Rachaneekorn Tammachote

Characterization of PKD Protein-Positive Exosome-Like Vesicles

Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1+ vesicles surround cilia in Pkhd1(del2/del2) mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene (BBS3). Our proteomic analysis confirmed that cleavage of polycystin-1 and fibrocystin occurs in vivo, in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in fibrocystin. In vitro, these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cells in a rapid and highly specific manner. These data suggest that PKD proteins are shed in membrane particles in the urine, and these particles interact with primary cilia.

Ciliary and centrosomal defects associated with mutation and depletion of the Meckel syndrome genes MKS1 and MKS3

Meckel syndrome (MKS) is a lethal disorder characterized by renal cystic dysplasia, encephalocele, polydactyly and biliary dysgenesis. It is highly genetically heterogeneous with nine different genes implicated in this disorder. MKS is thought to be a ciliopathy because of the range of phenotypes and localization of some of the implicated proteins. However, limited data are available about the phenotypes associated with MKS1 and MKS3, and the published ciliary data are conflicting. Analysis of the wpk rat model of MKS3 revealed functional defects of the connecting cilium in the eye that resulted in lack of formation of the outer segment, whereas infertile wpk males developed spermatids with very short flagella that did not extend beyond the cell body. In wpk renal collecting duct cysts, cilia were generally longer than normal, with additional evidence of cells with multiple primary cilia and centrosome over-duplication. Kidney tissue and cells from MKS1 and MKS3 patients showed defects in centrosome and cilia number, including multi-ciliated respiratory-like epithelia, and longer cilia. Stable shRNA knockdown of Mks1 and Mks3 in IMCD3 cells induced multi-ciliated and multi-centrosomal phenotypes. These studies demonstrate that MKS1 and MKS3 are ciliopathies, with new cilia-related eye and sperm phenotypes defined. MKS1 and MKS3 functions are required for ciliary structure and function, including a role in regulating length and appropriate number through modulating centrosome duplication.

Epitope-Tagged Pkhd1 Tracks the Processing, Secretion, and Localization of Fibrocystin

Mutations in the PKHD1 gene, which encodes fibrocystin, cause autosomal recessive polycystic kidney disease (ARPKD). Unfortunately, the lack of specific antibodies to the mouse protein impairs the study of splicing, post-translational processing, shedding, and temporal and spatial expression of endogenous fibrocystin at the cellular and subcellular level. Here, we report using a knock-in strategy to generate a null Pkhd1 strain and a strain that expresses fibrocystin along with two SV5-Pk epitope tags engineered in-frame into the third exon, immediately C-terminal to the signal-peptide cleavage site in a poorly conserved region. By 6 mo of age, the Pkhd1-null mouse develops massive cystic hepatomegaly and proximal tubule dilation, whereas the mouse with epitope-tagged fibrocystin has histologically normal liver and kidneys at 14 mo. Although Pkhd1 was believed to generate many splice forms, our western analysis resolved fibrocystin as a 500 kD product without other forms in the 15-550 kD range. Western analysis also revealed that exosome-like vesicles (ELVs) secrete the bulk of fibrocystin in its mature cleaved form, and scanning electron microscopy identified that fibrocystin on ELVs attached to cilia. Furthermore, the addition of ELVs with epitope-tagged fibrocystin to wild-type cells showed that label transferred to primary cilia within 5 min. In summary, tagging of the endogenous Pkhd1 gene facilitates the study of the glycosylation, proteolytic cleavage, and shedding of fibrocystin.

A common and two novel GBA mutations in Thai patients with Gaucher disease

Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the glucocerebrosidase (GBA) gene, leading to a deficiency of lysosomal β-glucosidase and accumulation of glycosphingolipids in macrophages. We studied five Thai families with GD (four with GD type 1 and one with GD type 2). Using long-template PCR, PCR using specific primers for the functional gene, direct sequencing of all coding regions of GBA and restriction enzyme digestions, all 10 mutant alleles were successfully identified. The common c.1448T>C (p.L483P or L444P) mutation was identified in 60% of mutant alleles. Of the two patients homozygous for the p.L483P (L444P) mutation, one died from hepatic failure at age 16 years and the other died from sepsis at age 12 years. This p.L483P (L444P) mutation was found in four different haplotypes, suggesting that it was a recurrent mutation, not caused by a founder effect. Two novel mutations, a missense (c.1204T>C, p.Y402H), and a termination codon mutation (c.1609T>C, p.X537A) were found. Studies to determine the molecular pathomechanism of the p.X537A mutation, the first of its kind in this gene, showed that it decreased the amount of protein being expressed and the enzymatic activity, while it was still correctly localized.

Primary hyperoxaluria type 1 and brachydactyly mental retardation syndrome caused by a novel mutation in AGXT and a terminal deletion of chromosome 2

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disorder caused by mutations in the alanine:glyoxylate aminotransferase (AGXT) gene, located on chromosome 2q37. Mutant AGXT leads to excess production and excretion of oxalate, resulting in accumulation of calcium oxalate in the kidney, and progressive loss of renal function. Brachydactyly mental retardation syndrome (BDMR) is an autosomal dominant disorder, caused by haploinsufficiency of histone deacetylase 4 (HDAC4), also on chromosome 2q37. It is characterized by skeletal abnormalities and developmental delay. Here, we report on a girl who had phenotypes of both PH1 and BDMR. PCR‐sequencing of the coding regions of AGXT showed a novel missense mutation, c.32C>G (p.Pro11Arg) inherited from her mother. Functional analyses demonstrated that it reduced the enzymatic activity to 31% of the wild‐type and redirected some percentage of the enzyme away from the peroxisome. Microsatellite and array‐CGH analyses indicated that the proband had a paternal de novo telomeric deletion of chromosome 2q, which included HDAC4. To our knowledge, this is the first report of PH1 and BDMR, with a novel AGXT mutation and a de novo telomeric deletion of chromosome 2q.

Association between ADAM12 polymorphism and knee osteoarthritis in Thai population

BACKGROUND Osteoarthritis (OA), a common degenerative joint disorder in the elderly, is characterized by the destruction of articular cartilage, bony outgrowths at joint margins, and synovitis. The objective of this study was to evaluate whether there is an association between the ADAM12 (rs3740199) polymorphism and susceptibility to knee OA in a Thai population. METHODS Genomic deoxyribonucleic acid (DNA) was isolated from 200 Thai knee OA patients and 200 healthy controls. High resolution melting analysis was used to detect ADAM12 polymorphisms. The melt profile of all DNA samples was generated on the CFX96™ real-time polymerase chain reaction system and analyzed by Precision Melt Analysis™ software. The genotype distributions and allele frequencies of ADAM12 were compared between groups using the StatCalc program. RESULTS The significant associations were shown from the C allele (OR=2.10, 95% CI=1.16-3.79, P=0.008) and the CC genotype (OR=4.28, 95% CI=1.21-15.72, P=0.01) in male knee OA patients. No significant association was observed in female patients. CONCLUSION The rs3740199 in ADAM12 was associated with knee OA susceptibility in Thai male patients, and individuals with the CC genotype carried the highest risk when compared with the GG and GC genotypes. CLINICAL RELEVANCE The rs3740199 polymorphism of the ADAM12 gene can potentially be used to determine genetically high-risk subgroup of knee osteoarthritis and to better understand the pathogenesis of knee osteoarthritis.

VDR and TNFSF11 polymorphisms are associated with osteoporosis in Thai patients

Determining molecular markers for osteoporosis may be valuable for improving the quality of life of affected elderly patients by aiding in early detection and disease management. In the present study, the association between single nucleotide polymorphisms (SNPs) of the vitamin D receptor (VDR) and tumour necrosis factor superfamily number 11 (TNFSF11) genes and the susceptibility of developing osteoporosis was investigated in a Thai female cohort. The study group consisted of 105 Thai postmenopausal patients diagnosed with osteoporosis and 132 healthy Thai postmenopausal female volunteers. DNA extracted from blood samples was used to genotype the VDR and TNFSF11 genes using polymerase chain reaction-restriction fragment length polymorphism and sequencing analysis. For VDR, the frequencies of the genotypes TT, CT and CC for the TaqI SNP (rs731236) were 87.88, 11.36 and 0.76%, respectively, in the control group, while in the osteoporosis cohort were 92.38, 5.71 and 1.91%, respectively. For the FokI SNP (rs2228570), the frequencies of the genotypes CC, CT and TT were 31.06, 55.30 and 13.64%, respectively, in the control group, and in the osteoporosis group were 29.52, 43.81 and 26.67%, respectively. For BsmI SNP (rs1544410), the frequencies of the genotypes GG, GA and AA were 78.03, 18.94 and 3.03%, respectively, in control group, and in the osteoporosis group were 80.95, 18.10 and 0.95%, respectively. The significant risk of osteoporosis associated with the FokI SNP was determined. The odds ratio (95% confidence interval) was 2.30 (1.14-4.69; P=0.01) among patients with osteoporosis with TT as the susceptibility genotype. For TNFSF11, the frequencies of the genotypes TT, CT and CC for the -290C>T SNP (rs9525641) in the control group were 36.36, 50.76 and 12.88%, respectively, while in the osteoporosis group were 31.43, 56.19 and 12.38%, respectively. For the -643C>T SNP (rs9533156), the frequencies of the genotypes TT, CT and CC in the control group were 35.61, 48.48 and 15.91%, respectively, while in the osteoporosis group were 32.38, 55.24 and 12.38%, respectively. For the -693G>C SNP (rs9533155), the frequencies of the genotypes CC, CG, and GG in the control group were 39.39, 46.97 and 13.64%, respectively, and in the osteoporosis group were 36.19, 53.33 and 10.48%, respectively. No significant associations of the TNFSF11 SNPs with osteoporosis were determined; however, it was notable that the GCT haplotype of TNFSF11 may be a protective haplotype for osteoporosis. Therefore, it was concluded that the SNP FokI of VDR may be a potential molecular biomarker for the development of osteoporosis in Thai females.