Lan-Szu Chou

Genotype–phenotype correlation in hereditary hemorrhagic telangiectasia: Mutations and manifestations

Hereditary hemorrhagic telangiectasia (HHT) is a genetically heterogeneous vascular dysplasia with multiple telangiectases and arteriovenous malformations and it is caused by mutations in endoglin gene (ENG) (HHT1) and activin A receptor type II‐like 1 gene (ACVRL1) (HHT2). We evaluated 111 patients with HHT from 34 families by history, examination, screening for vascular malformations, and sequencing of both genes. We found mutations in 26 of the 34 kindreds (76%) analyzed—54% were in ENG and 46% were in ACVRL1. Mutations in ACVRL1 cluster largely in exons 7 and 8, but ENG mutations were widely distributed within that gene. We found that epistaxis had an earlier onset in patients with HHT1 than those with HHT2, but the severity by middle ages was similar. Pulmonary arteriovenous malformations were more frequent and on the average of larger size in HHT1. Hepatic vascular malformations were more common in patients with HHT2. Cerebral arteriovenous malformations were more common in patients with HHT1, but spinal arteriovenous malformations were seen only in patients with HHT2. Truncating mutations in ENG were associated with more affected organs and more severe hemorrhaging than were missense mutations. We conclude that HHT2 has a later onset than HHT1 and the former may disproportionately involve smaller vessels in tissues with more significant vascular remodeling.

A Comparison of High-Resolution Melting Analysis With Denaturing High-Performance Liquid Chromatography for Mutation Scanning Cystic Fibrosis Transmembrane Conductance Regulator Gene as a Model

High-resolution melting analysis (HRMA) was compared with denaturing high-performance liquid chromatography (dHPLC) for mutation scanning of common mutations in the cystic fibrosis transmembrane conductance regulator gene. We amplified (polymerase chain reaction under conditions optimized for melting analysis or dHPLC) 26 previously genotyped samples with mutations in exons 3, 4, 7, 9, 10, 11, 13, 17b, and 21, including 20 different genotypes. Heterozygous mutations were detected by a change in shape of the melting curve or dHPLC tracing. All 20 samples with heterozygous mutations studied by both techniques were identified correctly by melting (100% sensitivity), and 19 were identified by dHPLC (95% sensitivity). The specificity of both methods also was good, although the dHPLC traces of exon 7 consistently revealed 2 peaks for wild-type samples, risking false-positive interpretation. Homozygous mutations could not be detected using curve shape by either method. However, when the absolute temperatures of HRMA were considered, G542X but not F508del homozygotes could be distinguished from wild type. HRMA easily detected heterozygotes in all single nucleotide polymorphism (SNP) classes (including A/T SNPs) and 1- or 2-base-pair deletions. HRMA had better sensitivity and specificity than dHPLC with the added advantage that some homozygous sequence alterations could be identified. HRMA has great potential for rapid, closed-tube mutation scanning.

DNA Macroarray Profiling of Lactococcus lactis subsp. lactis IL1403 Gene Expression during Environmental Stresses

ABSTRACT This report describes the use of an oligonucleotide macroarray to profile the expression of 375 genes in Lactococcus lactis subsp. lactis IL1403 during heat, acid, and osmotic stress. A set of known stress-associated genes in IL1403 was used as the internal control on the array. Every stress response was accurately detected using the macroarray, compared to data from previous reports. As a group, the expression patterns of the investigated metabolic genes were significantly altered by heat, acid, and osmotic stresses. Specifically, 13 to 18% of the investigated genes were differentially expressed in each of the environmental stress treatments. Interestingly, the methionine biosynthesis pathway genes (metA-metB1 and metB2-cysK) were induced during heat shock, but methionine utilization genes, such as metK, were induced during acid stress. These data provide a possible explanation for the differences between acid tolerance mechanisms of L. lactis strains IL1403 and MG1363 reported previously. Several groups of transcriptional responses were common among the stress treatments, such as repression of peptide transporter genes, including the opt operon (also known as dpp) and dtpT. Reduction of peptide transport due to environmental stress will have important implications in the cheese ripening process. Although stress responses in lactococci were extensively studied during the last decade, additional information about this bacterium was gained from the use of this metabolic array.

A Comparison of High-Resolution Melting Analysis With Denaturing High-Performance Liquid Chromatography for Mutation Scanning

High-resolution melting analysis (HRMA) was compared with denaturing high-performance liquid chromatography (dHPLC) for mutation scanning of common mutations in the cystic fibrosis transmembrane conductance regulator gene. We amplified (polymerase chain reaction under conditions optimized for melting analysis or dHPLC) 26 previously genotyped samples with mutations in exons 3, 4, 7, 9, 10, 11, 13, 17b, and 21, including 20 different genotypes. Heterozygous mutations were detected by a change in shape of the melting curve or dHPLC tracing. All 20 samples with heterozygous mutations studied by both techniques were identified correctly by melting (100% sensitivity), and 19 were identified by dHPLC (95% sensitivity). The specificity of both methods also was good, although the dHPLC traces of exon 7 consistently revealed 2 peaks for wild-type samples, risking false-positive interpretation. Homozygous mutations could not be detected using curve shape by either method. However, when the absolute temperatures of HRMA were considered, G542X but not F508del homozygotes could be distinguished from wild type. HRMA easily detected heterozygotes in all single nucleotide polymorphism (SNP) classes (including A/T SNPs) and 1-or 2-base-pair deletions. HRMA had better sensitivity and specificity than dHPLC with the added advantage that some homozygous sequence alterations could be identified. HRMA has great potential for rapid, closed-tube mutation scanning.

Unlabeled oligonucleotide probes modified with locked nucleic acids for improved mismatch discrimination in genotyping by melting analysis

With a frequency of 1 in 1000 bp, single nucleotide polymorphisms (SNPs) are used to study complex inherited diseases (1,2). A universal concern in genotyping techniques is that rare variants may interfere. Melting analysis addresses this concern and is a homogeneous and simple method for genotyping (3,4). For example, the 2-probe/2-fluorophore system HybProbe

The Development of Next-Generation Sequencing Assays for the Mitochondrial Genome and 108 Nuclear Genes Associated with Mitochondrial Disorders

Sanger sequencing of multigenic disorders can be technically challenging, time consuming, and prohibitively expensive. High-throughput next-generation sequencing (NGS) can provide a cost-effective method for sequencing targeted genes associated with multigenic disorders. We have developed a NGS clinical targeted gene assay for the mitochondrial genome and for 108 selected nuclear genes associated with mitochondrial disorders. Mitochondrial disorders have a reported incidence of 1 in 5000 live births, encompass a broad range of phenotypes, and are attributed to mutations in the mitochondrial and nuclear genomes. Approximately 20% of mitochondrial disorders result from mutations in mtDNA, with the remaining 80% found in nuclear genes that affect mtDNA levels or mitochondrion protein assembly. In our NGS approach, the 16,569-bp mtDNA is enriched by long-range PCR and the 108 nuclear genes (which represent 1301 amplicons and 680 kb) are enriched by RainDance emulsion PCR. Sequencing is performed on Illumina HiSeq 2000 or MiSeq platforms, and bioinformatics analysis is performed using commercial and in-house developed bioinformatics pipelines. A total of 16 validation and 13 clinical samples were examined. All previously reported variants associated with mitochondrial disorders were found in validation samples, and 5 of the 13 clinical samples were found to have mutations associated with mitochondrial disorders in either the mitochondrial genome or the 108 nuclear genes. All variants were confirmed by Sanger sequencing.

Relationship of arginine and lactose utilization by Lactococcus lactis ssp. lactis ML3

The arginine deiminase (ADI) pathway exists in many microorganisms and is one of the main characteristics that distinguish between Lactococcus lactis ssp. lactis and ssp. cremoris. The ADI pathway converts arginine to produce ammonia, carbon dioxide, and ATP. Induction of this pathway in relation to energy production is not well characterized. The presence of arginine changed the metabolism of strain ML3 in the media containing different levels of lactose. The interaction between lactose and arginine utilization changed the pH of the growth medium in a cyclic pattern. Transcriptional analysis showed that the first enzyme in the ADI pathway (arginine deiminase, arcA) was induced by pH rather than arginine addition. Analysis of the ratio arginine to lactose led to a statistical model that linked lactose, arginine, external pH, and the ADI pathway.

Complete Gene Scanning by Temperature Gradient Capillary Electrophoresis Using the Cystic Fibrosis Transmembrane Conductance Regulator Gene as a Model

Many inherited diseases involve large genes with many different mutations. Identifying a wide spectrum of mutations requires an efficient gene-scanning method. By differentiating thermodynamic stability and mobility of heteroduplexes from heterozygous samples, temperature gradient capillary electrophoresis (TGCE) was used to scan the entire coding region of the cystic fibrosis transmembrane conductance regulator gene. An initial panel (29 different mutations) showed 100% agreement between TGCE scanning and previously genotyped results for heterozygous samples. Different peak patterns were observed for single base substitutions and base insertions/deletions. Subsequently, 12 deidentified clinical samples genotyped as wild type for 32 mutations were scanned for the entire 27 exons. Results were 100% concordance with the bidirectional sequence analysis. Ten samples had nucleotide variations including a reported base insertion in intron 14b (2789 + 2insA) resulting in a possible mRNA splicing defect, and an unreported missense mutation in exon 20 (3991 G/A) with unknown clinical significance. This methodology does not require labeled primers or probes for detection and separation through a temperature gradient eliminates laborious temperature optimization required for other technologies. TGCE automation and high-throughput capability can be implemented in a clinical environment for mutation scanning with high sensitivity, thus reducing sequencing cost and effort.

Detection of Large Rearrangements in the Cystic Fibrosis Transmembrane Conductance Regulator Gene by Multiplex Ligation-Dependent Probe Amplification Assay When Sequencing Fails to Detect Two Disease-Causing Mutations

AIMS Most of the over 1600 mutations and sequence variants identified to date in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are point mutations or small deletions/insertions detectable by conventional sequencing. However, large rearrangements (deletions, duplications, or insertion/deletion mutations) have recently been reported to constitute 1-2% of CFTR mutations. The CFTR sequencing protocol at ARUP Laboratories interrogates the coding regions of all 27 exons and all intron/exon boundaries of the gene. This study was undertaken to determine whether testing for large gene rearrangements could improve the mutation detection rate. RESULTS Nine cases with abnormal quantitative pilocarpine iontophoresis sweat chloride (SC) values (>60 mEq/L) and 20 cases with borderline SC levels (40-60 mEq/L) with only one or no mutations detected by the ARUP 32 mutation panel, including the 23 mutations recommended by American College of Medical Genetics (ACMG) for carrier screening, followed by sequencing, were tested using a multiplex ligation-dependent probe amplification (MLPA) assay. MLPA analysis identified one deletion among nine patients with SC >60 who had previously been tested with sequencing. None of the cases with borderline SC levels showed rearrangements. CONCLUSION The MLPA assay for detection of large rearrangements may be valuable in individuals with positive SC levels where one or no mutations have been identified by sequencing.

Multiplex genotyping by melting analysis of loci-spanning probes: β-globin as an example

Multiplexing genotyping technologies usually require as many probes as genetic variants. Oligonucleotides that span multiple loci--loci spanning probes (LSProbes)--hybridize to two or more noncontiguous DNA sequences present in a template and can be used to analyze multiple variants simultaneously. The intervening template sequence, omitted in the LSProbe, creates a bulge-loop during binding. Melting temperatures of the probe, monitored by fluorescence reading are specific to the presence or absence of the mutations. We previously described LSProbes as a molecular haplotyping tool and apply here the principle to genotype simultaneously three mutations of the beta-globin gene responsible for the corresponding hemoglobinopathies. Analysis with both labeled and unlabeled LSProbes demonstrate that the four possible alleles studied (WT, HbS, HbC, and HbE) are identifiable by the specific melting temperatures of the LSProbes. This demonstrates that, in addition to their haplotyping capabilities, LSProbes are able to genotype in a single step, loci 58 nucleotides apart.