Use of cell-free fetal nucleic acids in maternal blood for prenatal diagnosis: the reality of this scenario in Brazil

Abstracts

A descoberta de ácidos nucleicos fetais livres no plasma de gestantes possibilitou o desenvolvimento de novos testes de diagnóstico pré-natal não invasivo para a determinação do sexo e do Rh fetal. Esses testes foram implantados no sistema de saúde pública de diversos países da Europa há mais de cinco anos. As novas possibilidades de aplicação diagnóstica dessas tecnologias são a detecção de aneuploidias cromossômicas fetais, de doenças monogênicas fetais e de distúrbios relacionados com a placenta, temas pesquisados intensivamente por diversos grupos ao redor do mundo. O objetivo deste estudo é expor a situação brasileira no âmbito de pesquisa e utilização clínica dos testes disponíveis comercialmente que utilizam esses marcadores moleculares plasmáticos, ressaltando as vantagens, tanto econômicas quanto de segurança, que os testes não invasivos têm em relação aos atualmente utilizados em nosso sistema de saúde pública.

Diagnóstico não invasivo; pré-natal; ácidos nucleicos livres; DNA fetal; plasma materno; Brasil

REVIEW ARTICLE

^IMSc in Sciences, Medical School, Universidade de São Paulo (USP); PhD Student, Department of Obstetrics and Gynecology, Medical School, USP, São Paulo, SP, Brazil

^IIPhD in Biological Sciences, USP; Professor, Virology Laboratory, Instituto de Medicina Tropical, USP; Chief, Department of Molecular Biology, Fundação Pró-sangue/Hemocentro de São Paulo; Research Consultant, Banco de Sangue do Hospital Albert Einstein, São Paulo, SP, Brazil

^IIIProfessors of Obstetrics, Department of Obstetrics and Gynecology, USP, São Paulo, SP, Brazil

^IVMSc in Biology, USP; Assistant Physician, Clínica Obstétrica do Hospital das Clínicas, USP, São Paulo, SP, Brazil

Correspondence to

SUMMARY

The discovery of cell-free fetal nucleic acids in the plasma of pregnant women has allowed the development of new, noninvasive prenatal diagnostic tests for the determination of fetal gender and Rh. These tests have been implemented in the public health system in several countries of Europe for over five years. The new possibilities for diagnostic use of these technologies are the detection of fetal chromosomal aneuploidies, monogenic fetal disorders, and placental-related disorders, subjects that have been intensively studied by several groups around the world. The aim of this review was to assess the Brazilian research and clinical scenarios regarding the utilization of commercially available tests that use these plasma markers, stressing the advantages, both economic and safety-related, that non-invasive tests have when compared to those currently used in the Brazilian public health system.

Keywords: Noninvasive diagnosis; prenatal care; free nucleic acids; fetal DNA; maternal plasma; Brazil.

INTRODUCTION

The development and improvement of noninvasive diagnostic tests are consistently pursued goals in medicine. The discovery of cell-free fetal DNA in the blood of pregnant women¹, associated with the extreme sensitivity of molecular biology techniques that can detect very small amounts of nucleic acids, has enabled the development of new assays for noninvasive prenatal diagnosis (PND).

Research shows that cell-free fetal DNA comprises about 6% of the total amount in maternal circulation², and is completely eliminated after the delivery³ (half-life of 16 minutes). These nucleic acids are released mainly by apoptosis of the trophoblast cells that form the placenta^4-6. Their isolation from maternal blood is relatively simple, inexpensive, and adaptable to the routine of clinical laboratories.

The main technical difficulty of this type of analysis is the differentiation between cell-free fetal DNA and cellfree maternal DNA, which is present in plasma at higher amounts than the former; this differentiation is only possible when fetal genes or DNA segments with sequences that are different from the maternal genotype are analyzed.

In addition to DNA, messenger RNAs (mRNAs)7 and microRNAs (short sequences of RNAs with post-transcriptional regulatory function)⁸ of fetal origin also circulate in the maternal plasma. The analysis of these types of nucleic acids has an advantage over DNA analysis, as it allows the evaluation of fetal gene expression, which may reflect not only genetic characteristics (DNA) of the fetus, but momentary physiological situations^9,10.

The aim of this review was to assess the Brazilian scenario regarding noninvasive prenatal diagnostic testing, emphasizing tests that are already available in clinical practice in several countries, and to explore what is still possible to develop from the analysis of cell-free fetal nucleic acids (cffNA) in the plasma of pregnant women.

DIAGNOSTIC TESTS USED IN PRENATAL ROUTINE ASSESSMENT

FETAL GENDER

Detection of Y chromosome sequences in maternal plasma indicates that the gender of the fetus is male, as normal women do not have the Y chromosome in their genome. In pregnant women in whom these sequences are not detected, the fetus is presumed female. Fetal gender determination was the first clinically available test that used analysis of fetal nucleic acids in maternal blood¹¹.

Most available tests detect the gender-determining region (SRY) of chromosome Y¹², although other Ychromosome- specific sequences have been described, such as DYS^13-15, DYZ¹⁶, and DAZ¹⁷.

Over the years, the test has been improved, currently reaching over 99% of sensitivity and specificity after the 6^th week of gestation, and reaching over 99.9% after the 8^th week. There are some tools that ensure the safety of the test, such as the use of internal controls to prevent falsenegative results (false evaluation of female fetal gender)^18.

Aside from the curiosity to know the fetal gender, the main clinical application of fetal gender determination is to aid in the investigation of genetic X-chromosomelinked diseases^19,20, such as Duchene's muscular dystrophy and hemophilia, where the confirmation of the female gender excludes the possibility of the fetus carrying such diseases, or even to indicate the treatment of metabolic disorders associated with ambiguous genitalia, such as congenital adrenal hyperplasia (CAH)¹².

In countries such as China, this testing is prohibited for ethical reasons²¹, unless the knowledge of fetal gender offers the possibility of intrauterine treatment. Some European countries offer the test for free within the public health system for the investigation of gender-related diseases²². In Brazil, this test was first described by Levi et al. in 2003²³. Currently several private laboratories in Brazil offer the test commercially to families who wish to know the fetal gender from the seventh week of pregnancy.

With technological development, the costs of fetal gender determination have decreased²⁴, making testing more accessible, and opening new perspectives of management for X-chromosome-linked genetic diseases, as previously described, including through the Unified Brazilian Health System (Sistema Único de Saúde - SUS).

FETAL RH

Noninvasive genetic fetal RhD genotype testings in RhD-negative mothers through detection of free fetal DNA in maternal plasma was first demonstrated in 1998 by Lo et al.²⁵. Since then, more robust methods have been developed, and currently the test is offered in the public health system of many countries in Europe (such as Belgium, United Kingdom, France, the Netherlands, Germany, Spain) with high accuracy (sensitivity of 100% and specificity of 98.6%)²⁶. The test has been studied by some groups in Brazil^27,28, and one of them was able to demonstrate its applicability in highly mixed race populations such as that of Brazil, with 100% accuracy²⁹.

In RhD-negative pregnant women, there is a 16% chance of sensitization to RhD antigen when carrying a RhD-positive fetus, and the consequent development of hemolytic disease of the newborn³⁰.

There is a prophylaxis against the maternal immune response, which consists of the administration of anti-D immunoglobulin, which binds and neutralizes fetal RhD antigens. In Brazil, this treatment is performed in all RhD-negative pregnant women during pregnancy, regardless of fetal genotype. As 40% of RhD-negative pregnant women carry fetuses that are also RhD negative, prophylaxis in these cases is unnecessary. Additionally, the product is obtained from human blood (plasma-derived), with a theoretical risk of spreading infection and with the risk of sensitivity reactions³¹.

Currently, few private laboratories provide this test commercially in Brazil. The implementation of the test in the Brazilian public health system is extremely important to prevent the risks associated with anti-D immunoglobulin prophylaxis. It would also be economical, as it would eliminate the costs of unnecessary prophylaxis and the testing for monitoring maternal sensitization, as demonstrated in other countries³².

NEW POSSIBILITIES OF USE

CHROMOSOMAL ANEUPLOIDIES

There are two major lines of research on tests for noninvasive PND assays for fetal chromosomal aneuploidy. One is focused on the analysis of cell-free fetal DNA through extremely sophisticated techniques³³ such as digital polymerase chain reaction (PCR)³⁴ (counting each DNA molecule) and mass sequencing (simultaneous sequencing of millions of DNA molecules)^35,36, allowing, in theory, for a precise quantification of the small variations in the total free DNA (maternal + fetal), consequent to the presence of extra genetic material or lack thereof in relation to normal, depending on the aneuploidy. The two major limitations to the practical implementation of this technique are its high cost and the complexity of the involved in this technology, including sophisticated bioinformatics software, with the diagnosis being achieved only in experimental situations, where it required a very large initial blood volume and proportions of free fetal DNA that are not practically feasible.

The other line of research focuses on the analysis of fetal gene expression, in which fetal-specific mRNAs (mainly expressed by placental tissue) are isolated from maternal plasma. The test is based on the single nucleotide polymorphism (SNP)/mRNA approach, where the ratio between the alleles of SNPs located in genes expressed by placental tissue allows the detection of extra copies of the chromosome where the investigated genes are located^37-39. The frequency of heterozygous polymorphisms varies among different populations, so the test should use polymorphisms that are informative for each ethnic group.

Three major studies that investigated the effectiveness of tests that analyze fetal DNA using mass sequencing were recently published, demonstrating high sensitivity and specificity^40-42. However, as these tests were based on extremely expensive and sophisticated technologies that do not allow simultaneous analysis of many samples (low efficiency), and with a long delay to release the results, there is no possibility of applying them in individual and less specialized clinical laboratories. These tests would have to be performed in major centers that have the technology, at high costs.

Some experts indicate the SNP/mRNA approach as the most promising for noninvasive PND of chromosomal aneuploidies, as the methodology is simple enough to be employed in small clinical laboratories, the costs are reasonable, and the diagnosis is achieved using samples of maternal plasma with high yield, 92% sensitivity, and 100% specificity⁴³. Large-scale studies are needed to confirm this hypothesis.

A private North-American company has been offering the test commercially in this country since March, 2012, promising results in ten days, based on the recently published results of a prospective study using mass sequencing of fetal DNA in maternal plasma, conducted with 532 samples, which reached 100% specificity in the most prevalent trisomies⁴⁴. In Brazil, no studies have been published on noninvasive diagnosis of fetal chromosomal aneuploidy to date.

MONOGENIC DISEASES

The investigation of monogenic diseases with high prevalence, such as thalassemia in populations of Mediterranean origin, and cystic fibrosis in populations of European origin, should be, in theory, part of routine prenatal research. In cases of rare monogenic diseases, such as Huntington's disease, investigation should be conducted when there is a family history of the disease.

The noninvasive PND of monogenic diseases with dominant paternal inheritance is possible due to the absence of the disease-causing allele in the maternal genome, and, so far, has been performed in at least one pregnancy for the following diseases: Huntington's disease^45-47, achondroplasia^48-50, myotonic dystrophy⁵¹, and Apert syndrome⁵².

Autosomal recessive diseases are often difficult to diagnose noninvasively, as there is still no way to distinguish between identical maternal and paternal alleles. Therefore, the cell-free fetal DNA in maternal plasma can only be used to determine the carrier status of the fetus in compound heterozygotes, by detecting the disease allele inherited from the father (in cases where more than one allele is known for such disease), and when the maternal and paternal alleles are different. To date, the carrier status of the fetus has been determined using cell-free fetal DNA in the following recessive genetic diseases, in at least one pregnancy: cystic fibrosis^53-55, β-thalassemia⁵⁶, α-thalassemia 1⁵⁷, congenital adrenal hyperplasia⁵⁸, and hemophilia⁵⁹ in some European countries. There have been no studies or case reports in Brazil using cffNA for the noninvasive diagnosis of autosomal recessive diseases, or monogenic diseases of paternal inheritance.

PLACENTAL-RELATED DISORDERS

Recent literature has proposed that cffNA can be used to predict disorders whose etiology is based on poor placental development, as this is associated with increased apoptosis of the trophoblast cells that form the outermost placental layer⁵.

Several groups have associated an increase in the amount of cffNA in maternal plasma with disorders whose etiology seems to be related to poor placental development. Some examples are preeclampsia^60-62, HELLP syndrome⁶³, fetal growth restriction^64,65, premature birth^66,67, placenta accreta⁶⁸, placenta inccreta⁶⁹, hyperemesis gravidarum⁷⁰, and polyhydramnios⁷¹. In some cases, it has been verified that there is an increase in cffNA levels before symptom onset, and that these levels correlate with disease severity.

The major limitation of using cffNA as a marker of these placental disorders is that many of them have a multifactorial etiology, or are often unknown. It is first necessary to elucidate the nature of these diseases, as well as establish a normality curve of cffNA concentration in normal pregnancies, so that, later, the analysis in affected pregnancies can be carried out.

CONCLUSION

Noninvasive determination of fetal gender and Rh is well established and has been used clinically for more than five years by several countries in Europe^72-74. In Brazil, these tests have limited use due to their high costs. Their implementation in the public health system would be extremely important as an alternative to invasive procedures, as the latter bring risks during pregnancy and ultimately are outdated when compared to the safety and accuracy of the new tests. Furthermore, the use of noninvasive tests can be advantageous from an economic viewpoint, as the value of a noninvasive test tends to be lower than that of invasive tests, and the early result prevents unnecessary Rh prophylaxis costs in Rh-negative pregnant women and therapy for CAH in male fetuses.

Brazil has a great potential for the development of pioneering and innovative PND research, as recent data from the Brazilian Institute of Geography and Statistics (Instituto Brasileiro de Geografia e Estatística - IBGE) show that there are 2.5 million births per year in the country and an even larger number of pregnancies, both healthy and affected by different complications. This potential should be explored by the research groups specializing in each type of pregnancy complications (such as preeclampsia, fetuses with aneuploidy, monogenic diseases, among others), so that the results of these studies are given back to society as safer and more effective tests and made available in the public health system.

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Use of cell-free fetal nucleic acids in maternal blood for prenatal diagnosis: the reality of this scenario in Brazil

Renata Moscolini Romão^I; José Eduardo Levi^II; Mário Henrique Burlacchini de Carvalho^III; Rossana Pulcineli Vieira Francisco^III; Antônio Gomes de Amorim Filho^IV; Marcelo Zugaib^V

Publication Dates

• Publication in this collection
  17 Oct 2012
• Date of issue
  Oct 2012

History

• Received
  30 Jan 2012
• Accepted
  20 May 2012