Matthew W. Snyder

Noninvasive Whole-Genome Sequencing of a Human Fetus

Sequencing of cell-free fetal-derived DNA from maternal plasma provides a noninvasive way to predict the fetal genome sequence. Not Your Mother’s Genome There are more than 3000 single-gene (Mendelian) disorders that are individually rare but collectively affect ~1% of births. Currently, only a few specific disorders are screened for during pregnancy, and definitive diagnosis requires invasive procedures such as amniocentesis. An ideal prenatal genetic diagnostic would noninvasively screen for all Mendelian disorders early in pregnancy. Exploiting the observation that ~10% of DNA floating freely in a pregnant woman’s plasma originates from the fetus she carries, several groups have developed DNA sequencing–based tests for conditions such as trisomy 21, the genetic cause of Down syndrome. Although these tests may readily detect gross abnormalities such as an extra copy of an entire chromosome, the noninvasive determination of a complete fetal genome sequence has remained out of reach. Here, Kitzman et al. reconstruct the whole-genome sequence of a human fetus using samples obtained noninvasively during the second trimester, including DNA from the pregnant mother, DNA from the father, and “cell-free” DNA from the pregnant mother’s plasma (a mixture of the maternal and fetal genomes). A big challenge for the authors was to be able to predict which genetic variants were passed from mother to fetus, because the overwhelming majority of DNA in the pregnant mother’s plasma derives from her genome rather than that of the fetus. To overcome this problem, the authors applied a recently developed technique to resolve the mother’s “haplotypes”—groups of genetic variants residing on the same chromosomes—and then used these groups to accurately predict inheritance. Another challenge was the identification of new mutations in the genome of the fetus. The authors demonstrate that, in principle, such mutations can be sensitively detected and triaged for validation. Although these methods must be refined and their costs driven down, this study hints that comprehensive, noninvasive prenatal screening for Mendelian disorders may be clinically feasible in the near future. Analysis of cell-free fetal DNA in maternal plasma holds promise for the development of noninvasive prenatal genetic diagnostics. Previous studies have been restricted to detection of fetal trisomies, to specific paternally inherited mutations, or to genotyping common polymorphisms using material obtained invasively, for example, through chorionic villus sampling. Here, we combine genome sequencing of two parents, genome-wide maternal haplotyping, and deep sequencing of maternal plasma DNA to noninvasively determine the genome sequence of a human fetus at 18.5 weeks of gestation. Inheritance was predicted at 2.8 × 106 parental heterozygous sites with 98.1% accuracy. Furthermore, 39 of 44 de novo point mutations in the fetal genome were detected, albeit with limited specificity. Subsampling these data and analyzing a second family trio by the same approach indicate that parental haplotype blocks of ~300 kilo–base pairs combined with shallow sequencing of maternal plasma DNA is sufficient to substantially determine the inherited complement of a fetal genome. However, ultradeep sequencing of maternal plasma DNA is necessary for the practical detection of fetal de novo mutations genome-wide. Although technical and analytical challenges remain, we anticipate that noninvasive analysis of inherited variation and de novo mutations in fetal genomes will facilitate prenatal diagnosis of both recessive and dominant Mendelian disorders.

Copy-Number Variation and False Positive Prenatal Aneuploidy Screening Results

Investigations of noninvasive prenatal screening for aneuploidy by analysis of circulating cell-free DNA (cfDNA) have shown high sensitivity and specificity in both high-risk and low-risk cohorts. However, the overall low incidence of aneuploidy limits the positive predictive value of these tests. Currently, the causes of false positive results are poorly understood. We investigated four pregnancies with discordant prenatal test results and found in two cases that maternal duplications on chromosome 18 were the likely cause of the discordant results. Modeling based on population-level copy-number variation supports the possibility that some false positive results of noninvasive prenatal screening may be attributable to large maternal copy-number variants. (Funded by the National Institutes of Health and others.).

Large-scale genomic sequencing of extraintestinal pathogenic Escherichia coli strains

Large-scale bacterial genome sequencing efforts to date have provided limited information on the most prevalent category of disease: sporadically acquired infections caused by common pathogenic bacteria. Here, we performed whole-genome sequencing and de novo assembly of 312 blood- or urine-derived isolates of extraintestinal pathogenic (ExPEC) Escherichia coli, a common agent of sepsis and community-acquired urinary tract infections, obtained during the course of routine clinical care at a single institution. We find that ExPEC E. coli are highly genomically heterogeneous, consistent with pan-genome analyses encompassing the larger species. Investigation of differential virulence factor content and antibiotic resistance phenotypes reveals markedly different profiles among lineages and among strains infecting different body sites. We use high-resolution molecular epidemiology to explore the dynamics of infections at the level of individual patients, including identification of possible person-to-person transmission. Notably, a limited number of discrete lineages caused the majority of bloodstream infections, including one subclone (ST131-H30) responsible for 28% of bacteremic E. coli infections over a 3-yr period. We additionally use a microbial genome-wide-association study (GWAS) approach to identify individual genes responsible for antibiotic resistance, successfully recovering known genes but notably not identifying any novel factors. We anticipate that in the near future, whole-genome sequencing of microorganisms associated with clinical disease will become routine. Our study reveals what kind of information can be obtained from sequencing clinical isolates on a large scale, even well-characterized organisms such as E. coli, and provides insight into how this information might be utilized in a healthcare setting.

Noninvasive fetal genome sequencing: a primer

We recently demonstrated whole genome sequencing of a human fetus using only parental DNA samples and plasma from the pregnant mother. This proof‐of‐concept study demonstrated how samples obtained noninvasively in the first or second trimester can be analyzed to yield a highly accurate and substantially complete genetic profile of the fetus, including both inherited and de novo variation. Here, we revisit our original study from a clinical standpoint, provide an overview of the scientific approach, and describe opportunities and challenges along the path toward clinical adoption of noninvasive fetal whole genome sequencing.

Non-invasive fetal genome sequencing: Opportunities and challenges†

We recently predicted the whole genome sequence of a human fetus using samples obtained non-invasively from the pregnant mother and the father. [Kitzman et al., 2012] This advance raises the possibility that it may soon be possible to perform genome-wide prenatal genetic testing without an invasive procedure early in pregnancy. Such a test would substantially broaden the scope of fetal genetic results that could be available prenatally. Non-invasive fetal genome sequencing (NIFGS) does not inherently raise new ethical issues, or those that cannot be addressed within the existing framework of medical bioethics. Indeed, many of the same issues have been raised by the introduction of other prenatal testing / screening technologies, now in wide use, and again more recently by the introduction of whole genome sequencing for clinical diagnosis. [Sayres et al., 2011, Schmitz et al., 2009, Ravitsky, 2009, Benn et al., 2009, Tabor et al., 2011, Berg et al., 2011] However, the ethical issues are, somewhat, magnified by the possibility of NIFGS and compounded by controversies surrounding elective pregnancy termination, rights of individuals with disabilities, and eugenics. Accordingly, the prospect of successful NIFGS, even on a research basis, is likely to generate considerable controversy and debate about the acceptability of developing such technologies, much less if and how they should be used. We view this response as very positive because it provides all stakeholders and the broader public in general with the opportunity to carefully consider and deliberate these issues in what we would hope is a thoughtful and balanced way. As NIFGS becomes technically tractable and increasingly cost-effective, and as an acceptable false positive/false negative profile is achieved, one population for which it might be of great benefit may be pregnant women who are currently offered invasive prenatal diagnostic testing. Such women are typically at risk for genetic conditions based on screening results or family history, and NIFGS would likely reduce if not eliminate adverse outcomes from invasive testing for most of these women. The expanded use of NIFGS would present several advantages and challenges. Broader use of NIFGS might lead to the greater detection of Mendelian disorders in families who would not otherwise have been offered prenatal testing, as well as families who might have refused invasive testing because of risks to the pregnancy and fetus. NIFGS could augment or even replace current approaches to neonatal screening as most such disorders are autosomal or X-linked recessive (e.g., hypothyroidism and congenital hearing loss are only sometimes Mendelian). Prenatal identification of disorders now found in neonatal screening would afford for earlier parental education, diminished false positives and the accompanying costs of retesting and parental anxiety and earlier therapeutic interventions. Earlier detection of such disorders would also foster improved prenatal care, pregnancy and delivery management and/or postnatal intervention. For example, 90% of genetic variants in SCNA1 that cause seizure disorders are de novo, and identification by NIFGS could allow for diagnosis before the onset of seizures and consideration of appropriate precautions and/or pharmacological treatment. [Marini et al., 20011] Similarly, 50% of mutations causing Multiple Endocrine Neoplasia 2B are spontaneous, and earlier identification of these mutations could prompt prophylactic thyroidectomy and improve outcomes. [Carlson et al., 1994] The availability of NIFGS could increase the utilization of prenatal testing, and in turn increase rates of elective termination, both for disorders for which testing is currently available and for the wide arrange of disorders and traits for which testing would be newly available. [Tischler et al., 2011] On the other hand, NIFGS might also make pregnancy termination safer, less costly, and less traumatic as it could be performed early in gestation. Broad use of NIFGS might result in increased societal pressure for pregnant women to undergo screening and terminate any fetus suspected to have a Mendelian condition. This could reverse important and continuing social progress towards civil rights and social support for people and families with disabilities. In addition, this societal pressure might threaten parental autonomy over reproductive decision-making. Broader use of NIFGS might also create or magnify social stigmas or inequities. NIFGS would likely remain expensive and may not be reimbursable by insurance in the short-term. This might exaggerate disparities between people who can easily afford access and those who cannot. If access is limited to those who can afford it, it is possible that a disproportionate number of lower income families could suffer from the higher rates of morbidity and mortality of invasive testing. In the extreme scenario, children with Mendelian conditions would be disproportionately born to lower income families that could not afford NIFGS. Such a disparity would likely further stigmatize many of these conditions and exaggerate existing disparities in access to healthcare and benefits for these populations. Another key issue raised by NIFGS is that it represents a substantially more comprehensive test for Mendelian disorders with a known cause, and will identify variants that are beyond the scope of conventional prenatal screening and diagnosis. Specifically, variants will be identified that indicate increased risk for developing adult onset conditions. This is not unique to NIFGS: in fact this is an ongoing challenge in pediatric clinical genetic testing. [Wilfond et al., 2009] Such information may be irrelevant or inappropriate to return for the benefit of the fetus/future child, but may have direct implications for the health of the parent, and therefore provide indirect benefit to any current or future children. However, if NIFGS is more broadly implemented, the scope of the results identified and the number of individuals affected may increase substantially. This will further overwhelm the existing infrastructure for providing genetic counseling. As with other applications of whole-genome sequencing, NIFGS will identify variants of ambiguous clinical utility in genes known to be associated with both pediatric and adult complex disease. For example, Kitzman et al. found a de novo novel missense variant in ACMSD, a gene in which common variants have been associated with Parkinson disease by genome-wide association. [Klitzman et al., 2012, International Parkinson Disease Genomics Consortium et al., 2011] This variant causes substitution of a highly conserved amino acid residue, but in the absence of compelling evidence of its role in Parkinson disease or other conditions, its detection is of limited clinical value. While this is no different than the challenge of interpreting WGS information in general, pregnancy might be a particularly vulnerable time in which to receive this information and parents might feel compelled to give more credence to the information than it warrants. There are several other important issues that require consideration. Will the non-invasive nature of this test, combined with the enhanced detection of Mendelian disorders, lead to a substantial increase in the number of women who consider prenatal diagnosis? How will the medical community meet the challenge of providing genetic counseling to address the complex nature of the information that may be identified? These concerns raise the possibility that some women may not be able to provide adequate informed consent, or may proceed with actions such as terminations without complete understanding of the test results or the prognosis for various rare Mendelian disorders. If NIFGS allows the creation of a record of a child’s whole genome prior to its birth, what should happen to that data? Should it be stored as part of the child’s medical record, with the possibility for future updating, analysis and mining for medically relevant information? Or should it be destroyed? Who should make this decision and have control over the data? As with many new technologies, NIFGS will be accompanied by many ethical and social challenges. We think that it is imperative that these questions and issues be discussed and addressed by a diverse group of stakeholders, as well as through collection of empirical data on stakeholder perspectives and concerns. Much can be learned from the history of the implementation of other prenatal testing approaches, such as amniocentesis and CVS, as well as the ongoing debates about pediatric genetic testing and return of results from whole genome sequencing. [Rapp, 2000]

Copy-Number Variation and False Positive Prenatal Aneuploidy Screening Results: The Authors Reply

To the Editor: The study by Snyder et al. (April 23 issue)1 highlights some shortcomings of massively parallel shotgun sequencing methods in noninvasive prenatal screening. They used a method that does not target specific regions of chromosomes 18 and 21, and they provided evidence to show that maternal copy-number variants may have interfered with the assessment of fetal aneuploidy. Two aspects of the design of targeted assays such as Digital Analysis of Selected Regions (DANSR)2 reduce the effect of maternal copy-number variants in assessment of the risk of fetal aneuploidy. First, they target chromosome locations that have not been observed to harbor common copy-number variants. Second, the Fetal-fraction Optimized Risk of Trisomy Evaluation (FORTE) algorithm dynamically compares chromosome quantitation with the estimated fetal fraction for each assay and masks assays of outlier regions.3 In Figure 1, we show four examples in which appropriate low-risk reports were generated with the use of the FORTE algorithm despite the presence of rare maternal copy-number variants ranging from 1.4 to 7.4 Mb. Christopher Kingsley, Ph.D. Eric Wang, Ph.D. Arnold Oliphant, Ph.D. Ariosa Diagnostics San Jose, CA No potential conflict of interest relevant to this letter was reported.

Genome wide association with quantitative resistance phenotypes in Mycobacterium tuberculosis reveals novel resistance genes and regulatory regions

Drug resistance is threatening attempts at tuberculosis epidemic control. Molecular diagnostics for drug resistance that rely on the detection of resistance-related mutations could expedite patient care and accelerate progress in TB eradication. We performed minimum inhibitory concentration testing for 12 anti-TB drugs together with Illumina whole genome sequencing on 1452 clinical Mycobacterium tuberculosis (MTB) isolates. We then used a linear mixed model to evaluate genome wide associations between mutations in MTB genes or noncoding regions and drug resistance, followed by validation of our findings in an independent dataset of 792 patient isolates. Novel associations at 13 genomic loci were confirmed in the validation set, with 2 involving noncoding regions. We found promoter mutations to have smaller average effects on resistance levels than gene body mutations in genes where both can contribute to resistance. Enabled by a quantitative measure of resistance, we estimated the heritability of the resistance phenotype to 11 anti-TB drugs and identify a lower than expected contribution from known resistance genes. We also report the proportion of variation in resistance levels explained by the novel loci identified here. This study highlights the complexity of the genomic mechanisms associated with the MTB resistance phenotype, including the relatively large number of potentially causative or compensatory loci, and emphasizes the contribution of the noncoding portion of the genome.

Noninvasive fetal genome sequencing: a primer: Noninvasive fetal genome sequencing: a primer

We recently demonstrated whole genome sequencing of a human fetus using only parental DNA samples and plasma from the pregnant mother. This proof‐of‐concept study demonstrated how samples obtained noninvasively in the first or second trimester can be analyzed to yield a highly accurate and substantially complete genetic profile of the fetus, including both inherited and de novo variation. Here, we revisit our original study from a clinical standpoint, provide an overview of the scientific approach, and describe opportunities and challenges along the path toward clinical adoption of noninvasive fetal whole genome sequencing.

Noninvasive fetal genome sequencing

We recently demonstrated whole genome sequencing of a human fetus using only parental DNA samples and plasma from the pregnant mother. This proof‐of‐concept study demonstrated how samples obtained noninvasively in the first or second trimester can be analyzed to yield a highly accurate and substantially complete genetic profile of the fetus, including both inherited and de novo variation. Here, we revisit our original study from a clinical standpoint, provide an overview of the scientific approach, and describe opportunities and challenges along the path toward clinical adoption of noninvasive fetal whole genome sequencing.