DNA polymorphisms of lipase related genes

Abstract

We previously determined sequenced the PNLIP gene encoding pancreatic lipase in cell lines of subjects with clinical deficiency of pancreatic lipase (MIM 246600) and found no putative disease-causing mutations. As part of the ongoing analysis of the genomic DNA of these subjects, we now report the development of genomic amplification primers to sequence the coding regions of CLPS, CEL, PLRP1, and PLRP2, encoding pancreatic co-lipase, carboxyl-ester lipase, and pancreatic-lipase-related proteins-1 and -2, respectively. Whereas we found no putative disease-causing missense or nonsense mutations in these samples, we discovered a total of 13 common polymorphisms (12 single nucleotide polymorphisms) in these four genes. Genotypes of these polymorphisms may be useful in future association analyses.

Introduction

Pancreatic lipase deficiency (PLD; MIM 246600) is characterized by malabsorption of long chain triglyceride fatty acids, failure to thrive in infancy and childhood, and the absence of pancreatic lipase (PL) secretion after secretin stimulation (Sheldon 1964; Rey et al. 1966). We have previously shown that the PNLIP gene encoding PL is not mutated in PLD (Hegele et al. 2001). Other candidate genes for PLD include those with related function, such as CLPS and CEL, which encode pancreatic co-lipase (Sims and Lowe 1992) and carboxyl-ester lipase (Lidberg et al. 1992), respectively, and with related structures, such as PLRP1 and PLRP2, which encode PL-related proteins-1 and -2, respectively (Giller et al. 1992). We sequenced these four candidate genes in subjects with PLD. Whereas no putative disease-causing mutations were discovered, we found several common polymorphisms that we further characterized in normal subjects.

Materials and methods

Study subjects

Cell lines of PL-deficient probands

Lymphoblasts from four PLD patients were purchased from Coriell Cell Repositories (Camden, N.J.). These subjects have been previously reported (Hegele et al. 2001). Briefly, subject GM13278 was a 3-year-old Caucasian female with chronic diarrhea, failure to thrive, and complete deficiency of PL activity following secretin stimulation. Subject GM13322 was a 2-year-old Hispanic male with failure to thrive, developmental abnormalities, and complete deficiency of PL activity following secretin stimulation. Subject GM13343 was a 5-year-old Caucasian male with chronic diarrhea, failure to thrive, and complete deficiency of PL activity following secretin stimulation. Subject GM13344 was a 2-year-old Caucasian male with failure to thrive, developmental abnormalities, and complete deficiency of PL activity following secretin stimulation. No family data were available for any subject.

Normal controls

Samples from a DNA archive of 50 normal Caucasian subjects were used to determine polymorphism allele frequencies. Five of these normal control DNA samples were included in the screening experiments. Samples were available from 50 Chinese and 50 African subjects for additional genotyping of polymorphisms.

Cell culture and genomic DNA isolation

Lymphoblasts were cultured in minimal essential medium (Life Technologies, Rockville, Md.) supplemented with 20% heat-inactivated fetal bovine serum at 37°C in an atmosphere of 5% CO₂ and 95% air. Genomic DNA from cultured lymphoblasts was obtained by using a commercial method, following the manufacturer's instructions (Puregene, Gentra Systems, Minneapolis, Minn.). Genomic DNA from neonatal samples was prepared as described (Hegele et al. 2001).

Screening candidate genes for DNA variants

In order to amplify CLPS, CEL, PLRP1, and PLRP2 coding regions and intron-exon boundaries from genomic DNA, primer sets were developed by using NCBI sequences. Gene names, OMIM numbers, National Center for Biotechnology Information (NCBI) numbers, primer sequences, and product sizes are shown in Table 1. Primers were each designed to anneal at 60°C, which allowed for the use of a single amplification apparatus for all reactions. Amplification conditions were: 94°C for 5 min, followed by 30 cycles comprising 30 s each at 94°C, 60°C, and 72°C, ending with a single 10-min extension step at 72°C. All samples were directly sequenced in a Prism 377 Automated DNA sequencer (PE Applied Biosystems, Mississauga, Canada).

Table 1. Amplification primers for genes encoding lipase-related proteins (MIM Mendelian Inheritance in Man, NCBI National Center for Biotechnology Information)

Genotyping candidate gene polymorphisms

Rapid genotyping methods included: (1) detection of length polymorphism either by gel electrophoresis of amplified fragments for insertion/deletion polymorphisms or by digestion with a restriction endonuclease followed by gel electrophoresis for polymorphisms that altered a recognition site; (2) the use of a fluorescent allele-specific detection method (SNaPSHOT, PE Applied Biosystems, Mississauga, Canada); (3) the use of electropherograms of direct sequencing reactions. The details of the methods used for each specific polymorphism are shown in Table 2.

Table 2. Polymorphisms in genes encoding lipase-related proteins (c. cDNA sequence nucleotide number, IVS intron, PAGE polyacrylamide gel electrophoresis, ins insertion, rs1049125 identification number in National Center for Biotechnology Information SNP database)

Statistical analysis

SAS version 6.12 (SAS Institute, Cary, N.C.) was used for all statistical analyses. Chi-square analysis was used to test deviations of genotype frequencies from those predicted by the Hardy-Weinberg equation. The nominal level of statistical significance for all analyses was P<0.05.

Results

Identification of candidate gene polymorphisms

Genomic DNA from the cell lines from the four PLD subjects and from five normal Caucasian controls were screened by using the amplification primers from Table 1 and the amplification program described above. The polymorphisms detected in the screening experiments are shown in Table 2. Frequencies of the minor allele of each polymorphism in 50 Caucasians are shown in Table 2, in addition to frequencies of specific alleles in 50 Chinese and 50 African subjects. None of the observed genotype frequencies deviated from the expectations according to the Hardy-Weinberg equation.

Discussion

We report: (1) primer sets that amplify the entire sequence of CLPS, CEL, PLRP1, and PLRP2, encoding pancreatic co-lipase, carboxyl-ester lipase, and pancreatic-lipase-related proteins-1 and -2, respectively; (2) no mutations in these four genes in subjects with PLD; (3) a total of 13 common polymorphisms, 12 being single nucleotide polymorphisms (SNPs), in these four genes. Of the 12 SNPs, 11 appear not to have been previously reported, whereas only PLRP1 L461P has been cited in the NCBI SNP database (no. rs1049125). The PLRP1 IVS8 +20C→T SNP was first identified in subject GM13322 and appears to be specific for subjects of African origin, suggesting that subject GM13322 might have had African admixture. The PLRP2 W357X SNP is also of particular interest since it is a common nonsense polymorphism that predicts premature truncation, by ~24%, of the gene product, whose full length form is expressed and has lipase activity (Giller et al. 1992). However, in the absence of evidence of expression, functional data, or additional clinical correlation, the relevance of such a truncated gene product is uncertain. The common SNPs in these genes may prove to be helpful in future association and/or pharmacogenetic studies.