BIO 2064 MolDiag Human Diseases March 2023 UPD

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MOLECULAR DIAGNOSTICS: BIO 2064
Molecular Detection of Human Diseases

Abnormality of chromosomes
• Autosomes: 23 pairs, 44 autosomes, 2 sex chromosomes
• Diploid, Polyploidy, and Aneuploidy
• Trisomy (n+1)
• Monosomy (n-1)
• Mosaicism: Early segregation errors (Ex: 47, XXY, 47, XYY, 45, X)
• Chromosomal abnormality :Detection through karyotyping and FISH
• Structural mutation or alternation: chromosomal breakage, and reunion
causes by chemicals, radiation, and etc. Chromosomal translocation is common in cancer cases
• Chromosomal mutations could result in spontaneous abortion (50%) or stillbirths (5%)
Patterns of Inheritance
• Gain of function mutation
• Loss of function mutation
• Dominant Negative mutation
• Autosomal-Dominant: One Parent: 50% chance of inheritance in offspring
• Autosomal Recessive: 25% chance of inheritance in off-springs
• X-linked /sex-linked via X-chromosome
• Penetrance Versus Expressivity
https://www.ncbi.nlm.nih.gov/books/NBK22090/figure/A671/?report=objectonly
Patterns of Inheritance:Dominant Negative

Examples of dominant negative:
• TP53
• Tyrosine kinase receptor
• Mutated protein blocks the function
of normal protein through dimerization or tetra-merization
Reference: Buckingham, L., & Flaws, M. L. (2012). Molecular Diagnostics: Fundamentals, Methods and
Ref: http://www.nature.com/nchembio/journal/v7/n5/images_article/nchembio.546-F7.jpg
Clinical Applications. 2nd Edition. F. A. Davis Company, Philadelphia, USA
Abnormality of chromosomes:Examples
Down Syndrome
Ref: http://www.wellcomecollection.org/idoccache/25cfd53a-60fb-4e36-95c8-46935021802a_1_0.jpg
Single Gene -Disorders

Disease Protein Type of Mutation Detection
Involved protein
Muscular Dystrophy Dystrophin (DMD), Structural Deletion RFLP+Southern Blot,
Xp12.2) Multiplex PCR,
Linkage analysis
Sickle cell anemia Hemoglobin Beta (11p Structural Missense PCR-RFLP,

15.5) sequencing
Hypercholesteremia Low-Density Cell surface Deletion, Sequencing, PCR

Lipoprotein Receptor receptor point mutation
(19p13.2)
Li-Fraumeni syndrome TP53 (17p13) Transcription Missene Sequencing, SSCP,
factor, Tumor DGGE
suppressor gene
Phenylketonuria Phenylalanine Metabolic Enzyme Missense, deletion Direct sequencing
Hydroxylase (12q24.1)
Highlighted Molecular Diagnostic methods

Linkage Analysis:
1. Mapping of gene mutation based on known locus, example STR
2. PCR STR to identify STR that is linked to affected individual
3. Identify STR that is consistently associated with the gene
mutation of affected individuals


 Tay-Sachs Disease: Mutation in HEXA gene which encodes beta-hexosaminidase A, an enzyme
presents in Lysosome, that helps to breakdown sphingo-lipids, Build-up of Sphingo-lipid is toxic to
neuron, leading to loss of neuromuscular function in affected infant/children
Blood Disorder
 Leiden Factor V mutation:
gene encodes for Factor V, a protein participates in clotting process
• When Factor V is mutated (Leiden Mutation), increase risk of clotting is expected. Example,
increase risk of deep vein thrombosis
Sequence Specific Primer PCR

PCR-RFLP Clinical Applications. 2nd Edition. F. A. Davis Company, Philadelphia, USA

Clotting Cascade
Damaged surface and tissue can trigger clotting

Ref: http://theprintedword.newartriot.net/eoblair/blood/clotting_cascade.gif
• Mutations of F8 gene result in inhibition of clotting process: Hemophilia

• F8 gene is located at X chromosome
Ref: http://ghr.nlm.nih.gov/gene/F8
• Mutations of F8 gene result in inhibition of clotting process: Hemophilia

• F8 gene is located at X chromosome, and F8 mutations affect males
http://ommbid.mhmedical.com/data/Books/ommbid/ch172fg2.gif
Ref: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1299132/pdf/5-7400221.pdf
1. Mutation or delete of Dystrophin gene results in Duchene Muscular Dystrophy (DMD)

2. Dystrophin links cytoskeleton and muscle fibers to sarcolemma cell membrane
through Dystrophin associated protein complex
3. Mutation or dystrophy gene results in loss of neuromuscular function
4. Dystrophin is a sex-linked protein
Dystrophin Mutations
• Deletions of Dystrophin gene
can be detected by Southern Blot
and sequence specific primer PCR
• Gene deletion results in loss of
function of Dystrophin protein
http://www.nature.com/nrg/journal/v14/n6/full/nrg3460.html#f2
Hemochromatosis: Over-absorption of iron from food
• Over-absorption of iron can lead to liver, and skin damage as well as heart disease and diabetes
• Mutations in HFE gene encodes for hemochromatosis type I protein
• PCR RFLP analysis of HFE mutation
Mutations in CFTR (Cystic Fibrosis)
• CFTR forms channels and regulates flow of sodium and chloride ions in the lung
• Mutations in CFTR affect CFTR function, leading to fluid accumulation in the lung
• CFTR mutations can be identified through SSCP, SSP-PCR, and PCR-RFLP
Summary
• Human diseases are results of gene and chromosomal abnormality (mutation, trisomy and etc)
• Patterns of mutation inheritance includes gain of function, loss of function, dominant negative, and etc
• Sex linked diseases (DMD and Hemophilia) due to mutation/deletion of Dystrophin, and Factor VIII affect males
population
• Gene mutation of Leiden Factor V increases risk of deep vein thrombosis
• Factor VIII mutations affect blood clotting, leading to hemophilia
• Receptor mutations of HFE and CFTR genes affect iron absorption, and flow of sodium Chloride, respectively
• Detection of the human disease mutations can be carried out through SSCP, SSP-PCR, PCR-RFLP,
Southern blot, direct sequencing, and FISH.

BIO 2064 MolDiag Human Diseases March 2023 UPD

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BIO 2064 MolDiag Human Diseases March 2023 UPD

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Restricted/Do Not Distribute/JT

MOLECULAR DIAGNOSTICS: BIO 2064

Molecular Detection of Human Diseases

MOLECULAR DIAGNOSTICS: BIO 2064

MOLECULAR DIAGNOSTICS: BIO 2064

MOLECULAR DIAGNOSTICS: BIO 2064

Patterns of Inheritance:Dominant Negative

MOLECULAR DIAGNOSTICS: BIO 2064

MOLECULAR DIAGNOSTICS: BIO 2064

Single Gene -Disorders

Sickle cell anemia Hemoglobin Beta (11p Structural Missense PCR-RFLP,

Hypercholesteremia Low-Density Cell surface Deletion, Sequencing, PCR

MOLECULAR DIAGNOSTICS: BIO 2064

Highlighted Molecular Diagnostic methods

Clinical Applications. 2nd Edition. F. A. Davis Company, Philadelphia, USA

MOLECULAR DIAGNOSTICS: BIO 2064

Sequence Specific Primer PCR

PCR-RFLP Clinical Applications. 2nd Edition. F. A. Davis Company, Philadelphia, USA

Damaged surface and tissue can trigger clotting

• Mutations of F8 gene result in inhibition of clotting process: Hemophilia

• Mutations of F8 gene result in inhibition of clotting process: Hemophilia

MOLECULAR DIAGNOSTICS: BIO 2064

1. Mutation or delete of Dystrophin gene results in Duchene Muscular Dystrophy (DMD)

Hemochromatosis: Over-absorption of iron from food

Mutations in CFTR (Cystic Fibrosis)

MOLECULAR DIAGNOSTICS: BIO 2064

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