Genotyping single nucleotide polymorphisms by MALDI mass spectrometry in clinical applications

Clinical Biochemistry 38 (2005) 335 – 350

Review

Genotyping single nucleotide polymorphisms by MALDI mass spectrometry in clinical applications Jfrg Tost, Ivo Glynne Gut* Centre National de Ge´notypage, Baˆtiment G2, 2 Rue Gaston Cre´mieux, CP 5721, 91057 Evry Cedex, France Received 13 July 2004; received in revised form 22 November 2004; accepted 9 December 2004 Available online 23 January 2005

Abstract Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has become one of the most powerful and widely applied technologies for SNP scoring and determination of allele frequencies in the post-genome sequencing era. Although different strategies for allele discrimination combined with MALDI were devised, in practice only primer extension methods are nowadays routinely used. This combination enables the rapid, quantitative, and direct detection of several genetic markers simultaneously in a broad variety of biological samples. In the field of molecular diagnostics, MALDI has been applied to the discovery of genetic markers, that are associated with a phenotype like a disease susceptibility or drug response, as well as an alternative means for diagnostic testing of a range of diseases for which the responsible mutations are already known. It is one of the first techniques with which whole genome scans based on single nucleotide polymorphisms were carried out. It is equally well suited for pathogen identification and the detection of emerging mutant strains as well as for the characterization of the genetic identity and quantitative trait loci mapping in farm animals. MALDI can also be used as a detection platform for a range of novel applications that are more demanding than standard SNP genotyping such as mutation/polymorphism discovery, molecular haplotyping, analysis of DNA methylation, and expression profiling. This review gives an introduction to the application of mass spectrometry for DNA analysis, and provides an overview of most studies using SNPs as genetic markers and MALDI mass spectrometric detection that are related to clinical applications and molecular diagnostics. Further, it aims to show specialized applications that might lead to diagnostic applications in the future. It does not speculate on whether this methodology will ever reach the diagnostic market. D 2005 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Mass spectrometry; Matrix-assisted laser desorption/ionization; MALDI; Single nucleotide polymorphism; SNP; Genotyping; High throughput Quantification; ; Haplotyping

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . MALDI mass spectrometry . . . . . . . . . . . . . . . . . MALDI and SNPs . . . . . . . . . . . . . . . . . . . . . . Methods based on primer extension for allele discrimination Single base primer extension (SBE) . . . . . . . . . . . . . Multiple base primer extension . . . . . . . . . . . . . . . Nucleotide depletion genotyping assay (NUDGE) . . . . . . Data interpretation . . . . . . . . . . . . . . . . . . . . . . Applications using MALDI mass spectrometric detection . . Genotyping in pharmacogenetics. . . . . . . . . . . . . . . Disease association studies with MALDI mass spectrometric

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* Corresponding author. E-mail address: [email protected] (I.G. Gut). 0009-9120/$ - see front matter D 2005 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2004.12.005

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Asthma. . . . . . . . . . . . . . . . . . . . . . . . . . Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . Cardiovascular diseases . . . . . . . . . . . . . . . . . Obesity. . . . . . . . . . . . . . . . . . . . . . . . . . Various . . . . . . . . . . . . . . . . . . . . . . . . . . Scans of candidate regions and whole genome scans . . MALDI-based diagnostic assays . . . . . . . . . . . . . Molecular haplotyping . . . . . . . . . . . . . . . . . . Bacterial and viral typing by MALDI mass spectrometry Genotyping in animals and plants . . . . . . . . . . . . Mutation and polymorphism discovery . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction DNA research has come a long way in the last 50 years. Rosalind Franklin’s X-ray analysis of a DNA molecule [1], from which Watson and Crick derived the structural model of DNA with its two chains and its helical nature [2], laid the foundations to high profile projects such as the human genome sequencing project, which has now been announced as completed [3]. Supported by this enormous amount of reference data, attention is now turning towards the elucidation of gene function and regulation. It is hoped that a better understanding of the biological processes will help to unravel the complex genetic traits of, for example, multifactorial disorders. Understanding the genetic basis of human variation is a vital goal in biomedical research. Alterations in gene sequences, expression levels, and protein structure and function have been associated with many types of disease. Genetic variations such as polymorphisms and mutations influence transcription into RNA as well as the following translation into proteins. In recent years, research has focussed on single nucleotide polymorphisms (SNPs) as genetic markers because of their simpler mutational dynamics and greater prevalence compared to microsatellites. SNPs are single base changes that occur at a specific position in a genome. By definition, the less frequent allele has an abundance of 1% or greater, otherwise it is referred to as mutation [4]. In diploid species, like humans, SNPs are usually biallelic (a SNP has two alleles and only in very rare cases three or four alleles). On average, one SNP is found every 300 to 1000 bases in humans and they are estimated to represent as much as 90% of all genetic variations [5]. As part of the draft sequence, 1.42 million SNPs were reported [6] and public databases now contain more than 6 million mapped human SNPs [3]. Only a small portion of them lie within coding regions (cSNPs) and an even smaller percentage is responsible for amino acid changes in expressed proteins. However, SNPs in non-coding regions can still affect gene regulation, influence mRNA stability and conformation, and the quantity and quality of expressed gene product. To

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geneticists, the fact that a marker can be lodged directly in the genomic region of interest, for example, a candidate gene, has great value. An allele of an SNP can constitute a genetic risk factor as it may increase susceptibility to certain diseases. Due to their binary nature, SNPs are fairly easy to genotype and the interpretation of the readout can be automated. The field of SNP genotyping methods and highthroughput versions thereof has been reviewed extensively in the past few years [7–9]. The diverse approaches fulfill criteria such as accuracy of SNP detection, sensitivity to score SNPs using a small amount of template, throughput capacity, flexibility of the procedure, and cost-effectiveness. SNP genotyping methods that use matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry for detection are among the most powerful and reliable [10].

MALDI mass spectrometry Mass spectrometry and particularly matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has revolutionized the instrumental analysis of biomolecules [11,12]. In the last years, MALDI has become a standard tool for the identification and quantification of proteins and protein-based biomarker profiling and proteomic pattern analysis [13]. The analysis of protein-based biomarkers in clinical diagnosis has recently been reviewed [14,15] and this review will therefore solely concentrate on the potentials and demonstrated applications of MALDI mass spectrometry for nucleic acid-based diagnosis. For analysis by MALDI mass spectrometry, analytes are embedded into a matrix of small organic molecules and deposited on the metal surface of a target plate. The matrices, which are in a more than 1000-fold excess, are usually aromatic acids with a chromophore absorbing at the emission wavelength of the laser—commonly N2 (k = 337 nm). Part of the absorbed energy is transferred to the analyte. This process–coined soft ionization–greatly facilitates the production of intact, mainly single charged gas-phase ions of large,

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non-volatile, and thermally labile biomolecules. The events occurring in the desorption plume are not yet fully understood and despite its widespread use, the development of new matrices or other improvements has remained an empirical process. The ions are transferred electrostatically into a mass spectrometer in which they are separated from each other and the matrix molecules, most commonly by time-of-flight (TOF) measurement by drifting through a field-free region. The ion’s flight time is thereby proportional to the square root of its mass-to-charge ratio. The TOF analyzer gained widespread popularity due to its simplicity, robustness, and sensitivity over a large mass range. DNA analysis by MALDI mass spectrometry has almost exclusively been carried out with TOF analyzers. However, analysis of nucleic acids by MALDI mass spectrometry has some drawbacks, such as that it is extremely sensitive to the presence of metal contaminants due to the multiple negatively charged sugar phosphate backbone which is susceptible to adduct formation causing peak broadening and reduction of resolution, sensitivity, and accuracy. As cations such as sodium and potassium are highly abundant in buffers for molecular biology reactions, stringent purification procedures need to be applied. Additional problems for MALDI analysis of nucleic acids are fragmentation, especially depurination, and a decreased sensitivity with increased size of the analyte. This restricts the routine analysis to small oligonucleotides up to a length of 30 nucleotides. Therefore, DNA analysis by MALDI mass spectrometry turned from initial efforts from replacing gelbased sequence analysis to the problem of mutation detection.

MALDI and SNPs Rapid, precise, and cost-effective high-throughput methods are required to perform the large-scale screens necessary for the discovery of genetic markers involved in the etiology and pathophysiology of multifactorial disorders and for the identification of potential markers for clinical diagnosis, prognosis, and monitoring. Traditional methods based on molecular biology techniques often lack essential features for clinical settings such as accuracy, automation, and throughput. Mass spectrometry-based methods for SNP genotyping have been continuously improved and MALDI-MS is now one of the most automated and efficient detection platforms, very price competitive when used at high throughput, and among those delivering results of highest accuracy and reliability [10]. Mass spectrometry assays based on primer extension reactions outcompete most other methods in terms of robustness, accuracy, reproducibility, and success rates when directly compared [16,17]. The same platform can also be applied to the analysis of DNA methylation analysis [18], expression profiling [19], and proteomics [13], making the mass spectrometer one of the most versatile tools in the post-genome sequencing era. All methods for SNP genotyping combine two elements: first, the generation of an allele-specific product, and second

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the analysis thereof. Currently, four principles exist for the generation of the allele-specific products: hybridization, ligation, cleavage, and primer extension. All of the mentioned methods for allele discrimination have been combined with MALDI analysis (Table 1). Methods that were applied to the identification and analysis of SNPs that might contribute to a disease or drug side effect predisposition and might therefore be of diagnostic and clinical relevance are reviewed in the following. Technical details of most genotyping assay using mass spectrometric detection are described in detail in a recent exhaustive review [10], to which the interested reader is referred to for more details. As most investigations use assays based on primer extension reactions, this review concentrates on the different applications of these assays.

Methods based on primer extension for allele discrimination Primer extension [20] is the most widely applied method for allele distinction for SNP genotyping and point mutation analysis and has been combined with a broad range of detection platforms [8]. Incorporation of a complementary nucleotide by a DNA polymerase distinguishes more accurately between the two alleles than the different thermal stability of hybridizing allele-specific probes [21]. Primer extension assays are flexible, robust, and well suited for high-throughput applications. Several primer extension assays that use MALDI as the detection platform have been developed. In all of these assays, the DNA target sequence is amplified by PCR. Remaining dNTPs and primers are removed from the reaction by various methods, because these compounds would be undesirable and interfere in the following primer extension reaction. A primer for primer extension anneals with its 3V-terminal base immediately upstream of the SNP on the target sequence. Discrimination of the alleles is achieved by essentially three different strategies: single base primer extension (SBE), multiple base primer extension, and nucleotide depletion. Each allele product of a SNP is detected at a specific mass due to the addition of nucleotides that naturally differ in mass (SBE) or to the addition of a different number of bases of specific mass (multiple base primer extension). After the allelespecific reaction, samples need to be conditioned prior to the analysis step and sample preparation is of crucial importance to obtain satisfactory results. Components of enzyme buffers such as detergents, stabilizers, and glycerol as well as salts can severely interfere with MALDI analysis. This process can be carried out by various methods, such as reversed phase purification, ethanol precipitation, and streptavidin-coated magnetic beads. Ion exchange beads allow a homogeneous assay format and are suitable for automation of this step [22,23]. Modified sugar-phosphate backbones can be charge neutralized by chemical means and render it insensitive to adduct formation [24]. An important feature for the analysis by MALDI is the generation of small

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Table 1 Overview: Methods for allele discrimination and assays with MALDI mass spectrometric detection Method for allele Method distinction

Purification

Mass range, m/z Main advantage

Hybridization

Oligonucleotide ligation Cleavage

Primer extension

PNA Monitored nuclease selection assay Nuclease protection assay Ligase chain reaction

Magnetic beads None

2000–4000 5000–8000

ZipTips Magnetic beads

3000–5000 N9000

Restriction enzymes

Desalting columns

Invader assay

Magnetic beads

PinPoint assay

ZipTips

PROBE assay

Magnetic beads

Homogenous MassEXTEND assay Solid Phase Capturable Single Base Extension assay VSET assay

Ion-exchange resin

GenoLINK assay

Magnetic beads

4000–8000

GenoSNIP assay

Magnetic beads

1000–2000

GOOD assay Nucleotide depletion assay

none MultiScreen plates

1000–2000 4000–8000

Magnetic beads

Main disadvantage

Simple principle Prize of PNAs Cheap, robust enzyme Time-consuming analysis

Reference [134,135] [136]

Cheap, robust enzyme Prize of PNAs Exponential product Low enzymatic specificity amplification 10,000–100,000 Simple principle Not universally applicable, expensive enzymes 1000–2000 No PCR, isothermal Large amount of DNA reaction consumed 4000–9000 Accurate, rapid data Mass difference between accumulation, A/T may not be resolved multiplexing 4000–8000 Accurate, rapid data Solid-phase purification accumulation 4000–8000 High-throughput, Cost of instrumentation rapid, accurate set-up

[137] [138]

4000–8000

Solid-phase purification

[28,37]

Purification

[40]

Solid-phase purification Solid-phase purification

Bruker Saxonia GmbH [29]

Modification chemistry Purification

[26,35] [42]

Ethanol precipitation 4000–8000

products (3 to 25 bases). Duplex structures of extension primers and templates dissociate under MALDI conditions so that only the short extension primer and primer extension products are detected.

Single base primer extension (SBE) For single base primer extension reaction (Fig. 1a), the primer anneals immediately next to the polymorphic site and is extended with a single nucleotide, using terminating dideoxynucleotides (ddNTPs) and a DNA polymerase. This approach is applied in the PinPoint assay [25], commercialized by Applied Biosystems as the Sequazymek PinPoint SNP Typing Kit, the GOOD assay [26,27], the solid phase capturable single base extension (SPC-SBE) assay [28] and the genoSNIP assay [29]. Analysis of some SNPs may be difficult by SBE assays as the smallest mass difference between two nucleotides (A and T) is only 9 Da, which is demanding to resolve in the typically used mass range of 4000 to 9000 Da. Several strategies were reported to enhance separation of the alleles. In the classical PinPoint assay, it is the use of tags [30] or cleavable extension primers [31] which can then achieve multiplexing levels of up to a 15-plex reaction [32,33]. The degree of multiplexing is also limited by the design of site-

Accurate, rapid data accumulation, high multiplexing Accurate, rapid data accumulation Accurate, rapid data accumulation Increased sensitivity, small products No purification Accurate, rapid data accumulation

[139–141] [142] [25,32]

[38] [23]

specific extension probes that must be accommodated in the mass range of the mass spectrometer. The GOOD assay circumvents the resolution problems by a chemical modification strategy (charge tagging, [34]) which increases signal intensity to that of peptides and reduces the extension products to a core-sequence of four to five nucleotides that contain the allele information [35]. In a variant of the GOOD assay, SBE is carried out with photocleavable extension primers [36]. Photocleavable extension primers are also the key point of the genoSNIP assay [29]. These strategies bring the masses of the allele-specific extension products into a mass range, where sensitivity and resolution are improved. In contrast to the other assays, the SPC-SBE assay uses biotinylated ddNTPs and only extended oligonucleotides are retained during sample conditioning thereby increasing the possibility for multiplexing as no signals corresponding to non-extended primers are detected [37].

Multiple base primer extension To enhance the clear assignment of the product peaks in the mass spectrum, alleles are spaced further apart by using a mixture of deoxynucleotides (dNTPs) and dideoxynucleotides (ddNTPs) (Fig. 1b). The oligonucleotide for the primer extension reaction anneals again immediately adjacent to a

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Fig. 1. Schematic representation of the three different modes, in which primer extension is performed with MALDI mass spectrometric detection (demonstrated using a virtual C/T SNP) with corresponding mass spectra. (a) Single base primer extension reaction as described for the PinPoint assay using four ddNTPs [25], (b) multiple base primer extension as described for the MassEXTEND assay [23], in this case, one dGTP is added to the three other ddNTPs, and (c) nucleotide depletion genotyping assay [42], for which extension is carried out in the presence of only three dNTPs, the fourth one is depleted.

polymorphism; one allele is extended by only one base while the second one by two or more. Termination occurs at the first nucleobase in the template complementary to a ddNTP. This strategy generates products with a mass difference of at least 300 Da which facilitates allele discrimination. This concept is used in the PROBE assay [38] the MassEXTEND assay [23,39] (Sequenom), the VSET assay [40], and the GenoLink assay (Bruker Saxonia Analytik GmbH, Leipzig. Germany). The MassEXTEND assay is the result of continuous improvement of the original PROBE assay running on a highly automated, high-throughput platform, available for different dimensions of throughput. The homogeneous MassEXTEND uses ion-exchange resin for sample conditioning prior to analysis, avoiding biotinylated primers and immobilization as used in the PROBE assay. Nanoliter quantities of the samples are transferred onto silicon chips precharged with 3-hydroxypicolinic acid (HPA) as matrix by piezoelectric pipetting [41]. As the entire preparation is volatilized with a few laser pulses, the need for bsweetQ spots of the HPA matrix is avoided and the spot-to-spot reproducibility increases. Multiplex primer extension reactions of up to eight-plexes have been reported and four-plex can be used routinely. Most of the below-described studies have used

the PROBE/MassEXTEND assay as Sequenom offers a turn-key solution with chemistries, sample preparation robotics, a dedicated MALDI mass spectrometer and supporting software (MassARRAY system). This facilitates the genotyping procedure and makes it accessible to non mass spectrometrists.

Nucleotide depletion genotyping assay (NUDGE) Decode Genetics very recently presented a variant of a multiple base primer extension assay (Fig. 1c) [42]. The primer is designed to anneal with its 3Vterminus two bases upstream of the polymorphic site. The allele discrimination is carried out in the presence of three deoxynucleotides, the fourth one, which is complementary to one allele of the SNP, is depleted. Termination occurs thus for one allele at the polymorphic position while for the other one at the first nucleobase in the template complementary to the depleted dNTP. This strategy allows reducing costs as the DNA polymerase from the PCR is sufficiently active to perform the allele-differentiating step and does not have to be a special DNA polymerase as no ddNTPs have to be incorporated. However, non-proof reading (exonuclease

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deficient) polymerases should be used to prevent degradation of the oligonucleotide used for primer extension.

Data interpretation The interpretation of the mass spectrometric data is nowadays largely automated and comes as part of the software with most of the commercially available mass spectrometers. For DNA analysis, initially, peak identification is carried out followed by labeling and assignment. For peak identification, signal to noise is determined. If a given threshold is surpassed, a signal is identified. Thereafter the maximum of the peak is determined which is then labeled. Peak intensity at the maximum is measured from the baseline. For mutation analysis, the alleles have to have different masses. The calling of the alleles relies on a peak at a discreet known mass exceeding an intensity threshold. The aim of the allele-distinction method is to well separate the two alleles of a polymorphism by providing good mass separation. This facilitates calling. A peak outside the discreet mass window is disregarded while the presence of a peak in the discreet mass window is used for analysis. One of the great strengths of mass spectrometry lies in that the actual allele product is analyzed rather than, for example, a red or green fluorescent tag emission that is relayed back to the allele. Any error in manipulation is very easy to pinpoint because the brightQ allele masses are not obtained. Modern instruments can be operated with external calibration (no internal size standards are required). Calibration is stable for months and variation of mass peaks tends to be b0.05%. For diagnostic applications, it makes sense to establish multiplex assays testing various polymorphisms simultaneously. Interpretation of multiplexes is as easy as simplex tests and signals from different test can be used as internal controls for handling and quality of reagents.

Applications using MALDI mass spectrometric detection MALDI-TOF mass spectrometry has evolved as a mature device for the analysis of genetic markers in the postgenome sequencing era. Applications of MALDI mass spectrometry relevant to human health and disease can be roughly classified into five main categories: (1) pharmacogenetic and pharmacogenomic profiling, (2) genotyping for detection of an association between genetic marker and a disease, (3) diagnostic testing, (4) traceability in the animal husbandry industry to ensure consumer’s health, and (5) bacterial and viral typing.

Genotyping in pharmacogenetics There is considerable interest for pharmacokinetic and pharmacodynamic studies in the analysis of normal func-

tional variants caused by single nucleotide exchanges in genes encoding drug metabolizing, transporting, or target enzymes. These SNPs are estimated to account for up to 95% of inter-individual variability leading to a difference in drug metabolism and effect [43]. The promise is that patients will be medicated as a function of their genetic profile assessing individual responses to pharmacologic agents and thereby minimizing the risk of adverse drug effects and loss of time in therapy. Mass spectrometric detection has so far only been used in a few applications. SNPs in the flavin-containing monooxygenase form 3 (FMO3) gene are implicated in abnormal drug metabolism and the allele frequencies of five common SNPs discovered in the coding and adjacent regions were determined in populations of different ethnic background using the MassEXTEND assay and combined with functional studies for assessing altered enzyme activity [44,45]. Combinations of SNPs (haplotypes) in the h2-adrenergic receptor gene correlate with the response to asthma treatment using agonists such as albuterol [46]. Several multiplex primer extension assays have been devised analyzing the haplotype tagging genetic variants on a variety of detection platforms. MALDI mass spectrometric analysis has been combined with the solid phase capturable primer extension assay in a triplex format [47]. Haplotypes of this gene have also been directly determined by mass spectrometry using an allelespecific PCR amplification and multiplex primer extension based on the GOOD assay (see below, [48]).

Disease association studies with MALDI mass spectrometric detection MALDI mass spectrometry has been used for the detection of possible association between genetic variants and a disease. Thereby the tested marker can be either the disease causing variant itself or be in linkage disequilibrium with a genetic factor causing the disease, which is so far undetected. Table 2 presents an overview of most studies so far published. However, most of these studies were carried out on a small number of samples that might be insufficient to detect the subtle effects in complex disease [49]. It should also be kept in mind that these are probably only part of the association studies carried out with MALDI mass spectrometry as negative associations are far more difficult to publish when no technical improvements are presented at the same time.

Asthma Asthma is a chronic inflammatory disease of the airways and is the most common chronic childhood disease in industrialized countries. Several genome scans have been carried out identifying numerous candidate genes [50]. Some of them have been fine typed using mass spectrometry as

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Table 2 Association studies carried out using MALDI mass spectrometry Gene

Disease

Individuals cases/controls

SNPs

Assay

Ref.

MDR1 MDR1 IL1RN ADAM33 ADAM33 IL110 IL1RN STAT6 Folate/homocysteine metabolism CETP Various pathways PNMT Calpain 10 PPAR g2 GHSR SLC6A14 Interleukin gene cluster 6p21 20q13 Genome wide

Acute lymphoblastic leukemia Colorectal cancer Ankylosing spondylitis Asthma Asthma Asthma Asthma Asthma Cardiovascular diseases Cardiovascular diseases Cardiovascular diseases Hypertension Diabetes Diabetes Obesity Obesity Malaria susceptibility Various Type 2 diabetes Morbidity

80 45/45 394/500 48/597 587 families 415 (97 families) 762 (trios) 474 (108 sib pair families) 460 Pools (400/400) 14/19 307/266 781/822 522/413 454/329 837/968 716/1080 5 pools 4 pools + individual genotyping 2 pools

1 1 3 15 17 1 39 12 12 15 39 2 3 1 2 2 8 534 291 6500

PROBE PROBE MassEXTEND MassEXTEND MassEXTEND PROBE PROBE PROBE PinPoint MassEXTEND MassEXTEND MassEXTEND MassEXTEND PROBE MassEXTEND MassEXTEND MassEXTEND MassEXTEND MassEXTEND MassEXTEND

[62] [63] [69] [55] [56] [52] [53] [51] [66] [76] [64] [65] [61] [59] [68] [67] [70] [77] [78] [79]

For reasons of simplification, numbers of separately analyzed cases and controls sample sets such as replications may have been merged. For abbreviations of gene names, see text.

analysis tool. SNPs in the signal transducer and activator of transcription 6 (STAT6) gene were analyzed by the PROBE assay [51]. Genotypes correlated with IgE serum levels, but no clear implication in the pathology of asthma was found. An SNP in the interleukin 10 promoter (IL10_C-592A) analyzed with the PROBE assay showed a possible association ( P = 0.036) in combination with eosinophil cell counts [52]. Several SNPs in the anti-inflammatory cytokine interleukine-1 receptor antagonist gene (IL1RN) were associated with asthma in a German population using the PROBE assay [53]. Associations were replicated by the authors in an independent second sample set (Italian population) confirming their findings. The association of SNPs in the gene of a disintegrin-like and metalloproteinasecontaining protein 33 (ADAM33) with asthma [54], was replicated in a family based study and a case-control association study showing significant association of 3 out of 15 SNPs with the phenotype [55]. However, these results could not be reproduced in a recent study using a North American population analyzing fifteen SNPs in the ADAM33 gene by the MassEXTEND assay [56]. These results might indicate that the causative gene/polymorphism is nearby but not in the ADAM33 locus as a haplotype but not a single SNP was found to be correlated and linkage disequilibrium is high in this chromosomic region.

[57]. A previously identified polymorphism in the peroxisome proliferator-activated receptor g2 gene (PPAR g2Pro12Ala), that has been consistently associated with a decreased risk for type 2 diabetes and diabetes-related traits [58], was tested in a population of 935 Finish samples using the PROBE assay confirming the hypothesis [59]. Association of another candidate gene, calpain 10, previously identified in Mexican Americans with T2DM [60], was not connected in a large Finnish population as analyzed by the MassEXTEND assay [61].

Cancer Genetic alterations from point mutations and SNPs, to insertions and deletions, to chromosomal translocations play a causal role in the process of malignant transformation. The possible correlation between an SNP (C3435T) in the multidrug resistance gene and overexpression of the corresponding mRNA in acute lymphoblastic leukemia has been tested in a small study using the PROBE assay [62], but no significant relationship was found. The frequency of the same SNP, previously brought into relation with altered cellular drug uptake in colorectal cancer, was also not increased in tumors compared to matched normal controls in another small study from the same laboratory and could not be correlated with survival rate [63].

Diabetes Type 2 diabetes mellitus (T2DM) is a heterogeneous disease that is caused by both multiple interacting genetic and environmental factors. Several recent genome scans studies have identified potential major susceptibility loci

Cardiovascular diseases Cardiovascular diseases are among the leading causes of mortality in industrialized countries. 39 SNPs in 29

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candidate genes of five different pathways that may be related to cardiovascular disease were genotyped in 19 Japanese individuals with myocardial infarction and 14 controls in up to a quadruplex format using the MassARRAY platform [64]. Significant differences in allele frequencies were found for three SNPs in the transgelin gene, the hepatocyte growth factor, and the plasminogen activator inhibitor-1 gene, respectively, and further three SNPs showed weak associations. A possible association between two polymorphisms in the promoter region of the phenylethanolamine N-methyltransferase (PNMT) gene and essential hypertension was investigated in several ethnic groups [65]. One SNP showed significant association in the African American population, but not in the Greek or American White study group; however, only a small number of samples was analyzed. Meyer et al. set up a robust twelve-plex assay based on the PinPoint format for the simultaneous analysis of polymorphisms in eight genes related to homocysteine and folate metabolism which might be correlated to an increased risk for cardiovascular disease [66]. However, no biological data is given in the publication.

Obesity The solute carrier family 6 number 14 (SLC6A14) gene was identified as a candidate gene for obesity by microsatellite linkage analysis and subsequent SNP genotyping in a small sample cohort analyzed by pyrosequencing. Two SNPs in the 3V untranslated region of this gene, which is potentially involved in the regulation of appetite, showed significant association in this first association study. One of them was subsequently confirmed in a larger sample cohort using the homogeneous MassEXTEND assay [67]. 2 SNPs in the GH secretagogue receptor (GHSR) gene, involved in body weight regulation, were screened for association with obesity, but no conclusive evidence was found for the involvement of polymorphisms in its regulatory function [68].

Various The interleukin-1 receptor antagonist gene (IL1RN) had been identified by microsatellite linkage scans as a candidate gene for ankylosing spondylitis, a chronic painful disease affecting the spine that may lead to stiffness of the back. Three SNPs in exons 4 and 6 were examined using the MassEXTEND assay and the two SNPs in exon 6 and particularly one haplotype were found to significantly associated with the syndrome [69]. Genes of the interleukin-1 cluster were also investigated for a susceptibility to malaria in a two-stage case-control study [70]. However, only a small genetic effect for these genes was found; one SNP showed a barely significant association between mild

and severe malaria while the other one distinguished mild malaria form from controls.

Scans of candidate regions and whole genome scans The above-described applications concentrated on one or a few candidate polymorphisms that were postulated to be implicated in a particular pathogenesis. These studies therefore needed some a priori knowledge or at least a hypothesis. To screen for so far unknown associations, pooling of DNA samples in a pool of cases and a pool of controls was proposed as a means to reduce the number of genotypes necessary to detect the subtle effects of genetic variations in multifactorial diseases [71]. To detect differences between pools, accurate determination of their respective allele frequencies is required, thus a quantitative detection. Several approaches based on MALDI mass spectrometry have been devised in the last years, permitting a determination of the allele ratios with accuracy better than 5% independent of pool size for polymorphisms with a minor allele frequency of at least 10% [72– 75]. These quantitative features of MALDI mass spectrometry have been applied predominantly to SNP validation in a population studied, but some association studies have been performed using directly pooled DNA samples. 15 SNPs in the cholesteryl ester transfer protein (CETP) gene were compared between two pools consisting of 400 individuals with either high or low high-density lipoprotein cholesterol levels [76]. 14 out of the 15 SNPs showed a significant association confirming previous finding, but more importantly cutting down the 12,000 reactions necessary for individual genotyping to only 150 reactions including five replicates per pool and marker. In the first megabase scan using a MALDI mass spectrometric detection (PROBE/MassEXTEND), 534 SNPs in a region spanning 25 Mb on chromosome 6p21 comprising the major histocompatibility complex were analyzed in pooled patient samples for a variety of complex disease such as Crohn’s disease, schizophrenia, asthma, and diabetes (type 1) [77]. For all diseases, many known associations were confirmed and some others were found for the first time. The search of a susceptibility locus for type two diabetes on chromosome 20q13 was entirely carried out by the MassEXTEND assay [78]. SNPs were validated by quantitative MALDI mass spectrometry with a minimum frequency of 5%. 381 SNPs (291 successful) in a 10-Mb interval were genotyped in pools of cases and controls. 21 SNPs displaying differences in allele frequency were then used for follow-up individual genotyping. A 400-kb region was selected for fine typing increasing the number of SNPs by capturing all haplotype blocks in this region. Eight SNPs showed association to type 2 diabetes in a 60-kb region spanning the promoters 1 and 2 of the hepatocyte nuclear

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factor-4a, especially one SNP 1.3 kb downstream of promoter 2. Whole genome scans analyzing tens or hundreds of thousands of SNPs are the most powerful methods to detect genetic risk variants in a hypothesis-free approach although important issues of study design such as the number of samples and SNPs analyzed and statistical problems of multiple testing are still under discussion. Sequenom scanned 6500 SNPs in 5000 genes in pools of several hundred age stratified Caucasians in a morbidity gene discovery study [79]. In contrast to conventional casecontrol association studies, only healthy individuals were screened expecting a decrease of a disease susceptibility allele with age due to morbidity and mortality. Around 50 SNPs showed a significant allele frequency change with age. Among them, an SNP in the dual-specific A kinaseanchoring protein (D-AKAP2) gene showed the strongest correlation with a negative health prognosis, which may be related to cardiac dysfunction. Sequenom claims to have carried out twelve genomewide association studies for a variety of clinical phenotypes and quantitative traits including several types of cancer, diabetes, obesity, hypertension, and schizophrenia [80,81]. 25,000 to 85,000 SNPs were selected from a panel of 130,000 SNPs identified and verified in a Caucasian population. These studies discovered numerous novel candidate genes and replicated previous findings, and results are said now to be further verified and regions on interest subjected to fine typing. None of these studies has yet been published, but this preliminary information show the potential of MALDI mass spectrometry-based genotyping and its possible application to whole genome scans. Another study for a genome-wide scan in pooled DNA samples from 230 cases and 400 controls for the detection of risk factors of a cerebrovascular disease phenotype in children with sickle cell disease was recently announced also using the MassARRAY platform [82]. This screen is accompanied by a case/control candidate gene study analyzing 28 SNPs in 20 genes involved in a number of systems and pathways related to stroke risk in the individual samples. The MassARRAY system was also chosen as one of two high-throughput SNP genotyping platforms for the 10 million SNP genotypes in the GenomEUtwin project, which aims at identifying common diseases by combining whole genome scans (microsatellites), dense mapping (microsatellites/SNPs), and candidate gene genotyping (SNPs) in monozygotic twins [83].

MALDI-based diagnostic assays Potential diagnostic assays for the analysis of mutations that are routinely tested for in clinical laboratories have been devised in the last years. Preventive screening of patients and carriers demands methods with high reliability and very high quality; MALDI mass spectrometry is among those

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[73]. Methods, currently applied for the detection of known genetic variants and risk alleles in clinical diagnosis, are sometimes time consuming and/or rely on secondary physical properties of oligonucleotides such as mobility or hybridization kinetics that may incorrectly call heterozygotes as homozygotes. Genotyping by MALDI mass spectrometry would therefore provide significant health care benefits. However, to our knowledge, no mass spectrometry DNA assays for currently applied clinical DNA tests have obtained FDA approval. However, highthroughput analysis of genetic risk variants may allow the screening of entire populations and change medicine from a crisis-driven intervention to predictive medicine [84]. Cystic fibrosis is the one of the most common inherited disease affecting about 1 of 3300 Caucasian infants. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. 3% of Caucasians are heterozygous carriers of a defective gene. Routine clinical diagnosis is currently carried out by the sweat chloride test or by an immunoreactive test for the level of trypsin followed by DNA testing [85]. One of the first published potential applications of the PROBE assay was the analysis of five common mutations in the CFTR gene in cystic fibrosis patients by MALDI mass spectrometry [38]. Recently, a panel of the 100 most common mutations analyzed by multiplexed PROBE assays was demonstrated using multiplex PCR amplification for target enrichment. These assays also identified unrecognized homozygous and compound heterozygous mutations [86] demonstrating the potential of this detection platform as an alternative means for DNA testing. An assay for the analysis of the four most common mutations (single nucleotide exchanges and deletions) in the adenomatous polyposis coli (APC) gene, that predispose to familial adenomatous polyposis (FAP), was proposed as an early means for the identification of a genetic susceptibility to colon cancer [87]. Mass spectrometry-based genotyping would provide a rapid alternative tool to the currently applied methods for detection and tumor profiling of colon adenomas and carcinomas [88]. Loss-of-function mutations occur in many types of cancers in the p53 tumor suppressor gene. A multiplex assay based on the solid phase capturable primer extension assay was set up to analyze thirty potential mutation sites simultaneously in three exons of this gene including the most frequently mutated codons [89]. This is also the highest so far demonstrated multiplex factor for a homogeneous MALDI mass spectrometric assay and would provide an alternative to direct fluorescent sequencing, which is so far considered the gold standard for mutation screening. Mutation in the RET proto-oncogene have been found in most cases of familial medullary thyroid carcinoma and nearly all multiple endocrine neoplasia. Genetic testing is nowadays routinely carried out for family members at risk to detect inherited RET mutations with subsequent possibility of a prophylactic thyroidectomy [90]. The PROBE assay has very early been proposed as a means

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for genetic screening for these mutations as demonstrated for two mutations on codon 634 [91]. The solid phase capturable primer extension assay was demonstrated on the genotyping of two disease associated SNPs (C282Y; H63D) in the human hereditary hemochromatosis (HHC) gene HFE [28], that lead to iron overload and result potentially in liver failure, depression, and diabetes in its homozygote or compound heterozygote constellation. Hereditary hemochromatosis is phenotypically detected by biochemical assays measuring serum transferring saturation and ferritin levels, and genetic testing is carried out in case of suspicion which can be done by a variety of assays [92]. Because of the high prevalence (1/400 Caucasians), high-throughput genetic screening approaches would be important to detect affected individuals early in the pathogenesis. Point mutations in the globin genes lead to either structural variants of hemoglobin or to a decreased expression of one of the chains leading to various forms of thalassemia, which can be detected with many methods, predominantly based on electrophoretic migration behavior of proteins or DNA testing with allele-specific probes/ primers or single strand conformation polymorphism analysis [93]. The MassARRAY technology was demonstrated as capable of distinguishing unambiguously the most common variants [94]. An assay was also devised for the simultaneous analysis of two highly prevalent polymorphisms in the blood clotting factors FV (G1691A, Factor V Leiden mutation) and prothrombin FII (G20210A) that are associated with a significantly increased risk for venous thrombosis (fourfold for heterozygotes and 50- to 100-fold for homozygotes for the factor V Leiden polymorphism, [95]). MALDI-MS would provide in this case an alternative method for the screening of these clinically important SNPs, which is currently carried out by either FDA-approved functional tests or PCR-based methods [96]. Alternative assays for the analysis of genetic risk factors such as the factor V Leiden G1691A, the prothrombin G20210A, and the methylentetrahydrofolate reductase C667T polymorphism were shown using the genoSNIP assay [29]. Individual variability in the Toll-like receptors (TLRs) recognizing lipopolysaccharides (endotoxin) fingerprints of microbial pathogens may mediate an altered immune response. Therefore, two assays for individual risk stratification of patients vulnerable to endotoxin-induced sepsis were developed using either the TaqMan technology or an MALDI assay based on the PROBE concept [97]. In contrast to the TaqMan assay, no incorrect genotype calls were obtained by mass spectrometry. Alzheimer’s disease is the most common form of dementia. The only sure diagnosis of Alzheimer’s disease is due to the neuropathological findings at autopsy. In lateonset and sporadic Alzheimer’s disease, the detection of the apolipoprotein E e4 allele can substantiate the clinical diagnosis of the disease. Other indicative tests in clinical

practice comprise the quantitative measurement of several protein biomarkers in cerebrospinal fluid [98]. The first demonstration of a cycled primer extension reaction of the PROBE assay was performed on the detection of the APOE e4 allele [99].

Molecular haplotyping Haplotypes are specific combinations of genetic variants located on an allele. The multiallelic nature and higher level of heterozygosity increases the information content of haplotypes compared to individual, biallelic SNPs. There is growing evidence that haplotype structure, rather than individual SNPs is the determinant of phenotypic consequences and has greater power to track any unobserved, but evolutionary linked, variable site [100,101]. In some studies, haplotypes conferring significantly to the risk for a complex disorder could be assigned while a single causal variant eluded definitive identification. Haplotypes may therefore also be more appropriate for pharmacogenetic assessment [46]. Knowledge of the haplotype structure of a region of interest allows decreasing the number of markers that need to be genotyped in association studies. Only those are genotyped that distinguish the different haplotypes from each other (htSNPs, [102]). Haplotypes are in practice mainly inferred from genotype data using mathematical algorithms as most techniques for molecular haplotyping are tedious, time consuming, or prone to errors. However, this procedure may be unacceptable in clinical practice due to the inherent statistical uncertainty. Recently, two methods for the physical determination of the phase of SNPs were devised. Fragments of up to 4 kb in length are amplified by allele-specific PCR using heterozygous boundary positions for anchoring the allele-specific primers. The alleles of the SNPs contained in these haploid fragments are subsequently determined by a multiplexed primer extension reaction and mass spectrometric detection exploiting the multiplexing potential and resolving power of MALDI MS [48]. The second approach uses dilution to a statistical level of a single molecule on which the SNP alleles are then analyzed by the MassEXTEND assay [103]. While the first method is restricted to the analysis of relatively small amplificates (such as haplotype block boundaries) and analysis of larger fragments would necessitate walking from one fragment to the next, the second method relies heavily on template integrity and is very sensitive to contaminations.

Bacterial and viral typing by MALDI mass spectrometry Identification of bacterial and viral pathogens is of utmost importance in clinical practice. In contrast to most protein or whole cell based identification methods, assays based on the genotypic identification do not require

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culturing of the bacteria prior to analysis. This reduces time for analysis greatly and makes the assay also applicable to strains that have not been cultured in a laboratory before (which are probably most strains). This analysis can be carried out on various detection platforms [104]. A mass spectrometry assay based on multiple base primer extension was investigated as a rapid alternative to the widely applied multilocus sequence typing [105] for the differentiation of meningococcal strains [106]. The analysis of one single nucleotide polymorphism in the fumarate hydrase gene allowed the distinction between hypervirulent and other strains of Neisseria meningitides. Drug sensitivity to first line treatments against malaria was carried out by genotyping four SNPs in the dihydrofolate reductase gene of the Malaria transmitting parasite Plasmodium falciparum using the PROBE assay [107]. Sensitivity of the devised mass spectrometric assay was increased compared to fluorescent sequencing which did not detect all variants in mixed infections in about a third of the cases. A quantitative MassEXTEND assay was applied to the quantitative monitoring of the broad dynamic spectrum of different mutant strains in natural and recombinant populations of live RNA viruses and their evolution over time [108]. As live attenuated viruses are used as vaccines against, for example, mumps, this assay enables testing of genetic stability and therefore consistency of the product. The quantitative feature of MALDI mass spectrometry permits also the screening for the emergence of drugresistant viral strains in a mixed population. The great diversity of bacterial strains and the high incidence of novel mutations giving rise to new strains somewhat limit the use of conventional genotyping assays for the analysis of single nucleotide differences between strains. Most of the recently developed assays for genotypic identification of bacteria therefore apply approaches based on different fragmentation strategies that enable not only the analysis of known sequence variants, but also the detection of new or so far unknown mutations. These assays are described below in the paragraph dealing with mutation discovery.

Genotyping in animals and plants Applications of SNP genotyping are not restricted to studies in humans. Essentially the same assays as above described are used in animals and plants. Large-scale SNP mapping studies have been at least carried out in two model organisms by MALDI mass spectrometry. Haplotype patterns have recently been defined in eight inbred mouse strains [109]. 1240 of the mainly in silico derived SNPs were validated by establishing MassEXTEND assays and integrated into a genetic map which is accessible through SNPview (www.gnf.org/SNP). 400 quadruplex genotyping assays were developed for rapid whole genome scans.

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In the widely used model plant, Arabidopsis thaliana, a set of multiplex (1–3) SNP genotyping assays by MALDI mass spectrometry spanning the entire genome with an average spacing of 1.15 Mbp was recently developed [110] to identify economically interesting QTLs in plants by highthroughput comparative studies. In farm animals, genotyping studies are used for the determination of genetic risk factors in genotype/phenotype association studies, identification of quantitative trait loci, for example, to improve breeding capacities, for animal identification and traceability purposes. The PROBE/ MassEXTEND assay was applied to the identification of SNPs in the neonatal Fc receptor (FCGRT) gene [111] and the beta 2-microglobulin (B2M) gene [112] in a multibreed panel of 96 cattle. Two haplotypes constructed from five SNPs of FCGRT and one haplotype constructed of 12 SNPs of B2M were found to be correlated with decreased immunoglobulin G uptake from the mother in neonatal calves and thus associated with increased calf mortality and morbidity. Assays based on the Sequenom technology were also set up for various other polymorphisms such as in the bovine bone morphogenetic protein receptor-1B, a candidate gene for ovulation rate in cattle [113], an SNP in the porcine MADH1 gene, a region harboring a potential quantitative trait locus for uterine capacity [114]. Comparative mapping using the MassARRAY platform identified four potential candidate genes for quantitative trait loci involved in different features of reproduction and meat quality growth on the porcine chromosome 10 [115]. MALDI mass spectrometry (PROBE assay) has also been applied to SNP fine map the haplotype structure in the interleukin 8 locus potentially associated with host defense against bacterial invasion [116]. For the detection of the transmissible spongiform encephalopathy in sheep and cattle, an assay for the analysis of polymorphism in the bovine prion protein gene (Prnp) for screening of animals with increased susceptibility to bovine spongiform encephalopathy was developed [117] and we have set up a GOOD assay for the detection of scrapie analyzing four polymorphisms in the SPrP gene (unpublished). For the unique determination of the genetic identity of animals and their parents, a minimal set of 32 SNPs in US beef cattle was defined and typed with the PROBE assay [118]. A set of similar size was developed suitable for the identification and traceability of animals of most known breeds using the GOOD assay (Planc¸on et al., manuscript in preparation).

Mutation and polymorphism discovery Originally, mass spectrometry was thought of as an alternative to conventional Sanger sequencing [119]. While the principle was demonstrated very early [120], the readlength is still restricted to less than 50 nucleotides in routine due to problems with resolution, fragmentation, and adduct

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formation. Gel- and capillary-based methods are on the other side well established for the analysis of fragments up to 1000 nucleotides. Sequencing by mass spectrometry in a clinically important context was demonstrated by the resequencing of exons 5–8 of the tumor suppressor gene p53 by overlapping primer walking using 21 primers for a total read-length of 670 bases [121]. Several assays based on cleavage strategies have recently been devised for comparative sequence analysis besides conventional re-sequencing of a region of interest, which becomes expensive and inefficient in a highthroughput application. In these methods, characteristic patterns of fragmentation are created instead of sequence ladders, which permit the detection of known and so far unknown mutations. Mutations are detected as mass shifts of peaks, missing or additional peaks in the fingerprints. These methods are ideally suited for the identification of bacterial strains, detection of newly emerging sequence variants, and monitoring of genetic changes in strains of clinical importance. One strategy consists of transcribing the DNA sequence into RNA which can be easily analyzed by MALDI as it is more stable in the desorption/ionization step in the mass spectrometer due to a stabilizing effect of the 2V hydroxyl group of the sugar moiety and results in reduced fragmentation of the analyte by depurination [122]. PCR primers contain at their 5V end promoter sequences for either T3, T7, or Sp6 RNA polymerase, which are used after PCR for an in vitro transcription [123–125]. Subsequent RNase T1 treatment base-specifically cleaves the transcript at every G position, while RNase A cleaves 3Vof pyrimidines. The two complementary strands are transcribed in different reactions to maximize chances to detect any mutation that would not be found in a single reaction due to mass degeneracy of fragments or mass differences below the resolution of the mass spectrometer. This assay was applied to the genotypic identification of mycobacterial strains through the amplification, transcription, and basespecific cleavage of the 16S rRNA distinguishing all 21 tested strains [126]. Testing of an additional locus (such as gyrB) would further improve the differentiation between closely related strains with identical 16S rRNA sequences and would provide a rapid alternative to currently applied methods [127]. This assay can also be used for re-sequencing of nucleic acids by base-specific cleavage after all four bases [23,128]. An RNAse specific reaction for C residues and cleavage reaction specific for U residues are carried out in separate wells and G- and A-specific reactions are simply performed on the reverse strands using the C- and U-specific reactions. The replacement of one dNTP in the case of pyrimidinespecific cleavage (RNase A) makes this reaction specific for the respective other pyrimidine base. SNPs are automatically identified by a software (SpectroCLEAVE) that reports mass shifts, missing and/or additional peaks [129]. The interrelation between missing peaks and creation of addi-

tional peaks re-enforces the accuracy and minimizes the risk of false negatives. Another enzymatic possibility for fragmentation is the replacement of dTTP by dUTP during PCR amplification. After strand separation, fragments for MALDI analysis are created by incubation with uracil-DNA-glycosylase which creates abasic sites and subsequent alkaline and heat treatment is used to cleave the sugar phosphate backbone at the abasic sites. This method has been demonstrated for the detection of a polymorphism in the Interleukin 12 gene [130]. This strategy was also applied to genotypic identification 16S rRNA of different (cultured and uncultured) bacterial strains [131]. Chemical methods provide an alternative means for the generation of fragments with the advantage of being less influenced by slight changes of reaction parameters than enzymes. Early approaches based on Maxam-Gilbert sequencing or hydrazin/anilin cleavage suffered however from unspecific and/or incomplete cleavage reactions. We developed an approach based on the incorporation of NTPs (ribonucleotides) during PCR amplification replacing one of the dNTPs. Fragments are generated by simple alkali backbone cleavage at the ribo-bases of the PCR product (Mauger, F. et al., manuscript in preparation). In an alternative strategy, chemically labile P3V-N5V-phosphoamidate nucleobases are incorporated during DNA amplification, that are subsequently cleaved under slightly acidic conditions [132]. This strategy was further optimized and recently validated for SNP identification in several target genes [133].

Concluding remarks MALDI mass spectrometry has proven in the last years to be a reliable detection platform for high-throughput screening of SNPs and point mutations, and publications using MALDI mass spectrometry for clinical questions have multiplied in the last 2 years. MALDI mass spectrometry will play a significant role in the next years for genomewide discovery of variations underlying disease susceptibility, assessment of drug sensitivity, and other clinically relevant phenotypes. Very recently developed assays for the discovery of unknown mutations have broadened its potential application for clinical diagnosis. Although no FDA or similar approval has yet been assigned to a mass spectrometry-based assay, this probably is only a question of time judging by the number of potential diagnostic applications and assays using MALDI mass spectrometry that have already been shown. Demands may quite likely already have been submitted. MALDI mass spectrometry has the potential to become one important platform in clinical diagnosis as it is very flexible and applicable to genetic and epigenetic diagnostics, expression profiling, and quantitative proteomics. On the other hand, the current generation of mass spectrometers has not yet reached the degree of push-button

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operation that other diagnostic systems offer. The evolution of mass spectrometry for DNA analysis in the past 10 years has been very impressive and the last step to an instrument requiring minimal specialized expertise is not very big. The technology has established itself well in the DNA variation research community. A further critical issue for the success of this method is the access to PCR for diagnostic applications which is still in the hands of one key player. This is a problem other DNA analysis methods trying to enter diagnostics also face.

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