R3HDM1 haploinsufficiency is associated with mild intellectual disability.

R3HDM1 (R3H domain containing 1) is an uncharacterized RNA-binding protein that is highly expressed in the human cerebral cortex. We report the first case of a 12-year-old Japanese male with haploinsufficiency of R3HDM1. He presented with mild intellectual disability (ID) and developmental delay. He had a pericentric inversion of 46,XY,inv(2)(p16.1q21.3)dn with breakpoints in intron 19 of R3HDM1 (2q21.3) and the intergenic region (2p16.1). The R3HDM1 levels in his lymphoblastoid cells were reduced to approximately half that of the healthy controls. However, the expression of MIR128-1, in intron 18 of R3HDM1, was not affected via the pericentric inversion. Knockdown of R3HDM1 in mouse embryonic hippocampal neurons suppressed dendritic growth and branching. Notably, the Database of Genomic Variants reported the case of a healthy control with a 488-kb deletion that included both R3HDM1 and MIR128-1. miR-128 has been reported to inhibit dendritic growth and branching in mouse brain neurons, which directly opposes the novel functions of R3HDM1. These findings suggest that deleting both R3HDM1 and MIR128-1 alleviates the symptoms of the disease caused by loss-of-function mutations in R3HDM1 only. Thus, haploinsufficiency of R3HDM1 in the patient may be the cause of the mild ID due to the genetic imbalance between R3HDM1 and MIR128-1.

Squamous Cell Carcinoma Antigen 2 (SCCA2, SERPINB4): An Emerging Biomarker for Skin Inflammatory Diseases.

Squamous cell carcinoma antigens 1 and 2 (SCCA1 and 2, SERPIN B3 and B4), members of the ovalbumin serpin (ov-serpin)/clade B serpin family, were originally discovered as tumor-specific antigens and are used as tumor markers for various kinds of squamous cell carcinomas. Recently, our understanding of the underlying mechanisms of how SCCA1/2 enhance tumor growth has greatly increased. Moreover, it has been shown that SCCA1/2 are involved in the pathogenesis of several inflammatory diseases: asthma, psoriasis, and atopic dermatitis (AD). IL-22 and IL-17, signature cytokines of type 17 inflammation, as well as IL-4 and IL-13, signature cytokines of type 2 inflammation, both of which are positively correlated with the pathogenesis of psoriasis and allergic diseases, respectively, can induce expression of SCCA1/2 in airway epithelial cells and/or keratinocytes, leading to high expression of SCCA1/2 in these diseases. Based on these findings, several trials have been performed to examine the potential of applying SCCA1/2 to biomarkers for these diseases. The findings show that SCCA2 is useful to aid diagnosis, estimate clinical severity and disease type, and assess responses to treatment in psoriasis and AD. These results suggest that SCCA2 has emerged as a novel biomarker for skin inflammatory diseases.

Phenotype-genotype correlations of PIGO deficiency with variable phenotypes from infantile lethality to mild learning difficulties.

Inherited GPI (glycosylphosphatidylinositol) deficiencies (IGDs), a recently defined group of diseases, show a broad spectrum of symptoms. Hyperphosphatasia mental retardation syndrome, also known as Mabry syndrome, is a type of IGDs. There are at least 26 genes involved in the biosynthesis and transport of GPI-anchored proteins; however, IGDs constitute a rare group of diseases, and correlations between the spectrum of symptoms and affected genes or the type of mutations have not been shown. Here, we report four newly identified and five previously described Japanese families with PIGO (phosphatidylinositol glycan anchor biosynthesis class O) deficiency. We show how the clinical severity of IGDs correlates with flow cytometric analysis of blood, functional analysis using a PIGO-deficient cell line, and the degree of hyperphosphatasia. The flow cytometric analysis and hyperphosphatasia are useful for IGD diagnosis, but the expression level of GPI-anchored proteins and the degree of hyperphosphatasia do not correlate, although functional studies do, with clinical severity. Compared with PIGA (phosphatidylinositol glycan anchor biosynthesis class A) deficiency, PIGO deficiency shows characteristic features, such as Hirschsprung disease, brachytelephalangy, and hyperphosphatasia. This report shows the precise spectrum of symptoms according to the severity of mutations and compares symptoms between different types of IGD.

Elevated Na+/H+ exchanger-1 expression enhances the metastatic collective migration of head and neck squamous cell carcinoma cells.

Cancer cells can migrate as collectives during invasion and/or metastasis; however, the precise molecular mechanisms of this form of migration are less clear compared with single cell migration following epithelial-mesenchymal transition. Elevated Na+/H+ exchanger1 (NHE1) expression has been suggested to have malignant roles in a number of cancer cell lines and in vivo tumor models. Furthermore, a metastatic human head and neck squamous cell carcinoma (HNSCC) cell line (SASL1m) that was isolated based on its increased metastatic potential also exhibited higher NHE1 expression than its parental line SAS. Time-lapse video recordings indicated that both cell lines migrate as collectives, although with different features, e.g., SASL1m was much more active and changed the direction of migration more frequently than SAS cells, whereas locomotive activities were comparable. SASL1m cells also exhibited higher invasive activity than SAS in Matrigel invasion assays. shRNA-mediated NHE1 knockdown in SASL1m led to reduced locomotive and invasive activities, suggesting a critical role for NHE1 in the collective migration of SASL1m cells. SASL1m cells also exhibited a higher metastatic rate than SAS cells in a mouse lymph node metastasis model, while NHE1 knockdown suppressed in vivo SASL1m metastasis. Finally, elevated NHE1 expression was observed in human HNSCC tissue, and Cariporide, a specific NHE1 inhibitor, reduced the invasive activity of SASL1m cells, implying NHE1 could be a target for anti-invasion/metastasis therapy.

Clinical, biochemical and metabolic characterisation of a mild form of human short-chain enoyl-CoA hydratase deficiency: significance of increased N-acetyl-S-(2-carboxypropyl)cysteine excretion.

BACKGROUND: Short-chain enoyl-CoA hydratase-ECHS1-catalyses many metabolic pathways, including mitochondrial short-chain fatty acid ß-oxidation and branched-chain amino acid catabolic pathways; however, the metabolic products essential for the diagnosis of ECHS1 deficiency have not yet been determined. The objective of this report is to characterise ECHS1 and a mild form of its deficiency biochemically, and to determine the candidate metabolic product that can be efficiently used for neonatal diagnosis. METHODS: We conducted a detailed clinical, molecular genetics, biochemical and metabolic analysis of sibling patients with ECHS1 deficiency. Moreover, we purified human ECHS1, and determined the substrate specificity of ECHS1 for five substrates via different metabolic pathways. RESULTS: Human ECHS1 catalyses the hydration of five substrates via different metabolic pathways, with the highest specificity for crotonyl-CoA and the lowest specificity for tiglyl-CoA. The patients had relatively high (∼7%) residual ECHS1 enzyme activity for crotonyl-CoA and methacrylyl-CoA caused by the compound heterozygous mutations (c.176A>G, (p.N59S) and c.413C>T, (p.A138V)) with normal mitochondrial complex I-IV activities. Affected patients excrete large amounts of N-acetyl-S-(2-carboxypropyl)cysteine, a metabolite of methacrylyl-CoA. CONCLUSIONS: Laboratory data and clinical features demonstrated that the patients have a mild form of ECHS1 deficiency harbouring defective valine catabolic and ß-oxidation pathways. N-Acetyl-S-(2-carboxypropyl) cysteine level was markedly high in the urine of the patients, and therefore, N-acetyl-S-(2-carboxypropyl)cysteine was regarded as a candidate metabolite for the diagnosis of ECHS1 deficiency. This metabolite is not part of current routine metabolic screening protocols, and its inclusion, therefore, holds immense potential in accurate diagnosis.

Clinical characterization and identification of duplication breakpoints in a Japanese family with Xq28 duplication syndrome including MECP2.

Xq28 duplication syndrome including MECP2 is a neurodevelopmental disorder characterized by axial hypotonia at infancy, severe intellectual disability, developmental delay, mild characteristic facial appearance, epilepsy, regression, and recurrent infections in males. We identified a Japanese family of Xq28 duplications, in which the patients presented with cerebellar ataxia, severe constipation, and small feet, in addition to the common clinical features. The 488-kb duplication spanned from L1CAM to EMD and contained 17 genes, two pseudo genes, and three microRNA-coding genes. FISH and nucleotide sequence analyses demonstrated that the duplication was tandem and in a forward orientation, and the duplication breakpoints were located in AluSc at the EMD side, with a 32-bp deletion, and LTR50 at the L1CAM side, with "tc" and "gc" microhomologies at the duplication breakpoints, respectively. The duplicated segment was completely segregated from the grandmother to the patients. These results suggest that the duplication was generated by fork-stalling and template-switching at the AluSc and LTR50 sites. This is the first report to determine the size and nucleotide sequences of the duplicated segments at Xq28 of three generations of a family and provides the genotype-phenotype correlation of the patients harboring the specific duplicated segment.

The spectrum of ZEB2 mutations causing the Mowat-Wilson syndrome in Japanese populations.

Mowat-Wilson syndrome (MWS) is a multiple congenital anomaly syndrome characterized by moderate or severe intellectual disability, a characteristic facial appearance, microcephaly, epilepsy, agenesis or hypoplasia of the corpus callosum, congenital heart defects, Hirschsprung disease, and urogenital/renal anomalies. It is caused by de novo heterozygous loss of function mutations including nonsense mutations, frameshift mutations, and deletions in ZEB2 at 2q22. ZEB2 encodes the zinc finger E-box binding homeobox 2 protein consisting of 1,214 amino acids. Herein, we report 13 nonsense and 27 frameshift mutations from 40 newly identified MWS patients in Japan. Although the clinical findings of all the Japanese MWS patients with nonsense and frameshift mutations were quite similar to the previous review reports of MWS caused by nonsense mutations, frameshift mutations and deletions of ZEB2, the frequencies of microcephaly, Hirschsprung disease, and urogenital/renal anomalies were small. Patients harbored mutations spanning the region between the amino acids 55 and 1,204 in wild-type ZEB2. There was no obvious genotype-phenotype correlation among the patients. A transfection study demonstrated that the cellular level of the longest form of the mutant ZEB2 protein harboring the p.D1204Rfs*29 mutation was remarkably low. The results showed that the 3'-end frameshift mutation of ZEB2 causes MWS due to ZEB2 instability.

Pathogenicity evaluation of variants of uncertain significance at exon-intron junction by splicing assay in patients with Mowat-Wilson syndrome.

High-throughput sequencing has identified vast numbers of variants in genetic disorders. However, the significance of variants at the exon-intron junction remains controversial. Even though most cases of Mowat-Wilson syndrome (MOWS) are caused by heterozygous loss-of-function variants in ZEB2, the pathogenicity of variants at exon-intron junction is often indeterminable. We identified four intronic variants in 5/173 patients with clinical suspicion for MOWS, and evaluated their pathogenicity by in vitro analyses. The minigene analysis showed that c.73+2T>G caused most of the transcripts skipping exon 2, while c.916+6T>G led to partial skipping of exon 7. No splicing abnormalities were detected in both c.917-21T>C and c.3067+6A>T. The minigene analysis reproduced the splicing observed in the blood cells of the patient with c.73+2T>G. The degree of the exon skipping was concordant with the severity of MOWS; while the patient with c.73+2T>G was typical MOWS, the patient with c.916+6T>G showed milder phenotype which has been seldom reported. Our results demonstrate that mRNA splicing assays using the minigenes are valuable for determining the clinical significance of intronic variants in patients with not only MOWS but also other genetic diseases with splicing aberrations and may explain atypical or milder cases, such as the current patient.

Characterization of a de novo balanced t(4;20)(q33;q12) translocation in a patient with mental retardation.

CHD6 is an ATP-dependent chromatin-remodeling enzyme, which has been implicated as a crucial component for maintaining and regulating chromatin structure. CHD6 belongs to the largest subfamily, subfamily III (CHD6-9), of the chromodomain helicase DNA (CHD-binding protein) family of enzymes (CHD1-9). Here we report on a female patient with a balanced translocation t(4;20)(q33;q12) presenting with severe mental retardation and brachydactyly of the toes. We identified the translocation breakpoint in intron 27 of CHD6 at 20q12, while the 4q33 breakpoint was intergenic. Northern blot analysis demonstrated the CHD6 mRNA in the patient's lymphoblastoid cells was decreased to ∼50% of the control cells. To investigate the cellular mechanism of diseases resulting from decreased CHD subfamily III proteins, we knocked down CHD6 or CHD7 by RNA interference in HeLa cells and analyzed chromosome alignment. The both CHD6- and CHD7-knockdown cells showed increased frequency of misaligned chromosomes on metaphase plates. Moreover, an elevated frequency of aneuploidy, the major cause of miscarriages and mental retardation, was observed in patients with CHD6 and CHD7 haploinsufficiency. These results suggest that CHD6 and CHD7 play important roles in chromatin assembly during mitosis and that mitotic delay and/or impaired cell proliferation may be associated with pathogenesis of the diseases caused by CHD6 or CHD7 mutations.

Clinical and genetic characterization of a patient with SOX5 haploinsufficiency caused by a de novo balanced reciprocal translocation.

Lamb-Shaffer syndrome (OMIM: 616803) is a neurodevelopmental disorder characterized by developmental delay, mild to moderate intellectual disability, speech delay, and mild characteristic facial appearance caused by SOX5 haploinsufficiency on chromosome 12p12.1. There are clinical variabilities among the patients with genomic alterations, such as intragenic deletions, a point mutation, and a chromosomal translocation of t(11;12)(p13;p12.1), in SOX5. We report herein a 5-year-old Japanese male with a de novo balanced reciprocal translocation t(12;20)(p12.1;p12.3) presenting a mild intellectual disability, speech delay, characteristic facial appearance, and autistic features. We determined the translocation breakpoints of the patient to be in intron 4 of SOX5 and the intergenic region in 20p12.3 via FISH and nucleotide sequence analyses. Thus, the present patient has SOX5 haploinsufficiency affecting 2 long forms of SOX5 and is the second reported case of Lamb-Shaffer syndrome caused by a de novo balanced reciprocal translocation. This report confirmed that haploinsufficiency of the 2 long forms of SOX5 presents common clinical features, including mild intellectual disability and autistic features, which could be useful for the clinical diagnosis of Lamb-Shaffer syndrome.

Novel mutation in HPRT1 causing a splicing error with multiple variations.

Lesch-Nyhan disease (LND) is a rare X-linked recessive disorder caused by deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT), encoded by the HPRT1. To date, nearly all types of mutations have been reported in the whole gene; however, duplication mutations are rare. We here report the case of a 9-month-old boy with LND. He showed developmental delay, athetosis, and dystonic posture from early infancy, but no self-injurious behaviors. Hyperuricemia was detected, and his HPRT enzyme activity in erythrocytes was completely deficient. A novel duplication mutation (c.372dupT, c.372_374 TTT > c.372_375 TTTT) was identified in exon 4 of the HPRT1, which causes aberrant splicing. This is the third case of a duplication mutation in the HPRT1 that causes splicing error.

Metastatic low-grade endometrial stromal sarcoma of the sigmoid colon three years after hysterectomy.

A 49-year-old woman, who had undergone hysterectomy for low-grade endometrial stromal sarcoma (ESS) 3 years ago, presented with a 2-wk history of lower abdominal pain. Barium enema and sigmoidoscopy disclosed a polypoid submucosal tumor. Histopathologic features of biopsy specimens from the lesion were similar to those of the resected uterine ESS. Under the diagnosis of metastatic ESS of the sigmoid colon, sigmoidectomy was performed. Microscopic examination demonstrated dense proliferation of spindle cells with little nuclear atypia, which were sometimes arranged in whorled pattern around abundant arterioles. Mitotic count is below 1 in 10 high-power fields. Immunohistochemically, the neoplastic cells were strongly positive for vimentin, estrogen receptor and progesterone receptor but negative for alpha-smooth muscle actin, S-100 protein and CD34. Thus, a final diagnosis of low-grade ESS metastasis to the sigmoid colon was made. Her postoperative course was uneventful and hormonal therapy with progestational agents is entertained.

Clinical variability in a Japanese hereditary lymphedema type I family with an FLT4 mutation.

Hereditary lymphedema type I (Milroy disease) is a rare autosomal dominant disease resulting from mutations of FLT4 encoding the vascular endothelial growth factor receptor-3. Patients develop edema of the legs and feet, resulting in chronic swelling of the lower extremities from the neonatal period. Here we report a Japanese family with 10 affected members of five generations of hereditary lymphedema type I. We identified a previously reported missense mutation of G857R in one allele of FLT4 from three affected individuals of three generations, the mother of whom presented only hemi-lymphedema of the left foot. Thus, the clinical features of hereditary lymphedema type I caused by a FLT4 mutation are heterogeneous and it would be appropriate to consider FLT4 mutations even in a patient with hemi-lymphedema of the foot.

Plasma adiponectin plays an important role in improving insulin resistance with glimepiride in elderly type 2 diabetic subjects.

OBJECTIVE: We investigated the effect of glimepiride, a third-generation sulfonylurea hypoglycemic agent, on insulin resistance in elderly patients with type 2 diabetes, in connection with plasma adiponectin and 8-epi-prostagrandin F2alpha (8-epi-PGF2alpha), an oxidative stress marker. RESEARCH DESIGN AND METHODS: A total of 17 elderly patients with type 2 diabetes received 12 weeks of treatment with glimepiride. Homeostasis assessment model of insulin resistance (HOMA-IR), homeostasis assessment model of beta-cell function, HbA(1c), C-peptide in 24-h pooled urine (urine CPR), and plasma concentrations of 8-epi-PGF2alpha, tumor necrosis factor-alpha (TNF-alpha), plasminogen activator inhibitor type 1, and adiponectin were measured at various times. The metabolic clearance rate of glucose (MCR-g) was also assessed by a hyperinsulinemic-euglycemic clamp. RESULTS: After 8 weeks of glimepiride treatment, significant reductions were observed in HbA(1c) (from 8.4 +/- 1.9 to 6.9 +/- 1.0%), HOMA-IR (from 2.54 +/- 2.25 to 1.69 +/- 0.95%), and plasma TNF-alpha concentrations (from 4.0 +/- 2.0 to 2.6 +/- 2.5 pg/ml). MCR-g was significantly increased from 3.92 +/- 1.09 to 5.73 +/- 1.47 mg. kg(-1). min(-1). Plasma adiponectin increased from 6.61 +/- 3.06 to 10.2 +/- 7.14 micro g/ml. In control subjects, who maintained conventional treatment, no significant changes were observed in any of these markers. CONCLUSIONS: Glimepiride remarkably improved insulin resistance, suggested by a significant reduction in HOMA-IR, an increase in MCR-g, and a reduction in HbA(1c) without changing extrapancreatic beta-cell function and urine CPR. Increased plasma adiponectin and decreased plasma TNF-alpha may underlie the improvement of insulin resistance with glimepiride.

[Occurrence of upper gastrointestinal tract disease after Helicobacter pylori eradication].

Helicobacter pylori infection is recognized to be a pathogen of various gastroduodenal disease. Eradication therapy of H. pylori reduces the recurrence of gastro-duodenal ulcer, improves histological gastritis, and is suggested to act a certain role in protection against gastric carcinogenesis. Although, several studies show uncomfortable results arise after H. pylori infection was cured. These studies suggest that gastro-esophageal reflux disease (GERD) and gastro-duodenal erosion may increase after successful eradication of H. pylori. Recently, adenocarcinoma of the gastric cardia and esophagus increase in incidence. Reflux esophagitis and Barrett's esophagus are recognized as precancerous lesion of esophageal adenocarcinoma. It is uncertain the association of newly occurrence of GERD after H. pylori eradication and increase of esophageal adenocarcinoma. GERD may cause adenocarcinoma development, though long term observations is necessary after H. pylori eradication.

Hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiencies: HPRT1 mutations in new Japanese families and PRPP concentration.

Mutation of hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch-Nyhan syndrome, which is characterized by hyperuricemia, severe motor disability, and self-injurious behavior, or HPRT-related gout with hyperuricemia. Four mutations were detected in two Lesch-Nyhan families and two families with partial deficiency since our last report. A new mutation of G to TT (c.456delGinsTT) resulting in a frameshift (p.Q152Hfs*3) in exon 3 has been identified in one Lesch-Nyhan family. In the other Lesch-Nyhan family, a new point mutation in intron 7 (c.532+5G>T) causing splicing error (exon 7 excluded, p.L163Cfs*4) was detected. In the two partial deficiency cases with hyperuricemia, two missense mutations of p.D20V (c.59A>T) and p.H60R (c.179A>G) were found. An increase of erythrocyte PRPP concentration was observed in the respective phenotypes and seems to be correlated with disease severity.

Clinical and biochemical characterization of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) deficiency that causes Leigh-like disease and ketoacidosis.

3-Hydroxyisobutyryl-CoA hydrolase (HIBCH) deficiency is an autosomal recessive disorder characterized by episodes of ketoacidosis and a Leigh-like basal ganglia disease, without high concentrations of pyruvate and lactate in the cerebrospinal fluid. Only 4 cases of HIBCH deficiency have been reported. However, clinical-biochemical correlation in HIBCH deficiency by determining the detailed residual enzyme activities has not yet been elucidated. Here, we report a case of two Japanese siblings with HIBCH deficiency carrying a new homozygous missense mutation (c.287C > A, [p.A96D]) at the substrate-binding site. A transfection study using HIBCH expression vectors harboring wild type or 4 reported mutations, including the newly identified mutation (p.A96D, p.Y122C, p.G317E, and p.K74Lfs*13), revealed a correlation between residual HIBCH activities and the severity of the disease. All HIBCH mutants, except p.K74Lfs*13, showed residual enzyme activity and only the patient with p.K74Lfs*13 had congenital anomalies. p.G317E showed only low enzyme activity (~ 3%) of that of wild-type HIBCH. Although p.A96D had approximately 7 times higher enzyme activity than p.G317E, patients with p.A96D died during childhood. These findings are essential for clinical management, genetic counseling, and specific meal and concomitant drug considerations as part of the treatment for patients with HIBCH deficiency.

Identification and characterization of splicing variants of PLEKHA5 (Plekha5) during brain development.

PLEKHA5 (pleckstrin homology domain-containing protein family A, member 5) belongs to the PLEKHA family (PLEKHA1-6); however, the properties of this protein remain poorly characterized. We have identified and characterized two forms of PLEKHA5 mRNA. The long form of PLEKHA5 (L-PLEKHA5) contains 32 exons, encodes 1282 amino acids, and is specifically expressed in the brain; the short form of PLEKHA5 (S-PLEKHA5) is generated by alternative splicing of L-PLEKHA5, contains 26 exons, encodes 1116 amino acids, and is ubiquitously expressed. Both forms of the protein contain putative Trp-Trp (WW) and pleckstrin homology (PH) domains and are located mainly in the cytosol. Developmental and age-dependent expression studies in the mouse brain have shown that Plekha5 is the most abundantly expressed protein at E13.5 with S-Plekha5 dominancy. L-Plekha5 levels increased gradually with the decrease in total Plekha5 levels; moreover, L-Plekha5 became the dominant protein at E17.5, maintaining its dominance throughout adulthood. Protein-lipid overlay assays have indicated that the PH domain of PLEKHA5 specifically interacts with PI3P, PI4P, PI5P, and PI(3,5)P2. These results suggest that the S- to L-conversion of PLEKHA5 (Plekha5) may play an important role in brain development through association with specific phosphoinositides.

Molecular analysis of X-linked inborn errors of purine metabolism: HPRT1 and PRPS1 mutations.

Mutations of two enzyme genes, HPRT1 encoding hypoxanthine guanine phosphoribosyltransferase (HPRT) and PRPS1 encoding a catalytic subunit (PRS-I) of phosphoribosylpyrophosphate synthetase, cause X-linked inborn errors of purine metabolism. Analyzing these two genes, we have identified three HPRT1 mutations in Lesch-Nyhan families following our last report. One of them, a new mutation involving the deletion of 4224 bp from intron 4 to intron 5 and the insertion of an unknown 28 bp, has been identified. This mutation resulted in an enzyme polypeptide with six amino acids deleted due to abnormal mRNA skipping exon 5. The other HPRT1 mutations, a single base deletion (548delT, 183fs189X), and a point mutation causing a splicing error (532+1G>A, 163fs165X) were detected first in Japanese patients but have been reported in European families. On the other hand, in the analysis of PRPS1, no mutation was identified in any patient.