Molecular Diagnosis ofα⁰-Thalassemia Through Urine DNA: A Novel DNA Source to Facilitate Prevention Programs in Remote Geographical Areas

Abstract

We assessed whether urinary DNA sediment was a feasible sample type for the molecular diagnosis of α-thalassemia (α-thal) mutations. Urine samples (5-10 mL) were collected from 218 male and female volunteers. The cells were centrifuged, and DNA was isolated according to the protocol of a commercial DNA isolation kit. Detection of the α(0)-thal [Southeast Asian (- -(SEA)) and - -(THAI)] deletions was performed using quantitative real-time polymerase chain reaction (q-PCR), in addition to conventional gap-PCR. The results revealed that DNA extracted from urinary sediment presented an average DNA content of 11.2 ± 5.5 ng/µL, and the 260/280 ratio indicative of DNA purity, was 1.2 ± 0.2. The overall q-PCR threshold cycle was 31.2 ± 2.3. The melting temperature for the - -(SEA) deletion was 87.3 ± 0.1 °C, while that of the wild type sequence was 92.5 ± 0.2 °C. There were 16 (7.3%) α(0)-thal SEA genotypes detected. These results were in agreement with those of the conventional gap-PCR and blood DNA analyses. Thus, DNA from urinary sediment can be efficiently used for the molecular diagnosis of α(0)-thal mutations. This approach allows for rapid diagnosis, is non invasive, and could be useful for preventing Hb Bart's (γ4) hydrops fetalis syndrome.

Detecting HbE Gene Using DNA Extracted from Urine Sediments by Chelex-plus-Heating Technique

“…The urine sediments were able to provide the DNA for molecular diagnosis in several fields, including SEA-a thalassemia 1, as mentioned before. 12 In the study of Suwannakhon et al, however, the commercial DNA extraction kit was used to extract DNA from the urine sediment. In the present study, we attempted to apply Chelex-100 resin and heat to extract genomic DNA from urine sediments.…”

“…In 2013, Ghatak et al 11 prepared the DNA from urine sediments and showed that this DNA was ready for PCR reaction. In 2015, Suwannakhon et al 12 were successful in using the urine sediment as a source of the genomic DNA for genotyping a-thalassemia 1 [Southeast Asian (SEA) type]. Recently, Ng et al 9 successfully prepared DNA from urine sediment used for Short Tandem Repeat (STR) genotyping.…”

Fast-Track Strategy for the Prevention of Hb Bart’s Hydrops Fetalis Syndrome

“…7,9 In brief, the PCR mixtures (25 µL) included 5 µl of DNA, 1X PCR buffer, 1.5 µM MgCl 2 , 200 µM of each dNTP, 2 µM of SYTO9, 1 unit of Platinum ® Taq DNA polymerase, and 0.2 µM of each primer of P1, P2, P3, TF, and TR. Thermal cycling was performed on the platform of a Bio-Rad CFX96 real-time system (Bio-Rad Laboratories, California, USA) beginning at 94°C for two minutes to activate the Taq DNA polymerase, followed by 40 cycles of denaturing at 94°C for 15 s, annealing at 64°C for 15 s, and extension at 72°C for 20 s. Fluorescence was measured on the SYBR channel (533 nm) at the end of each cycle.…”