block 1 biochem final

Epigenetics

Changes in gene expression, how much is made

2 main epigenetic mechanisms

DNA cytosine methylation and histone modification (acetylation and methylation)

What is the effect of histone acetylation

Neutralizes positive (basic) charges, causes weaker DNA-histone interactions

Histone acetylation is associated with which chromatin state

Euchromatin (open, active)

What is genome imprinting

Parent specific silencing of chromosomal regions

Paternal micro deletion of chromo 15 leads to

Prader-Willi syndrome

Maternal micro deletion of chromo 15 leads to

Angelman syndrome

Symptoms of prader-Willis (POHP)

Obesity/Overeating, hypogonadism

Angelman HAILS

Hand flapping, ataxia, intellectual disability, laughter, seizures

How can uniparental disomy cause Prader-Willis

2 maternal chromo 15

How can uniparental disomy cause Angelman

2 paternal chromo 15

What repeat expansion causes Fragile X

CGG expansion in FMR1 gene

How does Fragile X become epigenetic

Expanded CGG repeats lead to heavy methylation and silencing of FMR1

Cancer cells demonstrate what methylation pattern

Global hypomethylation and tumor suppressor hypermethylation

5-azacytidine

DNA methyltransferase inhibitor used as demethylating agent (leukemia)

Vorinostat

Histone deacetylase (HDAC) inhibitor for T cell lymphoma

What evidence shows epigenetics change over lifetimes

Mono twin studies showing Inc methylation differences with age

What is RFLP

Restriction fragment length polymorphism; DNA variation causing diff restriction fragment sizes

What 2 genetic changes produce RFLPs

SNPs and VNTRs

How do SNPs create RFLPs

By creating or abolishing restriction enzyme sites

How do VNTRs create RFLPs

Different number of tandem repeats produce fragments of different lengths

SNP-RFLPs are primarily used for what

Disease markers and gene mapping

VNTR-RFLPs are primarily used for what

DNA fingerprinting, forensics, and paternal testing

What restriction enzyme site is lost in sickle cell

Mstll site

In sickle cell, the mutant allele produces what RFLP pattern

Larger fragment bc mstll cannot cut

How can PCR diagnose sickle cell

Amplify b-globin region then digest with Mstll

What are ASO probes

Allele-specific oligonucleotide proves that bind only exact DNA sequence matches

Why are ASO probes used for sickle cell

Disease is caused by one specific nucleotide mutation

What gene is mutated in cystic fibrosis

CFTR

Most common cystic fibrosis mutation

F508 (in frame causing loss of phenylalanine)

Why can PCR and ASO probes diagnose cystic fibrosis

Mutation is highly specific and common

Why aren’t PCR and ASO probes used for PKU

PKU has over 400 different mutations through PAH gene

How is prenatal PKU diagnosed

Link disease to family specific RFLP marker

What is followed in PKU testing

A linked marker, not actual mutation

What disease is caused by CTG trinucleotide repeat expansion in DMPK

Myotonic dystrophy

Why can RFLP analysis diagnose repeat expansions

Expansion changes fragment size

Anticipation appears in

Huntingtons and myotonic dystrophy

What DNA feature forms the basis of paternity testing

VNTR polymorphisms

What modern forensic approach uses large SNP databases and family trees

Forensic genetic genealogy

What is gene therapy

Introduction of genetic material to treat disease

Does gene therapy replace abnormal genes

No, it adds a normal copy

Why does gene augmentation work best for autosomal recessive diseases

Adding one functional copy restores normal protein production

Somatic vs germline gene therapy.

Somatic is patient specific, germline is inherited

What type of gene therapy is used in humans

Somatic gene therapy only

What is ex vivo therapy

Cells removed, modified outside of body, reinfused

What is in vivo therapy

Gene delivery directly into patient tissues

Two essential components of successful gene therapy?

Therapeutic gene and delivery vector

Why are viruses commonly used as vectors

They naturally deliver DNA into cells

Major advantage of retroviral vectors?

Stable integration into host genome

Major disadvantage of retroviral vectors?

Insertional mutations

What cells do retroviruses infect

Dividing cells

Major advantage of adenovirus vectors?

Infect non dividing cells

Major disadvantage of adenovirus vectors

Strong immune response and transient expression

Do adenoviral DNA integrate into genome?

No, it remains episomal

3 major gene therapy strategies?

Gene augmentation, targeted cell killing, and targeted inhibition of gene expression

Which gene therapy strategy is used against cancer

Targeted killing of specific cells

Which gene therapy strategy might be useful for Huntingtons

Targeted inhibition of gene expression

What was first successful human gene therapy

ADA-deficient SCID treatment

How was ADA-SCID treated?

Patient lymphocytes modified ex vivo using retroviral ADA vector and reinfused

Major complications of early ADA gene therapy?

Some patients developed leukemia due to insertional mutations

components of CRISPR/ Cas 9?

Cas9 nuclease and guide RNA

What does Cas9 do

Creates targeted DNA strand breaks

What repair pathway commonly creates knockout mutations after Cas9 cleavage

NHEJ (nonhomo)

Which repair pathway allows precise editing

HR (homo)

NHEJ vs HR

NEHJ is error prone, HR is precise

High yield CRISPR sickle cell example

Ex vivo editing of BCL11A increased fetal hemoglobin (HbF)