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PubMed2001

Identification of three novel mutations in the USH1C gene and detection of thirty-one polymorphisms used for haplotype analysis.

Usher syndrome (USH) is a clinically and genetically heterogeneous autosomal recessive disorder in which sensorineural hearing loss is associated with retinitis pigmentosa. Usher syndrome type 1, the most severe form, is characterized by profound congenital deafness, vestibular dysfunction, and prepubertal onset of retinitis pigmentosa. Six different USH1 genes have so far been mapped, of which two have already been identified. MYO7A, encoding the unconventional myosin VIIA, underlies USH1B. Recently, the USH1C gene was shown to encode harmonin, a PDZ domain-containing protein. A previous screening of 18 unrelated USH1 patients, without a detected MYO7A mutation, for the three USH1C mutations described to date had demonstrated the presence of the 238-239insC mutation in the heterozygous state in four of them. A complete USH1C mutation screening in these four carriers of the 238-239insC mutation resulted in the detection of the second mutation in all the individuals, and the identification of three novel mutations, namely two splice site mutations (IVS1+1G>T and IVS5+1G>A) and a nonsense mutation (R31X). Thirty-one polymorphisms were detected in the USH1C gene. We observed that the E519D substitution is non-pathogenic, which is of particular interest for molecular diagnosis. Our analysis indicated that all the carriers of the 238-239insC mutation share a common haplotype. A different common haplotype was found in the two IVS1+1G>T carriers. Future studies of additional carriers and non-carriers should document the here proposed founder effect of these two mutations.

Abnormalities, Multiple↗
PubMed2001

Molecular defects underlying the Kell null phenotype.

Expression of the Kell blood group system is dependent on two proteins, Kell and XK, that are linked by a single disulfide bond. Kell, a type II membrane glycoprotein, is a zinc endopeptidase, while XK, which has 10 transmembrane domains, is a putative membrane transporter. A rare phenotype termed Kell null (Ko) is characterized by the absence of Kell protein and Kell antigens from the red cell membrane and diminished amounts of XK protein. We determined the molecular basis of eight unrelated persons with Ko phenotypes by sequencing the coding and the intron-exon splice regions of KEL and, in some cases, analysis of mRNA transcripts and expression of mutants on the cell surface of transfected cells. Six subjects were homozygous: four with premature stop codons, one with a 5' splice site mutation, G to A, in intron 3, and one with an amino acid substitution (S676N) in exon 18. Two Ko persons with premature stop codons had identical mutations in exon 4 (R128Stop), another had a different mutation in exon 4 (C83Stop), and the fourth had a stop codon in exon 9 (Q348Stop). Two Ko persons were heterozygous for two mutations. One had a 5' splice site mutation (G to A) in intron 3 of one allele that caused aberrant splicing and exon skipping, and the other allele had an amino acid substitution in exon 10 (S363N). The other heterozygote had the same amino acid substitution in exon 10 (S363N) in one allele and a premature stop codon in exon 6 (R192Stop) in the other allele. The S363N and S676N mutants, expressed in 293T cells, were retained in a pre-Golgi compartment and were not transported to the cell surface, indicating that these mutations inhibit trafficking. We conclude that several different molecular defects cause the Kell null phenotype.

Adult↗
PubMed2006

Partial expression defect for the SCN5A missense mutation G1406R depends on splice variant background Q1077 and rescue by mexiletine.

Mutations in the cardiac Na(+) channel gene SCN5A cause loss of function and underlie arrhythmia syndromes. SCN5A in humans has two splice variants, one lacking a glutamine at position 1077 (Q1077del) and one containing Q1077. We investigated the effect of splice variant background on loss of function and rescue for G1406R, a mutation reported to cause Brugada syndrome. Mutant and wild-type (WT) channels in both backgrounds were transfected into HEK-293 cells and incubated for up to 72 h with and without mexiletine. At 8 h, neither current nor cell surface expression was observed for the mutant in either background, but both were present in WT channels. At 24 h, small (<10% compared with WT) currents were noted and accompanied by cell surface expression. At 48 h, current density was approximately 40% of WT channels for the mutant in the Q1077del variant background but remained at <10% of WT channels in Q1077. Current levels were stable by 72 h. Coexpression with beta(1)- or beta(3)-subunits or insertion of the polymorphism H558R in the background did not significantly affect current expression. Mexiletine restored current density of the mutant channel in both backgrounds to nearly WT levels. The mutant channels also showed a negative shift in inactivation, slower recovery, and enhanced slow inactivation, consistent with a loss of function phenotype. These data show that a trafficking defect may be partial and time dependent and may differ with the splice variant background. Also, expression defects and gating abnormalities may contribute to loss of function for the same mutation.

Alternative Splicing↗
PubMed2003

Identification of the first non-Jewish mutation in familial Dysautonomia.

Familial Dysautonomia is an autosomal recessive disease with a remarkably high carrier frequency in the Ashkenazi Jewish population. It has recently been estimated that as many as 1 in 27 Ashkenazi Jews is a carrier of FD. The FD gene has been identified as IKBKAP, and two disease-causing mutations have been identified. The most common mutation, which is present on 99.5% of all FD chromosomes, is an intronic splice site mutation that results in tissue-specific skipping of exon 20. The second mutation, R696P, is a missense mutation that has been identified in 4 unrelated patients heterozygous for the major splice mutation. Interestingly, despite the fact that FD is a recessive disease, normal mRNA and protein are expressed in patient cells. To date, the diagnosis of FD has been limited to individuals of Ashkenazi Jewish descent and identification of the gene has led to widespread diagnostic and carrier testing in this population. In this report, we describe the first non-Jewish IKBKAP mutation, a proline to leucine missense mutation in exon 26, P914L. This mutation is of particular significance because it was identified in a patient who lacks one of the cardinal diagnostic criteria for the disease-pure Ashkenazi Jewish ancestry. In light of this fact, the diagnostic criteria for FD must be expanded. Furthermore, in order to ensure carrier identification in all ethnicities, this mutation must now be considered when screening for FD.

Child, Preschool↗
PubMed2000

Hereditary spastic paraplegia caused by mutations in the SPG4 gene.

Autosomal dominant hereditary spastic paraplegia (AD-HSP) is a genetically heterogeneous neurodegenerative disorder characterised by progressive spasticity of the lower limbs. The SPG4 locus at 2p21-p22 accounts for 40-50% of all AD-HSP families. The SPG4 gene was recently identified. It is ubiquitously expressed in adult and foetal tissues and encodes spastin, an ATPase of the AAA family. We have now identified four novel SPG4 mutations in German AD-HSP families, including one large family for which anticipation had been proposed. Mutations include one frame-shift and one missense mutation, both affecting the Walker motif B. Two further mutations affect two donor splice sites in introns 12 and 16, respectively. RT-PCR analysis of both donor splice site mutations revealed exon skipping and reduced stability of aberrantly spliced SPG4 mRNA. All mutations are predicted to cause loss of functional protein. In conclusion, we confirm in German families that SPG4 mutations cause AD-HSP. Our data suggest that SPG4 mutations exert their dominant effect not by gain of function but by haploinsufficiency. If a threshold level of spastin were critical for axonal preservation, such threshold dosage effects might explain the variable expressivity and incomplete penetrance of SPG4-linked AD-HSP.

Adenosine Triphosphatases↗
PubMed2004

Trans-splicing of a mutated glycosylasparaginase mRNA sequence by a group I ribozyme deficient in hydrolysis.

RNA reprogramming represents a new concept in correcting genetic defects at the RNA level. However, for the technique to be useful for therapy, the level of reprogramming must be appropriate. To improve the efficiency of group I ribozyme-mediated RNA reprogramming, when using the Tetrahymena ribozyme, regions complementary to the target RNA have previously been extended in length and accessible sites in the target RNAs have been identified. As an alternative to the Tetrahymena model ribozyme, the DiGIR2 group I ribozyme, derived from a mobile group I intron in rDNA of the myxomycete Didymium iridis, represents a new and attractive tool in RNA reprogramming. We reported recently that the deletion of a structural element within the P9 domain of DiGIR2 turns off hydrolysis at the 3' splice site (side reaction) without affecting self-splicing [Haugen, P., Andreassen, M., Birgisdottir, A.B. & Johansen, S.D. (2004) Eur. J. Biochem. 271, 1015-1024]. Here we analyze the potential of the modified ribozyme, deficient in hydrolysis at the 3' splice site, for application in group I ribozyme-mediated trans-splicing of RNA. The improved ribozyme catalyses both cis-splicing and trans-splicing in vitro of a human glycosylasparaginase mRNA sequence with the same efficiency as the original DiGIR2 ribozyme, but without detectable levels of the unwanted hydrolysis.

Aspartylglucosylaminase↗
PubMed1994

Suppression of mammalian 5' splice-site defects by U1 small nuclear RNAs from a distance.

One of the earliest events in the process of intron removal from mRNA precursors is the establishment of a base-pairing interaction between U1 small nuclear (sn) RNA and the 5' splice site. Mutations at the 5' splice site that prevent splicing can often be suppressed by coexpression of U1 snRNAs with compensatory changes, but in yeast, accurate splicing is not restored when the universally conserved first intron base is changed. In our mammalian system as well, such a mutation could not be suppressed, but the complementary U1 caused aberrant splicing 12 bases downstream. This result is reminiscent of observations in yeast that aberrant 5' splice sites can be activated by U1 snRNA from a distance. Using a rapid, qualitative protein expression assay, we provide evidence that 5' splice-site mutations can be suppressed in mammalian cells by U1 snRNAs with complementarity to a range of sequences upstream or downstream of the site. Our approach uncouples in vivo the commitment-activation step of mammalian splicing from the process of 5' splice-site definition and as such will facilitate the genetic characterization of both.

Alternative Splicing↗
PubMed2004

Molecular screening of ALK1/ACVRL1 and ENG genes in hereditary hemorrhagic telangiectasia in France.

Hereditary hemmorrhagic telangiectasia (HHT, or Osler-Rendu-Weber syndrome) is an autosomal dominant disease characterized by arteriovenous malformations, affecting 1 out of 10,000 individuals in France. The disease is caused by mutations of two genes: ENG and ALK1 (ACVRL1). We screened the coding sequence of ENG and ALK1 in 160 unrelated French index cases. A germline mutation was identified in 100 individuals (62.5%). A total of 36 mutations were found in ENG, including three nonsense mutations, 19 small insertions/deletions leading to a frameshift, two inframe deletions, seven missense mutations, and five intronic or splice-site mutations. Of the 36 mutations, 33 were novel mutations. A total of 64 mutations were found in ALK1, including six nonsense mutations, 28 small insertions/deletions leading to a frameshift, one inframe deletion, 27 missense mutations, and two intronic or splice-site mutations. Of the 64 mutations, 27 were novel mutations. Mutations were found in most parts of the coding sequence for both genes, except ALK1 exon 5 and ENG exons 12 to 14. Missense mutations in ALK1 were more frequent in exons 7, 8, and 10. ENG cDNA was sequenced for three intronic mutations: c.689+2T>C produced an abnormal transcript excluding exon 5, c.1103+3_1103+8del activated a cryptic splice site 22 bp upstream, and c.1428G>A produced two abnormal transcripts, one including intron 11 and the other excluding exon 10. Although most of the mutations were private, some recurrent mutations in ALK1 were of particular interest. Mutation c.1112_1113dupG (p.Gly371fsX391) was found in 17 unrelated individuals sharing a common haplotype, strongly suggesting a founder effect related to the concentration of patients previously reported in a specific French region (Rhône-Alpes). Three missense mutations involved the same codon: c.1231C>T (p.Arg411Trp), c.1232G>C (p.Arg411Pro), and c.1232G>A (p.Arg411Gln) were found in seven, two, and one patients, respectively. Haplotype analysis was in favor of both a founder effect and a mutation hot-spot.

Activin Receptors, Type I↗
PubMed2001

Detection of mutations in the COL4A5 gene by SSCP in X-linked Alport syndrome.

Alport syndrome is a progressive renal disease leading to chronic renal failure, which often is accompanied by sensorineural deafness and ophthalmological signs in the form of anterior lenticonus. The X-linked form of the disease is caused by mutations in the COL4A5 gene encoding the alpha5-chain of type IV-collagen. We performed mutation analysis of the COL4A5 gene by PCR-SSCP analysis of each of the 51 exons with flanking intronic sequences in 81 patients suspected of X-linked Alport syndrome including 29 clear X-linked cases, 37 cases from families with a pedigree compatible with X-linked inheritance, and 15 isolated cases. We found a mutation detection rate of 52% (42/81) (58% in males and 21% in females), and 69% (20/29) in families who clearly demonstrated X-linked inheritance. Thirty-six different mutations were found in 42 patients comprising 16 missense mutations, seven frameshifts, three in-frame deletions, four nonsense mutations, and six splice site mutations. Twenty-two of the mutations have not previously been reported. Furthermore, we found one non-pathogenic amino acid substitution, one rare variant in a non-coding region, and one polymorphism with a heterozygosity of 28%. Three de novo mutations were found, two of which were paternal and one of maternal origin.

Codon, Nonsense↗
PubMed1996

Relationship between COL4A5 gene mutation and distribution of type IV collagen in male X-linked Alport syndrome. Japanese Alport Network.

The renal immunohistochemical distribution of collagen IV chains was studied with a monoclonal antibody series recognizing the alpha 1(IV) to alpha 6(IV) chains in nine males with X-linked Alport syndrome whose COL4A5 mutation had been already identified. Two patients had a deletional mutation, six patients had a missense mutation and one patient had a splicing site mutation. The alpha 3(IV) to alpha 6(IV) chains were completely absent in the renal basement membrane of the two patients with a deletional mutation. On the contrary, in four of six patients with a missense mutation (substitution of a glycine within collagenous domain), antigenecity of the alpha 3(IV) to alpha 5(IV) chains was recognized in the glomerular basement membrane although it was weak. In addition, one of the remaining patients showed a normal histochemical pattern of all type IV collagen chains, while the rest one showed completely absent of the alpha 3(IV) to alpha 5(IV) chains at the same pattern of deletional mutation. One patient with a splice site mutation showed complete absence of the alpha 3(IV) to alpha 5(IV) chains from the glomerular basement membrane, but weak staining of the alpha 5(IV) and alpha 6(IV) chains from the Bowman's capsular basement membrane. Our observations indicated that there is variety in the staining of the alpha 3(IV) to alpha 6(IV) antibodies among male patients with COL4A5, mutations.

Adolescent↗
PubMed2002

Mutation screening of the fibrillin-1 (FBN1) gene in 76 unrelated patients with Marfan syndrome or Marfanoid features leads to the identification of 11 novel and three previously reported mutations.

Mutations in the gene encoding fibrillin-1 (FBN1) cause Marfan syndrome (MFS) and other related connective tissue disorders. In this study we performed SSCP to analyze all 65 exons of the FBN1 gene in 76 patients presenting with classical MFS or related phenotypes. We report 7 missense mutations, 3 splice site alterations, one indel mutation, one nonsense mutation and two mutations causing frameshifts: a 16bp deletion and a single nucleotide insertion. 5 of the missense mutations (Y1101C, C1806Y, T1908I, G1919D, C2251R) occur in calcium-binding Epidermal Growth Factor-like (EGFcb) domains of exons 26, 43, 46 and 55, respectively. One missense mutation (V449I) substitutes a valine residue in the non-calcium-binding epidermal growth factor like domain (EGFncb) of exon 11. One missense mutation (G880S) affects the "hybrid" motif in exon 21 by replacing glycine to serine. The 3 splice site mutations detected are: IVS1-1G>A in intron 1, IVS38-1G>A in intron 38 and IVS46+5G>A in intron 46. C628delinsK was identified in exon 15 leading to the substitution of a conserved cysteine residue. Furthermore two frameshift mutations were found in exon 15 (1904-1919del ) and exon 63 (8025insC) leading to premature termination codons (PTCs) in exon 17 and 64 respectively. Finally we identified a nonsense mutation (R429X) located in the proline rich domain in exon 10 of the FBN1 gene. Y1101C, IVS46+5G>A and R429X have been reported before.

Adolescent↗
PubMed1980

Mutations affecting RNA splicing and the interaction of gene expression of the yeast mitochondrial loci cob and oxi-3.

In Saccharomyces cerevisiae strains KL14-4A and 777-3A, four intervening sequences of 1900 (l alpha beta), 1400 (l beta gamma), 1300 (l gamma delta) and 650 bp (l delta epsilon) separate the five coding sequences (alpha-epsilon) of the structural gene (cob) for cytochrome b. Its major transcript is an 18S RNA (2200 nucleotides) which is likely to be the functional mRNA. The lengths of a series of larger transcripts and their hybridization with probes specific for different intervening sequences are consistent with their being intermediates in a splicing process which generates 18S RNA from a giant primary transcript (greater than or equal to 7.5 kb) covering the whole cob region. There is no absolute order of splicing. The intervening sequence l alpha beta is excised in two stages. The first generates a stable 10S RNA, coded for by sequences immediately downstream of the 18S RNA coding segment alpha. The function of this RNA is unknown. Its excision is an early step in processing, whereas excision of the remainder of l alpha beta is a late event. We have studied four cytochrome b-deficient mutants. These map in intervening sequences and are splicing-defective. They accumulate 22S-28S RNAs which contain one or more intervening sequences. The l alpha beta mutants synthesize long, novel polypeptides, antigenically related to cytochrome b, possibly as a result of read-through into the intervening sequences. Several cob mutants also display alterations in their transcripts of oxi-3, the locus which codes for cytochrome c oxidase subunit I. This indicates that interactions between cob and oxi-3 exist at the level of RNA processing.

Cytochromes↗
PubMed1998

Dihydropyrimidine dehydrogenase pharmacogenetics in patients with colorectal cancer.

Individuals with a deficiency in the enzyme dihydropyrimidine dehydrogenase (DPD) may experience severe life-threatening toxicity when treated with 5-fluorouracil (5-FU). As routine measurement of enzyme activity is not practical in many clinical centres, we have investigated the use of DNA mutation analysis to identify cancer patients with low enzyme levels. We have identified two new mutations at codons 534 and 543 in the DPD cDNA of a patient with low enzyme activity and screened the DNA from 75 colorectal cancer patients for these mutations and the previously reported splice site mutation (Vreken et al, 1996; Wei et al, 1996). In all cases, DPD enzyme activity was also measured. The splice site mutation was detected in a patient (1 out of 72) with low enzyme activity whereas mutations at codons 534 (2 out of 75) and 543 (11 out of 23) were not associated with low enzyme activity. These studies highlight the need to combine DPD genotype and phenotype analysis to identify mutations that result in reduced enzyme activity.

Adult↗
PubMed2007

The phenotypic expression of three MSH2 mutations in large Newfoundland families with Lynch syndrome.

To compare the phenotypic expression of three different MSH2 mutations causing Lynch syndrome, 290 family members at 50% risk of inheriting a mutation were studied. Two truncating mutations of the MSH2 gene have been identified in Newfoundland: an exon 8 deletion in five families (N=74 carriers) and an exon 4-16 deletion in one family (N=65 carriers). The third mutation was an intron 5 splice site mutation resulting in deletion of exon 5 in RNA and occurred in 12 families (N=151 carriers). Age to onset of first cancer, first colorectal cancer (CRC), first extracolonic cancers and death were compared. By age 60, 89% of family members with the intron 5 mutation, 81% with the exon 8 deletion, and 85% with the exon 4-16 deletion had developed cancer. For all three mutations males had a higher age-related risk of CRC and death compared to females. In the intron 5 splice site mutation carriers, the number of transitional cell cancers of the urinary tract was significantly lower and time to first ovarian cancer was significantly higher than in the carriers of the genomic deletions. The incidence of CRC in MSH2 mutation carriers, exposed to the same environment, is not modified by the specific mutation, although there is a suggestion that type of mutation may influence development of some extracolonic cancers.

Adult↗
PubMed2005

Conversion analysis for mutation detection in MLH1 and MSH2 in patients with colorectal cancer.

CONTEXT: The accurate identification and interpretation of germline mutations in mismatch repair genes in colorectal cancer cases is critical for clinical management. Current data suggest that mismatch repair mutations are highly heterogeneous and that many mutations are not detected when conventional DNA sequencing alone is used. OBJECTIVE: To evaluate the potential of conversion analysis compared with DNA sequencing alone to detect heterogeneous germline mutations in MLH1, MSH2, and MSH6 in colorectal cancer patients. DESIGN, SETTING, AND PARTICIPANTS: Multicenter study with patients who participate in the Colon Cancer Family Registry. Mutation analyses were performed in participant samples determined to have a high probability of carrying mismatch repair germline mutations. Samples from a total of 64 hereditary nonpolyposis colorectal cancer cases, 8 hereditary nonpolyposis colorectal cancer-like cases, and 17 cases diagnosed prior to age 50 years were analyzed from June 2002 to June 2003. MAIN OUTCOME MEASURES: Classification of family members as carriers or noncarriers of germline mutations in MLH1, MSH2, or MSH6; mutation data from conversion analysis compared with genomic DNA sequencing. RESULTS: Genomic DNA sequencing identified 28 likely deleterious exon mutations, 4 in-frame deletion mutations, 16 missense changes, and 22 putative splice site mutations. Conversion analysis identified all mutations detected by genomic DNA sequencing--plus an additional exon mutation, 12 large genomic deletions, and 1 exon duplication mutation--yielding an increase of 33% (14/42) in diagnostic yield of deleterious mutations. Conversion analysis also showed that 4 of 16 missense changes resulted in exon skipping in transcripts and that 17 of 22 putative splice site mutations affected splicing or mRNA transcript stability. Conversion analysis provided an increase of 56% (35/63) in the diagnostic yield of genetic testing compared with genomic DNA sequencing alone. CONCLUSIONS: The data confirm the heterogeneity of mismatch repair mutations and reveal that many mutations in colorectal cancer cases would be missed using conventional genomic DNA sequencing alone. Conversion analysis substantially increases the diagnostic yield of genetic testing for mismatch repair mutations in patients diagnosed as having colorectal cancer.

Adaptor Proteins, Signal Transducing↗
PubMed1999

A series of mutations in the dihydropteridine reductase gene resulting in either abnormal RNA splicing or DHPR protein defects. Mutations in brief no. 244. Online.

Five novel mutations are described which result in the rare hyperphenylalaninemia DHPR-deficiency. Three of these are located at different intron/exon boundaries within the DHPR gene, and disrupt the maturation of the DHPR transcript such that little full-length mRNA can be detected by RT-PCR. Each mutation alters a conserved nucleotide within the splice site consensus sequence, and results in the skipping of an exon and, in one case, the activation of an inappropriate splicing signal. Two further mutations are missense mutations resulting in a non-conservative amino acid change within the DHPR protein (L14P and G17V) and are associated with a severe phenotype.

Alternative Splicing↗
PubMed1999

Functional characterization of truncated growth hormone (GH) receptor-(1-277) causing partial GH insensitivity syndrome with high GH-binding protein.

We have previously reported a novel heterozygous donor splice site mutation in intron 9 of the GH receptor (GHR) gene in Japanese siblings who showed partial GH insensitivity and high serum GH-binding protein (GHBP) levels. This mutation caused the splicing abnormality and produced the truncated GHR consisting of 277 amino acids (GHR-277), which lacked most of the intracellular domain of GHR, including both boxes 1 and 2. In this study, we have characterized the function of GHR-277 expression in COS-7 and CHO cells in vitro. Scatchard analysis revealed that GHR-277 possessed approximately 1.5 times higher affinity to GH and twice the number of binding sites compared to wild-type full-length GHR (GHR-fl). The GHBP level in culture medium of GHR-277-expressing cells was approximately 3 times higher than that in GHR-fl-expressing cells. Interestingly, the ligand-induced internalization of GHR-277 was significantly reduced compared with that of GHR-fl. Moreover, in GH-induced tyrosine phosphorylation of signal transducer and activator of transcription-5 (STAT5), GHR-277 exerted a dominant negative effect when GHR-277 and GHR-fl were cotransfected. These in vitro data would well explain the clinical characteristics in our patients showing high serum GHBP levels and development of short stature despite a heterozygous mutation of the GHR gene.

Adolescent↗