0.5 Mb Array as a First‐Line Prenatal Cytogenetic Test in Cases without Ultrasound Abnormalities and Its Implementation in Clinical Practice

Using whole‐genome array testing instead of karyotyping in prenatal diagnosis for all indications may be desirable because of the higher diagnostic yield and shorter reporting time. The goal of this research was finding the optimal array resolution that could replace routine prenatal karyotyping in cases without ultrasound abnormalities, for example, referred for advanced maternal age or abnormal first trimester screening. As variants of unknown clinical significance (VOUS), if reported, might complicate decision‐making about continuation of pregnancy, such an optimal array resolution should have a high abnormality detection rate and reveal a minimal amount of VOUS. The array data of 465 fetuses were retrospectively evaluated with several resolution levels, and the Decipher microdeletion/microduplication syndrome list was reviewed to assess what could be theoretically missed with a lower resolution. A 0.5‐Mb resolution showed a high diagnostic yield potential and significantly minimized the number of VOUS. Based on our experience, we recommend genomic SNP array as a first‐tier test in prenatal diagnosis. The resolution should be chosen based on the indication. In cases of fetal ultrasound abnormalities or intrauterine fetal death (IUFD), high‐resolution analysis should be done. In other cases, we advise replacing karyotyping by SNP array analysis with 0.5 Mb resolution.     

Combined G-banded karyotyping and multiplex ligation-dependent probe amplification for the detection of chromosomal abnormalities in fetuses with congenital heart defects北大核心CSCD

Objective To assess the value of G-banded karyotyping in combination with multiplex ligation-dependent probe amplification (MLPA) as a tool for the detection of chromosomal abnormalities in fetuses with congenital heart defects. Methods The combined method was used to analyze 104 fetuses with heart malformations identified by ultrasonography. Abnormal findings were confirmed with chromosomal microarray analysis (CMA). Results Nineteen (18%) fetuses were found to harbor chromosomal aberrations by G-banded karyotyping and MLPA. For 93 cases, CMA has detected abnormalities in 14 cases including 10 pathogenic copy number variations (CNVs) and 4 CNVs of uncertain significance (VOUS). MLPA was able to detect all of the pathogenic CNVs and 1 VOUS CNV. Conclusion Combined use of G-banded karyotyping and MLPA is a rapid, low-cost and effective method to detect chromosomal abnormalities in fetuses with various heart malformations.     

Low-pass genome sequencing versus chromosomal microarray analysis: implementation in prenatal diagnosis

PurposeEmerging studies suggest that low-pass genome sequencing (GS) provides additional diagnostic yield of clinically significant copy-number variants (CNVs) compared with chromosomal microarray analysis (CMA). However, a prospective back-to-back comparison evaluating accuracy, efficacy, and incremental yield of low-pass GS compared with CMA is warranted.MethodsA total of 1023 women undergoing prenatal diagnosis were enrolled. Each sample was subjected to low-pass GS and CMA for CNV analysis in parallel. CNVs were classified according to guidelines of the American College of Medical Genetics and Genomics.ResultsLow-pass GS not only identified all 124 numerical disorders or pathogenic or likely pathogenic (P/LP) CNVs detected by CMA in 121 cases (11.8%, 121/1023), but also defined 17 additional and clinically relevant P/LP CNVs in 17 cases (1.7%, 17/1023). In addition, low-pass GS significantly reduced the technical repeat rate from 4.6% (47/1023) for CMA to 0.5% (5/1023) and required less DNA (50 ng) as input.ConclusionIn the context of prenatal diagnosis, low-pass GS identified additional and clinically significant information with enhanced resolution and increased sensitivity of detecting mosaicism as compared with the CMA platform used. This study provides strong evidence for applying low-pass GS as an alternative prenatal diagnostic test.     

染色体微阵列分析对于胎儿十二指肠梗阻的检测价值

目的:探讨染色体微阵列分析(chromosome microarray analysis,CMA)对于胎儿十二指肠梗阻(duodenal obstruction,DO)的检测价值。方法:选取51例超声提示存在DO的胎儿,将其分为单纯组和合并其他异常组。对其进行CMA检测,并随访所有病例的妊娠结局。结果:在51例胎儿中共...     

Prenatal diagnosis study using array comparative genomic hybridization for genotype-phenotype correlation in 772 fetuses

ObjectiveThe aim was to evaluate the main indications for prenatal diagnosis, the prevalence of abnormal copy number variations (CNVs), correlate them with clinical findings, analyze the prevalence of VUS, report the rare variants found and additionally highlight the clinical importance of microarray-based comparative genomic hybridization (aCGH) in prenatal diagnosis.Study designWe retrospectively analyzed a cohort of 772 fetuses with indication for genetic study in two tertiary hospitals, in a 9-years-period, using aCGH.ResultsOur results demonstrated 8.3 % (6.4–10.5 %, 95 % CI) detection rate of pathogenic CNVs. Within this group, the main indication was structural malformations (57 %) mainly involving central nervous system, skeletal and cardiac systems. Pathogenic results in cases with multiple malformations were higher than in cases with isolated anatomical system malformations showing statistical significant differences (p < 0.001). The second indication where we found more pathogenic CNVs was increased nuchal translucency (5–6.4 mm). In fact, the rate of pathogenic CNVs did not show significant differences between structural and non-structural malformations (p > 0.001), highlighting the relevance of genetic study by aCGH also in cases with no structural malformations.A total of 217 fetuses with CNVs classified as VUS were identified, mainly involving chromosomes X, 1 and 16.ConclusionOur findings demonstrate 4.9 % (4.2–5.6 %, 95 % CI) increased in the diagnostic yield using aCGH compared to the use of conventional karyotype alone, confirming that the aCGH can improve the accuracy of prenatal diagnosis. Our survey provides a full genotype-phenotype analysis that can be clinically useful for the classification of variants in the context of prenatal setting, helping to provide a better reproductive genetic counselling.     

无创产前检测对于胎儿染色体拷贝数变异的检测价值

目的探讨无创产前检测(non-invasive prenatal testing,NIPT)对于胎儿染色体拷贝数变异(copy number variations,CNVs)的检测价值。方法收集18661例接受NIPT检测的孕妇的临床资料,为提示胎儿携带CNVs的孕妇提供羊水染色体核型或/及染色体微阵列分析,并分析结果的一致性。结果在全部样本中,NIPT共提示21三体58例、18三体18例、13三体19例、18/21三体1例。88例孕妇接受了介入性产前诊断,59例的染色体微阵列分析结果与NIPT一致,符合率为67.05%。NIPT还检出CNVs 37例,其中19例(微缺失15例、微重复4例)接受了介入性产前诊断,14例的验证结果与NIPT一致,符合率为73.68%。结论NIPT检测具有较高的灵敏度和准确性,对检测提示的CNVs应考虑进行介入性产前诊断,追溯其亲代起源,为临床提供指导。     

High‐resolution molecular karyotyping in patients with developmental delay and/or multiple congenital anomalies in a clinical setting

Wincent J, Anderlid B‐M, Lagerberg M, Nordenskjöld M, Schoumans J. High‐resolution molecular karyotyping in patients with developmental delay and/or multiple congenital anomalies in a clinical setting. Microarray‐based comparative genomic hybridization (array‐CGH) enables genomewide investigation of copy‐number changes at high resolution and has recently been implemented as a clinical diagnostic tool. In this study we evaluate the usefulness of high‐resolution arrays as a diagnostic tool in our laboratory and investigate the diagnostic yield in the first 160 patients who were clinically referred for investigation of developmental delay (DD)/multiple congenital anomalies (MCA). During this period both 38K BAC‐arrays and 244K oligonucleotide‐arrays were used. Copy‐number variations (CNVs) not previously reported as normal variants were detected in 22.5% of cases. In 13.1% the aberrations were considered causal to the phenotype and in 9.4% the clinical significance was uncertain. There was no difference in diagnostic yield between patients with mild, moderate or severe DD. Although the effective resolution of the 244K oligonucleotide‐arrays was higher than the 38K BAC‐array, the diagnostic yield of both platforms was approximately equal and no causal aberrations <300 kb were detected in this patient cohort. We experienced that increasing the resolution of a whole genome screen in the diagnostic setting has its drawback of detecting an increased number of CNVs with uncertain contribution to a phenotype. Based on our experiences, array‐CGH is recommended as the first‐step analysis in the genetic evaluation of patients with DD and/or MCA.     

Application of chromosomal microarray in the evaluation of abnormal prenatal findings

We performed karyotype and array comparative genomic hybridization (aCGH) analyses on 177 prenatal samples, including 162 (92%) samples from fetuses with sonographic anomalies. Overall 12 fetuses (6.8%) had abnormal karyotype and 42 (23.7%) fetuses had abnormal microarray results: 20 (11.3%) with pathogenic copy number variations (CNVs), 16 with CNVs of uncertain clinical significance, 4 with CNVs establishing carrier status for recessive, X‐linked, or susceptibility to late onset dominant disease, and two CNVs with pseudomosaicism due to in vitro cultural artifacts. For 23 pregnancies (13%), aCGH contributed important new information. Our results highlight the interpretation challenges associated with CNVs of unclear significance, incidental findings, as well as technical aspects. Array CGH analysis significantly improved the detection of genomic imbalances in prenatal diagnosis of pregnancies with structural birth defects.     

单核苷酸多态性芯片与染色体核型分析在唐氏筛查高风险孕妇产前诊断中的比较研究

目的:探讨单核苷酸多态性芯片(SNP array)在唐氏筛查高风险孕妇胎儿染色体分析中的应用价值。方法:选取312例因唐氏筛查高风险的孕妇,行羊膜腔穿刺术后获得羊水,对羊水进行G显带核型分析和SNP array检测,比较核型分析与SNP array检测结果,并按年龄分组比较拷贝数变异(CNVs)的发生率差别。结果:核型分析和SNP array均准确发现2例21三体(0.64%),6例核型分析提示染色体平衡重组(1.92%)的样本经SNP array分析证实不存在重排片段重复或缺失。在303例核型正常的胎儿羊水细胞中,SNP array检测发现176例CNVs,其中良性CNVs 106例,临床意义不明确的CNVs(VOUS)61例,新发CNVs(de novo CNVs)9例,未发现已知的致病性CNVs。唐氏筛查高风险组与唐氏筛查高风险合并高龄组CNVs的分布差别无统计学意义(P0.05)。此外,本研究中首次报道14种CNVs。结论:SNP array可进一步确定核型分析的平衡易位是否存在染色体微缺失/重复。在核型正常的胎儿中,SNP array可检测出大量拷贝数异常,发现14种新的CNVs但现有数据库无法判断其临床意义,需进一步研究确认。此外,孕妇年龄对胎儿基因组中新发CNVs的发生率无明显影响。     

两例2p15-p16.1微缺失综合征的产前诊断

目的明确2例畸形流产患儿染色体拷贝数变异,分析引起2p15-p16.1缺失综合征畸形表型的相关基因以及关键区域。方法应用染色体微阵列分析(chromosomal microarray analysis,CMA)技术对畸形流产儿全基因组拷贝数目进行检测,并用计算机软件及生物信息学方法进行分析。结果CMA检测到2例患儿的染色体2p15-16.1区段有约255 kb的DNA拷贝数变异,2例患儿表型符合2p15-p16.1微缺失综合征遗传特征,缺失区域包含XPO1和USP34基因。结论2pl5近端73 kb的片段(chr2:61659957〜61733075,hg19)可能是引起2p15-p16.1微缺失综合征畸形表型的关键区域之一,XPO1和USP34为该缺失综合征的候选基因。     

Detection of Clinically Relevant Copy Number Variants with Whole‐Exome Sequencing

Copy number variation (CNV) is a common source of genetic variation that has been implicated in many genomic disorders. This has resulted in the widespread application of genomic microarrays as a first‐tier diagnostic tool for CNV detection. More recently, whole‐exome sequencing (WES) has been proven successful for the detection of clinically relevant point mutations and small insertion–deletions exome wide. We evaluate the utility of short‐read WES (SOLiD 5500xl) to detect clinically relevant CNVs in DNA from 10 patients with intellectual disability and compare these results to data from two independent high‐resolution microarrays. Eleven of the 12 clinically relevant CNVs were detected via read‐depth analysis of WES data; a heterozygous single‐exon deletion remained undetected by all algorithms evaluated. Although the detection power of WES for small CNVs currently does not match that of high‐resolution microarray platforms, we show that the majority (88%) of rare coding CNVs containing three or more exons are successfully identified by WES. These results show that the CNV detection resolution of WES is comparable to that of medium‐resolution genomic microarrays commonly used as clinical assays. The combined detection of point mutations, indels, and CNVs makes WES a very attractive first‐tier diagnostic test for genetically heterogeneous disorders.     

Efficient detection of chromosome imbalances and single nucleotide variants using targeted sequencing in the clinical setting

We evaluated an approach to detect copy number variants (CNVs) and single nucleotide changes (SNVs), using a clinically focused exome panel complemented with a backbone and SNP probes that allows for genome-wide copy number changes and copy-neutral absence of heterozygosity (AOH) calls; this approach potentially substitutes the use of chromosomal microarray testing and sequencing into a single test. A panel of 16 DNA samples with known alterations ranging from megabase-scale CNVs to single base modifications were used as positive controls for sequencing data analysis. The DNA panel included CNVs (n = 13) of variable sizes (23 Kb to 27 Mb), uniparental disomy (UPD; n = 1), and single point mutations (n = 2). All DNA sequence changes were identified by the current platform, showing that CNVs of at least 23 Kb can be properly detected. The estimated size of genomic imbalances detected by microarrays and next generation sequencing are virtually the same, indicating that the resolution and sensitivity of this approach are at least similar to those provided by DNA microarrays. Accordingly, our data show that the combination of a sequencing platform comprising focused exome and whole genome backbone, with appropriate algorithms, enables a cost-effective and efficient solution for the simultaneous detection of CNVs and SNVs.     

Genomic disorders 20 years on—mechanisms for clinical manifestations

Genomic disorders result from copy‐number variants (CNVs) or submicroscopic rearrangements of the genome rather than from single nucleotide variants (SNVs). Diverse technologies, including array comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) microarrays, and more recently, whole genome sequencing and whole‐exome sequencing, have enabled robust genome‐wide unbiased detection of CNVs in affected individuals and in reportedly healthy controls. Sequencing of breakpoint junctions has allowed for elucidation of upstream mechanisms leading to genomic instability and resultant structural variation, whereas studies of the association between CNVs and specific diseases or susceptibility to morbid traits have enhanced our understanding of the downstream effects. In this review, we discuss the hallmarks of genomic disorders as they were defined during the first decade of the field, including genomic instability and the mechanism for rearrangement defined as nonallelic homologous recombination (NAHR); recurrent vs nonrecurrent rearrangements; and gene dosage sensitivity. Moreover, we highlight the exciting advances of the second decade of this field, including a deeper understanding of genomic instability and the mechanisms underlying complex rearrangements, mechanisms for constitutional and somatic chromosomal rearrangements, structural intra‐species polymorphisms and susceptibility to NAHR, the role of CNVs in the context of genome‐wide copy number and single nucleotide variation, and the contribution of noncoding CNVs to human disease.     

Combined array CGH plus SNP genome analyses in a single assay for optimized clinical testing

In clinical diagnostics, both array comparative genomic hybridization (array CGH) and single nucleotide polymorphism (SNP) genotyping have proven to be powerful genomic technologies utilized for the evaluation of developmental delay, multiple congenital anomalies, and neuropsychiatric disorders. Differences in the ability to resolve genomic changes between these arrays may constitute an implementation challenge for clinicians: which platform (SNP vs array CGH) might best detect the underlying genetic cause for the disease in the patient? While only SNP arrays enable the detection of copy number neutral regions of absence of heterozygosity (AOH), they have limited ability to detect single-exon copy number variants (CNVs) due to the distribution of SNPs across the genome. To provide comprehensive clinical testing for both CNVs and copy-neutral AOH, we enhanced our custom-designed high-resolution oligonucleotide array that has exon-targeted coverage of 1860 genes with 60 000 SNP probes, referred to as Chromosomal Microarray Analysis – Comprehensive (CMA-COMP). Of the 3240 cases evaluated by this array, clinically significant CNVs were detected in 445 cases including 21 cases with exonic events. In addition, 162 cases (5.0%) showed at least one AOH region >10 Mb. We demonstrate that even though this array has a lower density of SNP probes than other commercially available SNP arrays, it reliably detected AOH events >10 Mb as well as exonic CNVs beyond the detection limitations of SNP genotyping. Thus, combining SNP probes and exon-targeted array CGH into one platform provides clinically useful genetic screening in an efficient manner.     

Chromosomal microarray in a highly consanguineous population: diagnostic yield,utility of regions of homozygosity,and novel mutations

Chromosomal microarray (CMA) has significantly improved diagnosing copy number variations (CNVs). Single nucleotide polymorphism (SNP) arrays confer additional utility in detecting regions of homozygosity (ROH). Investigating ROH for genes associated with recessive disorders for follow‐up sequencing can aid in diagnosis. In this study, we performed a retrospective review of clinical and molecular data for 227 individuals from a highly consanguineous population who previously had a CMA. Pathogenic CNVs were identified in 32 (14%) cases; ROH suggesting uniparental disomy (UPD) in three (1%) cases, and an additional 25 (11%) individuals were diagnosed with recessive disorders caused by mutations in ROH candidate genes, thereby increasing the CMA diagnostic yield to 26%. Among the 25 individuals with recessive diseases, 18 had novel mutations in 16 genes (ASPM, SPINK5, QARS, MEGF10, SPATA7, GMPPA, ABCA4, SRD5A2, RPGRIP1L, MET, SLC12A6, ALDH1A3, TNFRSF11A, FLNB, PHGDH, and FKBP10) including five with phenotypic expansion.     

Copy-number variants in clinical genome sequencing: deployment and interpretation for rare and undiagnosed disease

PurposeCurrent diagnostic testing for genetic disorders involves serial use of specialized assays spanning multiple technologies. In principle, genome sequencing (GS) can detect all genomic pathogenic variant types on a single platform. Here we evaluate copy-number variant (CNV) calling as part of a clinically accredited GS test.MethodsWe performed analytical validation of CNV calling on 17 reference samples, compared the sensitivity of GS-based variants with those from a clinical microarray, and set a bound on precision using orthogonal technologies. We developed a protocol for family-based analysis of GS-based CNV calls, and deployed this across a clinical cohort of 79 rare and undiagnosed cases.ResultsWe found that CNV calls from GS are at least as sensitive as those from microarrays, while only creating a modest increase in the number of variants interpreted (~10 CNVs per case). We identified clinically significant CNVs in 15% of the first 79 cases analyzed, all of which were confirmed by an orthogonal approach. The pipeline also enabled discovery of a uniparental disomy (UPD) and a 50% mosaic trisomy 14. Directed analysis of select CNVs enabled breakpoint level resolution of genomic rearrangements and phasing of de novo CNVs.ConclusionRobust identification of CNVs by GS is possible within a clinical testing environment.     

染色体微阵列产前诊断8q13.3微缺失一例

目的应用染色体微阵列分析(chromosome microarray analysis,CMA)技术对1例超声结构异常胎儿进行全基因组拷贝数变异(copy number variations,CNVs)检测,探讨CMA在超声结构异常胎儿产前诊断中的意义。方法应用常规G显带染色体核型分析胎儿及其父母的染色体核型,应用CMA技术分析胎儿及其父母的CNVs。结果G显带核型分析显示胎儿核型与母亲一致,为46,XN,t(8;11)(q21.2;q13)mat,父亲核型正常;父母CMA检测结果均未见异常;胎儿的检测结果为arr[GRCh37]8q13.3(71314082-73322915)×1,提示一条8号染色体的8q13.3区域发生2.00 Mb缺失。结论超声结构异常胎儿染色体核型分析检出的平衡易位,需借助CMA等技术进一步确定是否存在微缺失微重复。     

Global analysis of uniparental disomy using high density genotyping arrays

Background: Uniparental disomy (UPD), the inheritance of both copies of a chromosome from a single parent, has been identified as the cause for congenital disorders such as Silver-Russell, Prader-Willi, and Angelman syndromes. Detection of UPD has largely been performed through labour intensive screening of DNA from patients and their parents, using microsatellite markers. Methods: We applied high density single nucleotide polymorphism (SNP) microarrays to diagnose whole chromosome and segmental UPD and to study the occurrence of continuous or interspersed heterodisomic and isodisomic regions in six patients with Silver-Russell syndrome patients who had maternal UPD for chromosome 7 (matUPD7). Results: We have devised a new high precision and high-throughput computational method to confirm UPD and to localise segments where transitions of UPD status occur. Our method reliably confirmed and mapped the matUPD7 regions in all patients in our study. Conclusion: Our results suggest that high density SNP arrays can be reliably used for rapid and efficient diagnosis of both segmental and whole chromosome UPD across the entire genome.     

High-resolution SNP arrays in mental retardation diagnostics: how much do we gain?

We used Affymetrix 6.0 GeneChip SNP arrays to characterize copy number variations (CNVs) in a cohort of 70 patients previously characterized on lower-density oligonucleotide arrays affected by idiopathic mental retardation and dysmorphic features. The SNP array platform includes ∼900 000 SNP probes and 900 000 non-SNP oligonucleotide probes at an average distance of 0.7 Kb, which facilitates coverage of the whole genome, including coding and noncoding regions. The high density of probes is critical for detecting small CNVs, but it can lead to data interpretation problems. To reduce the number of false positives, parameters were set to consider only imbalances >75 Kb encompassing at least 80 probe sets. The higher resolution of the SNP array platform confirmed the increased ability to detect small CNVs, although more than 80% of these CNVs overlapped to copy number ‘neutral'' polymorphism regions and 4.4% of them did not contain known genes. In our cohort of 70 patients, of the 51 previously evaluated as ‘normal'' on the Agilent 44K array, the SNP array platform disclosed six additional CNV changes, including three in three patients, which may be pathogenic. This suggests that about 6% of individuals classified as ‘normal'' using the lower-density oligonucleotide array could be found to be affected by a genomic disorder when evaluated with the higher-density microarray platforms.     

Recurrent copy number variations as risk factors for autism spectrum disorders: analysis of the clinical implications

Chromosomal microarray analysis (CMA) is currently considered a first‐tier diagnostic assay for the investigation of autism spectrum disorders (ASD), developmental delay and intellectual disability of unknown etiology. High‐resolution arrays were utilized for the identification of copy number variations (CNVs) in 195 ASD patients of Greek origin (126 males, 69 females). CMA resulted in the detection of 65 CNVs, excluding the known polymorphic copy number polymorphisms also found in the Database of Genomic Variants, for 51/195 patients (26.1%). Parental DNA testing in 20/51 patients revealed that 17 CNVs were de novo, 6 paternal and 3 of maternal origin. The majority of the 65 CNVs were deletions (66.1%), of which 5 on the X‐chromosome while the duplications, of which 7 on the X‐chromosome, were rarer (22/65, 33.8%). Fifty‐one CNVs from a total of 65, reported for our cohort of ASD patients, were of diagnostic significance and well described in the literature while 14 CNVs (8 losses, 6 gains) were characterized as variants of unknown significance and need further investigation. Among the 51 patients, 39 carried one CNV, 10 carried two CNVs and 2 carried three CNVs. The use of CMA, its clinical validity and utility was assessed.