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BLGY1232 > Human Genetics 2 > Flashcards

Human Genetics 2 Flashcards

1
Q

Analysis of Haplotypes in Pedigrees

A
  • enables mapping of candidate genes
  • if we find that a conditions segregates with a particular section of the chromosome then we know where to start looking for the target gene
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2
Q

Identification of Rare Recombinant Haplotypes

A
  • a haplotype may describe the cosegregation of several polymorphic markers
  • by looking at recombinant haplotypes in affected and unaffected individuals it is possible to narrow down the length of the chromosome that the target gene could be in
  • the gene will be somewhere within a limit defined by the recombination break points
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3
Q

What is the next step after identification of recombinant haplotypes?

A
  • two closely linked SNP markers may still be very distant in the underlying genomic sequence
  • there may be many candidate genes in the identified range
  • to locate the underlying gene more closely it is sometimes possible to genotype more families with respect to additional families within the initial recombination interval
  • once the range has been narrowed down to ~300kb, the target section of DNA is sequenced
  • the sequence is compared with the genomic database to identify which genes are present and candidate ‘disease’ loci
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4
Q

Genetic Mapping of Polymorphic Markers With Respect to Inherited Conditions Requires That…

A

i) the condition shows clear Mendelian inheritance and high penetrance
ii) we have sufficiently large families with a genetic history of the condition
iii) we can identify recombinants within these families that allow us to locate a candidate gene

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5
Q

Are all conditions single gene traits?

A
  • many inherited conditions are not single gene traits
  • incidence and/or severity of the condition may depend on a number of different susceptibility loci at different positions that make different contributions
  • for these conditions, association studies are used to identify contributing genes
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6
Q

Single Gene vs Multiple Gene Traits

Single Gene Traits

A
  • can easily identify which chromosome carries the susceptibility locus
  • linkage analysis
  • relatively easy to find the target gene
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7
Q

Single Gene vs Multiple Gene Traits

Multiple Gene Traits

A
  • genes could be anywhere in the genome
  • must examine polymorphic markers throughout the genome to identify markers that segregate with the condition
  • this requires thousands of individuals
  • compare segregation in an affected population with a control group (unaffected)
  • looking for polymorphisms shared by the affected individuals that aren’t found in the unaffected individuals
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8
Q

International HapMap Project

A

-set up to develop a haplotype map of the human genome, the HapMap, which will describe the common patterns of DNA sequence variation

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9
Q

How many SNPs are there?

A
  • in the human population there are ~10 million SNPs

- the rarer SNP allele in each case has a frequency of at least 1% in the population so qualifies as a polymorphism

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10
Q

SNPs and Haplotypes

A
  • alleles of SNPs close together in the genome tend to be inherited together
  • a set of associated SNP alleles in a region of a chromosome can be described as a haplotype
  • most chromosomal regions have only a few common haplotypes (greater than 5% frequency) which account for most of the variation from person to person in a population
  • a chromosomal region may contain many SNPs, nut only a few “tag” SNPs can provide most of the information on the pattern of genetic variation in the region as SNP alleles that are close enough together will always segregate together
  • so if you know the SNP allele at a particular locus you know which SNP alleles usually segregate with it at surrounding SNP loci
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11
Q

Haplotype Maps

A
  • provides a map of chromosomes indicating where common haplotypes (linked groups of SNPs) are located along each chromosome and indicates which SNPs are used as tag SNPs to identify the different haplotypes at each locus
  • frequency of “haplotype alleles” can be determined for the general population
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12
Q

Uses of Haplotype Maps

A
  • for different groups (geographic, ethnic, …) which each share a common ancestry, certain haplotypes are more frequent than others, this means that haplotype analysis can be used to determine ancestry of populations
  • we can also compare a group of people with a disease to a group of people without
  • chromosomal regions where the two groups differ in their haplotype frequencies might contain genes affecting the disease
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13
Q

Starburst Chart

A
  • used for haplotype frequency population structure analysis
    1) genotype everybody
    2) analyse individuals in population for haplotypes specific to genomic regions
    3) indicated the number of people sharing the same haplotype by a circle whose diameter relates to that number
    4) members of the population who differ at a single marker are indicated by subsidiary circles
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14
Q

Reproductive Fitness and Selection

Example Study in Asia

A
  • 2132 men across central Asia had their Y chromosome genotypes for 32 polymorphic markers
  • a conserved haplotype was found in a large group
  • based on known mutation rates (no recombination on the Y chromosome) the origin of this haplotype can be dated to c. 1000 years ago
  • a single male line probably originating in Mongolia (as this is where the haplotype is most frequent) has spread in the last 1000 years to represent ~8% of males in a region stretching from China to Uzbekistan
  • most convincing source of this is Genghis Khan and his male relatives
  • an estimated 16 million men (~0.5% of the world’s total) now carry this haplotype
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15
Q

Association Studies Between Haplotypes and Disorders

A
  • multiple susceptibility genes requires you to search the whole genome
  • use the HapMap data to select the minimum number of tag SNPs that will allow you to haplotype the entire genome
  • this reduces the number of SNPs to cover from 10 million to ~300000
  • but this is still a lot, SNP chips (a type of microarray chip) are used to sequence many SNPs at the same time
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16
Q

Microarray Chips

A
  • surfaces with DNA single stranded sequences bonded to them
  • can be hybridised with complimentary sequences
  • the single stranded sequences on the chip are called ‘probes’ or ‘features’
  • the features are robotically arrayed on the surface in an ordered high density grid
  • the features will hybridise with a complimentary sequence if it is washed over the chip
  • if the complimentary sequences washed over are tagged with a fluorescent label then they can be detected
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17
Q

Affymetrix SNP-chip

A
  • one of the most common SNP chips
    1) isolate DNA from the individual to be genotyped, shear it to generate small fragments and label the fragments
    2) hybridise to the chip
    3) the temperature is increase slightly so that only perfectly matched sequences can hybridise
    4) mis-matched sequences are washed off
    5) the chip is analysed by a micro-scanner that detects and records fluorescence at each position on the chip
    6) the genotype of any individual can be scored for 600 000 different loci simultaneously on one chip
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18
Q

Whole Genome Association to Identify Gene Underlying Common Disorders
Study Information

A
  • in 2007 the results of a whole genome association study were reported by the Wellcome Trust
  • 7 different disorders were studied:
  • -Bipolar Disorder
  • -Coronary Artery Disease
  • -Hypertension
  • -Rheumatoid Arthritis
  • -Type 1 Diabetes
  • -Type 2 Diabetes
  • -Crohn’s Disease
  • 2000 sufferers of each disorder and a control group of 3000 healthy individuals were genotyped for 500 000 tag SNPs
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19
Q

Whole Genome Association to Identify Gene Underlying Common Disorders
Results

A
  • alleles more frequent in the affected population than the control group were identified
  • auto immune diseases showed associations with the same regions
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20
Q

Whole Genome Association to Identify Gene Underlying Common Disorders
Chron’s Disease and Type 1 Diabetes

A
  • both auto immune conditions
  • showed association with the same markers on chromosome 18
  • these SNPs highlight the T cell tyrosine phosphate gene, a negative regulator of inflammatory reponses
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21
Q

Whole Genome Association to Identify Gene Underlying Common Disorders
Bipolar Disorder

A
  • Symptoms: episodic recurrent mood swings from elation to depression
  • strong genetic component
  • sibling recurrence risk = 7-10 (7-10 x greater risk of a sibling of a sufferer also having the condition compared to another member of the population)
  • current definition of BD is based on a psychiatric appraisal of clinical features, there is currently no objective test
  • this study is one of the first indications of genetic polymorphisms associated with the condition
22
Q

Biobank

A
  • UK Biobank Project
  • long term survey of genetic and environmental contributions to health
  • collecting medical information, lifestyle information and genetic data from 500 000 UK volunteers aged 40 to 69
23
Q

Inherited Disorders…

A
  • inherited disorders are a consequence of our genetic information
  • we can’t change our genes
  • we can monitor what we pass on to the next generation
24
Q

Genetic Counselling

A

-provides information and options for carriers of alleles causing disorders

25
Q

Genetic Screening in Newborns

General Principles

A
  • condition screened for should be relatively common
  • condition should be treatable, with early intervention leading to positive outcomes
  • test should be accurate
26
Q

Phenylketonuria

A
  • autosomal recessive condition
  • frequency of 1 in 15000 in UK
  • caused by a mutation in phenylalanine hydroxylase
  • if untreated progressive mental retardation occurs
  • early diagnosis and implementation of low-phenylalanine diet prevents this
27
Q

Universal Screening in UK

A
  • NHS universal screening of all infants at 5 days old, usually with a heel prick blood test
  • tests for:
  • -Phenylketonuria
  • -Congenital Hypothrodism
  • -Sickle Cell Disease
  • -Cystic Fibrosis
  • -Medium Chain Acyl-CoA Dehydrogenase Deficiency
  • -Duchenne Muscular Dystrophy
  • -Homocystinuria
  • -Tyrosinaemia
28
Q

Screening For a Wider Range of Genetic Disorders

A
  • we now have the technology to screen for a wider range of genetic disorders
  • -is there any point testing with no suitable intervention
  • -how knowledge of susceptibility to a late onset disease affects patient
  • -would you want to know
  • -when would you want to know
29
Q

Prenatal Genetic Screening

Amniocentesis

A
  • amniocentesis samples amniotic fluid to collect foetal cells for analysis
  • test done at 14-20 weeks
30
Q

Prenatal Genetic Screening

Chronic Villus

A
  • chronic villus sampling collects placental cells

- test done at 10-15 weeks

31
Q

Prenatal Genetic Screening

A
  • commonly used for screening chromosomal abnormalities e.g. Down’s Syndrome
  • amniocentesis and chronic villus testing
  • invasive so there is some risk to the foetus
  • enables intervention before birth
  • provides parents with information, should they terminate the pregnancy/make preparations for birth etc.
32
Q

Preimplantation Genetic Diagnosis

A
  • associated with IVF
  • single cell can be removed from a ~4 cell stage blastocyst (early embryo) and genotype the DNA by PCR assay
  • used to screen alleles for single-gene disorders
  • also used to check embryos generated by IVF to ensure there are no chromosomal abnormalities
  • many ethical issues:
  • -IVF typically generates multiple embryos, what happens to the unwanted ones?
  • -what traits should parents be allowed to chose?
33
Q

Charlie Whitaker

A
  • born with Diamond Blacktan Anaemia (DBA) which prevents the body from making red blood cells
  • in the short term DBA can be managed with blood transfusions
  • can be permanently cured with a bone marrow transplant
  • his parents and sister did not carry compatible antigens
  • they selected an embryo from IVF with complimentary antigens, umbilical stem cells were collected and used for transplant
  • this was in 2003 and they had to fly to the US to do this as permission was refused by the UK HFEA
34
Q

Megan Matthews

A
  • born with Fanconi Anaemia, a DNA repair defect causing bone marrow failure
  • first child in the UK to be cured by a ‘saviour sibling’
  • following IVF, 3 day old embryos were tested for a genetic match
  • umbilical cord blood and bone marrow used for successful transplant
  • but Fanconi Anaemia is a general DNA repair disorder, Megan may suffer other consequences in tissues not replaced by bone marrow transplant
35
Q

Developing Effective Treatments for Human Disorders

A
  • we have to know how a condition develops throughout the life of the affected individuals
  • in many cases, we lack knowledge of the early stages as they may be asymptomatic, so the affected person isn’t medically assessed until symptoms appear
36
Q

Knockout Mice

A
  • now we can make transgenic mice that have been genetically engineered to contain defective genes and monitor progress throughout development
  • but, although mice and humans are both mammals, not all disorders progress in the same way in different species
37
Q

Genetic Engineering of Mice

A

1) embryonic stem cells derived from the inner cell mass (ICM) of the blastocyst are withdrawn, these cells are pluripotent
2) cells are cultured on a growth medium
3) DNA is delivered into the cells by electroporation, the inserted DNA is made up of sequences from either end of the gene of interest with a resistance gene in the middle.
4) a transgene homologous with a host gene can replace it by homologous recombination
5) select for resistance to obtain only transformed cells
6) inject transformed cells into a recipient embryo ensuring they become part of the inner cell mass
7) implant into surrogate mother

38
Q

Genetically Engineering Knockout Mice

e.g. White and Black Mice

A
  • cells taken from white mouse embryo and engineered
  • engineered cells injected into blastocysts from black mice
  • some of the offspring are mostly black, some are mostly white, some are a mixture of black white they are chiameras
  • chiamera mice are crossed with inbred black mice
  • if the transgenic cells develop as part of the germline in the chiameras then some gametes carry the wildtype allele and some the transgene
  • gametes with the wildtype allele will produce black offspring when crossed with the black mouse
  • gametes with the transgene will produce white offspring that are heterozygous for the wildtype and knocked out allele
  • white offspring can then be inbred to produce homozygous transgenic offspring
39
Q

Leptin

A
  • small polypeptide hormone
  • regulates adipose tissue formation and is involved in controlling appetite and metabolism
  • mice in which the leptin gene has been knocked out become grossly obese
40
Q

Huntingdons

A

-transgenic mice expressing a human Huntingdons disease CAG expansin show abnormal behaviour, ataxia and degeneration

41
Q

Gene Therapy

A
  • a number of experimental treatments are being investigated based on providing a wildtype allele to a homozygote with two defective copies of the gene
  • this requires a sufficient number of cells in the affected organ/tissue to take up and express the gene
  • for success DNA delivery must be highly efficient
42
Q

Gene Therapy

Viruses

A
  • very effective at attaching to cells and delivering genetic material
  • viral vectors have been widely used
43
Q

Gene Therapy

Cystic Fibrosis

A
  • adeno-associated viruses (AAV) can deliver wildtype CFTR genes to lung epithelial tissue
  • AAV are weak, non-pathogenic and only weakly immunogenic
  • continual doses are required as DNA isn’t integrated into host genome, the DNA is only transcribed in the cells
44
Q

Severe Combined Immunodeficiency

Description

A

-failure to male both T and B cells of the immune system

45
Q

Severe Combined Immunodeficiency

Causes

A
  • a number of genes can mutate to cause SCID
  • most common is X-linked SCID or X-SCID caused by a mutation in CD132 gene encoding a common component of interleukin receptors required for cell proliferation and differentiation
  • the second most common cause us ADA deficiency, adenosinedeaminase is required for dNTP production in lymphocytes
  • there are many other causal mutations
46
Q

Severe Combined Immunodeficiency

Treatment

A
  • bone marrow transplant is the best treatment but is limited by the availability of tissue matched donors
  • in the case that a transplant is not possible or fails, individuals have to be kept isolated to prevent infection that the immune system would not be able to fight off
47
Q

Severe Combined Immunodeficiency

David Vetter

A
  • David Vetter had the genotype XY and his wife was XsX
  • their first son was XsY and died of X-SCID
  • their second son was also born with X-SCID
  • they also has a daughter but neither her or the parents were a tissue match
  • but in 1984 after improvements in unmatched transplantation bone marrow from the sister was transplanted
  • this was initially successful but the transplanted cells were carrying a virus and the second son also died
48
Q

Gene Therapy as an Alternative to Bone Marrow Transplant

A

1) withdraw bone marrow cells from affected individuals
2) deliver a transgene consisting of a normal version of the affected gene
3) culture bone marrow cells in vitro to select a clone of cells containing and expressing the correct gene
4) return the cells to the patient, after whole body irradiation to kill existing bone marrow cells

49
Q

Severe Combined Immunodeficiency

Gene Therapy

A
  • bone marrow cells removed from patient
  • sequence for CD132 protein is engineered into the RNA genome of a retrovirus
  • the virus is used to infect bone-marrow cells and delivers its genome
  • reverse transcriptase converts the viral RNA genome into DNA which is able to integrate into the host cell genome
  • cells now contain a normal copy of the gene and can be returned to the patient
  • transgenic cells should then replace the patients lymphocytes with normal cells capable of mounting an immune response
50
Q

Gene Therapy

Pros and Cons

A
  • gene therapy for SCID has been trialled since 1990
  • first trials conducted with 2 girls with ADA deficiency, they were given engineered transplants over a 2 year period
  • they are still alive and continue to retain engineered cells but also require a back up therapy enzyme injection
  • in 2000, 2 boys with X-SCID treated with retrovirally delivered wildtype CD132 gene, 10 months after treatment they were thriving and 8 more patients were treated
  • in 2003 2 of the patients died of leukaemia, in each case the integrated transgene had inserted adjacent to a tumour suppressor gene affecting its function
  • further trials were halted and since then more of the patients have developed leukaemia

BLGY1232 (19 decks)

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