Effectiveness of Prophylactic Laparoscopic Surgery for Benign Adnexal Mass during Pregnancy: A Single-center Retrospective Study.

Objectives: This study aimed to evaluate the effectiveness of prophylactic laparoscopic surgery for avoiding adnexal torsion in pregnant women with benign adnexal masses. Materials and Methods: This report contains two analyses, each for a different group of patients. Analysis 1: Surgical and pregnancy outcomes were examined among the 126 cases who underwent laparoscopic assisted cystectomy for adnexal masses during pregnancy in our hospital between January 2001 and December 2020. Analysis 2: The incidence of adnexal torsion during pregnancy was evaluated among the cases with adnexal masses ≥5 cm who opted for conservative follow-up in our hospital between January 2011 and December 2020. Results: In analysis 1, the most common pathological diagnosis was a mature cystic teratoma (76.2%). The mean gestational age at surgery was 13.1 ± 1.3 weeks. No cases were converted to laparotomy and oophorectomy. Regarding delivery outcomes, 97.4% of cases went on to have full-term deliveries. In Analysis 2, the incidence of adnexal mass ≥5 cm that did not resolve spontaneously during pregnancy was 89 cases (0.8%). The frequency of malignancy was 3 cases (0.03%). In 28 cases who opted for conservative treatment, 5 (17.9%) underwent emergency surgery for adnexal torsion. Conclusion: Prophylactic surgery for benign adnexal masses during pregnancy can be performed laparoscopically and preserved ovarian functions. In pregnant women with adnexal masses that do not resolve spontaneously, planning laparoscopic surgery is considered beneficial for complications, such as adnexal torsion.

Changes in use and outcomes after fibrinogen concentrate insurance coverage for critical obstetrical hemorrhage: a nationwide questionnaire survey in Japan.

Fibrinogen concentrate (FC) for acquired hypofibrinogenemia associated with critical obstetrical hemorrhage (COH) was covered by public medical insurance in September 2021 in Japan. We aimed to investigate changes in the policy of FC use and its effect on COH after insurance coverage. A primary survey covering September 2020 to August 2021 and a secondary survey covering September 2021 to August 2022 were conducted at 428 higher-level medical facilities. We investigated the policy of FC use in transfusion strategy and the maternal outcomes in COH. Among the hospitals that responded to both surveys, the number of facilities that use FC increased from 51.5 (101/196) to 78.6% (154/196) (P < 0.0001). The number of COH cases treated using FC increased from 14.3 to 24.3% (P < 0.0001) and that transfused with ≥ 10 units of red blood cells (RBCs) decreased from 36.8 to 29.8% (P = 0.001). The incidence of pulmonary edema reduced by 3.7-2.0% (P = 0.021), and transfusion-induced allergy by 1.9-0.7% (P = 0.008). No changes were observed in the incidence of thromboembolism, arterial embolization, or hysterectomy. The increased use of FC after insurance coverage led to changes in the transfusion strategy, which may be associated with decreases in transfusions of RBCs, pulmonary edema, and transfusion-induced allergies.

Investigation of optimal weight gain during pregnancy: A retrospective analysis of the Japanese perinatal registry database.

AIM: This study aimed to determine the weight gain during pregnancy that minimizes the predicted probability of various perinatal adverse events according to the pre-pregnancy body mass index (BMI) and make recommendations for optimal weight gain in Japan. METHODS: The Japan Society of Obstetrics and Gynecology perinatal database for 2015-2017 was used. From the 719 723 deliveries included in this database, parturients with underlying diseases or missing data were excluded, and 419 114 deliveries were analyzed. A questionnaire survey was also conducted to weigh each perinatal adverse event. For each of the nine outcomes, a restricted cubic spline model was made to estimate the association between the "expected gestational weight gain at 40 weeks" and the outcome risk. RESULTS: Since the classes of medical facilities were generally the same, weights were assigned according to the mean of the questionnaires rather than by the class of the facility. For each pre-pregnancy BMI, the weight gains during pregnancy that minimized the predicted probability of various adverse perinatal events were 12-15, 10-13, 7-10, and upper limit of 5 kg for the underweight, normal-weight, obese 1, and obese ≥2 groups, respectively. CONCLUSIONS: The weight gain during pregnancy that minimizes the predicted probability of various perinatal adverse events according to the pre-pregnancy BMI was established.

Risk Factors for Alcohol Consumption after Starting Assisted Reproductive Technology Treatment among Japanese Women: Japan-Female Employment and Mental Health in Assisted Reproductive Technology (J-FEMA) Study.

AIMS: To determine the association between drinking habits and social factors among women undergoing assisted reproductive technology (ART) treatment in Japan. METHODS: The study participants who provided answers for the questionnaire concerning alcohol consumption were 1017 female patients undergoing ART treatment were enrolled in the Japan-Female Employment and Mental Health in assisted reproductive technology (J-FEMA) study between August and December 2018. Patient characteristics, including demographic, clinical, and socioeconomic status, were assessed using a self-administered questionnaire which was distributed only once during the period, regardless of their first or follow-up examination. We defined current drinkers who drank ≥46 g of ethanol per week as the habitual drinking group. The risk factors for habitual drinking were analyzed using multivariable-adjusted logistic regression. RESULTS: The proportion of habitual drinkers was 15.5% in this study population. The multivariable-adjusted odds ratios (95% confidence interval) for habitual drinking were 2.27 (0.99-5.21) for women aged ≥35 years versus those <35 years, 4.26 (1.98-9.16) for women having partners who currently drink compared to those with partners without current drinking, 1.84 (1.08-3.12) for women without a history of childbirth versus those with, and 1.77 (1.00-3.14) for working women compared with those not working. CONCLUSIONS: In our study, habitual drinking among women undergoing ART treatment was significantly associated with older age, no history of childbirth, partner's current drinking status, and working.

Gene expression signatures associated with chronic endometritis revealed by RNA sequencing.

Introduction: Chronic endometritis (CE) is a persistent inflammatory condition of the endometrium characterized by the infiltration of plasma cells in the endometrial stroma. CD138 immunohistochemistry is considered to improve the CE diagnosis rate. Methods: Using the number of CD138-positive cells equal or greater than five as a diagnostic criterion for CE, we identified 24 CE and 33 non-CE cases among women with infertility. We conducted RNA-sequencing analysis for these 57 cases in total as an attempt to elucidate the molecular pathogenesis of CE and to search for new biomarkers for CE. Results and Discussion: By comparing CE and non-CE groups, we identified 20 genes upregulated in the endometria of CE patients, including 12 immunoglobulin-related genes and eight non-immunoglobulin genes as differentially expressed genes. The eight genes were MUC5AC, LTF, CAPN9, MESP1, ACSM1, TVP23A, ALOX15, and MZB1. By analyzing samples in the proliferative and secretory phases of the menstrual cycle separately, we also identified four additional non-immunoglobulin genes upregulated in CE endometria: CCDC13 by comparing the samples in the proliferative phase, and OVGP1, MTUS2, and CLIC6 by comparing the samples in the secretory phase. Although the genes upregulated in CE may serve as novel diagnostic markers of CE, many of them were upregulated only in a limited number of CE cases showing an extremely high number of CD138-positive cells near or over one hundred. Exceptionally, TVP23A was upregulated in the majority of CE cases regardless of the number of CD138-positive cells. The upregulation of TVP23A in the endometria of CE cases may reflect the pathophysiology of a cell-type or cell-types intrinsic to the endometrium rather than the accumulation of plasma cells. Our data, consisting of clinical and transcriptomic information for CE and non-CE cases, helped us identify gene expression signatures associated with CE.

Rare FGFR fusion genes in cervical cancer and transcriptome-based subgrouping of patients with a poor prognosis.

BACKGROUND: Although cervical cancer is often characterized as preventable, its incidence continues to increase in low- and middle-income countries, underscoring the need to develop novel therapeutics for this disease.This study assessed the distribution of fusion genes across cancer types and used an RNA-based classification to divide cervical cancer patients with a poor prognosis into subgroups. MATERIAL AND METHODS: RNA sequencing of 116 patients with cervical cancer was conducted. Fusion genes were extracted using StarFusion program. To identify a high-risk group for recurrence, 65 patients who received postoperative adjuvant therapy were subjected to non-negative matrix factorization to identify differentially expressed genes between recurrent and nonrecurrent groups. RESULTS: We identified three cases with FGFR3-TACC3 and one with GOPC-ROS1 fusion genes as potential targets. A search of publicly available data from cBioPortal (21,789 cases) and the Center for Cancer Genomics and Advanced Therapeutics (32,608 cases) showed that the FGFR3 fusion is present in 1.5% and 0.6% of patients with cervical cancer, respectively. The frequency of the FGFR3 fusion gene was higher in cervical cancer than in other cancers, regardless of ethnicity. Non-negative matrix factorization identified that the patients were classified into four Basis groups. Pathway enrichment analysis identified more extracellular matrix kinetics dysregulation in Basis 3 and more immune system dysregulation in Basis 4 than in the good prognosis group. CIBERSORT analysis showed that the fraction of M1 macrophages was lower in the poor prognosis group than in the good prognosis group. CONCLUSIONS: The distribution of FGFR fusion genes in patients with cervical cancer was determined by RNA-based analysis and used to classify patients into clinically relevant subgroups.

A missense variant at the RAC1-PAK1 binding site of RAC1 inactivates downstream signaling in VACTERL association.

RAC1 at 7p22.1 encodes a RAC family small GTPase that regulates actin cytoskeleton organization and intracellular signaling pathways. Pathogenic RAC1 variants result in developmental delay and multiple anomalies. Here, exome sequencing identified a rare de novo RAC1 variant [NM_018890.4:c.118T > C p.(Tyr40His)] in a male patient. Fetal ultrasonography indicated the patient to have multiple anomalies, including persistent left superior vena cava, total anomalous pulmonary venous return, esophageal atresia, scoliosis, and right-hand polydactyly. After birth, craniofacial dysmorphism and esophagobronchial fistula were confirmed and VACTERL association was suspected. One day after birth, the patient died of respiratory failure caused by tracheal aplasia type III. The molecular mechanisms of pathogenic RAC1 variants remain largely unclear; therefore, we biochemically examined the pathophysiological significance of RAC1-p.Tyr40His by focusing on the best characterized downstream effector of RAC1, PAK1, which activates Hedgehog signaling. RAC1-p.Tyr40His interacted minimally with PAK1, and did not enable PAK1 activation. Variants in the RAC1 Switch II region consistently activate downstream signals, whereas the p.Tyr40His variant at the RAC1-PAK1 binding site and adjacent to the Switch I region may deactivate the signals. It is important to accumulate data from individuals with different RAC1 variants to gain a full understanding of their varied clinical presentations.

Normative change with gestation in fetal intraventricular pressure difference with color M-mode Doppler echocardiography.

AIM: The intraventricular pressure difference (IVPD) is the pressure difference in early diastole from the base to the apex of the ventricle. It is a useful marker for evaluating diastolic function because of its role as a suction force. This study investigated the changes in total and segmental IVPDs in normal fetuses throughout gestation to obtain normative data equations. METHODS: One hundred thirty-seven healthy pregnant women at 12-40 weeks of gestation were prospectively enrolled to evaluate IVPD. The color M mode was performed, and the image was evaluated using our own code to calculate the IVPD. Segmental IVPD was divided into mid to apex and base. Pearson's correlation coefficient was used to evaluate this relationship. RESULTS: There was a significant, positive relationship between IVPD and gestational age in both ventricles (right ventricle [RV]: r = 0.800, left ventricle [LV]: r = 0.818). As for segmental IVPD, basal and mid-apical IVPD also increased with gestation in both ventricles (RV: basal, r = 0.627; mid-apical, r = 0.705; LV: basal r = 0.758; mid-apical, r = 0.756). IVPG, which was calculated as IVPD/ventricular length, also showed a weak, positive relationship with gestation in both ventricles (RV r = 0.351, p < 0.001; LV r = 0.373, p < 0.001). CONCLUSION: The total and segmental IVPDs significantly increased linearly through time.

Neonatal outcomes of congenital diaphragmatic hernia in full term versus early term deliveries: A systematic review and meta-analysis.

This systematic review and meta-analysis aimed to review the optimal timing of delivery at term for neonates with prenatally diagnosed congenital diaphragmatic hernia (CDH). We reviewed the literature up to December 19, 2022 using MEDLINE and the Cochrane Library databases. The inclusion criteria were original articles, comparative studies of CDH neonates delivered at an early term (37-38 weeks of gestation) and at full term (39 weeks of gestation or later), and comparative studies investigating outcomes of CDH neonates. Six studies met the inclusion criteria, including 985 neonates delivered at an early term and 629 delivered at full term. The cumulative rate of survival to discharge showed no significant difference between CDH neonates delivered at an early term (395/515; 76.7%) or at full term (345/467; 73.9%) (risk ratio [RR] 1.01; 95% confidence interval [CI], 0.89-1.16; p = 0.85). Furthermore, the number of neonates requiring oxygen therapy at discharge was not significantly different between CDH neonates delivered at an early term (32/370; 8.6%) and at full term (14/154; 9.1%) (RR, 0.99; 95% CI, 0.36-2.70; p = 0.99). Therefore, the optimal timing of delivery at term for neonates with CDH remains unclear.

Comparison of the incidence of fetal prolonged deceleration after induction of labor analgesia between dural puncture epidural and combined spinal epidural technique: a pilot study.

BACKGROUND: Abnormal cardiotocogram (CTG) tracing may appear after induction of neuraxial labor analgesia. Non-reassuring fetal status (NRFS) indicated by severely abnormal tracings, such as prolonged deceleration (PD) or bradycardia, can necessitate immediate operative delivery. Combined spinal epidural analgesia (CSEA) is known to result in more frequent abnormal tracings than epidural analgesia (EA); however, the corresponding data related to dural puncture epidural (DPE) are unclear. We aimed to evaluate the rates of incidence of severe abnormal CTG after induction of DPE and CSEA. METHODS: In this study of nulliparous women with full-term pregnancy, data for the DPE intervention group were prospectively collected, while those for the CSEA control group were obtained from medical records. Neuraxial analgesia was performed with cervical dilation ≤ 5 cm, administering initial epidural dosing of 15 mL of 0.125% levobupivacaine with fentanyl 2.5µg/mL for DPE, and intrathecal 0.5% bupivacaine 2.5 mg (0.5ml), fentanyl 10 µg (0.2ml), and 1.3 mL of saline for CSEA. The primary outcome was the incidence of PD, defined as a fetal heart rate reduction ≥ 15 bpm below the baseline and with a lowest value < 80 bpm, and lasting for ≥ 2 min but < 10 min (fetal heart rate < 80 bpm does not have to last for ≥ 2 min), within 90 min after induction of neuraxial labor analgesia. RESULTS: A total of 302 patients were analyzed, with 151 in each group. The incidence of PD after DPE induction was significantly lower than that after CSEA induction (4.0% vs. 14.6%, P = 0.0015, odds ratio = 0.243, 95% confidence interval = 0.095-0.617). CONCLUSION: DPE appears to be a safer method compared to CSEA for neuraxial labor analgesia in the early stages of labor for nulliparous women. TRIAL REGISTRATION: UMIN-CTR: UMIN000035153 . Date registered: 01/01/2019.

Cervical Cerclage to Prevent Intrauterine Balloon Prolapse.

Intrauterine balloon prolapse sometimes occurs, and the intrauterine balloon must be reinserted. Furthermore, intrauterine balloon tamponade (IBT) failure can necessitate additional invasive procedures. We report a case of cervical cerclage with IBT for placenta previa with a cervical dilation. In our case, emergency cesarean section was performed at 35 + 4 weeks of gestation because of persistent hemorrhage. During the operation, we performed IBT to prevent further postpartum hemorrhage. However, immediately after the operation, uterine cervical dilatation was 6 cm, which resulted in cervical dilation and prolapse of the intrauterine balloon. Therefore, we performed cervical cerclage using absorbable sutures with IBT and blood transfusion. We speculated that the intrauterine balloon might have induced cervical canal ripening during the operation. Our case suggested that cervical cerclage with IBT is a useful method to prevent intrauterine balloon prolapse in cases with cervical dilation.

Utility of molecular subtypes and genetic alterations for evaluating clinical outcomes in 1029 patients with endometrial cancer.

BACKGROUND: We investigated the utility of a molecular classifier tool and genetic alterations for predicting prognosis in Japanese patients with endometrial cancer. METHODS: A total of 1029 patients with endometrial cancer from two independent cohorts were classified into four molecular subtype groups. The primary and secondary endpoints were relapse-free survival (RFS) and overall survival (OS), respectively. RESULTS: Among the 265 patients who underwent initial surgery, classified according to immunohistochemistry, patients with DNA polymerase epsilon exonuclease domain mutation had an excellent prognosis (RFS and OS), patients with no specific molecular profile (NSMP) and mismatch repair protein deficiency had an intermediate prognosis, and those with protein 53 abnormal expression (p53abn) had the worst prognosis (P < 0.001). In the NSMP group, mutant KRAS and wild-type ARID1A were associated with significantly poorer 5-year RFS (41.2%) than other genomic characteristics (P < 0.001). The distribution of the subtypes differed significantly between patients with recurrence/progression and classified by sequencing (n = 764) and patients who underwent initial surgery (P < 0.001). Among patients with recurrence/progression, 51.4% had the opportunity to receive molecular targeted therapy. CONCLUSIONS: A molecular classifier is a useful tool for determining prognosis and eligibility for molecularly targeted therapy in patients with endometrial cancer.

Risk of fetal undergrowth in the management of gestational diabetes mellitus in Japan.

AIMS/INTRODUCTION: In Japan, the increasing frequency of underweight among women of reproductive age and the accompanying increase in the rate of low birth weight (LBW) are social issues. The study aimed to establish a prospective registry system for gestational diabetes mellitus (GDM) in Japan and to clarify the actual status of GDM according to the International Association of Diabetes and Pregnancy Study Groups (IADPSG) criteria. MATERIALS AND METHODS: Pregnant women with gestational diabetes mellitus and those in the normal glucose tolerance (NGT) group were enrolled in the Diabetes and Pregnancy Outcome for Mother and Baby study from October 2015. Pregnant women with positive glucose screening in early and mid-to-late pregnancy underwent a 75 g oral glucose tolerance test by gestational week 32. Gestational diabetes mellitus was diagnosed according to IADPSG criteria. Women with a positive glucose screening test at mid-to-late pregnancy but NGT were enrolled as references (NGT group). Treatment for gestational diabetes mellitus and maternal and neonatal pregnancy data were prospectively collected on outcomes. RESULTS: In total 1,795 singleton pregnancies (878 women with GDM and 824 NGT women) were analyzed. The risk of LBW and small-for-gestational age in the GDM group was significantly higher than in the NGT group. A similar relationship was found for LBW risk in the non-overweight/obese group but not in the overweight/obese group. CONCLUSIONS: We established a prospective GDM registry system in Japan. In the management of GDM in Japan, suppression of maternal weight gain may be associated with reduced fetal growth, especially in non-overweight/obese women with GDM; however, further investigation is required.

Current state of management of struma ovarii and preoperative imaging features: A retrospective case series study of 18 patients at a single institution.

AIM: Even though 95% of struma ovarii are benign, it is often overtreated because of the difficulty to distinguish it from malignancy. In this study, our aim was to evaluate the current state of the preoperative diagnosis and the selection of the surgical procedure, and to improve preoperative diagnostic accuracy by retrospectively reviewing imaging findings. METHODS: We retrospectively reviewed the clinical course and imaging characteristics of 18 patients who were diagnosed postoperatively with struma ovarii, pathologically, at our institution between 2015 and 2021. RESULTS: The preoperative diagnoses included benign ovarian tumor in eight cases, borderline in four cases, and malignant in six cases. None of the cases were diagnosed as struma ovarii preoperatively. Of the seven patients who had confirmed a desire for future childbearing, four patients were suspected for borderline or malignant tumor preoperatively, and underwent abdominal adnexectomy. In patients without a desire for childbearing, laparoscopic surgery was performed in only 45% of the patients whose preoperative diagnosis was benign. On magnetic resonance imaging (MRI), 54% of the cases showed enhanced solid components, which is characteristic of malignant tumors, but diffusion restriction was observed in only 11%. On computed tomography (CT), 78% of the cases showed a high attenuation lesion reflecting thyroid tissue. CONCLUSIONS: Struma ovarii is difficult to distinguish from malignancy preoperatively, making the choice of surgical approach complicated. A comprehensive evaluation of diffusion-weighted MRI and CT findings may improve the accuracy of preoperative diagnosis of struma ovarii.

Distal arthrogryposis in a girl arising from a novel TNNI2 variant inherited from paternal somatic mosaicism.

TNNI2 at 11p15.5 encodes troponin I2, fast skeletal type, which is a member of the troponin I gene family and a component of the troponin complex. Distal arthrogryposis (DA) is characterized by congenital limb contractures without primary neurological or muscular effects. DA is inherited in an autosomal dominant fashion and is clinically and genetically heterogeneous. Exome sequencing identified a causative variant in TNNI2 [NM_003282.4:c.532T>C p.(Phe178Leu)] in a Japanese girl with typical DA2b. Interestingly, the familial study using Sanger sequencing suggested a mosaic variant in her healthy father. Subsequent targeted amplicon-based deep sequencing detected the TNNI2 variant with variant allele frequencies of 9.4-17.7% in genomic DNA derived from peripheral blood leukocytes, saliva, hair, and nails in the father. We confirmed a disease-causing variant in TNNI2 in the proband inherited from her asymptomatic father with its somatic variant. Our case demonstrates that careful clinical and genetic evaluation is required in DA.

Cat-Ear-Line: A Sonographic Sign of Cortical Development?

OBJECTIVES: Diagonal echogenic lines outside the lateral ventricle have often been observed in the anterior coronal planes of the normal fetal brain by neurosonography. We have observed abnormal shapes of these echogenic lines in cases of malformation of cortical development (MCD). We named the ultrasound finding "cat-ear-line" (CEL). This study aimed to examine how and when CEL develops in normal cases compared with MCD cases. METHODS: We retrospectively examined the fetal brain volume dataset acquired through transvaginal 3D neurosonography of 575 control cases and 39 MCD cases from 2014 to 2020. We defined CEL as the hyperechogenic continuous lines through subplate (SP) and intermediate zone (IZ), pre-CEL as the lines that existed only within the SP, and abnormal CEL as a mass-like or mosaic shadow-like structure that existed across the SP and IZ. All fetuses in the MCD group had some neurosonographic abnormalities and were ultimately diagnosed with MCD. RESULTS: The CEL was detected in 97.9% (369/377) of the control group from 19 to 30 weeks. The CEL visualization rate of the MCD group in the same period was 40.0% (14/35) which was significantly lower than that of the control group (P < .001). CONCLUSIONS: From this study, it appears that the CEL is an ultrasound finding observed at and beyond 19 weeks in a normally developing fetus. In some MCD cases, pre-CEL at and beyond 19 weeks or abnormal CEL was observed. Maldeveloped CEL at mid-trimester may help identify cases at-risk of subsequent MCD.

Guidelines for obstetrical practice in Japan: Japan Society of Obstetrics and Gynecology and Japan Association of Obstetricians and Gynecologists 2020 edition.

The 2017 clinical guidelines for obstetrical practice by the Japan Society of Obstetrics and Gynecology and the Japan Association of Obstetricians and Gynecologists were revised and published as the 2020 edition (in Japanese). The aim of these guidelines is to present appropriate standard obstetric diagnosis and management procedures that have reached consensus among Japanese obstetricians. The 2020 guidelines include 113 clinical questions and an appendix, followed by answers (CQ&A; originally 112 in the 2017 edition), a discussion, list of references, and some tables and figures presenting common problems and questions encountered in obstetrical practice. Each answer comes with a recommendation level of A, B, or C and has been prepared based principally on evidence or a consensus among Japanese obstetricians in situations where "evidence" is weak or lacking. Answers with a recommendation level of A or B represent current standard care practices in Japan. All 113 CQ&As and the appendix are presented here to promote a better understanding of the current standard care practices for pregnant and lactating women in Japan.

A novel FLNA variant in a fetus with skeletal dysplasia.

Otopalatodigital spectrum disorder (OPDSD) is characterized by variable phenotypes, including skeletal dysplasia, and is caused by pathogenic variants in filamin A-encoding FLNA. FLNA variants associated with lethal OPDSD primarily alter the CH2 subdomain of the ABD of FLNA. Herein, we report a novel FLNA mutation in a fetus with severe skeletal dysplasia in a pregnant multigravida female with a history of repeated miscarriages and terminations.

Pathogenic variants detected by RNA sequencing in Cornelia de Lange syndrome.

Recent studies suggest that transcript isoforms significantly overlap (approximately 60%) between brain tissue and Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs). Interestingly, 14 cohesion-related genes with variants that cause Cornelia de Lange Syndrome (CdLS) are highly expressed in the brain and LCLs. In this context, we first performed RNA sequencing of LCLs from 22 solved (with pathogenic variants) and 19 unsolved (with no confirmed variants) CdLS cases. Next, an RNA sequencing pipeline was developed using solved cases with two different methods: short variant analysis (for single-nucleotide and indel variants) and aberrant splicing detection analysis. Then, 19 unsolved cases were subsequently applied to our pipeline, and four pathogenic variants in NIPBL (one inframe deletion and three intronic variants) were newly identified. Two of three intronic variants were located at Alu elements in deep-intronic regions, creating cryptic exons. RNA sequencing with LCLs was useful for identifying hidden variants in exome-negative cases.