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,TURKEYedundar@cu.edu.tr

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.