Upload
miguel-boone
View
216
Download
2
Tags:
Embed Size (px)
Citation preview
High Resolution Melting Analysis for Genotyping Cell Free Fetal DNA
Ebru Dündar YENİLMEZ, Duygu DÜZGÜNCE, Abdullah TULİ
Çukurova University, Faculty of Medicine, Biochemistry Department, Saricam /Balcali, 01330, Adana,[email protected]
Materials and Methods
Maternal blood (5mL) was collected from 50 women at 7-12
weeks of gestation who undergo to CVS. Plasma was separated
by centrifugation at 1600 g and then 16000 g within 1 hour. Fetal
DNA was extracted from plasma with QIAamp blood mini kit
according to manufacturer instructions.
HRM analysis was used to identify HbS mutation of plasma
fetal DNA. Light Cycler was used for high resolution melting
assay of 50 impregnate plasmas, Hemoglobin S/C Toolset kit for
LightCycler was used according to manufacturer instructions3.
Quantitative fluorescent polymerase chain reaction assay (QF-
PCR) and Short Tandem Repeat (STR) markers analyzed with
AMPISTR kit by manufacturers instructions to detect fetal alleles
in the corresponding maternal plasma samples, when the
maternal and fetal DNA was HbS carrier.
In order to confirm the data of fetal DNA, CVS was analyzed
by both HRM and traditional methods; restriction fragment length
polymorphism (RFLP) and amplification refractory mutation
system (ARMS)5.
IntroductionSickle cell anemia (SCA) is one of the most common human
autosomal recessive disorders and is prevalent in the
Çukurova region of southern Turkey. The incidence of sickle
cell trait is 10.0% in the Çukurova region. Prenatal diagnosis
of SCA has been available by conventional prenatal
diagnostic procedure such as chorionic villus sampling
(CVS) in our prenatal diagnosis centre since 1992 . This
invasive procedure carries a risk of miscarriage about 1-2%.
Because of this risk, cell free fetal DNA in maternal plasma
is being increasingly studied in pregnancy in recent years.
The discovery of fetal DNA in maternal plasma has opened
up new opportunities in non-invasive prenatal diagnosis.
Detecting the mutation with cell free fetal DNA non-
invasively is an important goal of prenatal diagnosis.
High Resolution Melting analysis (HRM) combined with real-
time PCR was introduced in 1997. It is a new method for
DNA analysis especially for genotyping and mutation
scanning. Fluorescence analysis of DNA melting is more
sensitive than invasive methods and only nanogram
amounts of sample are needed.
Aim of The StudyThe aim of this study was detection of SCA mutation (HbS)
by HRM analysis in fetal DNA from maternal plasma.
Results
In our study, we used HRM analysis to detect HbS in fetal DNA as an alternative to invasive
techniques (ARMS, RFLP). Wild type control and homozygous patients presented a clearly
distinguishable difference in Tm (56 and 63°C, respectively). Samples which were analyzed
heterozygous for HbS, showed Tm peaks values between wild type and the mutant samples
(Figure A, B and C).
In 28 fetal samples with heterozygous HbS, the parents did not share the same mutations so that
the paternal mutation in maternal plasma would indicate the presence of paternal mutation in the
fetus. When the fetus carries the same genotype as the mother it was difficult to seperate fetal and
maternal DNA. We used STR analysis to see different loci that comes from the father .
A. Mother is beta thalassemia trait, father is
SCA trait (AS) and the fetus observed
heterozygous for SCA (AS).
B. Mother is beta thalassemia trait (AA),
father is SCA trait (AS) and the fetus
observed heterozygous for SCA (AS).
C. Homozygous wild type (AA) control and
the fetus observed homozygous mutant for
(SS).
ConclusionSickle cell anemia is a severe, lifelong disease that can be accurately diagnosed at prenatal period
by invasive methods such as CVS. Because the procedure carries a risk of miscarriage the
development of new non-invasive techniques using fetal DNA and fetal cells in maternal circulation
has been initiated. The scarce amount of fetal DNA within total DNA in maternal circulation makes
non-invasive prenatal diagnosis for single gene disorders difficult. In our study, we used HRM
analysis combined with real-time PCR, which can be operated with slight amount of DNA, to
diagnose sickle cell mutation prenatally at fetus. The HRM results were confirmed by traditional
techniques (RFLP, ARMS) at CVS samples. The samples which carried the same genotype with
mother were analyzed by STR. In 72% of the samples, paternally inherited fetal alleles detected by
STR were found to be informative and identification of loci different from mother helped the
differentiation of fetus from mother.
HRM analysis combined with real-time PCR has become an alternative method to classical PCR
procedures because of its being more practical, easier to perform, cost effective and less time-
consuming. Detecting SCA at the samples using CVS gives result with 100% accuracy by HRM
analysis. If maternal fetal DNA carries same mutation with mother, HRM analysis cannot always
discriminate between fetus and mother. In order to obtain absolute results with fetal DNA either
usage of different STR markers specific to the disease studied or designation of more specific
probes are suggested.
Acknowledgements
This project was funded by Çukurova University Science Foundation Grant (TF2005D2).
References1.Lo YMD, Tein MSC, Lau TK et al Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am. J of Human Genet. 1998; 62: 768-775.2.Marziliano N, Pele E, Minuti B et al. Melting temperature assay for a UGT1A gene variant in Gilbert Syndrome. Clin.Chem. 2003; 46(6): 853-60.3. Herrmann MG, Dobrowolski SF, Wittwer CT. Rapid b-globin genotyping by multiplexing probe melting temperature and color. Clin.Chem. 2000;46:425-28. 4.Wittwer CT, Reed GH, Gundri CN et al. High resolution genotyping by amplicon melting analysis using LCGreen. Clin.Chem. 2003; 46(6): 853-60.5.Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res, 1989; 17:2503-2516.
Genotype Fetus
Hb AA – wild type 12
Hb AS – heterozygous 28
Hb SS – mutant 10
Heterozygous (AS) father
Heterozygous (AS) fetus
Mother (AA) Heterozygous (AS) fetus
Homozygous mutant fetus(SS)
Homozygous (AA) wild type
In our study, we tried to use HRM analysis with real-
time PCR to genotype the fetus from plasma DNA for
HbS. Table1 shows the samples we established as wild
type, heterozygous and mutant in fetal DNA by HRM
analysis.
Table 1. Sample genotypes by HRM analysis.