Allison Uribe Hincapie. Seminario: Biología molecular. Tercer semestre, Medicina, Universidad Pontificia Bolivariana.

1. ALLISON URIBE HINCAPIE.
TERCER SEMESTRE.
UNIVERSIDAD PONTIFICIA BOLIVARIANA.

2. Autosomal recessive inborn error of the urea cycle caused by mutations in
the gene encoding the arginosuccinate synthetase (ASS1) enzyme.
CITRULLINEMIA TYPE I (CTLN1).
As a consequence of the enzymatic
failure citrulline accumulates in the
blood and urine and the amino acid
arginine is not synthesized, which thus,
without being essential, acquires such a
condition and forces it to be used in the
nutritional management of the disorder
This disease is characterized clinically by
hyperammonemia, progressive lethargy
and poor diet.

3. In the article identified two novel variants and a hypomorphic non-
coding variant in ASS1 and validated the pathogenicity using functional
studies.
1. C.649-651del (p.P217del).
2. 5′UTR variant (c.-4C>T).
3. C.1048C>T (p.Q350*).
4. C.649_651del (p.P217del).
5. C.1048C>T (p.Q350*).
6. C.-4C>T.
VARIANTS IN THE ASS1 GENE.

4. Functional identification of two novel variants
and a hypomorphic variant in ASS1 from
patients with Citrullinemia type I.
GENERAL OBJECTIVE:

5. 2.5 Real-time quantitative PCR.
Fundament: Enzymatic in vitro
amplification of particular DNA
sequence that enables records the
kinetics of the reaction and the
measurement of the amount of DNA
synthesized at each moment and
the quantification of bands of
nucleic acids.
Why is this test useful in the study?
Evaluate the transcription level of
variants and expression of TBP and
HPRT between groups.
METHODS:
2.6 Western blot.
Fundament: Proteins from cell
extracts are separated by gel
electrophoresis according to their
size, transferred to a filter, which is
incubated with antibodies attached
to the filter that react with the
protein of interest.
Why is this test useful in the study?
Levels of mutant proteins and their
similarity between variants.

6. METHODS:
2.7 Immunofluorescence analysis.
Fundament: It is based on the addition
of specific antibodies on extensions of
clinical samples made on a slide. The
antibody used is labeled with a
fluorescent substance (fluorochromes).
Why is this test useful in the study?
Results demonstrated that all three
mutant proteins remained evenly
distributed in the cytoplasm without
abnormal aggregation or significant
alteration to the subcellular
localization.
2.8 Enzyme-linked immunosorbent
assay (ELISA).
Fundament: A technique in which an
antibody or antigen is identified using
an antigen or antibody to reveal an
antigen-antibody reaction. It uses a
substance bound to an enzyme that
acts on a substrate producing a
colored solution.
Why is this test useful in the study?
Determine the activity ASS1 enzyme in
mutations

7. RESULTS:
Figure 1.
Figure 2.

8. RESULTS:
Figure 4.

9. DISCUSSION:

10. CONCLUSIONS:
Molecular engineering made it possible to evaluate the sequence of
chromosome 9, the only basis for protein synthesis of the ASS1
enzyme and to find mutations associated with CTLN1.
Molecular methods allowed the measurement of the enzymatic
activity of ASS1 and correlated this finding with the genotypic and
phenotypic presentation of the disease in patients with CTLN1.
The application of molecular technologies allows a fast and
accurate diagnosis of CTLN1, opening the door to significant
improvements in all areas of health care.