Diana W. Bianchi

Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome.

Griscelli syndrome (GS, MIM 214450), a rare, autosomal recessive disorder, results in pigmentary dilution of the skin and the hair, the presence of large clumps of pigment in hair shafts and an accumulation of melanosomes in melanocytes. Most patients also develop an uncontrolled T-lymphocyte and macrophage activation syndrome (known as haemophagocytic syndrome, HS), leading to death in the absence of bone-marrow transplantation. In contrast, early in life some GS patients show a severe neurological impairment without apparent immune abnormalities. We previously mapped the GS locus to chromosome 15q21 and found a mutation in a gene (MYO5A) encoding a molecular motor in two patients. Further linkage analysis suggested a second gene associated with GS was in the same chromosomal region. Homozygosity mapping in additional families narrowed the candidate region to a 3.1-cM interval between D15S1003 and D15S962. We detected mutations in RAB27A, which lies within this interval, in 16 patients with GS. Unlike MYO5A, the GTP-binding protein RAB27A appears to be involved in the control of the immune system, as all patients with RAB27A mutations, but none with the MYO5A mutation, developed HS. In addition, RAB27A-deficient T cells exhibited reduced cytotoxicity and cytolytic granule exocytosis, whereas MYO5A-defective T cells did not. RAB27A appears to be a key effector of cytotoxic granule exocytosis, a pathway essential for immune homeostasis.

Genome-Wide Fetal Aneuploidy Detection by Maternal Plasma DNA Sequencing

OBJECTIVE: To prospectively determine the diagnostic accuracy of massively parallel sequencing to detect whole chromosome fetal aneuploidy from maternal plasma. METHODS: Blood samples were collected in a prospective, blinded study from 2,882 women undergoing prenatal diagnostic procedures at 60 U.S. sites. An independent biostatistician selected all singleton pregnancies with any abnormal karyotype and a balanced number of randomly selected pregnancies with euploid karyotypes. Chromosome classifications were made for each sample by massively parallel sequencing and compared with fetal karyotype. RESULTS: Within an analysis cohort of 532 samples, the following were classified correctly: 89 of 89 trisomy 21 cases (sensitivity 100%, 95% [confidence interval] CI 95.9–100), 35 of 36 trisomy 18 cases (sensitivity 97.2%, 95% CI 85.5–99.9), 11 of 14 trisomy 13 cases (sensitivity 78.6%, 95% CI 49.2–99.9), 232 of 233 females (sensitivity 99.6%, 95% CI 97.6 to more than 99.9), 184 of 184 males (sensitivity 100%, 95% CI 98.0–100), and 15 of 16 monosomy X cases (sensitivity 93.8%, 95% CI 69.8–99.8). There were no false-positive results for autosomal aneuploidies (100% specificity, 95% CI more than 98.5 to 100). In addition, fetuses with mosaicism for trisomy 21 (3/3), trisomy 18 (1/1), and monosomy X (2/7), three cases of translocation trisomy, two cases of other autosomal trisomies (20 and 16), and other sex chromosome aneuploidies (XXX, XXY, and XYY) were classified correctly. CONCLUSION: This prospective study demonstrates the efficacy of massively parallel sequencing of maternal plasma DNA to detect fetal aneuploidy for multiple chromosomes across the genome. The high sensitivity and specificity for the detection of trisomies 21, 18, 13, and monosomy X suggest that massively parallel sequencing can be incorporated into existing aneuploidy screening algorithms to reduce unnecessary invasive procedures. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, www.clinicaltrials.gov, NCT01122524. LEVEL OF EVIDENCE: II

Impact of maternal age on obstetric outcome

OBJECTIVE: To estimate the effect of maternal age on obstetric outcomes. METHODS: A prospective database from a multicenter investigation of singletons, the FASTER trial, was studied. Subjects were divided into 3 age groups: 1) less than 35 years, 2) 35–39 years, and 3) 40 years and older. Multivariable logistic regression analysis was used to assess the effect of age on outcomes after adjusting for race, parity, body mass index, education, marital status, smoking, medical history, use of assisted conception, and patients study site. RESULTS: A total of 36,056 women with complete data were available: 28,398 (79%) less than 35 years of age; 6,294 (17%) 35–39 years; and 1,364 (4%) 40 years and older. Increasing age was significantly associated with miscarriage (adjusted odds ratio [adjOR]2.0 and 2.4 for ages 35–39 years and age 40 years and older, respectively), chromosomal abnormalities (adjOR 4.0 and 9.9), congenital anomalies (adjOR 1.4 and 1.7), gestational diabetes (adjOR 1.8 and 2.4), placenta previa (adjOR 1.8 and 2.8), and cesarean delivery (adjOR 1.6 and 2.0). Patients aged 35–39 years were at increased risk for macrosomia (adjOR 1.4). Increased risk for abruption (adjOR 2.3), preterm delivery (adjOR 1.4), low birth weight (adjOR 1.6), and perinatal mortality (adjOR 2.2) was noted in women aged 40 years and older. CONCLUSION: Increasing maternal age is independently associated with specific adverse pregnancy outcomes. Increasing age is a continuum rather than a threshold effect. LEVEL OF EVIDENCE: II-2

PCR Quantitation of Fetal Cells in Maternal Blood in Normal and Aneuploid Pregnancies

Fetal cells in maternal blood are a noninvasive source of fetal genetic material for prenatal diagnosis. We determined the number of fetal-cell DNA equivalents present in maternal whole-blood samples to deduce whether this number is affected by fetal karyotype. Peripheral blood samples were obtained from 199 women carrying chromosomally normal fetuses and from 31 women with male aneuploid fetuses. Male fetal-cell DNA-equivalent quantitation was determined by PCR amplification of a Y chromosome-specific sequence and was compared with PCR product amplified from known concentrations of male DNA run simultaneously. The mean number of male fetal-cell DNA equivalents detected in 16-ml blood samples from 90 women bearing a 46,XY fetus was 19 (range 0-91). The mean number of male fetal-cell DNA equivalents detected in 109 women bearing a 46,XX fetus was 2 (range 0-24). The mean number of male fetal-cell DNA equivalents detected when the fetus was male compared with when the fetus was female was highly significant (P = .0001). More fetal cells were detected in maternal blood when the fetus was aneuploid. The mean number of male fetal-cell DNA equivalents detected when the fetal karyotype was 47,XY,+21 was 110 (range 0.1-650), which was significantly higher than the number of male fetal-cell DNA equivalents detected in 46,XY fetuses (P = .0001). Feto-maternal transfusion of nucleated cells appears to be influenced by fetal karyotype. The sixfold elevation of fetal cells observed in maternal blood when the fetus had trisomy 21 indicates that noninvasive cytogenetic diagnosis of trisomy 21 should be feasible.

DNA Sequencing versus Standard Prenatal Aneuploidy Screening

BACKGROUND In high-risk pregnant women, noninvasive prenatal testing with the use of massively parallel sequencing of maternal plasma cell-free DNA (cfDNA testing) accurately detects fetal autosomal aneuploidy. Its performance in low-risk women is unclear. METHODS At 21 centers in the United States, we collected blood samples from women with singleton pregnancies who were undergoing standard aneuploidy screening (serum biochemical assays with or without nuchal translucency measurement). We performed massively parallel sequencing in a blinded fashion to determine the chromosome dosage for each sample. The primary end point was a comparison of the false positive rates of detection of fetal trisomies 21 and 18 with the use of standard screening and cfDNA testing. Birth outcomes or karyotypes were the reference standard. RESULTS The primary series included 1914 women (mean age, 29.6 years) with an eligible sample, a singleton fetus without aneuploidy, results from cfDNA testing, and a risk classification based on standard screening. For trisomies 21 and 18, the false positive rates with cfDNA testing were significantly lower than those with standard screening (0.3% vs. 3.6% for trisomy 21, P<0.001; and 0.2% vs. 0.6% for trisomy 18, P=0.03). The use of cfDNA testing detected all cases of aneuploidy (5 for trisomy 21, 2 for trisomy 18, and 1 for trisomy 13; negative predictive value, 100% [95% confidence interval, 99.8 to 100]). The positive predictive values for cfDNA testing versus standard screening were 45.5% versus 4.2% for trisomy 21 and 40.0% versus 8.3% for trisomy 18. CONCLUSIONS In a general obstetrical population, prenatal testing with the use of cfDNA had significantly lower false positive rates and higher positive predictive values for detection of trisomies 21 and 18 than standard screening. (Funded by Illumina; ClinicalTrials.gov number, NCT01663350.).

Maternal thyroid hypofunction and pregnancy outcome.

OBJECTIVE: To estimate whether maternal thyroid hypofunction is associated with complications. METHODS: A total of 10,990 patients had first- and second-trimester serum assayed for thyroid-stimulating hormone (TSH), free thyroxine (freeT4), and antithyroglobulin and antithyroid peroxidase antibodies. Thyroid hypofunction was defined as 1) subclinical hypothyroidism: TSH levels above the 97.5th percentile and free T4 between the 2.5th and 97.5th percentiles or 2) hypothyroxinemia: TSH between the 2.5th and 97.5th percentiles and free T4 below the 2.5th percentile. Adverse outcomes were evaluated. Patients with thyroid hypofunction were compared with euthyroid patients (TSH and free T4 between the 2.5th and 97.5th percentiles). Patients with and without antibodies were compared. Multivariable logistic regression analysis adjusted for confounders was used. RESULTS: Subclinical hypothyroidism was documented in 2.2% (240 of 10,990) in the first and 2.2% (243 of 10,990) in the second trimester. Hypothyroxinemia was documented in 2.1% (232 of 10,990) in the first and 2.3% (247 of 10,990) in the second trimester. Subclinical hypothyroidism was not associated with adverse outcomes. In the first trimester, hypothyroxinemia was associated with preterm labor (adjusted odds ratio [aOR] 1.62; 95% confidence interval [CI] 1.00–2.62) and macrosomia (aOR 1.97; 95% CI 1.37–2.83). In the second trimester, it was associated with gestational diabetes (aOR 1.7; 95% CI 1.02–2.84). Fifteen percent (1,585 of 10,990) in the first and 14% (1,491 of 10,990) in the second trimester had antithyroid antibodies. When both antibodies were positive in either trimester, there was an increased risk for preterm premature rupture of membranes (P=.002 and P<.001, respectively). CONCLUSION: Maternal thyroid hypofunction is not associated with a consistent pattern of adverse outcomes. LEVEL OF EVIDENCE: II

Microchimerism of presumed fetal origin in thyroid specimens from women: a case-control study

BACKGROUND Some so-called autoimmune diseases in women might be alloimmune and represent a chronic graft-versus-host response attributable to transplacentally acquired fetal cells. Thyroid disease is more common in women than men, and post partum exacerbation of thyroiditis is common. Our aim was to investigate whether there is an association between fetal cell microchimerism and thyroid disease in women. METHODS Surgical specimens were obtained from 29 women who underwent thyroidectomy for various thyroid disorders. Control specimens were taken from clinically and histologically normal thyroids obtained at necropsy from eight women who died from unrelated conditions. Medical records and pregnancy histories were reviewed. Fluorescence in-situ hybridisation analysis was done with probes specific for X and Y chromosomes. Slides were examined with a fluorescence microscope to detect the presence of male cells-with one X and one Y signal in the nucleus-among maternal cells containing two X signals. FINDINGS Male cells were seen in thyroid sections from 16 patients but not in those from controls (p=0.01). Male cells (1-165 per slide) were seen individually or in clusters in all thyroid diseases and were not restricted to inflammatory thyroid diseases. In one patient with a progressively enlarging goitre, we noted fully differentiated male thyroid follicles closely attached to and indistinguishable from the rest of the thyroid. INTERPRETATION Our findings suggest a relation between fetal cell microchimerism and thyroid disease. Furthermore, fetal stem cells might be capable of differentiation into mature thyroid follicles in their mothers with favourable environmental and developmental factors.

Assisted reproductive technology and pregnancy outcome.

OBJECTIVE: To determine whether the use of assisted reproductive technology (ART) is associated with an increase in chromosomal abnormalities, fetal malformations, or adverse pregnancy outcomes. METHODS: A prospective database from a large multicenter investigation of singleton pregnancies, the First And Second Trimester Evaluation of Risk trial, was examined. Subjects were divided into 3 groups: no ART use, use of ovulation induction (with or without intrauterine insemination), and use of in vitro fertilization (IVF). Multivariate logistic regression analysis was used to assess association between ART and adverse pregnancy outcomes (significance of differences was accepted at P < .05). RESULTS: A total of 36,062 pregnancies were analyzed: 34,286 (95.1%) were spontaneously conceived, 1,222 (3.4%) used ovulation induction, and 554 (1.5%) used IVF. There was no association between ART and fetal growth restriction, aneuploidy, or fetal anomalies after adjustment for age, race, marital status, years of education, prior preterm delivery, prior fetal anomaly, body mass index, smoking history, and bleeding in the current pregnancy. Ovulation induction was associated with a statistically significant increase in placental abruption, fetal loss after 24 weeks, and gestational diabetes after adjustment. Use of IVF was associated with a statistically significant increase in preeclampsia, gestational hypertension, placental abruption, placenta previa, and risk of cesarean delivery. CONCLUSION: Patients who undergo IVF are at increased risk for several adverse pregnancy outcomes. Although many of these risks are not seen in patients undergoing ovulation induction, several adverse pregnancy outcomes are still increased in this group. There was no increased incidence of fetal chromosomal or structural abnormalities in the women who used any type of ART compared with the women who conceived spontaneously. LEVEL OF EVIDENCE: II-2

Noninvasive Detection of Fetal Subchromosome Abnormalities via Deep Sequencing of Maternal Plasma

The purpose of this study was to determine the deep sequencing and analytic conditions needed to detect fetal subchromosome abnormalities across the genome from a maternal blood sample. Cell-free (cf) DNA was isolated from the plasma of 11 pregnant women carrying fetuses with subchromosomal duplications and deletions, translocations, mosaicism, and trisomy 20 diagnosed by metaphase karyotype. Massively parallel sequencing (MPS) was performed with 25-mer tags at approximately 10(9) tags per sample and mapped to reference human genome assembly hg19. Tags were counted and normalized to fixed genome bin sizes of 1 Mb or 100 kb to detect statistically distinct copy-number changes compared to the reference. All seven cases of microdeletions, duplications, translocations, and the trisomy 20 were detected blindly by MPS, including a microdeletion as small as 300 kb. In two of these cases in which the metaphase karyotype showed additional material of unknown origin, MPS identified both the translocation breakpoint and the chromosomal origin of the additional material. In the four mosaic cases, the subchromosomal abnormality was not demonstrated by MPS. This work shows that in nonmosaic cases, it is possible to obtain a fetal molecular karyotype by MPS of maternal plasma cfDNA that is equivalent to a chromosome microarray and in some cases is better than a metaphase karyotype. This approach combines the advantage of enhanced fetal genomic resolution with the improved safety of a noninvasive maternal blood test.

Fetal cells in the maternal circulation: feasibility for prenatal diagnosis

Over the past 25 years the utilization of prenatal diagnosis by expectant couples and their physicians has expanded due primarily to two trends: smaller family size, with an increased emphasis on assurance of the ‘normalcy’ of each child, and advancing parental age. In the United States it is currently considered ‘standard of care’ to offer prenatal cytogenetic diagnosis to pregnant women who will be 35 years or older at the time of delivery (American College of Obstetricians and Gynecologists, 1996). Such cytogenetic diagnoses are facilitated by obtaining fetal nucleated cells via an invasive technique such as chorionic villus sampling (CVS) or amniocentesis. 35 years of age was established as the threshold for these procedures because the liveborn incidence of an infant with the most common autosomal aneuploidy, trisomy 21, is roughly equal to the chance of a miscarriage secondary to these procedures (approximately 1 in 250). Despite the safety and accuracy of these techniques, to date there has been little impact upon the incidence of trisomy 21, which is still close to 1 per 1000 live births. A major reason for this is that prenatal diagnostic techniques are directed towards a minority of pregnant women. Although, individually, older pregnant women are at increased risk for having a baby with trisomy 21, as a group they are having a relatively small fraction of the total births. 80% of the newborn infants with Down syndrome are born to women under age 35, who are not offered the invasive prenatal diagnostic techniques because the risk of a procedural complication is greater than the incidence of Down syndrome in a given fetus. Therefore, over the past decade, increased attention has been paid to noninvasive techniques of screening for fetal trisomy 21 that can be offered to all pregnant women. Trisomy 21 is used as a benchmark because it is the most common liveborn aneuploidy associated with mental retardation and serious congenital anomalies. At present, screening for trisomy 21 consists initially of assessing the maternal age, because the risk of fetal chromosomal abnormalities due to nondisjunction increases as maternal age advances. In addition, second-trimester maternal serum screening for proteins such as human chorionic gonadotropin (hCG), oestriol, and alphafetoprotein (AFP) has been incorporated into routine obstetric care. Results are measured in absolute values, expressed in terms of multiples of the median, and interpreted as a mathematical risk for fetal trisomy 21. Women who have a test result that indicates a fetal Down syndrome risk of greater than 1 in 270 are offered amniocentesis. Because of the large-scale success of the serum-screening programmes which have been employed in the United States, Europe and Asia, research has been directed towards improving both their sensitivity and specificity. Current tests detect 60–70% of the cases of trisomy 21 with a calculated false positive rate of 5% (Wald et al, 1988). Addition of another marker such as dimeric inhibin A raises the sensitivity to 75% (Wald et al, 1996). More recently, a study measuring serum markers expressed during the first trimester, b-hCG and pregnancy-associated plasma protein A (PAPP-A), concluded that the sensitivity of screening at 10– 13 weeks of gestation is nearly as good as the secondtrimester test (Haddow et al, 1998). An independent and newer method to screen for fetal trisomy 21 is the sonographic assessment of a fluid-filled space at the back of the fetal neck known as the nuchal translucency (NT) measurement. An increased NT measurement over that expected for gestational age is associated with an increased risk of fetal chromosome and cardiac abnormalities (Nicolaides et al, 1992; Hyett et al, 1997). In a study of 96 127 singleton pregnancies, the combination of maternal age and NT measurement enabled detection of 77% of the fetuses with trisomy 21 in the 5% of patients with the largest NTs (Snijders et al, 1998). However, by recommending amniocentesis or CVS following a positive screening test, 30 invasive tests are required to find one affected fetus. According to an editorial that accompanied the above study, antenatal screening research for Down syndrome is likely to place more emphasis on lowering the false-positive rate to 1% or less by discovering new markers and reconfiguring existing screening techniques (Haddow, 1998). It is into the existing context of noninvasive screening for Down syndrome that fetal cells in maternal blood must be placed. Successful isolation of fetal cells from maternal blood represents a source of fetal chromosomes or DNA obtained non-invasively by maternal venepuncture. This test could be used as a primary screen, a secondary screen designed to be used in concert with the aforementioned screening tests (to reduce the 5% false-positive rate) (Farina & Bianchi, 1998), or, ultimately, as a diagnostic test. This review will summarize the key areas related to this field: the clinical implications of this test for the clinical practice of obstetrics and gynaecology, the surprising insights into the biology of pregnancy that have come from the study of fetal cells in maternal blood, and the technical challenges associated with rare event cell separation. British Journal of Haematology, 1999, 105, 574–583