High-resolution DNA Melting Analysis Research Articles

DNA extraction optimization and authentication of Vaccinium berries and their products by high-resolution DNA melting analysis

Food adulteration has been an issue in the food industry for many decades. The market for natural and healthy products has been expanding and therefore the value of raw ingredients harvested from natural sources has increased, and, in some cases, it has led to adulteration of such high-valued products. Berry fruits and products from different species of the genus Vaccinium have been gaining worldwide popularity because of their nutraceutical benefits. However, there are increasing reports of adulteration or misidentification of high-value wild berries with their cultivated counterparts. During recent years, DNA-based methods for the authentication of commercial products have gained popularity, especially regarding species identification. Based on the availability of their chloroplast genome information, we developed DNA markers to differentiate lingonberry from cranberry as well as to discriminate European bilberry from North American blueberry. By using Bar-HRM analysis (DNA barcoding combined with High Resolution Melting), the markers were tested on plant DNA as well as DNA extracted from diverse berry products. The developed markers were able to discriminate between species and, moreover, detect potential adulteration in products made from these berry species. Subsequently, the Bar-HRM result was verified by amplicon sequencing. To conclude, the developed molecular markers represent a valuable tool for the berry food industry.

A method for quick and efficient identification of cichlid species by high resolution DNA melting analysis of minibarcodes

Freshwater bodies are key in supporting aquatic and terrestrial life. Ecological balance of freshwater habitats is very vulnerable, hence, often significantly disrupted by climatic changes and anthropogenic acts. In Israel, due to its relatively arid climate, many freshwater resources have been disrupted and still are under great pressure. The Sea of Galilee is the largest surface freshwater body in the Middle East and a habitat to unique populations of several fishes, including six cichlid species. Studies on the ecology of these fish and their conservation require effective monitoring tools. In this study, a simple and efficient molecular method was developed to identify the species of these lake cichlids using high resolution melting analysis of mini DNA barcodes. The species of an individual sample can be identified by a single tube PCR reaction. This assay successfully identified sequence differences both among and within species. Here, this method identified the species for 279 small cichlid fry that could not be morphologically identified, allowing to estimate relative species abundance and map their distribution in time and location. The results are key to understand not only the ecology of young stages but also their recruitment potential to adult fish populations and their sustainability. This method can be readily implemented in further ecological studies and surveys related to these species, in the lake and its surroundings, as a tool to enhance understanding and protection of these species.

High-Resolution Melting (HRM) for rapid MLST analysis of Neisseria meningitidis

The genetic characterization of meningococcal isolates is extremely important for the epidemiological monitoring of meningococcal disease, through the identification of circulating epidemic clones, with the purpose of supporting specific actions of Health Surveillance to contain outbreaks. The objective of this work is to determine a strategy for the epidemiological control of Neisseria meningitidis (Nm) through the detection of genetic signatures of Multilocus Sequence Typing (MLST) genes, by the method of high-resolution DNA melting analysis (qPCR-HRM), to identify the main hypervirulent clones circulating in the country. We analyzed 65 cc103 strains, 19 cc11, 38 cc32 and 8 cc41/44 and 17 were not associated to a specific cc. For the abcZ gene a total of 112 strains were tested, 79 for adk and gdh genes, 87 for aroE, 27 for fumC and 70 strains for pdhC gene. The results obtained were compared and validated with nucleotide sequencing. The percentage of correct allele detection for each clonal complex ranged between 77% and 100%. After an active search in PubMLST, it was found that by inserting results from at least 4 alleles in the MLST database, it is possible to determine the clonal complex of 99% to 100% of the deposited samples. The results obtained in this study suggest that it is possible to identify Nm clonal complexes by a combination analysis of melting curves (TM) of four constitutional genes included in the MLST scheme by qPCR-HRM.

Decreased promoter methylation of E-cadherin gene due to overexpression of TET enzyme by vitamin C in MCF-7 cell line

BackgroundE-cadherin (CDH1) is a well-known tumor suppressor gene. The loss of CDH1 gene function caused by genetic or epigenetic alterations may consequently result in tumorigenesis. Vitamin C is an essential factor for epigenetic regulation and its great furnished potential in epigenetic therapy in cancer patients. ObjectiveThe present study aims to evaluate the effect of vitamin C on promoter methylation of the CDH1 gene and its possible correlation with the expression of CDH1 in the MCF-7 cell line. MethodsCDH1 expression was studied by reverse transcription-polymerase chain reaction (RT–PCR) analysis in MCF-7 cell line that was treated with different concentrations of vitamin C. TET enzyme activity was measured by ELISA. Promoter methylation of CDH1 was studied by Methylation-Sensitive High-Resolution Melting (MS-HRM) analysis of bisulfite-modified DNA. ResultsOur results showed that vitamin C increase not only TET enzyme activity but also TET gene overexpressed. In the following, cause to CDH1 gene promoter demethylation and upregulation of CDH1 gene in MCF-7 cell line. ConclusionCancer cells may respond to vitamin C induces CDH1 expression by promoter demethylation due to TET enzyme overexpression.

Development of High-Resolution DNA Melting Analysis for Simultaneous Detection of Potato Mop-Top Virus and Its Vector, Spongospora subterranea, in Soil.

In this study, a set of duplex reverse transcription PCR (RT-PCR)-mediated high-resolution DNA melting (HRM) analyses for simultaneous detection of potato mop-virus (PMTV) and its protist vector, Spongospora subterranea f. sp. subterranea (Sss), was developed. The infestation of soil by PMTV was detected with a tobacco-based baiting system. Total RNA extracted from the soil led to successful RT-PCR gel electrophoresis detection of both PMTV and Sss. To facilitate more efficient detection, newly designed primer pairs for PMTV RNA species (i.e., RNA-Rep, RNA-CP, and RNA-TGB) were analyzed together with the existing Sss primers via real-time RT-PCR. The resulting amplicons exhibited melting profiles that could be readily differentiated. Under duplex RT-PCR format, all PMTV and Sss primer combinations led to successful detection of respective PMTV RNA species and Sss in the samples by HRM analyses. When the duplex HRM assay was applied to soil samples collected from six fields at four different sites in New Brunswick, Canada, positive detection of PMTV or Sss was found in 63 to 100% samples collected from fields in which PMTV-infected tubers had been observed. In contrast, the samples from fields where neither PMTV- nor Sss-infected tubers had been observed resulted in negative detection by the assay. Bait tobacco bioassay for PMTV and Sss produced similar results. Of the soil samples collected from PMTV-infested fields, 63 to 83% and 100% led to PMTV and Sss infections in the bait tobacco plants, respectively, whereas no PMTV- or Sss-infected plants were obtained from soil samples collected from PMTV- and Sss-free fields.

Epigenetic changes in FOXO3 and CHEK2 genes and their correlation with clinicopathological findings in myelodysplastic syndromes

Objectives/backgroundMyelodysplastic syndromes (MDSs) are a heterogeneous disease in terms of clinical course and response to therapy. Epigenetic changes are the primary mechanism of MDS pathogenesis. FOXO3 and CHEK2 genes play significant roles in normal cellular mechanisms and are also known as tumor suppressor genes. We aimed to clarify the correlation of epigenetic changes in these genes with clinicopathologic findings in MDS. MethodsA total of 54 newly diagnosed MDS patients referred to Shariati and Firouzgar Hospitals (Tehran, Iran) were included in the study from 2013 to 2015, comprising the following cases: 26 with refractory cytopenia with unilineage dysplasia, 10 with refractory cytopenia with multilineage dysplasia, four refractory anemia with excess blasts-1 (RAEB-1), 11 refractory anemia with excess blasts-2 (RAEB-2), and three MDS associated with isolated deletion (5q-). Risk groups were determined according to the Revised International Prognostic Scoring System (IPSS-R). The methylation status of CHEK2 and FOXO3 promoters were determined by methylation-sensitive high-resolution melting analysis of sodium bisulfite-converted DNA. Expressions of CHEK2, FOXO3, and GAPDH were measured by quantitative real-time polymerase chain reaction and fold changes were calculated using the ΔΔCT method. ResultsStatistical analysis revealed no promoter methylation of CHEK2 and FOXO3 in healthy control specimens. FOXO3 promoter methylation was associated with high-risk World Health Organization subgroups (p = .017), high-risk IPSS-R (p = .007), high-risk cytogenetics (p = .045), and more than 5% blasts in bone marrow (p = .001). CHEK2 promoter methylation was correlated with more than 5% blasts in bone marrow (p = .009). ConclusionsPromoter methylation of CHEK2 and especially FOXO3 is associated with adverse clinicopathological findings and disease progression in MDS.

DNA Mini-Barcoding: A Derived Barcoding Method for Herbal Molecular Identification.

In recent years, the demand for natural herbal products (NHP) has increased; however, the quality of these products is difficult to confirm due to the lack of a comprehensive quality control system. Traditional methods are not effective in detecting processed ingredients. DNA barcoding is an established technique that has been used for more than 10 years. This technique uses short standard sequences (generally 200–600 bp) to identify species. While a complete DNA barcode is difficult to obtain from NHP due to DNA degradation, mini-barcoding is a complementary tool to identify species in NHP. DNA mini-barcoding uses smaller DNA segments for polymerase chain reaction amplification and can be applied to identify species rapidly. The present review summarizes the development and application of DNA mini-barcodes over recent years and discusses the limitations of this technique. This review also compares mini-barcoding and meta-barcoding, a technique using universal polymerase chain reaction primers to simultaneously amplify multiple DNA barcodes and identify many species in a single environmental sample. Additionally, other detection methods that can be combined with mini-barcodes, such as nucleotide signatures, high-resolution DNA melting analysis, and gold nanoparticles, are discussed. DNA mini-barcoding can fill the gaps left by other methods in the field of herbal molecular identification.

New SNP Markers to Identify Culex Complex Species (Diptera: Culicidae) by High-Resolution DNA Melting Analysis

The members from Culex pipiens complex are the primary vector of various arboviruses including West Nile virus and Eastern equine encephalitis in the United States. Within Cx. pipiens complex, there are three biotypes that differ largely in habitat, bloodmeal preference, mating behavior, and overwintering strategy. The three biotypes are Cx. pipiens form molestus (F.) (Diptera: Culicidae), Cx. pipiens f. pipiens (L.) (Diptera: Culicidae), and Cx. pipiens quinquefasciatus (Diptera: Culicidae) (also known as Cx. quinquefasciatus). It is clear that additional genetic markers, molecular markers based on single nucleotide polymorphisms (SNPs), were identified from the Cx. pipiens complex species. Genomic DNA was extracted from adult females of f. pipiens and f. molestus and was amplified by polymerase chain reactions. Thirty out of the 100 primer pairs showed amplification and were used for SNP identification. All thirty genes contained biotype-specific SNPs: 10 were located on the Cx. pipiens complex genetic map from previous genetic studies. We also tested a high-resolution DNA melting analysis as a biotype identification method by examining the SNPs in the two genes (CPIJ005487 and CPIJ002074). Our method provides a high confidence for biotype determination among the three Cx. pipiens complex mosquitoes.Data deposition: NCBI SNP Database (ssSNP accession nos.: ss947848635-ss947848878).

The shortcut strategy for beta thalassemia prevention.

We propose antenatal blood tests using high-resolution DNA melting (HRM) analysis for beta thalassemia mutation detection after hemoglobin A2 estimation as a modified strategy for the identification of beta thalassemia at-risk couples. Antenatal blood samples of 1,115 couples were transferred from the antenatal care clinic. Hemoglobin A2 was quantified, and proportions ≥3.5% were further assessed for beta thalassemia mutation using HRM analysis. Twelve types of beta thalassemia mutations, including hemoglobin E, were identified. There were 23 couples who were detected as at-risk. All at-risk couples were identified within 7 working days after sample receipt. Prenatal diagnosis revealed 6 affected fetuses. One fetus was homozygous CD17 (AT), and five fetuses exhibited beta0 – thalassemia/hemoglobin E disease. These results were consistent with the outcomes calculated using the Hardy-Weinberg equation. Antenatal blood tests for mutation detection using high-resolution DNA melting analysis after hemoglobin A2 estimation is a feasible laboratory method for the recruitment of couples with a fetus that is at risk for beta thalassemia. This modified strategy is cost-effective and may be beneficial for use in a beta thalassemia prevention program.

Molecular Markers to Select for the j-2–mediated Jointless Pedicel in Tomato

The jointless pedicel trait of tomato conferred by the j-2 gene is widely used in processing markets for stem-free removal of fruit to accommodate mechanized harvest. Although current utilization of j-2 for fresh-market tomato breeding is limited, interest in this trait may increase as breeders seek to address high labor costs through the development of mechanically harvestable cultivars for the fresh market. Yet, the introduction of this trait into new market classes heavily relies on phenotypic selection because there are presently no high-throughput methods available to genotype j-2. Reliable, high-throughput molecular markers to genotype the presence/absence of j-2 for selective breeding were developed. The molecular markers described here use the high-resolution DNA melting analysis (HRM) genotyping with single-nucleotide polymorphism (SNP) and derived cleaved amplified polymorphic sequence (dCAPS)–based genotyping. Two separate HRM-based markers target the j-2 on chromosome 12 or a linked sequence region 3.5 Mbp apart from the gene, and a dCAPS marker resides on the latter. We demonstrate the association between each marker and the jointless pedicel phenotype using segregating populations of diverse filial generations in multiple genetic backgrounds. These markers provide a useful resource for marker-assisted selection of j-2 in breeding populations.

High-resolution melting analysis (HRM) for mutational screening of Dnajc17 gene in patients affected by thyroid dysgenesis.

Congenital hypothyroidism is a frequent disease occurring with an incidence of about 1/1500 newborns/year. In about 75% of the cases, CH is caused by alterations in thyroid morphogenesis, defined "thyroid dysgenesis" (TD). TD is generally a sporadic disease but in about 5% of the cases a genetic origin has been demonstrated. Previous studies indicate that Dnajc17 as a candidate modifier gene for hypothyroidism, since it is expressed in the thyroid bud, interacts with NKX2.1 and PAX8 and it has been associated to the hypothyroid phenotype in mice carrying a single Nkx2.1 and Pax8 genes (double heterozygous knock-out). The work evaluates the possible involvement of DNAJC17 in the pathogenesis of TD. High-resolution DNA melting analysis (HRM) and direct sequencing have been used to screen for mutations in the DNAJC17 coding sequence in 89 patients with TD. Two mutations have been identified in the coding sequence of DNAJC17 gene, one in exon 5 (c.350A>C; rs79709714) and one in exon 9 (c.610G>C; rs117485355). The last one is a rare variant, while the rs79709714 is a polymorphism. Both are present in databases and the frequency of the alleles is not different between TD patients and controls. DNAJC17 mutations are not frequently present in patients with TD.

LNA Thymidine Monomer Enables Differentiation of the Four Single-Nucleotide Variants by Melting Temperature.

High-resolution melting (HRM) analysis of DNA is a closed-tube single-nucleotide polymorphism (SNP) detection method that has shown many advantages in point-of-care diagnostics and personalized medicine. While recently developed melting probes have demonstrated significantly improved discrimination of mismatched (mutant) alleles from matched (wild-type) alleles, no effort has been made to design a simple melting probe that can reliably distinguish all four SNP alleles in a single experiment. Such a new probe could facilitate the discovery of rare genetic mutations at lower cost. Here we demonstrate that a melting probe embedded with a single locked thymidine monomer (tL) can reliably differentiate the four SNP alleles by four distinct melting temperatures (termed the "4Tm probe"). This enhanced discriminatory power comes from the decreased melting temperature of the tL·C mismatched hybrid as compared to that of the t·C mismatched hybrid, while the melting temperatures of the tL-A, tL·G and tL·T hybrids are increased or remain unchanged as compared to those of their canonical counterparts. This phenomenon is observed not only in the HRM experiments but also in the molecular dynamics simulations.

Locked Nucleic Acid Thymine Monomer Probe Identifies Four Single-Nucleotide Variants by Melting Temperature

High-resolution melting (HRM) analysis of DNA is a closed-tube SNP detection method that has shown many advantages in clinical laboratory, point-of-care diagnostics, and personalized medicine. While recently developed melting probes have significantly improved the discrimination of mismatched (mutant) alleles from matched (wild-type) alleles, no one attempts to design a simple probe that can reliably distinguish mismatched alleles among themselves. Such a new probe could facilitate the discovery of rare genetic mutations at lower cost.

Molecular Diagnostics of Copper-Transporting Protein Mutations Allows Early Onset Individual Therapy of Menkes Disease

Menkes disease is a severe X-linked recessive disorder caused by a defect in the ATP7A gene, which encodes a membrane copper-transporting ATPase. Deficient activity of the ATP7A protein results in decreased intestinal absorption of copper, low copper level in serum and defective distribution of copper in tissues. The clinical symptoms are caused by decreased activities of copper-dependent enzymes and include neurodegeneration, connective tissue disorders, arterial changes and hair abnormalities. Without therapy, the disease is fatal in early infancy. Rapid diagnosis of Menkes disease and early start of copper therapy is critical for the effectiveness of treatment. We report a molecular biology-based strategy that allows early diagnosis of copper transport defects and implementation of individual therapies before the full development of pathological symptoms. Low serum copper and decreased activity of copperdependent mitochondrial cytochrome c oxidase in isolated platelets found in three patients indicated a possibility of functional defects in copper-transporting proteins, especially in the ATPA7 protein, a copper-transporting P-type ATPase. Rapid mutational screening of the ATP7A gene using high-resolution melting analysis of DNA indicated presence of mutations in the patients. Molecular investigation for mutations in the ATP7A gene revealed three nonsense mutations: c.2170C>T (p.Gln724Ter); c.3745G>T (p.Glu1249Ter); and c.3862C>T (p.Gln1288Ter). The mutation c.3745G>T (p.Glu1249Ter) has not been identified previously. Molecular analysis of the ATOX1 gene as a possible modulating factor of Menkes disease did not reveal presence of pathogenic mutations. Molecular diagnostics allowed early onset of individual therapies, adequate genetic counselling and prenatal diagnosis in the affected families.

Natural allelic variation in blueberry TERMINAL FLOWER 1

AbstractA blueberry (Vacciniumsp.) germplasm collection was screened for allelic variation inTERMINAL FLOWER 1(TFL1) that could be used in breeding flowering or architecture traits. TFL1 has been found to repress the transition from vegetative to reproductive growth in diverse plant species, with mutations leading to altered flowering and form. TheVcTFL1gene sequence was determined from the draft genome sequence of diploidV. corymbosumline W8520.VcTFL1is a member of a PEBP gene family and it could be distinguished from its family members by sequence comparison with PEBP family genes from other plants. High-resolution DNA melting analysis of 160Vacciniumaccessions detectedVcTFL1exons that differed in sequence from the W8520 control. DNA sequence analysis confirmed the presence of single nucleotide polymorphisms (SNPs) and identified haplotypes of tetraploid accessions. A total of 18 SNP sites were detected inVcTFL1coding sequences of theVacciniumgermplasm screened. A SNP causing an alanine-to-valine change in exon 4 (A159V) was determined by multiple bioinformatic tools to be deleterious toVcTFL1function. A diploidV. corymbosumaccession heterozygous for theVcTFL1mutation was identified as a candidate for breeding novel traits for blueberry.

BRCA1 Gene Mutation Screening for the Hereditary Breast and/or Ovarian Cancer Syndrome in Breast Cancer Cases: a First High Resolution DNA Melting Analysis in Indonesia.

Specific patterns of the hereditary breast and ovarian cancer (HBOC) syndrome are related to mutations in the BRCA1 gene. One hundred unrelated breast cancer patients were interviewed to obtain clinical symptoms and signs, pedigree and familial history of HBOC syndrome related cancer. Subsequently, data were calculated using the Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) risk prediction model. Patients with high score of BOADICEA were offered genetic testing. Eleven patients with high score of BOADICEA, 2 patients with low score of BOADICEA, 2 patient's family members and 15 controls underwent BRCA1 genetic testing. Mutation screening using PCR-HRM was carried out in 22 exons (41 amplicons) of BRCA1 gene. Sanger sequencing was subjected in all samples with aberrant graph. This study identified 10 variants in the BRCA1 gene, consisting of 6 missense mutations (c.1480C>A, c.2612C>T, c.2566T>C, c.3113A>G, c.3548 A>G, c.4837 A>G), 3 synonymous mutations (c.2082 C> T, c.2311 T> C and c.4308T>C) and one intronic mutation (c.134+35 G>T). All variants tend to be polymorphisms and unclassified variants. However, no known pathogenic mutations were found.

Identification and mutagenesis of disease susceptibility genes of Petunia hybrida

Hybrid petunia cultivars are susceptible to infection by powdery mildew (Oidium longipes) and potyviruses (potato Y virus, petunia flower mottle virus) during commercial vegetative propagation. Plant proteins encoded by susceptibility genes are required by powdery mildew and potyviruses to infect plant hosts. To examine the potential of developing genetic resistance to these pathogens, petunia orthologs of the susceptibility genes MLO, eIF4E, and eIF(iso)4E were identified and characterized. High-resolution DNA melting analysis was conducted with a population of chemically-mutagenized petunia plants to identify mutations in these genes. Among 865 M2 families, point mutations were detected in coding regions of PhMLO1, but not eIF4E or eIF(iso)4E. One of the point mutations caused a glycine to glutamate substitution that was predicted to have a deleterious effect on PhMLO function. This study demonstrated the potential of TILLING for the development of pathogen resistance in petunia through the loss of susceptibility gene function.

High-Resolution DNA Melting Analysis in Plant Research.

Genetic and genomic studies provide valuable insight into the inheritance, structure, organization, and function of genes. The knowledge gained from the analysis of plant genes is beneficial to all aspects of plant research, including crop improvement. New methods and tools are continually being developed to facilitate rapid and accurate mapping, sequencing, and analyzing of genes. Here, I review the recent progress in the application of high-resolution melting (HRM) analysis of DNA, a method that allows detecting polymorphism in double-stranded DNA by comparing profiles of melting curves. Use of HRM has expanded considerably in the past few years as the method was successfully applied for high-throughput genotyping, mapping genes, testing food products and seeds, and other areas of plant research.

Identification of Polymorphic Gnpat As a Risk Factor for Porphyria Cutanea Tarda

Symptoms of porphyria cutanea tarda (PCT) resolve when iron stores are depleted by phlebotomy, and a sequence variant of HFE (C282Y) that increases iron absorption by reducing hepcidin expression is a risk factor for PCT. These observations suggest that PCT is an iron dependent disease and that factors that affect iron homeostasis influence the risk of developing the PCT. Recently, a polymorphic variant (D519G) of GNPAT was shown to be enriched in male patients with type I hereditary hemochromatosis (HFE C282Y homozygotes) who presented with a high iron phenotype [McLaren CE, et al. Hepatology Aug;62(2):429-39 2015]. Available evidence suggests that like HFE C282Y, GNPAT D519G increases iron absorption by reducing expression of hepcidin. Therefore, we investigated the prevalence GNPAT D519G in patients with PCT. The study population consisted of 247 patients with PCT. High-resolution DNA melting analysis and Taqman SNP assays were used to identify HFE (C282Y) and GNPAT (D519G) allelic variants, respectively and mutations in uroporphyrinogen decarboxylase (UROD) were identified by nucleotide sequencing. Patients with mutant UROD were categorized as familial PCT (F-PCT) (n=87, 36.0%) and those with wild-type UROD were categorized as sporadic PCT (S-PCT) (n=155, 64.0%). GNPAT D519G was significantly enriched in the patient population (prevalence 22.1%, p<0.001) compared to the general population (prevalence 13.6%) as was HFE C282Y (prevalence of 19.2% in the study population compared to 1.5% in the general population, p<0.001). The relative risk of GNPAT D519G was significantly increased in patients with F-PCT compared to those with S-PCT (odds ratio 1.81, CI 1.06-3.08) whereas the relative risk of HFE C282Y in the F-PCT was lower than in the S-PCT population (odds ratio 0.82, CI 0.46-1.47). Together, these observations suggest that GNPAT D519G, is more likely to require a cofactor (e. g., mutant UROD) to produce PCT whereas HFE C282Y functions as an independent PCT risk factor. This difference may be a consequence of the relative potency of GNPAT D519G (weak) and HFE C282Y (strong) with respect to effects on iron homeostasis. Our studies have identified a new risk factor (polymorphic GNPAT) for PCT that adds support for the involvement of aberrant iron metabolism in the pathobiology of PCT. DisclosuresNo relevant conflicts of interest to declare.

Is Klotho F352V Polymorphism the Missing Piece of the Bone Loss Puzzle in Renal Transplant Recipients?

Background: Bone disorders are next to cardiovascular problems in frequency in renal transplant (RT) recipients. Reduction in 1,25-dihydroxycholecalciferol (1,25D) levels is among the reasons causing bone loss in these patients. Klotho (KL) serves as a co-receptor for fibroblast growth factor 23 (FGF23), and functions in vitamin D metabolism. KL polymorphisms have been identified in several studies, and phenylalanine to valine substitution at amino acid position 352 seemed to be important to KL function. We investigated KL F352V polymorphism and its relation with 1,25D levels in RT recipients. Methods: The study included 25 RT recipients (8 female, 17 male) and 26 (14 female, 12 male) healthy control subjects who were wild (FF) phenotypes in terms of KL F352V polymorphism. RT recipients with (FV, n = 11) and without (FF, n = 14) a heterozygote polymorphism were determined with high resolution DNA melting analysis of KL F352V polymorphism. Serum 1,25D levels were measured using the RIA method. Results: RT recipients with FV phenotype had significantly lower 1,25D levels (17.58 ± 18.38 pg/ml) compared to recipients with FF phenotype (44.91 ± 24.68 pg/ml) and control subjects (28.24 ± 12.13 pg/ml). 1,25D levels in RT recipients with FF phenotype were significantly higher than control subjects. Conclusions: KL F352V polymorphism may increase the expression of FGF23 co-receptor, KL protein and thus may decrease renal expression of 1α-hydroxylase, and/or stimulate 24-hydroxylase in RT recipients. The resultant decrease 1,25D levels may participate in bone loss in these patients.