Deficiency in pigment.         * Poikilocytes: Teardrop-shaped cells.         * Basophilic stippling: Spotty basophils.         * Microcytosis: Smaller RBCs.         * Radiology: "Hair-on-end" skull and osteoporotic hands due to bone marrow extension.

• Common Mutations in Greek Cypriots ($98.4\%$ of cases):     * c.93-21G>A (Intron 1): $79.8\%$     * c.92+6T>C (Intron 1): $5.5\%$     * c.92+1G>A (Intron 1): $5.1\%$     * c.316-106C>G (Intron 1): $5.1\%$     * $p.Gln39X$ (Exon 2): $2.9\%$

• Case Results:     * Spiros: Carrier for $p.Gln39X$ (determined by Allele-Specific PCR).     * Elena: Carrier for c.316-106C>G (determined by Restriction Enzyme Digestion, showing bands at $406\,bp$, $300\,bp$, and $106\,bp$).

Clinical Application: Leber Hereditary Optic Neuropathy (Case 6 - Fletcher Family)

• Patient Profile: Frank Fletcher, age $22$.

• Disease Profile (LHON):     * Genetic Basis: Mutations in $mtDNA$ affecting Complex I of the mitochondria, leading to dysfunctional ATP production.     * Variants: $18$ SNPs associated with LHON; $5$ cause disease outright.     * Major European Mutations:         1. $G11778A \rightarrow p.Arg340His$ ($ND4$ protein)         2. $G3460A \rightarrow p.Ala52Tyr$ ($ND1$ protein)         3. $T14484C \rightarrow p.Met64Val$ ($ND6$ protein)

• Testing Results:     * Frank tested negative for $G11778A$ via restriction enzyme digestion (using $SfaNI$ or $MaeIII$).     * Testing for $G3460A$ was performed via Pyrosequencing.     * Result: Frank has the $G3460A$ mutation (Sequence: $GGTGTCA$ vs Control: $GGCGTCA$).

Gene Scanning and Deletion Analysis

• Gene Scanning Methods: Used for scanning exons for unknown mutations before sequencing (now less common due to affordable sequencing).     * Heteroduplex Detection: Uses dHPLC or melt curve analysis. Heteroduplexes (mismatches formed in heterozygotes during cooling) melt at lower temperatures than homoduplexes. Fluorescent dyes binding to double-stranded DNA (dsDNA) signal the denaturing process.     * Single-strand Conformation Polymorphism (SSCP): PCR products are denatured and snap-cooled; single-stranded DNA (ssDNA) hybridizes to itself, forming different conformations based on sequence.

• Multiplex Ligation-Dependent Probe Amplification (MLPA):     * Function: Combines hybridization and ligation to quantify copy number changes (deletions/duplications).     * Mechanism:         1. Probes join with DNA ligase only if they match the template $100\%$.         2. All probes share standard sequences for a single pair of universal primers.         3. "Stuffer" sequences of varying lengths ensure each PCR product has a unique size for identification via electrophoresis.     * Case 4 (Davies Family) Update: Martin was known to have a deletion. MLPA was used for his mother (Lisa) and sister (Jessica) because MLPA provides quantitative results (half-peaks indicate carrier status) which standard PCR cannot provide for females with two X chromosomes.

DNA Sequencing Technologies

• Sanger Sequencing (The Ultimate Test):     * Components: Start with amplified DNA, use a single primer (one strand), and add normal deoxynucleotides ($dNTPs$) and fluorescently labeled dideoxynucleotides ($ddNTPs$).     * Mechanism: $ddNTPs$ lack the $OH$ group required to join with the phosphate of the next nucleotide, causing chain termination.     * Analysis: Capillary electrophoresis reads fragment sizes and colors to produce a chromatogram (trace).

• Pyrosequencing (Sequencing via Synthesis):     * Mechanism: Bases added one at a time (G > C > T > A). If a base is incorporated, pyrophosphates are released, driving an enzyme cascade.     * Four Enzyme Mix:         1. DNA Polymerase: Synthesizes the sequence.         2. Sulfurylase: Converts pyrophosphate to ATP.         3. Luciferase: Uses ATP to trigger light emission from Luciferin.         4. Apyrase: Breaks down unincorporated $dNTPs$ before the next cycle.     * Output: Peak height on the trace indicates the number of specific bases added.

• Next-Generation Sequencing (NGS):     * Allows sequencing of whole genomes, exomes, RNA, or custom panels.     * Includes pyrosequencing, sequencing by synthesis, sequencing by ligation, and ion semiconductor sequencing.

Clinical Application: Intellectual Disability (Case 11 - Kowalski Family)

• Patient Profile: Karol, age $3$, displaying slow development, hypotonia, unique facial/body hair features, and partial loss of the corpus callosum.

• Exome Sequencing: Targets only exons (the protein-coding part of DNA).     * Rationale: Exome is < 2\% of the $3\text{ billion}$ base genome but contains $85\%$ of known disease variants.     * Workflow: Construct shotgun library $\rightarrow$ Hybridization/Pulldown $\rightarrow$ Sequencing $\rightarrow$ Mapping/Alignment to reference genome (approx. $100\times$ coverage).

• Karol’s Results:     * Initially found $16,400$ variants; filtered to $410$ unique variants ($397$ Het, $13$ Hom).     * Filtering Order: Nonsense/Frameshift $\rightarrow$ Splicing $\rightarrow$ Missense $\rightarrow$ Pathogenicity check.     * Final Discovery: A de novo nonsense mutation c.3304C>T ($p.Q1102X$) in the $ARID1B$ gene on chromosome $6$.     * Confirmation: Comparison with parents (Kamil and Klaudia) confirmed the mutation was present only in Karol.