Two mutations within the coding sequence of the phenylalanine hydroxylase gene

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

CpG hotspot causes second mutation in codon 408 of the phenylalanine hydroxylase gene

Summary A new mutation has been identified in exon 12 of the gene encoding phenylalanine hydroxylase at codon 408. The single base change from guanine to adenine changes the amino acid arginine to glutamine; thus, the mutation is defined as R408Q. This codon is the site of a mutation known to causes phenylketonuria. Both these mutations are located at the same CpG site.     

Phenylalanine hydroxylase deficiency caused by a single base substitution in an exon of the human phenylalanine hydroxylase gene

A novel restriction fragment length polymorphism in the phenylalanine hydroxylase (PAH) locus generated by the restriction endonuclease MspI was observed in a German phenylketonuria (PKU) patient. Molecular cloning and DNA sequence analyses revealed that the MspI polymorphism was created by a T to C transition in exon 9 of the human PAH gene, which also resulted in the conversion of a leucine codon to a proline codon. The effect of the amino acid substitution was investigated by creating a corresponding mutation in a full-length human PAH cDNA by site-directed mutagenesis followed by expression analysis in cultured mammalian cells. Results demonstrate that the mutation in the gene causes the synthesis of an unstable protein in the cell corresponding to a CRM- phenotype. Together with the other mutations recently reported in the PAH gene, the data support previous biochemical and clinical observations that PKU is a heterogeneous disorder at the gene level.     

A de novo nonsense mutation in exon 28 of the neurofibromatosis type l (NF1) gene

We have screened a total of 105 unrelated patients with neurofibromatosis type l (NF1) for mutations in exon 28 of the NF1 gene using heteroduplex analysis and single strand conformation polymorphism analysis. One novel mutation has been identified and characterised. This mutation involves a 13-bp deletion (AAACTGGCTGAGC or AACTGGCTGAGCA) from base position 5077 (or 5078) to 5089 (or 5090) of the cDNA coding sequence. This alteration leads to a reading frame shift with a premature amber termination signal (TAG) at codon 1694. In addition, there is a change from lysine to threonine at codon 1693. The truncated gene product is estimated to be 1125 amino acid residues shorter than the predicted normal protein (2818 amino acids).     

A second transthyretin mutation at position 33 (Leu/Phe) associated with familial amyloidotic polyneuropathy

Genomic DNA was isolated from peripheral blood lymphocytes of a patient with familial amyloidotic polyneuropathy (FAP) and the transthyretin (TTR) gene examined for sequence mutations. Polymerase chain reaction was used to asymmetrically amplify the TTR exons. Direct DNA sequencing of the PCR product revealed a C for T mutation at the first base of codon 33 located in exon 2 of one transthyretin gene. This resulted in a substitution of leucine for phenylalanine at position 33. Exons 3 and 4 were examined and found to be normal. The mutation creates a novel DdeI restriction site at the point of the mutation.     

Computational and experimental analyses reveal previously undetected coding exons of the KRIT1 (CCM1) gene

A notable difficulty in annotating genomic sequence is identifying the correct start codon in a gene. An important such case has been found with KRIT1, the cerebral cavernous malformation type 1 (CCM1) gene. Analysis of human and mouse genomic sequence encompassing the region containing KRIT1/Krit1 using exon/gene-prediction and comparative alignment programs revealed putative exons upstream of the previously described first exon. These additional candidate exons show significant matches to mouse and human ESTs that are contiguous with and extend upstream from the previously designated 5' end of the KRIT1 cDNA sequence. RT-PCR and 5'RACE experiments confirm the presence of four additional upstream coding exons that encode an additional 207 amino acids. Importantly, a novel frameshift mutation in one of these newly identified KRIT1 exons has been found in a CCM1 family. These data establish the authentic KRIT1 amino acid sequence and suggest that the additional KRIT1 exons may harbor mutations in other CCM1 families. In addition, these results provide another example of the utility of rigorous computational and comparative sequence analysis for refining gene structure.     

Seven novel Tay-Sachs mutations detected by chemical mismatch cleavage of PCR-amplified cDNA fragments.

Total RNA was isolated from cultured fibroblasts from 12 unrelated patients with Tay-Sachs disease, an autosomal recessive disorder due to beta-hexosaminidase A deficiency. beta-Hexosaminidase mRNA was amplified by cDNA-PCR in four overlapping segments spanning the entire coding sequence. In two patients, abnormal size cDNA-PCR fragments in which exons were removed resulted from splicing mutations that were characterized at the genomic DNA level: both were G to A transitions, at the first position of intron 2 and at the fifth position of intron 4. Five other mutations have been identified by cDNA-PCR chemical mismatch analysis and direct sequencing of an amplified fragment containing the mismatch site. One missense mutation alters the codon for Ser210 to Phe in exon 6 and the other one alters the codon for Arg504 to Cys in exon 13. A 3-bp deletion results in the deletion of a phenylalanine residue in exon 8. Two nonsense mutations in exon 3 (Arg137 to stop) and in exon 11 (Arg393 to stop) are associated with a marked decrease of mRNA abundance, probably because they result in mRNA instability. Three of the six single base mutations involve the conversion of a CpG dinucleotide in the sense strand to TpG. These results demonstrate the extreme molecular heterogeneity of mutations causing Tay-Sachs disease. The procedure described in this paper allows the rapid detection of any type of mutation, except those impairing the promoter function. Applicable even to patients with splicing or nonsense mutations and very low mRNA abundance, it has therefore a potentially broad application in human genetics, for both diagnostic and fundamental purposes.     

Misreading of the argI message in Escherichia coli

It has previously been shown that either phenylalanine codon, UUU or UUC, could be misread as leucine during phenylalanine starvation, if the codons encoded residue 8 of the Escherichia coli argI gene product, ornithine transcarbamylase (OTC). However, no leucine misincorporation was detected when either of these same codons encoded residue 3. Here we report that leucine misincorporation can be directed by a UUU codon for residue 3 of OTC during phenylalanine starvation, if the argI gene has been mutated so that the codon preceding the UUU has been changed from the rarely used glycine codon GGG to the more commonly used GGC.     

Premature chromosome condensation is induced by a point mutation in the hamster RCC1 gene.

At the nonpermissive temperature, premature chromosome condensation (PCC) occurs in tsBN2 cells derived from the BHK cell line, which can be converted to the Ts+ phenotype by the human RCC1 gene. To prove that the RCC1 gene is the mutant gene in tsBN2 cells, which have RCC1 mRNA and protein of the same sizes as those of BHK cells, RCC1 cDNAs were isolated from BHK and tsBN2 cells and sequenced to search for mutations. The hamster (BHK) RCC1 cDNA encodes a protein of 421 amino acids homologous to the human RCC1 protein. In a comparison of the base sequences of BHK and BN2 RCC1 cDNAs, a single base change, cytosine to thymine (serine to phenylalanine), was found in the 256th codon of BN2 RCC1 cDNA. The same transition was verified in the RCC1 genomic DNA by the polymerase chain reaction method. BHK RCC1 cDNA, but not tsBN2 RCC1 cDNA, complemented the tsBN2 mutation, although both have the same amino acid sequence except for one amino acid at the 256th codon. This amino acid change, serine to phenylalanine, was estimated to cause a profound structural change in the RCC1 protein.     

Linkage and mutational analysis of familial Alzheimer disease kindreds for the APP gene region

A large number of familial Alzheimer disease (FAD) kindreds were examined to determine whether mutations in the amyloid precursor protein (APP) gene could be responsible for the disease. Previous studies have identified three mutations at APP codon 717 which are pathogenic for Alzheimer disease (AD). Samples from affected subjects were examined for mutations in exons 16 and 17 of the APP gene. A combination of direct sequencing and single-strand conformational polymorphism analysis was used. Sporadic AD and normal controls were also examined by the same methods. Five sequence variants were identified. One variant at APP codon 693 resulted in a Glu-->Gly change. This is the same codon as the hereditary cerebral hemorrhage with amyloidosis-Dutch type Glu-->Gln mutation. Another single-base change at APP codon 708 did not alter the amino acid encoded at this site. Two point mutations and a 6-bp deletion were identified in the intronic sequences surrounding exon 17. None of the variants could be unambiguously determined to be responsible for FAD. The larger families were also analyzed by testing for linkage of FAD to a highly polymorphic short tandem repeat marker (D21S210) that is tightly linked to APP. Highly negative LOD scores were obtained for the family groups tested, and linkage was formally excluded beyond theta = .10 for the Volga German kindreds, theta = .20 for early-onset non-Volga Germans, and theta = .10 for late-onset families. LOD scores for linkage of FAD to markers centromeric to APP (D21S1/S11, D21S13, and D21S215) were also negative in the three family groups. These studies show that APP mutations account for AD in only a small fraction of FAD kindreds.     

一中国脑海绵状血管瘤家系中发现krit1基因新的缺失突变

脑海绵状血管瘤（CCM）是多定位于中枢神经系统的一种脑部血管异常,少数在皮肤和视网膜处有并发症。依据致病基因在染色体上的不同位置分为CCM1、CCM2和CCM33种类型。目前,CCM1、CCM2和CCM3的致病基因已经被克隆,分别为krit1、MGC4607和细胞程序性死亡10基因（PDCD10）。利用连锁分析发现内蒙古的一个家系属于CCM1,突变检测发现患者CCM1基因（krit1）第9内含子和第10外显子拼接位点处存在一“GTA”缺失,该突变导致终止密码子提前出现,产生截短蛋白。实验结果支持krit1为CCM1致病基因。     

The genetic basis of Cowden’s syndrome: three novel mutations in PTEN/MMAC1/TEP1

Cowden’s syndrome (CS) is an autosomal dominant disorder associated with an increased risk of developing benign and malignant tumors in a variety of tissues, including the skin, thyroid, breast and brain. Women with CS are felt to have an increased risk of developing breast cancer, and virtually all women with CS develop bilateral fibrocystic disease of the breast. Recently, a series of germline mutations have been identified from CS families in a gene known as PTEN/MMAC1/TEP1. In this study, we used heteroduplex analysis and direct sequencing analysis and identified three novel germline mutations in the PTEN/MMAC1/TEP1 coding sequence from unrelated individuals with CS. We report a de novo transition (T→C) at nucleotide 335 in exon 5. This missense mutation resulted in a leucine to proline (CTA to CCA) change at codon 112. We also describe a novel splice site mutation (801+2T→G) in intron 7 that caused exon skipping in PTEN/MMAC1/TEP1 mRNA. The third mutation we report is a missense mutation, consisting of a transition (T→C) at nucleotide 202 in exon 3, resulting in a tyrosine to histidine (TAC to CAC) change at codon 68. Finally, we also detected a rare polymorphism in exon 7 of the PTEN/MMAC1/TEP1 coding sequence. These data confirm the observation that mutations of the PTEN/MMAC1/ TEP1 coding sequence are responsible for at least some cases of CS, and further define the spectrum of mutations in this autosomal dominant disorder. Received: 21 October 1997 / Accepted: 15 December 1997     

In vitro characterization of a human calcitonin receptor gene polymorphism

Calcitonin is a 32 amino acid peptide hormone that inhibits bone resorption by stimulating calcitonin receptors (CTR) located on the surfaces of osteoclasts. A polymorphism at nucleotide 1340 of the human calcitonin receptor gene (CALCR) lies within the coding region and has the potential to change the amino acid at codon 447 from leucine to proline. In the present study, we scanned the coding region, portions of the 5'-flanking and 3'-flanking sequences, and the intron-exon boundaries of the human CALCR gene for additional polymorphisms, and determined the frequency of the codon 447 polymorphism in several ethnic groups. Because a leucine to proline change has the potential for significant structural alteration, receptor genes encoding either leucine or proline at residue 447 were transiently expressed in COS-7 cells to determine the binding and functional consequences of this polymorphism. Our complete polymorphism scan of the CALCR gene identified 11 polymorphic sites in the gene and confirmed the presence of the previously identified nucleotide T1340C (codon 447) polymorphism. Ten of the 11 polymorphisms were single nucleotide polymorphisms (SNPs). For the codon 447 polymorphism, the prevalence of the TT genotype (leucine) was 59% in Caucasians, 27% in African-Americans, 0% in Asians, and 20% in Hispanics. The presence of this SNP appears to have no statistically significant difference with the receptor's ability to bind calcitonin or signal when activated with the hormone.     

Direct protein sequencing of wheat mitochondrial ATP synthase subunit 9 confirms RNA editing in plants

RNA editing, a process that results in the production of RNA molecules having a nucleotide sequence different from that of the initial DNA template, has been demonstrated in several organisms using different biochemical pathways. Very recently RNA editing was described in plant mitochondria following the discovery that the sequence of certain wheat and Oenothera cDNAs is different from the nucleotide sequence of the corresponding genes. The main conversion observed was C to U, leading to amino acid changes in the deduced protein sequence when these modifications occurred in an open reading frame. In this communication we show the first attempt to isolate and sequence a protein encoded by a plant mitochondrial gene. Subunit 9 of the wheat mitochondrial ATP synthase complex was purified to apparent homogeneity and the sequence of the first 32 amino acid residues was determined. We have observed that at position 7 leucine was obtained by protein sequencing, instead of the serine predicted from the previously determined genomic sequence. Also we found phenylalanine at position 28 instead of a leucine residue. Both amino acid conversions, UCA (serine) to UUA (leucine) and CUC (leucine) to UUC (phenylalanine), imply a C to U change. Thus our results seem to confirm, at the protein level, the RNA editing process in plant mitochondria.     

Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid

Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation.