[Small duct primary sclerosing cholangitis diagnosed after fever and liver disorder: a case report].

A woman in her 10s presented to our hospital with persistent fever and liver disorder and was admitted. It was considered that her fever was due to infectious, hematological, and collagen diseases; however, these diseases were excluded. Upper and lower gastrointestinal endoscopy revealed gastritis and indicated inflammatory bowel disease involvement. Neither bile duct stricture nor bile duct wall thickening was observed in the imaging. Thus, liver biopsy was performed due to worsening liver disorder. A diagnosis of small duct primary sclerosing cholangitis was made based on the findings of edematous enlargement of the portal tracts, neutrophilic infiltration, and destruction of the interlobular bile ducts. Furthermore, liver biopsy is helpful in diagnosing unknown liver disorders, even if no abnormality in the bile duct is observed on imaging.

Clinical usefulness of the ablative margin assessed by magnetic resonance imaging with Gd-EOB-DTPA for radiofrequency ablation of hepatocellular carcinoma.

BACKGROUND & AIMS: The aim of this study was to investigate the feasibility of ablative margin (AM) grading by magnetic resonance imaging (MRI) with Gd-EOB-DTPA administered prior to radiofrequency ablation (RFA), and to identify factors for achieving a sufficient AM and predictors for local tumor progression. METHODS: A total of 124 hepatocellular carcinomas (HCCs) were treated by RFA after Gd-EOB-DTPA administration. MRI and enhanced CT were performed within seven hours and one month after RFA. The AM assessment was categorized using three grades: AM (+), low-intensity area with continuous high-intensity rim; AM zero, low-intensity area with discontinuous high-intensity rim; and AM (-), low-intensity area extends beyond the high-intensity rim. Patients were followed and local tumor progression was observed. RESULTS: AM (+), AM zero, AM (-), and indeterminate were found in 34, 33, 26, and 31 nodules, respectively. The overall agreement rate between MRI and enhanced CT for the diagnosis of AM was 56.8%. The κ coefficient was 0.326 (p<0.001), indicating moderate agreement. Multivariate logistic regression analysis showed that a significant factor for the achievement of AM (+) on MRI was no contiguous vessels. The cumulative local tumor progression rates (0% at 1, 2, and 3 years) in 33 AM (+) nodules were significantly lower than those (3.6%, 11.5%, and 18.3% at 1, 2, and 3 years respectively) in 32 AM zero nodules. A multivariate Cox proportional hazards model identified tumor size as an independent predictor for local tumor progression. CONCLUSION: Gd-EOB-DTPA-MRI enabled an early assessment of RFA effectiveness in the majority ofHCC nodules. Local tumor progression was not detected in AM (+) nodules during the follow-up.

Comprehensive genotyping analysis of single nucleotide polymorphisms responsible for beef marbling in Japanese Black cattle.

BACKGROUND: Beef marbling is considered a desirable trait in the meat industry. Therefore, understanding the genetic factors that cause marbling is important. Previously, we performed a genome-wide association study to examine genetic factors associated with beef marbling in Japanese Black cattle and identified a candidate region between 10-30 Mbp on chromosome 7. We verified the effect of the SNPs in this region on beef marbling using linkage disequilibrium block analysis. We narrowed down the candidate region to a range of 15.8-16.1 Mbp. In this study, we comprehensively detected all of the SNPs in this region and verified their effects on beef marbling. RESULTS: Genome resequencing using four animals exhibiting high beef marbling standard (BMS) and four with low BMS revealed a total of 1,846 polymorphisms within the candidate region. Based on the annotation, we selected 13 SNPs exhibiting a moderate impact, as no high-impact SNPs were detected. All of the SNPs represented missense polymorphisms and were located in the following seven genes: RDH8, ANGPTL6, DNMT1, MRPL4, ICAM1, ICAM3, and ICAM5. Finally, we determined the effects of these SNPs on the BMS of a Japanese Black cattle population (n = 529). Analysis of variance revealed that the five SNPs were located in genes encoding the intercellular adhesion molecules (ICAM1, ICAM3, and ICAM5), and showed a highly significant association compared with the remainder (p < 0.01). The lowest p-value was observed for ICAM3_c.739G > A (p = 1.18E-04). Previous studies have suggested that intercellular adhesion molecules (ICAM) may be an upstream factor that regulates adipocyte differentiation. Therefore, considering the polymorphism and putative gene function, we suggest that ICAM1 is potentially responsible for beef marbling. c.470C > G and/or c.994G > A on ICAM1 may be responsible for this quantitative trait locus. CONCLUSIONS: Promising SNP candidates responsible for beef marbling were identified using extensive polymorphism verification in a previously reported QTL region. We aim to elucidate the mechanism of beef marbling in future studies by investigating how these polymorphisms alter protein structure and function.

Estimation of genetic parameters for visceral diseases of fattening Japanese Black cattle in Shimane Prefecture, Japan.

In this study, the genetic parameters of major visceral diseases were estimated using the postmortem inspection records of 9057 fattening Japanese Black cattle in Shimane Prefecture, Japan, and the genetic correlation between visceral diseases and carcass traits was analyzed. There were six visceral diseases with a prevalence of 5% or higher, namely, pleurisy, pneumonia, bovine abdominal fat necrosis (BFN), rumenitis, hemorrhagic hepatitis, and perihepatitis. Variance components were estimated using the Gibbs sampling method, and the heritability of the visceral disease ranged from 0.07 to 0.49 for perihepatitis and BFN, respectively. Significant negative genetic correlations were identified between pleurisy and rib thickness (-0.32), BFN and carcass weight (-0.29), and BFN and rib eye area (-0.22). No significant genetic correlation was observed among the visceral diseases. The least squares analysis of variance suggested that some visceral diseases decrease the value of carcass traits. In particular, carcass weight and rib eye area in individuals with BFN were 11.7 kg and 1.87 cm2 lower than those of healthy cattle, respectively. Thus, it was inferred that genetic factors were involved in the visceral diseases of fattening Japanese Black cattle in Shimane Prefecture.

The SNP in the promoter region of the bovine ELOVL5 gene influences economic traits including subcutaneous fat thickness.

Genetic analyses have contributed to improvements of economically important traits derived from adipose tissue such as fatty acid composition in beef. Elongation of very long chain fatty acids (ELOVL) genes encode for the enzymes that play an important role in elongation of long-chain fatty acids. In this study, we aimed to discover genetic polymorphisms of ELOVL gene family in cattle populations to develop genetic markers. As a result, five synonymous mutations were detected in the coding regions of the ELOVL1, ELOVL2, ELOVL3 and ELOVL5 genes. In addition, six mutations were identified in promoter region of the ELOVL5. Two of five mutations in the promoter region of ELOVL5 were expected to alter the ELOVL5 expression and influence the economic traits, because of the high synteny of the region which was essential for activation of Elovl5 in mouse. Therefore, we performed association analysis between the genotypes and traits and our result revealed that T allele of g.-110T>C in ELOVL5 gene promoter indicated significantly thinner subcutaneous fat thickness (TT, 2.39 cm; CT, 2.35 cm) than that of C allele (CC, 2.68 cm) in a Japanese Black population. Our results suggest that the g.-110T>C is a useful genetic marker for the breeding in beef cattle.

The SNPs in the ACACA gene are effective on fatty acid composition in Holstein milk.

Fatty acid composition is an important economic trait for both dairy and beef cattle and controlled by genetic factors. Candidate genes controlling fatty acid composition may be found in fat synthesis and metabolism pathways. Acetyl-CoA carboxylase is the flux-determining enzyme in the regulation of fatty acid synthesis in animal tissues. One of two isozymes of this enzyme, acetyl-CoA carboxylase-α (ACACA), catalyses the first committed step of fatty acid synthesis in mammalian cytosol, leading to the biosynthesis of long-chain fatty acids. In the current study, the sequence comparison of the coding sequence (CDS) and two promoter regions (PIA and PIII) in bovine ACACA gene was performed between Japanese Black and Holstein cattle to detect nucleotide polymorphisms influencing fatty acid composition in milk and beef. Five single nucleotide polymorphisms (SNPs) were identified in the CDS region, 28 SNPs in the PIA region and three SNPs in the PIII region. Association study revealed that CCT/CCT type of PIII_#1, #2/PIA_#26 indicated a higher percentage of C14:0 in the milk of the Holstein cattle than CCT/GTC type (p = 0.050) and that a difference of the percentage of C16:0 was observed between CCT/CCT and GTC/GTC type (p = 0.023). CDS_#2 T/T type indicated a higher percentage of C18:0 than T/C type (p = 0.008). In addition, the Japanese Black cattle with CC/GT type of PIII_#1, #2 showed a higher percentage of C18:2 in the meat than those with GT/GT type (p = 0.025). Since PIII is the promoter specific to mammary gland during lactation, the altered expression of the ACACA gene owing to the SNPs in the PIII region may influence the fatty acid composition in the milk.

Sampling method for estimating neutral allele frequency in a pedigreed population.

The estimated allele frequency at neutral DNA marker loci is a fundamental parameter for establishing a conservation scheme for a set of livestock breeds. In this study, we propose a novel 'minimum distance (MD) method' for estimating neutral allele frequencies, which minimizes the error by the use of pedigree information. Using computer simulation and actual microsatellite data for a pedigreed cattle population, the performance of the proposed method was compared with that of conventional random sampling (RND). MD manifests a promising superiority to RND, irrespective of the sample size and the depth of pedigree. It is theoretically demonstrated that the error of the estimates depends on both the sample size and the distance of founder allele frequencies between the sampled and the non-sampled individuals. MD samples individuals so as to minimize the latter source of estimation error.

Chronic Active Epstein-Barr Virus Infection Indistinguishable from Autoimmune Hepatitis: A Case Report.

Chronic active Epstein-Barr virus (CAEBV) infection is a rare disease, mainly affecting children, typically characterized by persistent infectious mononucleosis (IM)-like symptoms. We describe an adult case of CAEBV without IM-like symptoms, which was indistinguishable from autoimmune hepatitis (AIH). A 60-year-old woman with liver damage was diagnosed with AIH (International Diagnostic Score: 16 points). She had been treated with prednisolone for three years; however, her transaminases had never normalized. She was admitted for another liver biopsy due to repeated high fevers and worsening of her liver damage over two months. Her EBV-DNA copy number was 2.9 × 104 copies/µg DNA, and EBV-encoded small RNA1-positive lymphocytic infiltration was observed in both the present and previously collected (three years ago) liver tissue samples. This case implies that hepatic involvement in a CAEBV without IM-like symptoms is difficult to distinguish from AIH and may be misdiagnosed. In some steroid resistant AIH cases, evaluating for CAEBV may be valuable.

Verification of Candidate SNP Effects Reveals Two QTLs on BTA7 for Beef Marbling in Two Japanese Black Cattle Populations.

In our previous study, we used genome resequencing to detect all candidate polymorphisms within a quantitative trait loci (QTL) region for beef marbling reported previously at 10-30 Mbp on bovine chromosome 7, and we selected 6044 polymorphisms as candidate quantitative trait nucleotides (QTNs). In the present study, we aimed to identify quantitative trait genes (QTGs) and QTNs in this QTL region by verifying the effect of SNPs on beef marbling in two Japanese Black cattle populations using a Dynamic Array integrated fluidic circuit. In total, 96 selected SNPs were genotyped in 441 and 529 animals in Hyogo and Miyazaki cattle populations, respectively. The most significant p-values were detected in a SNP in a splice region of ALDH7A1 (SNP93_ALDH7A1; p = 3.46 × 10-5) in Hyogo cattle and a missense polymorphism of intercellular adhesion molecule-1 (ICAM1) (SNP37_ICAM1; p = 3.33 × 10-4) in Miyazaki cattle. Interestingly, SNP93_ALDH7A1 was not significant (p = 0.459) in Miyazaki cattle, and SNP37_ICAM1 showed a weakly significant association (p = 0.043) in Hyogo cattle. Thus, each population would likely have different QTGs and QTNs for beef marbling in the QTL region. In the Hyogo population, it was not possible to determine the accurate range of the linkage disequilibrium (LD) block in LD block analysis because of a strong LD structure throughout the assessed region. In Miyazaki cattle, however, an LD block containing SNP37_ICAM1 had a range of 15.8-16.1 Mbp, suggesting that QTNs would be located within this region. The functions of 19 genes in the LD block were investigated. ICAM1 is known to play an important role in adipocyte differentiation; given this function and the effect of amino acid substitution, SNP37_ICAM1 was identified as a promising candidate QTN for beef marbling. Further research on the effect of SNP37_ICAM1 on adipocyte differentiation is expected to provide insights into the mechanism underlying beef marbling formation.

A subpopulation of mouse esophageal basal cells has properties of stem cells with the capacity for self-renewal and lineage specification.

The esophageal epithelium is a prototypical stratified squamous epithelium that exhibits an exquisite equilibrium between proliferation and differentiation. After basal cells proliferate, they migrate outward toward the luminal surface, undergo differentiation, and eventually slough due to apoptosis. The identification and characterization of stem cells responsible for the maintenance of the esophageal epithelium remains elusive. Here, we employed Hoechst dye extrusion and BrdU label-retaining assays to identify in mice a potential esophageal stem cell population that localizes to the basal cell compartment. The self-renewing capacity of this population was characterized using a clonogenic assay and a 3D organotypic culture model. The putative esophageal stem cells were also capable of epithelial reconstitution in vivo in direct esophageal epithelial injury models. In both the 3D organotypic culture and direct mucosal injury models, the putative stem cells gave rise to undifferentiated and differentiated cells. These studies therefore provide a basis for understanding the regenerative capacity and biology of the esophageal epithelium when it is faced with injurious insults.

Effect of Five Polymorphisms on Percentage of Oleic Acid in Beef and Investigation of Linkage Disequilibrium to Confirm the Locations of Quantitative Trait Loci on BTA19 in Japanese Black Cattle.

Five polymorphisms associated with the percentage of oleic acid (C18:1) in beef fat were previously reported on bovine chromosome 19 in different Japanese Black cattle populations. This study aimed to verify the effects of these five polymorphisms on C18:1 using the same Japanese Black cattle population and conduct linkage disequilibrium (LD) analysis in order to determine the locations of the quantitative trait loci (QTLs). We genotyped the five polymorphisms (SREBP1 c.1065 + 83 (84bp indel), STARD3 c.1187 C > T, GH c.379 C > G, FASN g.841 G > C, and FASN g.16024 A > G) in two populations, which were bred in Hyogo and Gifu Prefectures, Japan (n = 441 and 443, respectively) in order to analyze their effects on C18:1 using analysis of variance (ANOVA). In the Hyogo population, SREBP1 c.1065 + 83 and STARD3 c.1187 C > T were significantly associated with C18:1 (p < 0.001). Meanwhile, FASN g.841 G > C, FASN g.16024 A > G, and GH c.379 C > G were significantly associated with C18:1 (p < 0.01) in the Gifu population. LD analysis was subsequently conducted to detect the range of the QTLs, which ranged from 32.2 to 46.4 Mbp and from 47.8 to 52.1 Mbp in the Hyogo and Gifu populations, respectively. In conclusion, this study confirmed the existence of QTLs on BTA19 and divided the candidate region for each QTL based on LD coefficients. These results could contribute to efficient searches for responsible genes and polymorphisms for fatty acid composition.

Estimation of genetic parameters for carcass grading traits, image analysis traits, and monounsaturated fatty acids in Japanese Black cattle from Hyogo Prefecture.

Genetic parameters for carcass grading traits, image analysis traits, and monounsaturated fatty acid (MUFA) percentages were estimated in 29,942 Japanese Black cattle from Hyogo Prefecture. The analyzed traits included five carcass grading traits, two image analysis traits, fat area ratio and fineness index, and two MUFA traits, one measured in intermuscular fat using near-infrared spectroscopy (NIRS) and the other in intramuscular fat using gas chromatography (GC). The heritability estimates of image analysis traits and MUFA were moderate to high, ranging from 0.395 to 0.740, and it was considered that they could be improved simultaneously with carcass grading traits because no severe genetic antagonism was observed. Although the heritability of the NIRS-based intermuscular MUFA was slightly lower than that of the GC-based intramuscular MUFA, the genetic correlation between the two methods was as high as 0.804. These results indicate that the NIRS method can be used as an alternative evaluation procedure to predict MUFA in intramuscular fat in the longissimus muscle.

A novel biomarker TERTmRNA is applicable for early detection of hepatoma.

BACKGROUNDS: We previously reported a highly sensitive method for serum human telomerase reverse transcriptase (hTERT) mRNA for hepatocellular carcinoma (HCC). alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) are good markers for HCC. In this study, we verified the significance of hTERTmRNA in a large scale multi-centered trial, collating quantified values with clinical course. METHODS: In 638 subjects including 303 patients with HCC, 89 with chronic hepatitis (CH), 45 with liver cirrhosis (LC) and 201 healthy individuals, we quantified serum hTERTmRNA using the real-time RT-PCR. We examined its sensitivity and specificity in HCC diagnosis, clinical significance, ROC curve analysis in comparison with other tumor markers, and its correlations with the clinical parameters using Pearson relative test and multivariate analyses. Furthermore, we performed a prospective and comparative study to observe the change of biomarkers, including hTERTmRNA in HCC patients receiving anti-cancer therapies. RESULTS: hTERTmRNA was demonstrated to be independently correlated with clinical parameters; tumor size and tumor differentiation (P < 0.001, each). The sensitivity/specificity of hTERTmRNA in HCC diagnosis showed 90.2%/85.4% for hTERT. hTERTmRNA proved to be superior to AFP, AFP-L3, and DCP in the diagnosis and underwent an indisputable change in response to therapy. The detection rate of small HCC by hTERTmRNA was superior to the other markers. CONCLUSIONS: hTERTmRNA is superior to conventional tumor markers in the diagnosis and recurrence of HCC at an early stage.

Effect of DNA markers on the fertility traits of Japanese Black cattle for improving beef quantity and quality.

Carcass traits have been efficiently improved by recent selection using DNA markers in beef cattle. Additionally, DNA markers might have an effect on other traits such as fertility traits; therefore attention should also be paid to such pleiotropic effects. However, the effects of the markers on both carcass and fertility traits have never been evaluated in the same population, since they are generally measured in different populations. The objective in the current study was to discuss effectiveness of DNA markers developed for carcass traits through investigation of their effects on carcass and fertility traits in a population. We genotyped six markers SCD V293A, FASN g.841G>C, PLAG1 g.49066C>G, NCAPG I442M, DGAT1 K232A, and EDG1 g.1471620G>T in a Japanese Black cattle population ( n = 515 ). To investigate their effects on carcass and fertility traits, we performed statistical analysis (ANOVA and the Tukey-Kramer honestly significant difference (HSD) test). In the results, three of six markers, SCD V293A, NCAPG I442M, and EGD1 g.1471620G>T, were significantly associated with both carcass and fertility traits. Remarkably, the same allele for each marker had positive effects on both traits, suggesting that we would be able to simultaneously improve them using these markers in this population. However, previous studies reported that the effects of DNA markers could differ among populations. Therefore, it is necessary to confirm the effect of the marker in each population before it is used for improvement.

Estimation of genetic parameters of defective appearances in Japanese Black heifer calves in Kagoshima.

Defective appearances, including white spotting (WS), tongue defect (TD), and nipple defect (ND), in Japanese Black cattle potentially lead to economic losses to farmers in Japan. We estimated genetic parameters of defective appearances using 553,433 records of Japanese Black heifer calves housed in the Kagoshima Prefecture. Variance and covariance were estimated using the Gibbs sampling algorithm. The estimated heritability ranged from 0.29 for TD to 0.76 for WS. Percent breeding value (%BV) estimates indicated high variation in WS and ND among sires, reflecting higher heritability. Furthermore, there was a positive linear relationship between the %BV estimate of a sire and the mean incidence rate of each defect in his female offsprings. TD was positively associated with other defects. Therefore, genetic factors strongly affect the incidence of defective appearances in Japanese Black cattle.

Detection of candidate polymorphisms around the QTL for fat area ratio to rib eye area on BTA7 using whole-genome resequencing in Japanese Black cattle.

In our previous study, we performed genome-wide association study (GWAS) to identify the genomic region associated with Fat area ratio to rib eye area (FAR) and detected a candidate in BTA7 at 10-30 Mbp. The present study aims to comprehensively detect all polymorphisms in the candidate region using whole-genome resequencing data. Based on whole-genome resequencing of eight animals, we detected 127,090 polymorphisms within the region. Of these, 31,945 were located within the genes. We further narrowed the polymorphisms to 6,044 with more than five allele differences between the high and low FAR groups that were located within 179 genes. We subsequently investigated the functions of these genes and selected 170 polymorphisms in eight genes as possible candidate polymorphisms. We focused on SLC27A6 K81M as a putative candidate polymorphism. We genotyped the SNP in a Japanese Black population (n = 904) to investigate the effect on FAR. Analysis of variance revealed that SLC27A6 K81M had a lower p-value (p = .0009) than the most significant SNP in GWAS (p = .0049). Although only SLC27A6 K81M was verified in the present study, subsequent verification of the remaining candidate genes and polymorphisms could lead to the identification of genes and polymorphisms responsible for FAR.

Partial analysis of hepatitis B virus DNA from hepatocellular carcinoma showing negative hepatitis B virus surface antigen: an analysis of two Japanese cases.

It has been reported that hepatitis B virus (HBV) DNA is detected in serum and/or liver in patients with hepatocellular carcinoma (HCC) without HBsAg. To adress this issue, we analyzed HBV genome in 2 HCC cases without HBsAg. The DNA from serum from patients with HCC was amplified with a nested PCR, and 'a' determinant of S region, core promoter region and precore region were sequenced. The first case, a 50 years-old male, was negative for HBsAg and HBeAg, and positive for anti-HBs, anti-HBe and anti-HBc. Viral load of HBV in serum was 4.0 log genome equivalent/ml by TMA assay, and was 1.1 X 105 copy/ml by real-time PCR system. A nucleotide analysis of the common 'a' determinant of S gene showed that the 5 first amino acids of 'a' determinant, CTIPA, were changed to CKTCTTPA. The second case, a 76 years-old male, was positive for anti-HBe, but negative for HBsAg, anti-HBs, HBeAg and anti-HBc. No missense or nonsense mutations were seen in 'a' determinant of S region. Viral load of serum HBV was < 3.7 log genome equivalent/ml by TMA assay, but was 2.4X103 copy/ml by real-time PCR system. The results of the present study suggest that the mechanisms of HBsAg loss are diverse among HCC patients without HBsAg, and that an analysis of HBV genome is a useful tool to dissolve molecular mechanisms losing HBs antigenicity.

The non-synonymous mutation in bovine SPP1 gene influences carcass weight.

Meat quality in beef cattle is controlled by genetic factors. SPP1 (secreted phosphoprotein 1) gene, coding a multifunctional cytokine with diverse biological functions, is the candidate gene influencing carcass traits. In this study, we tried to discover DNA polymorphisms associated with beef quality in bovine SPP1 gene, so that two SNPs (single nucleotide polymorphisms) in the promoter region and one SNP in the CDS (coding sequence) region were identified. Although the formers were predicted to alter SPP1 expression, they did not show any effects on the traits. On the contrary, statistical analysis revealed that g.58675C > T, a non-synonymous mutation from threonine to methionine in the conservative region, had a significant effect on carcass weight. Carcass weight of the animals with C/T allele (473.9 ± 6.0 kg) was significantly heavier than that of the C/C homozygotes (459.2 ± 2.8 kg). Because SPP1 gene functions in skeletal muscle cells as a positive regulator, the non-synonymous mutation might influence muscle development and remodeling, resulting in increased carcass weight of the C/T animals. Our results indicate that the SNP can be applied as a DNA marker for the improvement of beef cattle.

Whole-genome resequencing to identify candidate genes for the QTL for oleic acid percentage in Japanese Black cattle.

In our previous study, we detected a QTL for the oleic acid percentage (C18:1) on BTA9 in Japanese Black cattle through a genome-wide association study (GWAS). In this study, we performed whole-genome resequencing on eight animals with higher and lower C18:1 to identify candidate polymorphisms for the QTL. A total of 39,658 polymorphisms were detected in the candidate region, which were narrowed to 1993 polymorphisms within 23 genes based on allele differences between the high and low C18:1 groups. We subsequently selected three candidate genes, that is, CYB5R4, MED23, and VNN1, among the 23 genes based on their function in fatty acid metabolism. In each candidate gene, three SNPs, that is, CYB5R4 c.*349G > T, MED23 c.3700G > A, and VNN1 c.197C > T, were selected as candidate SNPs to verify their effect on C18:1 in a Japanese Black cattle population (n = 889). The statistical analysis showed that these SNPs were significantly associated with C18:1 (p < 0.05), suggesting that they were candidates for the QTL. In conclusion, we successfully narrowed the candidates for the QTL by detecting possible polymorphisms located within the candidate region. It is expected that the responsible polymorphism can be identified by demonstrating their effect on the gene's function.