TOPIC 11 - genetics Flashcards
what are the common factors in diseases caused more by genetics?
- rare
- genetics simple
- unifactorial
- high recurrence rate
what are the common factors in diseases caused by more by environment?
- common
- genetics complex
- multifactorial
- low recurrence rate
what are fully penetrant conditions?
environmental factors have no effect, mutations cause all problems
- usually due to single gene defect
what are low penetrance conditions?
genes have a small influence part, in addition to other genetic and environmental factors, in determining disease susceptibility
- usually due to multifactorial genes
give an example of multifactorial condition
MS- multiple sceleorsis
where genetic factors play a major part in determining susceptibility, but each individual factor has a very low penetrance
what are the 5 classifications of genetic disorders?
- multifactorial
- single gene
- chromosomal- chromosomes being lost and inserted onto other chromosomes
- mitochondrial (inheritance)
- somatic mutations (cancer)
what is a single gene/monogenic Mendelian disorder
mutations in single genes (often causing loss of function)
- dominant/recessive pedigree patterns (Mendelian inheritance)
- can affect structural proteins, enzymes, receptors, transcription factors
what are multifactorial diseases
variants in genes causing alteration of function and the effect of the environment
- “environmental” influences (eg. drugs, infections) + genetic predisposition = susceptibility to a disease
- variants in genes cause alteration of function
- one organ system affected
what are ‘variants’ in genes
2 or more types of forms in genetic variant in polygenic inheritance
- can change phenotypes and cause 2 different types of functions
what are chromosomal disorders?
- lots of info is gained or lost
= chromosomal imbalance causes alteration in gene dosage
-thousands of genes may be involved
multiple organ systems affected at multiple stages in gestation. - usually de novo (trisomies, deletions, duplications)
- in rare cases, can be inherited (translocations)
what is down syndrome/ trisomy 21
- too much gene dosage for chromosome 21
- a syndrome so collection of features
what are the phenotypes for people with down syndrome
- round face
- protruding tongue
- upslanting palpebral fissures
- epicanthic folds
- developmental delay
what are the ultrasound features of someone with trisomy 21
- short femurs
- nuchal translucency
- echogenic bowel
- choroid plexus cyst
- sandal gap, single palmar crease
what are micro deletions in chromosomes
- chromosomal abnormality
- chromosomal region is lost
how are microdeltions in chromosomes detected
too small to be observed microscopically
•identified by use of specific molecular cytogenetic techniques
what is DiGeorge syndrome
•DiGeorge syndrome- microdeletion of chromosome 22 : small mouth, prominent nose, congenital heart defects
what causes Williams-Beuren syndrome-WBS
microdeletion (about 26 genes from the long arm of chromosome 7)
what are the phenotypic traits of WBS
bright eyes stellate irides wide mouth upturned nose long philtrum flattened nasal bridge
what are the personality traits and congenital malformations of WBS
heart defects (heart murmurs, supravalvular aortic stenosis),
typical facies
overly sociable ("cocktail party" type personality)what are single gene disorders also known as
mendelian genetics
what is a dominant single gene disorder
heterozygous with one copy of alterd gene are affected
what is a recessive single gene disorder
homozygous with 2 copies of altered gene are affected
what is a X linked single gene disorder
males with one copy of altered gene on X chromosome affected
what kind of disorders pose a high risk to relatives?
single gene disorders
how do single gene disorders occur
spontaneously or from family inheritance
-some isolated cases due to new dominant mutations
what are symptoms of huntingtons disease
- Ataxia- inability to have voluntary movement or motion
- advanced stages = involuntary movements of head and face
NB- loss of degenerative tissue in white matter
what are the 2 diseases associated with Cholesterol deposition in patients heterozygous for familial hypercholesterolemia
- Tendon xanthomata,(deposition of yellow bodies with lipids and cholesterol in them)
- corneal arcus
what kind if disease is CF
autosomal recessive condition
what is required for CF patients
frequent hospital admissions/monitoring
physiotherapy
constant medications
how is CF treated
treatment with enzymes and nebulisers
how do we test for Duchenne muscular dystrophy
- do biopsy
- use brown stain- Gomori trichrome stain
- Normal muscle shows a regular architecture of cells with dystrophin (brown stain) on all the outer membranes
- if no brown staining= muscular dystrophy
what is autosomal dominant inheritance?
heterozygous with one copy of mutated gene are affected
-onset usually later on in life
what is autosomal recessive inheritence
homozygotes with 2 copies of mutated gene are effected
-loss of function, more severe, present early on,eg.CF
what is X linked inheritence
males with one copy of mutated gene on X chromosome are affected
what is mitochondrial inheritance?
Mutations in mitochondrial genome
During fertilisation all from oocyte = maternal disease
what do mitochondrial diseases cause in general
Mutations- defects in energy usage or productions
present in brain or eyes early on
what is Leber hereditary optic neuropathy
-caused by mitochondrial disfunction
-reduction in vision
(eyes are susceptible tissue and vunerable to environment and genetic changes)
In an autosomal recessive disorder, what is the chance that healthy sister is carrier if brother has disease?
2/3
what happens in dominantly inherited familial cancer syndromes?
both alleles of a gene become inactivated in a particular somatic cell leading to loss of control of growth and unchecked cell proliferation
are cancers usually sporadic or familial
sporadic
in multifactorial inheritance does the effect of each gene have a major impact?
no- effect of each gene is small as effects are polygenic (+ environment)
how many organ systems are affected in multifactorial inheritance?
one organ system
what Q are asked in clinical genetics?
- What? - establishing the diagnosis- often initial diagnosis and then another diagnosis to confirm
- How/Why?- determining the mode of inheritance
- Again? - calculating the risk-will this happen to another child
- Choices? - discussing the options, treatment? Therapeutic intervention? Prognosis?
how do you make a diagnosis in clinical genetics
-History
-Family- happened in family before
-Medical- make medical diagnosis
-Examination
-Investigations- testing hypothesis
-Chromosomes- testing chromosomes
-DNA
Biochemical- )cheap)
in pedigree diagrams what is the symbol for a male
square
in pedigree diagrams what is the symbol for a female
circle
in pedigree diagrams what is the symbol for an unaffected individual
empty sqaure/circle
in pedigree diagrams what is the symbol for a affected individual
filled in symbol
in pedigree diagrams what is the symbol for a dead person
diagonal line through symbol
in pedigree diagrams what is the symbol for a miscarrge
triangle
what extra info could you use on a pedigree diagram
• record names, dates of birth, and maiden names
what kind of inheritance disorders are miscarriages and still births more common in
chromosomal abnormalities
what is consanguinity
being related
in pedigree diagrams what is the symbol for a person whos sex is unkown
diamond
in pedigree diagrams why would you draw a double line
union of consanguineous couple
in pedigree diagrams what is the symbol for stillborn of unknown sex
diamond with ‘SB’ written by the side
in pedigree diagrams what is the symbol for a spontaoues abortion
triangle
in pedigree diagrams what is the symbol for a theraputic abortion
triangle with a line through it
what is Genetic heterogeneity
- deafness
- AD/AR/X linked/mitochondrial= many genes mutated giving rise to same consequence- deafness
what does penetrance means in terms of disease
how severe it is in individual (eg. different severities of huntingtons disease)
what does variable expressivity mean in terms of disease
how expressive it is
(eg. different expresivities of neurofibromatosis)
when the phenotype is expressed
what is NF ,neurofibromatosis
Mutations in NF1
in germ line= huge range symptoms
in somatic cells= cancer
how do we diagnose NF type 1
checking for the characteristic symptoms of the condition
2 or more of the following symptoms:
-six or more café au lait spots/pigmentation spots or (coffee-coloured skin patches) that are larger than 5mm in children, or 15mm in adults
- freckles under the arms or around the groin
- two or more neurofibromas (bumps on or under the skin), or one plexiform neurofibroma (a neurofibroma that develops where multiple branches of nerves come together)
- a tumour on the optic nerve (an optic glioma), which rarely causes symptoms or affects sight
t-wo or more tiny brown spots in the iris (the coloured part of the eye). These are known as Lisch nodules
- bone defects, such as bowing of the lower leg
- a family history of NF1
do we get a wide range of phenotypes with polygenic disorders
range of phenotypes associated with certain disease
some genes can cause more or less expressivity
same gene defect and can get different phenotypes
what are symptoms of huntingtons disease?
- Early signs and symptoms: irritability, depression, small involuntary movements, poor coordination, and trouble learning new information or making decisions
- involuntary jerking or twitching movements known as chorea- movements more pronounced as disease progresses
- changes in personality and a decline in thinking and reasoning abilities
when does huntingtons disease begin to show?
30-40s
usually live about 15 to 20 years after signs and symptoms begin
what happens in the genome with HT disease when its inherited
altered HTT gene is passed from one generation to the next, the size of the CAG trinucleotide repeat often increases in size. A larger number of repeats is usually associated with an earlier onset of signs and symptoms. This phenomenon is called anticipation
what is gentic councelling
process of communication and education which addresses concerns relating to the development and/or transmission of a hereditary disorder
what do we (as doctors) consider and talk about in genetic councelling
•non-directive •non-judgemental •bereavement choices and options •carrier testing •presymptomatic diagnosis •prenatal diagnosis •risk calculations (Mendelian, empirical or Bayesian?)
why might screening of the whole population be justifiable?
- if intervention available e.g. PKU (5 – 10 live births per year)
- if early diagnosis reduces morbidity or mortality e.g. FH
what is amniocentesis
- sample taken from amnion layer
- later stage of pregnancy
- look at cells shed from foetus in amnion for mutations
what are the complications with amniocentesis
- very invasive
- professional must be very experienced
- requires 2 professionals
- risk of miscarriage
what 2 ways do we test for mutations in a foetus
chorionic villus sampling
amniocentesis
what happens in G0 stage of cell cyle
cellular contents excluding the chromosomes are duplicated
what happens in the S phase of cell cyle
each of the 46 chromosomes is duplicated by the cell
what happens in the G2 stage of cell cyle
any errors in chormosme duplication are repaired
what are the last 2 stages of the cell cycle
- mitosis
- cytokinesis
what happens in prophase of mitosis
chromatin condenses into chromosomes
what happens in metaphase stage of mitosis
nuclear envelope disappears chromosmes align at equatorial plate
what happens in anapahse stage of mitosis
sister chromatids seperate
centromeres divide
telophase/cytokinseasis?
chromatin expands
cytoplasm divides
what does ‘inter’ mean
between
what does ‘pro’ mean
before
what does ‘meta’ mean
after
beyond
what does ‘ana’ mean
up
what does ‘telo’ mean
far off
distant
why does meiosis happen
-reduction division to 23 chromosomes per gamete so we dont get anaploidy
-ensures that every gamete is genetically unique (reassortment of genes) by:
crossing-over
independent segregation of chromosomes
what is the basic mechanism of meosis
- each homologue (“chromosome”) replicates to give two sister chromatids
- the maternal and paternal homologues pair together
- exchange of material between non-sister chromatids by crossing-over (recombination)
- two successive cell divisions producing four daughter cells
what is chiasma
visible cytologically
are the physical manifestations of crossing-over
in oogenesis how many of 4 daughter cells produces a mature oocyte
only one
other form polar bodies
does crossing over always happen correctly
Sometimes recombination between illegitimate chromosomes – between non homologous chromosomes
-chromosomes are similar but not identical
what is the mechanism of crossing over
- a homologous pair of paternal chromosomes
- during meiosis 1 each chromosome duplicates producing 2 sister chromatids
- crossing over happens
- in meiosis 2 they seperate
all chromosomes always condensed?
- no chromosomes only condensed during metaphase stage
–only time in the cell cycle when the chromosomes are visible
–DNA has already been copied (ie there is twice as much DNA in the cell as there is at interphase)
what do we stain chromosomes with
trypsin digestion of chromosomes followed by DNA staining with Giemsa
= get bright field G banding
how are chromosomes sorted?
between their banding and size
what does the centromere do?
jpoins
drags the chromatids apart in metaphase
what do the dark bands show + features?
- contain condensed chromatids (pick up more dye)
- G bands
- replicate late
- AT-rich
what are features of the light band
- replicate early in S phase
- less condensed chromatin
- transcriptionally active gene
- GC-rich
what is at the top and bottom of a chromosome and whats its function
teleomere:
DNA and protein cap ensures replication of the tip and tethers to nuclear membrane
what is the p arm of the chromosome
short arm
‘petit’
what is the q arm of the chromosome
long arm
what is a chromosome
very long continuous piece of DNA,1.7 meters: contains genes in a linear order
other than genes what are chromosomes made out of
RNA, histone proteins and non-histone proteins as packaging
what do chromosome abnormalities cause?
- altering the amounts of products of the genes involved
- altered amounts may cause anomalies directly or may alter the balance of genes acting in a pathway
–often affect many organ systems at once
whats trisomies
3 copies of gene
what is monosomy
one copy of gene
what does de novo mean
spontaneous events that happen in the germ line
what kind of mutations usually cause numerical abnormalities in the chromosomes
de novo/ spontaeous mutations
= non disjunction:
aneuploidy monosomy
polyploidy
what kind of mutations usually cause structural abnormalities in the chromosomes
also usually de novo
but can be inherited
= translocations
=repetitive DNA sequences
examples of structural abnormalitie diseases in chromosomes
translocations: reciprocal Robertsonian (centric fusion)
important in repetitive DNA sequences
deletions
duplications
inversions
what is mosaicism?
different cell lines (occurs post-zygotically during mitosis)
can get diff cell linages and constitutions within same developing embryo
What is karyotyping?
key physical landmarks are used to distinguish the chromosomes from each other
what does a karyotype represent?
cytogentic characteristics of the individual person and is an arrangement of the chromosomes in descending order of size
what number chromosome is the largest?
1
how is each pair of chromosome distinguished
individual banding pattern
what is down syndrome a result of?
sporadic de novas
what type of cells do you use for karyotyping?
- get actively dividing cells (eg. tumour cells, tissue culture cells, gonadal tissue, bone marrow)
- most observations done on small B lymphocyte but special techniques needed as these normally dividing cells
- need to stop cells dividing in M phase
what is the most widely used banding technique?
G-banding or Giemsa banding
what are the 2 types of chromosome abnormality we can have?
can be of either number (aneuploidy) or structure
what 2 main features do the specimens have that are used for chromosome abnormality?
- specimens contain spontaneously proliferating cell
(ed. bone marrow, lymph nodes, solid tumours, chorionic villi ) - specimens routinely cultured in the laboratory
( blood lymphocytes, tissue biopsies/fibroblasts from skin, amniotic fluid samples, long term CVS)
how long are lymphocytes cultures for?
3 days
how long are amniotic cells, fibroblasts, CVS, and other fetal tissues are cultured for?
10 days
what does G banding involve?
- G-banding involves trypsin digestion of chromosomes followed by DNA staining with Giemsa; G bands stain darkly with Giemsa
- Stop cells dividing in M phase
what can we use G banding for?
identifying every chromosome
how are chromosomes classified?
topography (shape and size)
what factors effect chromosome classification?
- size
- centromere position (telocentric, acrocentric, metacentric)
- chromomere and heterochromatin patterns
- high resolution band patterns produced by different stains
how are genes highlighted?
by DNA probes using fluorescence in situ hybridisation
- only used for specific test
how does fluoresce in situ hybridization-FISH work?
- all about binding of DNA to other DNA strand
- put chromosome on microscopic slide
- denature a bit of your DNA molecule to separate the strand by using alkali or heat
- meanwhile make complementary probe labelled with fluorescent marker
- hybridise probe to denatured chromosome
- fluorescent strand binds to DNA target with H bonding (fl
at what stage of the mitosis is the chromome used in FISH extracted?
metaphase
so there are 2 chromatids
what are olfactory receptor homologues?
- subtelomeric (pseudogenes)
- there are many clusters of FISH probes for olfactoey receptor homologues in the genome
- these are fluroscent R banding
how is FISH used for diagnosis of microdeletion syndrome Williams Beuren syndrome- WBS
- marker on chromosome specific for chromosome 7
- chromosome detects specific analoges from mum and dad
- sequence of FISH specific to microdeletion identifies loss of chromosome on that chromosome
what do we use FISH for
confirm diagnosis of microdeltions not make a new diagnosis
identify numerical and structural abnormalities
what is spectral karyotyping (SKY)/chromosome painting?
Spectral karyotyping and multicolour-FISH paint each human chromosome in one of 24 colours:
- labelling of individual chromosome = painting probes using various combinations of flurochromes
- add Cot-1 DNA ( which is enriched in repetitive sequences)to probe mixture with differently labelled chromosome painting probes
- by binding to repetitive sequences in fluorescently tagged probes = suppresses their hybridisation to target chromosomes
- metaphase chromosome prep
- hybridise, detect and analyse using interferometer or multiple flurencesnt dyes
what is spectral karyotyping (SKY)/chromosome painting used for?
When looking a heteroploidy in cancers – important way for diagnosis
what does FISH analyse?
chromosomes at the DNA or gene level of resolution-not looking at overall banding, patterns/histones ect
on chromosomes at which point in mitosis cell cycle is FISH used?
performed on dividing (metaphase) and non-dividing (interphase) cells
what are the 3 types of FISH probe
- repetitive sequences, including those at the centromere of a chromosome;
- DNA segments that will bind to and cover the entire length of a particular chromosome (“chromosome paint”)
- DNA segments from specific genes or regions on a chromosome that have been previously mapped or identified.
what is comparative genome hybridation- CGH?
technique reveals the loss or gain of chromosomal regions in test samples (for example, derived from a tumour) relative to normal controls
how does CGH work?
- DNA in the test and reference samples is labeled with green and red fluorochromes
- allowed to compete for hybridization sites on either metaphase chromosomes or an array of BAC (bacterial artificial chromosome) clones that represent thousands of small DNA segments distributed across the genome
- Areas on the chromosome, or spots on the array, that are more green than average are present in extra copies in the test sample; those that are more red than average are deleted in the test sample
what are the uses of cytogenetics?
- pregnant women >35 years have an increased risk of aneuploidy such as Down syndrome or trisomy 21
- pregnant women with serum screening results indicative of trisomy 21
- children with phenotypic problems and/or developmental delay
- couples with reproductive problems- eg. Previous miscarrages
- families with known translocations etc
- microdeletion syndromes
- diagnosis, prognosis and monitoring of many cancers
what is the mutation that happens in sickle cell anemia?
- mutational change to the DNA sequence of a gene, in this case in the molecule of beta-haemoglobin
- T-A base pair is replaced by A-T
- changes the sequence of the mRNA (A to U)
- glutamic acid ——> valine
in what 2 scenarios would you use genetic testing?
- we already know the identity of the mutation:
simple analytical methods (oligonucleotide ligation assay)
-we don’t know the identity of the mutation: discovery methods (esp. karyotyping and DNA sequencing)
what are the underlying technologies used in genetic testing?
- PCR
- DNA testing
what kind of genomic DNA is used in PCR?
- White blood cells (peripheral leukocytes)
- Buccal cells from smear/mouthwash
- Fresh tissues (tumour, post mortem)
- Paraffin-embedded tissue blocks
- Hair root
- Semen
- Egyptian mummies and other archaeological specimens
- Microorganisms
what is the name of the DNA strand created in PCR
cDNA
complementary DNA
what are oligonucleotide primers/oligos?
- Custom-made to your specified sequence by chemical synthesis
- Choice of the primer sequence is essential to ensure the specificity of the PCR reaction (Primers must bind to a unique sequence)
what factors should be considers when designing primers?
- length: usually ~20 nucleotides (range is 15 to 30)
- base composition
- melting temperature
what are thermostable DNA polymerases?
- Isolated from thermophilic bacteria that require thermostable enzymes for survival at high temperatures
what is Taq polymerase?
- Purified from Thermus aquaticus bacteria
- Optimum activity at 75°C
- Stable at 95°C
- Recombinant protein is now mass-produced from gene cloned and expressed in E. coli
using PCR what genetic diagnosis can we make?
-sickle cell anaemia
-thalassaemias
-cystic fibrosis
-Huntington disease
familial cancer syndromes
-muscular dystrophies
-tissue typing eg. HLA alleles for bone marrow transplantation
-presymptomatic diagnosis:
-carrier identification in at-risk families
-prenatal diagnosis
what is the oligonucleotide ligation assay (OLA)
- DNA mutation analysis
- more specific and sensitive test
- cheap
- determine product sizes by gel electrophoresis
- Many OLA probes of different sizes and colour
how does OLA work?
- complementary OLA1 strand made to the normal allele
- complementary OLA2 strand made to mutant allele
- complementary strands made for both normal and mutant allele in fragments = DNA ligase joins them together
- wheres theres mismatch of fragments ligase wont recognise = no ligation
why would ligation fail for alleles?
due to the mis-match: the existence of a point mutation at the site of ligation interferes with oligonucleotide ligation, resulting in no ligation product
what does OLA do
- only gives answers to Q already know
used to confirm - Rapid way of identifying mutations for which the mutation is already known (to be pathogenic )
how does chain termination occur?
- after complemtary strand produced using oligonucleotide primer
and catalysed by a DNA polymerase which requires deoxynucleotides (dATP, dCTP, dGTP and dTTP) to form the new strand - small amount of each dideoxynucleotide is also incorporated into the reaction mix (ddTTP etc.)
-DNA pol does not discriminate between dNTPs and ddNTPs, so the ddNTP is incorporated into the growing DNA strand - some dTTP replaced buy dideoxyTTP
- BUT ddNTP/ dideoxyTTP lacks the 3’hydroxyl group that is needed to form a phosphodiester bond with the next nucleotide phosphate
= each strand will terminate randomly
how does gel electrophoresis work?
- strands are of different sizes = can be separated by gel
- longer strands move slower = nearer start
- shorter strands move faster = nearer end
what is a nonsense mutation?
codon change to premature stop codon
what is MEGF10 ?
regulator of satellite cell myogenesis (formation of muscular tissue)
what do mutations in MEGF10 cause?
- early onset myopathy (affects the muscles that control voluntary movement in the body)
- areflexia (muscles don’t respond to stimuli)
- respiratory distress and dysphagia (swallowing difficulties)
- people doe very early on
what is the key principle in diagnosing genetic disease?
- detect changes in the sequnece of the gene
- if tis a unique mutation would have to use more expensive technique
- sequencing determines the exact position of the mutation within the gene
- determines the type of the mutation (including single base changes)
What DNA is sequenced by clonal sequencing?
PCR products
what is a syndrome?
a collection of features
what is trisomy 18?
edward’s syndrome
- 1 in 3000 births
- multiple malformations
- clenched hand with overlapping fingers
- will not survive long
what is trisomy 13?
patau syndrome
- 1 in 5000 births
- multiple malformations
- midline structures mainly affected (incomplete lobation of brain, cleft lip, heart disease)
- will not survive long
are autosomal or sex chromosome number anomalies more common?
sex chromosome
list the potential abnormal gametes
sperm with xy or 0
ova with xx, xxx or 0
what is klinefelter syndrome?
- males with xxy
- 1 in 850
- infertility
- poor development of secondary sex characteristics
- gynaecomastia and osteoperosis
- hypogonadism
- tall
what is turner syndrome?
- women with x
- 1 in 3000 (99% lost spontaneously in pregnancy)
- short stature, wide carrying angle of arms
- amenorrhoea (ovaries involute before birth)
- congenital heart disease (20%)
what is prader-willi syndrome?
microdeletion 15q13
what is cat eye syndrome?
22 inverted duplication (inv dup(22)(q11))
what are the two types of chromosomal translocations?
centric fusion (robertsonian) reciprocal
what is centric fusion?
breakage of two acrocentric chromosomes (13, 14, 15, 21, and 22) at or close to their centromeres —> fusion of long arms and short arms lost
what does acrocentric mean?
chromosome with centromere very close to one end (13, 14, 15, 21, and 22)
what is reciprocal fusion?
breakage of two non-homologous chromosomes with exchange of the fragments
what three different ways can downs syndrome be caused?
- trisomy 21 95%
- roberstonian translocation (4%)
- mosaicism (1%)
what is an acquired cytogenetic abnormality?
a genetic change associated with a neoplastic or cancer disease process
- a translocation between 9 and 22 occurs in 90% of patients with CML
what is CML?
chronic myelogenous leaukemia
