Accurate Molecular Diagnosis of Gaucher Disease Using Clinical Exome Sequencing as a First-Tier Test.

Stefania Zampieri,Antonio Barbato,Silvia Cattarossi,Maurizio Scarpa,Agata Fiumara,Giovanni Ciana,Andrea Dardis,Eleonora Pavan,Paolo Peruzzo

doi:10.3390/ijms22115538

Abstract

Gaucher disease (GD) is an autosomal recessive lysosomal disorder due to beta-glucosidase gene (GBA) mutations. The molecular diagnosis of GD is complicated by the presence of recombinant alleles originating from a highly homologous pseudogene. Clinical exome sequencing (CES) is a rapid genetic approach for identifying disease-causing mutations. However, copy number variation and recombination events are poorly detected, and further investigations are required to avoid mis-genotyping. The aim of this work was to set-up an integrated strategy for GD patients genotyping using CES as a first-line test. Eight patients diagnosed with GD were analyzed by CES. Five patients were fully genotyped, while three were revealed to be homozygous for mutations that were not confirmed in the parents. Therefore, MLPA (multiplex ligation-dependent probe amplification) and specific long-range PCR were performed, and two recombinant alleles, one of them novel, and one large deletion were identified. Furthermore, an MLPA assay performed in one family resulted in the identification of an additional novel mutation (p.M124V) in a relative, in trans with the known p.N409S mutation. In conclusion, even though CES has become extensively used in clinical practice, our study emphasizes the importance of a comprehensive molecular strategy to provide proper GBA genotyping and genetic counseling.

Highlights

The GBA gene (OMIM #606463) encodes for the lysosomal hydrolase acid b-glucocerebrosidase (GCase), a 497-amino acid membrane glycoprotein of 65 kDa responsible for the breakdown of the glycolipid glucosylceramide to ceramide and glucose, in association with the cofactor Saposin C
Bi-allelic pathogenic variants in the GBA gene cause Gaucher disease (GD), a rare inherited disorder in which the deficient activity of GCase leads to the progressive storage of glucosylceramide and other glycosphingolipids within lysosomes, resulting in multiorgan system disease [1]
Clinical exome sequencing (CES) was performed on eight unrelated patients affected by GD

Summary

Introduction

The GBA gene (OMIM #606463) encodes for the lysosomal hydrolase acid b-glucocerebrosidase (GCase), a 497-amino acid membrane glycoprotein of 65 kDa responsible for the breakdown of the glycolipid glucosylceramide to ceramide and glucose, in association with the cofactor Saposin C. The GBA gene consists of 11 exons and 10 introns, covering approximately 7.6 kb on chromosome 1q21. 16 kb downstream of the functional GBA gene, a nonfunctional highly homologous pseudogene (GBAP1) that shows a similar exon–intron organization [10] has been identified. Both nonreciprocal and reciprocal recombination events occur due to the high degree of homology and the close proximity between GBAP1 and GBA, promoting the formation of complex recombinant alleles [11,12]

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