Michael Bogwitz

Copy number variation and transposable elements feature in recent, ongoing adaptation at the Cyp6g1 locus.

The increased transcription of the Cyp6g1 gene of Drosophila melanogaster, and consequent resistance to insecticides such as DDT, is a widely cited example of adaptation mediated by cis-regulatory change. A fragment of an Accord transposable element inserted upstream of the Cyp6g1 gene is causally associated with resistance and has spread to high frequencies in populations around the world since the 1940s. Here we report the existence of a natural allelic series at this locus of D. melanogaster, involving copy number variation of Cyp6g1, and two additional transposable element insertions (a P and an HMS-Beagle). We provide evidence that this genetic variation underpins phenotypic variation, as the more derived the allele, the greater the level of DDT resistance. Tracking the spatial and temporal patterns of allele frequency changes indicates that the multiple steps of the allelic series are adaptive. Further, a DDT association study shows that the most resistant allele, Cyp6g1-[BP], is greatly enriched in the top 5% of the phenotypic distribution and accounts for ∼16% of the underlying phenotypic variation in resistance to DDT. In contrast, copy number variation for another candidate resistance gene, Cyp12d1, is not associated with resistance. Thus the Cyp6g1 locus is a major contributor to DDT resistance in field populations, and evolution at this locus features multiple adaptive steps occurring in rapid succession.

Cis-Regulatory Elements in the Accord Retrotransposon Result in Tissue-Specific Expression of the Drosophila melanogaster Insecticide Resistance Gene Cyp6g1

Transposable elements are a major mutation source and powerful agents of adaptive change. Some transposable element insertions in genomes increase to a high frequency because of the selective advantage the mutant phenotype provides. Cyp6g1-mediated insecticide resistance in Drosophila melanogaster is due to the upregulation of the cytochrome P450 gene Cyp6g1, leading to the resistance to a variety of insecticide classes. The upregulation of Cyp6g1 is correlated with the presence of the long terminal repeat (LTR) of an Accord retrotransposon inserted 291bp upstream of the Cyp6g1 transcription start site. This resistant allele (DDT-R) is currently at a high frequency in D. melanogaster populations around the world. Here, we characterize the spatial expression of Cyp6g1 in insecticide-resistant and -susceptible strains. We show that the Accord LTR insertion is indeed the resistance-associated mutation and demonstrate that the Accord LTR carries regulatory sequences that increase the expression of Cyp6g1 in tissues important for detoxification, the midgut, Malpighian tubules, and the fat body. This study provides a significant example of how changes in tissue-specific gene expression caused by transposable-element insertions can contribute to adaptation.

The genetic basis of resistance to diazinon in natural populations of Drosophila melanogaster.

Isofemale strains of Drosophila melanogaster were established from single inseminated females collected from populations along the east coast of Australia. Strains were tested for resistance to the organophosphorus insecticide diazinon at larval and/or adult stages of the life cycle. Considerable phenotypic variation was observed within and between population samples but there was no association between collection site of a sample and resistance status. Adult and larval resistance levels were uncorrelated. Resistance levels in adults were low (2-fold) and polygenically based. Larval resistance levels, due to single genes (or gene complexes) on chromosomes II and III, were significant (15-fold). Evidence indicates that the gene on chromosome II is Cyp6g1.

Germline SDHC mutation presenting as recurrent SDH deficient GIST and renal carcinoma

Sir, In October 2011 a 33-year-old female underwent resection of a 30 mm gastric gastrointestinal stromal tumour (GIST) and 22 mm liver metastasis with a mitotic count of 7 per 50 high power fields (HPF). Her history was remarkable for resections for three previous malignancies. In 1990 she underwent a gastric resection followed by adjuvant adriamycin and DTIC chemotherapy for a 140 mm multifocal ‘epithelioid leiomyosarcoma’ with a mitotic count of 4 per 50 HPF. In 2000 at the age of 22 she underwent a right nephrectomy for a 65 mm renal tumour for which the pathological diagnosis was ‘renal carcinoma of unclassifiable type’. In 2008 at the age of 30 she underwent thyroidectomy for papillary thyroid carcinoma. The family history was not suggestive of an inherited cancer syndrome. The patient’s sister and parents were asymptomatic and cancer free. The extended family history included a paternal aunt with colorectal carcinoma diagnosed at age 49, an additional paternal aunt with leukaemia at age 50 and two maternal aunts diagnosed with cervical cancer at ages 38 and 42. Initial genetic investigations including karyotype, p53 sequencing/MLPA and whole genome SNP array were negative. The pathology of the GIST with its accompanying liver metastasis from 2011 was reviewed. On the basis of its onset in young adulthood, gastric location, multilobulated growth pattern, absence of KIT or PDGFR mutations but diffuse strong positive staining for both KIT and DOG1 and predominance of epithelioid or plump spindled cells (Fig. 1), a diagnosis of succinate dehydrogenase (SDH) deficient GIST was suggested. The diagnosis was confirmed with immunohistochemistry for SDHB which showed negative staining of the neoplastic cells with positive staining in the non-neoplastic cells including the gastric epithelium and blood vessels (arrows in Fig. 1B,D). Additional immunohistochemistry for SDHA was positive – essentially excluding SDHA mutation. The slides from the gastric leiomyosarcoma resected in 1990 were reviewed and also showed typical features of succinate dehydrogenase deficient GIST, not an unexpected finding given that GIST (particularly in children) was only an emerging entity in the late 1980s and early 1990s and most tumours classified as leiomyosarcoma at that time or previously would now be classified as GIST. On review, the previously unclassifiable renal tumour showed identical morphology to that recently described in SDH related renal carcinoma (Fig. 2). Again immunohistochemistry for SDHB was negative with positive staining in the non-neoplastic cells including benign tubules and endothelial cells (arrows in Fig. 2B,D). The papillary carcinoma demonstrated typical morphology and showed normal positive staining for SDHB, suggesting it was more likely an incidental finding than a bona fide part of syndromic disease. A presumptive diagnosis of hereditary phaeochromocytomaparaganglioma (PGL) syndrome due to germline mutation of one

Alternative splicing removes an Ets interaction domain from Lozenge during Drosophila eye development

Physical and functional characteristics of the RUNX family of transcription factors are conserved between vertebrates and the Drosophila protein Lozenge. The runt-homology domain responsible for DNA binding and also the C-terminus are both nearly identical between the two proteins. The mammalian and fly proteins heterodimerize with a non-DNA binding partner protein to form a core binding factor essential for gene regulation during cell differentiation. The mammalian protein RUNX1 (AML1/PEBP2αB) interacts with the transcription factor Ets-1 to increase DNA binding and transactivation potential. Alternative splicing of the mammalian RUNX1 removes a domain required for this cooperative transactivation. In this work we determine the structure of the lozenge transcription unit and map 21 mutations. We show that the lozenge transcript is alternatively spliced during eye development to remove an Ets interaction domain. Emphasis is placed on Pointed the Drosophila homolog of the vertebrate Ets-1 protein; both Lozenge and Pointed proteins are needed for the activation of prospero expression. We use site-directed mutagenesis and yeast two-hybrid analysis to show that conserved amino acids within the alternate Lozenge exon are important for interaction with Pointed. Furthermore, the ectopic expression of Lozenge is sufficient to rescue Prospero expression in the presence of the Pointed competitor, YanACT. We show that both lozenge isoforms are expressed during eye development and that the relative ratio of the transcripts for the two isoforms is sensitive to changes in Ras activity. We suggest that during eye development, Lozenge isoforms function in divergent roles, either interacting with Pointed on downstream targets or by functioning independently to establish distinct cell fates.

Hereditary haemorrhagic telangiectasia, an Australian cohort: clinical and investigative features

The present study aims to describe the phenotypic features of patients with hereditary haemorrhagic telangiectasia (HHT) seen at Royal Melbourne Hospital, Victoria, Australia, and to customise a protocol for surveillance of patients with HHT.

The Cardiac Genetics Clinic: a model for multidisciplinary genomic medicine

Objectives: To describe patient characteristics, standard operating procedure, and uptake of genetic testing at the multidisciplinary Cardiac Genetics Clinic (CGC) at the Royal Melbourne Hospital during its first 6 years.

A cohort study of Gorlin syndrome with emphasis on standardised phenotyping and quality of life assessment

Gorlin syndrome (nevoid basal cell carcinoma syndrome) is a rare genetic predisposition to basal cell carcinomas (BCC), keratocysts of the jaw and calcification of the falx cerebri among other clinical features. With the advent of sonic hedgehog inhibitors for the treatment of BCC, it is timely to establish a cohort of individuals with Gorlin syndrome and collect standardised phenotypic information on these individuals. Moreover, the health‐related quality of life (QoL) in individuals with Gorlin syndrome is not well studied.

Absence of renal phenotype in hereditary haemorrhagic telangiectasia: Brief Communications

Hereditary haemorrhagic telangiectasia is characterised by abnormal blood vessel formation, producing telangiectasia and arteriovenous malformations in multiple organs. Information regarding possible renal involvement in hereditary haemorrhagic telangiectasia is limited. This study assessed renal structure and function in 11 patients with genetically confirmed diagnosis and known arteriovenous malformations in lung, liver, gastrointestinal tract or brain. All had significant current or past epistaxis. Despite the vascularity of the kidneys, we found no evidence of renal involvement. This observation warrants further consideration.