J Inherit Metab Dis (2012) 35:531–540 DOI 10.1007/s10545-011-9406-5 ORIGINAL ARTICLE Deletion of GPIHBP1 causing severe chylomicronemia Jonathan J. Rios & Savitha Shastry & Juan Jasso & Natalie Hauser & Abhimanyu Garg & André Bensadoun & Jonathan C. Cohen & Helen H. Hobbs Received: 18 July 2011 /Revised: 20 September 2011 /Accepted: 22 September 2011 /Published online: 19 October 2011 # The Author(s) 2011. This article is published with open access at Springerlink.com Abstract Lipoprotein lipase (LPL) is a hydrolase that Asian Indian boy who had severe chylomicronemia at 2 cleaves circulating triglycerides to release fatty acids to months of age. Array-based copy-number analysis of his the surrounding tissues. The enzyme is synthesized in genomic DNA revealed homozygosity for a 17.5-kb parenchymal cells and is transported to its site of action on deletion that included GPIHBP1. A 44-year-old aunt with the capillary endothelium by glycophosphatidylinositol a history of hypertriglyceridemia and pancreatitis was also (GPI)-anchored high-density lipoprotein-binding protein 1 homozygous for the deletion. A bolus of intravenously (GPIHBP1). Inactivating mutations in LPL; in its cofactor, administered heparin caused a rapid increase in circulating apolipoprotein (Apo) C2; or in GPIHBP1 cause severe LPL and decreased plasma triglyceride levels in control hypertriglyceridemia. Here we describe an individual with individuals but not in two GPIHBP1-deficient patients. complete deficiency of GPIHBP1. The proband was an Thus, short-term treatment with heparin failed to attenuate the hypertriglyceridemia in patients with GPIHBP1 deficiency. The increasing resolution of copy number microarrays and Communicated by: Ertan Mayatepek their widespread adoption for routine cytogenetic analysis is Competing interest: None declared. likely to reveal a greater role for submicroscopic deletions in Electronic supplementary material The online version of this article Mendelian conditions. We describe the first neonate with (doi:10.1007/s10545-011-9406-5) contains supplementary material, complete GPIHBP1 deficiency due to homozygosity for a which is available to authorized users. deletion of GPIHBP1. J. J. Rios : J. Jasso : N. Hauser : A. Garg : J. C. Cohen (*) : H. H. Hobbs (*) Eugene McDermott Center for Human Growth and Development, Introduction University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA Type I hyperlipoproteinemia is a rare, autosomal recessive e-mail: [email protected] disorder characterized by the accumulation of large e-mail: [email protected] triglyceride-rich lipoproteins in the circulation (chylomi- cronemia), which leads to the development of eruptive S. Shastry : A. Garg : J. C. Cohen Division of Nutrition and Metabolic Diseases, Center for Human xanthomas, failure to thrive, and recurrent pancreatitis Nutrition, University of Texas Southwestern Medical Center, (Brunzell and Deeb 2001). The disorder is caused by Dallas, TX, USA mutations that disrupt the lipolytic processing of triglyceride- rich lipoproteins, including inactivating mutations in genes A. Bensadoun Division of Nutritional Sciences, Cornell University, encoding lipoprotein lipase (LPL) (Havel and Gordon 1960), Ithaca, NY, USA the enzyme that hydrolyzes circulating triglycerides; its cofactor, APOC2 (Breckenridge et al. 1978); a lipase- H. H. Hobbs specific chaperone known as lipase maturation factor 1 Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, (Peterfy et al. 2007); and the protein that anchors the enzyme Dallas, TX, USA to the endothelial surface, glycophosphatidylinositol 532 J Inherit Metab Dis (2012) 35:531–540 (GPI)-anchored high-density lipoprotein-binding protein Sequence variants were verified by manually inspecting 1 (GPIHBP1) (Beigneux et al. 2007). GPIHBP1 was chromatograms. originally identified as a high-density lipoprotein (HDL)- binding protein (Ioka et al. 2003), but the subsequent Genotyping and copy number variation (CNV) detection Array- finding that Gpihbp1 knockout mice have severe hyper- based single nucleotide polymorphism (SNP) genotyping triglyceridemia revealed an essential role for the protein in was performed on genomic DNA from proband 1 using the the action of LPL at capillary endothelial surfaces (Beigneux human 1-M Beadchip (Illumina, San Diego, CA, USA) et al. 2007). LPL is synthesized primarily by adipocytes, according to the manufacturer’s Infinium II Assay protocol. skeletal myocytes, and cardiomyocytes and is secreted into Briefly, 750 ng of genomic DNA was amplified, enzymat- the extracellular space where it binds proteoglycans (Brunzell ically fragmented, and precipitated with isopropanol, as and Deeb 2001). GPIHBP1 binds LPL in the capillary described (Illumina’s Infinium II Assay). The fragmented subendothelial spaces and translocates the enzyme to the genomic DNA was hybridized with an Illumina Human 1-M luminal surfaces of endothelial cells (Davies et al. 2010). In beadchip that contained 50-mer probes attached to a silicon- mice lacking GPIHBP1, heparin-releasable LPL activity is based array that interrogates 1,072,820 SNP. A single-base reduced (Beigneux et al. 2007). GPIHBP1 appears to extension reaction was performed to label the annealed DNA function as a platform for lipolysis of circulating triglycerides in an allele-specific manner. The hybridized array was (Beigneux et al. 2007). scanned using the Illumina BeadArray Reader to image the Human GPIHBP1 is located on the distal long arm of laser-excited fluorophores for each probe. Data was analyzed chromosome 8 and encodes a 184-amino-acid protein that using the Beadstudio Genotyping Module (v3.3.7). Individual contains three domains: N-terminal highly acidic, lympho- probe log ratios [logR = log2 (sample intensity/expected cyte antigen (Ly6), and C-terminal to which the glyco- reference intensity)] were plotted by their genomic positions phosphatidylinositol is attached (Beigneux et al. 2009a). using the Genome Browser in the Illumina Genome Viewer Mutations in the acidic and Ly6 domains interfere with the (v3.2.9). A smoothing line was generated using a 200-kb ability of GPIHBP1 to bind LPL and chylomicrons moving average window to enhance visualization of the (Beigneux et al. 2009c; Gin et al. 2008). Several mutations copy number gains and losses in the Genome Viewer. in GPIHBP1 gene have been identified in patients with Probe-intensity data was analyzed using Partek Genomic chylomicronemia (Table 1). Here, we describe the first Suite software (Partek, St. Louis, MO, USA). Briefly, copy reported patient with a complete absence of GPIHBP1 due number was estimated from intensity values relative to to a deletion of GPIHBP1 and assess the therapeutic baseline copy number calculated from array results of seven efficacy of heparin in this individual and in a second unrelated individuals with other genetic disorders. Genomic patient with hypertriglyceridemia who was homozygous for segmentation algorithms were used to identify genomic missense mutation c.203 G >A (p.Cys68Tyr) in GPIHBP1. regions that differed significantly in signal intensity. Genotype data was imported into Partek Genomic Suite software and analyzed using a Hidden Markov Model Materials and methods (Churchill 1989) to identify regions of homozygosity. For comparison, we examined genotype data from an offspring of Study participants The study was approved by the Institu- a second-cousin mating and from six European Americans tional Review Board of the University of Texas Southwestern who were offspring of nonconsanguinious matings. Size Medical Center, USA. Written informed consent was obtained distributions of the regions of homozygosity are represented from all participants prior to collecting venous blood after a asaquantileplot. 12-h fast. Genomic DNA was extracted using the Gentra Puregene Blood Kit (Qiagen, Valencia, CA,USA) or the Easy- GPIHBP1 deletion mapping and pedigree analysis To DNA kit (Invitrogen, Carlsbad, CA,USA) following the define the deletion breakpoints, oppositely oriented oligo- manufacturers’ protocol. Fasting plasma lipids and lipopro- nucleotides complementary to sequences flanking the teins were measured by Quest Diagnostics. putative deletion were used to amplify the deletion junction. The primers used to amplify the junction were as follows: Sequencing coding regions of candidate genes The exons forward, 5′-GCATCAGGTATGAAGAGTTTGG-3′ and re- of LPL, APOC2, APOA5, and LMF1 were amplified from verse, 5′-CAGGGTCTGTGTGGAAGAGG-3′. 50 ng of genomic DNA using the polymerase chain reaction (PCR) and oppositely oriented oligonucleotides Heparin infusion Fasting blood samples were collected that flank the exons of these genes (Supplementary Table 1). from each patient from a heparin lock. A total of 100 U/kg Amplification products were sequenced using dye- heparin was infused intravenously (APP Pharmaceuticals, terminator chemistry and an ABI 3730xl DNA analyzer. Schaumburg, IL, USA). Venous blood samples were then J Inherit Metab Dis (2012) 35:531 Table 1 Clinical characteristics of patients with GPIHBP1 deficiency Reference Wang and Beigneux Olivecrona Franssen Coca-Prieto Charriere This report Hegele 2007 et al. (2009b) et al. (2010) et al. (2010) et al. (2011) et al. (2011) – 540 Mutation in G56R/G56R G56R/G56R Q115P/Q115P C65S/C68G C65S/C68G C65S/C68G C65Y/C65Y C68Y/C68Y C89F/Deletion G175RG175R C68Y/C68Y Deletion/ GPIHBP1 deletion Current Age 47/F 52/M 33/M 40/F 35/M