SUPPLEMENTAL METHODS GALC Assay The GALC NBS assay has been described.1 All steps were performed at room temperature and ambient pressure except where otherwise noted. Briefly, GALC assay solution (pH 4.4) containing synthetic substrate (D-galactosyl-β1-1’-N-octanoyl-D-erythro-sphingosine; GALC-S) and internal standard (N-decanoyl-D-erythro-sphingosine; GALC-IS) is added to each well containing a DBS specimen. Plates are incubated at 37ºC for 15-22 hour (hr) with orbital shaking. Reactions are quenched with 200 µL methanol-ethyl acetate (1:1 v/v). Sample aliquots are transferred to deep well plates and liquid-liquid extraction is performed with 400 µl ethyl acetate and 400 µl house-deionized water. After centrifugation, 150 µl of the top (organic) layer is transferred to a flat-bottom microtiter plate and solvent is removed with an air stream. The residue is dissolved in 100 µL of 5 mM ammonium formate in acetonitrile-water (4:1 v/v) or, since January 2009, in methanol-water (4:1 v/v). Samples are then analyzed by MS/MS. Beginning December 30, 2013 the GALC assay was multiplexed with a C26:0 assay for X-linked adrenoleukodystrophy (X-ALD). This addition required us to change the methanol strength of the sample extracts that are tested, from 80:20 methanol:water to a 99:1 methanol:water matrix. An additional function was added to the MS/MS method to analyze the multiple reaction monitoring (MRM) channels required for X-ALD testing. The limit of detection for this assay has been determined to be 0.24 µmol/L/hr and the coefficient of variation is <20%. Molecular Analysis DNA is extracted from duplicate 3-mm DBS punches using the CASM method.2 Protocols for deletion assays, sequencing and melting curve analysis have evolved slightly through time; current protocols are shown for each. 44 Deletion Assays PCR primer sets for amplification across the breakpoint of the 30-kb deletion,3,4 and a 7kb deletion extending from the end of exon 7 into exon 8, are listed in Supplemental Table 1. PCRs are carried out using LightCycler DNA Master HybProbe mix (Roche Applied Science, Indianapolis, IN), MgCl2 (Roche), TaqStart Antibody (Clontech Laboratories, Mountain View, CA), primers (Integrated DNA Technologies, Coralville, IA), 2 µl undiluted DNA, and PCR grade water to a final volume of 40 µl. Reactions are run using a touchdown PCR program consisting of a 5 min initial denaturation at 95°C; 5 cycles of (95°C for 20 s, annealing at 62˚C for 20 s, and elongation at 72°C for 40 s); followed by 10 cycles of (95°C for 20 s, 61˚C for 15 s, 72°C for 40 s); then 30 cycles of (95°C for 20 s, 60˚C for 10 s, 72°C for 40 s); and a final extension for 5 min at 72°C. PCR specifications are listed in Supplemental Table 1. Detection of the 7-kb deletion uses three primers in a single PCR reaction, and detection of the 30-kb deletion is carried out by running separate wild type and mutant PCR reactions. The two reactions targeting the 30-kb deletion region are mixed before running PCR products for both assays on a 1% agarose gel to distinguish by size. A DNA extraction control (wild type), a heterozygous control and a no template control (NTC) are included with each run. GALC Sequencing Eighteen primer sets encompassing the GALC promoter and coding region are listed in Supplemental Table 1. PCR for sequencing reactions is carried out using LightCycler DNA Master HybProbe mix, MgCl2, TaqStart Antibody, forward and reverse primers, 2.5 µl DNA (diluted 1:1.6 [v/v] in water), and PCR grade water to a final volume of 30 µl. Specifications are listed in Supplemental Table 1. The same cycling conditions are used for all 18 reactions. The touchdown PCR program includes a 5 min initial denaturation at 95°C; five consecutive 45 programs with denaturation at 95°C for 15 s, annealing for 30 s; and extension at 72°C for 15 s. The five programs include, in order, 5 cycles with annealing at 62˚C, 5 cycles with annealing at 60˚C, 5 cycles with annealing at 58˚C, 10 cycles with annealing at 54˚C, and 15 cycles with annealing at 52˚C. PCR products are confirmed on 1% agarose gels prior to cycle sequencing. PCR products (5 µl each) are treated with 2 µl ExoSAP-IT (US Biochemicals, Cleveland, OH). Purified PCR products are cycle-sequenced using Applied Biosystems BigDye Terminator v3.1 Cycle Sequencing Ready Reaction (at 1/16th manufacturer recommended concentration; ABI; Applied Biosystems, Foster City CA) and applied to Centri-Sep spin columns (Princeton Separations, Adelphia, NJ) on an ABI 3730 Genetic Analyzer. The test sample is run in duplicate, and each run includes a wild type DNA extraction control and NTC. Test plates for amplification and sequence analysis are prepared in advance. Sequences are analyzed by comparison to the reference sequence (accession number D86181.2) using ABI SeqScape v2.5 software. Melting curve analysis Melting curve analysis is used to rapidly assess polymorphism content (p.R168C, p.I546T, and p.D232N), and four pathogenic mutations initially thought to be common among KD patients (p.G270D, c.1424delA, p.T513M, and p.Y551S; Supplemental Table 2). Probe sets are synthesized by TIB MolBiol (Adelphi, NJ). PCR is carried out using LightCycler DNA Master HybProbe mix, MgCl2, TaqStart Antibody, primers, 1 µl undiluted DNA, and PCR grade water to a final volume of 30 µl. Cycling is performed using GALC sequencing parameters listed above. Melt curve analysis is carried out by denaturing at 95˚C for 30 s, annealing at 35˚C for 30 s, and melted to 80˚C at 0.1˚C/s. Specifications are listed in Supplemental Table 2. After PCR, 9 µl of PCR product and 0.5 µl each probe is transferred to a glass capillary for melting curve analysis on a LightCycler (Roche). 46 Screen Positive Follow-up Algorithm Infants with one mutation are referred, primarily, to minimize the chance that a second mutation has gone undetected,5 but also to allow genetic counseling and possibly additional testing for carrier families. Prior to 2010, infants with GALC activity ≤8% DMA were referred regardless of mutation status, but these infants are no longer immediately referred because the first five infants in this category carried only polymorphisms, and the delay due to second tier molecular analysis has been minimized. At the SCC, blood samples are taken for diagnostic GALC testing, identity and HLA testing, if necessary. The diagnostic enzymatic test utilizes 3Hlabeled galactosylceramide in sonicated leukocytes isolated from heparinized blood.6 Activity from diagnostic testing is expressed as nmol/hr/mg protein. KD risk assignments are based on the GALC activities of screen positive infants measured using this conventional diagnostic assay, and were agreed upon by the NYS KD Consortium.7 Infants with enzyme activity between 0-0.15 are considered to be at high risk; 0.16-0.29 as moderate risk; 0.3-0.5 as low risk; and >0.5 are not considered to be at risk (henceforth, no risk). In 2012, the low risk category was eliminated, and infants with GALC 0.3-0.5 are now classified as no risk, unless they have two potentially disease-associated mutations, in which case they are classified as moderate risk. Infants with two variants/mutations can be classified as low/no risk if enzyme activity is within the defined ranges, above.7 HLA testing is performed if genotype and/or diagnostic lab enzyme activity is indicative of a high risk early infantile case. Identity testing is completed by the newborn screening program to ensure that the blood samples (original newborn specimen and sample collected from baby at follow-up) are from the same infant. To establish mutation phase, parent samples are collected, sent to the screening lab and tested for the mutations/variants carried by the infant. Families of infants considered at risk are offered entry in a registry sponsored by the Hunter’s Hope Foundation under a research protocol. 47 References 1. Orsini JJ, Morrissey MA, Slavin LN, et al. Implementation of newborn screening for Krabbe disease: population study and cutoff determination. Clin Biochem. 2009;42(9):877-84. 2. Saavedra-Matiz CA, Isabelle JT, Biski CK, et al. Cost-Effective and Scalable DNA Extraction Method from Dried Blood Spots. Clin Chem. 2013;59(7):1045-51. 3. Rafi MA, Luzi P, Chen YQ, Wenger DA. A large deletion together with a point mutation in the GALC gene is a common mutant allele in patients with infantile Krabbe disease. Hum Mol Genet. 1995;4(8):1285-9. 4. Luzi P, Rafi MA, Wenger DA. Characterization of the large deletion in the GALC gene found in patients with Krabbe disease. Hum Mol Genet. 1995;4(12):2335-8. 5. Tanner AK, Chin ELH, Duffner PK, Hegde M. Array CGH improves detection of mutations in the GALC gene associated with Krabbe disease. Orphanet J Rare Dis. 2012;7(1):38. 6. Wenger DA, Sattler M, Clark C, McKelvey H. An improved method for the identification of patients and carriers of Krabbe’s disease. Clin Chim Acta. 1974;56(2):199-206. 7. Duffner PK, Caggana M, Orsini JJ, et al. Newborn screening for Krabbe disease: the New York State model. Pediatr Neurol. 2009;40(4):245-52. 48