A novel mutant (transthyretin Ile-50) related to amyloid polyneuropathy. Single-strand conformation polymorphism as a new genetic marker.

DNA sequence polymorphisms in transthyretin (TTR) genes were investigated by single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction products. The amplified DNA fragments that encode each exon of the normal TTR gene showed two bands, representing the two complementary single strands of DNA. In one patient with amyloid polyneuropathy, the exon 3 DNA showed a unique, aberrant migration pattern. Direct sequencing analysis of the amplified exon 3 revealed a single base change (G-to-T), resulting in a novel amino acid substitution (Ser-50----Ile). We also present the SSCP patterns for five known Japanese TTR variants.     

Detection of sequence variations in the adenomatous polyposis coli (APC) gene using denaturing high-performance liquid chromatography

We have evaluated the usefulness of denaturing high performance liquid chromatography (dHPLC) for scanning the adenomatous polyposis coli (APC) gene for point mutations, small deletions, and insertions. Our assay consists of 28 sets of primers to amplify the 15 exons of the APC gene. All PCR reactions were amplified simultaneously using the same reaction conditions in a 96-well format and then analyzed by dHPLC, using empirically determined optimum temperatures for partial fragment denaturation. Previously studied DNA specimens from 47 familial adenomatous polyposis (FAP) patients were analyzed by dHPLC and all mutations were correctly identified and confirmed by sequence analysis. This approach identified a single-base substitution in exon 6 and a 2-bp insertion in exon 15 that initially had not been detected by single-strand conformational polymorphism (SSCP) analysis. A novel mutation in exon 15 of the APC gene, 2065delG (codon 689) that had previously been undetected by the protein truncation test (PTT) was also identified by dHPLC. We present our validation studies of dHPLC technology for APC gene analysis in terms of sensitivity and specificity and compare it to current standard scanning technologies including PTT, SSCP, and conformational sensitive gel electrophoresis (CSGE).     

Rapid identification of mutations in the glucocerebrosidase gene of Gaucher disease patients by analysis of single-strand conformation polymorphisms

To detect mutations in the glucocerebrosidase gene in Gaucher disease patients, we used the recently described technique of single-strand conformation polymorphism (SSCP) analysis in combination with selective amplification. We analyzed exon 8, 9, 10 and 11 of the glucocerebrosidase gene; these exons were sequentially amplified using the selectively amplified products as templates. We found variant SSCP patterns corresponding to the presence or absence of the 6433C mutation, which was detected by NciI digestion analysis, in exon 10. Furthermore, we detected four variant SSCP patterns in exon 8, 10 and 11. Sequencing analysis consistently revealed four single-base substitutions in the corresponding exons, three novel missense mutations (5409A, 6375G and 6682T) and one silent polymorphism (6594A). These mutations were found only in one patient; therefore, these findings have confirmed the marked genetic heterogeneity of Gaucher disease. SSCP analysis in combination with selective amplification is a rapid and sensitive procedure for the screening of the mutations in the glucocerebrosidase gene of patients with Gaucher disease.     

人肝细胞癌中抑癌基因PTEN／MMAC1／TEP1的突变分析

PTEN/MMAC1/TEP1 is a tumor suppressor gene. Its mutation has been found in several different types of human cancers. 34 primary human hepatocellular carcinomas have been examined for mutations in exon 5 and exon 8 of the PTEN gene. Exon 5 and exon 8 were amplified by polymerase chain reaction (PCR) with intronic primers and subjected to single strand conformation polymorphism (SSCP) analysis. SSCPs were found in 4 of the 34 hepatocellular carcinomas analyzed. Direct sequencing of the PCR products identified single base-pair substitutions in the four tumor DNA samples, two in intron 4 and two in exon 8. One of the base-pair substitution in exon 8 is a missense mutation, which changed codon 304 of PTEN protein from Cys to Gly. These data suggest that PTEN may be involved in the carcinogenesis and development of hepatocellular carcinoma.     

人肝细胞癌中抑癌基因PTEN/MMAC1/TEP1的突变分析

PTEN／MMAC1／TEP1(PTEN)是新近分离到的抑癌基因,在多种肿瘤中存在突变。我们检测了34例人肝细胞癌中PTEN基因第5外显子和第8外显子的突变。采用聚合酶链方法以内含子引物扩增第5和第8外显子,继之以单链构象多态性和测序技术分析PTEN基因突变。有4例肝细胞癌SSCP显示异常条带并经测序证实存在突变。2例发生于第4内含子,突变位点相同;另两例发生于第8外显子,其中1例碱基颠换导致PTEN蛋白产物304位半胱氨酸突变为甘氨酸。     

MHC-DRB exon 2 allele polymorphism in Indian river buffalo (Bubalus bubalis)

The polymorphism of the major histocompatibility complex (MHC) class II DRB gene of riverine buffalo (Bubalus bubalis) was studied. Second exon sequences from the buffalo DRB locus, homologous to the cattle DRB3 gene, were amplified and characterized. A combination of single strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) in a non-denaturing gel was used to identify new DRB second exon sequences. SSCP, HA and finally sequencing allowed the identification of 22 MHC-DRB exon 2 alleles from 25 unrelated Indian river buffalo. These are the first river buffalo DRB second exon sequences reported. A high degree of polymorphism in the sequences encoding the peptide binding regions was observed and some amino acid substitutions were found unique to the river buffalo.     

Mutation analysis of Taiwanese Wilson disease patients

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism, which is caused by mutation in copper-transporting ATPase (ATP7B). In the present study, we report a molecular diagnosis method to screen the WD chromosome in patients or in heterozygotic carriers in Taiwan. Exons 8, 11, 12, 13, 16, 17, and 18 of ATP7B are selected for the screening of mutations. The most common mutation, Arg778Leu or Arg778Gln, was first screened by PCR-RFLP then we combined single-stranded conformation polymorphism (SSCP) analysis followed by direct DNA sequencing on the DNA fragments with mobility shift on SSCP analysis. The diagnostic rate was compared with standard ATP7B whole gene sequencing analysis. Ten different mutations were identified among 29 WD patients; among them four were novel (Ala1168Pro, Thr1178Ala, Ala1193Pro, and Pro1273Gln). The false positive rates were tested against 100 normal individuals and listed as follows: exon 8: 5%; exon 11: 4%; exon 12: 6%; exon 13: 5%; exon 16: 5%; exon 17: 3%; exon 18: 4%. The Arg778Leu mutation exhibited the highest allelic frequency (43.1%). The detection rate of WD chromosomes is 65.52%, which is as sensitive as whole gene sequencing scanning. According to our results, WD chromosomes in Taiwan are predominantely located at exons 8, 11, 12, 13, 16, 17, and 18. The standard sequencing analysis on the entire gene is time consuming. We recommend screening these 7 exons first on those individuals who have a higher risk in having WD, before whole gene and promoter sequencing analysis in Taiwan.     

中国人先天性巨结肠RET基因突变特征研究

为了研究中国人先天性巨结肠(HD)RET基因突变特征,从分子水平探讨先天性巨结肠症(HD)的发病机理,揭示中国人群HD与RET基因突变的关系。我们应用聚合酶链反应-单链构象多态性(PCR—SSCP)-DNA测序方法,对50例HD患儿和30例无便秘正常对照者,进行RET基因第13外显子突变筛查,并对第13外显子突变阳性患儿家系作研究。结果发现在50例HD患儿中,有7例第13外显子扩增片段在SSCP分析时出现泳动变位。经DNA测序证实,有3种点突变类型(错义、同义和移码),第13外显子突变率为14％(7／50)。在7例第13外显子突变的患儿中,发现2例患儿的父亲存在与其子相同的突变。实验表明中国先天性巨结肠人群存在着RET基因突变,且以杂和性点突变为主,HD具有一定的遗传性。     

中国人先天性巨结肠RET基因突变特征研究

为了研究中国人先天性巨结肠(HD)RET基因突变特征,从分子水平探讨先天性巨结肠症(HD)的发病机理,揭示中国人群HD与RET基因突变的关系。我们应用聚合酶链反应-单链构象多态性(PCR-SSCP)-DNA测序方法,对50例HD患儿和30例无便秘正常对照者,进行RET基因第13外显子突变筛查,并对第13外显子突变阳性患儿家系作研究。结果发现在50例HD患儿中,有7例第13外显子扩增片段在SSCP分析时出现泳动变位。经DNA测序证实,有3种点突变类型(错义、同义和移码),第13外显子突变率为14%(7/50)。在7例第13外显子突变的患儿中,发现2例患儿的父亲存在与其子相同的突变。实验表明中国先天性巨结肠人群存在着RET基因突变,且以杂和性点突变为主,HD具有一定的遗传性。     

New mutant gene (transthyretin Arg 58) in cases with hereditary polyneuropathy detected by non-isotope method of single-strand conformation polymorphism analysis.

Single-strand conformation polymorphism (SSCP) was analyzed to detect a mutation in the transthyretin (TTR) gene from the mother and son showing polyneuropathy with carpal tunnel syndrome. DNA segments containing TTR coding sequence were amplified by polymerase chain reaction, heat denatured and electrophoresed on a neutral polyacrylamide gel. The single-stranded DNA fragments in the gel were transferred to a nylon membrane and hybridized with biotinylated TTR cDNA probe, followed with chemiluminescent DNA detection. The mobility shift was found in the fragments of exon 3 from the patients' DNA. Sequencing analyses of the exon 3 confirmed a T----G base change, resulting in a Leu 58----Arg substitution. TTR Arg 58 is the first mutant TTR gene that has been detected by SSCP analysis. The rapid and sensitive detection of new mutations at various sites on the TTR gene is hereafter possible by the present method in the facilities for non-radioactive experiments.     

Molecular evolution of the leptin exon 3 in some species of the family Canidae

The structure of the leptin gene seems to be well conserved. The polymorphism of this gene in four species belonging to the Canidae family (the dog (Canis familiaris) – 16 different breeds, the Chinese racoon dog (Nyctereutes procyonoides procyonoides), the red fox (Vulpes vulpes) and the arctic fox (Alopex lagopus)) were studied with the use of single strand conformation polymorphism (SSCP), restriction fragment length polymorphism (RFLP) and DNA sequencing techniques. For exon 2, all species presented the same SSCP pattern, while in exon 3 some differences were found. DNA sequencing of exon 3 revealed the presence of six nucleotide substitutions, differentiating the studied species. Three of them cause amino acid substitutions as well. For all dog breeds studied, SSCP patterns were identical.     

Molecular diagnostics for myelin proteolipid protein gene mutations in Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is a clinically heterogeneous, slowly progressive leukodystrophy. The recent detection of mutations in the myelin proteolipid protein (PLP) gene in several PMD patients offers the opportunity both to design DNA-based tests that would be useful in diagnosing a proportion of PMD cases and, in particular, to evaluate the diagnostic utility of single-strand conformation polymorphism (SSCP) analysis for this disease. A combination of SSCP analysis and direct sequencing of PCR-amplified DNA was used to screen for PLP mutations in 24 patients affected with leukodystrophies of unknown etiology. Two heretofore undescribed mutations in the PLP gene were identified, Asp202His in exon 4 and Gly73Arg in exon 3. The ease and efficiency of SSCP analysis in detecting new mutations support the utilization of this technique in screening for PLP mutations in patients with unexplained leukodystrophies.     

Four novel PEPD alleles causing prolidase deficiency.

Mutations at the PEPD locus cause prolidase deficiency (McKusick 170100), a rare autosomal recessive disorder characterized by iminodipeptiduria, skin ulcers, mental retardation, and recurrent infections. Four PEPD mutations from five severely affected individuals were characterized by analysis of reverse-transcribed, PCR-amplified (RT-PCR) cDNA. We used SSCP analysis on four overlapping cDNA fragments covering the entire coding region of the PEPD gene and detected abnormal SSCP bands for the fragment spanning all or part of exons 13-15 in three of the probands. Direct sequencing of the mutant cDNAs showed a G-->A, 1342 substitution (G448R) in two patients and a 3-bp deletion (delta E452 or delta E453) in another. In the other two probands the amplified products were of reduced size. Direct sequencing of these mutant cDNAs revealed a deletion of exon 5 in one patient and of exon 7 in the other. Intronic sequences flanking exons 5 and 7 were identified using inverse PCR followed by direct sequencing. Conventional PCR and direct sequencing then established the intron-exon borders of the mutant genomic DNA revealing two splice acceptor mutations: a G-->C substitution at position -1 of intron 4 and an A-->G substitution at position -2 of intron 6. Our results indicate that the severe form of prolidase deficiency is caused by multiple PEPD alleles. In this report we attempt to begin the process of describing these alleles and cataloging their phenotypic expression.     

Four New Mutations and Two Polymorphic Variants of the Low-Density Lipoprotein Receptor Gene in Familial Hypercholesterolemia Patients from St. Petersburg

In a collection of DNA samples from 100 unrelated patients with clinical features of familial hypercholesterolemia (FH), a search for mutations of exons 4 and 10 of the low-density lipoprotein (LDL) receptor gene was performed using heteroduplex and single-strand conformational polymorphism (SSCP) analyses followed by sequencing of amplified DNA fragments. Four new mutations of the LDL receptor gene were identified: C146R (c.499 T > C), A130P (c.451 G > C), G128G (c.477 T > C), and C188Y (c.626 G > A). Mutation A130P was assigned to the same chromosome with allele variant 447C. Two polymorphic sites in exon 10 of the LDL receptor gene (1413G/A and 1545C/T) were found in the Russian population for the first time. Based on the data obtained, familial hypercholesterolemia was confirmed in seven patients.     

Four new mutations and polymorphic variants of the low density lipoprotein receptor in patients with familial hypercholesterolemia in Saint Petersburg

In a collection of DNA samples from 100 unrelated patients with clinical features of familial hypercholesterolemia (FH), a search for mutations of exons 4 and 10 of the low-density lipoprotein (LDL) receptor gene was performed using heteroduplex and single-strand conformational polymorphism (SSCP) analyses followed by sequencing of amplified DNA fragments. Four new mutations of the LDL receptor gene were identified: C146R (c.499 T > C), A130P (c.451 G > C), G128G (c.477 T > C), and C188Y (c.626 G > A). Mutation A130P was assigned to the same chromosome with allele variant 447C. Two polymorphic sites in exon 10 of the LDL receptor gene (1413G/A and 1545C/T) were found in the Russian population for the first time. Based on the data obtained, familial hypercholesterolemia was confirmed in seven patients.     

Identification of a novel mutation in the ARSB gene that is frequent among Brazilian MPSVI patients

Mucopolysaccharidosis type VI, or Maroteaux-Lamy syndrome, is an autosomal recessive disease caused by the deficiency of arylsulfatase B (ARSB; N-acetyl-galactosamine-4-sulfatase, E.C.3.1.6.12), which is involved in the stepwise degradation of dermatan sulfate and chondroitin sulfate. The deficiency of this enzyme causes storage in the lysozomes and excretion in the urine of partially degraded dermatan sulfate. Twenty patients with MPSVI were analyzed, including 2 siblings. Genomic DNA from patients was extracted and amplified by PCR followed by analysis by single-strand conformation polymorphism (SSCP), which detects altered patterns in the single-stranded DNA. Amongst the patients analyzed for exon 8 of the ARSB gene, 5 patients presented an altered band pattern when compared to controls. After sequencing, we have detected a 23-bp deletion, extending from nucleotides 1,533 to 1,555, causing a frameshift and changing 2 amino acids before creating a premature stop codon at amino acid 514.     

Autosomal dominant retinal dystrophy (Rdy) in Abyssinian cats: exclusion of PDE6G and ROM1 and likely exclusion of Rhodopsin as candidate genes

Retinal dystrophy (Rdy) is an autosomal dominant photoreceptor dysplasia of Abyssinian cats and a model for autosomal dominant retinitis pigmentosa (ADRP) in man. We have pursued a candidate gene approach in the search for the causal mutation in Rdy. The genes RHO (encoding rhodopsin), ROM1 (encoding the structural retinal outer-membrane protein-1) and PDE6G (encoding the gamma subunit of the visual transduction protein cyclic guanosine monophosphate-phosphodiesterase) were polymerase chain reaction-amplified from normal feline genomic DNA. Leader, coding and 3' untranslated regions of each gene, and parts of introns were sequenced. Single-stranded conformation polymorphism (SSCP) analysis of Rdy-affected and normal cats was used to identify intragenic polymorphisms within ROM1 and PDE6G. DNA sequencing of all three genes in Rdy-affected cats was used to confirm results from SSCP. For both ROM1 and PDE6G polymorphisms identified by SSCP and sequencing showed disconcordance between the polymorphism and the disease phenotype within an Rdy disease pedigree. SSCP analysis of RHO performed across the 5' untranslated region, the entire coding sequence and the intron/exon boundaries in Rdy-affected and control cats failed to identify any intragenic polymorphisms that could be used for linkage analysis. DNA sequencing of these regions showed no differences between Rdy-affected and control cats. Mutations in ROM1 or in PDE6G are not causative of feline Rdy. The absence of potentially pathogenic polymorphisms in sequenced portions of the RHO gene makes it unlikely that a mutation in this gene is the cause of Rdy.     

Leishmania donovani: intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing

Four polymerase chain reaction (PCR)-based approaches were used to analyze diversity within 23 Sudanese isolates of Leishmania donovani. Methods compared were fingerprinting with single nonspecific primers, restriction analysis of the amplified ribosomal internal transcribed spacer (ITS) locus, single-stranded conformation polymorphism (SSCP), and sequencing of the ITS region. When PCR fingerprinting and restriction analysis of ITS were applied, highly similar fragment patterns were observed for all strains of L. donovani studied. The ITS1 locus gave five different SSCP profiles among the 23 Sudanese isolates, whereas the ITS2 locus was highly conserved with the exception of 1 isolate. Strains of L. donovani derived from other geographical areas were found to have different ITS2 patterns. SSCP analysis correlated well with results of DNA sequencing and confirmed that SSCP was able to detect genetic diversity at the level of a single nucleotide. SSCP had advantages over the other methods employed for investigation of sequence variation within the species L. donovani. There was no correlation between the form of clinical manifestation of the disease and the PCR fingerprinting, ITS-RFLP, or ITS-SSCP characteristics.     

SNP frequency and allelic haplotype structure of Beta vulgaris expressed genes

Based on EST sequences, fragments of 37 genes have been amplified and sequenced in two inbred lines of sugar beet. The rate of single nucleotide polymorphisms (SNP) corresponded to 1 every 130 bp, with an average (nucleotide diversity) value of 7.6×10^–3. When extrapolated to the whole sugar beet genome, randomly compared lines differ at 5.4×10⁶ SNPs in the genetic pool considered. In a wider search for SNP-related polymorphisms, 96 fragments of expressed genes were scanned with SSCP (single-strand conformation polymorphism) and heteroduplex (HA) analyses in 8 inbred lines. One SSCP or HA polymorphism was found every 1,470 bp of amplified DNA, corresponding to 5×10⁵ SSCP or HA loci in the whole genome. This frequency, 11 times lower than the SNP rate, was attributed to the high frequency of base pair substitution along the amplified fragment analysed electrophoretically. Therefore nucleotide variability was further studied by sequencing fragments of 10 genes in the same 8 lines. The results indicate that sugar beet alleles of expressed genes are very frequently organized as robust intragene haplotypes. In the 8 lines analysed, two haplotypes were identified for each of three gene fragments, three haplotypes for six gene fragments and four haplotypes for one gene fragment which is in good correspondence with the number of alleles detected by SSCP and HA analysis. In a cross between two lines, SSCP or HA alleles of expressed genes have 54% probability to be different.