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A R Lehmann

Publications and source records attributed to A R Lehmann.

At least 19 recordsLinked to original sources
PubMed2006

A novel mutation in the XPA gene associated with unusually mild clinical features in a patient who developed a spindle cell melanoma.

BACKGROUND: Xeroderma pigmentosum (XP) is an autosomal recessive disorder of, in most cases, defective nucleotide excision repair (NER) of ultraviolet radiation (UV)- and chemical-induced DNA damage. The condition is characterized by an increased sensitivity of the skin to UV radiation, with early development of pigmentary changes and premalignant lesions in sun-exposed areas of the skin, signs of photoageing and a greatly increased incidence from a young age of skin tumours including melanoma. Approximately 20% of patients with XP show neurological abnormalities of varying severity due to primary neuronal degeneration. Genetic analysis by somatic cell hybridization has led to the identification in the NER-defective form of XP of seven complementation groups, designated XP-A to XP-G. These complementation groups correspond to different proteins involved in the NER process. XP-A classically includes some of the most severely affected patients. OBJECTIVES: We describe a 61-year-old Punjabi woman with XP. Remarkably she had only mild cutaneous abnormalities, minimal neurological features and unusual longevity, and developed a malignant spindle cell melanoma. There are few previous reports of spindle cell melanoma associated with XP. To gain insight into the aetiology of these unusual features, we sought to analyse the DNA repair properties of the patient and identify the complementation group and the causative mutation in the defective gene. METHODS: Unscheduled DNA synthesis and the inhibition of RNA synthesis were measured. The complementation group was assigned by fusing the cells of our patient with XP cells of known complementation groups and determining the ability to carry out unscheduled DNA repair. Molecular analysis of the cDNA was carried out by polymerase chain reaction and DNA sequencing. RESULTS: Levels of DNA repair were extremely low and complementation analysis assigned the defect to the XP-A group. Sequencing of the XPA gene revealed a novel homozygous mutation of A-->G at the eighth nucleotide of intron 4 causing aberrant splicing and a nonfunctional truncated XP-A protein. However, a small amount of normally spliced mRNA was detected at <5% the level in normal cells. CONCLUSIONS: The small amount of normally spliced mRNA detected may be sufficient to explain the relatively mild clinical features in our patient.

Afghanistan↗
PubMed2006

Clinical and cellular ionizing radiation sensitivity in a patient with xeroderma pigmentosum.

XP14BR is a cell line derived from a xeroderma pigmentosum (XP) patient from complementation group C. The patient was unusual in presenting with an angiosarcoma of the scalp, treated by surgical excision and radiotherapy. Following 38 Gy in 19 fractions with 6 MEV electrons, a severe desquamation and necrosis of the underlying bone ensued, and death followed 4 years later. The cell line was correspondingly hypersensitive to the lethal effects of gamma irradiation. We had previously shown that this sensitivity could be discriminated from that seen in ataxia-telangiectasia (A-T). The cellular response to ultraviolet radiation below 280 nm (UVC) was characteristic of XP cells, indicating the second instance, in our experience, of dual cellular UVC and ionizing radiation hypersensitivity in XP. We then set out to evaluate any defects in repair of ionizing radiation damage and to verify any direct contribution of the XPC gene. The cells were defective in repair of a fraction of double strand breaks, with a pattern reminiscent of A-T. The cell line was immortalized with the vector pSV3neo and the XPC cDNA transfected in to correct the defect. The progeny derived from this transfection showed the presence of the XPC gene product, as measured by immunoblotting. A considerable restoration of normal UVC, but not ionizing radiation, sensitivity was observed amongst the clones. This differential correction of cellular sensitivity is strong evidence for the presence of a defective radiosensitivity gene, distinct from XPC, which is responsible for the clinical hypersensitivity to ionizing radiation. It is important to resolve how widespread ionizing radiation sensitivity is amongst XP patients.

Cell Death↗
PubMed2003

The fidelity of HPV16 E1/E2-mediated DNA replication.

Human papillomaviruses (HPV) are causative agents in a variety of human diseases; for example over 99% of cervical carcinomas contain HPV DNA sequences. Often in cervical carcinoma the HPV genome is integrated into the host genome resulting in unregulated expression of the viral transforming proteins E6 and E7. Therefore viral integration is a step toward HPV-induced carcinogenesis. Integration of the HPV genome could occur following double-strand DNA breaks that could arise during viral DNA replication. We investigated the fidelity of HPV 16 E1- and E2-mediated DNA replication of non-damaged and UVC-damaged templates in a variety of cell lines with different genetic backgrounds; C33a (derived from an HPV-negative cervical carcinoma), XP30RO (deficient in the by-pass polymerase eta (poleta)), XP30eta (expressing a restored wild-type poleta), XP12RO (nucleotide excision repair defective), and MRC5 (derived from a 14-week-old human fetus). The results demonstrate that the fidelity of E1- and E2-mediated DNA replication is reflective of the genetic background in which the assays are carried out. For example, restoring poleta to the XP30 cell line results in a 3-fold drop in the number of mutants obtained following replication of a UVC-damaged template. A relatively high percentage of the mutant-replicated molecules arise as a result of genetic rearrangement. This is the first time such studies have been carried out with an HPV replication system, and the results are discussed in the context of the HPV life cycle and what is known about HPV genomes in human cancers.

Blotting, Southern↗
PubMed2001

Mutations in the general transcription factor TFIIH result in beta-thalassaemia in individuals with trichothiodystrophy.

The transcription factor TFIIH is involved in both basal transcription and DNA repair. Mutations in the XPD helicase component of TFIIH can result in the diverse clinical features associated with xeroderma pigmentosum (XP) and trichothiodystrophy (TTD). It is generally believed that the multi-system abnormalities associated with TTD are the result of a subtle deficiency in basal transcription. However, to date, there has been no clear demonstration of a defect in expression of any specific gene in individuals with these syndromes. Here we show that the specific mutations in XPD that cause TTD result in reduced expression of the beta-globin genes in these individuals. Eleven TTD patients with characterized mutations in the XPD gene have the haematological features of beta-thalassaemia trait, and reduced levels of beta-globin synthesis and beta-globin mRNA. All these parameters were normal in three patients with XP. These findings provide the first evidence for reduced expression of a specific gene in TTD. They support the hypothesis that many of the clinical features of TTD result from inadequate expression of a diverse set of highly expressed genes.

Cells, Cultured↗
PubMed2001

Two individuals with features of both xeroderma pigmentosum and trichothiodystrophy highlight the complexity of the clinical outcomes of mutations in the XPD gene.

The xeroderma pigmentosum group D (XPD) protein is a subunit of transcription factor TFIIH with DNA helicase activity. TFIIH has two functions, in basal transcription and nucleotide excision repair. Mutations in XPD that affect DNA repair but not transcription result in the skin cancer-prone disorder, xeroderma pigmentosum (XP). If transcription is also affected, the result is the multi-system disorder trichothiodystrophy (TTD), in which there is no skin cancer predisposition, or in rare cases, XP combined with Cockayne syndrome. Up till now there have been no reports of combined clinical features of XP and TTD. We have now identified two patients with some features of both these disorders. One of these, XP189MA, a 3-year-old girl with sun sensitivity, mental and physical developmental delay, has XPD mutations not previously reported, and barely detectable levels of nucleotide excision repair. The other, XP38BR, a 28-year-old woman with sun sensitivity, pigmentation changes and skin cancers typical of XP, has a mutation that has been identified previously, but only in TTD patients with no features of XP. The level of repair of UV damage in XP38BR is substantially higher than that in other patients with the same mutation. With both patients, polarized light microscopy revealed a 'tiger-tail' appearance of the hair, and amino acid analysis of the hair shafts show levels of sulfur-containing proteins intermediate between those of normal and TTD individuals. Our findings highlight the complexities of genotype-phenotype relationships in the XPD gene.

Adult↗
PubMed2001

Domain structure, localization, and function of DNA polymerase eta, defective in xeroderma pigmentosum variant cells.

DNA polymerase eta carries out translesion synthesis past UV photoproducts and is deficient in xeroderma pigmentosum (XP) variants. We report that poleta is mostly localized uniformly in the nucleus but is associated with replication foci during S phase. Following treatment of cells with UV irradiation or carcinogens, it accumulates at replication foci stalled at DNA damage. The C-terminal third of poleta is not required for polymerase activity. However, the C-terminal 70 aa are needed for nuclear localization and a further 50 aa for relocalization into foci. Poleta truncations lacking these domains fail to correct the defects in XP-variant cells. Furthermore, we have identified mutations in two XP variant patients that leave the polymerase motifs intact but cause loss of the localization domains.

Amino Acid Sequence↗
PubMed2001

Xeroderma pigmentosum and related disorders: defects in DNA repair and transcription.

The genetic disorders xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD) are all associated with defects in nucleotide excision repair (NER) of DNA damage. Their clinical features are very different, however, XP being a highly cancer-prone skin disorder, whereas CS and TTD are cancer-free multisystem disorders. All three are genetically complex, with at least eight complementation groups for XP (XP-A to -G and variant), five for CS (CS-A, CS-B, XP-B, XP-D, and XP-G), and three for TTD (XP-B, XP-D, and TTD-A). With the exception of the variant, the products of the XP genes are proteins involved in the different steps of NER, and comprise three damage-recognition proteins, two helicases, and two nucleases. The two helicases, XPB and XPD, are components of the basal transcription factor TFIIH, which has a dual role in NER and initiation of transcription. Different mutations in these genes can affect NER and transcription differentially, and this accounts for the different clinical phenotypes. Mutations resulting in defective repair without affecting transcription result in XP, whereas if transcription is also affected, TTD is the outcome. CS proteins are only involved in transcription-coupled repair, a subpathway of NER in which damage in the transcribed strands of active genes is rapidly and preferentially repaired. Current evidence suggests that they also have an important but not essential role in transcription. The variant form of XP is defective in a novel DNA polymerase, which is able to synthesise DNA past UV-damaged sites.

Cockayne Syndrome↗
PubMed2001

DNA polymerase eta is an A-T mutator in somatic hypermutation of immunoglobulin variable genes.

To determine whether DNA polymerase eta plays a role in the hypermutation of immunoglobulin variable genes, we examined the frequency and pattern of substitutions in variable VH6 genes from the peripheral blood lymphocytes of three patients with xeroderma pigmentosum variant disease, whose polymerase eta had genetic defects. The frequency of mutation was normal but the types of base changes were different: there was a decrease in mutations at A and T and a concomitant rise in mutations at G and C. We propose that more than one polymerase contributes to hypermutation and that if one is absent, others compensate. The data indicate that polymerase eta is involved in generating errors that occur predominantly at A and T and that another polymerase(s) may preferentially generate errors opposite G and C.

Animals↗
PubMed2001

Characterization of a novel human SMC heterodimer homologous to the Schizosaccharomyces pombe Rad18/Spr18 complex.

The structural maintenance of chromosomes (SMC) protein encoded by the fission yeast rad18 gene is involved in several DNA repair processes and has an essential function in DNA replication and mitotic control. It has a heterodimeric partner SMC protein, Spr18, with which it forms the core of a multiprotein complex. We have now isolated the human orthologues of rad18 and spr18 and designated them hSMC6 and hSMC5. Both proteins are about 1100 amino acids in length and are 27-28% identical to their fission yeast orthologues, with much greater identity within their N- and C-terminal globular domains. The hSMC6 and hSMC5 proteins interact to form a tight complex analogous to the yeast Rad18/Spr18 heterodimer. In proliferating human cells the proteins are bound to both chromatin and the nucleoskeleton. In addition, we have detected a phosphorylated form of hSMC6 that localizes to interchromatin granule clusters. Both the total level of hSMC6 and its phosphorylated form remain constant through the cell cycle. Both hSMC5 and hSMC6 proteins are expressed at extremely high levels in the testis and associate with the sex chromosomes in the late stages of meiotic prophase, suggesting a possible role for these proteins in meiosis.

Amino Acid Sequence↗
PubMed2000

Identical mutations in the CSB gene associated with either Cockayne syndrome or the DeSanctis-cacchione variant of xeroderma pigmentosum.

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are two hereditary disorders in which photosensitivity is associated with distinct clinical and cellular phenotypes and results from genetically different defects. We have identified the primary molecular alteration in two patients in whom clinical manifestations strongly reminiscent of a severe form of XP were unexpectedly associated with the CS cellular phenotype and with a defect in the CSB gene. Sequencing of the CSB -coding region in both cDNA and genomic DNA showed that these patients had identical alterations to those in a patient with the clinical features of the classical form of CS. These data, together with fluorescence in situ hybridization analysis, demonstrated that the two siblings with XP as well as the CS patient were homozygous for the same CSB mutated allele, containing a silent C2830T change and a nonsense mutation C2282T converting Arg735 to a stop codon. The finding that the same inactivating mutation underlies different pathological phenotypes indicates that there is no simple correlation between the molecular defect and the clinical features. Therefore, alterations in the CSB gene give rise to the same repair defect at the cellular level but other genetic and/or environmental factors determine the pathological phenotype.

Cells, Cultured↗
PubMed2000

Targeted disruption of the cell-cycle checkpoint gene ATR leads to early embryonic lethality in mice.

Checkpoints of DNA integrity are conserved throughout evolution, as are the kinases ATM (Ataxia Telangiectasia mutated) and ATR (Ataxia- and Rad-related), which are related to phosphatidylinositol (PI) 3-kinase [1] [2] [3]. The ATM gene is not essential, but mutations lead to ataxia telangiectasia (AT), a pleiotropic disorder characterised by radiation sensitivity and cellular checkpoint defects in response to ionising radiation [4] [5] [6]. The ATR gene has not been associated with human syndromes and, structurally, is more closely related to the canonical yeast checkpoint genes rad3(Sp) and MEC1(Sc) [7] [8]. ATR has been implicated in the response to ultraviolet (UV) radiation and blocks to DNA synthesis [8] [9] [10] [11], and may phosphorylate p53 [12] [13], suggesting that ATM and ATR may have similar and, perhaps, complementary roles in cell-cycle control after DNA damage. Here, we report that targeted inactivation of ATR in mice by disruption of the kinase domain leads to early embryonic lethality before embryonic day 8.5 (E8.5). Heterozygous mice were fertile and had no aberrant phenotype, despite a lower ATR mRNA level. No increase was observed in the sensitivity of ATR(+/-) embryonic stem (ES) cells to a variety of DNA-damaging agents. Attempts to target the remaining wild-type ATR allele in heterozygous ATR(+/-) ES cells failed, supporting the idea that loss of both alleles of the ATR gene, even at the ES-cell level, is lethal. Thus, in contrast to the closely related checkpoint gene ATM, ATR has an essential function in early mammalian development.

Alleles↗
PubMed2000

A novel SMC protein complex in Schizosaccharomyces pombe contains the Rad18 DNA repair protein.

In Schizosaccharomyces pombe, rad18 is an essential gene involved in the repair of DNA damage produced by ionizing radiation and in tolerance of UV-induced DNA damage. The Rad18 protein is a member of the SMC (structural maintenance of chromosomes) superfamily, and we show that, like the other SMC proteins in condensin and cohesin, Rad18 is a component of a high-molecular-weight complex. This complex contains at least six other proteins, the largest of which is Spr18, a novel SMC family member closely related to Rad18, and likely to be its heterodimeric partner. SMC proteins have ATP-binding domains at the N- and C-termini, and two extended coiled-coil domains separated by a hinge in the middle. We show that the N-terminal ATP-binding domain of Rad18 is essential for all functions, and overexpression of an N-terminal mutant has a dominant-negative effect. We have identified an important mutation (S1045A) near the C-terminus of Rad18 that separates its repair and essential roles. Potential models for the role of the Rad18-Spr18 complex during DNA repair are discussed.

Adenosine Triphosphatases↗
PubMed2000

Mutations in the XPC gene in families with xeroderma pigmentosum and consequences at the cell, protein, and transcript levels.

Xeroderma pigmentosum (XP)-C is one of the more common complementation groups of XP, but causative mutations have thus far been reported for only six cases (S. G. Khan et al., J. Investig. Dermatol., 115: 791-796, 1998; L. Li et al., Nat. Genet., 5: 413-417, 1993). We have now extended this analysis by investigating the genomic and coding sequence of the XPC gene, the level of expression of the XPC transcript and the status of the XPC protein in 12 unrelated patients, including all of the 8 Italian XP-C cases identified thus far and in 13 of their parents. Eighteen mutations were detected in the open reading frame of the XPC gene, 13 of which are relevant for the pathological phenotype. The mutations are distributed across the gene, with no indication of any hotspots or founder effects. Only 1 of the 13 relevant changes is a missense mutation, the remainder causing protein truncations as a result of nonsense mutations (3), frameshifts (6), deletion (1) or splicing abnormalities (2). These findings indicate that the XPC gene is not essential for cell proliferation and viability and that mutations causing minor structural alterations may not give an XP phenotype and may not, therefore, be identified clinically. XP13PV was the only patient carrying a missense mutation (Trp690Ser on the paternal allele). This was also the only patient in which the XPC transcript was present at a normal level and the XPC protein was detectable, although at a lower than normal level. No quantitative alterations in the transcript or protein levels were detected in the XP-C heterozygous parents. However, the expression of the normal allele predominated in all of them, except the father of XP13PV, which suggests the existence of a possible mechanism for monitoring the amount of the XPC protein.

Adolescent↗
PubMed2000

UV damage causes uncontrolled DNA breakage in cells from patients with combined features of XP-D and Cockayne syndrome.

Nucleotide excision repair (NER) removes damage from DNA in a tightly regulated multiprotein process. Defects in NER result in three different human disorders, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS). Two cases with the combined features of XP and CS have been assigned to the XP-D complementation group. Despite their extreme UV sensitivity, these cells appeared to incise their DNA as efficiently as normal cells in response to UV damage. These incisions were, however, uncoupled from the rest of the repair process. Using cell-free extracts, we were unable to detect any incision activity in the neighbourhood of the damage. When irradiated plasmids were introduced into unirradiated XP-D/CS cells, the ectopically introduced damage triggered the induction of breaks in the undamaged genomic DNA. XP-D/CS cells thus have a unique response to sensing UV damage, which results in the introduction of breaks into the DNA at sites distant from the damage. We propose that it is these spurious breaks that are responsible for the extreme UV sensitivity of these cells.

Base Pairing↗
PubMed2000

The cancer-free phenotype in trichothiodystrophy is unrelated to its repair defect.

The DNA repair-deficient genetic disorders xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) can both result from mutations in the XPD gene, the sites of the mutations differing between the two disorders. The hallmarks of XP are multiple pigmentation changes in the skin and a greatly elevated frequency of skin cancers, characteristics that are not seen in TTD. XP-D and most TTD patients have reduced levels of DNA repair, but some recent reports have suggested that the repair deficiencies in TTD cells are milder than in XP-D cells. We reported recently that inhibition of intracellular adhesion molecule-1 (ICAM-1) expression by UVB irradiation was similar in normal and TTD cells but increased in XP-D cells, suggesting a correlation between ICAM-1 inhibition and cancer proneness. In the first part of the current work, we have extended these studies and found several other examples, including XP-G and Cockayne syndrome cells, in which increased ICAM-1 inhibition correlated with cancer proneness. However, we also discovered that a subset of TTD cells, in which arg112 in the NH2-terminal region of the XPD protein is mutated to histidine, had an ICAM-1 response similar to that of XP-D cells. In the second part of the work, we have shown that TTD cells with this specific NH2-terminal mutation are more sensitive to UV irradiation than other TTDs, most of which are mutated in the COOH-terminal region, and are indistinguishable from XP-D cells in cell killing, incision breaks, and repair of cyclobutane pyrimidine dimers. Because the clinical phenotypes of these patients do not obviously differ from those of TTDs with mutations at other sites, we conclude that the lack of skin abnormalities in TTD is independent of the defective cellular responses to UV. It is likely to result from a transcriptional defect, which prevents the skin abnormalities from being expressed.

Cell Line↗
PubMed2000

Molecular methods for the detection of mutations.

We report the results of a collaborative study aimed at developing reliable, direct assays for mutation in human cells. The project used common lymphoblastoid cell lines, both with and without mutagen treatment, as a shared resource to validate the development of new molecular methods for the detection of low-level mutations in the presence of a large excess of normal alleles. As the "gold standard, " hprt mutation frequencies were also measured on the same samples. The methods under development included i) the restriction site mutation (RSM) assay, in which mutations lead to the destruction of a restriction site; ii) minisatellite length-change mutation, in which mutations lead to alleles containing new numbers of tandem repeat units; iii) loss of heterozygosity for HLA epitopes, in which antibodies can be used to direct selection for mutant cells; iv) multiple fluorescence-based long linker arm nucleotides assay (mf-LLA) technology, for the detection of substitutional mutations; v) detection of alterations in the TP53 locus using a (CA) array as the target for the screening; and vi) PCR analysis of lymphocytes for the presence of the BCL2 t(14:18) translocation. The relative merits of these molecular methods are discussed, and a comparison made with more "traditional" methods.

Base Sequence↗