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Phytohemagglutinin stimulation of lymphocytes improves mutation analysis of carbamoylphosphate synthetase 1


Kretz, Rita; Hu, Liyan; Wettstein, Véronique; Leiteritz, Dana; Häberle, Johannes (2012). Phytohemagglutinin stimulation of lymphocytes improves mutation analysis of carbamoylphosphate synthetase 1. Molecular Genetics and Metabolism, 106(3):375-378.

Abstract

Carbamoylphosphate synthetase 1 (CPS1) is the first enzyme of the urea cycle. CPS1 deficiency is a rare autosomal-recessively inherited disorder that can lead to life-threatening hyperammonemia. Since there is no reliable biochemical marker for this disease, diagnosis relies on molecular means which is often done by RNA-based mutation analysis. Skin fibroblasts have been frequently used as a source of RNA while peripheral blood cells do not yield sufficient amounts of specific RNA. To avoid the costly and laborious use of cultured fibroblasts, we tried to use stimulated lymphocytes as an alternative. This was effectively achieved by short-term culture of full heparin blood in the presence of phytohemagglutinin. Hereby, subsequent reverse transcriptase-PCR of the CPS1 transcript became feasible and allowed to detect 16 different mutations (10 missense, 3 deletions, 2 nonsense, 1 duplication; 7 novel mutations) in 14 consecutive patients with CPS1 deficiency. When compared to retrospective data on cultured fibroblasts, the adapted method allowed substantial shortening of the median time to diagnosis (24 days versus 122 days, respectively). Besides disease causing mutations, we detected CPS1 transcript variants including one cryptic exon in RNA from lymphocytes with higher frequency than in RNA from fibroblasts. This underlines that all mutations found in RNA need to be confirmed by DNA sequencing. In conclusion, the presented approach improves the diagnostics of CPS1 deficiency. Besides the shortened time to diagnosis, the method is of particular importance for confirmation of findings of next generation sequencing and gene chips.

Abstract

Carbamoylphosphate synthetase 1 (CPS1) is the first enzyme of the urea cycle. CPS1 deficiency is a rare autosomal-recessively inherited disorder that can lead to life-threatening hyperammonemia. Since there is no reliable biochemical marker for this disease, diagnosis relies on molecular means which is often done by RNA-based mutation analysis. Skin fibroblasts have been frequently used as a source of RNA while peripheral blood cells do not yield sufficient amounts of specific RNA. To avoid the costly and laborious use of cultured fibroblasts, we tried to use stimulated lymphocytes as an alternative. This was effectively achieved by short-term culture of full heparin blood in the presence of phytohemagglutinin. Hereby, subsequent reverse transcriptase-PCR of the CPS1 transcript became feasible and allowed to detect 16 different mutations (10 missense, 3 deletions, 2 nonsense, 1 duplication; 7 novel mutations) in 14 consecutive patients with CPS1 deficiency. When compared to retrospective data on cultured fibroblasts, the adapted method allowed substantial shortening of the median time to diagnosis (24 days versus 122 days, respectively). Besides disease causing mutations, we detected CPS1 transcript variants including one cryptic exon in RNA from lymphocytes with higher frequency than in RNA from fibroblasts. This underlines that all mutations found in RNA need to be confirmed by DNA sequencing. In conclusion, the presented approach improves the diagnostics of CPS1 deficiency. Besides the shortened time to diagnosis, the method is of particular importance for confirmation of findings of next generation sequencing and gene chips.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Children's Hospital Zurich > Medical Clinic
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:July 2012
Deposited On:20 Feb 2013 13:49
Last Modified:05 Apr 2016 16:31
Publisher:Elsevier
ISSN:1096-7192
Publisher DOI:https://doi.org/10.1016/j.ymgme.2012.04.011
PubMed ID:22575620

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