Module 1 - Genetics of Obesity Flashcards
What is a genome? (3)
- genetic material of an organism
- hereditary information
- “coding and non-coding info
C-value paradox
organisms complexity not correlated with genome size
humans:
25,000 genes (small - coding region)
2.9 billion base pairs (large - includes non-coding)
Use genomic information for…
4 P’s
- prediction
- prevention
- personalization
- participation
Personalized medicine (nutrition) vs precision medicine (nutrition)
Personalized
- not feasible
Precision
- considers similar individuals
- targets specific need (ex. low iron absorption)
Common (older) method for sequencing genome
Sanger method (1980) aka dideoxy method
- amplify DNA
- 4 reacting mixtures
- primed DNA to be sequenced, DNA polymerase, nucleotides, 1 of 4 labeled chain terminating nucleotide
- chain build and ends at random
- gel electrophoresis determines length (smallest travels furthest - read bottom to top - smallest to largest - 5’ to 3’)
DNA name
deoxyribonucleic acid
2 groups who sequenced the genome
- goal
- primary difference between approaches
Public Consortium (human genome project)
- sequence ALL 3billion BP using BACs
- coding and non-coding
Private Consortium (celera genomics)
- sequence all genes shotgun method
- coding only
what is a BAC
bacterial artificial clone
- circular bacterial plasmid and gene of interest ligated (bond together)
of BPs used in shotgun and BAC
BAC = 150,000bp Shotgun = 2000bp, 10000bp
BAC approach advantages (2)
1 - Reduce chance to misassemble because location is better known
2 - sequencing step is quick, dont need to sequence both ends of DNA
BAC approach disadvantages (2)
1 - laborious and time consuming
2 - bioinformatically heavy
Shotgun approach advantages (1)
experimentally quicker
shotgun approach disadvantages (2)
1 - experimentally quicker (sequenced from both ends)
2 - major problems dealing with “repeat sequences”
BAC method DNA sequencing steps
1) Form BACs
- approximately 150,000bp
- 30,000 formed)
2) create BAC library (amplify in bacteria / ecoli)
3) sequence landmarks (200-500bp)
- dna postal codes
- overlap and create a map
4) form M13 library
- smaller pieces fit into specific BAC
5) sanger sequencing of M13
6) sequence alignment
Shotgun method of DNA sequencing steps
1) restriction enzymes cut DNA into 2000 and 10,000 bp
2) form base plasmid library
* skip steps 3 and 4
5) sanger sequencing
- dont know which piece belongs to which gene or chromosome
6) sequence alignment
- align 2000bp to 10,000bp
- huge problems with repeats - requires powerful computer
Importantance of studying DNA (4)
- inter-individual variability in disease phenotype and response to intervention (pharmacogenomics/nutrigenomics)
- infuence gene expression and function (gene dosage/ more copies)
- genes underlying common diseases (genetic targets for pharma - SNPs)
- how disease is passed between generations (transgenerational effect)
% genetic component of obesity
40-80% (strong!)
best method to study if environmental impact depends on genes
Twin studies (genetically identical)
- intervention, control for environmental factors
- similar impact on twins, huge variance between individuals
microscopic structural variations in genomes (2)
aneuploidy
- abnormal # of chromosomes
heteromorphism
- visible region of a chromosome that varies in size or morphology
small-scale variations in the genome (5)
1) non-coding DNA (junk DNA)
2) copy number variants (CNVs) and segmental duplications (SDs)
3) inversions
- DNA segment reverse orientation to rest of chromosome
4) translocations
- DNA portion changes position w/o changing sequence
5) single nucleotide polymorphisms (SNPs)
- single bp changes
non-coding dna regulation of protein-coding dna (3)
1) non-coding functional RNA’s (tRNAs, rRNAs, siRNAs, etc)
2) promotor/repressor seq.
3) sequence recognition during meiosis (centromeres)
Difference between segmental duplications and copy-number varients
SDs spread out
CNV side by side
potential issue when studying SDs and CNVs
reference genomes are only as good as the people in that pool
- highly variable between populations, geographically, etc
how do CNVs and SDs arise?
recombination within a chromosome
recombination between non-homologous chromosomes (maternal/paternal)
*happens in part due to repeat sequences in the genome
SD size
> 1kb
- 90% sequence identity (not perfect match but very similar)
- constitutes 5% of genome
SD location (is it random?)
spread over a persons genome
- not random
- 2% of SDs on chrome 3, 12% on chromosome 22
- still unclear why
Cause of SD formation
Replication errors or non-allelic homologous recombination (high sequence similarity but are not alleles)
- what determines where they occur still unclear
SD direct effects on phenotype
gene expression (dosage sensitive genes) - specifically genes involved with immunity (mean not random
SD indirect effect on phenotype
structural rearrangements within chromosomes
- can be positive or negative
major example of SD
- how does it work
CCL31L
- increases # cause reduction in susceptibility to HIV infection and progression of AIDS
- protein product competes with HIV binding site (CCR5 - chemokine receptor type 5)
*controversial
CCL31L - SD
- HIV-1/AIDS more common in smaller # of duplications
- why might # of duplications be higher in a places with higher disease prevalence?
possible adaptation due to environmental pressures
- reason still unclear
- controversial
CNV size
- proportion of genome?
1kb - 1mb
- constitutes 12% of the genome
CNV location
CNVs found side by side on chromosomes
CNV contribution to variations in genome
randomly selected genomes
- differ by atleast 1% due to CNVs
- relevance
- humans are 99% similar (not 99.9% - old theory)
- very common in human genome
cause of CNV formation
replication errors or non-allelic homologous recombination (high sequence similarity but not alleles)
- what determines where they occur still unclear
- same as SDs
Distribution and frequency of CNVs
prevalent across genome
- 10% of protein coding genes have CNVs
CNV direct effect on phenotypes
effect gene expression (dosage sensitive genes)
- enriched in regions where genes are involved with immune function and defence responses to bacteria
provide redundancy
- organisms can evolve new or modified functions
- 2 copies, 1 mutates – HUGE effect
- multiple copies, 1 mutates – small/no effect
- translocation, deletion or duplication events can affect exons, proteins aquire diff structure and function
CNV major example
AMY1 (alpha amylase)
- high # reported in populations consuming high carbohydrate (starch) diets
- more copies, more amylase in saliva to break down starch
- relevance
- shows potential effect of environmental pressures of #
CNVs involved with obesity
**AMY1 - alpha amylase
Others:
Chromosome 4
- UCP1 (thermogenesis)
- IL5 (inflammation)
Chromosome 16
- large CNV regions (28 genes) rare varient
- duplications – extreme leanness
- deletions – morbid obesity
Effect of large copy # of AMY1 (alpha amylase)
more copies, more protein, lower BMI
- found in saliva and blood
- link between ability to digest and body weight
Imprinted genes
- sex specific imprints (methylation)
- only receive one copy of gene
- certain genes always silenced from mom, others from dad during formation of egg and sperm
- silenced throughout life, reset sex-specific imprints and totipotency during egg/sperm formation
- necessary for normal development
Imprinted genes effect on health risk
protection from diploidy no longer exists
- mutation in the only copy, gene no longer expressed
- ex. Prader-Willi syndrome
Genetic conflict theory (imprinting)
- males try to maximize use of maternal resources so offspring become stronger and healthier
- females minimize use of maternal resources so all offspring survive
- imprinting (methylation) causes battle for nutrients
Imprinted genes and obesity
- controls resetting of parental imprints during gametogenesis (chromosome 15)
- equal occurrence in males, females and all ethnicities
- Prader-Willi Syndrome (PWS)
- Angelman Syndrome (AS)
- together most common cause for life threatening obesity in children
Pradaer Willi syndrome (PWS)
- cause (gene)
- clinical signs
4Mb deletion/silencing of ‘paternal’ chromosome 15 (70% of cases)
- multiple genes in this region
- SNRPN (gene encoding component of mRNA splicing)
- obesity, excessive hunger, hypergonadism, sleep apnea, behavioural problems, mild/moderate retardation
remember: P - paternal
Angelman Syndrome (AS)
- cause (gene)
- clinical signs
Deletion UBE3A on ‘maternal’ chromosome 15 (70% of cases)
- ubiquitin pathway
- severe mental retardation, absence of speech, smaller heads
- but no obesity**
Allele
alternate form of a gene at a given locus on a chromosome
Single Nucleotide Polymorphisms (SNPs)
- amount and frequency
10million SNPs within human population
- 1 every 300 nucleotides
- most frequent source of polymorphic changes
- variation based on geographic location
- 14000 people only 200,000 SNPs
- can tell with 50% accuracy where people are from (Europe study)
SNPs associated with obesity
FTO and MC4R
- 6 others less significant
- 8 SNPs together explain
FTO gene
- highly expressed in hypothalamus
- anorexigenic (appetite surpressing)
- demethylase (animal / in vitro)
- potential adipocyte development (white vs brown fat) and thermogenesis
SNP ‘risk’ allele
- weighed 3kg more on average
- consumed 200kcal more on average
Direct to consumer (DTC) genetic tests vs traditional predictors
- as predictors of obesity
- SNP (FTO or top 32) vs family
childhood obesity and parental obesity prediction
- 75% accuracy
genetic prediction (FTO or top 32)
- 57% accuracy
relevance**
- more SNPs found that associate with obesity, but
