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New genetic testing in prenatal diagnosis
Seminars in Fetal & Neonatal Medicine xxx (2013) 1e6
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New genetic testing in prenatal diagnosis Natalia Babkina, John M. Graham Jr. * Medical Genetics Institute, CedarseSinai Medical Center and Division of Medical Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
s u m m a r y Keywords: Chromosomal microarray Exome sequencing Next-generation sequencing Non-invasive prenatal screening Preimplantation genetic testing
Determining a genetic diagnosis prenatally permits patients to make informed reproductive decisions and to be counseled about possible fetal outcomes. Therefore, it is important for the provider to be aware of the spectrum of genetic conditions and to use appropriate testing modality to obtain specific diagnosis. This article reviews genetic techniques available for prenatal diagnosis such as preimplantation genetic testing, chromosomal microarray, non-invasive prenatal screening, and next-generation sequencing. Chromosomal microarray has emerged as the first diagnostic test for evaluation of multiple congenital anomalies and developmental delay as most of the next-generation sequencing methods do not detect copy-number variants (CNVs). Exome sequencing and whole genome sequencing are timeconsuming, so if this needs to be done to obtain an accurate genetic diagnosis, allow sufficient time. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Prenatal diagnosis plays an essential role in contemporary obstetrical care, and with proper planning, it can be available for pregnancies at risk for chromosomal and single-gene disorders. Standard chromosome testing has been available since the 1960s [1] and has been commonly used in the prenatal setting, when a fetus has abnormal findings on ultrasound. Prenatal genetic testing has now become much more sophisticated with an improved level of resolution. Standard chromosome testing is being superseded by the use of chromosomal microarrays. Most forms of prenatal diagnosis require invasive procedures for fetal-sample collection and therefore, although considered safe, these procedures involve a risk of fetal loss. The presence of cell-free fetal DNA in maternal circulation has been used as a basis for the development of non-invasive prenatal testing [2]. Application of the latest technologies, such as next-generation sequencing, which features both high sensitivity and accuracy, is allowing for more accurate preconception counseling when there is a previously affected relative, thereby broadening the scope of prenatal diagnosis to include many diseases that are both paternally and maternally inherited. Determining genetic diagnosis prenatally permits patients to make informed reproductive decisions and to be counseled about possible fetal outcomes, management options and recurrence risks. Therefore, it is important for the physician to be aware of the
* Corresponding author. Address: 8700 Beverly Blvd, PACT Suite 400, Los Angeles, CA 90048, USA. Tel.: þ1 310 423 9909; fax: þ1 310 423 2080. E-mail address: [email protected] (J.M. Graham).
full spectrum of genetic conditions, and to use appropriate testing and referrals to genetic healthcare providers in order to obtain a specific diagnosis. 2. Preconception counseling The prevalence of paternal and maternal conditions that are relevant to pregnancy outcomes varies according to many factors, such as parental age, ethnicity, medical history, and family history. Family history plays a critical role in assessing the risk of inherited medical conditions and single-gene disorders [3]. In general practice, the family history can be obtained using a questionnaire or a three-generation pedigree. A family history screening allows stratification of the risk level. Also, the use of a family history screening has been shown to increase the likelihood of detecting a patient at high risk of developing an inherited medical condition by 20%, compared with medical record review alone [4]. Family history of developmental delay, congenital malformations, or other constellation of clinical findings suggestive of a genetic condition requires thorough evaluation. If a disorder in the individual’s family has been identified as having a genetic cause, it may be possible to test parents to determine the risk for having an affected child. For example, a family history of known genetic conditions, such as TayeSachs or cystic fibrosis, should prompt testing for at least the person with an affected relative. It is paramount to identify the mutation in an affected individual, so prenatal diagnosis or preimplantation genetic diagnosis in cases of in-vitro fertilization (IVF) can be performed in order to test specifically for an identified familial mutation and decrease the risk of affected pregnancy. A history of recurrent pregnancy loss should
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Please cite this article in press as: Babkina N, Graham Jr. JM, New genetic testing in prenatal diagnosis, Seminars in Fetal & Neonatal Medicine (2013), http://dx.doi.org/10.1016/j.siny.2013.10.005
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prompt testing of both parents for chromosomal translocations. A parent with a balanced translocation identified by standard chromosome analysis can produce normal, balanced or unbalanced gametes that result in normal, balanced or unbalanced fetus. Recent data show that apparently balanced chromosomal rearrangements are associated with an abnormal phenotype in 6.7% of cases that may be due to cryptic genomic imbalances or to the disruption of genes at the breakpoints [5]. Whereas some genetic conditions that affect pregnancy outcomes are easily identified early in life, others are not and may require additional diagnostic testing. Up until about 10 years ago, w65e70% of patients with congenital malformations remained undiagnosed, including many patients with multifactorial defects (e.g. non-syndromic cleft lip with or without cleft palate or neural tube defects) or polygenic defects (e.g. Hirschsprung disease or hypospadias), 15e25% were thought to be genetic or genomic, and 10% were thought to be environmentally determined [6]. Previously, the diagnostic yield for children with intellectual disability varied between 50% and 80%, with 17e47% having a genetic/genomic cause and 20e50% remaining undiagnosed [7]. With the advent of high-resolution chromosomal microarrays, an additional 10e15% of such patients receive a genomic diagnosis (which is often a sporadic occurrence in an otherwise normal family) [8,9]. As the use of exome sequencing has increased over the last 2 years, most laboratories are reporting an additional 25e40% success rate in obtaining a genetic diagnosis (some of which are also sporadic). The practical implication of all these recent developments is that with modern genomic/genetic diagnostic techniques, up to 70e80% of patients can receive a genetic/genomic diagnosis, with an additional 10% attributed to environmental influences and maternal conditions. Multifactoral defects such as isolated congenital heart defects or orofacial clefts have had a large number of genetic and environmental influences identified, but genetic diagnosis for multifactoral defects is not currently available for clinical use. Fortunately, recurrence risks are relatively low, in the 3e5% range, and this recurrence risk can be reduced by up to 50% through use of folic acid prior to conception and during early pregnancy (covered elsewhere in this volume). Exome sequencing can be quite useful for polygeneic or conditions manifesting genetic heterogeneity. Ancestry influences the probability of being a carrier of many disorders that affect pregnancy. Typically, there is no family history of the condition as the carriers are asymptomatic. For example, using the current recommended mutation panel, negative carrier testing for cystic fibrosis in a Caucasian couple would result in a different chance of having an affected child than negative testing in an African-American couple since the carrier rate and the type of mutations are different in these two populations. Parental age is an important risk factor for adverse pregnancy outcomes. The risk of chromosomal non-disjunction increases with increasing maternal age that translates into higher risk of trisomy 21, 13 and 18. Recent data emphasize the importance of paternal age as a risk factor for new dominant mutations. The male germline accumulates point mutations due to replication errors and reduced activity of repair enzymes, strands mispairing of short tandem repeats and longer exposure to environmental mutagens [10]. In addition, in human sperm DNA is more methylated than oocyte DNA, which may account for the greater number of paternally derived point mutations occurring within a CpG dinucleotide [11]. Because of the large number of cell divisions during spermatogenesis, the mutation rate for base substitutions is much higher in men than in women, and increases with paternal age. The risk for de-novo autosomal dominant mutations calculated by Friedman was 0.3e0.5% among the offspring of fathers aged >40 years [12]. Recently, with the use of whole-genome sequencing the increase in the rate of de-novo mutations has been estimated to be two
mutations per year [13]. The conditions most strongly associated with advanced paternal age are those caused by point mutations in the FGFR2, FGFR3 and RET genes and include Pfeiffer syndrome, Crouzon syndrome, Apert syndrome, achondroplasia as well as multiple endocrine neoplasia type 2A (MEN2A) and multiple endocrine neoplasia type 2B (MEN2B) [14]. Certain dominant conditions such as neurofibromatosis type 1 (NF-1) caused by point mutations or small deletions show a lesser association with paternal age. Some genetic conditions such as Noonan syndrome, Apert syndrome and MEN2B demonstrate germline mosaicism as a result of selection in male germline stem cells that explains an association of these conditions with advanced paternal age [15]. There is also a growing body of evidence that advanced paternal age is associated with an increased risk for complex disorders such as certain congenital anomalies, schizophrenia, and autism spectrum disorders [16,17]. For autosomes and sex chromosomes, there is no strong evidence that aneuploidy is significantly increased in newborns as paternal age increases but two possible exceptions are trisomy 21 and Klinefelter syndrome as recent data suggest a paternal effect, either acting alone or in combination with a maternal age effect [18]. Therefore, genetic risk stratification based on family history, ethnicity and parental age is paramount for preconception counseling. Increased awareness of the importance of using family history as a screening tool, and of the value of preventive measures and increased surveillance, can improve the outcomes. The availability of advanced technology allows for the identification of genetic etiologies in many conditions for which specific diagnostic testing is currently available. 3. Preimplantation genetic testing Preimplantation genetic testing comprises all types of genetic testing performed on the embryos obtained from an IVF cycle and was first described by Handyside et al. [19] when the sex of the embryo was determined in two cases with a history X-linked genetic disorders. Preimplantation genetic testing is divided into two categories: preimplantation genetic screening (PGS) and preimplantation genetic diagnosis (PGD). PGS is performed on the embryos obtained from the parents with presumed normal karyotypes. Chromosomal aneuploidy is a major factor in implantation failure and spontaneous abortions. It was demonstrated that half of the embryos produced in vitro had chromosomal abnormalities that significantly decreased the implantation rate [20]. PGS is used as an embryo screening method for aneuploidy, as the morphological analysis is not reliable for aneuploidy prediction. The most common method used for PGS is analysis of the embryonic cells on day 3 after fertilization with fluorescent in-situ hybridization (FISH). Fast turnaround times and high accuracy are the advantages of PGS with FISH but the limited number of chromosomes that can be evaluated is a significant limitation. Prospective trials of PGS by FISH have not demonstrated any improvement in pregnancy rates [21]. Comprehensive aneuploidy screening using whole-genome array showed that aneuploidy may occur in any of the 23 pairs of chromosomes. Recent retrospective studies showed that PGS using genome-wide approach and testing for all 23 pairs of chromosomes could improve the pregnancy outcome in certain groups or patients. Specifically, in women aged >35 years with a history of recurrent pregnancy loss, PGS is associated with reduced first-trimester spontaneous abortion rate [22]. However, prospective randomized trials have not demonstrated a definitive benefit of PGS and therefore, currently, PGS is not recommended for routine use. PGD involves testing the embryos for a specific genetic disorder. PGD requires prior identification of the genetic cause of the
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condition in the family. Multiple conditions can be tested for, including cystic fibrosis [23], Huntington disease [24], achondroplasia [25] and many others. PGD was initially developed to identify the embryos that carried genes for serious, childhood-onset diseases. Recently, the American Society for Reproductive Medicine reported that PGD for adult-onset conditions is ethically justifiable when the condition is serious and there is no known intervention for the condition, or the available interventions are either inadequately effective or significantly burdensome, thus justifying testing for Huntington disease, Alzheimer disease, and cancer susceptibility genes (e.g. BRCA1 and -2) [26]. Depending on the nature of the condition, different methods can be used, such as FISH and chromosomal microarrays for CNVs and DNA sequencing for single-gene disorders with known mutation in the parent. Most commonly, the biopsy of the embryo is performed on day 3 or day 5 after fertilization. Blastomere stage biopsy on day 3 is associated with higher risk of damaging the embryo and misdiagnosis secondary to possible mosaicism [27], whereas trophectoderm biopsy performed at the blasctocyst stage on day 5 is associated with better results and more accurate diagnosis [22,27]. Although the risk of mosaicism at blastocyst stage is lower than at the blastomere stage, there still might be a discrepancy between results in the trophectoderm that further develops into placental tissue and the inner cell mass that becomes a fetus (e.g. confined placental mosaicism). Therefore, even with the availability of advanced techniques, the risk of embryo misdiagnosis exists, and it is currently recommended to offer chorionic villus sampling (CVS) or amniocentesis to rule out genetic diagnosis in the fetus, especially when there is a proven family history of a genetic condition. 4. Standard karyotype and chromosomal microarray Routine microscopic analysis of chromosomes (standard karyotype) has been used for more than 50 years to detect large chromosomal abnormalities such as translocations, deletions or duplications, but it cannot identify genomic imbalances <5 Mb. Submicroscopic deletions and duplications are identified in 15e20% of cases of autism and intellectual disability, thus chromosomal microarray became a first-line diagnostic test for the evaluation of congenital anomalies and intellectual disability postnatally [9]. Genomic microarrays provide a genome-wide screen for genomic imbalances at a high resolution, allowing for the detection of all known recurrent microdeletion and microduplication syndromes when using resolution of 400 kb for older arrays versus 7.7 kb for single nucleotide polymorphism (SNP) arrays. Current microarray technology also allows genome-wide detection of microscopic and submicroscopic copy number changes <100 kb. The use of DNA-based microarrays eliminates the need to use cell culture, decreases overall turnout time, and in general is less labor intensive [28,29]. Microarray technology is based on hybridization of labeled patient DNA with specific probes with known genomic coordinates. The measurement of signal intensity ratio of patient DNA to reference DNA from individual probes allows identification of gains or losses of chromosomal material. Proof of principle for the use of microarray in prenatal diagnosis was first described by Rickman et al. [28] DNA extracted from uncultured amniotic fluid cells was used for comparative genomic hybridization (CGH). Fetal DNA from CVS specimens has also been shown to be suitable for performing CGH. The feasibility of microarray analysis for prenatal detection of CNVs was demonstrated using different oligonucleotide array CGH platforms, as well as SNP arrays [30,31]. SNP arrays have additional oligonucleotide probes in order to provide more even coverage of the genome and improved detection of CNVs. The additional probes
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for SNP array also allow genotyping based on allele frequency and detection of copy-neutral runs of homozygosity, chromosomal mosaicism, and uniparental disomy. Although SNP arrays can detect uniparental isodisomy, parental parenatal samples are required for the detection of uniparental heterodisomy [31]. By design the microarrays can be further subdivided into targeted and whole-genome arrays. Targeted arrays have the most dense probe coverage in regions of the genome associated with known microdeletion/duplications syndromes or genes known to have clinical implications for human disease. Whole-genome arrays have evenly distributed probe coverage and a higher overall resolution. A number of prospective and retrospective studies testing the efficacy of microarray technique in detection of clinically significant chromosomal gains or losses during prenatal diagnosis have been performed. The additional diagnostic yield for clinically significant CNVs has ranged from 1.3% to 5% [29,30,32]. Most of the studies are small and include women at risk of having a fetus with chromosomal abnormalities based on evidence of structural defects detected on ultrasound. The largest prospective study includes 4406 women who underwent prenatal diagnosis secondary to advanced maternal age, abnormal screening results or abnormal findings on prenatal ultrasound. In samples with a normal karyotype, microarray analysis revealed clinically relevant deletions or duplications in 6% with a structural anomaly and 1.7% of those whose indications were advanced maternal age or positive screening results [33]. However, microarrays provide no information about the structure of the genome, so structural rearrangements that are copy number neutral, such as balanced translocations or inversions, cannot be detected by array and require the use of other techniques. Although inability to detect balanced rearrangements with array is considered a limitation, truly balanced translocations without gain or loss of material are less likely to be associated with a clinically significant phenotype in a neonate. However, apparently balanced rearrangements identified by chromosomal analysis are associated with a 6.7% risk of abnormal phenotype in a neonate that are usually caused by a genomic gain or loss at breakpoints, that are not detected with standard cytogenetic analysis and may be due to cryptic genomic imbalances or to the disruption of genes at the breakpoint. Array is useful not only in identifying the genomic imbalances at the breakpoints, but also in detecting unexpectedly complex rearrangements in other chromosomes. Schluth-Bolard et al. used next-generation sequencing to locate breakpoints at the molecular level in four patients with multiple congenital anomalies and/or intellectual deficiency, who were carrying apparently balanced chromosomal rearrangements and genomic imbalance excluded by microarray [5]. Thus, array CGH combined with next-generation sequencing is a powerful tool allowing rapid breakpoint cloning of apparently balanced chromosomal rearrangements at the molecular level. Systematic studies of apparently balanced chromosomal rearrangements with abnormal phenotype by array CGH have showed that the phenotype might occur due to genomic imbalances near or far from breakpoints. A recent metaanalysis estimated that 37% of two-breakpoint rearrangements are unbalanced [34]. Polyploidies such as triploidy or tetrapolidy cannot be detected by array CGH secondary to correction of DNA concentration; however, additional allelic information provided in SNP arrays improves polyploidy detection. High-level mosaicism can be diagnosed with arrays, but detection of low-level mosaicism is a potential limitation of microarrays. The capacity of array to detect low-level mosaicism depends on standard deviation and dynamic range. Several reports from the studies focused on postnatal evaluation suggested that array was comparable to conventional cytogenetic analysis in the detection rate of chromosomal mosaicism.
Please cite this article in press as: Babkina N, Graham Jr. JM, New genetic testing in prenatal diagnosis, Seminars in Fetal & Neonatal Medicine (2013), http://dx.doi.org/10.1016/j.siny.2013.10.005
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Concern for the potential risk for detection of the variants of unknown clinical significance that might result in parental anxiety is one of the limiting factors for the use of array technology prenatally. The reported incidence of variants of unknown significance varies from 0.5% to 2% depending on the array design and the availability of parental samples for comparison. In the studies that enrolled the fetuses with major malformations detected via prenatal ultrasound, the rate of the variants of unknown significance was reported as high as 12.2% [35]. A large prospective study that included women with advanced maternal age, abnormal screening results and/or abnormal findings on ultrasound reported an incidence of variants of unknown clinical significance of 3.4% among karyotypically normal cases [33]. However, with additional experience acquired over more recent years, only 1.5% cases remained in the category of variants of unknown clinical significance. 5. Non-invasive prenatal screening Non-invasive prenatal screening using fetal cell-free DNA from maternal blood became available with advancement of genomic technology and development of next-generation sequencing. The phenomenon of fetus-derived cell-free DNA circulating in a pregnant woman’s blood was first described in 1997 [2]. At present, it is widely accepted that apoptosis of the trophoblastic cells is the primary source of cell-free fetal DNA, which explains the high turnover of circulating DNA and its rapid clearance after delivery. In maternal serum up to 10% of DNA is of fetal origin [36]. Fetal cellfree DNA is present in maternal circulation in a quantity sufficient for testing from 10 weeks of gestation. Next-generation sequencing technology allows massive parallel sequencing of millions of amplified genetic fragments, and by using sophisticated bioinformatics analysis, it is possible to isolate fetal sequences from maternal sequence and to determine the dosage difference between fetal and reference sequences. Several studies demonstrated high sensitivity, high specificity, and low false-positive rates for detection of trisomy 13, 18, and 21 [37]. The detection rate of sex chromosome abnormalities is also high [38]. Overall, the detection rates are substantially higher than with maternal serum analytes, and are accompanied by a much lower (<1% false-positive rate) [39]. Recently, it has been shown that fetal RhD genotyping can be very accurately determined in all three trimesters using circulating cell-free fetal DNA in the maternal circulation [40]. Diagnosis of single-gene disorders is being validated for translation into clinical practice. In pregnancies at risk for an autosomal dominant disorder of paternal origin, the study of the paternal mutation or alleles in the maternal plasma could detect the fetal condition with regard to the pathology. Huntington disease [41], achondroplasia [42], and myotonic dystrophy [43] are autosomal dominant conditions that have been diagnosed through non-invasive prenatal testing. In couples with different mutations for a recessive disorder, the absence of the paternal mutation or detection of the normal paternal allele in maternal plasma can rule out the possibility of the fetus inheriting the disease. Detection of the paternal mutation increases the risk to the fetus from 1 in 4 to 1 in 2. Recent advances in the development of new technologies have detected both paternal and maternal mutations, and a genome-wide genetic map of the fetus has been constructed for the paternal and maternal haplotypes. Haplotype analysis in conjunction with targeted next-generation sequencing allowed non-invasive prenatal diagnosis for alpha- and betathalassemia by detecting paternally inherited alleles in the maternal plasma [44]. Other autosomal recessive disorders successfully diagnosed with non-invasive prenatal testing have included cystic fibrosis [45], propionic acidemia [46], congenital adrenal hyperplasia [47], and Leber congenital amaurosis [48].
Although the studies are promising, there are certain limitations for non-invasive prenatal screening. Sequence information identified through non-invasive prenatal screening is derived from the placenta, which raises the possibility of a false-positive result due to confined placental mosaicism, and similar to CVS, placental DNA may not reflect the true fetal DNA. The majority of studies on non-invasive prenatal screening include women aged >35 years who are at higher risk for aneuploidy. The data concerning noninvasive prenatal screening in low-risk populations are limited, but a decreased incidence of aneuploidy may be associated with lower positive predictive value. Also, prior knowledge about the prevalence of aneuploidies in the samples may have affected an analyst’s decisions about how to classify ambiguous test results. Current technology does not allow detection of chromosomal rearrangements and has not been validated for the majority of singlegene disorders and CNVs. In cases of abnormal findings detected via ultrasound, invasive testing may still be necessary. Non-invasive prenatal testing does not substitute for routine first- and secondtrimester screening, as it does not detect neural tube defects and has limitations in evaluation of structural abnormalities detected via ultrasound and in multiple gestations. As the clinical utility of cellfree DNA testing in the general population is unproven, professional organizations such as the American College of Obstetricians and Gynecologists, the Society for MaternaleFetal Medicine and the National Society of Genetic Counselors currently recommend cell-free DNA testing only for ‘high-risk pregnancies’ and they suggest that positive results be confirmed through invasive testing. 6. Whole-exome and whole-genome sequencing Next-generation sequencing has been used to identify causative genes in many Mendelian disorders. About 85% of known diseasecausing mutations occur within the 1% of the genome encoding for proteins. Therefore, whole exome sequencing represents a powerful method to cost-effectively capture the majority of proteincoding regions to identify single nucleotide variants (SNVs), and small insertion/deletions. It is now possible to non-invasively identify the fetal genome with the shotgun sequencing of maternal plasma DNA. Also, molecular counting of parental haplotypes and fetal exome capture has allowed exome screening of clinically relevant and deleterious alleles that were paternally inherited or were de novo [49]. There are data concerning the clinical diagnosis established by whole-genome sequencing of a prenatal sample e for example, a 13-day sequence and analysis pipeline detection of a disruption of CHD7 in a fetus with a suspected diagnosis of CHARGE syndrome who had multiple anomalies detected on ultrasound [50]. However, although comprehensive non-invasive whole-exome or whole-genome sequencing has significant potential, it also brings multiple concerns such as technical complexity, prolonged turnaround time, difficulty with bioinformatic interpretation, and ethical issues that limit its clinical use. Whole-exome or wholegenome sequencing is still considered impractical for routine prenatal care secondary to cost and turnaround time. Eventually, decreasing costs may allow personal genome sequencing to become available for routine analysis, but annotation and interpretation of variant information are essential to provide information that can be used to better manage an individual’s condition. The expanded use of non-invasive fetal exome or genome sequencing would present several advantages and challenges. Broader use might lead to the improved detection of Mendelian disorders in families who would not otherwise have been offered prenatal testing, as well as in families who might have refused invasive testing because of risk of fetal loss. However, the concern is that it will identify variants that are beyond the scope of
Please cite this article in press as: Babkina N, Graham Jr. JM, New genetic testing in prenatal diagnosis, Seminars in Fetal & Neonatal Medicine (2013), http://dx.doi.org/10.1016/j.siny.2013.10.005
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conventional prenatal screening and diagnosis, specifically variants that indicate increased risk for developing adult-onset conditions. The relevance of such information is controversial for a current pregnancy, but it may have significant implications for the health of the parent. Therefore, the broader implementation of non-invasive fetal genome sequencing will lead to wider application of the results and increased availability. 7. Conclusions Determining accurate genetic diagnosis prenatally permits families to make informed reproductive decisions. Therefore, it is important for the provider to be aware of the spectrum of potential genetic conditions, and to use appropriate testing modalities to obtain specific diagnosis.
Practice points The birth of a child with intellectual disabilities and/or congenital anomalies is an upsetting event for any family, so if a child is born with these problems, think ahead to the next pregnancy and try to ascertain the cause before the next pregnancy. It is best to perform SNP chromosomal microarray before attempting to sequence any gene(s) because many gene sequencing techniques do not detect CNVs. Exome sequencing and whole genome sequencing are time consuming (at least 6 months), so if this needs to be done to obtain a diagnosis that will provide accurate prenatal diagnosis, allow sufficient time.
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Balanced into array: genome-wide array analysis in 54 patients with an apparently balanced de novo chromosome rearrangement and a meta-analysis. Eur J Hum Genet 2011;19:1152e60. [35] D’Amours G, Kibar Z, Mathonnet G, Fetni R, Tihy F, Desilets V, et al. Wholegenome array CGH identifies pathogenic copy number variations in fetuses with major malformations and a normal karyotype. Clin Genet 2012;81: 128e41. [36] Lun FM, Chiu RW, Allen Chan KC, Yeung Leung T, Kin Lau T, Lo YMD. Microfluidics digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma. Clin Chem 2008;54:1664e72. [37] Palomaki GE, Kloza EM, Lambert-Messerlian GM, Haddow JE, Neveux LM, Ehrich M, et al. DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study. Genet Med 2011;13: 913e20. [38] Finning KM, Chitty LS. Non-invasive fetal sex determination: impact on clinical practice. Semin Fetal Neonatal Med 2011;13:69e75. [39] Simpson JL. Cell-free fetal DNA and maternal serum analytes for monitoring embryonic and fetal status. Fertil Steril 2013;99:1124e34. [40] Moise Jr KJ, Boring NH, O’Shaughnessy R, Simpson LL, Wolfe HM, Baxter JK, et al. Circulating cell-free fetal DNA for the detection of RHD status and sex using reflex fetal identifiers. Prenat Diagn 2013;33:95e101. [41] Gonzalez-Gonzalez MC, Garcia-Hoyos M, Trujillo-Tiebas MJ, Bustamante Aragones A, Rodriguez de Alba M, Diego Alvarez D, et al. Improvement in strategies for the non-invasive prenatal diagnosis of Huntington disease. J Assist Reprod Genet 2008;25:477e81.
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[42] Lim JH, Kim MJ, Kim SY, Kim HO, Song MJ, Kim MH, et al. Non-invasive prenatal detection of achondroplasia using circulating fetal DNA in maternal plasma. J Assist Reprod Genet 2010;28:167e72. [43] Amicucci P, Gennarelli M, Novelli G, Dallapiccola B. Prenatal diagnosis of myotonic dystrophy using fetal DNA obtained from maternal plasma. Clin Chem 2009;46:301e2. [44] Papasavva T, van Ijcken WF, Kockx CE, Van den Hout MC, Kountouris P, Kythreotis L, et al. Next generation sequencing of SNPs for non-invasive prenatal diagnosis: challenges and feasibility as illustrated by an application to b-thalassaemia. Eur J Hum Genet 2013;21:1403e10. [45] Bustamante-Aragones A, Gallego-Merlo J, Trujillo-Tiebas MJ, de Alba MR, Ugarte M, Ramos C. New strategy for the prenatal detection/exclusion of paternal cystic fibrosis mutations in maternal plasma. J Cyst Fibros 2008;7: 505e10.
[46] Bustamante-Aragones A, Pérez-Cerdá C, Pérez B, de Alba MR, Ugarte M, Ramos C. Prenatal diagnosis in maternal plasma of a fetal mutation causing propionic acidemia. Mol Genet Metab 2008;95:101e3. [47] Chiu RW, Lau TK, Cheung PT, Gong ZQ, Leung TN, Lo YM. Non invasive prenatal exclusion of congenital adrenal hyperplasia by maternal plasma analysis: a feasibility study. Clin Chem 2002;48:778e80. [48] Bustamante-Aragones A, Vallespin E, Rodriguez de Alba M, Trujillo-Tiebas MJ, Gonzalez-Gonzalez C, Diego-Alvarez D, et al. Early noninvasive prenatal detection of a fetal CRB1 mutation causing Leber congenital amaurosis. Mol Vis 2008;14:1388e94. [49] Fan C, Gu W, Wang J, Blumenfeld YJ, El-Sayed YY, Quake SR, et al. Noninvasive prenatal measurement of the fetal genome. Nature 2012;487(7407):320e4. [50] Talkowski ME, Ordulu Z, Pillalamarri V, Benson CB, Blumenthal I, Connolly S, et al. Clinical diagnosis by whole-genome sequencing of a prenatal sample. N Engl J Med 2012;367:2226e32.
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New genetic testing in prenatal diagnosis Natalia Babkina, John M. Graham Jr. * Medical Genetics Institute, CedarseSinai Medical Center and Division of Medical Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
s u m m a r y Keywords: Chromosomal microarray Exome sequencing Next-generation sequencing Non-invasive prenatal screening Preimplantation genetic testing
Determining a genetic diagnosis prenatally permits patients to make informed reproductive decisions and to be counseled about possible fetal outcomes. Therefore, it is important for the provider to be aware of the spectrum of genetic conditions and to use appropriate testing modality to obtain specific diagnosis. This article reviews genetic techniques available for prenatal diagnosis such as preimplantation genetic testing, chromosomal microarray, non-invasive prenatal screening, and next-generation sequencing. Chromosomal microarray has emerged as the first diagnostic test for evaluation of multiple congenital anomalies and developmental delay as most of the next-generation sequencing methods do not detect copy-number variants (CNVs). Exome sequencing and whole genome sequencing are timeconsuming, so if this needs to be done to obtain an accurate genetic diagnosis, allow sufficient time. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Prenatal diagnosis plays an essential role in contemporary obstetrical care, and with proper planning, it can be available for pregnancies at risk for chromosomal and single-gene disorders. Standard chromosome testing has been available since the 1960s [1] and has been commonly used in the prenatal setting, when a fetus has abnormal findings on ultrasound. Prenatal genetic testing has now become much more sophisticated with an improved level of resolution. Standard chromosome testing is being superseded by the use of chromosomal microarrays. Most forms of prenatal diagnosis require invasive procedures for fetal-sample collection and therefore, although considered safe, these procedures involve a risk of fetal loss. The presence of cell-free fetal DNA in maternal circulation has been used as a basis for the development of non-invasive prenatal testing [2]. Application of the latest technologies, such as next-generation sequencing, which features both high sensitivity and accuracy, is allowing for more accurate preconception counseling when there is a previously affected relative, thereby broadening the scope of prenatal diagnosis to include many diseases that are both paternally and maternally inherited. Determining genetic diagnosis prenatally permits patients to make informed reproductive decisions and to be counseled about possible fetal outcomes, management options and recurrence risks. Therefore, it is important for the physician to be aware of the
* Corresponding author. Address: 8700 Beverly Blvd, PACT Suite 400, Los Angeles, CA 90048, USA. Tel.: þ1 310 423 9909; fax: þ1 310 423 2080. E-mail address: [email protected] (J.M. Graham).
full spectrum of genetic conditions, and to use appropriate testing and referrals to genetic healthcare providers in order to obtain a specific diagnosis. 2. Preconception counseling The prevalence of paternal and maternal conditions that are relevant to pregnancy outcomes varies according to many factors, such as parental age, ethnicity, medical history, and family history. Family history plays a critical role in assessing the risk of inherited medical conditions and single-gene disorders [3]. In general practice, the family history can be obtained using a questionnaire or a three-generation pedigree. A family history screening allows stratification of the risk level. Also, the use of a family history screening has been shown to increase the likelihood of detecting a patient at high risk of developing an inherited medical condition by 20%, compared with medical record review alone [4]. Family history of developmental delay, congenital malformations, or other constellation of clinical findings suggestive of a genetic condition requires thorough evaluation. If a disorder in the individual’s family has been identified as having a genetic cause, it may be possible to test parents to determine the risk for having an affected child. For example, a family history of known genetic conditions, such as TayeSachs or cystic fibrosis, should prompt testing for at least the person with an affected relative. It is paramount to identify the mutation in an affected individual, so prenatal diagnosis or preimplantation genetic diagnosis in cases of in-vitro fertilization (IVF) can be performed in order to test specifically for an identified familial mutation and decrease the risk of affected pregnancy. A history of recurrent pregnancy loss should
1744-165X/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.siny.2013.10.005
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prompt testing of both parents for chromosomal translocations. A parent with a balanced translocation identified by standard chromosome analysis can produce normal, balanced or unbalanced gametes that result in normal, balanced or unbalanced fetus. Recent data show that apparently balanced chromosomal rearrangements are associated with an abnormal phenotype in 6.7% of cases that may be due to cryptic genomic imbalances or to the disruption of genes at the breakpoints [5]. Whereas some genetic conditions that affect pregnancy outcomes are easily identified early in life, others are not and may require additional diagnostic testing. Up until about 10 years ago, w65e70% of patients with congenital malformations remained undiagnosed, including many patients with multifactorial defects (e.g. non-syndromic cleft lip with or without cleft palate or neural tube defects) or polygenic defects (e.g. Hirschsprung disease or hypospadias), 15e25% were thought to be genetic or genomic, and 10% were thought to be environmentally determined [6]. Previously, the diagnostic yield for children with intellectual disability varied between 50% and 80%, with 17e47% having a genetic/genomic cause and 20e50% remaining undiagnosed [7]. With the advent of high-resolution chromosomal microarrays, an additional 10e15% of such patients receive a genomic diagnosis (which is often a sporadic occurrence in an otherwise normal family) [8,9]. As the use of exome sequencing has increased over the last 2 years, most laboratories are reporting an additional 25e40% success rate in obtaining a genetic diagnosis (some of which are also sporadic). The practical implication of all these recent developments is that with modern genomic/genetic diagnostic techniques, up to 70e80% of patients can receive a genetic/genomic diagnosis, with an additional 10% attributed to environmental influences and maternal conditions. Multifactoral defects such as isolated congenital heart defects or orofacial clefts have had a large number of genetic and environmental influences identified, but genetic diagnosis for multifactoral defects is not currently available for clinical use. Fortunately, recurrence risks are relatively low, in the 3e5% range, and this recurrence risk can be reduced by up to 50% through use of folic acid prior to conception and during early pregnancy (covered elsewhere in this volume). Exome sequencing can be quite useful for polygeneic or conditions manifesting genetic heterogeneity. Ancestry influences the probability of being a carrier of many disorders that affect pregnancy. Typically, there is no family history of the condition as the carriers are asymptomatic. For example, using the current recommended mutation panel, negative carrier testing for cystic fibrosis in a Caucasian couple would result in a different chance of having an affected child than negative testing in an African-American couple since the carrier rate and the type of mutations are different in these two populations. Parental age is an important risk factor for adverse pregnancy outcomes. The risk of chromosomal non-disjunction increases with increasing maternal age that translates into higher risk of trisomy 21, 13 and 18. Recent data emphasize the importance of paternal age as a risk factor for new dominant mutations. The male germline accumulates point mutations due to replication errors and reduced activity of repair enzymes, strands mispairing of short tandem repeats and longer exposure to environmental mutagens [10]. In addition, in human sperm DNA is more methylated than oocyte DNA, which may account for the greater number of paternally derived point mutations occurring within a CpG dinucleotide [11]. Because of the large number of cell divisions during spermatogenesis, the mutation rate for base substitutions is much higher in men than in women, and increases with paternal age. The risk for de-novo autosomal dominant mutations calculated by Friedman was 0.3e0.5% among the offspring of fathers aged >40 years [12]. Recently, with the use of whole-genome sequencing the increase in the rate of de-novo mutations has been estimated to be two
mutations per year [13]. The conditions most strongly associated with advanced paternal age are those caused by point mutations in the FGFR2, FGFR3 and RET genes and include Pfeiffer syndrome, Crouzon syndrome, Apert syndrome, achondroplasia as well as multiple endocrine neoplasia type 2A (MEN2A) and multiple endocrine neoplasia type 2B (MEN2B) [14]. Certain dominant conditions such as neurofibromatosis type 1 (NF-1) caused by point mutations or small deletions show a lesser association with paternal age. Some genetic conditions such as Noonan syndrome, Apert syndrome and MEN2B demonstrate germline mosaicism as a result of selection in male germline stem cells that explains an association of these conditions with advanced paternal age [15]. There is also a growing body of evidence that advanced paternal age is associated with an increased risk for complex disorders such as certain congenital anomalies, schizophrenia, and autism spectrum disorders [16,17]. For autosomes and sex chromosomes, there is no strong evidence that aneuploidy is significantly increased in newborns as paternal age increases but two possible exceptions are trisomy 21 and Klinefelter syndrome as recent data suggest a paternal effect, either acting alone or in combination with a maternal age effect [18]. Therefore, genetic risk stratification based on family history, ethnicity and parental age is paramount for preconception counseling. Increased awareness of the importance of using family history as a screening tool, and of the value of preventive measures and increased surveillance, can improve the outcomes. The availability of advanced technology allows for the identification of genetic etiologies in many conditions for which specific diagnostic testing is currently available. 3. Preimplantation genetic testing Preimplantation genetic testing comprises all types of genetic testing performed on the embryos obtained from an IVF cycle and was first described by Handyside et al. [19] when the sex of the embryo was determined in two cases with a history X-linked genetic disorders. Preimplantation genetic testing is divided into two categories: preimplantation genetic screening (PGS) and preimplantation genetic diagnosis (PGD). PGS is performed on the embryos obtained from the parents with presumed normal karyotypes. Chromosomal aneuploidy is a major factor in implantation failure and spontaneous abortions. It was demonstrated that half of the embryos produced in vitro had chromosomal abnormalities that significantly decreased the implantation rate [20]. PGS is used as an embryo screening method for aneuploidy, as the morphological analysis is not reliable for aneuploidy prediction. The most common method used for PGS is analysis of the embryonic cells on day 3 after fertilization with fluorescent in-situ hybridization (FISH). Fast turnaround times and high accuracy are the advantages of PGS with FISH but the limited number of chromosomes that can be evaluated is a significant limitation. Prospective trials of PGS by FISH have not demonstrated any improvement in pregnancy rates [21]. Comprehensive aneuploidy screening using whole-genome array showed that aneuploidy may occur in any of the 23 pairs of chromosomes. Recent retrospective studies showed that PGS using genome-wide approach and testing for all 23 pairs of chromosomes could improve the pregnancy outcome in certain groups or patients. Specifically, in women aged >35 years with a history of recurrent pregnancy loss, PGS is associated with reduced first-trimester spontaneous abortion rate [22]. However, prospective randomized trials have not demonstrated a definitive benefit of PGS and therefore, currently, PGS is not recommended for routine use. PGD involves testing the embryos for a specific genetic disorder. PGD requires prior identification of the genetic cause of the
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condition in the family. Multiple conditions can be tested for, including cystic fibrosis [23], Huntington disease [24], achondroplasia [25] and many others. PGD was initially developed to identify the embryos that carried genes for serious, childhood-onset diseases. Recently, the American Society for Reproductive Medicine reported that PGD for adult-onset conditions is ethically justifiable when the condition is serious and there is no known intervention for the condition, or the available interventions are either inadequately effective or significantly burdensome, thus justifying testing for Huntington disease, Alzheimer disease, and cancer susceptibility genes (e.g. BRCA1 and -2) [26]. Depending on the nature of the condition, different methods can be used, such as FISH and chromosomal microarrays for CNVs and DNA sequencing for single-gene disorders with known mutation in the parent. Most commonly, the biopsy of the embryo is performed on day 3 or day 5 after fertilization. Blastomere stage biopsy on day 3 is associated with higher risk of damaging the embryo and misdiagnosis secondary to possible mosaicism [27], whereas trophectoderm biopsy performed at the blasctocyst stage on day 5 is associated with better results and more accurate diagnosis [22,27]. Although the risk of mosaicism at blastocyst stage is lower than at the blastomere stage, there still might be a discrepancy between results in the trophectoderm that further develops into placental tissue and the inner cell mass that becomes a fetus (e.g. confined placental mosaicism). Therefore, even with the availability of advanced techniques, the risk of embryo misdiagnosis exists, and it is currently recommended to offer chorionic villus sampling (CVS) or amniocentesis to rule out genetic diagnosis in the fetus, especially when there is a proven family history of a genetic condition. 4. Standard karyotype and chromosomal microarray Routine microscopic analysis of chromosomes (standard karyotype) has been used for more than 50 years to detect large chromosomal abnormalities such as translocations, deletions or duplications, but it cannot identify genomic imbalances <5 Mb. Submicroscopic deletions and duplications are identified in 15e20% of cases of autism and intellectual disability, thus chromosomal microarray became a first-line diagnostic test for the evaluation of congenital anomalies and intellectual disability postnatally [9]. Genomic microarrays provide a genome-wide screen for genomic imbalances at a high resolution, allowing for the detection of all known recurrent microdeletion and microduplication syndromes when using resolution of 400 kb for older arrays versus 7.7 kb for single nucleotide polymorphism (SNP) arrays. Current microarray technology also allows genome-wide detection of microscopic and submicroscopic copy number changes <100 kb. The use of DNA-based microarrays eliminates the need to use cell culture, decreases overall turnout time, and in general is less labor intensive [28,29]. Microarray technology is based on hybridization of labeled patient DNA with specific probes with known genomic coordinates. The measurement of signal intensity ratio of patient DNA to reference DNA from individual probes allows identification of gains or losses of chromosomal material. Proof of principle for the use of microarray in prenatal diagnosis was first described by Rickman et al. [28] DNA extracted from uncultured amniotic fluid cells was used for comparative genomic hybridization (CGH). Fetal DNA from CVS specimens has also been shown to be suitable for performing CGH. The feasibility of microarray analysis for prenatal detection of CNVs was demonstrated using different oligonucleotide array CGH platforms, as well as SNP arrays [30,31]. SNP arrays have additional oligonucleotide probes in order to provide more even coverage of the genome and improved detection of CNVs. The additional probes
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for SNP array also allow genotyping based on allele frequency and detection of copy-neutral runs of homozygosity, chromosomal mosaicism, and uniparental disomy. Although SNP arrays can detect uniparental isodisomy, parental parenatal samples are required for the detection of uniparental heterodisomy [31]. By design the microarrays can be further subdivided into targeted and whole-genome arrays. Targeted arrays have the most dense probe coverage in regions of the genome associated with known microdeletion/duplications syndromes or genes known to have clinical implications for human disease. Whole-genome arrays have evenly distributed probe coverage and a higher overall resolution. A number of prospective and retrospective studies testing the efficacy of microarray technique in detection of clinically significant chromosomal gains or losses during prenatal diagnosis have been performed. The additional diagnostic yield for clinically significant CNVs has ranged from 1.3% to 5% [29,30,32]. Most of the studies are small and include women at risk of having a fetus with chromosomal abnormalities based on evidence of structural defects detected on ultrasound. The largest prospective study includes 4406 women who underwent prenatal diagnosis secondary to advanced maternal age, abnormal screening results or abnormal findings on prenatal ultrasound. In samples with a normal karyotype, microarray analysis revealed clinically relevant deletions or duplications in 6% with a structural anomaly and 1.7% of those whose indications were advanced maternal age or positive screening results [33]. However, microarrays provide no information about the structure of the genome, so structural rearrangements that are copy number neutral, such as balanced translocations or inversions, cannot be detected by array and require the use of other techniques. Although inability to detect balanced rearrangements with array is considered a limitation, truly balanced translocations without gain or loss of material are less likely to be associated with a clinically significant phenotype in a neonate. However, apparently balanced rearrangements identified by chromosomal analysis are associated with a 6.7% risk of abnormal phenotype in a neonate that are usually caused by a genomic gain or loss at breakpoints, that are not detected with standard cytogenetic analysis and may be due to cryptic genomic imbalances or to the disruption of genes at the breakpoint. Array is useful not only in identifying the genomic imbalances at the breakpoints, but also in detecting unexpectedly complex rearrangements in other chromosomes. Schluth-Bolard et al. used next-generation sequencing to locate breakpoints at the molecular level in four patients with multiple congenital anomalies and/or intellectual deficiency, who were carrying apparently balanced chromosomal rearrangements and genomic imbalance excluded by microarray [5]. Thus, array CGH combined with next-generation sequencing is a powerful tool allowing rapid breakpoint cloning of apparently balanced chromosomal rearrangements at the molecular level. Systematic studies of apparently balanced chromosomal rearrangements with abnormal phenotype by array CGH have showed that the phenotype might occur due to genomic imbalances near or far from breakpoints. A recent metaanalysis estimated that 37% of two-breakpoint rearrangements are unbalanced [34]. Polyploidies such as triploidy or tetrapolidy cannot be detected by array CGH secondary to correction of DNA concentration; however, additional allelic information provided in SNP arrays improves polyploidy detection. High-level mosaicism can be diagnosed with arrays, but detection of low-level mosaicism is a potential limitation of microarrays. The capacity of array to detect low-level mosaicism depends on standard deviation and dynamic range. Several reports from the studies focused on postnatal evaluation suggested that array was comparable to conventional cytogenetic analysis in the detection rate of chromosomal mosaicism.
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Concern for the potential risk for detection of the variants of unknown clinical significance that might result in parental anxiety is one of the limiting factors for the use of array technology prenatally. The reported incidence of variants of unknown significance varies from 0.5% to 2% depending on the array design and the availability of parental samples for comparison. In the studies that enrolled the fetuses with major malformations detected via prenatal ultrasound, the rate of the variants of unknown significance was reported as high as 12.2% [35]. A large prospective study that included women with advanced maternal age, abnormal screening results and/or abnormal findings on ultrasound reported an incidence of variants of unknown clinical significance of 3.4% among karyotypically normal cases [33]. However, with additional experience acquired over more recent years, only 1.5% cases remained in the category of variants of unknown clinical significance. 5. Non-invasive prenatal screening Non-invasive prenatal screening using fetal cell-free DNA from maternal blood became available with advancement of genomic technology and development of next-generation sequencing. The phenomenon of fetus-derived cell-free DNA circulating in a pregnant woman’s blood was first described in 1997 [2]. At present, it is widely accepted that apoptosis of the trophoblastic cells is the primary source of cell-free fetal DNA, which explains the high turnover of circulating DNA and its rapid clearance after delivery. In maternal serum up to 10% of DNA is of fetal origin [36]. Fetal cellfree DNA is present in maternal circulation in a quantity sufficient for testing from 10 weeks of gestation. Next-generation sequencing technology allows massive parallel sequencing of millions of amplified genetic fragments, and by using sophisticated bioinformatics analysis, it is possible to isolate fetal sequences from maternal sequence and to determine the dosage difference between fetal and reference sequences. Several studies demonstrated high sensitivity, high specificity, and low false-positive rates for detection of trisomy 13, 18, and 21 [37]. The detection rate of sex chromosome abnormalities is also high [38]. Overall, the detection rates are substantially higher than with maternal serum analytes, and are accompanied by a much lower (<1% false-positive rate) [39]. Recently, it has been shown that fetal RhD genotyping can be very accurately determined in all three trimesters using circulating cell-free fetal DNA in the maternal circulation [40]. Diagnosis of single-gene disorders is being validated for translation into clinical practice. In pregnancies at risk for an autosomal dominant disorder of paternal origin, the study of the paternal mutation or alleles in the maternal plasma could detect the fetal condition with regard to the pathology. Huntington disease [41], achondroplasia [42], and myotonic dystrophy [43] are autosomal dominant conditions that have been diagnosed through non-invasive prenatal testing. In couples with different mutations for a recessive disorder, the absence of the paternal mutation or detection of the normal paternal allele in maternal plasma can rule out the possibility of the fetus inheriting the disease. Detection of the paternal mutation increases the risk to the fetus from 1 in 4 to 1 in 2. Recent advances in the development of new technologies have detected both paternal and maternal mutations, and a genome-wide genetic map of the fetus has been constructed for the paternal and maternal haplotypes. Haplotype analysis in conjunction with targeted next-generation sequencing allowed non-invasive prenatal diagnosis for alpha- and betathalassemia by detecting paternally inherited alleles in the maternal plasma [44]. Other autosomal recessive disorders successfully diagnosed with non-invasive prenatal testing have included cystic fibrosis [45], propionic acidemia [46], congenital adrenal hyperplasia [47], and Leber congenital amaurosis [48].
Although the studies are promising, there are certain limitations for non-invasive prenatal screening. Sequence information identified through non-invasive prenatal screening is derived from the placenta, which raises the possibility of a false-positive result due to confined placental mosaicism, and similar to CVS, placental DNA may not reflect the true fetal DNA. The majority of studies on non-invasive prenatal screening include women aged >35 years who are at higher risk for aneuploidy. The data concerning noninvasive prenatal screening in low-risk populations are limited, but a decreased incidence of aneuploidy may be associated with lower positive predictive value. Also, prior knowledge about the prevalence of aneuploidies in the samples may have affected an analyst’s decisions about how to classify ambiguous test results. Current technology does not allow detection of chromosomal rearrangements and has not been validated for the majority of singlegene disorders and CNVs. In cases of abnormal findings detected via ultrasound, invasive testing may still be necessary. Non-invasive prenatal testing does not substitute for routine first- and secondtrimester screening, as it does not detect neural tube defects and has limitations in evaluation of structural abnormalities detected via ultrasound and in multiple gestations. As the clinical utility of cellfree DNA testing in the general population is unproven, professional organizations such as the American College of Obstetricians and Gynecologists, the Society for MaternaleFetal Medicine and the National Society of Genetic Counselors currently recommend cell-free DNA testing only for ‘high-risk pregnancies’ and they suggest that positive results be confirmed through invasive testing. 6. Whole-exome and whole-genome sequencing Next-generation sequencing has been used to identify causative genes in many Mendelian disorders. About 85% of known diseasecausing mutations occur within the 1% of the genome encoding for proteins. Therefore, whole exome sequencing represents a powerful method to cost-effectively capture the majority of proteincoding regions to identify single nucleotide variants (SNVs), and small insertion/deletions. It is now possible to non-invasively identify the fetal genome with the shotgun sequencing of maternal plasma DNA. Also, molecular counting of parental haplotypes and fetal exome capture has allowed exome screening of clinically relevant and deleterious alleles that were paternally inherited or were de novo [49]. There are data concerning the clinical diagnosis established by whole-genome sequencing of a prenatal sample e for example, a 13-day sequence and analysis pipeline detection of a disruption of CHD7 in a fetus with a suspected diagnosis of CHARGE syndrome who had multiple anomalies detected on ultrasound [50]. However, although comprehensive non-invasive whole-exome or whole-genome sequencing has significant potential, it also brings multiple concerns such as technical complexity, prolonged turnaround time, difficulty with bioinformatic interpretation, and ethical issues that limit its clinical use. Whole-exome or wholegenome sequencing is still considered impractical for routine prenatal care secondary to cost and turnaround time. Eventually, decreasing costs may allow personal genome sequencing to become available for routine analysis, but annotation and interpretation of variant information are essential to provide information that can be used to better manage an individual’s condition. The expanded use of non-invasive fetal exome or genome sequencing would present several advantages and challenges. Broader use might lead to the improved detection of Mendelian disorders in families who would not otherwise have been offered prenatal testing, as well as in families who might have refused invasive testing because of risk of fetal loss. However, the concern is that it will identify variants that are beyond the scope of
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conventional prenatal screening and diagnosis, specifically variants that indicate increased risk for developing adult-onset conditions. The relevance of such information is controversial for a current pregnancy, but it may have significant implications for the health of the parent. Therefore, the broader implementation of non-invasive fetal genome sequencing will lead to wider application of the results and increased availability. 7. Conclusions Determining accurate genetic diagnosis prenatally permits families to make informed reproductive decisions. Therefore, it is important for the provider to be aware of the spectrum of potential genetic conditions, and to use appropriate testing modalities to obtain specific diagnosis.
Practice points The birth of a child with intellectual disabilities and/or congenital anomalies is an upsetting event for any family, so if a child is born with these problems, think ahead to the next pregnancy and try to ascertain the cause before the next pregnancy. It is best to perform SNP chromosomal microarray before attempting to sequence any gene(s) because many gene sequencing techniques do not detect CNVs. Exome sequencing and whole genome sequencing are time consuming (at least 6 months), so if this needs to be done to obtain a diagnosis that will provide accurate prenatal diagnosis, allow sufficient time.
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Please cite this article in press as: Babkina N, Graham Jr. JM, New genetic testing in prenatal diagnosis, Seminars in Fetal & Neonatal Medicine (2013), http://dx.doi.org/10.1016/j.siny.2013.10.005