BS961

1
BS961

• BS961 Session 6 Transcriptomics/ Human Genetics

2
Objectives

• Explain the term transcriptome and describe the
  method and applications of microarray technology.
• Evaluate the range of genetic variation found in
  human populations.
• Explain the molecular approaches used to study
  human genetic variation.
• Describe and discuss the types of mutations
  underlying examples of human genetic diseases.
• Evaluate the methods and applications of gene
  testing in families and populations.

3
References Strachan and Read, Human Molecular
Genetics 3 Brown Genomes 3
4
Gene expression

• We have seen how we can mine the information
  generated through genome projects- finding
  possible functions for newly-identified genes.
• However, even if every gene in an organism can be
  identified and assigned a function, we still need
  to understand how the genome as a whole operates
  within the cell. The study of this can be called
  Functional Genomics.

5
Functional Genomics

• Which genes are transcriptionally active, which
  proteins (and variant forms of these proteins)
  are present, how do these interact and what
  effect is there on the metabolism of the cell?
• The questions are being addressed through new
  technologies.

6
Some important terms

• Genome
• The store of genetic material in an organism
  needed to construct and maintain a living example
  of that organism- most genomes are DNA, but
  several viruses have an RNA genome

7
(No Transcript)
8
Some important terms

• Transcriptome
• is the collection of RNA molecules derived from
  protein-encoding genes whose expression is
  required by the cell at a particular time
• transcriptomics is the name given to the analysis
  of the transcriptome

9

• to gain a complete picture of gene expression it
  is essential to also study proteins and their
  interactions
• Metodi Metodiev
• later in module
• Proteome
• Interactome

10
Transcriptome

• Analysed by cDNA or oligonucleotide arrays
• These can represent all the genes in an organism
• RNA from the cells to be studied is used to make
  labelled cDNA
• this is hybridised to the array to reveal how
  much is present

11
(No Transcript)
12
Transcriptomics

• By comparing the hybridization patterns, you can
  compare gene expression in two samples (diseased
  and healthy tissue or compare brain and muscle,
  for example)
• If the genome sequence is known, oligonucleotides
  representing each gene can be used DNA chips

13
(No Transcript)
14
(No Transcript)
15
(No Transcript)
16
(No Transcript)
17
(No Transcript)
18
(No Transcript)
19
(No Transcript)
20
(No Transcript)
21
(No Transcript)
22
(No Transcript)
23
(No Transcript)
24
Transcriptomics

• If the genome sequence is known, oligonucleotides
  representing each gene can be used DNA chips
• the DNA chips have large numbers of high-density
  oligonucleotides built up on the surface

25
Oligonucleotide array- chip
26
light catalyses addition of the next
nucleotide EXCEPT where chip is shielded
labelled RNA samples are hybridised to chips
and the signals read using fluorescence/laser
scanning microscopy
27
comparisons of expression Example 5 genes (A-E)
during a timecourse to measure expression at 1-7
hours statistical analysis of similarity
represented by the dendrogram
28
Applications of transcriptomics

• Understanding differences in gene expression
• in different tissues
• at different developmental stages
• in disease- both infectious and eg. cancer

29
human transcriptome analysis chromosome
11 length of blue bar corresponds to level of
expression (red off scale) boxed region
differences in expression in normal and cancerous
breast tissue
30
Whether tumours exhibit a luminal or
myoepithelial/basal phenotype has been correlated
with prediction and prognosis in breast cancer.
Global transcriptomes of normal myoepithelial and
luminal epithelial cells were, therefore,
compared to identify all transcripts that were
differentially expressed in these normal cell
types.
31
(No Transcript)
32
http//breast-cancer-research.com/content/8/5/R56
33

• microarray analysis in cancer
• diagnosis
• accurately determining the type of cancer and
  stage of disease
• treatment
• fingerprint to determine optimal treatment
  regimes will the tumour cells respond? to which
  drugs?

34
Studying genetic variation in man

35
Terminology of genetic variation

• Different variants of a gene are called alleles
• Genes showing variation are called polymorphic
• a gene with multiple alleles that can be
  distinguished (e.g. blood groups) can be used in
  gene mapping
• these are known as genetic markers

36
Development of human genetic markers

• Blood groups 1910-1960
• Serum proteins 1960-1975
• Number in genome 20-30 (very few)
• Fresh blood needed
• not used now

37
Development of human genetic markers

• HLA types 1970-
• Number in genome- 1 linked set
• Only 1 location
• used for transplantation or disease association
  with immune system

38
Development of human genetic markers

• DNA RFLP 1975-
• Number in genome gt105
• Only 2 alleles (present or absent)
• not only genes, other places in genome too

39
RFLPs

• Restriction enzymes cut DNA at specific places
  depending on the sequence
• Variation in DNA sequence (single base changes)
  leads to loss or gain of a restriction enzyme
  site

40
the polymorphic restriction enzyme site is
present in allele 1 and absent in allele 2
41
detection of restriction enzyme
polymorphism Southern blotting
42
detection of restriction enzyme polymorphism by
PCR primers flank the polymorphic site
43
Sickle cell anaemia disease mutation is an RFLP
44
SSLP

• Simple Sequence Length Polymorphism
• Variation in length of a repeat
• number in genome gt105
• many alleles
• Allele 1 TCTGAGAGAGGC
• Allele 2 TCTGAGAGAGAGAGGC
• Allele 2 is longer and gel electrophoresis of PCR
  product can distinguish the 2 alleles

45
(No Transcript)
46
PCR using primers flanking the polymorphism
detection using gel or capillary electrophoresis
47
Types of SSLP

• Minisatellites (DNA fingerprinting- length 25 bp)
  
• (Not randomly distributed in genome so less
  useful, also less feasible to automate typing)
• Microsatellites (length 2-4 bp)

48
Development of human genetic markers

• SNP 1998-
• Single nucleotide polymorphism

49

• Single nucleotide polymorphism
• limited variation-usually only 2 alleles
• (in theory could have 4)
• advantage of very large number in genome gt106
• Typed on a large scale by chip technology (fast,
  cheap, reproducible)

50
(No Transcript)
51
match gives a signal mismatch no hybridisation
no signal
52
other methods are based on using a SNP at the end
of the oligonucleotide probe (or primer)
53
ligation one long fragment no ligation two
short fragments
detect by capillary electrophoresis
54
Amplification Refractory Mutation System match-PC
R product no match-no product
55
SNP detection- can be automated and high
throughput

• Many 100,000s of probes can be synthesised on a
  DNA chip
• The sample to be probed is allowed to bind
• The signal from the chip is read and decoded by a
  fluorescence microscope detector attached to a
  computer

56

• SNPs
• allow very detailed genetic maps to be developed
• enable population studies of common diseases
  which have a genetic component

57

• Genetic diseases, inheritance and genetic testing

58
Types of Mutation

• 1. Single base substitutions
• Silent- change codon NOT amino acid
• Mis-sense- changed amino acid
• Nonsense- stop codon introduced
• Intron splice sites

59
normal splice site
60
splice site mutated
intron sequence retained coding sequence
disrupted frameshift likely
61
Types of Mutation

• 2. addition or removal of bases
• Deletions
• Insertions and duplications
• both can cause
• Frameshifts- short form of protein made

62
Frameshifts
normal protein loss/gain of a base in a single
codon
reading frame disrupted All codons after the
mutation are misread
63
Frameshifts
64
mutations in regulatory and promoter sequences
can affect gene expression
65
Mutations can be grouped into two types

• Loss of function
• Gain of function

66
heterozygous for loss of function allele
usually leads to recessive inheritance
67
Loss of function

• Point mutations- e.g. cystic fibrosis ?F504,
  G551D
• Deletions e.g. Duchenne muscular dystrophy

68
Gain of Function

• Over expression e.g. Charcot-Marie-Tooth disease
  (neuropathy)
• Novel substrate a-1 antitrypsin Pittsburgh allele
  (mutation causes change from elastase to thrombin
  recognition, leading to a bleeding disease)

69
TRINUCLEOTIDE REPEAT EXPANSION

• Neurodegenerative disorders caused by CAG
  expansion in coding region
• e.g. Huntingtons disease (HD)
• polyglutamine tracts seen in Huntingtin
• (5 similar disorders autosomal dominant, late
  onset, progressive diseases showing dementia,
  ataxia, chorea but different cells affected in
  each)

70
(No Transcript)
71
TRINUCLEOTIDE REPEAT EXPANSION

• Fragile X is the commonest genetic cause of
  mental retardation
• non-staining gap seen on X chromosome
• Fragile sites caused by CCG expansion in promoter
  /5UTR
• FMR1 gene silenced by methylation of promoter

72
Autosomal dominant
Every generation ? Has 1 in 2 (50) chance of
being affected
73
Autosomal recessive
Autosomal recessive
Affected children of carrier (unaffected)
parents ? Has 1 in 2 (50) chance of being
affected
74
Autosomal dominant
X-linked recessive
Female carriers have affected sons ? 1 in 4
chance OFFSPRING affected 1 in 2 chance male
child affected (1 in 2 chance female child
carrier)
75
Methods and Examples

• (IMPORTANT these tests can only be used where
  there are a limited number of known mutations or
  the mutation in the family has been defined)
• Note the links to the last lecture
• 1. Presence or absence of a restriction enzyme
  site (e.g. Sickle cell anaemia) detected by size
  of band on gel- rare to find one corresponding to
  disease allele

76
Sickle cell anaemia disease mutation is an RFLP
77
Methods and Examples

• 2. PCR
• Allele specific PCR (primer designed so it will
  only bind to the mutant sequence) e.g. cystic
  fibrosis
• Deletion detection by PCR (amplify individual
  exons check for presence of all exons)
• e.g. Duchenne muscular dystrophy

78

• Multiplex deletion screen for dystrophin
• samples from 10 patients with Duchenne muscular
  dystrophy
• PCR primers designed so that each exon gives a
  different sized PCR product

79
Interpretation solid lines- exons definitely
deleted dotted lines- deletion may include
untested exon
(samples 7 and 9 - deletions of exons not
examined here)
80
Methods and Examples

• 2.PCR
• Repeat expansion detection (amplify region
  containing repeat and look at size)
• e.g. Huntingtons disease

81
Huntington disease Fragment of gene containing
the (CAG)n repeat amplified by PCR separated by
gel electrophoresis
82
(No Transcript)
83
(No Transcript)
84
Prenatal diagnosis of an affected fetus
85
Methods and Examples

• 3. Southern blot- laborious, expensive, lots of
  DNA required
• Used for fragile X disease (repeats too large to
  amplify by PCR)
• 4. Sequencing (time consuming)
• New methods being developed based on GENE CHIPS,
  potentially very quick and high-throughput

86
Applicability

• These methods are commonly used where there is
  known to be a genetic disease in a family.
  Testing is accompanied by genetic counselling.
• Prenatal diagnosis parents may decide on
  elective abortion
• Nowadays increasingly pre-implantation embryo
  testing, with IVF
• Some populations may use testing to determine
  carrier status before marriage (Tay-Sachs in
  Jewish community) or before having children

87
Requirements for population screening

• A positive test must lead to some useful action
• The whole programme must be socially and
  ethically acceptable
• The test must have high sensitivity and
  specificity
• The benefits must outweigh its costs

88
(No Transcript)
89
Some Currently Available DNA-Based Gene Tests
Alpha-1-antitrypsin deficiency (AAT emphysema
and liver disease) Amyotrophic lateral sclerosis
(ALS Lou Gehrig's Disease progressive motor
function loss leading to paralysis and death)
Alzheimer's disease (APOE late-onset variety
of senile dementia) Ataxia telangiectasia (AT
progressive brain disorder resulting in loss of
muscle control and cancers) Gaucher disease (GD
enlarged liver and spleen, bone degeneration)
Inherited breast and ovarian cancer (BRCA 1 and
2 early-onset tumors of breasts and ovaries)
Hereditary nonpolyposis colon cancer (CA
early-onset tumors of colon and sometimes other
organs) Central Core Disease (CCD mild to
severe muscle weakness) Charcot-Marie-Tooth
(CMT loss of feeling in ends of limbs)
Congenital adrenal hyperplasia (CAH hormone
deficiency ambiguous genitalia and male
pseudohermaphroditism) Cystic fibrosis (CF
disease of lung and pancreas resulting in thick
mucous accumulations and chronic infections)
Duchenne muscular dystrophy/Becker muscular
dystrophy (DMD severe to mild muscle wasting,
deterioration, weakness) Dystonia (DYT muscle
rigidity, repetitive twisting movements) Emanuel
Syndrome (severe mental retardation, abnormal
development of the head, heart and kidney
problems) Fanconi anemia, group C (FA anemia,
leukemia, skeletal deformities) Factor V-Leiden
(FVL blood-clotting disorder) Fragile X
syndrome (FRAX leading cause of inherited mental
retardation) Galactosemia (GALT metabolic
disorder affects ability to metabolize galactose)
Hemophilia A and B (HEMA and HEMB bleeding
disorders) Hereditary Hemochromatosis (HFE
excess iron storage disorder) Huntington's
disease (HD usually midlife onset progressive,
lethal, degenerative neurological disease)
Marfan Syndrome (FBN1 connective tissue
disorder tissues of ligaments, blood vessel
walls, cartilage, heart valves and other
structures abnormally weak) Mucopolysaccharidosis
(MPS deficiency of enzymes needed to break down
long chain sugars called glycosaminoglycans
corneal clouding, joint stiffness, heart disease,
mental retardation) Myotonic dystrophy (MD
progressive muscle weakness most common form of
adult muscular dystrophy) Neurofibromatosis type
1 (NF1 multiple benign nervous system tumors
that can be disfiguring cancers)
Phenylketonuria (PKU progressive mental
retardation due to missing enzyme correctable by
diet) Polycystic Kidney Disease (PKD1, PKD2
cysts in the kidneys and other organs) Adult
Polycystic Kidney Disease (APKD kidney failure
and liver disease) Prader Willi/Angelman
syndromes (PW/A decreased motor skills,
cognitive impairment, early death) Sickle cell
disease (SS blood cell disorder chronic pain
and infections) Spinocerebellar ataxia, type 1
(SCA1 involuntary muscle movements, reflex
disorders, explosive speech) Spinal muscular
atrophy (SMA severe, usually lethal progressive
muscle-wasting disorder in children) Tay-Sachs
Disease (TS fatal neurological disease of early
childhood seizures, paralysis) Thalassemias
(THAL anemias - reduced red blood cell levels)
Timothy Syndrome (CACNA1C characterized by
severe cardiac arrhythmia, webbing of the fingers
and toes called syndactyly, autism)
Some Currently Available DNA-Based Gene Tests
Alpha-1-antitrypsin deficiency (AAT emphysema
and liver disease) Amyotrophic lateral sclerosis
(ALS Lou Gehrig's Disease progressive motor
function loss leading to paralysis and death)
Alzheimer's disease (APOE late-onset variety
of senile dementia) Ataxia telangiectasia (AT
progressive brain disorder resulting in loss of
muscle control and cancers) Gaucher disease (GD
enlarged liver and spleen, bone degeneration)
Inherited breast and ovarian cancer (BRCA 1 and
2 early-onset tumors of breasts and ovaries)
Hereditary nonpolyposis colon cancer (CA
early-onset tumors of colon and sometimes other
organs) Central Core Disease (CCD mild to
severe muscle weakness) Charcot-Marie-Tooth
(CMT loss of feeling in ends of limbs)
Congenital adrenal hyperplasia (CAH hormone
deficiency ambiguous genitalia and male
pseudohermaphroditism) Cystic fibrosis (CF
disease of lung and pancreas resulting in thick
mucous accumulations and chronic infections)
Duchenne muscular dystrophy/Becker muscular
dystrophy (DMD severe to mild muscle wasting,
deterioration, weakness) Dystonia (DYT muscle
rigidity, repetitive twisting movements)
90
Emanuel Syndrome (severe mental retardation,
abnormal development of the head, heart and
kidney problems) Fanconi anemia, group C (FA
anemia, leukemia, skeletal deformities) Factor
V-Leiden (FVL blood-clotting disorder) Fragile
X syndrome (FRAX leading cause of inherited
mental retardation) Galactosemia (GALT
metabolic disorder affects ability to metabolize
galactose) Hemophilia A and B (HEMA and HEMB
bleeding disorders) Hereditary Hemochromatosis
(HFE excess iron storage disorder) Huntington's
disease (HD usually midlife onset progressive,
lethal, degenerative neurological disease)
Marfan Syndrome (FBN1 connective tissue
disorder tissues of ligaments, blood vessel
walls, cartilage, heart valves and other
structures abnormally weak) Mucopolysaccharidosis
(MPS deficiency of enzymes needed to break down
long chain sugars called glycosaminoglycans
corneal clouding, joint stiffness, heart disease,
mental retardation) Myotonic dystrophy (MD
progressive muscle weakness most common form of
adult muscular dystrophy)
91
Neurofibromatosis type 1 (NF1 multiple benign
nervous system tumors that can be disfiguring
cancers) Phenylketonuria (PKU progressive
mental retardation due to missing enzyme
correctable by diet) Polycystic Kidney Disease
(PKD1, PKD2 cysts in the kidneys and other
organs) Adult Polycystic Kidney Disease (APKD
kidney failure and liver disease) Prader
Willi/Angelman syndromes (PW/A decreased motor
skills, cognitive impairment, early death)
Sickle cell disease (SS blood cell disorder
chronic pain and infections) Spinocerebellar
ataxia, type 1 (SCA1 involuntary muscle
movements, reflex disorders, explosive speech)
Spinal muscular atrophy (SMA severe, usually
lethal progressive muscle-wasting disorder in
children) Tay-Sachs Disease (TS fatal
neurological disease of early childhood
seizures, paralysis) Thalassemias (THAL anemias
- reduced red blood cell levels) Timothy
Syndrome (CACNA1C characterized by severe
cardiac arrhythmia, webbing of the fingers and
toes called syndactyly, autism)
92
Breast cancer

• http//ornl.gov/sci/techresources/Human_Genome/med
  icine/genetest.shtml
• http//www.cancer.gov/cancertopics/factsheet/Risk/
  BRCA

93

• Summary of the key points
• BRCA1 and BRCA2 are human genes that belong to a
  class of genes known as tumor suppressors.
  Mutation of these genes has been linked to
  hereditary breast and ovarian cancer
• A woman's risk of developing breast and/or
  ovarian cancer is greatly increased if she
  inherits a deleterious (harmful) BRCA1 or BRCA2
  mutation. Men with these mutations also have an
  increased risk of breast cancer.
• Both men and women who have harmful BRCA1 or
  BRCA2 mutations may be at increased risk of other
  cancers.
• Genetic tests are available to check for BRCA1
  and BRCA2 mutations. A blood sample is required
  for these tests, and genetic counseling is
  recommended before and after the tests.

94

• If a harmful BRCA1 or BRCA2 mutation is found,
  several options are available to help a person
  manage their cancer risk
• Federal and state laws help ensure the privacy of
  a persons genetic information and provide
  protection against discrimination in health
  insurance and employment practices
• Many research studies are being conducted to find
  newer and better ways of detecting, treating, and
  preventing cancer in BRCA1 and BRCA2 mutation
  carriers. Additional studies are focused on
  improving genetic counseling methods and
  outcomes. Our knowledge in these areas is
  evolving rapidly
• Also
• http//www.cancerhelp.org.uk/type/breast-cancer/ab
  out/risks/breast-cancer-genes