Theoretical expectations for deletional mutations in Duchenne muscular dystrophy.
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Chromosome Deletion↗
Biomedical subjects
F Giannelli
Publications and source records attributed to F Giannelli.
A factor IX mutation, verified by direct genomic sequencing, causes haemophilia B by a novel mechanism.
A novel factor IX gene mutation (factor IX London 2) has been characterized. This causes severe crm+ haemophilia B as the patient's plasma shows normal factor IX antigen level and less than 1% clotting activity. Sequence analysis of the entire cloned coding and promoter regions revealed a single point mutation: a G----A transition at position 31,119. This region of the patient's DNA was amplified in vitro by the polymerase chain reaction and the nucleotide change was confirmed by direct sequencing of the amplified products. The mutation results in the substitution of the arginine at position 333 by glutamine. This arginine residue is absolutely conserved in the catalytic domain of normal human and bovine factor IX, X and prothrombin. The substitution by glutamine causes the loss of a positive charge from the surface of the factor IX London 2 protein. This mutation pinpoints a previously unknown, functionally critical feature of factor IX which may be involved in substrate or co-factor binding.
Base Sequence↗
Xeroderma pigmentosum complementation group F in a non-Japanese patient.
Genetic complementation studies allowed assignment of a 22-year-old-white woman to the rare complementation group of classic, excision-defective xeroderma pigmentosum (group F cell strain), previously reported only in patients from Japan. She manifested mild cutaneous changes, with no tumors and normal sensitivity to monochromatic ultraviolet irradiation. Unscheduled deoxyribonucleic acid synthesis in cultured fibroblasts (XP126LO) after irradiation with germicidal ultraviolet light was reduced to 13% of control values during the first 2 hours and rose to 45% of normal by 7 to 8 hours.
Adult↗
Partial deletion by illegitimate recombination of the factor IX gene in a haemophilia B family with two inhibitor patients.
The inhibitor phenotype occurs in six haemophilia B patients in the UK and results from development of antibodies by the patients to administered factor IX. We have analysed a partial factor IX gene deletion (London 1) in a family with two inhibitor patients. The deletion results in retention of the first five exons which code for the light chain of factor IXa, and removal of 23 kb of DNA starting 704 bp 3' of the fifth exon and terminating 10.3 kb 3' of the last exon. The 5' break is at residue -113 of an Alu repeat. No significant homology exists between the 5' and 3' termini, but a 9 bp region of complementarity is found 23 bp and 60 bp from the 5' and 3' terminus, respectively. At the cloned deletion junction a new 16 bp sequence contributes a DraI site that is also found in the genomic DNA of the two patients and a heterozygous relative. The deletion is an example of illegitimate recombination and it is proposed that such deletions occur principally during DNA replication. Loss of the 3' sequences involved in the maturation of mRNA probably results in no factor IX production. Immunological studies show that the index patient's antibodies bind both to epitopes coded by deleted and by non-deleted segments of the gene.
Base Sequence↗
Xeroderma pigmentosum complementation group G--report of two cases.
Genetic complementation studies allowed assignment of a brother (XP124LO) and sister (XP 125LO), aged 14 and 12 years respectively, to the rare complementation group of classical xeroderma pigmentosum (XP), XP-G. Both patients manifested only mild cutaneous changes, with no UV-induced skin tumours, although abnormal sensitivity to UVB wavelengths was demonstrated by irradiation monochromator skin testing. Physical and neurological development was normal. Measurement of UV-induced unscheduled DNA synthesis in cultured fibroblasts showed reduction of repair synthesis to 14% and 16% of normal in XP124LO and XP125LO, respectively. This contrasts with a reduction to 5% of normal in previously described group G patients, XP2BI and XP3BR, who had correspondingly severe cutaneous and neurological manifestations.
Adolescent↗
Genetic heterogeneity of X-linked mental retardation with fragile X. Association of tight linkage to factor IX and incomplete penetrance in males.
X-linked mental retardation with fragile X or Martin-Bell Syndrome (MBS) is a frequent cause of mental retardation. So far segregation analysis of MBS in pedigrees ascertained by different, incomplete criteria has produced results, difficult to interpret, which suggest genetic complexity (Sherman et al. 1985). Biochemical and cell biological studies have failed to provide an assay for genetic heterogeneity in MBS and linkage analysis is the only available method. Such analysis, however, is complicated by the incomplete penetrance of the disease in males and the variable penetrance and expression of the defect in heterozygous females. We have used a new approach to test the heterogeneity of recombination between MBS and the coagulation factor IX gene or the anonymous probe 52A in a group of nine families who have sought genetic counselling at Guy's Hospital. We find that both our families alone and our families plus apparently complete samples of pedigrees reported in the literature, separate into two groups: one tightly and one loosely linked to factor IX. In the combined family sample these represent respectively 0.3 and 0.7 of the total and show recombination fractions of 0.0-0.15 and 0.25-0.5. Furthermore, the families with non-penetrant carrier males show tighter linkage to factor IX than the others, thus confirming the suggestion of a systematic difference among MBS families in the recombination between the disease and the factor IX locus. By contrast, no significant differences were found in the recombination between 52A and factor IX in the two groups of MBS families or in these families versus those with Hunter syndrome examined in our laboratory. The causes of the linkage heterogeneity we describe are not known. At least two alternatives can be considered: The existence of two MBS loci or differences in the recombination between a single MBS locus and the factor IX gene. The association between incomplete penetrance and tight linkage to factor IX as well as the discontinuous variation in recombination fraction we have observed seem to favour the former alternative.
Factor IX↗
Genetics of Hunter syndrome: carrier detection, new mutations, segregation and linkage analysis.
We have investigated 31 families segregation for Hunter Syndrome in order to advance our understanding of the genetics of this disease. The hair root test for the diagnosis of carriers of Hunter Syndrome was improved by the adoption of a new diagnostic index that distinguishes between carrier and normal females better than previous methods of analysis. One hundred and eleven female relatives of the affected children were tested by such procedures. This showed that seven out of 31 mothers were not carriers (22.6%), thus suggesting a small deficit of new mutation relative to the expectation that 33% of lethal, recessive alleles arise anew in a population at equilibrium at a sex-linked locus with equal mutation rates in male and female gametogenesis. The difference, however, is not statistically significant. The age of the parents of new mutants was slightly but significantly raised. Nevertheless, the independent increase in the age of the fathers of new female mutants was not statistically significant. Finally, a deficit of affected males was observed. This was significant and suggests the possibility of intrauterine loss of some affected males. Linkage analysis between the Hunter Syndrome locus, three polymorphisms in the Factor IX gene and the anonymous polymorphic probes 52A and DX13 showed that the Hunter locus is fairly closely linked to DX13, and hence distal to the Factor IX gene, while no linkage was observed with the 52A polymorphic site. The maximum lod score for the linkage between factor IX and the Hunter Syndrome locus was 0.424 at theta = 0.25; and that for the linkage between the Hunter Syndrome locus and DX13 was 3.01 at theta = 0.1.
Age Factors↗
DNA maintenance and its relation to human pathology.
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Aging↗
Prenatal diagnosis of Cockayne's syndrome.
Cockayne's syndrome (CS) was diagnosed prenatally by examination of amniotic cells cultured in vitro. RNA synthesis after irradiation with ultraviolet light was abnormal in cells from a fetus with CS but not in cells from a fetus which was normal. The procedure is simple and rapid and the outcome of the test in two cases (one positive, one negative) was unambiguous.
Amniotic Fluid↗
Characterisation and use of an intragenic polymorphic marker for detection of carriers of haemophilia B (factor IX deficiency).
DNA from 33 healthy White subjects was analysed with a 2 X 5 kilobase subgenomic DNA probe derived from the gene for coagulation factor IX, containing the exon "d" region of that gene. Intragenic Taq I restriction-fragment length polymorphism was revealed, with allelic frequencies estimated at 0 X 65 and 0 X 35 (SE = 0 X 06), also detectable by a cDNA probe. The genomic DNA probe is technically superior to the cDNA probe and has been used in three families with haemophilia B (factor IX deficiency). The polymorphic marker segregates with the deleterious mutation, allowing the identification or exclusion of carriers. The allelic frequencies of the Taq I polymorphism are virtually ideal. Therefore, such a polymorphism should be helpful both in genetic counselling of approximately 40% of affected families and in prenatal diagnosis.
Alleles↗
The gene structure of human anti-haemophilic factor IX.
The mRNA sequence of the human intrinsic clotting factor IX (Christmas factor) has been completed and is 2802 residues long, including a 29 residue long 5' non-coding and a 1390 residue long 3' non-coding region, but excluding the poly(A) tail. The factor IX gene is approximately 34 kb long and we define, by the sequencing of 5280 residues, the presumed promoter region, all eight exons, and some intron and flanking sequence. Introns account for 92% of the gene length and the longest is estimated to be 10 100 residues. Exons conform roughly to previously designated protein regions, but the catalytic region of the protein is coded by two separate exons. This differs from the arrangement in the other characterized serine protease genes which are further subdivided in this region.
Amino Acid Sequence↗
Actinic reticuloid--an idiopathic photodermatosis with cellular sensitivity to near ultraviolet radiation.
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Bloom Syndrome↗
Cellular hypersensitivity to UV-A: a clue to the aetiology of actinic reticuloid?
Fibroblasts cultured from six patients with actinic reticuloid (AR) showed striking cytopathic changes and inhibition of RNA synthesis after exposure to near-ultraviolet radiation that had no effect on normal and other photosensitive cell strains, such as those of xeroderma pigmentosum and Bloom syndrome. An abnormal pattern of DNA fragmentation was observed after doses insufficient to cause cytopathic effects. These results suggest a cellular defect in the prevention or repair of some damage caused by free radicals and other photoproducts. In order to explain the pathogenesis of AR, it is proposed that a deficiency in the cellular mechanisms dealing with oxygen radicals leads to the establishment of a vicious circle favouring the persistence of a lymphohistiocytic infiltrate and hence the chronic clinical course characteristic of the disease.
Cells, Cultured↗
The effect of aphidicolin on the rate of DNA replication and unscheduled DNA synthesis of Bloom syndrome and normal fibroblasts.
Ultraviolet radiation induced more unscheduled DNA synthesis (UDS) in ten Bloom syndrome (BS) fibroblast strains than in control cells, but this difference could be suppressed by aphidicolin treatment in at least nine BS strains. Aphidicolin, 1 and 5 micrograms/ml, were required to inhibit by 30% the UDS of BS and control cells respectively, but the DNA replication of BS cells did not prove abnormally sensitive to such an inhibitor. These findings are discussed in relation to current knowledge of the action of aphidicolin and hypotheses of the metabolic defect in BS.
Aphidicolin↗
Differences in patterns of complementation of the more common groups of xeroderma pigmentosum: possible implications.
Xeroderma pigmentosum of groups A, C and D shows complementation differing in kinetics, dependence on the dose of wild-type alleles and dependence on protein synthesis. Such differences suggest that XP-A, -C and -D carry mutations at different loci. The product of the first of these loci (factor A) is present in significant excess in normal fibroblasts, seems to turn over rapidly and may be a dimer or higher polymer. The products of the other two loci (factors C and D) do not seem to be present in significant excess in the cytoplasm of normal fibroblasts, but factor C may accumulate abnormally in XP-D. Factors C and D turn over slowly (D more than C) and they do not move freely from the cell nucleus. Factors A and C, at least, seem to act directly and not via gene regulation.
Cell Nucleus↗
[Electrocardiographic changes in accidental hypothermia].
An 83-year-old woman was found unconscious several hours after she had fallen and fractured her lower limbs in a very cold cellar. On admission she was in shock and had metabolic acidosis, anemia and hypokalemia; her axillary and rectal temperature was 23 degrees C. Her initial ECG showed atrial fibrillation with slow ventricular response and a prominent J wave on the left precordial leads. These changes reverted to normal when body temperature returned to 37 degrees C. Moreover a transient, hypothermia-associated increase of QRS voltage was noted.
Accidents, Home↗
Effect of incubation temperature on the frequency of sister chromatid exchanges in Bloom's syndrome lymphocytes.
The effect of incubation temperature on the frequency of sister chromatid exchange (SCE) has been studied in blood cultures from three Bloom's syndrome (BS) patients, three controls, and three BS heterozygotes. All cell types show slight increases of SCE at 39 degrees C while at 35 degrees C and 32 degrees C, SCE is reduced considerably in BS and slightly increased in normal cells. Prolonging lymphocyte culture to 140 h and adding BUdR for the last two S periods causes a similar decrease in the percentage of SCE in normal and BS cells but, while the latter show a further reduction if they are incubated at 32 degrees C during BUdR labelling, the normal cells show an increase. Therefore, BS and control lymphocytes respond similarly to changes in incubation time and differently to changes in incubation temperature. The possibility that the discrepant behaviour of the BS and control cultures may be due to different growth kinetics of their B and T lymphocytes has been discussed but considered unlikely. Since low temperature lengthens the cell cycle, it has been suggested that our findings and those published by others on co-cultivation experiments (except those of Tice et al. 1978) can be explained by assuming that slow growth reduces SCE in BS cells. This, and unpublished observations (Giannelli et al. 1981), suggest that some imbalance in the factors responsible for DNA replication may exist in BS and possibly account for the high level of SCE.
Bloom Syndrome↗
Tendency to high levels of UVR-induced unscheduled DNA synthesis in Bloom syndrome.
The unscheduled DNA synthesis (UDS) induced by ultraviolet radiations (UVR) in fibroblasts from 5 patients with Bloom Syndrome (BS) has been studied. Since often a high proportion of BS cells has large, probably polyploid nuclei, care was taken to select cells of normal size. Furthermore, UDS was usually measured over constant nuclear areas by a photometric method and each cell strain was tested on several different occasions. This showed that each BS cell strain had levels of UDS exceeding control values, on average, by 19-29%. The difference between the UDS of BS and control cells did not change with UVR doses from 5 to 100 J . m-2 but, unirradiated BS cells showed no evidence of "spontaneous" UDS. Also erythemal UVR (greater than 295 nm) elicited excessive UDS in the BS cell strain which was exposed to such radiations. When BS and control fibroblasts were incubated at low (32.5 degrees C) and high temperature (40.5 degrees C) before and after UVR doses of 5, 25 and 100 J . m-2, it was found that at the lowest dose the temperature of incubation did not modify the difference in UDS between normal and BS cells while, at the highest dose, BS cells incubated at 32.5 degrees C did not show more UDS than the controls. Finally, BS and control fibroblasts were fused with xeroderma pigmentosum (XP) cells of complementation groups C and D, which are complemented slowly by their partners, and it was than found that BS cells may transfer their tendency to high levels of UVR-induced UDS to their fusion partners. In keeping with these findings, UV-irradiated fibroblasts from BS heterozygotes seemed to show more UDS than normal cells. The anomalous behaviour of BS cells exposed to UVR is unexplained but trivial factors such as differences in cell geometry do not seem to account for our findings. Therefore, since an abnormally small endogenous pool of thymidine and "spontaneous" UDS in BS were not observed, it seems possible that the greater UDS performed during the first 1.5 h of repair by BS cells may be due to: greater numbers of damaged sites repaired; greater incorporation of thymidine per site repaired; or finally, both. In fact, the cell-fusion experiments suggest that BS cells may contain a diffusible factor which influences at least the initial rate of UDS. If such a factor were the product of the BS allele it could be argued that the BS mutations are not amorph and that the BS gene product may compete with that of the normal allele and modify the initial rate of UDS induced by UVR. It is hoped that our observations and their may possible interpretations will stimulate further work on BS.
Cell Line↗