Identification and molecular characterization of six novel mutations in the UDP-N-acetylglucosamine-1-phosphotransferase gamma subunit (GNPTG) gene in patients with mucolipidosis III gamma.
Mucolipidosis type III (MLIII) is an autosomal recessive disorder affecting lysosomal hydrolase trafficking. In a study of 10 patients from seven families with a clinical phenotype and enzymatic diagnosis of MLIII, six novel GNPTG gene mutations were identified. These included missense (p.T286M) and nonsense (p.W111X) mutations and a transition in the obligate AG-dinucleotide of the intron 8 acceptor splice site (c.610-2A>G). Three microdeletions were also identified, two of which (c.611delG and c.640_667del28) were located within the coding region whereas one (c.609+28_610-16del) was located entirely within intron 8. RT-PCR analysis of the c.610-2A>G transition demonstrated that the change altered splicing, leading to the production of two distinct aberrantly spliced forms, viz. the skipping of exon 9 (p.G204_K247del) or the retention of introns 8 and 9 (p.G204VfsX28). RT-PCR analysis, performed on a patient homozygous for the intronic deletion (c.609+28_610-16del), failed to detect any GNPTG RNA transcripts. To determine whether c.609+28_610-16del allele-derived transcripts were subject to nonsense-mediated mRNA decay (NMD), patient fibroblasts were incubated with the protein synthesis inhibitor anisomycin. An RT-PCR fragment retaining 43 bp of intron 8 was consistently detected suggesting that the 33-bp genomic deletion had elicited NMD. Quantitative real-time PCR and GNPTG western blot analysis confirmed that the homozygous microdeletion p.G204VfsX17 had elicited NMD resulting in failure to synthesize GNPTG protein. Analysis of the sequences surrounding the microdeletion breakpoints revealed either intrinsic repetitivity of the deleted region or short direct repeats adjacent to the breakpoint junctions. This is consistent with these repeats having mediated the microdeletions via replication slippage and supports the view that the mutational spectrum of the GNPTG gene is strongly influenced by the properties of the local DNA sequence environment.
Persichetti
Emanuele
E
Dipartimento di Medicina Interna, Università degli Studi di Perugia, Perugia, Italy.
Chuzhanova
Nadia A
NA
Dardis
Andrea
A
Tappino
Barbara
B
Pohl
Sandra
S
Thomas
Nick S T
NS
Rosano
Camillo
C
Balducci
Chiara
C
Paciotti
Silvia
S
Dominissini
Silvia
S
Montalvo
Anna Lisa
AL
Sibilio
Michela
M
Parini
Rossella
R
Rigoldi
Miriam
M
Di Rocco
Maja
M
Parenti
Giancarlo
G
Orlacchio
Aldo
A
Bembi
Bruno
B
Cooper
David N
DN
Filocamo
Mirella
M
Beccari
Tommaso
T
D000293:Adolescent; D000328:Adult; D017398:Alternative Splicing / Q000235:genetics; D001483:Base Sequence; D002648:Child; D018389:Codon, Nonsense / Q000235:genetics; D005260:Female; D005347:Fibroblasts / Q000201:enzymology / Q000473:pathology; D005838:Genotype; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009081:Mucolipidoses / Q000201:enzymology* / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D020125:Mutation, Missense / Q000235:genetics; D021122:Protein Subunits / Q000235:genetics*; D022821:RNA Splice Sites / Q000235:genetics; D020871:RNA Stability / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics*
D018389:Codon, Nonsense; D021122:Protein Subunits; D022821:RNA Splice Sites; D012333:RNA, Messenger; D017855:Transferases (Other Substituted Phosphate Groups); C504791:GNPTG protein, human