Thomas A. Randall

Serological, genomic, and structural analyses of the major mite allergen Der p 23

Der p 23 was recently identified in a European population as a major allergen and potentially a chitin binding protein.

Genomic Assortative Mating in Marriages in the United States

Assortative mating in phenotype in human marriages has been widely observed. Using genome-wide genotype data from the Framingham Heart study (FHS; number of married couples = 989) and Health Retirement Survey (HRS; number of married couples = 3,474), this study investigates genomic assortative mating in human marriages. Two types of genomic marital correlations are calculated. The first is a correlation specific to a single married couple “averaged” over all available autosomal single-nucleotide polymorphism (SNPs). In FHS, the average married-couple correlation is 0.0018 with p = 3×10−5; in HRS, it is 0.0017 with p = 7.13×10−13. The marital correlation among the positively assorting SNPs is 0.001 (p = .0043) in FHS and 0.015 (p = 1.66×10−24) in HRS. The sizes of these estimates in FHS and HRS are consistent with what are suggested by the distribution of the allelic combination. The study also estimated SNP-specific correlation “averaged” over all married couples. Suggestive evidence is reported. Future studies need to consider a more general form of genomic assortment, in which different allelic forms in homologous genes and non-homologous genes result in the same phenotype.

Genomic, RNAseq, and Molecular Modeling Evidence Suggests That the Major Allergen Domain in Insects Evolved from a Homodimeric Origin

The major allergen domain (MA) is widely distributed in insects. The crystal structure of a single Bla g 1 MA revealed a novel protein fold in which the fundamental structure was a duplex of two subsequences (monomers), which had diverged over time. This suggested that the evolutionary origin of the MA structure may have been a homodimer of this smaller subsequence. Using publicly available genomic data, the distribution of the basic unit of this class of proteins was determined to better understand its evolutionary history. The duplication and divergence is examined at three distinct levels of resolution: 1) within the orders Diptera and Hymenoptera, 2) within one genus Drosophila, and 3) within one species Aedes aegypti. Within the family Culicidae, we have found two separate occurrences of monomers as independent genes. The organization of the gene family in A. aegypti shows a common evolutionary origin for its monomer and several closely related MAs. Molecular modeling of the A. aegypti monomer with the unique Bla g 1 fold confirms the distant evolutionary relationship and supports the feasibility of homodimer formation from a single monomer. RNAseq data for A. aegypti confirms that the monomer is expressed in the mosquito similar to other A. aegypti MAs after a blood meal. Together, these data support the contention that the detected monomer shares similar functional characteristics to related MAs in other insects. An extensive search for this domain outside of Insecta confirms that the MAs are restricted to insects.

New Insights into Cockroach Allergens

Purpose of ReviewThis review addresses the most recent developments on cockroach allergen research in relation to allergic diseases, especially asthma.Recent FindingsThe number of allergens relevant to cockroach allergy has recently expanded considerably up to 12 groups. New X-ray crystal structures of allergens from groups 1, 2, and 5 revealed interesting features with implications for allergen standardization, sensitization, diagnosis, and therapy.SummaryCockroach allergy is strongly associated with asthma particularly among children and young adults living in inner-city environments, posing challenges for disease control. Environmental interventions targeted at reducing cockroach allergen exposure have provided conflicting results. Immunotherapy may be a way to modify the natural history of cockroach allergy and decrease symptoms and asthma severity among sensitized and exposed individuals. The new information on cockroach allergens is important for the assessment of allergen markers of exposure and disease, and for the design of immunotherapy trials.

Emergence and evolution of Zfp36l3.

In most mammals, the Zfp36 gene family consists of three conserved members, with a fourth member, Zfp36l3, present only in rodents. The ZFP36 proteins regulate post-transcriptional gene expression at the level of mRNA stability in organisms from humans to yeasts, and appear to be expressed in all major groups of eukaryotes. In Mus musculus, Zfp36l3 expression is limited to the placenta and yolk sac, and is important for overall fecundity. We sequenced the Zfp36l3 gene from more than 20 representative species, from members of the Muridae, Cricetidae and Nesomyidae families. Zfp36l3 was not present in Dipodidae, or any families that branched earlier, indicating that this gene is exclusive to the Muroidea superfamily. We provide evidence that Zfp36l3 arose by retrotransposition of an mRNA encoded by a related gene, Zfp36l2 into an ancestral rodent X chromosome. Zfp36l3 has evolved rapidly since its origin, and numerous modifications have developed, including variations in start codon utilization, de novo intron formation by mechanisms including a nested retrotransposition, and the insertion of distinct repetitive regions. One of these repeat regions, a long alanine rich-sequence, is responsible for the full-time cytoplasmic localization of Mus musculus ZFP36L3. In contrast, this repeat sequence is lacking in Peromyscus maniculatus ZFP36L3, and this protein contains a novel nuclear export sequence that controls shuttling between the nucleus and cytosol. Zfp36l3 is an example of a recently acquired, rapidly evolving gene, and its various orthologues illustrate several different mechanisms by which new genes emerge and evolve.

Are dust mite allergens more abundant and/or more stable than other Dermatophagoides pteronyssinus proteins?

To the Editor: Understanding the characteristics that differentiate allergens from nonallergens remains a perplexing question in allergy research. General features such as protein stability and abundance from source are frequently cited as likely determinative factors. However, rigorous statistical comparisons of allergens versus nonallergens on genomic and proteomic scales are lacking. In this study, the house dust miteDermatophagoides pteronyssinus (DP) proteome was evaluated using RNA-seq methods as a proxy to assess the abundance of all proteins in this source. In addition, the thermodynamic stabilities of 656 nonallergens and 19 allergens were evaluated using a combined chemical denaturation and mass spectrometry approach. The results showed that when expression and stability are considered in combination, the allergens are a statistically different population from other DP proteins. The allergens are more stable and more highly expressed. The combination of high levels of transcription and stability for the major allergens Der p 1, Der p 2, and Der p 23 is rare (<2% of all DP proteins) but not unique. Although it has been hypothesized that allergens are the abundant and/or stable proteins from an allergen’s source, experimental evidence to support or refute such hypotheses is lacking. To address the question of allergen abundance, the transcriptome of the house dust mite DP was analyzed with RNA-seq technologies (see this article’s Online Repository at www.jacionline.org). All 19 DP allergens in the official Allergen Nomenclature database of theWorld Health Organization and the

The Draft Genome Assembly of Dermatophagoides pteronyssinus Supports Identification of Novel Allergen Isoforms in Dermatophagoides Species

Background Dermatophagoides pteronyssinus (DP) and Dermatophagoides farinae (DF) are highly similar disease-associated mites with frequently overlapping geographic distributions. A draft genome of DP was assembled to identify candidate allergens in DP homologous to those in DF, investigate allergen isoforms, and facilitate comparisons with related Acari. Methods PacBio and Illumina whole genome sequencing was performed on DP. Assembly and reconstruction of the genomes were optimized for isoform identification in a heterogeneous population. Bioinformatic analyses of Acari genomes were peformed. Results The predicted size of the DP nuclear genome is 52.5 Mb. A predicted protein set of 19,368 proteins was identified, including all 19 currently recognized allergens from this species. Orthologs for 12 allergens established for DF were found. The population of DP mites showed a high level of heterozygosity that allowed the identification of 43 new isoforms for both established and candidate allergens in DP, including a new isoform for the major allergen Der p 23. Reanalyzing the previous DF data assuming heterozygosity, 14 new allergen isoforms could be indentified. Some new isoforms were observed in both species suggesting that these isoforms pre-dated speciation. The high quality of both genomes allowed an examination of synteny which showed many allergen orthologs are physically clustered but with species specific exon/intron structures. Comparative genomic analyses with other Acariformes mites showed that most of the allergen homologs are widely conserved within this Superorder. Conclusions Candidate allergens in DP were identified to facilitate future serological studies. While DP and DF are highly similar genetically, species-specific allergen isoforms exist to facilitate molecular differentiation.

The Limitations of Existing Approaches in Improving MicroRNA Target Prediction Accuracy

MicroRNAs (miRNAs) are small (18-24 nt) endogenous RNAs found across diverse phyla involved in posttranscriptional regulation, primarily downregulation of mRNAs. Experimentally determining miRNA-mRNA interactions can be expensive and time-consuming, making the accurate computational prediction of miRNA targets a high priority. Since miRNA-mRNA base pairing in mammals is not perfectly complementary and only a fraction of the identified motifs are real binding sites, accurately predicting miRNA targets remains challenging. The limitations and bottlenecks of existing algorithms and approaches are discussed in this chapter.A new miRNA-mRNA interaction algorithm was implemented in Python (TargetFind) to capture three different modes of association and to maximize detection sensitivity to around 95% for mouse (mm9) and human (hg19) reference data. For human (hg19) data, the prediction accuracy with any one feature among evolutionarily conserved score, multiple targets in a UTR or changes in free energy varied within a close range from 63.5% to 66%. When the results of these features are combined with majority voting, the expected prediction accuracy increases to 69.5%. When all three features are used together, the average best prediction accuracy with tenfold cross validation from the classifiers naïve Bayes, support vector machine, artificial neural network, and decision tree were, respectively, 66.5%, 67.1%, 69%, and 68.4%. The results reveal the advantages and limitations of these approaches.When comparing different sets of features on their strength in predicting true hg19 targets, evolutionarily conserved score slightly outperformed all other features based on thermostability, and target multiplicity. The sophisticated supervised learning algorithms did not improve the prediction accuracy significantly compared to a simple threshold based approach on conservation score or combining the results of each feature with majority agreements. The targets from randomly generated UTRs behaved similar to that of noninteracting pairs with respect to changes in free energy. Availability of additional experimental data describing noninteracting pairs will advance our understanding of the characteristics and the factors positively and negatively influencing these interactions.

Proteases of Dermatophagoides pteronyssinus

Since the discovery that Der p 1 is a cysteine protease, the role of proteolytic activity in allergic sensitization has been explored. There are many allergens with proteolytic activity; however, exposure from dust mites is not limited to allergens. In this paper, genomic, transcriptomic and proteomic data on Dermatophagoides pteronyssinus (DP) was mined for information regarding the complete degradome of this house dust mite. D. pteronyssinus has more proteases than the closely related Acari, Dermatophagoides farinae (DF) and Sarcoptes scabiei (SS). The group of proteases in D. pteronyssinus is found to be more highly transcribed than the norm for this species. The distribution of protease types is dominated by the cysteine proteases like Der p 1 that account for about half of protease transcription by abundance, and Der p 1 in particular accounts for 22% of the total protease transcripts. In an analysis of protease stability, the group of allergens (Der p 1, Der p 3, Der p 6, and Der p 9) is found to be more stable than the mean. It is also statistically demonstrated that the protease allergens are simultaneously more highly expressed and more stable than the group of D. pteronyssinus proteases being examined, consistent with common assumptions about allergens in general. There are several significant non-allergen outliers from the normal group of proteases with high expression and high stability that should be examined for IgE binding. This paper compiles the first holistic picture of the D. pteronyssinus degradome to which humans may be exposed.