Identification of 22 novel BTK gene variants in B cell deficiency with hypogammaglobulinemia.

X-linked agammaglobulinemia (XLA) is an inborn error of immunity caused by pathogenic variants in the BTK gene, resulting in impaired B cell differentiation and maturation. Over 900 variants have already been described in this gene, however, new pathogenic variants continue to be identified. In this report, we describe 22 novel variants in BTK, associated with B cell deficiency with hypo- or agammaglobulinemia in male patients or in asymptomatic female carriers. Genetic data was correlated with BTK protein expression by flow cytometry, and clinical and family history to obtain a comprehensive assessment of the clinico-pathologic significance of these new variants in the BTK gene. For one novel missense variant, p.Cys502Tyr, site-directed mutagenesis was performed to determine the impact of the sequence change on protein expression and stability. Genetic data should be correlated with protein and/or clinical and immunological data, whenever possible, to determine the clinical significance of the gene sequence alteration.

Expression, purification and microscopic characterization of human ATP-binding cassette sub-family B member 7 protein.

The ATP-binding cassette sub-family B member 7 (ABCB7) is a membrane transport protein located on the inner membrane of mitochondria, which could be involved in the transport of heme from the mitochondria to the cytosol. ABCB7 also plays a central role in the maturation of cytosolic iron-sulfur (Fe/S) cluster-containing proteins, and mutations can cause a series of mitochondrial defects. X-linked sideroblastic anemia and ataxia (XLSA-A) is a rare cause of early onset ataxia, which may be overlooked due to the usually mild asymptomatic anemia. The genetic defect has been identified as a mutation in the ABCB7 gene at Xq12-q13. Here, we report the expression, purification and the 2D projections derived from negatively stained electron micrographs of recombinant H. sapiens ABCB7 (hABCB7), paving the way from an atomic structure determination of ABCB7.

Impact of amino acid substitution in the kinase domain of Bruton tyrosine kinase and its association with X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is a rare disease that affects the immune system, characterized by a serial development of bacterial infection from the onset of infantile age. Bruton tyrosine kinase (BTK) is a non-receptor cytoplasmic kinase that plays a crucial role in the B-lymphocyte maturation. The altered expression, mutation and/or structural variations of BTK are responsible for causing XLA. Here, we have performed extensive sequence and structure analyses of BTK to find deleterious variations and their pathogenic association with XLA. First, we screened the pathogenic variations in the BTK from a pool of publicly available resources, and their pathogenicity/tolerance and stability predictions were carried out. Finally, two pathogenic variations (E589G and M630K) were studied in detail and subjected to all-atom molecular dynamics simulation for 200 ns. Intramolecular hydrogen bonds (H-bonds), secondary structure, and principal component analysis revealed significant conformational changes in variants that support the structural basis of BTK dysfunction in XLA. The free energy landscape analysis revealed the presence of multiple energy minima, suggests that E589G brings a large destabilization and consequently unfolding behavior compared to M630K. Overall, our study suggests that amino acid substitutions, E589G, and M630K, significantly alter the structural conformation and stability of BTK.

Clinical characteristics and prenatal diagnosis for 22 families in Henan Province of China with X-linked agammaglobulinemia (XLA) related to Bruton's tyrosine kinase (BTK) gene mutations.

BACKGROUND: X-linked agammaglobulinaemia (XLA) is a rare immunodeficiency disease for which recurrent severe infection is the major clinical symptom. BTK is the main causative gene, with X chromosome recessive inheritance. However, the mutations reported to date do not fully explain the disorder. METHODS: We detected the percentage of CD19+ B cells and serum immunoglobulin (IgG, IgA, and IgM) levels by flow cytometry and rate scatter immunoturbidimetry, and investigated the BTK mutation profile in 22 XLA patients using Sanger sequencing and real-time PCR . RESULTS: We evaluated the clinical symptoms of 22 XLA patients and investigated genetic mutations present, identifying six novel mutations in the BTK gene: 2 missense mutations (c.23G > T and c.112 T > C), 2 frameshift mutations (c.522_523insC and c.1060delA), 1 large deletion (deletion of exon 2 to 5), and 1 splice-site mutation (c.1631 + 2 T > C). Prenatal diagnoses were performed in six families (F10, F11, F15, F18, F20 and F21), with the following results: the male fetus in Family 10 (F10) did not carry the c.922_923delGA mutation; the male fetus in Family 15 (F15) did not carry the c.1631 + 1G > T splicing mutation; the female fetus in Family 20 (F20) did not carry the c.1931 T > C mutation; the female fetus in Family 21 (F21) did not carry the large deletion mutation. Hence, these four fetuses are not likely to develop XLA. Male fetuses with c.1060delA and c.1684C > T mutations were identified in Family 11 and Family 18, respectively. The pregnant woman in F18 chose to terminate the pregnancy, whereas the pregnant woman in F11 chose to continue the pregnancy. CONCLUSION: We confirmed the diagnosis of 22 XLA patients from 22 unrelated families and detected six new pathogenic mutations. Prenatal diagnosis was performed in six families. Early genetic diagnosis and routine lifelong immunoglobulin replacement therapy can prevent and treat infections in XLA children, saving their lives.

Bruton tyrosine kinase deficiency augments NLRP3 inflammasome activation and causes IL-1ß-mediated colitis.

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-ß or anakinra, an inhibitor of IL-1ß 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.

A novel Bruton tyrosine kinase gene variation was found in an adult with X-linked agammaglobulinemia during blood cross-matching prior to surgical operation.

AIMS/OBJECTIVES: To investigate the underlying molecular mechanism of the patient's ABO typing discrepancy. BACKGROUND: ABO typing discrepancy was frequently seen in patients due to different causes. In this study, ABO typing discrepancy was found in a 24-year-old man with arthralgia, whose forward ABO grouping was O and reverse ABO grouping was AB. Primary immunodeficiency disease was speculated in this patient, especially X-linked agammaglobulinemia (XLA). METHODS: Immunoglobulins of all isotypes were detected using a specific protein analyser. Lymphocyte subgroups were analysed by flow cytometry. All 19 exons and boundaries of BTK gene were amplified by polymerase chain reaction (PCR), and all PCR products were sequenced by a DNA analyser. BTK protein in the leukocytes and platelets was detected by Western blot. RESULTS: No B lymphocytes could be detected in the peripheral blood of the patient. A novel BTK gene variation, c.817G>T, in the exon 9 of BTK gene was discovered. No BTK protein expression could be detected in the leukocytes and platelets of the patient. CONCLUSIONS: XLA could be occasionally discovered by ABO typing discrepancy in some cases because of the deficiency of reciprocal IgM anti-A and/or anti-B antibodies in the serum of the patient. Humoral immunodeficiency is one of the causes of ABO typing discrepancy.

GLRX5 mutations impair heme biosynthetic enzymes ALA synthase 2 and ferrochelatase in Human congenital sideroblastic anemia.

Non-syndromic microcytic congenital sideroblastic anemia (cSA) is predominantly caused by defective genes encoding for either ALAS2, the first enzyme of heme biosynthesis pathway or SLC25A38, the mitochondrial importer of glycine, an ALAS2 substrate. Herein we explored a new case of cSA with two mutations in GLRX5, a gene for which only two patients have been reported so far. The patient was a young female with biallelic compound heterozygous mutations in GLRX5 (p.Cys67Tyr and p.Met128Lys). Three-D structure analysis confirmed the involvement of Cys67 in the coordination of the [2Fe2S] cluster and suggested a potential role of Met128 in partner interactions. The protein-level of ferrochelatase, the terminal-enzyme of heme process, was increased both in patient-derived lymphoblastoid and CD34+ cells, however, its activity was drastically decreased. The activity of ALAS2 was found altered and possibly related to a defect in the biogenesis of its co-substrate, the succinyl-CoA. Thus, the patient exhibits both a very low ferrochelatase activity without any accumulation of porphyrins precursors in contrast to what is reported in erythropoietic protoporphyria with solely impaired ferrochelatase activity. A significant oxidative stress was evidenced by decreased reduced glutathione and aconitase activity, and increased MnSOD protein expression. This oxidative stress depleted and damaged mtDNA, decreased complex I and IV activities and depleted ATP content. Collectively, our study demonstrates the key role of GLRX5 in modulating ALAS2 and ferrochelatase activities and in maintaining mitochondrial function.

Level of residual enzyme activity modulates the phenotype in phosphoglycerate kinase deficiency.

OBJECTIVE: To study the variable clinical picture and exercise tolerance of patients with phosphoglycerate kinase (PGK) 1 deficiency and how it relates to residual PGK enzyme activity. METHODS: In this case series study, we evaluated 7 boys and men from 5 families with PGK1 deficiency. Five had pure muscle symptoms, while 2 also had mild intellectual disability with or without anemia. Muscle glycolytic and oxidative capacities were evaluated by an ischemic forearm exercise test and by cycle ergometry. RESULTS: Enzyme levels of PGK were 4% to 9% of normal in red cells and 5% to10% in muscle in pure myopathy patients and 2.6% in both muscle and red cells in the 2 patients with multisystem involvement. Patients with pure myopathy had greater increases in lactate with ischemic exercise (2-3 mmol/L) vs the 2 multisystem-affected patients (<1 mmol/L). Myopathy patients had higher oxidative capacity in cycle exercise vs multisystem affected patients (≈30 vs ≈15 mL/kg per minute). One multisystem-affected patient developed frank myoglobinuria after the short exercise test. CONCLUSIONS: This case series study of PGK1 deficiency suggests that the level of impaired glycolysis in PGK deficiency is a major determinant of phenotype. Lower glycolytic capacity in PGK1 deficiency seems to result in multisystem involvement and increased susceptibility to exertional rhabdomyolysis.

The phenotypic spectrum of germline YARS2 variants: from isolated sideroblastic anemia to mitochondrial myopathy, lactic acidosis and sideroblastic anemia 2.

YARS2 variants have previously been described in patients with myopathy, lactic acidosis and sideroblastic anemia 2 (MLASA2). YARS2 encodes the mitochondrial tyrosyl-tRNA synthetase, which is responsible for conjugating tyrosine to its cognate mt-tRNA for mitochondrial protein synthesis. Here we describe 14 individuals from 11 families presenting with sideroblastic anemia and YARS2 variants that we identified using a sideroblastic anemia gene panel or exome sequencing. The phenotype of these patients ranged from MLASA to isolated congenital sideroblastic anemia. As in previous cases, inter- and intra-familial phenotypic variability was observed, however, this report includes the first cases with isolated sideroblastic anemia and patients with biallelic YARS2 variants that have no clinically ascertainable phenotype. We identified ten novel YARS2 variants and three previously reported variants. In vitro amino-acylation assays of five novel missense variants showed that three had less effect on the catalytic activity of YARS2 than the most commonly reported variant, p.(Phe52Leu), associated with MLASA2, which may explain the milder phenotypes in patients with these variants. However, the other two missense variants had a more severe effect on YARS2 catalytic efficiency. Several patients carried the common YARS2 c.572 G>T, p.(Gly191Val) variant (minor allele frequency =0.1259) in trans with a rare deleterious YARS2 variant. We have previously shown that the p.(Gly191Val) variant reduces YARS2 catalytic activity. Consequently, we suggest that biallelic YARS2 variants, including severe loss-of-function alleles in trans of the common p.(Gly191Val) variant, should be considered as a cause of isolated congenital sideroblastic anemia, as well as the MLASA syndromic phenotype.

Unusual Presentations of Dystrophinopathies in Childhood.

X-linked recessive mutations in the dystrophin gene are one of the most common causes of inherited neuromuscular disorders in humans. Duchenne muscular dystrophy, the most common phenotype, and Becker muscular dystrophy are often recognizable by certain clinical features; however, less frequent presentations require a higher degree of suspicion. In this article, we describe a series of 6 children (4 boys, 2 girls) referred to a tertiary pediatric neuromuscular clinic for isolated elevated creatine kinase levels (range: 720-7000 IU/L) identified on initial assessment for otherwise unexplained transaminase elevations (n = 2), a social communication disorder (n = 3), and exertional myalgia and/or rhabdomyolysis (n = 1). There was no preceding family history of neuromuscular disease. One boy had an additional history of severe cerebral palsy and cyclical vomiting, and 1 girl had a history of maternal hepatitis C. There was no significant weakness at presentation, and the majority remained stable over a prolonged period of follow-up (age range at last follow-up: 9-16 years). All 6 children were found to carry dystrophin gene mutations resulting in milder phenotypes. This series highlights that dystrophinopathies may not uncommonly present with features distinct from the classic Duchenne muscular dystrophy and Becker muscular dystrophy phenotypes in both boys and girls. Pediatricians should be aware of such atypical presentations to initiate a timely and adequate diagnostic process. Establishing the correct genetic diagnosis of a dystrophinopathy is important to allow appropriate genetic counseling, to implement relevant surveillance and management strategies, and to avoid unnecessary investigations in search of an incorrect alternative diagnosis.

Isoniazid inhibits human erythroid 5-aminolevulinate synthase: Molecular mechanism and tolerance study with four X-linked protoporphyria patients.

Mutations in the C-terminus of human erythroid 5-aminolevulinate synthase (hALAS2), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, are associated with two different blood disorders, X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP). XLSA-causing mutations yield hALAS2 variants with decreased activity, while XLPP-causing mutations result in a gain-of-function of hALAS2. There are no specific treatments for XLPP. Isonicotinic acid hydrazide (isoniazid, INH), an antituberculosis agent, can cause sideroblastic anemia as a side-effect, by limiting PLP availability to hALAS2, via inhibition of pyridoxal kinase or reaction with pyridoxal to form pyridoxal isonicotinoyl hydrazone. We hypothesized that INH also binds and directly inhibits hALAS2. Using fluorescence-activated cell sorting and confocal fluorescence microscopy, we demonstrate that INH reduces protoporphyrin IX levels in HeLa cells expressing either wild-type hALAS2 or XLPP variants. In addition, PLP and pyridoxamine 5'-phosphate (PMP) reversed the cellular inhibition of hALAS2 activity by INH. Steady-state kinetic analyses with purified hALAS2 indicated that INH directly inhibits the enzyme, noncompetitively or uncompetitively, with an apparent Ki of 1.2µM. Circular dichroism spectroscopy revealed that INH triggered tertiary structural changes in hALAS2 that altered the microenvironment of the PLP cofactor and hampered the association of PLP with apo-hALAS2. Treatment of four XLPP patients with INH (5mg·kg-1·day-1) over a six-month period was well tolerated but without statistically significant modification of PPIX levels. These results, taken together, permit us to further an INH inhibition kinetic mechanism for ALAS, which suggests the possible use of INH-derived drugs in treating patients with XLPP and potentially other protoporphyrin-accumulating porphyrias.

BTK Signaling in B Cell Differentiation and Autoimmunity.

Since the original identification of Bruton's tyrosine kinase (BTK) as the gene defective in the primary immunodeficiency X-linked agammaglobulinemia (XLA) in 1993, our knowledge on the physiological function of BTK has expanded impressively. In this review, we focus on the role of BTK during B cell differentiation in vivo, both in the regulation of expansion and in the developmental progression of pre-B cells in the bone marrow and as a crucial signal transducer of signals downstream of the IgM or IgG B cell antigen receptor (BCR) in mature B cells governing proliferation, survival, and differentiation. In particular, we highlight BTK function in B cells in the context of host defense and autoimmunity. Small-molecule inhibitors of BTK have very recently shown impressive anti-tumor activity in clinical studies in patients with various B cell malignancies. Since promising effects of BTK inhibition were also seen in experimental animal models for lupus and rheumatoid arthritis, BTK may be a good target for controlling autoreactive B cells in patients with systemic autoimmune disease.

625 kb microduplication at Xp22.12 including RPS6KA3 in a child with mild intellectual disability.

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.

Human Erythroid 5-Aminolevulinate Synthase Mutations Associated with X-Linked Protoporphyria Disrupt the Conformational Equilibrium and Enhance Product Release.

Regulation of 5-aminolevulinate synthase (ALAS) is at the origin of balanced heme production in mammals. Mutations in the C-terminal region of human erythroid-specific ALAS (hALAS2) are associated with X-linked protoporphyria (XLPP), a disease characterized by extreme photosensitivity, with elevated blood concentrations of free protoporphyrin IX and zinc protoporphyrin. To investigate the molecular basis for this disease, recombinant hALAS2 and variants of the enzyme harboring the gain-of-function XLPP mutations were constructed, purified, and analyzed kinetically, spectroscopically, and thermodynamically. Enhanced activities of the XLPP variants resulted from increases in the rate at which the product 5-aminolevulinate (ALA) was released from the enzyme. Circular dichroism spectroscopy revealed that the XLPP mutations altered the microenvironment of the pyridoxal 5'-phosphate cofactor, which underwent further and specific alterations upon succinyl-CoA binding. Transient kinetic analyses of the variant-catalyzed reactions and protein fluorescence quenching upon binding of ALA to the XLPP variants demonstrated that the protein conformational transition step associated with product release was predominantly affected. Of relevance is the fact that XLPP could also be modeled in cell culture. We propose that (1) the XLPP mutations destabilize the succinyl-CoA-induced hALAS2 closed conformation and thus accelerate ALA release, (2) the extended C-terminus of wild-type mammalian ALAS2 provides a regulatory role that allows for allosteric modulation of activity, thereby controlling the rate of erythroid heme biosynthesis, and (3) this control is disrupted in XLPP, resulting in porphyrin accumulation.

The structure of phosphoinositide phosphatases: Insights into substrate specificity and catalysis.

Phosphoinositides (PIs) are a group of key signaling and structural lipid molecules involved in a myriad of cellular processes. PI phosphatases, together with PI kinases, are responsible for the conversion of PIs between distinctive phosphorylation states. PI phosphatases are a large collection of enzymes that are evolved from at least two disparate ancestors. One group is distantly related to endonucleases, which apply divalent metal ions for phosphoryl transfer. The other group is related to protein tyrosine phosphatases, which contain a highly conserved active site motif Cys-X5-Arg (CX5R). In this review, we focus on structural insights to illustrate current understandings of the molecular mechanisms of each PI phosphatase family, with emphasis on their structural basis for substrate specificity determinants and catalytic mechanisms. This article is part of a Special Issue entitled Phosphoinositides.

Biochemical and cellular analysis of Ogden syndrome reveals downstream Nt-acetylation defects.

The X-linked lethal Ogden syndrome was the first reported human genetic disorder associated with a mutation in an N-terminal acetyltransferase (NAT) gene. The affected males harbor an Ser37Pro (S37P) mutation in the gene encoding Naa10, the catalytic subunit of NatA, the major human NAT involved in the co-translational acetylation of proteins. Structural models and molecular dynamics simulations of the human NatA and its S37P mutant highlight differences in regions involved in catalysis and at the interface between Naa10 and the auxiliary subunit hNaa15. Biochemical data further demonstrate a reduced catalytic capacity and an impaired interaction between hNaa10 S37P and Naa15 as well as Naa50 (NatE), another interactor of the NatA complex. N-Terminal acetylome analyses revealed a decreased acetylation of a subset of NatA and NatE substrates in Ogden syndrome cells, supporting the genetic findings and our hypothesis regarding reduced Nt-acetylation of a subset of NatA/NatE-type substrates as one etiology for Ogden syndrome. Furthermore, Ogden syndrome fibroblasts display abnormal cell migration and proliferation capacity, possibly linked to a perturbed retinoblastoma pathway. N-Terminal acetylation clearly plays a role in Ogden syndrome, thus revealing the in vivo importance of N-terminal acetylation in human physiology and disease.

Mutations in TRNT1 cause congenital sideroblastic anemia with immunodeficiency, fevers, and developmental delay (SIFD).

Mutations in genes encoding proteins that are involved in mitochondrial heme synthesis, iron-sulfur cluster biogenesis, and mitochondrial protein synthesis have previously been implicated in the pathogenesis of the congenital sideroblastic anemias (CSAs). We recently described a syndromic form of CSA associated with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD). Here we demonstrate that SIFD is caused by biallelic mutations in TRNT1, the gene encoding the CCA-adding enzyme essential for maturation of both nuclear and mitochondrial transfer RNAs. Using budding yeast lacking the TRNT1 homolog, CCA1, we confirm that the patient-associated TRNT1 mutations result in partial loss of function of TRNT1 and lead to metabolic defects in both the mitochondria and cytosol, which can account for the phenotypic pleiotropy.

Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model.

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.

Muscle involvement in Dent disease 2.

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.

Phosphoglycerate kinase deficiency due to a novel mutation (c. 1180A>G) manifesting as chronic hemolytic anemia in a Japanese boy.

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 ß-strand L. Because ß-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.