Analysis of spontaneous,gamma ray- and ethylnitrosourea-induced hprt mutants in HL-60 cells with multiplex PCR

AIM: To explore the molecular spectra and mechanism of human hypoxanthine guanine phosphoribosyl transferase (hprt) gene mutation induced by ethyluitrosourea (ENU) and 60Co γ-rays.METHODS: Independent human promyelocytic leukemia cells (HL-60) mutants at the hprt locus were isolated from untreated, ethyluitrosourea (ENU) and 60Co γ-ray-exposed cells, respectively, and verified by two-way screening. The genetic changes underlying the mutation were determined by multiplex polymerase chain reaction (PCR) amplification and electrophoresis technique.RESULTS: With dosage increased, survival rate of plated cell reduced (in the group with dosage of ENU with 100-200μg/ml, P＜0.01; in the group with dosage of 60Co γ-ray with 2-4 Gy, P＜0.05) and mutational frequency increased (in the group of ENU 12.5-200.0 μg/ml, P＜0.05; in the group of 60Co γ-ray with 1-4 Gy, P＜0.05) significantly. In the 13spontaneous mutants analyzed, 92.3 % of mutant clones did not show any change in number or size of exon, a single exon was lost in 7.7 %, and no evidence indicated total gene deletion occurred in nine hprt exons. However, deletions were found in 79.7 % of ENU-induced mutations (62.5-89.4 %,P＜0.01) and in 61.7 % of gamma-ray-induced mutations (28.6-76.5 %, P＜0.01). There were deletion mutations in all 9 exons of hprt gene and the most of induced mutations were chain deletion with multiplex exons (97.9 % in gammaray-induced mutants, 88.1% in ENU-induced mutants).CONCLUSION: The spectra of spontaneous mutations differs completely from that induced by EUN or 60Co γ-ray.Although both ENU and γ-ray can cause destruction of genetic structure, mechanism of mutagenesis between them may be different.     

Characterization of five partial deletions of the factor VIII gene.

Hemophilia A is an X-linked disorder of coagulation caused by a deficiency of factor VIII. By using cloned DNA probes, we have characterized the following five different partial deletions of the factor VIII gene from a panel of 83 patients with hemophilia A: (i) a 7-kilobase (kb) deletion that eliminates exon 6; (ii) a 2.5-kb deletion that eliminates 5' sequences of exon 14; (iii) a deletion of at least 7 kb that eliminates exons 24 and 25; (iv) a deletion of at least 16 kb that eliminates exons 23-25; and (v) a 5.5-kb deletion that eliminates exon 22. The first four deletions are associated with severe hemophilia A. By contrast, the last deletion is associated with moderate disease, possibly because of in-frame splicing from moderate disease, possibly because of in-frame splicing from adjacent exons. None of those patients with partial gene deletions had circulating inhibitors to factor VIII. One deletion occurred de novo in a germ cell of the maternal grandmother, while a second deletion occurred in a germ cell of the maternal grandfather. These observations demonstrate that de novo deletions of X-linked genes can occur in either male or female gametes.     

Two new large deletions in the low density lipoprotein receptor (LDLR) gene not revealed by PCR-based molecular diagnosis of familial hypercholesterolemia

In the French Canadian population six mutations appear to be responsible for about 85% of FH cases. Two of these mutations are large deletions. The most prevalent deletion is a >15 kb deletion of the promoter and first exon; the second, a 5 kb deletion that removes exons 2 and 3. The high frequency of these deletions in the French Canadian population has been attributed to a founder effect. Other mutations are present in the population but at a much lower prevalence. We recently identified two new large deletions in FH patients of French Canadian descent. Carriers of the new deletions were identified because of an unusual pattern of band migration on Southern blots. We have identified and sequenced the deletions' boundaries. The first deletion covers 3813 bp and removes exons 7 and 8. The second deletion covers 5994 bp and removes exons 3-6. These deletions have not been previously reported. They would have been missed if a PCR-based method had been used instead of Southern blot analysis.     

c-kit proto-oncogene exon 8 in-frame deletion plus insertion mutations in acute myeloid leukaemia

Genomic DNA from 60 cases of acute myeloid leukaemia (AML) was screened for mutations in the c-kit gene. DNA from all 21 exons was subjected to polymerase chain reaction (PCR) amplification and analysis by conformation sensitive gel electrophoresis (CSGE); exons showing altered CSGE patterns were then sequenced. Mutations were identified only in those patients with inv(16) (3/7 cases) or t(8;21) (1/2 cases) and comprised three in-frame deletion plus insertion mutations (exon 8) and one point mutation (exon 10, GTA --> ATA, Val530Ile). Exons 8 and 10 were then analysed in 31 further cases of inv(16) (n = 14) and t(8;21) (n = 17), revealing four additional exon 8 in-frame deletion plus insertion mutations, all of which were in cases of inv(16). All exon 8 in-frame deletion plus insertion mutations (n = 7) involved the loss or replacement of the codon for Asp419 which is highly conserved cross species and is located in the receptor's extracellular domain. The high frequency of the c-kit proto-oncogene exon 8 deletion plus insertion mutations in AML suggests an essential role for this region of the receptor's extracellular domain. The association with inv(16) invites speculation as to the link between these two changes in the pathogenesis of AML.     

Characterization of three new deletions at the 5′ end of the HPRT structural gene

Summary In a panel of seven unrelated HPRT-deficient patients three partial deletions of the 5 end of the HPRT structural gene were identified by Southern blot analysis. The deletions could be defined as the loss of exons 1–3, exons 2–3 and exon 3 respectively. In two of the deletion mutations aberrant restriction fragments occurred.Presented at a symposium on inherited metabolic diseases at Brno in 1989.     

Rearrangements in the LDL receptor gene in Dutch familial hypercholesterolemic patients and the presence of a common 4 kb deletion

DNA samples from 53 unrelated Dutch patients with familial hypercholesterolemia (FH) were screened for rearrangements in the gene for the LDL receptor (LDLR) by Southern analysis. Four different mutations have been detected by hybridisation of BglII digested genomic DNA with an exon 10-14 containing cDNA probe. The mutations are defined by a 7 kb insertion near exon 11, a partial gene duplication encompassing exons 9-12, a 4 kb deletion of exons 7 and 8 and an 0.4 kb deletion comprising the 5'-part of exon 16. These four different rearrangements in the LDLR gene account for 17% of the mutations in the Dutch FH population sample. Interestingly, the 4 kb deletion was detected in 5 unrelated FH patients (9.5%) and appeared to be identical to the deletion previously described (Russell, D.W. et al., Arteriosclerosis, 9 (Suppl. I) (1989) I-8; Russell, D.W. et al., Cold Spring Harbor Symp. Quant. Biol., 51 (1987) 401). in an FH patient of Dutch origin. This suggests that the 4 kb deletion is a common mutation in the Dutch FH population.     

Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery

Duchenne muscular dystrophy (DMD), the commonest form of muscular dystrophy, is caused by lack of dystrophin. One of the most promising therapeutic approaches is antisense-mediated elimination of frame-disrupting mutations by exon skipping. However, this approach faces two major hurdles: limited applicability of each individual target exon and uncertain function and stability of each resulting truncated dystrophin. Skipping of exons 45-55 at the mutation hotspot of the DMD gene would address both issues. Theoretically it could rescue more than 60% of patients with deletion mutations. Moreover, spontaneous deletions of this specific region are associated with asymptomatic or exceptionally mild phenotypes. However, such multiple exon skipping of exons 45-55 has proved technically challenging. We have therefore designed antisense oligo (AO) morpholino mixtures to minimize self- or heteroduplex formation. These were tested as conjugates with cell-penetrating moieties (vivo-morpholinos). We have tested the feasibility of skipping exons 45-55 in H2K-mdx52 myotubes and in mdx52 mice, which lack exon 52. Encouragingly, with mixtures of 10 AOs, we demonstrated skipping of all 10 exons in vitro, in H2K-mdx52 myotubes and on intramuscular injection into mdx52 mice. Moreover, in mdx52 mice in vivo, systemic injections of 10 AOs induced extensive dystrophin expression at the subsarcolemma in skeletal muscles throughout the body, producing up to 15% of wild-type dystrophin protein levels, accompanied by improved muscle strength and histopathology without any detectable toxicity. This is a unique successful demonstration of effective rescue by exon 45-55 skipping in a dystrophin-deficient animal model.     

伴食管平滑肌瘤的Alport综合征基因突变特征

目的：认识我国伴食管平滑肌瘤的Alport综合征患者的基因型特点。方法：对l例中国汉族伴食管平滑肌瘤的Alport综合征男性患者应用PCR方法扩增COL4A5基因的外显子l、2和COL4A6基因的外显子l(部分序列)、2及3，同时应用Southem杂交的方法进行DNA重组分析。结果：经PCR扩增和Southem杂交的方法揭示患者具有累及COL4A5和COL4A6两个基因5’端的大片段缺失，缺失范围可能至少包括COL4A5基因的外显子l、COL4A6基因的外显子l～2以及两基因间的启动子，估计此大片段缺失的断点在COL4A5基因的内含子l至COL4A6基因的内含子2中。结论：首次发现中国汉族中伴食管平滑肌瘤的Alport综合征患者具有COL4A5和COL4A6两个基因的缺失突变，该突变有可能累及C0L4A6基因的内含子2。同时亦提示具有COL4A5和COL4A6两个基因突变伴弥漫性平滑肌瘤表型的Alport综合征患者与COL4A6基因突变位置有关。     

Selective amplification of exons 3 and 8 of the human growth hormone receptor (hGHR) gene based on newly identified intron sequences.

The gene for human growth hormone receptor (hGHR) consists of at least 10 exons, and the corresponding protein is encoded in exons 2-10 which span at least 87 kbp of chromosome 5. Failure to amplify exons 3 and 8 of the hGHR gene from Japanese subjects with the previously reported primers prompted us to determine intron sequences flanking exon 3 and those flanking exon 8 of the hGHR gene, and novel intron sequences flanking exons 3 and 8 of the hGHR gene were identified. We designed new oligonucleotide primers based on these sequences, and successfully amplified DNA fragments encompassing exon 3 and those encompassing exon 8 of the hGHR gene. Since all of the 50 Japanese and the two Caucasians had the very same intron sequences which were different from the previously reported ones, it is more likely that the previously reported sequences were simply wrong than that there exist polymorphic differences in the intron sequences among different ethnic populations.     

Genetic instability and the etiology of somatic PIG-A mutations in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a hematologic disorder characterized by acquired PIG-A gene mutations that lead to defective bioassembly of glycosylphosphatidylinositol (GPI) anchors and the absence of GPI-linked surface proteins. As the etiology of these acquired PIG-A gene mutations is unknown, we hypothesized that patients with PNH have overall genetic instability and acquire somatic mutations throughout their genome. We first analyzed microsatellite sequences and found equivalent size variation using DNA from GPI-negative granulocytes compared with the DNA of paired GPI-positive B cell lines or normal granulocytes. We next quantitated the frequency of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene locus, and found 1 PNH patient with a large increase in hprt mutant frequency (256.7 x 10(-6) vs. 27.8 +/- 19.9 x 10(-6) for normal adults) that was confirmed on 4 independent blood samples. We also quantitated "illegitimate" VDJ genetic recombination events between the T cell receptor V gamma and J beta gene loci, and found a second PNH patient with a large increase (43.5 events per microgram of DNA vs. 1.3 +/- 0.8 events per microgram of DNA for normal adults), confirmed on 4 independent DNA samples. Both of these PNH patients are young females with no history of aplastic anemia. Our data show that PNH patients can have increased numbers of acquired somatic mutations in gene loci distinct from PIG-A. These data suggest that genetic instability may be associated with the development of PIG-A mutations that lead to the clinical picture of PNH.     

Gastrointestinal stromal tumors with KIT exon 11 deletions are associated with poor prognosis

BACKGROUND & AIMS: Gain-of-function mutations in the KIT receptor tyrosine kinase gene and rare mutations in the platelet-derived growth factor receptor alpha (PDGFRA) gene are important events in gastrointestinal stromal tumor (GIST) development. Different mutations are reportedly associated with distinctive phenotypes and possibly clinical behavior. We investigated the correlation among mutation type, phenotype, and clinical course in a preimatinib, population-based series of GIST with long-term follow-up. METHODS: Genomic DNA from 177 GIST patients was analyzed for KIT exons 9, 11, 13, and 17 and PDGFRA exons 12 and 18 mutations using denaturating high-performance liquid chromatography and bidirectional sequencing. RESULTS: KIT exon 11 mutations were detected in 101 of 177 GIST (61 deletions, 23 missense mutations, and 17 duplications); wild-type (WT) KIT and PDGFRA were detected in 63; KIT exon 9 and exon 17 mutations in 6 and 1, respectively; and PDGFRA exons 12 and 18 mutations in 3 each. GIST >5 cm vs GIST 相似文献   

Primary GH insensitivity '(Laron syndrome) caused by a novel 4 kb deletion encompassing exon 5 of the GH receptor gene: effect of intermittent long-term treatment with recombinant human IGF-I

OBJECTIVE: GH insensitivity syndrome (GHIS; Laron syndrome) is clinically characterized by severe postnatal growth failure and very low serum levels of IGF-I despite increased secretion of GH. This mainly autosomal recessive syndrome is clinically indistinguishable from isolated GH deficiency (IGHD). Fifty-one different mutations in the GH receptor (GHR) gene have been discovered, whereas only three deletions causing the disorder have been reported so far. In this report, we describe a consanguineous family from Sri Lanka with a novel deletion of 4097 bp in length encompassing exon 5. SUBJECTS AND METHODS: Parents of normal phenotype presented their second child (boy) to our clinic at the age of 7 months with severe growth retardation and the clinical features of IGHD (58 cm, -6.1 standard deviation score (SDS); 5.7 kg, -3.4 SDS). Assessment, however, revealed GHIS with absent GH-binding protein. Thereafter, the patient received intermittent recombinant human IGF-I (rhIGF-I; 80 microg/kg twice daily) treatment prepubertally for 5.5 years. Genomic DNA was extracted for genetic analysis and each exon was PCR amplified individually. Further, in order to amplify the GHR gene from exon 4 to 6, Expand Long Template PCR (Roche) was carried out. In addition, RNA isolation and RT-PCR were performed. RESULTS: Separate PCRs of each of the exons of the GHR gene revealed that exon 5 in the patient was missing. Thereafter, "Long PCR" from exons 4 to 6 revealed a 4097 bp deletion encompassing exon 5, in a homozygous state in the patient and in a heterozygous state in both parents. RT-PCR analysis revealed an exact absence of exon 5 resulting in a frameshift, leading to a stop codon in exon 6, which predicts a truncated, non-functional GHR protein. CONCLUSION: Fifty-one different mutations within the GHR gene causing GHIS have been reported so far. In contrast, only three deletions within the GHR gene are known. We describe a patient suffering from GHIS caused by a novel 4 kb deletion of the GHR gene encompassing exon 5 and, additionally, we focus on the effect of intermittent rhIGF-I treatment during prepuberty.     

Factor VIII gene deletions in haemophilia A patients in Czechoslovakia

Genomic DNA from 90 Czechoslovak haemophilia A patients from 81 pedigrees was analysed by Southern blotting and hybridization with factor VIII cDNA probes. Three partial deletions of the factor VIII gene were identified and characterized: a 4.8 kilobase (kb) deletion eliminating exon 10 in one patient with severe haemophilia A without inhibitor, a 6.1 kb deletion eliminating the 3' part of intron 13 and the 5' part of exon 14 in two related severe haemophiliacs, but only one of them produced inhibitor, and a 4.6 kb deletion eliminating the 3' part of intron 13 and the 5' part of exon 14 in a severe haemophiliac with high-titre inhibitor. Besides these three deletions, three different restriction site variants without apparent loss of DNA sequence were found.     

Structure of the human immunoglobulin C epsilon 2 gene, a truncated pseudogene: implications for its evolutionary origin.

Cloning of the overlapping DNA fragments together with Southern hybridization experiments showed the organization of the human C epsilon and C alpha gene cluster as 5'-C epsilon 2-14 kilobases-C alpha 1----C epsilon 1-13 kilobases-C alpha 2-3'. Comparison of the nucleotide sequences of the C epsilon 1 and C epsilon 2 genes revealed that four deletions have taken place in the C epsilon 2 gene and its flanking regions. The three deleted regions in the 5' side of the C epsilon 2 gene are partially filled with shorter inserted sequences. One of them has removed the CH1 and CH2 exons and a portion of the epsilon switch (S epsilon) region. The S epsilon region and the CH4 exon still retain the functional structures, whereas the CH3 exon has been inactivated by deleting its 5' intervening sequence necessary for splicing. The tetranucleotide T-G-G-G (or T-G-G-C), which is usually found in close proximity of the class-switch recombination sites in mouse myelomas, is located 5' to the three deletion sites. The results imply that the mechanism responsible for the heavy chain class-switch recombination might be relevant to the evolutionary mechanism of creation of the truncated C epsilon 2 gene. The other deletion in the 3' flanking region of the C epsilon 2 gene may be due to slipped mispairing of the short direct repeat (C-C-C-C-C) at both ends.     

Therapy-related acute myeloid leukemia-like MLL rearrangements are induced by etoposide in primary human CD34+ cells and remain stable after clonal expansion

Rearrangements involving the MLL gene on chromosome band 11q23 are a hallmark of therapy-related acute myeloid leukemias following treatment with topoisomerase II poisons including etoposide. Therapy-related and de novo genomic translocation breakpoints cluster within a well-characterized 8.3-kb fragment of MLL. Repair of etoposide-stabilized DNA topoisomerase II covalent complexes may initiate MLL rearrangements observed in patients. We used a culture system of primary human hematopoietic CD34+ cells and inverse polymerase chain reaction to characterize the spectrum of stable genomic rearrangements promoted by etoposide exposure originating within an MLL translocation hotspot in therapy-related leukemia. Alterations to the region were observed at a readily detectable frequency in etoposide-treated cells. Illegitimate repair events after minimal repair included MLL tandem duplications and translocations, with minor populations of deletions or insertions. In stably repaired cells that proliferated for 10 to 14 days, the significant majority of illegitimate events were MLL tandem duplications, and several deletions, inversions, insertions, and translocations. Thus, etoposide promotes specific rearrangements of MLL consistent with the full spectrum of oncogenic events identified in leukemic samples. Although etoposide-initiated rearrangements are frequent, only a small subset of translocations occurs in cells that proliferate significantly.     

A novel large deletion and single nucleotide insertion in the Wiskott–Aldrich syndrome protein gene

Deletion mutations of WAS are relatively rare and the precise localization of large deletions in the genome has rarely been described in previous studies. We report here a 5‐month‐old boy with a large deletion mutation in WAS that completely abolished protein expression. To localize the deletion, a 2816‐bp‐length sequence that spans between exons 9 and 12 was amplified. PCR amplification of the patient's sample revealed a single band of about 1 kb in contrast to the 2816‐bp‐amplicon in the control. Genomic DNA sequencing of the patient revealed a 1595‐bp‐deletion and an adenine insertion (g.5247_6841del1595insA). This large deletion of WAS resulted in partial loss of exon 10 and intron 11, and a complete loss of intron 10 and exon 11.     

Hypoxanthine-guanine phosphoribosyltransferase reporter gene mutation for analysis of in vivo clonal amplification in patients with HTLV type 1-associated Myelopathy/Tropical spastic paraparesis

We tested a surrogate selection approach utilizing mutation at a reporter gene [hypoxanthine-guanine phosphoribosyltransferase (hprt)] as a probe for in vivo cell division, for detection of clonal T cell expansion in human T lymphotropic (HTLV-1) carriers. Peripheral blood samples from HTLV-1-infected individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) were tested to determine the hprt mutant frequency (Mf). Wild-type and hprt mutant T cell clones were isolated, and clonal identity determined by multiplex PCR and DNA sequencing of T cell receptor (TCR) variable region beta-chain (TCR BV) and third complementarity determining regions (CDR3). Seven samples from HAM/TSP patients were tested, and Mfs were within the normal range for adults (mean 11.3 x 10(-6), max 22.4 x 10(-6), min 5.6 x 10(-6)). The frequency of HTLV-1 infection in wild-type and hprt mutant T cells from HAM/TSP patients was determined to identify enrichment in the mutant fraction of cells. This analysis was performed on 196 isolates from 6 individuals with HAM/TSP. In each case, there is enrichment for virally infected cells in the hprt mutant fraction of isolates. Ten mutant and eight wild-type isolates from sample LS42A (Mf 8.4 x 10(-6)) were tested for clonality by TCR BV PCR and sequencing. Of the 10 hprt mutants, there were two in vivo-expanded clones (four isolates with two identical TCRs, or 80% unique TCR sequences). These studies may provide new insights into the precise mechanism of HTLV-1 leukemogenesis, and aid in the study of mutator phenotypes generated by a combination of Tax-mediated in vivo expansion and mutagenesis.     

脊髓性肌萎缩症的基因研究

目的 研究我国Ⅰ～Ⅳ型脊髓性肌萎缩症（SMA）患者运动神经元生存基因（SMN）及神经细胞凋亡抑制蛋白（NAIP）基因外显子的缺失情况,以探讨此两种基因与SMA表型之间的关系。方法 应用PCR法检测45例Ⅰ～Ⅳ型SMA患者、30例表型正常的SMA直系亲属及30例正常对照的SMIN基因第7、8号外显子和NAIP基因第5、6号外显子缺失情况。结论 7例Ⅰ型和Ⅱ型SMA患者中6例纯合缺失SMN基因外显子7和8,1例纯合缺失外显子7而保留外显子8;8例Ⅲ型SMA患者仅1例缺失外显子7和8,余7例均无SMN基因的缺失;成人型（Ⅳ型）SMA未检测到SMN基因缺失;45例Ⅰ～Ⅳ型SMA患者均未检测到NAIP基因外显子5和（或）6的缺失。结论 Ⅰ型、Ⅱ型SMA可通过SMN基因第7、8号外显子的检测进行确诊,Ⅲ型SMA患者SMN基因缺失率低,故通过检测SMN基因7、8外显子进行基因诊断尚需谨慎,Ⅳ型SMA未检测到SMN基因缺失,发病可能与SMN基因缺失无关;NAIP基因在SMA发病中的作用尚不清楚。