Patrick R. Blackburn

Clonal competition with alternating dominance in multiple myeloma

Emerging evidence indicates that tumors can follow several evolutionary paths over a patients disease course. With the use of serial genomic analysis of samples collected at different points during the disease course of 28 patients with multiple myeloma, we found that the genomes of standard-risk patients show few changes over time, whereas those of cytogenetically high-risk patients show significantly more changes over time. The results indicate the existence of 3 temporal tumor types, which can either be genetically stable, linearly evolving, or heterogeneous clonal mixtures with shifting predominant clones. A detailed analysis of one high-risk patient sampled at 7 time points over the entire disease course identified 2 competing subclones that alternate in a back and forth manner for dominance with therapy until one clone underwent a dramatic linear evolution. With the use of the Vk*MYC genetically engineered mouse model of myeloma we modeled this competition between subclones for predominance occurring spontaneously and with therapeutic selection.

The CRISPR system - Keeping zebrafish gene targeting fresh

We are entering a new era in our ability to modify and edit the genomes of model organisms. Zinc finger nucleases (ZFNs) opened the door to the first custom nuclease-targeted genome engineering in the late 1990s. However, ZFNs remained out of reach for most research labs because of the difficulty of production, high costs, and modest efficacy in many applications. Transcription activator-like effector nucleases (TALENs) were built upon a DNA binding system discovered in a group of plant bacterial pathogens and broadened custom nuclease technology, showing significant improvements in both targeting flexibility and efficiency. Perhaps most importantly, TALENs are open source and easy to produce, providing zebrafish laboratories around the world with affordable tools that can be made in-house rapidly, at low cost, and with reliably high activity. Now a new system for targeted genome engineering derived from the CRISPR/Cas system in eubacteria and archaea promises to simplify this process further. Together, these tools will help overcome many of the bottlenecks that have constrained gene targeting in zebrafish, paving the way for advanced genome engineering applications in this model teleost.

No evidence of XMRV in prostate cancer cohorts in the Midwestern United States

BackgroundXenotropic murine leukemia virus (MLV)-related virus (XMRV) was initially identified in prostate cancer (PCa) tissue, particularly in the prostatic stromal fibroblasts, of patients homozygous for the RNASEL R462Q mutation. A subsequent study reported XMRV antigens in malignant prostatic epithelium and association of XMRV infection with PCa, especially higher-grade tumors, independently of the RNASEL polymorphism. Further studies showed high prevalence of XMRV or related MLV sequences in chronic fatigue syndrome patients (CFS), while others found no, or low, prevalence of XMRV in a variety of diseases including PCa or CFS. Thus, the etiological link between XMRV and human disease remains elusive. To address the association between XMRV infection and PCa, we have tested prostate tissues and human sera for the presence of viral DNA, viral antigens and anti-XMRV antibodies.ResultsReal-time PCR analysis of 110 PCa (Gleason scores >4) and 40 benign and normal prostate tissues identified six positive samples (5 PCa and 1 non-PCa). No statistical link was observed between the presence of proviral DNA and PCa, PCa grades, and the RNASEL R462Q mutation. The amplified viral sequences were distantly related to XMRV, but nearly identical to endogenous MLV sequences in mice. The PCR positive samples were also positive for mouse mitochondrial DNA by nested PCR, suggesting contamination of the samples with mouse DNA. Immuno-histochemistry (IHC) with an anti-XMRV antibody, but not an anti-MLV antibody that recognizes XMRV, sporadically identified antigen-positive cells in prostatic epithelium, irrespectively of the status of viral DNA detection. No serum (159 PCa and 201 age-matched controls) showed strong neutralization of XMRV infection at 1:10 dilution.ConclusionThe lack of XMRV sequences or strong anti-XMRV neutralizing antibodies indicates no or very low prevalence of XMRV in our cohorts. We conclude that real-time PCR- and IHC-positive samples were due to laboratory contamination and non-specific immune reactions, respectively.

Activation of P-TEFb by Androgen Receptor-Regulated Enhancer RNAs in Castration-Resistant Prostate Cancer

The androgen receptor (AR) is required for castration-resistant prostate cancer (CRPC) progression, but the function and disease relevance of AR-bound enhancers remain unclear. Here, we identify a group of AR-regulated enhancer RNAs (e.g., PSA eRNA) that are upregulated in CRPC cells, patient-derived xenografts (PDXs), and patient tissues. PSA eRNA binds to CYCLIN T1, activates P-TEFb, and promotes cis and trans target gene transcription by increasing serine-2 phosphorylation of RNA polymerase II (Pol II-Ser2p). We define an HIV-1 TAR RNA-like (TAR-L) motif in PSA eRNA that is required for CYCLIN T1 binding. Using TALEN-mediated gene editing we further demonstrate that this motif is essential for increased Pol II-Ser2p occupancy levels and CRPC cell growth. We have uncovered a P-TEFb activation mechanism and reveal altered eRNA expression that is related to abnormal AR function and may potentially be a therapeutic target in CRPC.

FusX: A rapid one-step transcription activator-like effector assembly system for genome science

Transcription activator-like effectors (TALEs) are extremely effective, single-molecule DNA-targeting molecular cursors used for locus-specific genome science applications, including high-precision molecular medicine and other genome engineering applications. TALEs are used in genome engineering for locus-specific DNA editing and imaging, as artificial transcriptional activators and repressors, and for targeted epigenetic modification. TALEs as nucleases (TALENs) are effective editing tools and offer high binding specificity and fewer sequence constraints toward the targeted genome than other custom nuclease systems. One bottleneck of broader TALE use is reagent accessibility. For example, one commonly deployed method uses a multitube, 5-day assembly protocol. Here we describe FusX, a streamlined Golden Gate TALE assembly system that (1) is backward compatible with popular TALE backbones, (2) is functionalized as a single-tube 3-day TALE assembly process, (3) requires only commonly used basic molecular biology reagents, and (4) is cost-effective. More than 100 TALEN pairs have been successfully assembled using FusX, and 27 pairs were quantitatively tested in zebrafish, with each showing high somatic and germline activity. Furthermore, this assembly system is flexible and is compatible with standard molecular biology laboratory tools, but can be scaled with automated laboratory support. To demonstrate, we use a highly accessible and commercially available liquid-handling robot to rapidly and accurately assemble TALEs using the FusX TALE toolkit. Together, the FusX system accelerates TALE-based genomic science applications from basic science screening work for functional genomics testing and molecular medicine applications.

Case report: 5 year follow-up of adult late-onset mitochondrial encephalomyopathy with lactic acid and stroke-like episodes (MELAS)

Mitochondrial encephalomyopathy with lactic acid and stroke-like episodes (MELAS) is a multisystem mitochondrial disorder that typically presents in childhood. We describe the follow-up of a patient who was diagnosed with late-onset MELAS at the age of 49. Her clinical course includes sensorineural hearing loss, seizures, and multiple episodes of stroke-like metabolic crises. Molecular genetic testing on whole blood revealed 31% heteroplasmy of a m.3243A > G variant in the mtDNA, the causative variant in approximately 80% of MELAS cases. The original diagnostic criteria for MELAS required the onset of stroke-like episodes prior to 40 years of age but this case and others demonstrate that onset may be delayed in certain individuals. Therefore, MELAS should be included in the differential diagnosis of stroke-like episodes in patients of any age.

Holocarboxylase synthetase deficiency pre and post newborn screening.

Holocarboxylase synthetase deficiency is an autosomal recessive disorder of biotin metabolism resulting in multiple carboxylase deficiency. The typical presentation described in the medical literature is of neonatal onset within hours to weeks of birth with emesis, hypotonia, lethargy, seizures, metabolic ketolactic acidosis, hyperammonemia, developmental delay, skin rash and alopecia. The condition is screened for by newborn screening (NBS) tandem mass spectroscopy by elevated hydroxypentanoylcarnitine on dried blood spots. Urine organic acid profile may demonstrate elevated lactic, 3-OH isovaleric, 3-OH propionic, 3-MCC, methylcitric acids, and tiglylglycine consistent with loss of function of the above carboxylases. Here we describe a cohort of patients, 2 diagnosed pre-NBS and 3 post-NBS with broad differences in initial presentation and phenotype. In addition, prior to the advent of NBS, there are isolated reports of late-onset holocarboxylase synthetase deficiency in the medical literature, which describe patients diagnosed between 1 and 8 years of life, however to our knowledge there are no reports of late-onset HCLS being missed by NBS. Also we report two cases, each with novel pathogenic variants HCLS, diagnosed at age 3 years and 21 months respectively. The first patient had a normal newborn screen whilst the second had an abnormal newborn screen but was misdiagnosed as 3-methylcrotonylcarboxylase (3-MCC) deficiency and subsequently lost to follow-up until they presented again with severe metabolic acidosis.

Maple syrup urine disease: mechanisms and management

Maple syrup urine disease (MSUD) is an inborn error of metabolism caused by defects in the branched-chain α-ketoacid dehydrogenase complex, which results in elevations of the branched-chain amino acids (BCAAs) in plasma, α-ketoacids in urine, and production of the pathognomonic disease marker, alloisoleucine. The disorder varies in severity and the clinical spectrum is quite broad with five recognized clinical variants that have no known association with genotype. The classic presentation occurs in the neonatal period with developmental delay, failure to thrive, feeding difficulties, and maple syrup odor in the cerumen and urine, and can lead to irreversible neurological complications, including stereotypical movements, metabolic decompensation, and death if left untreated. Treatment consists of dietary restriction of BCAAs and close metabolic monitoring. Clinical outcomes are generally good in patients where treatment is initiated early. Newborn screening for MSUD is now commonplace in the United States and is included on the Recommended Uniform Screening Panel (RUSP). We review this disorder including its presentation, screening and clinical diagnosis, treatment, and other relevant aspects pertaining to the care of patients.

Molecular modeling and molecular dynamic simulation of the effects of variants in the TGFBR2 kinase domain as a paradigm for interpretation of variants obtained by next generation sequencing

Variants in the TGFBR2 kinase domain cause several human diseases and can increase propensity for cancer. The widespread application of next generation sequencing within the setting of Individualized Medicine (IM) is increasing the rate at which TGFBR2 kinase domain variants are being identified. However, their clinical relevance is often uncertain. Consequently, we sought to evaluate the use of molecular modeling and molecular dynamics (MD) simulations for assessing the potential impact of variants within this domain. We documented the structural differences revealed by these models across 57 variants using independent MD simulations for each. Our simulations revealed various mechanisms by which variants may lead to functional alteration; some are revealed energetically, while others structurally or dynamically. We found that the ATP binding site and activation loop dynamics may be affected by variants at positions throughout the structure. This prediction cannot be made from the linear sequence alone. We present our structure-based analyses alongside those obtained using several commonly used genomics-based predictive algorithms. We believe the further mechanistic information revealed by molecular modeling will be useful in guiding the examination of clinically observed variants throughout the exome, as well as those likely to be discovered in the near future by clinical tests leveraging next-generation sequencing through IM efforts.

Silent Tyrosinemia Type I Without Elevated Tyrosine or Succinylacetone Associated With Liver Cirrhosis and Hepatocellular Carcinoma

Tyrosinemia type I (TYRSN1, TYR I) is caused by fumarylacetoacetate hydrolase (FAH) deficiency and affects approximately one in 100,000 individuals worldwide. Pathogenic variants in FAH cause TYRSN1, which induces cirrhosis and can progress to hepatocellular carcinoma (HCC). TYRSN1 is characterized by the production of a pathognomonic metabolite, succinylacetone (SUAC) and is included in the Recommended Uniform Screening Panel for newborns. Treatment intervention is effective if initiated within the first month of life. Here, we describe a family with three affected children who developed HCC secondary to idiopathic hepatosplenomegaly and cirrhosis during infancy. Whole exome sequencing revealed a novel homozygous missense variant in FAH (Chr15(GRCh38):g.80162305A>G; NM_000137.2:c.424A > G; NP_000128.1:p.R142G). This novel variant involves the catalytic pocket of the enzyme, but does not result in increased SUAC or tyrosine, making the diagnosis of TYRSN1 problematic. Testing this novel variant using a rapid, in vivo somatic mouse model showed that this variant could not rescue FAH deficiency. In this case of atypical TYRSN1, we show how reliance on SUAC as a primary diagnostic test can be misleading in some patients with this disease. Augmentation of current screening for TYRSN1 with targeted sequencing of FAH is warranted in cases suggestive of the disorder.