genetics and molecular biology

1. M.Prasad Naidu
MSc Medical Biochemistry,
Ph.D.Research Scholar

2. Classes of Biomolecules
Affected in Disease
 All classes of biomolecules found in cells are affected
in structure, function, or amount in one or another
disease
 Can be affected in a primary manner (e.g., defect in
DNA) or secondary manner (e.g., structures, functions,
or amounts of other biomolecules)

3. Rate of Biochemical
Alterations
 Biochemical alterations that cause disease may
occur rapidly or slowly
 Cyanide (inhibits cytochrome oxidase) kills within a
few minutes
 Massive loss of water and electrolytes (e.g., cholera)
can threaten life within hours
 May take years for buildup of biomolecule to affect
organ function (e.g., mild cases of Niemann-Pick
disease may slowly accumulate sphingomyelin in liver
and spleen)

4. Deficiency or Excess of
Biomolecules
 Diseases can be caused by deficiency or excess of
certain biomolecules
 deficiency of vitamin D results in rickets, excess
results in potentially serious hypercalcemia
 Nutritional deficiencies
 primary cause - poor diet
 secondary causes - inadequate absorption,
increased requirement, inadequate utilization,
increased excretion

5. Organelle Involvement
 Almost every cell organelle has been involved in
the genesis of various diseases

6. Different Mechanisms, Similar
Effect Different biochemical mechanisms can produce
similar pathologic, clinical, and laboratory findings
 The major pathological processes can be produced by
a number of different stimuli
 e.g., fibrosis of the liver (cirrhosis) can result from
chronic intake of EtOH, excess of copper (Wilson’s
disease), excess of iron (primary hemochromatosis),
deficiency of α1-antitrypsin, etc.
 different biochemical lesions producing similar end
point when local concentration of a compound
exceeds its solubility point (excessive formation or
decreased removal) → precipitation to form a calculus
 e.g., calcium oxalate, magnesium ammonium phosphate, uric
acid, and cystine may all form renal stone, but accumulate for
different biochemical reasons

7. Genetic Diseases
 Many disease are determined genetically
 Three major classes: (1) chromosomal disorders, (2)
monogenic disorders (classic Mendelian), and (3)
multifactorial disorders (product of multiple genetic
and environmental factors)

8. Genetic Diseases
 Polygenic denotes disorder caused by multiple
genetic factors independently of environmental
influences
 Somatic disorders - mutations occur in somatic
cells (as in many types of cancer)
 Mitochondrial disorders - due to mutations in
mitochondrial genome

9. Chromosomal Disorders
 Excess or loss of chromosomes, deletion of part
of a chromosome, or translocation
 e.g., Trisomy 21 (Down syndrome)
 Recognized by analysis of karyotype
(chromosomal pattern) of individual (if
alterations are large enough to be visualized)
 Translocations important in activating
oncogenes
 e.g., Philadelphia chromosome - bcr/abl)

10. Monogenic Disorders
 Involve single mutant genes
 Classification:
(1) autosomal dominant - clinically evident if one
chromosome affected (heterozygote)
 e.g., Familial hypercholesterolemia
(2) autosomal recessive - both chromosomes
must be affected (homozygous)
 e.g., Sickle cell anemia
(3) X-linked - mutation present on X
chromosome
 females may be either heterozygous or homozygous
for affected gene
 males affected if they inherit mutant gene

11. Multifactorial Disorders
 Interplay of number of genes and environmental
factors
 pattern of inheritance does not conform to classic
Mendelian genetic principles
 due to complex genetics, harder to identify
affected genes; thus, less is known about this
category of disease
 e.g., Essential hypertension

12. Inborn Error of Metabolism
 A mutation in a structural gene may affect the
structure of the encoded protein
 If an enzyme is affected, an inborn error of
metabolism may result
 A genetic disorder in which a specific enzyme is
affected, producing a metabolic block, that may
have pathological consequences

13. Inborn Error of Metabolism
 A block can have three results:
(1) decreased formation of the product (P)
(2) accumulation of the substrate S behind the block
(3) increased formation of metabolites (X, Y) of the
substrate S, resulting from its accumulation
 Any one of these three results may have
pathological effects
S → P Increased S → Decreased P
E
Normal Block
↑
Increased X,Y
*E

14. Inborn Error of Metabolism
 Phenylketonuria - mutant enzyme is usually
phenylalanine hydroxylase
 synthesize less tyrosine (often fair skinned), have ↑
plasma levels of Phe, excrete ↑phenylpyruvate and
metabolites
 If structural gene for noncatalytic protein affected by
mutation can have serious pathologic consequences
(e.g., hemoglobin S)
Increased phenylalanine → Decreased tyrosine
Block
↑
Increased phenylpyruvic acid
*E

15. Genetic Linkage Studies
 The more distant two genes are from each other on the
same chromosome, the greater the chance of
recombination occurring between them
 To identify disease-causing genes, perform linkage
analysis using RFLP or other marker to study inheritance
of the disease (marker)

16. Genetic Linkage Studies
• Simple sequence repeats (SSRs), or
microsatellites, small tandem repeat
units of 2-6 bp are more informative
polymorphisms than RFLPs; thus
currently used more

17. Methods to clone disease genes
 Functional approach
 gene identified on basis of biochemical defect
 e.g., found that phenotypic defect in HbS was
Glu→Val, evident that mutation in gene encoding
β-globin
 Candidate gene approach
 genes whose function, if lost by mutation, could
explain the nature of the disease
 e.g., mutations in rhodopsin considered one of the
causes of blindness due to retinitis pigmentosa

18. Methods to clone disease genes
 Positional cloning
 no functional information about gene product,
isolated solely by it chromosomal position
(information from linkage analysis
 e.g., cloning CF gene based on two markers that
segregated with affected individuals
 Positional candidate approach
 chromosomal subregion identified by linkage studies,
subregion surveyed to see what candidate genes
reside there
 with human genome sequenced, becoming method of
choice

19. Identifying defect in disease
gene
 Once disease gene identified, still can be arduous
task identifying actual genetic defect
Mutations in CFTR gene
Structure of CFTR gene and
deduced protein

20. Ethical Issues
 Once genetic defect identified, no treatment options
may be available
 Will patients want to know?
 Is prenatal screening appropriate?
 Will identification of disease gene
affect insurability?
• e.g., Hungtington’s disease - mutation due to trinucleotide
(CAG) repeat expansion (microsatellite instability)
– normal individual (10 to 30 repeats)
– affected individual (38 to 120) - increasing length of
polyglutamine extension appears to correlate with ↑ toxicity

21. Molecular Medicine
 Knowledge of human genome will aid in the
development of molecular diagnostics, gene
therapy, and drug therapy

22. Gene expression in diagnosis
 Diffuse large B-cell lymphoma (DLBCL),
a disease that includes a clinically and
morphologically varied group of tumors
that affect the lymph system and blood.
Most common subtype of non-
Hodgkin’s lymphoma.
 Performed gene-expression profiling
with microarray containing 18,000
cDNA clones to monitor genes involved
in normal and abnormal lymphocyte
development
 Able to separate DLBCL into two
categories with marked differences in
overall patient survival.
 May provide differential therapeutic
approaches to patients

23. Treatment for Genetic Diseases
 Treatment strategies
(1) correct metabolic consequences of disease by
administration of missing product or limiting
availability of substrate
 e.g., dietary treatment of PKU
(2) replace absent enzyme or protein or to increase its
activity
 e.g., replacement therapy for
hemophilia
(3) remove excess of stored
compound
 e.g., removal of iron by periodic
bleeding in hemochromatosis
(4) correct basic genetic abnormality
 e.g., gene therapy

24. Gene Therapy
 Only somatic gene therapy is permissible in
humans at present
 Three theoretical types of gene therapy
 replacement - mutant gene removed and replace
with a normal gene
 correction - mutated area of affected gene would
be corrected and remainder left unchanged
 augmentation - introduction of foreign genetic
material into cell to compensate for defective
product of mutant gene (only gene therapy
currently available)

25. Gene Therapy
 Three major routes of delivery of genes into humans
(1) retroviruses
 foreign gene integrates at random sites on chromosomes,
may interrupt (insertional mutagenesis) the expression of
host cell genes
 replication-deficient
 recipient cells must be
actively growing for
integration into genome
 usually performed ex vivo

26. Gene Therapy
(2) adenoviruses
 replication-deficient
 does not integrate into host cell genome
 disadvantage: expression of transgene gradually
declines requiring additional treatments (may
develop immune response to vector)
 treatment in vivo, vector can be introduced into
upper respiratory tract in aerosolized form
(3) plasmid-liposome complexes

27. Gene Therapy
 Conclusions based on recent gene therapy trials
 gene therapy is feasible (i.e., evidence for expression
of transgene, and transient improvements in clinical
condition in some cases
 so far it has proved safe (only inflammatory or
immune reactions directed toward vector or some
aspect of administration method rather than toward
transgene
 no genetic disease cured by this method
 major problem is efficacy, levels of transgene product
expression often low or transient

28. Genetic Medicines
 Antisense oligonucleotides
 complementary to specific mRNA
sequence
 block translation or promote
nuclease degradation of mRNA,
thereby inhibit synthesis of protein
products of specific genes
 e.g., block HIV-1 replication by
targeting gag gene
 Double-stranded DNA to form
triplex molecule