Mutation analysis of PAH gene and characterization of a recurrent deletion mutation in Korean patients with phenylketonuria

Phenylketonuria (PKU; MIM 261600) is an autosomal recessive metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH; EC 1.14.16.1). Point mutations in the PAH gene are known to cause PKU in various ethnic groups, and large deletions or duplications account for up to 3% of the PAH mutations in some ethnic groups. However, a previous study could not identify approximately 14% of the mutant alleles by sequence analysis in Korean patients with PKU, which suggests that large deletions or duplication might be frequent causes of PKU in Koreans. To test this hypothesis, we performed multiplex ligation-dependent probe amplification (MLPA) for the identification of uncharacterized mutant alleles after PAH sequence analysis of 33 unrelated Korean patients with PKU. Bi-directional sequencing of the PAH exons and flanking intronic regions revealed 27 different mutations, including four novel mutations (two missense and two deletion mutations), comprising 57/66 (86%) mutant alleles. MLPA identified a large deletion that encompassed exons 5 and 6 in four patients, another large deletion that extended from exon 4 to exon 7 in one patient, and a duplication of exon 4 in one patient. Chromosomal walking characterized the deletion breakpoint of the most common large deletion that involved exons 5 and 6 (c.456_706+138del). The present study shows that the allelic frequency of exon deletion or duplication is 9% (6/66) in Korean PKU patients, which suggests that these mutations may be frequent causes of PKU in Korean subjects.     

Identification of deletion and duplication genotypes of the PMP22 gene using PCR-RFLP, competitive multiplex PCR, and multiplex ligation-dependent probe amplification: a comparison

We evaluated the efficacy of PCR-RFLP, competitive multiplex PCR, and a commercially available system of multiplex ligation-dependent probe amplification (MLPA) for the determination of deletion and duplication genotypes of the PMP22 gene. We compared the methods for efficiency, sensitivity, and specificity. We determined the gene dosage of the PMP22 gene via PCR-RFLP, competitive multiplex PCR, and MLPA. To demonstrate the sensitivity and accuracy of these three methods, a total of 185 samples from 42 patients with hereditary neuropathy with liability to pressure palsies (HNPP), 57 patients with Charcot-Marie-Tooth disease type 1A (CMT1A), and 86 unaffected individuals, were analyzed. Molecular diagnosis by PCR-RFLP was performed on all 185 samples; 24 HNPP deletions and 33 CMT1A duplications were identified. In contrast, 25 HNPP deletions and 38 CMT1A duplications were identified correctly using competitive multiplex PCR and MLPA. Six samples were incorrectly identified by PCR-RFLP (one HNPP deletion and five CMT1A duplications). Competitive multiplex PCR and MLPA demonstrated reliability and relative speed compared to PCR-RFLP; they were superior to PCR-RFLP for gene dosage quantification. Multiplex PCR and MLPA should be the methods of choice for detection of deletion and duplication genotypes in molecular genetic diagnoses.     

Exon deletions of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemics

A consistent finding of many studies describing the spectrum of mutant phenylalanine hydroxylase (PAH) alleles underlying hyperphenylalaninemia is the impossibility of achieving a 100% mutation ascertainment rate using conventional gene-scanning methods. These methods include denaturing gradient gel electrophoresis (DGGE), denaturing high performance liquid chromatography (DHPLC), and direct sequencing. In recent years, it has been shown that a significant proportion of undetermined alleles consist of large deletions overlapping one or more exons. These deletions have been difficult to detect in compound heterozygotes using gene-scanning methods due to a masking effect of the non-deleted allele. To date, no systematic search has been carried out for such exon deletions in Italian patients with phenylketonuria or mild hyperphenylalaninemia. We used multiplex ligation- dependent probe amplification (MLPA), comparative multiplex dosage analysis (CMDA), and real-time PCR to search for both large deletions and duplications of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemia patients. Four deletions removing different phenylalanine hydroxylase (PAH) gene exons were identified in 12 patients. Two of these deletions involving exons 4-5-6-7-8 (systematic name c.353-?_912 + ?del) and exon 6 (systematic name c.510-?_706 + ?del) have not been reported previously. In this study, we show that exon deletion of the PAH gene accounts for 1.7% of all mutant PAH alleles in Italian hyperphenylalaninemics.     

Use of multiplex PCR and CE for gene dosage quantification and its biomedical applications for SMN, PMP22, and alpha-globin genes

Many genetic diseases are caused by the presence of point mutations, small insertions, and deletions in respective genes, and the number of diseases known to be caused by deletions and duplications involving large DNA genomes is increasing. These changes lead to underexpression or overexpression of the gene, according to changes in gene dosage. The methods for the detection of point mutations, small insertions, and deletions are well established, but the detection of larger genomic deletions or duplications is more difficult. Due to the lack of efficient and technically feasible protocols for gene dosage quantification, we describe a diagnostic protocol employing a combination of available methods. The efficient and accurate gene dosage quantification platform is combined with multiplex PCR and CE, and applied to detect dosages of several genes, including SMN, PMP22, and alpha-globin genes. The reliability of this novel methodology shows that it is a relatively speedy and low-cost procedure and a significant tool for genetic diagnosis. Its sensitivity and specificity for identifying deletion and duplication genotypes approach 100%. Moreover, once we establish this powerful system, we will further apply this technique to the rapid detection of trisomy syndromes and microdeletion syndromes, including trisomy 13, Down syndrome, DiGeorge syndrome, and others.     

Multiplex quantitative foodborne pathogen detection using high resolution CE-SSCP coupled stuffer-free multiplex ligation-dependent probe amplification

Sensitive multiplex detection methods for foodborne pathogens are important in controlling food safety, and detection of genetic markers is accepted to be one of the best tools for sensitive detection. Although CE technology offers great potential in terms of sensitive multiplex detection, the necessary amplification is confined to markers sharing common primers such as the 16S rRNA gene. For precise and sensitive detection, pathogen-specific genes are optimal markers. Although multiplex ligation-dependent probe amplification (MLPA) is appropriate for amplification of specific markers, the requirement for stuffers, to ensure length-dependent separation on CE, is a major obstacle in detection of foodborne pathogens. In the present study, we developed stuffer-free MLPA using high-resolution CE-SSCP to sensitively detect ten foodborne pathogens. The probe set for MLPA prior to CE-SSCP analysis was designed for species-specific detection. After careful optimization of each MLPA step, to ensure that CE-SSCP analysis was informative, we found that all ten pathogens could be reliably identified; the limits of detection were 0.5-5 pg of genomic DNA, and more than 100-fold increase could be quantitatively determined. Thus, MLPA-CE-SSCP is a sensitive and reliable technique for pathogen detection.     

BRCA1 mutation screening using restriction endonuclease fingerprinting-single-strand conformation polymorphism in an automated capillary electrophoresis system

Efficient mutation scanning techniques are needed for the rapid detection of novel disease-associated mutations and rare-sequence variants of putative importance. The large size of the breast cancer 1 gene (BRCA1) and the many mutations found throughout its entire coding sequence make screening for mutations in this gene particularly challenging. We have developed a method for screening exon 11 of the BRCA1 gene based on restriction enzyme digestion of fluorescence-labeled polymerase chain reaction (PCR) products followed by single-strand conformation polymorphism (SSCP) using an automated capillary electrophoresis system, denoted capillary restriction endonuclease fingerprinting (REF)-SSCP electrophoresis. Using this strategy on a control set of samples, we were able to detect 17 of 18 known sequence alterations. The method was then applied to screen 73 Norwegian females with family histories of breast and/or ovarian cancer. A total of 172 sequence alterations were detected, including substitutions, insertions, and deletions. One novel substitution of unknown function was identified. Sequencing of all samples negative in the capillary REF-SSCP system gave no additional mutations confirming the high sensitivity of the described methodology. Capillary REF-SSCP electrophoresis appeared as a technically convenient technique, requiring amplification of fewer PCR fragments than traditional SSCP. The novel strategy allows high-throughput mutation scanning without radioactive labeling and polyacrylamide gel electrophoresis (PAGE).     

Stuffer‐free multiplex ligation‐dependent probe amplification based on conformation‐sensitive capillary electrophoresis: A novel technology for robust multiplex determination of copy number variation

Developing diagnostic tools based on the application of known disease/phenotype‐associated copy number variations (CNVs) requires the capacity to measure CNVs in a multiplex format with sufficient reliability and methodological simplicity. In this study, we developed a reliable and user‐friendly multiplex CNV detection method, termed stuffer‐free MLPA‐CE‐SSCP, that combines a variation of multiplex ligation‐dependent probe amplification (MLPA) with CE‐SSCP. In this variation, MLPA probes were designed without the conventionally required stuffer sequences. To separate the similar‐sized stuffer‐free MLPA products, we adopted CE‐SSCP rather than length‐dependent conventional CE analysis. An examination of the genomic DNA from five cell lines known to vary in X‐chromosome copy number (1–5) revealed that copy number determinations using stuffer‐free MLPA‐CE‐SSCP were more accurate than those of conventional MLPA, and the CV of the measured copy numbers was significantly lower. Applying our system to measure the CNVs on autosomes between two HapMap individuals, we found that all peaks for CNV targets showed the expected copy number changes. Taken together, our results indicate that this new strategy can overcome the limitations of conventional MLPA, which are mainly related to long probe length and difficulties of probe preparation.     

Screening for genetic mutations. A review

A point mutation of a nucleotide within a single gene can have a profound effect on a specific organ and/or the entire human body. DNA sequences associated with human diseases may differ from the corresponding normal sequences by single nucleotide mutations or by large alterations such as deletions, insertions, duplications, or translocations of DNA segments or entire chromosomes. As a result of the heterogeneity of DNA alterations and genetic mutations, various screening approaches are required to detect these alterations. However, methods which facilitate the detection of large mutations in the genome are typically insensitive to point mutations, whereas methods which detect point mutations are not appropriate to detect large alterations within the genome. Since there is no single perfect method to screen for unknown mutations, combinations of these methods may be necessary for accurate genetic diagnosis. The applications of polymerase chain reaction (PCR) technology to genomic screening have made rapid and accurate genetical diagnosis possible. Furthermore, recent developments in the technology of DNA microarrays have opened the way for high throughput sequence analysis by hybridization, which shows great potential in both molecular biology and medicine in the near future.     

Multiplex polymerase chain reaction analysis of UV-A- and UV-B-induced delayed and early mutations in V79 Chinese hamster cells

We previously reported that approximately 10% of V79 Chinese hamster fibroblast populations clonally derived from single cells immediately after irradiation with either ultraviolet B (UV-B, 290-320 nm, mainly 311 nm) or ultraviolet A (UV-A, 320-400 nm, mainly 350-390 nm) radiation exhibit genomic instability. The instability is revealed by relatively high mutation frequencies in the hypoxanthine phosphoribosyl transferase (hprt) gene up to 23 cell generations after irradiation. These delayed mutant clones exhibited higher levels of oxidative stress than normal cells. Therefore, persistently increased oxidative stress has been proposed as a mechanism for UV-induced genomic instability. This study investigates whether this mechanism is reflected in the deletion spectrum of delayed mutant clones. Eighty-eight percent of the delayed mutant clones derived from UV-A-irradiated populations were found to have total deletion of the hprt gene. Correspondingly, 81% of UV-A-induced early mutations (i.e. detected shortly after irradiation) also had total deletions. Among delayed UV-B-induced mutant clones, 23% had total deletions and 8% had deletion of one exon, whereas all early UV-B events were either point mutations or small deletions or insertions. In conclusion, the multiplex polymerase chain reaction deletion screen showed that there were explicit differences in the occurrence of large gene alterations between early and delayed mutations induced by UV-B radiation. For UV-A radiation the deletion spectra were similar for delayed and early mutations. UV-A radiation is, in contrast to UV-B radiation, only weakly absorbed by DNA and probably induces mutation almost solely via production of reactive oxygen species. Therefore, the present results support the hypothesis that persistent increase in oxidative stress is involved in the mechanism of UV-induced genomic instability.     

Quantitative polymerase chain reaction and microchip electrophoresis to detect major rearrangements of the low-density lipoprotein receptor gene causing familial hypercholesterolemia

A variety of rearrangements in the low-density lipoprotein receptor (LDLR) gene cause severe forms of familial hypercholesterolemia (FH). However, current methods for searching these abnormalities in FH samples, e.g., Southern and Northern Blot, are labor-intensive and not routinely used by diagnostic laboratories. We developed a simpler approach based on the quantitative polymerase chain reaction (PCR) amplification of part or all gene's coding sequences by a series of multiplex amplifications comprising three nonadjacent gene sections plus a fourth section used as an internal reference. Thereafter, the analysis of these PCR products by microchip electrophoresis revealed either deletions or duplications in the investigated gene sections through the simple comparison of electropherograms obtained from mutant and control samples. This required primers leading to well-resolved peaks with minimal size differences among coamplified products and PCR conditions allowing a linear quantitative response to template amount variations as those caused by duplication or deletion of specific gene sections. Also, the inclusion of exon 17 amplification product as an internal reference in each multiplex PCR allowed the normalization of quantitative results by dividing the area of each amplified section by the area of exon 17. The comparison of these ratios calculated from 10 carriers of 6 LDLR known rearrangements with those obtained from 14 control samples showed that gross deletions roughly halved and duplications doubled the ratio values of exons involved in the mutation. This allowed to distinguish gross mutations from sample-to-sample differences that reached at maximum 8% variation over mean values.     

Colorimetric quantification of mRNA expression in rare tumour cells amplified by multiple ligation-dependent probe amplification

An enzyme-linked oligonucleotide assay (ELONA) for quantification of mRNA expression of five genes involved in breast cancer, extracted from isolated rare tumour cells and amplified by multiplex ligation-dependent probe amplification (MLPA) is presented. In MLPA, a multiplex oligonucleotide ligation assay is combined with a PCR reaction in which all ligation products are amplified by use of a single primer pair. Biotinylated probes complementary to each of the target sequences were immobilised on the surface of a streptavidin-coated microtitre plate and exposed to single-stranded MLPA products. A universal reporting probe sequence modified with horseradish peroxidase (URP–HRP) and complementary to a universal primer used during the MLPA step was further added to the surface-bound duplex as a reporter probe. Simultaneous addition of anchoring probe and target, followed by addition of reporter probe, rather than sequential addition, was achieved with no significant effect on sensitivity and limits of detection, but considerably reduced the required assay time. Detection limits as low as 20 pmol L⁻¹, with an overall assay time of 95 min could be achieved with negligible cross-reactivity between probes and non-specific targets present in the MLPA-PCR product. The same MLPA-PCR product was analysed using capillary electrophoresis, the technique typically used for analysis of MLPA products, and good correlation was observed. The assay presented is easy to carry out, relatively inexpensive, rapid, does not require sophisticated instrumentation, and enables quantitative analysis, making it very promising for the analysis of MLPA products.     

Multiplex identification of sepsis‐causing Gram‐negative pathogens from the plasma of infected blood

Early and accurate detection of bacterial pathogens in the blood is the most crucial step for sepsis management. Gram‐negative bacteria are the most common organisms causing severe sepsis and responsible for high morbidity and mortality. We aimed to develop a method for rapid multiplex identification of clinically important Gram‐negative pathogens and also validated whether our system can identify Gram‐negative pathogens with the cell‐free plasm DNA from infected blood. We designed five MLPA probe sets targeting the genes specific to major Gram‐negative pathogens (uidA and lacY for E. coli, ompA for A. baumannii, phoE for K. pneumoniae, and ecfX for P. aeruginosa) and one set targeting the CTX‐M group 1 to identify the ESBL producing Gram‐negative pathogens. All six target‐specific peaks were clearly separated without any non‐specific peaks in a multiplex reaction condition. The minimum detection limit was 100 fg of pathogen DNA. When we tested 28 Gram‐negative clinical isolates, all of them were successfully identified without any non‐specific peaks. To evaluate the clinical applicability, we tested seven blood samples from febrile patients. Three blood culture positive cases showed E. coli specific peaks, while no peak was detected in the other four culture negative samples. This technology can be useful for detection of major sepsis‐causing, drug‐resistant Gram‐negative pathogens and also the major ESBL producing Gram‐negatives from the blood of sepsis patients in a clinical setting. This system can help early initiation of effective antimicrobial treatment against Gram‐negative pathogens for sepsis patients, which is very crucial for better treatment outcomes.     

Microarray-based detection of Korean-specific <Emphasis Type="Italic">BRCA1</Emphasis> mutations

A reliable multiplex assay procedure to detect human genetic mutations in the breast cancer susceptibility gene BRCA1 using zip-code microarrays and single base extension (SBE) reactions is described. Multiplex PCR amplification was performed to amplify the genomic regions containing the mutation sites. The PCR products were then employed as templates in subsequent multiplex SBE reactions using bifunctional primers carrying a unique complementary zip sequence in addition to a mutation-site-specific sequence. The SBE primers, terminating one base before their mutation sites, were extended by a single base at a mutation site with a corresponding biotin-labeled ddNTP. Hybridization of the SBE products to zip-code microarrays was followed by staining with streptavidin–Cy3, leading to successful genotyping of several selected BRCA1 mutation sites with wild-type and heterozygote mutant samples from breast cancer patients. This work has led to the development of a reliable DNA microarray-based system for the diagnosis of human genetic mutations. Cheulhee Jung and Seong-Chun Yim contributed equally to this work.     

A novel high-resolution chipCE assay for rapid detection of EGFR gene mutations and amplifications in lung cancer therapy by a combination of fragment analysis, denaturing CE and MLPA

There is a growing interest in evaluating molecular markers as predictors of response to new generation of targeted cancer therapies. One of such areas is biological therapy targeting epidermal growth factor receptor gene (EGFR) in lung cancer. The testing of tumor tissue is focused on specific EGFR mutations and EGFR gene amplification, since tumors exhibiting positivity of either of the two marker types are highly sensitive towards the treatment. Although traditional methods of DNA sequencing and fluorescence in situ hybridization are still in use for the detection of EGFR mutations and gene amplification, respectively, there is a need for new dedicated techniques with the primary emphasis on simplicity, sensitivity, speed and cost effectiveness. The main purpose of this work was to integrate diverse assays for both EGFR tests onto a single platform to eliminate the need for different instruments and separate processing. We demonstrate a chip capillary electrophoresis (chipCE) application for EGFR mutation detection by a combination of fragment analysis and denaturing CE along with multiplex ligation-dependent probe amplification (MLPA) for evaluation of EGFR amplification. All separations are carried out in denaturing sieving polymer on a modified Bioanalyzer 2100 chipCE instrument running at temperatures of up to 65°C. The main strength of the resulting high-resolution chipCE application is in its simplicity, speed of analysis and minimal amount of sample required for complete testing of EGFR status. Such an approach could potentially fit medium throughput laboratories providing molecular pathology services for clinical oncologists with fast turnaround times and limited consumption of tissue material.     

Capillary electrophoresis for analysis of deletion and duplication in exon 44–55 of Duchenne muscular dystrophy gene

In this study, a genotyping CGE method was established for analysis of Duchenne muscular dystrophy (DMD) gene deletions and duplications in exon 44–55. A total of 12 DMD exons (exon 44–55) and 2 internal standard gene fragments were simultaneously amplified by using a universal multiplex PCR (UMPCR) and determined by CGE. The conditions of UMPCR and CGE were optimized, including the kinds of polymerase, temperatures in UMPCR, separation matrix, separation temperature, and voltage. Finally, the separation was performed by 1.2% poly(ethylene oxide) in 1× TBE buffer at ?6 kV and 25°C. After validation, our results showed the peak patterns for differentiation of genetic deletion or duplication in 27 DMD patients and normal subjects, according to the peak height ratios by comparison of two internal standard peaks. Among the 27 subjects, 23 cases are deletion type and four are duplication type. The data of two patients analyzed by this CGE‐PCR method were different from that of multiplex ligation dependent probe amplification method, and the sequencing results demonstrated that our results were correct. This UMPCR‐CGE method was considered better than the multiplex ligation dependent probe amplification method. Furthermore, this method can be used for eugenics in clinical applications.     

A Law of Mutation: Power Decay of Small Insertions and Small Deletions Associated with Human Diseases

Indels in evolutionary studies are rapidly decayed obeying a power law. The present study analyzed the length distribution of small insertions and deletions associated with human diseases and confirmed that the decay pattern of these small mutations is similar to that of indels when the mutation datasets are large enough. The describable decay pattern of somatic mutations may have application in the evaluation of varied penetrance of different mutations and in association study of gene mutation with carcinogenesis.     

Rapid mutation detection in complex genes by heteroduplex analysis with capillary array electrophoresis

Mutational analysis of large multiexon genes without prevalent mutations is a laborious undertaking that requires the use of a high-throughput scanning technique. The Human Genome Project has enabled the development of powerful techniques for mutation detection in large multiexon genes. We have transferred heteroduplex analysis (HA) by conformation-sensitive gel electrophoresis of the two major breast cancer (BC) predisposing genes, BRCA1 and BRCA2, to a multicapillary DNA sequencer in order to increase the throughput of this technique. This new method that we have called heteroduplex analysis by capillary array electrophoresis (HA-CAE) is based on the use of multiplex-polymerase chain reaction (PCR), different fluorescent labels and HA in a 16-capillary DNA sequencer. To date, a total of 114 different DNA sequence variants (19 insertions/deletions and 95 single-nucleotide substitutions - SNS) of BRCA1 and BRCA2 from 431 unrelated BC families have been successfully detected by HA-CAE. In addition, we have optimized the multiplex-PCR conditions for the colorectal cancer genes MLH1 and MSH2 in order to analyze them by HA-CAE. Both genes have been amplified in 13 multiplex groups, which contain the 35 exons, and their corresponding flanking intronic sequences. MLH1 and MSH2 have been analyzed in nine hereditary nonpolyposis colorectal cancer patients, and we have found six different DNA changes: one complex deletion/insertion mutation in MLH1 exon 19 and another five SNS. Only the complex mutation and one SNS may be classified as cancer-prone mutations. Our experience has revealed that HA-CAE is a simple, fast, reproducible and sensitive method to scan the sequences of complex genes.     

Diagnostic mutational analysis of MECP2 in Korean patients with Rett syndrome

Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder affecting 1 per 10,000-15,000 female births worldwide. The disease-causing gene has been identified as MECP2 (methyl-CpG-binding protein 2). In this study, we performed diagnostic mutational analysis of the MECP2 gene in RTT patients. Four exons and a putative promoter of the MECP2 gene were analyzed from the peripheral blood of 43 Korean patients with Rett syndrome by PCR-RFLP and direct sequencing. Mutations were detected in the MECP2 gene in approximately 60.5% of patients (26 cases/43 cases). The mutations consisted of 14 different types, including 9 missense mutations, 4 nonsense mutations and 1 frameshift mutation. Of these, three mutations (G161E, T311M, p385fsX409) were newly identified and were determined to be disease-causing mutations by PCR- RFLP and direct sequencing analysis. Most of the mutations were located within MBD (42.3%) and TRD (50%). T158M, R270X, and R306C mutations were identified at a high frequency. Additionally, an intronic SNP (IVS3+23C>G) was newly identified in three of the patients. IVS3+23C>G may be a disease-related and Korea-specific SNP for RTT. L100V and A201V are apparently disease-causing mutations in Korean RTT, contrary to previous studies. Disease-causing mutations and polymorphisms are important tools for diagnosing RTT in Koreans. The experimental procedures used in this study should be considered for clinical molecular biologic diagnosis.