Combined use of bacterial artificial chromosomes-on-beads with karyotype detection improves prenatal diagnosis.

BACKGROUND: This study evaluated the individual and combined diagnostic performance of the bacterial artificial chromosomes (BACs)-on-Beads (BoBs™) assay and conventional karyotyping for the prenatal detection of chromosomal abnormalities in pregnant women who were 35 or more years-old. METHOD: The primary outcome was concordance of any numerical, structural, or submicroscopic chromosomal abnormalities between BoBs™ and conventional karyotyping of amniotic fluid specimens from pregnant women at 17 to 22 weeks gestation. RESULTS: We examined samples from 4852 pregnant women. BoBs™ indicated that 4708 samples were normal (97.03%), and 144 were abnormal (2.97%); conventional karyotyping indicated that 4656 (95.96%) samples were normal and 196 (4.04%) were abnormal. The combined use of both methods indicated that 4633 of 4852 samples were normal (95.49%) and 219 of 4852 samples (4.51%) were abnormal. The kappa coefficient of the combined test was 0.70, indicating substantial consistency between BoBs™ and conventional karyotyping (95% CI = 0.65-0.76, P < 0.001). CONCLUSIONS: Our results indicate that the combined use of BoBs™ and conventional karyotyping detected more fetal abnormalities than either test alone.

[Diagnosis and reproductive guidance for a couple carrying a novel c.1893C>T mutation of the TECTA gene].

OBJECTIVE: To explore the molecular basis for an individual with postnatal deafness and provide genetic counseling for her family. METHODS: Following extraction of genomic DNA from peripheral blood samples, 127 genes associated with deafness were subjected to targeted capturing and next generation sequencing. Suspected mutation was verified by Sanger sequencing. RESULTS: The proband was found to carry a homozygous c.1893C>A mutation in the TECTA gene, which is located in the tectorial membrane of inner ear and may cause premature termination of translation of TECTA protein. In addition, two heterozygous mutations, c.13010C>T and c.12790G>A, were found in the USH2A gene. Whilst the former is likely to be pathogenic, the latter has unknown clinical significance. Further analysis suggested that all three mutations have derived from the parents of the proband. CONCLUSION: The homozygous c.1893C>A mutation of the TECTA gene probably underlies the proband's hearing loss which conformed to an autosomal recessive inheritance.

Assessment of efficacy of prenatal genetic diagnosis for fragile X syndrome using nested PCR.

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and the leading monogenic cause of autism spectrum disorder. It has previously been demonstrated that prenatal genetic diagnosis is efficient for the diagnosis of FXS. The present study investigated the diagnostic effects of nested polymerase chain reaction (PCR) for fragile X mental retardation 1 (FMR1) and expanded CGG repeats. It was demonstrated that the nested PCR assay rapidly measured the multi-copies of the FMR1 gene in individual samples. The nested PCR assay detected normal CGG repeat lengths and expanded CGG repeat lengths with a low occurrence of false positives. In addition, the nested PCR assay resulted in increased sensitivity and specificity for patients with FXS. Furthermore, the nested PCR assay identified the mutation and generated conclusive cases for FXS, indicating that this assay is beneficial for the diagnosis of FXS patients. In conclusion, these outcomes indicate that nested PCR assay is a reliable and easier method for diagnosis of FXS, which may be used for the diagnosis of FXS patients.

[Prenatal diagnosis of a tetrasomy 18p case using BACs-on-Beads technology and single nucleotide polymorphism array].

OBJECTIVE: To determine the origin of a supernumerary small marker chromosome found in a fetus using prenatal BACs-on-Beads (BoBs) and single nucleotide polymorphism array (SNP-array) assays. METHODS: The fetal sample was subjected to chromosomal karyotyping and BoBs analysis, and the results were validated with genome-wide scanning using a SNP microarray. RESULTS: The fetus was found to have a 47,XX,+mar karyotype. BoBs analysis indicated that there was an amplification between 18p11.32 and 18p11.21, which was verified by the SNP-array assay as a 18.3 Mb duplication occurring at 18p11.32q11.1. CONCLUSION: The karyotype of the fetus was determined as 47,XX,+der18(18p11.32?18q11.1::18q11.1?18p11.32). The duplication has involved important genes including SMCHD1, LPIN2 and TGIF1, which may result in severe malformations in the fetus.

[Detection of cell-free fetal DNA in maternal plasma for noninvasive prenatal screening of fetal chromosomal aneuploidies in women of advanced maternal age].

OBJECTIVE: To evaluate the efficiency of cell-free fetal DNA detection as a non-invasive prenatal screening (NIPS) method for women of advanced maternal age. METHODS: A total of 10 584 women of advanced maternal age who received NIPS were recruited from the Women's Hospital, Zhejiang University School of Medicine and Jiaxing Maternal and Child Health Hospital during February 2015 and September 2016. The pregnancy outcome was followed-up. The sensitivity, specificity, positive and negative predictive value of fetal chromosomal aneuploidy detected in NIPS were analyzed. And the relationship between maternal age and fetal common chromosomal aneuploidy was analyzed. RESULTS: The sensitivity, specificity, positive and negative predictive value of NIPS were 100.00%, 99.96%, 91.67%, 100.00% for trisomy 21, 100.00%, 99.93%, 68.18%, 100.00% for trisomy 18, and 100.00%, 99.97%, 25.00%, 100.00% for trisomy 13. High-risk rate and true positive rate of trisomy 21 were positively correlated with the maternal age (all P<0.01). There were significant differences in high-risk rate and true positive rate between 35-37 year old groups and 38-40 year old groups (all P<0.05). Such difference was also found in high-risk rate between 38-40 year old group and ≥ 41 year old group (P<0.05), but not in true positive rate between two groups (P>0.05). CONCLUSIONS: NIPS is effective for fetal chromosomal aneuploidy screening in women of advanced maternal age. For women under 38 years of age, NIPS is preferred; for women of 41 and above, invasive diagnostic methods are suggested; and for women between 38-41 years old, the option can be determined by themselves after risks and advantages were fully informed.

[Prenatal diagnosis of a Pallister-Killian syndrome case through analysis of a supernumerary chromosome using single nucleotide polymorphism array].

OBJECTIVE: To explore the origin of a supernumerary small marker chromosome (sSMC) in a fetus, and to assess the feasibility of single nucleotide polymorphism array (SNP-array) for prenatal diagnosis. METHODS: The fetal sample was subjected to karyotyping analysis. The identified sSMC was subjected to genome-wide scan using a SNP microarray chip. The results were validated with fluorescence in situ hybridization (FISH). RESULTS: The karyotype of the fetus was determined as 47,XX,+mar, which was verified by SNP microarray chip analysis as a 34.6 Mb duplication in 12p13.33p11.1. FISH analysis confirmed that the sSMC has originated from chromosome 12p. CONCLUSION: The karyotype of the fetus was determined as 47,XX,+i(12)(p10). Tetrasomy 12p is reported to be a marker for Pallister-Killian syndrome, which may result in multi-system anomalies. SNP-array analysis can simultaneously detect microdeletions and microduplications, which may be used for prenatal diagnosis of suspected cases.

[A case of Wolf-Hirschhorn syndrome diagnosed by single nucleotide polymorphism array].

OBJECTIVE: To explore the genetic causes for a child with multiple congenital malformations and epilepsy through analysis of copy number variations, and to correlate the genotype with the phenotype. METHODS: G-banding karyotyping was performed on the child and her parents. Single nucleotide polymorphisms array (SNP-array) was used to map the exact chromosomal breakpoints in the proband. The result was validated with fluorescence in situ hybridization (FISH). RESULTS: G banding analysis suggested that the proband had a karyotype of 46,XX,del(4)(p15), while both of his parents had a normal karyotype. SNP-array has identified a hemizygous deletion of 13.3 Mb on chromosome 4p16.3p15.33, which has been implicated in Wolf-Hirschhorn syndrome. FISH assay has confirmed the de novo origin of the deletion, with the karyotype and clinical phenotype of both parents taken into consideration. CONCLUSION: A case of Wolf-Hirschhorn syndrome has been diagnosed by clinical manifestation and karyotyping analysis. Compared with conventional karyotyping analysis, SNP-array has greater resolution and accuracy, and can provide useful information for genetic counseling.

A multicenter study of fetal chromosomal abnormalities in Chinese women of advanced maternal age.

OBJECTIVE: This study aimed to determine the rates of different fetal chromosomal abnormalities among women of advanced maternal age in China and to discuss the possible misdiagnosis risks of newer molecular techniques, for selection of appropriate prenatal screening and diagnostic technologies. MATERIALS AND METHODS: Second trimester amniocentesis and fetal karyotype results of 46,258 women were retrospectively reviewed. All women were ≥ 35 years old with singleton pregnancies. The rates of clinically significant chromosomal abnormalities (CSCAs), incidence of chromosomal abnormalities, and correlations with age were determined. RESULTS: From 2001 to 2010, the proportion of women of advanced maternal age undergoing prenatal diagnosis increased from 20% to 46%. The mean age was 37.4 years (range, 35-46 years). A total of 708 cases of CSCAs, with a rate of 1.53% were found. Trisomy 21 was the most common single chromosome abnormality and accounted for 55.9% of all CSCAs with an incidence of 0.86%. Trisomy 13, trisomy 18, and trisomy 21, the most common chromosome autosomal aneuploidies, accounted for 73.6% of all CSCAs, with a rate of 1.13%. As a group, the most common chromosomal aneuploidies (13/18/21/X/Y) accounted for 93.9% of all abnormalities, with a rate of 1.44%. The incidence of trisomy 21, trisomy 13/18/21 as a group, and 13/18/21/X/Y as a group was significantly greater in women aged 39 years and older (p < 0.001), but was not different between women aged 35 years, 36 years, 37 years, and 38 years. CONCLUSION: These findings may assist in genetic counseling of advanced maternal age pregnant women, and provide a basis for the selection of prenatal screening and diagnostic technologies.

[Diagnosis of a case with Williams-Beuren syndrome by single nucleotide polymorphism array].

OBJECTIVE: To explore the genetic cause for a child with mental retardation, developmental delay and multi-systemic developmental disorders by analyzing the copy number variations (CNVs) and correlating the genotype with the phenotype. METHODS: Routine G-banding was performed to analyze the karyotype of the patient and her parents. In addition, single nucleotide polymorphisms array (SNP-array) was used to determine the CNVs, which was confirmed by fluorescence in situ hybridization (FISH). RESULTS: No karyotypic abnormality was detected upon chromosome analysis. However, SNP-array has identified a de novo hemizygous deletion of 1673 kb on chromosome region 7q11.23, which has been associated with Williams-Beuren syndrome. The microdeletion was confirmed by FISH testing. CONCLUSION: A child with Williams-Beuren syndrome has been diagnosed by SNP-array and FISH. The de novo 7q11.23 microdeletion probably underlies the clinical manifestation of the patient. Compared with routine karyotype analysis, SNP-array is more useful for diagnosing children with multiple congenital anomalies with unclear etiology.

[Prenatal diagnosis of fetal chromosome aneuploidy by massively parallel genomic sequencing].

OBJECTIVE: To perform non-invasive prenatal diagnosis (NIPD) of chromosome aneuploidy by detecting free DNA in maternal peripheral plasma by massively parallel genomic sequencing and determine the feasibility of detecting cell-free fetal DNA in chromosomal copy. METHODS: The plasma samples from 1 264 gravidas were collected from February 2012 to July 2013 at our center. Those pregnant women with a gestational age of 13 to 33 weeks having high risks in serological screening or aged over 35 years or abnormal fetus on ultrasonography. The peripheral venous blood samples were drawn from pregnant women and plasma DNA was extracted for preparing a sequencing library. By using Illumina HiSeq2000, high-throughput sequencing was performed. Amnocentesis and karyotypic analysis were conducted for positive cases and those with negative results were followed up. RESULTS: By massively parallel genomic sequencing, 20 of them showed positive results. By the standard of chromosomal karyotypic analysis, there were 21 positive cases of 13 trisomy and the diagnostic accordance rate was 100%. Among 18 positive cases of 6 trisomy, there were amniocentesis (n = 5, except for one dead fetus), trisomy 18 (n = 4) and normal karyotype with placenta chimera (n = 1). One positive case of 13 trisomy was in accordance with karyotypic analysis. CONCLUSION: Massively parallel sequencing for NIPD may be used as further screening for pregnant women with high risks of serological screening. And this technology is highly consistent with karyotypic analysis in terms of sensitivity and specificity.

First trimester, second trimester, and integrated screening for Down's syndrome in China.

OBJECTIVE: To compare the effectiveness of first trimester, second trimester, and integrated screening for Down's syndrome. SETTING: Two prenatal diagnosis centres in China. METHODS: A total of 11,966 pregnant women (≥18 years) were screened over 21 months. First trimester screening (11-13 weeks) comprised measurement of serum free beta-human chorionic gonadotrophin (ß-hCG) and pregnancy-associated protein-A concentrations, and fetal nuchal translucency thickness. Second trimester screening (15-20 weeks) comprised measurement of ß-hCG and alpha fetoprotein concentrations. Computer software was used to calculate the risk of carrying a Down's syndrome fetus. RESULTS: The overall incidence of Down's syndrome was 0.2% (23/11,966). When the false-positive rate was fixed at 5%, detection rates for first trimester, second trimester, and integrated screening were 73.9%, 69.6%, and 82.6%, respectively. When the false-positive rate was fixed at 3%, detection/sensitivity rates for first trimester, second trimester, and integrated screening were 65.2%, 56.5%, and 73.9%, respectively. CONCLUSIONS: These findings suggest that integrated screening was the most effective means of screening for Down's syndrome in a Chinese population.

[First trimester and second-trimester integrated screening for Down's syndrome].

OBJECTIVE: To evaluate the effect of first and second-trimester integrated screening so as to provide an efficient screening protocol for Down's syndrome. METHODS: Using the dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA), the freeßhCG (beta human chorionic gonadotropin), PAPP-A (pregnancy associated plasma protein-A) and NT (nuchal translucency) value of type B ultrasound were assayed in the pregnancy serum during the first trimester (11-13W(+6) d) and free ßhCG and AFP (alpha fetoprotein) during the second trimester(15-20W(+6) d). By the risk calculation software, the risks during both trimesters and their integrated risk were calculated for each patient respectively. Amniocentesis and venepuncture were employed for diagnosing the high-risk patients (> 1/270). Electronic network follow-up was carried out after delivery. RESULTS: In a total of 4237 pregnant women, 98 were found to carry a high risk during the first trimester, 241 during the second trimester and 101 during the integrated screening respectively. And 2, 3 and 4 cases were diagnosed with Down's symptom at a detection rate of 50%, 75% and 100% and a detection efficiency of 1:50, 1:80 and 1:25 respectively. CONCLUSION: Integrated screening is superior to either the first or second-trimester screening. With a lower false positive rate and a higher detection rate, it reduces the chance of invasive puncture. Advanced type B ultrasonic technology is needed to improve the first-trimester diagnostic efficiency and to develop a better integrated screening protocol.

[Cytoskeleton rearrangements and induced apoptosis in human umbilical venous endothelial cell and WISH cell lines during the invasion of Staphylococcus aureus].

OBJECTIVE: To investigate the changes of cytoskeleton and induced apoptosis in human umbilical venous endothelial cells (HUVEC) and WISH cells during the invasion of Staphylococcus aureus. METHODS: S. aureus suspension was collected routinely and used to infect HUVEC and WISH cells for 10, 30, 60 and 120 min respectively. The cell-invading ability of S. aureus was observed by microscope and the rearrangement of cytoskeleton of these cells observed under fluorescent microscope. DAPI fluorescent staining and DNA agarose electrophoresis were performed to analyze the apoptosis in HUVEC cells induced by S. aureus. RESULTS: There was bacterial invasion after staphylococcus aureus was co-incubated with HUVEC and WISH cells for 10 min. The rates of infection were (54.9 +/- 2.4)% and (56.1 +/- 2.4)% at 60 min respectively. The ratios of F-actin rearrangements at 10 min, 30 min, 60 min and 120 min after the invasion of HUVEC and WISH cells with S. aureus were different at (54.7 +/- 2.8)%, (63.0 +/- 2.9)%, (71.0 +/- 2.6)%, (39.5 +/- 2.7)% and (63.3 +/- 2.6)%, (65.0 +/- 2.9)%, (77.0 +/- 2.4)% and (44.0 +/- 1.8)% respectively. The ratios of F-actin rearrangements at 10 min, 30 min and 60 min were higher than those at 120 min (P < 0.05). There was no change of microtubule observed in both cells at the same time. The apoptotic appearance was observed after the invasion of HUVEC cells with s. aureus at 60 min. CONCLUSION: S. aureus may invade the HUVEC and WISH cells through F-actin rearrangement. Apoptosis is induced in HUVEC cells at 60 min.