Louise H. Williams

Identification of Candidate Growth Promoting Genes in Ovarian Cancer through Integrated Copy Number and Expression Analysis

Ovarian cancer is a disease characterised by complex genomic rearrangements but the majority of the genes that are the target of these alterations remain unidentified. Cataloguing these target genes will provide useful insights into the disease etiology and may provide an opportunity to develop novel diagnostic and therapeutic interventions. High resolution genome wide copy number and matching expression data from 68 primary epithelial ovarian carcinomas of various histotypes was integrated to identify genes in regions of most frequent amplification with the strongest correlation with expression and copy number. Regions on chromosomes 3, 7, 8, and 20 were most frequently increased in copy number (>40% of samples). Within these regions, 703/1370 (51%) unique gene expression probesets were differentially expressed when samples with gain were compared to samples without gain. 30% of these differentially expressed probesets also showed a strong positive correlation (r≥0.6) between expression and copy number. We also identified 21 regions of high amplitude copy number gain, in which 32 known protein coding genes showed a strong positive correlation between expression and copy number. Overall, our data validates previously known ovarian cancer genes, such as ERBB2, and also identified novel potential drivers such as MYNN, PUF60 and TPX2.

Copy Number Analysis Identifies Novel Interactions Between Genomic Loci in Ovarian Cancer

Ovarian cancer is a heterogeneous disease displaying complex genomic alterations, and consequently, it has been difficult to determine the most relevant copy number alterations with the scale of studies to date. We obtained genome-wide copy number alteration (CNA) data from four different SNP array platforms, with a final data set of 398 ovarian tumours, mostly of the serous histological subtype. Frequent CNA aberrations targeted many thousands of genes. However, high-level amplicons and homozygous deletions enabled filtering of this list to the most relevant. The large data set enabled refinement of minimal regions and identification of rare amplicons such as at 1p34 and 20q11. We performed a novel co-occurrence analysis to assess cooperation and exclusivity of CNAs and analysed their relationship to patient outcome. Positive associations were identified between gains on 19 and 20q, gain of 20q and loss of X, and between several regions of loss, particularly 17q. We found weak correlations of CNA at genomic loci such as 19q12 with clinical outcome. We also assessed genomic instability measures and found a correlation of the number of higher amplitude gains with poorer overall survival. By assembling the largest collection of ovarian copy number data to date, we have been able to identify the most frequent aberrations and their interactions.

Are there any more ovarian tumor suppressor genes? A new perspective using ultra high-resolution copy number and loss of heterozygosity analysis.

Ovarian cancer is characterized by complex genetic alterations, including copy number loss and copy number‐neutral loss of heterozygosity (LOH). These alterations are assumed to represent the “second hit” of the underlying tumor suppressor gene (TSG), however, relative to the number of LOH hotspots reported, few ovarian TSGs have been identified. We conducted a high‐resolution LOH analysis using SNP arrays (500K and SNP6.0) of 106 primary ovarian tumors of various histological subtypes together with matching normal DNA. LOH was detected in at least 35% of samples on chromosomes 17, 19p, 22q, Xp, 13q, 8p, 6q, 4q, 5q, 1p, 16q, and 9q with a median minimal region of overlap of only 300 kb. Subtype‐specific differences in LOH frequency were noted, particularly for mucinous cases. We also identified 192 somatic homozygous deletions (HDs). Recurrent HDs targeted known TSGs such as CDKN2A (eight samples), RB1 (five samples), and PTEN (three samples). Additional recurrent HDs targeted 16 candidate TSGs near minimal regions of LOH on chromosomes 17, 13, 8p, 5q, and X. Given the importance of HDs in inactivating known genes, these candidates are highly likely to be ovarian TSGs. Our data suggest that the poor success of previous LOH studies was due to the inability of previous technology to resolve complex genomic alterations and distinguish true LOH from allelic imbalance. This study shows that recurrent regions of LOH and HD frequently align with known TSGs suggesting that LOH analysis remains a valid approach to discovering new candidates.

Genetic and Epigenetic Analysis of CHEK2 in Sporadic Breast, Colon, and Ovarian Cancers

Purpose: Germ-line variants in CHEK2 have been associated with increased breast, thyroid, prostate, kidney, and colorectal cancer risk; however, the prevalence of somatic inactivation of CHEK2 in common cancer types is less clear. The aim of this study was to determine if somatic mutation and/or epigenetic modification play a role in development of sporadic breast, colon, or ovarian cancers. Experimental Design: We undertook combined genetic and epigenetic analysis of CHEK2 in sporadic primary breast, ovarian, and colon tumors [all exhibiting chromosome 22q loss of heterozygosity (LOH)] and cancer cell lines. Expression of Chk2 was assessed by immunohistochemistry in 119 ovarian tumors. Results: Two novel germ-line variants were identified; however, none of the primary tumors harbored somatic mutations. Two CpG clusters previously implicated in CHEK2 silencing were investigated for evidence of hypermethylation. No methylation was detected at the distal CpG island. The proximal CpG cluster was methylated in all tumor and normal DNA, suggesting that this might not represent a true CpG island and is not relevant in the control of CHEK2 expression. Twenty-three percent of ovarian tumors were negative for Chk2 protein by immunohistochemistry, but there was no significant correlation between LOH across the CHEK2 locus and intensity of Chk2 staining (P = 0.12). Conclusions: LOH across the CHEK2 locus is common in sporadic breast, ovarian, and colorectal cancers, but point mutation or epigenetic inactivation of the retained allele is uncommon. Loss of Chk2 protein in ovarian cancer was not associated with allelic status, suggesting that inactivation does not occur as a consequence of haploinsufficiency.

An ENU-Induced Mutation of Cdh23 Causes Congenital Hearing Loss, but No Vestibular Dysfunction, in Mice

Mutations in the human cadherin 23 (CDH23) gene cause deafness, neurosensory, autosomal recessive 12 (DFNB12) nonsyndromic hearing loss or Usher syndrome, type 1D (characterized by hearing impairment, vestibular dysfunction, and visual impairment). Reported waltzer mouse strains each harbor a Cdh23-null mutation and present with hearing loss and vestibular dysfunction. Two additional Cdh23 mouse mutants, salsa and erlong, each carry a homozygous Cdh23 missense mutation and have progressive hearing loss. We report the identification of a novel mouse strain, jera, with inherited hearing loss caused by an N-ethyl-N-nitrosourea-induced c.7079T>A mutation in the Cdh23 gene. The mutation generates a missense change, p.V2360E, in Cdh23. Affected mice have profound sensorineural deafness, with no vestibular dysfunction. The p.V2360E mutation is semidominant because heterozygous mice have milder and more progressive hearing loss in advanced age. The mutation affects a highly conserved Ca(2+)-binding motif in extracellular domain 22, thought to be important for Cdh23 structure and dimerization. Molecular modeling suggests that the Cdh23(V2360E/V2360E) mutation alters the structural conformation of the protein and affects Ca(2+)-binding properties. Similar to salsa mice, but in contrast to waltzer mice, hair bundle development is normal in jera and hearing loss appears to be due to the loss of tip links. Thus, jera is a novel mouse model for DFNB12.

Identification of Three Novel Hearing Loss Mouse Strains with Mutations in the Tmc1 Gene

We report the identification of three new mouse models, baringo, nice, and stitch, with recessively inherited sensorineural deafness due to novel mutations in the transmembrane channel-like gene 1 (Tmc1). These strains were generated by N-ethyl-N-nitrosourea mutagenesis. DNA sequence analysis revealed changes in c.545A>G, c.1345T>C, and c.1661G>T, causing p.Y182C, p.Y449H, and p.W554L amino acid substitutions in baringo, nice, and stitch mutants, respectively. The mutations affect amino acid residues that are evolutionarily conserved across species. Similar to the previously reported Beethoven Tmc1 mutant, both p.Y182C and p.W554L are located outside a predicted transmembrane domain, whereas the p.Y449H mutation resides in the predicted transmembrane domain 4. Homozygous stitch-mutant mice have severe hearing loss at the age of 4 weeks and are deaf by the age of 8 weeks, whereas both baringo and nice mutants are profoundly deaf at the age of 4 weeks. None of the strains displays signs of vestibular dysfunction. Scanning electron microscopy revealed degeneration of outer hair cells in the basal region of baringo, nice, and stitch mutants. Immunolocalization studies revealed expression of TMC1 protein in the hair cells, spiral ganglion neurons, supporting cells, and stria ligament in the inner ear. Reduced levels of TMC1 protein were observed in the spiral ligament of mutants when compared with wild-type animals. These three allelic mutants provide valuable models for studying nonsyndromic recessive sensorineural hearing loss (DFNB7/11) in humans.

Annexin XI co-localises with calcyclin in proliferating cells of the embryonic mouse testis.

Mammalian sex determination relies on the expression of SRY, which triggers a tightly regulated cascade of gene expression leading to male differentiation. Many elements of this pathway remain to be identified. Here, we characterise Annexin XI (Anxa11), a gene whose major site of embryonic expression was within the undifferentiated and differentiating testis. Lower level expression was also observed in both sexes in the Müllerian and Wolffian ducts, the somitic dermamyotome, and the dorsal intermediate zone of the neural tube. Anxa11 transcripts were detected in the indifferent gonad from 10.5 days post coitum (dpc), becoming male specific as development proceeded. Expression was within the testis cords, initially in germ cells, and then in both Sertoli and germ cells. Annexin XI protein was seen in the testis cords from 12.5 dpc, localising to the cytoplasm of the Sertoli cells. Expression of calcyclin (S100a6), shown previously to interact with annexin XI in vitro, was also observed in proliferating cells of the embryonic testis, supporting a possible in vivo interaction. Developmental Dynamics 234:432–437, 2005.

Inner Ear Morphology Is Perturbed in Two Novel Mouse Models of Recessive Deafness

Human MYO7A mutations can cause a variety of conditions involving the inner ear. These include dominant and recessive non-syndromic hearing loss and syndromic conditions such as Usher syndrome. Mouse models of deafness allow us to investigate functional pathways involved in normal and abnormal hearing processes. We present two novel mouse models with mutations in the Myo7a gene with distinct phenotypes. The mutation in Myo7aI487N/I487N ewaso is located within the head motor domain of Myo7a. Mice exhibit a profound hearing loss and manifest behaviour associated with a vestibular defect. A mutation located in the linker region between the coiled-coil and the first MyTH4 domains of the protein is responsible in Myo7aF947I/F947I dumbo. These mice show a less severe hearing loss than in Myo7aI487N/I487N ewaso; their hearing loss threshold is elevated at 4 weeks old, and progressively worsens with age. These mice show no obvious signs of vestibular dysfunction, although scanning electron microscopy reveals a mild phenotype in vestibular stereocilia bundles. The Myo7aF947I/F947I dumbo strain is therefore the first reported Myo7a mouse model without an overt vestibular phenotype; a possible model for human DFNB2 deafness. Understanding the molecular basis of these newly identified mutations will provide knowledge into the complex genetic pathways involved in the maintenance of hearing, and will provide insight into recessively inherited sensorineural hearing loss in humans.

Stability of Intravenous Flucloxacillin Solutions Used for Hospital-in-the-home

Despite the uncertainty regarding flucloxacillins stability, flucloxacillin solutions are routinely used as 24‐hour ambulatory infusions for hospital‐in‐the‐home patients.

Eeyore: a novel mouse model of hereditary deafness.

Animal models that recapitulate human disease are proving to be an invaluable tool in the identification of novel disease-associated genes. These models can improve our understanding of the complex genetic mechanisms involved in disease and provide a basis to guide therapeutic strategies to combat these conditions. We have identified a novel mouse model of non-syndromic sensorineural hearing loss with linkage to a region on chromosome 18. Eeyore mutant mice have early onset progressive hearing impairment and show abnormal structure of the sensory epithelium from as early as 4 weeks of age. Ultrastructural and histological analyses show irregular hair cell structure and degeneration of the sensory hair bundles in the cochlea. The identification of new genes involved in hearing is central to understanding the complex genetic pathways involved in the hearing process and the loci at which these pathways are interrupted in people with a genetic hearing loss. We therefore discuss possible candidate genes within the linkage region identified in eeyore that may underlie the deafness phenotype in these mice. Eeyore provides a new model of hereditary sensorineural deafness and will be an important tool in the search for novel deafness genes.