Contrasting drug resistance phenotypes resulting from cytomegalovirus DNA polymerase mutations at the same exonuclease locus

BACKGROUND: Diverse mutations in the cytomegalovirus (CMV) DNA polymerase (pol) gene confer resistance to one or more of the antiviral drugs ganciclovir, foscarnet or cidofovir. The levels of resistance conferred by specific mutations are variable, ranging from insignificant resistance to triple-drug resistance. OBJECTIVES: Three pol mutations, I521T, P522A and P522L, detected in patients who received antiviral therapy for CMV infection, were studied by recombinant phenotyping to characterize their associated drug resistance. STUDY DESIGN: The individual mutations were transferred by homologous recombination into a reference CMV strain modified with a reporter gene and the drug concentrations required to reduce the reporter signal by 50% (IC50) were determined. RESULTS: The mutations I521T and P522A each conferred 3- to 4-fold increases in IC50 to both ganciclovir and cidofovir, while mutation P522L conferred no significant resistance to either drug. None of these mutations conferred foscarnet resistance. CONCLUSIONS: The resistance phenotypes of mutations I521T and P522A are as predicted from the known mutation P522S, but divergent results with P522L indicate that different amino acid substitutions at the same position may not have the same effect on drug resistance. New mutations must be individually validated for proper interpretation of genotypic resistance testing.     

Occurrence in vivo of sister chromatid exchanges at the same locus in successive cell divisions caused by nonrepairable lesions induced by gamma rays

The capacity of lesions induced by gamma radiation to produce sister chromatid exchanges (SCE) in successive divisions in mouse bone marrow cells in vivo was evaluated using a protocol for the three-way differentiation of sister chromatids. Evidence was obtained that exposure to gamma radiation induces DNA lesions that result in the formation of SCE at the same locus in two successive cell divisions. The relevance of this observation with respect to DNA repair and mutagenesis is discussed.     

Dominant and recessive forms of fibrochondrogenesis resulting from mutations at a second locus, COL11A2

Fibrochondrogenesis is a severe, recessively inherited skeletal dysplasia shown to result from mutations in the gene encoding the proα1(XI) chain of type XI collagen, COL11A1. The first of two cases reported here was the affected offspring of first cousins and sequence analysis excluded mutations in COL11A1. Consequently, whole-genome SNP genotyping was performed to identify blocks of homozygosity, identical-by-descent, wherein the disease locus would reside. COL11A1 was not within a region of homozygosity, further excluding it as the disease locus, but the gene encoding the proα2(XI) chain of type XI collagen, COL11A2, was located within a large region of homozygosity. Sequence analysis identified homozygosity for a splice donor mutation in intron 18. Exon trapping demonstrated that the mutation resulted in skipping of exon 18 and predicted deletion of 18 amino acids from the triple helical domain of the protein. In the second case, heterozygosity for a de novo 9?bp deletion in exon 40 of COL11A2 was identified, indicating that there are autosomal dominant forms of fibrochondrogenesis. These findings thus demonstrate that fibrochondrogenesis can result from either recessively or dominantly inherited mutations in COL11A2.     

Polymorphism at the HLA-E locus predates most HLA-A and -B polymorphism.

The extensive polymorphism of the classic class I antigens has been well described. In contrast, the nonclassic HLA antigens are distinguished by their low polymorphism. We examine here the HLA polymorphism of the HLA-E locus by examining the DNA sequence of cDNA from nine ethnically diverse individuals. From this analysis, we show that there is no polymorphism in the regions including exon 1 and from exon 4 to exon 8, the 3' untranslated exon. In exons 2 and 3, there are two base substitutions, one of which is at a replacement site and the other silent. The replacement substitution changes an arginine to a glycine at position 107, defining two alleles at the HLA-E locus. Using the PCR on exon 3 from genomic DNA and hybridization with oligonucleotide probes, we have examined 90 HLA-typed individuals to determine the relative frequency of the two alleles in the population and their association with the classical antigens. This analysis showed that these two alleles were present at nearly equal frequencies in the population. Surprisingly, both alleles were found in an essentially random association with all but one HLA-A and -B haplotype. The single exception was to the A1-B8 haplotype, which appeared to be linked to only one of the two alleles. One implication of this random association is that these HLA-E alleles may have existed before most of the HLA-A and B polymorphism. Thus, selection has maintained the HLA-E locus essentially unaltered during a time when considerable polymorphism was being selected for at the HLA-A and -B loci. This finding may also have important consequences in an unrelated bone marrow transplant, where it is predicted that 37% of HLA-A and -B matched donors are mismatched at the HLA-E locus.     

Classification of mutations at the HLA-A locus by use of the polymerase chain reaction

We investigated whether the polymerase chain reaction (PCR) could be used to determine the mechanism of mutation in lymphocyte clones mutated at the HLA-A locus. Three polymorphisms, at Factor XIIIA, D6S109, and intron 3 of the HLA-A gene, were used to study a series of clones previously characterised by Southern blotting (SB) at multiple loci on chromosome 6. For detection of loss of heterozygosity, the results of PCR and SB were concordant in 140 of 141 clones when polymorphism in the Factor XIIIA region was studied and in 144 of 145 clones when polymorphism in the HLA-A gene was studied. For classification of the mechanism of mutation, PCR and SB gave the same result in 88 of 92 clones (96%) when a combination of the HLA-A and Factor XIIIA polymorphisms was used and in 46 of 47 clones (98%) when a combination of the HLA-A and D6S109 polymorphisms was used. The results indicate that PCR provides a simple and reliable method for categorising mutations at the HLA-A locus as arising from mitotic recombination, deletion, or from presumptive minor changes within the gene. Rare events such as gene conversion, nondisjunction, or large deletions extending to the telomere will be misclassified. However, such events are rare for mutations at this locus. © 1993 Wiley-Liss, Inc.     

Homozygous and compound heterozygous mutations at the Werner syndrome locus

The Werner syndrome (WS) is a rare autosomal recessive progeroid disorder. The Werner syndrome gene (WRN) has recently been identified as a member of the helicase family. Four distinct mutations were previously reported in three Japanese and one Syrian WS pedigrees. The latter mutation was originally described as a 4 bp deletion spanning a spliced junction. It is now shown that this mutation results in a 4 bp deletion at the beginning of an exon. Nine new WRN mutations in 10 additional WS patients, both Japanese and Caucasian, are described. These include three compound heterozygotes (one Japanese and two Caucasian). The new mutations are located all across the coding region.      

The smallest teeth in the world are caused by mutations in the PCNT gene

We report a follow up study on two MOPD II Thai families with severe dental anomalies and hypoplastic alveolar bone. Striking dental anomalies comprise severe microdontia, opalescent and abnormally shaped teeth, and rootless molars. As a result of severe hypoplastic alveolar bone, most permanent teeth have been lost. Mutation analysis of PCNT revealed 2 novel mutations (p.Lys3154del and p.Glu1154X) and a recurrent mutation (p.Pro1923X). Teeth of the patient who carried a homozygous novel mutation of p.Glu1154X are probably the smallest ever reported. The sizes of the mandibular permanent incisors and all premolars were approximately 2-2.5 mm, mesiodistally. All previously reported, PCNT mutations have been described to cause premature truncation of the pericentrin protein. p.Lys3154del mutation was unique as it was pathogenic as a result of missing only a single amino acid. In situ hybridization of Pcnt shows its expression in the epithelium and mesenchyme during early stages of rodent tooth development. It is evident that PCNT has crucial role in tooth development. The permanent dentition is more severely affected than the one. This implies that PCNT appears to have more role in the development of the permanent dentition. As pericentrin is a critical centrosomal protein, the dental phenotype found in MOPD II patients is postulated to be the consequence of loss of microtubule integrity which leads to defective centrosome function.     

Two distinct phenotypes caused by two different missense mutations in the same codon of the VHL gene.

We have identified a family segregating von Hippel-Lindau (VHL) disease with a previously unreported T547A mutation in exon 1 of the VHL gene that causes a Tyr112 to Asn missense alteration in the protein. The mutation was identified by nucleotide sequencing and confirmed by restriction enzyme digestion. The mutation cosegregated with the disease in all five tested affected individuals from the extended family. The family consists of more than 100 at-risk individuals over seven generations. To date, we have identified 13 affected individuals of whom seven have had renal cell carcinoma and one has had a pheochromocytoma. No other case of a neuroendocrine tumor of the pancreas or adrenal gland (pheochromocytoma) was found or recognized retrospectively. Other manifestations in this family include retinal angioma and hemangioblastoma of the central nervous system. We also found the T547A mutation in three asymptomatic members of the family, ages 12, 19, and 20. Another mutation, T547C, which causes Tyr112 to His, has been seen at the same position and has been associated with VHL type 2A (pheochromocytoma, but no renal cell carcinoma) in two families with a total of 22 affected individuals [Chen F, Slife L, Kishida T, Mulvihill J, Tisherman SE, Zbar B, 1996: J Med Genet 33:716-717]. Thus, different amino acid changes at the same position can cause very distinct clinical phenotypes. It will be interesting to elucidate the functional differences that underlie the different phenotypes.     

Machado Joseph disease maps to the same region of chromosome 14 as the spinocerebellar ataxia type 3 locus.

Machado Joseph disease (MJD) is an autosomal dominantly inherited neuro-degenerative disorder primarily affecting the motor system. It can be divided into three phenotypes based on the variable combination of a range of clinical symptoms including pyramidal and extra-pyramidal features, cerebellar deficits, and distal muscle atrophy. MJD is thought to be caused by mutation of a single gene which has recently been mapped, using genetic linkage analysis, to a 29 cM region on chromosome 14q24.3-q32 in five Japanese families. A second disorder, spinocerebellar ataxia type 3 (SCA3), which has clinical symptoms similar to MJD, has also been linked to the same region of chromosome 14q in two French families. In order to narrow down the region of chromosome 14 which contains the MJD locus and to determine if this region overlaps with the predisposing locus for SCA3, we have performed genetic linkage analysis in seven MJD families, six of Portuguese/Azorean origin and one of Brazilian origin, using nine microsatellite markers mapped to 14q24.3-q32. Our results localise the MJD locus in these families to an 11 cM interval flanked by the markers D14S68 and AFM343vf1. In addition we show that this 11 cM interval maps within the 15 cM interval containing the SCA3 locus, suggesting that these diseases are allelic.     

Assembly and iron-binding properties of human frataxin, the protein deficient in Friedreich ataxia

Friedreich ataxia (FRDA) is an autosomal recessive degenerative disease caused by a deficiency of frataxin, a conserved mitochondrial protein of unknown function. Mitochondrial iron accumulation, loss of iron-sulfur cluster-containing enzymes and increased oxidative damage occur in yeast and mouse frataxin-depleted mutants as well as tissues and cell lines from FRDA patients, suggesting that frataxin may be involved in export of iron from the mitochondria, synthesis of iron-sulfur clusters and/or protection from oxidative damage. We have previously shown that yeast frataxin has structural and functional features of an iron storage protein. In this study we have investigated the function of human frataxin in Escherichia coli and Saccharomyces cerevisiae. When expressed in E.coli, the mature form of human frataxin assembles into a stable homopolymer that can bind approximately 10 atoms of iron per molecule of frataxin. The iron-loaded homopolymer can be detected on non-denaturing gels by either protein or iron staining demonstrating a stable association between frataxin and iron. As analyzed by gel filtration and electron microscopy, the homopolymer consists of globular particles of approximately 1 MDa and ordered rod-shaped polymers of these particles that accumulate small electron-dense cores. When the human frataxin precursor is expressed in S.cerevisiae, the mitochondrially generated mature form is separated by gel filtration into monomer and a high molecular weight pool of >600 kDa. A high molecular weight pool of frataxin is also present in mouse heart indicating that frataxin can assemble under native conditions. In radiolabeled yeast cells, human frataxin is recovered by immunoprecipitation with approximately five atoms of (55)Fe bound per molecule. These findings suggest that FRDA results from decreased mitochondrial iron storage due to frataxin deficiency which may impair iron metabolism, promote oxidative damage and lead to progressive iron accumulation.     

Induction of mutations at the thymidine kinase locus in CHO cells by restriction endonucleases

Induced mutation frequencies were measured at the tk locus (encoding for the enzyme thymidine kinase) following treatment of Chinese hamster ovary cells (CHO KI) with two restriction endonucleases (REs), PvuII and EcoRI, which generate 'blunt-ended' and 'cohesive-ended' DNA double-strand breaks (dsb), respectively. Electroporation was used to introduce these enzymes into the cells. Restriction endonucleases generating blunt-ended dsb have been shown to mimic the action of ionising radiation in causing chromosome aberrations, cell killing, mutations and oncogenic transformation. Here we show that the tk locus, PvuII induced an approximately 11-fold higher mutation frequency than EcoRI at the same enzyme concentrations. There are four PvuII and six EcoRI restriction sites in the Chinese hamster thymidine kinase gene. Hence the higher mutation induction by PvuII, despite the lower number of restriction sites than EcoRI in the tk gene, suggests that blunt-ended dsb represent more effective and critical mutagenic DNA lesions than the cohesive-ended type. In this respect, our results are similar to those we obtained previously for chromosomal aberrations and for cell killing. Results from the present study suggest that mutations could arise from unrepaired or misrepaired dsb possibly via induction of chromosomal deletions or stable exchanges between chromosomes.     

Somatic sequence variation at the Friedreich ataxia locus includes complete contraction of the expanded GAA triplet repeat, significant length variation in serially passaged lymphoblasts and enhanced mutagenesis in the flanking sequence.

The vast majority of Friedreich ataxia patients are homozygous for large GAA triplet repeat expansions in intron 1 of the X25 gene. Instability of the expanded GAA repeat was examined in 23 chromosomes bearing 97-1250 triplets in lymphoblastoid cell lines passaged 20-39 times. Southern analyses revealed 18 events of significant changes in length ranging from 69 to 633 triplets, wherein the de novo allele gradually replaced the original over 1-6 passages. Contractions and expansions occurred with equal frequency and magnitude. This behavior is unique in comparison with other large, non-coding triplet repeat expansions [(CGG)(n)and (CTG)(n)] which remain relatively stable under similar conditions. We also report a rare patient who, having inherited two expanded alleles, showed evidence of contracted GAA repeats ranging from nine to 29 triplets in DNA from two independent peripheral blood samples. The GAA triplet repeat is known to adopt a triplex structure, and triplexes in transcribed templates cause enhanced mutagenesis. The poly(A) tract and a 135 bp sequence, both situated immediately upstream of the GAA triplet repeat, were therefore examined for somatic mutations. The poly(A) tract showed enhanced instability when in cis with the GAA expansion. The 135 bp upstream sequence was found to harbor a 3-fold excess of point mutations in DNA derived from individuals homozygous for the GAA triplet repeat expansion compared with normal controls. These data are likely to have important mechanistic and clinical implications.     

CpG hotspot mutations at the LDL receptor locus are a frequent cause of familial hypercholesterolemia among South African Indians

Mutation analysis of genomic DNA samples obtained from seven unrelated South African Indians with familial hypercholesterolaemia (FH) revealed two novel and two recurrent missense mutations in the low density lipoprotein receptor (LDLR) gene. The novel mutations are transversions of C to G and A to T at nucleotide positions 1215 (N384K) and 2356 (S765C), respectively. The known mutations were detected in CpG dinucleotides at bases 661 and 682, respectively, in the mutation-rich exon 4 of the LDLR gene. Mutation D200Y was found in a single FH family, while mutation E207K was detected in two apparently unrelated Indian families on a new mutual haplotype. Analysis of published mutations including our new data has shown that more than 50% of the different LDLR gene mutations identified to date in South African Indians occur at CpG hotspots.     

Clinical report: Two patients with atelosteogenesis type I caused by missense mutations affecting the same FLNB residue

We present two patients with Atelosteogenesis Type I (AO type I) caused by two novel Filamin B (FLNB) mutations affecting the same FLNB residue: c.542G > A, predicting p.Gly181Asp and c.542G > C, predicting p.Gly181Arg. Both children had typical manifestations of AO type I, with severe rhizomelic shortening of the extremities, limited elbow and knee extension with mild webbing, pectus excavatum, broad thumbs with brachydactyly that was most marked for digits 3–5, dislocated hips and bilateral talipes equinovarus. Facial features included proptosis, hypertelorism, downslanting palpebral fissures, cleft palate, and retromicrognathia. The clinical course of one child was influenced by airway instability and bronchopulmonary dysplasia that complicated intubation and prevented separation from ventilator support. Respiratory insufficiency with tracheal hypoplasia, laryngeal stenosis, and pulmonary hypoplasia have all been described in patients with AO type I and we conclude that compromised pulmonary function is a major contributor to morbidity and mortality in this condition. © 2013 Wiley Periodicals, Inc.     

DFNA8/12 caused by TECTA mutations is the most identified subtype of nonsyndromic autosomal dominant hearing loss

The prevalence of DFNA8/DFNA12 (DFNA8/12), a type of autosomal dominant nonsyndromic hearing loss (ADNSHL), is unknown as comprehensive population-based genetic screening has not been conducted. We therefore completed unbiased screening for TECTA mutations in a Spanish cohort of 372 probands from ADNSHL families. Three additional families (Spanish, Belgian, and English) known to be linked to DFNA8/12 were also included in the screening. In an additional cohort of 835 American ADNSHL families, we preselected 73 probands for TECTA screening based on audiometric data. In aggregate, we identified 23 TECTA mutations in this process. Remarkably, 20 of these mutations are novel, more than doubling the number of reported TECTA ADNSHL mutations from 13 to 33. Mutations lie in all domains of the α-tectorin protein, including those for the first time identified in the entactin domain, as well as the vWFD1, vWFD2, and vWFD3 repeats, and the D1-D2 and TIL2 connectors. Although the majority are private mutations, four of them-p.Cys1036Tyr, p.Cys1837Gly, p.Thr1866Met, and p.Arg1890Cys-were observed in more than one unrelated family. For two of these mutations founder effects were also confirmed. Our data validate previously observed genotype-phenotype correlations in DFNA8/12 and introduce new correlations. Specifically, mutations in the N-terminal region of α-tectorin (entactin domain, vWFD1, and vWFD2) lead to mid-frequency NSHL, a phenotype previously associated only with mutations in the ZP domain. Collectively, our results indicate that DFNA8/12 hearing loss is a frequent type of ADNSHL.     

The molecular nature of mutations induced by adriamycin at the hprt locus of V79 cells

Adriamycin (AM), a widely used chemotherapeutic drug, induceda broad spectrum of gene mutations at the hprt locus of V79cells. The frequency and distribution of AM-induced deletionswas analyzed with multiplex polymerase chain reaction in twoV79 cell lines, which differed considerably in their spontaneousdeletion frequency. Among AM-induced mutants, deletions predominatedin both cell lines. Apart from total deletions of the hprt gene,partial deletions were found which were distributed all overthe hprt gene with breakpoints in nearly all introns. Underthe same experimental conditions, chromosome aberrations wereinduced by AM which mainly represented chromatid-type aberrations.Neither the induction of gene mutations nor the induction ofchromosome aberrations was enhanced by the repair inhibitor3-aminobenzamide. These results are discussed in the contextwith our earlier findings on bleomycin-induced mutations andit is suggested that at least two mechanisms lead to the formationof gene deletions. One of them seems to be associated with amisrepair process of frank DNA double-strand breaks and relatedto chromosome aberrations while the other is not. ¹To whom correspondence should be addressed