Comprehensive Mutation Screening in 55 Probands with Type 1 Primary Hyperoxaluria Shows Feasibility of a Gene- Based Diagnosis
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Comprehensive Mutation Screening in 55 Probands with Type 1 Primary Hyperoxaluria Shows Feasibility of a Gene- Based Diagnosis Carla G. Monico,* Sandro Rossetti,† Heidi A. Schwanz,‡ Julie B. Olson,* Patrick A. Lundquist,§ D. Brian Dawson,§ Peter C. Harris,† and Dawn S. Milliner* *Mayo Clinic Hyperoxaluria Center and †Department of Biochemistry and Molecular Biology, Division of Nephrology, and §Clinical Molecular Genetics Laboratory, Mayo Clinic College of Medicine, Rochester, Minnesota; and ‡Luther College, Decorah, Iowa Mutations in AGXT, a locus mapped to 2q37.3, cause deficiency of liver-specific alanine:glyoxylate aminotransferase (AGT), the metabolic error in type 1 primary hyperoxaluria (PH1). Genetic analysis of 55 unrelated probands with PH1 from the Mayo Clinic Hyperoxaluria Center, to date the largest with availability of complete sequencing across the entire AGXT coding region and documented hepatic AGT deficiency, suggests that a molecular diagnosis (identification of two disease alleles) is feasible in 96% of patients. Unique to this PH1 population was the higher frequency of G170R, the most common AGXT mutation, accounting for 37% of alleles, and detection of a new 3 end deletion (Ex 11_3UTR del). A described frameshift mutation (c.33_34insC) occurred with the next highest frequency (11%), followed by F152I and G156R (frequencies of 6.3 and 4.5%, respectively), both surpassing the frequency (2.7%) of I244T, the previously reported third most common pathogenic change. These sequencing data indicate that AGXT is even more variable than formerly believed, with 28 new variants (21 mutations and seven polymorphisms) detected, with highest frequencies on exons 1, 4, and 7. When limited to these three exons, molecular analysis sensitivity was 77%, compared with 98% for whole-gene sequencing. These are the first data in support of comprehensive AGXT analysis for the diagnosis of PH1, obviating a liver biopsy in most well-characterized patients. Also reported here is previously unavailable evidence for the pathogenic basis of all AGXT missense variants, including evolu- tionary conservation data in a multisequence alignment and use of a normal control population. J Am Soc Nephrol 18: 1905–1914, 2007. doi: 10.1681/ASN.2006111230 ype 1 primary hyperoxaluria (PH1; OMIM 259900) is a Southern blotting using an isolated full-length AGT cDNA rare but potentially life-threatening inborn error of me- probe determined that human AGT was encoded singly (3). T tabolism. Inherited deficiency of a liver-specific en- Early description of human AGT using protein A-gold im- zyme (alanine:glyoxylate aminotransferase [AGT]; E.C.2.6.1.44) munocytochemistry and isopycnic density gradient centrifuga- causes impaired glyoxylate metabolism in peroxisomes of hu- tion studies revealed a unique AGT-targeting defect: Mislocal- man hepatocytes (1). This autosomal recessive trait is invariably ization of 90% of the protein from peroxisomes to mitochondria characterized by marked hyperoxaluria with or without asso- in some patients with PH1 (4–6). Cloning followed by sequenc- ciated hyperglycolic aciduria, calcium oxalate urolithiasis or ing of human AGT cDNA that was isolated from livers of nephrocalcinosis, and progressive loss of renal function over patients with this peroxisome-to-mitochondria mistargeting time. phenotype showed three sequence variants in the coding re- In 1990, normal human AGT cDNA was isolated and se- gion (P11L, G170R, and I340M) (7). quenced (2) (GenBank X53414 and NM_000030). Characteriza- Of these, only G170R has been shown to be disease specific, tion and mapping of a genomic clone (designated as AGXT)to although P11L has been demonstrated to modify disease ex- the telomeric region of chromosome 2 (2q36–37) followed, with pression in vitro (8). In 2000, Lumb et al. (8) showed that ascertainment of a coding composition of 11 exons spread presence of P11L alone reduced the activity of AGT by a factor across 10 kb (GenBank M61755 to M61763 and M61833) (3). of 3, whereas coexpression with four of the most common mutations (G41R, G170R, F152I, and I244T) caused protein aggregation. These in vitro observations have been corrobo- Received November 10, 2006. Accepted March 14, 2007. rated by the fact that in patients with PH1 described so far, Published online ahead of print. Publication date available at www.jasn.org. three of these four mutations (G170R, F152I, and I244T) seem to Address correspondence to: Dr. Carla G. Monico, Mayo Clinic Hyperoxaluria segregate only in cis with P11L. It is postulated that inheritance Center and Departments of Internal Medicine and Pediatric and Adolescent of these common variants opposite P11L would not give rise to Medicine, Divisions of Nephrology and Pediatric Nephrology, Mayo Clinic Col- lege of Medicine, Rochester, MN 55902. Phone: 507-266-1045; Fax: 507-266-7891; the PH1 phenotype (8). E-mail: [email protected] Subsequent to detection of the P11L and I340M polymor- Copyright © 2007 by the American Society of Nephrology ISSN: 1046-6673/1806-1905 1906 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 1905–1914, 2007 phisms in patients with PH1 and mitochondrial AGT, Purdue et Grantham (16) while at the same time using evolutionary se- al. (9) also identified a closely linked 74-bp duplication in intron quence conservation and normal population data. 1 (IVS1 ϩ 74 bp). These three polymorphic variants (P11L, I340M, and IVS1 ϩ 74 bp) are now collectively referred to as the Materials and Methods “minor” allele of AGXT. A second normal haplotype of AGXT To determine the feasibility of using comprehensive mutation anal- that lacks these changes is recognized as the “major” allele. ysis for establishing a molecular-based diagnosis of PH1 and to expand Published frequencies for the minor AGXT allele in normal further on the heterogeneity of AGXT, we sequenced the entire coding populations range from approximately 2.3% in Chinese to as region of AGXT in 64 patients with PH1 from the Mayo Clinic Hyper- high as 28% in Saami (10). In PH1, the frequency of the minor oxaluria Center. A definitive diagnosis of PH1 was based on biochem- AGXT allele is higher (approximately 50%), attributed to the ical evidence along with hepatic enzyme analysis that documented AGT deficiency in the patient (n ϭ 48), an affected sibling (n ϭ 8), a first predilection for more common mutations (G170R, F152I, and cousin (n ϭ 1), or supporting molecular data (n ϭ 7). For our normal I244T) to segregate solely with this allele (11). control population, we screened 50 DNA samples of individuals of As of 2004, there were a total of 55 AGXT sequence variants predominantly European and North American descent. The study was reported in the Human Gene Mutation Database (www.hgm- approved by our institutional review board, and all participants pro- d.cf.ac.uk), 34 (approximately 62%) of which are missense or vided informed consent or assent. nonsense changes. The remaining mutations include six splic- Genomic DNA was extracted from peripheral blood leukocytes using ing changes, eight small deletions, four small insertions, one standard methods. The primer pairs and PCR reaction conditions that small insertion/deletion, and two large deletions. Fifty of these were used to amplify and sequence the 11 exons and exon-intron variants, many to date largely unclassified in terms of patho- boundaries of AGXT are listed in Table 1. Primer design was based on genicity, were recently summarized by Coulter-Mackie and the available published genomic sequence of AGXT (GenBank NT_005416). The promoter region of AGXT was not screened. For all Rumsby (12) in the single available review of published AGXT PCR reactions, we used 50 to 100 ng of genomic DNA, 5 to 10 pmol of sequence changes. In a separate report from these same au- reverse and forward primers, 0.25 U of AmpliTaq Gold (Applied Bio- thors, molecular analysis sensitivity was 62% for a large series systems, Foster City, CA), and 200 M dNTP (Invitrogen, Carlsbad, of 287 probands with liver biopsy–proven PH1, using restric- CA) in a total volume of 25 l, with addition of DMSO for optimization. tion enzyme-based screening for the now recognized three Amplification (94°C 30 s, Ta 58 to 62 °C 30 s, and 72°C 30 s for most common AGXT mutations (G170R, c.33_34insC, and denaturation, annealing, and extension steps, respectively) was per- I244T) (13). Detection of two mutant alleles was feasible in only formed in an MBS Satellite 0.2G Thermal Cycler (Applied Biosystems) 99 (34.5%) patients. for 25 to 30 cycles. PCR products were cleaned using ExoSAP IT, per the Given the disappointing results of this and earlier reports manufacturer’s (USB, Cleveland, OH) instructions. Sequencing was (14,15) regarding the application of limited mutation screening performed in both directions using the ABI PRISM 3700 DNA Analyzer (Applied Biosystems), and chromatograms were analyzed with the 4.5 using restriction enzyme digestion for the molecular diagnosis version of Sequencher Software (Gene Codes Corp., Ann Arbor, MI). of PH1, in this investigation, we assessed the diagnostic rele- Positive results were sequenced in duplicate, using a separately ampli- vance of performing whole-gene sequencing. In an effort to fied PCR product. establish the pathogenicity of all previously described and To screen for complex alleles (large deletions or insertions) and to newly discovered AGXT missense variants, we also report a increase the sensitivity of molecular analysis, we applied Luminex classification