Array CGH improves detection of mutations in the GALC gene associated with Krabbe disease

Abstract

Background: Krabbe disease is an autosomal recessive lysosomal storage disorder caused by mutations in the GALC gene. The most common mutation in the Caucasian population is a 30-kb deletion of exons 11 through 17. There are few other reports of intragenic GALC deletions or duplications, due in part to difficulties detecting them.

Methods and results: We used gene-targeted array comparative genomic hybridization (CGH) to analyze the GALC gene in individuals with Krabbe disease in whom sequence analysis with 30-kb deletion analysis identified only one mutation. In our sample of 33 cases, traditional approaches failed to identify two pathogenic mutations in five (15.2%) individuals with confirmed Krabbe disease. The addition of array CGH deletion/duplication analysis to the genetic testing strategy led to the identification of a second pathogenic mutation in three (9.1%) of these five individuals. In all three cases, the deletion or duplication identified through array CGH was a novel GALC mutation, including the only reported duplication in the GALC gene, which would have been missed by traditional testing methodologies. We report these three cases in detail. The second mutation remains unknown in the remaining two individuals (6.1%), despite our full battery of testing.

Conclusions: Analysis of the GALC gene using array CGH deletion/duplication testing increased the two-mutation detection rate from 84.8% to 93.9% in affected individuals. Better mutation detection rates are important for improving molecular diagnosis of Krabbe disease, as well as for providing prenatal and carrier testing in family members.

Figure 1

Case 1. A. Pedigree of the proband and parents. A filled symbol indicates the affected individual; symbols with a dot in the middle indicate carriers. GALC genotypes are given for each individual under the respective symbol. Sequence analysis with 30-kb deletion analysis identified one copy of a five-bp deletion in exon 16 in the proband and his mother. A second mutation was not identified. Array CGH analysis was performed on the proband’s father to identify the second mutation. B.GALC array CGH results for the father. A diagram of the GALC gene is given above the results with exon numbers indicated. The father carries a deletion of exon 8 of the GALC gene. (The presence of the deletion of exon 8 in the proband is inferred, as the proband passed away before deletion/duplication analysis was performed).

Figure 2

Case 2. A. Pedigree of the proband and parents. A filled symbol indicates the affected individual; symbols with a dot in the middle indicate carriers. GALC genotypes are given for each individual under the respective symbol. Sequence analysis with 30-kb deletion analysis identified one copy of the 30-kb deletion in the proband and her mother. A second mutation was not identified. Array CGH analysis was performed on the proband’s father to identify the second mutation, which was confirmed in the proband. B.GALC array CGH results for the father (top), mother (middle), and proband (bottom). A diagram of the GALC gene is given above the results with exon numbers indicated. The father carries a duplication of exons 11 through 14, while the mother carries the 30-kb deletion of exons 11 through 17. The combination of the duplication and deletion in the proband yields a neutral copy number for exons 11 through 14 (boxed region), since she has two copies of those exons (both from her father and none from her mother), while exons 15 through 17 are deleted (present in only one copy from her father).

Figure 3

Case 3. A. Pedigree of the proband and parents. A filled symbol indicates the affected individual; symbols with a dot in the middle indicate carriers. GALC genotypes are given for each individual under the respective symbol. Sequence analysis performed in another laboratory identified an apparently homozygous one nucleotide deletion mutation in the proband. One copy of the mutation was identified in the proband’s mother, but her father was negative. Array CGH analysis was performed on the proband’s father to identify a suspected deletion, which was confirmed in the proband. B.GALC array CGH results for the father (top) and proband (bottom). A diagram of the GALC gene is given above the results with exon numbers indicated. Both the father and the proband have one copy of a deletion of exons 1 through 6. The deletion inherited from her father made the mutation in exon 1 inherited from her mother appear homozygous in the proband by sequence analysis.

Figure 4

Algorithm for testing for Krabbe disease in affected individuals.