Ken Inoue

Genome-wide association study identifies HLA-DP as a susceptibility gene for pediatric asthma in Asian populations.

Asthma is a complex phenotype influenced by genetic and environmental factors. We conducted a genome-wide association study (GWAS) with 938 Japanese pediatric asthma patients and 2,376 controls. Single-nucleotide polymorphisms (SNPs) showing strong associations (P<1×10−8) in GWAS were further genotyped in an independent Japanese samples (818 cases and 1,032 controls) and in Korean samples (835 cases and 421 controls). SNP rs987870, located between HLA-DPA1 and HLA-DPB1, was consistently associated with pediatric asthma in 3 independent populations (P combined = 2.3×10−10, odds ratio [OR] = 1.40). HLA-DP allele analysis showed that DPA1*0201 and DPB1*0901, which were in strong linkage disequilibrium, were strongly associated with pediatric asthma (DPA1*0201: P = 5.5×10−10, OR = 1.52, and DPB1*0901: P = 2.0×10−7, OR = 1.49). Our findings show that genetic variants in the HLA-DP locus are associated with the risk of pediatric asthma in Asian populations.

Mutations in POLR3A and POLR3B Encoding RNA Polymerase III Subunits Cause an Autosomal-Recessive Hypomyelinating Leukoencephalopathy

Congenital hypomyelinating disorders are a heterogeneous group of inherited leukoencephalopathies characterized by abnormal myelin formation. We have recently reported a hypomyelinating syndrome characterized by diffuse cerebral hypomyelination with cerebellar atrophy and hypoplasia of the corpus callosum (HCAHC). We performed whole-exome sequencing of three unrelated individuals with HCAHC and identified compound heterozygous mutations in POLR3B in two individuals. The mutations include a nonsense mutation, a splice-site mutation, and two missense mutations at evolutionally conserved amino acids. Using reverse transcription-PCR and sequencing, we demonstrated that the splice-site mutation caused deletion of exon 18 from POLR3B mRNA and that the transcript harboring the nonsense mutation underwent nonsense-mediated mRNA decay. We also identified compound heterozygous missense mutations in POLR3A in the remaining individual. POLR3A and POLR3B encode the largest and second largest subunits of RNA Polymerase III (Pol III), RPC1 and RPC2, respectively. RPC1 and RPC2 together form the active center of the polymerase and contribute to the catalytic activity of the polymerase. Pol III is involved in the transcription of small noncoding RNAs, such as 5S ribosomal RNA and all transfer RNAs (tRNA). We hypothesize that perturbation of Pol III target transcription, especially of tRNAs, could be a common pathological mechanism underlying POLR3A and POLR3B mutations.

Transcription Factor Repression and Activation of the Human Acetylcholinesterase Gene

Acetylcholinesterase in man is encoded by a single gene, ACHE, located on chromosome 7q22. In this study, the transcription start sites and major DNA promoter elements controlling the expression of this gene have been characterized by structural and functional studies. Immediately upstream of the first untranslated exon of the gene are GC-rich sequences containing consensus binding sites for several transcription factors, including Sp1, EGR-1 and AP2. In vitro transcription studies and RNase protection analyses of mRNA isolated from human NT2/D1 teratocarcinoma cells reveal that two closely spaced transcription cap sites are located at a consensus initiator (Inr) element similar to that found in the terminal transferase gene. Transient transfection of mutant genes shows that removal of three bases of this initiator sequence reduces promoter activity by 98% in NT2/D1 cells. In vitro transcription studies and transient transfection of a series of 5′ deletion mutants of the ACHE promoter linked to a luciferase reporter show an Sp1 site at −71 to be essential for promoter activity. Purified Sp1 protein protects this site from DNase cleavage during in vitro footprinting experiments. A conserved AP2 consensus binding site, located between the GC box elements and the Inr, is protected by recombinant AP2 protein in DNase footprinting experiments, induces a mobility shift with AP2 protein and AP2-containing cell extracts, and fosters inhibition of transcription by AP2 as measured by transient transfection in mouse and human cell lines and in in vitro transcription reactions. These results indicate that AP2 functions as a repressor of human ACHE and mouse Ache transcription.

Disrupted SOX10 Regulation of GJC2 Transcription Causes Pelizaeus-Merzbacher-Like Disease

Mutations in the gap junction protein gamma‐2 gene, GJC2, cause a central hypomyelinating disorder; Pelizaeus‐Merzbacher‐like disease (PMLD; MIM311601). Using a homozygosity mapping and positional candidate gene approach, we identified a homozygous mutation (c.‐167A>G) within the GJC2 promoter at a potent SOX10 binding site in a patient with mild PMLD. Functionally, this mutation completely abolished the SOX10 binding and attenuated GJC2 promoter activity. These findings suggest not only that the SOX10‐to‐GJC2 transcriptional dysregulation is a cause of PMLD, but also that GJC2 may be in part responsible for the central hypomyelination caused by SOX10 mutations. ANN NEUROL 2010;68:250–254

Depletion of molecular chaperones from the endoplasmic reticulum and fragmentation of the Golgi apparatus associated with pathogenesis in Pelizaeus-Merzbacher disease

Background: Mutations of proteolipid protein 1 (PLP1) induce endoplasmic reticulum (ER) stress. Results: PLP1 mutants deplete some chaperones from the ER and induce fragmentation of the Golgi apparatus (GA). Conclusion: These changes affect clinical pathology in disease-causing mutations of PLP1. Significance: This work provides a novel insight involving global changes of organelles in pathogenesis of ER stress-related diseases. Missense mutations in the proteolipid protein 1 (PLP1) gene cause a wide spectrum of hypomyelinating disorders, from mild spastic paraplegia type 2 to severe Pelizaeus-Merzbacher disease (PMD). Mutant PLP1 accumulates in the endoplasmic reticulum (ER) and induces ER stress. However, the link between the clinical severity of PMD and the cellular response induced by mutant PLP1 remains largely unknown. Accumulation of misfolded proteins in the ER generally leads to up-regulation of ER chaperones to alleviate ER stress. Here, we found that expression of the PLP1-A243V mutant, which causes severe disease, depletes some ER chaperones with a KDEL (Lys-Asp-Glu-Leu) motif, in HeLa cells, MO3.13 oligodendrocytic cells, and primary oligodendrocytes. The same PLP1 mutant also induces fragmentation of the Golgi apparatus (GA). These organelle changes are less prominent in cells with milder disease-associated PLP1 mutants. Similar changes are also observed in cells expressing another disease-causing gene that triggers ER stress, as well as in cells treated with brefeldin A, which induces ER stress and GA fragmentation by inhibiting GA to ER trafficking. We also found that mutant PLP1 disturbs localization of the KDEL receptor, which transports the chaperones with the KDEL motif from the GA to the ER. These data show that PLP1 mutants inhibit GA to ER trafficking, which reduces the supply of ER chaperones and induces GA fragmentation. We propose that depletion of ER chaperones and GA fragmentation induced by mutant misfolded proteins contribute to the pathogenesis of inherited ER stress-related diseases and affect the disease severity.

Increased N-acetylaspartate in model mouse of Pelizaeus-Merzbacher disease.

To evaluate the N‐acetylaspartate (NAA) and N‐acetylaspartylglutamate (NAAG) biochemical pathways in the brain of myelin synthesis‐deficient (msd) mouse, a model of Pelizaeus‐Merzbacher disease (PMD).

Effect of curcumin in a mouse model of Pelizaeus–Merzbacher disease

PLP1 amino acid substitutions cause accumulation of misfolded protein and induce endoplasmic reticulum (ER) stress, causing Pelizaeus-Merzbacher disease (PMD), a hypomyelinating disorder of the central nerve system. Currently no effective therapy is available for PMD. Promoted by its curative effects in other genetic disease models caused by similar molecular mechanisms, we tested if curcumin, a dietary compound, can rescue the lethal phenotype of a PMD mouse model (myelin synthesis deficient, msd). Curcumin was administered orally to myelin synthesis deficit (msd) mice at 180 mg·kg(-1)·day(-1) from the postnatal day 3. We evaluated general and motor status, changes in myelination and apoptosis of oligodendrocytes by neuropathological and biochemical examination, and transcription levels for ER-related molecules. We also examined the pharmacological effect of curcumin in cell culture system. Oral curcumin treatment resulted in 25% longer survival (p<0.01). In addition, oligodendrocytes undergoing apoptosis were reduced in number (p<0.05). However, no apparent improvement in motor function, neurological phenotype, and myelin formation was observed. Curcumin treatment did not change the expression of ER stress markers and subcellular localization of the mutant protein in vitro and/or in vivo. Curcumin partially mitigated the clinical and pathological phenotype of msd mice, although molecular mechanisms underlying this curative effect are yet undetermined. Nonetheless, curcumin may serve as a potential therapeutic compound for PMD caused by PLP1 point mutations.

Patient-derived iPS cells for unveiling the molecular pathology of Pelizaeus-Merzbacher disease: a commentary on ‘Reduced PLP1 expression in induced pluripotent stem cells derived from a Pelizaeus–Merzbacher disease patient with a partial PLP1 duplication’

Patient-derived iPS cells for unveiling the molecular pathology of Pelizaeus-Merzbacher disease: a commentary on ‘Reduced PLP1 expression in induced pluripotent stem cells derived from a Pelizaeus–Merzbacher disease patient with a partial PLP1 duplication’

Modeling a complex neurocristopathy, PCWH, in a Sox10 BAC transgenic mouse

SOX10 is a transcription factor that is essential for the neural crest development and the myelin formation both in the PNS and CNS. SOX10 mutations are associated with two distinct neurocristopathies, Waardenburg-Hirschsprung disease (WS4) and peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome and Hirschsprung disease (PCWH), latter of which shows more complex and severe neurological phenotype. Clinical and molecular findings suggested that WS4 results form haploinsufficiency of SOX10, while PCWH is predicted to results from SOX10 acting as either dominant-negative or gain-of-function allele. Although model animals are available for WS4, no animal models for PCWH are currently present. To determine the molecular mechanisms for PCWH in vivo, we have founded BAC-transgenic mice (Tg) carrying a PCWH-causing mutant Sox10 construct. Tg showed abnormal motor coordination, circling behavior, hypopigmented coat color, perceptive deafness, and abnormal enlargement of colon. Microscopic examinations in nerve systems showed relatively normal migration and differentiation of oligodendrocyte and Schwann cells, but apparently delayed myelination in Tg mice. These findings suggested that the mutant Sox10 Tg mice mimic phenotypes observed in human patients with PCWH and thus serve as a model for PCWH.

Abnormal neuronal migration with ischemic brain injuries may cause cognitive dysfunction in extremely preterm infants

Heterozygous mutations in FOXG1 have been reported in patients with a congenital variant of Rett syndrome with varying brain malformations. FOXG1 encodes a winged helix transcriptional factor of the forkhead protein family which is mainly expressed in brain and regulates early steps in cortical development. The aim of this study was to search for mutations affecting the FOXG1 gene and to further delineate the clinical and brain imaging features of FOXG1-mutation positive patients. We report nine de novo mutations of FOXG1, including one previously described and eight novel alterations, consisting of two deletions, two chromosome rearrangements disrupting putative cis-regulatory elements, and five sequence changes among 189 patients tested. Adding these to prior reports, the mutational spectrum associated with FOXG1 mutations now includes six deletions and three other disruptions of chromosome 14q12, 12 null mutations (six nonsense, six frameshift), and only three missense mutations. Analysis of our nine patients and those in published reports demonstrates a complex constellation of clinical features including mild postnatal growth deficiency, severe postnatal microcephaly, severe mental retardation with absent language development, deficient social interactions such as poor eye contact resembling autism, combined stereotypies and frank dyskinesias with mixed features of athetosis, chorea and dystonia, epilepsy, poor sleep patterns, irritability in infancy, unexplained episodes of crying, recurrent aspiration, and gastroesophageal reflux. Brain imaging studies reveal simplified gyral pattern over the frontal lobes, reduced white matter volume, corpus callosum hypogenesis, and variable mild frontal pachygyria. While this phenotype overlaps both classic and congenital Rett syndrome, extensive clinical data demonstrates a distinctive and clinically recognizable phenotype. We conclude that de novo mutations in FOXG1 are a rare cause of autosomal dominant mental retardation in patients with microcephaly and various brain malformations that can include frontal pachygyria and abnormal corpus callosum.