Pharmacogenetics in Italy: current landscape and future prospects.

Pharmacogenetics investigates sequence of genes that affect drug response, enabling personalized medication. This approach reduces drug-induced adverse reactions and improves clinical effectiveness, making it a crucial consideration for personalized medical care. Numerous guidelines, drawn by global consortia and scientific organizations, codify genotype-driven administration for over 120 active substances. As the scientific community acknowledges the benefits of genotype-tailored therapy over traditionally agnostic drug administration, the push for its implementation into Italian healthcare system is gaining momentum. This evolution is influenced by several factors, including the improved access to patient genotypes, the sequencing costs decrease, the growing of large-scale genetic studies, the rising popularity of direct-to-consumer pharmacogenetic tests, and the continuous improvement of pharmacogenetic guidelines. Since EMA (European Medicines Agency) and AIFA (Italian Medicines Agency) provide genotype information on drug leaflet without clear and explicit clinical indications for gene testing, the regulation of pharmacogenetic testing is a pressing matter in Italy. In this manuscript, we have reviewed how to overcome the obstacles in implementing pharmacogenetic testing in the clinical practice of the Italian healthcare system. Our particular emphasis has been on germline testing, given the absence of well-defined national directives in contrast to somatic pharmacogenetics.

Hepatocyte Smoothened Activity Controls Susceptibility to Insulin Resistance and Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH), a leading cause of cirrhosis, strongly associates with the metabolic syndrome, an insulin-resistant proinflammatory state that disrupts energy balance and promotes progressive liver degeneration. We aimed to define the role of Smoothened (Smo), an obligatory component of the Hedgehog signaling pathway, in controlling hepatocyte metabolic homeostasis and, thereby, susceptibility to NASH. METHODS: We conditionally deleted Smo in hepatocytes of healthy chow-fed mice and performed metabolic phenotyping, coupled with single-cell RNA sequencing (RNA-seq), to characterize the role of hepatocyte Smo in regulating basal hepatic and systemic metabolic homeostasis. Liver RNA-seq datasets from 2 large human cohorts were also analyzed to define the relationship between Smo and NASH susceptibility in people. RESULTS: Hepatocyte Smo deletion inhibited the Hedgehog pathway and promoted fatty liver, hyperinsulinemia, and insulin resistance. We identified a plausible mechanism whereby inactivation of Smo stimulated the mTORC1-SREBP1c signaling axis, which promoted lipogenesis while inhibiting the hepatic insulin cascade. Transcriptomics of bulk and single Smo-deficient hepatocytes supported suppression of insulin signaling and also revealed molecular abnormalities associated with oxidative stress and mitochondrial dysfunction. Analysis of human bulk RNA-seq data revealed that Smo expression was (1) highest in healthy livers, (2) lower in livers with NASH than in those with simple steatosis, (3) negatively correlated with markers of insulin resistance and liver injury, and (4) declined progressively as fibrosis severity worsened. CONCLUSIONS: The Hedgehog pathway controls insulin sensitivity and energy homeostasis in adult livers. Loss of hepatocyte Hedgehog activity induces hepatic and systemic metabolic stress and enhances susceptibility to NASH by promoting hepatic lipoxicity and insulin resistance.

Deep learning-based quantification of NAFLD/NASH progression in human liver biopsies.

Non-alcoholic fatty liver disease (NAFLD) affects about 24% of the world's population. Progression of early stages of NAFLD can lead to the more advanced form non-alcoholic steatohepatitis (NASH), and ultimately to cirrhosis or liver cancer. The current gold standard for diagnosis and assessment of NAFLD/NASH is liver biopsy followed by microscopic analysis by a pathologist. The Kleiner score is frequently used for a semi-quantitative assessment of disease progression. In this scoring system the features of active injury (steatosis, inflammation, and ballooning) and a separated fibrosis score are quantified. The procedure is time consuming for pathologists, scores have limited resolution and are subject to variation. We developed an automated deep learning method that provides full reproducibility and higher resolution. The system was established with 296 human liver biopsies and tested on 171 human liver biopsies with pathologist ground truth scores. The method is inspired by the way pathologist's analyze liver biopsies. First, the biopsies are analyzed microscopically for the relevant histopathological features. Subsequently, histopathological features are aggregated to a per-biopsy score. Scores are in the identical numeric range as the pathologist's ballooning, inflammation, steatosis, and fibrosis scores, but on a continuous scale. Resulting scores followed a pathologist's ground truth (quadratic weighted Cohen's κ on the test set: for steatosis 0.66, for inflammation 0.24, for ballooning 0.43, for fibrosis 0.62, and for the NAFLD activity score (NAS) 0.52. Mean absolute errors on a test set: for steatosis 0.29, for inflammation 0.53, for ballooning 0.61, for fibrosis 0.78, and for the NAS 0.77).

The Loss of TBK1 Kinase Activity in Motor Neurons or in All Cell Types Differentially Impacts ALS Disease Progression in SOD1 Mice.

DNA sequence variants in the TBK1 gene associate with or cause sporadic or familial amyotrophic lateral sclerosis (ALS). Here we show that mice bearing human ALS-associated TBK1 missense loss-of-function mutations, or mice in which the Tbk1 gene is selectively deleted in motor neurons, do not display a neurodegenerative disease phenotype. However, loss of TBK1 function in motor neurons of the SOD1G93A mouse model of ALS impairs autophagy, increases SOD1 aggregation, and accelerates early disease onset without affecting lifespan. By contrast, point mutations that decrease TBK1 kinase activity in all cells also accelerate disease onset but extend the lifespan of SOD1 mice. This difference correlates with the failure to activate high levels of expression of interferon-inducible genes in glia. We conclude that loss of TBK1 kinase activity impacts ALS disease progression through distinct pathways in different spinal cord cell types and further implicate the importance of glia in neurodegeneration.

Changes in Albuminuria But Not GFR are Associated with Early Changes in Kidney Structure in Type 2 Diabetes.

BACKGROUND: In type 1 diabetes, changes in the GFR and urine albumin-to-creatinine ratio (ACR) are related to changes in kidney structure that reflect disease progression. However, such changes have not been studied in type 2 diabetes. METHODS: Participants were American Indians with type 2 diabetes enrolled in a clinical trial of losartan versus placebo. We followed a subset who underwent kidney biopsy at the end of the 6-year trial, with annual measurements of GFR (by urinary clearance of iothalamate) and ACR. Participants had a second kidney biopsy after a mean follow-up of 9.3 years. We used quantitative morphometric analyses to evaluate both biopsy specimens. RESULTS: Baseline measures for 48 participants (12 men and 36 women, mean age 45.6 years) who completed the study included diabetes duration (14.6 years), GFR (156 ml/min), and ACR (15 mg/g). During follow-up, glomerular basement membrane (GBM) width, mesangial fractional volume, and ACR increased, and surface density of peripheral GBM and GFR decreased. After adjustment for sex, age, ACR, and each morphometric variable at baseline, an increase in ACR during follow-up was significantly associated with increases in GBM width, mesangial fractional volume, and mean glomerular volume, and a decrease in surface density of peripheral GBM. Decline in GFR was not associated with changes in these morphometric variables after additionally adjusting for baseline GFR. CONCLUSIONS: In American Indians with type 2 diabetes and preserved GFR at baseline, increasing ACR reflects the progression of earlier structural glomerular lesions, whereas early GFR decline may not accurately reflect such lesions.

Renal compartment-specific genetic variation analyses identify new pathways in chronic kidney disease.

Chronic kidney disease (CKD), a condition in which the kidneys are unable to clear waste products, affects 700 million people globally. Genome-wide association studies (GWASs) have identified sequence variants for CKD; however, the biological basis of these GWAS results remains poorly understood. To address this issue, we created an expression quantitative trait loci (eQTL) atlas for the glomerular and tubular compartments of the human kidney. Through integrating the CKD GWAS with eQTL, single-cell RNA sequencing and regulatory region maps, we identified novel genes for CKD. Putative causal genes were enriched for proximal tubule expression and endolysosomal function, where DAB2, an adaptor protein in the TGF-ß pathway, formed a central node. Functional experiments confirmed that reducing Dab2 expression in renal tubules protected mice from CKD. In conclusion, compartment-specific eQTL analysis is an important avenue for the identification of novel genes and cellular pathways involved in CKD development and thus potential new opportunities for its treatment.

Profiling of Stem/Progenitor Cell Regulatory Genes of the Synovial Joint by Genome-Wide RNA-Seq Analysis.

Synovial joints suffer from arthritis and trauma that may be severely debilitative. Despite robust investigations in the roles of individual genes in synovial joint development and arthritis, little is known about global profiles of genes that regulate stem/progenitor cells of a synovial joint. The temporomandibular joint is a poorly understood synovial arthrosis with few clinical treatment options. Here, we isolated the articular and mature zones of the mandibular condyle by laser capture microdissection, performed genome-wide profiling, and analyzed molecular signaling pathways relevant to stem/progenitor cell functions. A total of 804 genes were differentially expressed between the articular and mature zones. Pathway analyses revealed 29 enriched signaling pathways, including the PI3K-Akt, Wnt, and Toll-like receptor signaling pathways that may regulate stem/progenitor cell homeostasis and differentiation into the chondrocyte lineage. Upstream regulator analyses further predicted potential upstream key regulators such as Xbp1, Nupr1, and Hif1a, and associated underlying mechanism networks were described. Among the multiple candidates of growth and transcriptional factors that may regulate stem/progenitor cells, we immunolocalized Sox9, Ihh, Frzb, Dkk1, Lgr5, and TGFß3 in the articular and mature zones. These findings provide a comprehensive genetic mapping of growth and transcriptional genes in the articular and mature zones of a synovial joint condyle. Differentially expressed genes may play crucial roles in the regulation of stem/progenitor cells in development, homeostasis, and tissue regeneration.

Distinct roles for motor neuron autophagy early and late in the SOD1G93A mouse model of ALS.

Mutations in autophagy genes can cause familial and sporadic amyotrophic lateral sclerosis (ALS). However, the role of autophagy in ALS pathogenesis is poorly understood, in part due to the lack of cell type-specific manipulations of this pathway in animal models. Using a mouse model of ALS expressing mutant superoxide dismutase 1 (SOD1G93A), we show that motor neurons form large autophagosomes containing ubiquitinated aggregates early in disease progression. To investigate whether this response is protective or detrimental, we generated mice in which the critical autophagy gene Atg7 was specifically disrupted in motor neurons (Atg7 cKO). Atg7 cKO mice were viable but exhibited structural and functional defects at a subset of vulnerable neuromuscular junctions. By crossing Atg7 cKO mice to the SOD1G93A mouse model, we found that autophagy inhibition accelerated early neuromuscular denervation of the tibialis anterior muscle and the onset of hindlimb tremor. Surprisingly, however, lifespan was extended in Atg7 cKO; SOD1G93A double-mutant mice. Autophagy inhibition did not prevent motor neuron cell death, but it reduced glial inflammation and blocked activation of the stress-related transcription factor c-Jun in spinal interneurons. We conclude that motor neuron autophagy is required to maintain neuromuscular innervation early in disease but eventually acts in a non-cell-autonomous manner to promote disease progression.

Bone morphogenetic protein and Notch signalling crosstalk in poor-prognosis, mesenchymal-subtype colorectal cancer.

The functional role of bone morphogenetic protein (BMP) signalling in colorectal cancer (CRC) is poorly defined, with contradictory results in cancer cell line models reflecting the inherent difficulties of assessing a signalling pathway that is context-dependent and subject to genetic constraints. By assessing the transcriptional response of a diploid human colonic epithelial cell line to BMP ligand stimulation, we generated a prognostic BMP signalling signature, which was applied to multiple CRC datasets to investigate BMP heterogeneity across CRC molecular subtypes. We linked BMP and Notch signalling pathway activity and function in human colonic epithelial cells, and normal and neoplastic tissue. BMP induced Notch through a γ-secretase-independent interaction, regulated by the SMAD proteins. In homeostasis, BMP/Notch co-localization was restricted to cells at the top of the intestinal crypt, with more widespread interaction in some human CRC samples. BMP signalling was downregulated in the majority of CRCs, but was conserved specifically in mesenchymal-subtype tumours, where it interacts with Notch to induce an epithelial-mesenchymal transition (EMT) phenotype. In intestinal homeostasis, BMP-Notch pathway crosstalk is restricted to differentiating cells through stringent pathway segregation. Conserved BMP activity and loss of signalling stringency in mesenchymal-subtype tumours promotes a synergistic BMP-Notch interaction, and this correlates with poor patient prognosis. BMP signalling heterogeneity across CRC subtypes and cell lines can account for previous experimental contradictions. Crosstalk between the BMP and Notch pathways will render mesenchymal-subtype CRC insensitive to γ-secretase inhibition unless BMP activation is concomitantly addressed. © 2017 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Unique Transcriptional Programs Identify Subtypes of AKI.

Two metrics, a rise in serum creatinine concentration and a decrease in urine output, are considered tantamount to the injury of the kidney tubule and the epithelial cells thereof (AKI). Yet neither criterion emphasizes the etiology or the pathogenetic heterogeneity of acute decreases in kidney excretory function. In fact, whether decreased excretory function due to contraction of the extracellular fluid volume (vAKI) or due to intrinsic kidney injury (iAKI) actually share pathogenesis and should be aggregated in the same diagnostic group remains an open question. To examine this possibility, we created mouse models of iAKI and vAKI that induced a similar increase in serum creatinine concentration. Using laser microdissection to isolate specific domains of the kidney, followed by RNA sequencing, we found that thousands of genes responded specifically to iAKI or to vAKI, but very few responded to both stimuli. In fact, the activated gene sets comprised different, functionally unrelated signal transduction pathways and were expressed in different regions of the kidney. Moreover, we identified distinctive gene expression patterns in human urine as potential biomarkers of either iAKI or vAKI, but not both. Hence, iAKI and vAKI are biologically unrelated, suggesting that molecular analysis should clarify our current definitions of acute changes in kidney excretory function.

ABC transporters and the proteasome complex are implicated in susceptibility to Stevens-Johnson syndrome and toxic epidermal necrolysis across multiple drugs.

Stevens-Johnson syndrome (SJS) and Toxic Epidermal Necrolysis (TEN) represent rare but serious adverse drug reactions (ADRs). Both are characterized by distinctive blistering lesions and significant mortality rates. While there is evidence for strong drug-specific genetic predisposition related to HLA alleles, recent genome wide association studies (GWAS) on European and Asian populations have failed to identify genetic susceptibility alleles that are common across multiple drugs. We hypothesize that this is a consequence of the low to moderate effect size of individual genetic risk factors. To test this hypothesis we developed Pointer, a new algorithm that assesses the aggregate effect of multiple low risk variants on a pathway using a gene set enrichment approach. A key advantage of our method is the capability to associate SNPs with genes by exploiting physical proximity as well as by using expression quantitative trait loci (eQTLs) that capture information about both cis- and trans-acting regulatory effects. We control for known bias-inducing aspects of enrichment based analyses, such as: 1) gene length, 2) gene set size, 3) presence of biologically related genes within the same linkage disequilibrium (LD) region, and, 4) genes shared among multiple gene sets. We applied this approach to publicly available SJS/TEN genome-wide genotype data and identified the ABC transporter and Proteasome pathways as potentially implicated in the genetic susceptibility of non-drug-specific SJS/TEN. We demonstrated that the innovative SNP-to-gene mapping phase of the method was essential in detecting the significant enrichment for those pathways. Analysis of an independent gene expression dataset provides supportive functional evidence for the involvement of Proteasome pathways in SJS/TEN cutaneous lesions. These results suggest that Pointer provides a useful framework for the integrative analysis of pharmacogenetic GWAS data, by increasing the power to detect aggregate effects of multiple low risk variants. The software is available for download at https://sourceforge.net/projects/pointergsa/.

Molecular differences between stromal cell populations from deciduous and permanent human teeth.

INTRODUCTION: Deciduous and permanent human teeth represent an excellent model system to study aging of stromal populations. Aging is tightly connected to self-renewal and proliferation and thus, mapping potential molecular differences in these characteristics between populations constitutes an important task. METHODS: Using specifically designed microarray panels, Real-Time Quantitative Polymerase Chain Reaction (RT q-PCR), Western blot, immunohistochemistry and siRNA-mediated knock down experiments, we have detected a number of molecules that were differentially expressed in dental pulp from deciduous and permanent teeth extracted from young children and adults, respectively. RESULTS: Among the differentially regulated genes, high-mobility group AT-hook 2 (HMGA2), a stem cell-associated marker, stood out as a remarkable example with a robust expression in deciduous pulp cells. siRNA-mediated knock down of HMGA2 expression in cultured deciduous pulp cells caused a down-regulated expression of the pluripotency marker NANOG. This finding indicates that HMGA2 is a pulpal stem cell regulatory factor. In addition to this, we discovered that several proliferation-related genes, including CDC2A and CDK4, were up-regulated in deciduous pulp cells, while matrix genes COL1A1, fibronectin and several signaling molecules, such as VEGF, FGFr-1 and IGFr-1 were up-regulated in the pulp cells from permanent teeth. CONCLUSIONS: Taken together, our data suggest that deciduous pulp cells are more robust in self- renewal and proliferation, whereas adult dental pulp cells are more capable of signaling and matrix synthesis.

An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex.

The major cell classes of the brain differ in their developmental processes, metabolism, signaling, and function. To better understand the functions and interactions of the cell types that comprise these classes, we acutely purified representative populations of neurons, astrocytes, oligodendrocyte precursor cells, newly formed oligodendrocytes, myelinating oligodendrocytes, microglia, endothelial cells, and pericytes from mouse cerebral cortex. We generated a transcriptome database for these eight cell types by RNA sequencing and used a sensitive algorithm to detect alternative splicing events in each cell type. Bioinformatic analyses identified thousands of new cell type-enriched genes and splicing isoforms that will provide novel markers for cell identification, tools for genetic manipulation, and insights into the biology of the brain. For example, our data provide clues as to how neurons and astrocytes differ in their ability to dynamically regulate glycolytic flux and lactate generation attributable to unique splicing of PKM2, the gene encoding the glycolytic enzyme pyruvate kinase. This dataset will provide a powerful new resource for understanding the development and function of the brain. To ensure the widespread distribution of these datasets, we have created a user-friendly website (http://web.stanford.edu/group/barres_lab/brain_rnaseq.html) that provides a platform for analyzing and comparing transciption and alternative splicing profiles for various cell classes in the brain.

The transcriptional regulatory network of proneural glioma determines the genetic alterations selected during tumor progression.

Proneural glioblastoma is defined by an expression pattern resembling that of oligodendrocyte progenitor cells and carries a distinctive set of genetic alterations. Whether there is a functional relationship between the proneural phenotype and the associated genetic alterations is unknown. To evaluate this possible relationship, we performed a longitudinal molecular characterization of tumor progression in a mouse model of proneural glioma. In this setting, the tumors acquired remarkably consistent genetic deletions at late stages of progression, similar to those deleted in human proneural glioblastoma. Further investigations revealed that p53 is a master regulator of the transcriptional network underlying the proneural phenotype. This p53-centric transcriptional network and its associated phenotype were observed at both the early and late stages of progression, and preceded the proneural-specific deletions. Remarkably, deletion of p53 at the time of tumor initiation obviated the acquisition of later deletions, establishing a link between the proneural transcriptional network and the subtype-specific deletions selected during glioma progression.

A molecular signature predictive of indolent prostate cancer.

Many newly diagnosed prostate cancers present as low Gleason score tumors that require no treatment intervention. Distinguishing the many indolent tumors from the minority of lethal ones remains a major clinical challenge. We now show that low Gleason score prostate tumors can be distinguished as indolent and aggressive subgroups on the basis of their expression of genes associated with aging and senescence. Using gene set enrichment analysis, we identified a 19-gene signature enriched in indolent prostate tumors. We then further classified this signature with a decision tree learning model to identify three genes--FGFR1, PMP22, and CDKN1A--that together accurately predicted outcome of low Gleason score tumors. Validation of this three-gene panel on independent cohorts confirmed its independent prognostic value as well as its ability to improve prognosis with currently used clinical nomograms. Furthermore, protein expression of this three-gene panel in biopsy samples distinguished Gleason 6 patients who failed surveillance over a 10-year period. We propose that this signature may be incorporated into prognostic assays for monitoring patients on active surveillance to facilitate appropriate courses of treatment.

Intricate interplay between astrocytes and motor neurons in ALS.

ALS results from the selective and progressive degeneration of motor neurons. Although the underlying disease mechanisms remain unknown, glial cells have been implicated in ALS disease progression. Here, we examine the effects of glial cell/motor neuron interactions on gene expression using the hSOD1(G93A) (the G93A allele of the human superoxide dismutase gene) mouse model of ALS. We detect striking cell autonomous and nonautonomous changes in gene expression in cocultured motor neurons and glia, revealing that the two cell types profoundly affect each other. In addition, we found a remarkable concordance between the cell culture data and expression profiles of whole spinal cords and acutely isolated spinal cord cells during disease progression in the G93A mouse model, providing validation of the cell culture approach. Bioinformatics analyses identified changes in the expression of specific genes and signaling pathways that may contribute to motor neuron degeneration in ALS, among which are TGF-ß signaling pathways.

Reprogramming of the microRNA transcriptome mediates resistance to rapamycin.

The mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation that is often deregulated in cancer. Inhibitors of mTOR, including rapamycin and its analogues, are being evaluated as antitumor agents. For their promise to be fulfilled, it is of paramount importance to identify the mechanisms of resistance and develop novel therapies to overcome it. Given the emerging role of microRNAs (miRNAs) in tumorigenesis, we hypothesized that miRNAs could play important roles in the response of tumors to mTOR inhibitors. Long-term rapamycin treatment showed extensive reprogramming of miRNA expression, characterized by up-regulation of miR-17-92 and related clusters and down-regulation of tumor suppressor miRNAs. Inhibition of members of the miR-17-92 clusters or delivery of tumor suppressor miRNAs restored sensitivity to rapamycin. This study identifies miRNAs as new downstream components of the mTOR-signaling pathway, which may determine the response of tumors to mTOR inhibitors. It also identifies potential markers to assess the efficacy of treatment and provides novel therapeutic targets to treat rapamycin-resistant tumors.

Early-stage epigenetic modification during somatic cell reprogramming by Parp1 and Tet2.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by using the pluripotency factors Oct4, Sox2, Klf4 and c-Myc (together referred to as OSKM). iPSC reprogramming erases somatic epigenetic signatures­as typified by DNA methylation or histone modification at silent pluripotency loci­and establishes alternative epigenetic marks of embryonic stem cells (ESCs). Here we describe an early and essential stage of somatic cell reprogramming, preceding the induction of transcription at endogenous pluripotency loci such as Nanog and Esrrb. By day 4 after transduction with OSKM, two epigenetic modification factors necessary for iPSC generation, namely poly(ADP-ribose) polymerase-1 (Parp1) and ten-eleven translocation-2 (Tet2), are recruited to the Nanog and Esrrb loci. These epigenetic modification factors seem to have complementary roles in the establishment of early epigenetic marks during somatic cell reprogramming: Parp1 functions in the regulation of 5-methylcytosine (5mC) modification, whereas Tet2 is essential for the early generation of 5-hydroxymethylcytosine (5hmC) by the oxidation of 5mC (refs 3,4). Although 5hmC has been proposed to serve primarily as an intermediate in 5mC demethylation to cytosine in certain contexts, our data, and also studies of Tet2-mutant human tumour cells, argue in favour of a role for 5hmC as an epigenetic mark distinct from 5mC. Consistent with this, Parp1 and Tet2 are each needed for the early establishment of histone modifications that typify an activated chromatin state at pluripotency loci, whereas Parp1 induction further promotes accessibility to the Oct4 reprogramming factor. These findings suggest that Parp1 and Tet2 contribute to an epigenetic program that directs subsequent transcriptional induction at pluripotency loci during somatic cell reprogramming.

The testis-specific double bromodomain-containing protein BRDT forms a complex with multiple spliceosome components and is required for mRNA splicing and 3'-UTR truncation in round spermatids.

Members of the BET (bromodomain and extra terminal motif) family of proteins have been shown to be chromatin-interacting regulators of transcription. We previously generated a mutation in the testis-specific mammalian BET gene Brdt (bromodomain, testis-specific) that yields protein lacking the first bromodomain (BRDT(ΔBD1)) and observed disrupted spermiogenesis and male sterility. To determine whether BRDT(ΔBD1) protein results in altered transcription, we analyzed the transcriptomes of control versus Brdt(ΔBD1/ΔBD1) round spermatids. Over 400 genes showed statistically significant differential expression, and among the up-regulated genes, there was an enrichment of RNA splicing genes. Over 60% of these splicing genes had transcripts that lacked truncation of their 3'-untranslated region (UTR) typical of round spermatids. We selected four of these genes to characterize: Srsf2, Ddx5, Hnrnpk and Tardbp. The 3'-UTRs of Srsf2, Ddx5 and Hnrnpk mRNAs were longer in mutant round spermatids and resulted in reduced protein levels. Tardbp was transcriptionally up-regulated and a splicing shift toward the longer variant was observed. All four splicing proteins were found to complex with BRDT in control and mutant testes. We thus suggest that, along with modulating transcription, BRDT modulates gene expression as part of the splicing machinery. These modulations alter 3'-UTR processing in round spermatids; importantly, the BD1 is essential for these functions.