Analyis of factor VIII mRNA reveals defects in everyone of 28 haemophilia A patients

Haemophilia A is a mutationally heterogeneous disease causedby defects in the large and complex factor VIII gene. Recentstudies examining the putative promoter, all exons and mostintron/exon boundaries have failed to detect mutations in halfthe patients with severe disease leading to hypotheses suchas mutations in remote controlling regions or even in genesother than factor VIII. We have amplified the factor VIII gene(putative promotor, coding region and poly-adenylation/cleavagesignal region) in 8 fragments from reverse transcribed mRNAand genomic DNA. Any mutation is then located by chemical mismatchdetection and characterised by direct sequencing. This rapidand efficient method has been fully successful and has revealedan unusual cluster of mutations causing severe disease. Of the28 patients we have reported, 5 had mild or moderate diseaseand all had a missense mutation. Twenty-three patients wereseverely affected and 13 of these had different detrimentalmutations that were fully characterised at the genomic DNA level.The remaining 10 patients all had mRNA with exon 22 not contiguousto exon 23. Since all exons were normal and so were the splicesites of intron 22, the mutation in these patients should bein the regions of intron 22 that were not screened. These resultsprove that all haemophilia A cases are due to mutations of thefactor VIII gene where, unexpectedly, intron 22 seems to bethe target of approximately 40% of the mutations causing severehaemophilia A.     

Cloning of a novel, anonymous gene from a megabase-range YAC and cosmid contig in the neurofibromatosis type 2/meningioma region on human chromosome 22q12

In order to permit detailed characterization of meningioma casesshowing deletions within chromosomal band 22q12 and furthersystematically clone genes located within this region, we establisheda genomic YAC and cosmid contig which encompasses a region inexcess of 1000 kb of 22q12. The YAC contig consists of 6 YACclones arranged Into 5 overlapping steps covering more than1100 kb. Two corresponding cosmid contigs consisting of 40 stepsof overlapping groups of cosmids encompasses 900–1000kb. This set of genomic clones provides a detailed physicalmap of this part of chromosome 22 and constitutes a basis forthe Isolation and characterization of genes that may be locatedwithin this chromosomal region. Employing the exon-amplificationmethod on two cosmids from the contig, we cloned a novel, anonymousgene, pK1.3, which potentially encodes a protein of 683 aminoacids with a predicted molecular weight of of 78.5 kD. Its 2.7kb mRNA is expressed ubiquitously. We estimated the genomicsize of this gene to 100 –150 kb, and it is located inthe Immediate centromeric vicinity of the neurofibromatosis2 (NF2) tumor suppressor gene.     

Prevalence of small rearrangements in the factor VIII gene F8C among patients with severe hemophilia A

Hemophilia A is a common X-linked bleeding disorder caused by various types of mutations in the factor VIII gene F8C. The most common intron 22-inversion is responsible for about 40% of the severe hemophilia A cases while large deletions, point mutations and small (less than 100 bp) deletions or insertions are responsible for the disease in the rest of patients. We report on nine novel (6 deletions, two indels and one partial duplication) and five recurrent small rearrangements identified in 15 German patients with severe hemophilia A, negative for the intron 22-inversion. c.2208-2214delTTATTAC/c.2207-2215insCTCTT and c.4665-4678del/c.4664-4678insAAGGAA identified in the present study are the first small indels described in the factor VIII gene. Our analyses suggest that the prevalence of this type of mutations (predominantly located in exon 14) among patients with severe phenotype and negative for the common intron 22-inversion, is about 30%. The correlation between these molecular defects and formation of factor VIII inhibitors as well as the parental origin of the de novo mutations are evaluated. Finally we show that denaturing HPLC (DHPLC) and classic heteroduplex analysis (HA) are able to detect these sequence alterations on 100% and could be preferred as a screening approach when analysing for mutations in factor VIII in severely affected patients.     

Mutational‐screening in the factor VIII gene resulting in the identification of three novel mutations,one of which is a donor splice mutation

Hemophilia A is an X‐linked bleeding disease caused by mutations in the coagulation factor VIII gene. The identification and characterization of pathogenic mutations allows the recognition of new mechanisms of functional disturbances of factor VIII. To screen for mutations exons 1‐26 of the factor VIII gene have been amplified genomically and analyzed by SSCP followed by direct sequencing of respective exons showing abnormal electrophoretic mobility on SSCP analysis. In the present study we report the detection of four mutations in the factor VIII gene, of which three are novel. The mutational analysis of a patient with severe hemophilia A has revealed that the a ®c transversion at position 3 of the donor‐splice‐site of intron 23 results in the skipping of exon 23. A novel nonsense mutation Q1778X in exon 16 of factor VIII gene has been identified in a second hemophilia A case. Furthermore two missense mutations have been ascertained: a novel, S183R, causing a mild phenotype of hemophilia A and R282H, previously described in association with severe hemophilia A. Hum Mutat 13:504, 1999. © 1999 Wiley‐Liss, Inc.     

Functional analysis of two novel splice site mutations of APOB gene in familial hypobetalipoproteinemia

Familial hypobetalipoproteinemia (FHBL) is a co-dominant disorder characterized by reduced plasma levels of low density lipoprotein cholesterol (LDL-C) and its protein constituent apolipoprotein B (apoB), which may be due to mutations in APOB gene, mostly located in the coding region of this gene. We report two novel APOB gene mutations involving the acceptor splice site of intron 11 (c.1471-1G>A) and of intron 23 (c.3697-1G>C), respectively, which were identified in two patients with heterozygous FHBL associated with severe fatty liver disease. The effects of these mutations on APOB pre-mRNA splicing were assessed in COS-1 cells expressing the mutant APOB minigenes.The c.1471-1G>A APOB minigene generated two abnormal mRNAs. In one mRNA the entire intron 11 was retained; in the other mRNA exon 11 joined to exon 12, in which the first nucleotide was deleted due to the activation of a novel acceptor splice site. The predicted products of these mRNAs are truncated proteins of 546 and 474 amino acids, designated apoB-12.03 and apoB-10.45, respectively. The c.3697-1G>C APOB minigene generated a single abnormal mRNA in which exon 23 joined to exon 25, with the complete skipping of exon 24. This abnormal mRNA is predicted to encode a truncated protein of 1220 amino acids, designated apoB-26.89.These splice site mutations cause the formation of short truncated apoBs, which are not secreted into the plasma as lipoprotein constituents. This secretion defect is the major cause of severe fatty liver observed in carriers of these mutations.     

Novel mutant mice secreting soluble CD4 without expression of membrane-bound CD4

Mutant mice derived from C57BR/cdJ mice were found to have a novel genetic defect in CD4 expression. Flow-cytometric analysis demonstrated that there were no CD4+ cells in either the thymus or the peripheral lymphoid organs of the mutant mice. Thymocytes of the mutant mice expressed an amount of CD4 mRNA comparable to normal mouse thymocytes, but the mutant CD4 mRNA was slightly smaller in size than normal CD4 mRNA. The sequence analysis of the mutant CD4 cDNA obtained from thymic RNA revealed that the defect in the CD4 expression was attributable to the deletion of the entire exon VIII, encoding a transmembrane domain of the CD4 molecule. Moreover, soluble CD4 was detected both in the culture supernatant of thymocytes and sera from mutant mice. The analysis of the genomic DNA sequence elucidated that one thymine was substituted for 14 base pairs at the junction between exon VIII and intron VIII in the mutant mice, which could possibly account for the alternative splicing of CD4 mRNA. These mutant mice showed reduced delayed-type hypersensitivity reactions against sheep red blood cells and antibody production against T-dependent antigen but not against T-independent antigen. Thus, these mutant mice have a novel defect in CD4 expression where CD4 mRNA is alternatively spliced to delete a transmembrane domain, giving rise to secretion of soluble CD4 instead of expression of membrane-bound CD4.     

Identification of two distinct intron elements involved in alternative splicing of beta-tropomyosin pre-mRNA

The rat beta-tropomyosin gene encodes two isoforms, termed skeletal muscle beta-tropomyosin and fibroblast last tropomyosim 1 (TM-1), via an alternative RNA processing mechanism. The gene contains 11 exons. Exons 1-5 and exons 8 and 9 are common to all mRNAs expressed from the gene. Exons 6 and 11 are used in fibroblasts, as well as smooth muscle, whereas exons 7 and 10 are used only in skeletal muscle. In the present studies we focused on the mutually exclusive internal alternative splice choice involving exon 6 (fibroblast-type splice) and exon 7 (skeletal muscle-type splice). We have identified two distinct elements in the intron, upstream of exon 7, involved in splice site selection. The first element is comprised of a polypyrimidine tract located 89-143 nucleotides upstream of the 3' splice site, which specifies the location of the lariat branchpoints used, 144-153 nucleotides upstream of exon 7. The 3' splice site AG dinucleotide has no role in selection of these branchpoints. The second element is comprised of intron sequences located between the polypyrimidine tract and the 3' splice site of exon 7. It contains an important determinant in alternative splice site selection, because deletion of these sequences results in the use of the skeletal muscle-specific exon in nonmuscle cells. We propose that the use of lariat branchpoints located far upstream from a 3' splice site may be a general feature of some alternatively excised introns, reflecting the presence of regulatory sequences located between the lariat branch site and the 3' splice site. The data also indicate that alternative splicing of the rat beta-tropomyosin gene is regulated by a somewhat different mechanism from that described for rat alpha-tropomyosin gene and the transformer-2 gene of Drosophila melanogaster.     

How are exons encoding transmembrane sequences distributed in the exon–intron structure of genes?

The exon–intron structure of eukaryotic genes raises a question about the distribution of transmembrane regions in membrane proteins. Were exons that encode transmembrane regions formed simply by inserting introns into preexisting genes or by some kind of exon shuffling? To answer this question, the exon‐per‐gene distribution was analyzed for all genes in 40 eukaryotic genomes with a particular focus on exons encoding transmembrane segments. In 21 higher multicellular eukaryotes, the percentage of multi‐exon genes (those containing at least one intron) within all genes in a genome was high (>70%) and with a mean of 87%. When genes were grouped by the number of exons per gene in higher eukaryotes, good exponential distributions were obtained not only for all genes but also for the exons encoding transmembrane segments, leading to a constant ratio of membrane proteins independent of the exon‐per‐gene number. The positional distribution of transmembrane regions in single‐pass membrane proteins showed that they are generally located in the amino or carboxyl terminal regions. This nonrandom distribution of transmembrane regions explains the constant ratio of membrane proteins to the exon‐per‐gene numbers because there are always two terminal (i.e., the amino and carboxyl) regions – independent of the length of sequences.     

Ten novel mutations in the HEXA gene in non-Jewish Tay -- Sachs patients

The heterogeneity of mutations causing Tay—Sachs diseasein non-Jewish populations requires efficient techniques allowingthe simultaneous screening for both known and novel mutations.ß-hexosaminidase mRNA isolated from cultured fibroblastsof 19 Tay-Sachs patients (7 with adult or late onset form ofthe disease and 12 with infantile Tay-Sachs disease) was amplifiedby cDNA—PCR in two overlapping segments spanning the entirecoding sequence. We used chemical mismatch cleavage (CMC), denaturinggradient gel electrophoresis (DGGE) and direct sequencing ofamplified fragments displaying a cleaved product or an alteredmelting behavior to screen the HEX A gene for mutations andto determine their distribution and frequency in the non-JewishTay—Sachs patients. These methods allowed us to identify31 out of 38 alleles studied (82%). In addition to 9 previouslydescribed mutations (the 4 bp insertion in exon 11, G to A transitionsat codons 170, 269, 482, 499 and 504, C to T transition at codon499 and 504 and a GT to AT transition at the donor site of intron9), we have identified 10 novel mutations. These include 1 donorsplice site defect in intron 6, 8 missense mutations at non-randomlydistributed conserved residues and a 2 bp deletion in exon 4.These results confirm the extreme molecular heterogeneity ofmutations causing Tay—Sachs disease in non-Jewish population.The strategy used should be profitable for identifying mutationsin large genes and for diagnostic purposes.