Identification of rare thalassemia variants using third-generation sequencing.

Routine PCR, Sanger sequencing, and specially designed GAP-PCR are often used in the genetic analysis of thalassemia, but all these methods have limitations. In this study, we evaluated a new third-generation sequencing-based approach termed comprehensive analysis of thalassemia alleles (CATSA) in subjects with no variants identified by routine PCR, Sanger sequencing, and specially designed GAP-PCR. Hemoglobin testing and routine PCR tests for 23 common variants were performed for 3,033 subjects. Then, Sanger sequencing and specially designed GAP-PCR were performed for a subject with no variants identified by routine PCR, no iron deficiency, and positive hemoglobin testing. Finally, the new CATSA method was conducted for the subjects with no variants identified by Sanger sequencing and specially designed GAP-PCR. In the 49 subjects tested by CATSA, eight subjects had variants identified. Sanger sequencing and independent PCR confirmed the CATSA result. In addition, it is the first time that Hb Lepore was identified in Hunan Province. In total, traditional methods identified variants in 759 of the 3,033 subjects, while CATSA identified additional variants in eight subjects. CATSA showed great advantages compared to the other genetic testing methods.

[Evaluation of performance of five bioinformatics software for the prediction of missense mutations].

OBJECTIVE: To study the prediction performance evaluation with five kinds of bioinformatics software (SIFT, PolyPhen2, MutationTaster, Provean, MutationAssessor). METHODS: From own database for genetic mutations collected over the past five years, Chinese literature database, Human Gene Mutation Database, and dbSNP, 121 missense mutations confirmed by functional studies, and 121 missense mutations suspected to be pathogenic by pedigree analysis were used as positive gold standard, while 242 missense mutations with minor allele frequency (MAF)>5% in dominant hereditary diseases were used as negative gold standard. The selected mutations were predicted with the five software. Based on the results, the performance of the five software was evaluated for their sensitivity, specificity, positive predict value, false positive rate, negative predict value, false negative rate, false discovery rate, accuracy, and receiver operating characteristic curve (ROC). RESULTS: In terms of sensitivity, negative predictive value and false negative rate, the rank was MutationTaster, PolyPhen2, Provean, SIFT, and MutationAssessor. For specificity and false positive rate, the rank was MutationTaster, Provean, MutationAssessor, SIFT, and PolyPhen2. For positive predict value and false discovery rate, the rank was MutationTaster, Provean, MutationAssessor, PolyPhen2, and SIFT. For area under the ROC curve (AUC) and accuracy, the rank was MutationTaster, Provean, PolyPhen2, MutationAssessor, and SIFT. CONCLUSION: The prediction performance of software may be different when using different parameters. Among the five software, MutationTaster has the best prediction performance.

[Paramyotonia congenita caused by a novel mutation of SCN4A gene in a Chinese family].

OBJECTIVE: To detect SCN4A gene mutation in a pedigree with paramyotonia congenita in order to facilitate genetic counseling and assisted reproduction. METHODS: Clinical data of the family was collected. DNA was extracted from peripheral blood samples. Potential mutation of the SCN4A gene was screened using PCR-Sanger sequencing. Potential mutation was detected in 3 affected relatives, 4 unaffected relatives and 100 unrelated healthy controls. Bioinformatics software was used to predict the effect of mutation on the protein function and conservation of the sequence at the mutation site across various species. RESULTS: A novel missense mutation c.4427T>C (p.Met1476Thr) was detected in the exon 24 of the SCN4A gene in the proband and other 3 affected relatives, but not in 4 unaffected relatives and 100 unrelated controls. Bioinformatic analysis indicated that the codon is highly conserved across various species, and that the mutation has caused damage to the structure and function of SCN4A protein. CONCLUSION: The c.4427 T>C (p.Met1476Thr) mutation of the SCN4A gene may contribute to the paramyotonia congenita. Detection of SCN4A gene mutation is an effective method for the diagnosis of paramyotonic congenita.

Using heteroclinic orbits to quantify topological entropy in fluid flows.

Topological approaches to mixing are important tools to understand chaotic fluid flows, ranging from oceanic transport to the design of micro-mixers. Typically, topological entropy, the exponential growth rate of material lines, is used to quantify topological mixing. Computing topological entropy from the direct stretching rate is computationally expensive and sheds little light on the source of the mixing. Earlier approaches emphasized that topological entropy could be viewed as generated by the braiding of virtual, or "ghost," rods stirring the fluid in a periodic manner. Here, we demonstrate that topological entropy can also be viewed as generated by the braiding of ghost rods following heteroclinic orbits instead. We use the machinery of homotopic lobe dynamics, which extracts symbolic dynamics from finite-length pieces of stable and unstable manifolds attached to fixed points of the fluid flow. As an example, we focus on the topological entropy of a bounded, chaotic, two-dimensional, double-vortex cavity flow. Over a certain parameter range, the topological entropy is primarily due to the braiding of a period-three orbit. However, this orbit does not explain the topological entropy for parameter values where it does not exist, nor does it explain the excess of topological entropy for the entire range of its existence. We show that braiding by heteroclinic orbits provides an accurate computation of topological entropy when the period-three orbit does not exist, and that it provides an explanation for some of the excess topological entropy when the period-three orbit does exist. Furthermore, the computation of symbolic dynamics using heteroclinic orbits has been automated and can be used to compute topological entropy for a general 2D fluid flow.

Identification of differentially expressed genes involved in laccase production in tropical white-rot fungus Polyporus sp. PG15.

Carbon sources and copper ion are the main influencing factors on the production of fungal laccase. To investigate the regulation of carbon source and copper ion in laccase production on the molecular level in tropical white-rot fungus PG15, a comparative analysis of gene expression patterns was performed by cDNA-amplified fragment length polymorphism (AFLP) technique. Selective amplifications with 120 primer combinations allowed the identification of 92 differentially expressed transcript-derived fragments (TDFs), ranging from 200 to 750 bp in size. The TDFs were from PG15 supplemented with different carbon sources and copper ion concentrations, majority of which downregulated laccase production. Twenty-one fragments that matched the database were functionally annotated and analyzed according to the up- and downregulation patterns identified by cDNA-AFLP. These fragments were probably involved in laccase production at the metabolism, signal transduction, transcription, or post-translation levels. This study provides the first catalog of genes involved in laccase production, together with their putatively functional annotations. These data provide potential candidates for improving laccase production in fungi by marker-assisted selection or genetic engineering.