Locus-specific PCR Research Articles

Novel rapid molecular diagnosis methods for comprehensive genetic analysis of 21-hydroxylase deficiency

BackgroundMolecular analysis of the CYP21A2 gene is highly important for understanding the aetiology of 21-hydroxylase deficiency (21-OHD). The aim of this study was to use a novel approach named CNVplex, together with the SNaPshot assay and direct sequencing, to identify CYP21A2 mutations efficiently and comprehensively. Targeted CYP21A2 mutation analysis was performed in 113 patients and 226 parents. Large rearrangements of CYP21A2 were characterized by CNVplex; twenty prevalent mutations, including nine common micro-conversions and eleven high-frequency mutations reported in the literature, were detected by SNaPshot; and rare mutations were investigated by direct sequencing.ResultsAmong the 113 21-OHD patients, 95.6% of the affected alleles were detected accurately by SNaPshot and CNVplex. Prevalent mutations were detected in 69.5% of the alleles; 62.4% of alleles contained pseudogene-derived micro-conversions, 1.8% contained nonpseudogene-derived mutations, and 5.3% contained complex variations resulting from multiple recombinations between CYP21A2 and CYP21A1P. Large rearrangements were identified in 27.0% of the alleles, including five types (CH-1, CH-3, CH-4, CH-5 and CH-8) of chimeric CYP21A1P/CYP21A2 genes. Two novel CYP21A2 haplotypes and four de novo CYP21A2 mutations were characterized. A rare haplotype with a c.955 C > T mutation in the duplicated CYP21A2 gene was found in 0.9% of the probands and 33.3% of the parents. In addition, four parents were also diagnosed with 21-OHD.ConclusionCNVplex and SNaPshot appear to be highly efficient and reliable techniques for use in a molecular diagnosis laboratory, and combined with direct sequencing based on locus-specific PCR, they might constitute a definitive way to detect almost all common and rare 21-OHD-related alleles.

Targeted long-read sequencing for comprehensive detection of CYP21A2 mutations in patients with 21-hydroxylase deficiency.

21-Hydroxylase deficiency (21-OHD) is caused by pathogenic CYP21A2 variations. CYP21A2 is arranged in tandem with its highly homologous pseudogene CYP21A1P; therefore, it is prone to mismatch and rearrangement, producing different types of complex variations. There were few reports on using only one method to detect different CYP21A2 variants simultaneously. Targeted long-read sequencing method was used to detect all types of CYP21A2 variants in a series of patients with 21-OHD. A total of 59 patients with 21-OHD were enrolled from Peking Union Medical College Hospital. Long-range locus-specific PCR and long-read sequencing (LRS) were performed to detect the pathogenic variants in CYP21A2. Copy-number variants of CYP21A2 were found in 25.4% of patients, including 5.1% with 3 copies of CYP21A2, 16.9% with 1 copy of CYP21A2, and 3.4% with 0 copy of CYP21A2. The remaining 74.6% of patients had 2 copies of CYP21A2. Pathogenic variants were identified in all 121 alleles of 59 patients. Specifically, single-nucleotide variants and small insertions/deletions (< 50bp) were detected in 79 alleles, of which conversed from CYP21A1P were detected in 63 alleles, and rare variants were found in the other 16 alleles. Large gene conversions (> 50bp) from pseudogene were detected in 10 alleles, and different chimeric genes (CYP21A1P/CYP21A2 or TNXA/TNXB) formed by large deletions were detected in 32 alleles. Of all variants, p.I173N was the most common variant (19.0%). Our study demonstrated that targeted long-read sequencing is a comprehensive method for detecting CYP21A2 variations, which is helpful for genetic diagnosis in 21-OHD patients.

Long-read sequencing: An effective method for genetic analysis of CYP21A2 variation in congenital adrenal hyperplasia

BackgroundThe sequence similarity between CYP21A2 gene and its inactive pseudogene CYP21A1P, and copy number variation (CNV) caused by unequal crossover, make it challenging to characterize the CYP21A2 gene through traditional methods. This study aimed to evaluate the clinical utility of the long-read sequencing (LRS) method in carrier screening and genetic diagnosis of congenital adrenal hyperplasia (CAH) by comparing the efficiency of the LRS method with the conventional multiplex ligation-dependent probe amplification (MLPA) plus Sanger sequencing approaches in CYP21A2 analysis. MethodsIn a retrospective study, full sequence analysis of the CYP21A2 and CYP21A1P was performed for three pedigrees through long-range locus-specific PCR followed by LRS based on the Pacific Biosciences (PacBio, California, USA) single-molecule real-time (SMRT) platform, and the results were compared with those obtained from next-generation sequencing (NGS)-based whole exome sequencing (WES) and the traditional methods of MLPA plus Sanger sequencing. ResultsThe LRS method successfully identified seven CYP21A2 variants, including three single nucleotide variants (NM_000500.9:c.1451G > C p.(Arg484Pro), c.293-13A/C > G (IVS2-13A/C > G), c.518 T > A p.(Ile173Asn)), one 111-bp polynucleotide insertion, one set of 3′URT variants (NM_000500.9:c.*368 T > C, c.*390A > G, c.*440C > T, c.*443 T > C) and two types of chimeric genes and straightforwardly depicted the inheritance patterns of these variants within families. Moreover, the LRS method enabled us to determine the cis–trans configuration of multiple variants in one assay, without the need to analyze additional family samples. Compared with traditional methods, this LRS method can achieve a precise, comprehensive and intuitive result in the genetic diagnosis of 21-hydroxylase deficiency (21-OHD). ConclusionThe LRS method is comprehensive in CYP21A2 analysis and intuitive in result presentation, which holds substantial promise in clinical application as a crucial tool for carrier screening and genetic diagnosis of CAH.

Short communication: diversity of endogenous avian leukosis virus subgroup E elements in 11 chicken breeds.

Avian leukosis virus subgroup E (ALVE) as a kind of endogenous retroviruses extensively exists in chicken genome. The insertion of ALVE has some effects on chicken production traits and appearance. Most of the work on ALVEs has been done with commercial breeds. We present here an investigation of ALVE elements in seven Chinese domestic breeds and four standard breeds. Firstly, we established an ALVE insertion site dataset by using the obsERVer pipeline to identify ALVEs from whole-genome sequence data of eleven chicken breeds, seven Chinese domestic breeds, including Beijing You (BY), Dongxiang (DX), Luxi Game (LX), Shouguang (SG), Silkie (SK), Tibetan (TB) and Wenchang (WC), four standard breeds, including White Leghorn (WL), White Plymouth Rock (WR), Cornish (CS), and Rhode Island Red (RIR). A total of 37 ALVE insertion sites were identified and 23 of them were novel. Most of these insertion sites were distributed in intergenic regions and introns. We then used locus-specific PCR to validate the insertion sites in an expanded population with 18~60 individuals in each breed. The results showed that all predicted integration sites in 11 breeds were verified by PCR. Some ALVE insertion sites were breeds specific, and 16 out of 23 novel ALVEs were found in only one Chinese domestic chicken breed. We randomly selected three ALVE insertions including ALVE_CAU005, ALVE_ros127, and ALVE_ros276, and obtained their insertion sequences by long-range PCR and Sanger sequencing. The insertion sequences were all 7525 bp, which were full-length ALVE insertion and all of them were highly homologous to ALVE1 with similarity of 99%. Our study identified the distribution of ALVE in 11 chicken breeds, which expands the current research on ALVE in Chinese domestic breeds.

Owl Monkey Alu Insertion Polymorphisms and Aotus Phylogenetics.

Owl monkeys (genus Aotus), or "night monkeys" are platyrrhine primates in the Aotidae family. Early taxonomy only recognized one species, Aotus trivirgatus, until 1983, when Hershkovitz proposed nine unique species designations, classified into red-necked and gray-necked species groups based predominately on pelage coloration. Recent studies questioned this conventional separation of the genus and proposed designations based on the geographical location of wild populations. Alu retrotransposons are a class of mobile element insertion (MEI) widely used to study primate phylogenetics. A scaffold-level genome assembly for one Aotus species, Aotus nancymaae [Anan_2.0], facilitated large-scale ascertainment of nearly 2000 young lineage-specific Alu insertions. This study provides candidate oligonucleotides for locus-specific PCR assays for over 1350 of these elements. For 314 Alu elements across four taxa with multiple specimens, PCR analyses identified 159 insertion polymorphisms, including 21 grouping A. nancymaae and Aotus azarae (red-necked species) as sister taxa, with Aotus vociferans and A. trivirgatus (gray-necked) being more basal. DNA sequencing identified five novel Alu elements from three different taxa. The Alu datasets reported in this study will assist in species identification and provide a valuable resource for Aotus phylogenetics, population genetics and conservation strategies when applied to wild populations.

Cebidae Alu Element Alignments and a Complex Non-Human Primate Radiation.

Simple SummaryRelationships among the small monkeys living in the Americas such as the squirrel monkeys, owl monkeys (the ‘night monkeys’), marmosets and capuchins (the ‘organ grinder’ monkeys) are still under debate. This study utilized multi-species DNA sequence alignments to investigate these relationships. Each alignment contained a unique Alu element insertion, a genetic marker considered ‘identical by descent’ such that the absence of the Alu element is the ancestral state. Alu element diversity reveals that the emergence and radiation of these different primate lineages was rapid and complex. The close relationship between squirrel monkey and capuchins was confirmed while the placement of owl monkey with respect to the marmoset remains unresolved. The results of this study will assist in research and conservation strategies for America’s monkeys.Phylogenetic relationships among Cebidae species of platyrrhine primates are presently under debate. Studies prior to whole genome sequence (WGS) availability utilizing unidirectional Alu repeats linked Callithrix and Saguinus as sister taxa, based on a limited number of genetic markers and specimens, while the relative positions of Cebus, Saimiri and Aotus remained controversial. Multiple WGS allowed computational detection of Alu-genome junctions, however random mutation and evolutionary decay of these short-read segments prevented phylogenetic resolution. In this study, WGS for four Cebidae genomes of marmoset, squirrel monkey, owl monkey and capuchin were analyzed for full-length Alu elements and each locus was compared to the other three genomes in all possible combinations using orthologous region sequence alignments. Over 2000 candidates were aligned and subjected to visual inspection. Approximately 34% passed inspection and were considered shared in their respective category, 48% failed due to the target being present in all four genomes, having N’s in the sequence or other sequence quality anomalies, and 18% were determined to represent near parallel insertions (NP). Wet bench locus specific PCR confirmed the presence of shared Alu insertions in all phylogenetically informative categories, providing evidence of extensive incomplete lineage sorting (ILS) and an abundance of Alu proliferation during the complex radiation of Cebidae taxa.

Comprehensive Analysis of Congenital Adrenal Hyperplasia Using Long-Read Sequencing.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder that has been included in newborn screening programs. Current approaches to gene testing for CAH are facing challenges because of the complexity of the CYP21A2 locus and genetic heterogeneity of the disease. A comprehensive analysis of CAH (CACAH) combining long-range locus-specific PCR and long-read sequencing (LRS) was developed to perform full sequence analysis of 5 common CAH candidate genes, including CYP21A2, CYP11B1, CYP17A1, HSD3B2, and StAR. In a blind retrospective study, the clinical utility of CACAH was evaluated in 37 samples by comparing to standard CAH testing using multiplex ligation-dependent probe amplification (MLPA) plus Sanger sequencing. Of the 37 clinical samples, a total of 69 pathogenic variants were identified, comprising 65 CYP21A2 variants, 2 HSD3B2 variants, and 2 CYP17A1 variants. For CYP21A2, the most frequent variant was c.518T > A (29.2%), followed by c.293-13C/A > G (21.5%). Compared with the current CAH testing using MLPA plus Sanger sequencing, the CACAH assay showed 100% specificity and 100% sensitivity, and precisely determined the junction sites of deletions/insertions and cis-trans configuration of multiple variants without analyzing family samples. Moreover, CACAH identified a case carrying 2 copies of CYP21A1 with the c.1451_1452delinsC variant on the same chromosome, which was not confirmed by MLPA plus Sanger sequencing. LRS-based CACAH can determine all genotypes of CAH accurately and reliably in one assay, presenting a comprehensive approach for CAH genetic diagnosis and carrier screening.

Recently Integrated Alu Elements in Capuchin Monkeys: A Resource for Cebus/Sapajus Genomics.

Capuchins are platyrrhines (monkeys found in the Americas) within the Cebidae family. For most of their taxonomic history, the two main morphological types of capuchins, gracile (untufted) and robust (tufted), were assigned to a single genus, Cebus. Further, all tufted capuchins were assigned to a single species, Cebus apella, despite broad geographic ranges spanning Central and northern South America. In 2012, tufted capuchins were assigned to their genus, Sapajus, with eight currently recognized species and five Cebus species, although these numbers are still under debate. Alu retrotransposons are a class of mobile element insertion (MEI) widely used to study primate phylogenetics. However, Alu elements have rarely been used to study capuchins. Recent genome-level assemblies for capuchins (Cebus imitator; [Cebus_imitator_1.0] and Sapajus apella [GSC_monkey_1.0]) facilitated large scale ascertainment of young lineage-specific Alu insertions. Reported here are 1607 capuchin specific and 678 Sapajus specific Alu insertions along with candidate oligonucleotides for locus-specific PCR assays for many elements. PCR analyses identified 104 genus level and 51 species level Alu insertion polymorphisms. The Alu datasets reported in this study provide a valuable resource that will assist in the classification of archival samples lacking phenotypic data and for the study of capuchin phylogenetic relationships.

Development of a high-resolution typing method for SLA-3, swine MHC class I antigen 3.

We developed a high-resolution and comprehensive typing method for swine leukocyte antigen 3 (SLA-3), an MHC class I gene, employing locus-specific genomic PCR followed by subsequent direct sequencing. A total of 292 individuals from nine pure, one cross-breed and six cell lines were successfully typed. A total of 21 SLA-3 alleles were identified, of which four were found to be novel alleles. However, the allelic diversity of SLA-3 was lower than that of previously reported class I genes, SLA-1 and -2. More SLA-3 alleles were observed in the Landrace and Yorkshire breeds than the other breeds. SLA-3*04:01 was identified in seven out of nine breeds and was the most widely distributed allele across all breeds. Therefore, the typing method reported in this study completes our efforts to develop high-resolution typing methods for major SLA molecules, facilitating the combined analysis of major SLA genes from field samples, which is important to understand the relationship between the adaptive immune responses against pathogens and the immunogenetic makeup of an individual.

Rats exhibit age-related mosaic loss of chromosome Y

Mosaic loss of the Y chromosome (LOY) is the most frequent chromosomal aberration in aging men and is strongly correlated with mortality and disease. To date, studies of LOY have only been performed in humans, and so it is unclear whether LOY is a natural consequence of our relatively long lifespan or due to exposure to human-specific external stressors. Here, we explored whether LOY could be detected in rats. We applied a locus-specific PCR and target sequencing approach that we used as a proxy to estimate LOY in 339 samples covering eleven tissues from young and old individuals. We detected LOY in four tissues of older rats. To confirm the results from the PCR screening, we re-sequenced 60 full genomes from old rats, which revealed that the Y chromosome is the sole chromosome with low copy numbers. Finally, our results suggest that LOY is associated with other structural aberrations on the Y chromosome and possibly linked to the mosaic loss of the X chromosome. This is the first report, to our knowledge, demonstrating that the patterns of LOY observed in aging men are also present in a rodent, and conclude that LOY may be a natural process in placental mammals.

Target-Specific Profiling of RNA m5C Methylation Level Using Amplicon Sequencing.

Mapping the position and quantifying the level of 5-methylcytosine (m5C) as a modification in different types of cellular RNA is an important objective in the field of epitranscriptomics. Bisulfite conversion has long been the gold standard for the detection of m5C in DNA, but it can also be applied to RNA. Here, we detail methods for bisulfite treatment of RNA, locus-specific PCR amplification, and detection of candidate sites by sequencing on the Illumina MiSeq platform.

Quantitative and Single-Nucleotide Resolution Profiling of RNA 5-Methylcytosine.

RNA has coevolved with numerous posttranscriptional modifications to sculpt interactions with proteins and other molecules. One of these modifications is 5-methylcytosine (m5C) and mapping the position and quantifying the level in different types of cellular RNAs and tissues is an important objective in the field of epitranscriptomics. Both in plants and animals bisulfite conversion has long been the gold standard for detection of m5C in DNA but it can also be applied to RNA. Here, we detail methods for highly reproducible bisulfite treatment of RNA, efficient locus-specific PCR amplification, detection of candidate sites by sequencing on the Illumina MiSeq platform, and bioinformatic calling of non-converted sites.

Clinical application of a novel next generation sequencing assay for CYP21A2 gene in 310 cases of 21- hydroxylase congenital adrenal hyperplasia from India.

Accurate diagnosis is required for management of Congenital adrenal hyperplasia (CAH). The conventional method for detection of mutations in the CYP21A2 gene is targeted capillary sequencing which is labor intensive and has limited multiplexing capability. Next generation sequencing (NGS) provides data with high sequence coverage and depth. Our objective was to develop an accurate NGS-based assay to characterize the mutation spectrum in CYP21A2 gene in Indian patients suspected to have 21-OH CAH. Cases with 21-OH CAH from 12 endocrine units across India were studied. DNA was extracted from proband's and parent's(subset) blood. Locus-specific long-range PCR and gel electrophoresis of amplicons was followed by NGS where no visible 30 kb homozygous/whole gene deletion was observed. Orthogonal confirmation was performed by capillary sequencing (ABI 3500) and Multiplex Ligation-dependent Probe Amplification (MLPA, MRC-Holland). PCR products were purified and individual libraries were pooled and sequenced (Illumina). Of the 310 CAH cases, biallelic mutations (pathogenic/ likely pathogenic variants involving both CYP21A2 gene copies) were detected in 256 (82.6%), heterozygous mutations in 13 (4.2 %), and none in 41 (13.2%). Most common mutation was c.293-13A/C>G (29.03%), followed by 30 kb deletion (18.24%). Thirty samples tested orthogonally (by capillary sequencing or MLPA) showed 100% concordance with NGS assay. Nine novel variants were identified. We have developed and validated a comprehensive NGS-based assay for detection of variants in CYP21A2 gene in patients with 21-OH CAH. We describe CYP21A2 mutation spectrum and novel variants in a large cohort of Indian patients with CAH.

Adapterama II: universal amplicon sequencing on Illumina platforms (TaggiMatrix).

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5′ end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

Abstract 4525: Modulating chemoresistance: Uncovering the role of mutant SMAD4R361H in colorectal cancer using PDO and PDX models

Abstract Mutations in the transforming growth factor-β (TGF-β) pathway occur frequently in colorectal cancers (CRC). TGF-β is integral for key cellular processes, including proliferation, migration, differentiation and apoptosis. TGF-β/SMAD4 controls the activation of the pathway. A previously described R361H mutation in SMAD4 has been correlated with decreased overall survival and is suspected to modulate chemoresistance. The aim of the present study is to generate CRISPR-engineered SMAD4R361H CRC organoids and to utilize them as in vitro and in vivo models to investigate the effect of SMAD4R361H mutation on drug response. We have established independent organoid (PDO) and matching patient-derived xenograft (PDX) models from 5 different regions of a chemo-naïve CRC. By targeted amplicon sequencing using a cancer hot spot gene panel, we identified the SMAD4R361H mutation in two of the 5 subpopulations, whereas three of the PDOs were SMAD4wt. Semi-automated compound screening revealed a strong resistance phenotype to afatinib, sapitinib and gefitinib as compared to the wild-type models in vitro. Similar drug response was observed in in vivo testing. To investigate the mechanism of drug resistance, we have engineered isogenic organoids (SMAD4R361H/CRISPR) from SMAD4wt organoids using CRISPR-Cas9 genome-editing. In brief, SMAD4wt organoids were transfected with an all-in-one spCas9-sgRNA-vector and a ssODN as repair template bearing the R361H point mutation. To increase homologous-directed repair, we co-transfected the organoids with a plasmid vector containing i53-bpA, an inhibitor of 53BP1. Single clones were then isolated and genotyped by locus specific PCR to confirm the particular R361H point mutation. We were able to achieve up to 50 % knock-in efficiency in these organoids. Utilizing CRISPR-Cas9, engineered SMAD4R361H/CRISPR organoids were established. These PDOs were subjected with both, native SMAD4wt and SMAD4R361H PDOs, to an in vitro drug screening, allowing for a direct comparison of their drug sensitivity. Based on these results, confirmatory in vivo experiments were designed to investigate how SMAD4 may be involved in modulating resistance in CRC organoids. These models have a prominent role in discovering the connection between genotype and phenotype in complex PDO and PDX models. While this approach is not limited to studying the role of the R361H mutation, it has a broad range of applications in understanding cancer biology, intra-tumor heterogeneity, drug response and may eventually lead to new clinical insights, including design of new therapeutic strategies. Citation Format: Ulrike Pfohl, Alessandra Silvestri, Dirk Schumacher, Karsten Boehnke, Johannes Haybaeck, Sanam Bashir, Ralf Kühn, Marlen Keil, Reinhold Schäfer, Wolfgang Walther, Christian Regenbrecht. Modulating chemoresistance: Uncovering the role of mutant SMAD4R361H in colorectal cancer using PDO and PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4525.

PS1539 A COMPARISON OF POST ALLOGENEIC STEM CELL TRANSPLANT CHIMERISM ANALYSIS METHODS: HIGH THROUGHPUT SNP GENOTYPING ANALYSED BY MALDI‐TOF MASS SPECTROMETRY VS. SHORT TANDEM REPEAT (STR) MARKER ANALYSIS

Background:Monitoring of donor chimerism levels is required in the post allogeneic stem cell transplant (SCT) setting to assess engraftment status. Chimerism levels can be assessed by a variety of methods including XY FISH (sex‐mismatch), microsatellite short tandem repeat (STR) analysis or single nucleotide polymorphism (SNP) genotyping by Q‐PCR. MALDI‐TOF Mass spectrometry (MS) is an alternative technique which can be used for high‐throughput SNP genotyping analysis for the purposes of post‐SCT monitoring. MALDI‐TOF MS offers rapid automated analysis and report generation compared to our existing STR methodology which requires expert analysis. We have evaluated MALDI‐TOF MS SNP genotyping as an alternative to STR analysis for chimerism status.Aims:1. To validate the use of the MALDI‐TOF MS SNP assay for clinical post‐SCT chimerism analysis across a range of transplant types (sibling, matched unrelated and double cord transplants). 2. To assess the accuracy and reproducibility of the assay across a range of defined recipient percentages using specifically prepared chimeric DNA samples. 3. To define the limits of sensitivity for detection of mixed chimerism. 4. To define the lowest DNA input concentration which permits detection of mixed chimerism.Methods:MALDI‐TOF MS SNP genotyping covers a panel of 92 SNPs. Locus specific multiplex PCR is followed by single base extension into the SNP site and detection with MALDI‐TOF MS. Mixed chimerism is assessed by analyzing the cumulative skew in genotyping data across informative SNPs. STR analysis comprises fluorescently tagged PCR of a panel of 16 highly polymorphic microsatellites. Relative peak height analysis of donor and recipient peaks is performed post‐PCR to give chimerism levels.114 post‐SCT clinical samples from 38 single donor and two double cord donor transplants were analyzed by both methods to assess the concordance of results obtained.Synthetic chimeras representing known % levels of donor and recipient DNA (1, 2, 5, 10, 30 and 50% recipient) were prepared and analyzed by both methods to assess accuracy and reproducibility across a range of recipient percentages and also to determine the limit of sensitivity for detection of mixed chimerism.DNA dilutions of the synthetic 2% recipient chimera were prepared in order to define the minimum acceptable DNA template concentration for detection of mixed chimerism in both assays.Results:Good concordance between methodologies was seen across all clinical samples tested, with greatest variation seen between 40–60% chimerism levels. Analysis of synthetic chimera samples also showed good concordance, with greatest variation seen at the expected 50% chimerism level.Analysis of the synthetic chimeras defined the sensitivity of detection for recipient DNA at 2% by both systems. The minimum input template DNA concentration for detection of mixed chimerism was shown to be 1ng/μl for MALDI‐TOF MS and 2ng/μl for STR analysis.Summary/Conclusion:The MALDI‐TOF MS SNP Assay gave highly comparable results to STR analysis for determination of post‐SCT chimerism levels and offered identical sensitivity to STR analysis for detection of mixed chimerism. The assay could reliably detect mixed chimerism at DNA concentrations as low as 1ng/μl. Results analysis and report generation is fully automated, taking less than 20 seconds per sample compared to STR analysis which takes 5–10 minutes of expert analysis per sample. We conclude that MALDI‐TOF MS SNP analysis offers a viable alternative to STR chimerism analysis with comparable accuracy and sensitivity and significantly increased analytical speed.

Genotyping of Sea Anemone during Early Development

Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the life of the animal. Following in vitro fertilization and de-jellying, zygotes are allowed to develop for 24 h at room temperature to reach the early- to mid-gastrula stage. The gastrula embryos are then placed on an agarose gel bed in a Petri dish containing seawater. Under the dissecting microscope, a tungsten needle is used to surgically separate an aboral tissue fragment from each embryo. Post-surgery embryos are then allowed to heal and continue development. Genomic DNA is extracted from the isolated tissue fragment and used as a template for locus-specific PCR. The genotype can be determined based on the size of PCR products or presence/absence of allele-specific PCR products. Post-surgery embryos are then sorted according to the genotype. The duration of the entire genotyping process depends on the number of embryos to be screened, but it minimally requires 4-5 h. This method can be used to identify knockout mutants from a genetically heterogeneous population of embryos and enables analyses of phenotypes during development.

Analysis of CYP21A2 gene mutation and phenotype in patients with 21-hydroxylase deficiency

Objective To investigate the spectrum of CYP21A2 gene mutation and the correlation between genotype and phenotype in patients with 21-hydroxylase deficiency in Tianjin and surrounding areas. Methods Genomic DNA was extracted from the peripheral blood samples of the proband. Locus-specific PCR, direct sequencing of PCR amplification products, and multiplex ligation-dependent probe amplification were applied to detect pathogenic gene CYP21A2 and the relationship between genotypes and phenotypes was analyzed. Results (1) Of 35 patients with 21-hydroxylase deficiency, 25 were classified as salt-wasting phenotype and 10 were simple virilizing phenotype. (2) 69 mutant alleles were detected in a total of 70 alleles in 35 patients. Only one mutant allele was detected in one patient. Two mutant alleles were detected in all other patients, with the mutation detection rate 98.6%. (3) A total of 6 types of mutations were detected, of which c. 293-13C /A>G (I2G) was the most common, accounting for 57.1% (40/70), followed by 18.6% (13/70) for large gene deletion or conversion, and 14.3% (10/70) for p. I173N. In addition, a novel mutation, c. 949C>T (p.R317X), which has not been reported previously, was detected as a pathogenic mutation. (4) Correlation analysis of genotype and phenotype in 35 children showed that the phenotype predicted by genotype was consistent with the actual salt-wasting phenotype in 31 children, and those in three children were inconsistent with the actual clinical phenotype. Conclusion The mutation characteristics of CYP21A2 gene in patients with 21-hydroxylase deficiency in Tianjin and surrounding areas are slightly different from those reported in other regions in China. A mutation c. 949C>T has not been reported, which enriches the mutation spectrum of CYP21A2 gene and provide the foundation for genetic counseling and prenatal diagnosis. Key words: 21-Hydroxylase deficiency; CYP21A2 gene; Mutation

Recently integrated Alu insertions in the squirrel monkey (Saimiri) lineage and application for population analyses

BackgroundThe evolution of Alu elements has been ongoing in primate lineages and Alu insertion polymorphisms are widely used in phylogenetic and population genetics studies. Alu subfamilies in the squirrel monkey (Saimiri), a New World Monkey (NWM), were recently reported. Squirrel monkeys are commonly used in biomedical research and often require species identification. The purpose of this study was two-fold: 1) Perform locus-specific PCR analyses on recently integrated Alu insertions in Saimiri to determine their amplification dynamics, and 2) Identify a subset of Alu insertion polymorphisms with species informative allele frequency distributions between the Saimiri sciureus and Saimiri boliviensis groups.ResultsPCR analyses were performed on a DNA panel of 32 squirrel monkey individuals for 382 Alu insertion events ≤2% diverged from 46 different Alu subfamily consensus sequences, 25 Saimiri specific and 21 NWM specific Alu subfamilies. Of the 382 loci, 110 were polymorphic for presence / absence among squirrel monkey individuals, 35 elements from 14 different Saimiri specific Alu subfamilies and 75 elements from 19 different NWM specific Alu subfamilies (13 of 46 subfamilies analyzed did not contain polymorphic insertions). Of the 110 Alu insertion polymorphisms, 51 had species informative allele frequency distributions between Saimiri sciureus and Saimiri boliviensis groups.ConclusionsThis study confirms the evolution of Alu subfamilies in Saimiri and provides evidence for an ongoing and prolific expansion of these elements in Saimiri with many active subfamilies concurrently propagating. The subset of polymorphic Alu insertions with species informative allele frequency distribution between Saimiri sciureus and Saimiri boliviensis will be instructive for specimen identification and conservation biology.

Homology-based enzymatic DNA fragment assembly-based illumina sequencing library preparation.

The current Illumina HiSeq and MiSeq platforms can generate paired-end reads of up to 2 x 250 bp and 2 x 300 bp in length, respectively. These read lengths may be substantially longer than genomic regions of interest when a DNA sequencing library is prepared through a target enrichment-based approach. A sequencing library preparation method has been developed based on the homology-based enzymatic DNA fragment assembly scheme to allow processing of multiple PCR products within a single read. Target sequences were amplified using locus-specific PCR primers with 8 bp tags, and using the tags, homology-based enzymatic DNA assembly was performed with DNA polymerase, T7 exonuclease and T4 DNA ligase. Short PCR amplicons can hence be assembled into a single molecule, along with sequencing adapters specific to the Illumina platforms. As a proof-of-concept experiment, short PCR amplicons (57–66 bp in length) derived from genomic DNA templates of field pea and containing variable nucleotide locations were assembled and sequenced on the MiSeq platform. The results were validated with other genotyping methods. When 5 PCR amplicons were assembled, 4.3 targeted sequences (single-nucleotide polymorphisms) on average were successfully identified within each read. The utility of this for sequencing of short fragments has consequently been demonstrated.