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

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Presentation on theme: "NYS Krabbe Newborn Screening Referrals NYMAC Albany, NY June 4-5, 2012 Carlos A. Saavedra-Matiz, M.D. Newborn Screening Program Wadsworth Center New York."— Presentation transcript:

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

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3 INVALID SPECIMENS

4 96-WELL PLATES ARE LABELED FOR USE EACH DAY

5 SPECIMENS ARE BUNDLED FOR REPEATS

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

7 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.

8  Late-Onset form (10-15%) - Variable clinical course from 6 months - 50 years - Weakness, vision loss, intellectual regression 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

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

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11 Internal Standard Product Artificial Substrate KRABBE DISEASE

12 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% Screen Negative Screen Positive Referral DNA testing 1 or more mutationsNo mutations

13 umol/L/hr Daily Mean

14 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.

15 p.I546T p.R168C p.Y551S 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

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

17 DNA SEQUENCE ANALYSIS  Exon 15  Gly559ValfsX10 (c.1675insT)

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

20 Referred infants - Risk categories GALC ACTIVITY RISK CATEGORY nmol/hr/mg protein (future: 0.2) High (future: 0.2 – 0.35) Moderate (future: eliminate) Low > 0.5 (future: >0.35) No Risk Courtesy of Dr. P. Duffner 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

21 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

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23 High Risk Children 1.p.R168C_g.30 Kb Del // p.I546T_p.X670Qext42 [TP] 9.9%0.01 nmol/hr/mg protein 2.p.R168C_g.30Kb Del // p.R168C_g.30 Kb Del [TP] 10.9%0.05 nmol/hr/mg protein 3.p.A5P _p.D232N_p.Y303C // p.A5P_p.D232N_p.Y303C 6.1%0.06 nmol/hr/mg protein 4.p.A5P_p.D232N_p.Y303C / p.I546T // p.D556fsX1 8.3%0.12 nmol/hr/mg protein 5.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

24 High Risk Children 7.c.-123_128del6_p.L618S // p.L618S 9.1%0.12 nmol/hr/mg protein 8.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 10.p.M101V_c G>C_p.A625T // p.M309V_p.I546T 4.7%0.03 nmol/hr/mg protein 11.c.147G>C_p.I546T // p.K85E_p.I546T 6.2%0.045 nmol/hr/mg protein 12.p.A5P _p.D232N_p.Y303C // p.T452I 4.9%0.05 nmol/hr/mg protein

25 Moderate Risk Children %ADM nmol/hr/mg protein Allele 1Allele 2 6.7%0.16p.A5P_p.D232N_p.Y303C 9.1%0.17c.535-1G>Ap.R53Q_p.I546T 9.7%0.18p.A5P_p.T96A_p.D232Np.A5P_p.D232N_p.Y303C 7.9%0.18p.M101V_c G>A_p.A625Tp.M101V_c G>A_p.A %0.18p.A5P_p.D232N_p.Y303C 9.4%0.18p.A247T_p.I546Tp.A5P_p.D232N_p.Y303C 8.0%0.20c.-123_128del6_p.L618S 11.5%0.20p.A5P_p.D232N_p.T513Mp.R168C_p.I546T 11.9%0.20p.G95Sp.R168C_p.I546T 10.4%0.20p.R168C_g.30Kb Delp.R168C_p.I546T 10.0%0.20p.G537R_c G>A_p.A625Tp.R168C_p.I546T 8.5%0.23p.A5P_p.D232N_p.Y303C 0.23p.A5P_p.T96A_p.D232Np.I546T_p.Y551S 8.3%0.24p.D445A_p.L618Sp.L618S 9.2%0.24p.A5P_p.D232N_p.Y303C 10.4%0.25p.A5P_p.D232N_p.Y303C 9.5%0.25p.A5P_p.D232N_p.Y303Cp.I546T_p.D556fsX1 10.3%0.25p.A5P_p.T96A_p.D232Np.M101V_c G>A_p.A625T 7.1%0.26p.R168C_g.30Kb Delp.R168C_p.I546T 8.9%0.28p.A5P_p.D232N_p.Y303C 9.1%0.28p.A5P_p.T96A_p.D232Np.A5P_p.D232N_p.Y303C 9.8%0.29p.R168C_g.30Kb Delp.P73L_p.I546T 9.8%0.29p.R168C_g.30Kb Delp.R168C_p.I546T 10.3%0.29p.A5P_p.T96A_p.D232Np.A5P_p.D232N_p.Y298del_p.I571T 10.9%0.30p.A5P_p.T96A_p.D232N

26 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 C>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

27 AlleleCarrier FreqAllele Freq p.I546T p.R168C p.D232N p.T96A g.30KbDel p.Y303C p.A625T

28 Krabbe Genotype (Additional Description) p.R168C-p.I546T Homozygous (Polys Only - No Referral) p.I546T Homozygous (Polys Only - No Referral) (Updated ) p.R168C-p.I546T Homozygous Enz Act <8% (Mom's only phased) c.-196T>C-c.-7G>C-p.A5P-p.T96A-p.D232N / c.-196T>C-c.-7G>C-p.A5P-p.T96A-p.D232N (Phased only Mom) c.-196T>C-c.-7G>C-p.A5P-p.D232N-p.Y303C Homozygous (Not phased) (Created ) p.R168C-p.I546T / p.R168C-p.I546T-c.*12G>A Enz Act <8% c.147G>C-p.I546T / p.R168C-p.I546T (090420) c.147G>C-p.I546T / p.I546T c.-335G>A / c.282C>T (p.D94D) / p.R168C-p.I546T / p.I546T c.-335G>A / p.R168C-p.I546T / p.I546T c.-335G>A-p.I546T / p.I546T c.-511C>T-p.I546T / p.I546T c A>G-p.I546T / p.R168C-p.I546T (Created ) c.-196T>C-c.-7G>C-p.A5P-p.D232N / p.I546T Polys Only (Not a referral – Created ) c.-196T>C-c.-7G>C-p.A5P-p.D232N ?-/ p.I546T Polys Only Enz Act <8% (Referral) One parent phased (Created ) c.-196T>C / c.-85C>T / c.-7G>C / p.A5P / p.R168C / p.D232N / p.I546T c.-335G>A / c.-59T>A / p.I546T Homozygous (Polys Only) (Created ) c.-335G>A-c.282C>T(p.D94D)-p.I546T / p.I546T (Polys Only-Updated090305) c.-335G>A-c.282C>T(p.D94D)-p.I546T // p.R168C-p.I546T (V3 = 3rd Case) c.-335G>A-c.282C>T(p.D94D)-p.I546T // p.R168C-p.I546T (V4-4thCase-Polys Only-Updated090205) c.-335G>A-c.282C>T(p.D94D)-p.I546T / p.R168C-p.I546T c.-335G>A-c.282C>T(p.D94D)-p.I546T / p.R168C-p.I546T (V2 = 2nd Case) c.-7G>C / p.A5P/ p.D232N / p.I546T/ p.R168C-p.I546T p.Arg168Cys_g.30-Kb Del // c.-335G>A_p.Gly360AspfsX2 (Created ) c.-196T>C--c.-7G>C--p.A5P--p.D232N--p.Y303C // p.A247T--p.I546T p.R380W-p.I546T // p.F491L-p.I546T p.V320M_p.R380W_p.I546T // p.I546T (Created ) c.-335G>A-p.E60K-p.I546T // c.-196T>C-c.-7G>C-p.A5P-p.T96A-p.D232N (Created ) p.I546T / p.A625T c.-335G>A - p.P73L(c.218C>T) - p.I546T/ p.R168C-g.30KbDel c.-196T>C-c.-7G>C-p.A5P-p.D232N-p.T452S-p.K568X / p.I546T c.-196T>C-c.-7G>C-p.A5P-p.T96A-p.D232N / p.M101V-c G>C-p.A625T c.-196T>C_c.-7G>C_p.A5P_p.D232N_p.T452S_p.G559Vfsx10 // p.V320M-p.Ile546Thr (Modified ) c.-196T>C_c.-7G>c_p.A5P_p.T96A_p.D232N // p.A563E (Created ) c.-196T>C--c.-115T>G--c.-7G>C--p.Ala5Pro--p.Arg53X--p.Asp232Asn // c.-196T>C--c.-7G>c--p.Ala5Pro--p.Thr96Ala--p.Asp232Asn c.-196T>C_c.-7G>C_p.Ala5Pro_p.Thr96Ala_p.Asp232Asn / c.-196T>C_c.-7G>C_p.Ala5Pro_p.Asp232Asn_p.Ser353Arg (Created ) c.-196T>C_c.-7G>c_p.A5P_p.T96A_p.D232N // c.-196T>C_c.-7G>c_p.D232N_p.Tyr303Cys (Created ) c.-196T>C-c.-7G>C-p.A5P-p.T96A-p.D232N / c.-335G>A-p.A247T-p.I546T c.-196T>C_c.-7G>c_p.A5P_p.D232N_p.Y303C // p.I546T_p.D556fsX1 (Created ) c.-196T>C_c.-7G>C_p.Ala5Pro_p.Asp232Asn_p.Thr452Ser_p.Gly559ValfsX10 // p.Val320Met_p.Ile546Thr p.I546-p.D556XT / c.-7G>C-p.A5P-p.D232N-p.Y303C c.-348C>T--c.-196T>C--c.-7G>C--p.A5P--p.D232N--p.Y303C / p.I546T / p.H375QX3 p.R168C-g.30KbDel / p.I546T (Phased) (Created ) p.R168C-g.30KbDel / p.I546T p.R168C-g.30KbDel / p.R168C-p.I546T p.A209E / p.I546T c.-335G>A / c.-196T>C / c.-7G>C / p.A5P-p.D232N-p.I546T / p.A247T / p.I546T c.-196T>C-c.-7G>C-p.A5P-p.D232N-p.A290T / p.I546T p.A44V-p.I546T / p.R168C-p.I546T p.A563E / p.I546T c.-335G>A-p.A247T-p.I546T // p.R168C-p.I546T p.A356S_p.I546T // p.R168C_p.I546T (Created ) p.R111X-p.I546T // p.R168C-p.I546T (Phased) (Created ) p.R168C-p.I546T / p.I546T p.R168C_p.I546T / p.I546T p.R204X // p.Ile546Thr p.R204X-p.I546T / p.I546T (Not phased) (Created ) p.R53Q--p.I546T // p.I546T p.N151S_p.I546T / c.-335G>A_p.I546T (Created ) p.N151S-p.I546T / p.R168C-p.I546T (Created ) p.N279I-p.I546T / p.R168C-p.I546T p.R168C-p.D171V-p.I546T // p.R168C-p.I546T p.D445A-p.L618S / p.L618S p.G268S-p.I546T // p.R168C-p.I546T (Created ) c.G307R / p.R168C-p.I546T (Created ) p.I546T-p.G559D / p.I546T (Created ) c.-335G>A_p.G95S // p.R168C_p.I546T (Created ) p.G95S / p.R168C-p.I546T (Created ) p.K83E-p.I546T / p.I546T p.Leu263X_p.Ile546Thr // p.Arg168Cys_p.Ile546Thr (Created ) p.L616P-p.I546T // p.R168C-p.I546T (Created ) p.M101V-c G>C-p.A625T / p.I546T (Not Phased) p.M101V-c G>C-p.A625T / p.I546T (Phased) p.M101V-c G>C-p.A625T / p.R168C-p.I546T p.M101V-c G>C-p.A625T / p.R168C-p.I546T (System) p.Met1?-p.I546T / p.I546T p.M101V-c G>C-p.A625T / p.R168C-p.I546T (Updated ) c.-335G>A - p.P73L(c.218C>T) - p.I546T / p.I546T c.-335G>A - p.P73L - p.I546T / p.R168C - p.I546T c.-335G>A - p.P73L - p.I546T / p.R168C - p.I546T (Second identical genotype-update090203) c.-335G>A-p.F498S-p.I546T / p.R168C-p.I546T (Created090622) Krabbe Genotype (Additional Description) p.R168C-p.I546T Homozygous (Polys Only - No Referral) p.I546T Homozygous (Polys Only - No Referral) (Updated ) p.R168C-p.I546T Homozygous Enz Act <8% (Mom's only phased) c.-196T>C-c.-7G>C-p.A5P-p.T96A-p.D232N / c.-196T>C-c.-7G>C-p.A5P-p.T96A-p.D232N (Phased only Mom) c.-196T>C-c.-7G>C-p.A5P-p.D232N-p.Y303C Homozygous (Not phased) (Created ) p.R168C-p.I546T / p.R168C-p.I546T-c.*12G>A Enz Act <8% c.147G>C-p.I546T / p.R168C-p.I546T (090420) c.147G>C-p.I546T / p.I546T c.-335G>A / c.282C>T (p.D94D) / p.R168C-p.I546T / p.I546T c.-335G>A / p.R168C-p.I546T / p.I546T c.-335G>A-p.I546T / p.I546T c.-511C>T-p.I546T / p.I546T c A>G-p.I546T / p.R168C-p.I546T (Created ) c.-196T>C-c.-7G>C-p.A5P-p.D232N / p.I546T Polys Only (Not a referral – Created ) c.-196T>C-c.-7G>C-p.A5P-p.D232N ?-/ p.I546T Polys Only Enz Act <8% (Referral) One parent phased (Created ) c.-196T>C / c.-85C>T / c.-7G>C / p.A5P / p.R168C / p.D232N / p.I546T c.-335G>A / c.-59T>A / p.I546T Homozygous (Polys Only) (Created ) c.-335G>A-c.282C>T(p.D94D)-p.I546T / p.I546T (Polys Only-Updated090305) c.-335G>A-c.282C>T(p.D94D)-p.I546T // p.R168C-p.I546T (V3 = 3rd Case) c.-335G>A-c.282C>T(p.D94D)-p.I546T // p.R168C-p.I546T (V4-4thCase-Polys Only-Updated090205) c.-335G>A-c.282C>T(p.D94D)-p.I546T / p.R168C-p.I546T c.-335G>A-c.282C>T(p.D94D)-p.I546T / p.R168C-p.I546T (V2 = 2nd Case) c.-7G>C / p.A5P/ p.D232N / p.I546T/ p.R168C-p.I546T p.Arg168Cys_g.30-Kb Del // c.-335G>A_p.Gly360AspfsX2 (Created ) c.-196T>C--c.-7G>C--p.A5P--p.D232N--p.Y303C // p.A247T--p.I546T p.R380W-p.I546T // p.F491L-p.I546T p.V320M_p.R380W_p.I546T // p.I546T (Created ) c.-335G>A-p.E60K-p.I546T // c.-196T>C-c.-7G>C-p.A5P-p.T96A-p.D232N (Created ) p.I546T / p.A625T c.-335G>A - p.P73L(c.218C>T) - p.I546T/ p.R168C-g.30KbDel c.-196T>C-c.-7G>C-p.A5P-p.D232N-p.T452S-p.K568X / p.I546T c.-196T>C-c.-7G>C-p.A5P-p.T96A-p.D232N / p.M101V-c G>C-p.A625T c.-196T>C_c.-7G>C_p.A5P_p.D232N_p.T452S_p.G559Vfsx10 // p.V320M-p.Ile546Thr (Modified ) c.-196T>C_c.-7G>c_p.A5P_p.T96A_p.D232N // p.A563E (Created ) c.-196T>C--c.-115T>G--c.-7G>C--p.Ala5Pro--p.Arg53X--p.Asp232Asn // c.-196T>C--c.-7G>c--p.Ala5Pro--p.Thr96Ala--p.Asp232Asn c.-196T>C_c.-7G>C_p.Ala5Pro_p.Thr96Ala_p.Asp232Asn / c.-196T>C_c.-7G>C_p.Ala5Pro_p.Asp232Asn_p.Ser353Arg (Created ) c.-196T>C_c.-7G>c_p.A5P_p.T96A_p.D232N // c.-196T>C_c.-7G>c_p.D232N_p.Tyr303Cys (Created ) c.-196T>C-c.-7G>C-p.A5P-p.T96A-p.D232N / c.-335G>A-p.A247T-p.I546T c.-196T>C_c.-7G>c_p.A5P_p.D232N_p.Y303C // p.I546T_p.D556fsX1 (Created ) c.-196T>C_c.-7G>C_p.Ala5Pro_p.Asp232Asn_p.Thr452Ser_p.Gly559ValfsX10 // p.Val320Met_p.Ile546Thr p.I546-p.D556XT / c.-7G>C-p.A5P-p.D232N-p.Y303C c.-348C>T--c.-196T>C--c.-7G>C--p.A5P--p.D232N--p.Y303C / p.I546T / p.H375QX3 p.R168C-g.30KbDel / p.I546T (Phased) (Created ) p.R168C-g.30KbDel / p.I546T p.R168C-g.30KbDel / p.R168C-p.I546T p.A209E / p.I546T c.-335G>A / c.-196T>C / c.-7G>C / p.A5P-p.D232N-p.I546T / p.A247T / p.I546T c.-196T>C-c.-7G>C-p.A5P-p.D232N-p.A290T / p.I546T p.A44V-p.I546T / p.R168C-p.I546T p.A563E / p.I546T c.-335G>A-p.A247T-p.I546T // p.R168C-p.I546T p.A356S_p.I546T // p.R168C_p.I546T (Created ) p.R111X-p.I546T // p.R168C-p.I546T (Phased) (Created ) p.R168C-p.I546T / p.I546T p.R168C_p.I546T / p.I546T p.R204X // p.Ile546Thr p.R204X-p.I546T / p.I546T (Not phased) (Created ) p.R53Q--p.I546T // p.I546T p.N151S_p.I546T / c.-335G>A_p.I546T (Created ) p.N151S-p.I546T / p.R168C-p.I546T (Created ) p.N279I-p.I546T / p.R168C-p.I546T p.R168C-p.D171V-p.I546T // p.R168C-p.I546T p.D445A-p.L618S / p.L618S p.G268S-p.I546T // p.R168C-p.I546T (Created ) c.G307R / p.R168C-p.I546T (Created ) p.I546T-p.G559D / p.I546T (Created ) c.-335G>A_p.G95S // p.R168C_p.I546T (Created ) p.G95S / p.R168C-p.I546T (Created ) p.K83E-p.I546T / p.I546T p.Leu263X_p.Ile546Thr // p.Arg168Cys_p.Ile546Thr (Created ) p.L616P-p.I546T // p.R168C-p.I546T (Created ) p.M101V-c G>C-p.A625T / p.I546T (Not Phased) p.M101V-c G>C-p.A625T / p.I546T (Phased) p.M101V-c G>C-p.A625T / p.R168C-p.I546T p.M101V-c G>C-p.A625T / p.R168C-p.I546T (System) p.Met1?-p.I546T / p.I546T p.M101V-c G>C-p.A625T / p.R168C-p.I546T (Updated ) c.-335G>A - p.P73L(c.218C>T) - p.I546T / p.I546T c.-335G>A - p.P73L - p.I546T / p.R168C - p.I546T c.-335G>A - p.P73L - p.I546T / p.R168C - p.I546T (Second identical genotype-update090203) c.-335G>A-p.F498S-p.I546T / p.R168C-p.I546T (Created090622)

29 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 KRABBE DISEASE GALC DATABASE New York State Newborn Screening Program Wadsworth Center, New York State Department of Health Mission Statement: To provide a resource for states considering screening for Krabbe disease Database Version Last Update # hits

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

31 ONGOING PROJECTS  Sequence analysis of confirmed cases  Sequence analysis of Whites, Blacks, Hispanics, Asians  Cloning of most common alleles for expression studies

32 GALC p.Lys612Asp (c.1836delA) / chr14q Del D M B M/D B B

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

34 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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