Evaluation of non‐invasive prenatal testing to detect chromosomal aberrations in a Chinese cohort

The aim of this study was to evaluate the clinical feasibility of non‐invasive prenatal testing (NIPT) to detect foetal copy number variations (CNVs). Next‐generation sequencing for detecting foetal copy number variations (CNVs) was performed on the collected samples from 161 pregnancies with ultrasound anomalies and negative NIPT results for aneuploidy. The performance of NIPT for detecting chromosome aberrations was calculated. The sensitivity and specificity of NIPT for detecting CNVs > 1 Mb were 83.33% and 99.34%; the PPV and negative predictive rate (NPV) were 90.91% and 98.68%. Non‐invasive prenatal testing can be performed to detect chromosomal aberrations in first trimester with high performance for CNVs, and occasional discordant cases are unavoidable.     

Noninvasive Detection of Fetal Subchromosome Abnormalities via Deep Sequencing of Maternal Plasma

The purpose of this study was to determine the deep sequencing and analytic conditions needed to detect fetal subchromosome abnormalities across the genome from a maternal blood sample. Cell-free (cf) DNA was isolated from the plasma of 11 pregnant women carrying fetuses with subchromosomal duplications and deletions, translocations, mosaicism, and trisomy 20 diagnosed by metaphase karyotype. Massively parallel sequencing (MPS) was performed with 25-mer tags at approximately 10⁹ tags per sample and mapped to reference human genome assembly hg19. Tags were counted and normalized to fixed genome bin sizes of 1 Mb or 100 kb to detect statistically distinct copy-number changes compared to the reference. All seven cases of microdeletions, duplications, translocations, and the trisomy 20 were detected blindly by MPS, including a microdeletion as small as 300 kb. In two of these cases in which the metaphase karyotype showed additional material of unknown origin, MPS identified both the translocation breakpoint and the chromosomal origin of the additional material. In the four mosaic cases, the subchromosomal abnormality was not demonstrated by MPS. This work shows that in nonmosaic cases, it is possible to obtain a fetal molecular karyotype by MPS of maternal plasma cfDNA that is equivalent to a chromosome microarray and in some cases is better than a metaphase karyotype. This approach combines the advantage of enhanced fetal genomic resolution with the improved safety of a noninvasive maternal blood test.     

孕中期超声联合无创产前基因筛查在检出染色体异常胎儿中的应用价值

摘要 目的：孕中期超声联合无创产前基因筛查（NIPT）在染色体异常胎儿检出中的应用价值。方法：选取2019年8月～2021年12月在石家庄市妇幼保健院产前检查的2000例孕中期孕妇,均接受超声检查和NIPT筛查。以羊水穿刺或引产后高通量测序结果为金标准,四格表法分析孕中期超声联合NIPT在染色体异常胎儿检出中的应用价值。结果：2000例孕中期孕妇中,超声检查共检出软指标异常37例,结构指标异常30例。NIPT筛查检出高风险孕妇17例,其中21-三体综合征11例、18-三体综合征6例。超声软指标和结构指标联合NIPT诊断胎儿染色体异常的灵敏度、特异度、阳性预测值、阴性预测值、漏诊率、误诊率、准确率分别为95.00%、99.95%、95.00%、99.95%、5.00%、0.05%、99.90%。结论：联合孕中期超声和NIPT可提高检出高风险染色体异常胎儿的灵敏度,降低漏诊率,对于早发现染色体异常胎儿具有重要价值,进而提高生育质量。     

超声心动图检查指标与胎儿染色体异常的相关性

摘要 目的：探究超声心动图检查指标与胎儿染色体异常之间的相关性。方法：选择2017年1月至2020年1月于我院接受产前超声心动图检查的980例高危产妇为研究对象,均对其开展超声心动图以及染色体核型检测,记录受检者染色体核型异常具体情况,筛选出染色体核型异常产妇（80例）作为研究组,设另900例染色体正常产妇为对照组,就两组产妇的超声心动图检查指标左心室Tei指数、右心室Tei指数以及颈项透明层厚度（NT）值差异进行比较,Spearman相关性分析探究上述超声心动图指标的相关性,最后绘制心动图指标对染色体异常的诊断ROC曲线图并计算AUC值。结果：（1）比较显示研究组胎儿的左心室Tei指数、右心室Tei指数和NT值均明显的高于对照组胎儿,组间差异具有统计学意义（P<0.05）;（2）相关性分析显示NT值同染色体异常胎儿的左心室Tei值、右心室Tei值均呈现明显的正相关联系（r=0.8927,r=0.9315,P<0.0001）;（3）ROC曲线绘制显示左心室Tei值、右心室Tei值和NT值对胎儿染色体异常的诊断AUC分别为0.9889、0.7574、0.7959（P<0.05）。结论：超声心动图检查指标同胎儿的染色体异常之间存在明显的关联性,可以考虑将超声心动图作为胎儿染色体异常的初筛手段,推广于临床应用。     

Clinical utility of chromosomal microarray analysis in invasive prenatal diagnosis

Novel methodologies for detection of chromosomal abnormalities have been made available in the recent years but their clinical utility in prenatal settings is still unknown. We have conducted a comparative study of currently available methodologies for detection of chromosomal abnormalities after invasive prenatal sampling.A multicentric collection of a 1-year series of fetal samples with indication for prenatal invasive sampling was simultaneously evaluated using three screening methodologies: (1) karyotype and quantitative fluorescent polymerase chain reaction (QF-PCR), (2) two panels of multiplex ligation-dependent probe amplification (MLPA), and (3) chromosomal microarray-based analysis (CMA) with a targeted BAC microarray. A total of 900 pregnant women provided informed consent to participate (94% acceptance rate). Technical performance was excellent for karyotype, QF-PCR, and CMA (~1% failure rate), but relatively poor for MLPA (10% failure). Mean turn-around time (TAT) was 7 days for CMA or MLPA, 25 for karyotype, and two for QF-PCR, with similar combined costs for the different approaches. A total of 57 clinically significant chromosomal aberrations were found (6.3%), with CMA yielding the highest detection rate (32% above other methods). The identification of variants of uncertain clinical significance by CMA (17, 1.9%) tripled that of karyotype and MLPA, but most alterations could be classified as likely benign after proving they all were inherited. High acceptability, significantly higher detection rate and lower TAT, could justify the higher cost of CMA and favor targeted CMA as the best method for detection of chromosomal abnormalities in at-risk pregnancies after invasive prenatal sampling.     

Pathogenic copy number variations are associated with foetal short femur length in a tertiary referral centre study

Shortened foetal femur length (FL) is a common abnormal phenotype that often causes anxiety in pregnant women, and standard clinical treatments remain unavailable. We investigated the clinical characteristics, genetic aetiology and obstetric pregnancy outcomes of foetuses with short FL and provided a reference for perinatal management of such cases. Chromosomal microarray analysis was used to analyse the copy number variations (CNV) in short FL foetuses. Of the 218 foetuses with short FL, 33 foetuses exhibited abnormal CNVs, including 19 with pathogenic CNVs and 14 with variations of uncertain clinical significance. Of the 19 foetuses with pathogenic CNVs, four had aneuploidy, 14 had deletions/duplications, and one had pathogenic uniparental diploidy. The 7q11.23 microdeletion was detected in three foetuses. The severity of short FL was not associated with the rate of pathogenic CNVs. The duration of short FL for the intrauterine ultrasound phenotype in foetuses carrying a pathogenic CNV was independent of the gestational age. Further, maternal age was not associated with the incidence of foetal pathogenic CNVs. Adverse pregnancy outcomes occurred in 77 cases, including termination of pregnancy in 63 cases, postnatal dwarfed foetuses with intellectual disability in 11 cases, and three deaths within 3 months of birth. Pathogenic CNVs closely related to foetal short FL were identified, among which the 7q11.23 microdeletion was highly associated with short FL development. This study provides a reference for the perinatal management of foetuses with short FL.     

Chromosomal microarray analysis for pregnancies with abnormal maternal serum screening who undergo invasive prenatal testing

Recently, chromosomal microarray analysis (CMA) has been implemented as a first-tier test in pregnancies with ultrasound anomalies. However, its application for pregnancies with abnormal maternal serum screening (AMSS) only is not widespread. This study evaluated the value of CMA compared to traditional karyotyping in pregnancies with increased risk following first- or second-trimester maternal serum screening. Data from 3973 pregnancies with referral for invasive prenatal testing following AMSS were obtained from April 2016 to May 2020. Routine karyotyping was performed and single nucleotide polymorphism array was recommended. The foetuses were categorized according to the indications as AMSS only (group A) and AMSS with ultrasound anomalies (group B). CMA was performed on 713 prenatal samples. The proportion of women opting for CMA testing in both groups increased over the years. The incremental yield of clinically significant findings for pregnancies with high risk of screening results was similar to that for the foetuses with ultrasound soft markers (P > 0.05), but significantly lower than that for the foetuses with structural anomalies (P < 0.05). The total frequencies of variants of unknown significance in groups A and B showed no significant difference (P > 0.05). CMA should be performed for pregnant women undergoing prenatal invasive testing due to AMSS, especially with high-risk results, regardless of ultrasound findings.     

Parental and chromosomal origin of unbalanced de novo structural chromosome abnormalities in man

We report the parental origin, and where possible the chromosomal origin of 115 de novo unbalanced structural chromosome abnormalities detectable by light microscopy. These consisted of 39 terminal deletions, 35 interstitial deletions, 8 rings, 12 duplications and 21 unbalanced translocations. In all categories the majority of abnormalities were of paternal origin, although the proportions varied from a high of 84% in the interstitial deletions and rings to a low of 58% in the duplications. Among the interstitial deletions and duplications, there were approximately equal numbers of intra- and interchromosomal abnormalities, while the majority of unbalanced translocations were isodisomic for the duplicated chromosome. The examination of the parental ages in the four main classes of abnormality showed terminal deletions of maternal origin to be associated with a significantly reduced maternal age. Thus, there is a clear propensity for structural chromosome abnormalities to occur in male germ cells, although the chromosomal origin seems similar irrespective of the parental origin.     

Dual‐color oligo‐FISH can reveal chromosomal variations and evolution in Oryza species

Fluorescence in situ hybridization using probes based on oligonucleotides (oligo‐FISH) is a useful tool for chromosome identification and karyotype analysis. Here we developed two oligo‐FISH probes that allow the identification of each of the 12 pairs of chromosomes in rice (Oryza sativa). These two probes comprised 25 717 (green) and 25 215 (red) oligos (45 nucleotides), respectively, and generated 26 distinct FISH signals that can be used as a barcode to uniquely label each of the 12 pairs of rice chromosomes. Standard karyotypes of rice were established using this system on both mitotic and meiotic chromosomes. Moreover, dual‐color oligo‐FISH was used to characterize diverse chromosomal abnormalities. Oligo‐FISH analyses using these probes in various wild Oryza species revealed that chromosomes from the AA, BB or CC genomes generated specific and intense signals similar to those in rice, while chromosomes with the EE genome generated less specific signals and the FF genome gave no signal. Together, the oligo‐FISH probes we established will be a powerful tool for studying chromosome variations and evolution in the genus Oryza.     

Rapid chromosomal evolution in the mesic four‐striped grass rat Rhabdomys dilectus (Rodentia,Muridae) revealed by mtDNA phylogeographic analysis

The mesic four‐striped grass rat Rhabdomys dilectus De Winton, 1897 is distributed in mesic regions of southern and eastern Africa. We carried out a molecular and chromosomal study of the northernmost populations of the species to provide insight into the subspecific boundaries identified within the species and to describe its genetic structure in Eastern Africa. Maximum likelihood, maximum parsimony and neighbour‐joining methods were used to construct phylogenetic relationships among all the haplotypes belonging to the large part of the species range. Times of divergences were estimated assuming a relaxed molecular clock with two calibration points. We identified three well‐supported clades within R. dilectus. One basal clade corresponding to Rhabdomys d. chakae (2n = 48) is found in South Africa. Two additional sister clades corresponding to R. d. dilectus (2n = 48 and 2n = 46) are allopatrically distributed in southern and northern parts of the species range. Genetic divergence among the three clades is relatively high (ranges 4.2–5.7%). A very divergent new karyotype 2n = 38, FNa = 60 was found in two high‐altitude populations on Mt. Meru and Mt. Kilimanjaro. The karyotype differences consist in three Robertsonian fusions and one whole‐arm reciprocal translocation. Interestingly, the mtDNA phylogeny does not match with the diploid numbers. In fact, the 2n = 38 specimens form a monophyletic group within a clade that includes specimens with the 2n = 46 karyotype that appears as paraphyletic. We estimated the new karyotype originated in peripatric condition during the last phases of the Pleistocene. This study confirms the importance of chromosomal analysis in detecting taxonomic units and cryptic diversity in rodents.     

Chromosomal mosaicism detected by karyotyping and chromosomal microarray analysis in prenatal diagnosis

To investigate the incidence and clinical significance of chromosomal mosaicism (CM) in prenatal diagnosis by G-banding karyotyping and chromosomal microarray analysis (CMA). This is a single-centre retrospective study of invasive prenatal diagnosis for CM. From 5758 karyotyping results and 6066 CMA results, 104 foetal cases with CM were selected and analysed further. In total, 50% (52/104) of foetal cases with CM were affected by ultrasound-detectable phenotypes. Regardless of whether they were singleton or twin pregnancies, isolated structural defects in one system (51.35%, 19/37 in singletons; 86.67%, 13/15 in twins) and a single soft marker (18.92%, 7/37 in singletons; 13.33%, 2/15 in twins) were the most common ultrasound anomalies. Mosaic autosomal trisomy (19.23%, 20/104) was the most frequent type, and its rate was higher in phenotypic foetuses (28.85%, 15/52) than in non-phenotypic foetuses (9.62%, 5/52). There was no difference in mosaic fractions between phenotypic and non-phenotypic foetuses based on specimen sources or overall classification. Discordant mosaic results were observed in 16 cases (15.38%, 16/104) from different specimens or different testing methods. Genetic counselling and clinical management regarding CM in prenatal diagnosis remain challenging due to the variable phenotypes and unclear significance. Greater caution should be used in prenatal counselling, and more comprehensive assays involving serial ultrasound examinations, different specimens or testing methods verifications and follow-up should be applied.     

A necdin/MAGE-like gene in the chromosome 15 autism susceptibility region: expression, imprinting, and mapping of the human and mouse orthologues

Background  

Proximal chromosome 15q is implicated in neurodevelopmental disorders including Prader-Willi and Angelman syndromes, autistic disorder and developmental abnormalities resulting from chromosomal deletions or duplications. A subset of genes in this region are subject to genomic imprinting, the expression of the gene from only one parental allele.     

彩色多普勒超声检查对胎儿颅内畸形筛查的应用价值及染色体异常分析

摘要 目的：探讨彩色多普勒超声检查对胎儿颅内畸形筛查的应用价值,并进行染色体异常分析。方法：选择2016年2月至2019年5月本院收治的进行胎儿颅内畸形筛查的高危孕妇120例,所有孕妇都给予彩色多普勒二维超声与三维超声筛查,对超声筛查异常者进行染色体异常分析,记录预后情况。结果：在120例孕妇中,二维超声诊断为胎儿颅内畸形12例,三维超声诊断为13例(预后都确诊为胎儿颅内畸形)。染色体核型筛查检出胎儿颅内畸形12例,其中21-三体综合征8例,18-三体综合征3例,13-三体综合征1例。确诊为胎儿颅内畸形的孕妇超声NT值都显著高于非胎儿颅内畸形孕妇,差异都有统计学意义(P<0.05)。孕妇选择终止妊娠10例,选择继续妊娠3例,继续妊娠3例胎儿都最终死亡。结论：产前彩色多普勒超声结合染色体核型在胎儿颅内畸形筛查中具有很高的价值,两者可互相补充,共同发挥诊断与预后评估价值。     

Non-invasive prenatal diagnosis by massively parallel sequencing of maternal plasma DNA

The presence of foetal DNA in the plasma of pregnant women has opened up new possibilities for non-invasive prenatal diagnosis. The use of circulating foetal DNA for the non-invasive prenatal detection of foetal chromosomal aneuploidies is challenging as foetal DNA represents a minor fraction of maternal plasma DNA. In 2007, it was shown that single molecule counting methods would allow the detection of the presence of a trisomic foetus, as long as enough molecules were counted. With the advent of massively parallel sequencing, millions or billions of DNA molecules can be readily counted. Using massively parallel sequencing, foetal trisomies 21, 13 and 18 have been detected from maternal plasma. Recently, large-scale clinical studies have validated the robustness of this approach for the prenatal detection of foetal chromosomal aneuploidies. A proof-of-concept study has also shown that a genome-wide genetic and mutational map of a foetus can be constructed from the maternal plasma DNA sequencing data. These developments suggest that the analysis of foetal DNA in maternal plasma would play an increasingly important role in future obstetrics practice. It is thus a priority that the ethical, social and legal issues regarding this technology be systematically studied.     

Evidence of compliance with and effectiveness of guidelines for noninvasive prenatal testing in China: a retrospective study of 189,809 cases

In China,the medical guidelines recommend performing noninvasive prenatal testing (NIPT) with caution for pregnant women aged 35 years or older.However,the Mother and Child Health Care Law suggests that all primiparous women whose age is older than 35 years undergo prenatal diagnosis.These two inconsistent suggestions/recommendations have made obstetricians confused about whether to offer NIPT to these older pregnant women.To face this issue and find out the solution we performed a retrospective study of 189,809 NIPT samples collected from 28 provincial-leveled administrative units in China.Of 1,564women with high-risk pregnancies who underwent NIPT,459 (29.3%) did not participate in follow-up.The compound sensitivity and specificity of NIPT for trisomies 21,18 and 13 detection was 99.1%(95%CI,98.0%-99.6%) and 99.9%(95%CI,98.8%-99.9%),respectively.In secundiparous women,NIPT showed high sensitivity and specificity similar to that in primiparous women.The observed risk for trisomies 21 and 18 significantly increased when the maternal age was 39 and older.After the publication of the current NIPT policy,the follow-up rate at our center was 97.9%;however,a large number of women are not in maternal and infant care networks nationwide,and that makes the follow-up rate outside our center relatively low.Our study shows that to balance the prevention of major aneuploidies and the limited resources for prenatal diagnosis,the cut-off age of 35for invasive prenatal diagnosis might be unnecessary.Although the NIPT guidelines are well written,how to practice it effectively,especially in less industrialized areas,is worth discussing.     

Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E

RNA sequencing studies have identified hundreds of non‐coding RNAs in bacteria, including regulatory small RNA (sRNA). However, our understanding of sRNA function has lagged behind their identification due to a lack of tools for the high‐throughput analysis of RNA–RNA interactions in bacteria. Here we demonstrate that in vivo sRNA–mRNA duplexes can be recovered using UV‐crosslinking, ligation and sequencing of hybrids (CLASH). Many sRNAs recruit the endoribonuclease, RNase E, to facilitate processing of mRNAs. We were able to recover base‐paired sRNA–mRNA duplexes in association with RNase E, allowing proximity‐dependent ligation and sequencing of cognate sRNA–mRNA pairs as chimeric reads. We verified that this approach captures bona fide sRNA–mRNA interactions. Clustering analyses identified novel sRNA seed regions and sets of potentially co‐regulated target mRNAs. We identified multiple mRNA targets for the pathotype‐specific sRNA Esr41, which was shown to regulate colicin sensitivity and iron transport in E. coli. Numerous sRNA interactions were also identified with non‐coding RNAs, including sRNAs and tRNAs, demonstrating the high complexity of the sRNA interactome.     

Proteomic profile determination of autosomal aneuploidies by mass spectrometry on amniotic fluids

Background  

Prenatal diagnosis of chromosomal abnormalities by cytogenetic analysis is time-consuming, expensive, and requires highly qualified technicians. Rapid diagnosis of aneuploidies followed by reassurance of women with normal results can be performed by molecular analysis of uncultured foetal cells. In the present study, we developed a proteomic fingerprinting approach coupled with a statistical classification method to improve diagnosis of aneuploidies, including trisomies 13, 18, and 21, in amniotic fluid samples.     

Different mutational function of low‐ and high‐linear energy transfer heavy‐ion irradiation demonstrated by whole‐genome resequencing of Arabidopsis mutants

Heavy‐ion irradiation is a powerful mutagen that possesses high linear energy transfer (LET). Several studies have indicated that the value of LET affects DNA lesion formation in several ways, including the efficiency and the density of double‐stranded break induction along the particle path. We assumed that the mutation type can be altered by selecting an appropriate LET value. Here, we quantitatively demonstrate differences in the mutation type induced by irradiation with two representative ions, Ar ions (LET: 290 keV μm^?1) and C ions (LET: 30.0 keV μm^?1), by whole‐genome resequencing of the Arabidopsis mutants produced by these irradiations. Ar ions caused chromosomal rearrangements or large deletions (≥100 bp) more frequently than C ions, with 10.2 and 2.3 per mutant genome under Ar‐ and C‐ion irradiation, respectively. Conversely, C ions induced more single‐base substitutions and small indels (<100 bp) than Ar ions, with 28.1 and 56.9 per mutant genome under Ar‐ and C‐ion irradiation, respectively. Moreover, the rearrangements induced by Ar‐ion irradiation were more complex than those induced by C‐ion irradiation, and tended to accompany single base substitutions or small indels located close by. In conjunction with the detection of causative genes through high‐throughput sequencing, selective irradiation by beams with different effects will be a powerful tool for forward genetics as well as studies on chromosomal rearrangements.     

Prenatal detection of aneuploidy and imbalanced chromosomal arrangements by massively parallel sequencing

Fetal chromosomal abnormalities are the most common reasons for invasive prenatal testing. Currently, G-band karyotyping and several molecular genetic methods have been established for diagnosis of chromosomal abnormalities. Although these testing methods are highly reliable, the major limitation remains restricted resolutions or can only achieve limited coverage on the human genome at one time. The massively parallel sequencing (MPS) technologies which can reach single base pair resolution allows detection of genome-wide intragenic deletions and duplication challenging karyotyping and microarrays as the tool for prenatal diagnosis. Here we reported a novel and robust MPS-based method to detect aneuploidy and imbalanced chromosomal arrangements in amniotic fluid (AF) samples. We sequenced 62 AF samples on Illumina GAIIx platform and with averagely 0.01× whole genome sequencing data we detected 13 samples with numerical chromosomal abnormalities by z-test. With up to 2× whole genome sequencing data we were able to detect microdeletion/microduplication (ranged from 1.4 Mb to 37.3 Mb of 5 samples from chorionic villus sampling (CVS) using SeqSeq algorithm. Our work demonstrated MPS is a robust and accurate approach to detect aneuploidy and imbalanced chromosomal arrangements in prenatal samples.