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.     

微阵列比较基因组杂交技术检测先天性心脏畸形胎儿的隐藏基因组拷贝数变异

目的 检测1例先天性主动脉弓离断和室间隔缺损胎儿的基因组拷贝数变异(copy number variations,CNVs),寻找其致病的遗传学证据,探讨微阵列比较基因组杂交技术(array-based comparative genomic hybridization,array-CGH)在分子细胞遗传诊断中应用的可行性.方法 对该患儿脐血及其父母外周血进行常规G显带核型分析,发现胎儿核型为46,XX,t(7;9)(q12;q21),双亲核型正常.进而应用array-CGH芯片对患儿进行全基因组高分辨率扫描分析,采用荧光原位杂交技术(fluorescence in situ hybridization,FISH)对新发现的CNVs进行实验验证.结果 array-CGH分析发现胎儿基因组存在1个病理性亚显微结构的拷贝数变异:del(22)(q11.2)(17 370 128～19 790 009,2.42 Mb).FISH实验结果验证了此22q11.2微缺失的存在.结论 隐藏的22q11.2微缺失可能是此胎儿致病的原因;染色体平衡易位的先天缺陷胎儿可能会含有位于重排断裂点区域之外的亚显微结构基因组拷贝数变异;微阵列比较基因组杂交具有高分辨率、高通量和高准确性等优点,适用于亚显微基因组拷贝数变异的检测.     

微阵列比较基因组杂交技术检测先天性心脏畸形胎儿的隐藏基因组拷贝数变异

目的 检测1例先天性主动脉弓离断和室间隔缺损胎儿的基因组拷贝数变异(copy number variations,CNVs),寻找其致病的遗传学证据,探讨微阵列比较基因组杂交技术(array-based comparative genomic hybridization,array-CGH)在分子细胞遗传诊断中应用的可行性.方法 对该患儿脐血及其父母外周血进行常规G显带核型分析,发现胎儿核型为46,XX,t(7;9)(q12;q21),双亲核型正常.进而应用array-CGH芯片对患儿进行全基因组高分辨率扫描分析,采用荧光原位杂交技术(fluorescence in situ hybridization,FISH)对新发现的CNVs进行实验验证.结果 array-CGH分析发现胎儿基因组存在1个病理性亚显微结构的拷贝数变异:del(22)(q11.2)(17 370 128～19 790 009,2.42 Mb).FISH实验结果验证了此22q11.2微缺失的存在.结论 隐藏的22q11.2微缺失可能是此胎儿致病的原因;染色体平衡易位的先天缺陷胎儿可能会含有位于重排断裂点区域之外的亚显微结构基因组拷贝数变异;微阵列比较基因组杂交具有高分辨率、高通量和高准确性等优点,适用于亚显微基因组拷贝数变异的检测.

Abstract：

Objective To detect the copy number variation (CNV) of a fetus with interrupted aortic arch and ventricular septal defect, in order to explore the underlying genetic causes of the congenital malformation, and investigate the feasibility of array-based comparative genomic hybridization (array-CGH)in molecular cytogenetic diagnosis. Methods The whole genome of the fetus with de novo apparently balanced translocations [46, XX, t ( 7 ; 9 ) ( q12 ; q21 ) ] diagnosed by G-banding was scanned and analyzed by array-CGH, and the copy number variation was confirmed by fluorescence in situ hybridization (FISH).Results A pathologic submicroscopic CNV ldel(22) (q11. 2) (17 370 128-19 790 009,-2. 42 Mb)] was identified and mapped by array-CGH. FISH test confirmed the microdeletion detected by array-CGH.Conclusion The cryptic 22q11.2 deletion might be the reason leading to the congenital malformation of the fetus. This study provides evidence that apparently balanced translocations classified by conventional cytogenetic techniques may host additional submicroscopic CNVs which are not located at the breakpoints.Due to the high-resolution, high-throughput and high-accuracy, array-CGH is considered to be a powerful tool for submicroscopic CNVs detection.     

微阵列比较基因组杂交技术检测先天性心脏畸形胎儿的隐藏基因组拷贝数变异

目的 检测1例先天性主动脉弓离断和室间隔缺损胎儿的基因组拷贝数变异(copy number variations,CNVs),寻找其致病的遗传学证据,探讨微阵列比较基因组杂交技术(array-based comparative genomic hybridization,array-CGH)在分子细胞遗传诊断中应用的可行性.方法 对该患儿脐血及其父母外周血进行常规G显带核型分析,发现胎儿核型为46,XX,t(7;9)(q12;q21),双亲核型正常.进而应用array-CGH芯片对患儿进行全基因组高分辨率扫描分析,采用荧光原位杂交技术(fluorescence in situ hybridization,FISH)对新发现的CNVs进行实验验证.结果 array-CGH分析发现胎儿基因组存在1个病理性亚显微结构的拷贝数变异:del(22)(q11.2)(17 370 128～19 790 009,2.42 Mb).FISH实验结果验证了此22q11.2微缺失的存在.结论 隐藏的22q11.2微缺失可能是此胎儿致病的原因;染色体平衡易位的先天缺陷胎儿可能会含有位于重排断裂点区域之外的亚显微结构基因组拷贝数变异;微阵列比较基因组杂交具有高分辨率、高通量和高准确性等优点,适用于亚显微基因组拷贝数变异的检测.     

Cryptic unbalanced translocation t(17;18)(p13.2;q22.3) identified by subtelomeric FISH and defined by array-based comparative genomic hybridization in a patient with mental retardation and dysmorphic features

Molecular cytogenetics allows the identification of cryptic chromosome rearrangements, which is clinically useful in mentally retarded and/or dysmorphic individuals with normal results from conventional cytogenetics analysis. We report on a 3-year-old girl with mental retardation, growth deficiency, speech delay, and dysmorphic features including hypertelorism, upslanting palpebral fissures, midfacial hypoplasia, and posteriorly rotated ears. The G-banding analysis showed a 46,XX,t(3;8)(q26.2;p21.1)mat karyotype. However, her clinical features were suggestive of the 18q syndrome. Subtelomeric FISH analysis revealed a der(18) translocated material from chromosome 17. Array-based comparative genomic hybridization (array-CGH) with subtelomeric BAC and PAC clones confirmed the abnormality and refined the breakpoints to 18q22.3-qter and 17p13.2-pter (deletion of 8.5 Mb and duplication of 3.9 Mb, respectively). This case demonstrates the diagnostic utility of combining conventional cytogenetics with molecular chromosome analyses for the identification of subtle chromosome abnormalities.     

t(9;11)(p22;q24) in a patient with acute monocytic leukemia

A case of t(9;11)(p22;q24) in a patient with acute monocytic leukemia is described. The difficulties of establishing the precise breakpoints involved in this emerging association are discussed.     

Fortuitous detection of a submicroscopic deletion at 1q25 in a girl with Cornelia-de Lange syndrome carrying t(5;13)(p13.1;q12.1) by array-based comparative genomic hybridization

We report on a 2-year-old Japanese girl with Cornelia-de Lange syndrome (CdLS) who had mental and growth retardation, together with characteristic facial anomalies and mild extremity malformations. She had a balanced chromosomal translocation, 46,XX,t(5;13)(p13.1;q12.1) de novo. Surprisingly, this was the same translocation that had provided a clue to the identification of a major causative gene for CdLS, NIPBL [Krantz et al., 2004; Tonkin et al., 2004]. Using fluorescence in situ hybridization (FISH), the breakpoint was confirmed to lie within NIPBL at 5p13.1. Furthermore, array-based comparative genomic hybridization (array-CGH) demonstrated a cryptic 1-Mb deletion harboring six known genes at 1q25-q31.1. A FISH analysis of her parents confirmed that the deletion was de novo. Although patients with interstitial deletions at 1q are rare, some of their features were similar to those observed in our patient, indicating that her clinical manifestations are likely to be affected by not only the disruption of NIPBL but also the concomitant microdeletion at 1q25-q31.1. The present case suggests that array-CGH can uncover cryptic genomic aberrations affecting atypical phenotypes even in well-known congenital disorders.     

1p and 3p deletions in meningiomas without detectable aberrations of chromosome 22 identified by comparative genomic hybridization

Meningioma is a common tumor of the meninges covering the central nervous system. Although generally a benign tumor, meningioma often recurs and is malignant in 5–10% of all cases. Loss of chromosome 22 loci, and specifically inactivation of the NF2 tumor suppressor gene, is considered one of several critical steps in the tumorigenesis of meningioma. However, cytogenetic and molecular investigations have failed to detect either aberrations of chromosome 22 or mutations in the NF2 gene in approximately 40% of all tumors, thus making it apparent that an alternative mechanism(s) is responsible for the development of a large fraction of meningiomas. This subset of meningiomas is not distinct with regard to clinical and histopathological features from tumors showing deletions on chromosome 22. It is, therefore, important to attempt the elucidation of molecular pathway(s) that may operate in the tumorigenesis of these tumors. We used comparative genomic hybridization (CGH) to identify regions of the genome other than chromosome 22, contributing to the development of meningioma. We analyzed 25 tumors that had undergone detailed LOH analysis on chromosome 22 and were shown to contain no detectable deletions. Two benign, malignancy grade I, meningiomas showed concurrent deletion of 1p and 3p. These results suggest that loss of both 1p and 3p may contribute to meningioma tumorigenesis. This may represent genetic changes that are alternative to deletions on chromosome 22. Genes Chromosomes Cancer 20:419–424, 1997. © 1997 Wiley-Liss, Inc.     

DNA copy number aberrations in intestinal-type gastric cancer revealed by array-based comparative genomic hybridization

Genomic instability can be divided into 2 categories: chromosomal instability (CIN) and microsatellite instability (MSI). CIN has been linked to aneuploidy and chromosomal aberrations, and high-level loss of heterozygosity (LOH-H) has been suggested to be an indicator of CIN. High-level MSI (MSI-H), which results from nonfunctional mismatch repair, has previously been suggested to be mutually exclusive with CIN. Four MSI-H and three LOH-H primary gastric tumors of intestinal histology were used for copy number analysis by array-based comparative genomic hybridization (aCGH) with 13,000 cDNA targets. The MSI-H group showed fewer gains (0-12, average 4.5) and losses (0-10, average 2.5) per tumor as compared to the LOH-H group (9-15 gains, average 11.6 and 1-6 losses, average 4). Two MSI-H tumors did not show any copy number changes and one showed only gains of whole chromosomes. The most common alterations were gains of 20q (5/7 samples), 1q, 8, and 10p (3/7 samples) and losses of 1p and 5p (3/7 samples). The minimal amplified regions in 1q and 20q were localized to 1q21.1 approximately q21.2, 1q21.3, 20q11.2, 20q13.12, and 20q13.3 approximately qter. No copy number change was found to be specific for MSI-H or LOH-H. The results suggest that the LOH-H phenotype revealed by microsatellite analysis predicts reliably copy number abnormalities on aCGH and that a subset of MSI-H and all LOH-H tumors share the CIN phenotype.     

Rapid array-based genomic characterization of a subtle structural abnormality: a patient with psychosis and der(18)t(5;18)(p14.1;p11.23)

Array-based copy number analysis has recently emerged as a rapid means of mapping complex and/or subtle chromosomal abnormalities. We have compared two such techniques, using bacterial artificial chromosome (BAC) and single nucleotide polymorphism (SNP) arrays in the evaluation of a 45-year-old woman with dysmorphic features, mental retardation, psychosis, and an unbalanced derivative chromosome 18, (46,XX, der(18)t(18;?)(p12;?)). Both array-based methods demonstrated that the additional material on chromosome 18 was of 5p origin. The 5p duplication mapped telomeric to 25.320 Mb (BAC array) and 25.607 Mb (SNP array), corresponding to the band 5p14.1. Both BAC and SNP arrays also showed a deletion involving chromosome 18p extending telomeric from 8.437 Mb (BAC array) and 8.352 Mb (SNP array), corresponding to the band 18p11.23. Molecular cytogenetic mapping using fluorescence in situ hybridization (FISH) supported the array findings and further refined the breakpoint regions, confirming that the BAC and SNP chips were both useful in this regard. Both case reports and linkage analyses have implicated these chromosomal intervals in psychosis. The array-based experiments were completed over the course of several days. While these methods do not eliminate the requirement for traditional fine-mapping, they provide an efficient approach to identifying the origin and extent of deleted and duplicated material in chromosomal rearrangements.     

Characterization of a de novo balanced translocation t(9;18)(p23;q12.2) in a patient with oculoauriculovertebral spectrum

We report a patient presenting with oculoauriculovertebral spectrum and a de novo balanced reciprocal translocation t(9;18)(p23;q12.2). Physical mapping of the translocation breakpoints by fluorescent in situ hybridization showed that the breakpoints are located in two regions encompassing gene deserts. An additional paternally inherited duplication in 18p11.23p11.31 was identified by array-CGH. We discuss the possible involvement of these chromosomal abnormalities in OAVS.     

Bipolar affective disorder partially cosegregates with a balanced t(9;11)(p24;q23.1) chromosomal translocation in a small pedigree

Analysis of an extended pedigree in which a balanced t(9;11)(p24;q23.1) translocation was found to cosegregate with bipolar affective disorder revealed that five of 11 translocation carriers had bipolar affective disorder and one carrier had unipolar depression. There were no affected individuals in the pedigree without the balanced translocation. We hypothesized that gene(s) or gene regulatory regions disrupted by the translocation might be contributing to the bipolar affective disorder in a dominant fashion. To test this hypothesis, we isolated the derivative chromosome 9 and derivative chromosome 11 in somatic cell hybrids and identified the nearest flanking markers on chromosome 9 (D9S230 and D9S2011E/HRFX3) and chromosome 11 (EST00652 and CRYA2). YAC contigs were constructed in the region of flanking markers for both chromosomes 9 and 11. Chromosome 11 breakpoint was localized within an 8-kb region in a small insert (100 kb) YAC. Chromosome 9 breakpoint was localized within approximately 2 Mb region. Several genes and ESTs including EST00652, CRYA2, DRD2, 5HTR3 on chromosome 11 and VLDLR and SLC1A1 on chromosome 9 were mapped within the vicinity of the breakpoint but were shown not to be disrupted by the translocation breakpoint. Although several possibilities exist regarding the role of the balanced translocation in developing bipolar affective disorder in this pedigree, including a chance cosegregation, identification of a disrupted gene or gene regulatory region with the help of physical mapping resources described in this study may help to identify the presence of a susceptibility gene for this disorder. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 81:81–91, 1998. © 1998 Wiley-Liss, Inc.     

应用微阵列比较基因组杂交技术精确诊断不平衡染色体畸变

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4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Affymetrix SNP 6.0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4例染色体不平衡畸变的诊断并且进行精确定位,其中对2例患者镜下染色体出现无法确定来源的额外条带进行了自身直接重复的确诊;对2例患者G显带无法识别的缺失合并重复的衍生染色体进行了精确诊断.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独特的高分辨率、高敏感性和高特异性,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大的应用价值.     

Inherited balanced translocation t(9;17)(q33.2;q25.3) concomitant with a 16p13.1 duplication in a patient with schizophrenia

We report two rare genetic aberrations in a schizophrenia patient that may act together to confer disease susceptibility. A previously unreported balanced t(9;17)(q33.2;q25.3) translocation was observed in two schizophrenia‐affected members of a small family with diverse psychiatric disorders. The proband also carried a 1.5 Mbp microduplication at 16p13.1 that could not be investigated in other family members. The duplication has been reported to predispose to schizophrenia, autism and mental retardation, with incomplete penetrance and variable expressivity. The t(9;17) (q33.2;q25.3) translocation breakpoint occurs within the open reading frames of KIAA1618 on 17q25.3, and TTLL11 (tyrosine tubulin ligase like 11) on 9q33.2, causing no change in the expression level of KIAA1618 but leading to loss of expression of one TTLL11 allele. TTLL11 belongs to a family of enzymes catalyzing polyglutamylation, an unusual neuron‐specific post‐translational modification of microtubule proteins, which modulates microtubule development and dynamics. The 16p13.1 duplication resulted in increased expression of NDE1, encoding a DISC1 protein partner mediating DISC1 functions in microtubule dynamics. We hypothesize that concomitant TTLL11‐NDE1 deregulation may increase mutation load, among others, also on the DISC1 pathway, which could contribute to disease pathogenesis through multiple effects on neuronal development, synaptic plasticity, and neurotransmission. Our data illustrate the difficulties in interpreting the contribution of multiple potentially pathogenic changes likely to emerge in future next‐generation sequencing studies, where access to extended families will be increasingly important. © 2011 Wiley‐Liss, Inc.     

Array-based comparative genomic hybridization facilitates identification of breakpoints of a novel der(1)t(1;18)(p36.3;q23)dn in a child presenting with mental retardation

Monosomy of distal 1p36 represents the most common terminal deletion in humans and results in one of the most frequently diagnosed mental retardation syndromes. This deletion is considered a contiguous gene deletion syndrome, and has been shown to vary in deletion sizes that contribute to the spectrum of phenotypic anomalies seen in patients with monosomy 1p36. We report on an 8-year-old female with characteristics of the monosomy 1p36 syndrome who demonstrated a novel der(1)t(1;18)(p36.3;q23). Initial G-banded karyotype analysis revealed a deleted chromosome 1, with a breakpoint within 1p36.3. Subsequent FISH and array-based comparative genomic hybridization not only confirmed and partially characterized the deletion of chromosome 1p36.3, but also uncovered distal trisomy for 18q23. In this patient, the duplicated 18q23 is translocated onto the deleted 1p36.3 region, suggesting telomere capture. Molecular characterization of this novel der(1)t(1;18)(p36.3;q23), guided by our clinical array-comparative genomic hybridization, demonstrated a 3.2 Mb terminal deletion of chromosome 1p36.3 and a 200 kb duplication of 18q23 onto the deleted 1p36.3, presumably stabilizing the deleted chromosome 1. DNA sequence analysis around the breakpoints demonstrated no homology, and therefore this telomere capture of distal 18q is apparently the result of a non-homologous recombination. Partial trisomy for 18q23 has not been previously reported. The importance of mapping the breakpoints of all balanced and unbalanced translocations found in the clinical laboratory, when phenotypic abnormalities are found, is discussed.     

B-cell lineage acute lymphoblastic leukemia with i(7q) and t(9;9)(p22;p24)

A 61-year-old Japanese male with acute lymphoblastic leukemia (ALL) is reported. Surface marker examinations revealed that the leukemic cells were of a B-cell lineage; the cells had common ALL antigens, Ia-like antigens, and B1 and B4 antigens. Chromosome analysis showed abnormalities of t(9;9)(p22;p24), as well as an isochromosome for the long arm of chromosome 7. The heritable fragile site at 9p was not detected in the peripheral blood cells of this patient at remission phase.     

Translocation (2;8)(p12;q24) associated with a cryptic t(12;21)(p13;q22) TEL/AML1 gene rearrangement in a child with acute lymphoblastic leukemia

We report a case of childhood acute lymphoblastic leukemia with the simultaneous occurrence of a t(2;8)(p12;q24) typically associated with mature B cell or Burkitt leukemia, and a t(12;21)(p13;q22) exclusively associated with pre-B cell ALL. The lymphoblasts were characterized as L2 morphology by the French-American-British classification. However, there were atypical morphologic findings for L2 ALL, including vacuolization in some cells. The lymphoblasts were periodic acid-Schiff positive and myeloperoxidase negative. Immunophenotypic analysis revealed that the majority of lymphoblasts were TdT+, CD10+, CD19+, CD20-, and cytoplasmic mu+. These features were consistent with an immature pre-B cell leukemia phenotype with some characteristics of a mature B-cell leukemia. A t(2;8)(p12;q24)(p12;q24), characteristic of mature B-cell leukemia or Burkitt type leukemia, was detected by conventional cytogenetics with no other cytogenetic abnormalities. However, diagnostic peripheral blood and bone marrow specimens demonstrated simultaneous occurrence of a cryptic t(12;21)(p13;q22) by both FISH and RT-PCR. The simultaneous occurrence of these translocations in a pediatric patient have implications for the pathogenesis of leukemias with t(2;8)(p12;q24) as well as t(12;21)(p12;q22). Analysis of additional cases of leukemia with translocations involving the MYC locus on 8q24 will be required to determine the frequency of association with the cryptic t(12;21)(p13;22), and the prognostic significance of the simultaneous occurrence of the translocations.     

Detection of multiple gene amplifications in glioblastoma multiforme using array-based comparative genomic hybridization

We have used a new method of genomic microarray to investigate amplification of oncogenes throughout the genome of glioblastoma multiforme (GBM). Array-based comparative genomic hybridization (array CGH) allows for simultaneous examination of 58 oncogenes/amplicons that are commonly amplified in various human cancers. Amplification of multiple oncogenes in human cancers can be rapidly determined in a single experiment. Tumor DNA and normal control DNA were labeled by nick translation with green- and red-tagged nucleotides, respectively. Instead of hybridizing to normal metaphase chromosomes in conventional comparative genomic hybridization (CGH), the probes of the mixed fluorescent labeled DNA were applied to genomic array templates comprised of P1, PAC, and BAC clones of 58 target oncogenes. The baseline for measuring deviations was established by performing a series of independent array CGH using test and reference DNA made from normal individuals. In the present study, we examined fourteen GBMs (seven cell lines and seven tumours) with CGH and array CGH to reveal the particular oncogenes associated with this cancer. High-level amplifications were identified on the oncogenes/amplicons CDK4, GLI, MYCN, MYC, MDM2, and PDGFRA. The highest frequencies of gains were detected on PIK3CA (64.3%), EGFR (57.1%), CSE1L (57.1%), NRAS (50%), MYCN (42.9%), FGR (35.7%), ESR (35.7%), PGY1 (35.7%), and D17S167 (35.7%). These genes are suggested to be involved in the GBM tumorigenesis.     

Mosaic tetrasomy 12p with triplication of 12p detected by array-based comparative genomic hybridization of peripheral blood DNA

A patient whose dysmorphism at birth was not diagnostic for Pallister-Killian syndrome (PKS) was found to have mosaic tetrasomy 12p by an array-based comparative genomic hybridization of peripheral blood DNA. He was determined to be mosaic for 46,XY,trp(12)(p11.2 --> p13) in cultured skin fibroblasts. His appearance was typical for PKS at 4 months of age.