Cryptic Splice Donor Site Research Articles

Detecting tissue-specific alternative splicing and disease-associated aberrant splicing of the PTCH gene with exon junction microarrays

Mutations in the human ortholog of Drosophila patched (PTCH) have been identified in patients with autosomal dominant nevoid basal cell carcinoma syndrome (NBCCS), characterized by minor developmental anomalies and an increased incidence of cancers such as medulloblastoma and basal cell carcinoma. We identified many isoforms of PTCH mRNA involving exons 1-5, exon 10 and a novel exon, 12b, generated by alternative splicing (AS), most of which have not been deposited in GenBank nor discussed earlier. To monitor splicing events of the PTCH gene, we designed oligonucleotide arrays on which exon probes and exon-exon junction probes as well as a couple of intron probes for the PTCH gene were placed in duplicate. Probe intensities were normalized on the basis of the total expression of PTCH and probe sensitivity. Tissue-specific regulation of AS identified with the microarrays closely correlated with the results obtained by RT-PCR. Of note, the novel exon, exon 12b, was specifically expressed in the brain and heart, especially in the cerebellum. Additionally, using these microarrays, we were able to detect disease-associated aberrant splicings of the PTCH gene in two patients with NBCCS. In both cases, cryptic splice donor sites located either in an exon or in an intron were activated because of the partial disruption of the consensus sequence for the authentic splice donor sites due to point mutations. Taken together, oligonucleotide microarrays containing exon junction probes are demonstrated to be a powerful tool to investigate tissue-specific regulation of AS and aberrant splicing taking place in genetic disorders.

A donor splice site mutation in intron 1 of CYBA, leading to chronic granulomatous disease

Chronic granulomatous disease (CGD) is a rare congenital disorder in which the patients' phagocytes fail to kill ingested microbes due to an inability to generate superoxide and other microbicidal oxygen derivatives. This inability is caused by mutations in one of the four components of the phagocyte-specific NADPH oxidase. A small subgroup of CGD patients has mutations in the CYBA gene that encodes the p22- phox subunit of the NADPH oxidase. This subunit forms, together with gp91- phox, a flavocytochrome b 558 heterodimer in the phagocyte plasma membrane. Expression of both subunits is required for normal expression of this heterodimer. Here, we report an autosomal recessive CGD patient with neutrophils that did not express flavocytochrome b 558 and did not generate superoxide upon activation. Analysis of genomic DNA revealed a 4-bp deletion at the exon-1/intron-1 boundary in CYBA (IVS1 + 4_7delAGTG). In the patient's cDNA, we found a low expression of an abnormal product, containing exon 1 extended by 79 nucleotides from intron 1, joined to exon 2. This extension is apparently caused by the activation of a cryptic donor splice site with a GT sequence at position 84–85 in intron 1. Both parents of the patient had the same mutation in their genomic DNA, in heterozygous form, but their cDNA contained exclusively the wild-type p22- phox cDNA sequence, indicating that the mutant mRNA was labile. This is, as far as we know, the first description of the molecular and clinical consequences of a donor splice site mutation in intron 1 of any gene reported so far.

Inhibiting farnesylation of progerin prevents the characteristic nuclear blebbing of Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that is characterized by dramatic premature aging and accelerated cardiovascular disease. HGPS is almost always caused by a de novo point mutation in the lamin A gene (LMNA) that activates a cryptic splice donor site, producing a truncated mutant protein termed "progerin." WT prelamin A is anchored to the nuclear envelope by a farnesyl isoprenoid lipid. Cleavage of the terminal 15 aa and the farnesyl group releases mature lamin A from this tether. In contrast, this cleavage site is deleted in progerin. We hypothesized that retention of the farnesyl group causes progerin to become permanently anchored in the nuclear membrane, disrupting proper nuclear scaffolding and causing the characteristic nuclear blebbing seen in HGPS cells. Also, we hypothesized that blocking farnesylation would decrease progerin toxicity. To test this hypothesis, the terminal CSIM sequence in progerin was mutated to SSIM, a sequence that cannot be farnesylated. SSIM progerin relocalized from the nuclear periphery into nucleoplasmic aggregates and produced no nuclear blebbing. Also, blocking farnesylation of authentic progerin in transiently transfected HeLa, HEK 293, and NIH 3T3 cells with farnesyltransferase inhibitors (FTIs) restored normal nuclear architecture. Last, treatment of both early- and late-passage human HGPS fibroblasts with FTIs resulted in significant reductions in nuclear blebbing. Our results suggest that treatment with FTIs represents a potential therapy for patients with HGPS.

RPGR is mutated in patients with a complex X linked phenotype combining primary ciliary dyskinesia and retinitis pigmentosa

Introduction: Primary ciliary dyskinesia (PCD) is a rare disease classically transmitted as an autosomal recessive trait and characterised by recurrent airway infections due to abnormal ciliary structure and function. To...

805. Effective Suicide Gene Therapy of Leukemia in a Novel Model of Retroviral Insertion

Insertional oncogenesis has become the major safety concern for clinical gene transfer into human hematopoietic stem cells, because random retroviral vector integration may activate cellular proto-oncogenes and lead to the emergence of hyperproliferative clones. One potential approach to prevent adverse events while retaining the clinical benefits is to include a suicide gene in the retroviral vector carrying the therapeutic gene; if needed, vector-driven proliferation could be eliminated by drug treatment. Our lab has developed a novel model for experimental insertional oncogenesis in mice to assess the effectiveness of suicide gene therapy. Delivery of a bicistronic MoMLV-based retroviral vector encoding the murine cellular oncogene c-kitD814V to the IL-3 dependent murine leukemia cell line Ba/F3 resulted in vector-driven autonomous cell proliferation after IL-3 withdrawal from the culture medium. The vector was designed to simultaneously express a suicide gene from an internal ribosome entry site (IRES). We tested three different suicide gene candidates: the wild type Herpes simplex virus thymidine kinase (HSV-TK); sr39TK, a mutant created by semi-random mutagenesis that has been reported to have a much higher affinity for Ganciclovir (GCV); and superTK, based on the sr39TK sequence with two silent point mutations introduced to eliminate cryptic RNA splice acceptor and donor site. We derived clonal cell lines from transduced Ba/F3 cells expressing c-kitD814V and either one of the three suicide genes. All cell lines were sensitive to GCV -induced cell death in vitro, and the Ba/F-sr39TK and Ba/F-superTK cells responded to GCV dosages approximately 10 fold lower than the Ba/F-HSV-TK cells. We also characterized GCV-sensitivity of heterogenous, non-selected transduced cell pools to account for suicide escape mutants that may develop due to partial vector loss, splicing events, gene rearrangements or epigenetic silencing. GCV treatment for 21 days of Ba/F3 cell pools transduced with HSV-TK or sr39TK resulted in emergence of GCV resistance; in contrast, proliferation was consistently inhibited by long-term GCV treatment of Ba/F3 cells transduced with superTK. The cell lines transduced to express c-kitD814V were leukemogenic in vivo when injected i.v. into immunocompetent, syngeneic Balb/c mice. Moreover, leukemogenicity was strictly dependent on vector insertion since mice challenged with non-transduced Ba/F3 cells did not develop leukemia. GCV treatment (50 mg/kg i.p.) of Balb/c on five consecutive days one week after challenge with 5,000 Ba/F-HSV-TK cells did not protect from leukemia development, and the mice succumbed to disease around day 21 post-challenge. In contrast, the same GCV treatment of mice injected with 5,000 Ba/F-sr39TK or Ba/F-superTK led to 100% survival 40 days post-challenge. Thus, in this model of vector-driven insertional oncogenesis, a suicide gene approach was effective for eliminating leukemia using modified HSV-TK variants with improved biological activity and diminished aberrant splicing.

Somatic and gonadal mosaicism in Hutchinson–Gilford progeria

We have studied a patient with Hutchinson-Gilford progeria (HGP). Sequence analysis of the LMNA gene demonstrated the presence of a c.1824 C > T (p.G608G) mutation, activating a cryptic splice donor site and leading to the formation of a truncated Lamin A protein. All molecularly characterized autosomal dominant HGP cases described so far result from de novo LMNA mutations, mostly originating on the paternal allele and are often linked with advanced paternal age. However, in our patient, the mutation was transmitted by the mother who showed somatic and germline mosaicism without HGP manifestations.

Potential Impact of a Single Nucleotide Polymorphism in the Hyaluronan Synthase 1 Gene in Waldenström's Macroglobulinemia

The hyaluronan synthase 1 (HAS1) gene encodes a plasma membrane protein that synthesizes hyaluronan, an extracellular matrix molecule. Previously, in patients with Waldenström's macroglobulinemia (WM), we detected upregulation of HAS1 transcripts and identified aberrant splice variants of this gene. Aberrant splicing of HAS1 results from activation of cryptic splice sites. In turn, activation of cryptic donor and acceptor splice sites can be promoted by mutations occurring upstream of these sites and/or at the branch point of slicing. We measured the frequency of the HAS1 833A/G polymorphism (ie, single-nucleotide polymorphism; SNP) in patients with WM and healthy donors. Additionally, HAS1 gene expression was evaluated in the same group of patients. Our observations so far suggest that HAS1 833A/G SNPs contribute to aberrant splicing of this gene; this idea is supported by the fact that 833A/G SNP is located on an exonic splicing enhancer motif. Based on the results obtained thus far, we speculate that individuals with HAS1 833G/G genotype are predisposed toward aberrant HAS1 splicing and expression of HAS1 variants, resulting in an enhanced risk of developing WM. Study of a larger group of patients and healthy donors is needed to confirm these speculations and to evaluate the prognostic significance of these findings.

A novel ancestral splicing mutation in the multidrug resistance protein 2 gene causes Dubin-Johnson syndrome in Ashkenazi Jewish patients

Dubin-Johnson syndrome (DJS) is an inherited disorder characterized by chronic conjugated hyperbilirubinemia due to the absence or dysfunction of the multidrug resistance protein 2 (MRP2). We previously identified two distinct ancestral mutations causing DJS in 22 unrelated Iranian and five unrelated Moroccan Jewish patients, respectively. In this study we identified and characterized the mutation causing DJS in Ashkenazi Jewish patients and assessed a possible founder effect. Sequencing of all 32 exons of the MRP2 gene identified a novel IVS8+4A-->G mutation in three unrelated homozygotes. Haplotype analysis using four intragenic dimorphisms disclosed a founder effect for the mutation. RT-PCR and real time PCR analysis of mRNA from one patient revealed three splice variants all leading to frameshifts and predicting premature termination codons. The main splice variant was a consequence of the use of a cryptic donor splice site inside exon 8. Liver biopsy in one patient revealed complete absence of MRP2 from the canalicular membrane of hepatocytes. In conclusion, our results provide strong evidence that an ancestral IVS8+4A-->G mutation causes DJS in Ashkenazi Jewish patients by abolishing normal splicing of intron 8 leading to aberrantly spliced products that predict truncation of MRP2.

Nonconsensus intronic mutations cause episodic ataxia

We discovered intronic mutations in two episodic ataxia type 2 (EA2) families: a four-nucleotide GAGT deletion at IVS41+(3-6) and a single nucleotide insertion (insT) at IVS24+3. We expressed minigenes harboring the mutations in cell lines to demonstrate exon skipping from the deletion mutation and the activation of a cryptic splice donor site from the insertion mutation. The identification of these disease-causing mutations expands the spectrum of EA2 mutations and emphasizes the importance of intronic sequences in regulating gene expression.

The mechanism of biogenesis and potential function of the two alternatively spliced mRNAs encoded by the murine Msx3 gene

The homeodomain-containing Msx3 gene, the newest member of the Msx family, encodes two mRNAs, with an unknown relationship to each other. To elucidate how Msx3 gene generates the two transcripts, we cloned their corresponding cDNAs from an E10.5 mouse embryo cDNA library. The alignment of sequences of the two Msx3-specific cDNAs with the corresponding regions of the genomic DNA revealed that read-through of the sequences preceding a cryptic splice donor site in the first intron of Max3 gene generated the longer transcript. The longer Msx3 mRNA (Msx3-l) contains 66 nucleotides spliced in frame that would encode a protein with 22 additional amino acids. These extra 22 amino acids are inserted between the residues 72 and 73, exactly 14 amino acids upstream of the homeodomain of the smaller Msx3 protein. In situ hybridization and competitive RT-PCR experiments revealed that both Msx3-s and Msx3-l mRNAs elicited similar spatio-temporal patterns of expression in the developing embryo, with maximal expression of both mRNAs occurring in the embryos between 8.5 and 12.5 days post-coitus. We found that while Msx3-s down-regulated Msx1 promoter in transfected C2C12 cells, co-expression of Msx3-l alone did not affect the activity of the Msx1 promoter. In contrast, a concomitant expression of Msx3-s and Msx3-l in the transfected C2C12 cells neutralized the repressive effect of Msx3-s on the Msx1 promoter. In transient expression assays, the repressive action of Msx3-s on Msx1 promoter could also be reversed by co-expression of exogenous Sp1. Our data indicate that the potential interactions among the protein products encoded by the alternately spliced Msx3 mRNAs and the putative constituents of transcriptional co-activators and co-repressors may have functional consequences in vivo.

Polymorphisms in the Hyaluronan Synthase 1 Gene May Be Predisposing Factors for Waldenstrom's Macroglobulinemia.

The hyaluronan synthase 1 gene (HAS1), which maps to chromosome location 19q13.4 encodes a plasma membrane protein, which synthesizes hyaluronan (HA), an extracellular matrix molecule. Previously, in WM patients we detected up-regulation of HAS1 transcripts and identified aberrant splice variants of this gene, termed HAS1Va, Vb, and Vc. In patients with multiple myeloma, expression of HAS1Vb either alone or in combination with HAS1 and other variants strongly correlates with poor survival (P=0.001). Our gene expression analysis of the HAS1 family members demonstrated that 76–97% of individual CD20+ IgM+ WM cells obtained from BM aspirates and from blood (PBMC) express HAS1Va or HAS1Vb aberrant splice variants, often in the absence of full length HAS1 transcripts. Aberrant splicing of HAS1 is the result of activation of cryptic splice sites, which lead to exon skipping and/or intron retention. In turn, activation of cryptic donor and acceptor splice sites of the gene can be promoted by the mutations occurring upstream of these sites and/or at the branch point of slicing. We measured the frequency of a known polymorphism in the HAS1 gene of 16 BM and 30 PB samples obtained from WM patients, in comparison with PBMC samples from 33 healthy donors. Our results indicate that in healthy individuals, the frequency of the two alleles each reached 50% perhaps reflecting stabilizing selection. The majority of healthy donors (61%) are heterozygous for the HAS1 gene polymorphism. In contrast, 92% of analyzed WM patients are homozygous for this same polymorphism. An expression analysis of HAS1 and variants in BM cells and PBMC obtained from the same group of WM patients demonstrated that only those patients who were homozygous for the HAS1 polymorphism express HAS1 and/or its variants. The few WM patients who are heterozygous for this polymorphism have no detectable expression of aberrantly spliced HAS1 variants or full length of HAS1, a phenotype identical to that of healthy donors. Thus, our observations so far suggest that polymorphisms in HAS1 may contribute to aberrations such as exon skipping and/or activation of a new cryptic splice site leading to aberrant splicing of HAS. Based on the results obtained thus far, we speculate that individuals who are homozygous for HAS1 polymorphism(s) are at enhanced risk of developing WM due to a predisposition towards aberrant HAS1 splicing and expression of HAS1 variants, with predicted oncogenic consequences. However, the study of a much larger number of patients and healthy donors is needed to confirm these speculations and to evaluate the prognostic significance of these findings.

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.

SKIP modifies gene expression by affecting both transcription and splicing

SKIP has been described as a transcriptional coregulator as well as a spliceosome component, but the relationship between these functions is not clear. We found that SKIP activated reporter gene expression from the basal promoters of viral origin. SKIP exhibited more prominent effect on the promoters with stronger activities, in an experiment employing a series of reporter constructs carrying different numbers of GC boxes. We also found that SKIP suppressed aberrant splicing at a cryptic splice donor site in the luciferase reporter gene. In addition, SKIP suppressed splicing of an extra intron created by a β-thalassemia mutation in the human β-globin gene. In the transfection experiment, an intronless reporter exhibited a higher level of expression, but was less significantly activated by SKIP, than the intron-containing reporter. These results indicate that SKIP affects gene expression by both transcriptional activation and regulation of pre-mRNA splicing.

An activated 5' cryptic splice site in the human ALG3 gene generates a premature termination codon insensitive to nonsense-mediated mRNA decay in a new case of congenital disorder of glycosylation type Id (CDG-Id).

A defect of the dolichyl-P-Man:Man5GlcNAc2-PP-dolichyl mannosyltransferase encoded by the ALG3 gene (alias NOT56L) causes congenital disorder of glycosylation type Id (CDG-Id). In this work, a new mutation in the ALG3 gene causing atypical splicing is described with characterization of expression levels and transcript stabilities of the different splice products. A silent mutation in exon 1 of the ALG3 gene (c.165C<T) resulted in a deletion in the corresponding transcripts (c.160_196del) due to the activation of a cryptic donor splice site. Expression studies revealed that negligible amounts of normal transcripts were present in the patient. The deletion in the ALG3 gene generated a premature termination codon (PTC) coding for an ALG3 protein truncated after the first N-terminal transmembranous domain (p.Val54fsX66). Nonsense mediated decay (NMD) of mRNA is a general mechanism for clearing of RNA molecules containing suitable PTCs. However, suppression of NMD using cycloheximide had no influence on ALG3 transcript levels, although the PTCs of the transcript fulfill the criteria for the initiation of NMD. The results presented in this work demonstrate that factors abrogating NMD of the ALG3 gene exists and that the ALG3 gene can serve as a valuable tool for further investigations of the regulation of NMD.

Dystrophinopathy caused by mid-intronic substitutions activating cryptic exons in the DMD gene

In the course of a mutation search performed by muscle dystrophin transcript analysis in 72 Duchenne and Becker Muscular Dystrophies (DMD/BMD) patients without gross gene defect, we encountered four unrelated cases with additional out-of-frame sequences precisely intercalated between two intact exons of the mature muscle dystrophin mRNA. An in silico search of the whole dystrophin genomic sequence revealed that these inserts correspond to cryptic exons flanked by one strong and one weak consensus splice site and located in the mid-part of large introns (introns 60, 9, 1M, and 62, respectively). In each case we identified an intronic point mutation activating the cryptic donor or acceptor splice site. The patients exhibited a BMD/intermediate phenotype consistent with the presence of reduced amounts of normally spliced transcript and normal dystrophin. The frequency of this new type of mutation is not negligible (6% of our series of 65 patients with ‘small’ mutations). It would be missed if the exploration of the DMD gene is exclusively performed on exons and flanking sequences of genomic DNA.

Identification and characterization of a novel splice-site mutation in a patient with Wiskott-Aldrich syndrome

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by immunodeficiency, eczema, and thrombocytopenia with small platelets. A wide spectrum of mutations in the Wiskott-Aldrich syndrome protein ( WASP) gene have been identified as causative of the disease. In the present paper, we report on a family with a boy affected by WAS, with a splice-site mutation caused by a T to G substitution in the +2 position of intron 6 (IVS6+2T>G). Expression studies performed in COS-7 and U-937 cells showed that the mutation affected the normal splicing process. As a consequence, an abnormally long transcript of 38 nucleotides is generated. Such missplicing is probably due to the activation of a cryptic splice donor site located 38 nt downstream of exon 6. The translation of such aberrant mRNA will produce a truncated protein with a premature stop at codon 190. Thus, a novel splice-site mutation is reported in a patient with a mild WAS phenotype.

Outcome of donor splice site mutations accounting for congenital afibrinogenemia reflects order of intron removal in the fibrinogen alpha gene (FGA)

Congenital afibrinogenemia (Mendelian Inheritance in Man #202400) is a rare, autosomal recessive disorder characterized by the complete absence of circulating fibrinogen. Our recent studies on the molecular basis of the disease showed that the most common genetic defect is a donor splice mutation in fibrinogen alpha gene (FGA) intron 4, IVS4+1G>T. Two other FGA donor splice mutations, in intron 1 (IVS1+3A>G) and intron 3 (IVS3+1_+4delGTAA), were identified in afibrinogenemia patients. Because it was impossible to directly study the effect of these mutations on mRNA splicing in patient hepatocytes, we used a transfected cell approach, which previously allowed us to show that the common IVS4 mutation causes afibrinogenemia due to the activation of multiple cryptic donor splice sites. In this study, analysis of the IVS3delGTAA mutation showed exon 3 skipping in 99% of transcripts and exons 2 and 3 skipping in 1% of transcripts. The different outcomes of these donor splice mutations appear to follow the model proposed in a study of fibrillar collagen genes, where donor splice mutations occurring in a rapidly spliced intron with respect to upstream introns lead in most cases to exon skipping, while mutations in later-spliced introns lead to intron inclusion or cryptic splice-site utilization. Indeed, we found that in FGA intron 3 was preferentially spliced first, followed by intron 2, intron 4, and intron 1.

Multiple RNA splicing and the presence of cryptic RNA splice donor and acceptor sites may contribute to low expression levels and poor immunogenicity of potential DNA vaccines containing the env gene of equine infectious anemia virus (EIAV)

The env gene is an excellent candidate for inclusion in any DNA-based vaccine approach against equine infectious anemia virus (EIAV). Unfortunately, this gene is subjected to mutational pressure in E. coli resulting in the introduction of stop codons at the 5′ terminus unless it is molecularly cloned using very-low-copy-number plasmid vectors. To overcome this problem, a mammalian expression vector was constructed based on the low-copy-number pLG338-30 plasmid. This permitted the production of full-length EIAV env gene clones (plcnCMV env) from which low-level expression of the viral surface unit glycoprotein (gp90) was detected following transfection into COS-1 cells. Although this suggested the nuclear export of complete env mRNA moieties at least two additional polypeptides of 29 and 20 kDa (probably Rev) were produced by alternative splicing events as demonstrated by the fact that their synthesis was prevented by mutational inactivation of EIAV env splice donor 3 (SD3) site. The plcnCMV env did not stimulate immune responses in mice or in horses, whereas an env construct containing an inactivated SD3 site (plcnCMVΔSD3) did induce weak humoral responses against gp90 in mice. This poor immunogenicty in vivo was probably not related to the inherent antigenicity of the proteins encoded by these constructs but to some fundamental properties of EIAV env gene expression. Attempts to modify one of these properties by mutational inactivation of known viral RNA splice sites resulted in activation of previously unidentified cryptic SD and slice acceptor sites.

Krit1 Missense Mutations Lead to Splicing Errors in Cerebral Cavernous Malformation

At least 40% of families affected with cerebral cavernous malformation have a mutation in Krit1. We previously identified two point mutations in Krit1 leading to changes in amino acids (D137G and Q210E) in two different families. Further RNA analysis reveals that both point mutations actually activate cryptic splice-donor sites, causing aberrant splicing and leading to a frameshift and protein truncation. To date, no simple missense mutations have been detected in Krit1.

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.