微阵列比较基因组杂交在临床细胞遗传诊断中的应用研究

目的探讨微阵列比较基因组杂交技术(array-based comparative genomic hybridization,array-CGH)在诊断不平衡染色体畸变中的应用价值。方法选取5例常规G显带染色体核型分析未能确诊的不平衡染色体畸变病例,按照标准的Cyto Scan 750K微阵列的操作手册进行杂交、洗涤及全基因组扫描,并通过相应的计算机软件分析结果。结果通过array-CGH技术分析,明确了所有5例染色体不平衡畸变的诊断并且进行精确定位,对2例G显带未识别的缺失合并重复的衍生染色体进行了精确诊断,并对1例镜下染色体出现无法确定来源的额外染色体条带进行确诊。结论 array-CGH技术在DNA水平上对染色体不平衡畸变的诊断具有敏感性、特异性和高分辨率,并且能够精确定位,对染色体疾病作出基因型—表型关系的诊断具有重要的应用价值。     

Array—CGH技术用于产前诊断可疑胎儿染色体异常

目的探讨微阵列比较基因组杂交技术（array—based comparative genomic hybridization,array—CGH）在产前诊断胎儿染色体异常中的应用价值。方法产前诊断发现4例常规G显带染色体核型分析不能明确的胎儿染色体异常,按照标准的array—CGH操作分析对这些病例进行全基因组检测。结果通过array—CGH技术分析,明确了4例胎儿可疑染色体异常的诊断并且进行精确定位,1例染色体部分缺失,1例正常,1例染色体部分重复,1例不平衡易位。结论array—CGH技术对产前诊断胎儿染色体异常具有高分辨率,能够精确定位异常片段,明确胎儿预后,对产前诊断具有重要应用价值。     

微阵列比较基因组杂交检测和分析一例46,X0,+der(?)胎儿的全基因组拷贝数变化

目的检测和分析一例46,X0,+der(?)胎儿的全基因组拷贝数变化(CNVs),确定胎儿的核型,探讨微阵列比较基因组杂交(array-CGH)在临床细胞遗传诊断中运用的可行性和优越性。方法对胎儿进行常规G显带染色体分析,应用array-CGH芯片进行全基因组高分辨率扫描和分析,RT-qPCR验证array-CGH的结果。结果G显带染色体分析显示胎儿的核型为46,X0,+der(?)。Array-CGH显示衍生染色体为Y染色体,且不存在CNVs;另外,共检测出了118个亚显微CNVs。RT-qPCR证明array-CGH的结果是准确的。结论与传统的细胞遗传分析方法相比,array-CGH具有高分辨率、高通量和高准确性等优点,为亚显微水平染色体畸变的检测提供了一种新型的强大的分析平台。     

微阵列比较基因组杂交技术诊断9p部分三体患儿一例

目的 确定1例生长发育迟缓、语言发育障碍患儿的核型,分析其染色体畸变与表型的相关性,探讨微阵列比较基因组杂交(array-based comparative genomic hybridization,array-CGH)在临床分子遗传学诊断中的应用及其优越性.方法 应用G显带对患儿及其父母进行核型分析,进一步采用array-CGH技术对患儿进行全基因组高分辨率扫描分析,确定其衍生染色体片段的来源.结果 G显带染色体分析显示患儿及其母亲均为inv(9)(p13q13)携带者,患儿13号染色体存在一衍生片段.array-CGH结果证实患儿衍生片段源自9号染色体短臂,确定为9p13.1-p24.3三体.患儿母亲array-CGH结果未见异常.结论 inv(9)(p13q13)与患儿异常表型无关,患儿的异常表型可归因于9p13.1-p24.3三体.同传统细胞遗传分析方法相比,array-CGH具有高分辨率和高精确性的优点.     

光谱核型分析联合微阵列比较基因组杂交诊断15号环状染色体综合征

目的 探讨联合应用光谱核型分析技术(spectral karyotyping,SKY)和微阵列比较基因组杂交技术(microarray-based comparative genomic hybridization,array-CGH)在诊断复杂疑难的环状染色体畸变中的价值.方法 对1例常规G显带染色体核型分析疑诊为46,XY,r(15)?的8岁男性生长发育迟缓患儿依次应用SKY及array-CGH技术常规进行制片杂交,并通过相应的显微摄像系统和计算机软件分析结果.结果 SKY技术明确了该患儿环状染色体来源于15号染色体,array-CGH技术明确患儿15q26.3末端存在约594 kb的缺失,染色体基因位点编码范围为99689349-100282878.结论 联合应用现代分子细胞遗传学技术可以从细胞到分子水平精确诊断复杂疑难的环状染色体病例,是常规染色体核型分析的有益补充,也有利于细胞遗传学向分子水平深入.     

微阵列比较基因组杂交技术诊断9q部分三体患者一例

目的对1例患者衍生的9号染色体进行基因组拷贝数分析,明确其遗传物质的来源并推测其发生机制。探讨微阵列比较基因组杂交技术(array-based comparative genomic hybridization,array-CGH)在临床分子遗传学诊断中的应用及其优越性。方法对患者外周血进行常规G显带分析,进一步应用array-CGH对患者进行全基因组扫描检测,应用Real-time Quantitative PCR对异常拷贝数区域进行验证检测,确定其衍生染色体片段的来源。结果患者外周血染色体核型分析提示为46,XX,der(9)(p?)。array-CGH分析提示患者9q22和9q34之间插入了16p13.3约3.68M的重复片段、14q32.3约3.37M的重复片段以及9q33.3约25Kb的重复片段。RT-q PCR证明array-CGH的结果是正确的。结论患者衍生的9号染色体来源于16p、14q及9q部分片段的重复,是导致患者多次流产的主要原因。array-CGH应用到临床具有高分辨、高通量和高准确性的优点,适用于全基因组拷贝数变异分析。     

产前诊断胎儿泌尿系统畸形与染色体1q21.1微缺失

目的 应用微阵列比较基因组杂交技术探讨胎儿先天性泌尿系统畸形的遗传学病因.方法 选取32例经产前超声检查提示发生不同程度泌尿系统畸形并且常规G显带核型分析方法未发现异常的胎儿病例及其父母的DNA,按照标准的Affymetrix cytogenetic 2.7M芯片的操作手册进行杂交、洗涤及全基因组扫描,应用配套的CHAS软件分析结果.结果 微阵列比较基因组杂交技术检测发现9例胎儿基因组发生了不平衡的拷贝数变异(copy number variations,CNVs),检出率为28％.其中4例CNVs遗传自亲代(12.5％);2例CNVs在相关数据库中提示在正常人基因组中存在(6％);3例是新发的致病性CNVs(9％),并且这3例胎儿样本均发生了染色体1q21.1微缺失和微重复,异常片段内包含与泌尿生殖系统功能密切相关的PDZK1基因.结论 先天性泌尿系统畸形胎儿基因组发生不平衡畸变的几率约为28％,其中致病性的基因组不平衡异常约占9％.染色体1q21.1区带DNA拷贝数改变是导致先天性泌尿系统畸形的病因之一,其致病机制可能与PDZK1基因的异常表达有关.     

5例环状染色体综合征的产前诊断和表型分析

目的通过对5例环状染色体综合征患者进行细胞遗传学分析,探讨高分辨率G显带核型分析和微阵列比较基因组杂交两种方法对环状染色体的诊断优势,并探讨5例环状染色体综合染色体缺失片段和定位于其中的基因与临床表型的关系。方法 2017年就诊于深圳市妇幼保健院的5例环状染色体综合征病例纳入研究。用染色体G带高分辨显带和微阵列比较基因组杂交技术对5例环状染色体进行识别与定位。结果病例1羊水染色体核型结果:45,XN,-18[13]/46,XN,r(18)(p11.2q22.3)[47],羊水微阵列比较基因组杂交结果:arr[GRCH37]18q22.3q23(70,063,358-78,013,728)x1;18p11.32p11.23(136,227-8,002,810)x1;18p11.23p11.22(8,013,797-8,877,061)x3。病例2脐血染色体核型结果:46,XN,rec(21)r(21q)dup(21q)(q11.2-q22.2)?,脐血微阵列比较基因组杂交结果:arr[GRCH37]21q11.2q22.3(15,016,486-47,044,951)x2～4;32MB.21q22.3(47,052,734-48,093,361)x1。病例3脐血染色体核型结果:45,XY,-21[14]/46,XN,r(21)[86],脐血微阵列比较基因组杂交结果:arr[GRCh37]21q11.2q22.3(15016486_47632178)x3[0.47];21q22.3(47632178_48093361)x1。病例4羊水染色体核型结果:46,XX,r(4)(p16q35),羊水微阵列比较基因组杂交结果:arr[GRCH37]4p16.3p16.1(68,345-8,721,580)x1;4q35.2(190,602,426-190,957,460)x1。病例5羊水染色体核型结果:mos45,X[46]/46,x,r(x)(p22.3q21.1)[34],羊水微阵列比较基因组杂交结果:Xq21.1q28(82119329_155233098)x1;Xp22.33p22.32(168551_5677733)x1;Xp22.32q21.1(5677733-82119329)x1[0.4]。结论 (1)环状染色体综合征患儿的临床特征与染色体区带缺失重复部位和大小相关。(2)G显带核型分析和微阵列比较基因组杂交诊断环状染色体各有优势:第一传统的G显带核型分析首先可判断是否存在嵌合的染色体核型;第二即使微阵列比较基因组结果提示染色体仅有长臂或短臂的缺失,也不能排除环状染色体的可能,亦需要传统的G显带染色体核型共同分析;第三微阵列比较基因组杂交能够精确基因组微小缺失和重复,利于基因组拷贝数变异与临床表型分析。因此联合G显带核型分析和微阵列比较基因组杂交对于环状染色体的诊断和遗传咨询具有指导意义。     

颅面畸形家系的胎儿产前诊断与分析

目的 探讨先天性颅面畸形胎儿的产前诊断技术.方法 选择产前检查B超显示胎儿具有颅面畸形的孕妇1例,采集孕妇羊水、外周血和其丈夫外周血样本,进行常规G显带核型分析.再采用比较基因组杂交技术(aaray comparative genomic hybridization,array-CGH)进行全基因组高分辨扫描和分析,逆转录荧光定量PCR方法对array-CGH结果进行验证.并在患儿出生后再进行重复检测确认.结果 G显带核型分析未见异常,array-CGH显示胎儿1p36.33区域有重复,长度约为722 kb,该片段中VWA1和PYGO2基因与软骨发育有关,定量PCR实验证实了比较基因组杂交的结果,拟诊为颅面畸形,胎儿出生后得到进一步确认.结论 应用比较基因组杂交技术,成功对1例先天性颅面畸形胎儿进行了产前诊断,并确定了两个与颅面骨发育有关的候选基因VWA1和PYGO2.     

荧光原位杂交联合微阵列比较基因组杂交分析一例原发性闭经患者的染色体畸变

目的分析1例原发性闭经患者的染色体畸变,探讨该患者原发性闭经的可能原因。方法采集临床已确诊的原发性闭经患者外周血,并抽提基因组DNA,进行荧光原位杂交和微阵列比较基因组杂交,分析染色体异常。结果微阵列比较基因组杂交显示患者染色体Xp22.31区域存在长1.637Mb片段的三倍体,Xp21.2-q21.1区域存在长52.156 Mb片段的重复片段。结论微阵列比较基因组杂交技术可以检测染色体微小畸变,值得临床推广应用。     

Genomic profiling of myeloid sarcoma by array comparative genomic hybridization

Myeloid sarcoma (MS) is a tumor mass of myeloblasts or immature myeloid cells occurring in an extramedullary site. In this study, seven cases of MS [stomach (1), testis (1), skin (2), and lymph node (3)] and 3 synchronous and 1 follow-up bone marrow (BM) samples were studied for genomic abnormalities using array comparative genomic hybridization (array-CGH). Array-CGH construction used approximately 5,400 bacterial artificial chromosome clones from the RPCI-11 library, spanning the human genome. Data were analyzed using the DNAcopy software and custom heuristics. All MS cases had genomic abnormalities detected by array-CGH. Unbalanced genomic abnormalities in five MS cases were confirmed by conventional cytogenetics (CC) and/or fluorescence in situ hybridization (FISH); these abnormalities included loss of 4q32.1-q35.2, 6q16.1-q21, and 12p12.2-p13.2 and gain of 8q21.2-q24.3, 8, 11q21-q25, 13q21.32-q34, 19, and 21. Array-CGH was also invaluable in identifying possible deletions, partner translocations, and breakpoints that were questionable by CC. The remaining two MS cases had genomic aberrations detected by array-CGH, but were not studied further by CC/FISH. Chromosome 8 was most commonly abnormal (3/7 cases). Identical genomic abnormalities were demonstrated in MS and in synchronous BM in two cases. These results demonstrate that array-CGH is a powerful tool to screen MS tissue for unbalanced genomic abnormalities, allowing identification of chromosome abnormalities when concurrent BM is nonanalyzable or nonleukemic.     

Microarray-based comparative genomic hybridization in the study of constitutional chromosomal abnormalities

Chromosomal aberrations are the first cause of mental impairment and dysmorphism. Rearrangements involving large chromosomal segments can be detected by standard chromosome analysis using GTG-banding, but this technique is not suited for the detection of small chromosome abnormalities. Array comparative genomic hybridisation (array-CGH) is a method used to detect segmental DNA copy number alterations. Recently, advances in this technology have enabled high-resolution examination for identifying genetic alterations and copy number variations on a genome-wide scale. This review describes the current genomic array platforms and CGH methodologies and highlights their applications for studying constitutional disease.     

SNP array-based copy number and genotype analyses for preimplantation genetic diagnosis of human unbalanced translocations

Preimplantation genetic diagnosis (PGD) for chromosomal rearrangements (CR) is mainly based on fluorescence in situ hybridisation (FISH). Application of this technique is limited by the number of available fluorochromes, the extensive preclinical work-up and technical and interpretative artefacts. We aimed to develop a universal, off-the-shelf protocol for PGD by combining single-nucleotide polymorphism (SNP) array-derived copy number (CN) determination and genotyping for detection of unbalanced translocations in cleavage-stage embryos. A total of 36 cleavage-stage embryos that were diagnosed as unbalanced by initial PGD FISH analysis were dissociated (n=146) and amplified by multiple displacement amplification (MDA). SNP CNs and genotypes were determined using SNP array. Epstein-Barr Virus-transformed cell lines with known CR were used for optimising the genomic smoothing (GS) length setting to increase signal to noise ratio. SNP CN analysis showed 23 embryos (64%) that were unbalanced in all blastomeres for the chromosomes involved in the translocation, 5 embryos (14%) that were normal or balanced in all blastomeres and 8 embryos (22%) that were mosaic. SNP genotyping, based on analysis of informative SNP loci with opposing homozygous parental genotypes, confirmed partial monosomies associated with inheritance of unbalanced translocation in surplus embryos. We have developed a universal MDA-SNP array technique for chromosome CN analysis in single blastomeres. SNP genotyping could confirm partial monosomies. This combination of techniques showed improved diagnostic specificity compared with FISH and may provide more reliable PGD analysis associated with higher embryo transfer rate.     

Array-CGH in a series of 30 patients with mental retardation, dysmorphic features, and congenital malformations detected an interstitial 1p22.2-p31.1 deletion in a patient with features overlapping the Goldenhar syndrome

Genosensor Array 300 (Abbott) is a multiplex platform for array-based comparative genomic hybridization that detects unbalanced genomic aberrations including whole chromosome gains/losses, microdeletions, duplications and unbalanced subtelomeric rearrangements. A series of 30 patients with unexplained mental retardation, dysmorphic features, congenital abnormalities and normal high resolution karyotype and FISH subtelomeric studies were analyzed using Genosensor Array 300 array-CGH. We identified a chromosomal aberration in one patient with an interstitial 1p31.1 deletion. FISH analysis with BACs specific probes of the 1p region confirmed the interstitial 1p22.2-p31.1 deletion. The patient was a 20-year-old man with short stature, facial dysmorphism including asymmetry, scoliosis, severe psychomotor delay and an epibulbar dermoid cyst. The phenotype was compatible with Goldenhar syndrome despite the absence of asymmetric ears. This observation is of interest since it could be a clue in the search for the genes responsible for Goldenhar syndrome. This study demonstrates the utility of the array-CGH technology in detecting interstitial deletions.     

High-resolution copy number analysis of paraffin-embedded archival tissue using SNP BeadArrays

High-density SNP microarrays provide insight into the genomic events that occur in diseases like cancer through their capability to measure both LOH and genomic copy numbers. Where currently available methods are restricted to the use of fresh frozen tissue, we now describe the design and validation of copy number measurements using the Illumina BeadArray platform and the application of this technique to formalin-fixed, paraffin-embedded (FFPE) tissue. In fresh frozen tissue from a set of colorectal tumors with numerous chromosomal aberrations, our method measures copy number patterns that are comparable to values from established platforms, like Affymetrix GeneChip and BAC array-CGH. Moreover, paired comparisons of fresh frozen and FFPE tissues showed nearly identical patterns of genomic change. We conclude that this method enables the use of paraffin-embedded material for research into both LOH and numerical chromosomal abnormalities. These findings make the large pathological archives available for genomic analysis, which could be especially relevant for hereditary disease where fresh material from affected relatives is rarely available.     

Detection of chromosomal imbalances in children with idiopathic mental retardation by array based comparative genomic hybridisation (array-CGH)

Chromosomal aberrations are a common cause of multiple anomaly syndromes that include growth and developmental delay and dysmorphism. Novel high resolution, whole genome technologies, such as array based comparative genomic hybridisation (array-CGH), improve the detection rate of submicroscopic chromosomal abnormalities allowing re-investigation of cases where conventional cytogenetic techniques, Spectral karyotyping (SKY), and FISH failed to detect abnormalities. We performed a high resolution genome-wide screening for submicroscopic chromosomal rearrangements using array-CGH on 41 children with idiopathic mental retardation (MR) and dysmorphic features. The commercially available microarray from Spectral Genomics contained 2600 BAC clones spaced at approximately 1 Mb intervals across the genome. Standard chromosome analysis (>450 bands per haploid genome) revealed no chromosomal rearrangements. In addition, multi-subtelomeric FISH screening in 30 cases and SKY in 11 patients did not detect any abnormality. Using array-CGH we detected chromosomal imbalances in four patients (9.8%) ranging in size from 2 to 14 Mb. Large scale copy number variations were frequently observed. Array-CGH has become an important tool for the detection of chromosome aberrations and has the potential to identify genes involved in developmental delay and dysmorphism. Moreover, the detection of genomic imbalances of clinical significance will increase knowledge of the human genome by performing genotype-phenotype correlation.     

Whole genome SNP arrays as a potential diagnostic tool for the detection of characteristic chromosomal aberrations in renal epithelial tumors.

Renal tumors with complex or unusual morphology require extensive workup for accurate classification. Chromosomal aberrations that define subtypes of renal epithelial neoplasms have been reported. We explored if whole-genome chromosome copy number and loss-of-heterozygosity analysis with single nucleotide polymorphism (SNP) arrays can be used to identify these aberrations and classify renal epithelial tumors. We analyzed 20 paraffin-embedded tissues representing clear cell, papillary renal and chromophobe renal cell carcinoma, as well as oncocytoma with Affymetrix GeneChip 10K 2.0 Mapping arrays. SNP array results were in concordance with known genetic aberrations for each renal tumor subtype. Additional chromosomal aberrations were detected in all renal cell tumor types. The unique patterns allowed 19 out of 20 tumors to be readily categorized by their chromosomal copy number aberrations. One papillary renal cell carcinoma type 2 did not show the characteristic 7/17 trisomies. Clustering using the median copy number of each chromosomal arm correlated with histological class when using a restricted set of chromosomes. In addition, three morphologically challenging tumors were analyzed to explore the potential clinical utility of this method. In these cases, the SNP array-based copy number evaluation yielded information with potential clinical value. These results show that SNP arrays can detect characteristic chromosomal aberrations in paraffin-embedded renal tumors, and thus offer a high-resolution, genome-wide method that can be used as an ancillary study for classification and potentially for prognostic stratification of these tumors.     

Gain of 9p due to an unbalanced rearrangement der(9;18): a recurrent clonal abnormality in chronic myeloproliferative disorders

Clonal aberrations leading to gain of 9p—mostly due to trisomy 9—are often reported in polycythemia vera. We report on four cases of chronic myeloproliferative disorders that demonstrated a new recurrent unbalanced rearrangement between chromosomes 9 and 18 leading to trisomy of 9p and a monosomy of 18p. This abnormality was confirmed with fluorescence in situ hybridization using chromosome painting and locus-specific probes. Three cases were diagnosed as polycythemia vera; one case presented with secondary acute myeloid leukemia following idiopathic osteomyelofibrosis. The prognostic impact of this unbalanced aberration and of gains of 9p in chronic myeloproliferative disorders remains to be clarified.