A novel splicing mutation in propionic acidemia associated with a tetranucleotide direct repeat in the PCCB gene

Propionic acidemia is an inborn error of organic acid metabolism caused by a deficiency of propionyl Coenzyme A (CoA) carboxylase. cDNAs sequenced from a subunit deficient Japanese patient (no. 187) showed an in-frame 57-bp deletion in one allele. Genomic DNA analysis revealed a four-nucleotide deletion of bases 3 to 6 in the 3 intron adjacent to the deleted exon, which disrupted the consensus 5 splice signal and caused exon skipping. This deletion removed one-half of a tetranucleotide direct repeat at the splice junction and presumably resulted from slipped mispairing.     

Mutation analysis of a Sandhoff disease patient in the Maronite community in Cyprus

Sandhoff disease occurs in the Christian Maronite community in Cyprus, a community that established over a thousand years ago. Nowadays, this community comprises less than 1% of the whole population, and has been culturally and socially isolated. Cultured fibroblasts from a patient from this inbred group showed a -hexosaminidase subunit mRNA of apparently the normal size but of reduced quantity. A mutational analysis of cDNA obtained by polymerase chain reaction amplification of mRNA showed a deletion of A at nt 76 (counted from A of the initiation codon, ATG). The deletion results in a frame shift and a premature termination within 20 amino acids from the N-terminus of the normal mature enzyme protein. The patient was homozygous for the deletion. The 5-end of the gene showed many discrepancies from the previously published sequence. We consider that these differences are probably polymorphisms of little functional significance, because the patient's fibroblasts generate decreased but stable mRNA and because some of these base changes were also found in the genes from control fibroblasts. An extensive evaluation of the prevalence of this mutant allele in this community is being initiated.     

Molecular genetic evidence for a founder effect in familial hypercholesterolemia among French Canadians

Summary Familial hypercholesterolemia (FH), at a prevalence of about 1 in 200 in the French-Canadian population, is caused by a 10-kb deletion in the low-density lipoprotein (LDL) receptor gene in 60% of French-Canadian FH heterozygotes. We genotyped 159 FH patients who carry this common mutation and 221 healthy French-Canadian controls for five DNA restriction fragment length polymorphisms (RFLPs) of the LDL receptor gene. The allele numbers for four of the five RFLPs differed significantly (P < 0.001) between FH patients and control subjects. Our results suggest that the 10-kb deletion carrier allele is associated with a single haplotype (called the B haplotype). In a family study including a patient homozygous for the 10-kb deletion, we showed that the B haplotype cosegregates with the deletion in affected members and that the B haplotype is also associated with the normal allele in some members of the family. We identified 15 different haplotypes for the normal allele in 10-kb deletion carrier FH heterozygotes. These results offer strong support, at a molecular level, for the hypothesis of a founder effect for the 10-kb deletion in the French-Canadian population.This work was presented in part at the meeting: Advances in Gene Technology: the molecular biology of human genetic disease, Miami, Florida, 1991     

The molecular defect in a family with mild atypical osteogenesis imperfecta and extreme joint hypermobility: Exon skipping caused by an 11-bp deletion from an intron in one COL1A2 allele

Summary We have investigated a family with an autosomal dominantly inherited connective-tissue defect causing extreme joint hypermobility, premature osteoporosis and late-onset fractures. Analysis of collagenous proteins from affected individuals showed a deletion in some 2(I) chains. Peptide mapping localized this to the CB peptide 2CB4, which covers the N-terminal one-third of the protein chain. Polymerase chain reaction amplification and sequencing of cDNA derived from this region of the mRNA identified á heterozygous deletion of the 54 by comprising exon 9. Similar analysis of the genomic DNA revealed an 11-bp deletion from bp3 to bp13 of IVS-9. This disrupts the consensus 5 splice signal (GTAAGT) and leads to exon skipping. In a family study of 13 affected and unaffected family members using both heteroduplex formation and direct analysis for the deletion, all of the affected, but no unaffected individuals, were found to carry the deletion. This generated a positive Lod score of 2.6 with the Liped programme.     

Identification of a deletion in the low density lipoprotein (LDL) receptor gene in a patient with familial hypercholesterolaemia

Summary DNA samples from 60 unrelated UK patients with familial hypercholesterolaemia (FH) were screened by Southern blot hybridisation to detect gross alterations in the low density lipoprotein (LDL) receptor gene. One patient was found to have a 2kb deletion in the 3 part of the gene. The deletion cosegregates with the FH phenotype in his family. This finding is compatible with the deletion being the cause of FH in this case and makes a presymptomatic test based on DNA analysis available for this family. The defects in most of the other patients are likely to be due to point mutations.     

Screening for mutations in exon 4 of the LDL receptor gene in a German population with severe hypercholesterolemia

A group of 218 patients with severe hypercholesterolemia (LDL cholesterol >260 mg/dl) living in the Cologne area were screened for mutations in the 3 half of exon 4 of the low density lipoprotein (LDL) receptor gene by the single-strand conformation polymorphism (SSCP) method. The analysed fragment was 242 bp in length and comprised approximately 6% of the coding region. In 11 patients an abnormal SSCP pattern was observed. Two of the abnormal fragment patterns were identical. The results of the SSCP screening could be confirmed by direct DNA sequencing. Three of the ten different mutations were previously described (3 bp deletion: codon 197; Asp₂₀₀Gly; Glu₂₀₇stop). Of the newly identified mutations there were two deletions, two insertions, one combined insertion and deletion mutation and two single base pair substitutions [1 bp deletion: G in codon 197; 37 bp deletion: T in codon 196–208 or AT in 196–207 and GA in codon 208; 18 bp insertion: codon 201–206; 8 bp insertion: codon 155–156 and GA in codon 157; 6 bp insertion (codon 196–197) and 5 bp deletion (codon 199, C in codon 198 and G in codon 198 or 200); Asp₂₀₀Tyr; Asp₂₀₃Val]. The 8-bp insertion was detected in a second unrelated individual. The analysis of the functional consequences of the mutations indicates that all mutations were causative of the LDL cholesterol elevation.     

Molecular detection of a translocation (Y;11)(q11.2;q24) in a 45,X male with signs of Jacobsen syndrome

Summary A 45,X karyotype was found in a boy with dysmorphic features, hypoglycaemia and pancytopenia. DNA analysis showed the presence of the Y-chromosomal DNA sequences SRY, ZFY, DYZ4, DYZ3 and DYS1. Using fluorescent in situ hybridization, we located DYZ4 and DYZ3 on chromosome llqter and concluded that a de novo translocation (Y;11)(q11.2;q24) with a deletion of 11q24qter and a deletion of Yq11.2Yqter were present; Jacobsen syndrome and azoospermia are associated with these deletions. Signs of Jacobsen syndrome were observed in the patient.     

Direct screening of a mitochondrial DNA deletion valuable for Amerindian evolutionary research

A rapid, simple restriction isotyping method is described for the direct screening of a previously characterized Amerindian mitochondrial DNA (mtDNA) marker consisting of a 6-bp Huetar deletion. In a sample of 31 maternally non-related Chibchan Amerindian Bribri from Costa Rica, this deletion was present in 74%, arguing for the usefulness of this private polymorphism for Amer-indian taxonomic research.     

The DNA gyrase of Escherichia coli participates in the formation of a spontaneous deletion by recA-independent recombination in vivo

Summary A system for detecting a spontaneous deletion in Escherichia coli was developed and the role of DNA gyrase in deletion formation was studied. A derivative of plac5, AM36, was isolated in which whole pBR322 DNA was inserted in the lacZ gene and 227 by of the lac gene duplicated at both sides of the pBR322 DNA. E. coli lac ^–strains lysogenized by AM36 had a Lac^– phenotype and segregated Lac^– revertants. Sequence analyses showed that the revertant was formed by a deletion that eliminated the inserted pBR322 DNA and one copy of the duplicated segments. The frequency of lac ^– revertant formation was independent of recA function, was increased by oxolinic acid, an inhibitor of DNA gyrase, but was not increased in a lysogen of a nalidixic acid-resistant derivative. The reversion frequencies of temperature sensitive mutants of gyrA gene are 10 to 100 times lower than that of the wild-type strain. These results indicate that the DNA gyrase of E. coli participated in the in vivo deletion formation resulting from the direct repeats.     

Alpha-thalassemia in the four major aboriginal groups in Taiwan

A total of 1309 unrelated blood samples from four major Taiwan aboriginal groups, including 522 of the Ami, 246 of the Bunun, 227 of the Atayal, and 214 of the Paiwan groups, were collected. Subjects with a mean corpuscular volume below 85 fl and Hb A2 values below 3.5% were further studied with Southern hybridization to determine the status of -globin genes. In the Ami, 43 (4.1%) chromosomes had -thalassemia 1 and 43 (4.1%) had -thalassemia 2. Of the 43 -thalassemia 1 chromosomes, 33 were of the Thailand, one of the Philippine, and nine of the Southeast Asian deletion. Of the 43 -thalassemia 2 chromosomes, 42 were of the type I rightward deletion and one was of leftward deletion. In the Bunun group, one chromosome (0.2%) was of the Thailand deletion and two (0.4%) were of type I rightward deletion. In the Atayal group, only one chromosome (0.2%) was of the Philippine deletion. In the Paiwan group, four chromosomes (0.9%) were of the Southeast Asian deletion and three (0.7%) were of the Thailand deletion. Among the four groups, the Ami had the highest prevalence of -thalassemia, which was also higher than that of the Chinese living in Taiwan.     

Sandhoff disease in Argentina: high frequency of a splice site mutation in the HEXB gene and correlation between enzyme and DNA-based tests for heterozygote detection

The level of -hexosaminidase activity in plasma and leukocytes and the frequency of three known HEXB mutations were studied in an Argentinean deme with high incidence of infantile Sandhoff disease. Two mutations were previously identified in one of two Sandhoff patients from the region, a splice mutation, IVS-2+1 GA, and a 4-bp deletion, CTTT_782–785. These mutations, and a 16kb deletion from the 5' end of the HEXB gene common in non-Argentineans, were screened in 9 Sandhoff patients (all unrelated), 24 obligate heterozygotes, 33 additional individuals belonging to families with affected members, and 64 randomly ascertained individuals from the high risk region. Of 31 independent alleles examined, including those of the two patients previously reported, 30 had the IVS-2 splice mutation and only the originally reported patient had the CTTT deletion. The 16-kb deletion was not observed. Further, among the 57 unaffected members of families with a previous history of Sandhoff disease, and absolute correlation was found between carrier diagnosis by enzyme assay of leukocytes and the DNA-based tests for mutation. One of the 64 controls was classified as a carrier by enzyme assay but did not have one of the three mutations screened. We conclude that a single mutation predominates in this Argentinean population and that the DNA-based test can be an effective supplement or alternative to enzyme-based testing.     

A Y/5 translocation in a 45,X male with cri du chat syndrome

Summary In a patient described as a 45,X male with cri du chat syndrome, combined cytogenetic and molecular methods revealed Y euchromatic material to be translocated onto the short arm of one chromosome 5, resulting in a chromosome der(5)(5qter5p14::Yp11.31Ypter). The translocated Y euchromatin comprised only the distal short arm including the pseudoautosomal region and the so-called deletion intervals 1 and 2. A review of 45,X males from the literature showed that; most of them carry a paternally transmitted Y/autosome translocations; resulting in various autosomal deletions. Depending on the segment concerned, the deletion led to congenital malformations.     

Magnification of rRNA gene number in a Neurospora crassa strain with a partial deletion of the nucleolus organizer

Some progeny from crosses between the Neurospora crassa translocation strain T(IL VL)OY321 and normal sequence N. crassa strains are duplication strains with a partial deletion of the nucleolus organizer. Despite the deletion, these progeny are viable and produce a functional nucleolus. Quantification of rRNA gene number in these deletion progeny demonstrated a significant loss of rRNA genes, down to 60% of the parental wild-type level. Initially, all of these reduced nucleolus organizer (RNO) strains demonstrated a reduction in the rate of mycelial elongation in growth tubes. After several vegetative growth cycles some progeny reverted to the normal growth phenotype, and also showed an increase in the number of rRNA genes to approximately that of the wild type.     

DNA sequence analysis of spontaneous histidine mutations in a polA1 strain of Escherichia coli K12 suggests a specific role of the GTGG sequence

Summary Spontaneously arising histidine mutations in an Escherichia coli K12 strain deficient for DNA polymerase I were analysed at the DNA sequence level. We screened approximately 150000 colonies and isolated 106 histidine auxotrophs. Of these, 98 were unstable hisC mutations; 12 representative mutants analysed were shown to have arisen by the excision of a single quadruplet repeat in the sequence 5-GCTGGCTGGCTGGCTG-3. Of the eight mutations at other sites, three hisA deletions and one hisD deletion occurred as a consequence of misalignment of tandemly repeated pentamers (hisD) or decamers (hisA). A single hisA point mutation was found to be a missense mutation. Two extended deletions, covering the his operon were not analysed. We could not identify the hisC deletion by sequencing. We conclude that polA1 is a strong imitator that induces mutations mostly of the minus frameshift and deletion type by a Streisinger-type of mispairing in repetitive DNA sequences. Finally, the possible role of a 5-GTGG-3 sequence and its inverted or direct complements, which are found in the vicinity of all the deletions and frameshifts, is discussed.     

A complete deletion of the factor IX gene and new TaqI variant in a hemophilia B kindred

Summary We report a hemophilia B kindred in which the proband has a complete deletion of the factor IX gene extending a minimum of 80 kilobase pairs (kb) 3 of the gene. This individual has severe factor IX deficiency with no detectable circulating factor IX protein. In common with one previous report, despite a total deletion of the factor IX gene, this patient has not developed antibodies to factor IX. The mother of the proband was found to have a new TaqI variant of the factor IX gene on the nondeletion-bearing X chromosome. The location of the altered TaqI site was found to be 5 of exon IV between residues 9731-9734 and does not affect the function of the factor IX protein. The familial natures of both the variant allele and the deletion were established. In addition a study of this kindred at the DXS99 locus demonstrated the first reported recombination event between this site and the factor IX gene.     

Cloning of the breakpoints of a deletion associated with choroideremia

Summary In order to characterize a previously described submicroscopic deletion encompassing (part of) the choroideremia (tapetochoroidal dystrophy: TCD) gene, we have cloned a 10.5-kb EcoRI fragment from the patient's DNA: this fragment carries the junction between both deletion endpoints (junction fragment). The distal portion of this fragment defines a new marker within, or just distal to the TCD gene. This marker has been employed to confirm the diagnosis in several affected family members, and to rule out carriership in a female at risk with conspicuous clinical signs.This work was presented in part at the 5th International Retinitis Pigmentosa Congress, Melbourne 1988     

Identification of a nonsense mutation in the amelogenin gene (AMELX) in a family with X-linked amelogenesis imperfecta (AIH1)

A family with X-linked amelogenesis imperfecta (XAI) is described in which the disease is associated with a nonsense mutation in exon 5 of the amelogenin gene. This mutation involves a single base deletion (CCCCCCC) in the exon in an affected male, his sister and his mother. The effect of this deletion is to alter the reading frame and to introduce an inappropriate TGA stop codon (an opal mutation) into the exonic sequence of the amelogenin gene immediately 3 of the mutation. The clinical features in the examined members of this family indicate that, in some individuals, the most noticeable defect is of enamel hypoplasia. In others, the hypoplastic changes are subtle and might have been overlooked on cursory examination; the most noticeable change is of enamel colour, indicating a degree of hypomineralisation. We propose that the amelogenin gene is implicated in both the formation of enamel of normal thickness and in the normal mineralisation process.     

Plasmid R46 provides a function that promotes recA-independent deletion, fusion and resolution of replicon

Summary We report that plasmid R46 provides a function which promotes recA-independent deletion, replicon fusion, and resolution of the fusion. R46 belongs to the incompatibility group N and specifies resistance to ampicillin, tetracycline, streptomycin and sulfonamide. Four kinds of deletion derivatives were observed by selection for susceptability to tetracycline from ampicillin-resistant clones. A common region, will be called region thereafter, was postulated to be involved in these deletions. The replicon fusion occurred by a conjugative mobilization of each derivative with plasmid R388. The fusion was suggested to contain both replicons linked at each junction by the sequence in the region in direct orientation. The resolution of the replicon fusion was found between two regions and a consequently generated, parental deletion derivative and an R388 derivative which gained one region. It is possible that the region contains one potential Insertion Sequence (IS) element. These events were also speculated to occur as a consequence of insertion of the potential IS onto the intramolecular or intermolecular target sequence, or reciprocal recombination between two potential IS elements.     

Leftward deletion α-thalassaemia in the Saudi Arabian population

Summary Restriction endonucleases Bam HI and Bgl II were used to investigate the molecular basis of the deletion type of -thalassaemia in the Saudi population. Four homozygous cases and six heterozygous cases of the leftward deletion type of -thalassaemia (-) were identified. So far, the leftward type of -thalassaemia has been identified mainly in the Asiatic population, while the rightward deletion is universally distributed. This paper reports for the first time the presence of leftward deletion in the Saudi population and discusses the possibility of a more universal distribution of leftward deletion.