Comprehensive Mutation Screening in 55 Probands with Type 1 Primary Hyperoxaluria Shows Feasibility of a - 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_3؅UTR 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 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 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 strategy that is based on the scheme developed by FlexMAP tag/anti-tag system technology (17,18) to the multiplex liga-

Table 1. Flanking primer pairs and annealing temperatures that were used to amplify the 11 exons and exon- intron boundaries of AGXT

Forward Primer Reverse Primer ͓Magnesium͔ Annealing Amplicon Exon Ј Ј Ј Ј Temp Length (5 to 3 ) (5 to 3 ) (mM) (°C) (bp) 1 CCGCAGCACAAGCACAGATAAG CCCCCGAGTGACCCCCACT 2.0 61 360 TGAGACCCAGGCTCCCCGCa 59 454 or 528 2 CTTGGGGAGGCGGGGAGCCT TTGAAGGATGGATCCAGGGCCAT 2.5 62 288 3 GGCTTCTACAGTGTGTGCG ACCAGTCTTCCCACCATTC 2.5 59 314 4 TTGGAAAAGCCCTTGTCCCG TTAGACTCAGCCCAGAGCAAAGAG 2.5 58 456 5 AGGCTCAGAAACCCCATC ATCGGCTGCTGGACGCAC 3.0 59 256 6 GGATTCGTGGTAGGGTCGTAGC AGAGGGTGGGCGGGAGAGC 2.0 61 338 7 GGACAGCCAGCGAGACTG TGGAGAATCCCTGAGTGC 2.5 58 261 8 GCTCTCCCTGGCAGACGAAG TGTCCTCTCTCCCCCACCCC 2.5 60 405 9 TGTCACTGCCCACCAGCG AGCCCCTCATCCTTCCCC 2.0 60 185 10 CTCCCCCGGCTCCTCTGGAA GAGGGGTGGCGGGTTTGGTT 2.5 62 295 11 GGGTGGGTGGTCCTCACTC GCAGGGTCTGTTTGCTCC 2.0 60 300

aA second set of primers were designed for detection of the IVS1 ϩ 74 bp insertion in intron 1 (amplicon ϩ duplication ϭ 528; amplicon Ϫ duplication ϭ 454). J Am Soc Nephrol 18: 1905–1914, 2007 Mutation Screening in Type 1 Primary Hyperoxaluria 1907

tion-dependent probe amplification (MLPA) technique initially de- scribed by Schouten et al. (19). In this method, two template-specific probes are designed to screen for gene copy changes in the genetic region of interest: A short probe (approximately 25 bp) that consists of template-specific sequence and universal primer, along with a longer probe (approximately 75 bp) that is made of template-specific sequence, universal primer, and “stuffer” sequence complementary to a selected FlexMap bead. Long probes are phosphorylated to carry out the liga- tion reaction. The selected intragenic and extragenic probe sequences are listed in Table 2. For the multiplexing steps, we used the commercially available MLPA Kit from MRC Holland (Amsterdam, The Netherlands). PCR was performed for 24 cycles (30 s at 95°C, 30 s at 60°C, and 60 s at 72°C) using Platinum Taq (Invitrogen). Bead hybridization followed, using 40 ␮l of FlexMap bead mix plus 10 ␮l of PCR product, for a 1-h incubation period at 37°C. We then added 25 ␮l of a streptavidin phycoerythrin/ tetramethyl-ammonium chloride solution to each reaction well (2 ␮lof Streptavidin, R-phycoerythrin conjugate [1 mg/ml] and 250 ␮lof1ϫ tetramethyl-ammonium chloride). This mixture was incubated at room Long Probe temperature for 15 min, and the number of FlexMap beads that suc- cessfully hybridized was counted with a Luminex LX100. Fluorescence intensity that was generated from each sample compared with control probes (expressed as a peak ratio) was then used to assess its copy number. In the experience of D.B.D. and P.A.L., this method has proved robust in quantifying gene dosage changes in patients with Niemann- Pick type C (17,18). To classify the new AGXT missense variants described here, we developed and applied a scoring system that is based on the matrix of Grantham (16) and Abkevich et al. (20). Overall scores are provided for untranslated region (UTR). GAL3ST2 is located approximately 1 Mb from

Ј all described and newly detected AGXT missense mutations. Finally, we used the 8.1.1 version of the Mac Vector program to generate a multiple sequence alignment of the following AGT orthologs (corre- gtcctttgtcgatactgg gtcctttgtcgatactgg gtcctttgtcgatactgg gtcctttgtcgatactgg gtcctttgtcgatactgg gtcctttgtcgatactgg gtcctttgtcgatactgg gtcctttgtcgatactgg gtcctttgtcgatactgg sponding GenBank accession numbers): Homo sapiens (BAA02632), Ca- TTAACCCACGGGGAGTCGTCCACCGGCGTGCTGCAGCCCCTTCTTTTACAATACTTCAATACAATC TGTGACCTGCCCACTGGCACACAGCTGGCACTGGCACACACCTGTCTTTTCAAATCAATACTCAACTTT GCAGTCCCCAGGCCATGAGCCTCCCGGGAATGTTTAATAAAGTCAACAATCTTTTACAATCAAATC TATCTGCATGGGCGCCTGCAGGCACTGGGGCTGCAGCTCTAATCTTACTACAAATCCTTTCTTT GATGGAGGGCTTGTGCCCCATCCCCAGGGCACTGCGCAGCGGGAGAGCTACAAACAAACAAACATTATCAA CTCTCACCTCTGTGTCCGCCCTGCTGGGAAATATTCCAGGCCTTTAATCCTTTATCACTTTATCA CCAGCTTCTTTCACTTACTGTTTTTGACACTCACATGTACCGTCGTGGGCTAATCTTCTATATCAACATCTTAC CTGTACCCGCTGGCTATGACTGGAGAGACATCGTCAGCTACGTCTATCTTCATATTTCACTATAAAC GCTTCCCTGAATTCACCTGTCTTGGCTTCGTTTCGGAAGTAGGGGCTTTTCATCTTTTCATCTTTCAAT a nis familiaris (XP_848328.1), Felis catus (CAA53527.1), Oryctolagus cunic- ulus (S24155), Rattus rattus (CAA29656.1), Mus musculus (AAH25799.1), Xenopus tropicalis (NP001006705.1), and Danio rerio (AAH76465.1). AGXT

Results Overall, our fully sequenced cohort consisted of 64 patients with PH1 (55 unrelated probands, eight affected siblings, and one first cousin). AGXT genotyping in the 55 unrelated pro- bands is listed in Table 3. The 21 newly discovered mutations (12 missense, three nonsense, three splice site, two microinser- tion/deletion, and one large deletion) are shown in boldface type. Direct sequencing of the entire AGXT coding region re- vealed two disease alleles in 52 (95%) patients and a single Short Probe pathogenic change in the remaining three. In the unrelated PH1 group as a whole, minor and major allelic frequencies were 58% 80 kb were designed 1 kb, 2 kb and 80 kb from the 3 (64 of 110 alleles) and 42% (46 of 110 alleles), respectively. Two additional AGXT sequence variants are worthy of spe- cial mention. The first is a T 3 C transition in exon 8 (c.836 T 3 C, I279T), which was detected in probands 31, 33, 43, and 46, acattttgctgccggtcaTGGAACACACAGCAGGTCCTCCACTGTG acattttgctgccggtcaGGACGCTCGGTGACTCGGAGCCGG acattttgctgccggtcaAAAACCCAGTGCCTTCCAAATGAGCT acattttgctgccggtcaACAAGCCAGTGCTGCTGTTC acattttgctgccggtcaGCCAGCACCGCGAGGCCGCGGCG acattttgctgccggtcaTCCGGCTTCCCACAGTCACCACTGTGG acattttgctgccggtcaGCACTGCCCCAAGAAGAAGC acattttgctgccggtcaAAGCCCATCCACCAATCCTCAC acattttgctgccggtcaGGATTTGGAGGCTGTTCCTACTCAG 2 kb, and 3 Ј the significance of which was not clear because it was not detected in any of the control subjects screened and was found in tandem with two disease alleles in probands 33, 43, and 46. 1 kb, 3 Ј Previous expression of this variant by Coulter-Mackie et al. (21)

MLPA probe sequences for exons 1, 4, 9, 10, and 11 of on the background of the major AGXT allele, however, yielded on chromosome 2. MLPA, multiplex ligation-dependent probe amplification. 2 kb 80 kb 1 kb Name Ј Ј Ј only a minimal effect on AGT catalytic activity. The second is 3 3 GAL3ST2 Exon 4 Exon 9 Exon 10 Exon 11 3 Exon 1

Probes 3 Ј 3

a an intronic change (IVS10–91 G A) in proband 42, which AGXT Table 2. segregated with disease in her affected pedigree and again was 1908 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 1905–1914, 2007

Table 3. AGXT genotyping in 55 unrelated PH1 probandsa

b Predicted Predicted Proband Diagnosis Allele 1 Effect Allele 2 Effect

1 to 7 1 c.508 G3A G170R c.508 G3A G170R 8 to 10 2 c.508 G3A G170R c.508 G3A G170R 11 to 13 1 c.508G3A G170R c.33_34insC Frameshift 14 1 c.508G3A G170R c.121G3A G41R 15 2 c.508G3A G170R c.121G3A G41R 16 to 17 1 c.508G3A G170R c.454T3A F152I 18 to 19 1 c.508G3A G170R c.466G3A G156R 20 to 21 1 c.508G3A G170R c.568G3A G190R 22 1 c.508G3A G170R c.603C3A D201Ec 23 1 c.508G3A G170R c.612C3A Y204Xc 24 2 c.508G3A G170R IVS4–1G3A Splice site c.508G3A G170R IVS7 ؉ 1G3C Splice site 1 25 c.508G3A G170R IVS7 ؉ 1G3A Splice site 1 26 27 to 28 1 c.508G3A G170R IVS7–1G3C Splice site 29 1 c.508G3A G170R IVS8–3C3G Splice site c.508G3A G170R IVS9 ؉ 1G3T Splice sitec 1 30 31 2 c.508G3A G170R X X 32 2 c.33_34insC Frameshift c.698G3T R233Lc 33 1 c.33_34insC Frameshift c.454T3A F152I 34 1 c.121 G3A G41R c.481G3A G161S 35 1 c.74 T3G L25R c.454T3A F152I 36 1 c.454T3A F152I c.139 G3A G47R 37 1 c.466 G3A G156R c.956C3T P319L 38 1 c.466 G3A G156R c.454T3A F152I 39 1 c.466 G3A G156R c.783 T3A H261Q 40 1 c.731 T3C I244T IVS8–3C3G Splice site 41 1 c.737 G3A W246X c.454T3A F152I 42 1 c.346 G3 A G116R c.603C3A D201E 43 1 c.414delGGT V139delc IVS8 ϩ 1G3T Splice site 44 1 c.33_34insC Frameshift IVS8 ϩ 1G3T Splice site 45 1 c.33_34insC Frameshift c.907 C3T Q303X 46 to 47 1 c.33_34insC Frameshift c.33_34insC Frameshift 48 1 c.757 T3C C253Rc c.757 T3C C253R 49 1 c.646 G3A G216R c.646 G3A G216R 50 1 c.731 T3C I244T c.731 T3C I244T 51 1 c.26C3A T9Nc c.26C3A T9N 52 1 c.77 T3C L26P c.77 T3C L26P c.473C3T S158Lc c.306_307 insTCCTGGTTG p.V102insVLV ؉ G103E 1 53 ؉ c.308 G3A IVS8 ϩ 1G3T Splice site Ex 11_3؅UTR del Truncating 1 54 55 1 c.33_34insC Frameshift X X

aPH1, type 1 primary hyperoxaluria; X, mutation not detected. New mutations are shown in boldface type. b1, hepatic enzyme analysis documenting AGT deficiency; 2, biochemical diagnosis (hyperoxaluria with or without hyperglycolic aciduria) and/or hyperoxaluria with VB6 response and supporting molecular analysis (two AGXT mutations). cPreviously published in a smaller series (37). detected in the setting of two other seemingly pathogenic that have been detected only in homozygous form (S205P and changes. G82E) in families of reportedly nonconsanguineous back- Of the 48 unrelated probands with PH1 and availability of grounds (25,26). hepatic enzyme analysis, two disease alleles were satisfactorily To exclude the possibility that large deletions may have gone detected in 46 (96%), and a single disease allele was detected in undetected in our purely homozygous probands for rare mu- two, yielding a sensitivity of 98% (94 of 96 alleles detected) for tations (48, 49, 51, and 52) and in probands with a singly this whole-gene sequencing approach. When limited to the identified mutation (31, 54, and 55), we strategically designed three exons with the highest mutation frequencies (exons 1, 4, MLPA probes (exons 1, 4, and 11) across the AGXT coding and 7; Table 4), the sensitivity of molecular analysis for the liver region. Haplotype analysis for probands 48, 49, 51, 52, and 54 is biopsy cohort was 77% (72 of 94 alleles detected). listed in Table 5. To date, only two large partial AGXT deletions (5Ј untrans- We did not detect changes in gene copy in any of these tested lated region [UTR] to IVS5 and 5Ј UTR to IVS7) and a single patients, with the exception of proband 54 (Figure 1), in whom case of segmental maternal isodisomy of 2q37.3 have been we successfully detected a new deletion that involved exon 11, published (22–24). A likelihood of hemizygosity is also sup- also confirmed separately in an affected sibling. Because the ported by a few other rare (frequency Ͻ5%) mutations in PH1 haplotype analysis in this patient suggested a potential partial J Am Soc Nephrol 18: 1905–1914, 2007 Mutation Screening in Type 1 Primary Hyperoxaluria 1909

Table 4. Exon-specific combined mutation frequencies A for the 48 unrelated probands with PH1 and availability of hepatic enzyme analysisa Exon 11

Alleles Combined c.1220 C 3 Exon Mutation ͓ ͔ (n % ) Frequency A

1 c.33_34insC 12 (13) 18/94 (19%) 3 Exon 10 c.121 G A (G41R) 2 (2) c.976 G 3 3

c.26 C A (T9N) 1 (1) A c.74 T3G (L25R) 1 (1) 3

c.77 T C (L26P) 2 (1) 52 G 3

3 ϩ

4 c.508 G A (G170R) 32 (35) 46/94 (49%) Intron 8 c.454 T3A (F152I) 7 (8) IVS8 c.466 G3A (G156R) 5 (5) c.473 C3T (S158L) 1 (1) G c.481 G4A (G161S) 1 (1) 3 44 A 3

7 c.731 T C (I244T) 3 (3) 8/94 (9%) Ϫ Intron 7 c.737 G3A (W246X) 1 (1) c.757 T3C (C253R) 2 (2) IVS7 IVS7 ϩ 1G3C 1 (1) T IVS7 ϩ 1G3A 1 (1) 45 C 3

a Ϫ

When sequencing is limited to the 3 AGXT exons (1, 4, Intron 7 and 7) with the highest mutation frequencies, the sensitivity of molecular analysis is 77%. Only the 94 detected alleles are IVS7 included in the analysis. Intronic changes are listed with their T corresponding amplicon. 17 C 3 ϩ Intron 6 deletion that affected intron 8 to the 3Ј end of AGXT (Table 5), IVS6 we designed six additional probes (exon 9, 10, 3Ј 1 kb, 3Ј 2 kb, A 3Ј 80 kb, and GAL3ST2) to delineate its extent. These added

probes verified that this new deletion encompasses solely exon Exon 6 11 of AGXT and that it extends Ͼ2 kb downstream from the 3Ј c.654 G 3 UTR of the gene (Figure 1). T The classification system that was developed here for AGXT (Table 6) represents the first attempt to gauge the pathogenicity 91 C 3 ϩ of all reported missense variants. Our control population data, Intron 4 including number of alleles tested and allelic frequencies for the IVS4 seven new (shown in boldface type) and described AGXT poly- T morphisms in comparison with PH1 is depicted in (Table 7). None of the new AGXT missense variants was detected in the 13 C 3 ϩ normal control population tested. The multisequence align- Intron 2 ment is shown in Figure 2. IVS2 T

Discussion a

Using this whole-gene sequencing approach, we show a sen- Exon 2 sitivity for molecular analysis of 98% in 48 liver biopsy–proven c.264 C 3 cases of PH1, an improvement of 36% over the previously published restriction enzyme–based screen for the three most 74 bp ϩ

common mutations in AGXT (G170R, c.33_34insC, and I244T) Intron 1

(13). Comprehensive mutation screening by direct sequencing IVS1 T therefore seems to be a satisfactory method for detection of 74-bp insertion; D, allele without 74-bp insertion.

sequence variation in AGXT and appropriate for molecular ϩ Exon 1 Haplotype analysis in the four seemingly homozygous probands for rare mutations and in proband 54 (patient with newly detected partial

diagnosis of PH1. Given the small size of the gene, molecular c.32 C 3 analysis is relatively inexpensive and easily achieved. Because d, allele 484951 TT52 CC54 CC CC CC dd DD DD DD DD TT CC CC CC CC TT CC CC CC CC CC TT CT CC TT GG GG GA GG GG TT CC CC CC CC TT CC CC CC CT GG GG GG AA GA AA AA GG GG AA GG AA AA AA AA AA CC CA CC AA of the wide range of mutation types detected (missense, non- a Patient gene deletion, Ex 11_3 Ј UTR del) sense, microinsertion/deletions, and splice variants), direct se- Table 5. 1910 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 1905–1914, 2007

Table 6. Classification of new and described AGXT missense variants

Sequence Variant Amino Acid Change Grantham Matrix Scorea MSA Conservationb Overall Score

Newly discovered AGXT missense variants (n ϭ 13) c.26 C3A T9N 65 (2) 0/8 (0) 2 c.74 T3G L25R 102 (4) 6/8 (4) 8 c.77 T3C L26P 98 (4) 8/8 (5) 9 c.139 G3A G47R 125 (4) 8/8 (5) 9 c.473 C3T S158L 145 (4) 8/8 (5) 9 c.481 G3A G161S 56 (1) 8/8 (5) 6 c.603 C3A D201E 45 (1) 8/8 (5) 6 c.646 G3A G216R 125 (4) 8/8 (5) 9 c.698 G3T R233L 102 (4) 8/8 (5) 9 c.757 T3C C253R 180 (5) 8/8 (5) 10 c.783 T3A H261Q 24 (1) 8/8 (5) 6 c.836 T3C I279T 89 (3) 5/8 (3) 6 c.956 C3T P319L 98 (4) 8/8 (5) 9 Previously described AGXT missense variants (n ϭ 23) c.2 T3C M1T 81 (3) 6/6 (3) 6 c.121 G3A G41R 125 (4) 8/8 (5) 9 c.122 G3T G41V 109 (4) 8/8 (5) 9 c.244 G3A G82R 125 (4) 8/8 (5) 9 c.245 G3A G82E 98 (3) 8/8 (5) 8 c.322 T3C W108R 101 (4) 8/8 (5) 9 c.336 C3A A112D 126 (4) 8/8 (5) 9 c.346 G3A G116R 125 (4) 4/8 (3) 7 c.454 T3A F152I 21 (1) 8/8 (5) 6 c.466 G3A G156R 125 (4) 8/8 (5) 9 c.508 G3A G170R 125 (4) 8/8 (5) 9 c.518 G3A C173Y 194 (5) 8/8 (5) 10 c.547 G3A D183N 23 (1) 8/8 (5) 6 c.560 C3T S187F 155 (4) 5/8 (3) 7 c.568 G3A G190R 125 (4) 8/8 (5) 9 c.613 T3C S205P 74 (2) 7/8 (4) 6 c.697 C3T R233C 180 (5) 8/8 (5) 10 c.698 G3A R233H 29 (1) 8/8 (5) 6 c.731 T3C I244T 89 (3) 3/8 (1) 4 c.865 C3T R289C 180 (4) 4/8 (3) 7 c.893 T3C L298P 98 (4) 6/8 (4) 8 c.1007 T3A V336D 152 (4) 6/8 (4) 8 c.1049 G3A G350D 94 (4) 8/8 (5) 9

aFor Grantham Matrix score (shown in parentheses), Ͻ60 ϭ 1 point; 61 to 78.3 ϭ 2 points; 78.4 to 93.4 ϭ 3 points; Ͼ93.4 ϭ 4 points; and any cysteine substitution ϭ 5 points. bFor MSA conservation score (shown in parentheses), absolute conservation across 8 species tested ϭ 5; conservation in six or more species tested ϭ 4; conservation in four or more species tested ϭ 3; conservation in one to three species tested ϭ 1; conservation in 0 species tested ϭ 0. Overall score Ն8 ϭ high probability of pathogenicity; 6 to 8 ϭ moderate probability of pathogenicity; Յ5 ϭ likely not pathogenic (i.e., polymorphism). MSA, multiple sequence alignment.

quencing of AGXT has the added benefit of contributing to our sequencing (of exons 1, 4, and 7) and then direct sequencing of molecular understanding of PH1, despite the high prevalence the remaining exon and exon-intron boundaries with addition of private mutations. of family studies when indicated seems to be a suitable ap- Even if sequencing is limited to the three exons that contain proach. Mutation analysis may also be targeted to a particular the more common mutations (1, 4, and 7), the sensitivity re- ethnicity for which there are known AGXT associations (e.g., mains considerably higher (77%) than the reported mutation- I244T in Spanish populations). A liver biopsy would then be specific restriction enzyme approach (62%) (13). For diagnostic required only when this molecular approach proves nondiag- purposes, a prioritization scheme that consists first of limited nostic. J Am Soc Nephrol 18: 1905–1914, 2007 Mutation Screening in Type 1 Primary Hyperoxaluria 1911

Figure 1. Multiplex ligation-dependent probe amplification analysis. The five control (breast cancer anti- resistance 3 [BCAR3], catenin, ␤1 [CTNNB], HIR histone cell regulation defective homolog A [HIRA], TNF receptor superfamily 7 [TNFRSF7], and dystrophin [DMD]) and nine experimental probes (AGXT exons 1, 4, 9, 10, 11, 3Ј 1 kb, 3Ј 2 kb, 3Ј 80 kb, and galactose-3-O- sulfotransferase 2 [GAL3ST2]) are listed sequentially above the selected patient panels (female control, male control, proband 54). For AGXT, probes 3Ј 1 kb, 3Ј 2 kb, and 3Ј 80 kb are located 1, 2, and 80 kb from the 3Ј untranslated region (UTR), respectively. Large gene rearrangements were not detected in probands 48, 49, 51, and 52 (data identical to controls not shown). In proband 54, a deletion that encompasses exon 11 and extends at least 2 kb from the 3Ј UTR is shown. For the control probes, BCAR3, ; CTNNB1, chromosome 3; TNFRSF7, chromosome 12; HIRA, chromosome 22; DMD, exon 11, gene dosage control, X-chromosome.

Both published reports (27–31) and data that were obtained To date, several mutations have been documented on exon 7 here confirm that less common mutations are also found on of AGXT (29), the most common of which is I244T, a founder these same exons (1, 4, and 7), substantiating the strategy to mutation in Spanish patients who originated from a small sequence these coding regions directly, in lieu of performing island of the Canary Islands called La Gomera (34), where its mutation-specific restriction enzyme–based assays. A second frequency is 92% (35). The reported frequency for I244T has exon 4 mutation in particular (F152I) has been reported in otherwise ranged from 6 to 9% (11,13). We detected I244T in sufficient frequency (6.6 to 19%) in two different PH1 popula- only three of the 110 unrelated alleles screened (frequency of tions (Dutch and Canadian) (23,28) and again here (6.3%), 2.7%). Both patients (probands 40 and 50) were of Spanish making it a worthwhile addition to the PH1 molecular diag- descent (from Spain and southwest United States). nostic service. G156R, a less common exon 4 mutation, previ- After exons 1, 4, and 7, exon 6 contained the next most ously reported in patients of Israeli Arab and Italian descent frequent number of mutant alleles (five of 110) in our PH1 (30,31), was also found here in a frequency of 4.5%. cohort. Sequence conservation in this part of the coding region We detected G170R, the most common mutation in PH1, in is essential to AGT catalytic activity because exon 6 contains the 41 of our 110 unrelated alleles (frequency of 37%). This allelic highly conserved Lys209 residue, which is critical for co-factor frequency most closely resembles that of a Dutch PH1 cohort of (pyridoxal phosphate) binding via Schiff base formation. A 33 patients (allelic frequency 43%) (28). The pathogenic basis single sequence change (S205P) has been reported on this exon, (peroxisome-to-mitochondria mistargeting) of this change has in a patient of Japanese descent (25). It is interesting that we been well established in vitro (8) and is supported by segrega- detected three new sequence changes (D201E, Y204X, and tion analysis (32) and by its absence in screened normal con- G216R) in this part of the AGXT coding region. G216R was also trols, both in this cohort and elsewhere (7). just reported in one other patient with PH1 and shown to The c.33_34insC microinsertion has been documented in peo- segregate with disease in the affected family (36). ple of various ethnicities (13,27,30), in frequencies (12 to 14%) Additional noteworthy observations that arose from this that qualify for the second most common AGXT mutation. In fully sequenced PH1 cohort included detection of a new 3-bp, our cohort, the frequency of c.33_34insC was comparable (11%). in-frame microdeletion (V139del) on exon 3 (proband 43), In addition to detection of c.33_34insC and other, less frequent bringing the total number of reported (12) AGXT microinser- changes that are located on exon 1, direct sequencing of this tion/deletions to 14. V139del is the first reported mutation for part of the coding region has the advantage of supplying P11L this exon, the smallest for AGXT, consisting of only 65 bp genotyping. Because of the strict association between P11L and (GenBank accession no. M61756). Direct sequencing also facil- IVS1 ϩ 74 bp documented in the past (9,11), the latter change itated detection of three new splice variants (probands 25, 26, has been used as a marker for the minor AGXT allele. The and 30), for an overall frequency of AGXT splice-site variants of recent recognition of a breakage in this linkage, documented in approximately 10%. an African population (33), underscores that IVS1 ϩ 74 bp may Excluding our homozygous probands for common mutations no longer be a suitable surrogate for P11L in certain popula- (probands 1 to 10, 46, 47, and 50), there were an additional four tions. apparently homozygous patients in our group (probands 48, 1912 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 1905–1914, 2007

Table 7. Frequencies of AGXT sequence variants detected in normal control subjects and in patients with PH1a

Allelic Frequencies Exon Sequence Variants Controls PH1

1 c.32 C3T (P11L) 18/90 (20%) 64/110 (58%) IVS1 ϩ 74 bp 12/50 (24%) 65/110 (59%) 2 c.264 C3T (A88A) 19/92 (21%) 63/108 (58%) (IVS2 ؉ 13 C3T 18/92 (20%) 63/106 (59% 3 None n ϭ 94 alleles (IVS4 ؉ 91 C3T 17/58 (29%) 22/102 (22% 4 5 None n ϭ 90 alleles 6 c.654 G3A (S218S) 8/90 (9%) 6/96 (6%) (IVS6 ؉ 17 C3T 16/90 (18%) 56/92 (61% 7 None n ϭ 90 alleles 8 c.836 T3C None (n ϭ 94) 5/104 (5%) IVS7–44 A3G 67/94 (71%) 93/104 (89%) IVS7–45 C3T 40/94 (43%) 60/104 (58%) (IVS8 ؉ 52 G3A 59/94 (63%) 77/104 (74% 9 None n ϭ 96 alleles 10 c.1020 A3G (I340M) 14/92 (15%) 64/108 (59%) IVS10–91 G3A None (n ϭ 92) 1/108 (Ͻ1%) 11 c.1220 C3A(3ЈUTR) 44/94 (47%) 23/108 (21%)

aThe corresponding amino acid change, when appropriate, is given in parentheses. New variants are shown in boldface type. Intronic changes are listed with their corresponding amplicon. For some amplicons, variants were not discernible.

49, 51, and 52). Despite the observed absence of heterozygosity we applied a similar strategy for classifying both our newly dis- across all of the described intragenic AGXT polymorphisms in covered and all previously described AGXT missense variants. four of these patients who were purely homozygous for rare Except for T9N, the overall scores that were calculated for mutations, we did not detect any large gene rearrangements, our 13 newly discovered missense variants suggest that all suggesting ancestral founder haplotypes rather than undetec- are likely pathogenic. Purely based on the Grantham matrix ted hemizygosity. We did, however, identify a new deletion score and sequence alignment data, T9N would not be pre- that involves the 3Ј end of AGXT in proband 54 (Ex11_3ЈUTR dicted to be pathogenic, but its absence in any of the tested del). The haplotype and MLPA data in this proband and her control subjects argues against its being a polymorphism. affected sibling suggest that this deletion extends from exon 11 Screening in a larger cohort of control subjects may prove to at least 2 kb downstream from the 3ЈUTR. The 3Ј deletion otherwise. On the basis of similarly derived data for the 23 end point is outside of the coding region; therefore, in contrast already described AGXT missense variants, all are predicted to an intragenic breakpoint whose effect may cause a frameshift to be pathogenic, whereas (intriguingly) I244T, the Spanish in the reading frame, the predicted effect of this new deletion is founder mutation, is not. These three methods (Grantham truncating. matrix score, multi-sequence alignment score, and normal Apart from common or well-studied mutations, interpreta- control population data) therefore can be taken as being tion and classification of the increasingly detected sequence complementary rather than exclusive and predictive rather variation in AGXT are difficult, especially for diagnostics. As than definitive. As such, confirmatory in vitro functional such, another goal of our investigation was to provide a clas- assays should be performed whenever feasible. sification scheme of pathogenicity. Missense variants in partic- ular, which make up the majority of the described unclassified variants in PH1, are notoriously problematic to characterize in Conclusion the absence of functional assays, segregation analysis, or nor- We report our experience with comprehensive AGXT muta- mal population frequencies. tion analysis in a cohort of 55 unrelated probands with a Recently, a scheme developed for the BRCA1 gene has become definitive diagnosis of PH1. Our data suggest that in a majority a model for classification of missense variants, taking into account of well-characterized patients with PH1 (i.e., those with avail- the scoring system of chemical differences between amino acids ability of complete biochemical data and a high clinical index of that initially was developed by Grantham (16) and sequence con- suspicion), a molecular diagnosis using direct sequencing is servation data that were taken from multiple sequence alignment feasible, having higher sensitivity (98%) than the current re- (20). Since such an approach had not yet been instituted for PH1, striction enzyme–based approach (62%) (13), even when lim- J Am Soc Nephrol 18: 1905–1914, 2007 Mutation Screening in Type 1 Primary Hyperoxaluria 1913

Figure 2. Multiple sequence alignment of alanine:glyoxylate aminotransferase (AGT) orthologs. Conserved amino acids are shown in black, similar amino acids are in gray, and mismatches are in white. A letter indicating the corresponding amino acid change for all newly discovered missense variants is shown directly above the Homo sapiens sequence. ited to the three exons with the highest mutation frequencies Human peroxisomal L-alanine:glyoxylate aminotransferase. (77%). Evolutionary loss of a mitochondrial targeting signal by point Furthermore, we herewith provide the first pathogenic classifi- mutation of the initiation codon. Biochem J 268: 517–520, 1990 cation scheme for all new and old AGXT variants of unknown 3. Purdue PE, Lumb MJ, Fox M, Griffo G, Hamon-Benais C, clinical significance via provision of evolutionary conservation Povey S, Danpure CJ: Characterization and chromosomal mapping of a genomic clone encoding human alanine: and normal control population data. Similar analyses of additional glyoxylate aminotransferase. Genomics 10: 34–42, 1991 AGXT missense variants that are detected in the future may prove 4. Cooper PJ, Danpure CJ, Wise PJ, Guttridge KM: Immunocy- useful in interpreting their functional significance. tochemical localization of human hepatic alanine:glyoxylate aminotransferase in control subjects and patients with pri- Acknowledgments mary hyperoxaluria type 1. J Histochem Cytochem 36: 1285– We kindly thank Dr. Christopher J. Ward for invaluable assistance 1294, 1988 with bioinformatics, our patients for their gracious participation, and 5. Danpure CJ, Jennings PR: Further studies on the activity and the National Institutes of Health (DK 64865 and DK 73354) and Oxalosis subcellular distribution of alanine:glyoxylate aminotransfer- and Hyperoxaluria Foundation for research funding. ase in the livers of patients with primary hyperoxaluria type 1. Clin Sci 75: 315–322, 1988 Disclosures 6. Danpure CJ, Cooper PJ, Wise PJ, Jennings PR: An enzyme None. trafficking defect in two patients with primary hyperoxaluria type 1: Peroxisomal alanine:glyoxylate aminotransferase re- routed to mitochondria. J Cell Biol 108: 1345–1352, 1989 References 7. Purdue PE, Takada Y, Danpure CJ: Identification of muta- 1. Danpure CJ, Jennings PR: Peroxisomal alanine:glyoxylate tions associated with peroxisome-to-mitochondrion mistar- aminotransferase deficiency in primary hyperoxaluria type I. geting of alanine/glyoxylate aminotransferase in primary hy- FEBS Lett 201: 20–24, 1986 peroxaluria type 1. J Cell Biol 111: 2341–2351, 1990 2. Takada Y, Kaneko N, Esumi H, Purdue PE, Danpure CJ: 8. Lumb MJ, Danpure CJ: Functional synergism between the 1914 Journal of the American Society of Nephrology J Am Soc Nephrol 18: 1905–1914, 2007

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