Detection of submicroscopic subtelomeric chromosome translocations: a new case study

Two sisters presented with multiple congenital abnormalities and developmental delay; abnormalities elsewhere in their extended family suggested that their father carried a balanced translocation. G-banded chromosome analysis showed apparently normal karyotypes. Fluorescence in situ hybridisation (FISH) with whole chromosome paints revealed no apparent abnormality in the father. However, further FISH studies, using multiple subtelomeric probes, demonstrated a derivative chromosome 16 in one sister. Subsequent studies showed that her sister also had a derivative 16 which had been inherited in an unbalanced form from their father, who carried a balanced reciprocal translocation between chromosomes 1 and 16. This report describes the detection of this submicroscopic translocation and the clinical findings in the two sisters.     

Unexpected retention and concomitant loss of subtelomeric regions in balanced chromosome anomalies by FISH

Florescence in situ hybridization (FISH) using subtelomeric probes has been useful in detecting cryptic telomeric chromosomal rearrangements. We report, for the first time, that cytogenetically visible chromosome rearrangements can occur between the subtelomeric and telomeric region in clinically normal individuals with balanced chromosome anomalies in which one of the breakpoints involves a terminal band region. Using FISH with subtelomeric probes, we observed in three cases with a balanced reciprocal translocations the retention and subsequent loss of subtelomeric regions. In one case with a paracentric inversion, there was a proximal relocation of a subtelomeric region. Because subtelomeric regions serve important roles in chromosome pairing, this retention and concomitant loss or relocation of a subtelomeric region could possibly further disrupt the complex meiotic configurations of these balanced chromosome rearrangements. This may then have an effect on gamete production, placing these individuals at a higher risk for miscarriages and/or abnormal outcomes for individuals with similar chromosome aberrations.     

Sequential chromosome abnormalities in B cell chronic lymphocytic leukemia: a study of 13 cases

The chromosomal constitution of stimulated lymphocytes in 13 patients with B cell chronic lymphocytic leukemia (B-CLL) were sequentially examined using polyclonal B cell activators (PBA), i.e., Epstein-Barr virus (EBV), lipopolysaccharide W from E. coli (LPS), pokeweed mitogen (PWM), and protein A from Staphylococcus aureus (PA). Of the 11 patients (44 samplings) with abnormal clones, 2 patients had only trisomy 12, 6 patients had trisomy 12 plus other clonal abnormalities, such as +8, +9, +16, +18, 6q-, 15q+, and t(4;15), and the remaining 3 cases had various clonal abnormalities other than trisomy 12, such as trisomy 3, 8, 20, 21, and insertion of #7 and #12. These findings suggest that even though trisomy 12 may be a common abnormality in B-CLL, various other abnormal clones may also be present in vivo for relatively long periods of time. It appears that stimulated lymphocytes in patients with previous therapy tend to show chromosome abnormalities more frequently than those in untreated patients.     

36例X染色体异常的遗传学分析

目的通过对36例女性性发育异常患者的核型分析,进一步探讨X染色体异常的遗传学效应。方法采用外周血淋巴细胞培养制片、染色体G显带技术进行核型分析。结果36例性发育异常患者中X染色体数目异常26例,结构异常10例。结论女性X染色体异常可导致性腺发育不全,身材矮小,原发闭经,月经紊乱等,两条完整的X染色体对女性性腺及体征的发育是十分重要的。     

69例产前诊断胎儿染色体异常病例的临床分析

目的分析孕期不同高危指征与胎儿染色体异常之间的关系,提高产前胎儿染色体异常检出率。方法对1973例高危孕妇利用孕中期羊膜腔穿刺进行胎儿染色体核型分析。结果1973例产前诊断病例共检出染色体异常核型69例,异常率为3.50％,其中三体综合征39例,占异常核型的56.52％;性染色体数目异常11例,占异常核型的15.94％;常染色体结构异常19例,占异常核型的27.54％。结论血清筛查高危、高龄及B超“软指标”异常等高危孕妇,应进一步检测胎儿染色体,以减少染色体异常患胎出生。     

染色体微阵列分析在超声异常胎儿产前诊断中的应用价值

目的探讨染色体微阵列分析技术(chromosomal microarray analysis,CMA)在超声异常胎儿产前诊断中的应用价值。方法选取B超提示异常的胎儿293例,包括结构异常168例及非结构异常125例。在排除常见的染色体异常核型后,对其羊水行CMA检测。结果CMA共检出致病性拷贝数变异(pathogenic copy number variants,pCNVs)16例,检出率为5.46%。在168例结构异常胎儿中检出pCNVs 10例,检出率为5.95%。在125例非结构异常胎儿中检出pCNVs 6例,检出率为4.80%。结论与传统的染色体核型分析相比,CMA可以提高超声异常胎儿染色体异常的检出率,可作为有效的产前诊断方法。     

Chromosome abnormalities in malignant melanoma: clinical significance of nonrandom chromosome abnormalities in 206 cases

We report the cytogenetic abnormalities from a series of 206 primary malignant melanoma specimens referred to a single institution. A total of 169 out of 206 unique cases had chromosome breakpoints. A previously described statistical method was used to detect nonrandom distribution of chromosome breakpoints at the level of chromosome regions. Nonrandom occurrence of chromosome breakpoints (indicating that the observed number of breaks significantly exceeded the expected number of breaks) was detected in 28 regions, suggesting a hierarchy of genetic abnormalities in melanoma. Clinical variables and tumor characteristics were analyzed for associations with the presence of any nonrandom chromosome breakpoints; with individual, nonrandomly involved chromosome regions; and with paired, nonrandomly involved chromosome regions. No nonrandomly involved chromosome regions or pairs of regions appeared to significantly affect survival. These results identify recurring, nonrandom chromosome abnormalities in malignant melanoma. These results suggest that recurring, nonrandom chromosome alterations play a key role in the etiology and/or progression of malignant melanoma and identify targets within the genome for molecular genetic studies.     

High-resolution analysis of the subtelomeric regions of human embryonic stem cells

The use of human embryonic stem cells (hESCs) in most applications is dependent on their undifferentiated proliferation in vitro. Recent studies have illustrated the possibility that chromosomal changes may occur in hESCs during in vitro propagation of these cells. However, no studies so far have screened for chromosomal abnormalities in hESCs using high-resolution techniques that can detect alterations on a few base-pair levels. We have used the recently developed multiplex ligation-dependent probe amplification procedure to analyze the possible occurrence of deletions or duplications in the subtelomeric regions of hESCs in early and late passages. In this study we show that no subtelomeric anomalies were detected in any of the nine hESC lines investigated, supporting the conclusion that hESCs, under appropriate conditions, maintain genomic stability during in vitro propagation.     

American College of Medical Genetics and Genomics technical standards and guidelines: microarray analysis for chromosome abnormalities in neoplastic disorders

Microarray methodologies, to include array comparative genomic hybridization and single-nucleotide polymorphism–based arrays, are innovative methods that provide genomic data. These data should be correlated with the results from the standard methods, chromosome and/or fluorescence in situ hybridization, to ascertain and characterize the genomic aberrations of neoplastic disorders, both liquid and solid tumors. Over the past several decades, standard methods have led to an accumulation of genetic information specific to many neoplasms. This specificity is now used for the diagnosis and classification of neoplasms. Cooperative studies have revealed numerous correlations between particular genetic aberrations and therapeutic outcomes. Molecular investigation of chromosomal abnormalities identified by standard methods has led to discovery of genes, and gene function and dysfunction. This knowledge has led to improved therapeutics and, in some disorders, targeted therapies. Data gained from the higher-resolution microarray methodologies will enhance our knowledge of the genomics of specific disorders, leading to more effective therapeutic strategies. To assist clinical laboratories in validation of the methods, their consistent use, and interpretation and reporting of results from these microarray methodologies, the American College of Medical Genetics and Genomics has developed the following professional standard and guidelines.Genet Med 2013:15(6):484–494     

Chromosome abnormalities in ovarian adenocarcinoma: I. Nonrandom chromosome abnormalities from 244 cases.

Cytogenetics provides important insights into the molecular pathogenesis of human cancers. Although extensive data exist on recurring cytogenetic abnormalities in hematologic cancers, data on individual solid tumor types remain limited. Previous studies of ovarian carcinoma indicated the presence of multiple, complex clonal chromosome abnormalities. Cytogenetics remains one of a few techniques capable of detecting these multiple, simultaneously occurring genetic abnormalities. We describe cytogenetic abnormalities from a series of 244 primary ovarian cancer specimens referred to a single institution. A total of 201/244 cases had fully characterized clonal chromosome abnormalities, of which 134 showed clonal chromosome breakpoints. We used a novel statistical technique to detect nonrandom chromosome breakpoints at the level of chromosome regions. Nonrandom occurrence of chromosome breakpoints was detected at regions 1p1*, 1q1*, 1p2*, 1q2*, 1p3*, 1q3, 3p1*, 1q4*, 6q1*, 6p2, 6q2, 7p1*, 7q1, 7p2*, 11p1*, 11q1, 11q2*, 12p1, 12q2*, 13p1, and 19q1. Simultaneous occurrence of multiple abnormalities was common. However, 120/134 cases had breakpoints at one or more of 13 commonly involved regions (*), suggesting a hierarchy of genetic abnormalities. Among clinical and tumor variables that predict patient survival, tumor grade was significantly associated with the presence of chromosome breakpoints. In additional studies, we show that nonrandom chromosome abnormalities are associated with impaired survival in ovarian cancer and that specific, nonrandomly involved chromosome regions retain significant effects on survival when analyses are controlled for important clinical variables. Additional specific chromosome abnormalities in this series are described, including chromosome gains and losses in near-diploid cases and homogeneously staining regions. These results suggest that recurring, nonrandom chromosome abnormalities are important in the pathogenesis and/or progression of ovarian cancers, and target areas of the genome for molecular genetic studies.     

Molecular characterisation of patients with subtelomeric 22q abnormalities using chromosome specific array-based comparative genomic hybridisation

The 22q13 deletion syndrome is associated with global developmental delay, absent or delayed speech, and generalised hypotonia. In this study, the size and nature of 22q13 deletions (n=9) were studied in detail by high-resolution chromosome specific array-based comparative genomic hybridisation (array CGH). The deletion sizes varied considerably between the different patients, that is, the largest deletion spanning 8.4 Mb with the breakpoint mapping to 22q13.2 and the smallest deletion spanning 3.3 Mb with the breakpoint mapping to 22q13.31. In one case, a unique subtelomeric 3.9 Mb deletion associated with a 2.0 Mb duplication of 22q13 was observed, adding to a growing number of similar cases identified for other chromosome ends. Remarkably, this patient had signs suggestive of retinitis pigmentosa, which has never been reported before in the 22q13 deletion syndrome. The identification of two pairs of recurrent proximal breakpoints on 22q13 suggests that these specific regions may be prone to recombination, due to yet unknown genome architectural features. In addition to the copy number changes on 22q13, a duplication of approximately 330 kb on 22q11.1 was observed and shown to be a genetic large-scale copy number variation without clinical consequences. The current study failed to reveal relationships between the clinical features and the deletion sizes. Global developmental delay and absent or severely delayed speech were observed in all patients, whereas hypotonia was present in 89% of the cases (8/9). This study underscores the utility of array CGH for characterising the size and nature of subtelomeric deletions, such as monosomy 22q13, and underlines the considerable variability in deletion size in the 22q13 deletion syndrome regardless of the clinical phenotype.     

Chromosome abnormalities in ovarian adenocarcinoma: II. Prognostic impact of nonrandom chromosome abnormalities in 244 cases

In a large series of ovarian carcinomas from 244 patients, 134 cases had chromosome rearrangements. We showed before that the pattern of chromosome breakpoints involved 21 separate chromosome regions nonrandomly and, in 90% of cases with breaks, the breakpoints occurred within 13 commonly involved regions. Log-rank and proportional hazards regression analyses showed that the aggregate presence of a chromosome breakpoint in any of 21 nonrandomly involved regions and breaks in 9 distinct regions (1p1, 1q2, 1p3, 3p1, 6p2, 11p1, 11q1, 12q2, and 13p1) were associated with reduced patient survival. Breakpoints in other areas of the genome, including other nonrandomly involved regions, were not associated with decreased survival. Because many cases had breakpoints in more than one nonrandomly involved region, proportional hazards regression was also used to analyze for effects of each nonrandomly involved region, controlling for effects of other regions. With this approach, only breakpoints within 1p1 and 3p1 retained independent, deleterious effects on survival. Similarly, when nonrandomly involved regions were entered into a proportional hazards model containing clinical variables associated with altered patient survival (tumor grade, tumor stage, and residual disease > 1 cm after resection), only 1p1 (P = 0.007) and 3p1 (P = 0.04) were associated with independent, negative effects on survival. These studies demonstrate that chromosome breakpoints within specific, nonrandomly involved chromosome regions are associated with impaired survival in ovarian cancers. Regions 1p1 and 3p1 are identified as areas of particular significance and are appropriate targets for analytical techniques such as SAGE and microarray analysis.     

FISH analysis for apparently simple terminal deletions of the X chromosome: Identification of hidden structural abnormalities

We report on fluorescence in situ hybridization (FISH) analysis in 30 mosaic or nonmosaic females diagnosed as having apparently simple terminal X deletions by standard G‐banding analysis. FISH studies for DXZ1, the Xp and Xq telomere regions, and the whole X chromosome painting were carried out for the 30 females, indicating rearranged X chromosomes with signal patterns discordant with terminal deletions in 6 cases: one dic(X)(DXZ1++) chromosome, two der(X)(qtel++) chromosomes, one Xq? (qtel+) chromosome, and two der(X)(ptel++) chromosomes. Additional FISH studies were performed for the 6 cases using probes defining 12 loci on the X chromosome, showing large Xp deletion and small Xp duplication in the dic(X)(DXZ1++) chromosome, partial Xp deletions and partial Xq duplications in the two der(X)(qtel++) chromosomes, an interstitial Xq deletion in the Xq? (qtel+) chromosome, and partial Xq deletions and partial Xp duplications in the two der(X)(ptel++) chromosomes. Clinical assessment of the 6 cases revealed tall and normal stature in the two mosaic cases with the der(X)(ptel++) chromosomes that were shown to be associated with SHOX duplication. The results suggest that unusual X chromosome rearrangements are often misinterpreted as simple terminal X deletions, and that FISH analysis is useful for precise structural determination and better genotype‐phenotype correlation of the X chromosome aberrations. © 2001 Wiley‐Liss, Inc.     

37例世界首报染色体异常核型及临床分析

目的探讨世界首报染色体异常与疾病的关系。方法自1984年1月～2011年3月,对4511例有不良孕产史、不孕症、习惯性流产、生育畸形、死胎、死产、智力低下、表型异常、有遗传病家族史等前来就诊和咨询者进行细胞遗传学检查。取受检者肝素抗凝外周血,无菌条件下进行淋巴细胞72h培养,常规G显带,计数30个分裂相,分析核型3个,异常者加倍分析。必要时行C和R显带分析。结果检出异常染色体533例,异常检出率为11.82%。经中国医学遗传中心鉴定,世界首报染色体异常核型37例,占染色体异常的6.94%。结论世界首报染色体异常核型的检出,可降低人群发病率,并为疾病基因筛选、定位提供重要线索。     

Identification of novel deletion regions on chromosome arms 2q and 6p in breast carcinomas by amplotype analysis

DNA fingerprinting by arbitrarily primed PCR (AP-PCR) was employed to identify molecular genetic alterations in 37 primary breast carcinomas. AP-PCR is a PCR-based technique that uses only one primer of arbitrary sequence that generates a molecular karyotype (amplotype) of tumors. The breast cancer amplotype generated with two arbitrary primers (MCG1 and Blue) showed a relatively high frequency (more than 20% of the tumors) of gains at chromosomes 1, 4, and 8, and of losses at chromosomes 2, 4, 6, 9, 10, 11, 13, and the X chromosome. We further analyzed the regions most commonly gained at chromosome 8 (47%) and lost at chromosomes 2 (38%) and 6 (49%) by determining the subchromosomal localization of the fingerprint bands from these chromosomes. The region of gain at chromosome 8 was mapped at 8q24.1, close to MYC. Band MCG1-A1 was assigned to chromosome band 2q22, and band Blue-J was assigned to 6p21. Common losses of these chromosomal regions have not been described for breast cancer. To map these deletion regions more precisely, we performed loss of heterozygosity (LOH) analysis by microallelotyping on 20 of the 37 cancers previously analyzed by AP-PCR and another additional 52 breast carcinomas. The results suggest that the regions at 2q21-24 and 6p21-23 may harbor novel tumor suppressor genes for breast cancer. LOH at 2q21-24 (D2S2304) was more frequent in high-grade tumors (59%) than in low-grade tumors (29%) (P = 0.03). This suggests that this genetic alteration may be associated with tumor progression and shows the power of the amplotype approach in detecting novel genetic alterations that are useful as clinical parameters of breast cancer.     

Immunohistochemical analysis of PTEN in endometrial carcinoma: a tissue microarray study with a comparison of four commercial antibodies in correlation with molecular abnormalities.

The tumor suppressor gene PTEN/MMAC1 is located on chromosome 10q23.3. Inactivation of PTEN, either by mutations, deletions, or promoter hypermethylation, has been identified in a wide variety of tumors. Inactivation of the two alleles of PTEN is required, because it is a tumor suppressor gene. Immunohistochemical staining may be an effective screening method to demonstrate the absence of the protein in tumors exhibiting PTEN inactivation. We studied a tissue microarray, constructed from paraffin-embedded blocks of 95 endometrial carcinomas, 38 of them previously evaluated for alterations in PTEN. We also studied cell blocks obtained from one PTEN-defective endometrial cancer cell line, after transfection with either a plasmid encoding wild-type PTEN or the empty vector. The tumor samples were tested with four different anti-PTEN commercial antibodies: a polyclonal antibody, the monoclonal antibody 28H6, the monoclonal antibody 10P03, and the monoclonal antibody 6.H2.1. Results were correlated with the presence of abnormalities in PTEN, as well as with the immunohistochemical expression of phosphorylated AKT. Antibody 28H6 produced a predominant nuclear staining, while the other three antibodies produced a predominant cytoplasmic staining. There was no significant correlation between the results obtained with the four antibodies. The monoclonal antibody 6.H2.1 was the only one that exhibited a correlation with the presence of molecular alterations in PTEN, and a statistically significant association with immunostaining for phosphorylated AKT (r=-0.249, P=0.037). The monoclonal antibody 10P03 exhibited an association with phospho-AKT that did not have statistical significance. Both 6.H2.1 and 10P03 antibodies stained PTEN-transfected cells, and were negative in the PTEN-deficient cell line blocks. The polyclonal antibody and the monoclonal antibody 28H6 produced positive staining in PTEN-deficient cell line blocks, suggesting nonspecific staining. The results indicate that monoclonal antibody 6.H2.1 may be a suitable alternative for tumors with inactivation of PTEN.