[BRCA2 Mutation Profile in a Proband with Hereditary Breast and Ovarian Cancer-Two Germline Pathogenic Variants Aligned in the Cis Position].

We report the first Japanese case of hereditary breast and ovarian cancer(HBOC)carrying 2 germline pathogenic variants (GPVs)in the BRCA2 gene. Genetic testing of the BRCA1 and BRCA2 genes was performed in a young woman with HBOC and 2 GPVs were identified in the BRCA2 gene. Since simultaneous GPVs in both parental alleles(ie, trans)in the BRCA2 gene is diagnostic of Fanconi anemia, which is characterized by bone marrow dysfunction and susceptibility to malignancy, we genetically tested her relatives. The same variants were revealed, and both variants were located in the cis position. For patients with multiple GPVs in the BRCA2 gene, we should consider genetic testing of the relatives to confirm whether the variants are located in the cis or trans position under appropriate genetic counseling.

Retrospective details of false-positive and false-negative results in non-invasive prenatal testing for fetal trisomies 21, 18 and 13.

OBJECTIVE: Maternal characteristics and neonatal outcomes associated with cell-free DNA (cfDNA) results were analysed retrospectively to assess the details of false-positive and false-negative results after initial blood sampling in non-invasive prenatal testing (NIPT). STUDY DESIGN: A multicentre retrospective study was performed for women undergoing NIPT who received discordant cfDNA results between April 2013 and March 2018. The NIPT data obtained using massive parallel sequencing were studied in terms of maternal background, fetal fraction, z-scores, invasive procedure results and neonatal outcomes after birth. RESULTS: Of the 56,545 women who participated in this study, 54 false-positive (0.095 %) and three false-negative (0.006 %) cases were found. Seven of the 54 false-positive cases (13.0 %) had vanishing twin on ultrasonography. Among the 18 false-positive cases of trisomy 18, confined placental mosaicism (CPM) was confirmed in three cases (16.7 %), while CPM was present in one of the three false-negative cases of trisomy 21. CONCLUSION: These data suggest that the incidence of women with false-positive or false-negative results is relatively low, that such false results can often be explained, and that vanishing twin and CPM are potential causes of NIPT failure. Genetic counselling with regard to false results is important for clients prior to undergoing NIPT.

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.

Qualitative investigation of the factors that generate ambivalent feelings in women who give birth after receiving negative results from non-invasive prenatal testing.

BACKGROUND: Women who receive negative results from non-invasive prenatal genetic testing (NIPT) may find that they later have mixed or ambivalent feelings, for example, feelings of accepting NIPT and regretting undergoing the test. This study aimed to investigate the factors generating ambivalent feelings among women who gave birth after having received negative results from NIPT. METHODS: A questionnaire was sent to women who received a negative NIPT result, and a contents analysis was conducted focusing on ambivalent expressions for those 1562 women who responded the questionnaire. The qualitative data gathered from the questionnaire were analyzed using the N-Vivo software package. RESULTS: Environmental factors, genetic counseling-related factors, and increased anticipatory anxiety, affected the feeling of ambivalence among pregnant women. Furthermore, pregnant women desired more information regarding the detailed prognosis for individuals with Down syndrome and living with them and/or termination, assuming the possibility that they were positive. CONCLUSIONS: Three major interrelated factors affected the feeling of ambivalence in women. Highlighting and discussing such factors during genetic counseling may resolve some of these ambivalences, thereby enhancing the quality of decisions made by pregnant women.

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.

Analysis of clinical characteristics of breast cancer patients with the Japanese founder mutation BRCA1 L63X.

Background: BRCA1 and BRCA2 are high-penetrance inherited genes; different founder mutations have been reported in various areas and races. By using trial registration data from the Japanese hereditary breast and ovarian cancer syndrome (HBOC) consortium, we aimed to explore the clinicopathological characteristics of breast cancer patients with the Japanese founder mutation BRCA1 L63X. Results: We found 88 BRCA1 carriers, 76 BRCA2 carriers, and one carrier of both BRCA1 and BRCA2. Of 46 independent BRCA1 mutations, the BRCA1 L63X mutation was detected in 26 patients. We observed a significant difference in the proportion of triple-negative breast cancer phenotype among 88.9%, 72.5%, and 26.8% of BRCA1 L63X mutation, BRCA1 mutation, and BRCA2 mutation carriers, respectively (p < .001). Additionally, significant differences were also observed in nuclear grade in the resultant breast cancer between the groups (p < .001). Conclusions: A high proportion of Japanese HBOC patients showed the BRCA1 L63X mutation, and the clinical characteristics of breast cancer in patients with this mutation might differ from those in patients with other BRCA1 or BRCA2 mutations, in terms of the subtype and nuclear grade of the resultant cancer. Methods: From 827 patients in the Japanese HBOC consortium through August 2015, patients with BRCA1/2 mutations were included in this study. We compared the clinicopathological features among patients with BRCA1 L63X mutation, other BRCA1 mutations, and BRCA2 mutations using Chi-square test.

Performance and outcomes of noninvasive prenatal testing for twin pregnancies in Japan.

AIM: The purpose of this study was to describe the characteristics of women with twin pregnancies who undergo noninvasive prenatal testing (NIPT) as well as the post-partum and neonatal outcomes of such cases in Japan. METHODS: The study population consisted of women who were pregnant with twins and who underwent NIPT using massively parallel sequencing (MPS) at Nagoya City University Hospital between April 2013 and June 2016. Questionnaires were completed pre-NIPT and post-partum. RESULTS: Among 4009 women who underwent NIPT during the study period, 75 women (1.9%) were pregnant with twins. Fifteen women (20%) experienced vanishing twin/intrauterine fetal deaths at <22 weeks, and 60 women (80%) had normal twin pregnancies at the time of genetic counseling for NIPT. The use of NIPT was correlated with increased proportions of women using assisted reproductive technology (ART). The test had a high performance, with a false-positive rate of 1.7% and no false negatives. CONCLUSION: In this study, NIPT had a high performance, with a false positive rate of 1.7% and no false negatives. When treating women with twin pregnancies, the efficacy of NIPT should be explained during genetic counseling. Further larger studies are required to assess the reliability and validity of NIPT in twin pregnancies.

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.

Self-perceptions from people with Down syndrome in Japan.

Self-perception of people with Down Syndrome (DS) was originally studied in the United States in 2011; this study indicated that 99% of people with DS are happy with their lives. In this study, we investigated self-perceptions of people with DS in Japan and compared the results to the previous study made in the United States. The participants (n = 300) were 12 years old or older and members of Japan Down Syndrome Society (JDS). The questionnaire was collected between 30 October 2015 and 26 November 2015. Of 96(32%) responses, 76 (97%) agreed they are happy with their lives. We found that most people with DS in Japan also feel positive about themselves and their lives. Along with the start of Non-Invasive Prenatal Testing in Japan, DS draws an attention from Japanese people because maternal age has been increasing. This has resulted in the growth of the number of prenatal tests marketed in Japan. Therefore, we expect our results to be relevant to genetic counseling for the couples considering prenatal tests or parents raising their children with DS so that they have a clearer image about life with DS and can better visualize their life plans.

Correction: Genetic and clinical characteristics in Japanese hereditary breast and ovarian cancer: first report after establishment of HBOC registration system in Japan.

Correction to: Journal of Human Genetics advance online publication, 08 November 2017; https://doi.org/10.1038/s10038-017-0355-1.

Genetic and clinical characteristics in Japanese hereditary breast and ovarian cancer: first report after establishment of HBOC registration system in Japan.

The hereditary breast and ovarian cancer (HBOC) registration system of Japan was established by the Japanese HBOC Consortium. The first trial was registered in 2015 in four institutions to which some registration committee members belonged. We analyzed the information of 830 Japanese pedigrees, who underwent BRCA1/2 genetic testing, including mutation carriers with BRCA1 (N = 127) and BRCA2 (N = 115), and their families. The mutation-positive rate was 19.7%. Variants of uncertain significance were found in 6.5% of all individuals subjected to genetic testing for BRCA1/2. Compared to the United States, Japan had a higher mutation-positive rate in most categories, except for the groups with male breast cancer. Among the intrinsic subtypes of BRCA1-associated breast cancers, 75.8% were triple-negative. The incidence rate of contralateral breast cancer in BRCA1/2 mutation carriers was 0.99%/year. Among 240 mutation carriers, 26 and 62 patients underwent risk-reducing mastectomy (RRM) and risk-reducing salpingo-oophorectomy (RRSO), respectively; the respective frequencies of occult cancer were 7.1 and 3.2%. Metachronous breast cancer after RRM or peritoneal cancer after RRSO was not observed during the follow-up period. The nationwide registration system began last year and the system enables follow-up analysis in Japan.

Psychological distress in post-partum women after non-invasive prenatal testing (NIPT) in Japan.

AIM: The purpose of this study was to clarify the characteristics of psychological mental distress in post-partum women after non-invasive prenatal testing (NIPT) in Japan. METHODS: Psychological mental distress was assessed using the Kessler Psychological Distress Scale (K6). We compared patients with (i) low pre-NIPT K6 and low post-partum K6 scores (control group), and (ii) low pre-NIPT K6 and a high post-partum K6 scores (case group). RESULTS: Among the 697 women who underwent NIPT, 29 (4.2%) had low pre-NIPT K6 and high post-partum K6 scores (case group) and 668 (95.8%) had low pre-NIPT K6 and low post-partum K6 scores (control). Among women with negative NIPT findings, post-partum women with a high K6 score were compared to a control group of women with a low K6 score. Logistic regression analysis showed that primiparity (P = 0.007), low birthweight (P = 0.005) and use of intracytoplasmic sperm injection (P = 0.02) and assisted reproductive technology (P = 0.05) were significantly different between the groups. CONCLUSION: Even if women do not feel mental distress before NIPT, they may develop mental stress post-partum. In particular, primipara women who conceived through assisted reproductive technology (especially intracytoplasmic sperm injection) and gave birth to a low birthweight baby were more susceptible to developing post-partum distress. Thus, it is important to educate women that support is available, with consultation with other healthcare professionals during genetic counseling if necessary. Further studies are needed in order to determine the factors associated with post-partum mental distress.

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.

Background of couples undergoing non-invasive prenatal testing in Japan.

AIM: Our purpose was to assess the background of couples who were undergoing non-invasive prenatal testing (NIPT) in Japan. METHODS: The characteristics of 2486 women who had visited Nagoya City University Hospital for NIPT were compared with Japanese Demographic Trends as controls. The questionnaire included items regarding the maternal and paternal age, maternal age at marriage, age at first live birth, and conception mode. RESULTS: Compared with the controls, the percentage of women who were 4 or more years older than their partners was larger in the NIPT group (11.8% vs 6.5%). The maternal age at marriage, age at first live birth, and the duration between marriage and first birth tended to be greater in the NIPT group (32.6 years vs 29.3 years, 36.9 years vs 30.4 years, and 3.6 years vs 2.4 years, respectively), and the percentage of women who underwent assisted reproductive technology tended to be higher in the NIPT group (35-39 years: 21.2% vs 7.5%, 40-45 years: 36.2% vs 12.6%), compared with the controls. CONCLUSION: Knowing the specific backgrounds of couples who have undergone NIPT may be important for improving the quality of genetic counseling for NIPT.

Fetal cell-free DNA fraction in maternal plasma is affected by fetal trisomy.

The purpose of this noninvasive prenatal testing (NIPT) study was to compare the fetal fraction of singleton gestations by gestational age, maternal characteristics and chromosome-specific aneuploidies as indicated by z-scores. This study was a multicenter prospective cohort study. Test data were collected from women who underwent NIPT by the massively parallel sequencing method. We used sequencing-based fetal fraction calculations in which we estimated fetal DNA fraction by simply counting the number of reads aligned within specific autosomal regions and applying a weighting scheme derived from a multivariate model. Relationships between fetal fractions and gestational age, maternal weight and height, and z-scores for chromosomes 21, 18 and 13 were assessed. A total of 7740 pregnant women enrolled in the study, of which 6993 met the study criteria. As expected, fetal fraction was inversely correlated with maternal weight (P<0.001). The median fetal fraction of samples with euploid result (n=6850) and trisomy 21 (n=70) were 13.7% and 13.6%, respectively. In contrast, the median fetal fraction values for samples with trisomies 18 (n=35) and 13 (n=9) were 11.0% and 8.0%, respectively. The fetal fraction of samples with trisomy 21 NIPT result is comparable to that of samples with euploid result. However, the fetal fractions of samples with trisomies 13 and 18 are significantly lower compared with that of euploid result. We conclude that it may make detecting these two trisomies more challenging.

Chromosome abnormalities diagnosed in utero: a Japanese study of 28 983 amniotic fluid specimens collected before 22 weeks gestations.

To investigate the frequency and type of abnormal karyotype in Japan by amniocentesis before 22 weeks of gestation. We performed a retrospective analysis of 28 983 amniotic fluid specimens in a local population collected before 22 weeks gestations for fetal karyotyping. The incidence of abnormal karyotype was 6.0%. The main indication was advanced maternal age (AMA) of 35 years and older, which represented over half of the clinical indications. Abnormal karyotype was most frequently reported among the referrals for abnormal ultrasound findings (21.8%), followed by positive maternal serum screen results (5.3%). Three-fourths of abnormal karyotype was either autosomal aneuploidy (64.0%) or sex chromosome aneuploidy (11.6%). Abnormal karyotype was detected in 2.8% of pregnant women referred for AMA. Clinically significant abnormal karyotype increased with advancing maternal age. The frequency and type of abnormal karyotype detected by amniocentesis for various indications were determined. Amniocentesis was mainly performed among the referrals for AMA, which is a characteristic distribution of indications of Japan.

Maternal smoking and placental expression of a panel of genes related to angiogenesis and oxidative stress in early pregnancy.

OBJECTIVE: Maternal cigarette smoking is paradoxically associated with a decreased risk of developing preeclampsia. Since preeclampsia is thought to be associated with altered mechanisms of angiogenesis and oxidative stress, we aim to investigate the influence of maternal smoking on the early placental expression of a panel of genes related to angiogenesis and oxidative stress. MATERIAL AND METHODS: We collected villous tissue samples at 6-7 and 10-11 weeks of gestation from 31 women requesting surgical termination. Placental expression of the following genes were quantified by real-time PCR: vascular endothelial growth factor A (VEGFA), fms-like tyrosine kinase (Flt-1), soluble endoglin (sEng), placental growth factor (PlGF), heme oxygenase-1 (HMOX-1) and superoxide dismutase (SOD). Maternal smoking status was assessed by levels of serum cotinine. RESULTS: Placental expression of VEGFA was significantly higher in smoking women at 10-11 weeks of gestation compared with nonsmoking women at the same gestational age. There was no significant difference at 6-7 weeks of gestation. There was no variation in the expression of the other genes explored related to smoking status. CONCLUSIONS: Here we report that VEGFA placental expression was higher in smoking women at 10-11 weeks of gestation. Increased VEGFA expression in the early stages of pregnancy in smoking women might contribute to the decreased risk of developing preeclampsia.

Survey of second-trimester maternal serum screening in Japan.

AIM: Second-trimester maternal serum screening (MSS) is the predominant form of prenatal screening in Japan. We hypothesize that patient education and physician knowledge of Down syndrome (DS) screening are insufficient to provide adequate information regarding prenatal screening in Japan. MATERIAL AND METHODS: In-person or telephone interviews with medical personnel were conducted at 25 medical facilities utilizing Afp(4) (Esoterix Genetic Laboratories, LLC, a wholly-owned subsidiary of Laboratory Corporation of America Holdings) second trimester screening. We explored how medical facilities provided information about the MSS process to pregnant women. RESULTS: Before prenatal testing, 60% of medical facilities spent an average of less than 5 min per patient explaining the MSS process. The option of amniocentesis for chromosomal aneuploidy was explained at nearly all facilities, but only 60% explained the risk of amniocentesis. Ultrasound examination for open neural tube defects was explained only at half the facilities. Only five of 25 (20%) medical practitioners explained the possibility of having congenital disorders fetus. CONCLUSION: This study revealed that most obstetricians in Japan do not provide their patients with sufficient information about MSS.

Placental expression of microRNA-17 and -19b is down-regulated in early pregnancy loss.

OBJECTIVE: First, to determine if microRNA-17 and -19b are expressed in villous samples at early stages of pregnancy. Second, to determine whether placental expressions of these microRNAs along with their main targets (PTEN, CREB-1, TGFß-1 and TGFß-RII) are altered in early pregnancy loss. STUDY DESIGN: Expression levels of microRNAs and mRNA targets in villous samples from early pregnancy loss (n=11) and matched normal cases (n=20) by gestational age were determined by RT-PCR. RESULTS: Both microRNA-17 and -19b were expressed in all cases of normal pregnancy. They were significantly down-regulated (relative ratios: 0.35 and 0.34 respectively) in early pregnancy loss. Their main target, PTEN mRNA, was significantly up-regulated in early pregnancy loss (relative ratio: 2.6, 95%CI: 0.2-29.8). TGF-ß1, CREB-1 and TGFß-RII were not significantly different between the two groups. CONCLUSION: microRNA-17 and -19b are expressed in early stages of pregnancy. They are down-regulated in villous samples from early pregnancy loss. We suggest that these main members of the microRNA-17-92 cluster might be involved in placental invasion and its dysregulation might also be related to other conditions characterized by defective placentation.

Physiological changes in the pattern of placental gene expression early in the first trimester.

OBJECTIVE: To assess the physiological changes in the placental expression pattern of a panel of genes related to angiogenesis and oxidative stress during the early part of the first trimester of pregnancy. METHODS AND RESULTS: The expression of a selected panel of genes was quantified by reverse transcriptase-polymerase chain reaction in samples of villous trophoblasts obtained from women between 6 and 11 weeks of gestation undergoing elective artificial abortion. We found that the levels of messenger RNA (mRNA) expression of placental growth factor (PlGF), heme oxygenase 1(HO-1), and superoxide dismutase (SOD) increased significantly with gestational age (r = .37, P = .001; r = .24, P =.04; and r = .52, P < .001, respectively). Conversely, the mRNA expression level of fms-like tyrosine kinase 1 (FLT-1) decreased significantly (r = -.30, P = .009). CONCLUSION: During the early part of the first trimester of pregnancy, the placental gene expression levels of PlGF, HO-1, and SOD increase with gestational age, whereas the expression of FLT-1 decreases. The alteration in this pattern of gene expression in early pregnancy may therefore play an important role in placenta-related disorders such as preeclampsia.