Two Aldehyde Clearance Systems Are Essential to Prevent Lethal Formaldehyde Accumulation in Mice and Humans.

Reactive aldehydes arise as by-products of metabolism and are normally cleared by multiple families of enzymes. We find that mice lacking two aldehyde detoxifying enzymes, mitochondrial ALDH2 and cytoplasmic ADH5, have greatly shortened lifespans and develop leukemia. Hematopoiesis is disrupted profoundly, with a reduction of hematopoietic stem cells and common lymphoid progenitors causing a severely depleted acquired immune system. We show that formaldehyde is a common substrate of ALDH2 and ADH5 and establish methods to quantify elevated blood formaldehyde and formaldehyde-DNA adducts in tissues. Bone-marrow-derived progenitors actively engage DNA repair but also imprint a formaldehyde-driven mutation signature similar to aging-associated human cancer mutation signatures. Furthermore, we identify analogous genetic defects in children causing a previously uncharacterized inherited bone marrow failure and pre-leukemic syndrome. Endogenous formaldehyde clearance alone is therefore critical for hematopoiesis and in limiting mutagenesis in somatic tissues.

Epstein-Barr virus lytic gene BNRF1 promotes B-cell lymphomagenesis via IFI27 upregulation.

Epstein-Barr virus (EBV) is a ubiquitous human lymphotropic herpesvirus that is causally associated with several malignancies. In addition to latent factors, lytic replication contributes to cancer development. In this study, we examined whether the lytic gene BNRF1, which is conserved among gamma-herpesviruses, has an important role in lymphomagenesis. We found that lymphoblastoid cell lines (LCLs) established by BNRF1-knockout EBV exhibited remarkably lower pathogenicity in a mice xenograft model than LCLs produced by wild-type EBV (LCLs-WT). RNA-seq analyses revealed that BNRF1 elicited the expression of interferon-inducible protein 27 (IFI27), which promotes cell proliferation. IFI27 knockdown in LCLs-WT resulted in excessive production of reactive oxygen species, leading to cell death and significantly decreased their pathogenicity in vivo. We also confirmed that IFI27 was upregulated during primary infection in B-cells. Our findings revealed that BNRF1 promoted robust proliferation of the B-cells that were transformed by EBV latent infection via IFI27 upregulation both in vitro and in vivo.

Single-Cell Transcriptomic Analysis of Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis.

Epstein-Barr virus (EBV) infection can lead to infectious mononucleosis (EBV-IM) and, more rarely, EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), which is characterized by a life-threatening hyperinflammatory cytokine storm with immune dysregulation. Interferon-gamma (IFNγ) has been identified as a critical mediator for primary HLH; however, the detailed role of IFNγ and other cytokines in EBV-HLH is not fully understood. In this study, we used single-cell RNA sequencing to characterize the immune landscape of EBV-HLH and compared it with EBV-IM. Three pediatric patients with EBV-HLH with different backgrounds, one with X-linked lymphoproliferative syndrome type 1 (XLP1), two with chronic active EBV disease (CAEBV), and two patients with EBV-IM were enrolled. The TUBA1B + STMN1 + CD8 + T cell cluster, a responsive proliferating cluster with rich mRNA detection, was explicitly observed in EBV-IM, and the upregulation of SH2D1A-the gene responsible for XLP1-was localized in this cluster. This proliferative cluster was scarcely observed in EBV-HLH cases. In EBV-HLH cases with CAEBV, upregulation of LAG3 was observed in EBV-infected cells, which may be associated with an impaired response by CD8 + T cells. Additionally, genes involved in type I interferon (IFN) signaling were commonly upregulated in each cell fraction of EBV-HLH, and activation of type II IFN signaling was observed in CD4 + T cells, natural killer cells, and monocytes but not in CD8 + T cells in EBV-HLH. In conclusion, impaired responsive proliferation of CD8 + T cells and upregulation of type I IFN signaling were commonly observed in EBV-HLH cases, regardless of the patients' background, indicating the key features of EBV-HLH.

Updated mutational spectrum and genotype-phenotype correlations in ichthyosis patients with ABCA12 pathogenic variants.

Autosomal recessive congenital ichthyoses (ARCI) is a genetically heterogeneous condition that can be caused by pathogenic variants in at least 12 genes, including ABCA12. ARCI mainly consists of congenital ichthyosiform erythroderma (CIE), lamellar ichthyosis (LI) and harlequin ichthyosis (HI). The objective was to determine previously unreported pathogenic variants in ABCA12 and to update genotype-phenotype correlations for patients with pathogenic ABCA12 variants. Pathogenic variants in ABCA12 were detected using Sanger sequencing or a combination of Sanger sequencing and whole-exome sequencing. To verify the pathogenicity of a previously unreported large deletion and intron variant, cDNA analysis was performed using total RNA extracted from hair roots. Genetic analyses were performed on the patients with CIE, LI, HI and non-congenital ichthyosis with unusual phenotypes (NIUP), and 11 previously unreported ABCA12 variants were identified. Sequencing of cDNA confirmed the aberrant splicing of the variant ABCA12 in the patients with the previously unreported large deletion and intron variant. Our findings expand the phenotype spectrum of ichthyosis patients with ABCA12 pathogenic variants. The present missense variants in ABCA12 are considered to be heterogenous in pathogenicity, and they lead to varying disease severities in patients with ARCI and non-congenital ichthyosis with unusual phenotypes (NIUP).

Atomic view of cosolute-induced protein denaturation probed by NMR solvent paramagnetic relaxation enhancement.

The cosolvent effect arises from the interaction of cosolute molecules with a protein and alters the equilibrium between native and unfolded states. Denaturants shift the equilibrium toward the latter, while osmolytes stabilize the former. The molecular mechanism whereby cosolutes perturb protein stability is still the subject of considerable debate. Probing the molecular details of the cosolvent effect is experimentally challenging as the interactions are very weak and transient, rendering them invisible to most conventional biophysical techniques. Here, we probe cosolute-protein interactions by means of NMR solvent paramagnetic relaxation enhancement together with a formalism we recently developed to quantitatively describe, at atomic resolution, the energetics and dynamics of cosolute-protein interactions in terms of a concentration normalized equilibrium average of the interspin distance, [Formula: see text], and an effective correlation time, τc The system studied is the metastable drkN SH3 domain, which exists in dynamic equilibrium between native and unfolded states, thereby permitting us to probe the interactions of cosolutes with both states simultaneously under the same conditions. Two paramagnetic cosolute denaturants were investigated, one neutral and the other negatively charged, differing in the presence of a carboxyamide group versus a carboxylate. Our results demonstrate that attractive cosolute-protein backbone interactions occur largely in the unfolded state and some loop regions in the native state, electrostatic interactions reduce the [Formula: see text] values, and temperature predominantly impacts interactions with the unfolded state. Thus, destabilization of the native state in this instance arises predominantly as a consequence of interactions of the cosolutes with the unfolded state.

The Epstein-Barr Virus Enhancer Interaction Landscapes in Virus-Associated Cancer Cell Lines.

Epstein-Barr virus (EBV) persists in human cells as episomes. EBV episomes are chromatinized and their 3D conformation varies greatly in cells expressing different latency genes. We used HiChIP, an assay which combines genome-wide chromatin conformation capture followed by deep sequencing (Hi-C) and chromatin immunoprecipitation (ChIP), to interrogate the EBV episome 3D conformation in different cancer cell lines. In an EBV-transformed lymphoblastoid cell line (LCL) GM12878 expressing type III EBV latency genes, abundant genomic interactions were identified by H3K27ac HiChIP. A strong enhancer was located near the BILF2 gene and looped to multiple genes around BALFs loci. Perturbation of the BILF2 enhancer by CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) altered the expression of BILF2 enhancer-linked genes, including BARF0 and BALF2, suggesting that this enhancer regulates the expression of linked genes. H3K27ac ChIP followed by deep sequencing (ChIP-seq) identified several strong EBV enhancers in T/NK (natural killer) lymphoma cells that express type II EBV latency genes. Extensive intragenomic interactions were also found which linked enhancers to target genes. A strong enhancer at BILF2 also looped to the BALF loci. CRISPRi also validated the functional connection between BILF2 enhancer and BARF1 gene. In contrast, H3K27ac HiChIP found significantly fewer intragenomic interactions in type I EBV latency gene-expressing primary effusion lymphoma (PEL) cell lines. These data provided new insight into the regulation of EBV latency gene expression in different EBV-associated tumors. IMPORTANCE EBV is the first human DNA tumor virus identified, discovered over 50 years ago. EBV causes ~200,000 cases of various cancers each year. EBV-encoded oncogenes, noncoding RNAs, and microRNAs (miRNAs) can promote cell growth and survival and suppress senescence. Regulation of EBV gene expression is very complex. The viral C promoter regulates the expression of all EBV nuclear antigens (EBNAs), some of which are very far away from the C promoter. Another way by which the virus activates remote gene expression is through DNA looping. In this study, we describe the viral genome looping patterns in various EBV-associated cancer cell lines and identify important EBV enhancers in these cells. This study also identified novel opportunities to perturb and eventually control EBV gene expression in these cancer cells.

Juvenile Hemochromatosis With Non-transfused Hemolytic Anemia Caused by a De Novo PIEZO1 Gene Mutation.

Differential diagnosis of juvenile hemochromatosis along with hemolytic anemia is often difficult. We report a 23-year-old woman with macrocytic hemolytic anemia with iron overload. The patient showed high serum ferritin and transferrin saturation and low serum transferrin and ceruloplasmin. We also noticed stomatocytes in her blood smear, which was confirmed by scanning electron microscopy. Target gene sequencing identified a mutation in PIEZO1 (heterozygous c.6008C>A: p.A2003D). This mutation was reported previously in a family with dehydrated hereditary stomatocytosis (DHS1, [OMIM 194380]), but in the current case, it was identified to be a de novo mutation. We underscore DHS1 in the differential diagnosis of iron overload associated with non-transfused hemolytic anemia in children and young adults.

Single-cell RNA sequencing of intestinal immune cells in neonatal necrotizing enterocolitis.

PURPOSE: Necrotizing enterocolitis (NEC) causes fatal intestinal necrosis in neonates, but its etiology is unknown. We analyzed the intestinal immune response to NEC. METHODS: Using single-cell RNA sequencing (scRNA-seq), we analyzed the gene expression profiles of intestinal immune cells from four neonates with intestinal perforation (two with NEC and two without NEC). Target mononuclear cells were extracted from the lamina propria of the resected intestines. RESULTS: In all four cases, major immune cells, such as T cells (15.1-47.7%), B cells (3.1-19.0%), monocytes (16.5-31.2%), macrophages (1.6-17.4%), dendritic cells (2.4-12.2%), and natural killer cells (7.5-12.8%), were present in similar proportions to those in the neonatal cord blood. Gene set enrichment analysis showed that the MTOR, TNF-α, and MYC signaling pathways were enriched in T cells of the NEC patients, suggesting upregulated immune responses related to inflammation and cell proliferation. In addition, all four cases exhibited a bias toward cell-mediated inflammation, based on the predominance of T helper 1 cells. CONCLUSION: Intestinal immunity in NEC subjects exhibited stronger inflammatory responses compared to non-NEC subjects. Further scRNA-seq and cellular analysis may improve our understanding of the pathogenesis of NEC.

N6-methyladenosine Modification of Hepatitis B Virus RNA in the Coding Region of HBx.

N6-methyladenosine (m6A) is a post-transcriptional modification of RNA involved in transcript transport, degradation, translation, and splicing. We found that HBV RNA is modified by m6A predominantly in the coding region of HBx. The mutagenesis of methylation sites reduced the HBV mRNA and HBs protein levels. The suppression of m6A by an inhibitor or knockdown in primary hepatocytes decreased the viral RNA and HBs protein levels in the medium. These results suggest that the m6A modification of HBV RNA is needed for the efficient replication of HBV in hepatocytes.

Theory and Applications of Nitroxide-based Paramagnetic Cosolutes for Probing Intermolecular and Electrostatic Interactions on Protein Surfaces.

Solvent paramagnetic relaxation enhancement (sPRE) arising from nitroxide-based cosolutes has recently been used to provide an atomic view of cosolute-induced protein denaturation and to characterize residue-specific effective near-surface electrostatic potentials (ϕENS). Here, we explore distinct properties of the sPRE arising from nitroxide-based cosolutes and provide new insights into the interpretation of the sPRE and sPRE-derived ϕENS. We show that: (a) the longitudinal sPRE rate Γ1 is heavily dependent on spectrometer field and viscosity, while the transverse sPRE rate Γ2 is much less so; (b) the spectral density J(0) is proportional to the inverse of the relative translational diffusion constant and is related to the quantity ⟨r-4⟩norm, a concentration-normalized equilibrium average of the electron-proton interspin separation; and (c) attractive intermolecular interactions result in a shortening of the residue-specific effective correlation time for the electron-proton vector. We discuss four different approaches for evaluating ϕENS based on Γ2, J(0), Γ1, or ⟨r-6⟩norm. The latter is evaluated from the magnetic field dependence of Γ1 in conjunction with Γ2. Long-range interactions dominate J(0) and Γ2, while, at high magnetic fields, the contribution of short-range interactions becomes significant for J(ω) and hence Γ1; the four ϕENS quantities enable one to probe both long- and short-range electrostatic interactions. The experimental ϕENS potentials were evaluated using three model protein systems, two folded (ubiquitin and native drkN SH3) and one intrinsically disordered (unfolded state of drkN SH3), in relation to theoretical ϕENS potentials calculated from atomic coordinates using the Poisson-Boltzmann theory with either a r-6 or r-4 dependence.

EBV genome variations enhance clinicopathological features of nasopharyngeal carcinoma in a non-endemic region.

Nasopharyngeal carcinoma (NPC) is caused by infection with Epstein-Barr virus (EBV) and endemic in certain geographic regions. EBV lytic gene, BALF2, closely associates with viral reactivation and BALF2 gene variation, the H-H-H strain, causes NPC in endemic region, southern China. Here, we investigate whether such EBV variations also affect NPC in a non-endemic region, Japan. Viral genome sequencing with 47 EBV isolates of Japanese NPC were performed and compared with those of other EBV-associated diseases from Japan or NPC in Southern China. EBV genomes of Japanese NPC are different from those of other diseases in Japan or endemic NPC; Japanese NPC was not affected by the endemic strain (the BALF2 H-H-H) but frequently carried the type 2 EBV or the strain with intermediate risk of endemic NPC (the BALF2 H-H-L). Seven single nucleotide variations were specifically associated with Japanese NPC, of which six were present in both type 1 and 2 EBV genomes, suggesting the contribution of the type 2 EBV-derived haplotype. This observation was supported by a higher viral titer and stronger viral reactivation in NPC with either type 2 or H-H-L strains. Our results highlight the importance of viral strains and viral reactivation in the pathogenesis of non-endemic NPC.

TREC/KREC Newborn Screening followed by Next-Generation Sequencing for Severe Combined Immunodeficiency in Japan.

PURPOSE: The aim of this study is to evaluate the usefulness of T cell receptor excision circle (TREC) and/or kappa-deleting recombination excision circle (KREC) measurements integrated with diagnostic next-generation sequencing (NGS) analysis using a severe combined immunodeficiency (SCID) newborn screening (NBS) program. METHODS: TREC and/or KREC values were measured in 137,484 newborns between April 2017 and December 2021 using EnLite TREC (n = 80,791) or TREC/KREC kits (n = 56,693). For newborns with positive screening results, diagnostic NGS analysis was performed with a 349-gene panel to detect genetic mutations associated with primary immunodeficiencies (PIDs). RESULTS: A total of 145 newborns (0.11%) had abnormal TREC and/or KREC values, and a genetic diagnosis was established in 2 patients with SCID (1 in 68,742 newborns) (IL2RG-SCID and reticular dysgenesis) and 10 with non-SCID PIDs with T and/or B cell deficiencies (1 in 13,748 newborns) using NGS analysis. Furthermore, TREC values of 2849 newborns were measured and confirmed the significant correlation between the results of both TREC and TREC/KREC kits (P < 0.001) and naïve T cell counts. CONCLUSIONS: We performed the first large-scale TREC and TREC/KREC NBS programs in Japan. Our NBS programs followed by the diagnostic NGS analysis for newborns with abnormal TREC and/or KREC values are useful for the early identification and rapid molecular evaluation of not only SCID but also different non-SCID PIDs.

Epstein-Barr virus tegument protein BGLF2 in exosomes released from virus-producing cells facilitates de novo infection.

BACKGROUND: Viruses must adapt to the environment of their host cells to establish infection and persist. Diverse mammalian cells, including virus-infected cells, release extracellular vesicles such as exosomes containing proteins and miRNAs, and use these vesicles to mediate intercellular communication. However, the roles of exosomes in viral infection remain unclear. RESULTS: We screened viral proteins to identify those responsible for the exosome-mediated enhancement of Epstein-Barr virus (EBV) infection. We identified BGLF2 protein encapsulated in exosomes, which were released by EBV-infected cells. BGLF2 protein is a tegument protein that exists in the space between the envelope and nucleocapsid, and it is released into the cytoplasm shortly after infection. BGLF2 protein-containing exosomes enhanced viral gene expression and repressed innate immunity, thereby supporting the EBV infection. CONCLUSIONS: The EBV tegument protein BGLF2 is encapsulated in exosomes and released by infected cells to facilitate the establishment of EBV infection. These findings suggest that tegument proteins support viral infection not only between the envelope and nucleocapsid, as well as in extraviral particles such as exosomes. Video abstract.

Laser- and cryogenic probe-assisted NMR enables hypersensitive analysis of biomolecules at submicromolar concentration.

Solution-state NMR typically requires 100 µM to 1 mM samples. This limitation prevents applications to mass-limited and aggregation-prone target molecules. Photochemically induced dynamic nuclear polarization was adapted to data collection on low-concentration samples by radiofrequency gating, enabling rapid 1D NMR spectral acquisition on aromatic amino acids and proteins bearing aromatic residues at nanomolar concentration, i.e., a full order of magnitude below other hyperpolarization techniques in liquids. Both backbone H1-C13 and side-chain resonances were enhanced, enabling secondary and tertiary structure analysis of proteins with remarkable spectral editing, via the 13C PREPRINT pulse sequence. Laser-enhanced 2D NMR spectra of 5 µM proteins at 600 MHz display 30-fold better S/N than conventional 2D data collected at 900 MHz. Sensitivity enhancements achieved with this technology, denoted as low-concentration photo-CIDNP (LC-photo-CIDNP), depend only weakly on laser intensity, highlighting the opportunity of safer and more cost-effective hypersensitive NMR applications employing low-power laser sources.

Successful treatment of a novel type I interferonopathy due to a de novo PSMB9 gene mutation with a Janus kinase inhibitor.

BACKGROUND: Type I interferonopathies are a recently established subgroup of autoinflammatory diseases caused by mutations in genes associated with proteasome degradation or cytoplasmic RNA- and DNA-sensing pathways. OBJECTIVE: This study aimed to unveil the molecular pathogenesis of a patient with novel type I interferonopathy, for which no known genetic mutations have been identified. METHODS: We performed the whole-exome sequencing of a 1-month-old boy with novel type I interferonopathy. We also investigated proteasome activities using patient-derived B lymphoblastoid cell lines (LCLs) and normal LCLs transduced with the mutant gene. RESULTS: Whole-exome sequencing identified a de novo proteasome 20S subunit beta 9 (PSMB9) p.G156D mutation in the patient who developed fever, a chilblain-like skin rash, myositis, and severe pulmonary hypertension due to the hyperactivation of IFN-α. Patient-derived LCLs revealed reduced proteasome activities, and exogenous transduction of mutant PSMB9 p.G156D into normal LCLs significantly suppressed proteasome activities, and the endogenous PSMB9 protein was lost along with the reduction of other immunoproteasome subunits, PSMB8 and PSMB10 proteins. He responded to the administration of a Janus kinase inhibitor, tofacitinib, and he was successfully withdrawn from venoarterial extracorporeal membranous oxygenation. At age 7 months, he received an unrelated cord blood transplantation. At 2 years posttransplantation, he no longer required tofacitinib and experienced no disease recurrence. CONCLUSIONS: We present the case of a patient with a novel type I interferonopathy caused by a de novo PSMB9 p.G156D mutation that suppressed the wild-type PSMB9 protein expression. Janus kinase inhibitor and stem cell transplantation could be curative therapies in patients with severe interferonopathies.

De Novo Mutations Activating Germline TP53 in an Inherited Bone-Marrow-Failure Syndrome.

Inherited bone-marrow-failure syndromes (IBMFSs) include heterogeneous genetic disorders characterized by bone-marrow failure, congenital anomalies, and an increased risk of malignancy. Many lines of evidence have suggested that p53 activation might be central to the pathogenesis of IBMFSs, including Diamond-Blackfan anemia (DBA) and dyskeratosis congenita (DC). However, the exact role of p53 activation in each clinical feature remains unknown. Here, we report unique de novo TP53 germline variants found in two individuals with an IBMFS accompanied by hypogammaglobulinemia, growth retardation, and microcephaly mimicking DBA and DC. TP53 is a tumor-suppressor gene most frequently mutated in human cancers, and occasional germline variants occur in Li-Fraumeni cancer-predisposition syndrome. Most of these mutations affect the core DNA-binding domain, leading to compromised transcriptional activities. In contrast, the variants found in the two individuals studied here caused the same truncation of the protein, resulting in the loss of 32 residues from the C-terminal domain (CTD). Unexpectedly, the p53 mutant had augmented transcriptional activities, an observation not previously described in humans. When we expressed this mutant in zebrafish and human-induced pluripotent stem cells, we observed impaired erythrocyte production. These findings together with close similarities to published knock-in mouse models of TP53 lacking the CTD demonstrate that the CTD-truncation mutations of TP53 cause IBMFS, providing important insights into the previously postulated connection between p53 and IBMFSs.

Relationship between plasma rabbit anti-thymocyte globulin concentration and immunosuppressive therapy response in patients with severe aplastic anemia.

OBJECTIVES: Patients with acquired aplastic anemia (AA) without HLA-matched sibling donors or aged >40 years receive immunosuppressive therapy (IST) with anti-thymocyte globulin (ATG). We investigated the relationship between plasma rabbit ATG (r-ATG) concentration and IST response. METHODS: From May 2012 to October 2017, 81 patients with severe AA who required initial IST were included. A 1:1 block randomization was employed for 2.5 and 3.5 mg/kg doses of r-ATG. RESULTS: No significant difference in response rates was observed between the 2.5 and 3.5 mg/kg groups (63% vs. 58%, P = .894). Median r-ATG concentrations on days 14 and 28 after IST were 15.2 (0.0-97.7) and 1.8 (0.0-74.9 µg/mL), respectively. According to r-ATG concentration, response rates were significantly higher in the group with higher r-ATG concentration than in those with lower r-ATG concentration (day 14, 88% vs. 52%; P = .006 and day 28, 79% vs. 46%; P = .005). In multivariate analysis, higher r-ATG concentrations at day 28 were independent predictors of favorable response to IST at 6 months (odds ratio, 0.29; 95% confidence interval, 0.09-0.93; P = .037). CONCLUSIONS: The present data indicate that higher r-ATG concentration at day 28 resulted in improved IST response.

Oncogenesis of CAEBV revealed: Intragenic deletions in the viral genome and leaky expression of lytic genes.

Epstein-Barr virus (EBV) is a causative agent of infectious mononucleosis and several malignancies involving lymphocytes and epithelial cells. We recently reported genomic analyses of chronic active EBV infection (CAEBV), a proliferative disorder of T and/or NK cells, as well as other lymphoid malignancies. We found that T and/or NK cells undergoing clonal expansion in CAEBV patients gain somatic driver mutations as the disorder progresses. Investigation of the viral genome revealed viral genomes harboring intragenic deletions in the BamHI-rightward transcripts (BART) region and in essential lytic genes. Interestingly, we observed that these deletions resulted in leaky expression of viral lytic genes. This increased expression of viral lytic genes is reminiscent of the "pre-latent abortive lytic" state, in which a substantial number of lytic genes are produced for weeks in the absence of progeny production, which contributes to cell survival upon de novo infection. It has been known that EBV can choose either latent or lytic state, but this dualistic concept may need to be reconsidered, as our data suggest the presence of the third, intermediate state; leaky expression of lytic genes that does not lead to completion of the full lytic amplification cycle. Leaky expression of lytic genes likely contributes to the formation and maintenance of several types of EBV-associated tumors. We also presented significant circumstantial evidence suggesting that EBV infects lymphoid progenitor cells in CAEBV before differentiation into T and NK cells. Taken together, our new data shed light on oncogenesis of CAEBV and other EBV-associated malignancies.

A founder variant in the South Asian population leads to a high prevalence of FANCL Fanconi anemia cases in India.

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, predisposition to cancer, and congenital abnormalities. FA is caused by pathogenic variants in any of 22 genes involved in the DNA repair pathway responsible for removing interstrand crosslinks. FANCL, an E3 ubiquitin ligase, is an integral component of the pathway, but patients affected by disease-causing FANCL variants are rare, with only nine cases reported worldwide. We report here a FANCL founder variant, anticipated to be synonymous, c.1092G>A;p.K364=, but demonstrated to induce aberrant splicing, c.1021_1092del;p.W341_K364del, that accounts for the onset of FA in 13 cases from South Asia, 12 from India and one from Pakistan. We comprehensively illustrate the pathogenic nature of the variant, provide evidence for a founder effect, and propose including this variant in genetic screening of suspected FA patients in India and Pakistan, as well as those with ancestry from these regions of South Asia.

Quantitative Interpretation of Solvent Paramagnetic Relaxation for Probing Protein-Cosolute Interactions.

Protein-small cosolute molecule interactions are ubiquitous and known to modulate the solubility, stability, and function of many proteins. Characterization of such transient weak interactions at atomic resolution remains challenging. In this work, we develop a simple and practical NMR method for extracting both energetic and dynamic information on protein-cosolute interactions from solvent paramagnetic relaxation enhancement (sPRE) measurements. Our procedure is based on an approximate (non-Lorentzian) spectral density that behaves exactly at both high and low frequencies. This spectral density contains two parameters, one global related to the translational diffusion coefficient of the paramagnetic cosolute, and the other residue specific. These parameters can be readily determined from sPRE data, and then used to calculate analytically a concentration normalized equilibrium average of the interspin distance, ⟨r-6⟩norm, and an effective correlation time, τC, that provide measures of the energetics and dynamics of the interaction at atomic resolution. We compare our approach with existing ones, and demonstrate the utility of our method using experimental 1H longitudinal and transverse sPRE data recorded on the protein ubiquitin in the presence of two different nitroxide radical cosolutes, at multiple static magnetic fields. The approach for analyzing sPRE data outlined here provides a powerful tool for deepening our understanding of extremely weak protein-cosolute interactions.