NYS Krabbe Newborn Screening Referrals

NYMACAlbany, NY

June 4-5, 2012

Carlos A. Saavedra-Matiz, M.D.Newborn Screening Program

Wadsworth CenterNew York State Department of Health

INVALID SPECIMENS

96-WELL PLATES ARE LABELEDFOR USE EACH DAY

SPECIMENS ARE BUNDLED FOR REPEATS

Krabbe Disease

Galactocerebrosidase Deficiency

Globoid Cell Leukodystrophy (GLD)

Galactosylceramidase Deficiency

GALC Deficiency

Knud (Haraldsen) Krabbe (1885-1965)

Krabbe K: A new familial, infantile form of diffuse brain sclerosis. Brain 1916; 39: 74.

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.

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

New York State AssayPunch 3-mm specimen

Add assay solution reagent and incubate

Quench reaction (50/50 MeOH/EtAc)Liquid / liquid extraction (EtAc/H20)Remove organic phase (150 μL)

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 2496-well platesare testedeach day

Calculate activity/sample, daily mean activity, % of daily mean act/sample

Internal Standard

Product

Artificial Substrate

KRABBE DISEASE

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 PositiveReferral

DNA testing

1 or more mutations No mutations

umol/L/hrDaily Mean

3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0

0.36 12.0 11.6 11.3 10.9 10.6 10.3 10.0 9.7 9.5 9.2 9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.7 7.5 7.3 7.2

0.37 12.3 11.9 11.6 11.2 10.9 10.6 10.3 10.0 9.7 9.5 9.3 9.0 8.8 8.6 8.4 8.2 8.0 7.9 7.7 7.6 7.4

0.38 12.7 12.3 11.9 11.5 11.2 10.9 10.6 10.3 10.0 9.7 9.5 9.3 9.0 8.8 8.6 8.4 8.3 8.1 7.9 7.8 7.6

0.39 13.0 12.6 12.2 11.8 11.5 11.1 10.8 10.5 10.3 10.0 9.8 9.5 9.3 9.1 8.9 8.7 8.5 8.3 8.1 8.0 7.8

0.40 13.3 12.9 12.5 12.1 11.8 11.4 11.1 10.8 10.5 10.3 10.0 9.8 9.5 9.3 9.1 8.9 8.7 8.5 8.3 8.2 8.0

0.41 13.7 13.2 12.8 12.4 12.1 11.7 11.4 11.1 10.8 10.5 10.3 10.0 9.8 9.5 9.3 9.1 8.9 8.7 8.5 8.4 8.2

0.42 14.0 13.5 13.1 12.7 12.4 12.0 11.7 11.4 11.1 10.8 10.5 10.2 10.0 4.4 9.5 9.3 9.1 8.9 8.8 8.6 8.4

0.43 14.3 13.9 13.4 13.0 12.6 12.3 11.9 11.6 11.3 11.0 10.8 10.5 10.2 10.0 9.8 9.6 9.3 9.1 9.0 8.8 8.6

0.44 14.7 14.2 13.8 13.3 12.9 12.6 12.2 11.9 11.6 11.3 11.0 10.7 10.5 10.2 10.0 9.8 9.6 9.4 9.2 9.0 8.8

0.45 15.0 14.5 14.1 13.6 13.2 12.9 12.5 12.2 11.8 11.5 11.3 11.0 10.7 10.5 10.2 10.0 9.8 9.6 9.4 9.2 9.0

0.46 15.3 14.8 14.4 13.9 13.5 13.1 12.8 12.4 12.1 11.8 11.5 11.2 11.0 10.7 10.5 10.2 10.0 9.8 9.6 9.4 9.2

0.47 15.7 15.2 14.7 14.2 13.8 13.4 13.1 12.7 12.4 12.1 11.8 11.5 11.2 10.9 10.7 10.4 10.2 10.0 9.8 9.6 9.4

0.48 16.0 15.5 15.0 14.5 14.1 13.7 13.3 13.0 12.6 12.3 12.0 11.7 11.4 11.2 10.9 10.7 10.4 10.2 10.0 9.8 9.6

0.49 16.3 15.8 15.3 14.8 14.4 14.0 13.6 13.2 12.9 12.6 12.3 12.0 11.7 11.4 11.1 10.9 10.7 10.4 10.2 10.0 9.8

0.50 16.7 16.1 15.6 15.2 14.7 14.3 13.9 13.5 13.2 12.8 12.5 12.2 11.9 11.6 11.4 11.1 10.9 10.6 10.4 10.2 10.0

0.51 17.0 16.5 15.9 15.5 15.0 14.6 14.2 13.8 13.4 13.1 12.8 12.4 12.1 11.9 11.6 11.3 11.1 10.9 10.6 10.4 10.2

0.52 17.3 16.8 16.3 15.8 15.3 14.9 14.4 14.1 13.7 13.3 13.0 12.7 12.4 12.1 11.8 11.6 11.3 11.1 10.8 10.6 10.4

0.53 17.7 17.1 16.6 16.1 15.6 15.1 14.7 14.3 13.9 13.6 13.3 12.9 12.6 12.3 12.0 11.8 11.5 11.3 11.0 10.8 10.6

0.54 18.0 17.4 16.9 16.4 15.9 15.4 15.0 14.6 14.2 13.8 13.5 13.2 12.9 12.6 12.3 12.0 11.7 11.5 11.3 11.0 10.8

0.55 18.3 17.7 17.2 16.7 16.2 15.7 15.3 14.9 14.5 14.1 13.8 13.4 13.1 12.8 12.5 12.2 12.0 11.7 11.5 11.2 11.0

0.56 18.7 18.1 17.5 17.0 16.5 16.0 15.6 15.1 14.7 14.4 14.0 13.7 13.3 13.0 12.7 12.4 12.2 11.9 11.7 11.4 11.2

0.57 19.0 18.4 17.8 17.3 16.8 16.3 15.8 15.4 15.0 14.6 14.3 13.9 13.6 13.3 13.0 12.7 12.4 12.1 11.9 11.6 11.4

0.58 19.3 18.7 18.1 17.6 17.1 16.6 16.1 15.7 15.3 14.9 14.5 14.1 13.8 13.5 13.2 12.9 12.6 12.3 12.1 11.8 11.6

0.59 19.7 19.0 18.4 17.9 17.4 16.9 16.4 15.9 15.5 15.1 14.8 14.4 14.0 13.7 13.4 13.1 12.8 12.6 12.3 12.0 11.8

0.60 20.0 19.4 18.8 18.2 17.6 17.1 16.7 16.2 15.8 15.4 15.0 14.6 14.3 14.0 13.6 13.3 13.0 12.8 12.5 12.2 12.0

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.

p.I546T

p.R168C

p.Y551S

RT-FRET-PCRc.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

Molecular Analysis of GALC30 kb deletion

Normal

Mutant

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

p.T513M (1538C>T)

DNA SEQUENCE ANALYSIS

Exon 15

Gly559ValfsX10

(c.1675insT)

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 8Mount SinaiMontefiore

HSA 7

Referred infants - Risk categories

GALC ACTIVITY RISK CATEGORYnmol/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

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

Referred infants: After NBS Screening

• Confirmatory Enzyme Analysis (Dr. David A. Wenger)Consult with Geneticist/Child NeurologistDraw blood sample-HLA, Identity (WC)

• If enzyme test affirms Krabbe disease likely..ExamCSF proteinMRINerve 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

High Risk Children1. 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.Y303C6.1% 0.06 nmol/hr/mg protein

4. p.A5P_p.D232N_p.Y303C / p.I546T // p.D556fsX18.3% 0.12 nmol/hr/mg protein

5. p.A5P / p.D232N / p.Y303C // p.H375QfsX3 / p.I546T9.6% 0.07 nmol/hr/mg protein

6. p.A247T / p.I546T // p.A5P/ p.T96A / p.D232N5.0% 0.09 nmol/hr/mg protein

High Risk Children7. 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 Del7.6% 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.1786+5G>C_p.A625T // p.M309V_p.I546T4.7% 0.03 nmol/hr/mg protein

11. c.147G>C_p.I546T // p.K85E_p.I546T6.2% 0.045 nmol/hr/mg protein

12. p.A5P _p.D232N_p.Y303C // p.T452I4.9% 0.05 nmol/hr/mg protein

Moderate Risk Children%ADM nmol/hr/mg protein Allele 1 Allele 26.7% 0.16 p.A5P_p.D232N_p.Y303C p.A5P_p.D232N_p.Y303C9.1% 0.17 c.535-1G>A p.R53Q_p.I546T9.7% 0.18 p.A5P_p.T96A_p.D232N p.A5P_p.D232N_p.Y303C7.9% 0.18 p.M101V_c.1786+5G>A_p.A625T p.M101V_c.1786+5G>A_p.A62511.9% 0.18 p.A5P_p.D232N_p.Y303C p.A5P_p.D232N_p.Y303C9.4% 0.18 p.A247T_p.I546T p.A5P_p.D232N_p.Y303C8.0% 0.20 c.-123_128del6_p.L618S c.-123_128del6_p.L618S11.5% 0.20 p.A5P_p.D232N_p.T513M p.R168C_p.I546T11.9% 0.20 p.G95S p.R168C_p.I546T10.4% 0.20 p.R168C_g.30Kb Del p.R168C_p.I546T10.0% 0.20 p.G537R_c.1786+5G>A_p.A625T p.R168C_p.I546T8.5% 0.23 p.A5P_p.D232N_p.Y303C p.A5P_p.D232N_p.Y303C

  0.23 p.A5P_p.T96A_p.D232N p.I546T_p.Y551S8.3% 0.24 p.D445A_p.L618S p.L618S9.2% 0.24 p.A5P_p.D232N_p.Y303C p.A5P_p.D232N_p.Y303C

10.4% 0.25 p.A5P_p.D232N_p.Y303C p.A5P_p.D232N_p.Y303C9.5% 0.25 p.A5P_p.D232N_p.Y303C p.I546T_p.D556fsX1

10.3% 0.25 p.A5P_p.T96A_p.D232N p.M101V_c.1786+5G>A_p.A625T7.1% 0.26 p.R168C_g.30Kb Del p.R168C_p.I546T8.9% 0.28 p.A5P_p.D232N_p.Y303C p.A5P_p.D232N_p.Y303C9.1% 0.28 p.A5P_p.T96A_p.D232N p.A5P_p.D232N_p.Y303C9.8% 0.29 p.R168C_g.30Kb Del p.P73L_p.I546T9.8% 0.29 p.R168C_g.30Kb Del p.R168C_p.I546T

10.3% 0.29 p.A5P_p.T96A_p.D232N p.A5P_p.D232N_p.Y298del_p.I571T10.9% 0.30 p.A5P_p.T96A_p.D232N p.A5P_p.T96A_p.D232N

NOVEL MUTATIONSReferral 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

Allele Carrier Freq Allele Freq

p.I546T 0.88 0.58

p.R168C 0.61 0.37

p.D232N 0.40 0.23

p.T96A 0.24 0.13

g.30KbDel 0.15 0.08

p.Y303C 0.12 0.07

p.A625T 0.06 0.03

Krabbe Genotype (Additional Description)p.R168C-p.I546T Homozygous (Polys Only - No Referral)p.I546T Homozygous (Polys Only - No Referral) (Updated 090911)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 090515)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.I546Tc.-335G>A / c.282C>T (p.D94D) / p.R168C-p.I546T / p.I546Tc.-335G>A / p.R168C-p.I546T / p.I546Tc.-335G>A-p.I546T / p.I546Tc.-511C>T-p.I546T / p.I546Tc.861-10A>G-p.I546T / p.R168C-p.I546T (Created 090608)c.-196T>C-c.-7G>C-p.A5P-p.D232N / p.I546T Polys Only (Not a referral – Created 07-06-2010)

c.-196T>C-c.-7G>C-p.A5P-p.D232N ?-/ p.I546T Polys Only Enz Act <8% (Referral) One parent phased (Created 090430)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 090922)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.I546Tc.-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.I546Tp.Arg168Cys_g.30-Kb Del // c.-335G>A_p.Gly360AspfsX2 (Created 06-07-2010)c.-196T>C--c.-7G>C--p.A5P--p.D232N--p.Y303C // p.A247T--p.I546Tp.R380W-p.I546T // p.F491L-p.I546Tp.V320M_p.R380W_p.I546T // p.I546T (Created 07-23-2010)c.-335G>A-p.E60K-p.I546T // c.-196T>C-c.-7G>C-p.A5P-p.T96A-p.D232N (Created 091103)p.I546T / p.A625Tc.-335G>A - p.P73L(c.218C>T) - p.I546T/ p.R168C-g.30KbDelc.-196T>C-c.-7G>C-p.A5P-p.D232N-p.T452S-p.K568X / p.I546Tc.-196T>C-c.-7G>C-p.A5P-p.T96A-p.D232N / p.M101V-c.1786+5G>C-p.A625Tc.-196T>C_c.-7G>C_p.A5P_p.D232N_p.T452S_p.G559Vfsx10 // p.V320M-p.Ile546Thr (Modified 100304)

c.-196T>C_c.-7G>c_p.A5P_p.T96A_p.D232N // p.A563E (Created 07-19-2010)

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.Asp232Asnc.-196T>C_c.-7G>C_p.Ala5Pro_p.Thr96Ala_p.Asp232Asn / c.-196T>C_c.-7G>C_p.Ala5Pro_p.Asp232Asn_p.Ser353Arg (Created 07-29-2010)c.-196T>C_c.-7G>c_p.A5P_p.T96A_p.D232N // c.-196T>C_c.-7G>c_p.D232N_p.Tyr303Cys (Created 100401)c.-196T>C-c.-7G>C-p.A5P-p.T96A-p.D232N / c.-335G>A-p.A247T-p.I546Tc.-196T>C_c.-7G>c_p.A5P_p.D232N_p.Y303C // p.I546T_p.D556fsX1 (Created 100415)c.-196T>C_c.-7G>C_p.Ala5Pro_p.Asp232Asn_p.Thr452Ser_p.Gly559ValfsX10 // p.Val320Met_p.Ile546Thrp.I546-p.D556XT / c.-7G>C-p.A5P-p.D232N-p.Y303Cc.-348C>T--c.-196T>C--c.-7G>C--p.A5P--p.D232N--p.Y303C / p.I546T / p.H375QX3p.R168C-g.30KbDel / p.I546T (Phased) (Created 090820)p.R168C-g.30KbDel / p.I546Tp.R168C-g.30KbDel / p.R168C-p.I546Tp.A209E / p.I546Tc.-335G>A / c.-196T>C / c.-7G>C / p.A5P-p.D232N-p.I546T / p.A247T / p.I546Tc.-196T>C-c.-7G>C-p.A5P-p.D232N-p.A290T / p.I546Tp.A44V-p.I546T / p.R168C-p.I546Tp.A563E / p.I546Tc.-335G>A-p.A247T-p.I546T // p.R168C-p.I546Tp.A356S_p.I546T // p.R168C_p.I546T (Created 01-29-2010)p.R111X-p.I546T // p.R168C-p.I546T (Phased) (Created 090902)p.R168C-p.I546T / p.I546Tp.R168C_p.I546T / p.I546Tp.R204X // p.Ile546Thrp.R204X-p.I546T / p.I546T (Not phased) (Created 090810)p.R53Q--p.I546T // p.I546Tp.N151S_p.I546T / c.-335G>A_p.I546T (Created 100112)p.N151S-p.I546T / p.R168C-p.I546T (Created 091027)p.N279I-p.I546T / p.R168C-p.I546Tp.R168C-p.D171V-p.I546T // p.R168C-p.I546T p.D445A-p.L618S / p.L618Sp.G268S-p.I546T // p.R168C-p.I546T (Created 090429)c.G307R / p.R168C-p.I546T (Created 090421)p.I546T-p.G559D / p.I546T (Created 091005)c.-335G>A_p.G95S // p.R168C_p.I546T (Created 100108)p.G95S / p.R168C-p.I546T (Created 090427)p.K83E-p.I546T / p.I546Tp.Leu263X_p.Ile546Thr // p.Arg168Cys_p.Ile546Thr (Created 06-29-2010)p.L616P-p.I546T // p.R168C-p.I546T (Created 090715)p.M101V-c.1786+5G>C-p.A625T / p.I546T (Not Phased)p.M101V-c.1786+5G>C-p.A625T / p.I546T (Phased)p.M101V-c.1786+5G>C-p.A625T / p.R168C-p.I546Tp.M101V-c.1786+5G>C-p.A625T / p.R168C-p.I546T (System)p.Met1?-p.I546T / p.I546Tp.M101V-c.1786+5G>C-p.A625T / p.R168C-p.I546T (Updated 090616)c.-335G>A - p.P73L(c.218C>T) - p.I546T / p.I546Tc.-335G>A - p.P73L - p.I546T / p.R168C - p.I546Tc.-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 090911)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 090515)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.I546Tc.-335G>A / c.282C>T (p.D94D) / p.R168C-p.I546T / p.I546Tc.-335G>A / p.R168C-p.I546T / p.I546Tc.-335G>A-p.I546T / p.I546Tc.-511C>T-p.I546T / p.I546Tc.861-10A>G-p.I546T / p.R168C-p.I546T (Created 090608)c.-196T>C-c.-7G>C-p.A5P-p.D232N / p.I546T Polys Only (Not a referral – Created 07-06-2010)c.-196T>C-c.-7G>C-p.A5P-p.D232N ?-/ p.I546T Polys Only Enz Act <8% (Referral) One parent phased (Created 090430)c.-196T>C / c.-85C>T / c.-7G>C / p.A5P / p.R168C / p.D232N / p.I546Tc.-335G>A / c.-59T>A / p.I546T Homozygous (Polys Only) (Created 090922)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.I546Tc.-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.I546Tp.Arg168Cys_g.30-Kb Del // c.-335G>A_p.Gly360AspfsX2 (Created 06-07-2010)c.-196T>C--c.-7G>C--p.A5P--p.D232N--p.Y303C // p.A247T--p.I546Tp.R380W-p.I546T // p.F491L-p.I546Tp.V320M_p.R380W_p.I546T // p.I546T (Created 07-23-2010)c.-335G>A-p.E60K-p.I546T // c.-196T>C-c.-7G>C-p.A5P-p.T96A-p.D232N (Created 091103)p.I546T / p.A625Tc.-335G>A - p.P73L(c.218C>T) - p.I546T/ p.R168C-g.30KbDelc.-196T>C-c.-7G>C-p.A5P-p.D232N-p.T452S-p.K568X / p.I546Tc.-196T>C-c.-7G>C-p.A5P-p.T96A-p.D232N / p.M101V-c.1786+5G>C-p.A625T

c.-196T>C_c.-7G>C_p.A5P_p.D232N_p.T452S_p.G559Vfsx10 // p.V320M-p.Ile546Thr (Modified 100304)c.-196T>C_c.-7G>c_p.A5P_p.T96A_p.D232N // p.A563E (Created 07-19-2010)

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.Asp232Asnc.-196T>C_c.-7G>C_p.Ala5Pro_p.Thr96Ala_p.Asp232Asn / c.-196T>C_c.-7G>C_p.Ala5Pro_p.Asp232Asn_p.Ser353Arg (Created 07-29-2010)

c.-196T>C_c.-7G>c_p.A5P_p.T96A_p.D232N // c.-196T>C_c.-7G>c_p.D232N_p.Tyr303Cys (Created 100401)c.-196T>C-c.-7G>C-p.A5P-p.T96A-p.D232N / c.-335G>A-p.A247T-p.I546Tc.-196T>C_c.-7G>c_p.A5P_p.D232N_p.Y303C // p.I546T_p.D556fsX1 (Created 100415)

c.-196T>C_c.-7G>C_p.Ala5Pro_p.Asp232Asn_p.Thr452Ser_p.Gly559ValfsX10 // p.Val320Met_p.Ile546Thrp.I546-p.D556XT / c.-7G>C-p.A5P-p.D232N-p.Y303Cc.-348C>T--c.-196T>C--c.-7G>C--p.A5P--p.D232N--p.Y303C / p.I546T / p.H375QX3p.R168C-g.30KbDel / p.I546T (Phased) (Created 090820)p.R168C-g.30KbDel / p.I546Tp.R168C-g.30KbDel / p.R168C-p.I546Tp.A209E / p.I546Tc.-335G>A / c.-196T>C / c.-7G>C / p.A5P-p.D232N-p.I546T / p.A247T / p.I546Tc.-196T>C-c.-7G>C-p.A5P-p.D232N-p.A290T / p.I546Tp.A44V-p.I546T / p.R168C-p.I546Tp.A563E / p.I546Tc.-335G>A-p.A247T-p.I546T // p.R168C-p.I546Tp.A356S_p.I546T // p.R168C_p.I546T (Created 01-29-2010)p.R111X-p.I546T // p.R168C-p.I546T (Phased) (Created 090902)p.R168C-p.I546T / p.I546Tp.R168C_p.I546T / p.I546Tp.R204X // p.Ile546Thrp.R204X-p.I546T / p.I546T (Not phased) (Created 090810)p.R53Q--p.I546T // p.I546Tp.N151S_p.I546T / c.-335G>A_p.I546T (Created 100112)p.N151S-p.I546T / p.R168C-p.I546T (Created 091027)p.N279I-p.I546T / p.R168C-p.I546Tp.R168C-p.D171V-p.I546T // p.R168C-p.I546T p.D445A-p.L618S / p.L618Sp.G268S-p.I546T // p.R168C-p.I546T (Created 090429)c.G307R / p.R168C-p.I546T (Created 090421)p.I546T-p.G559D / p.I546T (Created 091005)c.-335G>A_p.G95S // p.R168C_p.I546T (Created 100108)p.G95S / p.R168C-p.I546T (Created 090427)p.K83E-p.I546T / p.I546Tp.Leu263X_p.Ile546Thr // p.Arg168Cys_p.Ile546Thr (Created 06-29-2010)p.L616P-p.I546T // p.R168C-p.I546T (Created 090715)p.M101V-c.1786+5G>C-p.A625T / p.I546T (Not Phased)p.M101V-c.1786+5G>C-p.A625T / p.I546T (Phased)p.M101V-c.1786+5G>C-p.A625T / p.R168C-p.I546Tp.M101V-c.1786+5G>C-p.A625T / p.R168C-p.I546T (System)p.Met1?-p.I546T / p.I546Tp.M101V-c.1786+5G>C-p.A625T / p.R168C-p.I546T (Updated 090616)c.-335G>A - p.P73L(c.218C>T) - p.I546T / p.I546Tc.-335G>A - p.P73L - p.I546T / p.R168C - p.I546Tc.-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)

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KRABBE DISEASE GALC DATABASENew York State Newborn Screening ProgramWadsworth Center, New York State Department of Health

Mission Statement: To provide a resource for states consideringscreening for Krabbe disease

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GeneTests: Krabbe disease

Clinical Information

Online Mendelian Inheritance in Man: Krabbe disease (#245200) Online Mendelian Inheritance in Man: Atypical Krabbe disease (#611722) PubMed Search for Krabbe Disease Literature Diagnostic GALC Enzyme Testing Laboratory ClinicalTrials.gov

GALC Sequence Information

Entrez Gene: GALC Sequence of mRNA coding for GALC Isoform A Precursor (Major Isoform) Amino Acid Sequence of Isoform A Precursor Sequence of mRNA coding for GALC Isoform B Precursor (Minor Isoform) Amino Acid Sequence of Isoform B Precursor GALC Variants Catalogued in dbSNP (NCBI) GALC Variants Genotyped in the HapMap Project GALC Mutations Catalogued in the Human Gene Mutation Database (Requires free registration)

Information for Parents, Family Members & Caregivers

Krabbe Disease Information Page (National Institute of Neurological Disorders & Stroke) Hunter's Hope Website United Leukodystrophy Foundation National Organization for Rare Disorders Genetic Alliance Genetics Home Reference: Krabbe Disease Newborn Screening for Krabbe Disease in New York: New York State Newborn Screening Program

ONGOING PROJECTS

Sequence analysis of confirmed cases

Sequence analysis of Whites, Blacks, Hispanics, Asians

Cloning of most common alleles for expression studies

GALC p.Lys612Asp (c.1836delA) / chr14q31.3-32.12 Del

D

M

B

M/DB

M/D

B

p.R168C (E5)

p.I546T (E17)

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.