Analysis of the Galactose-1-Phosphate Uridyltransferase Gene in Specimens Identified through Newborn Screening for Galactosemia Using the Original

Dried Blood Specimen and Hi-Res Melting

Steven F. Dobrowolski*, Jason T. McKinney*, Edwin W. Naylor+, Zhili Lin+, Kasinathan Muralidharan, Carol J. Saunders#, Fay Keune, Jesse Harbour*, James B. Gibson^, Clinton R. Ellingson*
* Idaho Technology, Salt Lake City, Utah, + Pediatrix Screening, Bridgeville, Pennsylvania, Dept. of Human Genetics, Emory University School of Medicine, Atlanta, Georgia, # The Childrens Mercy Hospitals and Clinics, Kansas City, Missouri, ^ Dept of Pediatrics, UAMS College of Medicine, Arkansas Childrens Hospital, Little Rock, Arkansas, Utah Dept. of Health, Newborn Screening Program, Salt Lake City, Utah

Abstract
Newborn screening for galactosemia identifies candidate patients. The newborn dried blood spot (DBS) is used to assay common mutations but is perceived as an inadequate sample to routinely support comprehensive gene analysis. Herein is demonstrated the utility of the DBS to support comprehensive analysis of the galactose-1-phosphate uridyltransferase (GALT) gene. The DBS provided amplification template to assay the GALT gene in specimens identified by newborn screening as having elevated galactose and/or reduced GALT enzyme activity. PCR is performed in the presence of the dye LCGreen Plus. Following PCR, dye-saturated product was assayed using Hi-Res Melting. Samples producing aberrant melting profiles were recovered and sequenced to determine the nucleotide variant. Thirtynine DBS specimens testing outside critical cut-offs in screening for galactosemia and one sample from a clinically diagnosed patient were evaluated for mutations in the GALT gene. Eighteen mutations were identified using Hi-Res Melting, of these 8 were novel: (D39Xfs (c17-18 del CC), Del D97 (c289-291 del AAC), M142K (c425 T>A), Y251S (c752 A>C, c753 C>T), R263G (c787 C>G), T284N (c851 C>A), M298V (c892 A>G), Y323C (c968 A>G)) and 10 were previously reported: (R67H (c200 G>A), S135L (c404 C>T), T138M (c413 C>T), F171S (c512 T>C), Q188R (c563 A>G), L195P (c584 T>C), R231C (c691 C>T), N314D (c940 A>G), R333W (c997 C>T), Q344K (c1030 C>A)). In addition, 4 previously reported polymorphisms (IVS4 -27 G>C, IVS5 24G>A, L218L (c652 C>T), H315H (c945 T>C)) as well as 4 novel polymorphisms (IVS3 del 23 - -28, IVS5 49 T>A, N282N (c864 C>T), T292T (c876 G>A)) were identified. A 3.2 mm punch from the newborn screening DBS provided sufficient template for comprehensive analysis of the GALT gene. High resolution melting localizes sequence variation allowing DNA sequencing to be targeted to required regions. Thermal denaturation is non-destructive enabling the same amplification product to be used as DNA sequencing template. Comprehensive analysis of the GALT gene including isolating DNA from DBS, PCR, Hi-Res Melting, and preparation of sequencing reactions was completed in 4 hours. Using DNA from the DBS and gene scanning with Hi-Res Melting is applicable to any disorder identified by newborn screening where molecular genetic analysis is useful to the diagnostic regimen.

Results
Figure 1 shows subtractive difference plots for exons 1-10. No samples showed variation in exon 11. Each panel contains 10 wild type profiles and those samples with deviant profiles. Initially sample DBS12 provided molecular data that indicated it to be homozygous for the F171S mutation but the biochemical genetic evidence was not consistent with this genotype (see Table 2). Figure 2 analyzes selected F171S-positive samples with an alternative primer set that flanks the exon 6 primers described in Table 1. The profile of DBS12 is unique among the F171S-positive specimens and sequencing identified the novel IVS5 49 T>A polymorphism that destabilized the forward primer described in Table 1 and led to allele specific amplification. Figure 3 shows the analysis of the N314D and H315H variants. Table 2 summarizes newborn screening and molecular genetic results.

Conclusions
Screening for galactosemia using biochemical genetic means is complicated by partial enzyme deficiency and carrier status range. These issues make molecular genetic evaluation valuable to triage false positives and identify candidate patients. The DBS is available for follow-up molecular genetic evaluation immediately after abnormal screening results are realized. Less than 20% of the DNA isolated from a single 3.2 mm punch was required to assay the GALT gene. Nine samples are from classical galactosemia patients (DBS1, 6, 9, 15, 21, 26, 28, 29,38). Biochemical genetic evidence was not available for specimens DBS1 and DBS38, but genotypes (Q188R/R333W and Q188R/S192N) are consistent with classic disease. Samples DBS6, 15, and 28 have biochemical genetic data and genotypes consistent with disease. DBS21 and 29 have 2 disease-causing mutations (DBS21 Q188R/R333W, DBS29 R67H/R231C), but their galactose levels were in the normal range. DBS26 has elevated galactose and normal enzyme activity. This sample is homozygous for F171S, which retains partial (7%) enzyme activity. Genotype analysis was helpful in clarifying the status of samples DBS21, 26 and 29, since the biochemical profiles were not consistent with what is typically observed in classical disease. Sample DBS9 has biochemical data consistent with classical disease yet only the Q188R mutation was identified. A second mutation in a region not interrogated by the genescanning panel is likely. In 4 specimens (DBS17, 31, 32, 34) no mutations were identified. Samples DBS31 and DBS32 had elevated galactose with normal enzyme activity. Elevated galactose could indicate the sample was collected after feeding, as a transient increase of galactose is possible when an infant is fed milk-based formula or breast milk. Defects in galactose-4-epimerase and galactose kinase are characterized by increased galactose but associated with different genes. In DBS34 the initial newborn screening sample had reduced GALT activity but a second showed GALT activity in the normal range. DBS17 had reduced GALT enzyme activity without identifiable mutations. Biochemical results were not confirmed but it is possible that DBS17 carries a mutation outside the region interrogated by the gene-scanning panel. The utility of the DBS to support comprehensive gene analysis is not fully appreciated. This study demonstrates the DBS provides a source of DNA to support comprehensive gene analysis. Using DBS for gene analysis would eliminate the necessity to collect, process, store, analyze, and track many of the additional specimens currently requested.

Figure 1. Subtractive difference plots analyzing GALT exons 1-10. Each panel assays 1 exon, are standardized to wild type, contains profiles from 10 wild type specimens and those specimens having deviant profiles.

Methods
Newborn screening for galactosemia and selection of specimens. Sample DBS1 was identified by clinical evaluation at the University of Arkansas for Medical Sciences. Blood was drawn and spotted on filter paper. Specimens DBS2-5 were identified by Utah Department of Health, Newborn Screening Program using the Perkin-Elmer Neonatal GALT kit according to manufacturers recommendation. Specimens DBS6-40 were identified at Pediatrix Screening (formerly NeoGen). Specimens DBS6-12 were assayed using a modified Beutler semiquantitative screening test to assay GALT enzyme activity and a modification of the Misuma semi-quantitative screening test was used to determine total galactose. Critical cut-offs for these assays were 30% of control activity and 10mg/dl respectively. Specimens DBS13-37, 39, and 40 were identified using the Astoria-Pacific continuous flow quantitative Spotcheck fluorometric assays to determine GALT enzyme activity and total galactose. Cut-offs for these quantitative assays were 60 M and 15 mg/dl respectively. Preparation of DNA from Dried Blood Spots. DNA was isolated from a 3.2 mm punch from the DBS using a manual adaptation of the automated procedure previously described; DNA quantification was unnecessary (1). Polymerase Chain reaction. PCR primers in Table 1 were designed using Genbank sequence M96264. Reactions were performed in 10 l using 1 l of DBS extract as amplification template, 1X PCR buffer (Idaho Technology) containing 2 mM MgCl2 (exons 2-11) or 3 mM MgCl2 (exon 1), 20 mM each dNTP, 1X LCGreen Plus dye, and Klen Taq polymerase (AB Peptides) complexed with TaqStart antibody (BD Biosciences). Amplification was performed in the LightCycler (Roche) using an initial denaturation at 94 C for 1 minute followed by 45 cycles of 94 C for 2 seconds, 62 C for 5 seconds, and 72 C for 2 seconds. Ramp speed was 20 C/second except between 62 C and 72 C, which was 1 C /second. Following amplification, products were denatured at 94 C for 30 seconds and re-annealed by ramping to 40 C. A second amplification product was used to resolve a discrepancy between biochemical genetic data and molecular genetic data in sample DBS12. The primers 5'-TGGGGAGTAACATTTCTGTTTC-3' and 5'-TCCTCTGTCCCATCCATTAATC-3' amplify a 242 bp fragment, flanking the product generated with the exon 6 primer set described in Table 1. Hi-Res Melting. Hi-Res Melting was performed in the HR-1 instrument (Idaho Technology). Fragments were melted at a 0.3 C/second ramp rate. Melting profiles were analyzed with HR-1 software using fluorescence normalization, temperature overlay, and conversion to subtractive difference plots as described (2,3). Samples with variant melting profile were recovered from the capillary and prepared for DNA sequencing. Analysis of N314D and H315H in Exon 10. Owing to the frequency that N314D and H315H are represented in the sample set, a probebased assay was used to identify these sequence variations as described (4,5). Figure 2. Selected specimens were assayed with a primer set flanking that described in Table 1 to assay exon 6. Subtractive difference plots evaluating 4 wild type specimens, DBS12, DBS23, DBS24, and DBS26 are shown.
DBS12 (F171S, IVS5 -24 G>A, IVS5 -49 T>A)

Table 2. Summary of biochemical and molecular genetic data. No newborn screening data is available for DBS1. Samples DBS2-5 were identified using the Perkin-Elmer Neonatal GALT kit. Samples DBS6-12 were assayed using a modified Beutler semi-quantitative screening test to determine GALT activity and the Misuma semi-quantitative screening test to determine galactose. DBS13-40 were assayed using the Astoria-Pacific continuous flow quantitative Spotcheck assay. The exon in which mutations or polymorphisms are located is in parenthesis. Classic = classic galactosemia, G/N = carrier of a pathogenic mutation, D/N = carrier of the N314D Duarte D2 variant, D/G = carries N314D Duarte D2 variant and a pathologic mutation, N/N = having neither a mutation or the N314D Duarte D2 variant, ED/GK? = possible deficiency in galactose-4-epimerase or galactose kinase
Specimen
DBS1 DBS2 DBS3 DBS4 DBS5 DBS6 DBS7 DBS8 DBS9 DBS10 DBS11 DBS12 DBS13 DBS14 DBS15 DBS16 DBS17 DBS18 DBS19 DBS20 DBS21 DBS22 DBS23 DBS24 DBS25 DBS26 DBS27 DBS28 DBS29 DBS30 DBS31 DBS32 DBS33 DBS34 DBS35 DBS36 DBS37 DBS38 DBS39 DBS40

Mutation #1
Q188R (6) S135L (5) F171S (6) fsD39X (1) delta D97 (3) Q188R (6) R263G (8) N314D (10) Q188R (6) M142K (5) delta D97 (3) F171S (6) N314D (10) N314D (10) Q188R (6) Y251S (8) Q188R (6) Q188R (6) T138M (5) Q188R (6) N314D (10) F171S (6) F171S (6) N314D (10) F171S (6) S135L (5) Q188R (6) R67H (2) Q188R (6)

Mutation #2
R333W (10)

Mutation #3

Polymorphisms

Total Gal
NA NA NA NA NA >30 15-30 <10 >30 15-30 >30 >30 3.8 2.6 69.7 21.4 2.5 2 4.2 0.9 2.8 3.5 24.4 6.4 1.5 79.3 2.2 58.8 8.8 7.2 26.5 20.2 1.5 3.1 11.7 1.6 4.9 NA 4.3 2.5

GALT Enzyme
NA 3.2 3.2 2.9 3.5 0% 50% 25% 0% 23-30% 10-12% 53% 31 . 9 59.8 20.5 41 42.5 44.7 53.9 36.3 13 52.4 134.7 26.9 41.5 80.8 29.3 28.2 9.5 34 131.2 214 40.3 51.3 51.7 50.7 54 NA 54.7 31

Putative Phenotype
Classic G/N G/N G/N G/N Classic D/G D/G Classic D/G D/G D/G D/N D/N Classic D/G N/N G/N G/N D/G Classic D/N G/N D /G D/N Classic G/N Classic Classic G/N ED/GK def? ED/GK def? G/N N/N D/G D/N G/N Classic D/N D/G

T292T (9)

R344K (10) N314D (10) Q344K (10) N314D (10) N314D (10) N314D (10)

IVS3 del -23-28 IVS5 -24G>A IVS5 -24G>A IVS5 -24G>A L218L (7) T292T (9), H315H (10) IVS5 -24 G>A, T292T (9), H315H (10) IVS5 -24 G>A, T292T (9), H315H (10) IVS5 -24G>A H315H (10) T292T (9), H315H (10) IVS5 -24G>A L218L (7), T292T (9), H315H (10) IVS5 -24G>A, T292T (9), H315H (10), 2 copies IVS5 -24 G>A, IVS5 -49 T>A, T292T (9), H315H (10) IVS5 -24G>A, T292T (9), H315H (10) H315H (10), 2 copies

Y323C (10)

L195P (7) N314D (10)

N314D (10) R333W (10)

N314D (10) F171S (6) M298V (9) R231C (8)

Q188R (6) F171S (6) N314D (10) S135L (5) Q188R (6) N314D (10) T284N (9) N314D (10)

Table 1. Primers, concentration, and fragment size to assay the GALT gene

Exon
1 2 3 4 5 6 7 8 9 10 11

Forward Primer
5'-TGGACGGAGAAAGTGAAAGGTGAG-3' 5'-GCTCTGAGGACTGATCTTGA-3' 5'-GCAGTGAGTGCTTCTAGC-3' 5'-GAGGGACTTCTGCTGCAGAGA-3' 5'-AGCCAAGCCCTACCTCTC-3' 5'-TCAGGAGGGAGTTGACTT-3' 5'-CCTGTTCTTCTCTGCTTTTG-3' 5'-CCTTGATGACTTCCTATCCATTC-3' 5'-CCAGGCTGAGAGTCAGG-3' 5'-CTCTCCCCACTGTCTCT-3' 5'-CCCAGCCCTTATCCTCCTTAAT-3'

Reverse Primer
5'-GCCCCAGCGAGTCCCTG-3' 5'-GCAGATGCAGGGCTCTA-3' 5'-GTCCCTCAAGAGGTCTAGC-3' 5-GAGTCTCCAACTCTGGTTTGAAAG-3' 5'-TGCCCATCCAGGGGAAG-3' 5 AGTGCTGGCTCAGACTC-3' 5' AGAAAGTTAGGGACTCCTTG-3; 5'-TCCATCCAGTGCCTAGC-3' 5'-GTGTTGGGGCTAAATATAGGTA-3 5'-GAGACGCCAGACTGTTC-3' 5'-GATTCAAGGCCCTGTGGT-3'

Concentration [uM]
0.4 0.4 0.4 0.4 0.2 0.4 0.4 0.4 0.4 0.4 0.4

Size (bp)
180 230 153 129 219 140 201 210 150 214 288

DBS24 (F171S, IVS5 -24 G>A) DBS23 (F171S)

T292T (9), H315H (10) H315H (10) 2 copies T292T (9), H315H (10) H315H (10), 2 copies IVS4 -27 G>C, T292T (9), H315H (10) L218L (7) T292T (9), H315H (10) IVS5 -24G>A, N288N (9), T292T (9), H315H (10) IVS5 -24G>A

S192N (7) N314D (10)

DBS26 (F171S homozygous)

Figure 3. Analysis of the N314D Duarte D2 variant and the H315H polymorphism using a probe-based specific assay. The following primers 5-CTCTCTTCTTTCTGTCAGGG-3 and 5-TTTCGTAGCCAACCATGAAT-3 generate a fragment that flanks the N314D and H315H sites. An oligonucleotide probe using SimpleProbe chemistry (Idaho Technology, Salt Lake City, UT) 5AGCTGCCAATGGTCCCAGT 3 forms a perfect hybrid to the N314D allele, has a single base mismatch with the wild type allele, and has a 2 base pair mismatch with the H315H allele.

Contact Information Steven F. Dobrowolski, Ph.D. steven.dobrowoslki@idahotech.com 801-736-6354 www.idahotech.com

References

Jason T. McKinney jasonm@idahotech.com 801-736-6354 www.idahotech.com

1. Heath EM, O'Brien DP, Banas R, Naylor EW, Dobrowolski SF. Optimization of an automated DNA purification protocol for neonatal screening. Arch Pathol Lab Med. 1999;123(12):1154-60. 2. Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ. High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem. 2003;49(6):853-60 3. Dobrowolski SF, McKinney JT, Amat di San Filippo C, Giak Sim K, Wilcken B, and Longo N. Validation of dye-binding/high-resolution thermal denaturation for the identification of mutations in the SLC22A5 gene. Hum Mutat. 2005;25(3):306-313 4. McKinney JT, Campbell M, Dobrowolski SF, Saunders C. Identification of GALT mutations using specific assays and gene scanning. Genetics in Medicine. 2004;6(4):356. 5. Dobrowolski SF, Banas RA, Suzow JG, Berkley M, Naylor EW. Analysis of common mutations in the galactose-1-phosphate uridyltransferase gene: new assays to increase the sensitivity and specificity of newborn screening for galactosemia. J Mol Diagn. 2003; (1):42-7.