CPC: Module 2.11

2.3.07
Fitness factors
• Body weight:
-BMI measurement in kg/m2.
-Diet.
• Cardiovascular fitness.
-CVS (cardiac output 5.30L/min).
§Heart rate.
§Stroke volume.
-Lungs (ventilation 5-25L/min).
§Vital capacity.
§Flow rate - altitude and erythropoietin.
• Skeletal muscle.
-Hypertrophy.
-Microtrauma.
-Glycogen.
• Mental attitude.
-Positive thinking.
-Endorphin effect.

Lung diseases affecting fitness - pulmonary dysfunction
• Pulmonary oedema (secondary to cardiac failure).
• Asthma.
• COPD.
-Emphysema.
-Chronic bronchitis.
• Pulmonary fibrosis.

Heart failure
• Starling's law.
• Oxygen use by:
-Heart itself.
-Other tissues.
• Possibility of reversibility.
-LVAD device.

Metabolic syndrome
• Group of risk factors in one person.
-Central obesity.
-Abnormal lipids (high triglycerides and low HDL cholesterol) → atheroma.
-BP >130/85.
-Insulin resistance.
-Prothrombotic blood state.
-Elevated CRP.

Type II diabetes mellitus
• Genetic factors (concordance).
• Abnormal glucose load response.
• Insulin resistance (?reduced transporter protein).
• Elevated, but eventually insufficient insulin.
• >80% patients obsess.
• Amylin deposition in islets late on.

Energy Expenditure, Exercise And Weight Loss

2.3.07

• Basal metabolic rate (BMR): basic level of metabolic activity required to stay alive.
• At cellular level:
-Pumping ions across membranes.
-Turnover of proteins etc.
• At organ level:
-Pumping blood.
-Respiration.
-Muscle tone.
• BMR closely regulated to amount of non-fat tissue in body.
• Measured after overnight fast, at comfortable temperature, with subject awake and resting.

Schofield equations - different for different age groups and gender

Total energy expenditure (TEE)
• During sleep, EE lower than BMR.
• All other time, EE>BMR.
• EE increased by:
-Performance of external work.
-Heat generated by work.
-Ingestion of meals (dietary-induced thermogenesis - DIT).
• TEE = BMR + physical activity + DIT.
• BMR usually largest component of TEE.
• To estimate TEE due to physical activity, multiply energy of each by time spent of each activity.
• PAL (physical activity level) = Ratio of overall daily TEE to BMR.

Mobilisation of fat stores
• Mismatch of energy supply to energy demand.
• Therefore, energy stores must be mobilised.
• Adequate carbohydrate intake in diet will partially maintain glycogen stores.
• Smaller meals mean absorptive period shorter:
-Insulin concentration decreased and glucagon concentration increased.
-Leads to activation of hormone-sensitive lipase.

Triglyceride NEFA (non-esterified fatty acids)
CO2/ketone bodies

Liver

Glycerol Triglyceride

Muscle
Co2
Kidney

Liver Glucose

• Extent to which this continues (and protein breakdown spared) depends upon:
-Size of energy gap.
-Duration of decreased intake.
-Maintenance of physical activity/energy expenditure.

Integrated view of metabolism: metabolic diary
1) Waking up: post-absorptive state
• Plasma glucose and insulin at lowest in 24-hour cycle.
• Plasma NEFA at highest.
• Glucose enters blood from liver glycogenolysis and hepatic gluconeogenesis.
• Blood glucose used by brain and red blood cells.
• Skeletal muscle mainly uses NEFA for energy.
• Some breakdown of protein in muscle due to low insulin.
• Some amino acids oxidised and amino group transferred to pyruvate → alanine → liver → gluconeogenesis.

A) On holiday - plenty of food, not much exercise
2) Breakfast - carbohydrate, protein and plenty of fat
• Glucose and amino acids increase in blood in 15-30 minutes.
• Plasma glucose will remain somewhat elevated for 3-4 hours.
• Plasma insulin concentration rises.
• Glucagon secretion falls.
• Glycogen metabolism switches to synthesis.
• Increased insulin, decreased lipase activity and decreased release of NEFA.
• Increased muscle uptake of glucose and amino acids → glucose oxidation, protein and glycogen synthesis.
• Arrival of chylomicron triacyl glycerol.
• Insulin stimulates lipoprotein lipase → fatty acid uptake → triacyl glycerol storage.
• 4 hours post-meal, fat storage at maximum.

3) Lunch.
• Insulin-stimulated processes primed.
• Reinforces pattern of storage.
• Plasma NEFAs remain suppressed.
• Glycogen synthesis continues.
• Storage of TAG in adipose tissue continuous.

4) Dinner.
• Virtually no break in storage of nutrients.
• At end of day, replete +++.

B) Energetic day and healthy diet
2) Breakfast - mainly carbohydrate with little fat
• Sharper rise in glucose and insulin.
• Release of NEFAs suppressed.
• Preservation of adipose tissue TAG stores.

3) Some gentle exercise (walk/cycle to work)
• Some glycogen breakdown.
• Increased heart rate and concentration.
• Blood flow to muscle increases, delivering more substrate.
• Skeletal muscles require more substrate.
• More glucose uptake from plasma.
• Increased sympathetic activity and adrenaline → fat mobilisation.
• Plasma glucose decreased → insulin decrease→ less conservation of fat.
• Less suppression of glucagon by glucose.
• Change from high insulin: glucagon ratio with intense substrate storage, to lower ratio with less storage and diversion of substrate to muscle.

4) Lunch (low-fat)
• Metabolic system less primed for storage.
• Afternoon walk will divert more substrate into oxidisation.
• At end of day, less substrate will have been stored, and more oxidised.

Summary
• Storage of energy regulated.
• Body's immediate needs met.
• Surplus stored in integrated way.
• Integration for utilisation - energy available for activity as required.

Energy Balance And Body Composition

26.2.07

Body weight = Fat + Fat-Free Mass
0.900g/cm3 1.100g/cm3

Estimating total body fat: whole body density (typical figure: 1.024g/cm3). Methods:
• Underwater weighing - gold standard.
• Skinfolds - not direct measures of body fat - callipers - four sites:
-Biceps.
-Triceps.
-Subscapular.
-Iliac.
• Total body volume.

Estimating total body fat
• Total body water: inverse relationship between body water and fat.
-FFM = TBW/0.73.
• Total body potassium: gives idea of FFM - radioactive K40.
-Women: FFM = TBK/60.
-Men: FFM = TBK/66.
• Bioimpedance: more fat, greater resistance to electrical climate - if subject dehydrated, poor result.
• Ultrasound.
• X-rays.
• Dual energy x-rays (DeXA).
• MRI scans.
• CAT scans.

Estimating lean body mass
• Total body potassium (K40).
• Total body nitrogen: in vivo neutron activation analysis.

Estimating carbohydrate stores:
• Muscle biopsy.

Assessing body composition using BMI
• BMI = Weight (kg) / Height2 (m2).
• Not direct measure of body fat, but useful.
• Cutoffs:
-<20: underweight.
-20-24: "normal."
-25-29: overweight.
-30+: obese.
• NB1: athletes.
• NB2: children - Cole et al (2000) BMJ 320; 1-6.
• NB3: it's not just how much fat - it's where!

Energy balance
• Intake - output = Δstore
Protein BMR (basal metabolic rate) glycogen
Fat HIF (heat increment of feeding) fat
Carbohydrates activity
Alcohol
• Metabolisable energy.
Protein Fat Carbohydrate Alcohol
4 9 3.75 7 kcal/g
17 39 16 27 MJ/g

Losing weight
• Negative energy balance:
-Decreased intake.
-Increased expenditure.
-Decreased intake AND increased expenditure.

What is feasible?
• Energy deficit of 4.2MJ (1,000kcal)/day for 3-6 months.
• -29MJ (7,000kcal)/week = 1kg loss/week.
• Total loss ~5.20kg.
• After: Lean (1998) Handbook of Weight Management.
• NICE Guidelines: December 2006.

Module 2.11

The Middle Manager's Resolution.
26.2.07 - 9.3.07.

Clinical Molecule Genetics

23.2.07

Molecular genetics laboratory
• Sample (blood, buccal cells, muscle biopsy, fixed tissue, CVS, amniotic fluid, hair roots).


• DNA.


• Analysis (PCR, DNA hybridisation, DNA sequencing).


• Result (interpretation - allele sizing, risk calculation, unknown mutation).


• Report.

Methods of DNA analysis
• DNA amplification (PCR).
• DNA hybridisation.
-Genomic Southern blotting.
-DNA Microarrays (DNA chips).
• DNA sequencing.

DNA Microarrays
• 100,000+ oligonucleotide "probes" on microchip.
• Uses include:
-Expression studies.
-Rapid linkage analysis - SNPs.
-SNP analysis for pharmacogenetics.
-DNA sequencing.

Types of mutation
• Large-scale rearrangements.
-Deletions, duplications.
-Inversions.
• Point mutations.
-Base substitution.
-Small deletion/insertion.
• Expanding Trinucleotide repeats.
• Imprinting.

Deletions
• Part/whole of gene.
-DMD - ~66% cases.
-SMA - 95% cases = homozygous deletion.
• Large deletions of several genes.

Duplications
• Part/whole of gene.
-HMSN Ia - ~70% cases.
• Often reciprocal to deletion.
-HMSN/HNPP.

Inversions
• Part/whole of gene.
-Haemophilia A.
• Often many genes.

Point mutations
• Base substitution (SNPs).
-Nonsense mutation (creates stop codon).
-Misssense mutation (changes one amino acid).
-Silent mutation (no effect on protein).
-Splice site mutation (leads to exon slipping/translation of intronic sequence).
-Regulatory region mutations.
-Frameshift mutation.
• Small deletion/insertion.
• Single base change in promoter.
• Can lead to increase/decrease in protein product.
• Leads to milder forms of genetic disease.
• Likely to be involved in polygenic disorders.

Pathogenesis: CF mutation types
Expanding trinucleotide repeats
• Usually polymorphic - tend to be neurological consequences.
• CAG = HD/SCAs/SBMA (affected range 40-120 repeats).
• CTG - Myotonic dystrophy.
• CGG - Fragile X.
• Inheritance:
-Autosomal dominant.
-Autosomal recessive.
X-linked recessive.
-X-linked - Fragile X.
• Location:
-Exonic.
-Intronic.
-Close to gene.

Imprinting
• Maternal and paternal copies of gene differentially imprinted.
• Very few genes imprinted.
• Mechanism is methylation - silences gene.
• Imprint lost in germ cells.
• Imprinting-related disorders include Prader-Willi and Angelman syndromes (15q 1.4).
• Uniparental disomy.

Molecular genetics - types of analysis
• Known mutation.
-E.g. common point mutation, deletion/duplication, triplet repeat expansion.
-Set up mutation-specific assay. Relatively easy.
• Unknown mutation.
-Screen for changes.
-DNA sequencing.
• Linkage analysis.
-Family studies using polymorphic markers linked to gene of interest.

DNA polymorphic markers
• Identity testing.
• Rapid chromosome aneuploidy screening.
• Linkage analysis.

Potential problems for DNA analysis
• Germ line and somatic mosaicism.
• Genetic heterogeity.
-E.g. retinitis pigmentosa >20 genes to date.
• Sensitivity of mutation detection.
-Size of gene, limits of current technology.
-Cryptic splice sites.
• Pathogenicity of missense mutations.
• Linkage: recombination/new mutation.

Future developments
• Proteomics/metabolomics.
• Genetics of common disease.
-Susceptibility/risk factors.
• Pharmacogenetics.
-Adverse reactions/tailored drug therapy.
• Oncology.
-Acquired mutations.
-Tumour expression profiling.
• Gene therapy.

New technologies
• Electronic hybridisation.
• Microarrays.
• Comparative Genome Hybridisation (CGH).
• Mass spectrometry (1 million SNPs per day).
• Individual genome sequence.

Variation In Response To Drugs

19.2.07

What is pharmacogenetics?

We all vary in response to…
• Environmental chemicals.
• Nutrients.
• Drugs…

• ~15% smokers develop lung cancer.
• 20% of alcoholics will develop liver cirrhosis.
• With medicines:
-Patients may respond well.
-Patients may not respond at all.
-Patients may respond adversely.

Variability is an issue for:
1. Drug efficacy.
2. Drug safety.

"One dose fits all" - variable efficacy
• 6.5% (n=1225) admissions due to adverse drug reactions.
• Seven 800-bed hospitals occupied by adverse drug reaction patients.
• Cost £446m per annum.
• Death in 0.15% - equivalent to 5700 deaths per year.

Definitions
• Pharmacogenetics = Study of genetic basis for difference between individuals in response to drugs.
• Pharmacogenomics = Used in wider sense.

"The right medicine for the right patient"
• Rapid sequencing for specific gene polymorphisms.
• Knowledge of genetic sequences of target genes coding for enzymes, ion channels etc.

• 3 billion base pairs contain all information necessary to make a human being.
• Similarities and differences: 99.9% same, 0.1% different.

Potential benefits
1. Drug efficacy.
2. Drug safety.
3. Pharmacoeconomics.
4. Drug development.

Continuous variability
• Some patients: usual response.
• Some patients: less-than-usual response.
• Some patients: more-than-normal response.
• Continuous distribution.

Factors
• Genetic and environmental.
• Multiple genes.
• Race.
• Sex.
• Diet.
• Weight.
• Etc…

Discontinuous variability
• Discrete proportion (large/small).
• Respond differently from rest.
• Most often single gene.
• Genetic polymorphism.
• Mutation >1% of population.
• Simple inheritance.
• Two or more discontinuous forms.

Some examples
• Cytochrome p450 oxidation.
• Acetylation.
• Porphyrias.
• Abacavir hypersensitivity.

CYP 2DG
• Defective oxidation.
• First identified in debrisoquine.
• Extensive metabolisers (most).
• Poor metabolisers.
• Intermediate metabolisers.
• Ultra-rapid metaboliser.
• Variability in drug concentrations.
• Ethnic variability (6% in white populations).
• Metoprolol.
• Codeine.
• Haloperidol.
• Flecainide.
• Nortriptyline.
• CYP 2C9.
• Warfarin.
• Most sulphonylureas.

Acetylator status
• Important route of metabolism.
• Rapid/slow acetylators.
• Ethnic variability again:
-90% Japanese rapid acetylators.
-50% Western rapid acetylators.

Porphyrias
• Congenital/acquired disturbances in haem biosynthesis.
• Concomitant environmental/genetic factors.
• Acute = Neurovisceral presentation.
• Cutaneous = Photosensitive skin lesions.

Acute intermittent porphyria
• Autosomal dominant.
• Variable penetrance.
• PBG deaminase = Deficient enzyme.
• Enormous number of mutations found to be associated with acute intermittent porphyria - family specific.
• No accurate calculation of frequency.
• Acute intermittent porphyria most common acute porphyria.


Provocative factors
• Alcohol.
• Many drugs.
• Barbiturates.
• Anticonvulsants.
• Oestrogens (including OCP).
• Infection/surgery.
• Low-carbohydrate diets/fasting.

Abacavir hypersensitivity
• Nucleoside reverse transcriptase inhibitor used in HIV disease.
• 5% patients develop hypersensitivity reaction within 6 weeks.
• Rechallenge results in more serious reaction.
• HLA-B*5701 = Genetic predisposing factor (odds ratio of 29).

Thioprine methyl transferase (TPMT)
• Metabolises azathioprine and 6-mercaptopurine.
• Critical pathway in haemopoietic tissue.
• Associated with severe haemopoietic toxicity.

Clinical Cytogenetics

19.2.07

What is cytogenetics?
• Study of chromosomal pathology.
• Chromosomes are protein/DNA macromolecules.
• They carry genes.
• When they mutate, they cause phenotypic changes - some are recognised as syndromes.
• Constitutional changes.
• Acquired changes.

Cell and tissue culture
• There are a variety of tissues received in the Cytogenic laboratory, which include:
-Blood - lymphocytes.
-Amniotic fluid - foetal cells.
-Chorionic villus.
-Bone marrow.
-Products of conception, skin and tumour tissue.
• Most cells and tissues need to be cultured - exception to this:
-Bone marrow cells.
-Trophoblasts seen in chorionic villus tissue.

Investigations on blood samples
• Neonates - know syndromes and unexplained dismorphism and/or congenital abnormalities.
• Childhood developmental/intellectual delay.
• Pubertal failure.
• Infertility.

Prenatal diagnosis
• Indications for prenatal diagnosis include Downs risk from sera screening.
• Abnormalities seen on ultrasound scan.
• Known carriers of single gene/chromosomal disorder.
• Parents who have had previously chromosome-affected child.

Methodologies
1. Cell culture: mitotic cell division stopped at metaphase.
2. Stain by G banding: molecular Cytogenic - special identity by FISH.
3. Microscopy: molecular genetic methods e.g. MLPA, RTFCR.
4. Microarrays.
5. Write detailed interpretative reports.

Cell and tissue culture
• Aspects to be considered in cell culture include:
-Sterility - aseptic techniques, biological safety cabinets, antibiotics.
-Culture vessels - plastic/glass.
-Culture media - provides nutrients, maintains pH.
-Temperature - 370C maintained using incubator.
-Culture environment - carbon dioxide and oxygen levels can be altered.
-Mitogens.
-Harvesting procedure.
-Stop cells growing at metaphase using colcemid.
-Hypotonic solution swells cells.
-Fixative (methanol/acetic acid).

Molecular cytogenics - fluorescence in-situ hybridisation (FISH)
• Hybridises probes with complementary sequences to target DNA.
• Procedure consists of:
-Denaturation.
-Hybridisation.
-Stringency washing.
-Visualisation.
• Extend cytogenetic analysis to non-dividing cells.

New technologies - arrays
• Array CGH, BAC arrays and oligonucleotide arrays.
• Arrays consist of specific DNA sequences, which may be spotted onto a glass slide/some other substrate.
• Hybridisation with target DNA then performed.

What might cause non-disjunction?
• Failure of chromosomes to separate at meiosis I/chromatids at meiosis II.
• Asynapsis = Failure of chromosomes to pair.
• Asynapsis can be deduced by observing that number of chiasma reduced/non-existent.
• Can be estimated by recombination frequency - known that older women (>35 years) have half number recombinations compared to younger women.

Viable trisomy conditions
• Trisomy 21 = Down's.
• Trisomy 13 = Patau.
• Trisomy 18 = Edwards.

Sex chromosome aneuploidy
• 45, X = Turner.
• 47, XXX = Triple X.
• 47, XXY = Klinefelter.
• 47, XYY.

X chromosome inactivation
• In normal female with 2Xs, one X inactivated.
• In male with Klinefelter, one X inactivated.

Chromosomal abnormalities (structural)
• Deletion.
• Duplication.
• Isochromosome.
• Inversion.
• Translocation.

Deletions
• Deletion with >2% of total haploid genome will result in lethal outcome.
• Smallest viable loss from chromosome ~4Mb.
• Therefore, large number of contiguous genes lost, resulting in intellectual retardation and congenital malformations.

Microdeletions
• Describes small chromosomal loss only detectable by FISH.
• Well-described syndromes include:
-Prader-Willi.
-Angelman.
-William's.
-Wolf Hirschorn.
-Cri-du-chat.
-DiGeorge.
-Miller Dicker.

Reciprocal translocations
• Associated with:
-Impaired spermatogenesis.
-Miscarriages.
-Congenital abnormalities due to segregation at meiosis, resulting in unbalanced gametes.
• Identified as exchange of chromosomal material between non-homologous chromosomes.
• Break points can be in short/long arm - generally in non-transcribing DNA.
• Therefore little, if any, phenotypic effect.

Robertsonian translocations
• Affects acrocentric chromosomes 13, 14, 15, 21 and 22.
• Associated with recurring miscarriage and impaired spermatogenesis.
• Mal-segregation can give rise to Down's if 21 involved or Patau's if 13 involved.

Inversions
• Paracentric gametes - normal/inversion/dicentric/acentric.
• Pericentric gametes normal, inversion, duplication/deletion.
• In large pericentric inversions, duplicated and deleted segments very small therefore, newborns may survive.

Haematological malignancies - acquired disorders
• Clonal expansion of neoplastic cells derived from (or resembling) normal haematological cells.
• In general, leukaemias and lymphomas classified according to stage of normal haematopoiesis at which cells appear to be blocked.

Bone marrow samples
• Bone marrow obtained via iliac crest puncture by haematologist.
• Reasons for referral include:
-Confirmation of diagnosis for chronic myeloid leukaemia.
-Acute myeloid leukaemia (M2, M3, M4).
• Indication of prognosis, particularly in childhood ALL.

CPC On Genetic Disease

16.2.07

Case 1
• Male aged 24.
• Family history of colon cancer.
• Been on screening since aged 10.
• Now colectomy.

Familial adenomatous polypoposis (FAP)
• Autosomal dominant.
• APC gene - tumour suppressor gene.
• Dominant inheritance, but woks as recessive gene.
• Compare RB gene (retinoblastoma).
• Clinically: polyps in large intestine increased during childhood.
• Malignancy inevitable by late 20s.

Normal
APC loss/mutation
Metaplastic polyp
Loss of DNA methylation
Adenoma
Ras mutation (12p)
Adenoma
Loss of DCC (18q)
Late adenoma
Loss of p53
Carcinoma


Other tumour suppressor genes
• NF-1: neuroblastoma.
• NF-2: Schwannoma, meningioma.
• RB: retinoblastoma, osteosarcoma.
• p53: Li Fraumeni syndrome.
• WT-1: Wilms tumour (renal).
• BRCA-1, 2: Breast, ovary carcinoma.

Case 2
• Female, aged 42, well.
• Patient's uncle died from ischaemic heart disease aged 62.
• Sudden death, therefore, autopsy.
• Liver looked nodular.
• Histology taken.

Next…
• Patient has liver investigations and biopsy.
• LFTs normal.
• Ferritin high, serum iron high.

Autosomal recessive
• Genetic Haemochromatosis.
• Example of "founder" gene mutation (800AD, Celtic population).
• Iron overload due to increased absorption.
• May exceed 50g total (normal <6g).
• Commoner/earlier in men (no blood loss).
• Carrier 1:9 in Northern Europe, homozygotes 1:220 (but penetrance only ~20%).

Haemochromatosis
• Iron in toxic excess (catalyses free radicals).
• Results in:
-Cirrhosis.
-Pancreatic fibrosis.
-Cardiomyopathy.
-Tumours, especially hepatocellular carcinoma.

Other founder mutations - all recessive
Mutation Heterozygote resists
CF Diarrhoea
Sickle cell Malaria
FV Leiden (clotting) Sepsis
ALDH2 (alcohol) Alcoholism (?)
Lactose tolerance Allows milk use
GHB2 (deafness, Middle East) Unknown

Case 3
• Ghanaian (black) male aged 23.
• Episodes of severe abdominal and joint pain.
• Bad enough to need opiates.
• Last attack precipitated by bronchitis.

Sickle cell anaemia
• Recessive (autosomal).
• Point mutation at position 6 of beta globin chain.
• West Africa.
• Homozygote - crises caused by hypoxia ± infection.
• Autosplenectomy.
• Danger from hypoxia.
• Danger from infection, especially pneumonia.
• Joint damage from sickle crises.

Case 4
• Male aged 37 with family history of early cardiac death.
• Father died aged 42.
• Uncle died aged 44.
• Patient overweight (BMI = 34).
• And has xanthelasmata.

Investigations
• Cholesterol 12 mmol.
• LDL 8 mmol.
• FBC and clotting normal.
• U+Es and LFTs normal.

Familial hypercholesterolaemia
• Heterozygote 1:500.
• Typical cholesterol >7.5 mmol.
• Risk of early heart disease and stroke.
• Homozygote much rarer, but more severely affected.
• Cholesterol can be as high as 30!

Case 5
• Male patient aged 41 develops heart failure.
• Tall, thin, blue sclerae, long spidery fingers, pectus excavatus, high arched palate.
• Valve replacement.
• Died suddenly 2 years later.

Marfan's syndrome
• Incidence 1:5000.
• 75% cases familial autosomal dominant.
• Remainder sporadic (new mutation).
• Defect in fibrillin-1: abnormal form due to missense mutations.
• Tall with long gingers and toes.
• Relatively shorter upper body and long legs.
• Double-jointed digits.
• Eye.
• CVS.
• Sudden death from CVS causes.

X-linked recessive: fragile X
• Causes learning difficulties.
• Next commonest after Down's.
• IQ 20-60. Long face, large jaw and ears.
• Macro-orchidism.
• ~20% carrier males normal, but can transmit disorder.
• ~50% carrier females not affected.
• FMR-1 gene at Xq27.3 has CGG repeats in 5' untranslated region.
• Normally 10-55 repeats (average 29).
• Transmitter males pass on permutation without much amplification.
• Carrier females pass on dramatically amplified mutation.
• Amplification probably occurs in oogenesis, but not spermatogenesis.

Future prospects
• Better understanding of diseases (known genetic and postulated environmental), with application of Human Genome Project.
• Genetic tailoring of drug therapy.
• Common drugs like statins and anti-inflammatories may benefit.
• Tailored cancer treatment.

Pathology Of Genetic Disease

16.2.07

Developments
• Human Genome Project.
-Polymorphisms.
-0.1% (3mBp) variable.
-Imprinting ?role.

Congenital and genetic
• Congenital: existing at or before birth - may not become apparent until later.
• Genetic: inherited.

Genetic diseases
• >1000 known to affect humans.
• Diseases with at least some genetic component will affect 2/3 of population during lifetime.

Mutations
• Different scales.
-Whole chromosome gain/loss.
§Monosomy/trisomy.
+Mostly not transmitted.
-Chromosome rearrangement.
-Gene level (submicroscopic).

Cytogenic autosomal disorders
• Trisomies:
-Down's syndrome (trisomy 21).
§Average incidence 1/700 (1/25 if mother >45 years).
§95% cases maternal origin.
§40% patients have congenital heart disease.
§Rate of acute leukaemia increases 10-20X.
§Many get Alzheimer's after age 40 years.
-Edwards (trisomy 18) and Patau's (trisomy 13) rarer.

Sex chromosome disorders
• Two most common:
-Kilnefelter's (47, XXY/variants), 1:500 male.
§Hypogonadism, infertility.
§Mild learning difficulties (not all).
§Effect of extra X probably mitigated by lyonisation.
-Turners (45, X), 1:2000 female births.
§Many (?all) mosaics with XX, XY, XXX etc.
§Hypogonadal, short stature, skin webs.
§IQ often in normal range.

Mutations
• Gene level.
-Deletion (partial/complete).
-Point mutation (single base).
-Insertions/deletions → frame shift.
§Base pair gain/loss.
-Mutations in promoter/enhancer regions.
§Non-coding, but can interfere with transcription.
§Trinucleotide repeats (usually C and G bases).
-Mutation/deletions of MMR genes (HNPCC).

Mendelian disorders: autosomal dominant
• Affect 50% children.
• Parent often affected.
• May be late onset of effect.
• Variable penetrance (incidence).
• Variable expressivity (effect).
• Enzymes not usually involved because ~50% remains (other copy).
• Loss of function due to:
-Regulatory proteins involved in feedback.
-Key structural proteins.
• Gain of function e.g. overexpression.
• Colon: FAP.
• Chemistry:
-Acute intermittent Porphyria.
-Familial hypercholesterolaemia.
• Bones:
-Marfan.
Ehlers-Danlos.
-Osteogenesis imperfecta.
-Achondroplasia.
• Renal: polycystic kidneys.
• Blood:
-Hereditary spherocytosis.
-Von Willebrand's.
• CNS:
-Huntington's disease.
-Von Recklinghausen.
-Myotonic dystrophy.
-Tuberous sclerosis.

Mendelian disorders: autosomal recessive
• Everybody carries 5-8 recessive harmful genes.
• Parents phenotypically normal, but 25% siblings affected.
• Usually complete penetrance.
• Early onset.
• Blood:
-Sickle cell.
-Thalassaemias.
• Bone:
-Some Ehlers-Danlos.
-Alkaptomina.
• Endocrine: congenital adrenal hyperplasia.
• CNS.
• Chemistry:
-CF.
-PKU.
-Galactosaemia.
-Homocystinuria.
-Lysosomal storage disorders.
-Alpha-1-anti-trypsin deficiency.
-Wilson's disease.
-Haemochromatosis.
-Glycogen storage diseases.

Sex chromosomes
• Y chromosome has:
-Genes related to spermatogenesis (with internal copies).
-A few genes homologous to those on X, but no syndromes known from these.
• X chromosome (in female) is randomly inactivated (lyonisation) - mosaic state.

X-linked (recessive) disorders
• Males affected:
-Females may be partly affected due to lyonisation.
-Males described as hemizygous as no paired Y-chromosome gene generally exists.
-Affected males transmit disorder to daughters as carriers, but not to sons.
• Chemistry:
-Diabetes insipidus.
-Lesch-Nyhan (uric acid).
• Muscle: Duchenne's.
• Blood:
-Haemophilia A and B.
-Chronic granulomatous disease.
-G-6-PD deficiency.
-Agammaglobulinaemia.
-Wiskott-Aldrich (immunodeficiency).
• CNS: fragile X syndrome.

Single gene disorders: effects
• Enzyme defects:
-Substrate build-up.
§Mucopolysaccharidoses.
§Lysosomal storage diseases.
-Lack of product.
§Albinism.
§A-1-A-T deficiency.
• Structural alterations in other proteins - haemoglobinopathies - sickle cell anaemia (not thalassaemias).
• Altered reaction to drugs.
• Defects in membrane receptors.

Multifactorial genetic disorders
• Interaction with environment likely.
• Could be 7-10 genes involved.
• Congenital malformations easily observable model.
-Cleft lip/palate.
-Heart.
• IHD.
• Hypertension.
• Diabetes mellitus (especially type II).
• Pyloric stenosis.
• Gout.

Non-Mendelian disorders
• Triplet repeats: fragile X.
• Mitochondrial genes:
-Maternal inheritance.
-37 genes (24 translating and 13 code for metabolic enzymes).
-Several conditions:
§Leber optic neuropathy.
§Mitochondrial myopathy.

Diagnosis, Risks, Inheritance And Genetic Testing

12.2.07

Making referral to clinical genetics
• Positive family history, ?syndrome.
• Worried, when asked, planning children.
• Tell family about referral.
• Diagnosis, family history, age, investigations so far.
• What family know already etc.

Huntington's disease
• Autosomal dominant, 50% risk of transmission.
• Variable age of (adult) onset: 30s-50s.
• Anticipation, especially from father.
• Gene (triplet repeat) on chromosome 4p.
• Involuntary movements, tremor, chorea.
• Change of personality, drive.
• Behavioural changes.
• Psychiatric symptoms.

Diagnostic and predictive tests
• In presence of symptoms.
• Unaffected, but wants to know.
• Testing of foetus during pregnancy (at-risk parent knows their status).
• Excluding risk of Huntington's disease in foetus (at-risk parent does not want to know their status).

Issues in predictive genetic testing
• Testing asymptomatic for late-onset conditions.
• To know or not to know?
• Is there useful intervention?
• Possible harmful psychological effects.
• Insurance and employment issues.
• Does not predict age of onset/severity etc.

X (sex)-linked inheritance
• Women = carriers.
• Men affected (knight's move).
• No male-to-male transmission.

What can the Human Genome Project deliver?
• Genetic diagnosis, understanding process.
• Novel treatments, gene therapy.
• Miscarriage, infertility, organ transplantation.
• Dementia, heart disease, cancer.
• Genetically-produced drugs, gene therapy.
• Stem cells for transplantation.
• Information, ethical and practical dilemmas.

• SNPs - single-nucleotide polymorphisms.
• UK Biobank.

Module 2.10

A Family Slowly Going Off Their Legs.
12.2.07 - 23.2.07.

The Case Of A Patient Undergoing Surgery

5.2.07

Preoperative anaesthetic visit
• Explain all details of perioperative period.
• Allay anxiety.
• Answer queries.
• Check appropriate investigations have been done.
• Prescribe premedication.
• Explain procedure.
• Detail possible methods of postoperative analgesia e.g. PCA, epidural, spinal.
• Explain risks.
• Explain post-operative procedure.

Preoperative anaesthetic assessment
• Any previous anaesthetic?
• Allergic reaction?
• Awareness.
• Anxiety.

Drug therapies
• Should they be continued?
• Is it optimal?
• Any drug allergies?

Anatomy of airway
• Is it abnormal?
• Any pathology?
• Can patient open mouth?
• Any airway infection?
• Check teeth.

Preoperative preparation - indications for:
• Physiotherapy.
• Anti-embolism stockings.
• Heparin.

Preoperative investigations
• Which of these does she need?
• FBC, U+Es, Ca, glucose.
• Cross-match.
• CXR.
• ECG.
• Lung function tests.
• Blood gas analysis.

Premedication
• Allay anxiety.
• Relieve pain.
• Anti-sialogogue.
• Appropriate for surgery e.g. drying agent for surgery on airway.

Premedicants
• Anxiolytics: benzodiazepines, trimeprazine.
• Analgesics: oral e.g. NSAIDs, paracetemol; parenteral e.g. opiates, NSAIDs.
• Anticholinergics: atropine.
• Antiemetics.

Preparation for theatre
• Starve:
-4 hours: no fluid.
-6 hours: no solid food.
• Consent, correct side.
• No artificial teeth, hairgrips, jewellery, make-up.
• Empty bowel and bladder.

Immediate postoperative care recovery room
• Adequate oxygenation (any facial deformities?).
• Adequate pain relief.
• Adequate fluid therapy.

Postoperative pain
• Minor surgery, day case.
-Simple oral analgesia e.g. paracetemol, Dihydrocodeine.
• Intermediate surgery.
-NSAIDs, IM opiates.

Major surgery: postoperative pain
• IV opiates: continuous infusion patient-controlled analgesia (PCA).
• Epidural analgesics: local anaesthetic agents e.g. lignocaine, bupivicaine; opioids analgesics e.g. diamorphine, morphine, fentanyl - must be preservative-free.
• Local nerve blocks e.g. intercostals nerve blocks for thoracic surgery, 3-in-1 block for hip surgery.
• Spinal analgesia for lower abdominal and lower limb surgery.

Postoperative care - risks of:
• Chest infection (is she a smoker?).
• Appropriate antibiotic cover.
• DVT, PE (any risk factors?).
• MI.

Postoperative chest infection
• Which is most common organism to cause it?
• Is there risk of transmission?
• Other therapies.
• Physiotherapy.
• Nebulisation.

Waiting Lists And Discharges - Does The NHS Need More Beds?

5.2.07

1997: impending crisis
• 1,132,200 patients on waiting lists for hospital admission.
• 30,000 patients waiting for >1 year.

What causes waiting lists?
• Under-funding.
• Rationing.
• Inefficiency.
• Vested interests.

Waiting pools, not lists
• Treatment pool.
• Assessment pool.
• Not referred pool.

1997 political focus on waiting lists.

Cancer plan 1997
• Fast track referrals.
• Maximum 2-week wait.
-Breast cancer by April 1999.
-All other cancers by 2001.

March 2000
• 100,000 reduction achieved.
• 18-month waits virtually eliminated.
• 50,000 waiting 12 months.
• 130,000 waiting >26 weeks.

NHS plan 2000
• Outpatient:
-Maximum wait reduced from 6 to 3 months.
-Average wait 5 months.
• Inpatient:
-Maximum wait reduced from 18 to 6 months.
-Average wait 5 months.

National Beds Enquiry 2000
• Numbers and distribution of beds.
• Factors determining usage of beds.
• 66% beds occupied by patients >65.
• Traditional role of NHS:
-Managing life's incidents.
-Acute care.
• Ageing population:
-Increased chronic disease.
-Proactive and ongoing care.

The response
• Accelerating discharge:
-Reform of hospital organisation.
-Support in the community.
• Reducing admissions.
-Alternative treatments.
-Support in the community.

• Absence of alternatives to acute care contributory factor to all categories of delay.

Delayed discharge from hospital
• Symptom and cause of:
-Poor bed management.
-Failure of communication between health and social care.

Problems with hospital discharge
• Delayed.
• Occurs too soon.
• Poorly-managed from patient/carer perspective.
• Transfer to unsafe environments.

Causes of delayed discharge
• Internal hospital factors:
-Timing of ward rounds.
-Waits for results.
-Delay in home assessment.
-Organisation of medication.
-Availability of transport.
• Coordination issues:
-Health services.
-Social care services.
-Other community services.
• Capacity and resource issues:
-Availability of rehabilitation places.
-Placement difficulty with care homes.
-Availability of home care provider.
• Patient/carer involvement:
-Failure to involve in decision-making.
-Limited choice of care options.

Improving discharge performance
• Discharge = process, not isolated event.
• Transfer from hospital to appropriate setting needs careful planning.
• Discharge planning should start before elective admissions and ASAP after emergency admissions.
• Patients and carers should be involved at all stages.

Improving services for vulnerable people

Intermediate care
• Range of integrated services to:
-Promote faster recovery from illness.
-Support timely discharge.
• In Liverpool:
-Emergency Response Team (ERT).
-Intermediate care team.
-ACTRITE (A+E assessment of COPD patients).
-Tracker nurses.
-Orthopaedic rehabilitation at home.

Patient journey through illness
• Many people fear experience of hospitalisation and loss of autonomy.
• Want to return to living normal lives ASAP: every effort should be made to help them do so.
• Acute hospitals should only be used for delivery of services that cannot be provided as effectively in home/social setting/community