Mutation at Intronic Repeats of the Ataxia-Telangiectasia Mutated (ATM) Gene and ATM Protein Loss in Primary Gastric Cancer with Microsatellite Instability

Ataxia-telangiectasia mutated (ATM) is a Ser/Thr protein kinase that plays a critical role in DNA damage-induced signaling and initiation of cell cycle checkpoint signaling in response to DNA-damaging agents such as ionizing radiation. We have previously reported the ATM protein loss by immunohistochemistry (IHC) in 16% of human gastric cancer (GC) tissue. We hypothesized that ATM gene intron mutations targeted by microsatellite instability (MSI) cause ATM protein loss in a subset of GC. We studied mononucleotide mutations at the intron of ATM gene, ATM IHC and MSI in GC. Ten human gastric cancer cell lines were studied for the ATM gene mutation at introns, RT-PCR, direct sequencing, and immunohistochemistry. GC tissues of 839 patients were analyzed for MSI and ATM IHC. Among them, 604 cases were analyzed for the ATM mutations at introns preceding exon 6, exon 10 and exon 20. Two human GC cell lines (SNU-1 and -638) showed ATM intron mutations, deletion in RT-PCR and direct sequencing, and ATM protein loss by IHC. The frequencies of ATM mutation, MSI, and ATM protein loss were 12.9% (78/604), 9.2% (81/882) and 15.2% (134/839), respectively. Analysis of associations among MSI, ATM gene mutation, and ATM protein loss revealed highly co-existing ATM gene alterations and MSI. ATM intron mutation and ATM protein loss were detected in 69.3% (52/75) and 53.3% (40/75) of MSI positive GC. MSI positivity and ATM protein loss were present in 68.4% (52/76) and 48.7% (37/76) of GC with ATM intron mutation. ATM mutation and ATM protein loss had characteristics of old age, distal location of tumor, large tumor size, and histologic intestinal type. Our study might be interpreted as that ATM gene mutation at intron might be targeted by MSI and lead to ATM protein loss in a selected group of GC.     

Analysis of lissencephaly-causing LIS1 mutations.

Mutations in the LIS1 gene may result in severe abnormalities of brain cortical layering known as lissencephaly. Most lissencephaly-causing LIS1 mutations are deletions that encompass the entire gene, therefore the mechanism of the disease is regarded as haploinsufficiency. So far, 13 different intragenic mutations have been reported: one point mutation, H149R; deletion of exon 9, which results in deleted acids Delta301-334; deletion of exon 4, which results in deleted amino acids Delta40-64; 10 mutations resulting in truncated proteins and one predicted to result in extra amino acids. We studied the consequences of the point mutation, deletion mutation and one of the reported truncations. In order to study LIS1 structure function, we introduced an additional point mutation and other truncations in different regions of the protein. The consequences of these mutations to protein folding were studied by gel filtration, sucrose density gradient centrifugation and measuring resistance to trypsin cleavage. On the basis of our results, we suggest that all truncation mutations and lissencephaly-causing point mutations or internal deletion result in a reduction in the amount of correctly folded LIS1 protein.     

Two Novel Mutations on Exon 8 and Intron 65 of COL7A1 Gene in Two Chinese Brothers Result in Recessive Dystrophic Epidermolysis Bullosa

Dystrophic epidermolysis bullosa is an inherited bullous dermatosis caused by the COL7A1 gene mutation in autosomal dominant or recessive mode. COL7A1 gene encodes type VII collagen – the main component of the anchoring fibrils at the dermal–epidermal junction. Besides the 730 mutations reported, we identified two novel COL7A1 gene mutations in a Chinese family, which caused recessive dystrophic epidermolysis bullosa (RDEB). The diagnosis was established histopathologically and ultrastructurally. After genomic DNA extraction from the peripheral blood sample of all subjects (5 pedigree members and 136 unrelated control individuals), COL7A1 gene screening was performed by polymerase chain reaction amplification and direct DNA sequencing of the whole coding exons and flanking intronic regions. Genetic analysis of the COL7A1 gene in affected individuals revealed compound heterozygotes with identical novel mutations. The maternal mutation is a 2-bp deletion at exon 8 (c.1006_1007delCA), leading to a subsequent reading frame-shift and producing a premature termination codon located 48 amino acids downstream in exon 9 (p.Q336EfsX48), consequently resulting in the truncation of 2561 amino acids downstream. This was only present in two affected brothers, but not in the other unaffected family members. The paternal mutation is a 1-bp deletion occurring at the first base of intron 65 (c.IVS5568+1delG) that deductively changes the strongly conserved GT dinucleotide at the 5′ donor splice site, results in subsequent reading-through into intron 65, and creates a stop codon immediately following the amino acids encoded by exon 65 (GTAA→TAA). This is predicted to produce a truncated protein lacking of 1089 C-terminal amino acids downstream. The latter mutation was found in all family members except one of the two unaffected sisters. Both mutations were observed concurrently only in the two affected brothers. Neither mutation was discovered in 136 unrelated Chinese control individuals. This study reveals novel disease-causing mutations in the COL7A1 gene.     

Genomic structure, chromosomal location, and mutation analysis of the human CDC14A gene.

Human CDC14A is a dual-specificity phosphatase that shares sequence similarity with the recently identified tumor suppressor, MMAC1/PTEN/TEP1. By radiation hybrid mapping, we localized CDC14A to chromosome band 1p21, a region that has been shown to exhibit loss of heterozygosity in highly differentiated breast carcinoma and malignant mesothelioma. We have mapped the exon-intron structure of CDC14A gene and found an in-frame ATG at 14 codons upstream of the previously reported start site (GenBank Accession No. AF000367). In screening a panel of 136 cDNAs from tumor cell lines for coding mutations, we have identified a 48-bp in-frame deletion in the cDNA of the breast carcinoma cell line, MDA-MB-436. This deletion is the result of an acceptor splice site mutation (AG to AT) in intron 12 that causes the skipping of exon 13 in the gene. Loss of expression of the wildtype allele in the same breast cell line supports the possibility that CDC14A may be a tumor suppressor gene that is targeted for inactivation during tumorigenesis.     

Simultaneous detection of multiple mutations in epidermal growth factor receptor based on fluorescence quenching of quantum dots

We have developed a simultaneous detection method for two common mutations in the epidermal growth factor receptor gene based on the fluorescence quenching phenomenon caused by aggregation of CdSe quantum dots. For detection of the in-frame deletion in exon 19 and the L858R point mutation in exon 21, water-soluble CdSe quantum dots with two sizes were functionalized using four different types of probe oligonucleotides. Addition of target oligonucleotides with the deletion mutation in exon 19 into the suspensions caused crosslinking-induced aggregation of green-emitting quantum dots, followed by the fluorescence quenching while that with the L858R point mutation resulted in aggregation of yellow-emitting quantum dots. In addition, targets with both deletion and point mutations caused aggregation of both green- and yellow-emitting quantum dots. This method allows a simultaneous detection of mutations in exon 19 and 21 of EGFR gene in a single experiment. We found that minimum mutant concentration that could be detected by this method was as low as 2% for deletion mutation, and 5% for point mutation. PCR products of EGFR gene were also used to confirm that our method could be used to detect mutation in amplified DNA fragments.     

Characterization of exon skipping mutants of the COP1 gene from Arabidopsis

The removal of introns from pre-mRNA requires accurate recognition and selection of the intron splice sites. Mutations which alter splice site selection and which lead to skipping of specific exons are indicative of intron/exon recognition mechanisms involving an exon definition process. In this paper, three independent mutants to the COP1 gene in Arabidopsis which show exon skipping were identified and the mutations which alter the normal splicing pattern were characterized. The mutation in cop1–1 was a G→A change 4 nt upstream from the 3′ splice site of intron 5, while the mutation in cop1–2 was a G→A at the first nucleotide of intron 6, abolishing the conserved G within the 5′ splice site consensus. The effect of these mutations was skipping of exon 6. The mutation in cop1–8 was G→A in the final nucleotide of intron 10 abolishing the conserved G within the 3′ splice site consensus and leading to skipping of exon 11. The splicing patterns surrounding exons 6 and 11 of COP1 in these three mutant lines of Arabidopsis provide evidence for exon definition mechanisms operating in plant splicing.     

Molecular characterization of a brown midrib3 deletion mutation in maize

The caffeic acid O-methyltransferase (COMT) gene plays an important role in the synthesis of lignin. We have used the polymerase chain reaction in conjuction with genomic analysis to characterize deletion mutations of this gene in maize. In addition, we have analyzed and compared regions of the COMT gene from three distinct heterotic groups. Both PCR and Southern analysis indicate that the active wild-type COMT gene can be polymorphic. We suggest that the intron domain of at least one heterotic inbred can contribute to the alteration of the wild-type gene. In addition, multiple deletion mutations have occurred at this locus. We have found a previously uncharacterized deletion mutation in which segments of both the intron and exon have been deleted and replaced by other sequences. Precise knowledge of its sequence has allowed us to develop an assay by which we can follow this mutation in a breeding program.     

Molecular analysis of X-linked inborn errors of purine metabolism: HPRT1 and PRPS1 mutations

Mutations of two enzyme genes, HPRT1 encoding hypoxanthine guanine phosphoribosyltransferase (HPRT) and PRPS1 encoding a catalytic subunit (PRS-I) of phosphoribosylpyrophosphate synthetase, cause X-linked inborn errors of purine metabolism. Analyzing these two genes, we have identified three HPRT1 mutations in Lesch-Nyhan families following our last report. One of them, a new mutation involving the deletion of 4224 bp from intron 4 to intron 5 and the insertion of an unknown 28 bp, has been identified. This mutation resulted in an enzyme polypeptide with six amino acids deleted due to abnormal mRNA skipping exon 5. The other HPRT1 mutations, a single base deletion (548delT, 183fs189X), and a point mutation causing a splicing error (532+1G>A, 163fs165X) were detected first in Japanese patients but have been reported in European families. On the other hand, in the analysis of PRPS1, no mutation was identified in any patient.     

A novel Phytochrome B allele in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> exhibits partial mutant phenotype: a short deletion in N-terminal extension reduces Phytochrome B activity

During analysis of an Arabidopsis thaliana line possessing a Phytochrome A epiallele (phyA’), a partial Phytochrome B-deficient phenotype was observed, consisting of elongated hypocotyls in seedlings grown under constant white light or red light (660 nm). The observed hypocotyls were twice the length (8 mm) of wild-type (4 mm), but approximately half the length of a null phyB-9 mutant (14 mm). Several analyses were performed to characterize this apparent partial phyB mutant. Sequencing of the entire exonic region revealed three point mutations that altered codon usage, and one in-frame 12 base pair deletion. Each of the point mutations has been described in other lines that display wild-type phenotype, and therefore their effect is thought to be minimal, if any. The N-terminal deletion of amino acids 9 through 12 (GGGR) is a unique mutation found in this line. This deletion most likely contributes to the phyB mutant phenotype by lowering the binding affinity of the active form of Phytochrome B (Pfr) with Phytochrome Interacting Factor 3 (PIF3).     

Nature and frequency of mutations in the argininosuccinate synthetase gene that cause classical citrullinemia

Citrullinemia is an autosomal recessive disorder caused by a genetic deficiency of argininosuccinate synthetase (ASS). So far 20 mutations in ASS mRNA have been identified in human classical citrullinemia, including 14 single base changes causing missense mutations in the coding sequence of the enzyme, 4 mutations associated with an absence of exons 5, 6, 7, or 13 in mRNA, 1 mutation with a deletion of the first 7 bases in exon 16 (which is caused by abnormal splicing), and 1 mutation with an insertion of 37 bases between the exon 15 and 16 regions in mRNA. In order to identify the abnormality in the ASS gene causing the exon 7 and 13 deletion mutations and the 37-base insertion mutation between exons 15 and 16 in mRNA, and to establish a DNA diagnostic test, we isolated and sequenced the genomic DNA surrounding each exon. The absence of exon 7 or 13 in ASS mRNA resulted from abnormal splicing caused by a single base change in the intron region: IVS-6^–2 (a transition of A to G at the second nucleotide position within the 3 splice cleavage site of intron 6) and IVS-13⁺⁵ (a transition of G to A at the fifth nucleotide position within the 5 splice cleavage site of intron 13), respectively. The IVS-6^–2 mutation resulted in the creation of an MspI restriction site. DNA diagnostic analysis of 33 Japanese alleles with classical citrullinemia showed that 19 alleles had the IVS-6^–2 mutation (over 50% of the mutated alleles in Japanese patients). It was thus confirmed that one mutation is predominant in Japan. This differs from the situation in the USA where there is far greater heterogeneity. The insertion mutation in mRNA on the other hand resulted from abnormal splicing caused by a 13-bp deletion at the splice-junction between exon 15 and intron 15. The deletion had a short direct repeat (CTCAGG) at the breakpoint junction and presumably resulted from slipped mispairing.     

A type II collagen mutation also results in oto-spondylo-megaepiphyseal dysplasia

Oto-spondylo-megaepiphyseal dysplasia (OSMED) is a skeletal dysplasia characterized by severe sensorineural hearing loss, enlarged epiphyses and early onset of osteoarthritis. COL11A2 has been reported as a causative gene for OSMED. We have identified a novel COL2A1 mutation at a splice-acceptor site within intron 10 (c.709–2A>G) in an OSMED patient. This mutation caused the skipping of exon 11, and of exons 11 and 13. These exon-skipping events are presumed to cause an in-frame deletion of the triple helical region of the COL2A1 product. Thus, our findings highlight the genetic heterogeneity of OSMED and extend the phenotypic spectrum of type II collagenopathy, as well as confirming the overlap between type II and type XI collagenopathies.     

BRCA1 mutations in African Americans

The breast cancer predisposing gene, BRCA1, was analyzed for germline mutations in 45 African American families at high-risk for hereditary breast cancer. Patients were considered high-risk if they had a family history of the disease, early onset breast cancer, bilateral breast cancer, or breast and ovarian cancer. The entire BRCA1 coding and flanking intron regions have been examined by single stranded conformation polymorphism analysis followed by sequencing of variant bands. Eleven different BRCA1 germline mutations/variations were identified in 7 patients from the 45 high-risk families. Two pathogenic, protein-truncating mutations were detected in exon 11. A ten base pair tandem duplication, 943ins10, was present in a woman with breast and ovarian cancer whose first-degree relatives had prostate cancer. A four base pair deletion, 3450del4, was detected in a breast cancer patient with five cases of breast cancer in the family; two of the proband's sisters with breast cancer also carried the same mutation. Four amino acid substitutions (Lys1183Arg, Leu1564Pro, Gln1785His, and Glu1794Asp) and four nucleotide substitutions in intron 22 (IVS22+78 C/A, IVS22+67 T/C, IVS22+8 T/A and IVS22+7 T/C) were observed in patients and not in control subjects. One early onset breast cancer patient carried five distinct BRCA1 variations, two amino acid substitutions and three substitutions in intron 22. An amino acid substitution in exon 11, Ser1140Gly, was identified in 3 different unrelated patients and in 6 of 92 control samples. The latter probably represents a benign polymorphism. Electronic Publication     

Molecular characterization of two deletion events involving Alu-sequences, one novel base substitution and two tentative hotspot mutations in the hypoxanthine phosphoribosyltransferase (HPRT) gene in five patients with Lesch-Nyhan syndrome

Mutations identified in the hypoxanthine phosphoribosyltransferase (HPRT) gene of patients with Lesch-Nyhan (LN) syndrome are dominated by simple base substitutions. Few hotspot positions have been identified, and only three large genomic rearrangements have been characterized at the molecular level. We have identified one novel mutation, two tentative hot spot mutations, and two deletions by direct sequencing of HPRT cDNA or genomic DNA from fibroblasts or T-lymphocytes from LN patients in five unrelated families. One is a missense mutation caused by a 610C→T transition of the first base of HPRT exon 9. This mutation has not been described previously in an LN patient. A nonsense mutation caused by a 508C→T transition at a CpG site in HPRT exon 7 in the second patient and his younger brother is the fifth mutation of this kind among LN patients. Another tentative hotspot mutation in the third patient, a frame shift caused by a G nucleotide insertion in a monotonous repeat of six Gs in HPRT exon 3, has been reported previously in three other LN patients. The fourth patient had a tandem deletion: a 57-bp deletion in an internally repeated Alu-sequence of intron 1 was separated by 14 bp from a 627-bp deletion that included HPRT exon 2 and was flanked by a 4-bp repeat. This complex mutation is probably caused by a combination of homologous recombination and replication slippage. Another large genomic deletion of 2969 bp in the fifth patient extended from one Alu-sequence in the promoter region to another Alu-sequence of intron 1, deleting the whole of HPRT exon 1. The breakpoints were located within two 39-bp homologous sequences, one of which overlapped with a well-conserved 26-bp Alu-core sequence previously suggested as promoting recombination. These results contribute to the establishment of a molecular spectrum of LN mutations, support previous data indicating possible mutational hotspots, and provide evidence for the involvement of Alu-mediated recombination in HPRT deletion mutagenesis. Received: 21 April 1998 / Accepted: 16 July 1998     

Variability of biochemical and clinical phenotype in X-linked liver glycogenosis with mutations in the phosphorylase kinase PHKA2 gene

X-linked liver glycogenosis (XLG) resulting from phosphorylase kinase (Phk) deficiency is one of the most common forms of glycogen storage disease. It is caused by mutations in the gene encoding the liver isoform of the Phk α subunit (PHKA2). In the present study, we address the issue of phenotypic and allelic heterogeneity in XLG. We have identified mutations in seven male patients. One of these patients represents the variant biochemical phenotype, XLG subtype 2 (XLG2), where Phk activity is low in liver but normal or even elevated in erythrocytes. He carries a K189E missense mutation, which adds to the emerging evidence that XLG2 is associated with missense mutations clustering at a few sites. Two patients display clinical phenotypes unusual for liver Phk deficiency, with dysfunction of the kidneys (proximal renal tubular acidosis) or of the nervous system (seizures, delayed cognitive and speech abilities, peripheral sensory neuropathy), respectively, in addition to liver glycogenosis. In the patient with kidney involvement, we have identified a missense mutation (P399S) and a trinucleotide deletion (2858del3) leading to the replacement of two amino acids by one new residue (N953/L954I), and a missense mutation has also been found in the patient with neurological symptoms (G1207W). These two cases demonstrate that PHKA2 mutations can also be associated with uncommon clinical phenotypes. Finally, in four typical XLG cases, we have identified three truncating mutations (70insT, R352X, 567del22) and an in-frame deletion of eight well-conserved amino acids (2452del24). Together, this study adds eight new mutations to the previously known complement of sixteen PHKA2 mutations. All known PHKA2 mutations but one are distinct, indicating pronounced allelic heterogeneity of X-linked liver glycogenosis with mutations in the PHKA2 gene. Received: 17 October 1997 / Accepted: 23 December 1997     

Comparative and genetic analysis of the porcine glucocerebrosidase (GBA) gene

The genomic sequence of the porcine (Sus scrofa) glucocerebrosidase (GBA) gene (5.7 kb), encoding glucocerebrosidase (glucosylceramidase; acid beta-glucosidase; EC 3.2.1.45), was determined and compared with human (Homo sapiens) GBA and GBAP (pseudogene). The porcine gene harbours 11 exons and 10 introns, and the genomic organization is identical with human GBA. The exon sequences, coding for signal peptide and mature protein, show 81% and 90% sequence identity, respectively, with the corresponding human GBA sequences. Short interspersed elements, SINEs (PREs), are present in introns 2, 4 and 7. There is no evidence of a pseudogene in pig. The deduced protein sequence of GBA consists of 39 amino acids of signal peptide (long form) and 497 amino acids of the mature protein; the latter shows 90% sequence identity with the human protein. Four polymorphisms were observed within the porcine gene: insertion/deletion of one of the two SINEs (PREs) in intron 2 (locus PREA); deletion of a 37- to 39-bp stretch in intron 4 (one direct repeat and 5′ end of PRE); deletion of a 47-bp stretch in the middle part of PRE in intron 4 (locus PREB); and single-base transition (C–T) in intron 6 (locus HaeIII–RFLP). GBA was assigned to chromosome 4q21 by FISH and was localized to the same region by linkage analysis and RH mapping, i.e., to the chromosome 4 segment where quantitative trait loci for growth and some carcass traits are located.