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NYS Krabbe Newborn Screening Referrals

NYS Krabbe Newborn Screening Referrals. Carlos A. Saavedra-Matiz , M.D. Newborn Screening Program Wadsworth Center New York State Department of Health. NYMAC Albany, NY June 4-5, 2012. INVALID SPECIMENS. 96-WELL PLATES ARE LABELED FOR USE EACH DAY .

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NYS Krabbe Newborn Screening Referrals

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  1. NYS Krabbe Newborn Screening Referrals Carlos A. Saavedra-Matiz, M.D. Newborn Screening Program Wadsworth Center New York State Department of Health NYMAC Albany, NY June 4-5, 2012

  2. INVALID SPECIMENS

  3. 96-WELL PLATES ARE LABELED FOR USE EACH DAY

  4. SPECIMENS ARE BUNDLED FOR REPEATS

  5. Krabbe Disease • Krabbe K: A new familial, infantile form of diffuse brain sclerosis. Brain 1916; 39: 74. • Galactocerebrosidase Deficiency • Globoid Cell Leukodystrophy (GLD) • Galactosylceramidase Deficiency • GALC Deficiency Knud (Haraldsen) Krabbe (1885-1965)

  6. Krabbe Disease • “Inherited metabolic disorder” • Autosomal recessive; Pan-ethnic • Carrier frequency ~ 1:150; Incidence 1:100,000 • Deficiency of the lysosomal enzyme galactocerebrosidase(GALC) • Mutations in GALC • Decreased ability to degrade galactolipids • Failure of myelination in the CNS and PNS • Pathology is limited to nervous system • Progressive neurologic deterioration and death • May be under-diagnosed • Cerebral palsy • Multiple sclerosis • At least two forms Source: Wenger, et al. In Scriver, 2001.

  7. Krabbe Disease – Clinical Features • Infantile form (85-90%) - Normal for first few months; develops feeding difficulties - Developmental delay, irritability and spasticity before 6 months of age - Death usually before 2 years • Late-Onset form (10-15%) - Variable clinical course from 6 months - 50 years - Weakness, vision loss, intellectual regression

  8. Punch 3-mm specimen Add assay solution reagent and incubate 19 hours Up to 24 96-well plates are tested each day New York State Assay Quench reaction (50/50 MeOH/EtAc) Liquid / liquid extraction (EtAc/H20) Remove organic phase (150 μL) Dry plates 1 hour Re-dissolve in MS suitable solvent (80/20 MeOH/H20) Analyze samples, 1.5 minutes per sample Calculate activity/sample, daily mean activity,% of daily mean act/sample

  9. KRABBE DISEASE Artificial Substrate Product Internal Standard

  10. Krabbe Screening: Cutoffs and Testing Algorithm All specimens tested for GALC activity < 20% of daily mean > 20% of daily mean Retested in duplicate (or more) Average of 3 samples ≤ 12% Average of 3 samples > 12% DNA testing 1 or more mutations No mutations Screen Positive Referral Screen Negative

  11. If activity ≤ 12% then perform DNA Testing • Mutated gene: GALC • Chromosome 14 • 17 exons • 75+ known mutations (finding many unknown variants) • 30 kb deletion is most common • Multiple activity-attenuating polymorphisms • Reduce activity by 80-90% Wenger, et al. In Scriver, 2001.

  12. p.I546T p.Y551S p.R168C RT-FRET-PCR c.602C>T (p.R168C) c.694G>A (p.694G>A) c.1637T>C (p.I546T) c.809G>A (p.G270D) c.1538C>T (p.T513M) c.1652A>C (p.Y551S) c.1424delA PCR-Agarose Gel 30Kb Deletion 7.4Kb Deletion

  13. Molecular Analysis of GALC 30 kb deletion • Reduce number of false positive screens • Predict phenotype (?) • Method: • Agarose gel for 2 common deletions • 30 kb • 7 kb • Probes for 7 common mutations/polymorphisms • Sequence all 17 exons and Promoter Region Normal Mutant p.T513M (1538C>T)

  14. DNA SEQUENCE ANALYSIS • Exon 15 • Gly559ValfsX10 (c.1675insT)

  15. New York State Specialty Care Programs Strong Memorial Hospital HSA 3 HSA 5 Crouse Memorial Hospital HSA 2 Albany Medical Center HSA 1 HSA 4 Women and Children’s Hospital of Buffalo HSA 6 Westchester Medical Center HSA 8 Mount Sinai Montefiore SUNY at Stony Brook HSA 7

  16. Referred infants -Risk categories GALC ACTIVITY RISK CATEGORY nmol/hr/mg protein 0.0 0.15 (future: 0.2) High 0.16 0.29 (future: 0.2 – 0.35) Moderate 0.3 0.5 (future: eliminate) Low > 0.5(future: >0.35)No Risk Note that these categories have been evaluated by the New York State Krabbe Consortium, and will be changed in near future. Conf EA Dr. David A. Wenger at Jefferson Medical College Courtesy of Dr. P. Duffner

  17. Referred infants: After NBS Screening • Confirmatory Enzyme Analysis (Dr. David A. Wenger) • Consult with Geneticist/Child Neurologist • Draw blood sample-HLA, Identity (WC) • If enzyme test affirms Krabbe disease likely.. • Exam • CSF protein • MRI • Nerve conduction, BAER, VER, and other studies • recommend periodic evaluation, depending on dx lab enzyme activity level. • A point system is used, if 4 or more points: • Consideration given to receive cord blood treatment

  18. High Risk Children • p.R168C_g.30 Kb Del // p.I546T_p.X670Qext42[TP] • 9.9% 0.01 nmol/hr/mg protein • p.R168C_g.30Kb Del // p.R168C_g.30 Kb Del [TP] • 10.9% 0.05 nmol/hr/mg protein • p.A5P _p.D232N_p.Y303C// p.A5P_p.D232N_p.Y303C • 6.1% 0.06 nmol/hr/mg protein • p.A5P_p.D232N_p.Y303C/ p.I546T // p.D556fsX1 • 8.3% 0.12 nmol/hr/mg protein • p.A5P / p.D232N / p.Y303C // p.H375QfsX3 / p.I546T • 9.6% 0.07 nmol/hr/mg protein • 6. p.A247T / p.I546T // p.A5P/ p.T96A / p.D232N • 5.0% 0.09 nmol/hr/mg protein

  19. High Risk Children 7. c.-123_128del6_p.L618S // p.L618S 9.1% 0.12 nmol/hr/mg protein p.R168C_g.30Kb Del // p.R168C_g.30Kb Del 7.6% 0.02 nmol/hr/mg protein [parents refused TP; symptomatic] 9. p.R168C_g.30Kb Del // p.G360DfsX2 [TP] 5.6% 0.12 nmol/hr/mg protein p.M101V_c.1786+5G>C_p.A625T // p.M309V_p.I546T 4.7% 0.03 nmol/hr/mg protein c.147G>C_p.I546T // p.K85E_p.I546T 6.2% 0.045 nmol/hr/mg protein p.A5P _p.D232N_p.Y303C // p.T452I 4.9% 0.05 nmol/hr/mg protein

  20. Moderate Risk Children

  21. NOVEL MUTATIONS Referral Group (02/10/12) p.R53Q (x7) p.M101V (x16) p.N151S (x3) p.R63C (x8) p.R53X p.D94D (x8) p.W115R (x2) p.G559D p.L616P p.P239H p.D556X (x2) p.A44V p.A209E p.A209T p.P73L (x5) p.V320M (x5) p.E60K (x2) p.M309V p.G360DfsX2 c.1786+5C>G (x17) p.X670Q (x3) p.K83E (x2) p.D445A c.*12C>A p.R111X (x2) p.A290T p.Y601X p.H375QfsX3 p.Met1? p.R63C (x8) p.A356S p.S353R p.L263X

  22. KRABBE DISEASE GALC DATABASE New York State Newborn Screening Program Wadsworth Center, New York State Department of Health GALC Database Summary Data Variant/Mutation Data Patients/Clinical Data GALC Sequence Data Search Submit Data Krabbe Disease GALC Gene Krabbe Disease Links References Disclaimer How to Cite this Database Contact Site Map Mission Statement: To provide a resource for states considering screening for Krabbe disease Database Version Last Update # hits

  23. Krabbe Disease Links Summary Data Page Search & Download Page News Page Variant/Mutation Data How to Cite this Database Page Patient/Clinical Data Krabbe Disease GALC Database HomePage Contributors Page GALC Sequence Page References Page Contact Info Page How to Submit Data Sequence Alignment Page Data Dictionary Page

  24. ONGOING PROJECTS • Sequence analysis of confirmed cases • Sequence analysis of Whites, Blacks, Hispanics, Asians • Cloning of most common alleles for expression studies

  25. GALC p.Lys612Asp (c.1836delA) / chr14q31.3-32.12 Del B M/D B M D M/D B

  26. p.R168C (E5) p.I546T (E17)

  27. Mutations in the galactocerebrosidase (GALC) gene cause globoid cell leukodystrophy (GLD or Krabbe disease). Krabbe is an autosomal recessive disease. This newborn has two mutations, three polymorphisms and two promoter region variants in the GALC gene. One copy each of the p.Arg63Cys (c.187C>T) and p.Tyr303Cys (c.908A>G) mutations were detected. The phenotype associated with the novel p.Arg63Cys mutation is unknown; however, a previous report of a mutation in the same amino acid, p.Arg63His, in two adolescent Krabbe sisters (in a different genetic background) suggests that p.Arg63Cys may be associated with the juvenile-onset form of the disease. The p.Tyr303Cys (c.908A>G) mutation has been associated with the late-onset form of the disease in a different genetic background. In general, late-onset forms of Krabbe are heterogeneous with a slow progression of clinical signs. One copy each of the allelic variants p.Ala5Pro (c.13G>C), p.Asp232Asn (c.694G>A) and p.Ile546Thr (c.1637T>C) were also detected. The p.Ala5Pro variant and p.Asp232Asn polymorphism virtually always occur together: p.Ala5Pro is not expected to affect GALC activity since it is located in the leader peptide, which is removed during processing and p.Asp232Asn reduces enzyme activity by 30-40%. The p.Ile546Thr polymorphism reduces GALC enzyme activity by 60-70%. In addition, this infant has one copy each of the c.-196T>C and c.-7G>C promoter region variants. No transcriptional effect is expected from these variants since they are not located at any of the putative GALC regulatory element binding sites. One allele in this baby consists of p.Arg63Cys_p.Ile546Thr and the second allele consists of c.-196T>C_c.-7G>C_p.Ala5Pro_p.Asp232Asn_p.Tyr303Cys. The reduction in GALC enzyme activity from each allele has not been quantified and the expected clinical course of this baby cannot be predicted. Each parent carries one of these alleles thus future pregnancies have a recurrence risk of 25%. Due to this genotype and the low GALC enzyme activity, referral to a metabolic specialist for evaluation, follow-up and genetic counseling is recommended. This test was performed using the polymerase chain reaction (PCR) to amplify across the breakpoint of the 30-kb deletion, the most common GALC gene mutation causing the classic infantile form of Krabbe disease. The PCR products were analyzed by agarose gel electrophoresis. Further analysis was completed by PCR followed by fluorescence-based sequence analysis of the promoter, all 17 exons, and intron/exon boundaries of the GALC gene.

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