Abstract. Permanent isolated proximal renal tubular acidosis (pRTA) with ocular abnormalities is a systemic disease involving short stature, isolated pRTA, mental retardation, and ocular abnormalities. Kidney Na + /HCO 3 - cotransporter (kNBC1) cDNA from peripheral lymphocytes from a patient with permanent isolated pRTA and bilateral glaucoma was screened, and a novel homozygous mutation, namely a cytosine-to-thymine transition at nucleotide 234, which resulted in the formation of a stop codon at codon 29, was identified. This homozygous mutation, Q29X, was identified in the unique 5′-end of the kNBC1 gene ( SLC4A4 ) of the patient. Cosegregation of this Q29X mutation with the disease and heterozygosity in the parents of the affected patient were observed. The absence of this mutation in 156 alleles from 78 Japanese individuals indicates that this mutation is directly related to the disease and is not a common DNA sequence polymorphism. This nonsense mutation predicts a truncated kNBC1 protein that lacks the 1007 amino acids of the carboxyl-terminus, and the effect on kNBC1 cotransport activity is likely to be a loss of function. In contrast, the pancreatic Na + /HCO 3 - cotransporter of the patient is not likely to be affected by this nonsense mutation. These results have implications for understanding the role of kNBC1 in the pathophysiologic processes of pRTA associated with ocular abnormalities and mental retardation.
Dent disease is an X-linked disorder characterized by low-molecular-weight (LMW) proteinuria, hypercalciuria, nephrocalcinosis, urolithiasis and renal dysfunction. Dent disease is caused by mutations in at least two genes, i.e. CLCN5 and OCRL1, and its genetic background and phenotypes are common among European countries and the USA. However, only few studies on Dent disease in Japan, which was originally called 'low-molecular-weight proteinuric disease', have been reported thus far. In this study, we analysed genetic background and clinical phenotype and laboratory data of 86 unrelated Japanese Dent disease patients. The results demonstrated that the genetic basis of Japanese Dent disease was nearly identical to those of Dent disease in other countries. Of 86 unrelated Japanese Dent patients, 61 possessed mutations in CLCN5 (Dent-1), of which 27 were novel mutations; 11 showed mutations in OCRL1 (Dent-2), six of which were novel, and the remaining 14 patients showed no mutations in CLCN5 or OCRL1 (Dent-NI). Despite the similarity in genetic background, hypercalciuria was detected in only 51%, rickets in 2% and nephrocalcinosis in 35%. Although the patients were relatively young, six patients (8%) showed apparent renal dysfunction. Japanese Dent disease has a wider clinical spectrum than Dent disease in Europe and the USA.
The X-linked form of hyper-IgM syndrome (HIGM1) is a rare disorder characterized by the inability of B cells to undergo isotype switch by a deficiency of CD40 ligand (CD40L) on activated T lymphocytes. The patients suffer from recurrent infections not only due to a lack of B lymphocyte activation but also due to defect of T lymphocyte functions. In addition, neutropenia is frequently accompanied by these symptoms. A patient with HIGM1, we experienced, suffered from recurrent infections and neutropenia. But he had a normal number of hematopoietic stem cell by the in vitro colony forming assay. CD34+ myeloid stem cell has been known to express CD40. We speculated by these facts that myeloid cell numbers are regulated by CD40-CD40L interaction.
Homozygous mutations in SLC4A4 , encoding the electrogenic Na + -HCO 3 − cotransporter NBCe1, have been known to cause proximal renal tubular acidosis (pRTA) and ocular abnormalities. In this study, we report two sisters with pRTA, ocular abnormalities, and hemiplegic migraine. Genetic analysis ruled out pathological mutations in the known genes for familial hemiplegic migraine, but identified a homozygous 65-bp deletion (Δ65bp) in the C terminus of NBCe1, corresponding to the codon change S982NfsX4. Several heterozygous members of this family also presented glaucoma and migraine with or without aura. Despite the normal electrogenic activity in Xenopus oocytes, the Δ65bp mutant showed almost no transport activity due to a predominant cytosolic retention in mammalian cells. Furthermore, coexpression experiments uncovered a dominant negative effect of the mutant through hetero-oligomer formation with wild-type NBCe1. Among other pRTA pedigrees with different NBCe1 mutations, we identified four additional homozygous patients with migraine. The immunohistological and functional analyses of these mutants demonstrate that the near total loss of NBCe1 activity in astrocytes can cause migraine potentially through dysregulation of synaptic pH.
Although it is assumed that most patients with autosomal dominant dopa-responsive dystonia (DRD) have a GTP cyclohydrolase I dysfunction, conventional genomic DNA sequencing of the gene (GCH1) coding for this enzyme fails to reveal any mutations in about 40% of DRD patients, which makes molecular genetic diagnosis difficult. We found a large heterozygous GCH1 deletion, which cannot be detected by the usual genomic DNA sequence analysis, in a three-generation DRD family and conclude that a large genomic deletion in GCH1 may account for some "mutation-negative" patients with dominantly inherited DRD.
Eight multi-drug resistant mutants (4.94%) were found in 162 clinical isolates of P. aeruginosa after exposure to norfloxacin at different concentrations (1/4, 1/2, 1, 2 and 4 MICs) and were investigated for the mechanisms of drug resistance.All the mutants were eight-times more resistant to norfloxacin than the respective parents, and showed the cross-resistance to the other fluoroquinolones. However, these mutants differed in the drug-resistant patterns; the 94-74 mutant was resistant to carbenicillin, ceftazidime and chloramphenicol, TA-15 mutant was resistant to imipenem, and the 93-183 was resistant to carbenicillin, ceftazidime and gentamicin.TA-16 mutant only showed a marked decrease in the bacterial uptake of norfloxacin. Profiles of the outer membrane proteins of the mutants were analyzed by SDS-PAGE method. The six mutants, except for TA-52 and 93-183 mutants, increased the intensity of bands in the 46 KD region. Three mutants (TA-15, TA-16 and 93-183) decreased the intensity of 44 KD (OMPE) and also the former two decreased the intensity of 22 KD (OMP G). The gryA mutations associated with fluoroquinolone-resistance were investigated for the eight mutants, and the 93-183 mutant showed to have the gyrA mutation caused by the alteration in the amino acid sequence of gyrA; Thr-83 (ACC) to Ile (ATC).The result of the present study indicate that multi-drug resistance of clinical isolates of P. aeruginosa develops through different types of mechanisms, and is not easily explained fully by the present results, and suggest that many factors attributed to the development of the resistances in those mutants remains to be clarified.
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