Joan H. M. Knoll

Inhibition of telomerase limits the growth of human cancer cells

Telomerase is a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, called telomeres. In most human somatic cells, telomerase expression is repressed, and telomeres shorten progressively with each cell division. In contrast, most human tumors express telomerase, resulting in stabilized telomere length. These observations indicate that telomere maintenance is essential to the proliferation of tumor cells. We show here that expression of a mutant catalytic subunit of human telomerase results in complete inhibition of telomerase activity, reduction in telomere length and death of tumor cells. Moreover, expression of this mutant telomerase eliminated tumorigenicity in vivo. These observations demonstrate that disruption of telomere maintenance limits cellular lifespan in human cancer cells, thus validating human telomerase reverse transcriptase as an important target for the development of anti-neoplastic therapies.

Angelman syndrome 2005: updated consensus for diagnostic criteria.

In 1995, a consensus statement was published for the purpose of summarizing the salient clinical features of Angelman syndrome (AS) to assist the clinician in making a timely and accurate diagnosis. Considering the scientific advances made in the last 10 years, it is necessary now to review the validity of the original consensus criteria. As in the original consensus project, the methodology used for this review was to convene a group of scientists and clinicians, with experience in AS, to develop a concise consensus statement, supported by scientific publications where appropriate. It is hoped that this revised consensus document will facilitate further clinical study of individuals with proven AS, and assist in the evaluation of those who appear to have clinical features of AS but have normal laboratory diagnostic testing.

Identification of Four Highly Conserved Genes between Breakpoint Hotspots BP1 and BP2 of the Prader-Willi/Angelman Syndromes Deletion Region That Have Undergone Evolutionary Transposition Mediated by Flanking Duplicons

Prader-Willi and Angelman syndromes (PWS and AS) typically result from an approximately 4-Mb deletion of human chromosome 15q11-q13, with clustered breakpoints (BP) at either of two proximal sites (BP1 and BP2) and one distal site (BP3). HERC2 and other duplicons map to these BP regions, with the 2-Mb PWS/AS imprinted domain just distal of BP2. Previously, the presence of genes and their imprinted status have not been examined between BP1 and BP2. Here, we identify two known (CYFIP1 and GCP5) and two novel (NIPA1 and NIPA2) genes in this region in human and their orthologs in mouse chromosome 7C. These genes are expressed from a broad range of tissues and are nonimprinted, as they are expressed in cells derived from normal individuals, patients with PWS or AS, and the corresponding mouse models. However, replication-timing studies in the mouse reveal that they are located in a genomic domain showing asynchronous replication, a feature typically ascribed to monoallelically expressed loci. The novel genes NIPA1 and NIPA2 each encode putative polypeptides with nine transmembrane domains, suggesting function as receptors or as transporters. Phylogenetic analyses show that NIPA1 and NIPA2 are highly conserved in vertebrate species, with ancestral members in invertebrates and plants. Intriguingly, evolutionary studies show conservation of the four-gene cassette between BP1 and BP2 in human, including NIPA1/2, CYFIP1, and GCP5, and proximity to the Herc2 gene in both mouse and Fugu. These observations support a model in which duplications of the HERC2 gene at BP3 in primates first flanked the four-gene cassette, with subsequent transposition of these four unique genes by a HERC2 duplicon-mediated process to form the BP1-BP2 region. Duplicons therefore appear to mediate genomic fluidity in both disease and evolutionary processes.

The γ-aminobutyric acid receptor γ3 subunit gene (GABRG3) is tightly linked to the α5 subunit gene (GABRA5) on human chromosome 15q11–q13 and is transcribed in the same orientation

Abstract GABAA receptors are heterooligomeric ligand-gated ion channels that mediate the effect of the inhibitory neurotransmitter γ-aminobutyric acid. The GABAA receptors consist of at least 15 different receptor subunits that can be classified into 5 subfamilies (α, β, γ, δ, ϱ) on the basis of sequence similarity. Chromosomal mapping studies have revealed that several of the GABAA receptor subunit genes appear to be organized as clusters. One such cluster, which consists of the GABAA receptor β3 (GABRB3) and α5 (GABRA5) subunit genes, is located in chromosome 15q11–q13. It is shown here that the GABAA receptor γ3 subunit gene (GABRG3) also maps to this region. Lambda and P1 phage clones surrounding both ends of GABRG3 were isolated; the clones derived from the 5′ end of GABRG3 were linked to an existing phage contig spanning the 3′ end of GABRA5. The two genes are located within 35 kb of each other and are transcribed in the same orientation.

In Situ Hybridization to Metaphase Chromosomes and Interphase Nuclei

In situ hybridization is used to determine the chromosomal map location and the relative order of genes and DNA sequences within a chromosomal band. It can also be used to detect aneuploidy, gene amplification, and subtle chromosomal rearrangements. Fluorescence in situ hybridization (FISH), probably the most widely used method, is described in the first basic protocol. Two support protocols are provided to amplify weak fluorescent signals obtained in FISH. Nonisotopic probes can also be detected by enzymatic reactions using horseradish peroxidase or alkaline phosphatase, as described in alternate protocols. Nonisotopic labeling of DNA probes by nick translation is described in a support protocol. The order of closely spaced FISH probes along chromosomes in interphase nuclei can be determined. A basic protocol for isotopic in situ hybridization (IISH) with 3H is provided followed by a support protocol for preparation of autoradiographic emulsion.

A 9-year-old Male with a Duplication of Chromosome 3p25.3p26.2: Clinical Report and Gene Expression Analysis

We describe a 9‐year‐old male referred for genetic evaluation for Prader‐Willi syndrome (PWS). PWS is the most common genetically defined cause of life‐threatening obesity and results from a functional loss of paternally expressed genes from the chromosome 15q11‐q13 region. The patient presented with pervasive developmental disorder, delayed speech, and rapid onset of obesity at age 4 years, all features similar to PWS. However, chromosome 15q11‐q13 methylation testing and fragile X studies were normal. GTG‐banding and fluorescence in situ hybridization (FISH) with whole chromosome 3 paint probe (WCP3) and a chromosome 3p subtelomeric probe suggested a duplication of 3p25.3p26.2, a finding supported by comparative genomic hybridization (CGH). This region of chromosome 3p contains genes which contribute to obesity and behavioral problems, most notably, ghrelin (GHRL), an oxytocin receptor (OXTR), solute carrier family six members (gamma‐aminobutyric acid (GABA) neurotransmitter transporters, SLC6A1 and SLC6A11), and peroxisome proliferator‐activated receptor gamma (PPARG). To characterize these obesity and behavior related genes in our subject, we performed quantitative RT‐PCR and compared expression levels with similarly aged male subjects (four non‐obese males, four obese males, and four PWS males—two with 15q11‐q13 deletions and two with maternal disomy 15). Our studies suggest increased expression of several genes in the 3p duplication region, including GHRL and PPARG, which may contribute to the phenotypic features in our 3p duplication subject.

The syntenic relationship between the critical deletion region for the Prader-Willi/Angelman syndromes and proximal mouse chromosome 7

The deleted region of the proximal long arm of human chromosome 15, common to a large group of patients with the Prader-Willi and Angelman syndromes, has recently been defined. We have mapped to the mouse genome segments homologous to human probes found within and flanking this deletional region. These probes define a region of conserved synteny on proximal chromosome 7 of the mouse. Because the Prader-Willi and Angelman syndromes are postulated to result from genomic imprinting within the common deletion, these probes may define a genomically imprinted region on mouse chromosome 7.

Sequence-Based, In Situ Detection of Chromosomal Abnormalities at High Resolution

We developed single copy probes from the draft genome sequence for fluorescence in situ hybridization (scFISH) which precisely delineate chromosome abnormalities at a resolution equivalent to genomic Southern analysis. This study illustrates how scFISH probes detect cryptic and subtle abnormalities and localize the sites of chromosome rearrangements. scFISH probes are substantially shorter than conventional recombinant DNA‐derived probes, and Cot1 DNA is not required to suppress repetitive sequence hybridization. In this study, 74 single copy sequence probes (>1,500 bp) have been developed from ≥100 kb genomic intervals associated with either constitutional or acquired disorders. Applications of these probes include detection of congenital microdeletion syndromes on chromosomes 1, 4, 7, 15, 17, 22 and submicroscopic deletions involving the imprinting center on chromosome 15q11.2q13. We demonstrate how hybridization with multiple combinations of probes derived from the Smith‐Magenis syndrome interval on chromosome 17 identified a patient with an atypical, proximal deletion breakpoint. A similar multi‐probe hybridization strategy has also been used to delineate the translocation breakpoint region on chromosome 9 in chronic myelogenous leukemia. Probes have also been designed to hybridize to multiple cis paralogs, both enhancing the chromosomal target size and detecting chromosome rearrangements, for example, by splitting and separating a family of related sequences flanking an inversion breakpoint on chromosome 16 in acute myelogenous leukemia. These novel strategies for rapid and precise characterization of cytogenetic abnormalities are feasible because of the sequence‐defined properties and dense euchromatic organization of single copy probes.

The oxytocin receptor gene (OXTR) localizes to human chromosome 3p25 by fluorescence in situ hybridization and PCR analysis of somatic cell hybrids

The human oxytocin receptor regulates parturition and myometrial contractility, breast milk let-down, and reproductive behaviors in the mammalian central nervous system. Kimura et al. recently identified a human oxytocin receptor cDNA by means of expression cloning from a human myometrial cDNA library. To elucidate further the molecular mechanisms that regulate oxytocin receptor gene expression and to define the expected Mendelian inheritance of possible human disease states, we must determine the number of genes, their localization, and their organization and structure. We summarize below our data indicating that the human oxytocin receptor gene is localized to 3p25 and exists as a single copy in the haploid genome. 9 refs., 2 figs.

In situ Hybridization and Detection Using Nonisotopic Probes

Nonisotopic in situ hybridization can be used to determine the cellular location and relative levels of expression for specific transcripts within cells and tissues. RNA in specimen preparations is hybridized with a biotin‐ or digoxigenin‐labeled probe, which is generally detected by fluorescence or enzymatic methods. Fluorescence in situ hybridization (FISH), probably the most widely used method, is described here, along with amplification of weak FISH signals. Nonisotopic probes can also be detected by enzymatic reactions using horseradish peroxidase or alkaline phosphatase, as described here. Curr. Protoc. Mol. Biol. 79:14.7.1‐14.7.17.