Linda Rodgers

Tumour evolution inferred by single-cell sequencing

Genomic analysis provides insights into the role of copy number variation in disease, but most methods are not designed to resolve mixed populations of cells. In tumours, where genetic heterogeneity is common, very important information may be lost that would be useful for reconstructing evolutionary history. Here we show that with flow-sorted nuclei, whole genome amplification and next generation sequencing we can accurately quantify genomic copy number within an individual nucleus. We apply single-nucleus sequencing to investigate tumour population structure and evolution in two human breast cancer cases. Analysis of 100 single cells from a polygenomic tumour revealed three distinct clonal subpopulations that probably represent sequential clonal expansions. Additional analysis of 100 single cells from a monogenomic primary tumour and its liver metastasis indicated that a single clonal expansion formed the primary tumour and seeded the metastasis. In both primary tumours, we also identified an unexpectedly abundant subpopulation of genetically diverse ‘pseudodiploid’ cells that do not travel to the metastatic site. In contrast to gradual models of tumour progression, our data indicate that tumours grow by punctuated clonal expansions with few persistent intermediates.

Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method.

We developed a method for immunoaffinity purification of Saccharomyces cerevisiae adenylyl cyclase based on creating a fusion with a small peptide epitope. Using oligonucleotide technology to encode the peptide epitope we constructed a plasmid that expressed the fusion protein from the S. cerevisiae alcohol dehydrogenase promoter ADH1. A monoclonal antibody previously raised against the peptide was used to purify adenylyl cyclase by affinity chromatography. The purified enzyme appeared to be a multisubunit complex consisting of the 200-kilodalton adenylyl cyclase fusion protein and an unidentified 70-kilodalton protein. The purified protein could be activated by RAS proteins. Activation had an absolute requirement for a guanine nucleoside triphosphate.

The contribution of de novo coding mutations to autism spectrum disorder

Whole exome sequencing has proven to be a powerful tool for understanding the genetic architecture of human disease. Here we apply it to more than 2,500 simplex families, each having a child with an autistic spectrum disorder. By comparing affected to unaffected siblings, we show that 13% of de novo missense mutations and 43% of de novo likely gene-disrupting (LGD) mutations contribute to 12% and 9% of diagnoses, respectively. Including copy number variants, coding de novo mutations contribute to about 30% of all simplex and 45% of female diagnoses. Almost all LGD mutations occur opposite wild-type alleles. LGD targets in affected females significantly overlap the targets in males of lower intelligence quotient (IQ), but neither overlaps significantly with targets in males of higher IQ. We estimate that LGD mutation in about 400 genes can contribute to the joint class of affected females and males of lower IQ, with an overlapping and similar number of genes vulnerable to contributory missense mutation. LGD targets in the joint class overlap with published targets for intellectual disability and schizophrenia, and are enriched for chromatin modifiers, FMRP-associated genes and embryonically expressed genes. Most of the significance for the latter comes from affected females.

Inferring tumor progression from genomic heterogeneity

Cancer progression in humans is difficult to infer because we do not routinely sample patients at multiple stages of their disease. However, heterogeneous breast tumors provide a unique opportunity to study human tumor progression because they still contain evidence of early and intermediate subpopulations in the form of the phylogenetic relationships. We have developed a method we call Sector-Ploidy-Profiling (SPP) to study the clonal composition of breast tumors. SPP involves macro-dissecting tumors, flow-sorting genomic subpopulations by DNA content, and profiling genomes using comparative genomic hybridization (CGH). Breast carcinomas display two classes of genomic structural variation: (1) monogenomic and (2) polygenomic. Monogenomic tumors appear to contain a single major clonal subpopulation with a highly stable chromosome structure. Polygenomic tumors contain multiple clonal tumor subpopulations, which may occupy the same sectors, or separate anatomic locations. In polygenomic tumors, we show that heterogeneity can be ascribed to a few clonal subpopulations, rather than a series of gradual intermediates. By comparing multiple subpopulations from different anatomic locations, we have inferred pathways of cancer progression and the organization of tumor growth.

A family of human phosphodiesterases homologous to the dunce learning and memory gene product of Drosophila melanogaster are potential targets for antidepressant drugs.

We have isolated cDNAs for four human genes (DPDE1 through DPDE4) closely related to the dnc learning and memory locus of Drosophila melanogaster. The deduced amino acid sequences of the Drosophila and human proteins have considerable homology, extending beyond the putative catalytic region to include two novel, highly conserved, upstream conserved regions (UCR1 and UCR2). The upstream conserved regions are located in the amino-terminal regions of the proteins and appear to be unique to these genes. Polymerase chain reaction analysis suggested that these genes encoded the only homologs of dnc in the human genome. Three of the four genes were expressed in Saccharomyces cerevisiae and shown to encode cyclic AMP-specific phosphodiesterases. The products of the expressed genes displayed the pattern of sensitivity to inhibitors expected for members of the type IV, cyclic AMP-specific class of phosphodiesterases. Each of the four genes demonstrated a distinctive pattern of expression in RNA from human cell lines.

DBC2, a candidate for a tumor suppressor gene involved in breast cancer

A previously uncharacterized gene, DBC2 (deleted in breast cancer), was cloned from a homozygously deleted region at human chromosome 8p21. DBC2 contains a highly conserved RAS domain and two putative protein interacting domains. Our analyses indicate that DBC2 is the best candidate tumor suppressor gene from this region. It lies within the epicenter of the deletions and is homozygously deleted in 3.5% (7/200) of breast tumors. Mutation analysis of DBC2 led to discovery of two instances of somatic missense mutations in breast tumor specimens, whereas no missense mutations were found in other candidates from the region. Unlike other genes in the region, expression of DBC2 is often extinguished in breast cancer cells or tissues. Moreover, our functional analysis revealed that DBC2 expression in breast cancer cells lacking DBC2 transcripts causes growth inhibition. By contrast, expression of a somatic mutant discovered in a breast cancer specimen does not suppress the growth of breast cancer cells.

Cloning and characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein

Adenylyl cyclase from S. cerevisiae contains at least two subunits, a 200 kd catalytic subunit and a subunit with an apparent molecular size of 70 kd, which we now call CAP (cyclase-associated protein). We cloned a cDNA encoding CAP by screening a yeast cDNA expression library in E. coli with antisera raised against the purified protein. The cDNA contained an open reading frame capable of encoding a 526 amino acid protein that is not homologous to any sequences in the current data bases. Adenylyl cyclase activity in membranes from cells that lacked CAP was not stimulated by RAS2 proteins in vitro. These results suggest that CAP is required for at least some aspects of the RAS-responsive signaling system. Mutants lacking CAP had four additional phenotypes that appear to be unrelated to effects of the RAS/adenylyl cyclase pathway: the inability to grow on rich medium (YPD), temperature sensitivity on minimal medium, sensitivity to nitrogen starvation, and a swollen cell morphology.

ran1+ controls the transition from mitotic division to meiosis in fission yeast.

SummaryWe have investigated the genetic and physiological control of meiosis in fission yeast. Nutritionally depleted h+/h− diploid cells become irreversibly commited to meiosis immediately prior to the initiation of premeiotic S phase. Premeiotic DNA synthesis requires matP+, matM+, mei2+ and mei3+ but not the mitotic cell cycle control gene, cdc2+, ran1+ is an essential gene, loss of which provokes sexual conjugation, premeiotic DNA synthesis, pseudo-meiosis and the sporulation of haploid cells. Our experiments suggest that sexual differentiation is achieved physiologically by the inhibition of ran1+ activity in a two-step process. In the first step, partial inhibition of ran1+ in starved haploid cells, leads to cell cycle arrest in G1 followed by sexual conjugation. In the second step, a pathway requiring the matP+, matM+ and mei3+ genes of the newly-formed zygote, further inhibits ran1+ and thereby commits the cell to meiosis. mei2+ is required for meiotic commitment after full inhibition of ran1+. ran1+ is normally essential for vegetative cell reproduction but is inessential in cells which have abnormally high levels of cAMP-dependent protein kinase. We propose that the ran1+ gene encodes a highly controlled protein kinase which shares key substrates with cAMP-dependent protein kinase.

Genome-wide copy number analysis of single cells

Copy number variation (CNV) is increasingly recognized as an important contributor to phenotypic variation in health and disease. Most methods for determining CNV rely on admixtures of cells in which information regarding genetic heterogeneity is lost. Here we present a protocol that allows for the genome-wide copy number analysis of single nuclei isolated from mixed populations of cells. Single-nucleus sequencing (SNS), combines flow sorting of single nuclei on the basis of DNA content and whole-genome amplification (WGA); this is followed by next-generation sequencing to quantize genomic intervals in a genome-wide manner. Multiplexing of single cells is discussed. In addition, we outline informatic approaches that correct for biases inherent in the WGA procedure and allow for accurate determination of copy number profiles. All together, the protocol takes ∼3 d from flow cytometry to sequence-ready DNA libraries.

byr2, a Schizosaccharomyces pombe gene encoding a protein kinase capable of partial suppression of the ras1 mutant phenotype

Schizosaccharomyces pombe contains a single gene, ras1, which is a homolog of the mammalian RAS genes. ras1 is required for conjugation, sporulation, and normal cell shape. ras1 has been previously identified as ste5. We report here a gene we call byr2 that can encode a predicted protein kinase and can partially suppress defects in ras1 mutants. ras1 mutant strains expressing high levels of byr2 can sporulate competently but are still defective in conjugation and abnormally round. byr2 mutants are viable and have normal shape but are absolutely defective in conjugation and sporulation. byr2 is probably identical to ste8. In many respects, byr2 resembles the byr1 gene, another suppressor of the ras1 mutation, which has been identified previously as ste1. Our data indicate that if ras1, byr2, and byr1 act along the same pathway, then the site of action for byr2 is between the sites for ras1 and byr1.