Keith Bauer

The Molecular Anatomy of Spontaneous Germline Mutations in Human Testes

The frequency of the most common sporadic Apert syndrome mutation (C755G) in the human fibroblast growth factor receptor 2 gene (FGFR2) is 100–1,000 times higher than expected from average nucleotide substitution rates based on evolutionary studies and the incidence of human genetic diseases. To determine if this increased frequency was due to the nucleotide site having the properties of a mutation hot spot, or some other explanation, we developed a new experimental approach. We examined the spatial distribution of the frequency of the C755G mutation in the germline by dividing four testes from two normal individuals each into several hundred pieces, and, using a highly sensitive PCR assay, we measured the mutation frequency of each piece. We discovered that each testis was characterized by rare foci with mutation frequencies 103 to >104 times higher than the rest of the testis regions. Using a model based on what is known about human germline development forced us to reject (p < 10−6) the idea that the C755G mutation arises more frequently because this nucleotide simply has a higher than average mutation rate (hot spot model). This is true regardless of whether mutation is dependent or independent of cell division. An alternate model was examined where positive selection acts on adult self-renewing Ap spermatogonial cells (SrAp) carrying this mutation such that, instead of only replacing themselves, they occasionally produce two SrAp cells. This model could not be rejected given our observed data. Unlike the disease site, similar analysis of C-to-G mutations at a control nucleotide site in one testis pair failed to find any foci with high mutation frequencies. The rejection of the hot spot model and lack of rejection of a selection model for the C755G mutation, along with other data, provides strong support for the proposal that positive selection in the testis can act to increase the frequency of premeiotic germ cells carrying a mutation deleterious to an offspring, thereby unfavorably altering the mutational load in humans. Studying the anatomical distribution of germline mutations can provide new insights into genetic disease and evolutionary change.

Mutation Scanning Using MUT-MAP, a High-Throughput, Microfluidic Chip-Based, Multi-Analyte Panel

Targeted anticancer therapies rely on the identification of patient subgroups most likely to respond to treatment. Predictive biomarkers play a key role in patient selection, while diagnostic and prognostic biomarkers expand our understanding of tumor biology, suggest treatment combinations, and facilitate discovery of novel drug targets. We have developed a high-throughput microfluidics method for mutation detection (MUT-MAP, mutation multi-analyte panel) based on TaqMan or allele-specific PCR (AS-PCR) assays. We analyzed a set of 71 mutations across six genes of therapeutic interest. The six-gene mutation panel was designed to detect the most common mutations in the EGFR, KRAS, PIK3CA, NRAS, BRAF, and AKT1 oncogenes. The DNA was preamplified using custom-designed primer sets before the TaqMan/AS-PCR assays were carried out using the Biomark microfluidics system (Fluidigm; South San Francisco, CA). A cross-reactivity analysis enabled the generation of a robust automated mutation-calling algorithm which was then validated in a series of 51 cell lines and 33 FFPE clinical samples. All detected mutations were confirmed by other means. Sample input titrations confirmed the assay sensitivity with as little as 2 ng gDNA, and demonstrated excellent inter- and intra-chip reproducibility. Parallel analysis of 92 clinical trial samples was carried out using 2–100 ng genomic DNA (gDNA), allowing the simultaneous detection of multiple mutations. DNA prepared from both fresh frozen and formalin-fixed, paraffin-embedded (FFPE) samples were used, and the analysis was routinely completed in 2–3 days: traditional assays require 0.5–1 µg high-quality DNA, and take significantly longer to analyze. This assay can detect a wide range of mutations in therapeutically relevant genes from very small amounts of sample DNA. As such, the mutation assay developed is a valuable tool for high-throughput biomarker discovery and validation in personalized medicine and cancer drug development.

SNP genotyping using alkali cleavage of RNA/DNA chimeras and MALDI time-of-flight mass spectrometry.

Single nucleotide polymorphisms (SNPs) are now widely used for many DNA analysis applications such as linkage disequilibrium mapping, pharmacogenomics and traceability. Many methods for SNP genotyping exist with diverse strategies for allele-distinction. Mass spectrometers are used most commonly in conjunction with primer extension procedures with allele-specific termination. Here we present a novel concept for allele-preparation for SNP genotyping. Primer extension is carried out with an extension primer positioned immediately upstream of the SNP that is to be genotyped, a complete set of four ribonucleotides and a ribonucleotide incorporating DNA polymerase. The allele-extension products are then treated with alkali, which results in the cleavage immediately after the first added ribonucleotide. In addition, to obtain fragments easily detectable by mass spectrometry, we have included a ribonucleotide in the primer usually at the fourth nucleotide from the 3′ terminus. The method was tested on four SNPs each with a different combination of nucleotides. The advantage over other mass spectrometry-based SNP genotyping assays is that this one only requires a PCR, a primer extension reaction with a universal extension mix and an inexpensive facile cleavage reaction, which makes it overall very cost effective and easy in handling.

DNA sequencing by MALDI-TOF MS using alkali cleavage of RNA/DNA chimeras

Approaches developed for sequencing DNA with detection by mass spectrometry use strategies that deviate from the Sanger-type methods. Procedures demonstrated so far used the sequence specificity of RNA endonucleases, as unfortunately equivalent enzymes for DNA do not exist and therefore require transcription of DNA into RNA prior to fragmentation. We have developed a novel, rapid and accurate concept for DNA sequencing using mass spectrometry and RNA/DNA chimeras and applied it to sequence mitochondrial DNA. Our method is based on the preparation of a chimeric RNA/DNA with a DNA polymerase that also incorporates ribonucleotides. Sequencing is carried out with one ribonucleotide (ATP, CTP or GTP) and the other three nucleotides in their deoxyribo-form. The product is treated with alkali, which cleaves 3′ of all ribonucleotides to form a terminal 3′ phosphate. Conditions have been streamlined so that molecular, biological and alkali cleavage conditions are compatible with matrix-assisted laser desorption/ionization time-of-flight (MALDI) mass spectrometric analysis. Fragment analysis by MALDI MS provides a sequence-specific fingerprint, which allows the identification of differences between a reference and another sequence. Due to the mass profile, the position and kind of the mutation can be assigned. These differences between signatures are indicative of known, unidentified, rare and private mutations. This novel DNA sequencing protocol was applied to sequence the hypervariable region 1 (HV1) of mitochondrial DNA in 22 individuals.

Next generation MUT-MAP, a high-sensitivity high-throughput microfluidics chip-based mutation analysis panel.

Molecular profiling of tumor tissue to detect alterations, such as oncogenic mutations, plays a vital role in determining treatment options in oncology. Hence, there is an increasing need for a robust and high-throughput technology to detect oncogenic hotspot mutations. Although commercial assays are available to detect genetic alterations in single genes, only a limited amount of tissue is often available from patients, requiring multiplexing to allow for simultaneous detection of mutations in many genes using low DNA input. Even though next-generation sequencing (NGS) platforms provide powerful tools for this purpose, they face challenges such as high cost, large DNA input requirement, complex data analysis, and long turnaround times, limiting their use in clinical settings. We report the development of the next generation mutation multi-analyte panel (MUT-MAP), a high-throughput microfluidic, panel for detecting 120 somatic mutations across eleven genes of therapeutic interest (AKT1, BRAF, EGFR, FGFR3, FLT3, HRAS, KIT, KRAS, MET, NRAS, and PIK3CA) using allele-specific PCR (AS-PCR) and Taqman technology. This mutation panel requires as little as 2 ng of high quality DNA from fresh frozen or 100 ng of DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Mutation calls, including an automated data analysis process, have been implemented to run 88 samples per day. Validation of this platform using plasmids showed robust signal and low cross-reactivity in all of the newly added assays and mutation calls in cell line samples were found to be consistent with the Catalogue of Somatic Mutations in Cancer (COSMIC) database allowing for direct comparison of our platform to Sanger sequencing. High correlation with NGS when compared to the SuraSeq500 panel run on the Ion Torrent platform in a FFPE dilution experiment showed assay sensitivity down to 0.45%. This multiplexed mutation panel is a valuable tool for high-throughput biomarker discovery in personalized medicine and cancer drug development.

High-specificity single-tube multiplex genotyping using Ribo-PAP PCR, tag primers, alkali cleavage of RNA/DNA chimeras and MALDI-TOF MS.

Here, we describe a high‐throughput, single‐tube, allele‐specific ribonucleotide analog pyrophosphorolysis‐activated polymerization (ribo‐PAP) PCR multiplex genotyping and resequencing method. An RNA/DNA chimeric PCR product is generated using genomic DNA as starting template, a panel of allele‐selective 5′‐tagged primers, a reverse primer, one nucleotide in the ribo‐form (90–100%), the other nucleotides in the deoxy‐form, a DNA polymerase capable of incorporating ribonucleotides, a suitable buffer and thermal cycling. The RNA/DNA chimeric PCR products are fragmented by treatment with alkali and analyzed by mass spectrometry. All allele‐selective primers have a 5′ repetitive motif where each repeat unit has a unique, distinct mass upon reverse copying and alkali fragmentation. The mass of the complement repeat fragment or flag identifies the primer or primers that were recruited in the ribo‐PAP PCR. The method readily identifies homozygous and heterozygous positions in simplex or duplex ribo‐PAP PCR. Many different tags can be analyzed simultaneously. The assay can genotype several SNPs in a single tube. It thus constitutes the simplest genotyping protocol with multiplex analysis. This novel genotyping and resequencing protocol was applied to different genomic loci: NOS1 and H19 in 30 individuals in simplex ribo‐PAP PCR and at two SLCO1B1 loci in 95 individuals in duplex ribo‐PAP PCR.

Ribo-polymerase chain reaction--a facile method for the preparation of chimeric RNA/DNA applied to DNA sequencing.

We describe ribo‐polymerase chain reaction (PCR), a method for the preparation of chimeric RNA/DNA. The RNA/DNA chimeric nucleic acids are generated directly from genomic DNA starting templates with two locus‐specific primers, three nucleotides in their deoxy form and the fourth in its ribo form, a DNA polymerase capable of incorporating ribo bases, a suitable buffer, and thermal cycling. We have applied ribo‐PCR to resequence DNA by directly fragmenting the RNA/DNA chimeras with alkali and analyzing the fragments by mass spectrometry (MS). Mass fingerprint is used to identify deviations from the reference sequence. This method readily detects homozygous sequence deviations as well as heterozygous positions directly from genomic DNA samples. With the high‐throughput capability of MS, this facile method is well suited for screening DNA sequences of limited regions of the genome in a large number of individuals. It can also be used to sequence multiple distant genomic loci in a single reaction. This novel ribo‐PCR resequencing protocol was applied to different genomic loci involving nitric oxide synthase 1 (NOS1) and H19 in 30 individuals and SLCO1B1 in 95 individuals. Hum Mutat 33:1010–1015, 2012.