Cynthia C. Morton

Replicating genotype-phenotype associations.

What constitutes replication of a genotype–phenotype association, and how best can it be achieved?

Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness

H+-ATPases are ubiquitous in nature; V-ATPases pump protons against an electrochemical gradient, whereas F-ATPases reverse the process, synthesizing ATP. We demonstrate here that mutations in ATP6B1, encoding the B-subunit of the apical proton pump mediating distal nephron acid secretion, cause distal renal tubular acidosis, a condition characterized by impaired renal acid secretion resulting in metabolic acidosis. Patients with ATP6B1 mutations also have sensorineural hearing loss; consistent with this finding, we demonstrate expression of ATP6B1 in cochlea and endolymphatic sac. Our data, together with the known requirement for active proton secretion to maintain proper endolymph pH, implicate ATP6B1 in endolymph pH homeostasis and in normal auditory function. ATP6B1 is the first member of the H+-ATPase gene family in which mutations are shown to cause human disease.

Disruption of the architectural factor HMGI-C: DNA-binding AT hook motifs fused in lipomas to distinct transcriptional regulatory domains

Lipomas are one of the most common mesenchymal neoplasms in humans. They are characterized by consistent cytogenetic aberrations involving chromosome 12 in bands q14-15. Interestingly, this region is also the site of rearrangement for other mesenchymally derived tumors. This study demonstrates that HMGI-C, an architectural factor that functions in transcriptional regulation, has been disrupted by rearrangement at the 12q14-15 chromosomal breakpoint in lipomas. Chimeric transcripts were isolated from two lipomas in which HMGI-C DNA-binding domains (AT hook motifs) are fused to either a LIM or an acidic transactivation domain. These results, identifying a gene rearranged in a benign neoplastic process that does not proceed to a malignancy, suggest a role for HMGI-C in adipogenesis and mesenchyme differentiation.

Unrepaired DNA breaks in p53-deficient cells lead to oncogenic gene amplification subsequent to translocations.

Amplification of large genomic regions associated with complex translocations (complicons) is a basis for tumor progression and drug resistance. We show that pro-B lymphomas in mice deficient for both p53 and nonhomologous end-joining (NHEJ) contain complicons that coamplify c-myc (chromosome 15) and IgH (chromosome 12) sequences. While all carry a translocated (12;15) chromosome, coamplified sequences are located within a separate complicon that often involves a third chromosome. Complicon formation is initiated by recombination of RAG1/2-catalyzed IgH locus double-strand breaks with sequences downstream of c-myc, generating a dicentric (15;12) chromosome as an amplification intermediate. This recombination event employs a microhomology-based end-joining repair pathway, as opposed to classic NHEJ or homologous recombination. These findings suggest a general model for oncogenic complicon formation.

Tumor-Associated Endothelial Cells with Cytogenetic Abnormalities

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Tumor blood vessels have been shown to differ from normal counterparts, for example, by changes in morphology. An important concept in tumor angiogenesis is that tumor endothelial cells are assumed to be genetically normal, although these endothelial cells are structurally and functionally abnormal. However, we hypothesized that given the phenotypic differences between tumor and normal blood vessels, there may be genotypic alterations as well. Mouse endothelial cells were isolated from two different human tumor xenografts, melanoma and liposarcoma, and from two normal endothelial cell counterparts, skin and adipose. Tumor-associated endothelial cells expressed typical endothelial cell markers, such as CD31. They had relatively large, heterogeneous nuclei. Unexpectedly, tumor endothelial cells were cytogenetically abnormal. Fluorescence in situ hybridization (FISH) analysis showed that freshly isolated uncultured tumor endothelial cells were aneuploid and had abnormal multiple centrosomes. The degree of aneuploidy was exacerbated by passage in culture. Multicolor FISH indicated that the structural chromosomal aberrations in tumor endothelial cells were heterogeneous, indicating that the cytogenetic alterations were not clonal. There was no evidence of human tumor-derived chromosomal material in the mouse tumor endothelial cells. In marked contrast, freshly isolated normal skin and adipose endothelial cells were diploid, had normal centrosomes, and remained cytogenetically stable in culture even up to 20 passages. FISH analysis of tumor sections also showed endothelial cell aneuploidy. We conclude that tumor endothelial cells can acquire cytogenetic abnormalities while in the tumor microenvironment.

Integration of cytogenetic landmarks into the draft sequence of the human genome

We have placed 7,600 cytogenetically defined landmarks on the draft sequence of the human genome to help with the characterization of genes altered by gross chromosomal aberrations that cause human disease. The landmarks are large-insert clones mapped to chromosome bands by fluorescence in situ hybridization. Each clone contains a sequence tag that is positioned on the genomic sequence. This genome-wide set of sequence-anchored clones allows structural and functional analyses of the genome. This resource represents the first comprehensive integration of cytogenetic, radiation hybrid, linkage and sequence maps of the human genome; provides an independent validation of the sequence map and framework for contig order and orientation; surveys the genome for large-scale duplications, which are likely to require special attention during sequence assembly; and allows a stringent assessment of sequence differences between the dark and light bands of chromosomes. It also provides insight into large-scale chromatin structure and the evolution of chromosomes and gene families and will accelerate our understanding of the molecular bases of human disease and cancer.

Diagnostic relevance of clonal cytogenetic aberrations in malignant soft tissue tumors

BACKGROUND Malignant soft-tissue tumors often present substantial diagnostic challenges. Chromosome aberrations that might be diagnostic have been identified in some types of soft-tissue tumors, but the overall frequency and diagnostic relevance of these aberrations have not been established. METHODS We attempted to determine the karyotypes of a series of 62 consecutive, unselected malignant spindle-cell or small round-cell soft-tissue tumors (from 46 adults and 16 children) after direct harvesting of cells or short-term culture. All tumors were examined independently by immunohistochemical staining in addition to routine light-microscopical evaluation, and all but two tumors were examined by electron microscopy. RESULTS Metaphases were obtained from 61 of the 62 tumors, and clonal chromosome aberrations were identified in 55 (89 percent). In the six tumors that yielded metaphases but lacked apparent clonal aberrations, the normal metaphases were found to originate from non-neoplastic stromal elements within the tumor specimens. Thus, all tumors in which karyotyping was successful contained clonal chromosome aberrations. Forty of 62 tumors (65 percent) contained clonal chromosome aberrations that either suggested or confirmed a specific diagnosis; in 15 of these tumors (24 percent of all tumors), the aberrations were important in establishing the final diagnosis. Cytogenetic analyses were particularly informative about small round-cell tumors from children: 8 of 14 round-cell tumors contained diagnostically important chromosome aberrations. Using the combined approaches of light and electron microscopy, immunohistochemistry, and cytogenetics, we established an unambiguous diagnosis for 60 of 62 tumors. CONCLUSIONS Cytogenetic analyses reveal clonal chromosome aberrations in virtually all malignant soft-tissue tumors. These clonal chromosome aberrations, particularly in small round-cell tumors in children, often have diagnostic relevance.

Mutations in a novel cochlear gene cause DFNA9, a human nonsyndromic deafness with vestibular dysfunction

DFNA9 is an autosomal dominant, nonsyndromic, progressive sensorineural hearing loss with vestibular pathology. Here we report three missense mutations in human COCH (previously described as Coch5b2), a novel cochlear gene, in three unrelated kindreds with DFNA9. All three residues mutated in DFNA9 are conserved in mouse and chicken Coch, and are found in a region containing four conserved cysteines with homology to a domain in factor C, a lipopolysaccharide-binding coagulation factor in Limulus polyphemus. COCH message, found at high levels in human cochlear and vestibular organs, occurs in the chicken inner ear in the regions of the auditory and vestibular nerve fibres, the neural and abneural limbs adjacent to the cochlear sensory epithelium and the stroma of the crista ampullaris of the vestibular labyrinth. These areas correspond to human inner ear structures which show histopathological findings of acidophilic ground substance in DFNA9 patients.

Hodgkin's Disease, Lymphomatoid Papulosis, and Cutaneous T-Cell Lymphoma Derived from a Common T-Cell Clone

BACKGROUND Lymphomatoid papulosis is a benign cutaneous eruption that in 10 to 20 percent of patients is associated with the development of lymphoma. The atypical cells of lymphomatoid papulosis histologically resemble the malignant cells of cutaneous T-cell lymphoma or the Reed-Sternberg cells of Hodgkins disease. We studied a patient in whom lymphomatoid papulosis developed in 1971, Hodgkins disease in 1975, and cutaneous T-cell lymphoma in 1985, to determine whether these diseases are clonally related. METHODS The T-cell-receptor alpha-chain gene was cloned and sequenced from a cell line derived from the advanced-stage cutaneous T-cell lymphoma, and the polymerase chain reaction was used to search for this rearrangement of the alpha-chain gene in tissues obtained earlier that were affected by Hodgkins disease or lymphomatoid papulosis. RESULTS The tumor-specific rearrangement of the alpha-chain gene was detected in the patients earlier tissues affected by lymphomatoid papulosis and Hodgkins disease, but not in control tissue, including uninvolved tissues from the staging laparotomy for Hodgkins disease. Cytogenetic studies revealed a translocation, t(8;9)(p22;p24), in cutaneous T-cell lymphoma lines and in a dermatopathic lymph node removed two years before the clinical onset of the cutaneous T-cell lymphoma. Immunohistochemical findings were consistent with an activated T-cell phenotype for the atypical cells of lymphomatoid papulosis, the Reed-Sternberg cells of Hodgkins disease, and the malignant cells of the T-cell lymphoma. CONCLUSIONS Lymphomatoid papulosis, Hodgkins disease, and cutaneous T-cell lymphoma can be derived from a single T-cell clone. A t(8;9) genetic translocation may be involved in the pathogenesis of lymphomatoid papulosis or its progression to malignant disease.

Novel ATP6V1B1 and ATP6V0A4 mutations in autosomal recessive distal renal tubular acidosis with new evidence for hearing loss

Autosomal recessive distal renal tubular acidosis (rdRTA) is characterised by severe hyperchloraemic metabolic acidosis in childhood, hypokalaemia, decreased urinary calcium solubility, and impaired bone physiology and growth. Two types of rdRTA have been differentiated by the presence or absence of sensorineural hearing loss, but appear otherwise clinically similar. Recently, we identified mutations in genes encoding two different subunits of the renal α-intercalated cell’s apical H+-ATPase that cause rdRTA. Defects in the B1 subunit gene ATP6V1B1, and the a4 subunit gene ATP6V0A4, cause rdRTA with deafness and with preserved hearing, respectively. We have investigated 26 new rdRTA kindreds, of which 23 are consanguineous. Linkage analysis of seven novel SNPs and five polymorphic markers in, and tightly linked to, ATP6V1B1 and ATP6V0A4 suggested that four families do not link to either locus, providing strong evidence for additional genetic heterogeneity. In ATP6V1B1, one novel and five previously reported mutations were found in 10 kindreds. In 12 ATP6V0A4 kindreds, seven of 10 mutations were novel. A further nine novel ATP6V0A4 mutations were found in “sporadic” cases. The previously reported association between ATP6V1B1 defects and severe hearing loss in childhood was maintained. However, several patients with ATP6V0A4 mutations have developed hearing loss, usually in young adulthood. We show here that ATP6V0A4 is expressed within the human inner ear. These findings provide further evidence for genetic heterogeneity in rdRTA, extend the spectrum of disease causing mutations in ATP6V1B1 and ATP6V0A4, and show ATP6V0A4 expression within the cochlea for the first time.