BMC Medical Genetics BioMed Central Technical advance Open Access Polymorphisms in the glucocerebrosidase gene and pseudogene urge caution in clinical analysis of Gaucher disease allele c.1448T>C (L444P) Justin T Brown*, Cora Lahey, Walairat Laosinchai-Wolf and Andrew G Hadd Address: Asuragen, Inc., Austin, Texas, USA Email: Justin T Brown* - [email protected]; Cora Lahey - [email protected]; Walairat Laosinchai- Wolf - [email protected]; Andrew G Hadd - [email protected] * Corresponding author Published: 03 August 2006 Received: 17 March 2006 Accepted: 03 August 2006 BMC Medical Genetics 2006, 7:69 doi:10.1186/1471-2350-7-69 This article is available from: http://www.biomedcentral.com/1471-2350/7/69 © 2006 Brown et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Gaucher disease is a potentially severe lysosomal storage disorder caused by mutations in the human glucocerebrosidase gene (GBA). We have developed a multiplexed genetic assay for eight diseases prevalent in the Ashkenazi population: Tay-Sachs, Gaucher type I, Niemann- Pick types A and B, mucolipidosis type IV, familial dysautonomia, Canavan, Bloom syndrome, and Fanconi anemia type C. This assay includes an allelic determination for GBA allele c.1448T>C (L444P). The goal of this study was to clinically evaluate this assay. Methods: Biotinylated, multiplex PCR products were directly hybridized to capture probes immobilized on fluorescently addressed microspheres. After incubation with streptavidin- conjugated fluorophore, the reactions were analyzed by Luminex IS100. Clinical evaluations were conducted using de-identified patient DNA samples. Results: We evaluated a multiplexed suspension array assay that includes wild-type and mutant genetic determinations for Gaucher disease allele c.1448T>C. Two percent of samples reported to be wild-type by conventional methods were observed to be c.1448T>C heterozygous using our assay. Sequence analysis suggested that this phenomenon was due to co-amplification of the functional gene and a paralogous pseudogene (ΨGBA) due to a polymorphism in the primer-binding site of the latter. Primers for the amplification of this allele were then repositioned to span an upstream deletion in the pseudogene, yielding a much longer amplicon. Although it is widely reported that long amplicons negatively impact amplification or detection efficiency in recently adopted multiplex techniques, this assay design functioned properly and resolved the occurrence of false heterozygosity. Conclusion: Although previously available sequence information suggested GBA gene/ pseudogene discrimination capabilities with a short amplified product, we identified common single-nucleotide polymorphisms in the pseudogene that required amplification of a larger region for effective discrimination. Page 1 of 9 (page number not for citation purposes) BMC Medical Genetics 2006, 7:69 http://www.biomedcentral.com/1471-2350/7/69 Background within ΨGBA. However, these designs are not predicted to Gaucher disease is a lysosomal storage disorder caused by be ideal for more recently developed techniques – such as mutations in the human glucocerebrosidase gene real-time PCR and suspension bead arrays – that are being (GBA)[1] (for a review, see Grabowski[2]). There is con- adopted by high-throughput clinical laboratories[3-5,8- siderable interest in clinical and research analysis of GBA. 14,21]. Assay methods have typically involved combinations of long-template PCR, gel electrophoresis, and southern Using publicly available genomic DNA sequences blotting [3-5]. While these approaches are effective, there [1,15,16], we developed multiplexed suspension bead is a drive to take advantage of the improvements offered array reagents to characterize alleles covering 8 diseases by multiplexed techniques[6]. A common analytical that are highly prevalent in the Ashkenazi Jewish popula- implementation of multiplexing involves the generation tion. The diseases assayed have a collective carrier fre- of multiple amplicons in a single PCR-based assay. Subse- quency of 1:6 in this population: Tay-Sachs, Gaucher type quent multiplex detection methods range from capillary I, Niemann-Pick types A and B, mucolipidosis type IV, electrophoresis to liquid-bead arrays[7,8]. Importantly, it familial dysautonomia, Canavan, Bloom syndrome, and is well established that amplicon length bears heavily on Fanconi anemia type C. While assays for these diseases amplification efficiency and that many recently adopted have generally involved multiple diagnostic platforms [3- multiplex techniques display a preference for smaller 5,22], the Signature Ashkenazi Carrier Panel (ACP) rea- amplicons [8-14]. Multiplex assays are thus often devel- gents allow for simultaneous genetic determinations of all oped from well-proven simplex PCR designs by reducing diseases. This report details our identification of common amplicon length. This and other technical changes present ΨGBA and GBA polymorphisms not reported in the liter- design challenges and can have unintended consequences ature. These results demonstrate the importance of clini- that are not readily revealed in the controlled environ- cal evaluation and assay redesign in reducing the false ment of the development laboratory. positive frequency in a multiplex genetic test. For example, in order to design new human genetic assays Methods with smaller amplicons, ample nucleotide sequence data Reagents and samples sets are required. However, the sequence information Solutions of 1M Tris-HCl, pH 8.0, 1X TE (10 mM Tris, 1 required for quality design may not always be readily mM EDTA, pH 8.0), 10 % SDS, Signature Amp Mix I and available, particularly in regard to the genomic diversity nuclease-free water were obtained from Ambion Diagnos- present in a given population. In the case of GBA, there are tics (Austin, TX). Uracil-N-Glycosylase (HK-UNG) was relatively few publicly available unique genomic DNA obtained from EpiCentre (Madison, WI). AmpliTaq Gold sequences[1,15,16]. Sequences derived from cDNA are and 10X Gold Buffer were obtained from Applied Biosys- more abundant[17] but are not so useful as a design aid tems (Foster City, CA). Deoxynucleotides were obtained for assays targeting genomic DNA. Such design challenges from Bioline (Randolf, MA). Tetramethyl ammonium in clinical molecular analysis of GBA are well estab- chloride (TMAC), sodium sarkosyl, and 2-(N-Mor- lished[5]. For example, a pseudogene (ΨGBA) exists pholino)ethanesulfonic acid (MES) were purchased from downstream of GBA that is 96% identical to the func- Sigma (St. Louis, MO). The MES was adjusted to pH 4.5 tional gene[1]. Although ΨGBA is expressed[18], the pres- by addition of 5 N NaOH then stored at 4C. Streptavidin- ence of various defects, including a 55-bp deletion in exon β-phycoerythrin (SA-PE) was obtained from ProZyme 9, predict that it encodes a non-functional protein[1]. (Alameda, CA). EDC, (1-ethyl-3-[3-dimethylaminopro- Interestingly, in all sequences reported to date ΨGBA also pyl] carbodiimide hydrochloride), was obtained from has an apparent defect paralogous to the 1448 T to C tran- Pierce (Rockford, IL). xMAP carboxylated microspheres sition (c.1448T>C) that codes for a leucine to proline sub- were obtained from Luminex (Austin, TX). Human stitution at position 444 (L444P) in GBA[1,15,16]. When genomic DNA samples obtained from the Coriell Institute found in the functional gene, the c.1448T>C mutation for Medical Research (Camden, NJ) were purchased as can cause disease[19] and has been demonstrated in one DNA isolated from cell lines. Oligonucleotides were syn- system to reduce enzymatic activity by 77%[20]. In order thesized by Biosearch Technologies (Novato, CA). All oli- to accurately perform genetic analysis of GBA, it is there- gonucleotides were reverse-phase cartridge purified and fore imperative to distinguish the functional gene from prepared as 100 μM stock solutions in deionized water. the pseudogene. Unfortunately, the c.1448T>C transition Residual patient genomic DNA samples were used in this falls in a region of very high identity between GBA and study. Samples were randomly selected and provided ΨGBA. Attempts to address this issue have involved assay without coding or identifying information to members of systems amenable to large amplicons, such as restriction the research team. Investigators were not provided with digest with gel electrophoresis, gene sequencing, or selec- previously determined genotypes until after testing was tive amplification using the upstream 55-bp deletion completed. In addition, the investigators did not have Page 2 of 9 (page number not for citation purposes) BMC Medical Genetics 2006, 7:69 http://www.biomedcentral.com/1471-2350/7/69 access to the laboratory information systems of the facili- then with 0.1% SDS, and then resuspended in 100 μL of ties. 1X TE buffer, pH 8.0. Coupled microspheres were stored at 4C in the dark. PCR amplification PCR reactions were prepared in 50-μL volumes using 5 Bead-array hybridization mM Mg2+, 5 U of AmpliTaq Gold, 0.1 U of HK-UNG, and A multiplex bead array containing c.1448T, c.1448T>C, amplification primers. Either 0.8 pmol of each primer and 43 additional capture probes was prepared in 1X GDL444P-US1 and GDL444P-DS1 or, after clinical evalu- hybridization buffer (3.0 M TMAC, 50 mM Tris-HCl, 4 ations and redesign, 1.6 pmol of each primer GDL444P- mM EDTA, and 0.1% Sarkosyl, pH 8.0) such that each of U6 and GDL444P-DS1 was included. PCR primers for the 45 beads was at a final concentration of 100 beads/uL. array detection and primers for sequencing are listed in Samples were analyzed by mixing 48 μL of the bead solu- Table 1. The final nucleotide mixture was 0.2 mM dATP, tion with a 2 μL portion of amplified products. The sam- dCTP, and dGTP, 0.05 mM dTTP, and 0.4 mM dUTP.