Primary hyperoxaluria: Comprehensive mutation screening of the disease causing genes and spectrum of disease-associated pathogenic variants.

The primary hyperoxalurias are rare disorders of glyoxylate metabolism. Accurate diagnosis is essential for therapeutic and management strategies. We conducted a molecular study on patients suffering from recurrent calcium-oxalate stones and nephrocalcinosis and screened primary hyperoxaluria causing genes in a large cohort of early-onset cases. Disease-associated pathogenic-variants were defined as missense, nonsense, frameshift-indels, and splice-site variants with a reported minor allele frequency <1% in controls. We found pathogenic-variants in 34% of the cases. Variants in the AGXT gene causing PH-I were identified in 81% of the mutation positive cases. PH-II-associated variants in the GRHPR gene are found in 15% of the pediatric PH-positive population. Only 3% of the PH-positive cases have pathogenic-variants in the HOGA1 gene, responsible to cause PH-III. A population-specific AGXT gene variant c.1049G>A; p.Gly350Asp accounts for 22% of the PH-I-positive patients. Pathogenicity of the identified variants was evaluated by in-silico tools and ACMG guidelines. We have devised a rapid and low-cost approach for the screening of PH by using targeted-NGS highlighting the importance of an accurate and cost-effective screening platform. This is the largest study in Pakistani pediatric patients from South-Asian region that also expands the mutation spectrum of the three known genes.

The association of urinary interferon-gamma inducible protein-10 (IP10/CXCL10) levels with kidney allograft rejection.

BACKGROUND: Interferon-gamma inducible protein-10 (IP-10/CXCL10) is a chemokine involved in the alloimmune response against kidney allograft. We aimed to investigate the association of urinary CXCL10 protein levels with rejection in renal transplant patients. METHODS: A total of 273 urine samples from (biopsy-proven) rejection and non-rejection patients and controls were included in this study. CXCL10 levels were analyzed for association with rejection. RESULTS: The data showed statistically significant differences in the CXCL10 levels between rejection vs. non-rejection (p < 0.001). Among the rejection groups, statistically significant differences for CXCL10 levels were found between ACR vs. NAD (p < 0.001), ACR vs. BLR (p = 0.019) and AVR vs. NAD (p = 0.009). Receiver Operating Characteristic (ROC) curve analysis of CXCL10 showed an area under the curve (AUC) of 0.74 with 72% sensitivity and 71% specificity at 27.5 pg/ml between rejection and non-rejection group. Kaplan-Meier curve analysis among different levels of CXCL10 showed a better rejection-free graft survival in patients with <100 pg/ml when compared to >200 pg/ml (38 ± 6 vs. 12 ± 1.0 weeks; log-rank p < 0.001) and 100-200 pg/ml (38 ± 6 vs. 22 ± 9 weeks; log-rank p = 0.442) concentration. CONCLUSION: The results indicate significantly increased levels of CXCL10 protein in the urine at the time of allograft rejection. This association of urinary CXCL10 protein levels with rejection could provide an additional tool for the non-invasive monitoring of allograft rejection.

Analysis of the glutathione S-transferase genes polymorphisms in the risk and prognosis of renal cell carcinomas. Case-control and meta-analysis.

BACKGROUND: The Glutathione S-transferases (GSTs) genes deletion polymorphisms have been associated with the progression of several cancers. The association studies between the 2 GSTs (GSTM1 and GSTT1) null polymorphisms with the susceptibility to renal cell carcinoma (RCC) have been inconclusive. Therefore, with the inclusion of our own data, we performed a comprehensive meta-analysis to assess the association between these 2 polymorphisms and the risk of RCC. METHODS: A systematic literature search was carried out for studies published in the PubMed, EMBASE, Cochrane library, and Google Scholar from 1997 to December 2014. Results were stated as pooled odds ratios (ORs) for nonparametric data after heterogeneity analysis with 95% CI using fixed effect or random effect model. RESULTS: We systematically selected 13 relevant studies after thorough searches from the databases. Data showed no association between the GSTM1 and the GSTT1 null genotypes and the risk of RCC (OR = 1.01; CI: 0.92-1.11; P = 0.89 for GSTM1 and OR = 1.14; CI: 0.91-1.42; P = 0.25 for GSTT1). No association was found when the data were stratified according to the geographical/ethnic basis, source of control, and the risk factor evaluation. Subgroup analysis of occupational exposure to pesticides showed an inverse association of the active genotypes of both GSTM1 and GSTT1 polymorphisms with the exposed group of RCC (P<0.00001 and P<0.00001, respectively). The combined null genotype of the GSTM1/GSTT1 significantly increased the susceptibility to RCC by 1.4-fold (P = 0.001). This association remained significant for the Asian populations in subgroup analysis (OR = 1.8; CI: 1.30-2.49; P = 0.0004). CONCLUSION: In conclusion, this meta-analysis suggests that the 2 GSTs deletion polymorphisms independently have no association with the risk of RCC. However, combination of both deletions increases the risk of developing the RCC.

Unique molecular alteration patterns in von Hippel-Lindau (VHL) gene in a cohort of sporadic renal cell carcinoma patients from Pakistan.

BACKGROUND: Renal cell carcinoma (RCC) is the most frequent form of kidney cancer in adults. Somatic mutations that inactivate the von Hippel-Lindau (VHL) gene are the most common cause of RCC. The frequencies of molecular changes in the VHL gene in RCCs vary among different populations. So far, a single chromosomal-based study has been reported from a South Asian population. This report presents, for the first time, the somatic changes and promoter hypermethylation in VHL in a cohort of 300 RCC patients from Pakistan. METHODS: To identify mutations in the VHL gene, direct DNA sequencing was carried out. Epigenetic silencing was investigated by using methylation-specific polymerase chain reaction. RESULTS: Our data showed molecular alterations in the VHL gene in 163 (54%) renal cell carcinoma patients. Somatic mutations were found in 87 (29%) patients and 35 novel mutations were identified. VHL promoter hyper-methylation analysis showed epigenetic changes in 106 (35%) out of 300 patients. Patients who had no evidence of molecular alterations in the VHL gene were significantly younger than patients who carried some molecular change. Molecular alterations in the VHL gene were not restricted to clear-cell RCCs (ccRCCs). CONCLUSIONS: This is the first report that identifies molecular aberrations in the VHL gene from a South Asian population. The frequency of somatic mutation is lower and that of promoter hypermethylation is higher when compared with data from other parts of the world. The data has important implications in the population-specific application of tailored preventive and therapeutic regimens in non-familial RCCs.

Association of the ACE-II genotype with the risk of nephrotic syndrome in Pakistani children.

Nephrotic syndrome is a common pediatric glomerular disease associated with heavy proteinuria. Since, the angiotensin converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism is a putative genetic risk factor for NS, in this study, ACE (I/D) polymorphism was analyzed in 268 NS and 223 control samples by a PCR-based method. The genotypic and allelic frequencies were determined and the association between ACE I/D polymorphism and NS was evaluated. The frequency distribution of the II, ID and DD genotypes was 82 (30.6%), 128 (47.8%) and 58 (21.6%) in the NS patients and 9 (4.0%), 171 (76.7%) and 43 (19.3%) in the control samples respectively. In the Pakistani pediatric NS population, the II genotypic and allelic frequencies were found to be significantly associated with the disease (OR=6.755; C.I=3-14.9). No significant association was found between this polymorphism and the response to standard steroid therapy. Thus, in contrast to reports from other parts of the world, the II genotype was found to be significantly associated with NS in the Indian and Malay populations and in the Pakistani population described here. To our knowledge, this is the first report from Pakistan describing the association of the ACE I/D polymorphism with pediatric NS. On the basis of these results, it is suggested that analysis of the ACE (I/D) polymorphism should be performed for the early diagnosis in the high risk NS patients in South Asia.

Association of a single-nucleotide polymorphism in the promoter region of the VEGF gene with the risk of renal cell carcinoma.

AIMS: Vascular endothelial growth factor (VEGF) protein plays an important role in tumor development and progression. Polymorphisms in the VEGF gene may lead to over- or underexpression of the protein and may be associated with either risk or progression of malignancy. The aim of this case-control study is to identify and quantify the correlation between VEGF polymorphisms and renal cell carcinoma (RCC). RESULTS: Restriction fragment length polymorphism methods were used for the analysis of VEGF polymorphisms at -2578 and +936 positions in the promoter and 3'-untranslated regions, respectively. The VEGF -2578 A-allele was associated with an increased risk of RCC (odds ratio: 1.6; 95% CI: 1.2-2.3) and A-carrier genotypes were strongly correlated (odds ratio: 2.7; 95% CI: 1.5-4.7) with higher risk. Comparison of VEGF +936 C/T polymorphism between patient and control groups revealed no association with renal carcinoma. Both VEGF -2578 C/A and VEGF +936 C/T polymorphisms showed no significant association with the histopathological parameters of RCC. CONCLUSIONS: This study shows that VEGF -2578 A-allele and A-carrier genotypes are associated with an increased risk of RCC. In groups with higher incidence of RCC, a screening test for this polymorphism may be recommended in conjunction with other established markers.