Hilary S. Gammill

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.

Effect of parity on fetal and maternal microchimerism: interaction of grafts within a host?

Small amounts of genetically foreign cells (microchimerism, Mc) traffic between a mother and fetus during pregnancy. Commonly, these grafts durably persist. For women, multiple naturally acquired Mc grafts can accrue, as they harbor Mc from their own mothers (maternal Mc, MMc) and subsequently acquire fetal Mc (FMc) through pregnancy. The nature of interactions between these naturally acquired grafts may inform, and be informed by, observations in transplantation, including the effect of noninherited maternal HLA antigens (NIMA) and double-unit cord blood transplantation (CBT). We asked whether FMc and MMc are impacted by the addition of new grafts as evaluated by increasing parity. Mc was identified by quantitative PCR for a nonshared polymorphism unique to the Mc source. Despite increasing sources of Mc, FMc did not increase with increasing parity. MMc concentration was significantly lower with increasing parity. The odds ratio for detection of MMc for 2 or more births compared with 1 birth was .11 (95% CI 0.03-0.42, P = .001). These observations suggest that interactions occur among naturally acquired grafts and are of interest in light of recent observations of graft-graft interaction resulting in predominance of 1 unit in double-unit CBT and the correlation of MMc with the NIMA effect.

Adverse Pregnancy Outcomes and Risk of Subsequent Rheumatoid Arthritis

Pregnancy and reproductive outcomes have been associated with altered risk of some autoimmune diseases, including rheumatoid arthritis (RA). We sought to determine whether prior pregnancy resulting in a low birth weight (LBW) infant or preterm birth is associated with a risk of subsequent RA in the mother.

Mode of delivery at periviable gestational ages: impact on subsequent reproductive outcomes

Abstract Aim: The aim of this study was to assess the risk of subsequent delivery complications after extremely preterm deliveries by initial (index) pregnancy mode of delivery (MOD): cesarean (CD) versus vaginal (VD). Methods: This is a retrospective, longitudinal cohort study using Washington State birth certificate data and International Classification of Diseases, Ninth Revision codes, 1989–2008, identifying women with deliveries 20–26 weeks’ gestation and linked subsequent deliveries. Index MOD was considered as a predictor of adverse subsequent maternal and neonatal outcomes, using t-test, χ2-test or Fisher’s exact test, and regression analysis. Results: Of 2472 women with periviable delivery and subsequent birth, index CD (n=386) and index VD (n=2086) showed similar risks of composite morbidity (16.1% vs. 15.4%, P=0.76) and subsequent hemorrhage (9.6% vs. 11.1%, P=0.39). Women with index CD were more likely than index VD to experience uterine rupture (1.8% vs. 0.1%, P<0.001), to deliver earlier (35.9 vs. 36.9 weeks, P<0.001), and to have lower birth weight (2736 vs. 3014 g, P<0.001) subsequently. Neonatal hospital charges and lengths of stay were also higher after index CD. Conclusions: MOD at extreme prematurity did not impact subsequent maternal hemorrhage or overall morbidity. However, CD was associated with substantial uterine rupture risk despite evidence of practice to avoid labor (lower birth weight and earlier delivery) in the subsequent pregnancy.

Microchimerism in women with recurrent miscarriage.

Miscarriage is the most common pregnancy complication, and recurrent miscarriage (3 or more consecutive pregnancy losses) affects 1–5% of couples. Maternal-fetal exchange and the persistence of exchanged material as microchimerism appears to be disrupted in complicated pregnancies. We recently conducted a longitudinal cohort study of microchimerism in women with recurrent miscarriage. Our initial data raise multiple questions that require further investigation. Here, we review our data from this recent study and provide additional information regarding microchimerism in the granulocyte cell layer. This area of investigation offers a unique window into early reproductive events, and future related studies have the potential to identify novel therapeutic approaches and insights into human evolution.

Fetal cellular microchimerism in miscarriage and pregnancy termination.

Fetal cells transfer to the mother during pregnancy and can persist long-term as microchimerism. Acquisition of microchimerism may also occur during pregnancy loss, either miscarriage or pregnancy termination. Because nearly half of all pregnancies end in loss, we recently investigated the magnitude of fetal cell transfer during pregnancy loss and whether obstetric clinical factors impacted cell transfer. Prospective measurement of fetal cellular microchimerism before and after miscarriage and termination of pregnancy demonstrated a significant transfer of fetal cells in these pregnancies, with higher concentrations of fetal microchimerism in pregnancy termination vs. miscarriage and in those that were managed surgically vs. medically. The frequency of pregnancy loss as a proportion of all pregnancies, and the overrepresentation of fetal genetic abnormalities in pregnancy loss suggest that the resultant acquisition of fetal microchimerism could have a unique and substantial impact on women’s health.

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.