Brandt Jones

Identification of a Herpes Simplex Labialis Susceptibility Region on Human Chromosome 21

BACKGROUND Most of the United States population is infected with either herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2, or both. Reactivations of HSV-1 infection cause herpes simplex labialis (HSL; cold sores or fever blisters), which is the most common recurring viral infection in humans. METHODS To investigate the possibility of a human genetic component conferring resistance or susceptibility to cold sores (i.e., a HSL susceptibility gene), we conducted a genetic linkage analysis that included serotyping and phenotyping 421 individuals from 39 families enrolled in the Utah Genetic Reference Project. RESULTS Linkage analysis identified a 2.5-Mb nonrecombinant region of interest on the long arm of human chromosome 21, with a multipoint logarithm of odds score of 3.9 noted near marker abmc65 (D21S409). Nonparametric linkage analysis of the data also provided strong evidence for linkage (P = .0005). This region of human chromosome 21 contains 6 candidate genes for herpes susceptibility. CONCLUSIONS The development of frequent cold sores is associated with a region on the long arm of human chromosome 21. This region contains several candidate genes that could influence the frequency of outbreaks of HSL.

Deep Sequencing for the Detection of Virus-Like Sequences in the Brains of Patients with Multiple Sclerosis: Detection of GBV-C in Human Brain

Multiple sclerosis (MS) is a demyelinating disease of unknown origin that affects the central nervous system of an estimated 400,000 Americans. GBV-C or hepatitis G is a flavivirus that is found in the serum of 1–2% of blood donors. It was originally associated with hepatitis, but is now believed to be a relatively non-pathogenic lymphotropic virus. Fifty frozen specimens from the brains of deceased persons affected by MS were obtained along with 15 normal control brain specimens. RNA was extracted and ribosomal RNAs were depleted before sequencing on the Illumina GAII. These 36 bp reads were compared with a non-redundant database derived from the 600,000+ viral sequences in GenBank organized into 4080 taxa. An individual read successfully aligned to the viral database was considered to be a “hit”. Normalized MS specimen hit rates for each viral taxon were compared to the distribution of hits in the normal controls. Seventeen MS and 11 control brain extracts were sequenced, yielding 4–10 million sequences (“reads”) each. Over-representation of sequence from at least one of 12 viral taxa was observed in 7 of the 17 MS samples. Sequences resembling other viruses previously implicated in the pathogenesis of MS were not significantly enriched in any of the diseased brain specimens. Sequences from GB virus C (GBV-C), a flavivirus not previously isolated from brain, were enriched in one of the MS samples. GBV-C in this brain specimen was confirmed by specific amplification in this single MS brain specimen, but not in the 30 other MS brain samples available. The entire 9.4 kb sequence of this GBV-C isolate is reported here. This study shows the feasibility of deep sequencing for the detection of occult viral infections in the brains of deceased persons with MS. The first isolation of GBV-C from human brain is reported here.

Genome-wide association study identifies variants at 16p13 associated with survival in multiple myeloma patients

Here we perform the first genome-wide association study (GWAS) of multiple myeloma (MM) survival. In a meta-analysis of 306 MM patients treated at UCSF and 239 patients treated at the Mayo clinic, we find a significant association between SNPs near the gene FOPNL on chromosome 16p13 and survival (rs72773978; P=6 × 10(-10)). Patients with the minor allele are at increased risk for mortality (HR: 2.65; 95% CI: 1.94-3.58) relative to patients homozygous for the major allele. We replicate the association in the IMMEnSE cohort including 772 patients, and a University of Utah cohort including 318 patients (rs72773978 P=0.044). Using publicly available data, we find that the minor allele was associated with increased expression of FOPNL and increased expression of FOPNL was associated with higher expression of centrosomal genes and with shorter survival. Polymorphisms at the FOPNL locus are associated with survival among MM patients.

STAT1 binds to the herpes simplex virus type 1 latency-associated transcript promoter

The authors hypothesized that environmental stimuli induce cytokines that act through an intracellular cascade, which includes signal transducers and activators of transcription (STATs), to change herpes simplex virus (HSV) gene expression, thereby inducing viral reactivation. The HSV type 1 (HSV-1) latency-associated transcript (LAT) gene regulates viral reactivation within neurons via an unknown mechanism. HSV-1 deletion mutants that are missing key portions of the LAT gene, particularly the 3′ region of the LAT promoter, do not reactivate normally in vivo. The authors hypothesized that STAT transcription factors may bind in this region to regulate viral reactivation. Electrophoretic mobility shift assay (EMSA) experiments were performed by incubating mouse trigeminal ganglion (TG) nuclear extracts with each of three overlapping sequences representing the 3′ region of the HSV-1 LAT promoter (referred to as oligos L1, L2, and L3). The ganglionic nuclear extracts bound specifically to oligos L1 and L3, but not L2. Oligos L1 and L3 contain predicted STAT binding sequences whereas L2 does not. Specific binding to oligo L3 (including the TATA box sequence) was supershifted by incubating with anti-STAT1 antibodies, but not by incubating with anti-STAT3 or anti-STAT5a antibodies. Specific L3 binding was reduced by competing with excess unlabeled STAT1 consensus sequences. These results indicate that STAT1, probably as part of a complex, is capable of binding to the LAT promoter on or near the TATA box. Further studies are required to determine if STAT1 is required for LAT expression in vivo. This work supports the hypothesis that interferons act through STAT1 to regulate the expression of HSV-1 LAT.

Signal Transducers and Activators of Transcription (Stat) Are Detectable in Mouse Trigeminal Ganglion Neurons

We studied signal transducers and activators of transcription (Stat) expression in mouse trigeminal ganglia (TG) to gain an understanding of herpes simplex virus (HSV) infection and reactivation. Mouse TG were harvested and were either frozen for Western blot analysis or preserved in 4% paraformaldehyde for subsequent immunohistochemistry study. The thawed specimens were homogenized, and nuclear/cytoplasmic extractions were performed for Western blots and immunoprecipitation. Immunohistochemistry showed that Stat1, Stat3, Stat4, Stat5b, and phosphotyrosine Stat3 localized to TG neurons, not surrounding satellite cells. Western blot of TG nuclear and cytoplasmic extracts confirmed the presence of these Stat at the appropriate molecular weights. Stat2 was undetectable in TG by these methods. Immunoprecipitation of TG nuclear extracts did not confirm the presence of Stat-Stat dimers in these specimens. These studies show that several Stat, including phosphotyrosine Stat3, are present in TG neurons, the site of HSV latency, where they could act upon latent viral DNA to effect reactivation.

Discordant Haplotype Sequencing Identifies Functional Variants at the 2q33 Breast Cancer Risk Locus

The findings from genome-wide association studies hold enormous potential for novel insight into disease mechanisms. A major challenge in the field is to map these low-risk association signals to their underlying functional sequence variants (FSV). Simple sequence study designs are insufficient, as the vast numbers of statistically comparable variants and a limited knowledge of noncoding regulatory elements complicate prioritization. Furthermore, large sample sizes are typically required for adequate power to identify the initial association signals. One important question is whether similar sample sizes need to be sequenced to identify the FSVs. Here, we present a proof-of-principle example of an extreme discordant design to map FSVs within the 2q33 low-risk breast cancer locus. Our approach employed DNA sequencing of a small number of discordant haplotypes to efficiently identify candidate FSVs. Our results were consistent with those from a 2,000-fold larger, traditional imputation-based fine-mapping study. To prioritize further, we used expression-quantitative trait locus analysis of RNA sequencing from breast tissues, gene regulation annotations from the ENCODE consortium, and functional assays for differential enhancer activities. Notably, we implicate three regulatory variants at 2q33 that target CASP8 (rs3769823, rs3769821 in CASP8, and rs10197246 in ALS2CR12) as functionally relevant. We conclude that nested discordant haplotype sequencing is a promising approach to aid mapping of low-risk association loci. The ability to include more efficient sequencing designs into mapping efforts presents an opportunity for the field to capitalize on the potential of association loci and accelerate translation of association signals to their underlying FSVs. Cancer Res; 76(7); 1916-25. ©2016 AACR.

Erratum: Genome-wide association study identifies variants at 16p13 associated with survival in multiple myeloma patients (Nature Communications (2015) 6 (7539) DOI: 10.1038/ncomms8539)

In this Article, members of the UCSF cohort who had been alive for longer than two years were inadvertently included in the data presented in Table 3. USCF/old treatments should have 109 patients with a hazard ratio of 3.35 and a P value of 0.00028 instead of the 124 patients with a hazard ratio of 3.37 and a P value of 0.00026. The USCF/new patients should have 187 patients with a hazard ratio of 3.57 and a P value of 0.0007 instead of the 208 patients with a hazard ratio of 3.62 and a P value of 0.0006. Finally, in the table legend, the first line should read ‘All models are adjusted for age, gender and principal components 1–3’. The exclusion of these individuals does not change the conclusions of the study. The correct version of Table 3 appears below.

Different Mechanisms Preserve Translation of Programmed Cell Death 8 and JunB in Virus-Infected Endothelial Cells

Objective—Translation initiation of eukaryotic mRNAs typically occurs by cap-dependent ribosome scanning mechanism. However, certain mRNAs are translated by ribosome assembly at internal ribosome entry sites (IRESs). Whether IRES-mediated translation occurs in stressed primary human endothelial cells (ECs) is unknown. Methods and Results—We performed microarray analysis of polyribosomal mRNA from ECs to identify IRES-containing mRNAs. Cap-dependent translation was disabled by poliovirus (PV) infection and confirmed by loss of polysome peaks, detection of eukaryotic initiation factor (eIF) 4G cleavage, and decreased protein synthesis. We found that 87.4% of mRNAs were dissociated from polysomes in virus-infected ECs. Twelve percent of mRNAs remained associated with polysomes, and 0.6% were enriched ≥2-fold in polysome fractions from infected ECs. Quantitative reverse transcription–polymerase chain reaction confirmed the microarray findings for 31 selected mRNAs. We found that enriched polysome associations of programmed cell death 8 (PDCD8) and JunB mRNA resulted in increased protein expression in PV-infected ECs. The presence of IRESs in the 5′ untranslated region of PDCD8 mRNA, but not of JunB mRNA, was confirmed by dicistronic analysis. Conclusion—We show that microarray profiling of polyribosomal mRNA transcripts from PV-infected ECs successfully identifies mRNAs whose translation is preserved in the face of stress-induced, near complete cessation of cap-dependent initiation. Nevertheless, internal ribosome entry is not the only mechanism responsible for this privileged translation.

Reframing Breast Cancer Molecular Subtypes: Modeling Expression Patterns with Orthogonal Dimensions

High-risk breast cancer pedigrees contain inherited risk variants, but underlying genetic complexities impair gene-finding. Focusing on particular tumor characteristics clustering in pedigrees may simplify these complexities. PAM50 gene expression in 1149 sporadic and familial breast tumors did not indicate intrinsic subtypes were enriched in non-BRCA1/2 pedigrees. However, a principal component approach revealed five common expression dimensions: three capturing key features of intrinsic subtypes; two novel and significantly enriched in pedigrees. Proof-of-concept gene-mapping using the novel dimensions identified a 0.5 Mb genomewide significant region at 12q15 (p=2.6x10-8) segregating to 8 breast cancer cases through 32 meioses. The region contains CNOT2, a gene controlling cell viability via the CCR4-NOT transcriptional regulatory deadenylase complex. These findings support the hypothesis that germline susceptibilities influence tumor characteristics and that expression dimensions partition genetic heterogeneity providing new avenues for germline genetic studies. More broadly, this approach may clarify heterogeneities impeding other domains exploring molecular characteristics.Complex diseases can be highly heterogeneous. To characterize molecular heterogeneity, feature selection methods are often used to identify genes that capture key expression differences in the transcriptome. These gene sets are used in prediction algorithms to define distinct subtypes. Molecular subtyping has been used extensively in cancers and found to be informative for clinical care, e.g., PAM50 (Prediction Analysis of Microarray 50) for breast cancer. However, many tissues do not fit neatly into a single archetypal subtype. We propose that expression diversity can be more comprehensively represented with multiple quantitative dimensions, and that improved methods to model heterogeneity will generate new discoveries. Here, we apply principal components analysis to PAM50 gene expression data from 911 population-based breast tumors and identify orthogonal dimensions. These dimensions not only recapitulate categorical breast intrinsic subtypes, but also include dimensions not previously recognized. Furthermore, while 238 familial breast tumors (non-BRCA1/2 high-risk pedigrees) were not significantly enriched by intrinsic subtype, two novel expression dimensions were highly enriched in the pedigrees. Proof-of-concept gene-mapping using these dimensions identified a 0.5Mb genomewide significant region at 12q15 (p = 2.6×10−8) segregating to 8 breast cancers through 32 meioses. The region contains CNOT2, a gene controlling cell viability via the CCR4-NOT transcriptional regulatory deadenylase complex. These findings suggest that the multiple dimension approach is a flexible and powerful method to characterize tissue expression within a defined feature set. Our results support the hypothesis that germline susceptibilities influence tumor characteristics and that expression dimensions partition genetic heterogeneity, providing new avenues for germline genetic studies.It is well-known that breast tumors exhibit different expression patterns that can be used to assign intrinsic subtypes - the PAM50 assay, for example, categorizes tumors into: Luminal A, Luminal B, HER2-enriched and Basal-like - yet tumors are often more complex than categorization can describe. We used 911 sporadic breast tumors to reparameterize expression from the PAM50 genes to five orthogonal tumor dimensions using principal components (PC). Three dimensions captured intrinsic subtype, two dimensions were novel, and all replicated in 945 TCGA tumors. By definition dimensions are independent, an important attribute for inclusion in downstream studies exploring effects of tumor diversity. One application where tumor subtyping has failed to provide impact is susceptibility genetics. Germline genetic heterogeneity reduces power for gene-finding. The identification of heritable tumor characteristics has potential to increase homogeneity. We compared 238 breast tumors from high-risk pedigrees not attributable to BRCA1 or BRCA2 to 911 sporadic breast tumors. Two PC dimensions were significantly enriched in the pedigrees (intrinsic subtypes were not). We performed proof-of-concept gene-mapping in one enriched pedigree and identified a 0.5 Mb genomewide significant region at 12q15 that segregated to the 8 breast cancer cases with the most extreme PC tumors through 32 meioses (p=2.6x10-8). In conclusion, our study: suggests a new approach to describe tumor diversity; supports the hypothesis that tumor characteristics are heritable providing new avenues for germline studies; and proposes a new breast cancer locus. Reparameterization of expression patterns may similarly inform other studies attempting to model the effects of tumor heterogeneity.

Identification of Novel Breast Tumor Expression Dimensions within the PAM50 and Discovery of a Breast Cancer Susceptibility Locus at 12q15

High-risk breast cancer pedigrees contain inherited risk variants, but underlying genetic complexities impair gene-finding. Focusing on particular tumor characteristics clustering in pedigrees may simplify these complexities. PAM50 gene expression in 1149 sporadic and familial breast tumors did not indicate intrinsic subtypes were enriched in non-BRCA1/2 pedigrees. However, a principal component approach revealed five common expression dimensions: three capturing key features of intrinsic subtypes; two novel and significantly enriched in pedigrees. Proof-of-concept gene-mapping using the novel dimensions identified a 0.5 Mb genomewide significant region at 12q15 (p=2.6x10-8) segregating to 8 breast cancer cases through 32 meioses. The region contains CNOT2, a gene controlling cell viability via the CCR4-NOT transcriptional regulatory deadenylase complex. These findings support the hypothesis that germline susceptibilities influence tumor characteristics and that expression dimensions partition genetic heterogeneity providing new avenues for germline genetic studies. More broadly, this approach may clarify heterogeneities impeding other domains exploring molecular characteristics.Complex diseases can be highly heterogeneous. To characterize molecular heterogeneity, feature selection methods are often used to identify genes that capture key expression differences in the transcriptome. These gene sets are used in prediction algorithms to define distinct subtypes. Molecular subtyping has been used extensively in cancers and found to be informative for clinical care, e.g., PAM50 (Prediction Analysis of Microarray 50) for breast cancer. However, many tissues do not fit neatly into a single archetypal subtype. We propose that expression diversity can be more comprehensively represented with multiple quantitative dimensions, and that improved methods to model heterogeneity will generate new discoveries. Here, we apply principal components analysis to PAM50 gene expression data from 911 population-based breast tumors and identify orthogonal dimensions. These dimensions not only recapitulate categorical breast intrinsic subtypes, but also include dimensions not previously recognized. Furthermore, while 238 familial breast tumors (non-BRCA1/2 high-risk pedigrees) were not significantly enriched by intrinsic subtype, two novel expression dimensions were highly enriched in the pedigrees. Proof-of-concept gene-mapping using these dimensions identified a 0.5Mb genomewide significant region at 12q15 (p = 2.6×10−8) segregating to 8 breast cancers through 32 meioses. The region contains CNOT2, a gene controlling cell viability via the CCR4-NOT transcriptional regulatory deadenylase complex. These findings suggest that the multiple dimension approach is a flexible and powerful method to characterize tissue expression within a defined feature set. Our results support the hypothesis that germline susceptibilities influence tumor characteristics and that expression dimensions partition genetic heterogeneity, providing new avenues for germline genetic studies.It is well-known that breast tumors exhibit different expression patterns that can be used to assign intrinsic subtypes - the PAM50 assay, for example, categorizes tumors into: Luminal A, Luminal B, HER2-enriched and Basal-like - yet tumors are often more complex than categorization can describe. We used 911 sporadic breast tumors to reparameterize expression from the PAM50 genes to five orthogonal tumor dimensions using principal components (PC). Three dimensions captured intrinsic subtype, two dimensions were novel, and all replicated in 945 TCGA tumors. By definition dimensions are independent, an important attribute for inclusion in downstream studies exploring effects of tumor diversity. One application where tumor subtyping has failed to provide impact is susceptibility genetics. Germline genetic heterogeneity reduces power for gene-finding. The identification of heritable tumor characteristics has potential to increase homogeneity. We compared 238 breast tumors from high-risk pedigrees not attributable to BRCA1 or BRCA2 to 911 sporadic breast tumors. Two PC dimensions were significantly enriched in the pedigrees (intrinsic subtypes were not). We performed proof-of-concept gene-mapping in one enriched pedigree and identified a 0.5 Mb genomewide significant region at 12q15 that segregated to the 8 breast cancer cases with the most extreme PC tumors through 32 meioses (p=2.6x10-8). In conclusion, our study: suggests a new approach to describe tumor diversity; supports the hypothesis that tumor characteristics are heritable providing new avenues for germline studies; and proposes a new breast cancer locus. Reparameterization of expression patterns may similarly inform other studies attempting to model the effects of tumor heterogeneity.