Internship Report

Detection of Mutation in Exons of HBB, HBA1, and HBA2 Genes of Thalassemia Patients Through Sanger Sequencing

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Thalassemia can be defined as a recessive autosomal blood disorder that mainly affects the
amount and normal function of hemoglobin that works primarily to carry oxygen throughout the
body. The low amount or absence of hemoglobin within the red blood cells can cause a tremendous
effect on the body as the RBCs could be degraded, creating an anemic condition for those who suffer
from the disease. In molecular terms, two types of Thalassemia have been defined according to the
gene involved: Alpha Thalassemia, caused by mutation either in HBA1 or HBA2 gene and Beta
Thalassemia, which is caused by mutation in HBB gene. This disease cannot be prevented as it
progresses through genetic markers inherited from the parents, making diagnosing the disease
essential to treat the patient before the condition becomes severe. By this means, this project aimed
to create an alternative diagnosis method to detect Thalassemia through optimization of PCR
reaction to determine the optimized condition for the primers to be compatible in detecting Alpha
and Beta Thalassemia variants on their specific exons and to confirm the mutation occurred in Alpha
and Beta Thalassemia variants using Sanger Sequencing method. The project was done in the
molecular diagnostic department of PT. Prodia Widyahusada, where the company aims to be the
leading center of Thalassemia diagnosis as the former leading center of Thalassemia diagnosis
(Eijkman Institute), was merged by the government into BRIN. Three types of samples were used,
starting from the negative control samples, Alpha Thalassemia suspect samples, and Beta
Thalassemia samples. Several lab techniques were conducted, starting from DNA extraction,
followed by PCR, gel electrophoresis, cycle sequencing, and Sanger Sequencing. The project to detect
Alpha Thalassemia was halted due to some setbacks that occurred, such as the unfinished
optimization of the long primers for both HBA1 and HBA2 for the nested PCR procedure due to the
limited time of the internship period. As for Beta Thalassemia, the analysis of mutations on HBB gene
was a success as several mutations were found on the Beta Thalassemia positive sample but not on
the control and Alpha Thalassemia samples. Several types of mutation were found, including
missense, nonsense, splice donor, substitution on the 3’ prime UTR, and frameshift variant, which
were confirmed through the analysis of DNA sequence and the database provided by Clinvar by NCBI
as well as genome browser called ensemble.org.

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Detail Information

Series Title

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Call Number

BM 22-019

Publisher

i3L, Jakarta : i3L, Jakarta., 2022

Collation

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Language

English

ISBN/ISSN

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Classification

NONE

Content Type

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Media Type

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Carrier Type

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Edition

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Subject(s)

Beta Thalassemia
Alpha Thalassemia
HBB
HBA1
HBA2
PCR
Sanger Sequencing
Mutations
DNA Sequences

Specific Detail Info

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Statement of Responsibility

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