Xavier T. Reveles

Molecular characterization of a patient with central nervous system dysmyelination and cryptic unbalanced translocation between chromosomes 4q and 18q.

We report on a 12‐year‐old boy who presented with delayed development and CNS dysmyelination. Genetic studies showed a normal 46,XY karyotype by routine cytogenetic analysis, and 46,XY.ish del(18)(q23)(D18Z1+, MBP−) by FISH using a locus‐specific probe for the MBP gene (18q23). Though the patient appeared to have normal chromosome 18s by repeated high resolution banding analysis, his clinical features were suggestive of a deletion of 18q. These included hearing loss secondary to stenosis of the external auditory canals, abnormal facial features, and foot deformities. FISH studies with genomic probes from 18q22.3 to 18qter confirmed a cryptic deletion which encompassed the MBP gene. In an attempt to further characterize the deletion, whole genome screening was conducted using array based comparative genomic hybridization (array CGH) analysis. The array CGH data not only confirmed a cryptic deletion in the 18q22.3 to 18qter region of approximately 7 Mb, it also showed a previously undetected 3.7 Mb gain of 4q material. FISH studies demonstrated that the gained 4q material was translocated distal to the 18qter deletion breakpoint. The 18q deletion contains, in addition to MBP, other known genes including CYB5, ZNF236, GALR1, and NFATC1, while the gained 4q material includes the genes FACL1 and 2, KLKB1, F11 and MTNR1A. The use of these combined methodologies has resulted in the first reported case in which array CGH has been used to characterize a congenital chromosomal abnormality, highlighting the need for innovative molecular cytogenetic techniques in the diagnosis of patients with idiopathic neurological abnormalities.

Detection of Recurrent Copy Number Loss at Yp11.2 Involving TSPY Gene Cluster in Prostate Cancer Using Array-Based Comparative Genomic Hybridization

Prostate cancer is the second leading cause of cancer deaths among American men. The loss of Y chromosome has been frequently observed in primary prostate cancer as well as other types of cancer. Earlier, we showed that introduction of the human Y chromosome suppresses the in vivo tumorigenicity of the prostate cancer cell line PC-3. To further characterize the Y chromosome, we have developed a high-density bacterial artificial chromosome (BAC) microarray containing 178 BAC clones from the human Y chromosome. BAC microarray was used for array comparative genomic hybridization on prostate cancer samples and cell lines. The most prominent observation on prostate cancer specimens was a deletion at Yp11.2 containing the TSPY tandem gene array. Out of 36 primary prostate tumors analyzed, 16 (44.4%) samples exhibited loss of TSPY gene copies. Notably, we observed association between the number of TSPY copies in the blood and the incidence of prostate cancer. Moreover, PC-3 hybrids with an intact Yp11.2 did not grow tumors in nude mice, whereas PC-3 hybrids with a deletion at Yp11.2 grew tumors in nude mice.

PHC3, a component of the hPRC-H complex, associates with E2F6 during G0 and is lost in osteosarcoma tumors

Polyhomeotic-like 3 (PHC3) is a ubiquitously expressed member of the polycomb gene family and part of the human polycomb complex hPRC-H. We found that in normal cells PHC3 associated with both hPRC-H complex components and with the transcription factor E2F6. In differentiating and confluent cells, PHC3 and E2F6 showed nuclear colocalization in a punctate pattern that resembled the binding of polycomb bodies to heterochromatin. This punctate pattern was not seen in proliferating cells suggesting that PHC3 may be part of an E2F6-polycomb complex that has been shown to occupy and silence target promoters in G0. Previous loss of heterozygosity (LoH) analyses had shown that the region containing PHC3 underwent frequent LoH in primary human osteosarcoma tumors. When we examined normal bone and human osteosarcoma tumors, we found loss of PHC3 expression in 36 of 56 osteosarcoma tumors. Sequence analysis revealed that PHC3 was mutated in nine of 15 primary osteosarcoma tumors. These findings suggest that loss of PHC3 may favor tumorigenesis by potentially disrupting the ability of cells to remain in G0.

Molecular characterization of 18p deletions: Evidence for a breakpoint cluster

Purpose: To determine the size and parental origin of the deletion in individuals with 18p− syndrome.Methods: Molecular and fluorescence in situ hybridization analyses of the pericentromeric region of chromosome 18 were performed on genomic DNA and chromosomes from study participants.Results: The majority of the breakpoints were located between markers D18S852 on 18p and D18S1149 on 18q, a distance of approximately 4 Mb. The parental origin of these deletions appears to be equally distributed, half maternally derived and half paternally derived.Conclusion: The distributions of both the size and parental origin of the 18p deletions support the presence of a breakpoint cluster in the 18p− syndrome.

Golli-MBP copy number analysis by FISH, QMPSF and MAPH in 195 patients with hypomyelinating leukodystrophies

The inherited disorders of CNS myelin formation represent a heterogeneous group of leukodystrophies. The proteolipoprotein (PLP1) gene has been implicated in two X‐linked forms, Pelizaeus‐Merzbacher disease (PMD) and spastic paraplegia type 2, and the gap junction protein α12 (GJA12) gene in a recessive form of PMD. The myelin basic protein (MBP) gene, which encodes the second most abundant CNS myelin protein after PLP1, presents rearrangements in hypomyelinating murine mutants and is always included in the minimal region deleted in 18q‐ patients with an abnormal hypomyelination pattern on cerebral MRI. In this study, we looked at the genomic copy number at the Golli‐MBP locus in 195 patients with cerebral MRI suggesting a myelin defect, who do not have PLP1 mutation. Although preliminary results obtained by FISH suggested the duplication of Golli‐MBP in 3 out of 10 patients, no abnormal gene quantification was found using Quantitative Multiplex PCR of Short Fluorescent fragments (QMPSF), Multiplex Amplifiable Probe Hybridization (MAPH), or another FISH protocol using directly‐labelled probes. Pitfalls and interest in these different techniques to detect duplication events are emphasised. Finally, the study of this large cohort of patients suggests that Golli‐MBP deletion or duplication is rarely involved in inherited defects of myelin formation.

Use of DNA sequencing analysis to confirm fungemia due to Trichosporon dermatis in a pediatric patient.

ABSTRACT This is the first reported case of human disease caused by Tricosporon dermatis, an organism recently transferred to the genus Trichosporon from Cryptococcus and now confirmed to be a human pathogen.

Chromosome 18 suppresses prostate cancer metastases

Loss of heterozygosity and allelic imbalance data has shown that there are two distinct regions of loss on chromosome 18q associated with the progression of prostate cancer (CaP). To investigate the functional significance of chromosome 18q loci in CaP, we utilized the technique of microcell-mediated chromosome transfer to introduce an intact chromosome 18 into the human prostate cancer cell line, PC-3. Three of the resulting hybrid lines were compared to the PC-3 cells in vitro and in vivo. The hybrid cell lines, containing an intact copy of the introduced chromosome 18, exhibited a substantial reduction in anchorage-dependent and independent growth in vitro. These hybrid cell lines also made smaller tumors in nude mice following subcutaneous injection compared to PC-3 cells. Because tumor growth was not completely eliminated by introduction of chromosome 18, we assessed the ability of the hybrids to metastasize to bone after intra-cardiac inoculation in a nude mouse model. Mice inoculated with PC-3 hybrids containing intact copies of chromosome 18 had significantly fewer bone metastases and dramatically improved survival compared to PC-3 cells. In addition, the introduction of chromosome 18 significantly reduced tumor burden in extraskeletal sites. This was not because of differences in growth rates because mice bearing hybrids were monitored for metastases over twice as long as mice bearing PC-3 cells. Taken together, these data suggest that chromosome 18 has a functional role in CaP to suppress growth and metastases. Identification of the responsible gene(s) may lead to molecular targets for drug discovery.

Molecular cytogenetics as a clinical test for prognostic and predictive biomarkers in newly diagnosed ovarian cancer

BackgroundThere is a clinical need for routinely available genomic biomarker testing in newly diagnosed ovarian cancer. In the current study we performed molecular cytogenetics using a validated array based comparative genomic hybridization (array CGH) assay to screen for the presence of predictive and prognostic biomarkers in archival diagnostic tissue from ovarian cancer patients. We hypothesized that biomarkers of high-risk disease would be detectable in tumor samples from patients with treatment refractory, advanced disease, and would be detected less frequently in tumor samples from patients with more favorable outcomes. In addition, we predicted that the use of a genome-wide copy number analysis (CNA) testing platform would enable us to identify novel potentially targetable chromosomal alterations of therapeutic significance in a percentage of cases.MethodsFormalin-fixed paraffin-embedded tissue (FFPE) tumor bank specimens were retrieved from the initial surgical resection for 18 ovarian cancer patients. Molecular cytogenetics was performed by array CGH for the detection of somatic chromosomal alterations associated with high-risk disease including amplifications of the CCNE1 and HER2 genes. Genomic risk stratification results were correlated with available clinical data. CGH data from each patient’s tumor genome was also surveyed for the presence of potentially targetable aberrations. Relevant therapeutic agents and open studies for investigational drugs were reported for each patient.ResultsHigh-risk genomic alterations were identified in 12/18 (67%) of cases and all patients with high-risk markers had advanced, treatment refractory disease. Three tumors with minimal genomic changes had no high-risk markers and were from patients with Stage I/II disease that had been completely resected and under surveillance for recurrence. Eleven patients (61%) had at least one potentially targetable genomic alteration including CCNE1, HER2, KRAS gene amplifications, and somatic BRCA1 and/or BRCA2 gene deletions. Bi-allelic PTEN gene deletion was detected in one patient’s tumor.ConclusionsClinical genomic profiling of ovarian tumors by array CGH augments pathologic grade and stage to help stratify newly diagnosed ovarian cancer into high and low-risk disease. This personalized genomic information can also help guide treatment planning and disease monitoring by identifying novel potentially targetable genomic alterations that can be used by clinicians to choose rational directed therapies for patients with chemo-resistant disease.