Genetic and histopathological analysis of transverse testicular ectopia without persistent Müllerian duct syndrome: two case reports.

BACKGROUND: Transverse testicular ectopia (TTE) is a rare anomaly in which both testes descend through a single inguinal canal into the same hemiscrotum. Although almost 20-50% of patients with TTE exhibit persistent Müllerian duct syndrome (PMDS) and many genetic analyses have been performed, no reports have described the genes contributing to TTE without PMDS. Here, we report two cases of TTE without PMDS using immunohistochemical staining and genetic analysis. CASE PRESENTATION: Two Asian patients with TTE without PMDS were subjected to orchiopexy. We performed testicular biopsies during operation and obtained blood samples before the operation. Testicular tissues were stained for c-kit, placental alkaline phosphatase (PLAP), and undifferentiated embryonic cell transcription factor 1 (UTF1) to evaluate the presence of intratubular malignant germ cells. Additionally, we performed polymerase chain reaction-based direct sequencing to identify single nucleotide polymorphisms in genes associated with regression of the Müllerian duct and testicular descent (that is, anti-Müllerian hormone [AMH], AMH receptor 2 [AMHR2], insulin-like 3 [INSL3], and relaxin family peptide receptor 2 [RXFP2]). The three-dimensional structures of proteins were predicted using SWISS-MODEL. In immunohistochemical analysis, c-kit and UTF1 were positive, whereas PLAP was negative in three testicular tissue samples from the two patients. These features were also detected on the unaffected side. In variant analysis, common missense variants in the AMH gene (g.365G>T; c.165G>T; p.Ser49Ile [rs10407022]) were observed. All variants in INSL3 and RXFP2 genes were intronic or silent. CONCLUSIONS: Because UTF1, a specific marker of spermatogonial stem cell activity, was expressed in both the affected and unaffected sides in the testicular tissues of two patients, the risk of malignancy may be high in these patients. Although the etiology of TTE without PMDS remains unclear, our variant analysis results were consistent with previous reports, and variants in the AMH gene (rs10407022) may contribute to the specific phenotype of TTE without PMDS.

Novel Risk Loci Identified in a Genome-Wide Association Study of Urolithiasis in a Japanese Population.

BACKGROUND: A family history of urolithiasis is associated with a more than doubling of urolithiasis risk, and a twin study estimating 56% heritability of the condition suggests a pivotal role for host genetic factors. However, previous genome-wide association studies (GWAS) have identified only six risk-related loci. METHODS: To identify novel urolithiasis-related loci in the Japanese population, we performed a large-scale GWAS of 11,130 cases and 187,639 controls, followed by a replication analysis of 2289 cases and 3817 controls. Diagnosis of urolithiasis was confirmed either by a clinician or using medical records or self-report. We also assessed the association of urolithiasis loci with 16 quantitative traits, including metabolic, kidney-related, and electrolyte traits (such as body mass index, lipid storage, eGFR, serum uric acid, and serum calcium), using up to 160,000 samples from BioBank Japan. RESULTS: The analysis identified 14 significant loci, including nine novel loci. Ten regions showed a significant association with at least one quantitative trait, including metabolic, kidney-related, and electrolyte traits, suggesting a common genetic basis for urolithiasis and these quantitative traits. Four novel loci were related to metabolic traits, obesity, hypertriglyceridemia, or hyperuricemia. The remaining ten loci were associated with kidney- or electrolyte-related traits; these may affect crystallization. Weighted genetic risk score analysis indicated that the highest risk group (top 20%) showed an odds ratio of 1.71 (95% confidence interval, 1.42 to 2.06) - 2.13 (95% confidence interval, 2.00 to 2.27) compared with the reference group (bottom 20%). CONCLUSIONS: Our findings provide evidence that host genetic factors related to regulation of metabolic and crystallization pathways contribute to the development of urolithiasis.

Genetic differences in C57BL/6 mouse substrains affect kidney crystal deposition.

We previously established an experimental model of calcium oxalate crystal deposition in the mouse kidney using C57BL/6 mice. C57BL/6J (B6J) and C57BL/6N (B6N) are two core substrains of C57BL/6 mice. B6J and B6N substrains have approximately the same genomic sequence. However, in whole-genome analyses, substrains have slight genetic differences in some genes. In this study, we used these substrains as kidney crystal formation models and compared their genetic backgrounds to elucidate the pathogenic mechanisms of kidney stone formation. Eight-week-old male B6J and B6N mice (n = 15 in each group) were administered 80 mg/kg glyoxylate for 12 days, and the amount of kidney crystal depositions was compared. The expression levels of six genes (Snap29, Fgf14, Aplp2, Lims1, Naaladl2, and Nnt) were investigated by quantitative polymerase chain reaction, and the protein levels were evaluated by western blotting and immunohistochemistry. The amount of kidney crystal depositions was significantly higher in B6J mice than in B6N mice on days 6 and 12. The expression of nicotinamide nucleotide transhydrogenase (Nnt) gene was significantly lower in B6J mice than in B6N mice. The expression of Nnt protein was observed only in B6N mice, and preferential high expression was seen in renal tubular epithelial cells. The results of this study provide compelling evidence that differences in mouse substrains affect kidney crystal deposition and that the absence of Nnt protein could be involved in crystal formation in B6J mice.

A replication study for three nephrolithiasis loci at 5q35.3, 7p14.3 and 13q14.1 in the Japanese population.

A previous genome-wide association study (GWAS) reported three novel nephrolithiasis-susceptibility loci at 5q35.3, 7p14.3 and 13q14.1. Here, we investigated the association of these loci with nephrolithiasis by using an independent Japanese sample set. We performed case-control association analysis using 601 patients with nephrolithiasis and 201 control subjects. We selected seven single-nucleotide polymorphisms (SNPs): rs12654812 and rs11746443 from 5q35.3 (RGS14-SLC34A1-PFN3-F12); rs12669187 and rs1000597 from 7p14.3 (INMT-FAM188B-AQP1); and rs7981733, rs1170155, and rs4142110 from 13q14.1 (DGKH (diacylglycerol kinase)), which were previously reported to be significantly associated with nephrolithiasis. rs12654812, rs12669187 and rs7981733 were significantly associated with nephrolithiasis after Bonferroni's correction (P=3.12 × 10(-3), odds ratio (OR)=1.43; P=6.40 × 10(-3), OR=1.57; and P=5.00 × 10(-3), OR=1.41, respectively). Meta-analysis of current and previous GWAS results indicated a significant association with nephrolithiasis (P=7.65 × 10(-15), 7.86 × 10(-14) and 1.06 × 10(-9), respectively). We observed a cumulative effect with these three SNPs; individuals with three or more risk alleles had a 5.9-fold higher risk for nephrolithiasis development than those with only one risk allele. Our findings elucidated the significance of genetic variation at these three loci in nephrolithiasis in the Japanese population.

The role of long-term loading of cholesterol in renal crystal formation.

We studied the effects of cholesterol load on urinary stone in rats receiving a standard diet or a high fat diet. Sixty male rats were randomized to two groups and were fed either a standard diet (SD group) or a high fat diet (HFD group) for 8 weeks. Then the two groups were further divided into four groups. SD group, HFD group, SD + EG group (with standard diet + ethylene glycol administration for two weeks), and HFD + EG group (with high fat diet + ethylene glycol administration). The starting date of EG administration was considered to be week 0. Twenty-four-hour urine samples were collected in week 0, week 1, and week 2, and oxalate excretion and citrate excretion were measured by capillary electrophoresis analyzer The excretion of phosphorus, magnesium, and creatinine for 24 hours was measured using an automated analyzer Serum sodium, potassium, chloride, calcium, phosphate, magnesium, creatinine, total cholesterol, triglyceride, HDL-cholesterol and glucose were determined using an automated analyzer The kidney tissues were obtained to perform hematoxyline-eosine staining and Pizzolato's staining to detect oxalate-containing crystals. The average body weight in HFD groups and HFD + EG group in week 0 was significantly higher than that of SD group and SD + EG group. The calcium oxalate crystal deposition was not observed in all groups in week 0. HFD + EG group in week 1 had sporadically calcium oxalate crystal deposition in renal distal tubular cells and tubular lumens. In week 2, the number of crystal deposition in HFD + EG group was increased remarkably. The crystals were slightly observed in SD + EG group in week 2. The excretion of urinary calcium and phosphate in HFD group and HFD + EG group was significantly higher than that of the SD group and SD + EG group in week 0. The amount of urinary citrate excretion in the SD group and SD + EG group showed a significantly higher value compared with that of the HFD group and HFD + EG group in week 0. The level of serum total cholesterol in the HFD group and HFD + EG group was higher compared to that in the SD group and SD + EG group. The serum triglyceride level was not significantly different in the four groups in week 0. Interestingly, the level of triglyceride of EG administration groups (SD + EG and HFD + EG group) was significantly higher than that in EG no-administration groups (SD group and HFD group) in week 1 and week 2. The serum glucose level in the HFD group and HFD + EG group was significantly higher than that in the SD group and SD + EG group in week 0. In week 2, the glucose level of EG administration groups (HDF + EG group and SD + EG group) was significantly lower than that of EG no-administration groups (HFD group and SD group). In conclusion, this result suggested that long-term loading of cholesterol could increase renal calcium stone formation.

Functional variants in ADH1B and ALDH2 coupled with alcohol and smoking synergistically enhance esophageal cancer risk.

BACKGROUND & AIMS: Esophageal squamous cell carcinoma (ESCC) is prevalent among Asian populations, with marked regional variations in incidence and mortality. Patients with ESCC have a very poor prognosis, but detection of ESCC at earlier stages could improve clinical outcome. Therefore, identification of epidemiologic factors that influence the development of ESCC would facilitate prevention and/or early detection of the disease. METHODS: We performed a 2-step genome-wide association study with subsequent replication analysis using a total of 1070 Japanese ESCC cases and 2836 controls. We also used logistic regression analysis to estimate the effect of gene-gene and gene-environmental interactions. RESULTS: We identified the significant associations of ESCC with 4q21-23 and 12q24 regions, which include nonsynonymous single nucleotide polymorphisms (SNP) in ADH1B (rs1229984, P = 6.76 x 10(-35)) and ALDH2 (rs671, P = 3.68 x 10(-68)) that were previously shown to be associated with ESCC susceptibility. Multiple logistic regression analysis revealed SNP rs671, rs1229984, alcohol drinking, and smoking as the independent risk factors for ESCC (odds ratios of 1.66, 1.85, 1.92, and 1.79, respectively). Moreover, individuals who had both genetic and lifestyle-related risk factors had a nearly 190 times higher risk of ESCC than those who had neither of these. CONCLUSIONS: We found 2 known functional variants involved in the metabolism of alcohol and tobacco by-products as the most significant risk factors for the development of ESCC in a Japanese population. The individuals carrying both risk genotypes have a higher baseline risk of ESCC that is substantially increased by 2 lifestyle risk factors.

A functional SNP in the NKX2.5-binding site of ITPR3 promoter is associated with susceptibility to systemic lupus erythematosus in Japanese population.

Systemic lupus erythematosus (SLE) is one of the common autoimmune diseases with complex genetic components. To identify a gene(s) susceptible to SLE, we performed a case-control association study using genome-wide gene-based single nucleotide polymorphisms (SNPs) in Japanese population. Here we report that an SNP (rs3748079) located in a promoter region of the inositol 1,4,5-triphosphate receptor type 3 (ITPR3) gene on chromosome 6p21 was significantly associated with SLE in two independent Japanese case-control samples [P=0.0000000178 with odds ratio of 1.88, 95% confidence interval (CI) of 1.51-2.35]. This particular SNP also revealed associations with rheumatoid arthritis (RA) (P=0.0084 with odds ratio of 1.23, 95% CI of 1.05-1.43) and with Graves' disease (GD) (P=0.00036 with odds ratio of 1.57, 95% CI of 1.22-2.02). We found the binding of NKX2.5 specific to the non-susceptible T allele in the region including this SNP. Furthermore, an SNP in NKX2.5 also revealed an association with SLE (P=0.0037 with odds ratio of 1.74, 95% CI of 1.19-2.55). Individuals with risk genotype of both ITPR3 and NKX2.5 loci have higher risk for SLE (odds ratio=5.77). Our data demonstrate that genetic and functional interactions of ITPR3 and NKX2.5 play a crucial role in the pathogenesis of SLE.

Congenital prepubic sinus: a variant of dorsal urethral duplication (Stephens type 3).

Abstract A 3-year-old boy was admitted for yellowish discharge from a tiny opening in the midline prepubic area on the dorsal base of the penis. No other symptom related to the genitourinary tract was noted. Sinuousgraphy showed a non-communicating sinus tract with the urinary tract. At surgical exploration, through a diamond-shaped incision, a 4.5 cm-long sinus was found that ended blindly as a fibrous cord at the anterior surface of the pubic symphisis. Histologically, it was lined by stratified squamous epithelium and surrounded by bundles of smooth muscle and collagen. According to Stephens' classification, the sinus appears to be a variant of dorsal urethral duplication of Stephens type 3.