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A number of patient-specific and leukemia-associated factors are related to the poor outcome in older patients with acute myeloid leukemia (AML). However, comprehensive studies regarding the impact of genetic alterations in this group of patients are limited. In this study, we compared relevant mutations in 21 genes between AML patients aged 60 years or older and those younger and exposed their prognostic implications. Compared with the younger patients, the elderly had significantly higher incidences of PTPN11, NPM1, RUNX1, ASXL1, TET2, DNMT3A and TP53 mutations but a lower frequency of WT1 mutations. The older patients more frequently harbored one or more adverse genetic alterations. Multivariate analysis showed that DNMT3A and TP53 mutations were independent poor prognostic factors among the elderly, while NPM1 mutation in the absence of FLT3/ITD was an independent favorable prognostic factor. Furthermore, the status of mutations could well stratify older patients with intermediate-risk cytogenetics into three risk groups. In conclusion, older AML patients showed distinct genetic alterations from the younger group. Integration of cytogenetics and molecular mutations can better risk-stratify older AML patients. Development of novel therapies is needed to improve the outcome of older patients with poor prognosis under current treatment modalities.

Glycogen-storage disease type II (GSDII; OMIM #232300), an autosomal recessive disorder caused by a deficiency of the glycogen hydrolysis enzyme acid alpha-glucosidase (acid GAA; acid maltase, EC. 3.2.10.20), results in the accumulation of glycogen in the lysosome. We performed a molecular genetic study on 29 patients with infantile-onset glycogen-storage disease type II (GSDII), 6 with juvenile-onset GSDII and one carrier for GSDII. Seventeen different mutations were identified among them; 8 were novel mutations: c.421C > A (p.L141M), c.872T > C (p.L291P), c.893A > C (p.Y298S), c.1375G > A (p.D459N), c.1437G > C (p.K479N), c.1509_1511del (p.A504del), c.1960T > C (p.S654P), and c.2174G > C (p.R725P). One of the mutations identified, c.2238G > C (p.W746C), which was a sequence change of unknown pathogenic significance causing diminished enzyme activity,was found homozygously in a juvenile-onset patient. We also found a juvenile-onset patient with homozygote c.1935C > A mutation which was frequently found in infantile-onset patients. In addition to mutations, we also identified 14 new polymorphisms in the acid alpha-glucosidase gene. The genotype/phenotype correlations indicated that c.2238G > C (p.W746C) is correlated with juvenile- onset GSDII and that c.872T > C (p.L291P) and c.1411_1414del (p.E471fsX5) are correlated with infantile-onset GSDII. Mutational analysis of GAA is useful in genetic counseling and prenatal diagnosis of the disease.

Mutations of the telomeric survival motor neuron gene (SMN1) are related to spinal muscular atrophy (SMA). However, no phenotype-genotype correlation has been observed since the SMN1 gene is lacking in the majority of patients affected with either the severe form (type I) or the milder forms (types II and III). Here, we analyze the SMN, NAIP and P44 genes in 132 Chinese SMA patients and their families. At least three types of normal allele, and four types of mutant allele were found in this study. The combination of one normal allele with one mutant allele resulted in carriers of different types, and the combination of different mutant alleles accounted for the different genotypes among different types of SMA. Deletions of mutant alleles can be further subgrouped into four types, which includes involving SMN1, SMN1 and NAIP(T) (telomeric portion of NAIP gene), SMN1 and NAIP(T) and P44(T) (telomeric portion of P44 gene), and SMN1 and SMN2 (centromeric portion of SMN gene). Some of the severe (type I) SMA cases correlated with the extent of deletions in the SMN, NAIP and P44 genes or the dosage of SMN gene when both SMN1 and SMN2 are deleted. We also found two novel point mutations, an A insertion at codon 8 (AGT-->AAGT) and an A substitution at codon 228 (TTA-->TAA).

Background: Crouzon syndrome is an autosomal dominant disorder causing premature fusion of the cranial suture. Mutations have been reported in exon IIIa or IIIc of the fibroblast growth factor receptor 2 (FGFR2) gene., Methods: In the present study, nine unrelated Crouzon syndrome patients were screened for mutations in the two exons of FGFR2 by polymerase chain reaction and direct sequencing., Results: Mutations were detected in 67% (6/9) of all cases. More than half the studied Crouzon patients carried a mutation resulting in either the loss or gain of a cysteine residue. A novel mutation, Tyr281Cys substitution, was discovered at exon IIIa., Conclusions: The mechanisms by which the same genotypes cause different phenotypes for each type of craniosynostosis syndrome in still uncertain. However, the molecular identification of the FGFR gene has made a great impact on the clinical classification of craniosynostosis syndromes; a new classification based on genotypes seems to be unavoidable.

The protein tyrosine kinase activity of c-src proto-oncogene product, pp60(c-src), is elevated in a number of human cancers, including colon cancer. Phosphorylation of human pp60(c-src) carboxy-terminal tyrosine 530 suppresses its kinase activity. A recent report suggested that the risk of colon cancer is higher for those who carry a C-->T transition mutation on codon 531 (Gln-531-->Amber-531) of src gene. This mutation caused a prematured translation termination and up-regulated the kinase activity. To examine whether this mutation could be a risk factor for colon carcinoma in the Chinese population, we used the same PCR-based assay to analyze src genotypes of 131 colon cancers and other various types of carcinoma. No mutation was detected in all specimens that were screened in this study. Thus, mutation at Gln-531 of src gene does not seem to be involved in the development of colon cancer in Chinese ethnicity.

Thanatophoric dysplasia (TD) is the most common form of lethal neonatal dwarfism with micromelic shortening of the limbs, macrocephaly, platyspondyly, and reduced thoracic cavity. R248C mutation in the extracellular domain of fibrobast growth factor receptor 3 (FGFR3) was common in TD type I. Two TD type I patients were examined for R248C mutation by use of restriction digestion and direct sequencing. The results showed that both patients carried R248C mutation. Because of the homogeneity of R248C mutation among different ethnic populations, all TD patients should be analysed using this PCR-based method presented in this work.

The 10q23.3 gene PTEN (phosphatase and Tensin homologue deleted on chromosome 10) or MMAC1 (mutated in multiple advanced cancers 1) was recently reported to undergo frequent mutation, including mutations and deletions in multiple advanced cancers. This study showed that the aberrant transcripts of this gene are frequently found in cancers of the digestive tract, paired non-cancerous tissues and normal peripheral mononuclear cells. Sequence analysis of the aberrant transcripts revealed three types of deletions: (i) a deletion junction with a splicing-like donor or acceptor sequence; (ii) several-base homology near or between the donor acceptor site at the deletion junction; and (iii) deletion with insertion. From these results, it is suggested that aberrant transcripts of PTEN/MMAC1 found by nested reverse transcription-polymerase chain reaction are a common (or natural) phenomenon unrelated to oncogenesis. The mechanism producing these aberrant transcripts needs further investigation. Using single-strand conformation polymorphism and direct sequencing to analyse for small base changes of the genomic DNA of the PTEN/MMAC1 gene revealed no point mutations or small base changes.

A mutant population of bamboo mosaic potexvirus (BaMV) was isolated after serial passage using Chenopodium quinoa plants. While the wild type inoculum induced indistinct chlorotic lesions, the mutant produced obvious lesions on C. quinoa although RNA accumulation of the mutant in Nicotiana benthamiana protoplasts was significantly reduced compared to wild type. Mutations were identified in the helicase-like domain. One RT-PCR-generated cDNA clone (designated pL1-33) representing the helicase-like region showed four nucleotide mutations encoding three amino acid changes that were shown to result in dramatically decreased viral accumulation. Independent analyses of the effects of these substitutions showed that nucleotide changes at position 1722 resulting in a leucine to proline switch and position 2129 resulting in a histidine to tyrosine switch had the greatest effect on viral accumulation. Combination of these two mutations resulted in a undetectable viral accumulation. We have identified that amino acids within the helicase domain but outside the universally conserved helicase-like motifs that play an important role in viral amplification.

beta-Thalassemia, a hematologic disorder characterized by the deficiency or the absence of beta-globin production, is the most widespread inherited disorder in the world; it is also common in Taiwan. We studied 38 patients in central Taiwan with beta-thalassemia major, using amplified created restriction site analysis for detection. On analysis, six different point mutations were found among 76 chromosomes, of which 32 chromosomes (42.1%) had a C to T substitution at nucleotide 654, 30 (40%) had frameshift codons 41/42 with four nucleotides (TCTT) deletion, 7 (9.2%) had an A to T substitution at codon 17, 3 (3.9%) had frameshift codons 71/72 (insertion of A), 2 (2.6%) had an A to G substitution at position -28, and 2 (2.6%) had frame-shift codons 27/28 (insertion of C). The first two mutations accounted for 62 of the 76 beta-thalassemia mutations in this study. As to mutations in each individual with beta-thalassemia major, the incidence of compound heterozygotes of two different mutations was higher than that of homozygotes of a single mutation (60% vs 40%). Compound heterozygotes of C to T substitution at nucleotide 654 of IVS-2 and frameshift codons 41/42 with four-nucleotide deletion was the most common pattern of beta-thalassemia mutations in each individual (23.7%). Our results were unique compared with those from similar studies performed in southern China. Frequencies of beta-thalassemia mutations found in the current study were assessed and compared with frequencies found in previous studies conducted in northern and southern Taiwan.

Wilson disease is an autosomal recessive disorder of copper metabolism. Mutation screening in Wilson disease has led to the detection of at least 89 disease-specific mutations. Some mutations appear to be population specific, while others are common to many populations. In this study, 38 Taiwanese patients with Wilson disease were screened using single-strand conformation polymorphism analysis, followed by direct DNA sequencing. We found 12 different mutations, six of which were novel. All our detected mutations were found to be in eight exons. Four mutations in three loci (Arg778Gln, Arg778Leu, Gly943Asp, and Pro992Leu) accounted for about 58% of the mutant alleles we detected. Using an RNA transcriptional assay, we confirmed that both of our detected splice-site mutations resulted in exon skipping.

Turnip yellow mosaic virus is a positive-strand RNA virus that produces light green or yellow-green mosaic symptoms in Chinese cabbage plants. We have characterized a strain that produces nearly uniform yellow-green chlorosis in systemically infected Chinese cabbage leaves. The increased symptom severity is due to the single nucleotide substitution U1888-->C, which results in a tyrosine to histidine substitution in the movement protein encoded by ORF-69. Coding by the overlapping ORF-206 is not affected. The mutation results in fourfold higher accumulations of viral products in systemically infected Chinese cabbage leaves but does not affect viral replication in isolated protoplasts. These results suggest that the increased viral yield and symptom severity result from improved viral spread in the host plant. These effects were specific to Chinese cabbage, since neither viral yield nor symptoms in turnips were affected by the U1888-->C mutation.

The 3' noncoding region of turnip yellow mosaic virus RNA includes an 82-nucleotide-long tRNA-like structure domain and a short upstream region that includes a potential pseudoknot overlapping the coat protein termination codon. Genomic RNAs with point mutations in the 3' noncoding region that result in poor replication in protoplasts and no systemic symptoms in planta were inoculated onto Chinese cabbage plants in an effort to obtain second-site suppressor mutations. Putative second-site suppressor mutations were identified by RNase protection and sequencing and were then introduced into genomic cDNA clones to permit their characterization. A C-57----U mutation in the tRNA-like structure was a strong suppressor of the C-55----A mutation which prevented both systemic infection and in vitro valylation of the viral RNA. Both of these phenotypes were rescued in the double mutant. An A-107----C mutation was a strong second-site suppressor of the U-96----G mutation, permitting the double mutant to establish systemic infection. The C-107 and G-96 mutations are located on opposite strands of one helix of a potential pseudoknot, and the results support a functional role for the pseudoknot structure. A mutation near the 5' end of the genome (G + 92----A), at position -3 relative to the initiation codon of the essential open reading frame 206, was found to be a general potentiator of viral replication, probably as a result of enhanced expression of open reading frame 206. The A + 92 mutation enhanced the replication of mutant TYMC-G96 in protoplasts but was not a sufficiently potent suppressor to permit systemic spread of the A + 92/G-96 double mutant in plants.

Single and multiple nucleotide substitutions have been introduced into the anticodon loop of the tRNA-like structure of turnip yellow mosaic virus (TYMV) genomic RNA. We studied the effects of these mutations on in vitro valylation and on replication in Chinese cabbage protoplasts and plants. Only those mutants capable of efficient and complete valylation showed efficient replication in protoplasts and gave rise to systemic symptoms in whole plants. Mutants that accepted valine inefficiently (in some cases Vmax/Km values were less than 10(-3) relative to wild-type values) replicated to levels 200- to 500-fold below wild-type levels in protoplasts (estimated on the basis of coat protein and genomic RNA levels). These mutants could not support systemic spread in plants. In one plant inoculated with TYMC-A55 RNA, which replicates poorly in protoplasts, systemic symptoms developed after a delay. The reversion in replication was accompanied by improved valine acceptance and the appearance of a U57 second-site mutation. Our results indicate a correlation between valine acceptance activity and viral yield. Possible roles for valylation are discussed, and the results are compared with those of similar studies with brome mosaic virus which suggested that tyrosylation is not crucial for brome mosaic virus replication (T. W. Dreher, A. L. N. Rao, and T. C. Hall, J. Mol. Biol. 206:425-438, 1989).