Kazuo Kubota

A rapid screening method to detect autosomal-dominant ectodermal dysplasia with immune deficiency syndrome.

7. Spergel JM, Paller AS. Atopic dermatitis and the atopic march. J Allergy Clin Immunol 2003;112(suppl):S118-27. 8. Tran DQ, Andersson J, Wang R, Ramsey H, Unutmaz D, Shevach EM. GARP (LRRC32) is essential for the surface expression of latent TGF-beta on platelets and activated FOXP31 regulatory T cells. Proc Natl Acad Sci U S A 2009;106: 13445-50. 9. Marenholz I, Bauerfeind A, Esparza-Gordillo J, Kerscher T, Granell R, Nickel R, et al. The eczema risk variant on chromosome 11q13 (rs7927894) in the population-based ALSPAC cohort: a novel susceptibility factor for asthma and hay fever. Hum Mol Genet 2011;20:2443-9.

TRAM Is Involved in IL-18 Signaling and Functions as a Sorting Adaptor for MyD88

MyD88, a Toll/interleukin-1 receptor homology (TIR) domain-containing adaptor protein, mediates signals from the Toll-like receptors (TLR) or IL-1/IL-18 receptors to downstream kinases. In MyD88-dependent TLR4 signaling, the function of MyD88 is enhanced by another TIR domain-containing adaptor, Mal/TIRAP, which brings MyD88 to the plasma membrane and promotes its interaction with the cytosolic region of TLR4. Hence, Mal is recognized as the “sorting adaptor” for MyD88. In this study, a direct interaction between MyD88-TIR and another membrane-sorting adaptor, TRAM/TICAM-2, was demonstrated in vitro. Cell-based assays including RNA interference experiments and TRAM deficient mice revealed that the interplay between MyD88 and TRAM in cells is important in mediating IL-18 signal transduction. Live cell imaging further demonstrated the co-localized accumulation of MyD88 and TRAM in the membrane regions in HEK293 cells. These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction. The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal. MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.

Functional assessment of the mutational effects of human IRAK4 and MyD88 genes.

Human interleukin-1 receptor-associated kinase 4 (IRAK4) deficiency and myeloid differentiating factor 88 (MyD88) deficiency syndromes are two primary immune-deficiency disorders with innate immune defects. Although new genetic variations of IRAK4 and MyD88 have recently been deposited in the single nucleotide polymorphism (SNP) database, the clinical significance of these variants has not yet been established. Therefore, it is important to establish methods for assessing the association of each gene variation with human diseases. Because cell-based assays, western blotting and an NF-κB reporter gene assay, showed no difference in protein expression and NF-κB activity between R12C and wild-type IRAK4, we examined protein-protein interactions of purified recombinant IRAK4 and MyD88 proteins by analytical gel filtration and NMR titration. We found that the variant of IRAK4, R12C, as well as R20W, located in the death domain of IRAK4 and regarded as a SNP, caused a loss of interaction with MyD88. Our studies suggest that not only the loss of protein expression but also the defect of Myddosome formation could cause IRAK4 and MyD88 deficiency syndromes. Moreover a combination of in vitro functional assays is effective for confirming the pathogenicity of mutants found in IRAK4 and MyD88-deficiency patients.

Molecular analysis of the binding mode of Toll/interleukin-1 receptor (TIR) domain proteins during TLR2 signaling

Toll-like receptor (TLR) signaling is initiated by the binding of various adaptor proteins through ligand-induced oligomerization of the Toll/interleukin-1 receptor (TIR) domains of the TLRs. TLR2, which recognizes peptidoglycans, lipoproteins or lipopeptides derived from Gram-positive bacteria, is known to use the TIR domain-containing adaptor proteins myeloid differentiating factor 88 (MyD88) and MyD88 adaptor-like (Mal). Molecular analyses of the binding specificity of MyD88, Mal, and TLR2 are important for understanding the initial defenses mounted against Gram-positive bacterial infections such as Streptococcus pneumoniae. However, the detailed molecular mechanisms involved in the multiple interactions of these TIR domains remain unclear. Our study demonstrates that the TIR domain proteins MyD88, Mal, TLR1, and TLR2 directly bind to each other in vitro. We have also identified two binding interfaces of the MyD88 TIR domain for the TLR2 TIR domain. A residue at these interfaces has recently been found to be mutated in innate immune deficiency patients. These novel insights into the binding mode of TIR proteins will contribute to elucidation of the mechanisms underlying innate immune deficiency diseases, and to future structural studies of hetero-oligomeric TIR-TIR complexes.

Characterization of NLRP3 Variants in Japanese Cryopyrin-Associated Periodic Syndrome Patients

The etiology of cryopyrin-associated periodic syndrome (CAPS) is caused by germline gene mutations in NOD-like receptor family, pryin domain containing 3 (NLRP3)/cold-induced autoinflammatory syndrome 1 (CIAS1). CAPS includes diseases with various severities. The aim of this study was to characterize patients according to the disease severity of CAPS. Five Japanese patients with four kinds of gene variations in NLRP3 were found and diagnosed as CAPS or juvenile idiopathic arthritis. Two mutations in NLRP3, Y563N and E688K, found in CAPS patients exhibit significant positive activities in the nuclear factor-κB reporter gene assay. Increased serum interleukin (IL)-18 levels were only observed in severe cases of CAPS. In mild cases of CAPS, the serum IL-18 levels were not increased, although lipopolysaccharide- or hypothermia-enhanced IL-1β and IL-18 production levels by their peripheral blood mononuclear cells were detectable. This series of case reports suggests that a combination of in vitro assays could be a useful tool for the diagnosis and characterization of the disease severity of CAPS.

De novo KIF1A mutations cause intellectual deficit, cerebellar atrophy, lower limb spasticity and visual disturbance

Recently, de novo KIF1A mutations were identified in patients with intellectual disability, spasticity and cerebellar atrophy and/or optic nerve atrophy. In this study, we analyzed a total of 62 families, including 68 patients with genetically unsolved childhood cerebellar atrophy, by whole-exome sequencing (WES). We identified five de novo missense KIF1A mutations, including only one previously reported mutation (p.Arg316Trp). All the mutations are located in the motor domain of KIF1A. In all patients, initial symptom onset was during the infantile period, and included developmental delay in three patients and gait disturbance in two. Thereafter, they showed gait disturbances, exaggerated deep tendon reflexes, cerebellar symptoms and cerebellar atrophy on brain magnetic resonance imaging. Four patients showed lower limb spasticity, upper limb clumsiness and visual disturbances. Nerve conduction study revealed peripheral neuropathy in three patients. This study further delineates clinical features of de novo KIF1A mutations. Genetic testing of KIF1A should be considered in children with developmental delay, cerebellar atrophy and pyramidal features.

The first report of Japanese patients with asparagine synthetase deficiency

BACKGROUND Asparagine synthetase (ASNS) deficiency was recently discovered as a metabolic disorder of non-essential amino acids, and presents as severe progressive microcephaly, intellectual disorder, dyskinetic quadriplegia, and intractable seizures. METHODS Two Japanese children with progressive microcephaly born to unrelated patients were analyzed by whole exome sequencing and novel ASNS mutations were identified. The effects of the ASNS mutations were analyzed by structural evaluation and in silico predictions. RESULTS We describe the first known Japanese patients with ASNS deficiency. Their clinical manifestations were very similar to reported cases of ASNS deficiency. Progressive microcephaly was noted during the prenatal period in patient 1 but only after birth in patient 2. Both patients had novel ASNS mutations: patient 1 had p.L145S transmitted from his mother and p.L247W which was absent from his mother, while patient 2 carried p.V489D and p.W541Cfs*5, which were transmitted from his mother and father, respectively. Three of the four mutations were predicted to affect protein folding, and in silico analyses suggested that they would be pathogenic. CONCLUSION We report the first two Japanese patients with ASNS deficiency. Disease severity appears to vary among patients, as is the case for other non-essential amino acid metabolic disorders.

Genetic variations in MyD88 adaptor-like are associated with atopic dermatitis

Toll-like receptors (TLRs) are important pathogen-associated molecular pattern recognition receptors involved in initiating immune responses. The adaptor protein MyD88 adaptor-like (Mal), involved in signaling downstream of TLRs, plays a crucial role in mediating NF-κB activation. The association of Mal polymorphisms with allergic diseases has not previously been defined. The objective of this study was to detect polymorphisms in the Mal gene and to investigate their association with allergic diseases. Mal gene polymorphisms were genotyped in 310 subjects. The functional effects of Mal variants were analyzed in vitro. One Mal polymorphism, c.303 G>A (Q101Q), was found at a significantly lower frequency in atopic dermatitis patients (p=0.016). Q101Q is in linkage disequilibrium with -103 A>G (rs1893352) and c.539 C>T (S180L) (rs8177374) in the HapMap database. The A allele of -103 A>G showed significantly reduced transcription of Mal compared with the G allele. In addition, three rare variants were identified in this study, c.394 G>A (E132K), c.428 G>A (R143Q) and c.570 G>C (E190D), and were shown to lead to loss-of-function of Mal. It is possible that gene polymorphisms in Mal could affect atopic dermatitis by influencing the innate immune system. We show that Q101Q, which is in linkage disequilibrium with -103 A>G and S180L, may play a protective role against atopic dermatitis. Furthermore, we propose that loss-of-function variants of Mal could predispose individuals to atopic dermatitis or other immunological disorders.

Congenital inner ear malformations without sensorineural hearing loss in children.

Inner ear malformations are frequently found in patients with congenital hearing loss. It has been reported that normal hearing is rare in patients with severe inner ear vestibular malformations. A 9-year-old boy had had complained of recurrent dizziness and disequilibrium for 2 months. Clinical and neuro-otological examinations showed peripheral involvement of the vestibular system, while audiological investigation was normal. High-resolution magnetic resonance imaging, with three-dimensional reconstruction, showed dysplasia of the bilateral lateral semicircular canals (LSCCs). Isolated vestibular malformation might not be as rare as previously thought, and should be examined by imaging of the temporal bone.

In Vitro Analysis of the Functional Effects of an NLRP3 G809S Variant with the co-Existence of MEFV Haplotype Variants in Atypical Autoinflammatory Syndrome

PurposeHereditary periodic fever syndromes have been considered monogenic diseases. However, some recent reports have described patients with co-existence of recurrent fever responsible genes. This study assessed whether a rare variant, found in Japanese children showing atypical autoinflammatory syndrome, located in the leucine-rich repeat domain of Nod-like receptor family, pyrin domain containing 3 (NLRP3) with co-existence of Mediterranean fever (MEFV) haplotype variants may contribute to a proinflammatory phenotype using a systematic approach.MethodsCytokine production in serum or from peripheral blood monocytes was measured by ELISA. DNA sequence analysis of genes including NLRP3, MEFV, mevalonate kinase (MVK), and tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A) were performed on patient samples. In vitro functional assays determined the effects of the NLRP3 variants and pyrin using NF-κB activation and speck formation assays.ResultsA heterozygous genetic variant of NLRP3, G809S, was found in samples from both patients. Additionally the previously reported heterozygous MEFV variants (P369S-R408Q or E148Q-P369S-R408Q) were also detected in both patients. Serum IL-1ra and sTNFR1 levels increased in the attack phase of the disease in both patients. The production levels of IL-1β from monocytes isolated from both cases were elevated following LPS and IFN-γ stimulation. The NLRP3 G809S variant demonstrated no increase of NF-κB activity following monosodium urate stimulation, whereas it significantly increased speck formation by interacting with apoptosis-associated speck-like protein with caspase recruitment domain.ConclusionsThe phenotype of atypical autoinflammatory disease in patients could be modified by a synergistic effect with two other variants of autoinflammatory-associated genes.