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PubMed2003

Analysis of GNAS1 and overlapping transcripts identifies the parental origin of mutations in patients with sporadic Albright hereditary osteodystrophy and reveals a model system in which to observe the effects of splicing mutations on translated and untranslated messenger RNA.

Albright hereditary osteodystrophy (AHO) is caused by heterozygous deactivating GNAS1 mutations. There is a parent-of-origin effect. Maternally derived mutations are usually associated with resistance to parathyroid hormone termed "pseudohypoparathyroidism type Ia." Paternally derived mutations are associated with AHO but usually normal hormone responsiveness, known as "pseudo-pseudohypoparathyroidism." These observations can be explained by tissue-specific GNAS1 imprinting. Regulation of the genomic region that encompasses GNAS1 is complex. At least three upstream exons that splice to exon 2 of GNAS1 and that are imprinted have been reported. NESP55 is exclusively maternally expressed, whereas exon 1A and XL alphas are exclusively paternally expressed. We set out to identify the parental origin of GNAS1 mutations in patients with AHO by searching for their mutation in the overlapping transcripts. This information would be of value in patients with sporadic disease, for predicting their endocrine phenotype and planning follow-up. In doing so, we identified mutations that resulted in nonsense-mediated decay of the mutant Gs alpha transcript but that were detectable in NESP55 messenger RNA (mRNA), probably because they lie within its 3' untranslated region. Analysis of the NESP55 transcripts revealed the creation of a novel splice site in one patient and an unusual intronic mutation that caused retention of the intron in a further patient, neither of which could be detected by analysis of the Gs alpha complementary DNA. This cluster of overlapping transcripts represents a useful model system in which to analyze the effects that mutant sequence has on mRNA-in particular, splicing-and the mechanisms of nonsense-mediated mRNA decay.

Alternative Splicing↗
PubMed2002

A novel aberrant splicing mutation of the PEX16 gene in two patients with Zellweger syndrome.

Human Pex16p, a peroxisomal membrane protein composed of 336 amino acids, plays a central role in peroxisomal membrane biogenesis. A nonsense mutation (R176ter) in the PEX16 gene has been reported in the case of only one patient (D-01) belonging to complementation group D of the peroxisome biogenesis disorders. We have now identified two patients belonging to group D (D-02 and D-03) whose fibroblasts were found to contain no peroxisomal membrane structure ghosts. Molecular analysis of the PEX16 gene revealed aberrant cDNA species lacking 65 bp, corresponding to exon 10 skipping caused by a splice site mutation (IVS10 + 2T -->C). Both patients, although unrelated, were homozygous for this mutation. This mutation changes the amino acid sequence starting from codon 298 and introduces a termination codon at codon 336. As a consequence, the cell's ability to membrane synthesis and protein import is disrupted, which implies that the changed C terminus of the Pex16p in these patients likely affects its function.

ATP-Binding Cassette Transporters↗
PubMed2002

Novel COL6A1 splicing mutation in a family affected by mild Bethlem myopathy.

Bethlem myopathy is an early-onset benign myopathy characterized by proximal muscular weakness and multiple flexion contractures. It is a dominantly inherited disorder associated with mutations in the three COL6 genes encoding type VI collagen. We detected a g-->a substitution at +1 position of COL6A1 intron 3 in a four-generation Italian family affected by a mild form of Bethlem myopathy. The mutation results in the activation of a cryptic splice donor site at the 3' end of exon 3, leading to the loss of 66 nucleotides and an "in-frame" deletion of 22 amino acids in the NH2-domain. Molecular analysis on fibroblasts of the propositus showed that the mutated mRNA was present and stable, but the mutated protein could not be detected. Western blot and immunofluorescence analyses showed a decreased level of collagen VI synthesis and deposition in fibroblasts of the propositus. Together, the results suggest that the mutated protein was highly unstable and rapidly degraded, and that the mild phenotype was caused by a reduced amount of normal collagen VI microfibrils. In addition, we demonstrated that lymphocytes can be used for the first mutation screening analysis of patients with Bethlem myopathy.

Adolescent↗
PubMed2004

A new HLA-B44 allele (B*44020102S) with a splicing mutation leading to a complete deletion of exon 5.

Using a combination of serology and polymerase chain reaction with sequence-specific primer (PCR-SSP), we have identified in a volunteer bone marrow donor a new HLA class I antigen within the B44 serotype. This human leukocyte antigen (HLA)-B44 variant was typed as 'blank' by microlymphocytotoxicity, whereas the B*44020101 allele was identified by PCR-SSP. A family study confirmed the Mendelian segregation of this blank antigen identified on one of the maternal haplotype transmitted to her child. The DNA sequence of B*44new, now referred to as B*44020102S, performed from the promoter region to the 3' untranslated region revealed a single nucleotide difference (A/G) compared to B*44020101 at the end of intron 4 in the acceptor-splicing site. This mutation leads to an incorrect splicing characterized by the deletion of exon 5 that encodes the transmembrane domain of the HLA antigen. Indeed, full-length complementary DNA sequencing revealed a complete absence of exon 5. Fluorescence-activated cell sorter analysis confirmed the absence of expression of HLA-B44 on the cell surface in the donor, compared to the HLA-B44 positive control. The isoelectric focusing analysis failed to reveal the presence of an HLA-B44 antigen in the donor, showing that no normal HLA-B*44020101 allele was synthesized. The new B*440201010102S allele is a soluble form of B44 without any detectable cell-surface expression. It can thus be considered as a soluble antigen, a form apparently inactive and unfit for antigen presentation.

3' Untranslated Regions↗
PubMed2005

A filamin A splice mutation resulting in a syndrome of facial dysmorphism, periventricular nodular heterotopia, and severe constipation reminiscent of cerebro-fronto-facial syndrome.

BACKGROUND: Mutations of the filamin A locus (FLNA) on Xq28 have been established in girls with periventricular nodular heterotopia and in patients with otopalatodigital and overlapping phenotypes, the pathogenesis of these phenotypes being thought to be quite distinct. To date only six male cases of periventricular nodular heterotopia (PVNH) have been reported and these almost invariably associated with severe neurological signs. METHODS AND RESULTS: We report a new phenotype of male PVNH, with relatively normal development, no epilepsy or other neurological abnormality, severe constipation, and facial dysmorphism and without a discernible skeletal phenotype. This phenotype is associated with a splice site mutation in FLNA c.1923C>T, resulting in the generation of both normal and aberrant mRNA. CONCLUSIONS: We postulate that the patient retains enough FLNA function to avoid the usual lethality associated with loss of function mutations in males and suggest that the severe constipation may be a clue to the molecular aetiology of other X linked conditions associated with severe constipation.

Abnormalities, Multiple↗
PubMed1999

Late infantile neuronal ceroid lipofuscinosis is due to splicing mutations in the CLN2 gene.

Late infantile neuronal ceroid lipofuscinosis, LINCL, is one of the most common pediatric neurodegenerative disorders. It is caused by mutations in the CLN2 gene, which encodes a lysosomal pepstatin-insensitive peptidase (LPIP). We have identified a novel mutation, T523-1G --> A, by molecular analyses of three unrelated LINCL cases. The mutation was found to affect a 3' intronic splicing acceptor site, resulting in an aberrant mRNA with an insertion of 146 bp of intronic sequence. This causes a frame shift, produces a nonfunctional truncated protein, and results in LINCL.

Adenine↗
PubMed1993

A splicing mutation in the alpha 5(IV) collagen gene of a family with Alport's syndrome.

DNA sequence analysis of the alpha 5(IV) collagen chain gene (COL4A5) was carried out between exon 47 and 51, which encode the noncollagenous (NC) domain, in eight Japanese families with Alport's syndrome. In one family with X-linked inheritance of the disease, a point mutation (G to C) was found at the 3' end of exon 49 in the COL4A5. This mutation converted the codon of a conserved methionine-1601 to the codon for isoleucine, and also altered the normal splicing process. The polymerase chain reaction (PCR) product amplified between exons 47 and 51 of cDNA in the affected male (hemizygote) of this family contained four fragments with various molecular weights, whereas that of a normal control contained one with the expected molecular weight. Sequence analysis of the PCR fragments of the male patient revealed various types of alternative splicing between the exons, reflecting the various sizes of PCR fragments. The PCR amplified product of the cDNA of the affected female (heterozygote), on the other hand, contained a fragment with the same molecular weight as the normal control. Sequence analysis of the PCR fragments of her cDNA revealed normal splicing and no point mutation at the 3' end of exon 49. These findings indicate that this point mutation at the consensus sequence not only converted the codon but also altered the splicing between these exons encoding the NC domain of the COL4A5. Resulting in missense of the alpha 5(IV) chain, changing a large portion of the carboxyl terminal crosslinking NC domain, this mutation can alter the normal structure of the type IV collagen network.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed2001

A recurrent RNA-splicing mutation in the SEDL gene causes X-linked spondyloepiphyseal dysplasia tarda.

Spondyloepiphyseal dysplasia tarda (SEDL) is a genetically heterogeneous disorder characterized by mild-to-moderate short stature and early-onset osteoarthritis. Both autosomal and X-linked forms have been described. Elsewhere, we have reported the identification of the gene for the X-linked recessive form, which maps to Xp22.2. We now report characterization of an exon-skipping mutation (IVS3+5G-->A at the intron 3 splice-donor site) in two unrelated families with SEDL. Using reverse transcriptase (RT)-PCR, we demonstrated that the mutation resulted in elimination of the first 31 codons of the open reading frame. The mutation was not detected in 120 control X chromosomes. Articular cartilage from an adult who had SEDL and carried this mutation contained chondrocytes with abundant Golgi complexes and dilated rough endoplasmic reticulum (ER). RT-PCR experiments using mouse/human cell hybrids revealed that the SEDL gene escapes X inactivation. Homologues of the SEDL gene include a transcribed retropseudogene on chromosome 19, as well as expressed genes in mouse, rat, Drosophila melanogaster Caenorhabditis elegans, and Saccharomyces cerevisiae. The latter homologue, p20, has a putative role in vesicular transport from ER to Golgi complex. These data suggest that SEDL mutations may perturb an intracellular pathway that is important for cartilage homeostasis.

Adult↗
PubMed1990

Inheritance of an RNA splicing mutation (G+ 1 IVS20) in the type III procollagen gene (COL3A1) in a family having aortic aneurysms and easy bruisability: phenotypic overlap between familial arterial aneurysms and Ehlers-Danlos syndrome type IV.

Inheritance of a single base mutation in the type III procollagen gene (COL3A1) was studied in a family with aortic aneurysms and easy bruisability. The mutation was a substitution of A for G+ 1 of intron 20 of the gene and caused aberrant splicing of RNA transcribed from the mutated allele. The phenotype in the family included aortic aneurysms that ruptured and produced death. It also included easy bruisability, but it did not include other characteristic features of Ehlers-Danlos syndrome type IV, such as ecchymoses, abnormal scarring, or prominent subcutaneous blood vessels. The data from the family, together with a review of other probands with mutations in the type III procollagen gene, indicated that there is phenotypic overlap between Ehlers-Danlos syndrome type IV and familial arterial aneurysms not associated with any overlap between Ehlers-Danlos syndrome type IV and familial arterial aneurysms not associated with any of the striking changes in skin originally cited as a characteristic feature of Ehlers-Danlos syndrome type IV. In addition, the results suggested that DNA tests for mutations in the type III procollagen gene may be useful to identify individuals predisposed to developing arterial aneurysms.

Adult↗
PubMed1986

Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells.

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.

Animals↗
PubMed2004

A homozygous splice mutation in the HSF4 gene is associated with an autosomal recessive congenital cataract.

PURPOSE: To map the locus and identify the gene causing autosomal recessive congenital cataracts in a large consanguineous Tunisian family. METHODS: DNA was extracted from blood samples from a large Tunisian family with an autosomal recessive, congenital, total white cataract. A genome-wide scan was performed with microsatellite markers. All exons and the splice sites of the HSF4 gene were sequenced in all members of the Tunisian family and in control individuals. RT-PCR was used to detect different transcripts of the HSF4 gene in the human lens. The transcripts were cloned in a TA cloning vector and sequenced. RESULTS: Two-point linkage analyses showed linkage to markers on 16q22 with a maximum lod score of 17.78 at theta = 0.01 with D16S3043. Haplotype analysis refined the critical region to a 1.8-cM (4.8-Mb) interval, flanked by D16S3031 and D16S3095. This region contains HSF4, some mutations of which cause the autosomal dominant Marner cataract. Sequencing of HSF4 showed a homozygous mutation in the 5' splice site of intron 12 (c.1327+4A-->G), which causes the skipping of exon 12. A more detailed study of the transcripts resulting from alternative splicing of the HSF4 gene in the lens is also reported, showing the major transcript HSF4b. CONCLUSIONS: This is the first report describing association of an autosomal recessive cataract with the HSF4 locus on 16q21-q22.1 and the first description of HSF4 splice variants in the lens showing that HSF4b is the major transcript.

Cataract↗
PubMed1991

A donor splice mutation and a single-base deletion produce two carboxyl-terminal variants of human serum albumin.

At least 35 allelic variants of human serum albumin have been sequenced at the protein level. All except two COOH-terminal variants, Catania and Venezia, are readily explainable as single-point substitutions. The two chain-termination variants are clustered in certain locations in Italy and are found in numerous unrelated individuals. In order to correlate the protein change in these variants with the corresponding DNA mutation, the two variant albumin genes have been cloned, sequenced, and compared to normal albumin genomic DNA. In the Catania variant, a single base deletion and subsequent frameshift leads to a shortened and altered COOH terminus. Albumin Venezia is caused by a mutation that alters the first consensus nucleotide of the 5' donor splice junction of intron 14 and the 3' end of exon 14, which is shortened from 68 to 43 base pairs. This change leads to an exon skipping event resulting in direct splicing of exon 13 to exon 15. The predicted Venezia albumin product has a truncated amino acid sequence (580 residues instead of 585), and the COOH-terminal sequence is altered after Glu-571. The variant COOH terminus ends with the dibasic sequence Arg-Lys that is apparently removed through stepwise cleavage by serum carboxypeptidase B to yield several forms of circulating albumin.

Amino Acid Sequence↗
PubMed1997

Three novel splice mutations in the PCCA gene causing identical exon skipping in propionic acidemia patients.

Propionyl-CoA carboxylase (PCC) is a mitochondrial, biotin-dependent enzyme involved in the catabolism of branched chain amino acids, odd chain fatty acids, and other metabolites. PCC consists of non-identical subunits, alpha and beta, encoded by the PCCA and PCCB genes, respectively. Inherited deficiency of PCC due to mutations in either the PCCA or the PCCB gene results in propionic acidemia (PA), a clinically heterogeneous disorder with a severe, often lethal, neonatal form, and a mild, later onset form. To characterize PCCA gene mutations responsible for PCC deficiency, we analyzed RT-PCR products obtained from cultured fibroblasts from Spanish PCC-alpha deficient patients. In three patients, smaller than normal PCR products were observed, and sequence analysis revealed the deletion of a 54-bp exon in the cDNA. Sequencing of genomic DNA from these three patients led to the identification of three novel mutations in the PCCA gene, two short deletions and one small insertion, adjacent to short direct repeats, and all of them affecting the consensus splice sites of the skipped exon. These mutations, 1771IVS-2del9, 1824IVS+3del4, and 1824IVS+3insCT, are the cause of the aberrant splicing of the PCCA pre-mRNA and result in an in-frame deletion of 54 nucleotides in the cDNA, probably leading to an unstable protein structure which is responsible for the lack of activity leading to PCC deficiency in these patients.

Amino Acid Metabolism, Inborn Errors↗