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
Address correspondence to: Dr. Carla G. Monico, Mayo Clinic Hyperoxaluria Center and Departments of Internal Medicine and Pediatric and Adolescent Medicine, Divisions of Nephrology and Pediatric Nephrology, Mayo Clinic College of Medicine, Rochester, MN 55902. Phone: 507-266-1045; Fax: 507-266-7891; E-mail: monico.carla{at}mayo.edu
Received for publication November 10, 2006.
Accepted for publication March 14, 2007.
Mutations in AGXT, a locus mapped to 2q37.3, cause deficiencyof liver-specific alanine:glyoxylate aminotransferase (AGT),the metabolic error in type 1 primary hyperoxaluria (PH1). Geneticanalysis of 55 unrelated probands with PH1 from the Mayo ClinicHyperoxaluria Center, to date the largest with availabilityof complete sequencing across the entire AGXT coding regionand documented hepatic AGT deficiency, suggests that a moleculardiagnosis (identification of two disease alleles) is feasiblein 96% of patients. Unique to this PH1 population was the higherfrequency of G170R, the most common AGXT mutation, accountingfor 37% of alleles, and detection of a new 3' end deletion (Ex11_3'UTR del). A described frameshift mutation (c.33_34insC)occurred with the next highest frequency (11%), followed byF152I and G156R (frequencies of 6.3 and 4.5%, respectively),both surpassing the frequency (2.7%) of I244T, the previouslyreported third most common pathogenic change. These sequencingdata indicate that AGXT is even more variable than formerlybelieved, with 28 new variants (21 mutations and seven polymorphisms)detected, with highest frequencies on exons 1, 4, and 7. Whenlimited to these three exons, molecular analysis sensitivitywas 77%, compared with 98% for whole-gene sequencing. Theseare the first data in support of comprehensive AGXT analysisfor the diagnosis of PH1, obviating a liver biopsy in most well-characterizedpatients. Also reported here is previously unavailable evidencefor the pathogenic basis of all AGXT missense variants, includingevolutionary conservation data in a multisequence alignmentand use of a normal control population.
Type 1 primary hyperoxaluria (PH1; OMIM 259900) is a rare butpotentially life-threatening inborn error of metabolism. Inheriteddeficiency of a liver-specific enzyme (alanine:glyoxylate aminotransferase[AGT]; E.C.2.6.1.44) causes impaired glyoxylate metabolism inperoxisomes of human hepatocytes (1). This autosomal recessivetrait is invariably characterized by marked hyperoxaluria withor without associated hyperglycolic aciduria, calcium oxalateurolithiasis or nephrocalcinosis, and progressive loss of renalfunction over time.
In 1990, normal human AGT cDNA was isolated and sequenced (2)(GenBank X53414 and NM_000030). Characterization and mappingof a genomic clone (designated as AGXT) to the telomeric regionof chromosome 2 (2q36–37) followed, with ascertainmentof a coding composition of 11 exons spread across 10 kb (GenBankM61755 to M61763 and M61833) (3). Southern blotting using anisolated full-length AGT cDNA probe determined that human AGTwas encoded singly (3).
Early description of human AGT using protein A-gold immunocytochemistryand isopycnic density gradient centrifugation studies revealeda unique AGT-targeting defect: Mislocalization of 90% of theprotein from peroxisomes to mitochondria in some patients withPH1 (4–6). Cloning followed by sequencing of human AGTcDNA that was isolated from livers of patients with this peroxisome-to-mitochondriamistargeting phenotype showed three sequence variants in thecoding region (P11L, G170R, and I340M) (7).
Of these, only G170R has been shown to be disease specific,although P11L has been demonstrated to modify disease expressionin vitro (8). In 2000, Lumb et al. (8) showed that presenceof P11L alone reduced the activity of AGT by a factor 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 corroborated by the factthat in patients with PH1 described so far, three of these fourmutations (G170R, F152I, and I244T) seem to segregate only incis with P11L. It is postulated that inheritance of these commonvariants opposite P11L would not give rise to the PH1 phenotype(8).
Subsequent to detection of the P11L and I340M polymorphismsin patients with PH1 and mitochondrial AGT, Purdue et al. (9)also identified a closely linked 74-bp duplication in intron1 (IVS1 + 74 bp). These three polymorphic variants (P11L, I340M,and IVS1 + 74 bp) are now collectively referred to as the "minor"allele of AGXT. A second normal haplotype of AGXT that lacksthese changes is recognized as the "major" allele. Publishedfrequencies for the minor AGXT allele in normal populationsrange from approximately 2.3% in Chinese to as high as 28% inSaami (10). In PH1, the frequency of the minor AGXT allele ishigher (approximately 50%), attributed to the predilection formore common mutations (G170R, F152I, and I244T) to segregatesolely with this allele (11).
As of 2004, there were a total of 55 AGXT sequence variantsreported in the Human Gene Mutation Database (www.hgmd.cf.ac.uk),34 (approximately 62%) of which are missense or nonsense changes.The remaining mutations include six splicing changes, eightsmall deletions, four small insertions, one small insertion/deletion,and two large deletions. Fifty of these variants, many to datelargely unclassified in terms of pathogenicity, were recentlysummarized by Coulter-Mackie and Rumsby (12) in the single availablereview of published AGXT sequence changes. In a separate reportfrom these same authors, molecular analysis sensitivity was62% for a large series of 287 probands with liver biopsy–provenPH1, using restriction enzyme-based screening for the now recognizedthree most common AGXT mutations (G170R, c.33_34insC, and I244T)(13). Detection of two mutant alleles was feasible in only 99(34.5%) patients.
Given the disappointing results of this and earlier reports(14,15) regarding the application of limited mutation screeningusing restriction enzyme digestion for the molecular diagnosisof PH1, in this investigation, we assessed the diagnostic relevanceof performing whole-gene sequencing. In an effort to establishthe pathogenicity of all previously described and newly discoveredAGXT missense variants, we also report a classification strategythat is based on the scheme developed by Grantham (16) whileat the same time using evolutionary sequence conservation andnormal population data.
To determine the feasibility of using comprehensive mutationanalysis for establishing a molecular-based diagnosis of PH1and to expand further on the heterogeneity of AGXT, we sequencedthe entire coding region of AGXT in 64 patients with PH1 fromthe Mayo Clinic Hyperoxaluria Center. A definitive diagnosisof PH1 was based on biochemical evidence along with hepaticenzyme analysis that documented AGT deficiency in the patient(n = 48), an affected sibling (n = 8), a first cousin (n = 1),or supporting molecular data (n = 7). For our normal controlpopulation, we screened 50 DNA samples of individuals of predominantlyEuropean and North American descent. The study was approvedby our institutional review board, and all participants providedinformed consent or assent.
Genomic DNA was extracted from peripheral blood leukocytes usingstandard methods. The primer pairs and PCR reaction conditionsthat were used to amplify and sequence the 11 exons and exon-intronboundaries of AGXT are listed in Table 1. Primer design wasbased on the available published genomic sequence of AGXT (GenBankNT_005416). The promoter region of AGXT was not screened. Forall PCR reactions, we used 50 to 100 ng of genomic DNA, 5 to10 pmol of reverse and forward primers, 0.25 U of AmpliTaq Gold(Applied Biosystems, Foster City, CA), and 200 µM dNTP(Invitrogen, Carlsbad, CA) in a total volume of 25 µl,with addition of DMSO for optimization. Amplification (94°C30 s, Ta 58 to 62 °C 30 s, and 72°C 30 s for denaturation,annealing, and extension steps, respectively) was performedin an MBS Satellite 0.2G Thermal Cycler (Applied Biosystems)for 25 to 30 cycles. PCR products were cleaned using ExoSAPIT, per the manufacturer’s (USB, Cleveland, OH) instructions.Sequencing was performed in both directions using the ABI PRISM3700 DNA Analyzer (Applied Biosystems), and chromatograms wereanalyzed with the 4.5 version of Sequencher Software (Gene CodesCorp., Ann Arbor, MI). Positive results were sequenced in duplicate,using a separately amplified PCR product.
Table 1. Flanking primer pairs and annealing temperatures that were used to amplify the 11 exons and exon-intron boundaries of AGXT
To screen for complex alleles (large deletions or insertions)and to increase the sensitivity of molecular analysis, we appliedLuminex FlexMAP tag/anti-tag system technology (17,18) to themultiplex ligation-dependent probe amplification (MLPA) techniqueinitially described by Schouten et al. (19). In this method,two template-specific probes are designed to screen for genecopy changes in the genetic region of interest: A short probe(approximately 25 bp) that consists of template-specific sequenceand universal primer, along with a longer probe (approximately75 bp) that is made of template-specific sequence, universalprimer, and "stuffer" sequence complementary to a selected FlexMapbead. Long probes are phosphorylated to carry out the ligationreaction. The selected intragenic and extragenic probe sequencesare listed in Table 2.
Table 2. MLPA probe sequences for exons 1, 4, 9, 10, and 11 of AGXTa
For the multiplexing steps, we used the commercially availableMLPA Kit from MRC Holland (Amsterdam, The Netherlands). PCRwas performed for 24 cycles (30 s at 95°C, 30 s at 60°C,and 60 s at 72°C) using Platinum Taq (Invitrogen). Beadhybridization followed, using 40 µl of FlexMap bead mixplus 10 µl of PCR product, for a 1-h incubation periodat 37°C. We then added 25 µl of a streptavidin phycoerythrin/tetramethyl-ammoniumchloride solution to each reaction well (2 µl of Streptavidin,R-phycoerythrin conjugate [1 mg/ml] and 250 µl of 1x tetramethyl-ammoniumchloride). This mixture was incubated at room temperature for15 min, and the number of FlexMap beads that successfully hybridizedwas counted with a Luminex LX100. Fluorescence intensity thatwas generated from each sample compared with control probes(expressed as a peak ratio) was then used to assess its copynumber. In the experience of D.B.D. and P.A.L., this methodhas proved robust in quantifying gene dosage changes in patientswith Niemann-Pick type C (17,18).
To classify the new AGXT missense variants described here, wedeveloped and applied a scoring system that is based on thematrix of Grantham (16) and Abkevich et al. (20). Overall scoresare provided for all described and newly detected AGXT missensemutations. Finally, we used the 8.1.1 version of the Mac Vectorprogram to generate a multiple sequence alignment of the followingAGT orthologs (corresponding GenBank accession numbers): Homosapiens (BAA02632), Canis familiaris (XP_848328.1), Felis catus(CAA53527.1), Oryctolagus cuniculus (S24155), Rattus rattus(CAA29656.1), Mus musculus (AAH25799.1), Xenopus tropicalis(NP001006705.1), and Danio rerio (AAH76465.1).
Overall, our fully sequenced cohort consisted of 64 patientswith PH1 (55 unrelated probands, eight affected siblings, andone first cousin). AGXT genotyping in the 55 unrelated probandsis listed in Table 3. The 21 newly discovered mutations (12missense, three nonsense, three splice site, two microinsertion/deletion,and one large deletion) are shown in boldface type. Direct sequencingof the entire AGXT coding region revealed two disease allelesin 52 (95%) patients and a single pathogenic change in the remainingthree. In the unrelated PH1 group as a whole, minor and majorallelic frequencies were 58% (64 of 110 alleles) and 42% (46of 110 alleles), respectively.
Table 3.AGXT genotyping in 55 unrelated PH1 probandsa
Two additional AGXT sequence variants are worthy of specialmention. The first is a T C transition in exon 8 (c.836 T C, I279T), which was detected in probands 31, 33, 43, and 46,the significance of which was not clear because it was not detectedin any of the control subjects screened and was found in tandemwith two disease alleles in probands 33, 43, and 46. Previousexpression of this variant by Coulter-Mackie et al. (21) onthe background of the major AGXT allele, however, yielded onlya minimal effect on AGT catalytic activity. The second is anintronic change (IVS10–91 G A) in proband 42, which segregatedwith disease in her affected pedigree and again was detectedin the setting of two other seemingly pathogenic changes.
Of the 48 unrelated probands with PH1 and availability of hepaticenzyme analysis, two disease alleles were satisfactorily detectedin 46 (96%), and a single disease allele was detected in two,yielding a sensitivity of 98% (94 of 96 alleles detected) forthis whole-gene sequencing approach. When limited to the threeexons with the highest mutation frequencies (exons 1, 4, and7; Table 4), the sensitivity of molecular analysis for the liverbiopsy cohort was 77% (72 of 94 alleles detected).
Table 4. Exon-specific combined mutation frequencies for the 48 unrelated probands with PH1 and availability of hepatic enzyme analysisa
To date, only two large partial AGXT deletions (5' untranslatedregion [UTR] to IVS5 and 5' UTR to IVS7) and a single case ofsegmental maternal isodisomy of 2q37.3 have been published (22–24).A likelihood of hemizygosity is also supported by a few otherrare (frequency <5%) mutations in PH1 that have been detectedonly in homozygous form (S205P and G82E) in families of reportedlynonconsanguineous backgrounds (25,26).
To exclude the possibility that large deletions may have goneundetected in our purely homozygous probands for rare mutations(48, 49, 51, and 52) and in probands with a singly identifiedmutation (31, 54, and 55), we strategically designed MLPA probes(exons 1, 4, and 11) across the AGXT coding region. Haplotypeanalysis for probands 48, 49, 51, 52, and 54 is listed in Table 5.
Table 5. Haplotype analysis in the four seemingly homozygous probands for rare mutations and in proband 54 (patient with newly detected partial gene deletion, Ex 11_3'UTR del)a
We did not detect changes in gene copy in any of these testedpatients, with the exception of proband 54 (Figure 1), in whomwe successfully detected a new deletion that involved exon 11,also confirmed separately in an affected sibling. Because thehaplotype analysis in this patient suggested a potential partialdeletion that affected intron 8 to the 3' end of AGXT (Table 5),we designed six additional probes (exon 9, 10, 3' 1 kb, 3' 2kb, 3' 80 kb, and GAL3ST2) to delineate its extent. These addedprobes verified that this new deletion encompasses solely exon11 of AGXT and that it extends >2 kb downstream from the3' UTR of the gene (Figure 1).
Figure 1. Multiplex ligation-dependent probe amplification analysis. The five control (breast cancer anti-estrogen 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, 3' 1 kb, 3' 2 kb, 3' 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 3' 1 kb, 3' 2 kb, and 3' 80 kb are located 1, 2, and 80 kb from the 3' 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 3' UTR is shown. For the control probes, BCAR3, chromosome 1; CTNNB1, chromosome 3; TNFRSF7, chromosome 12; HIRA, chromosome 22; DMD, exon 11, gene dosage control, X-chromosome.
The classification system that was developed here for AGXT (Table 6)represents the first attempt to gauge the pathogenicity of allreported missense variants. Our control population data, includingnumber of alleles tested and allelic frequencies for the sevennew (shown in boldface type) and described AGXT polymorphismsin comparison with PH1 is depicted in (Table 7). None of thenew AGXT missense variants was detected in the normal controlpopulation tested. The multisequence alignment is shown in Figure 2.
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.
Using this whole-gene sequencing approach, we show a sensitivityfor molecular analysis of 98% in 48 liver biopsy–provencases of PH1, an improvement of 36% over the previously publishedrestriction enzyme–based screen for the three most commonmutations in AGXT (G170R, c.33_34insC, and I244T) (13). Comprehensivemutation screening by direct sequencing therefore seems to bea satisfactory method for detection of sequence variation inAGXT and appropriate for molecular diagnosis of PH1. Given thesmall size of the gene, molecular analysis is relatively inexpensiveand easily achieved. Because of the wide range of mutation typesdetected (missense, nonsense, microinsertion/deletions, andsplice variants), direct sequencing of AGXT has the added benefitof contributing to our molecular understanding of PH1, despitethe high prevalence of private mutations.
Even if sequencing is limited to the three exons that containthe more common mutations (1, 4, and 7), the sensitivity remainsconsiderably higher (77%) than the reported mutation-specificrestriction enzyme approach (62%) (13). For diagnostic purposes,a prioritization scheme that consists first of limited sequencing(of exons 1, 4, and 7) and then direct sequencing of the remainingexon and exon-intron boundaries with addition of family studieswhen indicated seems to be a suitable approach. Mutation analysismay also be targeted to a particular ethnicity for which thereare known AGXT associations (e.g., I244T in Spanish populations).A liver biopsy would then be required only when this molecularapproach proves nondiagnostic.
Both published reports (27–31) and data that were obtainedhere confirm that less common mutations are also found on thesesame exons (1, 4, and 7), substantiating the strategy to sequencethese coding regions directly, in lieu of performing mutation-specificrestriction enzyme–based assays. A second exon 4 mutationin particular (F152I) has been reported in sufficient frequency(6.6 to 19%) in two different PH1 populations (Dutch and Canadian)(23,28) and again here (6.3%), making it a worthwhile additionto the PH1 molecular diagnostic service. G156R, a less commonexon 4 mutation, previously reported in patients of IsraeliArab and Italian descent (30,31), was also found here in a frequencyof 4.5%.
We detected G170R, the most common mutation in PH1, in 41 ofour 110 unrelated alleles (frequency of 37%). This allelic frequencymost closely resembles that of a Dutch PH1 cohort of 33 patients(allelic frequency 43%) (28). The pathogenic basis (peroxisome-to-mitochondriamistargeting) of this change has been well established in vitro(8) and is supported by segregation analysis (32) and by itsabsence in screened normal controls, both in this cohort andelsewhere (7).
The c.33_34insC microinsertion has been documented in peopleof various ethnicities (13,27,30), in frequencies (12 to 14%)that qualify for the second most common AGXT mutation. In ourcohort, the frequency of c.33_34insC was comparable (11%). Inaddition to detection of c.33_34insC and other, less frequentchanges that are located on exon 1, direct sequencing of thispart of the coding region has the advantage of supplying P11Lgenotyping. Because of the strict association between P11L andIVS1 + 74 bp documented in the past (9,11), the latter changehas been used as a marker for the minor AGXT allele. The recentrecognition of a breakage in this linkage, documented in anAfrican population (33), underscores that IVS1 + 74 bp may nolonger be a suitable surrogate for P11L in certain populations.
To date, several mutations have been documented on exon 7 ofAGXT (29), the most common of which is I244T, a founder mutationin Spanish patients who originated from a small island of theCanary Islands called La Gomera (34), where its frequency is92% (35). The reported frequency for I244T has otherwise rangedfrom 6 to 9% (11,13). We detected I244T in only three of the110 unrelated alleles screened (frequency of 2.7%). Both patients(probands 40 and 50) were of Spanish descent (from Spain andsouthwest United States).
After exons 1, 4, and 7, exon 6 contained the next most frequentnumber of mutant alleles (five of 110) in our PH1 cohort. Sequenceconservation in this part of the coding region is essentialto AGT catalytic activity because exon 6 contains the highlyconserved Lys209 residue, which is critical for co-factor (pyridoxalphosphate) binding via Schiff base formation. A single sequencechange (S205P) has been reported on this exon, in a patientof Japanese descent (25). It is interesting that we detectedthree new sequence changes (D201E, Y204X, and G216R) in thispart of the AGXT coding region. G216R was also just reportedin one other patient with PH1 and shown to segregate with diseasein the affected family (36).
Additional noteworthy observations that arose from this fullysequenced PH1 cohort included detection of a new 3-bp, in-framemicrodeletion (V139del) on exon 3 (proband 43), bringing thetotal number of reported (12) AGXT microinsertion/deletionsto 14. V139del is the first reported mutation for this exon,the smallest for AGXT, consisting of only 65 bp (GenBank accessionno. M61756). Direct sequencing also facilitated detection ofthree new splice variants (probands 25, 26, and 30), for anoverall frequency of AGXT splice-site variants of approximately10%.
Excluding our homozygous probands for common mutations (probands1 to 10, 46, 47, and 50), there were an additional four apparentlyhomozygous patients in our group (probands 48, 49, 51, and 52).Despite the observed absence of heterozygosity across all ofthe described intragenic AGXT polymorphisms in four of thesepatients who were purely homozygous for rare mutations, we didnot detect any large gene rearrangements, suggesting ancestralfounder haplotypes rather than undetected hemizygosity. We did,however, identify a new deletion that involves the 3' end ofAGXT in proband 54 (Ex11_3'UTR del). The haplotype and MLPAdata in this proband and her affected sibling suggest that thisdeletion extends from exon 11 to at least 2 kb downstream fromthe 3'UTR. The 3' deletion end point is outside of the codingregion; therefore, in contrast to an intragenic breakpoint whoseeffect may cause a frameshift in the reading frame, the predictedeffect of this new deletion is truncating.
Apart from common or well-studied mutations, interpretationand classification of the increasingly detected sequence variationin AGXT are difficult, especially for diagnostics. As such,another goal of our investigation was to provide a classificationscheme of pathogenicity. Missense variants in particular, whichmake up the majority of the described unclassified variantsin PH1, are notoriously problematic to characterize in the absenceof functional assays, segregation analysis, or normal populationfrequencies.
Recently, a scheme developed for the BRCA1 gene has become amodel for classification of missense variants, taking into accountthe scoring system of chemical differences between amino acidsthat initially was developed by Grantham (16) and sequence conservationdata that were taken from multiple sequence alignment (20).Since such an approach had not yet been instituted for PH1,we applied a similar strategy for classifying both our newlydiscovered and all previously described AGXT missense variants.
Except for T9N, the overall scores that were calculated forour 13 newly discovered missense variants suggest that all arelikely pathogenic. Purely based on the Grantham matrix scoreand sequence alignment data, T9N would not be predicted to bepathogenic, but its absence in any of the tested control subjectsargues against its being a polymorphism. Screening in a largercohort of control subjects may prove otherwise. On the basisof similarly derived data for the 23 already described AGXTmissense variants, all are predicted to be pathogenic, whereas(intriguingly) I244T, the Spanish founder mutation, is not.These three methods (Grantham matrix score, multi-sequence alignmentscore, and normal control population data) therefore can betaken as being complementary rather than exclusive and predictiverather than definitive. As such, confirmatory in vitro functionalassays should be performed whenever feasible.
We report our experience with comprehensive AGXT mutation analysisin a cohort of 55 unrelated probands with a definitive diagnosisof PH1. Our data suggest that in a majority of well-characterizedpatients with PH1 (i.e., those with availability of completebiochemical data and a high clinical index of suspicion), amolecular diagnosis using direct sequencing is feasible, havinghigher sensitivity (98%) than the current restriction enzyme–basedapproach (62%) (13), even when limited to the three exons withthe highest mutation frequencies (77%).
Furthermore, we herewith provide the first pathogenic classificationscheme for all new and old AGXT variants of unknown clinicalsignificance via provision of evolutionary conservation andnormal control population data. Similar analyses of additionalAGXT missense variants that are detected in the future may proveuseful in interpreting their functional significance.
We kindly thank Dr. Christopher J. Ward for invaluable assistancewith bioinformatics, our patients for their gracious participation,and the National Institutes of Health (DK 64865 and DK 73354)and Oxalosis and Hyperoxaluria Foundation for research funding.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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J. Am. Soc. Nephrol. 2007 18: 1617-1618.
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Abstract
Introduction
C transition in exon 8 (c.836 T 
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, 3' 1 kb, 3' 2 kb, 3' 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 3' 1 kb, 3' 2 kb, and 3' 80 kb are located 1, 2, and 80 kb from the 3' 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 3' UTR is shown. For the control probes, BCAR3, chromosome 1; CTNNB1, chromosome 3; TNFRSF7, chromosome 12; HIRA, chromosome 22; DMD, exon 11, gene dosage control, X-chromosome.