Arhinia, congenital absence of the nose, is a rare malformation. We present the third reported case of arhinia accompanied by hypogonadism and demonstrate that this is due to gonadotropin deficiency.
A 13-year-old Caucasian boy with congenital arhinia presented for evaluation of delayed puberty and micropenis. We examined genes known to be associated with hypogonadotropic hypogonadism for mutations and performed a chromosomal microarray to assess copy number variation.
No mutations in KAL1, FGFR1, PROK2, PROKR2, FGF8, CHD7 and GnRHR were identified in our patient and there were no copy number variations observed that would explain the phenotype. Though studies are limited in such patients, we suggest that hypogonadotropic hypogonadism is associated with arhinia and that the two entities likely result from a common genetic cause that affects early nasal development and gonadotropin-releasing hormone neuron formation or migration.
Obtaining a germ cell line is one of the most important steps in developing a transgenic or knockout mouse with a targeted mutated gene of interest. A common problem with this technology is that embryonic stem (ES) cells often lack, or are extremely inefficient at, germ line transmission.
To determine whether chromosomal anomalies are correlated with inefficient ES cell germ line transmission, we examined 97 constructed ES cell lines using conventional cytogenetic analysis, and fluorescence in situ hybridization (FISH). Chromosomal abnormalities occurred in 44 (45%) out of the 97 specimens analyzed: 31 specimens had trisomy 8 or mosaic trisomy 8, eight specimens had partial trisomy 8 resulting from unbalanced translocations, and five specimens had other chromosomal anomalies.
Our data suggest that chromosomal analysis is an important tool for improving the yield and quality of gene targeting experiments.
Mouse ES cells; Chromosomal aberrations; FISH; Mosaicism
Fluorescence in situ hybridization (FISH) tests provide promising molecular imaging biomarkers to more accurately and reliably detect and diagnose cancers and genetic disorders. Since current manual FISH signal analysis is low-efficient and inconsistent, which limits its clinical utility, developing automated FISH image scanning systems and computer-aided detection (CAD) schemes has been attracting research interests. To acquire high-resolution FISH images in a multi-spectral scanning mode, a huge amount of image data with the stack of the multiple three-dimensional (3-D) image slices is generated from a single specimen. Automated preprocessing these scanned images to eliminate the non-useful and redundant data is important to make the automated FISH tests acceptable in clinical applications. In this study, a dual-detector fluorescence image scanning system was applied to scan four specimen slides with FISH-probed chromosome X. A CAD scheme was developed to detect analyzable interphase cells and map the multiple imaging slices recorded FISH-probed signals into the 2-D projection images. CAD scheme was then applied to each projection image to detect analyzable interphase cells using an adaptive multiple-threshold algorithm, identify FISH-probed signals using a top-hat transform, and compute the ratios between the normal and abnormal cells. To assess CAD performance, the FISH-probed signals were also independently visually detected by an observer. The Kappa coefficients for agreement between CAD and observer ranged from 0.69 to 1.0 in detecting/counting FISH signal spots in four testing samples. The study demonstrated the feasibility of automated FISH signal analysis applying a CAD scheme to the automated generated 2-D projection images.
Fluorescence in situ hybridization (FISH); Automated FISH signal analysis; Computer-aided detection (CAD); Molecular imaging biomarker
The authors developed an integrated computer-aided detection (CAD) scheme for detecting and classifying metaphase chromosomes as well as assessing its performance and robustness. This scheme includes an automatic metaphase-finding module and a karyotyping module and it was applied to a testing database with 200 digital microscopic images. The automatic metaphase-finding module detects analyzable metaphase cells using a feature-based artificial neural network (ANN). The ANN-generated outputs are analyzed by a receiver operating characteristics (ROC) method and an area under the ROC curve is 0.966. Then, the automatic karyotyping module classifies individual chromosomes of this cell into 24 types. In this module, a two-layer decision tree-based classifier with eight ANNs established in its connection nodes was optimized by a genetic algorithm. Chromosomes are first classified into seven groups by the ANN in the first layer. The chromosomes in these groups are then separately classified by seven ANNs into 24 types in the second layer. The classification accuracy is 94.5% in the first layer. Six ANNs achieved the accuracy above 95% and only one had lessened performance (80.6%) in the second layer. The overall classification accuracy is 91.5% as compared to 86.7% in the previous study using two independent datasets randomly acquired from our genetic laboratory. The results demonstrate that our automated scheme achieves high and robust performance in identification and classification of metaphase chromosomes.
Metaphase chromosome; Karyotype; Computer-aided detection; Artificial neural network; Receiver operating characteristics
AIM: To investigate the genomic copy number alterations that may harbor key driver genes in gastric tumorigenesis.
METHODS: Using high-resolution array comparative genomic hybridization (CGH), we investigated the genomic alterations of 20 advanced primary gastric adenocarcinomas (seventeen tubular and three mucinous) of Chinese patients from the Jilin province. Ten matching adjacent normal regions from the same patients were also studied.
RESULTS: The most frequent imbalances detected in these cancer samples were gains of 3q26.31-q27.2, 5p, 8q, 11p, 18p, 19q and 20q and losses of 3p, 4p, 18q and 21q. The use of high-resolution array CGH increased the resolution and sensitivity of the observed genomic changes and identified focal genetic imbalances, which included 54 gains and 16 losses that were smaller than 1 Mb in size. The most interesting focal imbalances were the intergenic loss/homozygous deletion of the fragile histidine triad gene and the amplicons 11q13, 18q11.2 and 19q12, as well as the novel amplicons 1p36.22 and 11p15.5.
CONCLUSION: These regions, especially the focal amplicons, may harbor key driver genes that will serve as biomarkers for either the diagnosis or the prognosis of gastric cancer, and therefore, a large-scale investigation is recommended.
Array comparative genomic hybridization; Amplicon; Gastric adenocarcinoma; Oncogene; Fragile histidine triad
Gain-of-function mutations of tyrosine kinase FLT3 are frequently found in acute myeloid leukemia (AML). This has made FLT3 an important marker for disease diagnosis and a highly attractive target for therapeutic drug development. This study is intended to generate a sensitive substrate for assays of the FLT3 enzymatic activity.
We expressed in Escherichia coli cells a glutathione S-transferase (GST) fusion protein designated GST-FLT3S, which contains a peptide sequence derived from an autophosphorylation site of FLT3. The protein was used to analyze tyrosine kinase activity of baculovirus-expressed FLT3 and crude cell extracts of bone marrow cells from AML patients. It was also employed to perform FLT3 kinase assays for FLT3 inhibitor screening.
GST-FLT3S in solution or on beads was strongly phosphorylated by recombinant proteins carrying the catalytic domain of wild type FLT3 and FLT3D835 mutants, with the latter exhibiting much higher activity and efficiency. GST-FLT3S was also able to detect elevated tyrosine kinase activity in bone marrow cell extracts from AML patients. A small-scale inhibitor screening led to identification of several potent inhibitors of wild type and mutant forms of FLT3.
GST-FLT3S is a sensitive protein substrate for FLT3 assays. It may find applications in diagnosis of diseases related to abnormal FLT3 activity and in inhibitor screening for drug development.
Tyrosine kinase; FLT3; Activity assay; Inhibitor screening; Acute myeloid leukemia
Intravascular lymphoma is an aggressive and extremely rare extranodal lymphoma with neoplastic lymphoid cells confined exclusively within intravascular spaces. The histopathologic findings are subtle due to the rarity of the neoplastic cells in blood vessels. Clinical presentations are non-specific and focal space-occupying lesions or lymphoadenopathy are always lacking. It is a diagnostic challenge. Secondary hemophagocytic syndrome is uncommon and is typically associated with infection, malignancy, and suppressed immune states. Intravascular lymphoma has a strong association with hemophagocytic syndrome in Asian patients, the so-called "Asian variant", but not in Western patients. We report a case of intravascular B-cell lymphoma in a Caucasian patient associated with secondary hemophagocytic syndrome. The patient was diagnosed by core liver biopsy and successfully treated. This case demonstrates the importance of high index of suspicion and astute histopathologic examination in recognition of this unusual clinical and pathologic combination.
Intravascular lymphoma; hemophagocytic syndrome; liver; Asian variant; hepatosplenomegaly
Undifferentiated embryonal sarcoma of the liver (UESL) represents a heterogeneous group of tumors derived from mesenchymal tissues. Earlier cytogenetic studies in limited cases demonstrated that UESL is associated with a recurrent translocation t(11;19)(q11;q13.3-q13.4) or add(19)(q13.4). In this report, we present our array comparative genomic hybridization (aCGH), fluorescence in situ hybridization (FISH) findings, and a missense mutation of TP53 gene by DNA sequencing in a 19-year-old patient with UESL. The data were compared to laboratory findings reported by previous studies.
Undifferentiated embryonal sarcoma of the liver (UESL); Cytogenetic anomalies; FISH; aCGH; TP53 mutation
Recent studies suggest that cancer stem cells (CSCs) are responsible for cancer resistance to therapies. We therefore investigated how glioblastoma-derived CSCs respond to the treatment of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Neurospheres were generated from glioblastomas, characterized for CSC properties including self-renewal, cell differentiation and xenograft formation capacity, and analyzed for TRAIL-induced apoptosis, CASP8 genomic status, and caspase-8 protein expression. The neurosphere NSC326 was sensitive to TRAIL-induced apoptosis as evidenced by cell death and caspase-8, -3, and -7 enzymatic activities. In contrast, however, the neurosphere NSC189 was TRAIL-resistant. G-banding analysis identified five chromosomally distinguishable cell populations in the neurospheres. Fluorescence in situ hybridization revealed the variation of chromosome 2 copy number in these populations and the loss of CASP8 locus in 2q33-34 region in a small set of cell populations in the neurosphere. Immunohistochemistry of NSC189 cell blocks revealed the lack of caspase-8 protein in a subset of neurosphere cells. Western blotting and immunohistochemistry of human glioblastoma tumors demonstrated the expression of caspase-8 protein in the vast majority of the tumors as compared to normal human brain tissues that lack the caspase-8 expression. This study shows heterogeneity of glioblastomas and derived CSCs in the genomic status of CASP8, expression of caspase-8, and thus responsiveness to TRAIL-induced apoptosis. Clinic trials may consider genomic analysis of the cancer tissue to identify the genomic loss of CASP8 and use it as a genomic marker to predict the resistance of glioblastomas to TRAIL apoptosis pathway-targeted therapies.
Apoptosis; cancer stem cells; caspase-8; glioblastoma; TRAIL
Anogenital cancers and head and neck cancers are causally-associated with infection by high-risk human papillomavirus (HPV). The mechanism by which high-risk HPVs contribute to oncogenesis is poorly understood. HPV16 encodes three genes (HPV16 E5, E6, and E7) that can transform cells when expressed independently. HPV16 E6 and E7 have well-described roles causing genomic instability and unregulated cell cycle progression. The role of HPV16 E5 in cell transformation remains to be elucidated. Expression of HPV16 E5 results in enlarged, polyploid nuclei that are dependent on the level and duration of HPV16 E5 expression. Live-cell imaging data indicate these changes do not arise from cell-cell fusion or failed cytokinesis. The increase in nuclear size is a continual process that requires DNA synthesis. We conclude HPV16 E5 produces polyploid cells by endoreplication. These findings provide insight into how HPV16 E5 can contribute to cell transformation.
erbB-2 is amplified or overexpressed in approximately 30% of human breast cancers, and has been associated with poor prognosis and therapeutic resistance. Previous studies have suggested that erbB-2 overexpression in transgenic mice induces genomic instability; however, the patterns of genetic lesions vary with individual model systems. The development of mammary tumors in multiparous murine mammary tumor virus (MMTV)-erbB-2 transgenic mice is accelerated due to hormonal interactions which induce the overexpression of MMTV-mediated erbB-2. However, whether or not accelerated tumor development is associated with modified cytogenetic patterns remains to be determined. In this study, chromosomal changes were characterized in mammary tumor cells derived from multiparous MMTV-erbB-2 transgenic mice, and compared with tumor cells derived from control virgin mice. Immunohistochemistry and Western blotting were used to detect erbB-2 overexpression in mammary tissues. Each of the five tumors from the multiparous MMTV-erbB-2 transgenic mice was found to exhibit a marked chromosomal imbalance, compared with only one tumor with aberrant chromosomes among the five tumors from the control virgin mice. In particular, trisomy 5 and loss of the X chromosome were recurrent cytogenetic lesions in tumors from the parous mice, which is a novel pattern compared with previous studies. The elevated number of genetic lesions in tumors from parous mice, which were characterized by enhanced erbB-2 overexpression and increased receptor tyrosine kinase activation in the mammary glands, suggest a causal role for erbB-2 in the genomic instability present in these tumors. These data advance our understanding of erbB-2-mediated pathogenesis and underscore the role of cytogenetic alteration in this process.
erbB-2/Neu; genomic instability; breast cancer; transgenic mouse model
Systemic lupus erythematosus (SLE) is more common among women than men with a ratio of about 10 to 1. We undertook this study to describe familial male SLE within a large cohort of familial SLE. SLE families (two or more patients) were obtained from the Lupus Multiplex Registry and Repository. Genomic DNA and blood samples were obtained using standard methods. Autoantibodies were determined by multiple methods. Medical records were abstracted for SLE clinical data. Fluorescent in situ hybridization (FISH) was performed with X and Y centromere specific probes, and a probe specific for the toll-like receptor 7 gene on the X chromosome. Among 523 SLE families, we found five families in which all the SLE patients were male. FISH found no yaa gene equivalent in these families. SLE-unaffected primary female relatives from the five families with only-male SLE patients had a statistically increased rate of positive ANA compared to SLE-unaffected female relatives in other families. White men with SLE were 5 times more likely to have an offspring with SLE than were White women with SLE but there was no difference in this likelihood among Black men. These data suggest genetic susceptibility factors that act only in men.
Systemic lupus erythematosus; men; autoantibodies; genetics
The numerical and/or structural deviation of some chromosomes (i.e., monosomy and polysomy of chromosomes 3 and X) are routinely used as positive genetic biomarkers to diagnose cervical cancer and predict the disease progression. Among the available diagnostic methods to analyze the aneusomy of chromosomes 3 and X, fluorescence in situ hybridization (FISH) technology has demonstrated significant advantages in assisting clinicians to more accurately detect and diagnose cervical carcinoma at an early stage, in particular for the women at a high risk for progression of low-grade and high-grade squamous intra-epithelium lesions (LSIL and HSIL). In order to increase the diagnostic accuracy, consistency, and efficiency from that of manual FISH analysis, this study aims to develop and test an automated FISH analysis method that includes a two-stage scheme. In the first stage, an interactive multiple-threshold algorithm is utilized to segment potential interphase nuclei candidates distributed in different intensity levels and a rule-based classifier is implemented to identify analyzable interphase cells. In the second stage, FISH labeled biomarker spots of chromosomes 3 and X are segmented by a top-hat transform. The independent FISH spots are then detected by a knowledge-based classifier, which enables recognition of the splitting and stringy FISH signals. Finally, the ratio of abnormal interphase cells with numerical changes of chromosomes 3 and X is calculated to detect positive cases. The experimental results of four test cases showed high agreement of FISH analysis results between the automated scheme and the cytogeneticist’s analysis including 92.7% to 98.7% agreement in cell segmentation and 4.4% to 11.0% difference in cell classification. This preliminary study demonstrates that the feasibility of potentially applying the automatic FISH analysis method to expedite the screening and detecting cervical cancer at an early stage.
Fluorescence in situ hybridization (FISH); Automated FISH analysis; Cervical cancer; Computer-aided detection (CAD); Chromosomes 3 and X
JAK2V617F is found in the majority of patients with Ph- myeloproliferative neoplasms (MPNs) and has become a valuable marker for diagnosis of MPNs. However, it has also been found in many other hematological diseases, and some studies even detected the presence of JAK2V617F in normal blood samples. This casts doubt on the primary role of JAK2V617F in the pathogenesis of MPNs and its diagnostic value.
In the present study, we analyzed JAK2V617F positivity with 232 normal blood samples and 2663 patient blood, bone marrow, and amniotic fluid specimens obtained from a clinical genetics laboratory by using a simple DNA extraction method and a sensitive nested allele-specific PCR strategy.
We found JAK2V617F present in the majority (78%) of MPN patients and in a small fraction (1.8-8.7%) of patients with other specific hematological diseases but not at all in normal healthy donors or patients with non-hematological diseases. We also revealed associations of JAK2V617F with novel as well as known chromosomal abnormalities.
Our study suggests that JAK2V617F positivity is associated with specific hematological malignancies and is an excellent diagnostic marker for MPNs. The data also indicate that the nested allele-specific PCR method provides clinically relevant information and should be conducted for all cases suspected of having MPNs as well as for other related diseases.
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that predominantly affects women. Despite Klinefelter's syndrome (47,XXY) and SLE coexisting in isolated cases, no association has been established with SLE or any other autoimmune disease. Methods: Sex chromosome genotyping was performed in 981 SLE patients (213 were men). A first group of 843 SLE patients from 378 multiplex families and a second group of 138 men with non-familial SLE were evaluated. Fluorescent in situ hybridization (FISH) and karyotyping in transformed B cell lines enumerated chromosomes for selected cases.
Of 213 men with SLE, five had Klinefelter's syndrome (or 1 in 43). Four of them were heterozygous at X markers. FISH and karyotyping confirmed Klinefelter’s syndrome in the fifth. An overall rate of 235 47,XXY per 10,000 male SLE patients (95%CI: 77 to 539) was found, a dramatic increase over the known prevalence of Klinefelter's syndrome in an unselected population (17 per 10,000 live male births). Asking men with SLE about fertility was highly sensitive (100%) for Klinefelter’s syndrome. All 768 SLE women were heterozygous at X.
47,XXY Klinefelter's syndrome, often subclinical, is increased in men with SLE by ~14-fold, compared to its prevalence in men without SLE. Diagnostic vigilance for 47,XXY males in SLE is warranted. These data are the first to associate Klinefelter's syndrome with an autoimmune disease found predominantly in women. The risk of SLE in Klinefelter's syndrome is predicted to be similar to the risk in normal 46,XX women and ~14-fold higher than in 46,XY men, consistent with SLE susceptibility being partly explained by a X chromosome gene dose effect.
We developed and tested a new automated chromosome karyotyping scheme using a two-layer classification platform. Our hypothesis is that by selecting most effective feature sets and adaptively optimizing classifiers for the different groups of chromosomes with similar image characteristics, we can reduce the complexity of automated karyotyping scheme and improve its performance and robustness. For this purpose, we assembled an image database involving 6900 chromosomes and implemented a genetic algorithm to optimize the topology of multi-feature based artificial neural networks (ANN). In the first layer of the scheme, a single ANN was employed to classify 24 chromosomes into seven classes. In the second layer, seven ANNs were adaptively optimized for seven classes to identify individual chromosomes. The scheme was optimized and evaluated using a “training-testing-validation” method. In the first layer, the classification accuracy for the validation dataset was 92.9%. In the second layer, classification accuracy of seven ANNs ranged from 67.5% to 97.5%, in which six ANNs achieved accuracy above 93.7% and only one had lessened performance. The maximum difference of classification accuracy between the testing and validation datasets is <1.7%. The study demonstrates that this new scheme achieves higher and robust performance in classifying chromosomes.
Artificial neural network; genetic algorithm; karyotype; metaphase chromosome; training-testing-validation
Systemic lupus erythematosus (SLE) disproportionately affects females. Recent work demonstrates that men with Klinefelter's syndrome (47,XXY males) have a similar risk of developing SLE as do genotypic females. We present an unusual case of an African American family with two SLE affected individuals in which one of the SLE patients also has Turner's syndrome [46,X,del(X)(q13)]. While not definitive, this family raises interesting questions regarding the role of genes located on the X chromosome in the development of SLE. The paucity of case reports documenting the overlap of SLE with Turner's syndrome while there is and association of male SLE with Klinefelter's syndrome suggests a lower risk of SLE in Turner's females. These observations are consistent with a gene dose effect at X with two X chromosomes (46,XX or 47,XXY) conferring higher risk and one X chromosome (46,XY or 45,XO) conferring lower risk of SLE.
Genistein is a major soy isoflavone with multiple properties. The impact of soy/genistein on breast cancer is controversial. One of the issues is whether soy/genistein has a genotoxic effect at physiological concentrations. To address this question using an in vitro model, we first established MCF-10A/G0 and MCF-10A/G1 cell lines, which were MCF-10A cells exposed to 0.01% DMSO (as vehicle control), i.e. MCF-10A/G0, or 1 µM of genistein for three months, MCF-10A/G1, respectively. Chromosomal changes were compared between the two cell lines by routine G-banded chromosome analyses, regular CGH and oligo array-based CGH. After three months of exposure to genistein, the cell line MCF-10A/G1 showed a loss of a normal chromosome 8, a gain of extra chromosome 20, plus a loss of a chromosomal segment on the short arm of chromosome 9, which leads to a homozygous deletion of the tumor suppressor genes, INK4/p16 and INK4/p15. Our results suggest that long-term/low concentration exposure to genistein may have the potential to induce chromosomal imbalances. These genotoxic effects may work in concert with other factors to induce genetic lesions that contribute to soy/genistein associated risk.
Visual search and identification of analyzable metaphase chromosomes using optical microscopes is a very tedious and time-consuming task that is routinely performed in genetic laboratories to detect and diagnose cancers and genetic diseases. The purpose of this study is to develop and test a computerized scheme that can automatically identify chromosomes in metaphase stage and classify them into analyzable and un-analyzable groups. Two independent datasets involving 170 images are used to train and test the scheme. The scheme uses image filtering, threshold, and labeling algorithms to detect chromosomes, followed by computing a set of features for each individual chromosome as well as for each identified metaphase cell. Two machine learning classifiers including a decision tree (DT) based on the features of individual chromosomes and an artificial neural network (ANN) using the features of the metaphase cells are optimized and tested to classify between analyzable and un-analyzable cells. Using the DT based classifier the Kappa coefficients for agreement between the cytogeneticist and the scheme are 0.83 and 0.89 for the training and testing datasets, respectively. We apply an independent testing and a two-fold cross-validation method to assess the performance of the ANN-based classifier. The area under and receiver operating characteristic (ROC) curve is 0.93 for the complete dataset. This preliminary study demonstrates the feasibility of developing a computerized scheme to automatically identify and classify metaphase chromosomes.
Artificial neural network; Decision tree; Metaphase chromosomes; Microscopic digital images
Cleidocranial dysplasia (CCD) is an autosomal dominant skeletal dysplasia associated with cranial, clavicular, and dental anomalies. It is caused by mutations in the RUNX2 gene, which encodes an osteoblast-specific transcription factor and maps to chromosome 6p21. We report clinical and molecular cytogenetic studies in a patient with clinical features of CCD including wormian bones, delayed fontanel closure, hypoplastic clavicles and pubic rami, and supernumerary dentition. Additional abnormalities of bone growth and connective tissue, including easy bruisability, scarring, bleeding, joint hypermobility, and developmental delay were also observed. Molecular cytogenetic studies identified a de novo apparently balanced three-way translocation 46,XY,t(4;6;21)(p16;p21.1;q21). Further mapping revealed the breakpoint on 6p21 to be ∼50 kb upstream of exon 1 of the RUNX2 gene, with RUNX2 being intact on the derivative chromosome 6. We hypothesize that the proband's CCD has arisen from disruption of the developmentally regulated gene RUNX2 at the 6p21 breakpoint, due to a position effect mutation which may have altered the expression of the gene. Further studies might unravel a new regulatory element for RUNX2.
three-way chromosome translocation; cleidocranial dysplasia (CCD); fluorescence in situ hybridization (FISH); phenotype–genotype correlation