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PubMed2005

[Mutations and polymorphisms of the P gene associated with oculocutaneous albinism type II].

Oculocutaneous albinism typeII(OCA2), the most common type of albinism, is an autosomal recessive disorder. It is caused by mutations in the P gene, which is located on chromosome 15q11.1-q12 and divided into 24 exons and 23 introns. P gene codes for 838-amino-acid integral membrane protein with 12 putative transmembrane domains, but the exact function is not clear yet. There are at least 60 pathologic mutations and 43 non-pathologic polymorphisms have been found. Pathologic mutations include missense mutations, nonsense mutations, frameshift mutations and splice-sit mutations. But unlike TYR gene, most of P gene mutations are located on the C-terminal and don' t cluster in defined regions. It is difficult to define the pathologic mutations since many non-pathologic polymorphisms also lie in exons. Some non-pathologic missense mutations may be associated with phenotypic variation in normally pigmented individuals and need to further study.

Albinism, Oculocutaneous↗

PubMed2006

[Identification of two novel mutations of human blood coagulation factor V gene in a Chinese family with congenital factor V deficiency].

OBJECTIVE: To discover the mutations of human blood coagulation factor V (FV) gene in a Chinese family with congenital factor V deficiency, and to explore the molecular mechanism associated with the congenital factor V deficiency. METHODS: PCR and DNA sequencing were used to look for the FV gene mutations in the proband. And the novel mutation were testified by PCR restriction fragment length polymorphism technique or reverse DNA sequencing. One hundred healthy volunteers were chosen as controls at random. RESULTS: Two novel mutations were discovered in the FV gene of proband, which were the A1763C missense mutation in exon 11 and the splicing site mutation in the 3' terminal of intron 16 (G-->T). The pedigree analysis showed that the two mutations inherited from his parents respectively: the A1763C came from his father, and the G-->T from his mother. The A1763C missense mutation in exon 11 was not found in each of 100 healthy volunteers. CONCLUSION: The congenital deficiency of FV in the proband might be caused by the A1763C missense mutation in exon 11 and the splicing site mutation in the 3' terminal of intron 16, which jointly caused the proband to be a double heterozygote.

Base Sequence↗

PubMed1996

Type of mutation in the neurofibromatosis type 2 gene (NF2) frequently determines severity of disease.

The gene predisposing to neurofibromatosis type 2 (NF2) on human chromosome 22 has revealed a wide variety of different mutations in NF2 individuals. These patients display a marked variability in clinical presentation, ranging from very severe disease with numerous tumors at a young age to a relatively mild condition much later in life. To investigate whether this phenotypic heterogeneity is determined by the type of mutation in NF2, we have collected clinical information on 111 NF2 cases from 73 different families on whom we have performed mutation screening in this gene. Sixty-seven individuals (56.2%) from 41 of these kindreds revealed 36 different putative disease-causing mutations. These include 26 proposed protein-truncating alterations (frameshift deletions/insertions and nonsense mutations), 6 splice-site mutations, 2 missense mutations, 1 base substitution in the 3' UTR of the NF2 cDNA, and a single 3-bp in-frame insertion. Seventeen of these mutations are novel, whereas the remaining 19 have been described previously in other NF2 individuals or sporadic tumors. When individuals harboring protein-truncating mutations are compared with cases with single codon alterations, a significant correlation (P < .001) with clinical outcome is observed. Twenty-four of 28 patients with mutations that cause premature truncation of the NF2 protein, schwannomin, present with severe phenotypes. In contrast, all 16 cases from three families with mutations that affect only a single amino acid have mild NF2. These data provide conclusive evidence that a phenotype/genotype correlation exists for certain NF2 mutations.

Adolescent↗

PubMed1996

E-cadherin is inactivated in a majority of invasive human lobular breast cancers by truncation mutations throughout its extracellular domain.

We have analysed a series of 49 human breast cancers for mutations in the entire coding region plus flanking intron sequences of the E-cadherin gene. The tumours included 41 infiltrating lobular carcinomas, two infiltrating ducto-lobular carcinomas and six infiltrative ductal carcinomas. In the lobular carcinomas 23 different somatic mutations were detected, of which seven were insertions, 11 deletions, two nonsense mutations and three splice site mutations. The other tumours showed no detectable E-cadherin mutations. All the frameshift and nonsense mutations are expected to generate a secreted E-cadherin fragment instead of a transmembrane protein with cell adhesion activity. The majority of the mutations (21 of 23) were found in combination with loss of heterozygosity of the wild type E-cadherin locus (16q22.1), a hallmark of classical tumour suppressor genes. The mutations were scattered over the whole coding region and no hot spots could be identified. All mutations described here were previously unreported. In conclusion, we have identified up to now E-cadherin mutations in 27 of 48 (56%) infiltrating lobular breast carcinomas and in 0 of 50 breast cancers of other histopathological subtypes. These data provide strong evidence that frequent E-cadherin mutations are involved in the particular etiology of sporadic lobular breast cancers.

Binding Sites↗

PubMed1998

Assessment of sequence-based p53 gene analysis in human breast cancer: messenger RNA in comparison with genomic DNA targets.

The high prevalence of p53 mutations in human cancers and the suggestion from several groups that the presence or absence of p53 mutations might have both prognostic and therapeutic consequences point to the importance of optimal methods for p53 determination. Several strategies exploring this have been described, based either on mRNA or genomic DNA as a template. However, no comparative study on the reliability of the two templates has been performed. The principal aim of this study was to study the concordance of RNA- and DNA-based direct sequencing methods in detecting p53 mutations in breast tumors. In 100 tumors, 22 mutations were detected by both methods. Furthermore, one stop mutation, two splice-site mutations, and one intron alteration were found only by genomic sequencing. In addition, the comparative study suggests that cells with missense mutations have increased steady-state concentrations of p53-specific mRNA, in contrast to cells with a gene encoding a truncated protein.

Aneuploidy↗

PubMed2006

A 'nonsense' mutation leads to aberrant splicing of hMLH1 in a German hereditary non-polyposis colorectal cancer family.

Hereditary Non-polyposis Colorectal Cancer (HNPCC) is an autosomal dominant cancer predisposition syndrome caused by germline mutations in at least four genes encoding integral components of the cellular DNA mismatch repair (MMR) system. The spectrum of genetic alterations encompasses missense- and nonsense mutations, intronic mutations affecting splice donor or acceptor sites as well as small-scale deletions and insertions. We have identified a 'nonsense' mutation that activates a cryptic splice site generating an in frame deletion of the last 17 codons of exon1 of the hMLH1 gene causing HNPCC in a German family. We present a comprehensive genetic analysis of this family that demonstrates important aspects of HNPCC pathogenesis.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Rare autosomal recessive cardiac valvular form of Ehlers-Danlos syndrome results from mutations in the COL1A2 gene that activate the nonsense-mediated RNA decay pathway.

Splice site mutations in the COL1A2 gene of type I collagen can give rise to forms of Ehlers-Danlos syndrome (EDS) because of partial or complete skipping of exon 6, as well as to mild, moderate, or lethal forms of osteogenesis imperfecta as a consequence of skipping of other exons. We identified three unrelated individuals with a rare recessively inherited form of EDS (characterized by joint hypermobility, skin hyperextensibility, and cardiac valvular defects); in two of them, COL1A2 messenger RNA (mRNA) instability results from compound heterozygosity for splice site mutations in the COL1A2 gene, and, in the third, it results from homozygosity for a nonsense codon. The splice site mutations led to use of cryptic splice donor sites, creation of a downstream premature termination codon, and extremely unstable mRNA. In the wild-type allele, the two introns (IVS11 and IVS24) in which these mutations occurred were usually spliced slowly in relation to their respective immediate upstream introns. In the mutant alleles, the upstream intron was removed, so that exon skipping could not occur. In the context of the mutation in IVS24, computer-generated folding of a short stretch of mRNA surrounding the mutation site demonstrated realignment of the relationships between the donor and acceptor sites that could facilitate use of a cryptic donor site. These findings suggest that the order of intron removal is an important variable in prediction of mutation outcome at splice sites and that folding of the nascent mRNA could be one element that contributes to determination of order of splicing. The complete absence of pro alpha 2(I) chains has the surprising effect of producing cardiac valvular disease without bone involvement.

Adult↗

PubMed2005

Splicing of human immunodeficiency virus RNA is position-dependent suggesting sequential removal of introns from the 5' end.

Transcription of the HIV-1 genome yields a single primary transcript, which is alternatively spliced to >30 mRNAs. Productive infection depends on inefficient and regulated splicing and appears to proceed in a tight 5' to 3' order. To analyse whether sequential splicing is mediated by the quality of splice sites or by the position of an intron, we inserted the efficient beta-globin intron (BGI) into the 3' region or 5'UTR of a subgenomic expression vector or an infectious proviral plasmid. RNA analysis revealed splicing of the 3' BGI only if all upstream introns were removed, while splicing of the same intron in the 5'UTR was efficient and independent of further splicing. Furthermore, mutation of the upstream splice signal in the subgenomic vector did not eliminate the inhibition of 3' splicing, although the BGI sequence was the only intron in this case. These results suggest that downstream splicing of HIV-1 RNAs is completely dependent on prior splicing of all upstream intron(s). This hypothesis was supported by the mutation of the major 5' splice site in the HIV-1 genome, which completely abolished all splicing. It appears likely that the tight order of splicing is important for HIV-1 replication, which requires the stable production of intron containing RNAs, while splicing of 3' introns on incompletely spliced RNAs would be likely to render them subject to nonsense-mediated decay.

5' Untranslated Regions↗

PubMed1998

Mutations that alter RNA splicing of the human HPRT gene: a review of the spectrum.

The human HPRT gene contains spans approximately 42,000 base pairs in genomic DNA, has a mRNA of approximately 900 bases and a protein coding sequence of 657 bases (initiation codon AUG to termination codon UAA). This coding sequence is distributed into 9 exons ranging from 18 (exon 5) to 184 (exon 3) base pairs. Intron sizes range from 170 (intron 7) to 13,075 (intron 1) base pairs. In a database of human HPRT mutations, 277 of 2224 (12.5%) mutations result in alterations in splicing of the mRNA as analyzed by both reverse transcriptase mediated production of a cDNA followed by PCR amplification and cDNA sequencing and by genomic DNA PCR amplification and sequencing. Mutations have been found in all eight 5' (donor) and 3' (acceptor) splice sequences. Mutations in the 5' splice sequences of introns 1 and 5 result in intron inclusion in the cDNA due to the use of cryptic donor splice sequences within the introns; mutations in the other six 5' sites result in simple exon exclusion. Mutations in the 3' splice sequences of introns 1, 3, 7 and 8 result in partial exon exclusion due to the use of cryptic acceptor splice sequences within the exons; mutations in the other four 3' sites result in simple exon exclusion. A base substitution in exon 3 (209G-->T) creates a new 5' (donor) splice site which results in the exclusion of 110 bases of exon 3 from the cDNA. Two base substitutions in intron 8 (IVS8-16G-->A and IVS8-3T-->G) result in the inclusion of intron 8 sequences in the cDNA due to the creation of new 3' (acceptor) splice sites. Base substitution within exons 1, 3, 4, 6 and 8 also result in splice alterations in cDNA. Those in exons 1 and 6 are at the 3' end of the exon and may directly affect splicing. Those within exons 3 and 4 may be the result of the creation of nonsense codons, while those in exon 8 cannot be explained by this mechanism. Lastly, many mutations that affect splicing of the HPRT mRNA have pleiotropic effects in that multiple cDNA products are found.

Animals↗

PubMed2005

Spectrum of splicing errors caused by CHRNE mutations affecting introns and intron/exon boundaries.

BACKGROUND: Mutations in CHRNE, the gene encoding the muscle nicotinic acetylcholine receptor epsilon subunit, cause congenital myasthenic syndromes. Only three of the eight intronic splice site mutations of CHRNE reported to date have had their splicing consequences characterised. METHODS: We analysed four previously reported and five novel splicing mutations in CHRNE by introducing the entire normal and mutant genomic CHRNEs into COS cells. RESULTS AND CONCLUSIONS: We found that short introns (82-109 nucleotides) favour intron retention, whereas medium to long introns (306-1210 nucleotides) flanking either or both sides of an exon favour exon skipping. Two mutations are of particular interest. Firstly, a G-->T substitution at the 3' end of exon 8 predicts an R286M missense mutation, but instead results in skipping of exon 8. In human genes, a mismatch of the last exonic nucleotide to U1 snRNP is frequently compensated by a matching nucleotide at intron position +6. CHRNE intron 8 has a mismatch at position +6, and accordingly fails to compensate for the exonic mutation at position -1. Secondly, a 16 bp duplication, giving rise to two 3' splice sites (g.IVS10-9_c.1167dup16), results in silencing of the downstream 3' splice site. This conforms to the scanning model of recognition of the 3' splice site, which predicts that the first "ag" occurring after the branch point is selected for splicing.

Adult↗

PubMed1993

Alteration of splice site selection by an exon mutation in the human glycophorin A gene.

We report the identification in human glycophorin A gene of a novel exon mutation that affects splice site selection. DNA mapping showed that the mutation has abolished a unique MspI site marking the exon III-intron 3 splice junction (ACCG/GT). Genomic sequencing confirmed the occurrence of a single G-->A transition at the terminal nucleotide position of exon III. Analysis of the mRNA composition demonstrated a partial inactivation of the altered 5' splice site as well as skipping of exons involving the alternative use of other constitutive splice sites. The full-length transcript with the cognate A change encodes a variant glycophorin with an arginine replacing a glycine at position 59 and defining the ERIK epitope, whereas the exon III-deleted transcript specifies a shorter glycophorin carrying the Sta antigen. Also identified were the misspliced mRNA species with exon I-IV and exon I-V connections generated by selection of 5' splice sites far distant from the mutated site. Although having a correct translation frame, the predicted polypeptides of such exon-skipping products were not assembled on the erythrocyte membrane probably due to the severe truncation of the signal sequence required for targeting and translocation. These observations reveal a new mechanism for antigenic diversity of human glycophorins.

Alternative Splicing↗

PubMed1989

A 5' splice-region G----C mutation in exon 1 of the human beta-globin gene inhibits pre-mRNA splicing: a mechanism for beta+-thalassemia.

We have characterized a Mediterranean beta-thalassemia allele containing a sequence change at codon 30 that alters both beta-globin pre-mRNA splicing and the structure of the hemoglobin product. Presumably, this G----C transversion at position -1 of intron 1 reduces severely the utilization of the normal 5' splice site since the level of the Arg----Thr mutant hemoglobin (designated hemoglobin Kairouan) found in the erythrocytes of the patient is very low (2% of total hemoglobin). Since no natural mutations of the guanine located at position -1 of the CAG/GTAAGT consensus sequence had been isolated previously, we investigated the role of this nucleotide in the constitution of an active 5' splice site by studying the splicing of the pre-mRNA in cell-free extracts. We demonstrate that correct splicing of the mutant pre-mRNA is 98% inhibited. Our results provide further insights into the mechanisms of pre-mRNA maturation by revealing that the last residue of the exon plays a role at least equivalent to that of the intron residue at position +5.

Adult↗

PubMed2002

Mutation analysis of the spastin gene (SPG4) in patients in Germany with autosomal dominant hereditary spastic paraplegia.

Hereditary spastic paraplegias (HSP) comprise a genetically and clinically heterogeneous group of neurodegenerative disorders characterized by progressive spasticity and hyperreflexia of the lower limbs. Autosomal dominant hereditary spastic paraplegia 4 linked to chromosome 2p (SPG4) is the most common form of autosomal dominant hereditary spastic paraplegia. It is caused by mutations in the SPG4 gene encoding spastin, a member of the AAA protein family of ATPases. In this study the spastin gene of HSP patients from 161 apparently unrelated families in Germany was analyzed. The authors identified mutations in 27 out of the 161 HSP families; 23 of these mutations have not been described before and only one mutation was found in two families. Among the detected mutations are 14 frameshift, four nonsense, and four missense mutations, one large deletion spanning several exons, as well as four mutations that affect splicing. Most of the novel mutations are located in the conserved AAA cassette-encoding region of the spastin gene. The relative frequency of spastin gene mutations in an unselected group of German HSP patients is approximately 17%. Frameshift mutations account for the majority of SPG4 mutations in this population. The proportion of splice mutations is considerably lower than reported elsewhere.

Adenosine Triphosphatases↗

PubMed1998

A naturally occurring functional allele of the rice waxy locus has a GT to TT mutation at the 5' splice site of the first intron.

In cultivated rice two wild-type alleles, Wxa and Wxb, predominate at the waxy locus, which encodes granule-bound starch synthase. The activity of Wxa is 10-fold higher than that of Wxb at the level of both protein and mRNA. Wxb has a +1G to T mutation at the 5' splice site of the first intron. Sequence analysis of Wxb transcripts revealed that splicing occurs at the mutant AG/UU site and at two cryptic sites: the first is A/GUU, one base upstream of the original site and the second is AG/GU found approximately 100 bases upstream of the mutant splice site. We introduced single base mutations to the 5' splice sites of both Wxa and Wxb, fused with the gus reporter gene and introduced them into rice protoplasts. Analysis of GUS activities and transcripts indicated that a G to T mutation in Wxa reduced GUS activity and the level of spliced RNA. Conversely, a T to G mutation of Wxb restored GUS activity and the level of spliced RNA to that of wild-type Wxa. These results demonstrated that the low level expression of Wxb results from a single base mutation at the 5' splice site of the first intron. It is of interest that the Wxb allele of rice carrying the G to T mutation of intron 1 has been conserved in the history of rice cultivation because there is a low amylose content of the seed caused by this mutation.

Alleles↗

PubMed2000

Jagged1 (JAG1) mutation detection in an Australian Alagille syndrome population.

Alagille syndrome (AGS) is an autosomal dominant disorder characterized by abnormal development of the liver, heart, skeleton, eye, and face. Mutations in the Jagged1 gene (JAG1) have been found to result in the AGS phenotype and both protein truncating mutations and missense mutations have been identified. Using single stranded conformational polymorphism analysis we have screened 22 AGS affected individuals from 19 families for mutations within Jagged1. Twelve distinct Jagged1 mutations were identified in 15 (68.2%) of the 22 AGS cases, seven of which are novel. The mutations include three small deletions (25%), two small insertions (16.6%), three missense mutations (25%), two nonsense mutations (16.6%), and two splice-site mutations (16.6%). These mutations are spread across the entire coding sequence of the gene and most are localized to highly conserved motifs of the protein predicted to be important for Jagged1 function. One-half of the mutations found in this study are located between exons 9 and 12, a region constituting only 12% of the coding sequence. A splice-donor site mutation in intron 11 was shown to cause aberrant splicing of Jagged1 mRNA, consequently terminating translation prematurely in exon 12. The results of this study are consistent with the proposal that either haploinsufficiency for wild type Jagged1 and/or dominant negative effects produced by mutated Jagged1 are responsible for the AGS phenotype.

Alagille Syndrome↗

PubMed1994

Carbonic anhydrase II deficiency: single-base deletion in exon 7 is the predominant mutation in Caribbean Hispanic patients.

To date, three different structural gene mutations have been identified in patients with carbonic anhydrase II deficiency (osteopetrosis with renal tubular acidosis and cerebral calcification). These include a missense mutation (H107Y) in two families, a splice junction mutation in intron 5 in one of these families, and a splice junction mutation in intron 2 for which many Arabic patients are homozygous. We report here a novel mutation for which carbonic anhydrase II-deficient patients from seven unrelated Hispanic families were found to be homozygous. The proband was a 2 1/2-year-old Hispanic girl of Puerto Rican ancestry who was unique clinically, in that she had no evidence of renal tubular acidosis, even though she did have osteopetrosis, developmental delay, and cerebral calcification. She proved to be homozygous for a single-base deletion in the coding region of exon 7 that produces a frameshift that changes the next 12 amino acids before leading to chain termination and that also introduces a new MaeIII restriction site. The 27-kD truncated enzyme produced when the mutant cDNA was expressed in COS cells was enzymatically inactive, present mainly in insoluble aggregates, and detectable immunologically at only 5% the level of the 29-kD normal carbonic anhydrase II expressed from the wild-type cDNA. Metabolic labeling revealed that this 27-kD mutant protein has an accelerated rate of degradation. Six subsequent Hispanic patients of Caribbean ancestry, all of whom had osteopetrosis and renal tubular acidosis but who varied widely in clinical severity, were found to be homozygous for the same mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis, Renal Tubular↗

PubMed2006

Characterization of 17 new cases of X-linked chronic granulomatous disease with seven novel mutations in the CYBB gene.

OBJECTIVE: Molecular identification and clinical characterization of genetic mutations in patients with X-linked chronic granulomatous disease (CGD). PATIENTS AND METHODS: Genomic DNA from 17 male patients with proven X-linked CGD based on clinical history, clinical examination, and specific granulocyte function tests were amplified by polymerase chain reaction (PCR) for sequences of the CYBB gene encoding gp91-phox. Mutations in the resulting PCR products were identified by DNA sequencing. RESULTS: Sequence analysis revealed four deletions (453_454delAG; 802delC; 962delG; 993delG), one combined deletion/insertion/duplication [156_173delTCAGCACTGGCACTGGCC/174_175insT/175_176insCCTGCCTGAATTTCT(dupl187_200)]; one insertion (574_575insCCTCAT), four nonsense mutations (332G > A; 402C > T; 690C > T; 1340C>G), two missense mutations (933A > C; 1041A > C) and four potential splice-site mutations (5'intron1 gt-->at; 3'intron1ag-->at; 5'intron3 gtaag-->gtaaa; 5'intron4 gtaa-->ctaa). Seven of these mutations were indeed novel, whereas four mutations not previously reported to the X-CGDbase were found to be of the same type as database reports of unrelated families. The six remaining mutations have been reported previously to the X-CGDbase but have as yet not been described in detail. CONCLUSION: Our findings underline the great heterogeneity of mutations involving the CYBB gene. Neither a mutational hot spot in the gp91-phox gene nor a clear correlation between molecular defect and clinical manifestation in unrelated families could be demonstrated. Remarkable is a splice-site mutation (5'intron3 gtaag-->gtaaa) identified in a 40-year-old patient with late onset "adult" CGD and residual nicotinamide adenine dinucleotide phosphate reduced oxidase activity. The enormous delay of clinical symptoms of this particular mutation could be explained by an age-related variable sensitivity of the splicing machinery to the present splice-site mutation.

Adult↗

PubMed1993

Novel splicing, missense, and deletion mutations in seven adenosine deaminase-deficient patients with late/delayed onset of combined immunodeficiency disease. Contribution of genotype to phenotype.

We examined the genetic basis for adenosine deaminase (ADA) deficiency in seven patients with late/delayed onset of immunodeficiency, an underdiagnosed and relatively unstudied condition. Deoxyadenosine-mediated metabolic abnormalities were less severe than in the usual, early-onset disorder. Six patients were compound heterozygotes; 7 of 10 mutations found were novel, including one deletion (delta 1019-1020), three missense (Arg156 > His, Arg101 > Leu, Val177 > Met), and three splicing defects (IVS 5, 5'ss T+6 > A; IVS 10, 5'ss G+1 > A; IVS 10, 3'ss G-34 > A). Four of the mutations generated stop signals at codons 131, 321, 334, and 348; transcripts of all but the last, due to delta 1019-1020, were severely reduced. delta 1019-1020 (like delta 955-959, found in one patient and apparently recurrent) is at a short deletional hot spot. Arg156 > His, the product of which had detectable activity, was found in three patients whose second alleles were unlikely to yield active ADA. The oldest patient diagnosed was homozygous for a single base change in intron 10, which activates a cryptic splice acceptor, resulting in a protein with 100 extra amino acids. We speculate that this "macro ADA," as well as the Arg156 > His, Arg101 > Leu, Ser291 > Leu, and delta 1019-1020 products, may contribute to mild phenotype. Tissue-specific variation in splicing efficiency may also ameliorate disease severity in patients with splicing mutations.

Adenosine Deaminase↗