Lesson 23

Question 1

what four ways can DNA methylation cause human disorders?

Answer 1

1. damage on DNA that cause tumors or genetic diseases
2. aberrant silencing of gene expression causing genetic disorders
3. aberrant expression of specific genes (cancer)
4. diseases associated with imprinting Beck-Wiederman syndrome or Angelman syndrome

Question 2

what is one of the of the main damages to DNA that causes tumors or genetic disease?

Answer 2

damage resulting from deamination

Question 3

what do we mean when we say “aberrant gene expression”?

Answer 3

quite a large number of genes are affected by the process → oftentimes a mutation in a reader or writer

Question 4

what is Rett syndrome (RTT)?

Answer 4

a generic and progressive neurological disorder (rare) that mainly affects females

Question 5

describe the early, post natal phase of RTT:

Answer 5

appears symptom free: there are no issues detected - only detectable through prenatal genetic testing

Question 6

describe the second phase of RTT:

Answer 6

there a developmental stagnation followed by a regression phase → during this phase they start to be disinterested in the world outside (unresponsive) and can become violent (screaming) or lose all skills they have previously developed

Question 7

what happens to the brain during the regression phase?

Answer 7

the growth of the brain slows down and patient suffer from microcephaly

Question 8

what happens after the regression phase?

Answer 8

the disease stabilizes but severe neurological damage is already done and many are unable to express themselves or communicate

Question 9

what are some of the main clinical features of RTT?

Answer 9

severe ID, loss oc expressive language, loss of hands use, stereotypical hand movements. apraxia, breathing abnormalities, epilepsy, scoliosis, bone fragility, heart issues

Question 10

what is the first and second hallmark of RTT?

Answer 10

regression followed by loss of the use of their hands

Question 11

what chromosome is RTT linked to?

Answer 11

X → only in females

Question 12

describe the specific gene mutated in RTT:

Answer 12

gene is X-linked and encodes for methyl-CpG-binding protein 2 (MeCP2), a reader of DNA methylation that brings histone acetylases on DNA → is a transcriptional repressor that binds methylated DNA and silences gene
expression reading methylated CpGs without sequence specificity

Question 13

describe MeCP2:

Answer 13

highly abundant nuclear factor that is ubiquitously expressed (particularly in neural tissue) and in mature neurons appears t function as a chromatin condensing factor

Question 14

what type of disorder is RTT:

Answer 14

mongenic

Question 15

what disorders to MeCP2 mutations cause in males?

Answer 15

neonatal encephalotomy (severe intellectual disability, seizures, and breathing issues) and Rett syndrome (mother has mutation on one X and passes it to son, much less severe but presents symptoms)

Question 16

what type of mutation is RTT associated with?

Answer 16

loss of function

Question 17

what is an example of MeCP2 gain of function mutation?

Answer 17

MeCP2 duplication sundrome

Question 18

what is MeCP2 duplication syndrome?

Answer 18

the subjects have 2 or 3 copies of the wild type gene (more copies the worse the phenotype) - resembles RTT → intellectual disability, bad seizures, respiratory infections due to the fact that they cannot swallow properly due to breathing issues

Question 19

who is the carrier for MeCP2 duplicate syndrome?

Answer 19

the mother

Question 20

what three things is the clinical severity of RTT dependent on?

Answer 20

1. x-activation patterns
2. MeCP2 mutations
3. presence of modifier genes

Question 21

what are the two main types of MeCP2 mutations?

Answer 21

• premature stop codon and framshifts
• missense mutations in the TRD or CTD or methyl binding domain

Question 22

why is it easier to study KO MeCP2 in male mice?

Answer 22

they are hemixygous, therefore everything is more homogenous → the onset is earlier and more severe

Question 23

what is an issue with many pre-clinical studies being performed only on male mice?

Answer 23

because of the hormones, female mice behave diversely depending on which period they are in and this poses an issue for understanding the disorder

Question 24

what occurs in the brains of KO MeCP2 mice?

Answer 24

there is a reduced volume and it is believed this reduction is not given by the loss of cells, but is given by the fact that the neurons that are without MECP2 are smaller in soma, less arborized, they do have less spine in synaptic contacts and all of these leads to a brain
in which the number of cells seems to be the same with respect to a normal one, but everything has a higher densities, and this leads to a smaller brain

Question 25

what happens if there are less spines in the mouse brain?

Answer 25

there are going to be less synapse → this is a synaptogenesis disorder = there are less contacts and networks are not functioning properly

Question 26

what is there an imbalance between in KO MeCP2 brains?

Answer 26

imbalance between excitatory and inhibitory activity → imbalance changes depending on the brain district so there are regions that are hyperoxidated and regions that are hypooxidized

Question 27

what did BA inject at high dosages to go from no MeCP2 to normal MeCP2 expression in mice?

Answer 27

tamoxifen

Question 28

what are two dangers of transducing the new gene into the affected cells?

Answer 28

• the issue of getting good viruses with good diffusion in the brain and to be able to control the level of those dosages
• the general toxicity of the high dosage of MeCP2 the the virus could induce

Question 29

what are the goal of potential pharmacological approaches?

Answer 29

meant to modulate the molecular pathways disrupted by the impairment of MeCP2 activity

Question 30

what is MeCP2 also relevant for besides wild type neurons?

Answer 30

astrocytes → it has been proven that astrocytes secrete factor that are detrimental for the neurons, so we can try to understand what those facts are and how to isolate and delete them

Question 31

if a drug is able to be created for RTT, when will it most likely need to be given?

Answer 31

the patients entire life

Question 32

how do stem cells act in neurons?

Answer 32

function through bio standard mechanisms → if we transplant stem cells in a mouse that is affected by stroke or multiple sclerosis (conditions in which
there are stimuli that attract the cells), stem cells go to the brain and start to secrete factors that are useful for the brain itself

Question 33

what is lacking in Rett syndrome?

Answer 33

it is well known that a neutrophil factor is lacking →BDNF: but you cannot provide BDNF because it doesn’t cross the blood brain barrier

Question 34

if we treat KO neurons with NPCs the phenotype is rescued, what does this mean?

Answer 34

shows that NPCs are able to secrete something that has a positive effect on defective neurons → could be a good cure but in particular can be helpful for identifying which pathways might be effective

Question 35

what is Rett syndrome most likely caused by?

Answer 35

subtle activation or dysregulation of different genes (those patents who have too much “transcriptional noise” in their brains) → defined as a dominant disorder

Question 36

what is ICF syndrome?

Answer 36

immunodeficiency, centromere instability, and facial anomalies syndrome → rare genetic recessive disorder in which the mutated gene is mainly DNMT3B

Question 37

how are these patents diagnosed?

Answer 37

they present with multiple recurrent infections (the number of B cells is incredibly reduced) and it is possible to find aberrant chromosome shape

Question 38

what is DNMT3B?

Answer 38

a de novo methyl transferase that methylates the centromeric and peri-centromeric DNA, particularly chromosomes 1, 9, and 16

Question 39

how is ICF inherited?

Answer 39

inherited from the parents → from two healthy carriers and gain two mutated alleles

Question 40

where are the majority of the mutations in ICF patients?

Answer 40

most are in the catalytic domain: these are hypomorphic DNMT each they are function but greatly reduced - sometimes there are also mutations in the amino terminal portion of the protein

Question 41

what region is sometimes mutated in ICF patients?

Answer 41

PWWP → important in recognizing some specific post translational modification of histones

Question 42

what do we know for sure about ICF?

Answer 42

• there is a general reduction of methylation, so is
  not only the pericentromeric heterochromatin; in general, there is a reduction of 40% in these patients
  and this is occurring everywhere (in every region)
• we can see a decrease of
  methylation in different regions, and this leads to spurious transcription

→ we have a disorder in which we have an unscheduled transcription, the activation of non-coding RNAs and
microRNAs, transcriptional start sites that should not be used are activated: so, we have different defects in gene expression that leads to this phenotype

Question 43

how does chromatin tie into epigenetic pathways that cause cancer?

Answer 43

chromatin can express different transcriptional factors, DNA sequence mutations can affect those transcriptional factors and different epigenetic mechanisms might go to the affect the gene expression leading to cancer

Question 44

what two contradictory features does cancer generally have?

Answer 44

hypomethylation and hypermethylation

Question 45

where does hypomethylation in cancer usually affect?

Answer 45

generally affects regions where methylation mostly occurs = the regions relevant for genomic defense

Question 46

what two things can hypomethylation induce?

Answer 46

can either induce activation, and this is dangerous for genome stability, or it can activate sequences that can start to jump around or produce viruses

Question 47

where can hypomethylation occur?

Answer 47

oncogenes