ESHG Posters 22

Population screening for beta-thalassemia point mutations; development of a Micro-Array based Single Base Extension approach.

C. L. Harteveld¹, C. H. M. van Moorsel², I. F. A. C. Fokkema¹, M. Yavarian³, R. van Zwieten², M. de Haas², D. Roos², J. T. den Dunnen¹, P. C. Giordano¹;
¹Human and Clinical Genetics, Leiden University Medical Center, Leiden, NETHERLANDS, ²Sanquin Research at CLB, Amsterdam, NETHERLANDS, ³The Thalassemia Medical Centre, Medical Faculty, Bandar Abbas University, Pasteur Laboratory, Bandar Abbas, IRAN (ISLAMIC REPUBLIC OF).

Beta-thalassaemia is an autosomal recessive trait, which may occur at a high incidence in populations living in areas endemic for malaria. Carriership for this trait is believed to provide a selective advantage of survival after a Plasmodium falciparum infection, which is the inducer of malaria tropica. Homozygotes or compound heterozygotes, on the other hand, suffer from a severe microcytic hypochromic anemia known as beta-thalassaemia major, which results in early death (without proper treatment) or life-long transfusion and chelation therapy dependence. The majority of molecular defects causing beta-thalassemia are point mutations affecting beta-globin gene expression. The total number of different beta-thalassaemia causing mutations known to date is approximately 180. However a limited number of mutations occurs in different populations. Once the spectrum of beta-thalassemia-causing mutations is known for a certain population, a screening strategy for efficient diagnosis can be applied. The feasiblity of a micro-array based approach using Single Base Extension was tested in an immigrant-Dutch and an Iranian population from Hormozgan. A total of 18 different mutation-specific oligonucleotide primers were selected, covering approximately 90% of the Dutch and 86% of the Iranian beta-thalassemia mutations. The use of tagged-oligoprimers for mutation detection and subsequent hybridization to a generic micro-array containing covalently bound oligo-probes complemetary to the different tags, allows the simultaneous analysis of 27 frequently occurring single-base mutations in the beta-globin gene. We present the comparison of the results obtained by micro-array analysis and direct sequencing to discuss the feasibility of this approach.

H. Roomere¹, A. Meiel¹, N. Tõnisson², J. Zernant², A. Kurg¹, H. Pavel², G. Slavin², P. Hainaut³, A. Metspalu¹^,2;
¹Institute of Molecular and Cell Biology University of Tartu, Tartu, ESTONIA, ²Asper Biotech Ltd., Tartu, ESTONIA, ³International Agency for Research on Cancer, Lyon, FRANCE.

We have developed and evaluated APEX (arrayed primer extension) - based test for resequencing of TP53 tumour suppressor gene. Identification of TP53 gene mutations is important, because there is a correlation between the mutation type and the prognosis of cancer. Using the APEX test we can get full mutation data for the sequenced region of the gene at both DNA strands in a single assay.
A patient DNA sample is amplified, digested enzymatically, and annealed to arrayed primers, which promote sites for template-dependent DNA polymerase extension reaction using four fluorescently labelled dideoxy nucleotides. The TP53 gene test spans exons 2 to 9 plus introns 2 and 8. 98 individuals were analysed to obtain data on performance of the chip in a large-scale study. An average of 97,5% of the arrayed p53 gene sequence was identified from either sense or antisense strand and 81% from both strands. A common polymorphism in exon 4 (Arg72Pro) was found with minor allele frequency of 0.26.
Genomic DNA from 11 tumour samples was sequenced in a blind test. The results were predominantly concordant with TTGE (Temporal Temperature Gradient Electrophoresis) plus dideoxy sequencing. GenoramaTM imaging system and genotyping software were used for imaging and semiautomatic sequence analysis.
The fact that our assay can simultaneously perform mutation detection and correct identification of codon 72 status adds further weight to its usefulness as Arg72Pro polymorphism has recently been proposed to play a role in tumorigenesis.

Gene assembling, a new approach in mutation detection techniques - An application for BRCA genes scanning

V. R. Yassaee¹, A. Dalton²;
¹The University of Sheffield, Sheffield, UNITED KINGDOM, ²North Trent Molecular Genetics laboratory, Sheffield Children’s Hospital, UNITED KINGDOM.

Many disease susceptibility genes are large and consist of many exons in which point mutations are scattered throughout. There has been an increasing demand for rapid and accurate method for scanning of point mutations in BRCA genes particularly.
Meta-PCR is a new method for creating chimeric DNA molecules using a modified PCR reaction (Wallace et al 1999) that allows maximizing the length of sequence that can be scanned by downstream technique.
Here we present data to demonstrate the assembles of exons 2, 20,23 and 24 of the BRCA1 gene and their subsequently analysis by direct sequencing.
The BRCA1 exons 2 and 20 are hot spot regions that are known to harbour particularly deleterious mutations. In order to avoid missing any mutation in these two exons, the above four exons were assembled in the following order of preferences: 23 20, 2 and 24. However, the order of fragments can be predetermined by primer design.
We verified by direct sequencing that the order and sequence of the component exons in the Meta-PCR products were as predicted. Meta-PCR products from three previously ascertained heterozygotes for BRCA1 mutations were directly sequenced and gave the same sequence patterns.
Scanning of each exon of BRCA1 and BRCA2 genes individually represent a tedious task. Meta-PCR technique might be circumvents this problem and is likely to be useful for clinical molecular diagnostic laboratories, helping them to fulfil the demand for scanning of complex genetic disease at the lower cost.
v.r.yassaee@shef.ac.uk

Mutation Detection by High Throughput Single Strand Conformation Polymorphism using Automated Capillary Array Electrophoresis: Validation of Sensitivity.

P. S. Andersen¹, C. Jespersgaard¹, J. Vuust¹, M. Christiansen¹, L. A. Larsen²;
¹Statens Serum Institut, Copenhagen, DENMARK, ²University of Copenhagen, Copenhagen, DENMARK.

Detection of unknown mutations requires identification of any nucleotide substitution in a gene and is achieved through DNA sequencing. For large genes or large number of samples, this is a time-consuming process, necessitating methods that rapidly identify DNA fragments containing mutations, the exact nature of which may then be determined by DNA sequencing. One such screening method is Single Strand Conformation Polymorphism (SSCP) analysis, in which DNA fragments differing in only one nucleotide position may be separated on the basis of sequence-specific conformation of single stranded DNA. Polyacrylamide slab gel electrophoresis was originally employed for the single stranded DNA separation; recently, however, SSCP analysis carried out using capillary electrophoresis (CE-SSCP) has been introduced as a high-throughput screening method, and with the advent of multicapillary instruments, SSCP analysis using capillary array electrophoresis (CAE-SSCP) may increase the throughput several fold. In the present study, we have validated the mutation detection sensitivity of CAE-SSCP by constructing and analysing a panel of 68 mutants representing all types of substitutions as well as insertions and deletions in different sequence contexts in four exons from human genes. PCR amplicons, 150 to 300 bp in length, labelled with fluorescent dyes, were analysed by CAE-SSCP at three different temperatures (18, 25, and 35 °C) on a 16-channel ABI3100 Genetic Analyzer. Results were collected as data points and analysed with ABI Genescan and Genotyper software. The overall mutant detection level was found to be 96%, confirming the usefulness of this particular method for high throughput mutation detection.

Pyrosequence Analysis for Detection of Mutations Associated with Hereditary Hearing Loss in the Connexin 26 Gene and Mitochondrial DNA

A. Ferraris¹^,2, E. Rappaport³, S. Toth⁴, F. Lysholm⁴, J. Williamson⁴, R. Restagno⁵, B. Dallapiccola², P. Fortina³;
¹Department of Pediatrics, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA, ²Dipartimento di Medicina Sperimentale e Patologia, Universita' La Sapienza and Istituto CSS Mendel, Rome, ITALY, ³Department of Pediatrics, The Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA, ⁴Pyrosequencing, Inc., Westborough, MA, ⁵Laboratorio di Genetica Molecolare, Ospedale Infantile Regina Margherita, Torino, ITALY.

Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in Connexin 26 (CX26) gene account for about 20% of all cases of childhood deafness. This number approaches 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, A1555G, in the 12S rRNA gene, is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence information to an enzyme cascade system which generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing for detecting some of the common mutations causing HHL. Genomic DNA samples were collected from peripheral blood or dried blood spots using standard protocols. Mitochondrial DNA was coisolated with total genomic DNA. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT) and Asian (235delC, V37I) origins was confirmed by Pyrosequencing analysis. A total of 41 different mutations in CX26 gene and the mitochondrial A1555G mutation were also confirmed. Genotyping of up to 6 different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. In addition, tests of the ability to quantitate Mt DNA heteroplasmy were successful. Major advantages of the Pyrosequencing approach include high throughput genotyping of disease-causing mutations, accurate and reproducible results, and assay flexibility. Experimental conditions can be optimized for a high degree of standardization and cost-effectiveness.

Diagnostic Mutation Scanning Using Microtitre Array Capillary Electrophoresis Fluorescent SSCP

G. R. Taylor¹, D. Cuthbert-Heavens², A. Smith¹, C. F. Taylor², R. S. Charlton¹;
¹St James’s University Hospital, Leeds, UNITED KINGDOM, ²Cancer Research UK, Leeds, UNITED KINGDOM.

Mutation scanning can, in principle, provide a rapid screen of candidate exons or genes prior to a full sequence analysis, saving time and reducing costs. Single Stranded Conformational Polymorphism Analysis (SSCP) has been a popular method for scanning due to its simplicity (1). The only other method of comparable ease is Denaturing High Performance Liquid Chromatography (DHPLC)(2). We previously compared DHPLC with an improved Fluorescent SSCP (3). The two methods were of similar sensitivity. Here we report the adaptation of SSCP to a capillary array format the enables automated sampling and re-running of the same samples at different temperatures. Run times were reduced from 18 hours using the Applied Biosystems 377 to less than 1 hour using the 3100. We analysed 66 samples and controls from exons in hMSH2, hMLH1, p53, VHL and Menin using three different buffers (Tris-borate, Tris-taurine and Tris-MES) each at 5 temperatures. All mutations were detected, although to achieve this level of sensitivity we needed to use more than one buffer system. Some mutations were more "detectable" (that, seen under more conditions) than others. We used the MFOLD (http://bioinfo.math.rpi.edu/~zukerm/) single stranded DNA structure analysis program to investigate the detectability of various mutations with the aim of making SSCP more predictable.
1. Orita M et al (1989) Genomics 5 (4):874-879
2. Underhill PA et al (1997)Genome Research 7:996-1005
3. Ellis LA, Taylor CF, and Taylor GR (2000) Human Mutation 15:556-564.

Mutation detection assay for ABCR (ABCA4) Gene, based on Arrayed Primer Extension technology

M. Külm¹, K. Jaakson¹, S. Tarrikas¹, A. Hutchinson², R. L. Allikmets², A. Metspalu³;
¹Asper Biotech Ltd., Tartu, ESTONIA, ²Columbia University, New York, NY, ³Institute of Molecular and Cell Biology, University of Tartu, Estonian Biocentre, Tartu, ESTONIA.

The ABCR gene arrayed primer extension (APEX) assay was designed for the detection of over 300 variants currently described in ABCR (ABCA4) gene. Mutations in the gene are associated with at least five inherited retinal dystrophies: Stargardt disease (STGD), fundus flavimaculatus (FFM), cone-rod dystrophy (CRD), retinitis pigmentosa (RP) and age-related macular degeneration (AMD).
We have used Arrayed Primer Extension (APEX) technology for identification of ABCR gene mutations as an alternative to mutation detection based on SSCP plus dideoxy sequencing. The genomic DNA sample is amplified and annealed to arrayed primers, which promote sites for DNA polymerase extension reactions using four fluorescently labelled dideoxynucleotides. The oligonucleotide array scans a total of 349 mutation sites (the vast majority of mutations currently known in the ABCR gene) from both strands. The ABCR chip was validated with 150 STGD patients and 100 controls in a blind test. The same DNA samples were previously analysed with the SSCP technology. Differences in SSCP and chip results were controlled by sequencing. The APEX based technology determines the existing genetic variation with 97% efficiency.
The APEX method provides efficient tool for mutation and polymorphism analysis both for scientific and clinical research. The ABCR gene APEX assay can be applied as screening tool in ophthalmic genetics.

F. Joncourt, B. Neuhaus, K. Jostarndt, S. Kleinle, S. Gallati;
Human Molecular Genetics, Children's University Hospital, Berne, SWITZERLAND.

Two thirds of Duchenne and Becker (DMD/BMD) muscular dystrophy cases are due to deletions in the dystrophin gene on Xp21. While most deletions are readily detected in patients by PCR, the identification of female carriers in affected families remains difficult in some cases, e.g. when no informative markers are available in the deleted region.
Using the Light cycler technology we have developed a simple and rapid assay for the direct detection of carriers of exon 44, 45 and 49 deletions in the dystrophin gene. Primer pairs for each exon were designed to give specific and efficient PCR under identical conditions. The products were quantified using SybrGreen and the ratios between the potentially deleted exon and a non-deleted reference exon were calculated. We have analysed 16 known carriers and 16 non-carriers in 8 independent assays: in all cases the correct status was reliably detected (mean ratios: carriers 0.52, non carriers 1.11). There was no overlap between the ratios of carriers and non-carriers: carriers ranged from 0.33-0.69, non-carriers from 0.85-1.35.
We are currently adapting the assay to a number of other exons frequently deleted in the dystrophin gene and we are testing the possibility of adapting the protocol to other conditions involving gene deletions or duplications such as CMT/HNPP and NF. The method has the advantage that no specific labelled hybridisation probes are needed. It offers a reliable, rapid and inexpensive possibility for the detection of heterozygous carriers of deletions in the dystrophin as well as in a number of other genes.

A rapid, accurate and quantitative method for analysis of the methylation status of imprinted genes, using primer extension and IP RP HPLC.

M. M. Matin, D. P. Hornby;
Transgenomic Research laboratory, Department of Molecular Biology, University of Sheffield, Sheffield S10 2TN, UNITED KINGDOM.

We have developed a rapid, accurate and quantitative method for the detection of methylation differences at specific CpG sites based on bisulphite treatment of DNA followed by primer extension and ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC). The application of the method is illustrated by analysis of differentially imprinted alleles arising from Prader-Willi and Angelman syndromes.
In order to convert unmethylated cytosines to uracil, plasmid and genomic DNA samples were treated with sodium bisulphite and the targeted sequence was then amplified using oligodeoxynucleotide primers specific for the bisulphite-deaminated DNA. The PCR product(s) from this step was used as a template for a primer extension reaction and the products were subsequently analysed chromatographically using IP RP HPLC. This method eliminates the need to use restriction enzymes to determine the methylation status of the amplicon and also circumvents the need for radio labelling for the quantitative measurements. Finally, this method removes the need for nucleotide sequencing since it is not solely reliant on the presence or absence of one or more PCR products, as is the case with related methods.

Fluorescent analysis of microsatellite markers using a unique fluorescent primer : a simple technique validated on 55 CA or tetra repeats

M. Cossée, M. Guiot, S. Samimi, V. Biancalana;
Laboratoire de diagnostic génétique, Strasbourg, FRANCE.

Study of microsatellite (CA or tetra repeats) segregation around a locus is a tool widely used. Traditionally, such a study is performed with two flanking specific primers, one being labelled (radioactive or fluorescent) for detection. Use of radioactivity is inconvenient in a diagnostic setting, and implies an additional step of kination for each microsatellite analysed. For fluorescent analysis on automated sequencers, labelled primers are commercially available, but are rather costly and have a limited lifetime. This aspect is a real limitation when such primers are used infrequently for genetic counselling of very rare diseases.
For each microsatellite, we use its two unlabeled specific primers, and a unique fluorescent primer, in an single reaction. One of the specific flanking primers is tailed with a common 5’ sequence selected by Warner et al (1996) for the TP-PCR technique. Primer sequences were chosen in order to define the best combination between one of the specific primer and the other with the common tail. Detection is achieved with a fluoresceinated primer corresponding to this common sequence. In the early amplification cycles, specific primers give rise to a product with the tail. A particular molar ratio of primers ensures that the specific primer with the tail is exhausted in the early amplification cycles. This allows the fluoresceinated primer to amplify preferentially from the end of products from previous amplification rounds.
We have validated this strategy for 55 microsatellites, most of them on the X chromosome. We have tested successfully multiplex for up to 4 microsatellites.

R. Burgemeister, K. Schuetze;
PALM Microlaser Technologies, Bernried, GERMANY.

State-of-the-art molecular analyses need the handling with extremely pure samples to yield a good result. This is important e.g. for single cell genetic analyses (like in fetal cells in maternal blood, in preimplantation diganosis or in disseminated tumor cells), for microarray technologies and for single chromosome preparation. Thus pure sample preparations are indispensible for various fields in medicine and biology.
To obtain pure samples is one of the most thrilling tasks in modern molecular science. An up-to-date tool in this topic is Laser Microdissection combined with Laser Pressure Catapulting. This laser technology simply utilizes the force of focused laser light to eject a selected specimen from the object plane and to directly lift it into the cap of a routine microfuge tube. This completely non-contact Laser Pressure Catapulting technology avoids any danger of contamination with unwanted specimen. In every case where the comparison of different cell types (genetic, expressional or proteomic) is important for research or diagnosis, a precise differentiation between selected cells is mandatory.
Any kind of tissue from various sources (also archival histological samples or living cells) and even subcellular structures can be captured using this laser method. Wherever precise micromanipulation is required or where the procurement of homogenous samples is obligatory for the subsequent analysis of specific genetic or proteomic alterations, the PALM MicroLaser system is a key technology.

The GenoSNIP assay - A novel method for SNP genotyping by MALDI-TOF mass spectrometry

M. Kostrzewa¹, K. Fahr¹, T. Elßner¹, W. Pusch², J. Wurmbach³, T. Wenzel¹;
¹Bruker Saxonia Analytik GmbH, Leipzig, GERMANY, ²Bruker Daltonik GmbH, Bremen, GHANA, ³Bruker Daltonik GmbH, Bremen, GERMANY.

After completion of the Human Genome Project, intensive exploration of diversity in different individuals is one of the main objectives in human molecular genetics. In particular, analysis of single nucleotide polymorphisms (SNPs) is expected to have an enormous impact in future human medicine, e.g. by determination of risk factors for common diseases, causes of multifactorial diseases, and modulators of pharmacologic effects.
MALDI-TOF mass spectrometry has been shown to be one of the most valid methods for SNP genotyping. Combination of analysis speed, accuracy of results, cost-effectiveness, and automation capabilities makes MALDI-TOF one of the most promising SNP-typing technologies for medical research and diagnostics. On the other hand, drawbacks of the technique are complex sample preparation and high purification requirements before MALDI measurement.
Here, we present a novel method for MALDI SNP typing sample preparation, GenoSNIP. An UV-cleavable site is introduced into a primer which hybridizes adjacent to the polymorphic site. This photolinker creates an abasic site in the primer and does not prevent annealing to the target sequence. The primer is converted by single nucleotide extension to products specific for the corresponding alleles. Subsequently, these products are cleaved by UV light resulting in very small molecules, usually pentamers, which can be measured with very high sensitivity, accuracy, and resolution in a MALDI-TOF MS instrument. We present different designs of the GenoSNIP assay including a simple pipetting protocol which is fully compatible with the 384 microtiterplate format. The genotyping of several common genetic risk factors is demonstrated.

J. M. Sorenson¹, K. Gunning¹, G. Chappell¹, Q. Doan¹, P. Suri¹, R. Paul¹, A. Pradhan¹, B. Kshirsargar¹, G. Mason¹, P. Honebein², G. Denisov³, M. Schoppe⁴, A. Pronyaev⁴, L. Johnston-Dow¹;
¹Applied Biosystems, Foster City, CA, ²Systems Integration Solutions, San Francisco, CA, ³Paracel Inc., A Celera Business, Pasadena, CA, ⁴Group Logic, Arlington, VA.

The release of version 1.1 of SeqScapeS software marked the introduction of basecalling quality values and assembly-based SNP identification into a desktop comparative sequencing software tool. SeqScapeS software is compatible with Windows NTÒ/WindowsÒ 2000 OS and contains fully integrated basecalling, trimming, sequence assembly, alignment, and sequence comparison tools for fast and accurate sequence comparisons and variant identification. We present here a comparison of the features, ease of use, and robustness of algorithms between SeqScapeS software and other available comparative sequencing software tools. We also critically evaluate the accuracy of analysis results from SeqScapeS software version 1.1 versus SequencherS software. Highlights are presented of new algorithm features in development for the next version of SeqScapeS software, including the ability to detect and identify heterozygous frameshift mutations using direct sequencing.

Multiplex PCR of Short Fluorescent Fragments: a simple, fast and reliable method for the detection of heterozygous genomic rearrangements

T. Frebourg¹^,2, F. Charbonnier², F. Casilli¹, G. Raux¹, P. Saugier-Veber¹^,2, N. Drouot², M. Tosi¹;
¹INSERM EMI 9906, Faculty of Medicine, Rouen, FRANCE, ²Department of Genetics, CHU, Rouen, FRANCE.

The detection of heterozygous genomic deletions and duplications is technically difficult and represents a serious limitation to the complete diagnosis of many genetic diseases. We have developed Multiplex PCR of Short Fluorescent Fragments, a simple method which is based on: (i) the simultaneous amplification of several short genomic sequences using fluorescently labeled primers, (ii) the use of a limited number of cycles, (iii) the superposition of the fluorescent electropherograms and, (iv) comparison, between patients and controls, of the peaks representing the fluorescence of each amplicon. This method has already been adapted to the following genes: MSH2, MLH1, MSH6, C1NH, SMN, BRCA1 et RB1. It has already been included into our diagnostic routine of the HNPCC syndrome, of the hereditary forms of breast and ovarian cancer and of retinoblastoma and has improved the genetic counseling of spinal muscular atrophy. Moreover, this method has allowed us to characterize precisely the boundaries of a large number of heterozygous deletions or duplications. This method is much more sensitive and rapid than the Southern blot technique commonly used, is better suited than quantitative real time PCR to the analysis of genes containing large numbers of exons, and appears to be more flexible than the MAPH (Multiplex Amplifiable Probe Hybridization) method, particularly because it can be rapidly adapted to large numbers of genes.

DNA sequence analysis using ten-mer oligonucleotides microarray and allele-specific primer extension.

M. Teder¹, M. Nelis², L. Nagirnaja², A. Kurg², A. Metspalu²;
¹Estonian Biocentre, Tartu, ESTONIA, ²Institute of Molecular and Cell Biology, University of Tartu, Tartu, ESTONIA.

The aim of this project was to test 10-mer oligonucleotides for sequence analysis by arrayed primer extension (APEX) method in microarray format on glass slides.
Firstly, 256 oligonucleotides, consisting of constant GAATTC part in 5'end and a variable 3' end part, containing all possible tetramers, were chosen. Twenty synthetic oligonucleotides of 50bp length, containing EcoRI restriction site, were used as templates in this system. Reaction mixture for APEX contained all four fluorescently labeled ddNTP-s, thermostable DNA polymerase and denatured templates. During optimization different template concentrations were tested at temperatures ranging 20°C - 45°C. The nucleotide in the fifth position from the EcoRI site was correctly identified in all templates.
Secondly, to further improve APEX specificity with thermostable DNA polymerase, we tried to raise Tm's of 10-mers by including one, two or three LNA monomers into different positions of three oligonucleotides. The reactivity of original and modified oligonucleotides was tested in APEX and hybridization experiments at temperatures ranging 30°C - 50°C. Fluorescence signals were scanned by Array Scanner 428 and quantitated by ArrayPro Analyzer 4.0 software. Contrary to expectations, LNA oligonucleotides produced weaker signals than normal 10-mers in APEX reactions. At the same time LNA oligonucleotides gave equal or 2-6 times higher signals in hybridization. Probably LNA monomers interfere in DNA polymerase reaction with such oligonucleotides.
In conclusion, 10-mers could be effectively used for APEX method in oligonucleotide microarray format on glass slides. Addition of LNA monomers into DNA oligonucleotides did not improve performance of these oligonucleotides in APEX reactions.

Applications Of Genetic Analyzer (genescan) In The Diagnosis Of Genetic Diseases

M. García-Hoyos, M. J. Trujillo, C. González-González, J. Gallego, D. Cantalapiedra, C. Ramos, C. Ayuso;
Department of Genetics. Fundación Jiménez Díaz, Madrid, SPAIN.

Diagnostic methods for genetic diseases have suffered great development in the last years because of the technologic advances. One of these new methods is based in an automated genetic analysis by capillary electrophoresis.
The ABI Prims® 310 Genetic Analyzer (Applied Biosystems) make use of two software packages: Sequence Analysis and GeneScan.
The GeneScan program is used in our laboratory to analyse the fragments obtained after DNA amplification by a PCR using fluorescence-labelled primers:
Indirect studies: haplotypes analysis by small tandem repeat (STR) markers in 56 families.
- Duchenne/Becker Muscular Dystrophy.
- Prader Willi and Angelman syndromes.
- Charcot-Marie-Tooth 1A /Pressure Palsie (HNPP).
- XLRP.
Rapid detection of the major chromosome aneuploidies (13, 18, 21, X and Y) and fetal sex determination in 47 cases.
Direct studies: expansion of unstable repeats in 34 cases.
- Huntington´s disease.
- Myotonic dystrophy.
- Fragile X syndrome.
Similar results were obtained when compared with previous conventional methods.
Therefore GeneScan program allows a reliable and reproducible analysis.
Besides, other advantages of this technique are:
- More accuracy distinguishing homozygote from heterozygote status.
- More authomatic and easier technique.
- Avoiding the use of radioactive products.
This technology has led to the innovation and improvement of the diagnostic methods.

SeqScape™ Software: Further advances in Applied Biosystems' Sequence Comparative Analysis Tool for Variant Identification

K. M. C. Gunning, Q. C. Doan, L. A. Johnston-Dow, J. M. Sorenson;
Applied Biosystems, Foster City, CA.

In biological research, the end goal is obtaining a high quality result in a timely and cost effective manner. Applied Biosystems has developed more efficient and handsfree methods by which this sequence data can be obtained with the development and improvements to automated sequence analysis. Following in this vein, downstream applications are required to allow the researcher to fully analyze and understand the data generated. Further advances in data analysis with SeqScape™ software v1.1 now mean this goal is accomplished in an organized and competent way. The next revision of the software tool is in progress which includes improved functionality of the Reference Data Group, the use of discontinuous numbering, better definition of Intron/Exons and Primer locations as well as features to support haplotyping and ORF recognition. These, coupled with the implementation of library searching and tree drawing tools will benefit a larger range of comparative analysis researchers. Additionally, access control and an audit trail are becoming more important in the realm of pharmacogenomics. By providing these sophisticated capabilities the user achieves a more valuable and comprehensive analysis of their data and progresses faster to an effective outcome.

Predicting the Optimal PCR primer annealing temperature for use with the Transgenomic Optimase^TM polymerase.

C. Lowes¹, H. Lamb¹, K. O'Connor¹, J. Walter²;
¹Transgenomic Ltd, Newcastle, Northumberland, UNITED KINGDOM, ²Transgenomic Ltd, Crewe, Cheshire, UNITED KINGDOM.

Optimase^TM polymerase is a novel proof reading polymerase. The high fidelity of this enzyme finds particular applicability to mutation detection using Denaturing High Performance Liquid Chromatography (DHPLC). Empirical determination of the optimal primer annealing temperature (Ta) during PCR has been carried out using 29 different human genomic primer sets for Optimase polymerase (Transgenomic Inc), Ampli Taq Gold (Perkin Elmer) and Pfu (Stratagene) using optimal conditions for each enzyme and a gradient thermal cycler (Hybaid). The results from this work enable a simplified strategy for successful prediction of the primer annealing temperature to be determined. In summary prediction of the primer Tm using an equation that takes into account the monovalent salt concentration, primer length and GC composition is critical and is different for each enzyme buffer composition. In addition we indicate the necessity to distinguish between primer Tm and Ta. We show here for the Optimase^TMDNA polymerase an ideal where the Ta is 2-3^oC higher than the predicted Tm for optimal PCR amplification.

Demonstration of Hot start characteristics in Transgenomic Optimase^TM Polymerase

H. Lamb¹, C. Lowes¹, K. O'Connor¹, J. M. Walter²;
¹Transgenomic Ltd, Newcastle, Northumberland, UNITED KINGDOM, ²Transgenomic Ltd, Crewe, Cheshire, UNITED KINGDOM.

The use of so-called “Hot Start” techniques has been employed to improve specificity, sensitivity and yield of the polymerase chain reaction (PCR). Although efficient amplification by PCR enzymes is optimal at elevated temperatures there is a possibility that some amplification may occur during sample set up particularly if this procedure is carried out at room temperature. For this reason the choice of a “HotStart “ enzyme is thought to convey a significant advantage. Numerous permutations of this technology are available including omitting an essential component until the tubes are at 70oC, physically separating the components using a barrier material such as a wax plug and enzyme inhibition by the use of antibodies or chemical inactivation. We have isolated, characterised and developed a novel proof reading polymerase (Optimase^TM polymerase). This high fidelity DNA polymerase has been shown to have minimal activity at ambient temperature. We demonstrate the reality of routinely setting up PCR reactions at room temperature and also being able to store samples for twenty four hours, prior to cycling, with no adverse effects on yield, misincorporation rate or amplification specificity.

Multiplex capillary heteroduplex analysis (MCHA): A rapid and sensitive method for detection of mutations in Bardet-Biedl syndrome genes.

B. E. Hoskins, P. J. Scambler, P. L. Beales;
Institute of Child Health, London, UNITED KINGDOM.

Background
Six Bardet-Biedl syndrome loci (BBS1-6) have been mapped and three identified (BBS2, 4 & 6). Inheritance in some families is complex with multiallelic participation, making linkage unreliable. These factors hamper our ability to efficiently screen for mutations in BBS patients.
Aims
To develop a rapid and cost-effective mutation assay for BBS genes.
Methods
PCR amplification using fluorescent oligonucleotides for BBS2, 4 & 6 are denatured with like wild-type PCR products to generate potential heteroduplexes to be analysed on the MegaBACE 1000. Throughput can be greatly enhanced by multiplexing compatible fragments. Between 70 and 95 Bardet-Biedl pedigrees were analysed for the presence of heteroduplex formation.
Results
We adopted two approaches using MCHA; 1. “blind” screening of BBS pedigrees for mutations in BBS4 and BBS6 followed by sequencing. 2. screening for known mutations in pedigrees previously sequenced for BBS2 mutations.

We detected 25 changes in BBS4 and 18 changes in BBS6 subsequently revealed to be SNPs or novel mutations.
Screening of BBS2 revealed 6 changes including a two-base deletion and single base substitution, a 100% detection-rate.

Conclusions
MCHA is superior to slab gel HA in particular its ability to detect point mutations. The assay is simple and rapid with up to 96 samples analysed in 40 minutes permitting resolution of fragments up to 650bp thus reducing the overall number of analyses required. We have found adaptation of HA provides a rapid and accurate screening method for mutation detection in BBS.

Comparative sequence data analysis using SeqScape^TM software for high throughput mutation scanning in DNA diagnostic analyses

A. L. J. Kneppers, M. J. R. van der Wielen, D. J. Verbove, M. Losekoot, E. Bakker;
Leiden University Medical Center, Leiden, NETHERLANDS.

The identification of disease causing small mutations in human genetic diseases is generally performed using indirect mutation scanning techniques such as SSCA, DGGE or DHPLC and subsequent sequencing of PCR amplified products. Direct sequencing is relatively straightforward and therefore suitable for automation of the complete procedure from reaction setup to purification of PCR products and sequencing reaction. The availability of automated pipetting stations and large capacity DNA sequencers enables the use of this method for high throughput mutation scanning. However, a major drawback of this approach is the large amount of sequence data to be analyzed both efficiently and accurately. Furthermore, the available sequence analysis software is usually not developed for comparative mutational analysis and therefore not entirely suitable. Recently the SeqScape^TM software (Applied Biosystems) was introduced, allowing base calling, sequence assembly, alignment and comparison, all combined in one analysis. Prior to the analysis, for each amplicon a fragment specific analysis definition is setup containing analysis defaults, reference sequence and base variants previously identified. This results in clean sequence data and easy identification and characterization of homo- and heterozygous base variants and has led to a significant reduction in manual data editing in our laboratory. However, for correct identification of heterozygous deletion or insertion mutations, extended data management and quality assurance improvements are required. This approach has enabled us to standardize our mutation detection method and perform more than 2000 DNA diagnostic tests annually in over 700 amplicons and 50 different genes in an efficient and accurate procedure.

Automated screening by DHPLC detects LDLR mutations in FH patients from New Zealand

C. F. Bunn¹, P. George¹, A. Fellowes¹, C. Lintott², R. Scott², H. Walker³, J. Mann³;
¹Canterbury Health Laboratories, Christchurch, NEW ZEALAND, ²Lipid and Diabetes Research Group, Christchurch Hospital, Christchurch, NEW ZEALAND, ³Department of Human Nutrition, University of Otago, Dunedin, NEW ZEALAND.

Familial hypercholesterolaemia (FH) is a common inherited disease, with premature vascular disease occurring 10-20 years earlier than in polygenic hyperlipidaemia. FH is easily and effectively treated. Although founder effects occur, countries with heterogeneous populations have an array of mutations; thus a gene screening approach is the appropriate first step for mutation detection.
Methods previously used to screen for LDLR mutations lack sensitivity, therefore samples from patients in NZ are being screened for LDLR mutations by denaturing HPLC (DHPLC). DHPLC has numerous advantages: analysis is rapid, inexpensive and automated. Sensitivity and specificity range from 96-100%.
Seven patient samples plus controls are screened simultaneously in a largely automated process. After PCR setup in two microtitre plates by a Tecan robot, all exons are amplified under one set of conditions. Samples are then analysed by DHPLC and duplicate abnormal products sequenced by direct sequencing.
Eight novel mutations were characterised from 27 different mutations (in 52 patients). Seven novel mutations are in the EGF domain, with the eighth in the membrane-spanning domain. The majority of mutations are localised in the EGF domain and not the ligand-binding domain as previously reported.
The diversity of LDLR mutations highlights the importance of analyses that target both known and novel mutations, especially in heterogeneous populations. As effective clinical management of FH is aided by early diagnosis, mutation detection programmes must take into account the incidence of novel mutations. Automated setup for DHPLC appears ideally suited to LDLR mutation analysis in clinical and research settings.

Novel Sample Preparation Technology for Rapid, High-Throughput Purification of Genomic DNA from Blood and Tissue Culture Cells

F. M. Holder¹, D. Qu², F. Igoe², S. Kabachus², M. Smith²;
¹Whatman International Ltd., Maidstone, UNITED KINGDOM, ²Whatman Inc., Newton, MA.

With the explosive growth in disciplines such as genomics, diagnostics, transfusion medicine and human ID there is an increasing need for fast, simple and high-quality sample preparation systems. New developments in filter media are bringing about a revolution in DNA isolation, purification and storage procedures. Using our expertise in filtration technologies, Whatman has developed a range of novel and complementary systems for the purification of nucleic acids from whole blood and other sample types. These unidirectional vacuum-based systems offer maximum flexibility, enabling purification of DNA from up to 10 ml of blood in 30 minutes whilst minimising risks inherent in multiple-step extraction protocols. A new 96-well format is now available (Gen96) which is designed to process small volume blood samples (5-75 ml) and tissue culture cells (2500 – 1x10⁶cells per well) in under 30 minutes. Genomic DNA recoveries of up to 90% of total are observed and the gDNA yielded is of high quality, suitable for PCR based techniques including STR analysis. Gen96 incorporates patented FTAÔtechnology, lysing cells on contact. Once the sample has dried this technology protects the immobilised gDNA from degradation, bacterial colonisation, UV and free radical damage. Viruses and other microorganisms are inactivated, offering user safety and simple shipment of samples. The system is therefore ideal for remote sampling applications, with samples able to be stored at room temperature for months prior to extraction without loss of DNA integrity or performance.

High sensivity and specificity of denaturing high performance liquid chromatography (DHPLC) for mutation analysis of the FBN1 gene in patients with Marfan syndrome.

A. Mori¹, S. Ansaldi¹, M. Grasso¹, A. Pilotto¹, C. Lucchelli¹, L. Lanzarini², M. Diegoli³, L. Tavazzi², E. Arbustini¹;
¹Cardiovascular Pathol. and Molec. Diagn. - Res.Transplantation Lab. , IRCCS Policlinico S.Matteo, Pavia, ITALY, ²Cardiology Division, IRCCS Policlinico S.Matteo, Pavia, ITALY, ³Department of Pathology - University of Pavia, Pavia, ITALY.

Marfan syndrome is an autosomal dominant inherited disorder of the connective tissue that principally involves the cardiovascular,ocular and skeletal systems. The incidence is estimated to be 1:5000, with 25% sporadic cases. The leading cause of death is related to the cardiovascular involvement, in particular aortic root dilatation and rupture.
The disease is caused by alteration in FBN1 gene (65 exons, located at 15q15-q21.1). Causal mutations are scattered throughout the gene and are largely unique to individual families.
The FBN1 gene was analyzed in 29 unrelated patients suspected to be affected by Marfan syndrome. To develop an efficient and faster method capable of identify all possible mutations in this gene, we introduced DHPLC technology in the analysis of 25 exons in which mutations recur. We first analysed the FBN1 exons and exon-flanking non coding regions gene coding regions with automated sequencing of all 65 exons (ABI PE- 373 DNA Sequencer) to identify mutations and polymorphisms. Then, DHPLC analysis was carried out on the WaveTM DNA Fragment Analysis System (Transgenomic, Cheshire, UK). DNA fragment elution profiles were displayed using the Transgenomic WAVEMAKER-TM software. Chromatograms were analysed and amplified fragments showing alterations were re-confirmed by automated sequencing. Overall, by direct sequencing we indentify 19 variants (14 in coding regions and 5 in intronic sequences). A corresponding number of heteroduplex profiles was detected with DHPLC with 100% correspondence to the variant-containing regions previously identified by direct sequencing. Our results confirms that DHPLC is a highly sensitive and specific technology for DNA sequence variant detection.

APEX scaling up. Optimisation of the high throughput Arrayed Primer Extension based DNA variation analysis for accurate and affordable large-scale projects.

K. Kepp¹^,2, M. Remm³, E. Lõhmussaar¹^,2, U. Gerst-Talas²;
¹Institute of Molecular and Cell Biology, Tartu University, Tartu, ESTONIA, ²Asper Biotech Ltd., Tartu, ESTONIA, ³Estonian Biocentre, Tartu, ESTONIA.

The hybridisation of complementary strands of DNA is the underlying principle of all microarray-based techniques for the analysis of DNA variation. Arrayed Primer Extension based microarray technology puts Sanger sequencing, where a polymerase is used to extend one strand of DNA primed by other strands, on a chip. The oligonucleotides arrayed on a glass chip hybridise to the sample DNA, then a polymerase and fluorescent terminator nucleotides are added. Four colours are used, each corresponding to a DNA nucleotide. The arrayed oligonucleotides are synthesized so that they are just one nucleotide from the site of the expected mutation. Incorporation of the dye terminator nucleotide gives us an exact readout of what is at the site of the mutation. This dual selection feature gives the chip a higher signal-to-noise ratio for increased reading accuracy compared to the traditional hybridisation only chip.
Asper Biotech has been running routine custom assays with 3,000 different oligos on a chip, but we are nowhere near the limit. Along side with the substantial increase of the number of oligos of the chip, application of multiplex PCR of ultimate importance in reduction of the assay cost-per-SNP. We have devised proprietary software for prediction of matching and grouping the hundreds to thousands of primer pairs before the start of the wet lab experiments.
In the current poster presentation we demonstrate the performance of our in silico results through PCR lab to the chip, as well as piling up smaller subarrays to a large chip with increased complexity.

Deletion/Duplication Detection In The 79-Exon DMD-Gene Using A Porous Micro-array System.

M. E. Kalf¹, S. White¹, A. Chan², Y. Wu², G. van Ommen¹, M. Breuning¹, T. Raap¹, J. den Dunnen¹;
¹Leiden University Medical Center, Leiden, NETHERLANDS, ²PamGene, ‘s Hertogenbosch, NETHERLANDS.

DNA deletions and/or duplications are a frequent cause of genetic disease. Detecting these is a critical aspect of DNA diagnosis, yet due to technical problems this analysis is often not performed. We have tested Multiplex Amplifiable Probe Hybridization (MAPH) as a tool for detecting such mutations in the DMD-gene, which cause Duchenne and Becker Muscular Dystrophy (DMD/BMD). MAPH is a quantitative technique, with probes being recovered by PCR following hybridization to immobilized genomic DNA.
We have analyzed the PCR-products using micro-array technology, in particular porous microarrays (PamChip^TM). Compared to planar arrays, the PamChip^TM has several advantages, including the larger active surface area, the possibility to vary hybridisation stringency during analysis and significantly decreased hybridization times.
60-mer oligos specific for individual exons were spotted on the PamChip^TM substrate. Following MAPH the PCR products were fluorescently labeled and hybridized on the microarray. Sufficient signal was obtained within 10 minutes. By comparing spot intensities between controls and patients it was possible to detect deletions and duplications in both males and females, including one and two exon duplications that were missed by other techniques. Preliminary results show a high level of reproducibility, suggesting that even smaller changes might be detectable, including mosaic cases.
This proof-of principle has encouraged us to explore the possibility of expanding the number of probes used within one hybridization. The high throughput capabilities of the PamChip^TM should help laboratories to cope with the ever increasing number of genes which need to be screened for deletions or duplications.

A systematic mutation screening approach for syndromic and nonsyndromic forms of mental retardation in human Xp21.1-Xp11.23

J. Ramser¹, G. Wen², I. Martinez Garay³, M. Dufault¹, H. Hellebrand¹, S. Engert¹, C. Schwartz⁴, M. Platzer², A. Meindl¹;
¹Ludwig-Maximilians-University, Munich, GERMANY, ²Institute of Molecular Biotechnology, Jena, GERMANY, ³University of Valencia, Valencia, SPAIN, ⁴Greenwood Genetic Center, Greenwood, SC.

Apart from 7 syndromic forms of MRX which include Prieto and Renpenning, more than 20 non-syndromic MRX families can be mapped completely or partially to the Xp21.1-11.22 region. Therefore, we are establishing a detailed gene catalogue for a region flanked by markers DXS1237 and DXS146. This interval encompasses approximately 12 megabases and is under investigation via mapping and genomic sequencing at both the Sanger Centre (UK) and within our group. Exploring the genomic data, we have identified about 120 functional genes, including 65 known genes, 55 novel genes including transcripts with unknown function like e.g. the ones of the KIAA series, spliced ESTs and genes based on exon prediction only. In addition 31 pseudogenes have been found. Assuming that the region of interest is a representative part of the X chromosome due to its extreme variance in gene densities, we expect about 1000-1200 genes for the entire chromosome.
Of the 120 putative functional genes, 10 have been associated with genetic diseases, including one, TM4SF2, that is mutated in some families suffering from MR. Detailed expression studies, including in silico as well as wetlab experiments, are in process. Immobilization of the genes on membranes and subsequent RNA-hybridization approaches will allow the establishment of expression profiles in a wide variety of tissues. A systematic approach for mutation screening on the genomic and/or cDNA level has already been established and screening is underway for the Prieto and the Renpenning syndrome. Finally, cSNPs are being collected and documented.

K. M. C. Gunning, E. E. McIntyre, C. M. Wike, K. M. Chansky, B. F. Johnston, J. M. Bourey, A. M. Wheaton, C. L. Brown;
Applied Biosystems, Foster City, CA.

Applied Biosystems has always placed a large emphasis on continuous innovation. The 3100 system opened the doors for many researchers interested in speeding up their access to genetic information by providing a flexible platform that automated the process of genetic analysis. In order to keep pace with discovery, we have expanded this platform by further increasing the realm of possibilities, yet maintaining the quality and dependability one expects from Applied Biosystems. With the addition of the 80cm array for long read sequencing, and the 22 cm array for SNP analysis, we have increased the throughput of the instrument and allowed substantial cost savings to the investigator. When coupled with improvements to the Data Collection software the average investigator has acquired a more automated system, capable of allowing continuous operation for more than 24 hrs. We will discuss in detail how these features will expand your research capabilities and present a cost effective and handsfree solution, ready to decipher the unknown in molecular research.

J. A. Correa¹, S. Dudoit², D. R. Goldstein³;
¹Florida Atlantic University, Boca Raton, FL, ²UC Berkeley, Berkeley, CA, ³UCLA, Los Angeles, CA.

DNA microarrays are increasingly being used to study the molecular basis of complex diseases like cancer, often aiming to identify genes potentially influencing important clinical outcomes (e.g. survival). To identify genes, survival is modeled (Cox proportional hazards) as a function of sets of gene expression levels. In its simplest version, the modeling is done one gene at a time. Prioritizing results for follow-up requires a realistic assessment of the significance of a relevant test statistic for the thousands of simultaneous comparisons made with a typical microarray.
We have studied the performance of several multiple testing procedures for survival data (including Bonferroni, Westfall and Young maxT family-wise error rate (FWER) controlling procedure, and Benjamini and Hochberg false discovery rate (FDR) controlling procedure). We illustrate their use on two publicly available cancer microarray datasets: a melanoma dataset with survival and gene expression measurements for 15 individuals on 3613 genes; and a lymphoma dataset with 40 individuals and 4026 genes. Although there are several unadjusted p-values smaller than 0.01, none of the genes appears particularly promising once adjustment is made for multiple testing. The lack of significant findings with either the FWER or FDR controlling procedures could be due to the small sample size, but also points to limitations of current approaches. Firstly, FWER controlling procedures may simply be too stringent in some microarray applications. Secondly, FDR controlling procedures, while less conservative than FWER controlling procedures, need to be refined to take into account the joint distribution of the gene expression levels.

Construction of a microarray on 5q31-q33 region to identify genes controlling resistance or susceptibility to parasitic diseases as schistosomiasis and malaria.

C. Chevillard¹, B. Loriot², N. Crapoulet¹, R. Tagett², R. Houlgatte², C. NGuyen², A. Dessein¹;
¹INSERM U399, Marseille, FRANCE, ²INSERM U136, Marseille, FRANCE.

Schistosomiasis affects 200 million people and is a major public health problem. Our group performed studies on the causes of high infections in an endemic area of Brazil. Certain subjects appeared to to be predisposed to high infections whereas others always exhibited low infection in spite of high exposure. This suggested that host-specific factors were important in the control of infection. Using segregation and linkage analysis, it was shown that there was strong evidence for the control of infection by a major locus (SM1, located in the 5q31-q33 region which contains a number of gene that encode cytokines that play an important role in the regulation of immune response against parasites. Immunological studies performed on the same population showed that SM1 control is linked to the differentiation of the T helper cells into Th1 or Th2 lymphocytes. Furthermore, it was also reported that blood parasitemie in Plasmodium falciparum are controlled by a locus located in the same region. This region is containing a large number of genes. In order to facilitate the analysis of this locus we have developed a systematic expression level analysis of the 5q31-q33 interval by microarray technology. The target will be a I.M.A.G.E. clone set arrayed on nylon support. The probe will be labeled with 33P. Indeed combination of nylon array with 33P labeled radioactive probes provides 100 fold better sensitivity, making it possible to perform expression profiling experiments using submicrogram amounts of unamplified total RNA from small biological samples.

Genome-wide survey of genes associated with coronary atherosclerosis and aortic aneurysm using polymorphic microsatellite markers and microarray technology

J. Taguchi¹, G. Tamiya², K. Okamoto², S. Koide², S. Handa², H. Inoko²;
¹Tokai University, Hachiouji, Tokyo, JAPAN, ²Tokai University, Isehara, JAPAN.

The aim of this study is to exclusively identify genes related to coronary atherosclerosis and aortic aneurysm by genome-wide association analysis using polymorphic microsatellite markers. Microsatellite markers showed linkage disequilibria with disease-related alleles spanning from 100 to 200 kilobases (kb). For the purpose of genome-wide association studies, we finished identifying 30,000 polymorphic microsatellite markers from the human genome draft sequence which can cover whole genome. We then genotyped pooled DNA from 100 to 200 healthy Japanese individuals using those markers to confirm microsatellite polymorphism, allele frequency and heterozygosity. We have simultaneously started to apply those markers to conduct a genome-wide association study of atherosclerosis-susceptibility genes. We also investigated gene expression in aortic aneurysm tissue using GeneChip (Affymetrix) U95 sets.
We report here our findings to date of different susceptibility locations within the genome. For example, we found strong association in the area from 7q32.3 to 7q36.3 confirmed by two different normal and diseased DNA pools. By narrowing the area, we believe there should be unknown gene related to coronary atherosclerosis. In addition, we found about 4700 known genes expressed in aortic aneurysm tissue. Genes of osteopontin, apolipoprotein E, metalloproteinases and cathepsin D were highly upregulated. We believe the information gathered from DNA microarray technology would complement our genome-wide association studies. This combined approach should accelerate identification of atherosclerosis-related genes.

SNP analysis and linkage disequilibrium map of the human chromosome 22 using APEX arrays.

E. Lõhmussaar¹^,2, J. Zernant², R. Mägi², K. Kepp¹, T. Puurand², N. Tõnisson², I. Valvas², G. Slavin², R. Andreson¹^,2, A. Kurg¹, M. Remm³^,2, A. Metspalu¹^,2;
¹Institute of Molecular And Cell Biology, Tartu, ESTONIA, ²Asper Ltd., Tartu, ESTONIA, ³Estonian Biocentre, Tartu, ESTONIA.

Sequence variants in the human genome are responsible for the genetic component of disease, individuality and drug response. In order to find associations between SNPs and phenotype, large sample sets need to be genotyped with high-density markers. With chromosome 22 fully sequenced and a SNP map constructed across 22q, we have performed genotyping of Estonian and German samples and CEPH families with 1279 SNP markers. An array with 5200 oligonucleotides was designed to genotype each SNP twice from both DNA strands simultaneously using APEX technology. Allele frequencies, Hardy-Weinberg equilibrium and heterozygosities were calculated for each genotyped marker.
We have characterised the patterns of linkage disequilibrium and calculated D’ for the whole 33Mb of chromosome 22. Our results demonstrated that along the chromosome the pattern of LD is highly variable, where regions of high LD are interspersed with regions of little or no detectable LD. It appears that SNPs from public databases will need additional testing in order to find out useful SNPs in respect of genotyping technology. Construction of LD maps across the human genome and identifying haplotypes in individual genomic regions will facilitate the identification and characterization of genetic variants responsible for common complex diseases.

MassARRAYÔ Analysis of Fragmented Nucleic Acids: Applications in Typing, Sequence Validation, and Targeted SNP Discovery

C. P. Rodi¹, N. Storm², B. Darnhofer-Patel¹, R. Hartmer³, L. Leppin¹, S. Böcker¹, M. Denissenko¹, D. van den Boom¹;
¹Sequenom, Inc., San Diego, CA, ²Sequenom, Inc., Hamburg, GERMANY, ³Sequenom, Hamburg, GERMANY.

The MassARRAYÔ System is widely used to do high-throughput scoring of single nucleotide polymorphisms (SNPs) using the MassEXTENDÔ assay. A new application for high-throughput genetic analysis is presented which we call Fragmentation. Single-stranded nucleic acid is created and in four separate reactions fragmented at positions corresponding to each of the four bases. Using a reference sequence, such as the now available human genome, the precision, accuracy, and resolution of MALDI-TOF Mass Spectrometry allows one to definitively identify each resulting peak. Taken together, the collection of peaks creates a sort of biological barcode that supports high throughput typing. It also allows one to quickly and accurately perform sequence validation, key to many European patents. Recent software developments now also allow us to use this technology in high-throughput, targeted SNP discovery. In contrast to other techniques such as SSCP and dHPLC, in the majority of the cases Fragmentation not only detects SNPs, but can also definitively identify and locate the polymorphisms. This allows for the facile and quick design of a MassEXTEND assay for accurate, high-throughput SNP scoring.

Development and application of a cytochrome b SNP microarray to screen patients with mitochondrial complex III deficiency

U. A. Nuber¹, F. Erdogan¹, F. A. Hol², H. H. Ropers¹;
¹MPI for Molecular Genetics, Berlin, GERMANY, ²University Medical Center Nijmegen, Nijmegen, NETHERLANDS.

Complex III is the second enzyme in the electron transport chain of mitochondrial oxidative phosphorylation, and consists of several polypeptide subunits. One of these, cytochrome b, is encoded by mitochondrial (mt) DNA. So far, mutations in MTCYB and a nuclear encoded gene, BCS1L, have been found as cause of complex III deficiency. Patients with cytochrome b mutations may show widely varying clinical phenotypes, ranging from pure exercise intolerance to multisystem abnormalities, and the clinical findings also overlap with other mitochondrial diseases. These disorders are therefore best classified on the basis of pathogenic mtDNA variations. In order to systematically screen patients with biochemically determined or suspected complex III deficiency for single-nucleotide DNA variations, we have established a cytochrome b SNP microarray. This tool consists of covalently attached allele-specific primers representing all 90 SNPs and mutations of MTCYB found to date. In contrast to other SNP detection methods, only one PCR product comprising the complete MTCYB mtDNA and therefore all variations, is used as a target. The subsequent primer elongation protocol allows the typing of all 90 variations in one reaction. To test if this method is suitable for detecting heteroplasmy, we have mixed MTCYB DNA targets with known polymorphisms from two different samples. Material from approximately 70 patients is currently being analysed to detect novel cytochrome b phenotype-genotype correlations.
Our next aim will be the extension of this microarray to cover mtDNA encoded polypeptides of complex I.

Accurate SNP genotypes determined using a multiplex ligation/PCR based assay (OLA/PCR) in combination with a universal DNA microarray

M. H. Wenz, A. Bach, J. Day, F. de la Vega, E. Friedlander, K. Hayashibara, D. Holden, S. Johnson, R. Koehler, L. Leong, D. Maglasang, T. Paner, D. Ridzon, S. Short, P. Wu;
Applied Biosystems, Foster City, CA.

One of the expected consequences of the Human Genome Project is the practical utilization of extensive genetic diversity of populations. Public and private efforts have discovered polymorphic repeat sequences, as well as single nucleotide polymorphisms (SNPs) for genotyping. Several different databases now contain millions of candidate SNPs. In order to realize the full potential of SNP genotyping in large-scale population studies, sensitive, accurate and cost effective methods have to be established for scoring SNP genotypes. We have developed a ligation/PCR based assay (OLA/PCR) that uses a universal DNA microarray for SNP-specific assay product identification. Genomic DNA is interrogated with multiplex sets of ligation probes specific for selected SNP loci. After ligation, the resulting products are simultaneously amplified in a universal PCR reaction. The minimum single tube multiplex level has been shown to be ~50 loci, translating into a consumption of approximately 1 ng of gDNA per SNP genotype. We have assembled several multiplexes and tested these against 28 different CEPH DNA samples to demonstrate the feasibility of this approach. We will present data that describe the reproducibility and accuracy of this assay.