c

c 

m ? Field of study
a Molecular biology
ʹ DNA, genes and gene expression
b Human genetics
ʹ human variation and heredity
c Medical genetics
ʹ hereditary nature of human disease
d Clinical genetics
ʹ direct care, diagnosis and counseling of patients with genetic disease; focus on patient and family members
2 ? Clinical genetics
a m

- Tjio and Levan discovered correct human chromosome number (4, not 48)
b m
s
Cytogenetics
- structure and function of cell especially chromosomes
- chromosomes differentiated, chromosome breakpoints in translocation elucidated
- deletion named and understood
- deletion syndromes (DiGeorge and Prader-Willi syndrome) identified
Somatic cell genetic
- identify chromosome location of gene without sexual crossing
Biochemical genetics
- biochemical events involved in genetics
c m
8s
- molecular probes -> cytogenetics more accurate
d m

s till now
- diagnosis and treatment
- genetic screening
- genetic counseling
3 ? Genetic screening
- genetically test all relevant individuals in a defined population on an equitable basis
- objectives:
a ? Allow individuals to be better informed of genetic risks and reproductive options
b ? Prevent morbidity and alleviate suffering
- requirements:
a ? understand and calculate sensitivity, specificity, false positive, false negative, and positive predictive
value
b ? interpret negative test results
c ? understand type of genetic screening programmes and their aims
d ? be aware of genetic screening programme criteria
4 ? Genetic screening criteria
a ? Disease
- relatively common
- potentially serious effects which can be prevented
- can be treated early
- pregnancy can be offered to be terminated
b ? Test
- high sensitivity and specificity
- sensitivity -> proportion of cases which are detected (true positives)
- specificity -> proportion of true negatives obtained among non-disease patients
- positive predictive value -> proportion of individuals with positive test results with disease
- negative predictive value -> proportion of individuals with negative test results without the disease



  



 True positive (TP) False positive (FP) D




 False negative (FN) True negative (TN) D 
 
            
 c ? Programme
- offered fairly and equitably
- widely available
- morally acceptable by substantial proportion of population
- voluntary
- easily understood
- prior well-informed counseling
- cost-benefit factor
 ? Genetic counseling
- communication process that deals with the human problems associated with the occurrence, or risk of a
genetic disorder in a family
- to help individuals/family achieve:
a ? Comprehend the medical facts
b ? Appreciate how heredity contributes and the risk of recurrence
c ? nderstand the alternatives for dealing with the risk of recurrence
d ? Choose course of action based on risk, family goals, ethical and religious standards
e ? Make the best possible adjustments for the disorder
- consultands: person seeking genetic counseling
- ultimate decision must be based on patient͛s belief (nondirective counseling)
- nondirective counseling:
a consultands make the decision
b strong commitment essential
c every patient has different needs
d counselors must not throw in own bias
e directive counseling: patients told what his decision should be
- patients will need time to talk privately with others
- patients will need time to understand and come to terms with nature and consequences
- retrospective counseling: given after individual develops genetic disease/after couple have child with birth
defect
- prospective counseling: given in anticipation/being at risk for developing genetic disease; increase chance of
having normal child

 ? ndications for genetic counseling

a ? Mother/father much older
b ? Family history of genetic disease
c ? Genetic disease more prevalent in ethnic group
eg: whites ʹ cystic fibrosis; blacks ʹ sickle cell anemia; Ashkenazi jews ʹ Tay-Sachs disease;
Mediterraneans ʹ ɴ-thallasemia; Asians ʹ ɲ-thallasemia
d ? Woman planning for pregnancy taking medication that is teratogenic/exposed to occupational
chemicals/alcohol
e ? Woman successfully treated for phenylketonuria, having normal diet, high blood phenylalanine
f ? Woman with insulin-dependent diabetes mellitus
g ? Woman with no history of rubella
h ? Previous child with multiple congenital defect
i ? Consanguinity
j ? epeated pregnancy lost/infertility
k ? Before genetic testing, after results of genetic testing
l ? Follow-up of positive result
 ? Diagnosis
- accurate diagnosis important -> to give adequate counseling and risk assessment
- cause has to be identified
- procedure carried out:
a ? Complete history taking
b ? Physical examination
c ? Cytogenetic study
d ? Biochemical study
 e ? Biopsy
f ? adiograph
g ? CAT scans
h ? Sonograph
8 ? Causes of syndromes
a ? Mendelian
b ? Non-medelian (environmental, chromosomal, multifactorial, sporadic)

? Mendelian syndromes
a ? Autosomal dominant
- one non-functional/mutant allele
- both gender can transmit
- multiple generations
- males and females equally affected
- variability of clinical findings
- later/adult onset
- homozygotes more severely affected
- new mutations
- eg: Achondroplasia; Noonan͛s syndrome; Ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome;
Apert͛s syndrome
b ? Autosomal recessive
- two non-functional/mutant allele
- horizontal transmission
-affected by parental consanguinity
- male and female affected
- consistent degree of severity
- early onset
- new mutations rare
- higher frequency in certain ethnic groups
- eg: Ataxia-telangiectasia syndrome; Meckel-Gruber syndrome; hizomelic chondrodysplasia punctata
syndrome; adial aplasia thrombocytopenia syndrome (TA)
c ? -linked dominant
- one non-functional/mutant allele at -chromosome
- no male-to-male inheritance
- affected females usually milder
- affected males not affected sons, all daughters affected
- mimic autosomal dominant
-lethal in affected males
- increased miscarriage (male fetuses)
- eg: Fragile syndrome; ncontinentia pigmenti type 
d ? -linked recessive
- no male-to-male transmission
- males more frequently affected
- females carrier but unaffected
- affected males related through females
- new mutations from maternal grandfathers
- eg: Menkes͛ syndrome; Hunter͛s syndrome (mucopolysaccharidosis type , gargoylism)
e ?
-linked
- male-to-male transmission only
- increased infertility
- discrepancy between chromosomal and phenotypic gender
m ? Environmental causes of birth defect
- when fetus exposed to causing agents in utero
a ? Medications
- fetal dilantin syndrome: ingestion of dilantin/phenytoin (epilepsy) during mst trimester
- retinoic acid/accutane: treatment of acne
- fetal warfarin syndrome: ingestion of warfarin sodium/Coumadin any time
 b ? Drugs
- fetal alcohol syndrome: cause of mental retardation, completely preventable
c ? nfectious agents
- rubella
- toxoplasmosis
- cytomegalovirus
- syphilis
d ? Deformations
- equinovarus (club foot): lack of amniotic fluid
- facial asymmetry: normal, grow into normal proportion
e ? Maternal diseases
- women successfully treated for phenylketonuria, back to normal diet, have high blood phenylalanine->
growth deficiency
- important to continue low phenylalanine diet
f ? thers
- early amnion rupture-> sporadic
- early in pregnancy -> anencephaly, facial clefting
- later in pregnancy -> limb reduction, polydactyl
mm ? Chromosomal causes
a ? Autosomal chromosome syndromes
- Down͛s syndrome (trisomy 2m)
- Patau͛s syndrome (trisomy m3)
- Cri-du-chat syndrome (p-deletion)
b ? -chromosome syndromes
- Turner͛s syndrome (4, )
- Klinefelter͛s syndrome (4,
)
c ? Microdeletion syndromes
- Prader-Willi syndrome, del(mqmm-qm3)
- Angelman͛s syndrome, del(mqmm-qm3)
- DiGeorge syndrome, del(22qmm 2)
- Miller-Dieker syndrome, del(mpm3 3)
m2 ? Multifactorial causes
a ? pen neural tube defects
i ? Anencephaly
ii ? Spina bifida cystica (meningocele, myelomeningocele)
b ? Pyloric stenosis
c ? Cleft lip/palate
d ? Coronary artery disease
e ? Hypertension
6 
cc

m ? verview
- all inherited genetic disease caused by various types of mutations
- mutations: permanent changes in base sequence of DNA
-how gene product altered determines the clinical phenotype and type of inheritance pattern
- dominant: mutation will be clinically evident even if only one chromosome is affected (heterozygous)
- recessive: both chromosomes must be affected (homozygous) to be clinically evident
- -linked: mutation present on chromosome
2 ? Monogenic disorders
- single gene disorders
-  groups:
a autosomal recessive
b autosomal dominant
c -linked
d haploinsufficiency
e dominant inheritance of a predisposition to develop cancer
3 ? Autosomal recessive inheritance
- loss-of-function mutations
- disables the gene, NA or protein -> little/no functional product produced
- common
- both copies of gene dysfunctional
- one functional copy sufficient to preclude clinical symptom
- both parents carrier, risk of child affected is 2
- many disease results from mutation in gene that codes for proteins involved in cellular metabolism
- include enzymes for metabolism (carbohydrates, nucleotides, amino acids, lipids and organic acids), protein
for maintaining cell structure, hemostasis, transport, cell-cell communication, and growth control also
inactivated
4 ? Sickle cell anemia
- disorder of ɴ-globin gene due to Glu-to-Val missence mutation at position 
- HbS can bind oxygen, but less soluble in deoxygenated blood ->form rod-shaped polymers aggregate ->
distortion and sickling of cells -> cannot enter capillaries -> blockage and lack of blood flow -> BC destroyed
-> hemolytic anemia
- phenotype not obvious at birth, within first 2 years
- excess swelling of feet and hand, recurrent infections, pain, enlarged spleen
- homozygotes: affected, usually fatal
- heterozygotes: not affected, greater resistance to malarial infection
- variants according to geographical location: Cameroon, Senegal, Saudi-Asian
- HbA = ɲ2ɴ2, HbS = ɲ2S2
- non-molecular diagnosis: full blood count; sickle solubility test
- molecular diagnosis:
a haemoglobin electrophoresis + confirmation with HLPC -> different Hb migrate at different speeds
b restriction endonuclease cleavage site -> adenine-thymine substitution recognized by 6, normal allele
cut (m mkb fragment) while mutant allele not cut (m 3 kb fragment)
 ? Cystic fibrosis
- common and potentially fatal
- mucus accumulation obstructing respiratory pathways
- infections ( 
D  ) may lead to respiratory failure
- mucus may obstruct pancreatic ducts -> intestine and liver problems
- males infertile
- excessive salt loss in sweat
- with treatment, life prolonged till 2
-4
- cystic fibrosis gene: chromosome q3m 2, 2 exons, 23 kb, codes for   kb mNA, protein cystic fibrosis
transmembrane conductance regulator (CFT) that is m48 amino acids
- CFT function: cyclic adenosine monophosphate (cAMP)-regulated Cl- channel, with transmembrane
domains spanning cell membranes as channel pores; regulate activity of other ion channel (sodium channel,
calcium-activated chloride channel)
-  cystic fibrosis caused by 3bp deletion in exon m leads to loss of phenylalanine at position 8 in CFT
 - 3 cystic fibrosis caused by 3 additional mutations
- mutation of CF gene leads to:
a complete loss of protein
b defect in protein processing (ɷF8 mutation)
c defective regulation of channel
d defective conduction through channel
- non-molecular diagnosis: clinical presentation; sweat test
- molecular diagnosis: PC + ASH (allele specific oligonucleotide hybridization)
- for families with 2 risk, chorionic villi biopsy of fetus tested by mutation analysis
- newborn tested by cheek brushing
 ? Lysosomal storage disease
- lysosomes contain enzymes important for digesting macromolecules (glycoprotein, glycosaminoglycans,
glycolipids)
- removal of carbohydrate catalysed by sugar hydrolyses
- if hydrolyses defective, substrates accumulate
- glycolipids have sugars (galactose, glucose) linked to ceramide
- glycolipids release sugar and ceramides when hydrolysed
- mutations that results in defect in degradation enzymes leads to accumulation of substrates that are
inhibitory to cells
- Tay-Sachs disease and Gaucher͛s disease common among Ashkenazi Jews
 ? Tay-Sachs disease
- gangliosides broken down with sugar removed sequentially to produce ceramide core
- ganglioside GM2 degraded to ganglioside GM3 by hexosaminidase A
- in Tay-Sachs, hexosaminidase A defective -> GM2 accumulated
- mutation cause:
a complete loss of function (infantile form)
b reduced enzyme activity (later onset)
- in infantile form:
a children asymptomatic before 3 months
b after 3 months, mild motor weakness and progressive neurologic degeneration
c death by 2-4 years
d cherry-red spot in eye (precipitation of GM2 ganglioside)
- hexosaminidase A made up of ɲ and ɴ subunit, ɲ subunit mutated in Tay-Sachs disease
- ɲ subunit coded by 3 A gene on chromosome mq23-24, m4 exons, 3 kb
- mutations involved include:
a 4-bp insertion in exon mm (downstream stop codon)-> majority of carrier
b splicing mutation in intron m2 (aberrant mNA)
c Gly-to-Ser missence mutation in exon  -> in adult onset form
- molecular diagnosis:
a hexosaminidase A assays on synthetic substrates/GM2
b PC amplification + ASH
- treatment: management of infection and nutrition
- genetic counseling and prenatal diagnosis available for Jewish community
8 ? Gaucher͛s disease
- defect in ɴ-glucosidase (degrades glucocerebrosides in lysosome)
- accumulation of glucocerebroside -> enlargement of spleen and liver, lesions in bones
- vary from mild to severe (type , , )
- type 
a most common
b enlargement of spleen and liver, skeletal disease
c mildly affected adults to severely affected children
- ɴ-glucosidase
a breakdown glucosylceramide to glucose and ceramide
b membrane associated glycoprotein
c gene on chromosome mq2m, mm exons, 32 kb
- diagnosis
a enzymatic assay using peripheral blood cells, water-soluble substrates and protein antibody (cannot
 differentiate types of disease)
b molecular techniques: PC + ASH
-
 of alleles caused by 4 mutations:
a L444P in exon m (missence mutation)-> homozygotes have severe visceral and neurologic disease
b VS2+m (splicing mutation)
c 84GG (frameshift mutation)
d N3S in exon
(missence mutation) -> not at risk of neurologic disease

? Phenlyketonuria (PK)
- loss of function of phenylalanine hydroxylase (catalyse conversion of phenylalanie to tyrosine)
- phenylalanine hydroxylase gene: chromosome m2q22-q24,
kb, m3 exons, mNA 2 4kb
- 2 different mutations identified
- severe form have low/undetectable levels of enzyme
- milder forms caused by missence mutations -> protein produced but reduced activity
- mutations:
a VSm2ntm (deletion in exon m2 and stop mutation)
b m8Q (missence mutation)
c 2mQ (missence mutation)
d 48W (missence mutation)
m ? Autosomal Dominant
- change-of-function mutation
- defect in only one allele
- only if one parent is carrier, risk is 
- usually caused by missence mutation
- production of mutant protein with altered function
- more restricted as protein/NA remain stable although functionally abnormal
- expansion disorders: trinucleotide repeats at beginning/end at gene undergo expansion-> alter
protein/NA, protein/NA produced but function abnormal
- types of autosomal dominant inherited disorders
a Huntington͛s disease (Huntingtin)
b myotonic dystrophy (protein kinase)
c osteogenesis imperfect (collagen)
mm ? Haploinsufficiency
- amount of product produced by a single normal allele insufficient to prevent clinical phenotype
- heterozygotes for defective allele will express clinical phenotype
- homozygous state lethal
- Eg: Familial hypercholesterolemia (LDL)
m2 ? Predisposition to cancer
- tumor suppressor gene: encodes protein crucial for preventing abnormal cell growth that may form
tumours
- when single copy inactivated, individual inherit predisposition to develop cancer
- carrier parents have risk of 
- inheritance does not produce cancer
- when second allele inactivated, cell may develop cancer
- eg:
a retinoblastoma (pb)
b early onset breast, ovarian, prostate, colon cancer (BCAm)
c early onset breast cancer (BCA2)
d familial adenomatous polyposis (adenomatous polyposis coli)
e neurofibromatosis m (neurofibromin)
f Li-Fraumeni syndrome (tumour suppressor protein, p3)
g hereditary nonpolyposis colorectal cancer (DNA mismatch repair system)
m3 ? -linked inheritance
- genes located on -chromosome
- different inheritance patterns in males and females
- females have 2 copies of , so can be homozygous or heterozygous for defective gene
- males have only one copy of , so always homozygous
- eg: DMD (dystrophin), Fragile syndrome (FM m protein ʹ trinucleotide expansion)
- -linked recessive
a expressed in males who receive from mother
b expressed in females only if both mother and father are homozygous
c predominantly in males, rarely in females
- -linked dominant
a expressed in heterozygous females
b males cannot transmit to son, but transmit to all daughters
c when female affected,  chance that any male/female child will inherit disorder

c 6
c
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m ? Terms
a ? Monogenic
- phenotype due to mendelian inheritance of alleles at a single genetic loci
b ? ligogenic
- phenotype due to interaction of few loci (less than m genes)
- eg: Hirschsprung disease (requires both mutant genes on chromosome m3 and 2m)
c ? Polygenic
- phenotype due to many loci under control by several genes at once (more than

genes)
- eg: thallasemia
d ? Multifactorial
- phenotype due to many loci and environmental factors
- eg: heart disease, diabetes, hypertension, peptic ulcers
e ? Mixed
- phenotype due to a single loci with multifactorial modifications
2 ? Polygenic disease
- tend to run in families, but does not fit simple medelian patterns
- associated genes can eventually be located
- low heritability
- no single genetic factor responsible for the gene
- many genes and additional environmental causes involved
- incidence of disease dependent on a balance of risks
- balance between gene variants and environmental factors with positive and negative effects
- too many negative factors tip balance towards disease
- large-scale studies can help solate genes that contribute since individuals with alleles that predispose them
will be statistically more likely to be affected
- many disease identified by correlations between single nucleotide polymorphisms and complex disease in
large populations
3 ? Thallasemia
- actually a single-gene, autosomal recessive blood disease
- cell does not make enough of one globin chain in hemoglobin -> creation of abnormal hemoglobin ->
anemia
- independent segregation of ɲ and ɴ globin genes more accurately described as polygenic
- ɲ thalassemia -> mendelian autosomal recessive inheritance
- ɴ thalassemia -> more complicated, ɴ thalassemia subtype involves defect in ɲ globin gene, classified
polygenic
4 ? nflammatory bowel disease
- chronic relapsing intestinal inflammation
- modeled as -loci polygenic disorder
- exact number of genes and relative impact of each unknown
 ? Multifactorial diseases
- phenotype originates from genetic predisposition based on mutations on gene
- other genetic contributions from effects of modifying genes
- environment also contributes to trigger/influence the progression
- can occur in isolation (affected child but unaffected parents, no clear mendelian pattern)
- environmental factors can increase/decrease risk
- more frequently in one gender, but not sex limited
- concordance rates in monozygotic and dizygotic twins contradict mendelian proportions
- more frequent in specific ethnic group
- eg: Diabetes, psychiatric illness, alzheimer͛s disease, coronary artery disease, hypertension, mental
retardation, congenital malformation
 ? Type m Diabetes Mellitus (nsulin dependent diabetes mellitus)
- autoimmune destruction of pancreatic ɴ islet cells
- insulin not avaiable to regulate glucose metabolism
- a lot is known about genetic causes, less about environmental factors
- strongest genetic effect involves both class  and  gene at HLA locus (DDMm)
- VNT polymorphism associated with insulin gene (DDM2): 3 alleles, allele  increase risk, allele  decrease
 risk (through immune tolerance)
- DDM2 effect (m) < DDMm effect (42)
- DDM3- loci also implicated, but little is known
 ? Type 2 Diabetes Mellitus (non insulin dependent diabetes mellitus)
- less is known
- MD
(mature onset diabetes of young) shows autosomal dominant with high penetrance
- many mutations known to cause MD

a MD
m: hepatic nuclear factor 4ɲ
b MD
2: glucokinase
c MD
3: hepatic transcription factor m
d MD
4: insulin promoter factor m
- DNA screening limited as many mutations occur
- MD
should be confirmed as they do not require insulin treatment
8 ? Alzheimer͛s disease
- 2/3 of dementia cases
- m in -
years,  in >
 years
- early onset dementia ->  Alzheimer͛s disease
- <  Alzheimer͛s cases is autosomal dominant
- mutation found in 3 genes:
a ɴ amyloid precursor protein (APP) on chromosome 2m
b Presenilin m
c Presenilin 2
- abnormal accumulation contains:
a plaques (extracellular fibrillar aggregates of Aɴ42 peptide)
b neurofibrillary tangles comprising protein tau
- mutation in À gene increase Aɴ42 peptide generated -> highly amyloidogenic
- mutation in presenilin m and 2 genes causes proteolysis of ɴ amyloid precursors -> increase Aɴ42 peptides
- late onset Alzheimer͛s disease -> sporadic, more common, basis uknown
- involves variant of ApoE gene (ɸ2, ɸ3 and ɸ3 and ɸ4)
- one ApoE ɸ4 allele doubles the lifetime risk, not carrying reduce risk by 4
- DNA testing not done due to uncertainty, only in early onset cases/positive family history

? Coronary artery disease
- many factors increase risk: obesity, T2DM, high BP, high levels of LDL, cholesterol, gum disease
- run in families, but no clear mendelian pattern, can occur in isolation
- more often in men
- higher risk among African Americans than Caucasians/Asians
m ? Summary
- single gene disorder rare
- multifactorial disorders contribute bulk of modern public health concerns
- upsurge in interest due to Human Genome Project
6
c
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m ? Mosaicism
- combinations of pieces arranged in certain manner, no two pieces are truly the same
- cells undergo changes during development, may differ from another nearby group
- presence of genetically distinct cell populations within an organism
- caused by spontaneous DNA mutations, spontaneous reversion of mutation, epigenetic changes in
chromosomal DNA and chromosomal abnormalities
2 ? Germline mosaicism
- germ cells: reproductive cells which contributes to genesis of new organism, haploid
- 2 offspring show same autosomal dominant or -linked disease although no family history
- mutation unlikely as disease occurs in two/more siblings
- germline mosaicism more likely
- mutation occurred during embryonic development of one parent and affected germline but not somatic
cells -> parent unaffected
- increase recurrence risk for future offspring, cause complex patterns in autosomal inherited disease
3 ? Somatic mosaicism
- somatic cells: cells other than those destined to become germ cells, diploid
- caused by alterations of genes in specific somatic cells
- not transmitted to subsequent generations through the germline
- sudden appearance of abnormal form of gene in one part of body
-eg: cancers, congenital malformation, autoimmune disease
4 ? Germline vs Somatic Mosaicism
- pedigree and molecular analysis can combine to determine
- time of event leading to mosaicism determine outcome
- during development -> germ and somatic cells can be affected
- later in life -> affect either somatic of germ cells
- only in somatic cells -> phenotype depend on extend of mosaicism, will not be passed to offspring
- only in germ cells-> individual unaffected, offspring affected
 ? Cancer
- disease where cells grow abnormally and can become malignant
- genetic disease at the cellular level
- evidence of genetic component of cancer
a increased risk with genetic defect in DNA repair
b mutagens that mutate DNA causes cancer
c structural chromosomal arrangements predispose to tumour development
d malignant phenotype can be conferred on normal cells by gene transfer studies with oncogene
e malignant phenotype can be induced in vivo by gene manipulation
- mutations not inherited, but originate and accumulate in cells over lifetime
- oncogene: gene that contributes to formation of cancer
- viral oncogene (v-onc) have cellular homologues called cellular oncogene (c-onc)
- proto-oncogene is cellular oncogene that do not form tumour at native state
 ? Development of tumour
a initiation
- first mutagenic event that gives cell potential for unrestrained growth
b promotion
- receipt of mitogenic signal
- may be natural (death of nearby cell) or artificial (exposure to organic chemical)
c progression
- additional mutations
- acquisition of invasiveness
- tumour extend to surrounding
- interfere with normal function
- tumour grow in size, depends on angiogenesis
- metastasis: tumour migrate to new sites to establish secondary tumours
 ? Genetic basis of cancer
- focus on mutations that permit cell escape growth control
- 2 major classes of cancer-initiating genes
 a oncogenes (activate proliferation)
b tumour suppressor gene (inhibit cellular proliferation)
8 ? ncogene
- growth-promoting genes
- needed for cells to grow and divide
- associated with neoplastic proliferation following to a mutation
- mutations phenotypically dominant, only one copy is mutated
- first genes shown to induce cancer were in viruses
- most virus do not cause cancer
- some NA and DNA virus oncogenic
- retroviruses significant in research on genetic basis of cancer

? etroviruses
- NA tumour viruses
- cause of cancers, much less in humans
- 3 core genes:
and  (structural protein), (reverse trascriptase)
- fourth gene: onc (ability to induce tumour growth)
- transformation: acquisition by normal cells of neoplastic phenotype-> cells lose normal growth control
- 2 classes:
a acutely transforming viruses -> induce tumours within few weeks; transform cells in culture; eg ous
sarcoma virus (SV)
b weakly oncogenic viruses -> induce tumours after long latent period; rarely transform cells in culture; eg
Avian leukosis virus (ALV)
- comparison between SV and ALV-> discovery of oncogene
- SV contain genetic material for transformation (named  )
-  lies between
 gene and 3͛-LT
- fusion protein:
a replication competent -> virus that carries normal retrovirus genes + separate oncogene
b replication defective -> requires presence of helper virus; part of retroviral genome deleted and
transforming gene joined; strong promoter supports active transcription of oncogene-> fusion protein
- retroviral oncogene not required for viral replication
- all retroviral oncogenes have homologues in normal host cell DNA (proto-oncogenes)
- origin of retroviral oncogenes: retrovirus intergrated into host genome adjacent to proto-oncogene -> DNA
deletion/aberrant transcription and NA splicing -> fusion transcript produced -> packaged into virion
together with normal retroviral transcript -> recombination ->retrovirus can integrate the new genetic
combination into host genome
- genes in normal cells similar to retroviral oncogenes
- tumours could arise from spontaneous alterations without mediation of virus
- important to examine human tumours for alteration in known proto-oncogenes
- several alterations in proto-oncogenes -> converted to cellular oncogenes
- studied by transferring oncogene to suitable cells in culture
- tumour in culture exhibit:
a immortality: no limit of cell division if space and nutrient sufficient
b uncontrolled growth: escape from density-dependent growth inhibition
m ? Activation/rigin of ncogene
a Chromosomal translocation/rearrangement
- changing the postion of oncogenes can alter their function
- translocation of À
oncogene on chromosome
to chromosome 22 (breakpoint cluster region, BC) ->
Philadelphia chromosome -> hybrid À
transcript produce tyrosine kinase protein -> greater activity
than normal ABLm gene product -> part of signaling cascade involved in regulation of cell growth and gene
expression -> develop lymphoma -> chronic granulocytic (myelogenous) leukaemia (CML)
b Gene amplification
- tumour gradually becomes insensitive to drugs that was initially effective
- drug resistance
- eg: neuroblastoma (6 gene amplified 3x); resistance to methotrexate (hundreds of copies of
dihydrofolate reductase gene)
- amplified genes organized in tandem arrays as homogenous staining regions/double minute chromosomes
c Point mutation
 - single base pair change observed in different genes
- many malignancies
- site of mutations limited to codon m2,m3, m which code for binding of GTP/GDP
- failure of to convert GTP-  (active) to GDP- (inactive) produce excess stimulatory activity ->
unregulated cellular proliferation
d Viral insertion
- perturb proto-oncogene function
- eg: hepatitis B virus, hepatocellular carcinoma
mm ? Tumour suppressor gene
- anti-oncogenes
- role in normal cellular proliferation and differentiation
- prevent cells from multiplying
- loss of inactivation lead to neoplastic changes
- mutations tend to be genetic recessives, both copies inactivated
- both copies lost/inactivated -> unrestrained growth -> tumour may develop
- retinoblastoma (Bm): rare inheritable, retinal tumour in one/both eyes develop during early years, spread
along optic tract to brain, lethal, inherited/non-inherited forms, autosomal dominant
m2 ? Loss of growth control
a cells proliferating too much -> lost control of cell cycle
- cell cycle: one cell grows, become two cells
- after mitosis, long period Gm -> synthesize more macromolecules except DNA
- Gm followed by S -> DNA is replicated
- S followed by G2 -> growth phase without DNA replication
- finally M phase -> chromosome condense and separate by mitotic spindle into new nuclear membranes,
followed by division of cytoplasm into two daughter cells
- restriction  point -> decision to enter S phase and replicate DNA, made in Gm, regulated by cyclins, cyclin-
dependent kinases (cdk eg Bm) and interacting proteins
- mutations can lead to improper regulation and initiate tumour
b cells dying too slowly -> lost control of programmed cell death
- inherent capacity to commit suicide via regulated pathway of reactions
- eliminates surplus cells during development
- prime defence against cancer
- loss of cell death common in malignancy
- response to internal signs: blebbing of cell membrane, cell shrinkage, condensation of nucleus, digestion of
genome into fragments
m3 ? Breast cancer
- family history is a risk factor
-  attributed to BCAm and BCA2
- behaves like classic mendelian dominants with very high penetrance
- high predisposition in those with mutant allele due to loss of heterozygosity
- high probability remaining normal allele will be lost/inactivated -> initiate tumour
- carrier of mutant BCAm/2 alleles increased risk for ovarian cancer
- BCA2 mutations risk factor for male breast cancer
- loss of heterozygosity: process of losing one allele from cell already heterozygous, important mechanism
for cancer development in those with predisposing allele
a chromosome loss
b mitotic recombination
c independent second mutation
- BCA m and 2 large genes, widely expressed in human body
-  mutations -> genetic screening not practical as too many possibilities
- evidence BCAm and 2 involved in DNA repair
a associate with other DNA repair proteins
b associate with each other -> function in same pathway
c localized in nucleus
d role as coactivators/corepressors of transcription
e associate with TP3 (cell cycle regulation protein)
f protein levels change during cell cycle (low in early Gm, high in S)
m4 ? Summary
- cancer: genetic disease due to loss of cell growth control
- development: initiation -> promotion -> progression
- control of cell cycle: mutation in genes that control involved in tumour initiation/progression
- breast cancer: general development of cancer
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m ? Mitochondria
- every human cell contains hundreds of mitochondria
- cellular power plant
- descendants of prokaryotes, intracellular symbionts, remnants of complete bacterial genome, most
lost/transferred to eukaryotic nucleus
- contain hundreds of protein, all encoded by nuclear genes
- proteins for DNA NA synthesis, protein synthesis, fatty acid oxidation, citric acid cycle, mitochondrial
membrane, oxidative phosphorylation
- complex organelles
- two membranes and a cristae
- cellular energy production
- most ATP generated by oxidative phoshorylation
- NADH, FADH 2 (electron carriers) indirectly generate ATP synthesis by catabolism of carbohydrates, lipids
and proteins (pyruvates, fatty acids, amino acids -> acetyl CoA -> C2 and water via citric acic cycle -> NADH,
FADH2)
- respiratory chain: electron transferred stepwise to oxygen with simultaneous export of protons to inter
membrane space
- chemiosmotic gradient is the source of energy to make ATP from ADP and inorganic phosphate as
hydrogen ions pass through ATP synthase back into matrix
2 ? Mitochondrial genome
- genetic chimeras
- DNA
- m kb
- m3 polypeptides, 24 NAs (2 rNAs, 22 tNAs)
- small but essential
- high density of information: no introns, first nucleotide is last nucleotide of previous gene
- do not have similar universal genetic code: GA is tryptophan instead of termination codon
- NDm-: subunits of NADH dehydrogenase (complex )
- Cytb: cytochrome b (complex )
- C,  and : cytochrome oxidase (complex V)
- ATP, ATP8: ATP synthase
- tNA gene: carries amino acid
3 ? Synthesis and assembly of complex mitochondrial proteins
- hundreds of protein involved in mitochondrial structure and contents, nearly all encoded by nuclear gene
- different from cytoplasmic protein synthesis
- require special transport mechanisms to get protein from site of synthesis to within mitochondrial
membranes
- outer mitochondrial membrane freely permeable to small molecules which can pass through porin
- inner mitochondrial membrane impermeable to all but 2 and H2 (problem for function and assembly)
- substrates must be imported via translocases
- translocases, nucleic acid and protein synthesis apparatus synthesized on cytoplasmic ribosomes
- all contain short sequence of amino acid at beginning that targets mitochondria
- many chaperone proteins and assembly proteins help protein cross into mitochondrial membrane, do not
remain in mitochondria
- mitochondrial component contains both nucleus-encoded and mitochondrial-encoded polypeptides
4 ? Mitochondrial DNA mutations
- presence of separate genome within cytoplasmic organelle give rise to mutations
- mtDNA mutate mx faster than nuclear DNA
- due to:
a constant generation of reactive oxygen species
b lower fidelity
c less accurate DNA repair
d lack of histones on mtDNA makes them more accessible
- 2 types of disease:
a due to mutation in mitochondrial DNA
b due to mutation in nuclear DNA
 - as mitochondria do not undergo mitosis, mtDNA do not replicate together/in synchrony with nuclear
chromosomes
- mtDNA distribute randomly to daughter cells
- defects in mitochondrial genes -> reduced/abolished production of ATP
- inheritance maternal:
a sperm mitochondria does not enter fertilized ovum
b affected females transmit to all progeny
c affected males almost never transmit
- heteroplasmy: mitochondria not precisely distributed to daughter cells at cytokinesis -> complicating
- each cell contains hundreds of mitochondria with hundreds of mtDNA
- mutations affect some while others unaffected
- mutant:normal mtDNA ratio varies among offsprings, tissues and developmental stages within individuals
- clear cut pattern may not occur
- results in incomplete penetrance
- large degree of phenotypic variability
? Myoclonic epilepsy and ragged red fibre disease (MEF)
- caused by A->G transition at bp8344 in gene encoding tNA-lysine
- CNS abnormalities (epilepsy, deafness, dementia); skeletal and cardiac muscle deficiency
- severity and symptoms vary
- analysis on muscle biopsy:
a restriction enzyme cleavage site
b A->G transition correlates with MEF
c good correlation between severity of symptom and proportion of mutant DNA
? Deafness
- caused by A->G transition at bpm of m2S rNA
- treatment for bacterial infection with aminoglycoside antibiotics leads to loss of hearing
- 2 rNA genes in mitochondria code for synthesis of proteins in mitochondria
- aminoglycoside kill bacteria via interaction with ribosomes ->mismatching of aminoacyl-tNAs ->
accumulation of errors in bacterial protein -> good for infection control
- bacterial ribosomes quite similar to mitochondrial ribosomes
- some homoplasmic for A->G mutation at bpm -> result in binding of aminoglycoside to mutant rNA ->
interferes with translation within mitochondria -> loss of mitochondria function
- mitochondria protein synthesis in cochlea fails -> nerve cells die
- some showed mutation without exposure to antibiotics and had maternally inherited deafness-> complex
inheritance pattern
? Leber͛s hereditary optic neuropathy (LHN)
- caused by missence mutation at bpmm8 in ND4 subunit of complex 
- G->A substitution leads to arginine -> histidine at position 34 in ND4
- mutation leads to defect in oxidative phosphorylation
- most common inherited blindness in young males
- begins 2-24
- vision loss begins centrally and spreads peripherally
- decline in optic nerve
- abnormal cardiac function
8? Kearns-Sayre syndrome
- large deletion of several mitochondrial genes
- origin of DNA replication not deleted
- heteroplasmy, cell death due to total failure of essential mitochondrial functions
- neuromuscular symptoms: retinal degeneration, heart disturbance, dementia, seizures
- age of onset varies, worsens with time
- leads to death from respiratory failure/general systemic malfunction
- within individual, all deleted DNA have same deletion -> from single deletion event
- in non-dividing tissue, proportion of mtDNA containing deletion increases as smaller deletion-bearing
molecules replicate faster

? Nuclear DNA mutations
-
 of proteins encoded by nuclear genes
 - show mendelian inheritance
- most mitochondrial disorders attributable to nuclear genes
m ? Complex  (NADH-Q reductase)
- first and largest, 43 subunit,  encoded by mtDNA
- most common deficiency
- Leigh Syndrome (LS):
a early onset neurodegenerative
b psychomotor retardation, basal ganglia dysfunction
c fatal
d  nuclear genes defective
mm ? Complex  (succinate dehydrogenase)
- contributes electrons to complex 
- all subunits encoded by nuclear DNA
- Autosomal recessive Leigh Syndrome
a mutation in complex  gene
- Hereditary paraganglioma (autosomal dominant)
a mutations in 2 complex  protein
b benign tumours in head and neck
m2 ? Complex  (Q-cytochrome c reductase, cytochrome b and cm)
- catalyse transfer of electron from ubiquinone to cytochrome c
- mm subunits, m encoded by nuclear gene
- no mutations in nuclear genes implicated in oxidative phosphorylation defects
- mutation in mtDNA gene associated with myopathy
m3 ? Complex V (cytochrome oxidase, cytochrome a and a3)
- terminal complex
- transfer electrons from ferrocytochrome c to molecular oxygen
- 3 mitochondria-encoded subunits form core complex
- m additional nucleus-encoded subunits
- many genes contribute to structure/assembly -> C deficiency frequent cause of oxidative phosphorylation
defect
- heterogenous clinical feature: mutation partially suppress activity, tissue-specific difference in abundance,
heteroplasmy effect on different parts of body
m4 ? Complex V (ATP synthase)
- m subunits, 2 encoded by mtDNA
- ATP mutated in NAP (neuropathy, ataxia and retinitis pigmentosa) syndrome and Leigh syndrome
m ? Mendelian disorders with indirect effect on oxidative phosphorylation
a Friedreich͛s neurodegeneration disorder
- progressive neurodegeneration
- expansion of triplet repeat within intron in frataxin gene inhibits transcription
- reduced activity of mitochondrial enzymes with Fe-S centres (complex  and )
b Nuclear-mitochondrial communication disorder
- loss of integrity
- eg: progressive external opthalmoplegia (PE)
- autosomal dominant
- exercise intolerance after third ʹfourth decade of life
- four nuclear genes involved with mitochondrial DNA synthesis ->aberrant forms -> accumulation of
multiple mtDNA deletion
m ? Summary
- mitochondrial diseases: mitochondrial gene and nuclear gene
- mitochondrial mutation: maternal inheritance, heteroplasmy
- nuclear mutation: mendelian inheritance
- mutations in mitochondrial genes result in reduced/abolished ATP production

cc
  6c6 

m ? Paraoxonase m
- esterase
- physically attached to HDL
- hydrolyse aromatic esters (organophosphates, lactones)
- true physiological substance unknown
2 ? PN family
- 3 known members: 
- located on chromosome , q2m:22
- common evolutionary precursor -> share structural homology, located adjacent to one another
- PNm extensively studied -> role in hydrolyzing xenobiotics, implications in artherosclerosis
- PN2 and PN3 protective against oxidative stress
3 ? Molecular basis
- m
8 known SNPs in  gene
- SNPs found in non-coding introns, coding exons (
6and !") and regulatory regions (#$)
- influences the expression level of PNm
-
6(methionine/leucine) located in exon 3, !"(glutamine/arginine) located in exon 
- associated with accelerated artherosclerosis and premature cardiovascular disease
4 ? Active site of PNm
- !" cause m-fold decrease in PNm activity
- ! reshapes active site, improving positioning of paraoxon
 ? Biochemical structure of PNm
- active site feature catalytic calcium ion and a histiine dyad (Hismm and Hism34) -> mediate lactonase
activity
- role of calcium:
a maintain active site by participating directly or maintaining the appropriate conformation
b facilitate removal of diethyl phosphate, rendering phosphorus susceptible to nucleophilic attack
- hydrolytic active site mediate 2 major anti-artherogenic functions:
a protection of LDL from oxidation
b stimulation of HDL-mediated macrophage cholesterol efflux
 ? Artherogenesis
- arteries undergo gradual thickening of the intima causing decreased elasticity, narrowing and reduced
blood supply
- monocyte adhere to endothelial monolayer -> transmigrate to intima -> differentiate into macrophages ->
LDL undergo oxidative modification by arterial wall cells (macrophages) -> increased generation of oxidized
LDL, free radicals and reactive oxygen species -> oxidized LDL taken up by macrophage via scavenger
receptors -> deposition of cholesterol in arterial wall -> macrophage cholesterol accumulation -> foam cell
formation -> accelerate artherosclerosis
- inverse association between HDL and coronary heart disease
- plasma HDL level correlate with PNm level
- HDL provide amphipathic environment to shield hydrophobic N-terminal of PNm -> necessary for PNm to
interact effectively
- PNm preserves HDL function by inhibiting oxidation
- mechanism of protection traditionally focused on role of HDL as lipid transporter in reverse cholesterol
transport
- oxidative modification of LDL in artery wall also believed to be central in pathogenesis of artherosclerosis
 ? bjectives
- observe variation in gene frequencies in Malaysian population
- determine PNm activity in diabetic subjects with and without complications compared to healthy
individuals
- determine PNm status of individual
- identify discordance between genotype and phenotype
- identify predictors of PNm activity and complications in type 2 diabetes mellitus
8 ? Materials and method
- variation in gene frequencies determined using PC-based genotyping and two-substrate activity assay
- association of polymorphisms with complications in type 2 diabetes mellitus examined using association
tests
 - univariate analysis of PNm activity performed using general linear model
- multivariate analysis using paraoxonase and diazoxonase activity performed
- disconcordance between genotype and phenotype resolved by using DNA sequencing
- two-substrate activity assay: paraoxonase and diazoxonase activities determined using spectrophotometry
-> Pase catalyse paraoxon hydrolysis to liberate p-nitrophenol, DZase catalyse diazoxon hydrolysis to
liberate MHP -> two-dimensional plot used in ! genotyping and activity phenotyping
- extraction of DNA from EDTA whole blood
- PC-based genotyping:

bp PC product was amplified -> !has native À% restriction site at
codon m
2 (mutant-type, CGA, arginine) while !" does not (wild-type, CAA, glutamine) -> when cut
with À%, form 3 and 3
bp bands -> ! genotype determined as complete digestion (!) partial
digestion (!"), and no digestion (!"")
- PNm status: providing a functional assessment of plasma ! and plasma level of PNm,
encompassing the two polymorphic factors that most affect PNm activity
- results of PC-based genotyping were compared with activity phenotyping, repeated analyses until
concordance achieved, disconcordant results were sequenced
- DNA sequencing:

bp product was purified using MinElute kit -> sequenced by AB 33 DNA Analyser ->
procedure based on chain termination method (Sanger &m
) -> resultant sequence aligned using
BLAST against human genomic and transcript nucleotide sequence database-> highly similar sequence
obtain -> CAA at m
2 (exon ) = !""; CGA, peak of G entirely overlapping peak of A = !";
CGA, peak of G partially overlapping peak of A = !

? Demographic and physical characteristics
- gender: slightly higher PNm activity in male compared to female, not significantly different
- age: significant difference in age between diabetics and control -> limited time to recruit older controls ->
age may be confounding factor in analysis of PNm activity
m ? Clinical characteristics
- plasma LDL-C not significantly different between diabetics and control
- HDL-C significantly lower in diabetics with/without complication compared to controls
- triglycerides were higher in diabetics with complications compared to controls
- total cholesterol significantly lower in diabetics with complications compared to diabetics and controls
- PNm activity weakly correlated with HDL-C
mm ? PNm status
-  polymorphism and PNm activity:

and ! had highest PNm activity, 66 and !""
lowest PNm activity
-  polymorphism and lipid profile: no association
- PNm activity and ethnicity: ndians have high allele frequencies of 
6 polymorphism; significantly
lower PNm activity
- PNm activity and type 2 DM: PNm activity in diabetics with complications significantly lower than controls,
diabetic and CVD patients
- PNm activity and serum lipids: HDL-C had positive correlation with PNm activity, likely factor that
contributes towards variation in PNm activity
m2 ? PNm status and complications in type 2 DM
- allele frequencies of !" polymorphism equally distributed
- due to:
a small sample size
b results confounded by the different CVD complications
- age, BM and PNm activity associated with complications in type 2 DM
- ethnicity, diabetic status and HDL-C associated with PNm activity
- diabetic status and PNm activity mutually present as significant predictors independent of 
polymorphism
- PNm activity significantly lower in diabetics with complications -> PNm prevents LDL lipid peroxidation ->
protect against diabetic complications
- ! allele associated with high PNm activity, CVD patients had higher PNm activity compared to
controls -> contrary to expectations that high PNm activity confers high degree of protection against CVD ->
PNm active site requirements for its protective role against LDL oxidation which may not be identical to
PNm active site against synthetic substrates such as paraoxon
m3 ? Discordance in genotype and phenotype
- degree of concordance range between
8 -

3 (percentage error less than m)
- two-substrate enzymic assay fairly accurate
m4 ? Conclusion
- distribution of !"and !
6 genotype frequencies in 3 Malaysian ethnic groups showed
that Malay and Chinese ethnic groups showed higher frequency of  allele
-  allele considered a risk factor for CVD in some populations
- diabetics with CVD complications had lower PNm activity compared to diabetics without complications,
whereas polymorphism was equally distributed
- PNm status provided a functional assessment of plasma !alloforms, as well as plasma levels of
PNm activity
- P Nm status coupled with PC-based genotyping detected discrepancies between genotype and
phenotype due to possible mutations in  gene
- diabetic status and PNm activity were mutually present as significant predictors of independent of 
polymorphism
m ? eal time PC
- disadvantage of traditional PC
a time consuming
b poor precision
c low sensitivity
d low resolution
e non-automated
f size-based discrimination only
g results not numerical
h ethidium bromide not quantitative
i post PC processing
- advantage of real time PC
a collect data in exponential growth phase
b increase in fluorescent signal directly proportional to number of amplicons generated
c cleaved probes is permanent record of amplification of amplicon
d no post PC processing
e detection up to 2-fold change


cc c  6

cc

m ? Free radical
- atoms/molecules with one/more unpaired electrons
- types of free radicals:
a reactive oxygen species -> derived from molecular oxygen (2, a diradical)
b reactive nitrogen species -> derived from nitric oxide (N2, a radical)
- if 2 free radicals react -> neutralize each other
- if free radical react with stable molecule -> generation of more free radicals
- enables auto-catalytic chain reactions -> molecules converted into free radicals to propagate damages
- chemical bonds formed from sharing of 2 electrons
- many, but not all free radicals are chemically reactive -> find another electron to pair up via single-electron
transfers/free radical addition reactions
2 ? Sources of nitric oxide
- nitric oxide synthase in mammalian tissue:
a NS- -> neuronal
b NS- -> macrophage inducible
c NS- -> endothelial
- nitric oxide produced in vascular endothelium
- peroxynitrous (HN): strong oxidant with cytotoxic properties; can initiate lipid peroxidation and thiol
oxidation
- immune-stimulated macrophages produce N -> inhibition reduces microbiocidal and tumoricidal activities
- common gaseous free radical, critical in vascular physiology
3 ? Formation of xidants (Electron Transfer eactions)
- superoxide anion (2ͻ-, radical): addition of one electron to molecular oxygen
- hydrogen peroxide (H22, not radical): addition of 2 electrons to molecular oxygen/ addition of m electron
to superoxide anion
- hydroxyl radical (Hͻ, radical): one-electron reduction of hydrogen peroxide
4 ? eactive oxygen species
- derived initially from oxygen, some do not contain unpaired electrons in outermost orbit, hence not free
radical
- eg: H22, HCl, N
 ? Stimuli that form free radicals
- physiological
a respiration (superoxide, hydrogen peroxide, hypochlorous acid, nitric oxide)
b transition metals in free form (eg Fe2+, Cu+)
c body cells: endothelium (nitric oxide, nitrous oxide); macrophage (nitrous oxide); neurons (peroxy nitrite)
d aging
e phagocytosis
f oxidation of foods and endogenous compounds
g transportation of substance for energy production
- pathological
a radiation (H2 ± H+ + H-)
b metabolism of drugs
c transition metals (Fe2+, Cu+)
d ultraviolet rays
e emotional stress
 ? Action of free radicals
- act on cell membrane and organelle membranes -> cause cell injury and death via oxidative reaction
- free radicals = oxidants
- lipid peroxidation: polyunsaturated fatty acids in cell membrane more vulnerable -> increase cell
permeability -> calcium influx -> altered cell pH
- alter enzyme and receptor proteins -> cross-linking and fragmentation of protein, oxidation of amino acid ->
abnormal cell behavior
- fracturing of cell nucleus -> single strand DNA damage -> lethal (cell death, phagocytosis) / sublethal
(increased cell permeability, toxicity, mutation)
 ? Hydrogen peroxide
- not a free radical, little reactivity
 - can diffuse long distances crossing membrane
- react with transition metals by hemolytic cleavage -> yield highly reactive hydroxyl radical
- readily permeates membranes -> not compartmentalized
- Haber & Weiss-Fenton reactions:
a 2ͻ- + H22 ± 2 + ͻH + H- (hydrogen peroxide forms hydroxyl radical with superoxide anion)
b Fe2+ + H22 ± Fe3+ + ͻH + H- (hydrogen peroxide forms hydroxyl radical with ferrous ion)
c 2ͻ- + Fe3+ ± 2 + Fe2+ (superoxide anion and ferric ion reforms molecular oxygen and ferrous ion)
- hydroxyl radical is the strongest oxidizing agent, reacts indiscriminately with all organic molecules
- ferrous limits rate of reaction
- ferric recycled to ferrous by superoxide -> enable Fenton reaction to go on -> forms more hydroxyl
8 ? Biochemical reactivity of hydroxyl radical
a hydrogen abstraction to organic molecule
- ͻH + H ± ͻ + H2 (formation of organic radical)
- ͻ + 2 ± ͻ (reaction molecular oxygen in triplet ground state)
- ͻ + H ± ͻ + H (formation of second carbon radical)
b addition of hydroxyl to organic molecule
- ͻH +  ± ͻH
- ͻH + Fe3+ ± H + Fe2+ + H+ (form stable oxidized product)
- ͻH + 2 ± H + 2ͻ- + H+ (form stable oxidized product)
- ͻH + ͻH ± - + 2H2 (cross-linking of hydroxylated products)

? Sources of oxidants
a Enzymic reactions
- NADH oxidase
- NADPH-P4 reductase
- xanthine oxidase
b Cellular sources
- leukocyte and macrophages
- mitochondrial electron transfer
- microsomal mono-oxygenase
- endoplasmic reticulum
- plasma membrane
c Environmental sources
- pollutants
- V
- radiation
d Non radical
- glycolate oxidase
- acetyl-CoA oxidase
- D-amino acid oxidase
- NADH oxidase
- urate oxidase
e Enzymic generation
- monoamine oxidase
f Non-cellular sources
- Fenton reaction
- superoxide anion/hydrogen peroxide
m ? Sources of superoxide anion
a enzymic reaction
- NADH oxidase
- NADPH-P4 reductase
- xanthine oxidase
b Cellular sources
- leukocyte and macrophages
- mitochondrial electron transfer
- microsomal monooxygenase
c Environmental factors
 - ultraviolet light
- -rays
- toxic chemicals
- aromatic hydroxylamies
- aromatic nitro compounds
- insecticides
- chemotherapeutic agents
mm ? Sources of hydrogen peroxide
a Enzymic generation (non radical)
- glycolate oxidase
- acetyl-CoA oxidase
- D-amino acid oxidase
- NADH oxidase
- urate oxidase
- monoamine oxidase
b dismutation of 2ͻ- (radical)
- fusion of 2 superoxide anions
m2 ? xidative damae to lipid
- polyunsaturated lipids are susceptible to oxygen free radicals
- 3 steps
a initiation: abstraction of H from methylvinyl group to form carbon centred radical
b propagation: reaction with triplet oxygen -> form peroxy radical -> abstracts H from second fatty acid ->
form lipid hydroperoxide and another carbon radical
c amplification: presence of ferrous -> Fenton reaction -> form alkoxy radicals and degradation products
(aldehyde and hydrocarbon) -> chain reaction amplified
d termination: carbon/peroxy radicals cros-link to form conjugated products (not radicals)
m3 ? xidative damage to protein
- site-specific amino acid modification
- fragmentation of peptide chain
- aggregation of cross-linked products
- altered electrical charge
- increased susceptibility to proteolysis
- depends on primary, secondary or tertiary protein structure
- sulfur and thiol containing amino acids very susceptible
- oxidation may cause irreversible modifications
- specific examples:
a activated oxygen abstract H from cystein -> thiyl radical -> cross-link -> disulfide bridge
b oxygen add to methionine -> methionine sulfoxide derivatives
c oxidation of iron-sulfur centre by superoxide -> destroy enzyme function
d tryptophan oxidized -> cross-link -> bityrosine product
e His, Lys, Pro, Arg, Ser form carbonyl groups
f protein and sugar moiety cross-linking
m4 ? xidative damage to DNA
- hydrogen abstraction yields strand breaks
- hydroxyl addition add to deoxyguanosine (G) -> form 8-hydroxy-deoxyguanosine (8dG) -> evidence of free
radical attack
- induces numerous lesions -> cause deletions, mutations, and other lethal genetic effects
- base and sugar moieties are susceptible
- types of damages
a single strand breaks: oxidation of sugar moiety; Fenton reaction with metal catalysts bound to DNA
b cross-linking to protein: thymine-cystein adducts -> not readily repaired
c base degradation: 8-hydroxyguanosine Hydroxymethyl urea, urea, thymine glycol, thymine, adenine ring-
opened and adenine ring saturated products
- DNA obvious weak link in cell͛s ability to tolerate free radical attack due to:
a DNA effective n binding metals (Fenton reactions)
b less damage can be tolerated
 - reason why eukaryotic cells have compartmentalized DNA in nucleus, away from sites of redox cycling
- DNA repair enzymes may repair oxidative damages too
m ? Methods to evaluate free radical induced damages
a lipid
- lipid peroxidation products
- chemiluminescence-based detection using HPLC
- TBA test
- HPLC/spectrophotometric methods
- xylenol orange/F assay -> detecting authentic peroxides in LDL
b proteins
- carbonyl assay -> amino acid produce carbonyl after attack by S
- reaction with 2,4-dinitrophenylhydrazine -> measure carbonyl
- 3-nitrotyrosine (HPLC, GC-MS)
c DNA
- measure 8-hydroxyguanine using HPLC, GC-MS, radioactive technique
m ? Antioxidant defence
- any substance when present in low concentrations compare to oxidized substrate, significantly
delays/inhibits oxidation of that substrate
- antioxidant enzymes:
a superoxide dismutase
b catalase
c glutathione peroxidase
d glutathione reductase
- non-enzymic antioxidants
a endogenous: metal binding proteins, GSH, ubiquinol-m, urate, bilirubin, lipoic acid
b dietary: vitamin C, vitamin E, carotenoids, trace elements (selenium), flavanoids
- oxidative stress: imbalance between production of free radicals and their neutralization
a increase free radical production
b lowered antioxidant levels (compromised defense system)
-free radicals produced in normal processes through use of oxygen
- environmental pollutants and drugs cause free radical production
- free radicals can damage cells structure and function
- antioxidant protect cells against free radicals by scavenging
m ? xidative stress and hyperglycemia
- hyperglycemia cause increase polyol pathway, increase glucose autooxidation and increased protein
glycation
- lead to increased oxidative stress, lowered antioxidant defence and increased oxidative factors
- oxidative stress activate signaling pathways which leads to
a insulin resistance
b vasculopathy, retinopathy, neuropathy, nephropathy
c ɴ cell dysfunction
m8 ? xidative stress and Cancer
- carconigenesis: chronic oxidative stress (inflammation, genetic disorder, environment) ± diminished
antioxidant protection ± increased genetic instability ± new phenotypes (less apoptosis, more growth) ±
tumour progression
- cancer therapy: S-based therapy ± tumour contains apoptosis/oxidant resistant cells? ± No (tumour
regression)
- neutrophils, eosinophils, macrophage ± release oxidants ї continuous production may cause cancer at
site of production
- cancer therapy: depends on production of S for cytotoxicity ± tumour cells sensitivity affect treatment
success
6
c

m ? Filarial nematode infections
- -  '

 and 
 
effective diagnosis needed for drug treatment and control programmes
- problems of conventional diagnosis:
a inadequate sensivitiy
b long pre-patency
c inconvenience to patients
- alternatives
a immunoassay measuring antibody and parasite antigens
b molecular assays to detect parasite DNA
- -&'

 &: lymphatic filariasis, elephantiasis
- &: skin and eye lesions
- life cycle: infective larval stage transmitted from arthropods during blood meals -> mature in host, females
produce  
  ->  
  acquired by arthropod during blood meals -> develop into infective
larvae -> transmit again in next blood meal
- diagnosis:
a -&'

 &: microscopic detection in peripheral blood -> nocturnal periodicity, unequal
distribution
b &: microscopic detection in skin nodules, skin-snips incubated in saline -> low sensitivity
- cannot detect infection until many months/years after infection
2 ? Molecular applications in filarial nematode infection
a detection of antibody (ELSA)
- problem: antigens from parasite extensively cross-reactive; cross-reaction with other filarial infections
- solution: use species-specific antigens, monoclonal antibodies
b recombinant antigen (onchecerciasis)
- cDNA encoding antigens aspartyl protease inhibitor
- high sensitivity using ELSA
c recombinant antigen for lymphatic filariasis
- cloned antigen S P-m (low molecular weight)
- recognized from sera of microfilaraemic individuals
d rapid detection kit (Brugiaapid)
- Brugia infections
- recombinant antigens
e hybridization with DNA probe
- no longer popular
- replaced by PC-based assays
f PC-based assay
- species specific DNA probe to design primers: repetitive DNA (small, high copy number/ large, interspersed,
moderate to high copy number); multiple copy gene (tandem repeats)
- detection: agarose gel electrophoresis, blotting, hybridization, labeling
(radioactive/colorimetric/chemiluminescence), ELSA (biotin-labelled PC products)
3 ? Advantages of PC-based assays
a &
- improved sensitivity
- correlation between strain with epidemiologic pattern of blindness
- establishment and confirmation of vector-parasite transmission
- assess infection status after therapy
b -&'


identify parasite in mosquitoes and blood
c &malayi
- detection of parasite, phylogenetic relationships with other species
4 ? Parasitic protozoa infection
- microscopy is gold standard
- labour intensive
- protozoa may be morphologically similar, very small and occur in small numbers
- in vitro cultivation costly, slow, difficult
 - immunodiagnosis problematic due to cross-reactions and inconsistencies
-    
a cause of diarrhoeal illness in humans and livestock
b cannot grow in vitro
c microscopy time consuming and insensitive
d immunodiagnosis hampered by antigenic variability
e flow cytometry too costly
- advantage of PC
a sensitivity, easy, high thoughput
b useful for diagnosis and epidemiology
c direct detection
- diagnostic PC primer
a m8s rDNA gene: from sequence information, however gene is conserved so cross-reactive
b APD (rapid amplification of DNA): detect nucleotide sequence polymorphisms without knowing
sequence; species specific (derived from repetitive DNA sequence); diagnostic fragment can be eluted,
cloned and sequenced; tested as diagnostic primer
- problems of PC
a false positive/negative: avoid contamination
b faecal samples: contain PC inhibitors (bilirubin, bile salts, polysaccharides); DNA extraction must remove
traces of PC inhibitor
 ? Molecular applications in parasitic protozoa infection
a    : environmental and clinical detection
b (
 ': distinguish (&  and (&
c species (malaria)
- conventional diagnosis: labourous microscopic examination
- problems: low parasitemia, mixed infections
- epidemiological use to detect pyrimethamine resistance
- sample collection: blood spot on filter paper
- PC genotyping to distinguish relapse from new infection
d 

conventional diagnosis: serology, isolation by in vitro culture
- serology problem: most population positive for toxoplasma antibody
- molecular diagnosis: different conditions of toxoplasmosis
- molecular diagnosis problems: sensitivity, DNA extraction, inconsistency
 ? Molecular detection of parasitic nematodes
- conventional diagnosis: morphological feature of eggs, larvae, circulating immature parasites
- problem: distinguishing related species
- molecular approaches
a PC-FLP distinguish:
- dog and human isolates of D 
   
- animal hookworms )

 
 À
 

À
 
 
- zoonotic infections    
and    
b PC with specific primers
-
* 
 * 
  

c PC-hybridisation
- 
 
 ? Detection of parasites in food
a 

 host: warm blooded animals (definitive host: cats)
- cyst found in musculature of sheep, goat, pig, cattle
- PC primers: P3 gene, Bm gene
b   
   

- caused by ingesting raw/undercooked meat containing infective larvae
- most common in pork, beef and horse meat
- recombinant and synthetic antigen for ELSA: Ts-3, 4
, 3 kDa
- conventional antigen preparation require live parasites from rodents
 - problem: reproduce native glycoprotein in recombinants
- epidemiology: PC distinguish different species
8 ? Molecular epidemiology
- predictive significance to better define aetiology of disease
- understand causation of disease
- specific role of molecular epidemiology:
a determine the cause of parasitic disease (agents, host, environment)
b elucidate interactions and relationships between parasites at population level
c utilize information for solving problems related to control
- tools
a analyses of nucleic acid and protein
b PC-based approaches
c APD, FLP, direct sequencing
- useful properties of genome and protein
a ribosomal NA (m8S, , 8S, 28S gene)
b minisatellites and microsatellites
c rNA intergenomic spacer region
d loci affecting phenotype
e isoenzymes/alloenzymes
- applications
a determine phylogeny and evolutionary processes
b species discriminations (taxonomic status)
c intraspecific variants (population genetic structure, breeding system)
d tracking transmission (dynamics, symptoms-response to treatment correlation)
e ecological interactions with host
f genetic markers (phenotype-genetic difference correlation, transmission and markers)
6
c
m ? ntroduction
- virus initiate infection by attaching to specific receptors on surface of susceptible host cell
- receptors have role in entry and virus binding
- virus receptors: cellular surface protein which specifically bind viruses resulting in attachment of virus
particle to cell
- important to study
a major determinant of route of virus entry
b pathogenesis of virus
c design of drugs that inhibit virus-receptor interactions
- enterovirus m receptor
a single stranded NA virus
b Picornaviridae, human enterovirus A
c clinical spectrum: HFM disease, aseptic meningitis, acute flaccid paralysis, encephalomyelitis, pulmonary
oedema
2 ? Molecular method used to indentify virus receptor
a DNA transfection
b DNA cloning
c fusion PC
d in vitro transcription
e NA transfection
3 ? DNA transfection
- introducing exogenous donor DNA into recipient cells (human rhabdomyosarcoma cells, D)
- frequently used in clinical EVm isolation
- mouse L
2
cells not susceptible
- live EVm virus generated by viral NA transfection
- restriction occur early step of infection (attachment/internalization/viral uncoating)
- high genomic DNA extracted from D cells
- co-transfection: genomic DNA and plasmid (puromycin N-acetyltransferase gene) transfected into L
2

cells
- transient transfection: temporary but high expression of target gene; not necessarily integrated into host
chromosome; large number of sample analysed in short period; harvested m-4 days after transfection
- stable transfection: establish cell lines; transfected gene intergrated into chromosomal DNA; lower
efficiency; cotransfected with selectable genetic markers
- cotransformation: function and expression require stable transfection; 8 of cells transient transfection;
nonhomologous intermolecular recombination and ligation of DNA; stable cell lines isolated (with
intergrated transfected DNA); uptake, intergration and expression rare; stable transformants isolated by
selection of cells with new phenotype (antibiotic resistance)
4 ? Transfection methods:
a lipid mediated
b Calcium phosphate
- most widely used; inexpensive, simple and wide-range
- mixing DNA with calcium chloride -> add to buffered saline/phosphate -> incubate at room temperature ->
precipitate dispersed onto cultured cells -> taken up by endocytosis/phagocytosis -> coprecipitate escapes
from endosome/lysosome -> enters cytoplasm and transferred to nucleus -> transformant cells generated ->
screening susceptibility using infectious cDNA clone of EVm-GFP
c DEAE-dextran mediated
d Electroporation
 ? Generation of infectious cDNA clone of EVm
- discovering function of a gene
- find possible phenotypes derived from specific genetic sequence
- powerful tool for structural and functional studies of viral genome
- infectious transcripts generated in vitro with NA polymerase
- steps: DNA sequencing -> design primers, E,vector -> digestion, purification, ligation -> cloning into vector
-> transformation into E coli -> DNA extraction -> DNA sequencing -> in vitro transcription -> NA
transfection
 ? Determine ͛- and 3͛-end sequence of viral genome
a clone intro plasmid and sequence insert using universal primer (T, Mm3, SP)
b /3 ACE (rapid amplification cDNA end)
- require: adaptor primer, (dT)m adaptor primer, gene specific antisence primer, terminal transferase
enzyme
 ?  ACE:
- T-PC
- Purified
- Homopolymeric tail added to 3͛-end using terminal transferase (TdT)
- conditions for TdT:
a purified cDNA, buffer, dATP, TdT
b Poly-A tailing done at 3 C, m mins
c TdT reaction inactivated by heating for m mins,  C
d Amplification of poly-A tail done in L reaction with gene specific primer and (dt)m adaptor primer
e if not enough PC product, second PC carried out to obtain visible PC product
8 ? 3 ACE:
- T-PC with (dt)m adaptor primer
- Direct PC with adaptor and gene specific primer

? Strategy in construction of infectious cDNA clones
- mst PC
F: Notl-T-SK-EV-: E site, T promoter, viral genome (NT)
: SK-EV-2
82
-2nd PC
F: SK-EV283
: Sal-dt2: E site, poly T
- mst PC product digested with Not and Eco -> ligated into pSVAm4 -> DNA sequencing -> digested with
Eco and Sal
- 2nd PC product digested with Eco and Sal -> ligated into pCmm
-> DNA sequencing -> digested with
Eco and Sal
- ligation and transformation into E coli -> DNA sequencing -> in vitro reverse transcription and NA
transfection
m ? n vitro transcription
- synthesis of NA from DNA template
- require:
a purified linear DNA template containing promoter
b ribonucleotide triphosphates
c buffer system
d NA polymerase: DNA-dependent; specific promoter sequence; eg T, T3, SP
- antisense
a complementary to mNA
b NA use as probe for hybridization to mNA
c ͛-end of noncoding strand adjacent to +mG
- sense
a consensus sequence with mNA
b expression, structural or functional studies, NA quantitation using artificial sense strand NA
c ͛-end of coding strand must be adjacent/just downstream of +mG of promoter
- vector should be linearised downstream from promoter and inserted sequence to be transcribed
- after NA polymerase binds, separates two DNA strands and uses the 3͛->͛ strand as template for
synthesis of complementary ͛->3͛ NA strand
mm ? Contruction of EVm-GFP cDNA
- green fluorescence protein: from jellyfish (À +   ); most interesting protein; marker for gene
expression and intracellular process; stable to heat, extreme pH and chemical denaturants; continue to emit
fluorescence after fixation
m2 ? DNA microarray
- to identify putative human gene in 2 mouse cell line transcription analysis comparing 2 transformants and
untransfected mouse cells via human microarray analysis
- comparing 2 transformants and L
2
cells (untransfected cell lines)
 - m4 genes in Ltrm cells (mouse cell line m) and 33 genes in Ltr24 cells (mouse cell line 2) showed more
than 2 fold increase in expression in Ltrm/Ltr24 cells
m3 ? Preparation of expression vector encoding SCAB2, CCL2 or BCDN3
- to determine whether expression of SCAB2/CCL2/BCDN3 makes L
2
susceptible to EVm, L
2

transfected with plasmid encoding (pCA-SCAB2/pCA-CCL2/pCA-BCDN3) and infected with EVm-GFP
- steps: NA extraction -> T-PC -> ligated into plasmid -> transfect into L
2
cells -> selection of L
2

transformant with antibiotic -> dilution purified cells -> single clone
- L
2
cells did not become susceptible to EVm-GFP by transfections of pCA-CCL2/pCA-BCDN3
- results suggest SCAB2 sufficient to permit EVm infection of Ltrm cells
m4 ? Mammalian expression vector
- plasmid used to introduce specific gene into target cell
- once inside cell, protein encoded by gene produced by cellular-transcription and translation machinery
- vectors with antibiotic-resistance open reading frames allow identification via antibiotic selection




6 
 
m ? Neurological disorders
- affect the nervous system
- CNS diseases: Alzheimer͛s disease, Parkinson͛s disease, Huntington͛s disease, motor neuron disease
- PNS diseases: Charcot Marie Tooth
2 ? Facts
- one of
muscular dystrophy (a group of genetic, degenerative disease affecting voluntary muscles)
- progressive muscle wasting and weakness that begin with microscopic changes
- as muscles degenerate, muscle strength declines
- primarily affects males ( -linked)
- show signs as early as 3 years
- gradually weakens skeletal muscle of arms, legs and trunk
- by early teen, heart and respiratory muscles affected
- most die by 3s from heart/respiratory complications
- Becker͛s muscular dystrophy milder version of DMD: onset in teens/early adulthood, slower
- common disorder
- diagnosis aims: improve treatment, determine carrier status
- cell-based therapies promising
3 ? Diagnosing patient with DMD
a clinical examinations
- late walkers, trouble with stairs, awkward gait, Gower͛s manoeuvre
- EMG
- serum creatine phosphokinase concentration levels
- muscle biopsy from bicep/quadriceps
- histopathological analysis on dystrophin distribution (dystrophin staining)
b genetic analysis
- detect mutation in dystrophin by PC/sequencing
4 ? Discovery of causal gene
- from family pedigree -> -linked
- FSH -> identified dystrophin gene (largest human gene, 2 2Mb, transcript m 4kb, 
exons)
 ? Genetic testing for DMD/BMD
- mutations that occur:
a large deletions: - (
 frameshift) -> multiplex PC/MLPA detection kit
b point mutations/small deletions/insertions: 3-3
c duplications: -m -> multiplex PC
 ? MLPA
- probes:
a synthesis olionucleotide -bp (PC primer sequence
)
b Mm3-derived oligonucleotide -4bp (PC primer sequence )
- steps:
a denaturation
b hybridization
- MLPA probe mix added to denatured genomic DNA -> 2 parts hybridise to adjacent target sequence
c ligation
- probes ligated by thermostable ligase
d amplification
- universal primer pair used to amplify all ligated probes-> amplification product of each probe has unique
length
- only ligated matched probes are amplified
e separation and quantification by capillary electrophoresis
- each peak is amplification product of specific probe
- sample compared to control
- difference in relative peak indicates copy number change of target sequence (mutation)
 ? Carrier testing
a CK level
-  female DMD carrier have 2-mx normal CK level
- normal range = 22-m
/L
 b DMD gene dosage
- females have 2 chromosomes -> 2x DMD gene
- carrier with deletion: m copy have missing exons
- carrier with duplication: extra copy of exons
c mutation known -> screen female relatives for mutation
d mutation unknown -> screen gene dosage (southern blot/quantitative real-time PC)
e T-PC: carrier have higher Ct value (less template)
8 ? Possible DMD alternatives
a restoring function of dystrophin
b utilizing protein (utrophin) similar to dystrophin
c stem cells to replace damaged muscles

? trophin therapy
- muscles in developing babies contain utrophin (very similar to dystrophin)
- as babies mature, utrophin replaced with dystrophin in almost all muscle fibres
- EF turn down utrophin production in mature muscle
- reducing cellular EF -> increase utrophin production (cultured muscle cells and mice)
- artificial gene switch protein -> supercharge utrophin gene -> produce excess utrophin protein
m ? Stem cell therapy
- skeletal muscle repair by adult human mesenchymal stem cell from synovial membrane
- human stem cells treated mdx mice
mm ? eplace defective DMD gene with new one
- cDNA for DMD m4kb
- need artificial construct of gene
- transferred to nuclei and express appropriately
- need large vectors
- ealier limitations -> inadequate size of adenoviral vectors
- now have synthetic non-viral DNA plasmid
- phase m trial -> not very efficient in muscle tissue
m2 ? epair defective DMD gene
- BMD patients also have large deletion, but relatively mild phenotype
- as long as N and C terminals intact, able to link between extracellular matrix and cytoskeleton
- transfer of smaller DMD construct in progress
- DMD and BMD phenotype differ due to nature of mutation
- DMD: mutation cause stop codon/ deletion or duplication cause frameshift -> protein unstable
- BMD: deletion/duplications, but sequence still-in-frame, some have deletions similar to DMD
- crucially, BMD mutations involve splicing motifs which interfere with splice site selection -> induce
spontaneous exon skipping -> result in in-frame transcript
- Kobe patient: 2bp deletion in exon m
-> caused exon skipping -> contains exon-recognition site
(ES)/exon splicing enhancer (ESE) -> motif needed for exon inclusion
- artificially induce exon skipping -> S bindin to ESE essential for exon inclusion -> block ESE = skip exon ->
use antisense oligonucleotide to cover the ESE site -> S cannot bind properly -> in-frame DMD mNA
transcript
- antisense oligonucleotide (AN): synthetically manufactured; recognize ESEs of particular exon; antisense
sequence (efficient binding); have 2͛  ʹmethyl phosphorthinate group -> resist cellular endonuclease and
inhibit NaseH activity
- intramuscular injection of AN restores up to 2 dystrophin level, dystrophin detected at least 3 months
later
- different mutations require different AN, however some AN influence skipping of neighbouring exons
- sustained AN production: AN cloned into small nuclear NA -> delivered by retrovirus -> otherwise,
need repeated AN injection


6c
m ? ntroduction
- inherited disorder of haemoglobin synthesis -> alteration of globin chain production rate
- autosomal recessive
- any decrease in production of globin chains (ɲ,ɴ, ɷ, ɶ) will greatly hamper haemoglobin synthesis
- since 2 types of chain pair up to form normal HbA, decrease in production of one chain leads to excess of
the other type -> accumulation in cell -> unstable product cause cell destruction
- imbalance is hallmark of thalassemia
2 ?  thalassemia
- heterogeneous group of autosomal recessive disorder
- result from reduced (ɴ+ thalassemia) / absent (ɴ thalassemia) production of ɴ-globin chains
3 ? Clinical classification of ɴ-thalassemia
- 3 clinical haematological conditions:
a carrier state
b thalassemia intermedia
c thalassemia major (Cooley͛s anemia)
- ɴ thalassemia major results from homozygosity/compound heterozygosity (heterozygotes for both ɴ+ and ɴ
and two different form of either ɴ+ and ɴ ) of severe mutations in ɴ globin gene cluster
- reduces output/complete absence of ɴ globin
- unassembled excess ɲ globin precipitate -> defective erythroid precursor maturation, ineffective
erythropoeisis and shortened red-cell survival
- ɴ thalassemia carrier clinically asymptomatic, characterized by microcytosis, reduced Hb content,
increased  of HbA2 and inconsistent increase of HbF (fetal haemoglobin)
4 ? Pathophysiology of ɴ thalassemia
- marrow expansion -> deformities, osteopenia and focal defects, periarticular syndrome
- marrow hyperplasia -> increased iron absorption and deposition in tissues
- maintained ɶ chain production -> pair up with excess ɲ to form HbF -> reduce symptoms -> protect red
blood cells
 ?  globin gene cluster
- short arm of chromosome mm
- locus control region: promote erythroid-specific gene expression; coordinate developmental regulation of
gene
- Gɶ and Aɶ gene: produce ɶ globin chains
- ʘɴ gene: pseudogene; evolutionary remnant of previously active ɴ globin-like gene
 ? Molecular defect of ɴ-globin gene
- more than m inherited mutations affect structure/synthesis of ɲ and ɴ globin chain
- almost all mutations similar in principle, but different in pattern
- more than 2 molecular defect of ɴ globin gene reported
- vast majority of ɴ thalassemia caused by SNP or oligonucleotide addition/deletion that affect critical areas
- ɴ thalassemia rarely caused by gross gene deletion
- point mutations/frameshift mutation completely inactivate ɴ globin gene -> ɴ thalassemia
- mutation of initiation codon ATG, mutation affecting NA processing, mutation within intron/exon that
create alternate splice sites -> ɴ+ thalassemia
 ? ɲ thalassemia
- reduction/complete absence of ɲ globin gene expression
- classified according to severity:
a Hb Bart͛s hydrops fetalis syndrome
b HbH disease
c ɲ thalassemia trait
d silent carrier state
- classified according to molecular classification based on output of each ɲ globin gene chain that constitute
haploid pairs
a normal: ɲɲ/ɲɲ (4 ɲ globin gene)
b ɲ2-thalassemia: ɲɲ/-ɲ (m gene deletion, silent carrier)
c cis-ɲm-thalassemia: ɲɲ/-- (2 gene deletion, reduced globin, Chinese)
d trans-ɲm-thalassemia: ɲ-/ɲ- (2 gene deletion, reduced globin, Malay)
e HbH disease: --/ɲ- (3 gene deletion, more reduced globin)
 f Hb Bart͛s hydrops fetalis: --/-- (4 gene deletion, incompatible with life, death in utero)
- groups as ɲ and ɲ+ thalassemia indicating complete/partial termination of ɲ globin gene expression
- Hb Bart͛s hydrops fetalis
a hydropic, abnormal fetal development
b dies in utero ( months)/ still birth/ dies after birth
c maternal complications (placentomegaly, hypertension, difficult birth)
d 4ɲ gene deletion (--SEA/--SEA )
e  of hydropic fetus in Chinese
8 ? ɲ globin gene cluster
- encoded on chromosome m
- ͛-ɺ-ʘɺ-ʘɲ2-ʘɲm-ɲ2-ɲm-ɽ-3͛
- kb
- 2 highly homologous 4kb duplication units, maintained throughout evolution gene conversion and unequal
crossovers event

? Molecular defects of ɲ globin gene
- result from deletion/point mutation in ɲ globin gene/controlling sequence
- leads to reduced synthesis of ɲ chains
- more than
 caused by deletions of ɲ globin gene
- structural mutations rarely cause ɲ thalassemia
m ? Molecular classification of ɲ thalassemia
- normal individuals have 2 ɲ genes on each chromosome m (ɲɲ/ɲɲ)
- four possible manifestations, based on loss of m-4 ɲ globin gene
- wide spectrum of clinical disorder, severity related to number of ɲ globin gene deleted
- loss of m ɲ gene -> silent carrier, no detectable BC abnormalities, mild hypochromic microcystic anemia, at
risk of hydrops baby with HbH disease
- loss of 4 ɲ genes -> fetal death in utero due to no oxygen-delivering capacity
- loss of 3 ɲ genes -> relative excess of ɴ globins/non ɲ globins, clinically silent carriers, HbH, chronic
haemolytic anemia
- ɲ+ thalassemia: milder form of a thalassemia, have some ɲ chains produced
- ɲ thalassemia: complete absence of ɲ chain
mm ? Deletional ɲ thalassemia (ɲ thalassemia)
a ʹɲ2 : removes 2 kb region; ͛ of ʘɷm to codon m
b ʹMED: most common Meditteranean deletion; deletes ͛ of ʘɷm to ͛ of ɽ
c ʹTHA: 34-38kb deletion; deletes ͛ of ɺ to 3͛ of ɲm
d ʹFL: removes all ɲ-like globin genes
e ʹSEA : common in SEA among Chinese; deletes 2kb; deletes ʘɲ2 until ɽm, spare ɺ2 and ʘɺm globin genes;
most common deletion in Hb Bart͛s hydrops fetalis; carrier have microcytosis, hypochromia, anisocytosis and
reduced ɲ/ɴ synthesis ratio
m2 ? Non-deletional ɲ thalassemia
- Hb Constant Spring (HbCS) most common non-deletional ɲ thalassemia in SEA
- mutation in ɲ2 globin gene -> ɲ globin variant with 3m extra amino acid
- base substitution from T to C (TAA -> CAA) at codon m42 (termination codon of ɲ2 gene)
6


c
m ? DNA cloning research in bacterial system
- technique to reproduce DNA fragments
- 2 approach:
a cell based (plasmid/chromosomal DNA from cells) -> vector required to carry DNA fragment into host cell
b PC based (DNA are synthetically synthesized)
- steps in gene cloning
a DNA recombination -> DNA fragment inserted into vector via restriction enzyme
b Transformation -> recombinant DNA enters host cell (E coli) by treatment with CaCl2 to increase
transformation efficiency
c Selective amplification -> specific antibiotic added to kill E coli without protection; transformed E coli
protected by antibiotic-resistance gene which can inactivate specific antibiotic
d isolation of desired DNA clones
2 ? Preparation of research methodology
a mportance/significance of research towards solving biofilm problem
- highly relevant for human health because biofilms are implicated in numerous debilitatingͶand often
chronicͶdiseases and inflammations, including cystic fibrosis, tuberculosis, orthodontal disease, sinusitis
and some forms of heart disease
- the formation of a protective extracellular matrix which confers structural stability and contributes towards
the remarkable resistance of biofilms to antibiotics, the immune defences of a host organism or indeed any
compound that might be used to remove them from contaminated foods, machinery or medical devices?
- contamination of food and its packaging, and fouling water supplies
- the important question is how to dismantle biofilm structures when treating disease or contamination, and
the best source of inspiration might be the natural defences that other living organisms have evolved
- the ability to accommodate various individuals in hostile environments where they could not survive on
their own, whereby some members protect the community against external assaults
- role in breaking down sewage and decontaminating polluted sites, and are vital for the health of
ecosystems