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Results for “Splicing mutations”

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PubMed1990

Uroporphyrinogen decarboxylase: a splice site mutation causes the deletion of exon 6 in multiple families with porphyria cutanea tarda.

Uroporphyrinogen decarboxylase (URO-D) is a cytosolic heme-biosynthetic enzyme that converts uroporphyrinogen to coproporphyrinogen. Defects at the uroporphyrinogen decarboxylase locus cause the human genetic disease familial porphyria cutanea tarda. A splice site mutation has been found in a pedigree with familial porphyria cutanea tarda that causes exon 6 to be deleted from the mRNA. The intron/exon junctions on either side of exon 6 fall between codons, so the resulting protein is shorter than the normal protein, missing only the amino acids coded by exon 6. The shortened protein lacks catalytic activity, is rapidly degraded when exposed to human lymphocyte lysates, and is not detectable by Western blot analysis in lymphocyte lysates derived from affected individuals. The mutation was detected in five of 22 unrelated familial porphyria cutanea tarda pedigrees tested, so it appears to be common. This is the first splice site mutation to be found at the URO-D locus, and the first mutation that causes familial porphyria cutanea tarda to be found in more than one pedigree.

Amino Acid Sequence↗

PubMed1994

Lipoprotein lipase deficiency due to a 3' splice site mutation in intron 6 of the lipoprotein lipase gene.

In a patient with primary hyperchylomicronemia as a result of lipoprotein lipase (LPL) deficiency, we sequenced all translated exons and intron-exon boundaries of the LPL gene. We found a C-->A mutation in position -3 at the acceptor splice site of intron 6 which caused aberrant splicing. The major transcript showed a deletion of exons 6 through 9 and amounted to about 3% of the normal transcript of a healthy control individual. In addition to this major transcript, we found trace amounts of both a normally spliced LPL mRNA and a second aberrant transcript devoid of exon 7. On the same allele, we detected in the LPL gene of our patient four polymorphic variations, three of which have not as yet been described. A second patient from an unrelated family, but from the same geographic area, was also found to be homozygous for the same mutation. Of the relatives of the two probands studied, 11 were heterozygous and 5 were unaffected by the mutation. LPL activity in postheparin plasma was near zero in the probands and reduced in 4 of the 10 heterozygotes. A third hyperchylomicronemic patient from the same area was found to be a compound heterozygote who carried on one allele the 3' splice site mutation of intron 6 and on the other one an already described missense mutation resulting in Gly188-->Glu substitution.

Alleles↗

PubMed2003

A novel splicing acceptor mutation of the factor VIII gene producing skipping of exon 25.

A gross deletion in the factor VIII (FVIII) mRNA was determined by reverse transcriptase polymerase chain reaction (RT-PCR) for a patient with moderately severe hemophilia A. Sequencing of the RT-PCR product depicted a 177-bp deletion ranging from nucleotide (nt) 6724 to nt 6900 of FVIII cDNA, exactly corresponding to the whole exon 25. Further study of the genomic DNA revealed the presence of a single base pair substitution (G >A) at position -1 of intron 24. The absolute consensus AG doublet of the intron 24 splicing acceptor changed to AA. In the novel splice site mutation, exon 24 was erroneously spliced to exon 26, skipping exon 25. The FVIII antigen level was normal despite the markedly reduced functional activity. Since exon 25 corresponds to part of the C2 domain, we speculate that for this patient the aberrant C2 domain markedly reduces binding affinity of FVIII protein to the phospholipid membrane, thus severely impairing the protein function.

Adolescent↗

PubMed2003

Solitary thyroid nodule as presenting symptom of Pendred syndrome caused by a novel splice-site mutation in intron 8 of the SLC26A4 gene.

UNLABELLED: Thyroid nodules are a rare occurrence in children but represent an important clinical problem because of the possibility of malignancy. We report the case of a 4-year-old boy with sensorineural deafness, who presented with a painless mass in the right anterior cervical region. Cervical ultrasound demonstrated a solid nodule (1.4 x 2.5 x 1.7 cm) in the right thyroid lobe. Thyroid function tests revealed compensated hypothyroidism (free T4 1.0 ng/dl; TSH 57 mIU/l) with no detectable thyroid antibodies. A 99mTc thyroid scan showed a generalised slightly increased tracer retention (4.6%) with an enlarged right lobe, without distinct nodules. A fine-needle aspiration biopsy revealed normal follicular cells. The boy was treated with l-thyroxine which resulted in a complete clinical and sonographical disappearance of the nodule. A CT scan of temporal bones revealed a bilaterally enlarged vestibular aqueduct with Mondini malformation of the cochlea. The combination of all these symptoms suggested the diagnosis of Pendred syndrome (PDS), a disorder characterised by congenital sensorineural hearing loss and a variable degree of thyromegaly due to mutations in the SLC26A4/PDSgene. DNA analysis disclosed a so far unreported homozygous splice site mutation (1002-4 C>G) in intron 8 of the SLC26A4 gene confirming this diagnosis. CONCLUSION: a solitary thyroid nodule may therefore be another presenting symptom of thyroid involvement in Pendred syndrome

Carrier Proteins↗

PubMed2005

Identification of a novel splice site mutation of the CSPG2 gene in a Japanese family with Wagner syndrome.

PURPOSE: To investigate the genetic basis and clinical variability of Wagner syndrome, a rare, dominantly inherited vitreoretinopathy. METHODS: Clinical examination, linkage analysis, and mutational screening were performed in a large, three-generation, consanguineous Japanese family with Wagner syndrome. The effect of splice site mutation was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis with lymphoblastoid cell total RNAs generated from affected individuals. RESULTS: Ocular phenotypes of affected members included an empty vitreous with fibrillary condensations, avascular membrane, perivascular sheathing, and progressive chorioretinal dystrophy and were similar to those of the original Wagner syndrome family. All affected eyes examined exhibited pseudoexotropia with ectopic fovea. No systemic manifestations were observed. Genetic linkage confirmed disease segregation with the previously identified WGN1 locus on 5q13-q14. A heterozygous A-->G transversion at the second base of the 3'-acceptor splice site of intron 7 (c.4004-2 A-->G) of the chondroitin sulfate proteoglycan 2 (CSPG2) gene that cosegregated with the disease was identified. Results of RT-PCR analysis indicated that the c.4004-2 A-->G mutation activates a cryptic splice site, located 39 bp downstream from the authentic 3' splice acceptor site. CONCLUSIONS: This linkage study confirmed the genetic homogeneity of the Wagner syndrome. CSPG2 encodes versican, a large chondroitin sulfate proteoglycan, which, in vitreous, binds to hyaluronan and link protein and forms large aggregates that are important for maintaining structural integrity. Although the CSPG2 gene has been excluded as a candidate for causing Wagner syndrome, these data emphasize the necessity of further mutational screening in new families and careful functional characterization.

Adolescent↗

PubMed1995

Two alpha subunit donor splice site mutations cause human trifunctional protein deficiency.

Human trifunctional protein catalyzes three steps in mitochondrial beta-oxidation of fatty acids, including the long chain 3-hydroxyacyl-CoA dehydrogenase step. Deficiency of this heterocomplex, which contains 4 alpha and 4 beta subunits, causes sudden unexplained infant death, a Reye-like syndrome, cardiomyopathy, or skeletal myopathy. We determined the molecular basis of this deficiency in a patient with neonatal presentation and later sudden death using reverse transcription and PCR amplification of his alpha subunit mRNA. We demonstrated a universal deletion of exon 3 (71 bp) in his mRNA. This deletion causes a frameshift and very early premature termination. Amplification of genomic DNA demonstrated that the patient was a compound heterozygote with two different mutations in the 5' donor splice site following exon 3: a paternally inherited G to A transversion at the invariant position +1 and a maternally inherited A to G mutation at position +3. Both allelic mutations apparently cause exon 3 skipping, resulting in undetectable levels of alpha subunit protein, and complete loss of trifunctional protein. This is the initial molecular characterization of trifunctional protein deficiency.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1996

A novel splice-site mutation in the gamma subunit of the epithelial sodium channel gene in three pseudohypoaldosteronism type 1 families.

Pseudohypoaldosteronism type 1 (PHA1, OMIM 264350) is an uncommon inherited disorder characterized by salt-wasting and end-organ unresponsiveness to mineralocorticoids. A complete genome search using homozygosity mapping in eleven consanguineous families with PHA1 provided conclusive evidence of linkage with heterogeneity. The disease locus mapped to chromosome 16p12.2-13.11 in six families and to 12p13.1-pter in the other five families. These two chromosomal regions harbour the genes encoding the three subunits of the human amiloride sensitive epithelial sodium channel (hENaC): SCNN1B and SCNN1G on 16p and SCNN1A on 12p. Our linkage results have been further supported by the recent report of mutations in the alpha and beta subunit genes in PHA1 patients. We now report the identification of a 3' splice site mutation in SCNN1G (318-1 G-->A) in three families showing linkage to 16p. Abnormal splicing results with the production of two messenger RNAs, one arising from activation of an adjacent cryptic splice site and the other from skipping of the downstream exon. The two corresponding mutant gamma hENaC subunits are predicted to have three highly conserved amino acids in the extracellular domain replaced by a novel amino acid (KYS106-108-->N) and truncation from 649 to 134 amino acids respectively. These three families all originate from the Indian sub-continent and the probands have severe generalized PHA. They share a common haplotype which suggests the presence of a founder mutation in this sub-population.

Amino Acid Sequence↗

PubMed1997

A novel splice site mutation in the androgen receptor gene results in exon skipping and a non-functional truncated protein.

Mutations of the androgen receptor (AR) gene and protein are associated with complete androgen insensitivity syndromes (CAIS) in individuals with XY genotypes causing them to develop as phenotypic females. Splice site mutations of the AR gene are very rare and in this report we describe the consequences of a novel G --> A mutation at the exon 7/intron 7 splice junction of the AR gene that resulted in CAIS in two siblings. Reverse transcriptase-polymerase chain reaction (RT-PCR) of the AR transcript in patient's fibroblasts was performed and sequencing of the product showed omission of exon 7, with exon 6 being spliced directly to exon 8. This resulted in a shift of the reading frame and the introduction of a premature stop codon 10 amino acids into exon 8. Immunoblot analyses showed that the resultant AR protein was partially deleted in its C-terminal region and was approximately 1.5 kDa smaller than the wild type. This truncated AR was non-functional as it was unable to bind its physiological ligand (dihydrotestosterone) in androgen-binding assays. This is the first documentation of a point mutation in the AR gene which causes exon skipping and proves that the mutation is the cause of CAIS in our two subjects.

Androgens↗

PubMed1993

Splice site mutation in the human protein C gene associated with venous thrombosis: demonstration of exon skipping by ectopic transcript analysis.

Heterozygosity for a G-->C mutation converting the highly conserved Gln184 (CAG) to His (CAC) was identified at the last nucleotide of exon 7 of the protein C gene in two family members with deep vein thrombosis. As the nucleotide is a part of the 5 splice site of intron G, it was examined how the mutation affected splicing of protein C pre-mRNA. Relevant protein C cDNA fragments were amplified with polymerase chain reaction after reverse transcription of ectopic mRNA from peripheral blood lymphocytes. Southern blot analysis and nucleotide sequencing of these fragments showed a fragment (A) corresponding to correctly spliced mRNA originating from the normal allele and a fragment (B) corresponding to a truncated mRNA lacking exon 7, originating from the mutant allele. A third fragment (C) lacking exons 7 and 8 was identified in both affected and unaffected family members, as well as in normal controls. Analysis of human liver protein C mRNA indicated that the ectopic lymphocyte mRNA was qualitatively representative for the tissue-specific mRNA. In conclusion, evidence is provided showing that the mutation abolishes formation of correctly spliced mRNA. This agrees with the observation that the mutation results in a type 1 protein C deficiency.

Adult↗

PubMed1999

[A novel splice-site mutation in the STA gene in a Japanese patient with Emery-Dreifuss muscular dystrophy].

Emery-Dreifuss muscular dystrophy (EDMD) is an X-linked recessive or autosomal dominant progressive muscular dystrophy characterized by progressive muscle wasting and weakness with scapulo-humero-peroneal distribution, early contracture and cardiomyopathy with conduction block. The responsible gene for EDMD, designated as 'STA', has been mapped to Xq 28 and cloned. It encodes a serine-rich protein of 254-amino-acid, called 'emerin', localized in the inner nuclear rim. We performed genetic analysis of a 23-year-old male clinically diagnosed as EDMD and found a novel point mutation. Total RNA was extracted from skeletal muscle and reverse-transcription and polymerase chain reaction amplification was performed using a set of oligonucleotide primers between 5'-flanking site of exon 1 and exon 4. Our patient gave a smaller PCR product (about 30 bp) than normal control. The determined cDNA sequence revealed a deletion of 29 bp, spanning position 164 to 192 in exon 1. To clarify the mutant allele, we performed genomic DNA sequence. Genomic DNA sequence from the initiation of exon 1 to the upstream lesion of exon 2 confirmed a novel point mutation G to C, at nucleotide 197 in the donor splice site of intron 1. This point mutation may interfere with the correct splicing of the mRNA and cause frameshift, resulted in truncation of predicted protein by premature stop. We report a novel point mutation G to C, at nucleotide 197 in the intron 1 of STA gene corresponding the truncation of predicted protein, which differs from any of the previously reported mutations.

Adult↗

PubMed2003

An acceptor splice site mutation in HOXD13 results in variable hand, but consistent foot malformations.

HOXD13 is the most 5' of the HOXD cluster of homeobox genes in chromosome band 2q31.1. Heterozygous expansions of a polyalanine tract in HOXD13 are typically associated with synpolydactyly characterized by insertional digit duplication associated with syndactyly. We screened for mutations of HOXD13 in patients with a variety of limb malformations and identified a novel heterozygous mutation (758-2delA) in a three-generation family without the typical synpolydactyly phenotype in the hands, but with bilateral partial duplication of the 2nd metatarsals within the first web space of the feet. This mutation locates in the acceptor splice site of exon 2 and is predicted to cause failure of normal splicing of HOXD13. The foot abnormality in this family is similar to that described in two families by Goodman et al. [1998: Am. J. Hum. Genet. 63: 992-1000] in which different deletions of HOXD13 were reported. These findings together lend support to a distinct phenotype resulting from haploinsufficiency of HOXD13.

Base Sequence↗

PubMed2000

PCR based diagnosis of 21-hydroxylase gene defects in Slovak patients with congenital adrenal hyperplasia.

OBJECTIVE: To analyse 21-hydroxylase gene for 8 most common mutations in patients with salt-wasting type of congenital adrenal hyperplasia. METHODS: Allele specific PCR performed on 8 salt-wasting CAH patients and their 23 healthy relatives. RESULTS: Two patients were homozygous for 8 bp deletion in exon 3, while 6 patients were homozygous for intron 2 splice mutation. Mutant allele for splice mutation was found also in both parents of patients with this type of mutation. CONCLUSIONS: These preliminary results show that only two mutations, 8 bp deletion in exon 3 and splice mutation in intron 2, were present in this group of Slovak patients with salt-wasting type of congenital adrenal hyperplasia.

Adolescent↗

PubMed2002

XLA patients with BTK splice-site mutations produce low levels of wild-type BTK transcripts.

X-linked agammaglobulinemia is caused by mutations in the BTK gene, which result in a precursor B-cell differentiation arrest in the bone marrow and the absence of or strongly reduced B lymphocytes in blood. We identified a patient with a mild clinical phenotype, low numbers of B lymphocytes, and a splice-site mutation in the BTK gene. The precursor B-cell compartment in the bone marrow of this patient was almost identical to that in healthy children. Using real-time quantitative polymerase chain reaction, we were able to detect low levels of wild-type BTK transcripts in his granulocytes. Therefore, we speculated that wild-type BTK transcripts might be responsible for a milder clinical and immunological phenotype, as has been shown in several other diseases. Consequently, we quantified the expression of wild-type BTK transcripts in granulocytes of eight additional patients with splice-site mutations and compared their phenotypes with 17 patients with other types of BTK mutations. In these eight patients, the presence of low levels of wild-type BTK transcripts did not show a clear correlation with the percentage, absolute number, or immunophenotype of B lymphocytes nor with age or serum immunoglobulin levels at diagnosis. Nevertheless, we postulate that the presence of wild-type BTK transcripts can be one of the many factors that influence the clinical and immunological phenotype in X-linked agammaglobulinemia.

Adolescent↗

PubMed2001

Truncated typeXVII collagen expression in a patient with non-herlitz junctional epidermolysis bullosa caused by a homozygous splice-site mutation.

SUMMARY: Type XVII collagen (180-kDa bullous pemphigoid antigen) is a structural component of hemidesmosomes. Mutations in the type XVII collagen gene (COL17A1) have been established to be the molecular basis of non-Herlitz junctional epidermolysis bullosa (JEB-nH), an inherited skin blistering disorder. Here we report for the first time truncated type XVII collagen expression, caused by homozygosity for a COL17A1 donor splice-site mutation (4261+1 g --> c), which was identified by PCR amplification on genomic DNA. By RT-PCR and sequencing of cDNA derived from mRNA from the patient's cultured keratinocytes, we provide evidence of cryptic splicing and exon skipping, most abundantly of exon 52. JEB-nH patients with COL17A1 splice-site mutations resulting in an exon skip often have no immunologically detectable type XVII collagen. However, in our patient with the generalized atrophic benign epidermolysis bullosa subtype, a small amount of type XVII collagen was detectable in the skin, and immunoblotting of cultured keratinocytes revealed that the 180-kDa protein was 10 kDa shorter. We hypothesize that the function of this truncated type XVII collagen polypeptide, which is expressed at low levels, is impaired, explaining the JEB-nH phenotype.

Aged↗

PubMed2004

MNGIE with lack of skeletal muscle involvement and a novel TP splice site mutation.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive multisystem disorder caused by thymidine phosphorylase (TP) deficiency, resulting in severe gastrointestinal dysmotility and skeletal muscle abnormalities. A patient is reported with a classical MNGIE clinical presentation but without skeletal muscle involvement at morphological, enzymatic, or mitochondrial DNA level, though gastrointestinal myopathy was present. MNGIE was diagnosed by markedly raised plasma thymidine and reduced thymidine phosphorylase activity. Molecular genetic analysis showed a homozygous novel splice site mutation in TP. On immunohistochemical studies there was marked TP expression in the CNS, in contrast to what has been observed in rodents. It is important to examine the most significantly affected tissue and to measure TP activity and plasma thymidine in order to arrive at an accurate diagnosis in this condition.

Adolescent↗

PubMed2004

A novel splice-site mutation in the common gamma chain (gammac) gene IL2RG results in X-linked severe combined immunodeficiency with an atypical NK+ phenotype.

Mutations in the gene encoding the common gamma chain (gammac) of interleukin receptors 2, 4, 7, 9, 15 and 21 result in X-linked severe combined immunodeficiency (SCID-X1). Classically, this disease is characterised by an absence of T and NK cells, and near normal numbers of functionally deficient B cells (B(+), T(-), NK(-) phenotype). Atypical phenotypes have also been described, but relatively little is known about the mechanisms by which the underlying mutations impair gammac-dependent interleukin receptor signalling to produce these disease variants. Here we describe a novel splice-site mutation resulting in the presence of near normal numbers of functionally deficient NK cells (B(+), T(-), NK(+) phenotype), in a SCID-X1 infant who was subsequently treated by gene therapy. The mutation, c.468+3A>C affecting the third base of intron 3 in the IL2RG gene, was shown to result in the production of two aberrantly spliced gammac mRNA species and reduction of correctly spliced message to trace levels, consistent with failure to detect gammac on the surface of B and NK cells by FACS analysis. The infant's genotype-phenotype correlation supports the hypothesis that interleukin 15 receptor-mediated signalling is preferentially retained as the amount of cell surface gammac becomes limiting. The possible implications for immunological reconstitution following gene therapy are also discussed.

Base Sequence↗

PubMed2002

Insulin-like growth factor binding protein-3 generation as a measure of GH sensitivity.

A total of 198 subjects were randomized to either high-dose (0.05 mg/kg.d) or low-dose (0.025 mg/kg.d) GH for 7 d; the alternate dose was then received after a 2-wk washout period. Groups included in the study were: normal, GH-insensitive (GHI; homozygous for the E180 splice mutation); heterozygous GHI (carriers of the E180 splice mutation); GH-deficient; and idiopathic short stature. Serum IGF binding protein-3 (IGFBP-3) concentrations (collected on d 1, 5, and 8 of treatment weeks) were GH-dependent, with significant elevation by d 5 of treatment, regardless of dose, in all normal subjects. GHI subjects had low baseline IGFBP-3 and poor or no response to either low- or high-dose GH. Heterozygous subjects, however, did not differ from age-matched normals with regard to IGFBP-3 generation. All GH-deficient subjects had subnormal baseline concentrations of IGFBP-3; most, but not all, were able to generate levels into normal ranges by 8 d of therapy. Children with idiopathic short stature showed a better response in IGFBP-3 generation compared with that previously observed with IGF-I, reaching concentrations in normal range with either dose of GH, suggesting that any GHI in this group is relatively limited to IGF-I production. For the diagnosis of GHI, the highest sensitivity (100%) and specificity (92%) was found on d 8 of the high-dose GH-IGFBP-3 generation test. Failure to raise both IGF-I and IGFBP-3 lowered sensitivity to 82-86% with low-dose GH, and 86-91% with high-dose GH.

Adolescent↗

PubMed2005

Unconventional intronic splice site mutation in SCN5A associates with cardiac sodium channelopathy.

BACKGROUND: Mutations in the cardiac sodium channel, SCN5A, have been associated with one type of long-QT syndrome, with isolated cardiac conduction defects and Brugada syndrome. The sodium channelopathies exhibit marked variation in clinical phenotypes. The mechanisms underlying the phenotypical diversity, however, remain unknown. Exonic SCN5A mutations can be detected in 20% of Brugada syndrome patients. RESULTS: An intronic mutation (c.4810+3_4810+6dupGGGT) in the SCN5A gene, located outside the consensus splice site, was detected in this study in a family with a highly variable clinical phenotype of Brugada syndrome and/or conduction disease and in a patient with Brugada syndrome. The mutation was not found in a control panel of 100 (200 alleles) ethnically matched normal control subjects. We provide in vivo and in vitro evidence that the mutation can disrupt the splice donor site, activate a cryptic splice site, and create a novel splice site. Notably, our data show that normal transcripts can be also derived from the mutant allele. CONCLUSIONS: This is the first report of an unconventional intronic splice site mutation in the SCN5A gene leading to cardiac sodium channelopathy. We speculate that its phenotypical diversity might be determined by the ratio of normal/abnormal transcripts derived from the mutant allele.

Adolescent↗