Homozygosity for a non‐pseudogene complex glucocerebrosidase allele as cause of an atypical neuronopathic form of Gaucher disease

Gaucher disease (GD) is a lysosomal storage disorder due to a deficiency of glucocerebrosidase (EC 3.2.1.45). GD is a multisystem disorder associated with striking variation in its clinical manifestations, severity, and course. Clinical phenotypes, ranging from severely affected infants to asymptomatic adults have traditionally been divided into three major groups on the basis of central nervous system involvement: type 1, non-neuronopathic (MIM 230800); type 2 acute neuronopathic (MIM 230900), and type 3 subacute neuronopathic (MIM 231000) [Beutler and Grabowski, 2001]. Human glucocerebrosidase gene (GBA; GenBank-EMBL accession no. J03059) is located on chromosome 1q21 and contains 11 exons. A highly homologous pseudogene (cGBA) lies 16 kb downstream from the functional gene [Horowitz et al., 1989]. To date, more than 200mutations including point mutations, deletions, insertions, and splicing aberrations have been identified in theGBAgene; various complex alleles,which have presumably arisen from crossovers between the functional gene and the pseudogene have also been described [Horowitz and Zimran, 1994; Grabowski and Horowitz, 1997; Cormand et al., 2000; Koprivica et al., 2000; Stone et al., 2000; Filocamo et al., 2002]. Only a few complex non-pseudogenederived alleles were previously reported [Eyal et al., 1991; Grace et al., 1999; Hodanov et al., 1999; Filocamo et al., 2002]. We have recently characterized a new non-pseudogene complex mutant allele, D409HþH255Q, in homozygosis in a patient with an atypical clinical presentation of neurological GD. The patient is a 25-month-old girl from Albany. She was normal at birth and achieved early developmental milestones. At the age of 5 months hepatosplenomegaly was first noticed. Threemonths later the child was admitted to the local hospital due to massive hepatosplenomegaly, anemia, and thrombocytopenia. Neurological examination showed spasticity with persistent retroflexion of the neck, convergent strabism, oculomotor apraxia, and laryngeal stridor. GD was diagnosed on the basis of the lymphoblast glucocerebrosidase level, which was 0.9 nmol/mg/hr (n.v.1⁄4 30.5 17.9). A compassionate enzymatic replacement treatment at a dosage of 60 U/kg of imiglucerase every 2 weeks was started at the age of 13 months, with a reduction of the hepatosplenomegaly. She hadno significant neurological improvement, but the neurological disease showed a very slowprogression: at the age of 25 months she is able to sit alone, walk with support, and pronounce some words. EEG and visual evoked potentials are normal; but brainstem evoked potentials demonstrate abnormal truncal conduction. BrainMRI reveals abnormal signal in T2 and FLAIR at the level of the pons, cerebellum, periventricular areas, and basal ganglia. Molecular analysis of the GBA gene was performed on patient’s genomic DNA extracted from lymphoblasts using standard methods. Sequencing of the entire GBA gene of the patient showed the presence of the rare H255Q and the common D409H mutations in exons 7 and 10, respectively (Table I). As RFLP analysis revealed homozygosity for both mutations, it was likely that the D409H coexisted on the same allele with H255Q (Fig. 1). Since at that time the parents were not at hand for molecular testing, the assumed presence of the twomutations in cis was investigated by cloning long-PCR products of the entire GBA gene in pCR2.1 (Invitrogen, Carlsbad, CA, USA). After transformation, about 10 colonies were selected for a complete sequence analysis, which confirmed the double mutant allele. It is unclear how this allele carrying two mutations originated. It is unlikely to have derived froma recombination event since in contrast toD409H, the H255Q mutation was not present in the pseudogene. The mutant combination probably arose from two independent events, namely the rare transversion H255Q could have occurred in exon 7 on a pre-existing common D409H allele. Additionally, although consanguinity between the patient’s parents was not proved, a common origin of the nonpseudogene complex glucocerebrosidase might be speculated. A familial study of microsatellite markers flanking the gene could support this hypothesis. Prediction of the disease severity based on the patient’s genotype is limited due to rarity of such complex alleles. However, considering D409H and H255Q as individual mutations, a neuronopathic nature can be supposed for both. Expression studies have demonstrated that the D409H allele is severely compromised, having very low activity [Grace et al., 1994; Pasmanik-Chor et al., 1997] and has been found to be associated with neuronopathic involvement. Concerning the individual H255Qmutation, this was previously described in a Greek patient as a compound heterozygote with severe RecTL leading to type 2 disease [Stone et al., 2000]. Apparently, the interactive effects of the twomutations explain the atypical phenotype of our patient. In fact, this child has a phenotype intermediate between type 2 and type 3 GD with long survival, early neurological