Attitudes toward and current status of disclosure of secondary findings from next-generation sequencing: a nation-wide survey of clinical genetics professionals in Japan.

The management of secondary findings (SFs), which are beyond the intended purpose of the analysis, from clinical comprehensive genomic analysis using next generation sequencing (NGS) presents challenges. Policy statements regarding their clinical management have been announced in Japan and other countries. In Japan, however, the current status of and attitudes of clinical genetics professionals toward reporting them are unclear. We conducted a questionnaire survey of clinical genetics professionals at two time points (2013 and 2019) to determine the enforcement of the SF management policy in cases of comprehensive genetic analysis of intractable diseases and clinical cancer genome profiling testing. According to the survey findings, 40% and 70% of the respondents stated in the 2013 and 2019 surveys, respectively, that they had an SF policy in the field of intractable diseases, indicating that SF policy awareness in Japan has changed significantly in recent years. Furthermore, a total of 80% of respondents stated that their facility had established a policy for clinical cancer genome profiling testing in the 2019 survey. In both surveys, the policies included the selection criteria for genes to be disclosed and the procedure to return SFs, followed by recommendations and proposals regarding SFs in Japan and other countries. To create a better list of the genes to be disclosed, further examination is needed considering the characteristics of each analysis.

Attitudes of clinical geneticists and certified genetic counselors to genome editing and its clinical applications: A nation-wide questionnaire survey in Japan.

Genome editing of the human embryo using CRISPR/Cas9 has the potential to prevent hereditary diseases from being transmitted to the next generation. However, attitudes to this technology have not been examined sufficiently among the genetic professionals who will use it in the near future. We conducted a questionnaire survey of Japanese clinical geneticists and certified genetic counselors. Differences were observed between them in their recognition of this technology and impressions on its difficulty and cost. Both groups worried about misuse of it, with insufficient information and rules. As key elements for such rules, they considered ethics, safety, and purpose. Most disapproved of modifying physical traits as an enhancement, though they hoped for the treatment of severe diseases. At current clinical sites, they tended to adopt a prudent attitude by mentioning only the possibility of genome editing in the future. Academic policies and legislation are required, especially for application in human embryos, through a consensus of professionals and general citizens. Furthermore, professionals should maintain awareness of new developments and regularly reexamine attitudes for the ongoing development of more suitable rules, education systems, and clinical protocols. As preparation for changes, opportunities to address ethical issues and initiate discussions are also required.

Classification of factors involved in nonreportable results of noninvasive prenatal testing (NIPT) and prediction of success rate of second NIPT.

OBJECTIVE: To evaluate the reasons for nonreportable cell-free DNA (cfDNA) results in noninvasive prenatal testing (NIPT), we retrospectively studied maternal characteristics and other details associated with the results. METHODS: A multicenter retrospective cohort study in pregnant women undergoing NIPT by massively parallel sequencing (MPS) with failed cfDNA tests was performed between April 2013 and March 2017. The women's data and MPS results were analyzed in terms of maternal characteristics, test performance, fetal fraction (FF), z scores, anticoagulation therapy, and other details of the nonreportable cases. RESULTS: Overall, 110 (0.32%) of 34 626 pregnant women had nonreportable cfDNA test results after an initial blood sampling; 22 (20.0%) cases had a low FF (<4%), and 18 (16.4%) cases including those with a maternal malignancy, were found to have altered genomic profile. Approximately half of the cases with nonreportable results had borderline z score. Among the women with nonreportable results because of altered genomic profile, the success rate of retesting using a second blood sampling was relatively low (25.0%-33.3%). Thirteen (11.8%) of the women with nonreportable results had required hypodermic heparin injection. CONCLUSIONS: The classification of nonreportable results using cfDNA analysis is important to provide women with precise information and to reduce anxiety during pregnancy.

Fetal cell-free DNA fraction in maternal plasma for the prediction of hypertensive disorders of pregnancy.

OBJECTIVE: The purpose of this study is to compare the fetal fractions during non-invasive prenatal testing (NIPT) in singleton pregnancies according to gestational age and maternal characteristics to evaluate the utility of this parameter for the prediction of pregnancy complications including gestational diabetes mellitus (GDM) and hypertensive disorders of pregnancy (HDP). STUDY DESIGN: This study was a multicenter prospective cohort study. The present data were collected from women whose NIPT results were negative. The relationships between the fetal fractions and the gestational age, maternal weight and height, and incidences of miscarriage, preterm delivery, and pregnancy complications including GDM, HDP and placental abruption were assessed. RESULTS: A total of 5582 pregnant women with verified NIPT negative results were registered in the study. The demographic characteristics of the study populations were statistically analyzed, and the women with HDP tended to have a low fetal fraction in samples taken during early gestation. The area under the curve (AUC) in a receiver operating characteristic curve (ROC) analysis was 0.608 for women with HDP. CONCLUSION: A low fetal fraction on NIPT might be correlated with future HDP. However, predicting HDP during early pregnancy in women with a low fetal fraction might be difficult.

Inborn Errors of RNA Lariat Metabolism in Humans with Brainstem Viral Infection.

Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.

Current status of non-invasive prenatal testing in Japan.

AIM: The purpose of this study was to report the 3-year experience of a nationwide demonstration project to introduce non-invasive prenatal testing (NIPT) of maternal plasma for aneuploidy, and review the current status of NIPT in Japan. METHODS: Tests were conducted to detect aneuploidy in high-risk pregnant women, and adequate genetic counseling was provided. The clinical data, test results, and pregnancy outcomes were recorded. We discuss the problems of NIPT on the basis of published reports and meta-analyses. RESULTS: From April 2013 to March 2016, 30 613 tests were conducted at 55 medical sites participating in a multicenter clinical study. Among the 30 613 women tested, 554 were positive (1.81%) and 30 021 were negative (98.1%) for aneuploidy. Of the 289, 128, and 44 women who tested positive for trisomies 21, 18, and 13, respectively, and underwent definitive testing, 279 (96.5%), 106 (82.8%), and 28 (63.6%) were determined to have a true-positive result. For the 13 481 women with negative result and whose progress could be traced, two had a false-negative result (0.02%). The tests were performed on the condition that a standard level of genetic counseling be provided at hospitals. CONCLUSION: Here, we report on the 3-year nationwide experience with NIPT in Japan. It is important to establish a genetic counseling system to enable women to make informed decisions regarding prenatal testing. Moreover, a welfare system is warranted to support women who decide to give birth to and raise children with chromosomal diseases.

A survey on awareness of genetic counseling for non-invasive prenatal testing: the first year experience in Japan.

The purpose of this study is to summarize the results from a survey on awareness of genetic counseling for pregnant women who wish to receive non-invasive prenatal testing (NIPT) in Japan. As a component of a clinical study by the Japan NIPT Consortium, genetic counseling was conducted for women who wished to receive NIPT, and a questionnaire concerning both NIPT and genetic counseling was given twice: once after pre-test counseling and again when test results were reported. The responses of 7292 women were analyzed. They expressed high satisfaction with the genetic counseling system of the NIPT Consortium (94%). The number of respondents who indicated that genetic counseling is necessary for NIPT increased over time. Furthermore, they highly valued genetic counseling provided by skilled clinicians, such as clinical geneticists or genetic counselors. The vast majority (90%) responded that there was sufficient opportunity to consider the test ahead of time. Meanwhile, women who received positive test results had a poor opinion and expressed a low-degree satisfaction. We confirmed that the pre-test genetic counseling that we conducted creates an opportunity for pregnant women to sufficiently consider prenatal testing, promotes its understanding and has possibilities to effectively facilitate informed decision making after adequate consideration. A more careful and thorough approach is considered to be necessary for women who received positive test results.

[Postpartum hemorrhage successfully treated with recombinant factor VIIa in Glanzmann thromboasthenia].

Glanzmann thrombasthenia is an inherited hemorrhagic disorder characterized by severe reduction or absence of platelet aggregation in response to multiple physiologic agonists due to qualitative or quantitative abnormalities of platelet glycoprotein (GP) IIb/IIIa. Treatment of bleeding episodes may require platelet transfusion. However, repeated platelet transfusions may result in GPIIb/IIIa and/or HLA immunization, and development of platelet refractoriness. Recently, a number of reports have suggested that recombinant factor VIIa product (rFVIIa) may be a therapeutic alternative in these situations. We have used rFVIIa to treat a 34-year-old primigravida woman for postpartum bleeding. She had been diagnosed with Glanzmann thrombasthenia at 32 years of age. At 37 weeks gestation, she underwent elective caesarean section uneventfully with platelet transfusion. Nine days later, she developed vaginal bleeding and received twenty units of platelet concentrates every day, but bleeding persisted. Thereafter, 4.8 mg intravenous rFVIIa was administered three times, which immediately arrested the bleeding. These results demonstrate that rFVIIa is effective for severe bleeding in Glanzmann thrombasthenia patients, especially in those with antiplatelet antibodies and/or platelet transfusion refractoriness.

No correlation between the number of fetal nucleated cells and the amount of cell-free fetal DNA in maternal circulation either before or after delivery.

OBJECTIVES: We have determined the number of fetal nucleated cells and the concentration of cell-free fetal DNA in parallel in the same maternal blood samples either before or after delivery, and studied the relationship between these two. METHODS: Venous blood samples were taken at four points around delivery from ten women who had singleton male fetus with informed consent. The number of fetal nucleated cells having a Y chromosome specific signal treated by two-color fluorescence in situ hybridization technique was counted using maternal whole blood. The concentration of sex-determining region Y gene sequence was determined using real-time quantitative PCR. RESULTS: The number of fetal nucleated cells decreased after delivery, and some fetal cells were present 1 month after delivery. While cell-free fetal DNA decreased rapidly after delivery and became undetectable 1 day after delivery in eight out of ten cases. The number of fetal nucleated cells did not correlate with the concentration of cell-free fetal DNA in maternal circulation. CONCLUSION: The present study demonstrates that cell-free fetal DNA disappears very rapidly after delivery and fetal nucleated cells remain longer in maternal circulation, and that there is no correlation between these two either before or after delivery.

Increased plasma mRNAs of placenta-specific 1 (PLAC1) and glial cells-missing 1 (GCM1) in mothers with pre-eclampsia.

In this study we have investigated whether quantitative analysis of placental mRNAs in maternal plasma provides a way to monitor placental status. We measured plasma concentrations of human chorionic gonadotropin beta-subunit (betahCG) and human placental lactogen (hPL) mRNAs as previously reported mRNAs and pregnancy associated plasma protein A (PAPP-A), placenta-specific 1 (PLAC1) and glial cells-missing 1 (GCM1) mRNAs, which have not been measured during the course of normal pregnancy. Firstly, peripheral blood was obtained at various times from healthy pregnant women to clarify the time course of placental mRNAs. Secondly, blood was obtained from women with pre-eclampsia and gestational age-matched controls to examine whether placental mRNAs change in pre-eclampsia. Plasma was separated from these samples for extraction of RNA, followed by reverse transcription polymerse chain reaction analysis. Median concentrations of PLAC1 and GCM1 mRNA in plasma of pre-eclamptic subjects respectively were 1625 and 2141 copies/ml, significantly higher than 195 and 881 copies/ml, the values for controls (Mann-Whitney test, p<0.001). No significant difference was seen in hPL, betahCG, or PAPP-A mRNA concentration between pre-eclamptic and control groups. Plasma PLAC1 and GCM1 mRNAs appear promising as noninvasively measurable molecular markers for pre-eclampsia.