Cancer genes disfavoring T cell immunity identified via integrated systems approach.

Adoptive T cell therapies (ACT) have been curative for a limited number of cancer patients. The sensitization of cancer cells to T cell killing may expand the benefit of these therapies for more patients. To this end, we use a three-step approach to identify cancer genes that disfavor T cell immunity. First, we profile gene transcripts upregulated by cancer under selection pressure from T cell killing. Second, we identify potential tumor gene targets and pathways that disfavor T cell killing using signaling pathway activation libraries and genome-wide loss-of-function CRISPR-Cas9 screens. Finally, we implement pharmacological perturbation screens to validate these targets and identify BIRC2, ITGAV, DNPEP, BCL2, and ERRα as potential ACT-drug combination candidates. Here, we establish that BIRC2 limits antigen presentation and T cell recognition of tumor cells by suppressing IRF1 activity and provide evidence that BIRC2 inhibition in combination with ACT is an effective strategy to increase efficacy.

Physiological Demands of Common Occupational Tasks among Australian Police Officers: A Descriptive Analysis.

OBJECTIVES: The aim of this study was to investigate the physiological demands placed on Australian police officers carrying out common operational tasks. METHODS: Forty participants (n = 40) from an Australian police force (mean age = 33.58 ± 7.78 years, mean height = 177.70 ± 7.28 cm, mean weight = 85.68 ± 14.52 kg, mean years of service: 6.74 ± 6.29 years) were recruited through preidentified local area commands. Spanning nine police stations from the same Australian state, volunteers wore monitoring devices to collect physiological measures (heart rate, respiratory rate, and skin temperature) throughout the course of four consecutive shifts (two day shifts and two night shifts). Descriptive data were recorded and analyzed by task and changes in physiological measures. RESULTS: Of the 345 duty calls attended by participants, the four most commonly reported tasks were as follows: 'check bona fides' (n = 76; 22%), 'driving urgently' (n = 45; 13%), 'attending a domestic incident' (n = 37; 10%), and 'attending a concern for welfare' (n = 30; 8%). Mean percentages of maximum heart rates (%HRmax) were considered of very light exercise intensity and ranged from 47.11 (± 7.18) to 50.15 (± 9.35) % for checking bona fides through to driving urgently respectively. Fifteen percent of tasks attended had officers exceed 100 %HRmax (near maximal to maximal exercise intensity). Mean skin temperatures varied little (36.02-36.27°C) between tasks, while mean respiratory rates were lowest when attending a domestic incident and highest when driving urgently (22.56 ± 3.83 and 24.72 ± 6.12 breaths/min, respectively). CONCLUSION: Police officers experienced numerous physiological challenges ranging from an intensity of very light exercise through to near maximal and maximal exercise throughout their working day with occasions where their heart rates exceeded 100 %HRmax. These findings highlight the physiological stress associated with common occupational policing tasks, highlighting the importance of cardiovascular health in police officers and the need for cardiovascular monitoring and conditioning.

Using antagonistic pleiotropy to design a chemotherapy-induced evolutionary trap to target drug resistance in cancer.

Local adaptation directs populations towards environment-specific fitness maxima through acquisition of positively selected traits. However, rapid environmental changes can identify hidden fitness trade-offs that turn adaptation into maladaptation, resulting in evolutionary traps. Cancer, a disease that is prone to drug resistance, is in principle susceptible to such traps. We therefore performed pooled CRISPR-Cas9 knockout screens in acute myeloid leukemia (AML) cells treated with various chemotherapies to map the drug-dependent genetic basis of fitness trade-offs, a concept known as antagonistic pleiotropy (AP). We identified a PRC2-NSD2/3-mediated MYC regulatory axis as a drug-induced AP pathway whose ability to confer resistance to bromodomain inhibition and sensitivity to BCL-2 inhibition templates an evolutionary trap. Across diverse AML cell-line and patient-derived xenograft models, we find that acquisition of resistance to bromodomain inhibition through this pathway exposes coincident hypersensitivity to BCL-2 inhibition. Thus, drug-induced AP can be leveraged to design evolutionary traps that selectively target drug resistance in cancer.

Epigenetic silencing of tumor suppressor Par-4 promotes chemoresistance in recurrent breast cancer.

Tumor relapse is the leading cause of death in breast cancer, largely due to the fact that recurrent tumors are frequently resistant to chemotherapy. We previously reported that downregulation of the proapoptotic protein Par-4 promotes tumor recurrence in genetically engineered mouse models of breast cancer recurrence. In the present study, we examined the mechanism and functional significance of Par-4 downregulation in recurrent tumors. We found that epithelial-to-mesenchymal transition (EMT) promotes epigenetic silencing of Par-4 in recurrent tumors. Par-4 silencing proceeded through binding of the EMT transcription factor Twist to the Par-4 promoter, where Twist induced a unique bivalent chromatin domain. This bivalent configuration conferred plasticity at the Par-4 promoter, and Par-4 silencing could be reversed with pharmacologic inhibitors of Ezh2 and HDAC1/2. Using an epigenome editing approach to reexpress Par-4 by specifically reversing the histone modifications found in recurrent tumors, we found that Par-4 reexpression sensitized recurrent tumors to chemotherapy in vitro and in vivo. Upon reexpression, Par-4 bound to the protein phosphatase PP1, caused widespread changes in phosphorylation of cytoskeletal proteins, and cooperated with microtubule-targeting drugs to induce mitotic defects. These results identify Twist-induced epigenetic silencing of Par-4 as a targetable axis that promotes chemoresistance in recurrent breast cancer.

Expanding the phenotypic spectrum of GABRG2 variants: a recurrent GABRG2 missense variant associated with a severe phenotype.

Pathogenic missense and truncating variants in the GABRG2 gene cause a spectrum of epilepsies, from Dravet syndrome to milder simple febrile seizures. In most cases, pathogenic missense variants in the GABRG2 gene segregate with a febrile seizure phenotype. In this case series, we report a recurrent, de novo missense variant (c0.316 G > A; p.A106T) in the GABRG2 gene that was identified in five unrelated individuals. These patients were described to have a more severe phenotype than previously reported for GABRG2 missense variants. Common features include variable early-onset seizures, significant motor and speech delays, intellectual disability, hypotonia, movement disorder, dysmorphic features and vision/ocular issues. Our report further explores a recurrent pathogenic missense variant within the GABRG2 variant family and broadens the spectrum of associated phenotypes for GABRG2-associated disorders.

A novel SERPINA1 mutation causing serum alpha(1)-antitrypsin deficiency.

Mutations in the SERPINA1 gene can cause deficiency in the circulating serine protease inhibitor α(1)-Antitrypsin (α(1)AT). α(1)AT deficiency is the major contributor to pulmonary emphysema and liver disease in persons of European ancestry, with a prevalence of 1 in 2500 in the USA. We present the discovery and characterization of a novel SERPINA1 mutant from an asymptomatic Middle Eastern male with circulating α(1)AT deficiency. This 49 base pair deletion mutation (T379Δ), originally mistyped by IEF, causes a frame-shift replacement of the last sixteen α(1)AT residues and adds an extra twenty-four residues. Functional analysis showed that the mutant protein is not secreted and prone to intracellular aggregation.

STR sequence analysis for characterizing normal, variant, and null alleles.

DNA sequence variation is known to exist in and around the repeat region of short tandem repeat (STR) loci used in human identity testing. While the vast majority of STR alleles measured in forensic DNA laboratories worldwide type as "normal" alleles compared with STR kit allelic ladders, a number of variant alleles have been reported. In addition, a sequence difference at a polymerase chain reaction (PCR) primer binding site in the DNA template can cause allele drop-out (i.e., a "null" or "silent" allele) with one set of primers and not with another. Our group at the National Institute of Standards and Technology (NIST) has been sequencing variant and null alleles supplied by forensic labs and cataloging this information on the NIST STRBase website for the past decade. The PCR primer sequences and strategy used for our STR allele sequencing work involving 23 autosomal STRs and 17 Y-chromosome STRs are described along with the results from 111 variant and 17 null alleles.

Production and certification of NIST Standard Reference Material 2372 Human DNA Quantitation Standard.

Modern highly multiplexed short tandem repeat (STR) assays used by the forensic human-identity community require tight control of the initial amount of sample DNA amplified in the polymerase chain reaction (PCR) process. This, in turn, requires the ability to reproducibly measure the concentration of human DNA, [DNA], in a sample extract. Quantitative PCR (qPCR) techniques can determine the number of intact stretches of DNA of specified nucleotide sequence in an extremely small sample; however, these assays must be calibrated with DNA extracts of well-characterized and stable composition. By 2004, studies coordinated by or reported to the National Institute of Standards and Technology (NIST) indicated that a well-characterized, stable human DNA quantitation certified reference material (CRM) could help the forensic community reduce within- and among-laboratory quantitation variability. To ensure that the stability of such a quantitation standard can be monitored and that, if and when required, equivalent replacement materials can be prepared, a measurement of some stable quantity directly related to [DNA] is required. Using a long-established conventional relationship linking optical density (properly designated as decadic attenuance) at 260 nm with [DNA] in aqueous solution, NIST Standard Reference Material (SRM) 2372 Human DNA Quantitation Standard was issued in October 2007. This SRM consists of three quite different DNA extracts: a single-source male, a multiple-source female, and a mixture of male and female sources. All three SRM components have very similar optical densities, and thus very similar conventional [DNA]. The materials perform very similarly in several widely used gender-neutral assays, demonstrating that the combination of appropriate preparation methods and metrologically sound spectrophotometric measurements enables the preparation and certification of quantitation [DNA] standards that are both maintainable and of practical utility.

Identification of ectodysplasin-A receptor gene deletion at 2q12.2 and a potential autosomal MR locus.

Mental retardation (MR) is not a common feature observed in patients with classical ectodermal dysplasias (EDs). Several genes responsible for EDs and MR have been identified. However, the causation has yet to be identified in a significant number of patients with either ED or MR. Here, we have molecularly characterized a de novo balanced translocation t(1;6)(p22.1;p22.1) in a female patient who had mild features of ED with hypodontia, microcephaly, and cognitive impairment. Mapping of the translocation breakpoints in the patient revealed no obvious causative gene for either ED or MR. Whole genome array CGH analysis unveiled two novel submicroscopic deletions at 2q12.2 and 6q22.3, unrelated to the translocation in the patient. The 2q12.2 deletion contains a known ED gene, ectodysplasin-A receptor (EDAR), and the loss of one copy of this gene is considered to be responsible for the ectodermal phenotype in the patient. It is plausible that a potential autosomal MR gene deleted at 2q12.2 or 6q22.3 is likely the cause of the neurodevelopmental defects in the patient.

Analysis of mutations in father-son pairs with 17 Y-STR loci.

We have examined 389 father/son sample pairs from U.S. Caucasians, African Americans, Hispanics and Asians using the 17 Y-STR loci in the Yfilertrade mark kit and observed a total of 24 differences between father and son. Thirteen mutations resulted in the gain of a repeat in the son and 11 resulted in a loss of a repeat. All samples resulted in single repeat mutations except one sample which contained a two repeat loss at Y-GATA-H4. Furthermore, two different sample pairs were found to have two mutations. An African American sample pair had a mutation at DYS458 and a second at DYS635 and an Asian sample pair had mutations at DYS439 and Y-GATA-H4.

Epigenetic gene silencing in the Wnt pathway in breast cancer.

Breast cancer is one of the most common malignancies in women. Despite advances in treatment of endocrine-dependent tumors, the complete molecular basis of transformation is still unknown. What is clear is that a variety of genetic lesions and epigenetic modifications are present in the neoplasm. Disregulation of several signaling pathways is known to be associated with breast cancer development, among them is the wingless and integration site growth factor (Wnt) pathway. While genetic mutations of certain components of this pathway, such as APC, are significant contributing factors for colorectal cancers, they are typically not the predominate mechanism associated with breast cancer. Instead, it appears that DNA hypermethylation leads to aberrant regulation of the Wnt pathway in breast cancer, and as such, this review focuses on the epigenetic regulation of Wnt pathway components in breast cancer.

Allele frequencies for 27 Y-STR loci with U.S. Caucasian, African American, and Hispanic samples.

A total of 263 U.S. Caucasians, 260 African Americans and 140 U.S. Hispanics or a subset of 31 Caucasians, 32 African Americans, and 32 Hispanics were typed for 27 Y-chromosome short tandem repeat (Y-STR) markers: DYS444, DYS446, DYS449, DYS463, DYS485, DYS490, DYS495, DYS504, DYS505, DYS508, DYS520, DYS522, DYS525, DYS532, DYS533, DYS534, DYS540, DYS556, DYS557, DYS570, DYS575, DYS576, DYS594, DYS632, DYS635, DYS641, and DYS643. Allele frequencies for each locus are reported along with nomenclature based on sequence analysis.

Chromosomal duplications along the Y-chromosome and their potential impact on Y-STR interpretation.

Y-chromosome short tandem repeat (Y-STR) markers are being used as potential tools for distinguishing low levels of male DNA in the presence of excess female DNA as is present in many sexual assault samples. Usually single copy Y-STR loci produce a single amplicon in single source samples, and thus the observation of multiple peaks at such a locus could suggest to an analyst that a mixture of more than one male contributor is present in the tested sample. However, many regions of the Y-chromosome are duplicated or even triplicated in some individuals and this fact can thus complicate potential mixture interpretation. Reasons for the presence of duplications at multiple loci within a single sample are explored in the context of Y-STR marker location along the chromosome. True male-male mixtures commonly exhibit more than one locus-specific PCR product across multiple Y-STR loci that are not adjacent to one another on the Y-chromosome. In addition, duplicated loci typically possess alleles that differ by only a single repeat unit and possess similar peak heights.

Allele frequencies for 70 autosomal SNP loci with U.S. Caucasian, African-American, and Hispanic samples.

189 samples from 3 different U.S. sample groups Caucasian (74), African American (71) and Hispanic (44) were typed for 70 autosomal genetic markers. These 70 markers are bi-allelic (C/T) short nucleotide polymorphisms (SNPs). For each sample, the 70 SNP markers were typed in 11 unique 6-plexes and a single 4-plex PCR. A total of 10 of the 210 tests (70 loci x 3 populations) for Hardy-Weinberg equilibrium indicated a statistically significant result. In order to evaluate the minimum number of SNP loci needed to distinguish all 189 samples from one another, we ranked the loci according to their levels of observed heterozygosity and p-values obtained upon testing for Hardy-Weinberg equilibrium. The top 12 loci according to these ranking criteria were tabulated along with the number of unique genotypes observed when combining subsequent SNP markers. The 12 selected SNPs possessed an observed heterozygosity of >0.45 in all three populations examined and thus would be expected to exhibit more differences between samples. All of the 189 samples in this study were individualized with a subset of 12 SNP loci. However, it is likely that the addition of more than 12 SNP loci will be required to resolve larger sets of unrelated individuals from one another. By way of comparison, in these same 189 individuals all but one pair is resolved from one another with three of the traditional short tandem repeat (STR) loci possessing the highest heterozygosity values (D2S1338, D18S51, and FGA) run with the Identifiler kit. The final pair of unrelated samples could be resolved with the combination of 4 STR loci: D2S1338, D18S51, FGA, and VWA.

Child with De Novo t(1;6)(p22.1;p22.1) translocation and features of ectodermal dysplasia with hypodontia and developmental delay.

We report on a 6.5-year-old girl with a balanced translocation between the short arms of chromosomes 1 and 6. She was referred for genetics evaluation because of developmental speech delay and congenital absence of several deciduous and permanent teeth. She was very sensitive to noise (hyperacusis), had poor hair and nail growth, decreased sweating, and turned very red with high fever. She had microcephaly (head circumference at the second centile; weight and height were at 25th centile), short palpebral fissures, epicanthal folds, sparse eyelashes, large ears, partial anodontia, short finger and toenails, and dry skin. She had mild developmental delay. Family history was significant for learning problems in two paternal uncles, one paternal aunt, and several paternal cousins. Thyroid studies, calcium, phosphorus, and alkaline phosphatase levels were normal. Her karyotype was 46,XX,t(1;6)(p22.1;p22.2), and parental karyotypes were normal. This apparently balanced translocation may have resulted in either a submicroscopic loss or disruption of a gene or genes involved in ectodermal dysplasia. There are no reported cases of ectodermal dysplasia associated with this chromosome rearrangement.