Simultaneous detection of fetal aneuploidy, de novo FGFR3 mutations and paternally derived ß-thalassemia by a novel method of noninvasive prenatal testing.

OBJECTIVE: The aim is to develop a novel noninvasive prenatal testing (NIPT) method that simultaneously performs fetal aneuploidy screening and the detection of de novo and paternally derived mutations. METHODS: A total of 68 pregnancies, including 26 normal pregnancies, 7 cases with fetal aneuploidies, 7 cases with fetal achondroplasia or thanatophoric dysplasia, 18 cases with fetal skeletal abnormalities, and 10 cases with ß-thalassemia high risk were recruited. Plasma cell-free DNA was amplified by Targeted And Genome-wide simultaneous sequencing (TAGs-seq) to generate around 99% of total reads covering the whole-genome region and around 1%  covering the target genes. The reads on the whole-genome region were analyzed for fetal aneuploidy using a binary hypothesis T-score and the reads on target genes were analyzed for point mutations by calculating the minor allelic frequency of loci on FGFR3 and HBB. TAGs-seq results were compared with conventional NIPT and diagnostic results. RESULTS: In each sample, TAGs-seq generated 44.7-54 million sequencing reads covering the whole-genome region of 0.1-3× and the target genes of >1000×depth. All cases of fetal aneuploidy and de novo mutations of achondroplasia/thanatophoric dysplasia were identified with high sensitivities and specificities except for one false-negative paternal mutation of ß-thalassemia. CONCLUSIONS: TAGs-seq is a novel NIPT method that combines the fetal aneuploidy screening and the detection of de novo FGFR3 mutations and paternal HBB mutations.

[Genetic analysis of a family with 9q34.3 microdeletion and microduplication caused by abnormal chromosome balance structure].

OBJECTIVE: To perform prenatal diagnosis, pedigree analysis, and genetic counseling of a pregnant woman who gave birth to a child with Kleefstra syndrome. METHODS: Karyotype analysis, chromosomal microarray analysis (CMA), multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH) were used of peripheral blood and amniotic fluid to find causes. Recurrence risk assessment was performed later. RESULTS: The amniotic fluid sample showed a 9q34.3 microduplication of arr (hg19) 9q34.3 (140 168 806-141 020 389)× 3, which overlapped the 9q34.3 microdeletion region of proband. The pregnant woman was detected with a balanced translocation of ish, t(9;17)(9q34.3; qter) (9p+; 17p+,9q+, 17q+). No other abnormal results were found in the family. CONCLUSION: Offspring who share the same chromosome segment deletion or duplication are always from parent who carries balanced chromosomal structural aberration.

Single-Cell Sequencing Reveals the Relationship between Phenotypes and Genotypes of Klinefelter Syndrome.

Klinefelter syndrome (KS) is one of the most common congenital disorders of male infertility. Given its high heterogeneity in clinical and genetic presentation, the relationship between transcriptome, clinical phenotype, and associated co-morbidities seen in KS has not been fully clarified. Here, we report a 47,XXY Chinese male with infertility and analyzed the differences in gene expression patterns of peripheral blood mononuclear cells (PBMCs) with regard to a Chinese male and a female control with normal karyotype by single-cell sequencing. A total of 24,439 cells were analyzed and divided into 5 immune cell types (including B cells, T cells, macrophage cells, dendritic cells, and natural killer cells) according to marker genes. Using unsupervised dimensionality reduction and clustering algorithms, we identified molecularly distinct subpopulations of cells between the KS patient and both controls. Gene ontology enrichment analyses yielded terms associated with well-known comorbidities seen in KS as well as an affected immune system and type I diabetes mellitus. Based on our data, we identified several candidate genes which may be implicated in regulating the phenotype of KS. Overall, this analysis provides a comprehensive map of the cell types of PBMCs in a KS patient at the single-cell level, which will contribute to the prevention of comorbidity and improvement of the life quality of KS patients.

[Analysis of CYP21A2 gene mutation in one case of congenital adrenal hyperplasia].

CYP21A2 gene mutations in a child with congenital adrenal hyperplasia (CAH), and the child's parents, were detected in the study. The clinical features, treatment monitoring and molecular genetic mechanism of CAH are reviewed. In the study, DNA was extracted from peripheral blood samples using the QIAGEN Blood DNA Mini Kit; a highly specific PCR primer for CYP21A2 gene was designed according to the sequence difference between CYP2lA2 gene and its pseudogene; the whole CYP2lA2 gene was amplified with PrimeSTAR DNA polymerase (Takara), and the amplification product was directly sequenced to detect and analyze CYP2lA2 gene mutation. The child was clinically diagnosed with CAH (21-hydroxylase deficiency, 21-OHD) at the age of 36 days, and the case was confirmed by genetic diagnosis at the age of 1.5 years. The proband had a homozygous mutation at c.293-13C in the second intron of CYP21 gene, while the parents had heterozygous mutations. Early diagnosis and standard treatment of CAH (21-OHD) should be performed to prevent salt-wasting crisis and reduce mortality; bone aging should be avoided to increase final adult height (FAH), and reproductive dysfunction due to oligospermia in adulthood should be avoided. These factors are helpful for improving prognosis and increasing FAH. Investigating the molecular genetic mechanism of CAH can improve recognition and optimize diagnosis of this disease. In addition, carrier diagnosis and genetic counseling for the proband family are of great significance.

Clinical application of massively parallel sequencing-based prenatal noninvasive fetal trisomy test for trisomies 21 and 18 in 11,105 pregnancies with mixed risk factors.

OBJECTIVE: To report the performance of massively parallel sequencing (MPS) based prenatal noninvasive fetal trisomy test based on cell-free DNA sequencing from maternal plasma in a routine clinical setting in China. METHOD: The MPS-based test was offered as a prenatal screening test for trisomies 21 and 18 to pregnant women in 49 medical centers over 2 years. A total of 11,263 participants were recruited and the MPS-based test was performed in 11,105 pregnancies. Fetal outcome data were obtained after the expected date of confinement. RESULTS: One hundred ninety cases were classified as positive, including 143 cases of trisomy 21 and 47 cases of trisomy 18. With the karyotyping results and the feedback of fetal outcome data, we observed one false positive case of trisomy 21, one false positive case of trisomy 18 and no false negative cases, indicating 100% sensitivity and 99.96% specificity for the detection of trisomies 21 and 18. CONCLUSION: Our large-scale multicenter study proved that the MPS-based test is of high sensitivity and specificity in detecting fetal trisomies 21 and 18. The introduction of this screening test into a routine clinical setting could avoid about 98% of invasive prenatal diagnostic procedures.

Diagnostic value of whole-body magnetic resonance imaging for bone metastases: a systematic review and meta-analysis.

PURPOSE: To assess the overall diagnostic accuracy of whole-body magnetic resonance imaging (WB-MRI) in detecting bone metastases with a meta-analysis. MATERIALS AND METHODS: The MEDLINE, EMBASE, Cancerlit, and Cochrane Library databases were searched from January 1995 to September 2010 for studies evaluating the accuracy of WB-MRI in detecting bone metastases. Histopathologic analysis and/or close clinical and imaging follow-up for at least 6 months was assessed. Meta-analysis methods were used to pool sensitivity and specificity and to construct summary receiver-operating characteristics. RESULTS: A total of 11 studies with 495 patients who fulfilled all of the inclusion criteria were considered for the analysis. No publication bias was found. WB-MRI had a pooled sensitivity of 0.899 (95% confidence interval [CI], 0.845-0.939) and a pooled specificity of 0.918 (95% CI, 0.882-0.946). The subgroup without diffusion-weighted imaging (DWI) positive results had higher pooled specificity 0.961 (95% CI, 0.922-0.984) than the subgroup with DWI (P < 0.05). CONCLUSION: WB-MRI was an accurate, cost-effective tool in detecting bone metastases. WB-MRI without DWI may improve the specificity of detecting bone metastases. DWI seems to be a sensitive but rather unspecific modality for the detection of bone metastatic disease. High-quality prospective studies regarding WB-MRI in detecting bone metastases still need to be conducted.

Performance Evaluation of NIPT in Detection of Chromosomal Copy Number Variants Using Low-Coverage Whole-Genome Sequencing of Plasma DNA.

OBJECTIVES: The aim of this study was to assess the performance of noninvasively prenatal testing (NIPT) for fetal copy number variants (CNVs) in clinical samples, using a whole-genome sequencing method. METHOD: A total of 919 archived maternal plasma samples with karyotyping/microarray results, including 33 CNVs samples and 886 normal samples from September 1, 2011 to May 31, 2013, were enrolled in this study. The samples were randomly rearranged and blindly sequenced by low-coverage (about 7M reads) whole-genome sequencing of plasma DNA. Fetal CNVs were detected by Fetal Copy-number Analysis through Maternal Plasma Sequencing (FCAPS) to compare to the karyotyping/microarray results. Sensitivity, specificity and were evaluated. RESULTS: 33 samples with deletions/duplications ranging from 1 to 129 Mb were detected with the consistent CNV size and location to karyotyping/microarray results in the study. Ten false positive results and two false negative results were obtained. The sensitivity and specificity of detection deletions/duplications were 84.21% and 98.42%, respectively. CONCLUSION: Whole-genome sequencing-based NIPT has high performance in detecting genome-wide CNVs, in particular >10Mb CNVs using the current FCAPS algorithm. It is possible to implement the current method in NIPT to prenatally screening for fetal CNVs.

Noninvasive Fetal Trisomy (NIFTY) test: an advanced noninvasive prenatal diagnosis methodology for fetal autosomal and sex chromosomal aneuploidies.

BACKGROUND: Conventional prenatal screening tests, such as maternal serum tests and ultrasound scan, have limited resolution and accuracy. METHODS: We developed an advanced noninvasive prenatal diagnosis method based on massively parallel sequencing. The Noninvasive Fetal Trisomy (NIFTY) test, combines an optimized Student's t-test with a locally weighted polynomial regression and binary hypotheses. We applied the NIFTY test to 903 pregnancies and compared the diagnostic results with those of full karyotyping. RESULTS: 16 of 16 trisomy 21, 12 of 12 trisomy 18, two of two trisomy 13, three of four 45, X, one of one XYY and two of two XXY abnormalities were correctly identified. But one false positive case of trisomy 18 and one false negative case of 45, X were observed. The test performed with 100% sensitivity and 99.9% specificity for autosomal aneuploidies and 85.7% sensitivity and 99.9% specificity for sex chromosomal aneuploidies. Compared with three previously reported z-score approaches with/without GC-bias removal and with internal control, the NIFTY test was more accurate and robust for the detection of both autosomal and sex chromosomal aneuploidies in fetuses. CONCLUSION: Our study demonstrates a powerful and reliable methodology for noninvasive prenatal diagnosis.

[Cell-free fetal DNA detection in maternal plasma using real-time PCR and cycling probe technology for prenatal screening beta-thalassaemia major].

OBJECTIVE: To analyze cell-free fetal DNA in maternal plasma for prenatal screening of beta-thalassaemia major. METHODS: Six couples undergoing prenatal diagnosis of beta-thalassaemia (gestational age range 23-26 weeks) were enrolled in this study. The husbands were all carriers of the CD17 (A-->T) mutation, and the wives carried another beta-thalassaemia mutation. The allele-specific primers and two fluorescent cycling probes were synthesized for the detection of the CD17 (A-->T) mutation, using FAM and HEX fluorescence labeling, respectively. The cell-free fetal DNA in the maternal plasma was detected using real-time PCR, and the fetal genotype was confirmed by cord blood conventional prenatal diagnosis. RESULTS: In the 6 pregnancies, FAM and HEX fluorescent signals were detected in 3 maternal plasma samples; in the other 3 samples, only FAM fluorescent signals were detected, suggesting the absence of paternally derived CD17 (A-->T) mutation. CONCLUSION: Examination of cell-free fetal DNA in maternal plasma using real-time PCR and cycling probe technology can be effective means for prenatal screening of beta-thalassaemia major.