6Knud (Haraldsen) Krabbe Krabbe DiseaseKrabbe K: A new familial, infantile form of diffuse brain sclerosis. Brain 1916; 39: 74.Galactocerebrosidase DeficiencyGloboid Cell Leukodystrophy (GLD)Galactosylceramidase DeficiencyGALC DeficiencyKnud (Haraldsen) Krabbe( )
7Krabbe Disease “Inherited metabolic disorder” Autosomal recessive; Pan-ethnicCarrier frequency ~ 1:150; Incidence 1:100,000Deficiency of the lysosomal enzyme galactocerebrosidase (GALC)Mutations in GALCDecreased ability to degrade galactolipidsFailure of myelination in the CNS and PNSPathology is limited to nervous systemProgressive neurologic deterioration and deathMay be under-diagnosedCerebral palsyMultiple sclerosisAt least two formsSource: Wenger, et al. In Scriver, 2001.
8Krabbe 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 yearsLate-Onset form (10-15%)- Variable clinical course from 6 months - 50 years- Weakness, vision loss, intellectual regression
9Add assay solution reagent and incubate New York State AssayPunch 3-mm specimenAdd assay solution reagent and incubate19 hoursUp to 2496-wellplatesare testedeach dayQuench reaction (50/50 MeOH/EtAc)Liquid / liquid extraction (EtAc/H20)Remove organic phase (150 μL)Dry plates1 hourRe-dissolve in MS suitable solvent (80/20 MeOH/H20)Analyze samples, 1.5 minutes per sampleCalculate activity/sample, daily mean activity, % of daily mean act/sample
11KRABBE DISEASEArtificial SubstrateProductInternal Standard
12Krabbe Screening: Cutoffs and Testing Algorithm All specimens tested for GALC activity< 20% of daily mean> 20% of daily meanRetested in duplicate (or more)Average of 3 samples ≤ 12%Average of 3 samples > 12%DNA testing1 or more mutationsNo mutationsScreen PositiveReferralScreen Negative
14If activity ≤ 12% then perform DNA Testing Mutated gene: GALCChromosome 1417 exons75+ known mutations (finding many unknown variants)30 kb deletion is most commonMultiple activity-attenuating polymorphismsReduce activity by 80-90%Wenger, et al. In Scriver, 2001.
16Molecular Analysis of GALC Reduce number of false positive screensPredict phenotype (?)Method:Agarose gel for 2 common deletions30 kb7 kbProbes for 7 common mutations/polymorphismsSequence all 17 exons and Promoter Region30 kb deletionNormalMutantp.T513M (1538C>T)
19New York State Specialty Care Programs Strong Memorial HospitalHSA 3HSA 5Crouse Memorial HospitalHSA 2Albany Medical CenterHSA 1HSA 4Women and Children’s Hospital of BuffaloHSA 6Westchester Medical CenterHSA 8Mount SinaiMontefioreSUNY at Stony BrookHSA 7
20Referred infants - Risk categories GALC ACTIVITY RISK CATEGORYnmol/hr/mg protein(future: 0.2) High(future: 0.2 – 0.35) Moderate(future: eliminate) Low> 0.5 (future: >0.35) No RiskNote that these categories have been evaluated by the New York State KrabbeConsortium, and will be changed in near future.Conf EA Dr. David A. Wenger at Jefferson Medical CollegeCourtesy of Dr. P. Duffner
21Referred 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 studiesrecommend 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
29KRABBE DISEASE GALC DATABASE New York State Newborn Screening ProgramWadsworth Center, New York State Department of HealthGALC DatabaseSummary DataVariant/Mutation DataPatients/Clinical DataGALC Sequence DataSearchSubmit DataKrabbe DiseaseGALC GeneKrabbe Disease LinksReferencesDisclaimerHow to Cite this DatabaseContactSite MapMission Statement: To provide a resource for states consideringscreening for Krabbe diseaseDatabase VersionLast Update# hits
30Variant/Mutation Data Summary Data PageKrabbe Disease LinksSearch & Download PageNews PageVariant/Mutation DataHow to Cite this Database PagePatient/Clinical DataKrabbe Disease GALC Database HomePageContributors PageGALC Sequence PageReferences PageContact Info PageHow to Submit DataSequence Alignment PageData Dictionary Page
31ONGOING PROJECTS Sequence analysis of confirmed cases Sequence analysis of Whites, Blacks, Hispanics, AsiansCloning of most common alleles for expression studies
32GALC p.Lys612Asp (c.1836delA) / chr14q31.3-32.12 Del BM/DBMDM/DB
34Mutations 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.