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PubMed2001

Identification of mutations including de novo mutations in Korean patients with hypokalaemic periodic paralysis.

BACKGROUND: Hypokalaemic periodic paralysis (hypoPP) is an autosomal dominant disorder involving the abnormal function of ion channels and it is characterized by paralysis attacks of varying severity, accompanied by a fall in blood potassium levels. Linkage analysis showed that the candidate locus responsible for hypoPP was localized to chromosome 1q31-32, and this locus encoded the muscle dihydropyridine-sensitive calcium channel alpha(1)-subunit (CACNA1S). So far, three different mutations in CACNA1S gene have been identified in patients with hypoPP: Arg528His, Arg1239His and Arg1239Gly in Caucasian patients. However, there are few reports about the mutations of CACNA1S gene in other races. METHODS: In this study, four Korean families with five hypoPP patients were screened for mutations of CACNA1S gene with polymerase chain reaction-based restriction analysis and single-strand conformation polymorphism analysis. To determine the mode of inheritance, haplotype analysis was done with three microsatellite markers (D1S1726, CACNL1A3, and D1S1723). RESULTS: Arg528His mutation was detected in three families, and one family had no known mutations. Moreover, for the first time, we detected de novo Arg528His mutations in two out of three families with hypoPP. Haplotype analysis using three microsatellite markers (D1S1726, CACNL1A3, and D1S1723) suggested the occurrence of de novo Arg528His mutations in two of the three families with Arg528His mutation. CONCLUSIONS: Arg528His mutations of CACNA1S, including de novo Arg528His mutations, were found in Korean patients with hypoPP. These results imply that de novo mutation, in addition to non-penetrance, is one of the genetic mechanisms that can explain the previous clinical observation that hypoPP occurs sporadically without family history.

Adolescent↗
PubMed2005

Patterns of p53 mutations separate ovarian serous borderline tumors and low- and high-grade carcinomas and provide support for a new model of ovarian carcinogenesis: a mutational analysis with immunohistochemical correlation.

The infrequent association of serous borderline tumors (SBTs) with invasive serous carcinoma has led to the view that SBTs are unrelated to invasive serous carcinoma. Nonetheless, mortality associated with SBTs is generally attributed to malignant transformation, and traditionally these tumors have been designated as "carcinomas of low malignant potential." Previous immunohistochemical studies evaluating p53 expression and molecular genetic studies evaluating mutational status have reported that p53 overexpression and mutations are infrequent in SBTs and occur in as many as 50% to 80% of invasive serous carcinomas. The different methodologies for determining p53 status and the failure to correlate the findings with tumor grade make these studies difficult to interpret. The current study was undertaken to overcome these deficiencies and to reconcile the relationship of SBTs to invasive serous carcinoma by performing a morphologic, immunohistochemical, and molecular genetic analysis comparing SBTs with low- and high-grade serous carcinoma. The molecular genetic analysis used a highly stringent, carefully designed nucleotide-sequencing method. A total of 96 sporadic serous tumors including 25 SBTs (11 atypical proliferative serous tumors and 14 intraepithelial low-grade serous carcinomas [noninvasive micropapillary serous carcinomas, MPSCs]), 12 low-grade serous carcinomas (invasive MPSCs), and 59 high-grade serous carcinomas were analyzed for their p53 mutational status of exons 5 to 9. Functional mutations, defined as mutations resulting in the alteration of the structure of the encoded protein, were detected in 30 of 59 (50.8%) high-grade serous carcinomas and 1 (8.3%) of 12 low-grade invasive serous carcinomas compared with 2 (8%) of 25 SBTs, both of these in intraepithelial low-grade serous carcinomas (noninvasive MPSCs). The similar frequency of p53 mutations in SBTs and low-grade invasive serous carcinomas in contrast to the significantly higher frequency of p53 mutations in high-grade serous carcinomas (P < 0.0005) suggests a common lineage for SBTs and low-grade invasive serous carcinomas and supports the view that SBTs are unrelated to the usual type of invasive serous carcinoma, which is a high-grade neoplasm. Mutational status was also correlated with p53 immunoreactivity. Although p53 immunoreactivity is generally higher in those specimens containing mutant p53, immunostaining is neither sufficiently specific nor sensitive enough to predict p53 mutations. The molecular genetic findings confirm our hypothesis of dual pathways of serous carcinogenesis based on previous analyses of KRAS and BRAF mutations on the same set of cases in which KRAS and BRAF mutations were found in 60% of SBTs and low-grade serous carcinoma but not in high-grade serous carcinomas. Based on these studies, we have proposed a model of serous carcinogenesis in which SBTs are the precursors of low-grade serous carcinomas whereas the usual type of invasive serous carcinoma is a high-grade neoplasm that develops "de novo" from in situ alterations in epithelial inclusion cysts.

Base Sequence↗
PubMed1999

Mechanisms of mutation in nondividing cells. Insights from the study of adaptive mutation in Escherichia coli.

When populations of cells are subjected to nonlethal selection, mutations arise in the absence of cell division, a phenomenon that has been called "adaptive mutation." In a strain of Escherichia coli that cannot metabolize lactose (Lac-) but that reverts to lactose utilization (Lac+) when lactose is its sole energy and carbon source, the mutational process consists of two components. (1) A highly efficient, recombination-dependent mechanism giving rise to mutations on the F' episome that carries the Lac- allele; and (2) a less efficient, unknown mechanism giving rise to mutations elsewhere in the genome. Both selected and nonselected mutations arise in the Lac- population, but nonselected mutations are enriched in Lac+ mutants, suggesting that some Lac+ cells have passed though a transient period of increased mutation. These results have several evolutionary implications. (1) DNA synthesis initiated by recombination could be an important source of spontaneous mutation, particularly in cells that are not undergoing genomic replication. (2) The highly active mutational mechanism on the episome could be important in the horizontal transfer of variant alleles among species that carry and exchange conjugal plasmids. (3) A sub-population of cells in a state of transient mutation could be a source of multiple variant alleles and could provide a mechanism for rapid adaptive evolution under adverse conditions.

Adaptation, Biological↗
PubMed2006

Human immunodeficiency virus type 1 coreceptor switching: V1/V2 gain-of-fitness mutations compensate for V3 loss-of-fitness mutations.

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is mediated by the virus envelope binding to CD4 and the conformationally altered envelope subsequently binding to one of two chemokine receptors. HIV-1 envelope glycoprotein (gp120) has five variable loops, of which three (V1/V2 and V3) influence the binding of either CCR5 or CXCR4, the two primary coreceptors for virus entry. Minimal sequence changes in V3 are sufficient for changing coreceptor use from CCR5 to CXCR4 in some HIV-1 isolates, but more commonly additional mutations in V1/V2 are observed during coreceptor switching. We have modeled coreceptor switching by introducing most possible combinations of mutations in the variable loops that distinguish a previously identified group of CCR5- and CXCR4-using viruses. We found that V3 mutations entail high risk, ranging from major loss of entry fitness to lethality. Mutations in or near V1/V2 were able to compensate for the deleterious V3 mutations and may need to precede V3 mutations to permit virus survival. V1/V2 mutations in the absence of V3 mutations often increased the capacity of virus to utilize CCR5 but were unable to confer CXCR4 use. V3 mutations were thus necessary but not sufficient for coreceptor switching, and V1/V2 mutations were necessary for virus survival. HIV-1 envelope sequence evolution from CCR5 to CXCR4 use is constrained by relatively frequent lethal mutations, deep fitness valleys, and requirements to make the right amino acid substitution in the right place at the right time.

Amino Acid Sequence↗
PubMed1994

Nonsense but not missense mutations can decrease the abundance of nuclear mRNA for the mouse major urinary protein, while both types of mutations can facilitate exon skipping.

In an effort to understand the mechanisms by which nonsense codons affect RNA metabolism in mammalian cells, nonsense mutations were generated within the gene for the secretory major urinary protein (MUP) of mice. The translation of MUP mRNA normally begins within exon 1 and terminates within exon 6, the penultimate exon. Through the use of Northern (RNA) blot hybridization and assays that couple reverse transcription and PCR, a nonsense mutation within codon 50 of exon 2 or codon 143 of exon 5 was found to reduce the abundance of fully spliced, nuclear MUP mRNA to 10 to 20% of normal without an additional reduction in the abundance of cytoplasmic mRNA. In contrast, a nonsense mutation within codon 172 of exon 5 was found to have no effects on the abundance of MUP mRNA. These findings suggest that a boundary between nonsense mutations that do and do not reduce the abundance of nuclear mRNA exists within the exon preceding the exon that harbors the normal site of translation termination. In this way, the boundary is analogous to the boundary that exists within the penultimate exon of the human gene for the cytosolic enzyme triosephosphate isomerase. Assays for exon skipping, i.e., the removal of an exon as a part of the flanking introns during the process of splicing, reveal that 0.1, 2.0, and 0.1% of MUP mRNA normally lack exon 5, exon 6, and exons 5 plus 6, respectively. Relative to normal, the two nonsense mutations within exon 5 increase the abundance of RNA lacking exon 5 on average 20-fold and increase the abundance of RNA lacking exons 5 plus 6 on average 5-fold. Since only one of these nonsense mutations also reduces the abundance of fully spliced nuclear mRNA to 10 to 20% of normal, the two mechanisms by which a nonsense mutation can alter nuclear RNA metabolism must be distinct. The analysis of missense mutations within codons 143 and 172, some of which retain the nonsense mutation, indicates that the reduction in the abundance of fully spliced nuclear mRNA is dependent upon the premature termination of MUP mRNA translation, whereas skipping is attributable to nonsense mutation-mediated changes in exon 5 structure rather than to the premature termination of translation. The increase in exon 5 skipping by either the nonsense or missense mutations within codon 172 correlates with a decrease in the complementarity of exon 5 to U1 snRNA. This suggests that a 5' splice site may extend as far as 12 nucleotides into the upstream exon, which is, to our knowledge, the largest extension.

Animals↗
PubMed2003

A novel mutation of the calcium sensing receptor gene is associated with chronic pancreatitis in a family with heterozygous SPINK1 mutations.

BACKGROUND: The role of mutations in the serine protease inhibitor Kazal type 1 (SPINK1) gene in chronic pancreatitis is still a matter of debate. Active SPINK1 is thought to antagonize activated trypsin. Cases of SPINK1 mutations, especially N34S, have been reported in a subset of patients with idiopathic chronic pancreatitis. However, the inheritance pattern is still unknown. Some cases with N34S heterozygosity have been reported with and without evidence for CP indicating neither an autosomal recessive nor dominant trait. Therefore SPINK1 mutations have been postulated to act as a disease modifier requiring additional mutations in a more complex genetic model. Familial hypocalciuric hypercalcemia (FHH) caused by heterozygous inactivating mutations in the calcium sensing receptor (CASR) gene is considered a benign disorder with elevated plasma calcium levels. Although hypercalcemia represents a risk factor for pancreatitis, increased rates of pancreatitis in patients with FHH have not been reported thus far. METHODS: We studied a family with a FHH-related hypercalcemia and chronic pancreatitis. DNA samples were analysed for mutations within the cationic trypsinogen (N29I, R122H) and SPINK1 (N34S) gene using melting curve analysis. Mutations within CASR gene were identified by DNA sequencing. RESULTS: A N34S SPINK1 mutation was found in all screened family members. However, only two family members developed chronic pancreatitis. These patients also had FHH caused by a novel, sporadic mutation in the CASR gene (518T>C) leading to an amino acid exchange (leucine->proline) in the extracellular domain of the CASR protein. CONCLUSION: Mutations in the calcium sensing receptor gene might represent a novel as yet unidentified predisposing factor which may lead to an increased susceptibility for chronic pancreatitis. Moreover, this family analysis supports the hypothesis that SPINK1 mutations act as disease modifier and suggests an even more complex genetic model in SPINK1 related chronic pancreatitis.

Adult↗
PubMed2005

TP53 mutations in ovarian carcinomas from sporadic cases and carriers of two distinct BRCA1 founder mutations; relation to age at diagnosis and survival.

BACKGROUND: Ovarian carcinomas from 30 BRCA1 germ-line carriers of two distinct high penetrant founder mutations, 20 carrying the 1675delA and 10 the 1135insA, and 100 sporadic cases were characterized for somatic mutations in the TP53 gene. We analyzed differences in relation to BRCA1 germline status, TP53 status, survival and age at diagnosis, as previous studies have not been conclusive. METHODS: DNA was extracted from paraffin embedded formalin fixed tissues for the familial cases, and from fresh frozen specimen from the sporadic cases. All cases were treated at our hospital according to protocol. Mutation analyses of exon 2-11 were performed using TTGE, followed by sequencing. RESULTS: Survival rates for BRCA1-familial cases with TP53 mutations were not significantly lower than for familial cases without TP53 mutations (p = 0.25, RR = 1.64, 95% CI [0.71-3.78]). Median age at diagnosis for sporadic (59 years) and familial (49 years) cases differed significantly (p < 0.001) with or without TP53 mutations. Age at diagnosis between the two types of familial carriers were not significantly different, with median age of 47 for 1675delA and 52.5 for 1135insA carriers (p = 0.245). For cases > or = 50 years at diagnosis, a trend toward longer survival for sporadic over familial cases was observed (p = 0.08). The opposite trend was observed for cases < 50 years at diagnosis. CONCLUSION: There do not seem to be a protective advantage for familial BRCA1 carriers without TP53 mutations over familial cases with TP53 mutations. However, there seem to be a trend towards initial advantage in survival for familial cases compared to sporadic cases diagnosed before the age of 50 both with and without TP53 mutations. However, this trend diminishes over time and for cases diagnosed > or = 50 years the sporadic cases show a trend towards an advantage in survival over familial cases. Although this data set is small, if confirmed, this may be a link in the evidence that the differences in ovarian cancer survival reported, are not due to the type of BRCA1 mutation, but may be secondary to genetic factors shared. This may have clinical implications for follow-up such as prophylactic surgery within carriers of the two most frequent Norwegian BRCA1 founder mutations.

Adult↗
PubMed1999

BRCA1/BRCA2 germline mutations in locally recurrent breast cancer patients after lumpectomy and radiation therapy: implications for breast-conserving management in patients with BRCA1/BRCA2 mutations.

PURPOSE: Breast cancer patients treated conservatively with lumpectomy and radiation therapy (LRT) have an estimated lifetime risk of local relapse (ipsilateral breast tumor recurrence [IBTR]) of 10% to 15%. For breast cancer patients carrying BRCA1 or BRCA2 (BRCA1/2) mutations, the outcome of treatment with LRT with respect to IBTR has not been determined. In this study, we estimate the frequency of BRCA1/2 mutations in a study of breast cancer patients with IBTR treated with LRT. PATIENTS AND METHODS: Between 1973 and 1994, there were 52 breast cancer patients treated with LRT who developed an IBTR within the prior irradiated breast and who were willing to participate in the current study. From our database, we also identified 52 control breast cancer patients treated with LRT without IBTR. The control patients were individually matched to the index cases with respect to multiple clinical and pathologic parameters. Lymphocyte DNA specimens from all 52 locally recurrent patients and 15 of the matched control patients under age 40 were used as templates for polymerase chain reaction amplification and dye-primer sequencing of exons 2 to 24 of BRCA1, exons 2 to 27 of BRCA2, and flanking intron sequences. RESULTS: After LRT, eight (15%) of 52 breast cancer patients had IBTR with deleterious BRCA1/2 mutations. By age, there were six (40%) of 15 patients with IBTR under age 40 with BRCA1/2 mutations, one (9.0%) of 11 between ages 40 and 49, and one (3.8%) of 26 older than age 49. In comparison to the six (40%) of 15 of patients under age 40 with IBTR found to have BRCA1/2 mutations, only one (6.6%) of 15 matched control patients without IBTR and had a BRCA1/2 mutation (P =.03). The median time to IBTR for patients with BRCA1/2 mutations was 7.8 years compared with 4.7 years for patients without BRCA1/2 mutations (P =.03). By clinical and histologic criteria, these relapses represented second primary tumors developing in the conservatively treated breast. All patients with BRCA1/2 mutations and IBTR underwent successful surgical salvage mastectomy at the time of IBTR and remain alive without evidence of local or systemic progression of disease. CONCLUSION: In this study, we found an elevated frequency of deleterious BRCA1/2 mutations in breast cancer patients treated with LRT who developed late IBTR. The relatively long time to IBTR, as well as the histologic and clinical criteria, suggests that these recurrent cancers actually represent new primary breast cancers. Early onset breast cancer patients experiencing IBTR have a disproportionately high frequency of deleterious BRCA1/2 mutations. This information may be helpful in guiding management in BRCA1 or BRCA2 patients considering breast-conserving therapy.

Adult↗
PubMed1995

Three novel mutations of thyroid hormone receptor beta gene in unrelated patients with resistance to thyroid hormone: two mutations of the same codon (H435L and H435Q) produce separate subtypes of resistance.

We report three novel mutations of the thyroid hormone receptor beta (TR beta) gene in three unrelated Japanese patients with resistance to thyroid hormone (RTH). Patients A and B exhibited generalized resistance phenotype, while patient C displayed more pituitary-selective unresponsiveness. Direct sequencing of TR beta gene exon 10 disclosed novel point mutations in all three patients. A Phe to Ile (TTC-->ATC) substitution at codon 451, a His to Leu (CAT-->CTT) substitution at codon 435, and a His to Gln (CAT-->CAA) substitution at codon 435 were identified in patients A, B, and C, respectively. Sequencing of TR beta gene exons 5-9 as well as TR alpha gene exons 4-9 failed to detect any additional mutations. All three patients were heterozygous for respective mutations. The unaffected parents of patients A and B, having normal thyroid function, possessed no mutations of TR beta gene exon 10, indicating that the F451I and H435L mutations occurred de novo. The F451I mutation is located near the most frequent mutation site in the ligand 2 subdomain. The identical codon mutations H435L and H435Q, which lie at the extreme carboxyl-terminus of the dimerization subdomain near the 9th heptad, were found in clinically different subtypes of RTH: patient B with generalized resistance and patient C with pituitary-selective resistance, respectively. The mutations broaden the growing catalogue of the TR beta gene mutations that could cause different phenotypes, despite the defects at the same codon.

Adult↗
PubMed2004

DJ-1 (PARK7) mutations are less frequent than Parkin (PARK2) mutations in early-onset Parkinson disease.

BACKGROUND: Mutations in the Parkin gene (PARK2) are the most commonly identified cause of recessively inherited early-onset Parkinson disease (EOPD) but account for only a portion of cases. DJ-1 (PARK7) was recently reported as a second gene associated with recessively inherited PD with a homozygous exon deletion and a homozygous point mutation in two families. METHODS: To investigate the frequency of DJ-1 mutations, the authors performed mutational analysis of all six coding exons of DJ-1 in 100 EOPD patients. For the detection of exon rearrangements, the authors developed a quantitative duplex PCR assay. Denaturing high performance liquid chromatography analysis was used to screen for point mutations and small deletions. Further, Parkin analysis was performed as previously described. RESULTS: The authors identified two carriers of single heterozygous loss-of-function DJ-1 mutations, including a heterozygous deletion of exons 5 to 7 and an 11-base pair deletion, removing the invariant donor splice site in intron 5. Interestingly, both DJ-1 mutations identified in this study were found in the heterozygous state only. The authors also detected a polymorphism (R98Q) in 1.5% of the chromosomes in both the patient and control group. In the same patient sample, 17 cases were detected with mutations in the Parkin gene. CONCLUSIONS: Mutations in DJ-1 are less frequent than mutations in Parkin in EOPD patients but should be considered as a possible cause of EOPD. The effect of single heterozygous mutations in DJ-1 on the nigrostriatal system, as described for heterozygous changes in Parkin and PARK6, remains to be elucidated.

Adolescent↗
PubMed1996

Low frequency of p53 mutations in human thyroid tumours; p53 and Ras mutation in two out of fifty-six thyroid tumours.

OBJECTIVE: p53 is a well-known nuclear phosphoprotein encoded by a suppressor gene know to be mutated in various kinds of human tumours. A relationship between p53 gene mutation and tumour progression seems to be a common feature of several neoplasias. DESIGN: In order to investigate the role of p53 mutations in human thyroid tumours, DNA samples derived from fifty-six neoplastic tissues, ranging from benign adenomas to undifferentiated carcinomas, were examined for the presence of p53 gene mutations. METHODS: The analysis has been conducted using polymerase chain reaction (PCR) amplification of the exons 5-9 of the p53 gene followed by single strand conformation polymorphism (SSCP) and sequence analyses. RESULTS: One anaplastic carcinoma and one papillary carcinoma showed p53 gene mutations in exons 5 and 8, respectively. A cell line established from the papillary carcinoma showed the same mutation present in the original tumour. Both p53 mutations were heterozygous. The p53 positive samples were analysed for other genetic alterations frequently detected in human thyroid carcinomas (mutations of the RET, TRK, and ras oncogenes): both p53-mutated samples proved to be mutated at level of codon 13 of the c-Ki-ras gene. CONCLUSIONS: Our data confirm that p53 gene alterations are rare in well-differentiated thyroid tumours, that they are an important requirement for the establishment in culture of human thyroid carcinoma cell lines, and that they can be associated with other genetic alterations, namely ras mutations, in the malignant progression of thyroid tumours.

Base Sequence↗
PubMed2005

Why are phenotypic mutation rates much higher than genotypic mutation rates?

The evolution of genotypic mutation rates has been investigated in numerous theoretical and experimental studies. Mutations, however, occur not only when copying DNA, but also when building the phenotype, especially when translating and transcribing DNA to RNA and protein. Here we study the effect of such phenotypic mutations. We find a maximum phenotypic mutation rate, umax, that is compatible with maintaining a certain function of the organism. This may be called a phenotypic error threshold. In particular, we find a minimum phenotypic mutation rate, umin, with the property that there is (nearly) no selection pressure to reduce the rate of phenotypic mutations below this value. If there is a cost for lowering the phenotypic mutation rate, then umin is close to the optimum phenotypic mutation rate that maximizes the fitness of the organism. In our model, there is selective pressure to decrease the rate of genotypic mutations to zero, but to decrease the rate of phenotypic mutations only to a positive value. Despite its simplicity, our model can explain part of the huge difference between genotypic and phenotypic mutation rates that is observed in nature. The relevant data are summarized.

Animals↗
PubMed2003

[Relationship between mutation on precore region of integrated HBV DNA and p53 gene mutation in hepatocellular carcinoma].

BACKGROUND & OBJECTIVE: The chronic infection of hepatitis B virus (HBV) is a recognized risk factor for the development of hepatocellular carcinoma (HCC); however, the precise mechanism of carcinogenesis is unknown. In order to better understand the molecular mechanism of hepatocarcinogenesis, this study was designed to investigate the relationship between the gene mutation on precore region of integrated HBV DNA, the mutation of p53 gene and the development of HCC. METHODS: The integrated HBV DNA, gene mutation on precore region of HBV DNA and p53 gene mutation were detected in 80 specimens of HCC, paratumor cirrhosis, liver cirrhosis, and normal liver tissue using polymerase chain reaction (PCR) and single strand conformation polymorphism (SSCP) technique. RESULTS: (1) The positive rates of integrated HBV DNA in HCC tissues, paratumor cirrhosis tissues, cirrhotic liver tissues, and normal liver tissues were 96.43%, 96.43%, 66.67%, and 6.66%, respectively. There were significant differences of the positive rates between normal liver tissues group and each of the other three groups (P< 0.005). (2) The gene mutation rates on precore region of HBV DNA in the four groups were 70.37%, 48.15%, 25.00%, and 0%, respectively. There were significant differences of the gene mutation rates among the four groups (P< 0.05). (3) The mutation rate (57.14%)of p53 gene in HCC was significantly higher than that in liver cirrhosis (16.67%) (P< 0.05). (4) The p53 gene mutation was positively associated with positivity of integrated HBV DNA and mutation on precore region of integrated HBV DNA in different liver tissues. CONCLUSION: The HBV DNA integration and its gene mutation on precore region and p53 gene mutation probably play synergic roles in the development of HCC.

Adult↗
PubMed1991

Catalytic triad residue mutation (Asp156----Gly) causing familial lipoprotein lipase deficiency. Co-inheritance with a nonsense mutation (Ser447----Ter) in a Turkish family.

We studied the molecular basis of familial Type I hyperlipoproteinemia in two brothers of Turkish descent who had normal plasma apolipoprotein C-II levels and undetectable plasma post-heparin lipoprotein lipase (LPL) activity. We cloned the cDNAs of LPL mRNA from adipose tissue biopsies obtained from these individuals by the polymerase chain reaction and directional cloning into M13 vectors. Direct sequencing of pools of greater than 2000 cDNA clones indicates that their LPL mRNA contains two mutations: a missense mutation changing codon 156 from GAU to GGU predicting an Asp156----Gly substitution and a nonsense mutation changing the codon for Ser447 from UCA to UGA, a stop codon, predicting a truncated LPL protein that contains 446 instead of 448 amino acid residues. Both patients were homozygous for both mutations. Analysis of genomic DNAs of the patients and their family members by the polymerase chain reaction, restriction enzyme digestion (the GAT----GGT mutation abolishes a TaqI restriction site), and allele-specific oligonucleotide hybridization confirms that the patients were homozygous for these mutations at the chromosomal level, and the clinically unaffected parents and sibling were true obligate heterozygotes for both mutations. In order to examine the functional significance of the mutations in this family, we expressed wild type and mutant LPLs in vitro using a eukaryotic expression vector. Five types of LPL proteins were produced in COS cells by transient transfection: (i) wild type LPL, (ii) Asp156----Gly mutant, (iii) Ser447----Ter mutant, (iv) Gly448----Ter mutant, and (v) Asp156----Gly/Ser447----Ter double mutant. Both LPL immunoreactive mass and enzyme activity were determined in the culture media and intracellularly. Immunoreactive LPLs were produced in all cases. The mutant LPLs, Asp156----Gly and Asp156----Gly/Ser447----Ter, were devoid of enzyme activity, indicating that the Asp156----Gly mutation is the underlying defect for the LPL deficiency in the two patients. The two mutant LPLs missing a single residue (Gly448) or a dipeptide (Ser447-Gly448) from its carboxyl terminus had normal enzyme activity. Thus, despite its conservation among all mammalian LPLs examined to date, the carboxyl terminus of LPL is not essential for enzyme activity. We further screened 224 unrelated normal Caucasians for the Ser447----Ter mutation and found 36 individuals who were heterozygous and one individual who was homozygous for this mutation, indicating that it is a sequence polymorphism of no functional significance. Human LPL shows high homology to hepatic triglyceride lipase and pancreatic lipase.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue↗
PubMed1997

K-ras gene mutations in normal colorectal tissues from K-ras mutation-positive colorectal cancer patients.

K-ras gene mutations have been reported as early events in colorectal tumorigenesis, but their role in tumor initiation and development is still unclear. To analyze and compare K-ras mutational patterns between colorectal tissues at different stages of tumor progression in individual patients, 65 colorectal tissue samples, including carcinoma, adenoma, histologically normal mucosa, submucosal muscularis propria, and histologically normal mucosa distant from tumor, were obtained from 13 patients with colorectal cancer. In addition, normal mucosal tissues obtained from four normal individuals were analyzed. Each of the 13 tumors was shown previously to harbor a mutation in either codon 12 or 13 of the K-ras gene by direct sequencing. These tissues were reanalyzed, using the recently established mutant allele enrichment + denaturing gradient gel electrophoresis method, which can detect one mutant allele in 10(4)-10(5) normal alleles, thus allowing for the analysis of infrequent cells bearing mutations against the background of wild-type cells. No K-ras codon 12 mutation was detected by this method in the histologically normal mucosal tissues sampled at the margin of resection distant from the tumor or in those obtained from four normal individuals. On the other hand, these mutations were detected in 9 of 10 adenoma and 6 of 10 mucosa samples from 10 patients with known K-ras codon 12 mutations, and also in 2 of 3 carcinoma, 2 of 3 adenoma, and 1 of 3 mucosa samples obtained from 3 patients with known K-ras codon 13 mutations. Thus, K-ras codon 12 mutations were found to occur with a high frequency (53.8%) in histologically normal mucosa adjacent to tumors of patients with K-ras mutation-positive colorectal cancer, suggesting that they may be useful biomarkers for early detection of colorectal cancer. Furthermore, multiple K-ras mutations were found in tissues of nearly half of the 13 patients, indicating that distinct evolutionary subclones may be involved in the development of tumor in some patients with colorectal cancer.

Adenoma↗
PubMed1997

p53 mutations in head and neck cancer: new data and evaluation of mutational spectra.

It has been suggested that the frequency, type, and location of p53 mutations (mutational spectra) can be linked to specific exogenous and endogenous carcinogenic agents and processes. Squamous cell carcinoma of the head and neck (SCCHN) provides an excellent tumor model to evaluate the utility of the p53 mutational spectra, given that it has well-defined and strong risk factors (tobacco and alcohol). The purpose of this analysis was to establish the pattern of p53 mutations in SCCHN and evaluate this mutational spectrum in comparison to the spectra for other cancers with similar and different risk factors, including cancers of the esophagus, lung, and colon. p53 mutational data were obtained from head and neck tumors collected at the University of North Carolina Hospitals and the published literature. A total of 14 of 33 tumors from the University of North Carolina Hospitals (42%) were found to have a p53 mutation. The alterations included three transversions, seven transitions, two deletions, and two suspected codon 47 polymorphisms. In general, SCCHN and esophageal cancer share a similar mutational pattern in contrast to colon cancer. These two aerodigestive tract cancers were statistically different from lung cancer, despite sharing tobacco as a major risk factor. For example, G-->T transversions, a mutation type considered to be characteristic of exogenous DNA-damaging agents including tobacco smoke carcinogens, varied among tobacco-related cancer sites (14% SCCHN, 11% esophageal, and 31% lung) in contrast to colon cancer (6%). The comparison of mutational spectra for SCCHN and other cancers indicates that the effects of both tobacco and alcohol exposure may yield a pattern of p53 mutations that reflects elements of both exogenous and endogenous exposures.

Adult↗
PubMed2003

Fourteen novel OPA1 mutations in autosomal dominant optic atrophy including two de novo mutations in sporadic optic atrophy.

The OPA1 gene, encoding a dynamin-related GTPase that plays a role in mitochondrial biogenesis, is implicated in most cases of autosomal dominant optic atrophy (ADOA). Sixty-nine pathogenic OPA1 mutations have been reported so far. Most of these are truncating mutations located in the GTPase domain coding region (exons 8-16) and at the 3'-end (exons 27-28). We screened 44 patients with typical ADOA using PCR-sequencing. We also tested 20 sporadic cases of bilateral optic atrophy compatible with ADOA. Of the 18 OPA1 mutations found, 14 have never been previously reported. The novel mutations include one nonsense mutation, 3 missense mutations, 6 deletions, one insertion and 3 exon-skipping mutations. Two of these are de novo mutations, which were found in 2 patients with sporadic optic atrophy. The recurrent c.2708_2711delTTAG mutation was found in 2 patients with a severe congenital presentation of the disease. These results suggest that screening for OPA1 gene mutations may be useful for patients with optic atrophy who have no affected relatives, or when the presentation of the disease is atypical as in the case of early onset optic atrophy.

Alternative Splicing↗
PubMed1999

Comparison of complementary and genomic DNA sequencing for the detection of mutations in the HMBS gene in British patients with acute intermittent porphyria: identification of 25 novel mutations.

Acute intermittent porphyria (AIP) is a low-penetrant autosomal dominant disorder caused by mutations in the hydroxymethylbilane synthase (HMBS) gene. Direct detection of mutations is becoming the method of choice for the accurate identification of asymptomatic affected individuals within AIP families so that they can be advised to avoid drugs and other compounds that provoke the life-threatening acute neurovisceral crises that characterise the condition. We describe a prospective comparison of direct automated sequencing of cDNA (29 patients) or genomic DNA (28 patients) to identify HMBS mutations in 57 patients referred consecutively for mutational analysis; 39 different mutations were identified in 54 patients. The sensitivity of the cDNA and genomic DNA methods was 69% and 95%, respectively, indicating that analysis of genomic DNA provides a higher mutation detection rate. Thirty mutations were restricted to a single family; only one (R173W) occurred in more than three families. Of the mutations (6 missense, 8 splice defects, 10 frameshifts, 1 nonsense), 25 have not been reported previously. One novel mutation (344+33G-->T) was located in a putative intron splice enhancer in intron7. Our results define the extent of allelic heterogeneity and the types (41% missense; 59% truncating) and distribution (35% in exons 10, 12, 14) of HMBS mutations, for AIP in the United Kingdom.

DNA, Complementary↗