Molecular basis of group A xeroderma pigmentosum: A missense mutation and two deletions located in a zinc finger consensus sequence of the XPAC gene

Summary The molecular basis of group A xeroderma pigmentosum (XP) was investigated, and 3 mutations located in a zinc finger consensus sequence (nucleotide 313–387) of the XP group A complementing (XPAC) gene were identified in 2 Caucasian patients GM2990 and GM2009 who had typical symptoms of group A XP. The first mutation was a C deletion at nucleotide 374. Patient GM2990 was a homozygote for this mutation. The second mutation was a 5-bp deletion (CTTAT) at nucleotides 349–353. The third mutation was a G to T transversion at nucleotide 323 that alters the Cys-108 codon (TGT) to a Phe codon (TTT). Patient GM2009 was a compound heterozygote for the 5-bp deletion and the missense mutation. Both deletions introduce frameshifts with premature translation terminations resulting in instability of the XPAC mRNA and disruption of the putative zinc finger domain of the XPAC protein. The missense mutation also predicts disruption of the zinc finger domain of the XPAC protein. The expression study showed that the missense mutation does indeed causes loss of repair activity of the XPAC protein. We conclude that these 3 mutations are responsible for group A XP.     

Three nonsense mutations responsible for group A xeroderma pigmentosum.

The molecular basis of xeroderma pigmentosum (XP) group A was studied and 3 nonsense mutations of the XP-A complementing gene (XPAC) were identified. One was a nucleotide transition altering the Arg-228 codon (CGA) to a nonsense codon (TGA). This transition creates a new cleavage site for the restriction endonuclease HphI. Of 21 unrelated Japanese XP-A patients examined, 1 (XP39OS) was a homozygote for this mutation and 3 were compound heterozygotes for this mutation and for the splicing mutation of intron 3 reported previously which is the most common mutation in Japanese patients and creates a new cleavage site for the restriction endonuclease AlwNI. The second mutation was a nucleotide transition altering the Arg-207 codon (CGA) to a nonsense codon (TGA). A Palestinian patient (XP12RO) who had severe symptoms of XP was homozygous for this mutation. The third mutation was a nucleotide transversion altering the Tyr-116 codon (TAT) to a nonsense codon (TAA). This transversion creates a new cleavage site for the restriction endonuclease MseI. Of the Japanese patients, 2 with severe clinical symptoms had this mutant allele. One was a compound heterozygote for this mutation and for the splicing mutation, and the other was heterozygous for this mutation and homozygous for the splicing mutation. Although most XP-A patients such as XP12RO have severe skin symptoms and neurological abnormalities of the de Sanctis-Cacchione syndrome, patient XP39OS was an atypical XP-A patient who had mild skin symptoms and minimal neurological abnormalities. Our results suggest that the clinical heterogeneity in XP-A is due to different mutations in the XPAC gene. Moreover, our data indicate that almost all Japanese cases of XP-A are caused by one or more of the 3 mutations, i.e., the splicing mutation of intron 3 and the 2 nonsense mutations of codons 116 and 228. Therefore, by restriction fragment length polymorphism analysis of PCR-amplified DNA sequences using the 3 restriction enzymes described above, rapid and reliable diagnosis of XP-A can be achieved in almost all Japanese subjects including prenatal cases and carriers.     

Novel mutations of endothelin-B receptor gene in Pakistani patients with Waardenburg syndrome

Mutations in EDNRB gene have been reported to cause Waardenburg-Shah syndrome (WS4) in humans. We investigated 17 patients with WS4 for identification of mutations in EDNRB gene using PCR and direct sequencing technique. Four genomic mutations were detected in four patients; a G to C transversion in codon 335 (S335C) in exon 5 and a transition of T to C in codon (S361L) in exon 5, a transition of A to G in codon 277 (L277L) in exon 4, a non coding transversion of T to A at −30 nucleotide position of exon 5. None of these mutations were found in controls. One of the patients harbored two novel mutations (S335C, S361L) in exon 5 and one in Intronic region (−30exon5 A>G). All of the mutations were homozygous and novel except the mutation observed in exon 4. In this study, we have identified 3 novel mutations in EDNRB gene associated with WS4 in Pakistani patients.     

High prevalence of the point mutation in exon 6 of the xeroderma pigmentosum group A-complementing (XPAC) gene in xeroderma pigmentosum group A patients in Tunisia.

Xeroderma pigmentosum (XP) patients in Tunisia who belong to the genetic complementation group A (XPA) have milder skin symptoms than do Japanese XPA patients. Such difference in the clinical features might be caused by the difference in the site of mutation in the XP A-complementing (XPAC) gene. The purpose of this study is to identify the genetic alterations in the XPAC gene in the Tunisian XPA patients and to investigate the relationship between the clinical symptoms and the genetic alterations. Three sites of mutation in the XPAC gene have been identified in the Japanese XPA patients, and about 85% of them have a G-->C point mutation at the splicing acceptor site of intron 3. We found that six (86%) of seven Tunisian XPA patients had a nonsense mutation in codon 228 in exon 6, because of a CGA-->TGA point mutation, which can be detected by the HphI RFLP. This type of mutation is the same as those found in two Japanese XPA patients with mild clinical symptoms. Milder skin symptoms in the XPA patients in Tunisia than in those in Japan, despite mostly sunny weather and the unsatisfactory sun protection in Tunisia, should be due to the difference in the mutation site.     

Three novel mutations causing a truncated protein within the RP2 gene in Italian families with X-linked retinitis pigmentosa

X-linked retinitis pigmentosa (XLRP) results from mutations in a number of loci, including RP2 at Xp11.3, and RP3 at Xp21.1. RP2 and RP3 genes have been identified by positional cloning. RP2 mutations are found in about 10% of XLRP patients. We performed a mutational screening of RP2 gene inpatients belonging to seven unrelated families in linkage with the RP2 locus. SSCP analysis detected three conformation variants, within exon 2 and 3. Direct sequencing of exon 2, disclosed a G-->A transition at nucleotide 449 (W150X), and a G-->T transversion in position 547 (E183X). Sequence analysis of exon 3 variant revealed an insertion (853/854insG), leading to a frameshift. In this patient, we detected an additional sequence alteration (A-->G at nucleotide 848, E283G). Each mutation was co-segregating with the disease in the affected family members available for the study. These mutations are expected to introduce a stop codon within the RP2 coding sequence probably resulting in a truncated or unstable protein.     

Novel compound heterozygous mutations for lipoprotein lipase deficiency. A G-to-T transversion at the first position of exon 5 causing G154V missense mutation and a 5' splice site mutation of intron 8.

We systematically investigated the molecular defects causing a primary LPL deficiency in a Japanese male infant (patient DI) with fasting hyperchylomicronemia (type I hyperlipoproteinemia) and in his parents. Patient DI had neither LPL activity nor immunoreactive LPL mass in the pre- and post-heparin plasma. The patient was a compound heterozygote for novel mutations consisting of a G-to-T transversion at the first nucleotide of exon 5 [+1 position of 3' acceptor splice site (3'-ass) of intron 4] and a T-to-C transition in the invariant GT at position +2 of the 5' donor splice site (5'-dss) of intron 8 (Int8/5'-dss/t(+2)c). The G-to-T transversion, although affecting the 11 nucleotide of the 3'-consensus acceptor splice site, resulted in a substitution of Gly(154) to Val (G154V; GG(716)C(-->)GTC). The mutant G154V LPL expressed in COS-1 cells was catalytically inactive and hardly released from the cells by heparin. The Int8/5'-dss/t(+2)c mutation inactivated the authentic 5' splice site of intron 8 and led to the utilization of a cryptic 5'-dss in exon 8 as an alternative splice site 133 basepairs upstream from the authentic splice site, thereby causing joining of a part of exon 8 to exon 9 with skipping of a 134-bp fragment of exon 8 and intron 8. These additional mutations in the consensus sequences of the 3' and 5' splice sites might be useful for better understanding the factors that are involved in splice site selection in vivo.     

A missense mutation (Trp86----Arg) in exon 3 of the lipoprotein lipase gene: a cause of familial chylomicronemia.

We have investigated a patient of English ancestry with familial chylomicronemia caused by lipoprotein lipase (LPL) deficiency. DNA sequence analysis of all exons and intron-exon boundaries of the LPL gene identified two single-base mutations, a T----C transition for codon 86 (TGG) at nucleotide 511, resulting in a Trp86----Arg substitution, and a C----T transition at nucleotide 571, involving the codon CAG encoding Gln106 and producing Gln106----Stop, a mutation described by Emi et al. The functional significance of the two mutations was confirmed by in vitro expression and enzyme activity assays of the mutant LPL. Linkage analysis established that the patient is a compound heterozygote for the two mutations. The Trp86----Arg mutation in exon 3 is the first natural mutation identified outside exons 4-6, which encompass the catalytic triad residues.     

Comorbidity of GJB2 and WFS1 mutations in one family

It is rarely reported that two distinct genetic mutations affecting hearing have been found in one family. We report on a family exhibiting comorbid mutation of GJB2 and WFS1. A four-generation Japanese family with autosomal dominant sensorineural hearing loss was studied. In 7 of the 24 family members, audiometric evaluations and genetic analysis were performed. We detected A-to-C nucleotide transversion (c.2576G>C) in exon 8 of WFS1 that was predicted to result in an arginine-to-proline substitution at codon 859 (R859P), G-to-A transition (c.109G>A) in exon 2 of GJB2 that was predicted to result in a valine-to-isoleucine substitution at codon 37 (V37I), and C-to-T transition (c.427C>T) in exon 2 of GJB2 that was predicted to result in an arginine-to-tryptophan substitution at codon 143 (R143W). Two individuals who had heterozygosity of GJB2 mutations and heterozygosity of WFS1 mutations showed low-frequency hearing loss. One individual who had homozygosity of GJB2 mutation without WFS1 mutation had moderate, gradual high tone hearing loss. On the other hand, a moderate flat loss configuration was seen in one individual who had compound heterozygosity of GJB2 and heterozygosity of WFS1 mutations. Our results indicate that the individual who has both GJB2 and WFS1 mutations can show GJB2 phenotype.     

Novelty of Axin 2 and lack of Axin 1 gene mutation in colorectal cancer: a study in Kashmiri population

Colorectal cancer is (CRC) one of the leading causes of mortality and morbidity. Various genetic factors have been reported to be involved in the development of colorectal cancers including Axin gene. Axin, a major scaffold protein, plays an important role in various bio signaling pathways. We aim to study mutational pattern of Axin gene in colorectal cancer patients of Kashmiri population. The paired tumor and adjacent normal tissue specimens of 50 consecutive patients with CRC were used in our study. The DNA preparations were evaluated for the occurrence of Axin 1 and Axin 2 gene mutations by direct DNA sequencing. We analyzed exon 1a, 1b, 1c, 2, 4, 6, and 10 of Axin 1 and exon 7 of Axin 2. In this study, we found a novel mutation of G>T (GCT>TCT) transversion in exon 7 of Axin 2 gene at codon G695T (p.alanine >?serine) at a frequency of 6% (3/50). In the same exon of Axin 2 gene a single nucleotide polymorphism (SNP) was detected in codon L688L (CCT>CTT) at a frequency of 36% (18/50). In exon 1c of Axin 1 a SNP was detected at codon D726D (GAT>GAC) at a frequency of 62.5% (31/50). Both the SNPs were synonymous hence do not lead to change of amino acid. Although Axin 1 and Axin 2 gene mutations have been found to be involved in the development of colorectal cancers, it seems to be a relatively rare event in Kashmiri population. However, an interesting finding of this study is the novelty of Axin 2 gene mutations which may be a predisposing factor in ethnic Kashmiri population to CRC.     

Novel splice, missense, and nonsense mutations in the fumarylacetoacetase gene causing tyrosinemia type 1.

In six unrelated patients with hereditary tyrosinemia type 1 (HT1), three different disease-causing mutations were found by DNA sequencing. Two Pakistani patients, with acute and intermediate forms of HT1, were homozygous for a G192-->T mutation in the last nucleotide of exon 2. This caused aberrant splicing with partial intron 2 retention and premature termination. Three Turkish patients with chronic and intermediate forms of HT1 were homozygous for an A698-->T mutation substituting aspartic acid 233 with valine. A Norwegian patient with an intermediate clinical phenotype was heterozygous for G786-->A, introducing a TGA stop codon for Trp262 (W262X). Site-directed mutagenesis and expression in a rabbit reticulocyte lysate system demonstrated that the nonsense and missense mutations abolished fumarylacetoacetase activity and gave reduced amounts of a truncated and a full-length protein, respectively. Simple tests were established to identify the three mutations by restriction digestion of PCR-amplified genomic DNA. Among 30 additional HT1 patients investigated, 2 were found to be homozygous and 1 heterozygous for G192-->T. Two other patients were homozygous and one was heterozygous for W262X.     

Predisposition for cysteine substitutions in the immunoglobulin-like chain of FGFR2 in Crouzon syndrome

Four cases of Crouzon syndrome, one familial and three sporadic, were investigated for mutations in exon B of the fibroblast growth factor receptor 2 (FGFR2) gene. In the familial case, a mutation was found at codon 340 that exchanged tyrosine for histidine. Mutations at codon 342, detected in the three sporadic cases, replaced a cysteine by another amino acid. While three of the mutations have been described before, the fourth mutation, a CG transversion at codon 342 in one of the sporadic cases, has not been recognized previously. Compilation of all exon B mutations in Crouzon syndrome described to date revealed that 6 of the 8 sporadic and 2 of the 9 familial cases have mutations in codon 342. These mutations caused the substitution of cysteine for another amino acid. Given that a mutation in codon 342 was found in 8 out of 17 cases and that in 9 cases the mutation occurred at five additional positions, codon 342 of exon B of the FGFR2 gene may be predisposed to mutations in Crouzon syndrome.     

Influence of uvrB and pKM101 on the spectrum of spontaneous, UV- and gamma-ray-induced base substitutions that revert hisG46 in Salmonella typhimurium

Oligonucleotide probes were used to identify base substitutions in 1089 revertants of hisG46 in Salmonella typhimurium that arose spontaneously or following irradiation with UV- or gamma-rays. The hisG46 allele, carrying a mutant CCC codon (Pro) in place of the wild-type codon CTC (Leu69) reverted via 6 distinguishable mutational events--C to T transitions at codon sites 1 or 2, C to A or C to G transversions at codon site 1, C to A at codon site 2, and an extragenic suppressor mutation. The distribution of hisG46 revertants differed among treatments and was influenced by the DNA-repair capacity of the bacteria. Plasmid pKM101 enhanced the frequencies of both spontaneous and induced mutations; transversion events were enhanced more efficiently by pKM101 than were transition events. Compared to Uvr+ bacteria, Uvr- bacteria had higher frequencies of spontaneous and induced mutations; transition mutations were enhanced more efficiently than were transversion mutations. The influence of DNA-repair activities on the mutational spectra provides some insights on the origins of spontaneous and UV-induced mutations.     

Characterization of a novel oncogenic K-ras mutation in colon cancer

Activating mutations of RAS are frequently observed in subsets of human cancers, indicating that RAS activation is involved in tumorigenesis. Here, we identified and characterized a novel G to T transversion mutation of the K-ras gene at the third position of codon 19 (TTG) which substituted phenylalanine for leucine in 3 primary colon carcinomas. Biological and biochemical activity was examined using transformed NIH3T3 cells expressing mutant or wild-type K-ras. Transformants harboring the K-ras mutation at codon 19 showed proliferative capacity under serum-starved conditions, less contact inhibition, anchorage-independent growth, tumorigenicity in nude mice and elevation of active Ras-GTP levels. These results indicated that this novel mutation possesses high oncogenic activity.     

Identification of missense, nonsense, and deletion mutations in the GRHPR gene in patients with primary hyperoxaluria type II (PH2)

Primary hyperoxaluria type II (PH2) is a rare disease characterized by the absence of an enzyme with glyoxylate reductase, hydroxypyruvate reductase, and D-glycerate dehydrogenase activities. The gene encoding this enzyme (GRHPR) has been characterized, and a single mutation has been detected in four PH2 patients. In this report, we have identified five novel mutations. One nonsense mutation (C295T) results in a premature stop codon at codon 99. A 4-bp deletion mutation has been found in the 5' consensus splice site of intron D, resulting in a predicted splicing error. Three missense mutations have been detected, including a missense transversion (T965G) in exon 9 (Met322Arg), a missense transition (G494A) in the putative co-factor binding site in exon 6 (Gly165Asp), and a substitution of an adenosine for a guanine in the 3' splice site of intron G. The functional consequences of the missense transversion and transition mutations have been investigated by transfection of cDNA encoding the mutated protein into COS cells. Cells transfected with either mutant construct have no enzymatic activity, a finding that is not significantly different from the control (empty) vector (P<0.05). These results further confirm that mutations in the GRHPR gene form the genetic basis of PH2. Ten of the 11 patients that we have genotyped are homozygous for one of the six mutations identified to date. Because of this high proportion of homozygotes, we have used microsatellite markers in close linkage with the GRHPR gene to investigate the possibility that the patients are the offspring of related individuals. Our data suggest that two thirds of our patients are the offspring of either closely or distantly related persons. Furthermore, genotyping has revealed the possible presence of a founder effect for the two most common mutations and the location of the gene near the marker D9S1874.     

Two mutations within the coding sequence of the phenylalanine hydroxylase gene

Summary Two previously unidentified mutations at the phenylalanine hydroxylase locus were found during a study of the relationship between genotype and phenotype in phenylketonuria and hyperphenylalaninemia. One mutation eliminates the BamHI site in exon 7 and the other eliminates the HindIII site in exon 11 of the phenylalanine hydroxylase gene. They were suspected because of deviating restriction fragment patterns and confirmed by amplification, via the polymerase chain reaction, of exon 7 and exon 11, respectively, followed by digestion with the appropriate restriction enzyme. Direct sequencing of amplified mutant exon 7 revealed a G/C to T/A transversion at the first base of codon 272, substituting a GGA glycine codon for a UGA stop codon. Direct sequencing of amplified mutant exon 11 revealed a deletion of codon 364, a CTT leucine codon. The exon 7 mutation can be expected to result in a truncated protein and the exon 11 mutation in the elimination of an amino acid in the catalytic region of the enzyme. A patient who is a compound heterozygote for these two mutations has classical phenylketonuria. It is concluded that each of the two mutations leads to a profound loss of enzymatic activity. The segregation of these mutations with disease alleles in 4 and 2 families, respectively, supports the hypothesis that multiple mutations at the phenylalanine hydroxylase locus explain the variable phenylalanine tolerance in patients with phenylalanine hydroxylase deficiency.     

Mutagenic specificity of N-acetoxy-3-aminobenzanthrone, a major metabolically activated form of 3-nitrobenzanthrone, in shuttle vector plasmids propagated in human cells

3-Nitrobenzanthrone (3-NBA) is a potent environmental mutagen and a potential human carcinogen present in diesel exhaust and airborne particulates. N-acetoxy-3-aminobenzanthrone (N-Aco-ABA) has been shown to be a major reactive metabolite of 3-NBA, which mainly produces adducts with guanine and adenine in cellular DNA. Here we analyzed mutations induced by N-Aco-ABA using supF shuttle vector plasmids to elucidate the mutagenic specificity of 3-NBA in human cells. Base sequence analysis of more than 100 plasmids with supF mutations induced in wildtype and DNA repair-deficient XP cells revealed that the major mutation was base substitutions of which the majority (42 and 38%, respectively) were G:C to T:A transversions. The next major mutation was G:C to A:T and A:T to G:C base substitutions in wildtype and XP cells, respectively. The DNA polymerase stop assay using N-Aco-ABA-treated plasmids as a template showed that most stop signals, i.e., adducted sites, appeared at G:C sites. These results suggest that N-Aco-ABA binds preferably to guanine rather than adenine, and adducted adenine is repaired more efficiently by the nucleotide excision repair. Error-prone DNA polymerases could insert adenine at sites opposite to N-Aco-ABA-adducted guanine, which leads to G:C to T:A transversion. These findings could be very important to evaluate the human lung cancer risk of environmental 3-NBA.     

Congenital afibrinogenemia: intracellular retention of fibrinogen due to a novel W437G mutation in the fibrinogen Bbeta-chain gene

Congenital afibrinogenemia is a rare autosomal recessive coagulation disorder characterised by hemorrhagic manifestations of variable entity and by severe plasma fibrinogen deficiency. Among the 31 afibrinogenemia-causing mutations so far reported, only 2 are missense mutations and both are located in the fibrinogen Bbeta-chain gene. Direct sequencing of the fibrinogen gene cluster in two afibrinogenemic Iranian siblings revealed a novel homozygous T>G transversion in exon 8 (nucleotide position 8025) of the fibrinogen Bbeta-chain gene. The resulting W437G missense mutation involves a highly conserved amino acid residue, located in the C-terminal globular D domain. The role of the W437G amino acid substitution on fibrinogen synthesis, folding, and secretion was assessed by in vitro expression experiments in COS-1 cells, followed by qualitative and quantitative analyses of intracellular and secreted mutant fibrinogen. Results of both pulse-chase experiments and enzyme-linked immunosorbent assays demonstrated intracellular retention of the mutant W437G fibrinogen and marked reduction of its secretion. These data, besides elucidating the pathogenetic role of the W437G mutation in afibrinogenemia, underline the importance of the Bbeta-chain D domain in fibrinogen folding and secretion.     

A homozygous mutation in a novel zinc-finger protein, ERIS, is responsible for Wolfram syndrome 2

A single missense mutation was identified in a novel, highly conserved zinc-finger gene, ZCD2, in three consanguineous families of Jordanian descent with Wolfram syndrome (WFS). It had been shown that these families did not have mutations in the WFS1 gene (WFS1) but were mapped to the WFS2 locus at 4q22-25. A G-->C transversion at nucleotide 109 predicts an amino acid change from glutamic acid to glutamine (E37Q). Although the amino acid is conserved and the mutation is nonsynonymous, the pathogenesis for the disorder is because the mutation also causes aberrant splicing. The mutation was found to disrupt messenger RNA splicing by eliminating exon 2, and it results in the introduction of a premature stop codon. Mutations in WFS1 have also been found to cause low-frequency nonsyndromic hearing loss, progressive hearing loss, and isolated optic atrophy associated with hearing loss. Screening of 377 probands with hearing loss did not identify mutations in the WFS2 gene. The WFS1-encoded protein, Wolframin, is known to localize to the endoplasmic reticulum and plays a role in calcium homeostasis. The ZCD2-encoded protein, ERIS (endoplasmic reticulum intermembrane small protein), is also shown to localize to the endoplasmic reticulum but does not interact directly with Wolframin. Lymphoblastoid cells from affected individuals show a significantly greater rise in intracellular calcium when stimulated with thapsigargin, compared with controls, although no difference was observed in resting concentrations of intracellular calcium.