Whole genome sequencing of low input circulating cell-free DNA obtained from normal human subjects.

Cell-free DNA circulates in plasma at low levels as a normal by-product of cellular apoptosis. Multiple clinical pathologies, as well as environmental stressors can lead to increased circulating cell-free DNA (ccfDNA) levels. Plasma DNA studies frequently employ targeted amplicon deep sequencing platforms due to limited concentrations (ng/ml) of ccfDNA in the blood. Here, we report whole genome sequencing (WGS) and read distribution across chromosomes of ccfDNA extracted from two human plasma samples from normal, healthy subjects, representative of limited clinical samples at <1 ml. Amplification was sufficiently robust with ~90% of the reference genome (GRCh38.p2) exhibiting 10X coverage. Chromosome read coverage was uniform and directly proportional to the number of reads for each chromosome across both samples. Almost 99% of the identified genomic sequence variants were known annotated dbSNP variants in the hg38 reference genome. A high prevalence of C>T and T>C mutations was present along with a strong concordance of variants shared between the germline genome databases; gnomAD (81.1%) and the 1000 Genome Project (93.6%). This study demonstrates isolation and amplification procedures from low input ccfDNA samples that can detect sequence variants across the whole genome from amplified human plasma ccfDNA that can translate to multiple clinical research disciplines.

Development of the mammalian axial skeleton requires signaling through the Gα(i) subfamily of heterotrimeric G proteins.

129/SvEv mice with a loss-of-function mutation in the heterotrimeric G protein α-subunit gene Gnai3 have fusions of ribs and lumbar vertebrae, indicating a requirement for Gα(i) (the "inhibitory" class of α-subunits) in somite derivatives. Mice with mutations of Gnai1 or Gnai2 have neither defect, but loss of both Gnai3 and one of the other two genes increases the number and severity of rib fusions without affecting the lumbar fusions. No myotome defects are observed in Gnai3/Gnai1 double-mutant embryos, and crosses with a conditional allele of Gnai2 indicate that Gα(i) is specifically required in cartilage precursors. Penetrance and expressivity of the rib fusion phenotype is altered in mice with a mixed C57BL/6 × 129/SvEv genetic background. These phenotypes reveal a previously unknown role for G protein-coupled signaling pathways in development of the axial skeleton.

Spontaneous and irradiation-induced tumor susceptibility in BRCA2 germline mutant mice and cooperative effects with a p53 germline mutation.

Mutations in both p53 and BRCA2 are commonly seen together in human tumors suggesting that the loss of both genes enhances tumor development. To elucidate this interaction in an animal model, mice lacking the carboxy terminal domain of Brca2 were crossed with p53 heterozygous mice. Females from this intercross were then irradiated with an acute dose of 5 Gy ionizing radiation at 5 weeks of age and compared to nonirradiated controls. We found decreased survival and timing of tumor onsets, and significantly higher overall tumor incidences and prevalence of particular tumors, including stomach tumors and squamous cell carcinomas, associated with the homozygous loss of Brca2, independent of p53 status. The addition of a p53 mutation had a further impact on overall survival, incidence of osteosarcomas and stomach tumors, and tumor latency. The spectrum of tumors observed for this Brca2 germline mouse model suggest that it faithfully recapitulates some human disease phenotypes associated with BRCA2 loss. In addition, these findings include extensive in vivo data demonstrating that germline Brca2 and p53 mutations cooperatively affect animal survivals, tumor susceptibilities, and tumor onsets.

Mutant Brca2/p53 mice exhibit altered radiation responses in the developing mammary gland.

Appropriate balance between proliferation and apoptosis is critical for mammary gland development and is often altered during tumorigenesis. Carcinogens like radiation induce DNA damage and activate protective responses such as cell cycle arrest and apoptosis. We used mice carrying Brca2(-/-) and/or p53(-/-) mutations to evaluate the individual and combined effects of these genes on cell proliferation and apoptosis in the developing mammary gland. Mice were exposed to 5Gy of radiation or chamber exposure (controls) followed by injection with BrdU. Mammary glands were collected 6 h post-radiation exposure and evaluated for proliferation (BrdU) and apoptosis (TUNEL) in terminal end buds (TEB) and ducts. Under control conditions, the Brca2 mutation reduced proliferation and apoptosis in TEB but not ducts, whereas the p53 mutation reduced apoptosis in TEB and ducts but did not influence proliferation. Despite these alterations in proliferation and/or apoptosis, neither mutation, either individually or combined, significantly altered the overall balance between the two as measured by the proliferation to apoptosis ratio (growth index). Following irradiation, the Brca2 mutation had no significant effect on proliferation or apoptosis, whereas the p53 mutation resulted in reduced apoptosis in TEB and ducts but did not significantly influence proliferation. Neither mutation by itself altered the growth index in the TEB after irradiation although combined Brca2/p53 mutation caused significantly increased proliferation, reduced apoptosis, and an elevated growth index in TEB and ducts. These results reveal both independent and collaborative growth regulatory roles for Brca2 and p53 under normal and adverse environmental conditions. Additionally, we demonstrate the importance of gene-environment interactions by showing that Brca2- and p53-deficient mice can compensate for their genetic deficiencies under control conditions but not after exposure to radiation. We also demonstrate distinct spatial differences in the cellular functions of Brca2 and p53 and show that combined mutation of both genes is more detrimental than loss of either gene alone.

Cancer susceptibility of mice with a homozygous deletion in the COOH-terminal domain of the Brca2 gene.

Inherited mutations of the human BRCA2 gene confer increased risks for developing breast, ovarian, and several other cancers. Unlike previously described Brca2 knockout mice that display predominantly embryonic lethal phenotypes, we developed mice with a homozygous germ-line deletion of Brca2 exon 27 that exhibit a moderate decrease in perinatal viability and are fertile. We deleted this Brca2 COOH-terminal domain because it interacts directly with the Rad51 protein, contains a nuclear localization signal, and is required to maintain genomic stability in response to various types of DNA damage. These homozygous Brca2-mutant mice have a significantly increased overall tumor incidence and decreased survival compared with their heterozygous littermates. Virgin female mice homozygous for this Brca2 mutation also display an inhibition of ductal side branching in the mammary gland at 6 months of age. Given their substantial viability and cancer predisposition, these mutant mice will be useful to further define the role of the COOH-terminal Brca2 domain in tumorigenesis both in vivo and in vitro.