- Email: [email protected]
Cockayne syndrome pedigree
Mutation Research 385 Ž1997. 107–114
Heritable genetic alterations in a xeroderma pigmentosum group GrCockayne syndrome pedigree Richard T. Okinaka a,) , Ana V. Perez-Castro a , Anthony Sena b, Kevin Laubscher a , Gary F. Strniste a , Min S. Park a , Rudy Hernandez a , Mark A. MacInnes a , Kenneth H. Kraemer c a
c
Life Sciences DiÕision, LS-6, M888, Los Alamos National Laboratory, Los Alamos, NM 87545, USA b Department of Biology, Northern New Mexico College, Espanola, NM 87532, USA Laboratory of Molecular Carcinogenesis, National Cancer Institute, Building 37 Room 3E24, Bethesda, MD 20892, USA Received 17 February 1997; revised 26 May 1997; accepted 26 May 1997
Abstract A search for genetic alterations within the XPG gene has been conducted on skin and blood cells cultured from a newly characterized xeroderma pigmentosum ŽXP. patient ŽXP20BE.. This patient is the ninth known case that falls into the extremely rare XP complementation group G. Four genetic markers within the XPG gene Žincluding two polymorphisms. demonstrated the Mendelian distribution of this gene from the parents to the patient and to an unaffected sibling. The patient ŽXP20BE. inherited a G to T transversion from his father in exon 1 of the XPG gene that resulted in the conversion of a glutamic acid at codon 11 to a termination codon. The patient also inherited an XP-G allele from his mother that produces an unstable or poorly expressed message. The cause of the latter defect is still uncertain. In addition to these alterations, XP20BE cDNA contained an mRNA species with a large splicing defect that encompassed a deletion from exon 1 to exon 14. This splicing defect, however, appears to be a naturally occurring low-frequency event that results from abnormal splicing that occurs between certain conserved non-consensus splicing signals within the human XPG gene. q 1997 Elsevier Science B.V. Keywords: Xeroderma pigmentosum; Cockayne Syndrome
1. Introduction Nucleotide excision repair ŽNER. is a universal repair process that recognizes and removes ‘bulky’ adducts from DNA molecules w1x. The sequence of events involved in NER is now well-established in
) Corresponding author. Tel: q1 Ž505. 667-2743; fax: q1 Ž505. 665-3024; e-mail: [email protected]
bacterial systems w2x. In humans, the discovery implicating defects in NER as being responsible for the genetic disorder xeroderma pigmentosum ŽXP. w3x, provided the impetus for the recent cloning of many of the human, rodent and yeast genes involved in this process Žsee reviews in w1,4–6x.. There are 7 XP complementation groups ŽA–G. with varying clinical symptoms w7x. Purification and reconstitution of these gene products into a functional repair complex has since led to a working model that explains the mechanistic basis for NER in humans w1,4,8x.
0921-8777r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 9 2 1 - 8 7 7 7 Ž 9 7 . 0 0 0 3 1 - 1
R.T. Okinaka et al.r Mutation Research 385 (1997) 107–114
108
The xeroderma pigmentosum group G gene codes for an 1186 amino acid protein w9–11x that functions as a specific endonuclease that hydrolyzes the sixth phosphodiester bond on the 3X-side of a DNA lesion during the nucleotide excision-repair process w12,13x. Two inactivating mutations w14x have previously been identified in the XPG protein of a single patient who had mild XP symptoms without CS w15x. Although the molecular basis for the combined XPrCS phenotype in the XP syndrome is still not known, important clues may exist in the kinds and positions of mutations that may lie within the XP-G gene of patients who suffer from the various forms of this disease. In this study we attempt to establish the hereditary basis for the XPrCS phenotype in a XP-G patient ŽXP20BE. w16x with severe neurological symptoms of Cockayne syndrome in addition to XP Žthe XPrCS complex. and his family by screening for potentially inactivating genetic alterations within the XPG gene.
2. Materials and methods 2.1. Cells A family pedigree with an XP-GrCS designation w16x was characterized and a series of Epstein–Barr virus immortalized lymphoblast cell lines and skin fibroblast cultures ŽXP20BE, normal, mother, father, and daughter. ŽTable 1. were established at the Institute for Medical Research, Camden, NJ. The cells were transported to Los Alamos as coded samples.
Table 1 Cell lines examined Donor
Lymphoblast
Fibroblast
XP20BE ŽXPGrCS. Mother Father Daughter Normal
AG08802 AG08804A AG08806A AG13017 GM606
AG08803 AG08805 AG08807 y y
This table lists the EB virus-transformed lymphoblast cell lines and corresponding primary skin fibroblast cultures that were examined in this study.
2.2. Cell growth Lymphoblastoid cells were cultured in static suspension cultures in RPMI medium maintained in a CO 2 incubator at 378C and supplemented with 15% fetal calf serum Žcomplement heat-inactivated. and the antibiotics penicillin and streptomycin. Cells were usually passaged 1:3 or 4 Žvrv. and maintained at cell densities between 2 = 10 5 and 8 = 10 5 per ml. All fibroblast cultures were maintained under the same conditions in DMEM medium supplemented with 10% fetal calf serum. 2.3. DNA preparations Genomic DNA were isolated by standard phenolrchloroform extractions as described elsewhere. Poly A messenger RNA was prepared utilizing a ‘Fastrack mRNA Isolation Kit’ according to the manufacturer’s instructions ŽInvitrogen Corporation, San Diego, CA.. The XP-G cDNA was obtained by first-round cDNA synthesis utilizing random hexamer priming and reverse transcription with Superscript II as described by the manufacturer ŽGibco BRL, Gaithersburg, MD.. Overlapping DNA fragments for either denaturing gradient gel electrophoresis ŽDGGE. analysis or DNA sequencing were generated by standard PCR techniques or by amplification using a modification of a long PCR method w17x. The modified long PCR technique was utilized to amplify fragments of 1000 bp or greater. Each reaction mixture for long PCRs consisted of 5 ml, 10 = buffer w18x; 1 ml each, 30 mm primers, 1 ml 15 mM dNTPs, 0.25 ml Amplitaq enzyme Ž5 Urml, Cetus Corporation.; 0.25 ml Vent DNA polymerase diluted 1:5 Ž10 Urml, New England Biolabs.. PCR reactions were performed in a Cetus 9600 GenAmp system. The modified long PCR conditions were: 948C melt, 15 s, 608C annealing, 15 s and 728C synthesis, 1.5 min. The PCR products were analyzed by electrophoretic separation on 6% polyacrylamide gels. 2.4. Mutation detection Mutations within PCR generated fragments were detected by either DGGE as previously described
R.T. Okinaka et al.r Mutation Research 385 (1997) 107–114
w19x or by direct DNA sequence analysis. A DNA melting profile was obtained for the entire XPG cDNA by the use of a program designed by L. Lerman ŽMIT.. This melting profile was utilized to design a strategy whereby overlapping fragments of the XPG gene could be analyzed by DGGE analysis. In brief, with the exception of a GC-rich region at the 5X-end of the cDNA, the XPG cDNA does not contain ‘naturally’ occurring high melting-clamps and as a result an artificial ‘clamp’ was inserted into
Table 2 PCR and sequencing primers and DGGE clamps Forward primers
Location Ž1sAUG.
Reverse primers
Location
E11R T16 Rudy 2 T10 59.4R1 594R2 T8 594R4 594R6 T22 594R8b 594R10 E10-3F 594R9 T4 594R7 E14-F3 T2 594R3
y44 to y28 y27 to y8 114 to 131 425 to 445 466 to 483 768 to 786 787 to 807 1125 to 1143 1400 to 1418 1450 to 1470 1738 to 1758 2039 to 2058 2206 to 2224 2599 to 2516 2518 to 2537 2818 to 2835 2882 to 2900 2973 to 2992 3325 to 3340
T73 E53R 593F3B T11 E7-3R 593F5 593F7 593F9 T1 T20 593F10 T15 E71L 593F6 T5 E14-5R 593F11 593F2 T3 593F1
199 to 180 511 to 494 678 to 650 842 to 823 857 to 843 975 to 958 1227 to 1208 1491 to 1473 1796 to 1775 1954 to 1935 2140 to 2120 2635 to 2617 2653 to 2636 2808 to 2791 2941 to 2922 2961 to 2943 3072 to 3064 3388 to 3379 3655 to 3638 3635 to 3616
This table indicates the positions of primers utilized in this study. The pairing of PCR primers was dictated by specific needs. Initially four ;1-kb overlapping fragments were generated by PCR off of the cDNA from each cell line. These fragments were then utilized to generate smaller, nested fragments that could be analyzed by direct DNA sequencing or by DGGE analysis. GCclamps were constructed by synthesizing designated primers with X an additional 42-bp GC rich tail onto its 5 -region. The GC-rich sequence consisted of the following: CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCG CCG CCC GCG. Clamps were constructed with the following primer sequences: EX4-5F, 594R4, T-20, T5 forward and reverse, 593F2 forward and reverse, and three other sequences two reverse primers Ž1071–1052., Ž1931– 1913. and a forward primer at Ž1869–1889.. These clamping primers were utilized to generate 300- to 500-bp PCR fragments that could be utilized in DGGE analysis of the entire XP-G cDNA w19x.
109
each PCR product by synthesizing a 20-bp XPG homologous primer with a 42-bp ‘GC-rich’ extension. Each PCR product was mixed with a known wild-type sequence to generate readily detectable mismatched mutantrwild-type heteroduplexes in a properly designed DGGE system. Two of the three alterations in this study were initially detected by DGGE analysis and later confirmed by direct sequencing of PCR products obtained from genomic or cDNA analysis. The third alteration was detected by direct sequencing of the 5X region of the cDNA which contained a high melt domain that was not readily amenable to DGGE analysis. The GC-rich clamp sequence and primers appropriate to these studies are depicted in Table 2.
3. Results 3.1. Detection of two polymorphic markers in the XP-G pedigree To avoid confusion between the numbering systems utilized in the three published XPG gene cDNA sequences w9–11x we have designated the A in the first ATG codon as bp 1 in the cDNA. Two previously identified polymorphisms w14x were initially detected by DGGE of PCR fragments generated from the cDNA of the two cell lineages, XP20BE and GM606 Žnormal.. These two polymorphic markers, a transition from C to T at cDNA position 138 Žexon 2, His 46 to His 46 . and a G to C transversion at bp 3310 in the XPG cDNA sequence Žexon 15, Asp1104 to His1104 . were useful in establishing the inheritance pattern within this XP-G pedigree. The first alteration created a restriction endonuclease site Ž NcoI. and restriction analysis of genomic PCR fragments ŽFig. 1. revealed that the father is homozygous for C at this position Žno digestion. while three of the lymphoblast cell strains ŽXP20BE, mother and daughter. showed only a partial digestion Žheterozygosity. at this locus. The fifth cell strain ŽGM606, normal. was homozygous for T at this position and illustrates a complete digestion pattern. The same three lymphoblast cells Žthe mother, the daughter and XP20BE. were also heterozygous for the second polymorphism ŽG to C at cDNA bp 3310.
110
R.T. Okinaka et al.r Mutation Research 385 (1997) 107–114
position 31 Žexon 1., a G to T transversion mutation, ŽFig. 2.. This mutation resulted in a change of Glu11 from glutamic to the termination codon UAG. The wild-type sequence in this region contains the recognition sequences for the restriction endonuclease BpmI and this site is destroyed by the G to T nonsense mutation. Restriction digests of exon 1 PCR fragments indicated that DNA from three of the cell strains Žthe mother, the daughter, and the normal cells. were totally digested into two equal-sized fragments ŽFig. 2., while the fragments from XP20BE and the father were only partially digested. These results were an indication that both the patient and father possess one XPG allele with a stop codon in place of Glu11 of the protein.
Fig. 1. Distribution of a His 46 polymorphism within the pedigree. Note the position of the bp 138 C to T transition that creates a CCATGG recognition signal for restriction endonuclease NcoI at the position of His 46 . Polyacrylamide gene electrophoresis of genomic exon 2 fragments were obtained following NcoI digestion. Lane 1, MspI digest of pBR322; Lane 2, sibling, AG13017; Lane 3, mother, AG08804A; Lane 4, father, AG08806A; Lane 5, patient XP20BE, AG08802; Lane 6, normal, GM606. The father is homozygous for the CAC codon while the normal cell, GM606, is homozygous for the CAT codon.
as determined by direct sequencing of genomic exon 15 fragments Ždata not shown.. The mother, the daughter and XP20BE could be distinguished from the father’s and GM606’s genotype on the basis of these markers. 3.2. Inheritance of a nonsense mutation Because of its high melting properties, the 5X-region of the XPG gene was initially analyzed by direct sequencing. A preliminary screen of exon 1 from genomic DNA from three of these cell lines ŽXP20BE, normal, and the mother. revealed a heterozygous mutant allele in XP20BE at cDNA bp
Fig. 2. Distribution of the Glu11 to termination codon mutation within the pedigree. A G to T transversion at bp 31 was detected in the cDNA of XP20BE cells. This mutation destroyed a BpmI restriction endonuclease site CTGGAG. The exon 1 region of the family pedigree was analyzed by PCR amplification followed by BpmI digest of genomic exon 1 fragments. Lane 1, MspI digest of pBR322; Lane 2, sibling, AG13017; Lane 3, mother, AG08804A; Lane 4, father, AG08806A; Lane 5, patient XP20BE. Heterozygosity in AG08806A and XP20BE is an indication that these two cells contain the termination mutation at this position. These results were repeated on genomic DNA obtained from the corresponding fibroblast cells and revealed an identical pattern Ždata not shown..
R.T. Okinaka et al.r Mutation Research 385 (1997) 107–114
111
3.3. The distribution of polymorphisms and the nonsense mutation is consistent with Mendelian inheritance The results obtained from the analysis of the two polymorphisms and the nonsense mutation allowed us to determine the inheritance pattern of the pedigree. XP20BE cells contained the nonsense mutation and the two polymorphisms as heterozygous alleles. The father is the carrier for the termination mutation at bp 31. The mother and the daughter are heterozygous for both polymorphisms, but unlike the patient, XP20BE, neither of these cells contained the nonsense mutation. The normal cell line was homozygous for both polymorphisms and did not carry the nonsense mutation. 3.4. A naturally occurring truncated message A ‘long’ PCR w17x analysis of the patient cDNA Žspanning 3.2 kb. revealed a 500-bp truncated product suggesting a potential splice-site mutation within the genome of the mother’s inherited allele. Sequencing analysis of this fragment revealed that the smaller PCR product contained a sequence that is joined precisely between the splice-site junction of exon 1 and the splice-site junction of exon 14 Ždata not shown.. Sequence analysis of the truncated message from the patient also clearly indicated the surprising result that the sequence of codon 11 was for the nonsense mutation ŽTAG. of the father rather than the CAG of the mother. It was therefore concluded that the truncated PCR product in XP20BE was not caused by a defect in the maternal allele, but most likely the product of a natural splicing error that occurs with low frequency during the processing of the XPG gene Žsee w14x and discussion.. 3.5. Low leÕels of the mother’s cDNA in the patient Extensive analysis of the patient’s cDNA has not yet revealed a second potential inactivating mutation within the coding region of the XPG gene. However, the lack of the mother’s allele in the truncated message suggested that this allele may be either unstable or inefficiently expressed in the patient. This possibility was examined by comparison of genomic and reverse transcribed PCR products from
Fig. 3. Analysis of a polymorphic marker in mRNA and genomic preparations. cDNA and genomic fragments were PCR amplified utilizing cDNA and exon specific primers to generate fragments of approximately the same length. These products were generated from AG08804A, the mother, Žlanes 2 and 3., XP20BE Žlanes 4 and 5. and AG08806A, the father, Žlanes 6 and 7.. The samples were then restriction digested with NcoI and analyzed by polyacrylamide gel electrophoresis. Lanes 1 and 8 are MspI digests of pBR322. Lanes, 2, 4 and 6 represent PCRs generated from cDNAs and lanes 3, 5, and 7 represent PCRs generated from genomic DNA of the same cells. The father ŽAG08806A, lanes 6 and 7. is homozygous and does not contain the NcoI site. AG08804A Žthe mother. and XP20BE are both homozygous for the NcoI site and their genomic digests Žlanes 3 and 5. illustrate this point. Their cDNAs, however, appear to be under represented for the allele containing the NcoI site. Density measurements on a digital imaging system ŽAlpha Innotech Corporation, San Leandro, CA. indicates that the two restriction fragments in AG08804A and XP20BE are approximately 25% of the intensity of the uncut fragment.
the father, the mother and the patient ŽFig. 3.. The region surrounding exon 2 was PCR amplified from both genomic and cDNA preparations and then digested with NcoI. Since the mother’s inherited allele contains the restriction site for NcoI, comparison of these digestion products provides a measure of the relative efficiency of amplification of the mother’s and father’s alleles in the patient. These results clearly demonstrate that the parental alleles are present at relatively equal amounts in the genomic DNA of the patient, but that the level of the mother’s NcoI-positive allele is substantially reduced in the cDNA preparations of both the mother and the patient. Density comparisons of these bands indicate that the level of the mother’s allele in the patient is 25% Žor 1r8th of the total message. of the father’s allele in this particular experiment Ždata not shown.. These data suggest that the mother’s inherited allele is either unstable or poorly expressed in the patient
112
R.T. Okinaka et al.r Mutation Research 385 (1997) 107–114
and leads to reduced recovery of intact XPG mRNA from this allele during isolation of poly A message from these cells. The cause for this apparent defect is not yet clear.
4. Discussion This study demonstrates the Mendelian inheritance of four genetic markers from the parents to a xeroderma pigmentosum group G patient and a sibling. The patient was heterozygous for a silent C to T transition at bp 138 ŽHis 46 . in exon 2; acquiring the C allele from his father and the T allele from his mother. The patient was also heterozygous for a G to C transversion at bp 3310 in exon 15 ŽAsp1104 to His1104 .; acquiring the Asp1104 allele from his father and the His1104 allele from his mother. Both of these alterations do not appear to affect the function of the XPG gene w14x. The patient also acquired from his father a G to T transversion mutation at bp position 31 that resulted in the conversion of Glu11 in exon 1 to a nonsense or stop codon. It is reasonable to assume that a nonsense mutation producing a truncated protein containing only 11 amino acids would be inactive. The unscheduled DNA synthesis and post-UV colonyforming ability of XP20BE is very low w16x. A potentially inactivating mutation was not detected in the mother’s allele but an RFLP analysis of reverse transcribed mRNA preparations revealed that both the mother and the patient possess an XPG allele that is either unstable or poorly expressed. It is possible that these analyses could have missed a second inactivating mutation from the coding portion of the mother’s allele and that the ‘unstable’ message itself does not produce a fully functional protein. Alternatively, the ‘unstable’ XPG message from the mother could be producing a low level of the functional XPG protein in the patient. The extremely rare incidence of XP group G disease Žonly 8 other patients reported w15,16,20–26x. may reflect the critical nature of this gene; perhaps suggesting that these patients can only survive because of residual XPG function. The cause of this latter defect is still uncertain because the poor expression of the mother’s allele could be due to mutations in almost any part of the XPG gene.
In addition to detecting four alterations within the XP-G pedigree, these analyses also uncovered an unusually large deletion event caused by abnormal splicing from the first to the fourteenth exons. At first glimpse it appeared that this defect might be related to the maternally inherited defect in the patient. However, sequencing data clearly indicated that the splicing error in the patient was a product derived primarily from the father’s message and that this product most likely represents a minor, but normal, error in the processing of the XPG gene. Analysis of the genomic structure around this region reveals that the splicing signals in the region around exon 13 and 14 do not obey strict consensus rules Žw35x, unpublished observations.. It is concluded that these non-consensus splice-site structures contribute to a naturally occurring abnormal splicing of this region. It has previously been noted that a minor fraction of the XPG pre mRNA is also abnormally spliced to cause a 55 bp deletion between positions bp 1078–1132 in the XPG gene of normal individuals w14x. It was suggested that ‘leaky’ splicing may contribute to the low abundance of functional XPG mRNA. It might also be argued that these abnormal splicing events might be a mechanism by which the level of this protein may be controlled in mammals. Although only 9 XP-G patients have been identified world wide w15,16,20–26x, this syndrome displays a wide array of phenotypes ranging from the typical sunlight-skin sensitivity of the XP syndrome to the more pleiotropic characteristics of the XPrCS syndrome including diminished growth and mental retardation Žsee discussion in w16x.. Recent studies with cells derived from Cockayne syndrome or XP-G patients have demonstrated that these cells are deficient in the preferential repair of bulky adducts and of ionizing radiation-induced DNA damage w27,28x. These results led to the hypothesis that the XPrCS dual phenotype is caused by defects in XPG gene that affect two functions: nucleotide excision repair and a more generalized transcription-associated repair function. In addition to the endonuclease activity, the XPG protein also has an apparent ‘loose association’ with the transcription factor TFIIH w29,30x. The coupling of nucleotide excision-repair activity to transcription in XP complementation groups B, and D has led to the implication that a defect in transcription may be related to other human
R.T. Okinaka et al.r Mutation Research 385 (1997) 107–114
syndromes that are associated with these repair defects, such as the XPrCS complex and patients with the syndrome of trichothiodystrophy Žsulfur-deficient brittle hair, photosensitivity, mental retardation and ichthyosis. without skin cancer w27,31–34x. To account for the pleiotropic nature of XP-G patients, this model would predict that certain XPG mutations would affect both transcription and repair while others would only alter the nucleotide excision-repair function. XP20BE cells were derived from a sun-sensitive boy who had severe symptoms of CS with mild XP-type pigmentation. The XP20BE patient had severe XPrCS syndrome with dwarfism, retinal degeneration, microcephaly and mental impairment. He died at age 6 years with marked cachexia Žweight 14.5 lb. without skin cancer. While the clinical phenotype of the patient accentuated the CS features, the cellular phenotype was typical of XP complementation group G with less than 5% of normal post-UV unscheduled DNA synthesis and very low post-UV cell survival w16x. The pedigree analysis of patient XP20BE indicates that the father’s inherited nonsense mutation at bp 31 would fit the criteria for the XPrCS dual phenotype since a non-leaky mutation at this position would almost certainly eliminate any XPG function. Although it is not yet evident whether the mother’s inherited, ‘unstable’ allele has any functional activity, one possibility may be that residual XPG gene function may not be sufficient to fully compensate for both NER and transcription-associated repair in this cell.
References w1x A. Sancar, Excision repair in mammalian cells, J. Biol. Chem. 270 Ž1995. 15915–15918. w2x J.J. Lin, A. Sancar, ŽA.BC excinuclease: the Escherichia coli nucleotide excision repair enzyme, Mol. Microbiol. 6 Ž1992. 2219–2224. w3x J.E. Cleaver, Defective repair replication of DNA in xeroderma pigmentosum, Nature 218 Ž1968. 652–656. w4x A. Sancar, Mechanisms of DNA excision repair, Science 266 Ž1994. 1954–1956. w5x D. Bootsma, J.H.J. Hoeijmakers, The molecular basis of nucleotide excision repair syndromes, Mutation Res. Fundam. Mol. Mech. Mutagen. 307 Ž1994. 15–23. w6x J.H.J. Hoeijmakers, Human nucleotide excision repair syndromes: molecular clues to unexpected intricacies, Eur. J. Cancer ŽA. 30A Ž1994. 1912–1921.
113
w7x J.E. Cleaver, K.H. Kraemer, Xeroderma pigmentosum and Cockayne syndrome, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle ŽEds.., The Metabolic and Molecular Bases of Inherited Disease, 7th ed., McGraw-Hill, New York, 1995, pp. 4393–4419. w8x A. Aboussekhra, M. Biggerstaff, M.K.K. Shivji, J.A. Vilpo, V. Moncollin, V.N. Podust, M. Protic, U. Hubscher, J.-M. ¨ Egly, R.D. Wood, Mammalian DNA nucleotide excision repair reconstituted with purified protein components, Cell 80 Ž1995. 859–868. w9x M.A. MacInnes, J.A. Dickson, R.R. Hernandez, D. Learmonth, G.Y. Lin, J.S. Mudgett, M.S. Park, S. Schauer, R.J. Reynolds, G.F. Strniste, J.Y. Yu, Human ERCC5 cDNAcosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of Saccharomyces cereÕisiae and Schizosaccharomyces pombe, Mol. Cell. Biol. 13 Ž1993. 6393–6402. w10x D. Scherly, T. Nouspikel, J. Corlet, C. Ucla, A. Bairoch, S.G. Clarkson, Complementation of the DNA repair defect in xeroderma pigmentosum group G cells by a human cDNA related to yeast RAD2, Nature 363 Ž1993. 182–185. w11x T. Shiomi, Y. Harada, T. Saito, N. Shiomi, Y. Okuno, M. Yamaizumi, An ERCC5 gene with homology to yeast RAD2 is involved in group G xeroderma pigmentosum, Mutation Res. 314 Ž1994. 167–175. w12x J.C. Huang, D.L. Svoboda, J.T. Reardon, A. Sancar, Human nucleotide excision nuclease removes thymine dimers from X DNA by incising the 22nd phosphodiester bond 5 and the X 6th phosphodiester bond 3 to the photodimer, Proc. Natl. Acad. Sci. USA 89 Ž1992. 3664–3668. w13x D.L. Svoboda, J.S. Taylor, J.E. Hearst, A. Sancar, DNA repair by eukaryotic nucleotide excision nuclease. Removal of thymine dimer and psoralen monoadduct by HeLa cell-free extract and of thymine dimer by Xenopus laevis oocytes, J. Biol. Chem. 268 Ž1993. 1931–1936. w14x T. Nouspikel, S.G. Clarkson, Mutations that disable the DNA repair gene XPG in a xeroderma pigmentosum group G patient, Hum. Mol. Genet. 3 Ž1994. 963–967. w15x P.G. Norris, J.L. Hawk, J.A. Avery, F. Giannelli, Xeroderma pigmentosum complementation group G-report of two cases, Br. J. Dermatol. 116 Ž1987. 861–866. w16x S.I. Moriwaki, M. Stefanini, A.R. Lehmann, J.H.J. Hoeijmakers, J.H. Robbins, I. Rapin, E. Botta, B. Tanganelli, W. Vermeulen, B.C. Broughton, K.H. Kraemer, DNA repair and ultraviolet mutagenesis in cells from a new patient with xeroderma pigmentosum group G and Cockayne syndrome resemble xeroderma pigmentosum cells, J. Invest. Dermatol. 107 Ž1996. 647–653. w17x W.M. Barnes, PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates, Proc. Natl. Acad. Sci. USA 91 Ž1994. 2216–2220. w18x J.L. Yang, V.M. Maher, J.J. McCormick, Amplification and direct nucleotide sequencing of cDNA from the lysate of low numbers of diploid human cells, Gene 83 Ž1989. 347–354. w19x R.T. Okinaka, S.L. Anzick, A. Oller, W.G. Thilly, Analysis of large X-ray-induced mutant populations by denaturing gradient gel electrophoresis, Radiat. Res. 135 Ž1993. 212– 221.
114
R.T. Okinaka et al.r Mutation Research 385 (1997) 107–114
w20x M.J. Cheesbrough, P.D.C. Kinmont, Xeroderma pigmentosum – a unique variant with neurological involvement, Br. J. Dermatol. 99 Ž1978. 61. w21x W. Keijzer, N.G. Jaspers, P.J. Abrahams, A.M. Taylor, C.F. Arlett, B. Zelle, H. Takebe, P.D. Kinmont, D. Bootsma, A seventh complementation group in excision-deficient xeroderma pigmentosum, Mutation Res. 62 Ž1979. 183–190. w22x C.F. Arlett, S.A. Harcourt, A.R. Lehmann, S. Stevens, W.A. Ferguson-Smith, W.N. Morley, Studies of a new case of xeroderma pigmentosum ŽXP3BR. from complementation group G with cellular sensitivity to ionizing radiation, Carcinogenesis 1 Ž1980. 745–751. w23x M. Ichihashi, Y. Fujiwara, Y. Uehara, A. Matsumoto, A mild form of xeroderma pigmentosum assigned to complementation group G and its repair heterogeneity, J. Invest. Dermatol. 85 Ž1985. 284–287. w24x J. Jaeken, H. Klocker, H. Schwaiger, R. Bellmann, M. Hirsch-Kauffmann, M. Schweiger, Clinical and biochemical studies in three patients with severe early infantile Cockayne syndrome, Hum. Genet. 83 Ž1989. 339–346. w25x W. Vermeulen, J. Jaeken, N.G. Jaspers, D. Bootsma, J.H. Hoeijmakers, Xeroderma pigmentosum complementation group G associated with Cockayne syndrome, Am. J. Hum. Genet. 53 Ž1993. 185–192. w26x B.C.K. Hamel, A. Raams, A.R. Schuitema-Dijkstra, P. Simons, I. Van der Burgt, N.G.J. Jaspers, W.J. Kleijer, Xeroderma pigmentosum–Cockayne syndrome complex: a further case, J. Med. Genet. 33 Ž1996. 607–610. w27x S.A. Leadon, P.K. Cooper, Preferential repair of ionizing radiation-induced damage in the transcribed strand of an active human gene is defective in Cockayne syndrome, Proc. Natl. Acad. Sci. USA 90 Ž1993. 10499–10503. w28x P.K. Cooper, S.A. Leadon, Defective repair of ionizing
w29x
w30x
w31x w32x
w33x
w34x
w35x
radiation damage in Cockayne’s syndrome and xeroderma pigmentosum group G, Ann. New York Acad. Sci. 726 Ž1994. 330–332. C.-H. Park, D. Mu, J.T. Reardon, A. Sancar, The general transcription-repair factor TFIIH is recruited to the excision repair complex by the XPA protein independent of the TFIIE transcription factor, J. Biol. Chem. 270 Ž1995. 4896–4902. D. Mu, C.-H. Park, T. Matsunaga, D.S. Hsu, J.T. Reardon, A. Sancar, Reconstitution of human DNA repair excision nuclease in a highly defined system, J. Biol. Chem. 270 Ž1995. 2415–2418. P.C. Hanawalt, Transcription-coupled repair and human disease, Science 266 Ž1994. 1957–1958. D. Bootsma, G. Weeda, W. Vermeulen, H. Van Vuuren, C. Troelstra, P. Van der Spek, J. Hoeijmakers, Nucleotide excision repair syndromes: molecular basis and clinical symptoms, Philos. Trans. R. Soc. Lond. B. 347 Ž1995. 75–81. K. Takayama, E.P. Salazar, B.C. Broughton, A.R. Lehmann, A. Sarasin, L.H. Thompson, C.A. Weber, Defects in the DNA repair and transcription gene ERCC2ŽXPD. in trichothiodystrophy, Am. J. Hum. Genet. 58 Ž1996. 263–270. B.C. Broughton, A.F. Thompson, S.A. Harcourt, W. Vermeulen, J.H. Hoeijmakers, E. Botta, M. Stefanini, M.D. King, C.A. Weber, J. Cole, Molecular and cellular analysis of the DNA repair defect in a patient in xeroderma pigmentosum complementation group D who has the clinical features of xeroderma pigmentosum and Cockayne syndrome, Am. J. Hum. Genet. 56 Ž1995. 167–174. D.L. Ludwig, J.S. Mudgett, M.S. Park, A.V. Perez-Castro, M.A. MacInnes, Molecular cloning and structural analysis of the functional mouse genomic XPG gene, Mammal. Genome 7 Ž1996. 644–649.
Heritable genetic alterations in a xeroderma pigmentosum group GrCockayne syndrome pedigree Richard T. Okinaka a,) , Ana V. Perez-Castro a , Anthony Sena b, Kevin Laubscher a , Gary F. Strniste a , Min S. Park a , Rudy Hernandez a , Mark A. MacInnes a , Kenneth H. Kraemer c a
c
Life Sciences DiÕision, LS-6, M888, Los Alamos National Laboratory, Los Alamos, NM 87545, USA b Department of Biology, Northern New Mexico College, Espanola, NM 87532, USA Laboratory of Molecular Carcinogenesis, National Cancer Institute, Building 37 Room 3E24, Bethesda, MD 20892, USA Received 17 February 1997; revised 26 May 1997; accepted 26 May 1997
Abstract A search for genetic alterations within the XPG gene has been conducted on skin and blood cells cultured from a newly characterized xeroderma pigmentosum ŽXP. patient ŽXP20BE.. This patient is the ninth known case that falls into the extremely rare XP complementation group G. Four genetic markers within the XPG gene Žincluding two polymorphisms. demonstrated the Mendelian distribution of this gene from the parents to the patient and to an unaffected sibling. The patient ŽXP20BE. inherited a G to T transversion from his father in exon 1 of the XPG gene that resulted in the conversion of a glutamic acid at codon 11 to a termination codon. The patient also inherited an XP-G allele from his mother that produces an unstable or poorly expressed message. The cause of the latter defect is still uncertain. In addition to these alterations, XP20BE cDNA contained an mRNA species with a large splicing defect that encompassed a deletion from exon 1 to exon 14. This splicing defect, however, appears to be a naturally occurring low-frequency event that results from abnormal splicing that occurs between certain conserved non-consensus splicing signals within the human XPG gene. q 1997 Elsevier Science B.V. Keywords: Xeroderma pigmentosum; Cockayne Syndrome
1. Introduction Nucleotide excision repair ŽNER. is a universal repair process that recognizes and removes ‘bulky’ adducts from DNA molecules w1x. The sequence of events involved in NER is now well-established in
) Corresponding author. Tel: q1 Ž505. 667-2743; fax: q1 Ž505. 665-3024; e-mail: [email protected]
bacterial systems w2x. In humans, the discovery implicating defects in NER as being responsible for the genetic disorder xeroderma pigmentosum ŽXP. w3x, provided the impetus for the recent cloning of many of the human, rodent and yeast genes involved in this process Žsee reviews in w1,4–6x.. There are 7 XP complementation groups ŽA–G. with varying clinical symptoms w7x. Purification and reconstitution of these gene products into a functional repair complex has since led to a working model that explains the mechanistic basis for NER in humans w1,4,8x.
0921-8777r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 9 2 1 - 8 7 7 7 Ž 9 7 . 0 0 0 3 1 - 1
R.T. Okinaka et al.r Mutation Research 385 (1997) 107–114
108
The xeroderma pigmentosum group G gene codes for an 1186 amino acid protein w9–11x that functions as a specific endonuclease that hydrolyzes the sixth phosphodiester bond on the 3X-side of a DNA lesion during the nucleotide excision-repair process w12,13x. Two inactivating mutations w14x have previously been identified in the XPG protein of a single patient who had mild XP symptoms without CS w15x. Although the molecular basis for the combined XPrCS phenotype in the XP syndrome is still not known, important clues may exist in the kinds and positions of mutations that may lie within the XP-G gene of patients who suffer from the various forms of this disease. In this study we attempt to establish the hereditary basis for the XPrCS phenotype in a XP-G patient ŽXP20BE. w16x with severe neurological symptoms of Cockayne syndrome in addition to XP Žthe XPrCS complex. and his family by screening for potentially inactivating genetic alterations within the XPG gene.
2. Materials and methods 2.1. Cells A family pedigree with an XP-GrCS designation w16x was characterized and a series of Epstein–Barr virus immortalized lymphoblast cell lines and skin fibroblast cultures ŽXP20BE, normal, mother, father, and daughter. ŽTable 1. were established at the Institute for Medical Research, Camden, NJ. The cells were transported to Los Alamos as coded samples.
Table 1 Cell lines examined Donor
Lymphoblast
Fibroblast
XP20BE ŽXPGrCS. Mother Father Daughter Normal
AG08802 AG08804A AG08806A AG13017 GM606
AG08803 AG08805 AG08807 y y
This table lists the EB virus-transformed lymphoblast cell lines and corresponding primary skin fibroblast cultures that were examined in this study.
2.2. Cell growth Lymphoblastoid cells were cultured in static suspension cultures in RPMI medium maintained in a CO 2 incubator at 378C and supplemented with 15% fetal calf serum Žcomplement heat-inactivated. and the antibiotics penicillin and streptomycin. Cells were usually passaged 1:3 or 4 Žvrv. and maintained at cell densities between 2 = 10 5 and 8 = 10 5 per ml. All fibroblast cultures were maintained under the same conditions in DMEM medium supplemented with 10% fetal calf serum. 2.3. DNA preparations Genomic DNA were isolated by standard phenolrchloroform extractions as described elsewhere. Poly A messenger RNA was prepared utilizing a ‘Fastrack mRNA Isolation Kit’ according to the manufacturer’s instructions ŽInvitrogen Corporation, San Diego, CA.. The XP-G cDNA was obtained by first-round cDNA synthesis utilizing random hexamer priming and reverse transcription with Superscript II as described by the manufacturer ŽGibco BRL, Gaithersburg, MD.. Overlapping DNA fragments for either denaturing gradient gel electrophoresis ŽDGGE. analysis or DNA sequencing were generated by standard PCR techniques or by amplification using a modification of a long PCR method w17x. The modified long PCR technique was utilized to amplify fragments of 1000 bp or greater. Each reaction mixture for long PCRs consisted of 5 ml, 10 = buffer w18x; 1 ml each, 30 mm primers, 1 ml 15 mM dNTPs, 0.25 ml Amplitaq enzyme Ž5 Urml, Cetus Corporation.; 0.25 ml Vent DNA polymerase diluted 1:5 Ž10 Urml, New England Biolabs.. PCR reactions were performed in a Cetus 9600 GenAmp system. The modified long PCR conditions were: 948C melt, 15 s, 608C annealing, 15 s and 728C synthesis, 1.5 min. The PCR products were analyzed by electrophoretic separation on 6% polyacrylamide gels. 2.4. Mutation detection Mutations within PCR generated fragments were detected by either DGGE as previously described
R.T. Okinaka et al.r Mutation Research 385 (1997) 107–114
w19x or by direct DNA sequence analysis. A DNA melting profile was obtained for the entire XPG cDNA by the use of a program designed by L. Lerman ŽMIT.. This melting profile was utilized to design a strategy whereby overlapping fragments of the XPG gene could be analyzed by DGGE analysis. In brief, with the exception of a GC-rich region at the 5X-end of the cDNA, the XPG cDNA does not contain ‘naturally’ occurring high melting-clamps and as a result an artificial ‘clamp’ was inserted into
Table 2 PCR and sequencing primers and DGGE clamps Forward primers
Location Ž1sAUG.
Reverse primers
Location
E11R T16 Rudy 2 T10 59.4R1 594R2 T8 594R4 594R6 T22 594R8b 594R10 E10-3F 594R9 T4 594R7 E14-F3 T2 594R3
y44 to y28 y27 to y8 114 to 131 425 to 445 466 to 483 768 to 786 787 to 807 1125 to 1143 1400 to 1418 1450 to 1470 1738 to 1758 2039 to 2058 2206 to 2224 2599 to 2516 2518 to 2537 2818 to 2835 2882 to 2900 2973 to 2992 3325 to 3340
T73 E53R 593F3B T11 E7-3R 593F5 593F7 593F9 T1 T20 593F10 T15 E71L 593F6 T5 E14-5R 593F11 593F2 T3 593F1
199 to 180 511 to 494 678 to 650 842 to 823 857 to 843 975 to 958 1227 to 1208 1491 to 1473 1796 to 1775 1954 to 1935 2140 to 2120 2635 to 2617 2653 to 2636 2808 to 2791 2941 to 2922 2961 to 2943 3072 to 3064 3388 to 3379 3655 to 3638 3635 to 3616
This table indicates the positions of primers utilized in this study. The pairing of PCR primers was dictated by specific needs. Initially four ;1-kb overlapping fragments were generated by PCR off of the cDNA from each cell line. These fragments were then utilized to generate smaller, nested fragments that could be analyzed by direct DNA sequencing or by DGGE analysis. GCclamps were constructed by synthesizing designated primers with X an additional 42-bp GC rich tail onto its 5 -region. The GC-rich sequence consisted of the following: CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCG CCG CCC GCG. Clamps were constructed with the following primer sequences: EX4-5F, 594R4, T-20, T5 forward and reverse, 593F2 forward and reverse, and three other sequences two reverse primers Ž1071–1052., Ž1931– 1913. and a forward primer at Ž1869–1889.. These clamping primers were utilized to generate 300- to 500-bp PCR fragments that could be utilized in DGGE analysis of the entire XP-G cDNA w19x.
109
each PCR product by synthesizing a 20-bp XPG homologous primer with a 42-bp ‘GC-rich’ extension. Each PCR product was mixed with a known wild-type sequence to generate readily detectable mismatched mutantrwild-type heteroduplexes in a properly designed DGGE system. Two of the three alterations in this study were initially detected by DGGE analysis and later confirmed by direct sequencing of PCR products obtained from genomic or cDNA analysis. The third alteration was detected by direct sequencing of the 5X region of the cDNA which contained a high melt domain that was not readily amenable to DGGE analysis. The GC-rich clamp sequence and primers appropriate to these studies are depicted in Table 2.
3. Results 3.1. Detection of two polymorphic markers in the XP-G pedigree To avoid confusion between the numbering systems utilized in the three published XPG gene cDNA sequences w9–11x we have designated the A in the first ATG codon as bp 1 in the cDNA. Two previously identified polymorphisms w14x were initially detected by DGGE of PCR fragments generated from the cDNA of the two cell lineages, XP20BE and GM606 Žnormal.. These two polymorphic markers, a transition from C to T at cDNA position 138 Žexon 2, His 46 to His 46 . and a G to C transversion at bp 3310 in the XPG cDNA sequence Žexon 15, Asp1104 to His1104 . were useful in establishing the inheritance pattern within this XP-G pedigree. The first alteration created a restriction endonuclease site Ž NcoI. and restriction analysis of genomic PCR fragments ŽFig. 1. revealed that the father is homozygous for C at this position Žno digestion. while three of the lymphoblast cell strains ŽXP20BE, mother and daughter. showed only a partial digestion Žheterozygosity. at this locus. The fifth cell strain ŽGM606, normal. was homozygous for T at this position and illustrates a complete digestion pattern. The same three lymphoblast cells Žthe mother, the daughter and XP20BE. were also heterozygous for the second polymorphism ŽG to C at cDNA bp 3310.
110
R.T. Okinaka et al.r Mutation Research 385 (1997) 107–114
position 31 Žexon 1., a G to T transversion mutation, ŽFig. 2.. This mutation resulted in a change of Glu11 from glutamic to the termination codon UAG. The wild-type sequence in this region contains the recognition sequences for the restriction endonuclease BpmI and this site is destroyed by the G to T nonsense mutation. Restriction digests of exon 1 PCR fragments indicated that DNA from three of the cell strains Žthe mother, the daughter, and the normal cells. were totally digested into two equal-sized fragments ŽFig. 2., while the fragments from XP20BE and the father were only partially digested. These results were an indication that both the patient and father possess one XPG allele with a stop codon in place of Glu11 of the protein.
Fig. 1. Distribution of a His 46 polymorphism within the pedigree. Note the position of the bp 138 C to T transition that creates a CCATGG recognition signal for restriction endonuclease NcoI at the position of His 46 . Polyacrylamide gene electrophoresis of genomic exon 2 fragments were obtained following NcoI digestion. Lane 1, MspI digest of pBR322; Lane 2, sibling, AG13017; Lane 3, mother, AG08804A; Lane 4, father, AG08806A; Lane 5, patient XP20BE, AG08802; Lane 6, normal, GM606. The father is homozygous for the CAC codon while the normal cell, GM606, is homozygous for the CAT codon.
as determined by direct sequencing of genomic exon 15 fragments Ždata not shown.. The mother, the daughter and XP20BE could be distinguished from the father’s and GM606’s genotype on the basis of these markers. 3.2. Inheritance of a nonsense mutation Because of its high melting properties, the 5X-region of the XPG gene was initially analyzed by direct sequencing. A preliminary screen of exon 1 from genomic DNA from three of these cell lines ŽXP20BE, normal, and the mother. revealed a heterozygous mutant allele in XP20BE at cDNA bp
Fig. 2. Distribution of the Glu11 to termination codon mutation within the pedigree. A G to T transversion at bp 31 was detected in the cDNA of XP20BE cells. This mutation destroyed a BpmI restriction endonuclease site CTGGAG. The exon 1 region of the family pedigree was analyzed by PCR amplification followed by BpmI digest of genomic exon 1 fragments. Lane 1, MspI digest of pBR322; Lane 2, sibling, AG13017; Lane 3, mother, AG08804A; Lane 4, father, AG08806A; Lane 5, patient XP20BE. Heterozygosity in AG08806A and XP20BE is an indication that these two cells contain the termination mutation at this position. These results were repeated on genomic DNA obtained from the corresponding fibroblast cells and revealed an identical pattern Ždata not shown..
R.T. Okinaka et al.r Mutation Research 385 (1997) 107–114
111
3.3. The distribution of polymorphisms and the nonsense mutation is consistent with Mendelian inheritance The results obtained from the analysis of the two polymorphisms and the nonsense mutation allowed us to determine the inheritance pattern of the pedigree. XP20BE cells contained the nonsense mutation and the two polymorphisms as heterozygous alleles. The father is the carrier for the termination mutation at bp 31. The mother and the daughter are heterozygous for both polymorphisms, but unlike the patient, XP20BE, neither of these cells contained the nonsense mutation. The normal cell line was homozygous for both polymorphisms and did not carry the nonsense mutation. 3.4. A naturally occurring truncated message A ‘long’ PCR w17x analysis of the patient cDNA Žspanning 3.2 kb. revealed a 500-bp truncated product suggesting a potential splice-site mutation within the genome of the mother’s inherited allele. Sequencing analysis of this fragment revealed that the smaller PCR product contained a sequence that is joined precisely between the splice-site junction of exon 1 and the splice-site junction of exon 14 Ždata not shown.. Sequence analysis of the truncated message from the patient also clearly indicated the surprising result that the sequence of codon 11 was for the nonsense mutation ŽTAG. of the father rather than the CAG of the mother. It was therefore concluded that the truncated PCR product in XP20BE was not caused by a defect in the maternal allele, but most likely the product of a natural splicing error that occurs with low frequency during the processing of the XPG gene Žsee w14x and discussion.. 3.5. Low leÕels of the mother’s cDNA in the patient Extensive analysis of the patient’s cDNA has not yet revealed a second potential inactivating mutation within the coding region of the XPG gene. However, the lack of the mother’s allele in the truncated message suggested that this allele may be either unstable or inefficiently expressed in the patient. This possibility was examined by comparison of genomic and reverse transcribed PCR products from
Fig. 3. Analysis of a polymorphic marker in mRNA and genomic preparations. cDNA and genomic fragments were PCR amplified utilizing cDNA and exon specific primers to generate fragments of approximately the same length. These products were generated from AG08804A, the mother, Žlanes 2 and 3., XP20BE Žlanes 4 and 5. and AG08806A, the father, Žlanes 6 and 7.. The samples were then restriction digested with NcoI and analyzed by polyacrylamide gel electrophoresis. Lanes 1 and 8 are MspI digests of pBR322. Lanes, 2, 4 and 6 represent PCRs generated from cDNAs and lanes 3, 5, and 7 represent PCRs generated from genomic DNA of the same cells. The father ŽAG08806A, lanes 6 and 7. is homozygous and does not contain the NcoI site. AG08804A Žthe mother. and XP20BE are both homozygous for the NcoI site and their genomic digests Žlanes 3 and 5. illustrate this point. Their cDNAs, however, appear to be under represented for the allele containing the NcoI site. Density measurements on a digital imaging system ŽAlpha Innotech Corporation, San Leandro, CA. indicates that the two restriction fragments in AG08804A and XP20BE are approximately 25% of the intensity of the uncut fragment.
the father, the mother and the patient ŽFig. 3.. The region surrounding exon 2 was PCR amplified from both genomic and cDNA preparations and then digested with NcoI. Since the mother’s inherited allele contains the restriction site for NcoI, comparison of these digestion products provides a measure of the relative efficiency of amplification of the mother’s and father’s alleles in the patient. These results clearly demonstrate that the parental alleles are present at relatively equal amounts in the genomic DNA of the patient, but that the level of the mother’s NcoI-positive allele is substantially reduced in the cDNA preparations of both the mother and the patient. Density comparisons of these bands indicate that the level of the mother’s allele in the patient is 25% Žor 1r8th of the total message. of the father’s allele in this particular experiment Ždata not shown.. These data suggest that the mother’s inherited allele is either unstable or poorly expressed in the patient
112
R.T. Okinaka et al.r Mutation Research 385 (1997) 107–114
and leads to reduced recovery of intact XPG mRNA from this allele during isolation of poly A message from these cells. The cause for this apparent defect is not yet clear.
4. Discussion This study demonstrates the Mendelian inheritance of four genetic markers from the parents to a xeroderma pigmentosum group G patient and a sibling. The patient was heterozygous for a silent C to T transition at bp 138 ŽHis 46 . in exon 2; acquiring the C allele from his father and the T allele from his mother. The patient was also heterozygous for a G to C transversion at bp 3310 in exon 15 ŽAsp1104 to His1104 .; acquiring the Asp1104 allele from his father and the His1104 allele from his mother. Both of these alterations do not appear to affect the function of the XPG gene w14x. The patient also acquired from his father a G to T transversion mutation at bp position 31 that resulted in the conversion of Glu11 in exon 1 to a nonsense or stop codon. It is reasonable to assume that a nonsense mutation producing a truncated protein containing only 11 amino acids would be inactive. The unscheduled DNA synthesis and post-UV colonyforming ability of XP20BE is very low w16x. A potentially inactivating mutation was not detected in the mother’s allele but an RFLP analysis of reverse transcribed mRNA preparations revealed that both the mother and the patient possess an XPG allele that is either unstable or poorly expressed. It is possible that these analyses could have missed a second inactivating mutation from the coding portion of the mother’s allele and that the ‘unstable’ message itself does not produce a fully functional protein. Alternatively, the ‘unstable’ XPG message from the mother could be producing a low level of the functional XPG protein in the patient. The extremely rare incidence of XP group G disease Žonly 8 other patients reported w15,16,20–26x. may reflect the critical nature of this gene; perhaps suggesting that these patients can only survive because of residual XPG function. The cause of this latter defect is still uncertain because the poor expression of the mother’s allele could be due to mutations in almost any part of the XPG gene.
In addition to detecting four alterations within the XP-G pedigree, these analyses also uncovered an unusually large deletion event caused by abnormal splicing from the first to the fourteenth exons. At first glimpse it appeared that this defect might be related to the maternally inherited defect in the patient. However, sequencing data clearly indicated that the splicing error in the patient was a product derived primarily from the father’s message and that this product most likely represents a minor, but normal, error in the processing of the XPG gene. Analysis of the genomic structure around this region reveals that the splicing signals in the region around exon 13 and 14 do not obey strict consensus rules Žw35x, unpublished observations.. It is concluded that these non-consensus splice-site structures contribute to a naturally occurring abnormal splicing of this region. It has previously been noted that a minor fraction of the XPG pre mRNA is also abnormally spliced to cause a 55 bp deletion between positions bp 1078–1132 in the XPG gene of normal individuals w14x. It was suggested that ‘leaky’ splicing may contribute to the low abundance of functional XPG mRNA. It might also be argued that these abnormal splicing events might be a mechanism by which the level of this protein may be controlled in mammals. Although only 9 XP-G patients have been identified world wide w15,16,20–26x, this syndrome displays a wide array of phenotypes ranging from the typical sunlight-skin sensitivity of the XP syndrome to the more pleiotropic characteristics of the XPrCS syndrome including diminished growth and mental retardation Žsee discussion in w16x.. Recent studies with cells derived from Cockayne syndrome or XP-G patients have demonstrated that these cells are deficient in the preferential repair of bulky adducts and of ionizing radiation-induced DNA damage w27,28x. These results led to the hypothesis that the XPrCS dual phenotype is caused by defects in XPG gene that affect two functions: nucleotide excision repair and a more generalized transcription-associated repair function. In addition to the endonuclease activity, the XPG protein also has an apparent ‘loose association’ with the transcription factor TFIIH w29,30x. The coupling of nucleotide excision-repair activity to transcription in XP complementation groups B, and D has led to the implication that a defect in transcription may be related to other human
R.T. Okinaka et al.r Mutation Research 385 (1997) 107–114
syndromes that are associated with these repair defects, such as the XPrCS complex and patients with the syndrome of trichothiodystrophy Žsulfur-deficient brittle hair, photosensitivity, mental retardation and ichthyosis. without skin cancer w27,31–34x. To account for the pleiotropic nature of XP-G patients, this model would predict that certain XPG mutations would affect both transcription and repair while others would only alter the nucleotide excision-repair function. XP20BE cells were derived from a sun-sensitive boy who had severe symptoms of CS with mild XP-type pigmentation. The XP20BE patient had severe XPrCS syndrome with dwarfism, retinal degeneration, microcephaly and mental impairment. He died at age 6 years with marked cachexia Žweight 14.5 lb. without skin cancer. While the clinical phenotype of the patient accentuated the CS features, the cellular phenotype was typical of XP complementation group G with less than 5% of normal post-UV unscheduled DNA synthesis and very low post-UV cell survival w16x. The pedigree analysis of patient XP20BE indicates that the father’s inherited nonsense mutation at bp 31 would fit the criteria for the XPrCS dual phenotype since a non-leaky mutation at this position would almost certainly eliminate any XPG function. Although it is not yet evident whether the mother’s inherited, ‘unstable’ allele has any functional activity, one possibility may be that residual XPG gene function may not be sufficient to fully compensate for both NER and transcription-associated repair in this cell.
References w1x A. Sancar, Excision repair in mammalian cells, J. Biol. Chem. 270 Ž1995. 15915–15918. w2x J.J. Lin, A. Sancar, ŽA.BC excinuclease: the Escherichia coli nucleotide excision repair enzyme, Mol. Microbiol. 6 Ž1992. 2219–2224. w3x J.E. Cleaver, Defective repair replication of DNA in xeroderma pigmentosum, Nature 218 Ž1968. 652–656. w4x A. Sancar, Mechanisms of DNA excision repair, Science 266 Ž1994. 1954–1956. w5x D. Bootsma, J.H.J. Hoeijmakers, The molecular basis of nucleotide excision repair syndromes, Mutation Res. Fundam. Mol. Mech. Mutagen. 307 Ž1994. 15–23. w6x J.H.J. Hoeijmakers, Human nucleotide excision repair syndromes: molecular clues to unexpected intricacies, Eur. J. Cancer ŽA. 30A Ž1994. 1912–1921.
113
w7x J.E. Cleaver, K.H. Kraemer, Xeroderma pigmentosum and Cockayne syndrome, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle ŽEds.., The Metabolic and Molecular Bases of Inherited Disease, 7th ed., McGraw-Hill, New York, 1995, pp. 4393–4419. w8x A. Aboussekhra, M. Biggerstaff, M.K.K. Shivji, J.A. Vilpo, V. Moncollin, V.N. Podust, M. Protic, U. Hubscher, J.-M. ¨ Egly, R.D. Wood, Mammalian DNA nucleotide excision repair reconstituted with purified protein components, Cell 80 Ž1995. 859–868. w9x M.A. MacInnes, J.A. Dickson, R.R. Hernandez, D. Learmonth, G.Y. Lin, J.S. Mudgett, M.S. Park, S. Schauer, R.J. Reynolds, G.F. Strniste, J.Y. Yu, Human ERCC5 cDNAcosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of Saccharomyces cereÕisiae and Schizosaccharomyces pombe, Mol. Cell. Biol. 13 Ž1993. 6393–6402. w10x D. Scherly, T. Nouspikel, J. Corlet, C. Ucla, A. Bairoch, S.G. Clarkson, Complementation of the DNA repair defect in xeroderma pigmentosum group G cells by a human cDNA related to yeast RAD2, Nature 363 Ž1993. 182–185. w11x T. Shiomi, Y. Harada, T. Saito, N. Shiomi, Y. Okuno, M. Yamaizumi, An ERCC5 gene with homology to yeast RAD2 is involved in group G xeroderma pigmentosum, Mutation Res. 314 Ž1994. 167–175. w12x J.C. Huang, D.L. Svoboda, J.T. Reardon, A. Sancar, Human nucleotide excision nuclease removes thymine dimers from X DNA by incising the 22nd phosphodiester bond 5 and the X 6th phosphodiester bond 3 to the photodimer, Proc. Natl. Acad. Sci. USA 89 Ž1992. 3664–3668. w13x D.L. Svoboda, J.S. Taylor, J.E. Hearst, A. Sancar, DNA repair by eukaryotic nucleotide excision nuclease. Removal of thymine dimer and psoralen monoadduct by HeLa cell-free extract and of thymine dimer by Xenopus laevis oocytes, J. Biol. Chem. 268 Ž1993. 1931–1936. w14x T. Nouspikel, S.G. Clarkson, Mutations that disable the DNA repair gene XPG in a xeroderma pigmentosum group G patient, Hum. Mol. Genet. 3 Ž1994. 963–967. w15x P.G. Norris, J.L. Hawk, J.A. Avery, F. Giannelli, Xeroderma pigmentosum complementation group G-report of two cases, Br. J. Dermatol. 116 Ž1987. 861–866. w16x S.I. Moriwaki, M. Stefanini, A.R. Lehmann, J.H.J. Hoeijmakers, J.H. Robbins, I. Rapin, E. Botta, B. Tanganelli, W. Vermeulen, B.C. Broughton, K.H. Kraemer, DNA repair and ultraviolet mutagenesis in cells from a new patient with xeroderma pigmentosum group G and Cockayne syndrome resemble xeroderma pigmentosum cells, J. Invest. Dermatol. 107 Ž1996. 647–653. w17x W.M. Barnes, PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates, Proc. Natl. Acad. Sci. USA 91 Ž1994. 2216–2220. w18x J.L. Yang, V.M. Maher, J.J. McCormick, Amplification and direct nucleotide sequencing of cDNA from the lysate of low numbers of diploid human cells, Gene 83 Ž1989. 347–354. w19x R.T. Okinaka, S.L. Anzick, A. Oller, W.G. Thilly, Analysis of large X-ray-induced mutant populations by denaturing gradient gel electrophoresis, Radiat. Res. 135 Ž1993. 212– 221.
114
R.T. Okinaka et al.r Mutation Research 385 (1997) 107–114
w20x M.J. Cheesbrough, P.D.C. Kinmont, Xeroderma pigmentosum – a unique variant with neurological involvement, Br. J. Dermatol. 99 Ž1978. 61. w21x W. Keijzer, N.G. Jaspers, P.J. Abrahams, A.M. Taylor, C.F. Arlett, B. Zelle, H. Takebe, P.D. Kinmont, D. Bootsma, A seventh complementation group in excision-deficient xeroderma pigmentosum, Mutation Res. 62 Ž1979. 183–190. w22x C.F. Arlett, S.A. Harcourt, A.R. Lehmann, S. Stevens, W.A. Ferguson-Smith, W.N. Morley, Studies of a new case of xeroderma pigmentosum ŽXP3BR. from complementation group G with cellular sensitivity to ionizing radiation, Carcinogenesis 1 Ž1980. 745–751. w23x M. Ichihashi, Y. Fujiwara, Y. Uehara, A. Matsumoto, A mild form of xeroderma pigmentosum assigned to complementation group G and its repair heterogeneity, J. Invest. Dermatol. 85 Ž1985. 284–287. w24x J. Jaeken, H. Klocker, H. Schwaiger, R. Bellmann, M. Hirsch-Kauffmann, M. Schweiger, Clinical and biochemical studies in three patients with severe early infantile Cockayne syndrome, Hum. Genet. 83 Ž1989. 339–346. w25x W. Vermeulen, J. Jaeken, N.G. Jaspers, D. Bootsma, J.H. Hoeijmakers, Xeroderma pigmentosum complementation group G associated with Cockayne syndrome, Am. J. Hum. Genet. 53 Ž1993. 185–192. w26x B.C.K. Hamel, A. Raams, A.R. Schuitema-Dijkstra, P. Simons, I. Van der Burgt, N.G.J. Jaspers, W.J. Kleijer, Xeroderma pigmentosum–Cockayne syndrome complex: a further case, J. Med. Genet. 33 Ž1996. 607–610. w27x S.A. Leadon, P.K. Cooper, Preferential repair of ionizing radiation-induced damage in the transcribed strand of an active human gene is defective in Cockayne syndrome, Proc. Natl. Acad. Sci. USA 90 Ž1993. 10499–10503. w28x P.K. Cooper, S.A. Leadon, Defective repair of ionizing
w29x
w30x
w31x w32x
w33x
w34x
w35x
radiation damage in Cockayne’s syndrome and xeroderma pigmentosum group G, Ann. New York Acad. Sci. 726 Ž1994. 330–332. C.-H. Park, D. Mu, J.T. Reardon, A. Sancar, The general transcription-repair factor TFIIH is recruited to the excision repair complex by the XPA protein independent of the TFIIE transcription factor, J. Biol. Chem. 270 Ž1995. 4896–4902. D. Mu, C.-H. Park, T. Matsunaga, D.S. Hsu, J.T. Reardon, A. Sancar, Reconstitution of human DNA repair excision nuclease in a highly defined system, J. Biol. Chem. 270 Ž1995. 2415–2418. P.C. Hanawalt, Transcription-coupled repair and human disease, Science 266 Ž1994. 1957–1958. D. Bootsma, G. Weeda, W. Vermeulen, H. Van Vuuren, C. Troelstra, P. Van der Spek, J. Hoeijmakers, Nucleotide excision repair syndromes: molecular basis and clinical symptoms, Philos. Trans. R. Soc. Lond. B. 347 Ž1995. 75–81. K. Takayama, E.P. Salazar, B.C. Broughton, A.R. Lehmann, A. Sarasin, L.H. Thompson, C.A. Weber, Defects in the DNA repair and transcription gene ERCC2ŽXPD. in trichothiodystrophy, Am. J. Hum. Genet. 58 Ž1996. 263–270. B.C. Broughton, A.F. Thompson, S.A. Harcourt, W. Vermeulen, J.H. Hoeijmakers, E. Botta, M. Stefanini, M.D. King, C.A. Weber, J. Cole, Molecular and cellular analysis of the DNA repair defect in a patient in xeroderma pigmentosum complementation group D who has the clinical features of xeroderma pigmentosum and Cockayne syndrome, Am. J. Hum. Genet. 56 Ž1995. 167–174. D.L. Ludwig, J.S. Mudgett, M.S. Park, A.V. Perez-Castro, M.A. MacInnes, Molecular cloning and structural analysis of the functional mouse genomic XPG gene, Mammal. Genome 7 Ž1996. 644–649.