B cell acute lymphoblastic leukaemia (B-ALL) infrequently presents with undetectable or very low peripheral blood (PB) lymphoblasts, which may cause diagnostic difficulties. We report the clinicopathological features of paediatric patients with B-ALL who presented with low (<5%; n=31) PB lymphoblasts by flow cytometry. 15 patients presented with <1% PB lymphoblasts (range: 0.0%-0.9%), a level potentially undetectable by manual smear review and standard 100-cell count. Compared with patients presenting with ≥5% PB lymphoblasts (n=45), those with <5% had more frequent musculoskeletal symptoms, less frequent lymphadenopathy and hepatosplenomegaly, higher platelet counts, lower white blood cell counts and lower lactate dehydrogenase. The presence of low PB lymphoblasts was associated with favourable cytogenetics (ETV6::RUNX1 or high hyperdiploidy without IKZF1 deletion) and less frequent central nervous system involvement. Aleukemic/low PB lymphoblast presentation of B-ALL is associated with unique clinicopathological features that overlap with non-malignant disorders. A high degree of clinical suspicion and careful review of PB flow cytometry data are required for timely diagnosis.

Heme biosynthesis is tightly coordinated such that essential heme functions including oxygen transport, respiration, and catalysis are fully supplied without overproducing toxic heme precursors and depleting cellular iron. The initial heme biosynthetic enzyme, ALA synthase (ALAS), exhibits heme-induced degradation that is dependent on the mitochondrial AAA+ protease complex CLPXP, but the mechanism for this negative feedback regulation had not been elucidated. By biochemical reconstitution, we have discovered that POLDIP2 serves as a heme-sensing adaptor protein to deliver ALAS for degradation. Similarly, loss of POLDIP2 strongly impairs ALAS turnover in cells. POLDIP2 directly recognizes heme-bound ALAS to drive assembly of the degradation complex. The C-terminal element of ALAS, truncation of which leads to a form of porphyria (XLDPP), is dispensable for interaction with POLDIP2 but necessary for degradation. Our findings establish the molecular basis for heme-induced degradation of ALAS by CLPXP, establish POLDIP2 as a substrate adaptor for CLPXP, and provide mechanistic insight into two forms of erythropoietic protoporphyria linked to CLPX and ALAS.

Identifying pathogenic non-coding variants that contribute to Mendelian conditions remains challenging, as the functional impact of these variants on gene function is often unknown. We present IsoRanker, a long-read transcriptome sequencing-based framework that prioritizes functionally relevant variants by detecting genes and isoforms with outlier expression, allelic imbalance, and/or nonsense-mediated decay (NMD). We generated paired cycloheximide-treated and untreated fibroblast transcriptomes from 31 individuals (3 individuals with known transcript-altering rare variants and 28 individuals with unsolved conditions) and linked transcripts to phased long-read genomes. IsoRanker successfully recovered known transcript alterations in this cohort, and exploratory subsampling analyses suggested that their prioritization was largely preserved down to cohorts of 11 individuals and ∼5 million full-length transcripts per individual. Performance was dependent upon de novo isoform caller choice, particularly for NMD-sensitive and previously unannotated isoforms. Among 28 previously unsolved cases, IsoRanker deprioritized 8 out of 10 fibroblast-expressed candidate splice-site variants while nominating 4 new leads. In one individual, IsoRanker prioritized HARS1, revealing bi-allelic non-coding variants that together produced a partial HARS1 loss of function and informed targeted therapy in this individual. These findings support long-read, NMD-aware transcriptomics with IsoRanker as an effective approach for generating isoform-level functional evidence, improving classification of non-coding variants and supporting the diagnosis of individuals with rare genetic conditions.

ABSTRACT:Severe congenital neutropenia (SCN) is characterized by neutropenia, recurrent infections, and an increased leukemia risk. Multiple genetic defects that underlie SCN have been identified, but a genetic diagnosis is still lacking in a significant proportion of patients. In this study, we report 4 independent pedigrees with heterozygous variants in LCP1. Variants c.740-1G>T and c.740-20_744del produced the same alternatively spliced RNA product, causing an in-frame deletion (p.A247_E254del). Variant c.509C>T in the third pedigree produced p.S170L, and variant c.806T>C in the fourth pedigree produced p.L269P. Affected individuals suffered from neutropenia, poor or complete lack of response to granulocyte colony-stimulating factor (G-CSF) treatment, and variable degrees of lymphopenia, hypogammaglobulinemia, and monocytopenia. Patients with A247_E254del and p.L269P presented with tetraploid cells in the bone marrow, indicative of disturbed cytokinesis. In one of these kindreds, 2 individuals developed acute leukemia. G-CSF nonresponsiveness and defective cell cycling were repaired upon correction of the LCP1 A247_E254del variant in patient-derived induced pluripotent stem cells, supporting the monogenic origin of the disease. Indicative of their gain-of-function effect, both the A247_E254del and S170L variants increased F-actin bundling and the formation of abnormal protrusions. Single-cell transcriptome analysis of A247_E254del bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) showed deregulation of signaling pathways that control mitosis in multilineage and lymphoid-primed HSPC subsets. We concluded that activating LCP1 variants cause a new hematopoietic disorder with autosomal dominant inheritance. Depending on the consequences of the LCP1 variants in terms of protein structure, patients may suffer from G-CSF refractory severe neutropenia, lymphopenia, hypogammaglobulinemia, monocytopenia, and defective cytokinesis.

Peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) rarely occurs in pediatric and young adult populations, where little is known about its clinicopathologic and molecular features. We characterized 16 cases of PTCL, NOS diagnosed in patients ≤21 years old. Seven (44%) cases demonstrated SMARCB1/INI1 loss by immunohistochemistry and SMARCB1/INI1 alterations by next-generation sequencing, including biallelic SMARCB1/INI1 deletion (n = 4), stop codon variant with 1-copy deletion (n = 1) or copy-neutral loss-of-heterozygosity (n = 1), and frameshift variant (n = 1). One case demonstrated biallelic SMARCE1 alterations (a nonsense variant and copy-neutral loss-of-heterozygosity), which, to our knowledge, has not been previously described in a hematopoietic neoplasm. The SWI/SNF-intact group harbored pathogenic TET2, PTEN, EZH2, or TP53 variants. CDKN2A deletions were present in 3 of 7 SWI/SNF-deficient and 0 of 4 SWI/SNF-intact cases. Chromosome 22q11.2 alterations were present on karyotype in 2 of 3 SMARCB1/INI1-deficient cases. SWI/SNF deficiency was associated with intermediate-to-large cell cytomorphology, frequent mitotic (88%; P = .041) and apoptotic (88%) activity, Reed-Sternberg-like cells (63%), necrosis (50%), and fibrosis (50%). All cases expressed CD45 and CD43 at initial diagnosis. SWI/SNF-deficient cases predominantly demonstrated a CD4+/CD8-, TCRab, and PTCL-GATA3 phenotype, whereas SWI/SNF-intact cases predominantly demonstrated a CD8+/CD4-, TCRgd, and PTCL-TBX21 phenotype. A PTCL-TBX21 phenotype was more common in SWI/SNF-intact than in SWI/SNF-deficient cases (P = .041). Cytotoxic markers were expressed in 71% of SWI/SNF-deficient and 88% of SWI/SNF-intact cases. Decreased or absent CD3 expression characterized 88% of SWI/SNF-deficient and 13% of SWI/SNF-intact cases (P = .01). Moreover, 63% of SWI/SNF-deficient cases demonstrated decreased or absent expression of ≥3 pan-T-cell antigens, compared with 13% of SWI/SNF-intact cases. Treatment was heterogeneous. Primary treatment failure or relapse occurred in 4 of 8 SWI/SNF-deficient and 4 of 7 SWI/SNF-intact cases. The median overall survival and event-free survival were 48.7 and 47.5 months for the SWI/SNF-deficient group, and 16.4 and 8.9 months for the SWI/SNF-intact group (P = nonsignificant). Death due to disease or therapy-related complications occurred in 4 of 8 (50%) cases in the SWI/SNF-deficient group and 6 of 7 (86%) of cases in the SWI/SNF-intact group. Our findings expand the knowledge of the clinicopathologic and molecular features of pediatric PTCL and highlight SWI/SNF-deficient T-cell lymphoma as a biologically distinct type of PTCL that is associated with characteristic clinicopathologic features.

Mitochondrial endosymbiosis was a pivotal event in eukaryotic evolution, requiring core proteins to adapt to function both within the mitochondria and in the host cell. Here, we systematically profile the localization of protein isoforms generated by alternate start codon selection during translation. We identify hundreds of pairs of differentially-localized protein isoforms, many of which affect mitochondrial targeting and are essential for mitochondrial function. The emergence of dual-localized mitochondrial protein isoforms coincides with mitochondrial acquisition during early eukaryotic evolution. We further reveal that eukaryotes use diverse mechanisms-such as leaky ribosome scanning, alternative transcription, and paralog duplication-to maintain the production of dual-localized isoforms. Finally, we identify multiple isoforms that are specifically dysregulated by rare disease patient mutations and demonstrate how these mutations can help explain unique clinical presentations. Together, our findings illuminate the evolutionary and pathological relevance of alternative translation initiation, offering new insights into the molecular underpinnings of mitochondrial biology.

Introduction. Many genes relevant to iron metabolism include a transcribed cis-regulatory element called iron-responsive element (IRE) that couples their expression to the availability of intracellular iron. Canonical IREs consist of a conserved stem-loop structure in the 5‘ or 3‘ untranslated region (UTR) of these mRNAs. IREs are bound by two iron-regulatory proteins (IRP1 & IRP2) to either limit the translation of the targeted transcript or to stabilize it. Among the 5'UTR IRE- genes, ALAS2, the erythroid-specific aminolevulinate synthase, catalyzes the first step of heme biosynthesis. Loss-of-function and gain-of-function mutations cause X-linked Sideroblastic Anemia (XLSA) and X-linked Protoporphyria (XLPP). A deficiency in the last enzyme of the pathway, ferrochelatase (FECH), causes the most common Erythropoietic Protoporphyria (EPP). A single-family with protoporphyria has been described with a mutation in the mitochondrial unfoldase CLPX. In the mouse, targeted deletion of IRP2 also results in protoporphyria. Several lines of evidence suggest a role for Alas2 in modifying the severity of erythroid porphyrias. In 4 patients with congenital erythropoietic porphyria due to the same UROS genotype, an XLPP mutation was also found in the most severely affected individual. A mutation in the Alas2-IRE has been described as a modifier in the CLPX-family. Furthermore, increased levels of Alas2 mRNA have been reported in EPP patients. Lastly, other reports suggest that iron deficiency may be protective in protoporphyrias. Methods. We generated a mouse line lacking the loop of Alas2-IRE (Alas2ΔLOOP), which should uncouple ALAS2 expression from IRP-dependent regulation. We phenotyped hemizygous male Alas2ΔLOOP/Y mice from 1 to 8 weeks of age. We compared their phenotype to established mouse models of protoporphyria (EPP FECHm1Pas/m1Pas, XLPP Alas2Q548X/Y, and IRP2-/-) at baseline. We also challenged animals with iron deficiency and iron overload. Lastly, we investigated the severity of EPP animals in the presence or absence of Alas2-IRE. Results. (a) Phenotype. Alas2ΔLOOP/Y mice have a transitory protoporphyria that resolves nearly completely by 8 weeks of age. The RBC protoporphyric phenotype is similar to XLPP mice, but substantially less severe than the EPP, XLPP, and IRP2 models. (b) Iron status. EPP, XLPP, and Alas2ΔLOOP male animals were challenged with either iron deficiency or iron overload at weaning. In iron-poor conditions, all three models have an increase in erythroid PPIX, with the Alas2ΔLOOPanimals having by far the most dramatic change after 5 weeks (mean MFI PPIX [SD] for Alas2ΔLOOPiron-replete: 12.8[6.0] v. iron poor 752[374]). However, only the Alas2 mutants (XLPP, Alas2ΔLOOP) see a modest but prolonged increase in erythroid PPIX when injected once with iron dextran. (c) Disease modifier. The double mutant Alas2ΔLOOP/Y FECHm1Pas/m1Pas (EPP-ΔLOOP) accumulate more erythroid PPIX than the EPP animals at 8 weeks of age (MFI PPIX mean [SD] for Alas2ΔLOOP 18.8[8.9], EPP 128.6 [19.1], EPP-ΔLOOP 659.6[384.7]). The fraction of fluorocytes (erythroid cells in peripheral blood abnormally containing PPIX) is also increased (%mean [SD] for EPP 57.8[8.9], EPP-ΔLOOP 97.6[2.6]). XLPP animals lacking one allele of IRP2 (Alas2Q548X/Y IRP2wt/-) present a phenotype similar to the EPP-ΔLOOP animals while Alas2Q548X/Y IRP2-/- are not viable. Conclusion. Altogether, our results confirm that iron status strongly modifies the erythroid PPIX accumulation, particularly when Alas2 expression is uncoupled from the IRPs control. We also demonstrated that the IRE-IRP system plays a critical role in mitigating the severity of protoporphyria in the rodents, providing evidence that targeting the Alas2-IRE may have pharmacological benefits.

Variants in the mitochondrial genome (mtDNA) cause a diverse collection of mitochondrial diseases and have extensive phenotypic overlap with Mendelian diseases encoded on the nuclear genome. The mtDNA is not always specifically evaluated in patients with suspected Mendelian disease, resulting in overlooked diagnostic variants. Here, we analyzed a cohort of 6,660 rare disease families (5,625 genetically undiagnosed [84%]) from the Genomics Research to Elucidate the Genetics of Rare diseases (GREGoR) Consortium, as well as other rare disease cohorts. Using dedicated pipelines to address the technical challenges posed by the mtDNA-circular genome, variant heteroplasmy, and nuclear misalignment-we called single nucleotide variants, small insertions/deletions, and large mtDNA deletions from exome and/or genome sequencing data, in addition to RNA sequencing data when available. Diagnostic mtDNA variants were identified in 10 previously genetically undiagnosed families (1 large deletion, 8 reported pathogenic variants, and 1 previously unreported likely pathogenic variant), as well as candidate diagnostic variants in a further 11 undiagnosed families. In one additional undiagnosed proband, detection of >900 heteroplasmic variants provided functional evidence of pathogenicity to a de novo variant in the nuclear gene POLG (DNA polymerase gamma), responsible for mtDNA replication and repair. Overall, mtDNA variant calling from data generated by exome and genome sequencing-primarily for nuclear variant analysis-resulted in a genetic diagnosis for 0.2% of undiagnosed families affected by a broad range of rare diseases, as well as the identification of additional promising candidates in 0.2%.

X-linked sideroblastic anemia (XLSA) in carrier females of ALAS2 mutations is not uncommon. We describe unique features and genotype/phenotype correlations in XLSA females and evaluate the contributions of X-chromosome skewing and clonal hematopoiesis, emphasizing the importance of distinguishing it from myelodysplastic syndromes with ring sideroblasts.

Erythropoietic protoporphyria (EPP) is a genetic disorder typically resulting from decreased ferrochelatase (FECH) activity, the last enzyme in heme biosynthesis. Patients with X-linked protoporphyria (XLPP) have an overlapping phenotype caused by increased activity of 5-aminolevulinic acid synthase 2 (ALAS2), the first enzyme in erythroid heme synthesis. In both cases, protoporphyrin IX (PPIX) accumulates in erythrocytes and secondarily in plasma and tissues. Patients develop acute phototoxicity reactions upon brief exposure to sunlight. Some also experience chronic liver disease, and a small fraction develop acute cholestatic liver failure. Therapeutic options are limited, and none, save hematopoietic stem cell transplantation, directly targets erythroid PPIX accumulation. Bitopertin is an investigational orally available small-molecule inhibitor of the erythroid cell-surface glycine transporter GLYT1. We established the bitopertin PPIX inhibitory half-maximal effective concentration in a human erythroblast EPP model and confirmed a marked reduction of PPIX in erythroblasts derived from patients with EPP. We demonstrate that bitopertin also reduced erythrocyte and plasma PPIX accumulation in vivo in both EPP and XLPP mouse models. Finally, the reduction in erythroid PPIX ameliorated liver disease in the EPP mouse model. Altogether, these data support the development of bitopertin to treat patients with EPP or XLPP.

Pediatric cutaneous T-cell lymphoproliferative disorders encompass a diagnostically complex set of rare diseases of undefined pathogenesis, including mycosis fungoides (MF), lymphomatoid papulosis (LyP), and primary cutaneous anaplastic large cell lymphoma (pcALCL). In the pediatric population, these disorders are much less common than in adults, which has precluded systematic evaluation of their molecular pathogenesis. We report the clinicopathologic and molecular features of pediatric MF (n = 14, ages 5-17 years at diagnosis), LyP (n = 8, ages 4-17 years), and pcALCL (n = 2). Next-generation sequencing analysis (targeted 72-gene fusion panel, targeted 447-gene exome sequencing panel, and/or FoundationOneHeme) was performed. JAK2 fusions were detected in 64% (7/11) MF (PCM1::JAK2, ILF3::JAK2 [n = 2], ATXN2L::JAK2 [n = 2], and NUP214::JAK2 [n = 2]) by next-generation sequencing of available cases. TYK2 fusions were identified in 1 of 11 MF (novel LMNA::TYK2), 3 of 5 LyP (NPM1::TYK2 [n = 2], and novel RAN::TYK2), and 1 of 2 pcALCL (RAN::TYK2) cases tested. A novel NUP214::FRK fusion was observed in the other pcALCL. Fusions were in-frame and retained the kinase domain of the 3’ partner in all cases. MF cases demonstrated clonal TCR gene rearrangements (12/12 tested). Treatment was heterogeneous, although it usually included narrowband ultraviolet B phototherapy for MF and topical steroids for MF and LyP. We demonstrate that pediatric MF, LyP, and pcALCL harbor frequent tyrosine kinase gene fusions with enrichment of JAK2 and TYK2 fusions, genomic alterations that are diagnostically useful and may be amenable to targeted therapy.

Abstract Purpose: First-in-human Phase I study for advanced MUC1* positive breast cancer with autologous T cells engineered to express either a chimeric antigen receptor, huMNC2-CAR44 or huMNC2-CAR22, which specifically bind to a cleaved form of MUC1 (MUC1*); evaluate the safety and preliminary anti-tumor activity. Methods: MUC1* (muk 1 star) is the growth factor receptor form of MUC1, created by cleavage and release of the N-terminal portion of MUC1. The targeting antibody, huMNC2, only recognizes the conformational epitope created when MUC1 is cleaved by specific tumor-associated enzymes that are correlated with poor prognosis. huMNC2 does not bind to full-length MUC1, which is expressed on all normal epithelial cells. huMNC2-scFv was incorporated into huMNC2-CAR44, comprising a CD8 hinge and transmembrane region, 4-1BB costimulatory domain and wild-type CD3z. huMNC2-scFv was also incorporated into huMNC2-CAR22 wherein the hinge, transmembrane and co-stimulatory portions were derived from CD28 and CD3z bears the 1XX mutations to increase in vivo persistence. Inclusion criteria require that the patient’s tumor is at least 30% MUC1* positive and that patient has progressed through 2 or 3 prior therapies, while in the metastatic setting. Patients receive standard Cy/Flu lymphodepletion approximately 3-days before CAR T treatment, administered at 1 of 4 dose levels ranging from 3.3X10^5 up to 1.0X10^7 CAR+ T cells. Results: To date, 8 patients have been treated with huMNC2-CAR44. No patients experienced neuro toxicities. No off-target toxicities were observed. 3 patients experienced CRS Grade 1-3. In 6 of the 8 patients, side effects were non-existent or minimal. However, one patient experienced a Grade 5 SAE that was deemed to be possibly related to the treatment. Best responses include Partial Responses and Stable Disease at a low CAR-T dose. Greatest efficacy was observed for patients whose biopsy showed H Scores >120. Patients are currently being enrolled for treatment with huMNC2-CAR22, where the 1XX mutations are expected to increase in vivo persistence, durability of response and reduced incidence of CRS. Conclusions: These data support a conclusion that the MUC1* antibody, huMNC2, is safe and could have high therapeutic value as a CAR T treatment for solid tumors with moderate to high antigen density. As the huMNC2-CAR22 (1XX) trial proceeds, we will assess if patient responses mirror our animal results that show that the 1XX mutations confer increased persistence, reduced exhaustion and the ability to kill tumors with low antigen density. Citation Format: Cynthia Carol Bamdad, Joanne E. Mortimer, Yuan Yuan, Jennifer M. Specht, Andrew K. Stewart, Benoit J. Smagghe, Stephen C. Lin, Mark G. Carter, Tim W. Synold, Mark D. Fleming, Stanley R. Hamilton, Vishwas Parekh, Danica M. Walkley, Qing Liu-Michael, Kevin R. Yi, Jac-Leen S. Nash, Michael J. Nash, Stephen J. Forman. Phase I first-in-human MUC1* targeted autologous CAR T cells for the treatment of metastatic breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT096.

Inherited iron metabolism defects are possibly missed or underdiagnosed in iron-deficient endemic settings because of a lack of awareness or a methodical screening approach. Hence, we systematically evaluated anemia cases (2019 to 2021) based on clinical phenotype, normal screening tests (high-performance liquid chromatography, α gene sequencing, erythrocyte sedimentation rate, C-reactive protein, and tissue transglutaminase), and abnormal iron profile by targeted next-generation sequencing (26-gene panel) supplemented with whole-exome sequencing, multiplex ligation probe amplification/mitochondrial DNA sequencing, and chromosomal microarray. Novel variants in ALAS2, STEAP3, and HSPA9 genes were functionally validated. A total of 290 anemia cases were screened, and 41 (14%) enrolled for genomic testing as per inclusion criteria. Comprehensive genomic testing revealed pathogenic variants in 23 of 41 cases (56%). Congenital sideroblastic anemia was the most common diagnosis (14/23; 61%), with pathogenic variations in ALAS2 (n = 6), SLC25A38 (n = 3), HSPA9 (n = 2) and HSCB, SLC19A2, and mitochondrial DNA deletion (n = 1 each). Nonsideroblastic iron defects included STEAP3-related microcytic anemia (2/23; 8.7%) and hypotransferrenemia (1/23; 4.3%). A total of 6 of 22 cases (27%) revealed a non-iron metabolism gene defect on whole-exome sequencing. Eleven novel variants (including variants of uncertain significance) were noted in 13 cases. Genotype-phenotype correlation revealed a significant association of frameshift/nonsense/splice variants with lower presentation age (0.8 months versus 9 years; P < 0.01) compared with missense variants. The systematic evaluation helped uncover an inherited iron defect in 41% (17/41) of cases, suggesting the need for active screening and awareness for these rare diseases in an iron-deficient endemic population.

Protein lipoylation, a vital lysine post-translational modification, plays a crucial role in the function of key mitochondrial tricarboxylic acid cycle enzymatic complexes. In eukaryotes, lipoyl post-translational modification synthesis occurs exclusively through de novo pathways, relying on lipoyl synthesis/ transfer enzymes, dependent upon mitochondrial fatty acid and Fe-S cluster biosynthesis. Dysregulation in any of these pathways leads to diminished cellular lipoylation. Efficient restoration of lipoylation in lipoylation deficiency cell states using either chemical or genetic approaches has been challenging because of pathway complexity and multiple upstream regulators. To address this challenge, we explored the possibility that a bacterial lipoate protein ligase A (lplA) enzyme, which can salvage free lipoic acid bypassing the dependency on de novo synthesis, could be engineered to be functional in human cells. Overexpression of the engineered lplA in lipoylation null cells restored lipoylation levels, cellular respiration, and growth in low glucose conditions. Engineered lplA restored lipoylation in all tested lipoylation null cell models, mimicking defects in mitochondrial fatty acid synthesis (MECR KO), Fe-S cluster biosynthesis (BOLA3 KO), and specific [lipoyl synthase], and LIPT1 [lipoyl (octanoyl) transferase 1] KOs). Furthermore, we describe a patient with a homozygous c.212C>T variant LIPT1 with a previously uncharacterized roleukemia cells engineered to harbor this missense LIPT1 allele recapitulate the lipoylation-deficient phenotype and exhibit impaired proliferation in low glucose that is completely restored by engineered lplA. This synthetic approach offers a potential therapeutic strategy for treating lipoylation disorders.