Population screening Flashcards
define allele frequency?
the proportion of a given allele among all alleles at a give locus- usually given a s a percentage
What is an association study? See
statistaical method for determining the co-occurence of a phenotype with a genotype
- if there is an association an allele will be found with a specific phenotype in a population more often then predicted by chance
independent of linkage
what is a linkage study?
linkage atypically looks at a few families with the same trait and multiple affected
Uses micro satellites to narrow don candidate region that is associated with the trait and can be used to study variants with large effect
what is genetic linkage?
tendency of 2 genes that are located in close proximity to be inherited together. The further apart 2 genes the more chance there will be a recombination event between them preventing them being inherited together
what is epistasis?
interaction between non-allelic genes that influence phenotype
- modifier genes
- one gene modifies the expression of another
what is fitness?
ability for a population to survive and reproduce in a particular environment
what is gene flow?
flow of genetic info between different population of the same specie by mating and migration
what is gene pol?
full range of alleles within an breeding population
what is genetic drift?
change in allele frequency in a population over generation due to chance i.e. not driven by selective pressure
- can result in loss of certain alleles
- more common in small populations
what is genetic load?
difference between optimal and average member of due to deleterious gens in gene pool
what is genotype freq?
frequency of a specific genotype in a population
what is a GWAS study?
Studies looking at associations between alleles throughout the genome and phenotypes.
study lost of unaffected and affected control
use SNPs for high resolution coverage
They are effective in detecting common alleles that contribute to the inherited component of common multifactorial diseases. Typically, the alleles identified by this approach have modest effect sizes that cannot fully account for disease susceptibility.
define haplotype?
combination of linked alleles on a chromosome- can b used to track inheritance of an unknown genetic trait
what is the Hardy-Weinberg law?
according to H-W allele freq will remain constant in a population if there is no evolutionary influence i.e. no sex selection, genetic drift, genetic flow, no mutation
used to asses risk for individuals with genetic disease
what is linkage disequilibirum?
the co-occurrence of 2 alleles in a population more often then predicted by chance
An association can only be found between alleles and disease if the disease is in LD with a marker allele.
LD is often expressed as D. If D=0 there is no association between alleles and, in this case, the distribution of alleles in the population is as expected (randomly distributed) and dependent on the allelic frequency.
define species?
group of organisms hat can reproduce with each other to produce fertile offspring
what is the LOD score
- log of the odds
stat used in the analysis of linkage
LOD= 3 indicates that 2 loci are statistically linked
what is a map unit (µu)?
measure of genetic distance between 2 genes
crorresponds to recombination freq of 1% or 1 centimorgan (CM)
what is the minor and major allele freq?
frequency of the most and least common alleles of a given locus
what is a morgan unit?
unit used to express the ditance between 2 genes based on the liklihood of recombination between them- more distantly located genes with have a grreater chance of recmobination
1M = recombination possibility of 100% 1 dM (deciMorgan)- recombination value of 10& 1 cM - recombination value of 1%
what is the mutation rate?
number of mutation per biological unit e.g. per round of division, per gamete
what is the odds ratio?
odds of having the disease and disease allele divided by the odds of having the disease without the risk allele
in pop study basically cases with genotype over controls with genotype and the higher the OR the greater the risk associated with the disease allele
what is penetrance?
proportion of individuals with a specific allele that express the associated genetic trait
what is the relative risk?
estimated probability of having the disease when the patient carries the risk allele in an association study
what was the Myriad Vs Association of molecular pathology case of 2013?
Land mark case between Myriad genetics and the association of molecular pathology
Myriad was one of the first US companies to offer DTC testing and held a patent for BRCA1/2 testing. Until the case against the AMP in which the US supreme court ruled that natural DNA is not patentable
what is a a screening test? (who does it test and aims)
screening tests asymptomatic/presymptomatic people in the population at risk of a disease
aims to identify disease before the patient develops symptoms and provide early intervention for improved health outcomes- reduced mortality and/or morbidity
what types of population screening are there?
mass- newborn screening
targeted- lung disease in coal miners
opportunist- BP at routine GP appointment
pro-active- cervical screening in women >25yrs
what is genetic screening?
for early detection (pre-symptomatic) or esclusion of a genetic disease, genetic predispostion to a disease or if an individual carries a variant that may result in disease in their offspring
different to population screening as a targeted to specific populations e.g. CF testing in ovum/sperm donors
what are the WHO criteria for offering a screening programme for a specific condition?
Condition
- must pose a significant health burden either due to its severity or prevalence (or both)
- natural history and epidemiology must be known
Test
- safe, simple, effective, precise and validated
- distribution of results must be known and cut-offs determined for positive and negative results
- acceptable to the population
Treatment
- must be an effective treatment available with evidence that early intervention improves outcomes
- agreed policy and pathway for follow-up of +ve result determined and optimised before testing offered
Programme
- benefits outweigh harm
- cheaper then only treating patients when they become symptomatic
- improving current treatment pathways should be considered first
what is sensitivity, specificity, PPV and NPV
sensitivity= % true positives detected by the test specificity= % true negative PPV = TP/TP+FN NPV= TN/TN+FP
sensitivity is prioritised if there is
- an efective treatment
- confirmatory diagnostic test
- communicable disease
PPV can be improved by increasing the prevalence of the disease in the population being screened either by pre-screening or only screening a selected population
what are the advantages of screening?
better prognosis
less radical treatment required
reduced costs and resources
-ve result can provide reassurance
what are the disadvantages of screening?
false -ve may give fale reassurance
false +ve may result in unecessary treatment, anxiety and testing
longer morbidity if prognosis is unaltered
what is the current state of newborn screening in the UK and EU?
currently no active screening programmes but it has been variable approved pending inplimentation or undergoing pilot schemes across the EU
In the UK in 2019 the UK national screening committee refused to add SMA to the NBS programme at this point because of too little info on:
reliability of test
evidence of nusernisen in asymptomatic children
acceptability and pathways for families following a positive result
- Pilot scheme ongoing in UK
what is the UK national screening committee?
set up in 1993 and responsible for screengin policies and procedures in the UK
- ensure that introduction of new screening programmes is acceptable and scientifically backed
- review existing screening programmes and pathways to ensure they are
ESHG and ACMG have also published screening recommendations
what is the newborn screening programme?
bloodspot test offered to all neonates on day 5
- guthrie card test
9 conditions where early intervention is beneficial, there are effective treatment and the tests are reliable, sensitive and specific
CF Phenyketonuria (PKU) medium chain acyl coA dehydrogenase deficiency (MCCAD) Sickle cell congenital hypothyroidism maple syrup urine disease isovaeric acidemia glutaric aciduria type 1 homocysteinuria
what is the FASP programme?
Fetal anomlay screening programme
- detailed USS scan at ~ 20weeks gestation
- aims to identify chromosomal abn that are associated with fetal anomaly
- 1+ USS abn or NT>3.5mm = offered invasive testing (PCR and array- now includes trio WES)
what is the down syndrome screening programme?
biochemical screening to ID pregnancies at increased risk of T21 who can be offered invasive testing (>1 in 150)
combined screen 11-13+6 weeks
NT, BhCG, Mat age, PAPP-A = age asjusted risk bask on MOM
quadruple score - 14-20wks
less good for T14 and 18
BhCG, unconjugated estriol, a-fetoprotein and inhibin A
what is preconception screening?
preconceptions screening involves screening a couple for their risk of having offsspring affected by a recessive disorder. Best if carried out before couple gets pregnant as there are more options available e.g. PGD, in practice its often only carried out during pregnancy
carrier screening is available for CF in Australia and Italy
- SMA in US
- in cyprus, turkey, sardinia testing for B-thal is offered. In Cyprus testing is a requirement for getting a marriage licence as the carrier freq is so high the number of affected individuals would cripple the health service
what is expanded carrier testing?
panels covering common AR diseases- mainly available privately.
tend to only test for the most common mutations so individuals need appropriate counselling and the residual risk should be given
what are the findings and requirement of the ESHG and ACMG on expanded carrier testing?
- aim is to enable autonomous decision making
- need clear information available if there is reduced penetrance or variable expressivity
- provide residual risk figures
what expanded carrier testing is available in the UK?
not routinely available in the UK however testing is targeted to specific groups for example AKJ can be tested for 30 diseases with high prevalence
carrier testing also offered if there is a family history or 1 member of a couple is a known carrier
what are the potential benefits or limitations of population genetic screening for breast cancer?
study suggested that screening women with no family history may be cost effective due to earlier diagnosis
however ESHG states cost cannot be the only justification for population screening
- reduced penetrance
- other factors- diet, lifestyle also v relevant
- low penetrance of breast cancer variants in general pop without family history
- potential physical and psychological harm to patients- may have unnecessary surgery or chemoprevention and the lack of effective strategy for ovarian cancer may increase anxiety with no benefit
what are the 4 key ethical priniciples in medicine?
autonomy- pateint has the autonomy and choicde
beneficence- highest priority is th welfare of the patient and minimising harm to them
non-maleficence- prevent and minimise harm to the patient
justice- equity of access and patients should be treated fairly
what is the importance of non-directive counselling in genetics?
- Genetic information may affect the entire family.
- Genetic discoveries may be predictors of future adverse effects on an individual/family.
- Genetic information and choices made may affect future generations.
what are the key ethical issues surrounding preconception/prenatal screening?
reproductive autonomy- individuals have the right to make informed decisions therefore need informed consent but counselling should be non-directive
what are the potential benefits of population genetic screening (ESHG 2013)?
- pre-symptomatioc detection where there are options for prevention, early treatment
detection of carrier status will allow people to make informed reproductive decisions
improved reproductive decision making and autonomy
what are the potential issues with prenatal
- ethical issues surrounding consent- NBS or prenatal testing may ID carrier status which will affect that individuals reproductive decisions and they have not consented for so removes their right to autonomous decision making
- may result in anxiety if information cannot be used to make positive choices e.g. variant of variable penetrance
- compliance: offered by medical professional so individuals may feel obliged to have the test
- may be undue pressure on an individual (family, religious, partner) to make certain reproductive decisions
- disclosure of information- results for one family member may have implications for relative who have not consented to testing.
- need to ensure the information is not misused by insurers or employers
what are the potential issues of preconception testing? (6)
- may result in -ve stigma against people with the condition and could be considered a form of eugenics
- chosing not to have a child with a genetic condition could devalue those people that have it
- pressure on parents to not have a child with a condition or judged if they do for placing extra burden on health service
- fewer people born with the condition may reduce the funding for services and treatment of people with the condition
- most conditions have a residual risk even after carrier testing
how can the potential issues of preconception carrier testing be overcome (ESHG/ACMG)
- test for common variants rather than whole gene to prevent detection of VUS
- provide clear information on penetrance and onset of conditions in pre-test counselling
- screening programmes must also have well defined pathways for affected individuals to ensure they are not receiving fewer services
- sufficient support should be given to couples chosing to terminate or continue a pregnancy so they have the choice to do either
- voluntary participation, equity of access and assess the benefit in terms of improved reproductive decision making rather than a reduce birth rate of affected individuals
what are the future directions in genetic screening?
increased used of NGS- WES and WGS
what are the future directions in genetic screening? ctDNA
NGS is being applied to cell free tumour DNA obtained from blood samples- this could enable non-invasive screening on a population scale.
Benefits= earlier diagnosis and treatment should result in better outcomes and decreased disease burden
limitations= technical, test sensitivity, false positive and cost
what are the future directions in genetic screening? cffDNA
cffDNA is being used for NIPD for some single gene disorders and NIPT is in the process of being rolled out as an NHS test following review by the national screening committee and NHS england and the RAPID study
what are the future directions in genetic screening? WGS/WES
ACMG has recommened that IFs in a specific genes and encourage labs to actively look for variants in a list of 59 reportable genes including genes associated with breast cancer, lynch syndrome, Marfan syndrome and cardiomyopathy
this is a cost effective method but requires informed counselling and does not provide equity of access
what are the future directions in genetic screening? preconception carrier screening- benefits and thoughts
offered commercially currently - so access not equitsable as based on abilit to afford testing, widespread adoption could follow
likelihood a non-consanguineous couple having a child with a AR affected offspring is low and primary goal of increasing reproductive autonomy is hard to quantify so herd to justify the cost effectiveness ads a publicly funded service
further research such as a pilot study in the netherlands which is offering the service through GPs will provide more information on the potential benefits of such a national programme
what is newborn screening?
screening offered to all noenates on day 5 of life using a heel prick bloodspot on a gutrhie card
first developed for phneylketonuria in 1969 and is now pone of the largest screening programmes with >800,000 newborns screened in the UK each year
should we screen for more diseases?
There is pressure to screen for more conditions and the public is generally accepting of screening however new programmes need to be carefully evaluated before being introduced?
what issues have there been with prostrate cancer and neuroblastoma screening?
prostrate cancer screening can result in unnecssary surgery with side effects of incontinence and impotence as PSA is a poor marker for disease that will develop into cancer so unnessecary treatments carried out
neuroblastoma screening was suspended in 2004 because although superficially 90% of those screened survived compared to 50% not screened there was no decrease in mortality therefore it may be that the screen is identifying patients that will never progress to severe disease of is failing to detect patients who will develop serious disease
hard to remove screening once introduced as patients who have had the screen and been positive and survived may attribute this to the screen even if thee is no evidence to support their belief
how does screening differ to diagnostic testing?
test asymptomatic/ pre-symptomatic individuals
not diagnostic and may need a confirmatory test
what are the features of down syndrome?
LD, DD risk of early onset alzheimers VSD or other congenital heart defect facial dysmorphism hearing, opthalmic, muscoskeletal, GI and thryoir features increased risk of leukemia
what are the MOM for DS screening?
DS risk from serum makers and age is calculated bydetermining how different a womens results are from the ref pop mean of unaffected population at the same gestational age
serum marker conc for pateint/ serum marker mean for normal pop
used in a bayes calcuation to adjust the prior risk based on maternal age
what factors can affect DS screening serum markes
need to adjust for the following
- weight (increased weight can dilute markers)
- IVF
- smoking
- ethnicity
- accuracy of dating (gestational age)
what are the justifications for DS screening?
- significant LD and risk of severe congenital abn e.g. cardiac defects
- likely to require ongoing support and be a cost to society (health and social care) and unlikely to contribute to the economy
- most women who undergo screening and have a positive result chose to terminate indicating support from the population
- screening can provide earlier diagnosis (than 20 week FASP allowing earlier less invasive termination)
- even if people don’t chose to terminate the knowledge of the result provides time to adjust, and access support services
what are the issues with DS screening?
- poor understanding of risk in the general population and low risk screen does not mean no chance of T21
- fale +ves could result in pregnancy loss from invasive
- women may feel obliged to have screen, then invasive and terminate as offered by medical professionals
- high rate of natural pregnancy loss in T21 so this may result in unnecessary decisions and anxiety for a pregnancy that would be naturally lost anyway
- perception that if screening is available a life with T21 is less valid- people that chose to continue pregnancies may feel they are a burden on sociability and less valid
what is the aim of the newborn screening programme?
aims is to provide treatment to newborn ASAP to reduce/prevent potential severe phenotype
when should patients be contacted by if there is a positive screening result?
2 weeks for PKU
3 weeks for CHT and MCADD
4 weeks for CF
6 weeks for SCD
what are the WHO criteria for a screening test?
- significant health issue (severe or common)
- know the natural history of the disease and have defined cut off of affected or not. Assumption is all cases will progress to the most severe form
- effective treatment available and evidence that early intervention is beneficial
- known incidence in target pop e.g. UK
- test with high PPV and sensitivity and specificity available that is acceptable and suitable to screening (ethical, safe, simple)
- cost effective
what are the 9 conditions tested for in NBS- which are genetic and which are metabolic?
genetic
- sickle cell disease
- CF
- congenital hypothyroidism
- phenylketonuria
inborn errors of metablism MCADD medium chain acyl-coA dehydrogenase Isovaleric acidemia glutaric acidemia type 1 maple syrup urine disease homocystinuria
predominantly treated by long term dietary management to prevent toxic build up of causative metabolite- due to deficiency in metabolism and toxic build-up
what is the phenotype in PKU?
results in severe MR
what is the cause of PKU
AR genetic disease due to mutation in the DAH gene
phenyalanine dehydrogenase which converts phenylalanine to tyrosine resulting in a toxic build-up especially in the brain
how is PKU tested for?
NBS detects increased phenylalanine in the blood by tandem MS
How is PKU treated?
patients are put on a low phenylalanine diet which significatly reduces the risk of neurologic handicap but if diagnosed too late there may be no benefit as damage has already been done
what are the feature of congenital hypothyroidism
failure to thrive and don’r grow properly
physical and mental handicap
neonates may have a protuding tongue, jaundice, feeding difficulties and low hairline
what causes congenital hypothyroidism?
primary- agenesis or dysgenesis of the thyroid gland
secondary- deficient levels of TSH
genetically heterogeneous and associated win mutations in multiple gene - e.g. thyroid transcription factors
how is congenital hypothryoidism treated?
thyroxine supplementation
How CH screened for?
increased level of TSH- produced as abn thyrpid gland is not responding
what are the features of MCADD?
deficiency in fat metabolism and can result in death
what causes MCADD?
AR mutations in ADADM = build up medium chain fatty acids (C8) which the body can’t efficiently uses for gluconeogenesis resulting in low energy availability especially for the brain
How is MCADD screened for?
tandem mass spec for levels of medium chain FA C8 compared to C10
what is the treatment for MCADD?
dietry intervention- regularily eat to prevent need for gluconeogenesis
what are the features of sickle cell disease?
AR disease with het advantage so has high carrier freq in regions of world affected by malaria e.g. africa
in hom form RBCs are sickle shaped and cannot flexibly bend to fit through small capillaries resulting in damage to blood vessels and organs, chronic pain ans infection
How is sickle cell disease screened for?
by seperating HB protein by isoelctric focusing or high performance liquid chromatography- abn Hb protein will have a different mass charge ration (IEF) or elution time (HPLC)
genetic testing can also be carried out for to allow family testing
other clinically significant heamoglobinopathies are also screened for
what causes CF?
mutation in the CFTR gene
How CF screened for ?
uses combination of genetic testing and IRT
- IRT has a poor PPV on its onwn and is raised imediately after birth in all so need to wait till day 5 to test
- genetic testing alone with a panel would identify a high number of carriers which should be avioded in prenatal testing
- test for IRT - if raised test, re assay then for 4 most common CF mutations
- CF confirmed - no more testing
- carrier- test with full CF panel and may need to IRT again if still not second mut detected
- no mutation - IRT again
what are the features of CF?
mutlisystem disorder affecting
- GI tract
- fertility - BAVD
- respiratory tract- recurrent infections, disseminated bronchiecstasis
- failure to thrive
- pancreatitis
How can CF be treated?
early intervention with high energy diet, medicine and physiotherapy does not cure but improves symptoms and outcomes
what are the features of maple syrup urine disease?
urine smells of maple syrup
vomiting
lethargy
progressive neurologic deterioration
due to defect in keto acid dehydrogenase resulting in build up of leucine, iso-leucine and valine
how is maple syrup urine disease tested for?
tandem mass spec for leucine. iso-leucine and valine
what is the treatment for maple syrup urine disease?
low protein diet to prevent build up of amino aciod and thiamine
early intervention prevents neurologic damage
what are the causes and features of homocystinuria?
AR didease due to mutation in cystathione b-synthase = catabolism of methionine and toxic accumulation of homocyteine
results in skeletal abn and LD
How is homocystinuria tested for?
tandem MS for undeviated MRM methionine
How is homocystinuria treated?
very low methionie diet and supplmetation - without treatment patients die before 30yrs
what are the causes and features of Glutaric acidemia type 1?
accumulation of glutyaric acid due to deficiency in glutaryl co-a dehydrogenase
macrocephaly and metabolic and neurologic crisis
How is Glutaric acidemia type 1 screened for?
Tandem mass spec for glutaryl carnitine
How is Glutaric acidemia type 1 treated?
low protein lysine restricted diet with carnitine supplementation
How is isovaleric acidemia tested for?
Tandem mass spec for isovalerylcarnitine
confirmed by analysis of urine organic acids
what are the causes and features isovaleric acidemia?
defective catabolism of leucine results in toxic accumulation of isovaleric acid and its glycine and carnitine derivatives
vomiting, metabolic crisis, failure to thrive, coma and even NND
How is isovaleric acidemia treated?
long term dietary management
what are the features of inborn errors of metabolism?
Due to defects in metabolic pathways
classical symptoms are either due to an accumulation of a substrate which toxic to the body or a deficiency in a product
generally Ar or XLR
what are the presenting features if IEM?
Multisystem disorder can present with: failure to thrive and wight loss vomiting and lethargy cytopenia heart failure immunodeficiency hypotonia NDD, seizure and stroke organomeglay
How are IEM treated?
dietary restriction/management for disorders resulting from a toxic accumulation of products and supplementation for deficiency’s
what IEMs are tested for in the NBS programme?
medium chain acyl-coA dehydrogenase deficiency homocystinuria glutaric acidemia isovaleric acidemia maple syrup urine disease phnyketonuria
How are IEMs diagnosed?
traditionally tested for biochemically but this can be non specific e.g. czellweger syndrome can be cuased by multiple peroxisomal storage disorders
biochemical testing may also require invasive testing e.g. liver biopsy for glycogen storage disorders
so genetic testing is also carried out as this can:
- be used to identify carriers and inform prenatal testing or reproductive descisions
- can help indicate mutation specific therpay e.g. PTC read through
- provide prognostic information
- provide specific diagnosis
what type of IEM is zellweger syndrome?
disorder of peroxisome biogenesis cause by multiple PEX gene and 3 different types
what is the phenotype, inheritance and testing for zellweger syndrome?
severe and can result in death < 1 yr- affects development, hearing, eyes and liver
AR
biochemical test is for very long chain fatty acids
Give an example of a urea cycle deficiency disorder?, the gene and testing
ornithine transcarbamylase deficiency
due to mutations in the OTC gene
can test for decreased OTC enzyme deficiency
what is the role of the urea cycle and hence symptoms of ornithine transcarbamylase deficiency?
uear cycle is the primary mechnaism for removing waste nitrogen from protein turn over and metabolising nitrogenous compounds
deficiency result in hyperammonemia
Give an example of a disorder of cholesterol biosynthesis?
Semi-lemli-Opitz syndrome
due to a deficiency in 7DHC
7-dehydrocholesterol reductase
Give an example of a glycogen storage disorder
Pompe disease type II
hypotonia, cardiomegaly, FTT, respiratory distress and hearing loss
what causes pompes disease?
deficiency in GAA enzyme
acid gucosidase- essential for dregredation of glycogen to glucose in lysosomes and enzyme deficiency is biochemically diagnostic
what are lysosome storage disorders?
IEM due to the toxic build up of material in cells due to enzyme deficiencies so lysosomes cannot perform their normal functions
due to mutations in emzymes that act in the lysosomes or transporters
what is the role of the lysosome?
cellular membrane bound protein with an acidic content due to a H+ pump
- usually function to digest materials and symptoms arise from the build up of partially degraded substances in the lysosomes
what are the 3 main groups of lysosomal storage disorder and what material is accumulated?
mucopolysacharidoses- build up of glycosaminoglycans
oligosaccharidoses- build up of oligosaccharides
sphingolipodoses- build up up sphingolipids
what are the genetic features of lysosomal storage disorders?
AR or XLR
LOF mutations
increased risk of neurodegenerative disorders in carries (alzheimers and parkinsons reported)
is there NBS for lysosomal disorders?
Being considered as there is a better outcome if treatment started early
- screening offered in Israel for Tay sachs
- taiwan tests for Pompe and Fabrys
what are sphingolipidoses?
disorder of lipid storage
mutations in enzymes that break down sphingolipids - important for the CNS and form part of the myelin sheath
Give an exmaple of 3 sphingolipidoses and the inheritance
Fabrys- X linked
Gaucher - AR
Niemann Pick - AR
What are the mucopolysaccharidoses?
result in an accumulation of glycosaminoglycans which are very long chain fatty acids
due to a deficiency in the enzymes required to break them down
what are the common phenotypes of mucopolysaccharidoses?
affects skeletal muscle and cause neuronal dysfunction
Give an example of 3 MPS disorders
MPS II Hunter syndrome
MPS III San Fillipo syndrome
MPS IV Morquio syndrome
What are the oligosaccharidoses?
glycogen storage disorders
due to a deficiency in enzymes required to degrade olligosaccharides into gycoproteins and glycolipids
What are the clinical features of oligosaccharidoses?
affects skeletal muscle and cause neuronal dysfunction
Give an example of 2 oligosaccharidoses
Pompe disease
Battens disease (neuronal ceroid lipofuscinoses)
what genes are affected in the hemoglobinopathies?
cause by pathogenic variants in the genes that encode the globin genes
what are the genes for hemoglobin alpha and beta?
HB alpha is encoded by HBA1 and HBA2
found on chr 16 in 2 4kb gene clusters
have high sequence homology
1 HBB gene on 11p
what is the role of hemoglobin in the cell?
fe containing O2 transport metalloprotein in the red blood cells of vertebrates
what is the pathogenic of the hemoglobinopathies?
pathogenic variants result in reduced synthesis of the hB or an abnormal Hb structure.
this causes disease in the thalassemias but resulting in a chain imbalance between HBA and HBB
excess of a subunit rather than a lack of expression causes the clinical consequences in thalassemia
how common are hemoglobinopathies? why
have a heterozygote advantage and protect against severe malaria in childhood
therefore there are high levels in areas affected by Malaria e.g. Mediterranean (cyprus, turkey), middle East, Africa and Asia.
there are also regionally specific disorders and therefore ethnic origin may point to a diagnosis
what is a-thalassemia?
most common hemoglobinopathy
due to mutation in one of the HBA genes (HBA1/2)
defecit in a chain production results in an imbalance with B chain formation
what are the genetic causes of a thal?
deletion of 1 or both HBA genes accounts for 90%- most commonly sue to unequal corssing over betweeen the HBAS clusters on 2 chromosome 16’s due to the high sequence homology
remaining 10% are point mutations spread throughout the gene
what is the phenotype genotype relationship in a thal
the more copies of HBA that a missing the more severe the phenotype
how many a thal syndromes are there- what is the severity
type 1 have loss of 1 gene- symptomless carrier
type 2- loss of 2 genes- asymptomatic but may have macrocytic anemia on examination
type 3 - loss of 3 genes = affected with anemia, hemolysis and splenomegaly
- may require blood transfusions
- lack of HBA results in abn b-globin tetramers being formed
type 4- loss of all 4 genes
- severe and onset in utero, results in still birth of early NND
- early diagnosis may enable blood transfusion in utero but likely to still have abn
- associated with rec misc
- fetal form as HBA is expressed in the fetus
what causes B-thal?
due to a deficiency in B globin and there is a reduction in adult Hb in red blood cells
expression begins at birth when fetal gamme globin declines so not associated with fetal onset as with HBA type 4
what is b- thal major
resutls in anemia and hepatosplenomegaly
onset from 6 months and blood transfusions from 2 yrs
most severe form
can be treated with regular blood transfusion but risk of fe overload so also need to prescribe fe chelators to overcome this
what is b- thal intemedia
mild to mod anemia
generally normal life but may need occasional blood transfusions
what can modify the severity of b-thal
due to imbalance in a and b chains
so severity can be reduced with co-inheritance with A-thal
- variants that sustain gamma globin levels also decrease severity
what is sickle cell disease and what is it caused by?
AR disorder due to mutations in HBS
most commonly homozygosity ofr c.20A>T Glu7Val
results in RBCs with an abnormal rigid ‘sickle’ shape- means they cant pass through small capillaries resulting in vascular occlusion = sickle crisis
- bone and joint pain and necrosis
- organ damage
- anemai
- risk of infection
- stroke
How are hemoglobinopathies tested for?
full blood count
Hb pattern analysis using isolectric focusing, HLPC or capilliary electrophoresis
DNA testing is used to confirm the mutation so carrier testing and prenatal diagnosis can be offered.
what is the NHS sickle cell screening programme?
offers screening to all mothers as part of the antenatal care
- if mother is a carrier the father is also offered testing
- all pregnant women offered in high prevelance areas but in low prevelance areas only women idfentified as being at risk from a family origin questionnaire are tested
- aim is to offer all women testing by 10 weeks gestation and to report result by 13 weeks to allow pregnancy decision making
- involvement of genetic testing depends on the prev hem lab result and as a screening service is will not detect all possible deleterious outcomes.
How can hemoglobinopathiesbe treated?
blood transfusion with concurrent fe chelation
BM transplant for severe cases but risk of GVHD
research currently looking into using lentiviral vectors (as they can carry large transgenes and infect quiescent cells) for gene therapy.
- phase 1 trials are ongoing for SCD gene therapies
what are the features of bleeding disorders?
due to an inability to from blood clots - result in excessive bleeding, oozing or re-initiation of bleeding, easy bruising.
caused by a deficiency in platelets or clotting factors
what is von willebrand disease and the causative gene?
VWF is a bleeding disorder caused by mutation in the VWF gene at 2p13
what is the role of VWF
is essential for platelet depending homeostasis and carries factor VII (F8) protecting it from degredation to sites of vascular damage
what are the 3 types of VWF and their inheritance?
Type 1 - partial quantitative deficiency in VWF
- most mild disease and genetic testing is not generally indicated
- Ar or AD
Type 2 is a qualitative deficiecy with high penetrance and can be AR or AD
Type 3 is most severe and is a complete quantitative deficiency due to 2 null mutatnts
- AR
- genetic testing indicated and prenatal testing can be offered
what complicates genetic analysis of the VWF gene?
it is a very large gene and there is a highly homologous PVWF psuedoegen
what is the deficiency in heamophilia A, what is the inheritance and incidence?
Due to a deficiency in F8 Xq28
XLR
more common than hemophilia B 1 in 5000
how is hemophilia A diagnosed?
severity is defined by the plasma activity of the F8 coagulation factor
genetic testing first looks for a intron 22 or intron 1 inversion as this accounts for >50% of cases
- intron 22 tested for by inverse PCR with primers designed to only produce a product when the inversion is present
negative cases have further mutation screening by sequencing and MLPA
what are the clinical features of hemophilia ?
decrease in clotting activity =
joint and muscle bleeds, easy bruising
recurrent bleeding in joints causes synovitis
leading cause of death in hemorrhage in the CNS
female carried are generally asymptomatic but may show mild features due to skewed X intactivation. Homozygous females are rare but present with the same features as males
what is the deficiency in hemophilia B, inheritance and incidence?
deficiency in F9 xq27
XLR
less commmon than HA - 1 in 30,000
what are inherited thrombophilias associated with?
increased risk of venous or arteriol thrombosis
thrombosis is a predisposition to from blood clots inappropriately
give 2 examples of inherited thrombophilias?
Factor V leiden thrombophilia
Hereditaty antithrombin deficiency
Give 3 examples of bleeding disorder?
Von willebrand disease
hemophilia A
hemophilia B
what are the clinical features of factor V leiden thrombophilia?
thrombosis
characterised by a poor anticoagulant response to activated protein C
usually no thrombotic event until adult and some may never develop one
what causes factor V leiden thrombophilia
due to mutation in the Fv gene on 1q24
FV is a glycorpotein made in the liver. In response to injury it is activated and acts with other factors to from a complex which converts inactive prothombin to active thrombin = blood clots
mutations result in 10x slower inactivation so there is excessive thrombin production and risk of abnormal clotting
what is hereditary antithrombin deficiency
AD disorder resulting in increased risk of clood clots
typically DVT and pulmonary embolism
What is linkage analysis?
Maps heritable traits to their chromosomal location.
- start by identifying genetic markers, commonly SNPs or STRs, on a section of a chromosome and then narrowing the region down until the gene or gene variant of interest is identified
What is linkage analysis used for ?
- Gene discovery
- Family studies when the mutation is unknown- this assumes the locus i.e. the gene of the familial disease is known.
- Method should use several informative microsatellite markers flanking the locus of interest to identify the high-risk haplotype.
- Requires analysis of key family members to assign phase and the high-risk haplotype and informativity of markers.
- Recombination may occur, which can make interpretation more difficult – Bayes calculations may be required.
Give examples of the use of linkage in diagnostic testing?
- DMD – identify high-risk haplotype where no causative pathogenic variant has been identified but a clinical diagnosis of dystrophinopathy has been confirmed by muscle biopsy.
- SMA – identify the high-risk haplotype where a homozygous SMN1 deletion has been identified in an affected child, but a parent carries two copies of SMN1 on one allele.
- HD – indirect prenatal test
- PGD- haplotype analysis allows the same design to be applied to multiple families regardless of the underlying mutation
What is linkage analysis suited to, requirements and resolution?
- Suited to highly penetrant monogenic disorders with mendelian inheritance.
- Requires large, multi-generational families.
- Poor genetic resolution, centimorgan range.
- Likelihood of linkage calculated as a LOD score.
What is linkage disequlibrium?
The non-random association of alleles at two or more loci with a frequency greater than expected by chance.
- Due to natural selection or chance.
- Underpins association studies. An association can only be found between alleles and disease if the disease is in LD with a marker allele.
- When a disease mutation arises on a founder chromosome and not much time has elapsed since the mutational event, the disease mutation will be in linkage disequilibrium with alleles from loci close to the gene. Thus, linkage disequilibrium can be a powerful tool for genetic mapping.
- Loci in LD will often be genetically linked, but LD can occur even if loci are on different chromosomes.
What affects LD?
- Recombination
- gene conversion
- selection
- population structure
- new mutation
- genetic drift
- gene flow- The greater the allele frequency differences between populations the greater the LD created when populations join.
- population history
How is linkage expressed?
- LD is often expressed as D. If D=0 there is no association between alleles
- D=1 is complete LD
- Between 0 and 1 there is some linkage between the alleles
How does recombination affect LD?
Over time recombination between loci will gradually reduce LD as alleles that were shared on an ancestral chromosome are separated. It can therefore be harder to find LD in older populations. Areas of the genome with lower recombination rate can maintain LD for longer.
How does selection affect LD?
If there is a selective advantage to two alleles coexisting, LD between the alleles is more likely to be maintained whereas a negative selection can remove LD
How does population structure and history affect LD?
- Inbreeding and non-random mating will alter the expected allele distributions.
- Older populations have shorter regions of LD as more recombination’s have taken place
How does new mutation affect LD?
A high new mutation rate at a locus will make it hard to detect LD. Mutations will arise on different ancestral backgrounds, the phenotypic affect may be the same, but the underlying haplotypes will not.
What is genetic linkage?
The tendency of alleles that are closely located on a chromosome to be inherited together at meiosis.
Why are SNPS used to study linkage not STRs?
- They occur frequently so can check short chromosome segments for disequilibrium- although only bi-allelic they are more common than STRs and can be detected via gentypeing chips allowing 100’s to be genotyped at once
- They have a relatively low mutation rate so persist through the population
- They are easy to genotype on a large scale
- Phase must be assigned, this is generally done using family members. If not available computer algorithms can determine the most likely haplotype
What are the disadvantages of linkage?
- Need to identify many families with several affected generations- this is harder for late onset or diseases with a high mortality
- Linkage studies are less helpful for complex traits, such as diabetes where multiple genes are important in disease causation
How is linkage scored?
- Linkage analysis requires the scoring of meioses as recombinant or non-recombinant for the locus in question.
- If this can be done (i.e. there aren’t too many homozygotes and enough variation) the locus is described as informative for linkage.
- All results must be replicated to be credible.
- Failure to replicate linkage does not necessarily disprove the hypothesis as linkages will often involve weak effects.
What is parametric linkage analysis?
In Parametric linkage analysis a series of parameters need to be
- Mode of inheritance (dominant/recessive/X-linked etc)
- Gene frequencies
- Penetrance – the most difficult to specify (beware phenocopies)
When can parametric linkage analysis be used?
- Parametric linkage analysis is used for simple Mendelian disorders.
- Not suitable for complex disease such as diabetes or schizophrenia (no idea of gene frequencies or penetrance of any susceptibility alleles or sometimes mode of inheritance).
What is the impact of double recombination on linkage?
Double crossovers can involve 2 or more chromatids, but if the region crossing over is close to the gene, a second event in the immediate vicinity is unlikely as the first event creates a phenomenon called interference, which restricts further crossing over.
What is the Lod Score?
- Log of the odds that 2 loci are linked
- Used log as it makes the numbers easier to work with
- Lod scores can be added across families
What LOD score is significant to accepting or excluding linkage?
- Z= 3.0 is the threshold of significance for accepting linkage (with a 5% chance of falsely rejecting the null hypothesis).
- Linkage can be rejected at values of Z
What is autozygosity mapping?
- Autozygosity mapping is a form of linkage analysis used in consanguineous families.
- Autozygosity occurs when individuals are homozygous at a particular locus because the alleles are identical by descent (IBD)- inherited from a common ancestor.
What is non parametric linkage analysis?
Method of linkage analysis that does not require an inheritance model.
Non-parametric methods are widely assumed to be more robust than parametric methods, but is complicated when combining data from families of different sizes
What is the principle of non-parametric linkage analysis?
The region of the genome with the disease gene will be from a common ancestor by affected members of the family more frequently than would be expected by chance.
This is identified by analysis for shared segments- this can be in any affected family members but affected sib-pairs are most commonly used.
what is the difference between IBD and IBS?
IBD: alleles shared by affected relatives are both copies of one specific allele that was present in a recent ancestor. Must be demonstrable
Identity by state (IBS): alleles shared by affected relatives appear identical but common ancestry cannot be demonstrated
What is the method for affected sib pair non-parametric linkage analysis?
- Genotype large no, of SNPs/ STRs in sibs covering whole genome
- Look for markers that do not follow mendelian ratios (1:2:1 ratios - e.g. sharing of one allele is >½ and sharing of 2 alleles is >¼ )
- if parents are available can determine if region is IBD or IBS - Using the combined data from all the families the IBD/IBS frequencies at each locus can be determined and each tested to see if it deviates from the null hypothesis of simple Mendelian segregation. Thus identifying over the whole dataset, loci where there is a significant excess of sib pairs that are IBD.
- When regions of suggestive linkage are identified, the region will then usually be re-examined using more densely spaced markers in an independent data set.
What is an Association study?
Compare cases to controls to identify association (statistical relationship) between a particular allele/genotype and a genetic trait.
Essentially cases and controls are genotyped and allele frequency differences are determined. If a SNP is more common in cases than controls it is said to be associated with the disease. It is not necessarily causative itself but marks a region of the genome which influences the risk of disease.
What is the resolution of a GWA study?
Association studies have fine genetic resolution in the kilo base range.
How are GWA studies hypothesis free?
Investigate the entire genome. The approach is therefore said to be non-candidate-driven in contrast to gene-specific candidate-driven studies.
What are the 4 main causes of positive association?
- Chance.
- False linkage due to linkage disequilibrium between marker being studies and the true disease-causing variant.
- Bias in resulting from population stratification. Different genetic variants are known to occur with varying frequencies in different populations and ethnic groups, quite independent of the frequency of disease in these groups- confounding bias. Need to be aware that apparent association may just reflect shared population history
- True association- the genetic variant is important in disease causation.
What is the difference between a linkage study and association study?
Linkage investigates the relationship between transmission of a haplotype/locus and the expression of a disease trait in families
Association analysis investigates the relation between a specific allele and the disease/trait within population.
what are the advantages of association studies?
Gene-association studies can be used for
• Can identify association in complex diseases e.g., diabetes and hypertension – in linkage unless you have a VERY large family you need to combine data from multiple families. For polygenic disorders it is highly unlikely that every family will have the same collection of underlying genetic factors and so linkage results will conflict.
• Can be used to study rare diseases
• Can be used to test for a specific marker in a particular gene (individual SNPs).
• It can also be useful in investigating gene-gene or gene-environment interactions.
What assumptions are required for GWAS studies
- Case and control participants are drawn from the same population.
- Case participants are representative of all cases of the disease or limitations on diagnostic specificity and representativeness are clearly specified.
- Genomic and epidemiologic data are collected similarly in cases and controls.
- Differences in allele frequencies relate to the outcome of interest rather than differences in background population between cases and controls.
What is the power of a study?
The power of a study is the ability of a design to pick up associations accurately.
What determines the power of a study?
- frequency of the risk allele in the population
- relative risk conferred by the disease-associated allele
- LD between genotyped marker and true risk allele
- sample size and genetic heterogeneity of the sample population
What technologies can be used for GWAS studies?
Illumina HumanOmni5
Affymetrix Genome-Wide Human SNP array 6.0.
What can be done to increase the power of a study?
- Increasing the sample size and using carefully matched cases and controls
- To increase the number of participants and prevent re-genotyping many studies combine previous data sets in meta-analysis.
- To confirm associations a study can genotype further patients and controls for any those if interest that appear to have an association. This increases the power of thr study but is cheaper than fully genotyping more individuals and can help ID false positives.
What is the P value?
When an association is detected, the significance is calculated using P. If P=0.05 this would represent a 5% chance of a false result.
What P-value is used for GWAS?
The high number of SNPs genotyped for GWAS means the value is only considered significant if VERY low.
For examples if the p-value was 0.05 then if 1,000,000 SNPs were genotyped you would expect 50,000 to be false results. This is too high therefore a P-value of less than 5x10-8 is used.
GWAS Limitations:
- cannot test causality just association
- Prone to confounding variables – need a carefully matched case and control population
- Expensive
- Necessity for multiple testing correction makes statistical threshold for significance hard to reach.
- Need large studies to have sufficient power to identify association for rare or low penetrance variants.
- Missing heritability- most GWAS identify SNPs only conferring small effects- this has led to the belief that GWA studies were ultimately not worth the expenditure
- Design efforts made to reduce false-positive results, may result in overlooking a true association.
What are the future directions for GWAS?
The rapidly decreasing price of complete genome sequencing have also provided a realistic alternative to genotyping array-based GWA studies.
What are the benefits of GWAS to human health?
GWAS studies are laying the foundations for personalised medicine and a move from a one size fits all model to one that aims to customise strategies based on an individual’s genetics.
Genome-wide association studies have successfully identified genetic variations that contribute to risk of type 2 diabetes, Parkinson’s disease, heart disorders, obesity, Crohn’s disease and prostate cancer.
what are the cardiomyopathies?
diseases of the cardiac muscle not caused by coronary disease, high blood pressure or heart valves
due to inherited or sporadic variants in cardiac muscle proteins
phenotypic variability from asymptomatic to severe
what are the genetics of the cardiacmyopathies
AR, XLR, AD, mitochondrial
> 40 genes
TTN accounts for 1/3 inherited cases
give an example of 3 cardiomyopathies
dilated CM
Hypertrophic CM
Restricted HC
What are the cardiac arrythmias?
disorders of the hearts electricla system including tachycardia, bradycardia or arrythmia
Give and example of 2 cardiac arryhtmias
Long QT syndrome- egenes encogind potassium and sodium channels- e.g. KCNQ1
Brugada syndrome- SCN5A
