Functional Characterization of the Novel Mutation IVS 8 - Science Direct

M. Romano, et al.

Blood Cells, Molecules, and Diseases (2000) 26(3) June: 171–176 doi:10.1006/bcmd.2000.0293, available online at http://www.idealibrary.com on

Functional Characterization of the Novel Mutation IVS 8 (ⴚ11delC) (ⴚ14T>A) in the Intron 8 of the Glucocerebrosidase Gene of Two Italian Siblings with Gaucher Disease Type I Submitted 04/14/00 (Communicated by Ernest Beutler, M.D., 04/14/00)

Maurizio Romano,1,2 Giorgia M. Danek,3 Francisco E. Baralle,1 Raffaella Mazzotti,4 and Mirella Filocamo4 ABSTRACT: Gaucher disease, the most common glycolipid storage disease, can be caused by a large variety of mutations. We report here the identification and characterization of a novel mutation in the human glucocerebrosidase gene, IVS 8 (⫺11delC) (⫺14T⬎A), in two siblings with Gaucher disease type I which occurs within the 3⬘ end of intron 8. Both siblings were compound heterozygotes for the IVS 8 (⫺11delC) (⫺14T⬎A) mutation and for the c.626 G⬎C (R170P) substitution within exon 6. No mRNA species carrying the IVS 8 (⫺11delC) (⫺14T⬎A) mutation were detected by RT–PCR analysis of the RNA extracted from the patients’ fibroblasts. To study the possible effects of the IVS 8 (⫺11delC) (⫺14T⬎A) sequence alteration on the splicing of the proximal exon 9, we have established an in vitro system generating a minigene carrying the genomic region of human glucocerebrosidase spanning from exon 8 to exon 10. Transfections into the human Hep3B cell line of the wild-type construct resulted in the expression of mRNA with the glucocerebrosidase exons correctly spliced. On the contrary, transfections of the construct carrying the IVS 8 (⫺11delC) (⫺14T⬎A) mutation resulted in the expression of mRNA with an 11-bp insertion located between the end of exon 8 and the beginning of exon 9. These results indicated that the 5243T⬎A substitution created a new 3⬘ splice site 11 bp upstream of the wild-type one, leading to the incorporation into the mRNA of these extra 11 bases. Moreover, the new 3⬘ splice site created by this 5243T⬎A transversion was preferred over the wild-type one in 100% of cases. The in vitro studies suggest that, in the patients, the 11-bp inclusion causes a shift in the reading frame with the generation of a stop codon after codon 388 which undergoes early degradation. © 2000 Academic Press

INTRODUCTION

symptoms include splenomegaly, hepatomegaly, anemia and bone lesions. Type II is an infantile form with severe neurologic manifestations and type III is the juvenile-onset form characterized by a later onset of neurologic symptoms than in type II and a longer course (1). Cloning of the glucocerebrosidase gene (GBA) has allowed identification of more than 100 GD alleles (2, 3). Most of the mutations found within the glucocerebrosidase gene are single nucleotide substitutions, deletions or insertions resulting in amino acid substitutions or terminations. In vitro studies

Gaucher disease (GD) is an autosomal recessive disorder caused by the decreased levels of the lysosomal enzyme glucocerebrosidase (EC 3.2.1.45). The disease is characterized by a broad phenotypic heterogeneity. Three clinical types are distinguished on the basis of the absence (type I) or presence and progression (types II and III) of neurological involvement. Among the three types of GD, type I (MIM No. 230800) is the most prevalent form of the disorder and the common

Reprint requests to: Mirella Filocamo, Laboratorio Diagnosi Pre/Postnatale Malattie Metaboliche–Istituto G. Gaslini, Largo G. Gaslini, 16147 Genova, Italy. Fax: ⫹39-010-3776590. E-mail: [email protected]. 1 International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012, Trieste, Italy. 2 Department of Physiology and Pathology, University of Trieste, Via A. Fleming 22, 34127, Trieste, Italy. 3 Istituto Burlo Garofolo of Trieste, Via dell’Istria, 65/1-34145 Trieste, Italy. 4 Laboratorio Diagnosi Pre/Postnatale Malattie Metaboliche–Istituto G. Gaslini, Genova, Italy. 1079-9796/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved

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based on the heterologous expression of mutant forms of the GBA gene have been carried out to analyze the effects of such mutations on the activity of the enzyme (4). On the other hand, mutations within the splice junction whose characterization was not always possible have also been described (5). In this work, we describe the set up of a system to investigate the effects on splicing of a novel peculiar splicing mutation of the human GBA associated with the appearance of GD type I in two Italian siblings.

quencing and/or restriction fragmentation analysis, as previously described (7). The following primers were used for the mutation IVS 8 (⫺11delC) (⫺14T⬎A) Gau8 5⬘acctacactctctggggacc-3⬘ (forward) and Gau9 5⬘ctgtcgacaaagttacgcac-3⬘ (reverse), annealing temperature 66°C, extension time 60 s. The forward primer Gau5 5⬘-gcttctctcttcactacctt-3⬘ and the reverse primer Gau6 5⬘-tgggtgacagagagagagac-3⬘ (annealing temperature 57°C, extension time 60 s) were used for the R170P mutation.

MATERIALS AND METHODS

Nucleotide sequencing. Sequencing of PCR products was carried out directly or after bluntend-cloning into the pUC18 SmaI/BAP (Amersham Pharmacia Biotech, Buckinghamshire, UK). Manual sequencing was performed using T7 sequencing kit (Amersham Pharmacia Biotech). Automated sequencing was performed using the CEQ 2000 sequencer machine (Beckman– Coulter, Fullerton, CA).

Patients A clinical and enzymatic diagnosis of Gaucher disease was performed in a 2-year-old child and in his younger daughter. Their parents are of Italian origin and not consanguineous. Both patients had marked visceral enlargement and underwent splenectomy in infancy. They began the enzyme replacement therapy at the age 16 and 14, respectively. Presently the patients are a 23-yearold and a 21-year-old university students. The absence of neurological involvement supports the classification of type 1 Gaucher disease.

Constructs. An in vitro system has been set up to analyze the effects of this mutation on splicing following the strategy described in (8). The 741-bp DNA fragment of the human ␣1-globin gene spanning from exon 1 to exon 3 was amplified by PCR (95°C 1 min, 60°C 1 min, 72°C 1 min; 35 cycles) using primer 5⬘ ␣-glob-BamHI/ SacI (5⬘-tttggatccgagctcagagagaacccaccatggtg-3⬘) and 3⬘␣-glob-EcoRV-NotI/XbaI (5⬘-aaatctagagcggccgcttaacggtatttggaggtc agcacggtgctcacagaagccaggaagatatccaggga-3⬘). The PCR product was cut with SacI and NotI restriction enzymes and cloned into pBK-CMV expression vector (Stratagene, La Jolla, CA) and SacI/NotI digested, generating the pBK-␣-globin vector (Fig. 2). The GBA genomic region of the proband, spanning from exon 8 to exon 10 was amplified by PCR (95°C 1 min, 60°C 1 min, 72°C 2 min; 35 cycles) using primers 5⬘ Gau8 (5⬘-gtactgacagacccagaagca-3⬘) and 3⬘ Gau 10 (5⬘-ggtttagcacgaccacaaca-3⬘) and was then blunt-end cloned within the third exon of ␣-globin gene in of the pBK-␣globin EcoRV digested. The orientation and the identity of clones carrying the wild type or the IVS 8 (⫺11delC)

Molecular Biology Techniques DNA extraction. DNA was extracted from peripheral blood leukocytes and/or fibroblasts by standard laboratory methods. RNA extraction and RT–PCR. Total RNA was extracted from fibroblasts using the Chomczynski and Sacchi method (6) and poly(dT) cDNA was synthesized using M-MLV reverse transcriptase (GIBCO Brl, Grand Island, NY), according to the manufacturer’s instructions. Mutational analysis of GBA gene. The analysis was performed on appropriate GBA fragments amplified by PCR. The primers were chosen to amplify the glucocerebrosidase gene selectively from the pseudogene. The PCR products were screened by non isotopic SSCP (single strand conformation polymorphism) combined with se172

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(⫺14T-⬎A) allele was confirmed by sequencing of the entire minigenes. Transfections. Human hepatocarcinoma Hep3B cell line was growing in Dulbecco’s modified Eagle’s medium supplemented with 4.5 g/l glucose, 10% fetal calf serum, 50 ␮g/ml gentamicin, and 4 mM glutamine. The DNA used for transfections was purified with JetStar columns (Genomed, GmbH, Wielandstr., GE). Liposome-mediated transfections of 3 ⫻ 105 Hep3B cells were performed using DOTAP (Boehringer), according to the manufacturer’s instructions. Five micrograms of DNA of construct were used for each transfection and 200 ng of a plasmid carrying human growth hormone (hGH) under CMV promoter control were included as an internal control to normalize transfection efficiency. After 12 h the medium was replaced with fresh medium and 24 h later the cultures were terminated. The RNA was extracted by the Chomczynski and Sacchi method, while the medium was used to assay the hGH production by ELISA (hGH-ELISA, Boehringer Mannheim). Each transfection experiment was repeated three times.

FIG. 1. Nucleotide sequences of the clones encompassing the intron 8 – exon 9 genomic region of the normal (WT) and the mutant [IVS 8 (⫺11delC) (⫺14T-⬎A)] alleles of the male proband. Arrows show the position where the ⫺14T3 A substitution and the ⫺–11delC deletion occur. Curved arrow shows the starting point of exon 9, whose initial codons are reported.

transversion at cDNA position 626 that predicts the nonconservative replacement of Proline for Arginine at codon 170 (R170P); the second was the substitution of thymine at genomic nucleotide 5243 with an adenine and the deletion of cytosine at nucleotide 5246, located at the 3⬘ end of intron 8 of glucocerebrosidase gene and it was referred to as IVS 8 (⫺11delC) (⫺14T⬎A). The presence of mutation R170P in the heterozygous form was confirmed by MnlI RFLP analysis, since this mutation creates a new cleavage site of the restriction enzyme. Both siblings were compound heterozygotes for these mutations and subsequently, the genotype analysis of parents revealed that the father was heterozygous for the R170P substitution whereas the mother was heterozygous for the IVS 8 (⫺11delC) (⫺14T⬎A) intronic mutation. Afterward, the RT–PCR analysis of the mRNA extracted from the fibroblasts of the proband revealed only the presence of the allele carrying the R170P substitution.

Nomenclature. Mutation nomenclature is given according to Antonarakis (9). Nucleotide sequences are numbered from the upstream initiator ATG as proposed by Beutler and Gelbart (5). The amino acid is from the mature N-terminus. RESULTS Genetic Studies The scanning of the glucocerebrosidase gene of the proband by SSCP analysis revealed mobility shifts in two genomic regions, the first localized in the sequence spanning exon 6 and the second in the sequence spanning exon 9. To characterize the nature of these alterations at genetic level, the DNA fragments of the GBA gene encompassing the exon 6, the exon 9 and their flanking intronic sequences were amplified by PCR, cloned and sequenced. Two different mutations were found: the first was the G to C

In Vitro Studies To understand the possible effects of the IVS 8 (⫺11delC) (⫺14T⬎A) sequence alteration, we have considered the possibility that it could affect the correct splicing of exon 9: in fact, the 5243T⬎A substitution might create a new 3⬘ splice site (TTG⬎TAG) just 11 bases upstream of the wild-type splice site (Fig. 1). However, the 173


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FIG. 2. Diagram of the ␣-globin/glucocerebrosidase hybrid minigene cloned in pBK-CMV vector. White boxes, ␣-globin exons; gray boxes, glucocerebrosidase exons; lines, introns; 5⬘ Gau 8 and 3⬘ ␣-glob, primers used to amplify specifically the transfected minigenes; WT (wild-type) and IVS 8 (⫺11delC) (⫺14T-⬎A), the relevant sequences of the two alleles are shown.

initial RT–PCR analysis of the mRNA extracted from the proband’s fibroblasts did not show any aberrantly spliced glucocerebrosidase mRNA. Therefore, to prove that the IVS 8 (⫺11delC) (⫺14T⬎A) sequence alteration was a real mutation and not a simple polymorphism, and to study its possible effects on splicing, an in-vitro system was established, creating two minigenes with the sequences of human glucocerebrosidase gene relevant to analyze the consequences of that mutation. To do this, the DNA fragment of the GBA gene of the male proband, encompassing the genomic region from exon 8 to 10 was amplified by PCR and was then cloned within the third exon of ␣-globin gene of the pBK-␣-globin vector. Two constructs for expression in eukaryotic cells were generated, carrying either the wild-type or IVS 8 (⫺11delC) (⫺14T⬎A) allele (Fig. 2). After liposome-mediated transfections of Hep3B cell line, RNA extraction and poly-A cDNA synthesis, the splicing products originated specifically by the transfected minigenes were amplified by PCR using a forward primer annealing within the glucocerebrosidase exon 8 and a reverse primer annealing within the third exon of ␣-globin. Direct sequencing of the PCR products showed that the transfections with the wild-type construct generated mRNA with the glucocerebrosidase exons correctly spliced, whereas the IVS 8 (⫺11delC) (⫺14T⬎A) construct generated mRNA with an 11-bp insertion located between the end of exon 8 and the beginning of exon 9 (Fig. 3). The proportion of the 11-bp inclusion was 100%. These results indicate that the 5243T⬎A sub-

stitution creates a new 3⬘ splice site 11 bp upstream of the wild-type one, leading to the incorporation into the mRNA of these extra 11 bases. DISCUSSION To date, more than 100 different mutant alleles have been reported in patients with Gaucher disease including missense or nonsense point mutations, insertions, partial total gene deletions, complex alleles (3, 10 –12). On the other hand, only a limited number of splicing mutations have been reported, mostly related to alterations of the consensus splicing junctions (5). Therefore, the

FIG. 3. Nucleotide direct sequences of cDNAs deriving from the transfections of the WT (A) and IVS 8 (⫺11delC) (⫺14T-⬎A) (B) ␣-globin/glucocerebrosidase hybrid constructs. (A) Direct sequencing of PCR products of the WT construct shows the correct splicing between exon 8 and exon 9 of the glucocerebrosidase gene. (B) Direct sequencing of PCR products of the IVS 8 (⫺11delC) (⫺14T-⬎A) construct shows the presence of an 11-bp insertion between exons 8 and 9. 174

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FIG. 4. Nucleotide and deduced amino acid sequence of the wild-type cDNA and of the cDNA with the 11-bp insertion. The 11-bp insertion between exon 8 and 9 causes a shift in the reading frame with the generation of a stop codon after the codon 388 (IVS 8 (⫺11delC) (⫺14T-⬎A) cDNA). Uppercase: exon 8 and exon 9. Lowercase: 11 bp insertion of intron 8 between exon 8 and exon 9.

goes early degradation, similarly to the rapid nonsense-mediated decay described for other mRNA carrying nonsense or frameshift mutations (13–18). Then, with regards to the possible effects of the R170P substitution found on the other allele, this mutation was recently described by Germain et al. (19) and it is considered functionally relevant because it occurs at a position evolutionarily conserved in human and mouse glucocerebrosidase. Thus, the compound heterozygosity for the IVS 8 (⫺11delC) (⫺14T⬎A) and the R170P mutations, may explain the fairly severe form of type I disease of the patients with early onset of considerable hepatosplenomegaly. In absence of the most common GBA mutations, the prognostic significance of novel or rare disease alleles needs further investigations. Moreover, our approach may be useful to investigate the effect of the mutations possibly related to splicing of the GBA gene or of other genes when the availability of patients’ RNA is limited or null.

detection of the IVS 8 (⫺11delC) (⫺14T⬎A) sequence alteration within intron 8 of GBA gene in one GD patient out of 128 unrelated Italian patients screened for GBA mutations, prompted us to search more evidence that may prove the mutation is causative of disease. Intriguingly, the location of the alteration within the 3⬘ splice site of exon 9 suggested that the mutation could possibly affect the splicing of that exon. To address this hypothesis, initially the mRNA of the proband was analyzed to ascertain if the intronic mutation could have altered the splicing pattern the GBA mRNA, but no aberrantly spliced forms of the GBA mRNA were detected. Since this finding might be due to the instability of the mRNA arising from the IVS 8 (⫺11delC) (⫺14T⬎A) allele, it was necessary to establish a system to investigate the possible role of the intronic alteration on the splicing of proximal exon 9. Importantly, using such a system, only the mRNA species carrying the 11 bp-insertion was detected after transfection with the IVS 8 (⫺11delC) (⫺14T⬎A) construct, showing that the new 3⬘ splice site created by this mutation was preferred over the wild-type one in the 100% of cases. These in vitro results suggest that, in the patients, the 11 bp-inclusion causes a shift in the reading frame with the generation of a stop codon after the codon 388 (Fig. 4). Therefore, in vivo, it seems that the aberrantly-spliced mRNA under-

ACKNOWLEDGMENTS The cell lines used in this work were obtained from the collection “Cell Lines and DNA Bank from Patients Affected by Genetic Disease” supported by Italian Telethon grants. 175


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