Novel mutations and polymorphisms in genes causing hereditary hemorrhagic telangiectasia

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant vascular disorder caused by mutations in Endoglin (ENG) or activin receptor-like kinase-1 (ALK1, ACVRL1) genes. We performed molecular characterization in clinically affected probands of 31 HHT families and detected a total of 28 different mutations in the two genes, including four shared by more than one family. Twelve mutations were identified in the ENG gene, six of which were novel and comprised two nonsense mutations in exons 6 and 8, deletions in exons 5 and 11, and splice site mutations in exon 12 and intron 8. Eleven of sixteen mutations identified in the ALK1 gene were novel single base pair substitutions in exons 4, 7, 8, and 9. We also describe the first de novo ALK1 mutation that causes a previously unreported c.1133C>A substitution of a highly conserved residue (p.P378H). The proband and his two daughters, who also carried the familial mutation, all suffered from gastrointestinal (GI) bleeding. In addition, we report seven newly identified polymorphisms and summarize all known ones in both genes.     

An Alu retrotransposition-mediated deletion of CHD7 in a patient with CHARGE syndrome

CHD7 mutations account for about 60-65% among more than 200 CHARGE syndrome cases. When rare whole gene deletion cases associated with chromosomal abnormalities are excluded, all mutations of CHD7 reported to date have been point mutations and small deletions and insertions, rather than exonic deletions. To test whether exonic deletions represent a common pathogenic mechanism, we assessed exon copy number by using a recently developed method, the multiplex PCR/liquid chromatography assay (MP/LC). Multiple exons were amplified using unlabeled primers, then separated by ion-pair reversed-phase high-performance liquid chromatography, and quantitated by fluorescence detection using a post-column intercalation dye under the premise that the relative peak intensities for each target directly reflect exon copy number. By using MP/LC, we identified one CHARGE syndrome patient who had a de novo deletion encompassing exons 8-12 among 13 classic CHARGE patients in whom screening by denaturing high-performance liquid chromatography (DHPLC) failed to identify point mutations and small insertions/deletions in CHD7. This is the first CHARGE patient who was documented to have exonic deletion of CHD7. The deletion closely recapitulated the Alu-mediated inactivation of the human CMP-N-acetylneuraminic acid hydroxylase gene (CMP-Neu5Ac hydroxylase), which is regarded as a novel molecular mechanism in the evolution from non-human primates to humans. As demonstrated in this study, MP/LC is a promising method for characterizing exonic deletions, which are largely left unexamined in most routine mutation analysis.     

Deletion and insertion in vivo somatic mutations in the hypoxanthine phosphoribosyltransferase (hprt) gene of human T-lymphocytes

Deletion and insertion mutations have been found to be a major component of the in vivo somatic mutation spectrum in the hypoxanthine phosphoribosyltransferase (hprt) gene of T-lymphocytes. In apopulation of 172 healthy people (average age, 34; mutant frequency, 10.3 × 10⁻⁶), deletion/in sertion mutations constituted 41% (89) of the 217independent mutations, the remainder being base substitutions. Mutations were identified by multiplex PCR assay of genomic DNA for exon regions, by sequencing cDNA, or sequencing genomic DNA. The deletion and insertion mutations were divided among ±1 to 2 basepair (bp) frameshifts (14%, 30), small deletions and insertions of 3–200 bps (13%, 28), large deletions of one or more exons (12%, 27), and complex events (2%, 4). Frameshift mutations were dominated by −1 bp deletions (21 of 30). Exon 3 contained five frameshift mutations in the run of 6 Gs, the only site in the coding region with multiple frameshift mutations, possibly caused by strand dislocation during replication. Both end points were sequenced for 23 of the 28 small deletions/insertions including two tandem duplication events in exon 6. More small deletions (8/28), pos sibly mediated by trinucleotide repeats, occurred in exon 2 than in the other exons. Large deletions included total gene deletions (6), exon 2 + 3 dele tions (4), and loss of multiple (9) and single exons (8) in genomic DNA. The diverse mutation spectrum indicates that multiple mechanisms operated at many different sequences and provides a resource for examination of deletion mutation. Environ. Mol. Mutagen. 30:371–384, 1997 © 1997 Wiley-Liss, Inc.     

Deletions Overlapping VCAN Exon 8 Are New Molecular Defects for Wagner Disease

Wagner disease is a rare nonsyndromic autosomal‐dominant vitreoretinopathy, associated with splice mutations specifically targeting VCAN exon 8. We report the extensive genetic analysis of two Wagner probands, previously found negative for disease‐associated splice mutations. Next‐generation sequencing (NGS), quantitative real‐time PCR, and long‐range PCR identified two deletions (3.4 and 10.5 kb) removing at least one exon–intron boundary of exon 8, and both correlating with an imbalance of VCAN mRNA isoforms. We showed that the 10.5‐kb deletion occurred de novo, causing somatic mosaicism in the proband's mother who had an unusually mild asymmetrical phenotype. Therefore, exon 8 deletions are novel VCAN genetic defects responsible for Wagner disease, and VCAN mosaic mutations may be involved in the pathogenesis of Wagner disease with attenuated phenotype. NGS is then an effective screening tool for genetic diagnosis of Wagner disease, improving the chance of identifying all disease‐causative variants as well as mosaic mutations in VCAN.     

Endoglin gene mutations and polymorphisms in Italian patients with hereditary haemorrhagic telangiectasia

Autosomal-dominant hereditary haemorrhagic telangiectasia (HHT) is a genetically heterogeneous disease caused by mutations in at least two different loci. We screened for mutations in four Italian families where segregation studies showed clear evidence of linkage to the endoglin (ENG) locus. In addition, one sporadic case and three patients with pulmonary arteriovenous malformations, belonging to small nuclear families unsuitable for linkage analysis, were included in the screening. The proband from each family was investigated using single-strand conformation polymorphism and heteroduplex analysis; potential variants were sequenced. Four novel and one previously reported mutation were detected, as well as three new polymorphisms. The novel mutations included deletions in exon 1 (patient 581/02), exon 5 (patient 780/01) and exon 7 (patient 700/01), and a C-->T229 substitution in exon 3 (patient 462/02). When analysing patient 700/01 and his affected daughter, we encountered a mutant ENG allele with two mutations--a deletion in exon 7 and a substitution in exon 12--which converts isoleucine 575 into threonine, in a non-conserved region. Both mutations were absent in the two healthy sons of the patient, while the polymorphic variant in exon 12 was present in his healthy father. These results and haplotype-segregation studies suggest that a de novo deletion had occurred in the gamete of paternal origin. For the first time the parental germline in which a de novo HHT mutation occurred has been identified.     

Mutations causing defective splicing in the human hprt gene.

Ten intron mutations and one exon mutation giving rise to defective splicing in the human gene for hypoxanthine phosphoribosyl transferase (hprt) in T-lymphocytes have been characterized. The splicing mutants were detected by PCR amplification of hprt cDNA and direct sequencing. Nine of the mutants showed skipping of whole exons or parts of exons in the cDNA, one mutant had an inclusion of an intron sequence into the cDNA, and one mutant showed both inclusion of an intron sequence and skipping of exons as well as a normal cDNA. Genomic PCR and direct sequencing of the splice sites involved showed one deletion of three base pairs and 10 different single base alterations to be responsible for these splice alterations. One mutation in the last base pair of exon 6 causing skipping of the entire exon 6 was found, whereas an identical mutation in the last base pair of exon 2 caused no aberrant splicing. It was also found that a deletion mutation in the pyrimidine rich stretch of the acceptor site of intron 7 caused skipping of the entire exon 8, whereas a base substitution in the last base of intron 7 caused exclusion of only the first 21 base pairs of exon 8 as a result of the activation of a cryptic acceptor site in exon 8. The results show that many different types of mutations at several different sites can cause splicing errors in the hprt gene and that the sequence differences between the splice sites influence the possible spectrum of mutations in each site.     

Novel genomic insertion-- deletion in MLH1: possible mechanistic role for non-homologous end-joining DNA repair

Hereditary non-polyposis colorectal cancer (HNPCC) is an inherited cancer syndrome caused by a defect in the mismatch repair pathway. The majority of HNPCC mutations have been detected in MLH1 and MSH2. Most reported mutations are substitutions, small insertions and deletions, but standard methods of mutation analysis do not detect large rearrangements. It is now established that large deletions, insertions and rearrangements account for a significant proportion of MLH1 and MSH2 mutations. We report an unusual rearrangement resulting in the deletion of exons 6, 7 and 8 of MLH1, with the retention of part of intron 6 and insertions of two nucleotides each flanking the retained sequence. The 349-bp-retained sequence is made up of two closely spaced Alu sequences. The mutation was initially detected by protein truncation test and cDNA sequencing. Multiplex ligation-dependent probe amplification confirmed the deletion of three exons. PCR and sequencing were used to characterize the breakpoint. Despite the high density of Alu elements in MLH1, there is no homology at the deletion breakpoints or insertion junctions in this case to suggest that homologous recombination has occurred. We propose a mechanism involving non-homologous end joining to explain the occurrence of this complex deletion.     

Molecular nature of spontaneous mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in chinese hamster ovary cells

The hypoxanthine-guanine phosphoribosyltransferase (hprt) locus has been widely used as a selectable genetic marker for studies of mammalian cell mutagenesis. We report here the spontaneous mutation spectrum at the hprt locus in 64 independently isolated mutants of Chinese hamster ovary (CHO) cells. All nine hprt exons were simultaneously analyzed via multiplex polymerase chain reaction (PCR) for rapid detection of gene deletions or insertions. Structural point mutations were identified by direct sequence analysis of the PCR amplified cDNA. The molecular nature of RNA splicing errors and insertions was analyzed by solid-phase direct exon sequencing. Single base substitutions were found in 24 mutants (38%), of which 21 were missense and 3 were nonsense mutations. Transversions were about twice as frequent as transitions. Fifteen mutants (23%) had deletions involving either intragenic small fragments (2), single exons (9), or multiple exons (4). The majority of deletion breakpoints (71%) were located in regions surrounding exons 4, 5, and 6. RNA splicing mutations were observed in 15 mutants (23%) and affected exons 3–8; most (6/15) resulted in the loss of exon 7. Two insertion mutants, one with a 209 bp insert in exon 4 and the other with a 88 bp insert accompanied by a 24 bp deletion in exon 6, represent novel mutations reported for the first time in spontaneous mutants of the mammalian hprt gene. © 1995 Wiley-Liss, Inc.     

Small deletions in the type II collagen triple helix produce Kniest dysplasia

Kniest dysplasia is a moderately severe type II collagenopathy, characterized by short trunk and limbs, kyphoscoliosis, midface hypoplasia, severe myopia, and hearing loss. Mutations in the gene that encodes type II collagen (COL2A1), the predominant protein of cartilage, have been identified in a number of individuals with Kniest dysplasia. All but two of these previously described mutations cause in-frame deletions in type II collagen, either by small deletions in the gene or splice site alterations. Furthermore, all but one of these mutations is located between exons 12 and 24 in the COL2A1 gene. We used heteroduplex analysis to identify sequence anomalies in five individuals with Kniest dysplasia. Sequencing of the index patients' genomic DNA identified four new dominant mutations in COL2A1 that result in Kniest dysplasia: a 21-bp deletion in exon 16, an 18-bp deletion in exon 19, and 4-bp deletions in the splice donor sites of introns 14 and 20. A previously described 28-bp deletion at the COL2A1 exon 12–intron 12 junction, deleting the splice donor site, was identified in the fifth case. The latter three mutations are predicted to result in exon skipping in the mRNA encoded from the mutant allele. These data suggest that Kniest dysplasia results from shorter type II collagen monomers, and support the hypothesis that alteration of a specific COL2A1 domain, which may span from exons 12 to 24, leads to the Kniest dysplasia phenotype. Am. J Med. Genet. 85:105–112, 1999. Published 1999 Wiley-Liss, Inc.     

Nonclassical splicing mutations in the coding and noncoding regions of the ATM Gene: maximum entropy estimates of splice junction strengths

Ataxia-telangiectasia (A-T) is an autosomal recessive neurological disorder caused by mutations in the ATM gene. Classical splicing mutations (type I) delete entire exons during pre-mRNA splicing. In this report, we describe nine examples of nonclassical splicing mutations in 12 A-T patients and compare cDNA changes to estimates of splice junction strengths based on maximum entropy modeling. These mutations fall into three categories: pseudoexon insertions (type II), single nucleotide changes within the exon (type III), and intronic changes that disrupt the conserved 3' splice sequence and lead to partial exon deletion (type IV). Four patients with a previously reported type II (pseudoexon) mutation all shared a common founder haplotype. Three patients with apparent missense or silent mutations actually had type III aberrant splicing and partial deletion of an exon. Five patients had type IV mutations that could have been misinterpreted as classical splicing mutations. Instead, their mutations disrupt a splice site and use another AG splice site located nearby within the exon; they lead to partial deletions at the beginning of exons. These nonclassical splicing mutations create frameshifts that result in premature termination codons. Without screening cDNA or using accurate models of splice site strength, the consequences of these genomic mutations cannot be reliably predicted. This may lead to further misinterpretation of genotype-phenotype correlations and may subsequently impact upon gene-based therapeutic approaches.     

Identification of 36 novel Jagged1 (JAG1) mutations in patients with Alagille syndrome

Alagille syndrome (AGS) is an autosomal dominant disorder characterized by five major symptoms: cholestasis, vertebral deformity, heart malformations, ocular defects and peculiar facial appearance. The previously described Jagged1 (JAG1) gene on chromosome 20p12 has been identified as being responsible for AGS. JAG1 encodes a transmembrane protein acting as ligand for the evolutionarily conserved Notch signaling pathway. Here we report 36 novel mutations in the JAG1 gene. We identified 12 novel deletions, 4 insertions, 8 missense, 7 nonsense and 5 splice site mutations. All mutations map to the sequence encoding the extracellular part of the Jagged1 protein. The mutations spread over the entire gene with slightly increased rates in exons 2 to 6 and exon 23 and 24. Eight novel missense mutations map to the Delta-Serrate-Lag2 (DSL) domain and adjacent sequences which are important for ligand-receptor interaction. Inheritance was determined in 27 families. Sixteen mutations (55%) were de novo and eleven mutations (45%) were transmitted. Altogether 226 different JAG1 mutations have been described in association with AGS, including our novel 36 mutations. AGS variants are spread over the entire gene with only a few mutations in exon 26. A relatively high number of mutations are clustered in exons 2 to 6. This sequence region shows high interspecies conservation and encodes the Notch receptor-binding region (DSL domain).     

Deletions in the 3′ Part of the NFIX Gene Including a Recurrent Alu‐Mediated Deletion of Exon 6 and 7 Account for Previously Unexplained Cases of Marshall–Smith Syndrome

Marshall–Smith syndrome (MSS) is a very rare malformation syndrome characterized by typical craniofacial anomalies, abnormal osseous maturation, developmental delay, failure to thrive, and respiratory difficulties. Mutations in the nuclear factor 1/X gene (NFIX) were recently identified as the cause of MSS. In our study cohort of 17 patients with a clinical diagnosis of MSS, conventional sequencing of NFIX revealed frameshift and splice‐site mutations in 10 individuals. Using multiplex ligation‐dependent probe amplification analysis, we identified a recurrent deletion of NFIX exon 6 and 7 in five individuals. We demonstrate this recurrent deletion is the product of a recombination between AluY elements located in intron 5 and 7. Two other patients had smaller deletions affecting exon 6. These findings show that MSS is a genetically homogeneous Mendelian disorder. RT‐PCR experiments with newly identified NFIX mutations including the recurrent exon 6 and 7 deletion confirmed previous findings indicating that MSS‐associated mutant mRNAs are not cleared by nonsense‐mediated mRNA decay. Predicted MSS‐associated mutant NFIX proteins consistently have a preserved DNA binding and dimerization domain, whereas they grossly vary in their C‐terminal portion. This is in line with the hypothesis that MSS‐associated mutations encode dysfunctional proteins that act in a dominant negative manner.     

Germ line MLH1 and MSH2 mutations in Taiwanese Lynch syndrome families: characterization of a founder genomic mutation in the MLH1 gene

This multicenter study evaluated the mutation spectrum and frequencies of the MLH1 and MSH2 genes and determined the occurrence of large genomic deletions in 93 unrelated Taiwanese families that fulfilled the Amsterdam criteria II by denaturing high-performance liquid chromatography analysis, DNA sequencing for aberrant chromatograms, and multiplex ligation-dependent probe amplification analysis. In total, 38 pathogenic mutations (10 large deletions and 28 point mutations or small deletion/insertions) in the MSH2 or MLH1 gene were identified in 61 of the 93 families (66%). Three of the 10 large deletions and 14 of the 28 point mutations or small insertions/deletions have not been reported elsewhere. Three mutations in the MLH1 gene, the MLH1 c.1846_1848delAAG (5 families), deletion exons 11–15 (4 unrelated families), and MLH1 c.793C>T (13 unrelated families), accounted for 35% of all cases with pathogenic mutations. Haplotype analysis indicated that mutant c.793C>T alleles were derived from two distinct common founders that might be inherited from a single ancestor of presumably Chinese origin. As a mutation detection strategy for Taiwanese Lynch syndrome patients, we recommend that diagnosis starts with screening for large genomic deletions and continues by screening for common mutations in exons 10 and 16 of the MLH1 gene prior to searching for small mutations in the remaining exons.     

Disease-associated mutations in conserved residues of ALK-1 kinase domain

Activin receptor-like kinase-1 (ALK-1), the gene mutated in HHT type 2 (HHT2), is a serine/threonine kinase receptor type I of the TGF-beta superfamily, specifically expressed on endothelial cells. We established an HHT2 genotype in 16 families and report nine novel mutations. These include insertions and deletions of single base pairs in exons 3, 8 and 9, as well as nonsense mutations in exons 4 and 8 of ALK-1, which would lead to premature truncation and unstable mRNA or protein. Three novel missense mutations were identified in exons 7 and 8 of the kinase domain. Five previously reported substitutions were also observed in the families analyzed. Our results bring to 36, the number of mutations associated with HHT2, and are mostly found in exons 8 and 3 followed by exons 4 and 7. To ascertain the potential functional implications of the missense mutations in the ALK-1 kinase domain, we generated a model based on the three-dimensional structure of the homologous ALK-5 kinase domain. Our data reveal that the 11 missense mutations modify residues conserved among type I receptors and alter the polarity, charge, hydrophobicity and/or size of the substituted amino-acid and likely lead to misfolded and nonfunctional proteins.     

Small deletions in the type II collagen triple helix produce kniest dysplasia.

Kniest dysplasia is a moderately severe type II collagenopathy, characterized by short trunk and limbs, kyphoscoliosis, midface hypoplasia, severe myopia, and hearing loss. Mutations in the gene that encodes type II collagen (COL2A1), the predominant protein of cartilage, have been identified in a number of individuals with Kniest dysplasia. All but two of these previously described mutations cause in-frame deletions in type II collagen, either by small deletions in the gene or splice site alterations. Furthermore, all but one of these mutations is located between exons 12 and 24 in the COL2A1 gene. We used heteroduplex analysis to identify sequence anomalies in five individuals with Kniest dysplasia. Sequencing of the index patients' genomic DNA identified four new dominant mutations in COL2A1 that result in Kniest dysplasia: a 21-bp deletion in exon 16, an 18-bp deletion in exon 19, and 4-bp deletions in the splice donor sites of introns 14 and 20. A previously described 28-bp deletion at the COL2A1 exon 12-intron 12 junction, deleting the splice donor site, was identified in the fifth case. The latter three mutations are predicted to result in exon skipping in the mRNA encoded from the mutant allele. These data suggest that Kniest dysplasia results from shorter type II collagen monomers, and support the hypothesis that alteration of a specific COL2A1 domain, which may span from exons 12 to 24, leads to the Kniest dysplasia phenotype.     

Hereditary hemorrhagic telangiectasia: two distinct ENG deletions in one family

Wooderchak W, Gedge F, McDonald M, Krautscheid P, Wang X, Malkiewicz J, Bukjiok CJ, Lewis T, Bayrak‐Toydemir P. Hereditary hemorrhagic telangiectasia: two distinct ENG deletions in one family. Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by aberrant vascular development. Mutations in endoglin (ENG) or activin A receptor type II‐like 1 (ACVRL1) account for around 90% of HHT patients, 10% of those are large deletions or duplications. We report here the first observation of two distinct, large ENG deletions segregating in one pedigree. An ENG exon 4–7 deletion was observed in a patient with HHT. This deletion was identified in several affected family members. However, some affected family members had an ENG exon 3 deletion instead. These deletions were detected by multiplex ligation‐dependent probe amplification and confirmed by mRNA sequencing and an oligo‐CGH array. Linkage analysis revealed that one individual with the exon 3 deletion inherited the same chromosome from his mother who has the exon 4–7 deletion. This finding has important clinical implications because it shows that targeted family‐specific mutation analysis for exon deletions could have led to the misdiagnosis of some affected family members.     

Rapid detection by fluorescent multiplex PCR of exon deletions and duplications in the C1 inhibitor gene of hereditary angioedema patients

Hereditary angioedema (HAE) is due to a variety of defects in the C1 inhibitor gene (C1NH gene), including approximately 20% of partial deletions/duplications whose boundaries are usually within Alu repeats. To ensure complete molecular characterization of C1 inhibitor deficiencies a fluorescent multiplex assay was constructed to amplify simultaneously five exons of C1NH and an exon of the BRCA1 gene. PCR protocols were optimized for these amplicons (size range between 300 and 700 bp). Forward and reverse chimeric primers that carry strand-specific 5' tags of 16 nucleotides were used to ensure similar levels of PCR products for each amplicon in the multiplex. Data were analyzed by superposing fluorescent profiles of test and control DNA and by visually comparing the normalized peak levels of corresponding amplicons, rather than by calculating the ratios of peak areas. Tests on a collection of known defects, including five different Alu-mediated deletions and a partial duplication have validated this approach. In a study of 19 sporadic cases of HAE, of which four had failed to reveal mutations upon screening all exons by fluorescent chemical cleavage, three de novo deletions were diagnosed by using this multiplex PCR approach: a deletion of exon 4, a deletion of exons 5 and 6, and an apparently complete gene deletion. Besides being suitable for the initial DNA screening of the C1NH gene in HAE patients prior to screening for point mutations, this method can be easily adapted to complex genes for the screening of rearrangements.     

Concurrent CIC mutations, IDH mutations, and 1p/19q loss distinguish oligodendrogliomas from other cancers

Oligodendroglioma is characterized by unique clinical, pathological, and genetic features. Recurrent losses of chromosomes 1p and 19q are strongly associated with this brain cancer but knowledge of the identity and function of the genes affected by these alterations is limited. We performed exome sequencing on a discovery set of 16 oligodendrogliomas with 1p/19q co-deletion to identify new molecular features at base-pair resolution. As anticipated, there was a high rate of IDH mutations: all cases had mutations in either IDH1 (14/16) or IDH2 (2/16). In addition, we discovered somatic mutations and insertions/deletions in the CIC gene on chromosome 19q13.2 in 13/16 tumours. These discovery set mutations were validated by deep sequencing of 13 additional tumours, which revealed seven others with CIC mutations, thus bringing the overall mutation rate in oligodendrogliomas in this study to 20/29 (69%). In contrast, deep sequencing of astrocytomas and oligoastrocytomas without 1p/19q loss revealed that CIC alterations were otherwise rare (1/60; 2%). Of the 21 non-synonymous somatic mutations in 20 CIC-mutant oligodendrogliomas, nine were in exon 5 within an annotated DNA-interacting domain and three were in exon 20 within an annotated protein-interacting domain. The remaining nine were found in other exons and frequently included truncations. CIC mutations were highly associated with oligodendroglioma histology, 1p/19q co-deletion, and IDH1/2 mutation (p < 0.001). Although we observed no differences in the clinical outcomes of CIC mutant versus wild-type tumours, in a background of 1p/19q co-deletion, hemizygous CIC mutations are likely important. We hypothesize that the mutant CIC on the single retained 19q allele is linked to the pathogenesis of oligodendrogliomas with IDH mutation. Our detailed study of genetic aberrations in oligodendroglioma suggests a functional interaction between CIC mutation, IDH1/2 mutation, and 1p/19q co-deletion.     

Nitric oxide donors induce large-scale deletion mutations in human lymphoblastoid cells: implications for mutations in T-lymphocytes from arthritis patients.

Rheumatoid arthritis (RA) is an inflammatory disease in which high levels of reactive nitrogen oxygen species (RNOS) may be present in the affected joints. RNOS are known to produce small-scale mutational events (transitions, transversions, small insertions, and small deletions) but the ability of these compounds to cause deletion of large segments of genomic DNA has not been previously determined. To address this question, a human lymphoblastoid cell line (WIL2-NS) was exposed to nitric oxide (NO)-donating drugs and hypoxanthine phosphoribosyltransferase (hprt)-negative clones were selected and analyzed by multiplex-PCR. Large-scale deletions accounted for 60-80% of hprt mutations arising in drug-treated cultures compared to 12% in untreated cultures (P-values of 0.006 and 0.0001, respectively, in two experiments). Deletion mutations in untreated cultures affected exon 9, whereas 75% of drug-induced deletion mutations affected exons 2, 3, and 9, and the remainder were very large, ranging from 26 to 1200 kbp. To compare this spectrum of NO-induced mutations in a lymphoblastoid line to that arising in vivo in arthritis patients, T-cells from RA patients, osteoarthritis (OA) patients, and controls were cloned and similarly analyzed. We previously showed that the overall frequency of Hprt mutant clones from patients is appreciably elevated compared to that of control subjects. Large-scale hprt deletions (0.5 to >26 kb) were detected in mutant T-cell clones from both RA and OA patients and also from control subjects. A total of 54 mutant clones from 16 RA patients and 19 mutant clones from 6 OA patients were studied. Of these, 6 clones (from 3 RA and 1 OA patient) had suffered large-scale deletions. A total of 9 control subjects were studied and 62 mutant clones were obtained. Of these, 19 had suffered large-scale deletions, arising in 7 of 9 control subjects. In conclusion, (1) RNOS are capable of inducing large-scale deletion mutations in a human lymphoblastoid cell line and (2) large-scale deletion mutations were found in 10-30% of T-cell clones from RA and OA patients and controls, which we hypothesize may be induced by RNOS.