LAB Molecular Diagnosis of Mutation and Inherited Diseases

1. Translocation

2. Deletion

3. Insertion

4. Duplication

5. Inversion

6. Tandem Repeat Expansion

Types of Structural Variants:

1. Point Mutation

2. Frameshift Mutation

3. Missense Mutation

4. Nonsense Mutation

5. Splice Site Mutation

Types of Mutation:

Destructive (non-balanced)

These SV Types are :

• Insertion

• Deletion

• Interspersed Duplication

• Tandem Duplication

Non-Destructive (balanced)

These SV Types are :

• Inversion

• Translocation (inter- or intra- chromosome)

Structural Variants

• Changes occur in our genes, possibly in the sequence of our DNA

• The structure of the genetic segment is the one being changed in the chromosome

• Keen to excessive mutation

Mutation

• Smaller scale changes, possibly only in the DNA sequence.

Inherited disease

Structural Variants and Mutations has similarity because they are both the cause of ________?

Translocation

• From one chromosome, there is a segment break as it will attach to another chromosome.

• A chromosome arm can be longer compared to the other arm.

Deletion

• Loss of segment

• Chromosome or DNA segment shortens.

Insertion

• Insertion of DNA sequence or segment in a chromosome.

Tandem Duplication

• It is adjacent or it came from the original copy.

Interspersed Duplication

• Possible to be randomly be inserted or duplicated in the chromosome or DNA sequence. It is also not adjacent from the original copy.

Inversion

• This is where the segment of DNA happens. Inside the chromosome, the orientation gets flipped which is relative to the normal orientation of the chromosome or DNA sequence.

Tandem Repeat Expansion

• There is an expansion in the DNA sequence.

• It is known to be Dinucleotide or Nucleotide.

• It happens inside the genome.

Complex Rearrangements

• Combination of structural variant, possibly 2 or more in 1 variant chromosome.

Point Mutation

• Single nucleotide base can be inserted, changed, or deleted from another nucleotide base.

Ex: Sickle Cell Anemia

• HPB gene of the beta globulin protein, the beta glutamic acid is being changed.

Sickle Cell Anemia

• HPB gene of the beta globulin protein, the beta glutamic acid is being changed

Frameshift Mutation

• It can be insertion or deletion, it is being shift into the reading frame of the gene.

Ex: Cystic Fibrosis

• If frameshift mutation occurs, transport of chloride ions (page 371)

Cystic Fibrosis

• If frameshift mutation occurs, transport of chloride ions

Missense Mutation

• DNA changes wherein the codon is being affected. The codon codes for the different amino acids.

Example: Hypercholesterolemia

• The LDLR gene is affected as the clearance of LDL (the fats) is being impaired

Hypercholesterolemia

• The LDLR gene is affected as the clearance of LDL (the fats) is being impaired

Nonsense Mutation

• There are changes or the stop codon is being affected. We will have pre-mature stop codon.

Example: Duchenne Muscular Dystrophy

• DMD gene is being affected. (page 371)

Duchenne Muscular Dystrophy

• DMD gene is being affected

Splice Site Mutation

• The junction of the intron and exon sequence is being affected which also affects the mRNA splicing.

Example: Beta-Thalassemia

• HPB gene is affected as there would be abnormal splicing. Decrease or reduced functional hemoglobin, (page 376)

Beta-Thalassemia

• HPB gene is affected as there would be abnormal splicing. Decrease or reduced functional hemoglobin.

Diseases Caused by Genetic Mutation and Structural Variant

THESE ARE:

• Alpha-1 Antitrypsin Deficiency

• Cystic Fibrosis

• Duchenne Muscular Dystrophy

• Huntington’s Disease

• Sickle Cell Disease

• Thalassemia

• Tay-Sachs Disease

• Alcohol Dependence

• Charcot-Marie-Tooth Disease

• Cardiovascular Disease (CVD)

• Parkinson’s Disease

• Achondroplasia

• Duane’s Retraction Syndrome

• Fragile X Syndrome

• Hemophilia

• Marfan Syndrome

• Cri du chat Syndrome

• Dominant Optic Atrophy (DOA)

Achondroplasia

What Disease has this type of Gene Involved?

GENE: FGFR3 gene

Duane’s Retraction Syndrome

What Disease has this type of Gene Involved?

GENE: CHN1 gene

Fragile X Syndrome

What Disease has this type of Gene Involved?

GENE: FMR1 gene

Hemophilia A

What Disease has this type of Gene Involved?

GENE: F8 gene

Hemophilia B

What Disease has this type of Gene Involved?

GENE: F9 gene

Marfan Syndrome

What Disease has this type of Gene Involved?

GENE: FBN1 gene

Cri du chat Syndrome

What Disease has this type of Gene Involved?

GENE: Deletion of part of the short (p) arm of chromosome 5

Dominant Optic Atrophy (DOA)

What Disease has this type of Gene Involved?

GENE: OPA1 gene

Alpha-1 Antitrypsin Deficiency

What Disease has this type of Gene Involved?

GENE: SERPINA1 gene

Cystic Fibrosis

What Disease has this type of Gene Involved?

GENE: CFTR gene

Duchenne Muscular Dystrophy

What Disease has this type of Gene Involved?

GENE: DMD gene

Huntington’s Disease

What Disease has this type of Gene Involved?

GENE: HTT gene

Sickle Cell Disease

What Disease has this type of Gene Involved?

GENE: HBB gene

Thalassemia

What Disease has this type of Gene Involved?

GENE: HBB gene, HBA1 and HBA2 genes

Tay-Sachs Disease

What Disease has this type of Gene Involved?

GENE: HEXA gene

Alcohol Dependence

What Disease has this type of Gene Involved?

GENE: Multiple gene involved, including ADH1B and ALDH2 genes

Charcot-Marie-Tooth Disease

What Disease has this type of Gene Involved?

GENE: Multiple genes, including PMP22, MFN2, and others

Cardiovascular Disease (CVD)

What Disease has this type of Gene Involved?

GENE: APOE, LDLR

Parkinson’s Disease

What Disease has this type of Gene Involved?

GENE: PARK2, SNCA, LRRK2, and other genes

1. The current knowledge of the gene(s) associated with the disease in question.

2. Degree of Molecular Heterogeneity

There are 2 Factors to consider in Choice of testing, these are:

The current knowledge of the gene(s) associated with the disease in question.

These are part of what factor in choice of testing?

• Those for which the causative gene has been isolated and

• Those for which it has not.

  • We can know it by checking the family history.

  • If the person has been tested by the signs and symptoms.

  • We can have an initial diagnosis or presumption regarding the disease the patient faces.

  • Once the disease is isolated, we can use direct mutation analysis .

    • PCR

    • NGS

  • If hindi pa naiisolate or naiidentify what disease, we use unknown or non-isolated pack .

    • We use linkage analysis as we use a marker known as STR and SNP.

      • Short Tandem Repeats (STR)

      • Single Nucleotide Polymorphism (SNP)

• PCR

• NGS

• Once the disease is isolated, we can use Direct Mutation Analysis. And Examples are:

• Short Tandem Repeats (STR)

• Single Nucleotide Polymorphism (SNP)

If hindi pa naiisolate or naiidentify what disease, we use Unknown or Non-Isolated pack. And Examples are:

Degree of Molecular Heterogeneity

These are part of what factor in choice of testing?

• If there are multiple genes involved that cause the disease, usually pag mas maraming genes ang involved - mas maraming test ang gagawin.

• Direct Mutation Analysis

  • most preferred than the direct linkage analysis.

    • Not timing consuming since you already know the disease.

    • You can directly diagnose it.

Direct Mutation Analysis

• must preferred than the direct linkage analysis.

  • Not timing consuming since you already know the disease.

  • You can directly diagnose it.

1. PCR Amplification

2. Gel Electrophoresis

3. Restriction Enzyme Analysis

4. Dot Blot Hybridization

5. Microarray Analysis

Examples of Direct Mutation Analysis Application:

PCR Amplification

• To amplify our products.

• To produce multiple copies.

Gel Electrophoresis

• For reading the different fragment size of the DNA.

• We compare it to the control (normal or diseased)

• Interpretation: If there are changes in the band. There is a mutation in the patient.

  • Insertion - additional band compared dun sa control.

  • Deletion - missing band.

Restriction Enzyme Analysis

• We would see if the restriction enzyme would react - if there is a possibility that it contains patient DNA with restriction site.

• If the restriction enzyme would react with the restriction site through cleavage pattern.

Dot Blot Hybridization

• We use labelled oligonucleotides which are fluorescently labelled.

Microarray Analysis

• We can detect multiple mutations simultaneously.

• We use multiple probes to cover the whole genome as we can consider the ACGH or ECGH

1. Single-Strand Conformation Polymorphism (SSCP)

2. Denaturing Gradient Gel Electrophoresis (DGGE)

3. Denaturing High-Performance Liquid Chromatography (DHPLC)

Examples of Techniques used in Unknown Mutation:

• Chemical Base

• Heat Base

• Temperature Base

2 Methods for DDG:

DHPLC

• we use chromatography column

• We look for the difference in retention time (gaano katagal madedenature)

• We use PCR amplification.

  • Wild Type DNA - standard and reference.

  • Mutant

Heterocouplexes

• nagband yung wild type and mutant. Nahybridize sila pareho.

Homocouplexes

• it came from the original double stranded DNA.

1. Multiplex Ligation-Dependent Probe Amplification (MLPA)

2. Capillary Electrophoresis-Strand Conformation Polymorphism (CE-SCP)

3. Stuffer-free Multiplex CNV Detection Method

Examples of Techniques used in Detecting Structural Changes:

Multiplex Ligation-Dependent Probe Amplification (MLPA)

• It is a method used to detect copy number variations (CNVs) in the genome.

• It combines the advantages of multiplex PCR and capillary electrophoresis and can detect deletions, duplications, or insertions in specific genomic regions.

• Duchenne Muscular Dystrophy

• Prader-Willi Syndrome

• Angelman Syndrome

Diseases tested using MLPA include the following:

Conventional MLPA

The picture is the flow of what MLPA?

Stuffer-free MLPAS

The picture is the flow of what MLPA?

1. Array Comparative Genomic Hybridization (aCGH)

2. Next-Generation Sequencing (NGS)

Examples of Whole Genome Analysis Techniques:

Array Comparative Genomic Hybridization (aCGH)

• We use so many specific probes as it will cover the whole genome to detect.

• The probes will bind to the DNA.

• Data Analysis: if there would be fluorescence in the genome.

Next-Generation Sequencing (NGS)

• More rapid and cost-effective.

• Detects structural variation.

• Higher solution.

• We could analyse specific segment or site of the genome that we want to test.

Single-cell sequencing (SCS)

• is a cutting-edge molecular technique that allows for the analysis of individual cells at the genomic, transcriptomic, or epigenomic level.

• Unlike traditional bulk sequencing methods, which analyze populations of cells together, SCS provides insights into the genetic molecular characteristics of individual cells within a heterogenous sample.

1. Cancer Research

2. Embryonic Development

3. Clinical Diagnostics

4. Stem Cell Research

5. Epigenetics

Single Cell Sequencing is used in the ff: