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Bruton tyrosine kinase (BTK) is present in a wide variety of cells and may thus have important non-B cell functions. Here, we explored the function of this kinase in macrophages with studies of its regulation of the NLR family, pyrin domain-containing 3 (NLRP3) inflammasome. We found that bone marrow-derived macrophages (BMDMs) from BTK-deficient mice or monocytes from patients with X-linked agammaglobulinemia (XLA) exhibited increased NLRP3 inflammasome activity; this was also the case for BMDMs exposed to low doses of BTK inhibitors such as ibrutinib and for monocytes from patients with chronic lymphocytic leukemia being treated with ibrutinib. In mechanistic studies, we found that BTK bound to NLRP3 during the priming phase of inflammasome activation and, in doing so, inhibited LPS- and nigericin-induced assembly of the NLRP3 inflammasome during the activation phase of inflammasome activation. This inhibitory effect was caused by BTK inhibition of protein phosphatase 2A-mediated (PP2A-mediated) dephosphorylation of Ser5 in the pyrin domain of NLRP3. Finally, we show that BTK-deficient mice were subject to severe experimental colitis and that such colitis was normalized by administration of anti-IL-β or anakinra, an inhibitor of IL-1β signaling. Together, these studies strongly suggest that BTK functions as a physiologic inhibitor of NLRP3 inflammasome activation and explain why patients with XLA are prone to develop Crohn's disease.

We describe a previously unappreciated role for Bruton’s tyrosine kinase (BTK) in fungal immune surveillance against aspergillosis, an unforeseen complication of BTK inhibitors (BTKi) used for treating B cell lymphoid malignancies. We studied BTK-dependent fungal responses in neutrophils from diverse populations, including healthy donors, patients who were treated with BTKi, and X-linked agammaglobulinemia patients. Upon fungal exposure, BTK was activated in human neutrophils in a TLR2-, Dectin-1-, and FcγR-dependent manner, triggering the oxidative burst. BTK inhibition selectively impeded neutrophil-mediated damage to Aspergillus hyphae, primary granule release, and the fungus-induced oxidative burst by abrogating NADPH oxidase subunit p40 phox and GTPase RAC2 activation. Moreover, neutrophil-specific Btk deletion in mice enhanced aspergillosis susceptibility by impairing neutrophil function, not recruitment or lifespan. Conversely, GM-CSF partially mitigated these deficits by enhancing p47 phox activation. Our findings underline the crucial role of BTK signaling in neutrophils for antifungal immunity and provide a rationale for GM-CSF use to offset these deficits in patients who are susceptible.

Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome is an adult-onset inflammatory disorder caused by somatic UBA1 mutations in hematopoietic stem cells. UBA1 encodes a key enzyme that catalyzes protein ubiquitination. Clinically, VEXAS is characterized by systemic inflammation and hematologic abnormalities. Patient studies have hinted that the transition of UBA1 -mutated stem cells into proinflammatory myeloid precursors may propagate the manifestations of VEXAS syndrome. In this issue of the JCI , Dong and colleagues developed nine unique conditional knockout mouse strains and found that only neutrophil-specific Uba1 deletion reproduced VEXAS syndrome–like findings. The observed phenotype was at least in part due to inflammatory reprogramming and longer survival of the mutant neutrophils. In addition to deepening our mechanistic understanding of VEXAS syndrome pathogenesis, this work should provide a platform to pursue more targeted approaches to treatment.

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.

Transporters at the hepatic canalicular membrane are essential for the formation of bile and the prevention of cholestatic liver disease. One such example is ATP8B1, a P4-type ATPase disrupted in three inherited forms of intrahepatic cholestasis. Mutation of the X-linked mouse gene Atp11c, which encodes a paralogous P4-type ATPase, precludes B-cell development in the adult bone marrow, but also causes hyperbilirubinemia. Here we explore this hyperbilirubinemia in two independent Atp11c mutant mouse lines, and find that it originates from an effect on nonhematopoietic cells. Liver function tests and histology revealed only minor pathology, although cholic acid was elevated in the serum of mutant mice, and became toxic to mutant mice when given as a dietary supplement. The majority of homozygous mutant females also died of dystocia in a maternal genotype-specific manner. ATP11C therefore represents a multifunctional transporter, essential for adult B-cell development, the prevention of intrahepatic cholestasis, and parturition, and is a new candidate for genetically undiagnosed cases of cholestasis and dystocia in humans.

5‐Aminolevulinate synthase (ALAS) is the first and rate‐limiting enzyme of heme biosynthesis in humans, animals, other non‐plant eukaryotes, and α‐proteobacteria. It catalyzes the synthesis of 5‐aminolevulinic acid, the first common precursor of all tetrapyrroles, from glycine and succinyl‐coenzyme A (sCoA) in a pyridoxal 5′‐phosphate (PLP)‐dependent manner. X‐linked sideroblastic anemias (XLSAs), a group of severe disorders in humans characterized by inadequate formation of heme in erythroblast mitochondria, are caused by mutations in the gene for erythroid eALAS, one of two human genes for ALAS. We present the first crystal structure of homodimeric ALAS from Rhodobacter capsulatus (ALAS Rc ) binding its cofactor PLP. We, furthermore, present structures of ALAS Rc in complex with the substrates glycine or sCoA. The sequence identity of ALAS from R. capsulatus and human eALAS is 49%. XLSA‐causing mutations may thus be mapped, revealing the molecular basis of XLSA in humans. Mutations are found to obstruct substrate binding, disrupt the dimer interface, or hamper the correct folding. The structure of ALAS completes the structural analysis of enzymes in heme biosynthesis.

Background Dent disease, an X-linked recessive renal tubulopathy, is caused by mutations in either CLCN5 (Dent disease 1) or OCRL (Dent disease 2). OCRL mutations can also cause Lowe syndrome. In some cases it is difficult to differentiate Dent disease 1 and 2 on the basis of clinical features only without genetic tests. Several studies have shown differences in serum levels of muscle enzymes between these diseases. The aim of our study was to test the validity of these findings. Methods In total, 23 patients with Dent disease 1 (Group A), five patients with Dent disease 2 (Group B) and 19 patients with Lowe syndrome (Group C) were enrolled in our study. The serum levels of three muscle enzymes [creatine phosphokinase (CPK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST)], were measured. The levels of a hepatic enzyme, alanine aminotransferase (ALT), were also measured as a control. Results One patient in Group B had muscle hypoplasia of both upper extremities. The serum levels of all three muscle enzymes assayed were higher in Group B or C patients than in Group A patients. Serum ALT levels were normal in all three groups of patients. Conclusions The serum levels of muscle enzymes in patients with Dent disease can be used as a biomarker to predict genotypes, even though the patients do not have clinical symptoms of muscle involvement.

The development of novel fertilization treatments, including in vitro fertilization and intracytoplasmic injection, has made pregnancy possible regardless of the level of activity of the spermatozoa; however, the etiology of male-factor infertility is poorly understood. Multiple studies, primarily through the use of transgenic animals, have contributed to a list of candidate genes that may affect male infertility in humans. We examined single nucleotide polymorphisms (SNPs) as a cause of male infertility in an analysis of spermatogenesis-specific genes. We carried out the prevalence of SNPs in the coding region of phosphoglycerate mutase 4 (PGAM4) on the X chromosome by the direct sequencing of PCR-amplified DNA from male patients. Using RT-PCR and western blot analyses, we identified that PGAM4 is a functional retrogene that is expressed predominantly in the testes and is associated with male infertility. PGAM4 is expressed in post-meiotic stages, including spermatids and spermatozoa in the testes, and the principal piece of the flagellum and acrosome in ejaculated spermatozoa. A case-control study revealed that 4.5% of infertile patients carry the G75C polymorphism, which causes an amino acid substitution in the encoded protein. Furthermore, an assay for enzymatic activity demonstrated that this polymorphism decreases the enzyme's activity both in vitro and in vivo. These results suggest that PGAM4, an X-linked retrogene, is a fundamental gene in human male reproduction and may escape meiotic sex chromosome inactivation. These findings provide fresh insight into elucidating the mechanisms of male infertility.

Here, we report on a patient with a 625 kb duplication in Xp22.12, detected by array comparative genomic hybridization (CGH). The duplicated region contains only one gene, RPS6KA3, that results in partial duplication. The same duplication was present in his mother and his maternal uncle. This partial duplication inhibits the RPS6KA3 expression, mimicking the effect of loss-of-function mutations associated with Coffin-Lowry syndrome (CLS). The phenotype of the patient here presented is not fully evocative of this syndrome because he does not present some of the facial, digital and skeletal abnormalities that are considered the main diagnostic features of CLS. This case is one of the few examples where RPS6KA3 mutations are associated with a non-specific X-linked mental retardation.

Phosphoglycerate kinase (PGK) deficiency, a rare X-linked inherited disorder, manifests as various combinations of hemolytic anemia, neurological dysfunction, and myopathy. We report a Japanese boy with PGK deficiency presenting as chronic hemolytic anemia. The diagnosis of PGK1 deficiency was made at 11 months of age on the basis of low PGK enzyme activity (36.7 IU/g Hb; normal, 264–326 IU/g Hb) and the identification through PGK1 gene sequencing of a novel missense mutation: c. 1180A>G at exon 10. The mutation, which has been designated PGK-Aoto, results in a Thr394Ala amino-acid substitution at β-strand L. Because β-strand L plays an important role in the function of the hinge connecting the two domains of PGK, the Thr394Ala substitution may perturb this motion. At 3 years of age the patient has transfusion-dependent hemolytic anemia but no evidence of neuromuscular disease or developmental delay. Long-term follow-up will be needed to identify possible future clinical manifestations.