Unit 2 Human Genetics

what does nuclear transplant tell us about all cells within an organism

that they all share the same genome

who won a nobel prize for his nuclear transplant work, what was it about and when

john gurdon, somatic cell nuclear transfer, 2012

explain who dolly the sheep is and process used

first mammal to be cloned from adult somatic cell, collected oocyte, removed spindle, did nuclear transfer from somatic cell in different sheep, implanted oocyte back into first sheep, dolly born!

dolly the sheep birthday

july 5th, 1996

what was special about dolly the sheep

first mammal to be clones from adult somatic cell, 21 animal species have since been cloned

what did cloning experiments teach ua

somatic cells contain all the DNA required to form a new individual

cells undergo changes in gene expression that are ___ through cell division but do not alter DNA sequence

stable/heritable

epigenetics

heritable changes in gene expression that are not due to changes in DNA sequence

what can epigenetics do in terms of reproduction

fertilized egg can develop into an adult containing many cell and tissue types that carryout different functions

epigenetic mechanisms

dna methylation, histone modification, non-coding RNA

where does dna methylation occur

5 position of pyrimidine ring, at CpG dinucleotides in genome

what is CpG methylation

(cytosine phosphodiester guanine); associated with gene transcription repression

once established, DNA methylated stated is maintained through ___

cell division

what are DNA methyltransfereases

enzymes that add methyl groups to cytosine (CpG dinucleotides)

DNMT1 acts on what type of DNA?

hemi-methylated DNA (when one parental strand is methylated and the daughter strand isn’t); this is how DNA methylation patterns are inherited through cell divisions. It’s the basis of epigenetic memory, and it keeps cell identity and gene silencing stable

where is DNA methylation especially important and why

brain (TET) enzyme bc it regulates gene expression involved in key brain functions; also neurons don’t reproduce

in the life cycle of methylation, how is a cytosine methylated de novo and how is it maintained?

de novo through DNMT3A and DNMT3B, maintained through DNMT1

what is responsible for active demethylation?

TET1-3 enzymes oxidize 5mC to 5-hydroxymethylcytosine (5-hmC), which is further processed and eventually replaced with unmethylated cytosine; indepdendent of replication

what is responible for passive demethylation

If DNMT1 is absent or inhibited during replication, new strands stay unmethylated

CpG islands can be used for what

to methylate transposable elements so they don’t mess up the gene expression of other genes

more CpG dinucleotides in genome are ____

methylated

where are islands of unmethylated CpG dinucleotides located?

near gene promoters

how many CpG islands in human genome

28000

what percent of genes have CpG island near promoter

40%

CpG islands destabilize nucleosomes allowing what?

binding of proteins that promote a transcriptionally active chromatin site

how does CpG methylation work

1. block transcription factors

2. recruit transcriptional repressors

example of repressor and what disease its variant can cause

methyl-CpG binding protein 2, variant can cause Rett Syndrome which causes severe developmental delay

how do we know methylation patterns respond to environmental stimuli

in identical twins, methylation signatures are almost identical early in life and then they diverge with age for twins who live in different environments

techniques to detect dna methylation

MeDIP, Bisulfite sequencing, NGS sequencing

explain MeDIP

fragmenting genomic DNA, use antibody that binds with methylated dna and isolate it and sequence it and those are regions of genome where we have dna methylation

bisulfite sequencing

unmethylated cytosines converted to uracil via deanimation, and then converted back to dna (so ultimately C>T)

ngs sequencing that are reliable for methylation detection

PacBio and Oxford Nanopore sequencing

why identify dna methylated sequences?

to do case control studies to associate dna methylation signatures with disease or cellular phenotypes (suggestive of RNA regulation)

what is EpiSign

clinical methylation assay designed to identify epigenetic signatures associated with specific genetic disease

how does EpiSign work

negative gene panel/exome testing, if Greenwood Genetic center has episign for this disease, if epigenetic signature matches suspected disorder, we can to genome sequencing to detect causal variant

kabuki syndrome example with epigenetic signature

compared to controls, kabuki syndrome has more methylation at PC1; if there’s an individual that has been diagnosed w kabuki but their genetic signature doens’t align then prolly wrong diagnosis

genomic imprinting

process by which only one copy of a gene in an individual (either from mother or father) is expressed, while the other copy is suppressed

what is the primary mechanism that drives genomic imprinting

dna methylation

if only have genetic info from mother or father, it is considered ___

lethal

how many imprinted genes across genome

100 genes

if there is a gene on paternal chromosome that causes disease, what mechanisms can cause someone to have this disease

deletion on maternal chromosome, uniparental disomy from only paternal, imprinting defect

when can assume imprinted

if no family history of genetic disorder

if “imprinted on maternal” then mother is ___

silenced

how to determine if variant is from mother or father

isolate the variant gene and test for that variant in both parents

how to test using methylation-dependent retriction enzymes

digest dna from patient, use variant restriction enzymes, the parent with the variant will have allele remain intact, the parent without variant will have allele fragmented

how to use linked genetic variants to determine who the disease allele is coming from

just use parents’ DNA sequencing and if the variant near the pathogenic variant was inhertied from father, we know its paternal

another way to see if variant from mother or father

PacBio seuqencing, get their haplotypes

if we see a pedigree that doesn’t work logistically, we can assume that ___ occured

imprinting

what causes uniparental disomy

nondisjunction and then trisomy rescue or diploid cells outcompete triploid cells

nondisjunction in meiosis I causes

2 gametes with trisomy and 2 gametes with monosomy

nondisjunction during meiosis II causes

2 normal gametes (M+F in each), 1 trisomy gamete and 1 monosomy gamete

what is trisomy rescue

one of the trisomy chromosomes is randomly tossed out during mitosis, anaphase lag

heterodisomy vs isodisomy

Inheriting two different, non-identical homologous chromosomes from a single parent. vs same chromosome from single parent (causes recessive diseases)

how frequent is uniparental disomy

0.02%, chromosome 16 has more trisomy rescure events reported

why do we have more maternal UPD than paternal UPD

maternal meiosis has a much higher rate of chromosomal error (nondisjunction), particularly as maternal age increases

detecting isodisomic UPD

• exome/genome sequencing or chromosomal microarray

• Identify regions of homozygosity that span an entire chromosome

• Parent samples not needed

detecting heterodisomic UPD

• exome/genome sequencing or chromosomal microarray

• Identify chromosome regions with mendelian inheritance errors

• Parent samples are required

innate immune system

rapid initiation, germline encoded, physical and chemical barriers, complement proteins, phagocytic cells, highly conserved

adaptive immune system

slow initiation (over days), gene segments, specificity, diversity, memory, clonal expansion, specialization, nonreactivity to seld

examples of innate immune cells

mast cells, dendritic cells, phagocytes, NK cells, epithelial barriers

examples of adaptive immunity cells

B lymphocytes, T lymphocytes, effector T cells, antibodies

function of thymus

t cell school

function of complement system

see bacteria

function of lymph nodes

immune cell communication

function of bone marrow

immune cell synthesis

lymphocytes express which two receptors?

b cell and t cell

why is it significant that multiple different genes encode for different receptors?

bc it allows for variability and diversity; also a cell only survives if the receptors are functional

each lymphocyte has ___ copies of the same receptor

many

inborn error of immunity (IEI)

genetic disorders of the immune system, also known as primary immunodeficiency

what was one of the first discoveries of IEI

BTK (no b cell)

in one teaspoon of blood, how many t cells and neutrophil?

5 million T cell, 17.5 million neutrophils

warning signs of IEI

unctrolled inflammation, severe allergies, autoimmune, four or more infections in a year, just getting sick a lot

how do genomic discoveries help us

targetted therapies and improving our understanding of ummune system

how many monogenic disorders are now recognized

>500 single gene disorders and 67 novel monogenic defects

Hyper IgE syndrome when descirbed and when genetic discovery

1966 and then 2007

why know about genetic causes of IEI

targeted inhibitor, repurpose medications, absent T cells requires different treatments

when did we learn a lot about the function of thymus

1965 by description of DiGeorge Syndrome

what is Huamn Toll like receptor 3 for

essentisal for protection against herpes virus in the brrain, but was not observed in experimental models only when studying patients with the virus

how can transfusion therapy work

genetic advances ultimately led to successful gene therapy in ADA-deficient SCID

LRBA deficiency

immune dysregulation

what did they do to solve LRBA deificicy

repurposed rhumatoid arthisitis drugs to this disease

how to treat IEI

monitorying, antibody replacement therapy, stem cell transplant, gene therapy

most common genetic cause of SCID

X-linked, usually diagnosed before born, stem cell transplant can cure

severe combined immunodeficiency (SCID)

deficiency of T cells and sometimes other immune cells depending on genetic defect, rare, fatal without treatment, bone marrow transplant can be effective if performed prior to 3 months of age

when was X-SCID genetic diagnosis

1993

T cell receptor excision circle (TRECs)

newborn screen, detects specific DNA only found in newly generated T cells that have left the thymus, can take the dried blood spot that newborns have to detect TREC’s and if absent, then suggests that no T cells

what is a possible treatment for pt with abnormal TREC

thymic transplant where u remove the donor’s pre-T cells, and implant into reciepient’s thigh

issues with newborn screening for T cell lymphopenia

high number of false positives

NIAID centralized sequencing initiaitve

1505 individuals from 1000 families, many had prior genetic testing, molecular diagnosis was correlated with younger age, male sex, high number of organ systems involved

molecular diagnosis rate of NIAID

32.7%

exome is about ___ of the genome

1-1.5%

current methods use ___ sequencing

short read

what is translational genomics research

interdisciplinary field of study that focuses on turning basic scientific discoveries about the human genome into practical, evidence-based medical diagnosis

clinical diagnosis

based on clinical features, like epilepsy, intellectual disability, low platelet count

genetic diagnosis

much more specific with genetic testing, like trisomy 21

chromosomal microarray

can detect large insertions/deletions

benefits of genome sequencing

comprehensive, detects mitochondrial variation, detects most variant types, can do phenotype-independent analysis (so if it’s complex, it’s chill), discovery of new disease genes, sequence non-coding regions of genome, allows for later analysis

drawbacks of genome sequencing

lots of data, requiring non-trivial computational and storage requirements, complex analysis, more costly than other types of genetic testing, unexpected findings