MOLECULAR BIOLOGY:
Principles in Clinical Medicine
Dr. Sophia Green
Department of Pathology, UWI
November 2022
LEARNING OBJECTIVES
• Identify with molecular techniques
• Applicability of techniques:
• How can molecular techniques assist in clinical
medicine:
• Diagnosis
• Prognosis
• Treatment
MOLECULAR BIOLOGY
• Definition:
• Branch of science that deals with the
structure and function of
macromolecules which are essential to
life
• Macromolecules:
• Nucleic acids
• Proteins
• Spans all walks of biochemistry and
genetics
• Interplay/relationships
MOLECULAR BIOLOGY
• Human Genome Project
• DNA sequencing of the whole human genome
• Known sequenced genomes:
• Human
• ~22,300 protein-coding genes
• <7% protein families specific for vertebrates
• Animals
• Micro-organisms (fungus, bacteria, protozoa)
• Plants
•  ABILITY TO DETECT & IDENTIFY DNA/RNA
SEQUENCES
MOLECULAR BIOLOGY
• Applicability in clinical medicine is based on the ability
to:
① Manipulate DNA
 Dismantle into smaller more manageable strands
•

Locate & visualize genes
Reassemble the strands
② Make individual genes
MOLECULAR TOOLS
• Polymerase chain reaction
(PCR)*
• Electrophoresis
• Gel electrophoresis
• Blotting & probing
• Western blot, Southern blot
• DNA sequencing
• DNA microarrays
• In situ Hybridization
• FISH, CISH
• Restriction enzymes
MOLECULAR TOOLS
• Next-generation sequencing
(massive parallel sequencing)
• Whole genome sequencing
• Whole exome sequencing
APPLIC ATIONS IN CLINIC AL
MEDICINE
IDENTIFICATION
• Genetic diseases
• Neoplasia
• Infectious diseases
THERAPY
• Genetic
• Haemophilia, Thalassemia
• Neoplasia
• Identifying individuals
• Personalized medicine
• Gender
• Cancer immunotherapy
MOLECULAR DISEASE
• Definition:
• Any disease in which the pathogenesis can be traced to a
single chemical substance (usually a protein) which is
usually absent, abnormal in structure or function, or
produced in inadequate amounts
• Any change in DNA sequence
 MUTATION !!!
MUTATIONS
• Inherited or acquired
• Type:
• Large scale
• Number e.g. Trisomy 21 (Down’s Syndrome)
• Structure e.g. Duplication On Chr 17  CharcotMarie-Tooth disease
• Small scale
• Point mutations e.g. Sickle cell anemia
• Dynamic
• Tri- or tetra-nucleotide repeats
APPLIC ATIONS IN CLINIC AL
MEDICINE
• Pathogenesis
• Diagnosis & Prognosis
• Therapy
MOLECULAR DISEASE
• Example:
• Diagnosis of Sickle cell disease:
ELECTROPHORESIS
PCR
NEOPLASIA
• Cumulative mutations
• E.g.Vogelstein (APC
pathway) and colon cancer
• Mutations in
neoplasia/cancer:
• Clonal
• Germline vs. Somatic
• Genes:
• Oncogenes
• Tumour suppressor
• DNA repair
• Apoptosis
NEOPLASIA
• Clinical Applications:
• Clonality
• Lineage
• Aetiology
• Diagnosis
• Prognosis
• Treatment
NEOPLASIA: CLONALITY
• Atypical lymphoid proliferation:
• Lymphoma vs. Reactive
• Different antigens exposure result in different
immunoglobulin heavy chains (IgH) present on lymphocytes
• LYMPHOMA:
• IgH gene rearrangement is the same (clonal) in the
neoplastic lymphocytes
• REACTIVE LYMPHOCYTES:
• Each lymphocyte class have different rearrangements
• PCR  amplify IgH gene
NEOPLASIA: CLONALITY
• Example: Atypical lymphoid proliferation
• Results of IgH gene PCR followed by agarose gel
electrophoresis
Modified from: Netto & Saad,
2006
Patient 1: lymphoma
Patient 2: reactive lymphoid
proliferation
NEOPLASIA: AETIOLOGY
• Example: HPV serotyping in cervical neoplasia
NEOPLASIA: AETIOLOGY
• Example: HPV serotyping in cervical neoplasia
NEOPLASIA: DIAGNOSIS
• Example: Detection of t(9;22) in chronic myeloid leukaemia
Normal nucleus:
(CML) using FISH
2 red signals; 2 green signals
Nucleus with CML:
1 red signal; 1 green signal;
1 yellow signal (or fused red-green)
Modified from: Netto et al., 2003
NEOPLASIA: PROGNOSIS
• Example: mTOR pathway and panceatic neuroendocrine
cancer
NEOPLASIA: TREATMENT
• Targeted therapy
• Example: Breast cancer with amplification of
Her/2neu gene:
• Confirmed by FISH
• Green-labeled probe for chromosome 17 centromere
• Red/orange-labeled probe for Her/2neu gene on chr
17
• No amplification: 2 greens & 2 reds/oranges
• Amplification: 2 greens & multiple reds/oranges
NEOPLASIA: TREATMENT
(TARGETED THERAPY TRASTUZUMAB)
Detection of HER2/neu amplification using FISH:
Patient 1
No Amplification
Modified from: Netto & Saad, 2006
Patient 2
Amplification
INFECTIOUS DISEASES
• DNA or RNA sequences of the
organisms’ genome
• Commonly utilized tools:
• ISH
• PCR
• Applications:
• Diagnosis
• HPV and cervical cancer
• C. difficile and pseudomembranous
colitis
• Epidemiology:
• Schistosoma and haematuria
• Therapy
• Recombinant vaccines
• Cervarix & Gardasil
• Guided antibiotic therapy
• Multidrug resistant
Staphylococcus aureus
INFECTIOUS DISEASES
• Example: Diagnosis – determine whether a
biopsy of cervix uteri contains high-risk HPV,
using in situ hybridization
Positive for HPV DNA
Fluorescent-labeled probe
(pink)
Positive for HPV DNA
Chromagen-labeled probe
(brown)
INFECTIOUS DISEASES
• Example: Subtyping Schistosoma species in urine
IDENTIFICATION
• Short tandem repeats/Microsatellites
• Genomic DNA has multiple short segments of DNA motifs
• Vary from person to person
• Single locus can have may different alleles with different numbers of alleles
• Less variation from family member to family member
• The motifs are :
• 2-5 base pairs long
• Repeated in tandem number of times
• Commonly utilized tools:
• PCR
• Electrophoresis
• DNA sequencing
IDENTIFICATION
IDENTIFICATION
• Transplant
• Analysis of donor graft
• Quantify donor and recipient cells after allogeneic
bone marrow transplant
• Forensics
• Identify victim
• Identify perpetrator
• Parentage
IDENTIFICATION
• Example: Paternity determination. Alleged father excluded.
Modified from: Cheng & Zhang, 2008
IDENTITY DETERMINATION
• Example: Paternity determination. Alleged father –
inclusion.
Modified from: Cheng & Zhang, 2008
IDENTITY DETERMINATION
• JACKETS: Made in Jamaica
• May 25, 2011
• Wikileaks: 1/10
• Dr. Sonia King (UWI): 1/3
• Carigen (?published data): >1/4
GENE THERAPY
• Definition:
• The delivery of foreign nucleic acid polymers into a patients cell as
a mode of treatment so as to achieve some therapeutic effect
GENE THERAPY
• Clinical applications:
• Restore protein deficiency (quantitative or qualitative) caused by
mutation by inserting non-mutated gene
• Example SCD:
• Genetic correction of point mutation
GENE THERAPY
• Clinical Applications:
• Cancer immunotherapy
• Example vaccinia virus and colon cancer
SUMMARY
• Innumerable clinical applications and increasing
exponentially everyday
• Clinical applications: Molecular diseases,
Neoplasia, Infectious diseases, Forensics
• Roles: Diagnosis, Prognosis, Treatment (WGS –
personalized medicine)
• Gene therapy: Already being utilized and very
promising (esp. in cancer immmunotherapy)