Balanced chromosomal rearrangement (or balanced chromosome abnormality, BCA) is a common chromosomal structural variation. Next-generation sequencing has been reported to detect BCA-associated breakpoints with the aid of karyotyping. However, the complications associated with this approach and the requirement for cytogenetics information has limited its application. Here, we provide a whole-genome low-coverage sequencing approach to detect BCA events independent of knowing the affected regions and with low false positives. First, six samples containing BCAs were used to establish a detection protocol and assess the efficacy of different library construction approaches. By clustering anomalous read pairs and filtering out the false-positive results with a control cohort and the concomitant mapping information, we could directly detect BCA events for each sample. Through optimizing the read depth, BCAs in all samples could be blindly detected with only 120 million read pairs per sample for data from a small-insert library and 30 million per sample for data from nonsize-selected mate-pair library. This approach was further validated using another 13 samples that contained BCAs. Our approach advances the application of high-throughput whole-genome low-coverage analysis for robust BCA detection-especially for clinical samples-without the need for karyotyping.

MicroRNAs are a new class of non-proteincoding, small RNAs that function as tumor suppressors or oncogenes. They participate in diverse biological pathways and function as gene regulators. A G>C polymorphism (rs2910164), which is located in the sequence of miR-146a precursor, results in a change from G:U to C:U in its stem region. However, it remains largely unknown whether this single nucleotide polymorphism (SNP) may alter esophageal squamous cell carcinoma (ESCC) susceptibility. In the current study, we evaluated association between rs2910164 and ESCC susceptibility in a case-control study of 444 sporadic ESCC patients and 468 matched cancer-free controls in a Chinese Han population. Compared with rs2910164 variant genotype CC, genotype GG was associated with increased risk of ESCC (Odds Ratio, 2.39, 95% Confidence Interval, 1.36-4.20). In the smokers, the risk of rs2910164 GG genotype was more notable (Odds Ratio, 3.17, 95% Confidence Interval, 1.71-4.46). In the stratification analyses, we also found there was a strong correlation between rs2910164 C/G variant and the clinical TNM stage (P < 0.01). These findings suggest that this functional SNP in pre-miR-146a could contribute to ESCC susceptibility and clinical outcome.

Previous studies on esophageal squamous cell carcinoma (ESCC) indicated that it contains much dysregulation of microRNAs (miRNAs). DNA hypermethylation in the miRNA 5 0 regulatory region is a mechanism that can account for the downregulation of miRNA in tumors (Esteller, N Engl J Med 2008;358:1148-59). Among those dysregulated miRNAs, miR-203, miR-34b/c, miR-424 and miR-129-2 are embedded in CpG islands, as is the promoter of miR-34a. We investigated their methylation status in ESCC by bisulfite sequencing PCR (BSP) and methylation specific PCR (MSP). The methylation frequency of miR-203 and miR-424 is the same in carcinoma and in the corresponding non-tumor tissues. The methylation ratio of miR-34a, miR-34b/c and miR-129-2 is 66.7% (36/54), 40.7% (22/54) and 96.3% (52/54), respectively in ESCC, which are significantly higher than that in the corresponding non-tumor tissues(p < 0.01). Quantitative RT-PCR analysis in clinical samples suggested that CpG island methylation is significantly correlated with their low expression in ESCC, 5-aza-2 0 -deoxycytidine (DAC) treatment partly recovered their expression in EC9706 cell line. We conclude that CpG island methylation of miR-34a, miR-34b/c and miR-129-2 are frequent events and important mechanism for their low expression in ESCC. DNA methylation changes have been reported to occur early in carcinogenesis and are potentially good early indicators of carcinoma (Laird, Nat Rev Cancer 2003;3:253-66). The high methylation ratio of miR-129-2 indicated its potential as a methylation biomarker in early diagnosis of ESCC.

BackgroundThe identification of causative mutations is important for treatment decisions and genetic counseling of patients with disorders of sex development (DSD). Here, we designed a new assay based on targeted next-generation sequencing (NGS) to diagnose these genetically heterogeneous disorders.MethodsAll coding regions and flanking sequences of 219 genes implicated in DSD were designed to be included on a panel. A total of 45 samples were used for sex chromosome dosage validation by targeted sequencing using the NGS platform. Among these, 21 samples were processed to find the causative mutation.ResultsThe sex chromosome dosages of all 45 samples in this assay were concordant with their corresponding karyotyping results. Among the 21 DSD patients, a total of 11 mutations in SRY, NR0B1, AR, CYP17A1, GK, CHD7, and SRD5A2 were identified, including five single nucleotide variants, three InDels, one in-frame duplication, one SRY-positive 46,XX, and one gross duplication with an estimated size of more than 427,038 bp containing NR0B1 and GK. We also identified six novel mutations: c.230_231insA in SRY, c.7389delA in CHD7, c.273C>G in NR0B1, and c.2158G>A, c.1825A>G, and c.2057_2065dupTGTGTGCTG in AR.ConclusionsOur assay was able to make a genetic diagnosis for eight DSD patients (38.1 %), and identified variants of uncertain clinical significance in the other three cases (14.3 %). Targeted NGS is therefore a comprehensive and efficient method to diagnose DSD. This work also expands the pathogenic mutation spectrum of DSD.Electronic supplementary materialThe online version of this article (doi:10.1186/s12881-016-0286-2) contains supplementary material, which is available to authorized users.

MicroRNAs (miRNAs) are small, non-protein-coding RNAs that function as tumour suppressors or oncogenes. A single nucleotide change in the sequence of pre-miRNA can affect miRNA expression, so single-nucleotide polymorphisms (SNPs) in pre-miRNA may be biomarkers for biomedical applications. In this study, we performed a genetic association study between the SNP (rs11614913) in pre-miRNA-196a and esophageal squamous cell carcinoma (ESCC) susceptibility in a case-control study. We found that the homozygote CC of this SNP increased the risk of ESCC compared with the homozygote TT and the risk was more evident among smokers than non-smokers. Therefore, this functional SNP may be a biomarker for ESCC outcome.

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BackgroundDuring human pregnancy, placental trophectoderm cells release extracellular vesicles (EVs) into maternal circulation. Trophoblasts also give rise to cell-free DNA (cfDNA) in maternal blood, and has been used for noninvasive prenatal screening for chromosomal aneuploidy. We intended to prove the existence of DNA in the EVs (evDNA) of maternal blood, and compared evDNA with plasma cfDNA in terms of genome distribution, fragment length, and the possibility of detecting genetic diseases.MethodsMaternal blood from 20 euploid pregnancies, 9 T21 pregnancies, 3 T18 pregnancies, 1 T13 pregnancy, and 2 pregnancies with FGFR3 mutations were obtained. EVs were separated from maternal plasma, and confirmed by transmission electronic microscopy (TEM), western blotting, and flow cytometry (FACS). evDNA was extracted and its fetal origin was confirmed by quantitative PCR (qPCR). Pair-end (PE) whole genome sequencing was performed to characterize evDNA, and the results were compared with that of cfDNA. The fetal risk of aneuploidy and monogenic diseases was analyzed using the evDNA sequencing data.ResultsEVs separated from maternal plasma were confirmed with morphology by TEM, and protein markers of CD9, CD63, CD81 as well as the placental specific protein placental alkaline phosphatase (PLAP) were confirmed by western blotting or flow cytometry. EvDNA could be successfully extracted for qPCR and sequencing from the plasma EVs. Sequencing data showed that evDNA span on all 23 pairs of chromosomes and mitochondria, sharing a similar distribution pattern and higher GC content comparing with cfDNA. EvDNA showed shorter fragments yet lower fetal fraction than cfDNA. EvDNA could be used to correctly determine fetal gender, trisomies, and de novo FGFR3 mutations.ConclusionsWe proved that fetal DNA could be detected in EVs separated from maternal plasma. EvDNA shared some similar features to plasma cfDNA, and could potentially be used to detect genetic diseases in fetus.

Cell-free DNA (cfDNA) testing for common fetal trisomies (T21, T18, T13) is highly effective. However, the usefulness of cfDNA testing in detecting other chromosomal abnormalities is unclear. We evaluated the performance of cfDNA testing for genome-wide abnormalities, and analyzed the incremental yield by reporting extra abnormalities. We performed genome-wide cfDNA testing in 15,626 consecutive pregnancies prospectively enrolled in this study. cfDNA testing results were reported and counseling was given depending on the presence of extra chromosomal abnormalities. cfDNA testing identified 190 cases (1.2%) of chromosomal abnormalities including 100 common trisomies and 90 additional abnormalities. By expanding the cfDNA reporting range to genome-wide abnormalities, the false positive rate increased to 0.39% (P<0.001) and positive predictive value (PPV) was reduced to 65.58% (P=0.42). However, the detection yield increased from 0.44% to 0.65% (P=0.014), and cfDNA testing detected 38.61% (39/101) additional abnormalities with no ultrasound and biochemical screening findings. cfDNA testing outperformed biochemical screening by showing 60 times higher true positive rate and fewer false negative results. Genome-wide cfDNA testing significantly increased the diagnostic yield by detecting extra abnormalities, especially those without diagnostic indications. Genome-wide cfDNA testing has fewer false positive and false negative results compared with biochemical screening.