2. Introduction
AUTOPHAGY
Protection from cytotoxicity, pathogens and protein aggregates.
Helps metabolism recycling
Process:
ULK1 complex receives
signal
Formation of transient double
membrane bound structure
called phagophore.
Expansion: becomes an
autophagosome
LC3-II recruits the
cytoplasmic cargo so the
autophagosome can take it
Autophagosome fuses with endolysosomal
system: Hydrolitic enzymes degrade the
cargo
3. ATG-7
It encodes as an enzyme which activates ATG12.
Preautophagosomal phagophore is promoted by this gen.
It facilitates lipidation of the protein LC3-I.
4. General objective
Highlight the importance of the AGT7 gene in the autophagy process by the
study of five unrelated families with recessive ATG7 variants
cerebellar hipoplasia
Thin posterior corpus callosum
Ataxia
Musculoskeletal abnormalities
Development delay
Facial dysmorphism
6. Sequencing
methodology
With two pioneering methods, it has
some aspects in common and it is
classified into chemical and enzymatic
techniques.
CHEMICAL Chemical hydrolysis.
Original design aplicable to
DNA seqcuences (<250
nucleotides)
It has three stages.
ENZYMATIC DNA is not degrated.
Interruption of the
synthesis of a
complementary strand.
It is described in three
sections.
7. Immunoblot
Is a laboratory technique used to identify a
specific protein in tissue. It is useful for the
diagnosis of multiple pathologies, it is affordable
and effective
8. INMUNOFLUORESCENCE
Is a type of immunohistochemistry technique that utilizes
fluorophores to visualize various cellular antigens such as
proteins. It can be used to visualize the localization of
various cellular components within cells and tissues
9. results
Five families with ataxia, developmental delay and
ATG7 variants.
Circles: female
members
Squares: male
members
Shaded symbols:
affected members
Diamonds:
unknown
members
10. results
Biopsy indicated that ATG7 was undetectable.
ATG7 levels were severely decreased in the
patient´s myoblasts.
P62 high levels were found in the patient´s
myoblasts.
11. ✓ AGT7 protein was undetectable in patient 1 and was
decreased in patients 3 through 6 and 10
✓ P62 levels were increased
✓ B-actine was used as a loading control
results
Analyzed
proteins
Patients and control
+ With
- Without
12. results
control
Analyzed proteins
Wild type
and variants
of AGT7
Analyzed proteins
control
Densitometric analysis of the ratio of LC3 to
GAPDH, normalized to wild-type values.
Introduction of plasmids
encoding wild-type ATG7
into immortalized
fibroblasts from Patient 1
for 24 hours
13. Our comprehensive clinical investigation of these patients
consolidates the critical importance of basal autophagy in human
neural and musculoskeletal integrity. Many of the clinical features
observed in these patients are recapitulated in the conditional
Atg7-knockout mouse models, including brain abnormalities
Komatsu M, Wang Q
Komatsu M, Waguri S
It has also been shown that canonical autophagy can still
proceed in the absence of ATG proteins, albeit at a reduced rate as
a result of impaired inner autophagosomal membrane degradation
Tsuboyama K, Koyama-Honda I, Sakamaki Y, Koike M, Morishita H,
Mizushima N.
These findings strengthen our understanding of autophagy in
human disease and expand the spectrum of clinical phenotypes
and genetic loci associated with congenital autophagy-deficient
syndromes. Given that the perinatal lethality of Atg5-null mice
can be avoided through selective restoration of autophagy in the
nervous system.
Yoshii SR, Kuma A, Akashi T
DISCUSSION
14. Conclusions
Important cells processes and mechanisms
that are not available to our eyes or through
a microscope, such as autophagy, can be
studied by molecular techniques
The molecular techniques are fundamental for the
analysis and study of genetic diseases and consequently
it allows the improvement of the diagnosis with methods
such as immunoblotting and exome sequencing
Molecular techniques also give us a light of options to treat
genetic diseases so damaging as the one of the present study, for
example by methods such as genetic therapy
