Background: Developmental disorders (DDs) are early onset disorders affecting 5%–10% of children worldwide. Chromosomal microarray analysis detecting CNVs is currently recommended as the first-tier test for DD diagnosis. However, this analysis omits a high percentage of disease-causing single nucleotide variations (SNVs) that warrant further sequencing. Currently, next-generation sequencing can be used in clinical scenarios detecting CNVs, and the use of exome sequencing in the DD cohort ahead of the microarray test has not been evaluated. Methods: Clinical exome sequencing (CES) was performed on 1090 unrelated Chinese DD patients who were classified into five phenotype subgroups. CNVs and SNVs were both detected and analysed based on sequencing data. Results: An overall diagnostic rate of 41.38% was achieved with the combinational analysis of CNV and SNV. Over 12.02% of patients were diagnosed based on CNV, which was comparable with the published CMA diagnostic rate, while 0.74% were traditionally elusive cases who had dual diagnosis or apparently homozygous mutations that were clarified. The diagnostic rates among subgroups ranged from 21.82% to 50.32%. The top three recurrent cytobands with diagnostic CNVs were 15q11.2-q13.1, 22q11.21 and 7q11.23. The top three genes with diagnostic SNVs were: MECP2, SCN1A and SCN2A. Both the diagnostic rate and spectrums of CNVs and SNVs showed differences among the phenotype subgroups. Conclusion: With a higher diagnostic rate, more comprehensive observation of variations and lower cost compared with conventional strategies, simultaneous analysis of CNVs and SNVs based on CES showed potential as a new first-tier choice to diagnose DD.

OBJECTIVES: To determine the diagnostic and clinical utility of trio-rapid genome sequencing in critically ill infants. DESIGN: In this prospective study, samples from critically ill infants were analyzed using both proband-only clinical exome sequencing and trio-rapid genome sequencing (proband and biological parents). The study occurred between April 2019 and December 2019. SETTING: Thirteen member hospitals of the China Neonatal Genomes Project spanning 10 provinces were involved. PARTICIPANTS: Critically ill infants (n = 202), from birth up until 13 months of life were enrolled based on eligibility criteria (e.g., CNS anomaly, complex congenital heart disease, evidence of metabolic disease, recurrent severe infection, suspected immune deficiency, and multiple malformations). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Of the 202 participants, neuromuscular (45%), respiratory (22%), and immunologic/infectious (18%) were the most commonly observed phenotypes. The diagnostic yield of trio-rapid genome sequencing was higher than that of proband-only clinical exome sequencing (36.6% [95% CI, 30.1–43.7%] vs 20.3% [95% CI, 15.1–26.6%], respectively; p = 0.0004), and the average turnaround time for trio-rapid genome sequencing (median: 7 d) was faster than that of proband-only clinical exome sequencing (median: 20 d) (p < 2.2 × 10^–16). The metagenomic analysis identified pathogenic or likely pathogenic microbes in six infants with symptoms of sepsis, and these results guided the antibiotic treatment strategy. Sixteen infants (21.6%) experienced a change in clinical management following trio-rapid genome sequencing diagnosis, and 24 infants (32.4%) were referred to a new subspecialist. CONCLUSIONS: Trio-rapid genome sequencing provided higher diagnostic yield in a shorter period of time in this cohort of critically ill infants compared with proband-only clinical exome sequencing. Precise and fast molecular diagnosis can alter medical management and positively impact patient outcomes.

Genetic diseases are a leading cause of death in infants in the intensive care setting; therefore, rapid and accurate genetic diagnosis is desired. To validate 24-h trio-exome sequencing (TES), samples from probands and their parents were processed by the AmpliSeq /Ion S5XL platform in a hospital clinical laboratory. Infants from the intensive care unit (ICU) suspected of having a genetic disease were enrolled. Regular and 24-h TES using the Agilent SureSelect capture kit/Illumina platform were performed on all samples in parallel. Of 33 enrolled infants, 23 received positive results with rapid TES, and an additional two diagnoses were achieved with regular TES. Among the 23 diagnosed patients, 10 experienced changes in medical management, such as hematopoietic stem cell transplant. Ten diagnosed cases were discharged prior to receiving the regular TES results; six received timely symptom control, and four withdrew medical support. Rapid TES enabled faster time to diagnosis, which resulted in an overall decrease in length of hospital stay. The 24-h TES can serve as a rapid response tool for patients with suspected monogenic disorders and can guide clinical decision-making in urgent cases.

Background: NKX2-5, GATA4 and HAND1 are essential for heart development, however, little is known regarding their epigenetic regulation in the pathogenesis of tetralogy of fallot (TOF). Methods: Methylation levels were measured in three regions of NKX2-5 (M1: -1596 bp ~ -1374 bp, M2: -159 bp ~ 217 bp and M3: 1058 bp ~ 1524 bp), one region of GATA4 (M: -392 bp ~ 107 bp) and three regions of HAND1 (M1: -887 bp ~ -414 bp, M2: -436 bp ~ 2 bp and M3: 37 bp ~ 398 bp) using the Sequenom MassARRAY platform. QRT-PCR was used to analyze NKX2-5 and HAND1 mRNA levels in the right ventricular myocardium of TOF patients. Results: TOF patients had a significantly higher NKX2-5_M3 median methylation level than controls (41.65% vs. 22.18%; p = 0.0074; interquartile range [IQR]: 30.46%–53.35%, N = 30 and 20.07%–24.31%, N = 5; respectively). The HAND1_M1 median methylation level was also significantly higher in TOF patients than controls (30.05% vs. 17.54%; p = 0.0054; IQR: 20.77%–40.89%, N = 30 and IQR: 14.69%–20.64%; N = 6; respectively). The methylation statuses of NKX2-5_M1, NKX2-5_M2, GATA4_M, HAND1_M2 or HAND1_M3 were not significantly different in TOF patients compared to controls. The methylation values for NKX2-5_M3 were negatively correlated with mRNA levels (r = - 0.463, p = 0.010, N = 30) and there was a significant association between HAND1_M1 methylation status and mRNA levels (r = - 0.524, p = 0.003, N = 30) in TOF patients. Conclusions: Aberrant methylation statuses of the NKX2-5 gene body and HAND1 promoter regions are associated with the regulation of gene transcription in TOF patients and may play an important role in the pathogenesis of TOF.

Although a lower methylation level of whole genome has been demonstrated in Tetralogy of Fallot (TOF) patients, little is known regarding changes in specific gene DNA methylation profiles and the possible associations with TOF. In current study, the promoter methylation statuses of congenital heart defect (CHD) candidate genes were measured in order to further understand epigenetic mechanisms that may play a role in the development of TOF. The methylation levels of CHD candidate genes were measured using the Sequenom MassARRAY platform. QRT-PCR was used to analyze the mRNA levels of CHD candidate genes in the right ventricular myocardium of TOF cases and normal controls. Methylation status analysis was performed on the promoter regions of 71 CHD candidate genes (113 amplicons). We found significant differences in methylation status, between TOF cases and controls, in 26 amplicons (26 genes) (p < 0.05). Of the 26 amplicons, 17 were up regulated and 9 were down regulated. Additionally, 14 of them were located in the CpG islands, 7 were located in the CpG island shores, and 5 were covering the regions near the transcription start site (TSS). The methylation status was subsequently confirmed and mRNA levels were measured for 7 represented candidate genes, including EGFR, EVC2, NFATC2, NR2F2, TBX5, CFC1B and GJA5. The methylation values of EGFR, EVC2, TBX5 and CFC1B were significantly correlated with their mRNA levels (p < 0.05). Aberrant promoter methylation statuses of CHD candidate genes presented in TOF cases may contribute to the TOF development and have potential prognostic and therapeutic significance for TOF disease.

Background: The purpose of this study is to evaluate the prognostic value of TFPI-2 expression in breast cancer patients through examining the correlation between TFPI-2 expression and breast cancer clinicopathologic features. Methods: Immunohistochemical staining combined with digital image analysis was used to quantify the expression of TFPI-2 protein in breast tumor tissues. For evaluation of the prognostic value of TFPI-2 expression to each clinicopathologic factor, Kaplan-Meier method and COX’s Proportional Hazard Model were employed. Results: TFPI-2 expression was significantly correlated with tumor size, lymph node metastasis, histologic grade, clinical stage, and vessel invasion. More importantly, TFPI-2 expression was also associated with disease-free survival (DFS) of breast cancer patients. We found that patients with high TFPI-2 expression had longer DFS compared with those with low or negative expression of TFPI-2 (P <0.05, log-rank test). Cox’s regression analysis indicated that TFPI-2 expression, histologic grade, and vessel invasion might be significant prognostic factors for DFS, while TFPI-2 expression and histologic grade were the most significant independent predictors for tumor recurrence. Compared with the group with low/high TFPI-2 expression, the TFPI-2 negative group was more likely to have tumor relapse. The hazard ratio of DFS is 0.316 (P <0.01). Conclusions: Low or negative expression of TFPI-2 is associated with breast cancer progression, recurrence and poor survival outcome after breast cancer surgery. TFPI-2 expression in breast tumors is a potential prognostic tool for breast cancer patients.