Anthony R. Gregg

ACMG statement on noninvasive prenatal screening for fetal aneuploidy

Noninvasive assessment of the fetal genome is now possible using next-generation sequencing technologies. The isolation of fetal DNA fragments from maternal circulation in sufficient quantity and sizes, together with proprietary bioinformatics tools, now allows patients the option of noninvasive fetal aneuploidy screening. However, obstetric care providers must become familiar with the advantages and disadvantages of the utilization of this approach as analysis of cell-free fetal DNA moves into clinical practice. Once informed, clinicians can provide efficient pretest and posttest counseling with the goal of avoiding patient harm. It is in the public’s best interest that test results contain key elements and that laboratories adhere to established quality control and proficiency testing standards. The analysis of cell-free fetal DNA in maternal circulation for fetal aneuploidy screening is likely the first of major steps toward the eventual application of whole fetal genome/whole fetal exome sequencing.Genet Med 2013:15(5):395–398

Noninvasive prenatal screening for fetal aneuploidy, 2016 update: a position statement of the American College of Medical Genetics and Genomics.

Disclaimer: This statement is designed primarily as an educational resource for clinicians to help them provide quality medical services. Adherence to this statement is completely voluntary and does not necessarily assure a successful medical outcome. This statement should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed toward obtaining the same results. In determining the propriety of any specific procedure or test, the clinician should apply his or her own professional judgment to the specific clinical circumstances presented by the individual patient or specimen. Clinicians are encouraged to document the reasons for the use of a particular procedure or test, whether or not it is in conformance with this statement. Clinicians also are advised to take notice of the date this statement was adopted and to consider other medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.Noninvasive prenatal screening using cell-free DNA (NIPS) has been rapidly integrated into prenatal care since the initial American College of Medical Genetics and Genomics (ACMG) statement in 2013. New evidence strongly suggests that NIPS can replace conventional screening for Patau, Edwards, and Down syndromes across the maternal age spectrum, for a continuum of gestational age beginning at 9–10 weeks, and for patients who are not significantly obese. This statement sets forth a new framework for NIPS that is supported by information from validation and clinical utility studies. Pretest counseling for NIPS remains crucial; however, it needs to go beyond discussions of Patau, Edwards, and Down syndromes. The use of NIPS to include sex chromosome aneuploidy screening and screening for selected copy-number variants (CNVs) is becoming commonplace because there are no other screening options to identify these conditions. Providers should have a more thorough understanding of patient preferences and be able to educate about the current drawbacks of NIPS across the prenatal screening spectrum. Laboratories are encouraged to meet the needs of providers and their patients by delivering meaningful screening reports and to engage in education. With health-care-provider guidance, the patient should be able to make an educated decision about the current use of NIPS and the ramifications of a positive, negative, or no-call result.Genet Med 18 10, 1056–1065.

Expanded carrier screening in reproductive medicine-points to consider: a joint statement of the American College of Medical Genetics and Genomics, American College of Obstetricians and Gynecologists, National Society of Genetic Counselors, Perinatal Quality Foundation, and Society for Maternal-Fetal Medicine.

The Perinatal Quality Foundation and the American College of Medical Genetics and Genomics, in association with the American College of Obstetricians and Gynecologists, the Society for Maternal-Fetal Medicine, and the National Society of Genetic Counselors, have collaborated to provide education for clinicians and laboratories regarding the use of expanded genetic carrier screening in reproductive medicine. This statement does not replace current screening guidelines, which are published by individual organizations to direct the practice of their constituents. As organizations develop practice guidelines for expanded carrier screening, further direction is likely. The current statement demonstrates an approach for health care providers and laboratories who wish to or who are currently offering expanded carrier screening to their patients.

ACMG position statement on prenatal/preconception expanded carrier screening.

For years, clinicians have offered gene-by-gene carrier screening to patients and couples considering future pregnancy or those with an ongoing pregnancy early in gestation. Examples include ethnic-specific screening offered to Ashkenazi Jewish patients and panethnic screening for cystic fibrosis and spinal muscular atrophy. Next-generation sequencing methods now available permit screening for many more disorders with high fidelity, quick turnaround time, and lower costs. However, instituting these technologies carries with it perils that must be addressed. The basis for the selection of disorders on expanded carrier screening panels should be disclosed. The information provided about disorders with mild phenotypes, variable expression, low penetrance, and/or characterized by an adult onset should be complete and transparent, allowing patients to opt out of receiving these test results. Patients also must be made aware of the concept of residual risk following negative test results. Laboratories have a duty to participate in and facilitate this information transfer.Genet Med 2013:15(6):482–483

Perinatal Development of Endothelial Nitric Oxide Synthase-Deficient Mice

Abstract The purpose of this study was to evaluate the influence of endothelial nitric oxide synthase (eNOS) deficiency on fetal growth, perinatal survival, and limb development in a mouse model with a targeted mutagenesis of the Nos3 gene. Wild-type (Nos3+/+) and eNOS-deficient fetuses (Nos3−/−) were evaluated on Gestational Day (E)15 and E17, and newborn pups were observed on Day 1 of life (D1). The average term duration of pregnancy was 19 days. For the evaluation of postnatal development, a breeding scheme consisting of Nos3+/−× Nos3+/−and Nos3−/− × Nos3−/− mice was established, and offspring were observed for 3 wk. Southern blotting was used for genotyping. No significant differences in fetal weight, crown-rump lengths (CRL), and placental weight were seen between Nos3+/+ and Nos3−/− fetuses on E15. By E17, Nos3−/− fetuses showed significantly reduced fetal weights, CRL, and placental weights. This difference in body weight was also seen throughout the whole postnatal period. In pregnancies of Nos3−/− females, the average number of pups alive on D1 was significantly decreased compared to either E15 or E17. Placental histology revealed no abnormalities. On E15, E17, and D1, Nos3−/− fetuses demonstrated focal acute hemorrhages in the distal limbs in 0%, 2.6%, and 5.7%, respectively, of all mutant mice studied on the respective days. Bone measurements showed significantly shorter bones in the peripheral digits of hindpaws of Nos3−/− newborns. We conclude mice deficient for eNOS show characteristically abnormal prenatal and postnatal development including fetal growth restriction, reduced survival, and an increased rate of limb abnormalities. The development of this characteristic phenotype of eNOS-deficient mice dates back to the prenatal development during the late third trimester of pregnancy.

Analysis of guanidinoacetate and creatine by isotope dilution electrospray tandem mass spectrometry

Guanidinoacetate methyltransferase (GAMT) deficiency is a disorder of creatine metabolism characterized by low plasma creatine concentrations in combination with elevated guanidinoacetate (GAA) concentrations. Although rare, GAMT deficiency has been identified in children with seizures, extrapyramidal movements, developmental delay, myopathies and behavioral abnormalities. Treatment with creatine monohydrate has been proven to be effective. We describe an isotope dilution electrospray tandem mass spectrometry (ES-MS/MS) assay for the simultaneous determination of plasma GAA and creatine using multiple reaction monitoring (MRM), d(3)-creatine as the internal standard and derivatization of GAA and creatine as butyl-esters. We analysed plasma of 16 healthy adults and 20 healthy children as well as three affected children. Plasma GAA concentrations were 5.02+/-1.84 micromol/l (mean+/-S.D.) in adults, 3.91+/-0.76 micromol/l in children age 5-10 years and 11.57, 15.16, 14.36 micromol/l in children with GAMT deficiency. Plasma creatine concentrations were 34.7+/-15.25 micromol/l in adults, 58.96+/-22.30 micromol/l in children and 5.37, 8.15, 403.5 micromol/l in two untreated children and one treated child with GAMT deficiency, respectively. GAA can also be reliably measured from filter cards, which is sufficient to make the correct diagnosis while creatine is consistently falsely elevated probably secondary to liberation of red cell creatine. In nine healthy newborn infants, GAA concentrations from filter cards were 4.83+/-1.43 and 5.04+/-1.84 micromol/l in 16 healthy adults. We conclude that isotope dilution ES-MS/MS is ideal for rapid high-throughput diagnosis of GAMT deficiency both from plasma and filter paper cards. Using this technique neonatal screening is feasible for this treatable inborn error of creatine metabolism.

Limb reduction defects in endothelial nitric oxide synthase-deficient mice

Nitric oxide synthases are a family of enzymes capable of converting L-arginine to L-citrulline with the subsequent release of nitric oxide (NO). NO has been shown to have multiple biologic effects depending on the isoform responsible for its production and its tissue of origin. Murine endothelial nitric oxide synthase (eNOS) is encoded by Nos3, located on mouse chromosome 5. NO produced from this isoform causes vascular smooth muscle relaxation. Other investigators have shown that the administration of nonspecific inhibitors of nitric oxide synthases to pregnant rats induces limb reduction defects. However, mice deficient in Nos3 have not previously been noted to show such abnormalities. To explore the importance of eNOS during development, we produced mice deficient in eNOS using embryonic stem cell technology. Limb reduction defects were seen in approximately 10% of the null animals. We also observed increased neonatal loss of homozygous deficient pups. One functional copy of Nos3 eliminates the risk of limb defects observed in our mouse strain. These findings have implications for understanding genetic predisposition to sporadic limb reduction defects in humans.

Monitoring uterine activity during labor: a comparison of 3 methods

OBJECTIVE Tocodynamometry (Toco; strain gauge technology) provides contraction frequency and approximate duration of labor contractions but suffers frequent signal dropout, necessitating repositioning by a nurse, and may fail in obese patients. The alternative invasive intrauterine pressure catheter (IUPC) is more reliable and adds contraction pressure information but requires ruptured membranes and introduces small risks of infection and abruption. Electrohysterography (EHG) reports the electrical activity of the uterus through electrodes placed on the maternal abdomen. This study compared all 3 methods of contraction detection simultaneously in laboring women. STUDY DESIGN Upon consent, laboring women were monitored simultaneously with Toco, EHG, and IUPC. Contraction curves were generated in real-time for the EHG, and all 3 curves were stored electronically. A contraction detection algorithm was used to compare frequency and timing between methods. Seventy-three subjects were enrolled in the study; 14 were excluded due to hardware failure of 1 or more of the devices (n = 12) or inadequate data collection duration (n = 2). RESULTS In comparison with the gold-standard IUPC, EHG performed significantly better than Toco with regard to the Contractions Consistency Index (CCI). The mean CCI for EHG was 0.88 ± 0.17 compared with 0.69 ± 0.27 for Toco (P < .0001). In contrast to Toco, EHG was not significantly affected by obesity. CONCLUSION Toco does not correlate well with the gold-standard IUPC and fails more frequently in obese patients. EHG provides a reliable noninvasive alternative, regardless of body habitus.

An Endothelial Nitric Oxide Synthase Gene Polymorphism is Associated with Preeclampsia

OBJECTIVE We sought to test the hypothesis that a polymorphism of the endothelial nitric oxide synthase gene (NOS3) is associated with preeclampsia. METHODS We collected and performed polymerase chain reaction (PCR) on genomic DNA from pregnant patients with and without preeclampsia. Patient history and clinical course were evaluated. MAIN OUTCOME MEASURE(S) Frequency of the intron 4 polymorphism of NOS3 (designated allele A) among patients with preeclampsia compared with controls. Clinical features of patients with preeclampsia and the A allele compared with those patients with preeclampsia who did not have the A allele. RESULTS The frequency of the A allele was 0.10 among controls versus 0.39 among patients with preeclampsia (p < 0.01). The odds ratio of developing preeclampsia when at least one A allele was present was 6.5 [95% confidence interval (CI): 2.1-19.7]. After adjusting for ethnic variation, the odds ratio increased to 7.2 (95% CI: 2.0-25.5). Among patients with preeclampsia, systolic blood pressure at the time of admission was higher for patients with at least one A allele compared with patients homozygous for the B allele (168 versus 156 mm Hg; p = 0.03), independent of gestational age (p = 0.01). CONCLUSION These data provide evidence for an association between NOS3 and preeclampsia. In defined ethnic groups, this NOS3 may offer predictive information regarding the subsequent development of preeclampsia and its clinical course.

Polymorphisms within the interleukin-1β gene cluster and preeclampsia ☆

Objective To identify associations between polymorphisms within the interleukin-1β gene cluster, all of which increase protein expression, and preeclampsia. Methods We genotyped a Hispanic population (69 women with preeclampsia and 47 controls) for two polymorphisms of the interleukin-1β gene (promoter region and exon 5) and one polymorphism of the interleukin-1 receptor antagonist gene in intron 2. Clinical data were collected from medical records. Values are given as means or medians. Statistical power to identify a difference in occurrence of interleukin-1β promoter, interleukin-1β exon 5, and interleukin-1 receptor antagonist gene polymorphisms in women with preeclampsia compared with controls was 21%, 15.9%, and 30.9%, respectively. Results We found no association between any single polymorphism and occurrence of preeclampsia. Among women with preeclampsia, those with polymorphism of interleukin-1 receptor antagonist gene had higher mean systolic blood pressure (BP) at admission (178 ± 33.4 versus 159 ± 19.5 mmHg, P = .039). When all three polymorphisms combined were evaluated, women with preeclampsia and at least three mutant alleles (n = 8) had higher mean systolic BP at admission (182 ± 30 versus 160 ± 20.5 mmHg, P = .009) and increased alanine aminotransferase (67 [10–1024] versus 20 [3–407] IU/L, P = .04) and aspartate aminotransferase (119 [25–2239] versus 24 [4–489] IU/L, P = .002). At admission, BP in controls was independent of any polymorphism identified. Conclusion Although the power of this study was limited, our data do not support a role for polymorphisms of the interleukin-1β and interleukin-1 receptor antagonist genes in the pathogenesis of preeclampsia among Hispanic women. Our findings do suggest that polymorphisms within the gene cluster might influence severity of preeclampsia.