Genetic Variation and Sickle Cell Disease Severity: A Systematic Review and Meta-Analysis.

Importance: Sickle cell disease (SCD) is a monogenic disorder, yet clinical outcomes are influenced by additional genetic factors. Despite decades of research, the genetics of SCD remain poorly understood. Objective: To assess all reported genetic modifiers of SCD, evaluate the design of associated studies, and provide guidelines for future analyses according to modern genetic study recommendations. Data Sources: PubMed, Web of Science, and Scopus were searched through May 16, 2023, identifying 5290 publications. Study Selection: At least 2 reviewers identified 571 original, peer-reviewed English-language publications reporting genetic modifiers of human SCD phenotypes, wherein the outcome was not treatment response, and the comparison was not between SCD subtypes or including healthy controls. Data Extraction and Synthesis: Data relevant to all genetic modifiers of SCD were extracted, evaluated, and presented following STREGA and PRISMA guidelines. Weighted z score meta-analyses and pathway analyses were conducted. Main Outcomes and Measures: Outcomes were aggregated into 25 categories, grouped as acute complications, chronic conditions, hematologic parameters or biomarkers, and general or mixed measures of SCD severity. Results: The 571 included studies reported on 29 670 unique individuals (50% ≤ 18 years of age) from 43 countries. Of the 17 757 extracted results (4890 significant) in 1552 genes, 3675 results met the study criteria for meta-analysis: reported phenotype and genotype, association size and direction, variability measure, sample size, and statistical test. Only 173 results for 62 associations could be cross-study combined. The remaining associations could not be aggregated because they were only reported once or methods (eg, study design, reporting practice) and genotype or phenotype definitions were insufficiently harmonized. Gene variants regulating fetal hemoglobin and α-thalassemia (important markers for SCD severity) were frequently identified: 19 single-nucleotide variants in BCL11A, HBS1L-MYB, and HBG2 were significantly associated with fetal hemoglobin (absolute value of Z = 4.00 to 20.66; P = 8.63 × 10-95 to 6.19 × 10-5), and α-thalassemia deletions were significantly associated with increased hemoglobin level and reduced risk of albuminuria, abnormal transcranial Doppler velocity, and stroke (absolute value of Z = 3.43 to 5.16; P = 2.42 × 10-7 to 6.00 × 10-4). However, other associations remain unconfirmed. Pathway analyses of significant genes highlighted the importance of cellular adhesion, inflammation, oxidative and toxic stress, and blood vessel regulation in SCD (23 of the top 25 Gene Ontology pathways involve these processes) and suggested future research areas. Conclusions and Relevance: The findings of this comprehensive systematic review and meta-analysis of all published genetic modifiers of SCD indicated that implementation of standardized phenotypes, statistical methods, and reporting practices should accelerate discovery and validation of genetic modifiers and development of clinically actionable genetic profiles.

Clinical and genomic characterization of an ATRA-insensitive acute promyelocytic leukemia variant with a FNDC3B::RARB fusion.

The promyelocytic leukemia-retinoic acid receptor-α (PML::RARA) fusion is the hallmark of acute promyelocytic leukemia (APL) and is observed in over 95% of APL cases. RARA and homologous receptors RARB and RARG are occasionally fused to other gene partners, which differentially affect sensitivity to targeted therapies. Most APLs without RARA fusions have rearrangements involving RARG or RARB, both of which frequently show resistance to all-trans-retinoic acid (ATRA) and/or multiagent chemotherapy for acute myeloid leukemia (AML). We present a 13-year-old male diagnosed with variant APL with a novel FNDC3B::RARB in-frame fusion that showed no response to ATRA but responded well to conventional AML therapy. While FNDC3B has been identified as a rare RARA translocation partner in ATRA-sensitive variant APL, it has never been reported as a fusion partner with RARB and it is only the second known fusion partner with RARB in variant APL. We also show that this novel fusion confers an RNA expression signature that is similar to APL, despite clinical resistance to ATRA monotherapy.

Treatment of generalized infantile myofibromatosis with sorafenib and imatinib: A case report.

Infantile myofibromatosis (IM) is characterized by solitary musculoskeletal nodules presenting during infancy but can manifest as multiple lesions with visceral involvement. Multicentric IM with visceral involvement carries a high risk of mortality and there is no consensus on treatment. We present a case of a patient with multicentric IM and pulmonary involvement who progressed on several chemotherapeutic regimens and subsequently had a complete response to sorafenib and later imatinib. This report describes the novel use of sorafenib and imatinib to treat generalized IM and the role of continued tyrosine kinase inhibitor therapy to maintain remission.

Cyclin-Dependent Kinase CRK9, Required for Spliced Leader trans Splicing of Pre-mRNA in Trypanosomes, Functions in a Complex with a New L-Type Cyclin and a Kinetoplastid-Specific Protein.

In eukaryotes, cyclin-dependent kinases (CDKs) control the cell cycle and critical steps in gene expression. The lethal parasite Trypanosoma brucei, member of the phylogenetic order Kinetoplastida, possesses eleven CDKs which, due to high sequence divergence, were generically termed CDC2-related kinases (CRKs). While several CRKs have been implied in the cell cycle, CRK9 was the first trypanosome CDK shown to control the unusual mode of gene expression found in kinetoplastids. In these organisms, protein-coding genes are arranged in tandem arrays which are transcribed polycistronically. Individual mRNAs are processed from precursor RNA by spliced leader (SL) trans splicing and polyadenylation. CRK9 ablation was lethal in cultured trypanosomes, causing a block of trans splicing before the first transesterification step. Additionally, CRK9 silencing led to dephosphorylation of RNA polymerase II and to hypomethylation of the SL cap structure. Here, we tandem affinity-purified CRK9 and, among potential CRK9 substrates and modifying enzymes, discovered an unusual tripartite complex comprising CRK9, a new L-type cyclin (CYC12) and a protein, termed CRK9-associated protein (CRK9AP), that is only conserved among kinetoplastids. Silencing of either CYC12 or CRK9AP reproduced the effects of depleting CRK9, identifying these proteins as functional partners of CRK9 in vivo. While mammalian cyclin L binds to CDK11, the CRK9 complex deviates substantially from that of CDK11, requiring CRK9AP for efficient CRK9 complex formation and autophosphorylation in vitro. Interference with this unusual CDK rescued mice from lethal trypanosome infections, validating CRK9 as a potential chemotherapeutic target.

Dynein Light Chain LC8 Is Required for RNA Polymerase I-Mediated Transcription in Trypanosoma brucei, Facilitating Assembly and Promoter Binding of Class I Transcription Factor A.

Dynein light chain LC8 is highly conserved among eukaryotes and has both dynein-dependent and dynein-independent functions. Interestingly, LC8 was identified as a subunit of the class I transcription factor A (CITFA), which is essential for transcription by RNA polymerase I (Pol I) in the parasite Trypanosoma brucei. Given that LC8 has never been identified with a basal transcription factor and that T. brucei relies on RNA Pol I for expressing the variant surface glycoprotein (VSG), the key protein in antigenic variation, we investigated the CITFA-specific role of LC8. Depletion of LC8 from mammalian-infective bloodstream trypanosomes affected cell cycle progression, reduced the abundances of rRNA and VSG mRNA, and resulted in rapid cell death. Sedimentation analysis, coimmunoprecipitation of recombinant proteins, and bioinformatic analysis revealed an LC8 binding site near the N terminus of the subunit CITFA2. Mutation of this site prevented the formation of a CITFA2-LC8 heterotetramer and, in vivo, was lethal, affecting assembly of a functional CITFA complex. Gel shift assays and UV cross-linking experiments identified CITFA2 as a promoter-binding CITFA subunit. Accordingly, silencing of LC8 or CITFA2 resulted in a loss of CITFA from RNA Pol I promoters. Hence, we discovered an LC8 interaction that, unprecedentedly, has a basal function in transcription.

Mono-allelic VSG expression by RNA polymerase I in Trypanosoma brucei: expression site control from both ends?

Trypanosoma brucei is a vector borne, lethal protistan parasite of humans and livestock in sub-Saharan Africa. Antigenic variation of its cell surface coat enables the parasite to evade adaptive immune responses and to live freely in the blood of its mammalian hosts. The coat consists of ten million copies of variant surface glycoprotein (VSG) that is expressed from a single VSG gene, drawn from a large repertoire and located near the telomere at one of fifteen so-called bloodstream expression sites (BESs). Thus, antigenic variation is achieved by switching to the expression of a different VSG gene. A BES is a tandem array of expression site-associated genes and a terminal VSG gene. It is polycistronically transcribed by a multifunctional RNA polymerase I (RNAPI) from a short promoter that is located 45-60 kb upstream of the VSG gene. The mechanism(s) restricting VSG expression to a single BES are not well understood. There is convincing evidence that epigenetic silencing and transcription attenuation play important roles. Furthermore, recent data indicated that there is regulation at the level of transcription initiation and that, surprisingly, the VSG mRNA appears to have a role in restricting VSG expression to a single gene. Here, we review BES expression regulation and propose a model in which telomere-directed, epigenetic BES silencing is opposed by BES promoter-directed, activated RNAPI transcription.

A new strategy of RNA interference that targets heterologous sequences reveals CITFA1 as an essential component of class I transcription factor A in Trypanosoma brucei.

Conditional gene silencing by RNA interference in Trypanosoma brucei can be inconclusive if knockdowns are inefficient or have off-target effects. To enable efficient, specific silencing of single-copy genes in mammalian-infective, bloodstream form trypanosomes, we developed a system that targets the heterologous and functional Trypanosoma cruzi U2AF35 3' untranslated region (UTR) (Tc3) or, alternatively, the sequence of the PTP tag, which can be fused to any mRNA of interest. Two cell lines were created, single-marker Tc3 (smTc3) and smPTP, which conditionally express Tc3 and PTP double-stranded RNA (dsRNA), respectively. The system depends on manipulating both alleles of the gene of interest so that cells exclusively express the target mRNA as a fusion to one of these heterologous sequences. We generated allele integration vectors in which the C-terminal part of a gene's coding sequence can be fused to either heterologous sequence in a single cloning step. We first tested this system with CITFA7, which encodes a well-characterized subunit of the class I transcription factor A (CITFA), an essential factor for transcription initiation by RNA polymerase I. Targeting either Tc3 or PTP fused to the CITFA7 mRNA resulted in gene knockdowns that were as efficient and specific as targeting the endogenous CITFA7 mRNA. Moreover, application of this system to CITFA1, which could not be silenced by established methods, demonstrated that the gene encodes an essential CITFA subunit that mediates binding of the transcription factor complex to RNA polymerase I promoters.

Transcription by the multifunctional RNA polymerase I in Trypanosoma brucei functions independently of RPB7.

Trypanosoma brucei has a multifunctional RNA polymerase (pol) I that transcribes ribosomal gene units (RRNA) and units encoding its major cell surface proteins variant surface glycoprotein (VSG) and procyclin. Previous analysis of tandem affinity-purified, transcriptionally active RNA pol I identified ten subunits including an apparently trypanosomatid-specific protein termed RPA31. Another ortholog was identified in silico. No orthologs of the yeast subunit doublet RPA43/RPA14 have been identified yet. Instead, a recent report presented evidence that RPB7, the RNA pol II paralog of RPA43, is an RNA pol I subunit and essential for RRNA and VSG transcription in bloodstream form trypanosomes [18]. Revisiting this attractive hypothesis, we were unable to detect a stable interaction between RPB7 and RNA pol I in either reciprocal co-immunoprecipitation or tandem affinity purification. Furthermore, immunodepletion of RPB7 from extract virtually abolished RNA pol II transcription in vitro but had no effect on RRNA or VSG ES promoter transcription in the same reactions. Accordingly, chromatin immunoprecipitation analysis revealed cross-linking of RPB7 to known RNA pol II transcription units but not to the VSG ES promoter or to the 18S rRNA coding region. Interestingly, RPB7 did crosslink to the RRNA promoter but so did the RNA pol II-specific subunit RPB9 suggesting that RNA pol II is recruited to this promoter. Overall, our data led to the conclusion that RNA pol I transcription in T. brucei does not require the RNA pol II subunit RPB7.

Duration of oliguria and anuria as predictors of chronic renal-related sequelae in post-diarrheal hemolytic uremic syndrome.

Prior long-term retrospective studies have described renal sequelae in 25-50% of postdiarrheal hemolytic uremic syndrome (HUS) survivors, but the ability to predict the likelihood of chronic renal-related sequelae at the time of hospital discharge is limited. We surveyed 357 children in our HUS registry who survived an acute episode of post diarrheal HUS (D+HUS) and were without end-stage renal disease (ESRD) at the time of hospital discharge. Of the 357 patients surveyed, 159 had at least 1 year (mean 8.75 years) of follow-up. Of these, 90 individuals were identified as having had at least 1 day of oliguria, with 69 individuals having had at least 1 day of anuria. The incidences of renal-related sequelae [proteinuria, low glomerular filtration rate (GFR), and hypertension] were determined among experimental groups based on oliguria and anuria duration. One or more sequelae (e.g. proteinuria, low GFR, hypertension) was seen in 25 (36.2%) of those who had no recorded oliguria and 34 (37.8%) of those with no recorded anuria. The prevalence of chronic sequelae increased markedly in those with more than 5 days of anuria or 10 days of oliguria, with anuria being a better predictor than oliguria of most related sequelae. A particularly high incidence of hypertension was seen in patients with > 10 days of anuria (55.6%) in comparison with those with no anuria (8.9%) [odds ratio (OR) 12.8; 95% confidence interval (CI) 2.9-57.5]. Patients with > 10 days of anuria were also at substantially increased risk for low GFR and proteinuria (OR 35.2; 95% CI 5.1-240.5). These findings may help identify children who need periodic and extended follow-up after hospital discharge.