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Flashcards in Module 1 Deck (85):
1

Malnutrition

A state of nutrition in which deficiency, excess or imbalance of nutrients causes measurable adverse effects on body function and clinical outcome.
incorporates undernutirtion

2

Overnutrition

A state of nutrition in which there is an excess of energy stores, represented by body fatness, causing measurable adverse effects on body function and clinical outcome

3

Obesity

An excess of body fat associated with a BMI >30

4

Malnutrition universal screening tool (MUST)

Step 1: BMI score
>20 score is 0
18.5-20 score is 1
<18.5 score is 2

Step 2: Weight loss score
-unplanned weight loss in 3-6 months
<5% score is 0
5-10% score is 1
>10% score is 2

Step 3: Acute disease effect score - If patient is acutely ill and there has been or is likely to be no nutritional intake >5 days - score 2

Step 4: Overall risk

0 - Low risk (routine clinical care and repeat screening)

1 -Medium risk (observe - document intake, assess improvement, repeat screening)

2- High risk ( Refer to nutrition team, increase intake, monitor and review)

5

Nutritional assessment

Muscle wasting
Loss of fat
Oedema/ascites
(low albumin)
Arthropometry

6

Nutrients

Fats
Amino acids and protein
Carbohydrates
Electrolytes and water

Minerals
Trace elements
Vitamins

7

PCR

Technique in the lab used to make millions of copies of a particular section of DNA (amplify)

5 core ingredients:
1.DNA template to be copied
2.Primers
3.DNA nucleotide bases
4. Taq polymerase
5.Buffer to ensure right conditions

Thermal cycling process:
1. Denaturing (double-stranded DNA is heated to separate it into 2 single strands

2. Annealing (temperature lowered to enable DNA primers to attach to the template DNA)

3. Extending (temperature is raised and new strand of DNA is made by the Taq polymerase enzyme)

These three stages are repeated 20-40 times

After the PCR has been completed, a method called electrophoresis can be used to check the quantity and size of DNA fragments produced

8

Chain termination (sanger method)

DNA denatured
Template strand is used

4 reaction mixtures are set up
each one including DNA template, DNA polymerase, nucleotides, A small amount of a labelled chain terminating variant of one of the 4 nucleotides (different one in each flask)

DNA polymerase incorporates the chain terminating variant at random, eventually ending the chain at every possible nucleotide position over a few 1000 bases, products are run on a gel electrophoresis gel and sequence can be deduced by reading from the smallest to the largest piece.

Now largely automated

The larger the fragment the less separation and resolution

Maximum amount of DNA sequence that can reliably be determined in one run is 500-1000 bases (determined in pieces and assembled in silico)

9

Shotgun sequencing

Large and long sequences over short ones

A method to make short fragments of DNA from large sequences (Sonication or DNase digestion can be used for random shearing) and after to use Sanger methods -> then use overlapping methods to put it back together -> make map of chromosome

10

New DNA sequencing Technologies

Sequence by synthesis -Pyrosequencing

-Synthesis on base pair at a time with real time detection
-PicoTiterPlate (PTP) Device -> DNA capture beads and enzyme beads deposited in each well on plate
-then pyrosequencing

11

Sanger method

Gold standard - ~700 bases/read in 1 hour ~1,000,000 bases in 24 hours Labour intensive

valuable for small projects
but expensive and time consuming

12

Coeliac disease

A chronic small intestinal immune-mediated enteropathy precipitated by exposure to dietary gluten in genetically susceptible individuals
Ludwigsson et ak. Gut, 2013
Gluten sensitive enteropathy

13

Inherited predispositions to colorectal cancer

Mendelian dominant (FAP,Lynch syndrome)
Mendelian recessive
(MYH associated polyposis)
Rare variants - low penetrance e.g. APC T3920A
Common low penetrance variants (GWAS)

14

Familial risk of CRC

<5% dominant inheritance
15% variation risk of colorectal cancer due to heritable/ genetic factors

15

Inherited CRC Syndromes

Early age onset
Dominant or recessive inheritance
Polyps or Polyposis

16

FAP

Autosomal Dominant
Definition: Colonic adenomas >100
Colorectal cancer
10% no family history
Extra-colonic features

17

Adenomatous polyposis coli gene

APC is a tumour suppressor gene

Second hit inactivates normal allele

WNT pathway (Myc)

Occurs early in tumourigenesis (adenoma)

Aneuploid tumours (variable number of chromosomes)

Frequently mutated in sporadic CRC

18

APC genotype:phenotype

Polyposis : truncating mutations (1000 polyps)

Attenuated : 3' and 5' mutations (100 polyps)

Polyps : point mutation (e.g. T3920A) (few polyps)



Associations

Gardner's - osteomas, epidermoid cysts

Turcot's medulloblastomas

19

APC T3920A

Ashkenazi Jew
AAATAAAA to (A)^8
Creates unstable poly A tract
Somatic mutation during replication
Adenomatous colonic polyps
OR 2-3 colorectal cancer

20

Management of FAP

Referral to genetic /polyposis registry
Surgical management
Surveillance of ‘at risk’ family (dye spray)
Genetic testing
PGD (Preimplantation Genetic Diagnosis)
?role aspirin / COX 2 inhibitors

21

Hereditary non-polyposis colorectal cancer (HNPCC)

LYNCH syndrome

Inherited alteration of DNA mismatch repair genes

Increased risk of CRC and other tumours

Tumours are diploid with microsattelite instability



22

Lynch syndrome genes

MLH1
PMS2 (complex with MLH1)

MSH2
MSH6
EPCAM

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Lynch syndrome phenotype

Autosomal dominant
no polyposis
early age onset cancer
CRC predominantly right-sided
Endometrial, ovarian, small bowel, ureter, or renal pelvis

24

Genetic diagnosis of lynch syndrome

2-3% of CRC due to LS
NICE guidelines testing of all CRCs
Tumour testing for loss of expression MismatchRepair genes and or Micro-satellite instability

25

Management of Lynch syndrome

Identification at-risk family members
Colonoscopy surveillance
Presymptomatic genetic testing
Risk reducing surgery
Surveillance associated cancers
Chemoprevention studies
Preimplantation genetic testing

Microsatellite instabillity has very rapid progression to cancer

26

Micro-satellite Instability

Condition of genetic hyper mutability (predisposition to mutation) that results from impaired DNA mismatch repair (MMR)

The presence of MSI represents phenotypic evidence that MMR is not functioning normally

27

CAPP2 Aspirin in Lynch syndrome cancer risk

Reduced cancer risk at 55.7 months in carriers of hereditary colorectal cancer with 600 mg of Aspirin per day

28

Hamartomatous polyposis syndromes

Share autosomal dominant pattern of inheritance and are characterised by hamartomatous polyps (tumor-like growth) of the GI tract

Very rare

Peutz Jeghers - LKB1

Juvenile Polyposis - SMAD4(20%) and BMPR1(20%)

Cowden's - PTEN

Hereditary mixed P S - GREM

29

MYH associated polyposis

Autosomal recessive
DNA base excision repair gene
Repairs oxidative damage to DNA
G:C -> A:T transversions
multiple adenomas in bi-allelic genecarriers
Transversions in tumour DNA

30

Genetic pathways to colorectal tumourigenesis

Chromosomal instability (85%)
-APC gene mutations
-Aneuploid tumour

Microsatellite instabillity
- Methylation MLH1 gene promoter
-Diploid tumour

31

Types of genes associated CRC

Growth control
(APC, SMAD4, BMPR1A)

DNA repair
(mismatch repair and base excision repair)

32

Next-generation/ Parallel DNA Sequencing

Detection of single fluorescent bases as they are incorporated into growing DNA strand

Massive parallel sequencing

Long and short reads

Illumina Sequencing by Synthesis (SBS)


Germline whole-genome sequencing
Tumour sequencing
Diagnostic gene panels

33

Oxford-Illumina WGS500 project

Whole genome of adenoma sufferers and CRC sequenced.

10000 coding region variants with potential effect on protein sequence per person
APC x3 and MSH x1

34

POLE and POLD1

Mutation proofread domains
Defect in correction of mispaired bases
Tumours micosatellite stable
Large numbers of base substoitutions

35

Polymerase Proofreading-associated Polyposis (PPAP)

Multiple colorectal adenomas
Early onset CRC
Mutation in the polymerase genes POLE and POLD1

36

The cancer genome atlas

detailed genetic analysis of multiple SPORADIC CRCs

Non-hypermutable cancers (84%)
- Chromosome unstable/aneuploid
<1 mutation/10^6 bases

Hypermutable cancers (16%)
-micro-satellite unstable/ diploid
>100 mutations /10^6 bases

37

Hypermutable Cancers

3/4 hypermethylated
MLH1 promoter methylation
MSI

1/4 POLE mutations
Microsatellite stable

38

Genetic heterogeneity

Intra-tumour heterogeneity and branched evolution revealed by multi-region sequencing

39

Gene Panels

Targeted gene panels are ideal for analyzing specific mutations or genes that have suspected associations with disease

40

Personalised medicine/Immunotherapy

Hypermutable cancers
Somatic mutations lead to multiple new non-self immunogenic antigens
Tumours characterised by lymphocytic inflitrate
Possible targets for immunotherapy

41

Programmed Death 1 Pathway

Th1 lymphocytes cytotoxic immune response to cells displaying 'non-self' antigens

PD-1 immune checkpoint to prevent autoimmunity - Changed in tumour - T cells leave other cells alone - tumours avaoid immune attack

42

PD-1 blockade in MSI tumours

MSI tumours large number of somatic mutations and non-self immunogenic antigens

Pembrolizumab is an anti-programmed death 1 immune checkpoint inhibitor

Assess clinical response MSI colorectal tumours to immunotherapy

Very effective in small group of patients (MSI)

43

Phenotype

The physical characteristics of something living, especially those characteristics which can be seen

The visible characteristics of an organism resulting from the interaction between its genetic makeup and the environment

44

UC

Confined to colon
Superficial
Continuous
Always involves the rectum
M=F
No granulomas
Fistulae and strictures rare because not tansmural

Symptoms
Bleeding
Mucus
Urgency
Diarrhoae

45

Crohns

Anywhere from mouth to anus
Full thickness (transmural)
Patchy (skip lesions)
Often spares the rectum
F>M 1.5:1
Granulomas (60%)
Fistulae and strictures common because of transmural inflammation

Abdominal Pain
Weight Loss
Tiredness/Lethargy
Diarrhoea
Abdominal mass
Rarely blood

46

IBD is variable

Diagnosis is not always reliable

Response to treatment is variable

Severity is difficult to define

Disease extent (25% changes)

Complications are difficult to assess

Patients who end up having a colectomy -> There is a 10% chance in a change in diagnosis

47

Crohn's colitis vs UC

Isolated Crohn's Colitis behaves like UC

Isolated Crohn's colitis is genetically similar to UC
- associated with HLA-DRB1*0103

CD colitis patients with pANCA have a UC-like phenotype

48

Urinary metabonomics in UC and Crohns disease

Differences between CD and UC in Hippurate

Hippurate is linked to intestinal bacterial metabolism

These differences are apparent even between UC and Crohn's colitis

49

Dichotomy between two patient groups for both UC and CD (some respond to treatment others do not)

Corticosteroid therapy in UC
Infliximab in CD

50

Possible phenotypic groups of IBD

Response to treatment
Disease severity
Disease extent
Disease behaviour

51

Why is it important to define phenotype in IBD?

Allows insight into pathogenesis (particularly relating to genetic predisposition)

Allows us to define response to treatment

May allow prognostication

Big problem is phenotype stable?
CD has no stable phenotype ( fluctuates from inflammatory to structuring, to penetrating)

52

Problems with phenotyping in IBD

Defined by humans
The defined phenotypes may not represent biologically important categories
They may change with time

53

Despite the problems with phenotyping in IBD it offers

Improve patient care by offering the right patients the right treatment
Help plan treatment by improving prognostication
Offer insights into disease pathogenesis

54

Vienna and Montreal Classification for CD and UC

Not stable so not that good

55

Aetiology for IBD

Genetic: NOD2, HLA, ATG, I123R

Antibodies:
Anto-saccaromyces cervisiae (ASCA) - Crohn's disease
pANCA - UC

Other theories:
Unsure

56

Methods of investigating aetiology in IBD

Genetic studies:
Association, Linkage (down through generations), GWAS

Studies of bacterial flora:
16S ribosomal subunit RNA typing

Surrogate markers:
Antibodies – ASCA, ANCA, OmpC, Flagellin
Metabonomics

Mechanistic studies:
Il23 pathway, autophagy, NOD2

How do you stratify the results?

57

Phenotype determining Genes is IBD

Do not confer overall disease susceptibility
Do affect the clinical phenotype of the disease
e.g. extra-intestinal manifestations, disease location
May not be found on whole population studies
Detection requires precise clinical characterisation of patients

58

Extra-intestinal manifestations of IBD

Arthritis:
Axial – Ankylosing Spondylitis
Peripheral
Skin:
Erythema nodosum
Pyoderma gangrenosum
Eyes:
Anterior uveitis
Episcleritis/Iritis
Liver:
PSC
Autoimmune hepatitis

59

Linkage Disequilibrium

Genes far apart - Recombination likely -
Genes NOT inherited together

Genes close together - Recombination unlikely - Genes are inherited together = Linkage disequilibrium

60

The effects of linkage disequilibrium

Association studies detect associations not causal relationships

The associations found may be due to another gene located close by (linkage disequilibrium)

Studies of genes around those where associations are found are often worthwhile

61

Erythema Nodosum, AThritis, Uveitis in IBD

These EIM’s are all associated with genes in the HLA region
The genes that determine the individual EIM’s may be in linkage disequilibrium
This may account for the overlapping clinical syndromes seen in practice

62

Clinical applications of phenotyping

So far disappointingly small
Little helps to determine response to treatment
Response to IFX
High CRP
Colonic disease
Non-smoking
Poor prognosis in CD
Early diagnosis
Stricturing disease
Colonic disease

63

Risk factors for disabling disease course for CD

Age onset below 40
Disease location small bowel and colon
Perianal lesions
Requirement for steroid treatemnt with first flare

64

Risk factors for disabling disease course for CD

Age onset below 40
Disease location small bowel and colon
Perianal lesions
Requirement for steroid treatment with first flare

65

The burden of IBD

Lifelong chronic disease, often affecting young people

Affects 1.5 million Americans
and 2.2 million in Europe

66

Changes in global incidence of IBD

Northern Hemisphere:
Higher incidence rates of Crohn's disease in northern latitudes than southern latitudes

Southern Hemisphere:
Opposite with highest incidence in New Zealand

Higher incidence in northern parts of countries: France, Italy, Spain, Portugal, Scotland

67

Increasing Incidence of IBD in the East

Always considered disease of the West

Increasing Incidence of IBD in Africa, Saudi arabia, Korea, Hong Kong

(Increasing incidence in Asia parallels socioeconomic development)

68

IBD Population Movement

Study in Sweden:
First generation immigrants from low incidence countries continue to have low risk compared to native population
Second generation immigrants, risk rises towards that of the native country

Study in Canada:
Younger age at time of arrival to Canada increased risk to that of native country

Indicates that exposure to environment early in life is important

69

Environmental factors influencing IBD

Physical activity
Appendectomy
Smoking
Stress
Vitamin D UV exposure
Hygiene
Diet
Sleep
Medications
Microbiome
Genetic Susceptibillity

70

Smoking and IBD

Protective in UC
Later onset in UC and less need for immunosuppression
Risk of UC increased after 2-5 yrs of smoking cessation and remained elevated for 20 yrs

Damaging in CD
Earlier age of onset and more frequent need for immunosuppression
risk is proportional to the amount smoked and smoking is associated with a more aggressive disease

71

Reasons for IBD and smoking

Although nicotine has been considered a potential putative agent influencing CD and UC risk, cigarettes also are associated with the generation of free radicals and carbon monoxide which may play a mechanistic role.
Smoking may also affect the colonic mucus layer, modify cytokine production, modulate humoral and cellular immunity (innate and adaptive responses), reduce smooth muscle tone and activity, change gut permeability, and affect the microvasculature.

72

Appendicectomy and UC

UC - protective effect

Studies show lower risk of UC in those <20yrs age (in appendicectomy if there is aetiology)

Also showed more limited disease extent, fewer relapses, and less likely to require immunosuppressive therapy

Altered immune response that leads to appendicitis (or results from appendicectomy) is protective against pathogenesis of UC

Appendix has a reservoir of enteric bacteria that regulates host immune response

73

Appendicectomy and CD

Risk Ratio of developing CD is high first year after operation, then drops down to normal rates by 5 yrs.


74

Vitamin D

Increasingly being recognised as important in pathogenesis of CD

Studies have shown
Higher level of Vit D -> reduced risk of CD
No effect on CD
Lower Vit D levels in patients with CD ->increased risk of hospitalisation and surgery

75

Microbiota in gut

Affected by diet, hygiene, and antibiotics

Leads to a dysbiosis

Dysbiosis is a term for a microbial imbalance or maladaptation on or inside the body, such as an impaired microbiota.

76

Diet and Hygiene in IBD

The Hygiene Hypothesis

If raised in a sanitary environment, you are more likely to develop IBD

The Cold chain hypothesis

Rising incidence of CD corresponds to introduction of refrigeration in society
(Excessive host response to bacteria that survive in cold temperatures)

IBD avoid:
Diet
Sugars,
dietary fate, red meat, cheese, dairy, oils, chocolate, fast food, margarine

Should:
Dietary fibre, Fruit, Veggies

77

Microbiota in IBD

Altered composition of gut flora in IBD
-Reduction in microbial diversity
-Reduction in beneficial bacteria (Bacteroides, lactobacilli)
-Increase in pathogenic bacteria (mycobacterium avium paratuberculosis)

78

IL-17-Producing T-Helper Cells
involved in CD

Differentiate from Naive Th0 cells in the presence of Il-1, Il-23, TGF-B in combination with IL-6

TH17 potent effector of inflammation releases Il17 and Il22

Th1 also involved in CD

Th2 involved in UC

T reg cells express FOXP3 -> regulatory no inflammation

79

IL 17 THC

Microbiota important in differentiating what kind of T cells

Study in mice found normally raised mice to have much larger amounts of TH17, germ free mice had very few. When germ free mice where put in normal environment, TH17 started to increase.

FOXP3 was much higher in germ free mice

80

Effect of giving antibiotics to conventionally raised mice at birth and adult

Adult - gram positive antibiotic - significant reduction of TH17 cells (from birth the difference is even greater)

This shows how microbiota influences the immune cells you have

81

Environmental impact differs depending on genetic predisposition

Lots of genes causing
NOD2 most important

These basic genes can affect the immune system in various ways:
1. Barrier function
2. Innate immunity
3. Interaction between innate and adaptive immunity

82

ATG16L1 gene affects autophagy

Aotophagy:

Essential process for maintenance of cellular homeostasis

Damaged cellular components are targeted by protein adaptors
Engage autophagy machinery
Engulf waste and deliver to lysosome for degradation

Very important in IBD, NASH, Pancreatitis


Genetics affects autopghagy
all along

83

Aberrant autophagy

defective intestinal homeostasis

84

Environmental effect on gene function - ATG16L1

Created mice with hypomorphic ATG16L1
(abnormality same as in CD who are homozygous for that alleles)

Wild mice

ATG16L1 (abnormal)

when norovirus given - destroyed - effect of virus on genetically susceptible mice is very different to the effect on wild mice

Environmental facyor is different if you are genetically different

Hypomorphic mice - showed improvement with IBD treatment

85

Bioinformatics

Science is producing vast quantities of data about biological systems

Bioinformatics combines:

Organising this information WITH Understanding how it is related using a range of mathematical, statistical, and computational techniques