Dataset Information

Rapid detection of monogenic causes of childhood-onset steroid-resistant nephrotic syndrome.

ABSTRACT:

Background and objectives

In steroid-resistant nephrotic syndrome (SRNS), >21 single-gene causes are known. However, mutation analysis of all known SRNS genes is time and cost intensive. This report describes a new high-throughput method of mutation analysis using a PCR-based microfluidic technology that allows rapid simultaneous mutation analysis of 21 single-gene causes of SRNS in a large number of individuals.

Design, setting, participants, & measurements

This study screened individuals with SRNS; samples were submitted for mutation analysis from international sources between 1996 and 2012. For proof of principle, a pilot cohort of 48 individuals who harbored known mutations in known SRNS genes was evaluated. After improvements to the method, 48 individuals with an unknown cause of SRNS were then examined in a subsequent diagnostic study. The analysis included 16 recessive SRNS genes and 5 dominant SRNS genes. A 10-fold primer multiplexing was applied, allowing PCR-based amplification of 474 amplicons in 21 genes for 48 DNA samples simultaneously. Forty-eight individuals were indexed in a barcode PCR, and high-throughput sequencing was performed. All disease-causing variants were confirmed via Sanger sequencing.

Results

The pilot study identified the genetic cause of disease in 42 of 48 (87.5%) of the affected individuals. The diagnostic study detected the genetic cause of disease in 16 of 48 (33%) of the affected individuals with a previously unknown cause of SRNS. Seven novel disease-causing mutations in PLCE1 (n=5), NPHS1 (n=1), and LAMB2 (n=1) were identified in <3 weeks. Use of this method could reduce costs to 1/29th of the cost of Sanger sequencing.

Conclusion

This highly parallel approach allows rapid (<3 weeks) mutation analysis of 21 genes known to cause SRNS at a greatly reduced cost (1/29th) compared with traditional mutation analysis techniques. It detects mutations in about 33% of childhood-onset SRNS cases.

SUBMITTER: Lovric S

PROVIDER: S-EPMC4046728 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Publications

Rapid detection of monogenic causes of childhood-onset steroid-resistant nephrotic syndrome.

Lovric Svjetlana S Fang Humphrey H Vega-Warner Virginia V Sadowski Carolin E CE Gee Heon Yung HY Halbritter Jan J Ashraf Shazia S Saisawat Pawaree P Soliman Neveen A NA Kari Jameela A JA Otto Edgar A EA Hildebrandt Friedhelm F

Clinical journal of the American Society of Nephrology : CJASN 20140417 6

<h4>Background and objectives</h4>In steroid-resistant nephrotic syndrome (SRNS), >21 single-gene causes are known. However, mutation analysis of all known SRNS genes is time and cost intensive. This report describes a new high-throughput method of mutation analysis using a PCR-based microfluidic technology that allows rapid simultaneous mutation analysis of 21 single-gene causes of SRNS in a large number of individuals.<h4>Design, setting, participants, & measurements</h4>This study screened in ...[more]

PMID: 24742477

Similar Datasets

Project description:BACKGROUND:Few data exist for the genetic variants underlying the risk for steroid-sensitive nephrotic syndrome (SSNS) in children. The objectives of this study were to evaluate HLA-DQA1 and APOL1 variants as risk factors for SSNS in African American children and use classic HLA antigen types and amino acid inference to refine the HLA-DQA1 association. STUDY DESIGN:Case-control study. SETTING & PARTICIPANTS:African American children with SSNS or steroid-resistant nephrotic syndrome (SRNS) were enrolled from Duke University and centers participating in the Midwest Pediatric Nephrology Consortium. FACTOR:Genetic variants in HLA-DQA1 (C34Y [rs1129740]; F41S [rs1071630]) and APOL1 high-risk alleles. OUTCOMES:SSNS and SRNS. MEASUREMENTS:Direct sequencing for the HLA-DQA1 and APOL1 variants in 115 African American children (65 with SSNS and 50 with SRNS). Imputation of classic HLA alleles and amino acids was done in 363 South Asian children. RESULTS:The 2 HLA-DQA1 variants were significantly associated with SSNS in African American children (C34Y: P=5.7 × 10-11; OR, 3.53; 95% CI, 2.33-5.42; F41S: P=1.2 × 10-13; OR, 4.08; 95% CI, 2.70-6.28), but not with SRNS (C34Y: P=0.6; F41S: P=0.2). APOL1 high-risk variants were not associated with SSNS (P=0.5) but showed significant associations with SRNS (P=1.04 × 10-7; OR, 4.17; 95% CI, 2.23-7.64). HLA-DQA1*0201, HLA-DQB1*0201, and HLA-DRB1*0701 were the classic HLA alleles with the most significant associations with SSNS risk. The most significantly associated amino acid positions were HLA-DQ?1 56 and 76 (both P=2.8 × 10-7). Conditional analysis revealed that these variants most likely account for the observed association. LIMITATIONS:Modest sample size and limited statistical power to detect small to moderate effect sizes. Children studied may not be representative of all African American children in the United States. CONCLUSIONS:HLA-DQA1 is a risk locus for SSNS, but not SRNS, in African American children, consistent with its role in SSNS risk in children of European, Asian, and African ancestries. There is little evidence of a significant role for the APOL1 high-risk alleles in childhood SSNS in African American children. Refinement of the HLA-DQA1 association identified the critical classic HLA antigen types and amino acids of the HLA-DQ ?1 molecule.

Dataset Information

Rapid detection of monogenic causes of childhood-onset steroid-resistant nephrotic syndrome.

Background and objectives

Design, setting, participants, & measurements

Results

Conclusion

Publications

Rapid detection of monogenic causes of childhood-onset steroid-resistant nephrotic syndrome.

Similar Datasets

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