Distal renal tubular acidosis caused by tryptophan-aspartate repeat domain 72 (WDR72) mutations.

Hereditary distal renal tubular acidosis (dRTA) is a rare genetic disease that is caused by mutations in SLC4A1, ATP6V1B1, or ATP6V0A4. However, there are many families with hereditary dRTA in whom the disease-causing genes are unknown. Accordingly, we performed whole exome sequencing and genetic studies of the members of a family with autosomal recessive dRTA of an unknown genetic etiology. Here, we report compound heterozygous pathogenic variations in tryptophan-aspartate repeat domain 72 (WDR72) (c.1777A>G [p.R593G] and c.2522T>A [p.L841Q]) in three affected siblings of a family with dRTA. Both variants segregated with dRTA in the family and were not observed in normal control subjects. Homologous modeling and in silico mutagenesis indicated that R593G and L841Q alter the H-bond formations in the nearby residues, affecting the WDR72 protein structure. All these evidences indicate that the identified WDR72 variations were probably to have caused hereditary dRTA in the reported family. In addition, homozygous nonsense mutation (c.2686C>T [p.R896X]) was identified in another family, strongly supporting the causal role of WDR72 in dRTA. Based on our literature review, WDR72 mutations associated with dRTA have not been previously described. This is the first identification of pathogenic variations in WDR72 as a cause of hereditary dRTA.

Human monoclonal single-chain antibodies specific to dengue virus envelope protein.

UNLABELLED: Dengue virus (DENV) infection is an arthropod-borne disease with increasing prevalence worldwide. Attempts have been made to develop therapeutic molecules for treatment for DENV infection. However, most of potentially therapeutic DENV monoclonal antibody was originated from mouse, which could cause undesirable effects in human recipients. Thus, fully human antibody is preferable for therapeutic development. Human single-chain variable fragments (HuScFv) with inhibitory effect to DENV infection were generated in this study. HuScFv molecules were screened and selected from the human antibody phage display library by using purified recombinant DENV full-length envelope (FL-E) and its domain III (EDIII) proteins as target antigens for biopanning. HuScFv molecules were then tested for their bindings to DENV particles by indirect ELISA and immunofluorescent microscopy. EDIII-specific HuScFv exhibited neutralizing effect to DENV infection in Vero cells in a dose-dependent manner as determined by plaque formation and cell ELISA. Epitope mapping and molecular docking results concordantly revealed interaction of HuScFv to functional loop structure in EDIII of the DENV E protein. The neutralizing HuScFv molecule warrants further development as a therapeutic biomolecule for DENV infection. SIGNIFICANCE AND IMPACT OF THE STUDY: No approved vaccine and specific drug for dengue virus (DENV) infection are available; thus, their developments are urgently required. The human single-chain variable antibody fragments (HuScFv) specific to DENV envelope (E) protein are potential to be developed as therapeutic biomolecules. HuScFv that bound specifically to recombinant full-length DENV E (FL-E) and its domain III (EDIII) were generated and testified for its inhibitory effect in DENV infection. EDIII-specific HuScFv inhibited DENV infection in a dose-dependent manner and has potential to be further developed as a therapeutic biomolecule for DENV infection.

A novel splice-acceptor site mutation (IVS13-2A>T) of polycystic kidney disease 1 (PKD1) gene resulting in an RNA processing defect with a 74-nucleotide deletion in exon 14 of the mRNA transcript.

Autosomal dominant polycystic kidney disease (ADPKD) occurs mainly from mutations of polycystic kidney disease 1 (PKD1) gene. A novel mutation of the PKD1 gene due to a nucleotide substitution in splice-acceptor site of IVS13 (AG->TG) was identified by analyses of PKD1-cDNA and genomic DNA. The IVS13-2A>T substitution resulted in an inactivation of this splice site and utilization of cryptic splice acceptor site in exon 14, causing a 74-nucleotide deletion of this exon in the PKD1-mRNA transcript. The abnormal transcript was present ectopically in the patients' lymphocytes. The partial deletion of PKD1-mRNA leads to frameshift translation and introduces a termination signal at codon 1075. The truncated protein with about one quarter of the full-length polycystin-1 is most likely inactive. Thus, the effect of this mutation would be "loss-of-function" type. Allele specific amplification (ASA) was developed to detect the mutation in DNA samples of other family members. The mutation was present in 11 affected but absent in 13 unaffected family members, corresponding to the results of linkage analysis. In addition, it was not observed in DNA samples of 57 unrelated healthy individuals. Hum Mutat 15:115, 2000.

Frameshift mutations with severe and moderate clinical phenotypes in Thai hemophilia A patients.

Six frameshift mutations in exon 14 of the factor VIII gene were identified in Thai hemophilia A patients. Although all these mutations created premature stop codons and expected to cause severe disease, the molecular defects and clinical severity were in discrepancy in some patients. Four mutations (delT3490, delACAC3618-21, delGA4429-30, and delA4658) were found in the patients with the severe clinical phenotype while two (delA3629-37 and insA4372-9) were observed in the patients who had moderate severity, with FVIII:C of 4.2 and 2.8%. The frameshift mutations in these two patients were due to deletion and insertion of an 'A' nucleotide in the stretches of 9As and 8As in codons 1191-4 and 1439-41, respectively. This indicates that deletion or insertion in the stretches of poly A nucleotides in exon 14 of the factor VIII gene is a likely cause of the moderate clinical severity in some cases of Thai hemophilia A patients.

Mutations of the factor VIII gene in thai hemophilia A patients.

Hemophilia A is a common X-linked bleeding disorder caused by mutations in the coagulation factor VIII gene. The entire coding and essential sequences of the factor VIII gene were generated by a combination of genomic DNA amplification and long reverse transcription-polymerase chain reaction (long RT-PCR) using factor VIII transcripts prepared from lymphocytes. Mutations were then screened by non-radioactive single strand conformation polymorphism (SSCP) analysis and characterized by DNA sequencing. We have identified six potentially pathogenic mutations in the factor VIII gene in Thai hemophilia A patients, including two nonsense mutations (R-5X and R1966X), three missense mutations (D542Y, G1850V, and G2325C), and a 4-bp insertion (ACTA) at codon 2245. Three of these mutations (D542Y, G2325C, and 4-bp insertion) have never been previously reported, and the ins2245 is the first example of such insertion probably causing factor VIII elongation. R1966X, D542Y, G1850V, and 4-bp insertion were associated with a severe hemophiliac phenotype whereas R-5X and G2325C were observed in moderately affected patients. Mutations in the factor VIII gene in Thai hemophilia A patients are likely to be heterogeneous. This study represents the first attempt to further the understanding of the molecular basis of hemophilia A in Thai.

Long RT-PCR Amplification of the entire coding sequence of the polycystic kidney disease 1 (PKD1) gene.

Characterization of mutations of the PKD1 gene has been limited by the fact that three-fourths of this gene at its 5' end is homologous to sequences of at least three other genes on the same chromosome. We have therefore developed a method of long reverse transcription PCR for selective amplification of the entire coding sequence of the PKD1 gene from its mRNA. A PCR primer specific to the sequence in the 3' unique region of the PKD1 gene was synthesized for use coupled with a primer binding to sequence in the homologous region at a distance of about 13.6 kb apart. The commercial availability of RNase H-free reverse transcriptase for long cDNA synthesis and of an enzyme mixture containing Taq and Pfu DNA polymerases for long-range PCR have made this development possible. The long PCR product was proven to be derived from PKD1-mRNA. The results clearly indicated that the long PCR product contained the coding sequence derived from PKD1-mRNA. To our knowledge, this is the first report of a procedure that can reproducibly isolate full-length PKD1 coding sequence from its mRNA transcript, which will prove useful for screening and characterization of mutations in the PKD1 gene.

HLA-DR and -DQ DNA polymorphisms: new linkage relationships established by RFLP genomic typing in Polynesians and Melanesians.

Class II restriction fragment length polymorphisms (RFLPs) of DR beta, DQ beta, and DQ alpha loci were examined in Polynesians of the southwest Pacific and in non-Austronesian-speaking Melanesians from the Papua New Guinean Highlands. Polynesians, previously considered to have a restricted set of HLA-DR antigens, showed class II gene heterogeneity associated with DR2, DR5, DRw6, and DRw8 RFLPs. Furthermore, Melanesians and Polynesians share certain antigens such as DRw6 and DRw8, but the DR beta 2 genes associated with DRw6 and the DQ genes associated with DRw8 are population-specific and show little or no overlap. This study has shown that genetic analysis of closely linked polymorphic genes is a powerful anthropological tool and supports the view that Polynesians represent an independent colonizing group in the Pacific, rather than a group evolved from within Melanesia.

Distal renal tubular acidosis and high urine carbon dioxide tension in a patient with southeast Asian ovalocytosis.

Southeast Asian ovalocytosis (SAO) is the best-documented disease in which mutation in the anion exchanger-1 (AE1) causes decreased anion (chloride [Cl-]/bicarbonate [HCO3-]) transport. Because AE1 is also found in the basolateral membrane of type A intercalated cells of the kidney, distal renal tubular acidosis (dRTA) might develop if the function of AE1 is critical for the net excretion of acid. Studies were performed in a 33-year-old woman with SAO who presented with proximal muscle weakness, hypokalemia (potassium, 2.7 mmol/L), a normal anion gap type of metabolic acidosis (venous plasma pH, 7. 32; bicarbonate, 17 mmol/L; anion gap, 11 mEq/L), and a low rate of ammonium (NH4+) excretion in the face of metabolic acidosis (26 micromol/min). However, the capacity to produce NH4+ did not appear to be low because during a furosemide-induced diuresis, NH4+ excretion increased almost threefold to a near-normal value (75 micromol/L/min). Nevertheless, her minimum urine pH (6.3) did not decrease appreciably with this diuresis. The basis of the renal acidification defect was most likely a low distal H+ secretion rate, the result of an alkalinized type A intercalated cell in the distal nephron. Unexpectedly, when her urine pH increased to 7.7 after sodium bicarbonate administration, her urine minus blood carbon dioxide tension difference (U-B Pco2) was 27 mm Hg. We speculate that the increase in U-B Pco2 might arise from a misdirection of AE1 to the apical membrane of type A intercalated cells.

Increased erythrocyte superoxide dismutase activities in beta 0-thalassaemia/haemoglobin E and in haemoglobin H diseases.

Erythrocyte superoxide dismutase activities were measured in 45 subjects, 15 each of beta 0-thalassaemia/haemoglobin (Hb) E disease, Hb H disease, and normal. The erythrocyte superoxide dismutase activities were significantly higher in the patients with beta 0-thalassaemia/Hb E and Hb H diseases than in the normal subjects. The increase of erythrocyte superoxide dismutase activities is most likely due to abnormalities specific to thalassaemic red cells rather than an increased number of younger red cells for reticulocytes and nucleated red blood cells did not affect the enzyme activity. Patients with beta 0-thalassaemia/Hb E disease with lower haemoglobin concentration had significantly higher superoxide dismutase activities. In all 45 subjects haemoglobin concentrations and superoxide dismutase activities were inversely correlated (r = -0.60 (p less than 0.001)). This indicates that the amounts of superoxide generated in the red cells may, at least partly, determine severity of red cell damage and thus severity of disease; the increased superoxide dismutase activity in thalassaemia is a response to superoxide generated in greater amounts because of accumulation of excessive globin chains and iron in the red cells. The superoxide dismutase activities in Hb H disease, an alpha-thalassaemic disease, were found to be strikingly increased, higher than in beta 0-thalassaemic disease or other conditions.

Heterogeneity of beta thalassaemia in Thailand.

Beta thalassaemia in Thailand is heterogeneous. Clinical, genetical, haematological and globin chain biosynthetic studies were performed in seven beta-thalassaemia families. The results showed different gene combinations. These were alpha-thalassaemia/homozygous beta 0-thalassaemia, questionable double heterozygosity between a beta-thalassaemia and a silent beta-thalassaemia genes with low Hb F of unexplained cause, silent beta-thalassaemia/beta+ - or beta 0-thalassaemia, high Hb A2 high Hb F-beta-thalassaemia - a new mutant, mild beta+-thalassaemia/Hb E, and beta 0-thalassaemia/heterocellular HPFH or delta beta-thalassaemia associated with alpha-thalassaemia. Most of variability of clinical and haematological findings in these families is due to heterogeneity of the beta-thalassaemia and related genes.

Detection and typing of human papilloma virus DNAs in normal cervix, intraepithelial neoplasia and cervical cancer in Bangkok.

We detected and typed HPV-DNA by polymerase chain reaction (PCR) in cervico-vaginal lavages of 102 women with normal cervical cytology, 57 patients with cervical intraepithelial neoplasia (CIN), and 23 cervical cancer patients. HPV-DNA detection and typing by in situ hybridization were also performed in cervical biopsies from CIN lesions and cancers. Five percent of women with normal cervical cytology, 46% of CIN, and 61% of cervical cancer were positive for HPV-DNA. Of CIN cases with positive HPV-DNA, 69, 15, 8, 4 and 4% were HPV-16, -33, -18, -11 and -16/33 respectively. Of cervical cancer cases with positive HPV-DNA, 86% were HPV-16, 7% were HPV-16/33, 7% were HPV-18/31. HPV typing was performed in biopsies from 37 CIN and 18 cervical cancers by in situ hybridization. By this method, 38% of CIN were HPV-DNA positive, of which 71% were HPV-16 and 7% were each of HPV-11, -18, -31 and -33. Thirty-nine percent of cervical cancers were positive, of which 71% and 29% were HPV-16 and HPV-16/18 respectively.

Rapid detection and identification of dengue viruses by polymerase chain reaction (PCR).

A polymerase chain reaction (PCR) method using sets of newly designed primers for rapid detection and simultaneous identification of dengue virus serotypes was developed and tested. The test is based on two sets of primers specific within the envelope (E) and non-structural (NS1) regions of the dengue-virus genome. Two sets of universal primers that bind to two target sequences which are shared by all the four serotypes of the virus within the E and NS1 regions are used. The resulting products are further amplified by another pair of inner or nested universal primers, which also bind to another set of shared sequences within the E and NS1 regions, respectively. The nested PCR of both the E and NS1 regions can detect dengue virus of all the four serotypes at a sensitivity of 1 plaque forming unit (pfu) or less. For the identification of serotypes, a mixture of four pairs of serotype-specific primers, specific to the E region, was used. The primers have been designed to bind to serotype specific sequences within the regions flanked by the outer universal primers, and giving the amplified products of different sizes, each corresponds to one particular serotype (405 bp for Den1, 346 bp for Den2, 196 bp for Den3, and 143 bp for Den4). A protocol has been developed and successfully applied to detect dengue virus in cell-culture supernatants and patients sera. The technique is simple and rapid, capable of not only detecting the dengue virus but also identifying the serotypes of the virus in clinical specimens.

Molecular defect of PKD1 gene resulting in abnormal RNA processing in a Thai family.

Autosomal dominant polycystic kidney disease (ADPKD) is a common human autosomal disorder caused mainly by mutations of the PKD1 gene. In analysis of PKD1 transcripts by long RT-PCR and nested PCR procedures, we observed PKD1-cDNA fragments from three ADPKD siblings from the same family with a size approximately 250 base pairs (bp) shorter than normal. Further investigations showed that the PKD1 transcripts from these patients had been abnormally processed, the nucleotide sequence of exon 43 containing 291 nt was missing from the transcripts, which would result in an abnormal polycystin-1 with an in-frame deletion of 97 amino acids. This splicing defect did not result from a mutation that disrupted the splice donor or acceptor sites adjacent to exon 43 or the branch sites in flanking introns but was most likely due to 20-bp deletion observed in intron 43. The intronic deletion was present in 8 affected members but absent in 11 unaffected members, corresponding with the results of genetic linkage analysis using 5 polymorphic markers in the PKD1 region. Molecular diagnosis of PKD1 in this family could, therefore, be carried out by genomic DNA amplification to directly detect the PKD1 intronic deletion.

Tropical distal renal tubular acidosis: clinical and epidemiological studies in 78 patients.

BACKGROUND: Distal renal tubular acidosis (dRTA) caused by mutations of the SLC4A1 gene encoding the erythroid and kidney isoforms of anion exchanger 1 (AE1 or band 3) has a high prevalence in some tropical countries, particularly Thailand, Malaysia, the Philippines and Papua New Guinea (PNG). Here the disease is almost invariably recessive and can result from either homozygous or compound heterozygous SLC4A1 mutations. METHODS: We have collected and reviewed our own and published data on tropical dRTA to provide a comprehensive series of clinical and epidemiological studies in 78 patients. RESULTS: Eight responsible SLC4A1 mutations have been described so far, four of them affecting multiple unrelated families. With the exception of the mutation causing South-East Asian ovalocytosis (SAO), none of these mutations has been reported outside the tropics, where dRTA caused by SLC4A1 mutations is much rarer and almost always dominant, resulting from mutations that are quite different from those found in the tropics. SLC4A1 mutations, including those causing dRTA, may cause morphological red cell changes, often with excess haemolysis. In dRTA, these red cell changes are usually clinically recessive and not present in heterozygotes. The high tropical prevalence of dRTA caused by SLC4A1 mutations is currently unexplained. CONCLUSION: A hypothesis suggesting that changes in red cell metabolism caused by these mutations might protect against malaria is put forward to explain the phenomenon, and a possible mechanism for this effect is proposed.

Alpha zero- and beta zero-thalassemia in a Thai family: unusually mild homozygous beta zero-thalassemia without alpha-globin gene deletion.

Alpha-globin genes were analyzed by the direct method of DNA mapping using alpha- and zeta-globin specific probes in a Thai family in which the proposita was an unusually mild beta zero-thalassemia homozygote. alpha zero-Thalassemia was found to be segregating in the family, inherited from the proposita's father by one of her younger sisters. However, alpha zero-thalassemia was not detected by this DNA mapping in the proposita. The mild homozygous beta zero-thalassemia in this family may result from interactions of a non-deletion alpha-thalassemia, a gene responsible for high proteolytic activity permitting more balanced globin-chain levels, or from an unusually active hemoglobin F production in the proposita.

Genetic distance analysis using DNA polymorphisms in the alpha-globin gene cluster.

Published frequencies for alleles at the alpha- and zeta-globin gene loci within the alpha-globin gene cluster were used to calculate genetic distances between populations of South East Asia and the Pacific region. When analyses using the alpha-globin alleles, populations are grouped according to the endemicity of malaria while the groupings derived from the embryonic zeta-globin allele frequencies appear to be far less influenced by the incidence of malaria and probably reflect ancestral or chance relationships due to founder effects, migration and small population sizes.

Identification of a new mutation (Gly420Ser), distal to the active site, that leads to factor XIII deficiency.

The molecular defects of the factor XIII A subunit gene were studied in a patient with factor XIII deficiency. Mutation analysis was performed on amplified DNA from each exon of this gene by single-strand conformation polymorphism (SSCP) and DNA sequencing techniques. A substitution of guanine by adenine at nucleotide 1258 in exon 10 of the coagulation factor XIII A subunit gene has been identified in the patient. The mutation results in the replacement of Gly420 by Ser in the core domain of the enzyme. Restriction enzyme analysis of amplified exon 10 DNA confirmed that the patient was homozygous for this mutation. A family study revealed that the mutation was inherited from both parents, who were first cousins. The potential effects of the mutation were predicted by molecular modeling of the amino acid substitution within the coordinates of the crystal structure. The substitution occurred within the core domain of the enzyme at a residue completely conserved among all known members of the transglutaminase family. The model of the mutant protein suggests that although the substitution of Gly420 by Ser causes only minor readjustment of the residues and does not appear to be particularly deleterious in terms of structure, the mutation is, however, likely to decrease the molecule's ability to undergo the conformational change that is thought to be required for full transglutaminase activity. Our data strongly support the previously published information about the functional significance of the residues surrounding, but not forming, the catalytic pocket in the A subunit of factor XIII.

Detection of beta-thalassemia and hemoglobin E genes in Thai by a DNA amplification technique.

Enzymatic DNA amplification and polyacrylamide gel electrophoresis, which demonstrate different sizes of DNA fragments, were used to detect the common mutations causing beta-thalassemia and hemoglobin (Hb) E in Thai people. The 4-bp deletion at codons 41 and 42 can be detected directly by polyacrylamide gel electrophoresis and ethidium bromide staining. Whereas the nonsense mutations at codon 17 (AAG----TAG) and Hb E (GAG----AAG at codon 26) were detected after digestion of the amplified DNA with the enzymes MaeI and MnlI, respectively.

Extremely high frequencies of alpha-globin gene deletion in Madang and on Kar Kar Island, Papua New Guinea.

Extremely high frequencies of the deletion form of alpha(+)-thalassemia (-alpha/), as studied by the DNA mapping technique, were found in the population of Madang, a coastal province in the north of Papua New Guinea (PNG) and in the population of Kar Kar, an island situated near Madang. Ninety-seven percent of the population tested from Madang and 89% of that from Kar Kar Island were either alpha(+)-thalassemia heterozygotes or homozygotes. By contrast, no examples of the deletion form were detected in the Eastern Highlands of PNG. The haplotype frequencies of alpha(+)-thalassemia (-alpha/) in Madang and Kar Kar Island were found to be 81.33% and 66.67%, respectively. A more detailed analysis of the gene deletion revealed that in both populations 96% were of the 4.2 kilobase (kb) type and 4% were of the 3.7-kb type. Thus, this group is the only example in which the 4.2-kb deletion is predominant over 3.7-kb defect. The presence in high frequencies of alpha(+)-thalassemia in the coastal area of Madang and on the neighboring island, where malaria has long been holoendemic or hyperendemic, and its virtual absence from the nonmalarious highlands of PNG suggest the role of malaria as the selective factor in maintaining alpha(+)-thalassemia. If this selective pressure is still operating, and since alpha(+)-thalassemia has no apparent homozygous disadvantage, the abnormal haplotype (-alpha/) will be in the process of fixation in this population.