NOT ON EXAM -Suzie Chen 4: Sickle Cell + CFTR

fetal hemoglobin in fetal red blood cells transports oxygen from the mothers _______ ________ to the ______ and _______ of the fetus

blood stream

tissues and organs

what is the difference between fetal hemoglobin and adult hemoglobin in terms of which chains they consist of?

fetal = 2 alpha and 2 GAMMA
adult - 2 alpha and 2 BETA

is fetal hemoglobin produced before of after birth?

when do fetal hemoglobin levels begin to gradually decrease?

do adults have any fetal hemoglobin?

• BEFORE around 6 weeks into pregnancy

• 2-4 months down to 1% at 1 years old

• YES about 1%

what is an advantage of fetal hemoglobin?

it binds more STRONGLY to oxygen then adult hemoglobin

sickle cell anemia is most frequent in african/ caucasion individuals

sickle cell anemia is a single-point mutation of the codon for the _______th amino acid which is normally ______ but is mutated to ___________

african

6th (glutamic acid → valine)

anemia is caused by _________________________ leading to complications in the

• heart

• lung

• brain

• joints

inability of hemoglobin to carry sufficient oxygen

a __________test can be taken to check for sickled red blood cells

are newborns required to take blood tests?

where is the test done in adults? where is it in babies?

• hemoglobin S blood test

• YES

• blood taken from arm vein (adults) foot or finger (babies)

if you get a positive result in the hemoglobin S screening/ blood test does that mean you have sickle cell disease? what further tests can be taken?

NO it means you have at least one mutated gene need to take another test to see if both are mutated since sickle cell disease is recessive

you can check for a low red blood cell count (anemia)

_______ cuts normal B-globin gene at _____ sites producing ___ restriction fragments that have ____bp and ____bp

radioactively labeled ______ spans region of sickle cell mutation from site 1 to 2 (ONLY 1st 1150 fragment NOT 200)

• MSII

• 3

• 2

• 1150 and 200

• probe

which enzyme cleaves normal b-globulin in 3 sites producing 2 restriction fragments?

MstII

The sickle cell mutations result in loss of ______ site ____

MstII cuts mutated B-globin gene only at sites ___ and ____, producing a SINGLE larger restriction fragment of ____________ bp

• MstII site 2

• 1 and 3 1350bp

do the following patients have sickle cell disease according to the bands on their southern blot

• 2 bands - 1 at 1350bp and 1 at 1150bp

• one band at 1150 bp

• one band at 1350 bp

• NO they are heterozygous

• NO they are homozygous for the correct MstII which cuts 3 sites leading to 2 fragments one which is 1150bp long

• YES they are homozygous for the mutated MsII which only cuts 2 sites leading to one large fragment that is 1350 base pairs long

how does the southern blot work?

since DNA is _____ charged due to the OH group of the sugar it moves toward positive electrodes in the gel

shorter fragement (1150— normal) will be farther since it is smaller it can travel faster compared to a longer fragment (1350 — sickled)

how are we able to visualize the bands of 1350 base pairs and 1150 basepairs?

• negatively

• 1150 farther down

• PROBE spanning from site 1-2 (1500bp) will hybridize once transferred to a nitrocellulose membrane or nylon

when completing PCR of a normal sequence of sickle cell gene how many fragments are created? how many restriction enzymes are present?

when completing a PCR of a mutated sequence of sickle cell gene how many fragments are created? how many restriction enzymes present?

• 4 fragments 3 restriction enzymes

• 3 fragments 2 restriction enzymes

how large are each of the four fragments created in PCR of normal sickle cell gene ?

what do you expect to show up in electrophoresis after cleavage and staining?

256

201

181

88

how large are each of the four fragments created in PCR of MUTANT sickle cell gene ?

what do you expect to show up in electrophoresis after cleavage and staining?

256

201+ 181 = 382 (bc/ they were not cut only 2 restriction enzymes)

88

what fragment base pair numbers would you expect to see in a stained electrophoresis? how many bands would show up?

• homozygous AA (normal individual)

• heterozygous AS

• homozygous SS (sickle cell positive)

• 256 + 201+ 181 + 88 (4 BANDS)

• 256+201+181+382+88 (5 BANDS)

• 256+ 382+88 (3 BANDS)

do you have to use a probe when completing PRC and then electrophoresis to determine if a patient has sickle cell disease?

NO because PCR is determined by SPECIFIC primers

where as southern blots use genomic DNA so you have to use probes to distinguish between the ends

TREATMENT OF SICKLE CELL ANEMIA:

• Drug Treatment: allow more cells to _______the microcirculation before sickling

• Allogenic Transplant: use _____ _______ stem cells from a donor without sickle cell disease

• Ex. Vivo Gene Therapy: bone marrow cells are modified by adding the ___ _____ gene, using a retroviral vector with gene editing to REACTIVATE ______ or correct the disease mutation

• In Vivo Gene Therapy: _____ gene editing in patients could circumvent (eliminate) the need for transplantation of modified patient cells IF sufficient efficiency and safety can be achieved (replace mutated gene with ____ _____)

• drug = transit

• allogenic = bone marrow

• ex vivo= b-globin fetal hemoglobin

• direct wild type

• __________ was the first FDA-approved drug to treat sickle cell disease by inhibiting the polymerization of the ____- Conformation (deoxy form of hemoglobin— lack of oxygen)

• this drug induces the production of _______ ___________by an unknown mechanism, the amino acid sequences between _______ and _____ are different

• the second drug is ________, which to the R-conformation (fully _______ form) of HbS reducing the fiber-forming ____-conformation of HbS

• hydroxyurea T

• fetal hemoglobin HbF HbS

• voxelotor oxygenated T

correction of sickle cell disease at the MOLEULAR level through replacing a patient’s bone marrow with bone marrow from an unaffected tissue match donor

provide proof of concept that GENETIC MANIPULATION of DEFECTIVE BONE MARROW CELLS might be equally therapeutic (CRISPR)

what is a limitation?

allogenic transplant

hard to find match (only 10% have match)

FUTURE Treatment Options:

1. _______ transplant: genetic manipulation of the patient’s own stem cells then transplant them back into the patient

2. permanent integration of a therapeutic ______ gene together with key regulatory elements into the DNA of the stem cells became feasible with the development of a robust gene transfer system

3. this gene transfer system allowed for sustained endogenously regulated expression of __-globin that is sufficient to revert SCD in patients

4. recent progress in understanding the regulation of _____ switching from ____ to ______ that occurs in birth provide new approach towards SCD treatment

5. can target regulatory elements of ___- globin gene in ______

6. can silence RNA to reduce the expression of BCL11A a repressor of the ____- globin gene in HbF

1. autologous

2. HBB

3. beta

4. HbF HBF to adult hemoglobin

5. gamma Hb

PILL that treats sickle cell disease:

• hemoglobin ________ under low oxygen conditions causing the sickle cell shape

• high levels of ______ = less polymerized HbS

• fetal hemoglobin genes _____ and ______ encode the y-globin and the adult ___ gene encodes for b-globin

• HBG1 , HBG2, and HBB are regulated by a cluster of _____ and a ____ control region (LCR), to upregulate ______ expression and fil red blood cells with adult hemoglobin

• in adults, the _____ and ______ promotes are silenced through the transcription of _______

• HbF can be raised in adults through the downregulation of ______

• we don’t know how _______ increases fetal hemoglobin, it just does

• polymerizes

• HbF

• HBG1 and HBG2 HBB

• enhancers locus HBB

• HBG1 and HBG2 BCL11A

• BCL11A

• hydroxyurea

When Widely Interispersed Zinc finger (WIZ) is downregulated by a molecular glue degrader dWIZ-2 is

HbF expression increased or decreased and how?

increased

• does CFTR effect african/ europian populations?

• CFTR normally works as a _____ ____ _____. what is the consequence of its dysfunction?

• which organs are impacted the most by CFTR?

• european

• chloride ion channel

  mucous thickens water is not attracted by NaCl to thin mucus (accumulate in ducts)

• liver and pancreas

what causes mucus to be thickened in cystic fibrosis?

mucus becomes a site of bacterial infection

DNA from bacteria thickens mucus

class 1 cystic fibrosis is associated with NO functioning

CFTR

how many different class mutations exist for cystic fibrosis?

7

which three base pairs are deleted in cystic fibrosis?

which amino acid is lost in the process?

CCT

loss of Phe (TTT)

how can you tell if a patient has cystic fibrosis?

• sequence their DNA using Sanger method

• PCR with restriction enzymes

target gene mutations treat cystic fibrosis NOT CURE

• there are CFTR __________ available to improve the function of faulty proteins

• different combination of medication for children 6 months and older

• approved in patients with certain mutations in the CFTR gene. whats an example of a mutation?

• modulators

• F508del mutation

clinical appreciation of recombinant DNA techniques:

• ______ for treatment of diabetes

• factor 8 for hemophilia

• _____ to prevent hepatitis and covid

• ________ __________ therapy via IV infusions (ex. lysosomal storage disease)

• mutated _____ CANNOT degrade a specific substrate in lysosome causing buildup of toxic materials in lysosomal storage disease

• _________ disease is another example of using enzyme replacement therapy. mutations in glucocerebrosidase lead to buildup of lipids in spleen or liver causing muscle dysfunction and difficulty breathing/ seizures

• gene therapy

• insulin

• vaccines

• enzyme replacement therapy

• gauchers

what are two examples of diseases that can be treatment with recombinant enzyme replacement therapy?

what is the mutated enzyme needing to be replaced in each?

lysosomal storage disease - enzyme to degrade contents of lysosome

gauchers disease - glucocerebreosidase