Detektion von Sequenzvarianten mittels DHPLC : Etablierung als automatisiertes Verfahren zur direkten Mutationsanalyse bei autosomal-rezessiver polyzystischer Nierenerkrankung

Autosomal recessive polycystic kidney disease (ARPKD) is one of the most common genetic disorders of the kidney in childhood. Beside cystic alterations of both kidneys, ARPKD is associated with cystic malformation of the bile ducts and congenital fibrosis of the liver. The clinical spectrum ranges from intrauterine manifestation with prenatal demise to survival into adulthood. The very severe course of disease and the statistic risk of 25% for any newborn in affected families points out that reliable prenatal diagnosis is an important element of genetic counseling in this disease. Prenatal ultrasound examination must be considered as uncertain; linkage analysis has its limitations and is sometimes even impossible. In cases with diagnostic doubts prenatal testing is only feasible by direct mutation analysis. ARPKD is caused by mutations in the PKHD1 gene on chromosome 6p12 which extents over about 470 kb and includes at least 66 exons with a large number of alternatively spliced transcripts. The longest continuous open reading frame encodes a 4074 amino acid protein. In addition to the diagnostic advantages, mutation analysis of PKHD1 contributes to the understanding of the molecular causes of ARPKD. Because of its high sensitivity and the high throughput of samples DHPLC is an appropriate method for the analysis of large multi exon genes. In this study DHPLC was tested for detection of sequence variants in PKHD1. The sensitivity of DHPLC varies with several conditions, particularly with the temperature of analysis. As an application to predict DHPLC conditions we employed the Transgenomic Wavemaker software and used 64 samples with known mutations to determine the reachable detection rate. For 72 of a total of 77 PCR fragments mutation analysis could be established by DHPLC, the remaining five fragments should be examined by direct sequencing. Under the defined DHPLC conditions it succeeded to detect all 64 known samples, in order to reach a sensitivity of 100%. DHPLC analysis has proven to be an efficient technique to detect sequence variants in PKHD1.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2006

Author dc:creator

• Dornia, Christian

Contributors dc:contributor

• Zerres, Klaus

• info:eu-repo/classification/ddc/610
• Medizin
• denaturierende Hochdruckflüssigkeitschromatographie (DHPLC)
• autosomal-rezessive polyzystische Nierenerkrankung (ARPKD)
• detanurating high performance liquid chromatography (DHPLC)
• autosomal recessive polycystic kidney disease (ARPKD)
• polycystic kidney and hepatic disease

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-122473%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:60783