Development of an allele-specific real-time PCR assay for discrimination and quantification of p63 R279H mutation in EEC syndrome

The ectrodactyly-ectodermal dysplasiaclefting syndrome is a rare autosomal dominant disorder caused by heterozygous mutations in the p63 gene, a transcription factor belonging to the p53 family. The majority of cases of ectrodactyly-ectodermal dysplasia syndrome are caused by de novo mutations and are therefore sporadic in approximately 60% of patients. The substitution of arginine to histidine (R279H), due to a c.836G>A mutation in exon 7 of the p63 gene, represents 55% of the identified mutations and is considered a mutational hot spot. A quantitative and sensitive real-time PCR was performed to quantify both wild-type and R279H alleles in DNA extracted from peripheral blood and RNA from cultured epithelial cells. Standard curves were constructed for both wild-type and mutant probes. The sensitivity of the assay was determined by generating serial dilutions of the DNA isolated from heterozygous patients (50% of alleles mutated) with wild-type DNA, thus obtaining decreasing percentages of p63 R279H mutant allele (50%, 37.5%, 25%, 12.5%, 10%, 7.5%, 5%, 2.5%, and 0.0%). The assay detected up to 1% of the mutant p63. The high sensitivity of the assay is of particular relevance to prenatal diagnosis and counseling and to detect therapeutic effects of drug treatment or gene therapy aimed at reducing the amount of mutated p63. © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Observational and Dynamical Characterization of Main-belt Comet P/2010 R2 (La Sagra)

We present observations of the recently discovered comet-like main-belt object P/2010 R2 (La Sagra) obtained by Pan-STARRS1 and the Faulkes Telescope-North on Haleakala in Hawaii, the University of Hawaii 2.2 m, Gemini-North, and Keck I telescopes on Mauna Kea, the Danish 1.54 m telescope (operated by the MiNDSTEp consortium) at La Silla, and the Isaac Newton Telescope on La Palma. An antisolar dust tail is observed to be present from 2010 August through 2011 February, while a dust trail aligned with the object's orbit plane is also observed from 2010 December through 2011 August. Assuming typical phase darkening behavior, P/La Sagra is seen to increase in brightness by >1 mag between 2010 August and December, suggesting that dust production is ongoing over this period. These results strongly suggest that the observed activity is cometary in nature (i.e., driven by the sublimation of volatile material), and that P/La Sagra is therefore the most recent main-belt comet to be discovered. We find an approximate absolute magnitude for the nucleus of HR = 17.9 ± 0.2 mag, corresponding to a nucleus radius of ~0.7 km, assuming an albedo of p = 0.05. Comparing the observed scattering surface areas of the dust coma to that of the nucleus when P/La Sagra was active, we find dust-to-nucleus area ratios of Ad /AN = 30-60, comparable to those computed for fellow main-belt comets 238P/Read and P/2008 R1 (Garradd), and one to two orders of magnitude larger than for two other main-belt comets (133P/Elst-Pizarro and 176P/LINEAR). Using optical spectroscopy to search for CN emission, we do not detect any conclusive evidence of sublimation products (i.e., gas emission), finding an upper limit CN production rate of Q CN 100 Myr, suggesting that it is likely native to its current location and that its composition is likely representative of other objects in the same region of the main belt, though the relatively close proximity of the 13:6 mean-motion resonance with Jupiter and the (3,-2,-1) three-body mean-motion resonance with Jupiter and Saturn mean that dynamical instability on larger timescales cannot be ruled out.

Allele resolution of HLA-A using oligonucleotide probes in a two-stage typing strategy

High-resolution polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP) typing methods for HLA-A identification have been established. The four systems, which operate independently of each other, are intended for use as secondary typing systems following HLA-A identification with a medium-resolution PCR-SSOP technique. The systems, all using digoxigenin-labelled probes, are based on group specific amplifications for resolution of: i) HLA-A*29 & -A*33; ii) HLA-A*24 & -A*30; and iii) HLA-A*26, -A*25, -A*11, -A*34, -A*66 and -A*68 alleles, respectively. The fourth system, for the detection of HLA-A*02 alleles, is a modification of a previously reported PCR-SSOP subtyping system. The methods have been applied to individuals from the local bone marrow registry and HLA-A allele frequencies for the Northern Ireland population have been established.

Investigation of Genetic Variation in Scavenger Receptor Class B, Member 1 (SCARB1) and Association with Serum Carotenoids

Objective: To investigate association of scavenger receptor class B, member 1 (SCARB1) genetic variants with serum carotenoid levels of lutein (L) and zeaxanthin (Z) and macular pigment optical density (MPOD).
Design: A cross-sectional study of healthy adults aged 20 to 70.
Participants: We recruited 302 participants after local advertisement.
Methods: We measured MPOD by customized heterochromatic flicker photometry. Fasting blood samples were taken for serum L and Z measurement by high-performance liquid chromatography and lipoprotein analysis by spectrophotometric assay. Forty-seven single nucleotide polymorphisms (SNPs) across SCARB1 were genotyped using Sequenom technology. Association analyses were performed using PLINK to compare allele and haplotype means, with adjustment for potential confounding and correction for multiple comparisons by permutation testing. Replication analysis was performed in the TwinsUK and Carotenoids in Age-Related Eye Disease Study (CAREDS) cohorts.
Main Outcome Measures: Odds ratios for MPOD area, serum L and Z concentrations associated with genetic variations in SCARB1 and interactions between SCARB1 and gender.
Results: After multiple regression analysis with adjustment for age, body mass index, gender, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, smoking, and dietary L and Z levels, 5 SNPs were significantly associated with serum L concentration and 1 SNP with MPOD (P<0.01). Only the association between rs11057841 and serum L withstood correction for multiple comparisons by permutation testing (P<0.01) and replicated in the TwinsUK cohort (P = 0.014). Independent replication was also observed in the CAREDS cohort with rs10846744 (P = 2×10-4), an SNP in high linkage disequilibrium with rs11057841 (r2 = 0.93). No interactions by gender were found. Haplotype analysis revealed no stronger association than obtained with single SNP analyses.
Conclusions: Our study has identified association between rs11057841 and serum L concentration (24% increase per T allele) in healthy subjects, independent of potential confounding factors. Our data supports further evaluation of the role for SCARB1 in the transport of macular pigment and the possible modulation of age-related macular degeneration risk through combating the effects of oxidative stress within the retina.
Financial Disclosure(s): Proprietary or commercial disclosures may be found after the references. Ophthalmology 2013;120:1632–1640 © 2013 by the American Academy of Ophthalmology.

Five authors reply

We read with interest the comments offered by Drs. Hughes and Bradley (1) on our systematic review (2). Four single nucleotide polymorphisms (SNPs), rs9332739 and rs547154 in the complement component 2 gene (C2) and rs4151667 and rs641153 in the complement factor B gene (CFB), were pooled. Hughes and Bradley point out that we omitted the most common variant, rs12614. In fact, rs12614 is in high linkage disequilibrium (LD) with rs641153, which was included, and the major allele of both of these SNPs is in the range of 90% (population code, CEU, in the International HapMap Project (http://hapmap.ncbi.nlm.nih.gov/)). Moreover, our review was initiated in September 2010, at which point only 4 studies had published associations with rs12614, whereas 14 studies (n = 11,378) were available for rs641153. While it is true that both SNPs are better analyzed as a haplotype, these data were simply not available for pooling.
Hughes and Bradley also point out that we obtained and pooled new data that were not previously published. While it is recommended that contact with authors be completed as part of a comprehensive meta-analysis, we acknowledge that these additional data were not previously published and peer reviewed and, hence, do not have the same level of transparency. However, given that sample collections often increase over time and that the instrumentation for genotyping is continually improving, we thought that it would be advantageous to use the most recent information; this is a subjective decision.
We also agree that the allele frequencies given by Kaur et al. (3) were exactly opposite to those expected and were suggestive of strand flipping. However, we specifically queried this with the lead author on 2 separate occasions and were assured it was not.
Hughes and Bradley do make an interesting suggestion that SNPs in high LD should be used as a gauge of genotyping quality in HuGE reviews. This is an interesting idea but difficult to put into practice as the r2 parameter they propose as a measure of LD has some unusual properties. Although r2 is a measure of LD, it is also linked to the allele frequency; even small differences in allele frequencies between 2 linked SNPs can reduce the r2 dramatically. Wray (4) explored these effects and found that, at a baseline allele frequency of 10%, even a difference in allele frequency between 2 SNPs as small as 2% can drop the r2 value below 0.8. This degree of allele frequency difference is consistent with what could be expected for sampling error. Furthermore, when we look at 2 linked dialleleic SNPs, giving 4 possible haplotypes, the absence of 1 haplotype dramatically reduces r2, despite the 2 loci being in high LD as measured by D'. In fact, this is the situation for rs12614 and rs641153, where the low frequency of 1 haplotype means that the r2 is 0.01 but the D' is 1.
Hughes and Bradley also suggest consideration of genotype call rate restrictions as an inclusion criterion for metaanalysis. This would be more appropriate when focusing on genetic variants per se, as considered within the context of a genome-wide association study or other specific genetic analysis where large numbers of SNPs are evaluated (5).
The concerns raised by Hughes and Bradley reflect the limited ability of a meta-analysis based on summary data to tease out inconsistencies best identified at the individual level. We agree that SNPs in LD should be evaluated, but this will not necessarily be straightforward. A move to make genetic data sets publicly available, as in the Database of Genotypes and Phenotypes (http://www.ncbi.nlm.nih.gov/ gap), is a step in the right direction for greater transparency.

Minor allele frequency of myeloproliferative neoplasm mutations in the Irish blood donor population

Myeloproliferative neoplasms (MPNs) are rare diseases that include classic entities; polycythaemia vera, essential thrombocythaemia and primary myelofibrosis. In this short report, minor allele frequencies of common MPN mutations are compared between the Irish blood donor population and other populations of European descent using data from the Haplotype Map project. The Affymetrix array 6.0 platform was utilised identifying nine single nucleotide polymorphisms (SNPs) and six proxy SNPs. The variability of allele frequencies for MPN mutations could account for the different incidence rates seen between populations of European ancestry, giving a better understanding of the genetic predisposition to MPNs. 

Strain-transcending Fc-dependent killing of Plasmodium falciparum by merozoite surface protein 2 allele-specific human antibodies.

It is widely accepted that antibody responses against the human parasitic pathogen Plasmodium falciparum protect the host from the rigors of severe malaria and death. However, there is a continuing need for the development of in vitro correlate assays of immune protection. To this end, the capacity of human monoclonal and polyclonal antibodies in eliciting phagocytosis and parasite growth inhibition via Fcγ receptor-dependent mechanisms was explored. In examining the extent to which sequence diversity in merozoite surface protein 2 (MSP2) results in the evasion of antibody responses, an unexpectedly high level of heterologous function was measured for allele-specific human antibodies. The dependence on Fcγ receptors for opsonic phagocytosis and monocyte-mediated antibody-dependent parasite inhibition was demonstrated by the mutation of the Fc domain of monoclonal antibodies against both MSP2 and a novel vaccine candidate, peptide 27 from the gene PFF0165c. The described flow cytometry-based functional assays are expected to be useful for assessing immunity in naturally infected and vaccinated individuals and for prioritizing among blood-stage antigens for inclusion in blood-stage vaccines.

Mapping Bias Overestimates Reference Allele Frequencies at the HLA Genes in the 1000 Genomes Project Phase I Data.

Next-generation sequencing (NGS) technologies have become the standard for data generation in studies of population genomics, as the 1000 Genomes Project (1000G). However, these techniques are known to be problematic when applied to highly polymorphic genomic regions, such as the human leukocyte antigen (HLA) genes. Because accurate genotype calls and allele frequency estimations are crucial to population genomics analyses, it is important to assess the reliability of NGS data. Here, we evaluate the reliability of genotype calls and allele frequency estimates of the single-nucleotide polymorphisms (SNPs) reported by 1000G (phase I) at five HLA genes (HLA-A, -B, -C, -DRB1, and -DQB1). We take advantage of the availability of HLA Sanger sequencing of 930 of the 1092 1000G samples and use this as a gold standard to benchmark the 1000G data. We document that 18.6% of SNP genotype calls in HLA genes are incorrect and that allele frequencies are estimated with an error greater than ±0.1 at approximately 25% of the SNPs in HLA genes. We found a bias toward overestimation of reference allele frequency for the 1000G data, indicating mapping bias is an important cause of error in frequency estimation in this dataset. We provide a list of sites that have poor allele frequency estimates and discuss the outcomes of including those sites in different kinds of analyses. Because the HLA region is the most polymorphic in the human genome, our results provide insights into the challenges of using of NGS data at other genomic regions of high diversity.