Identification of novel DYNC2H1 mutations associated with short rib-polydactyly syndrome type III using next-generation panel sequencing.

Short rib-polydactyly syndrome type III (SRPS3) is a perinatal lethal skeletal disorder with polydactyly and multisystem organ abnormalities. While ultrasound of the fetus can detect skeletal abnormalities characteristic of SRPS3, the syndrome is often difficult to diagnose before birth. As SRPS3 is an autosomal recessive disorder, identification of the gene mutations involved could lead to the development of prenatal genetic testing as an accurate method of diagnosis. In this study, we describe genetic screening approaches to identify potential abnormalities associated with SRPS3. Karyotype analysis, array comparative genomic hybridization (aCGH), and next-generation panel sequencing were each performed on a fetus showing signs of the disorder, as well as on the mother and father. Karyotype and aCGH results revealed no abnormalities. However, next-generation panel sequencing identified novel mutations in the DYNC2H1 gene. The fetus was compound heterozygous for both a missense mutation c.8313A > T and a frameshift mutation c.10711_10714delTTTA in the DYNC2H1 gene, which were inherited from the mother and father, respectively. These variants were further confirmed using Sanger sequencing and have not been previously reported. Our study indicates the utility of using next-generation panel sequencing in screening for novel disease-associated mutations.

A rare case of trisomy 11q23.3-11q25 and trisomy 22q11.1-22q11.21.

Partial duplication of the long arm of chromosome 11 and the partial trisomy of 22q are uncommon karyotypic abnormalities. Here, we report the case of a 6-year-old girl who showed partial trisomy of 11q and 22q, as a result of a maternal balanced reciprocal translocation (11;22), and exhibited dysmorphic features, severe intellectual disability, brain malformations, and speech delay related to this unique chromosomal abnormality. Array comparative genomic hybridization (array CGH) revealed a gain in copy number on the long arm of chromosome 11, spanning at least 18.22 Mb. Additionally, there was a gain in copy number on the long arm of chromosome 22, spanning at least 3.46 Mb. FISH analysis using a chromosome 11 short arm telomere probe (11p14.2), a chromosome 11 long arm telomere probe (11q24.3), and a chromosome 22 long arm telomere probe (22q13.33) confirmed the origin of the marker chromosome. It has been confirmed by the State Key Laboratory of Medical Genetics of China that this is the first reported instance of the karyotype 47,XX, +der(22)t(11;22)(q23.3;q11.1)mat in the world. Our study reports an additional case that can be used to further characterize and delineate the clinical ramifications of partial trisomy of 11q and 22q.

Analysis of skin color change and related gene expression after crossing of Dongxiang black chicken and ISA layer.

This study evaluated the effects of the autosomal domi-nant Fm gene in conjunction with the sex-linked Id gene on skin color and related gene expression. Ten Dongxiang black cocks were selected to build ten families by mating 60 individuals of ISA B-line layers. The skin color of the F1 generation was observed at different time points. At 126 days, 36 chickens were slaughtered, and gene expression of TYRP1, TYRP2, MC1R, and EDNRB in breast skin was assessed by quantitative RT-PCR. The ratio of Dongxiang black chickens with white skin chicks in the F1 generation to that of non-white was 3:7 (HoFF: HeFf). At 126 days, all F1 generation cocks showed white skin (115/115), while the percentages of hens with black skin were 100% (HoFF, 27/27) and 53.75% (HeFf, 43/80). The change in skin color peaked between 42 and 84 days. The offspring of HoFF displayed significantly higher expres-sion of MC1R, compared with those of HeFf (P < 0.05). The "L" value of hen's skin was significantly lower, and TYRP1 and TYRP2 expres-sion was significantly higher (P < 0.05) than in cocks with the same Fm/fm genotype. These findings indicate the presence of homozygous and heterozygous Fm in Dongxiang black chickens, with the offspring of homozygous birds showing a higher percentage of black skin percentage. The expression of the four genes studied was correlated with skin color, with TYRP1 and TYRP2 representing the most suitable molecular markers.

Aspergillus fumigatus diffusates suppress polymorphonuclear neutrophil phagocytic functions and respiratory burst levels in hematopoietic stem cell transplantation patients.

Invasive aspergillosis (IA) is a severe infection that commonly occurs in immunocompromised patients after hematopoietic stem cell transplantation (HSCT). The present study explores the effect of Aspergillus fumigatus diffusates (AfDs) on phagocytic function and superoxide anion (O2(-)) burst levels in polymorphonuclear neutrophils (PMNs) from post-HSCT patients. A. fumigatus conidia with or without AfD were used to stimulate the PMN from healthy donor or HSCT patient for two hours. PMN morphology was visualized by scanning electron microscopy. The levels of respiratory burst O2(-) produced by the PMNs were determined by flow cytometry. PMN phagocytic rates and phagocytic indexes were observed and calculated using periodic acid-Schiff (PAS) staining under a light-field microscope. No difference was found between the PMN phagocytic rates, phagocytic indexes, or O2(-) respiratory burst levels in health donor PMNs following treatments of A. fumigatus conidia with or without AfD. However, significant inhibition of these indices was seen in the PMNs from HSCT patients following treatment of A. fumigatus conidia plus AfD, compared to that with conidium treatment alone (P < 0.05). Therefore, AfD significantly inhibited the phagocytic function of PMNs from HSCT patients, potentially through inhibition of intracellular respiratory burst levels during phagocytosis. This suggests that the reason underlying the greater susceptibility of HSCT patients to aspergillosis might be the existence of AfD in vivo during infection. Further research on the mechanisms by which AfD affects the phagocytic function of PMNs from HSCT patients is therefore of great significance for the prevention of IA.

Molecular cloning, characterization, and expression of Rab5B, Rab6A, and Rab7 from Litopenaeus vannamei (Penaeidae).

The Rab protein family belongs to a superfamily of ras-like GTP-binding proteins. Rab proteins regulate many steps of membrane trafficking. In this study, three Rab family members, Rab5B, Rab6A, and Rab7, designated LvRab5B, LvRab6A, and LvRab7, were cloned from Litopenaeus vannamei. The full-length cDNA sequences of LvRab5B, LvRab6A, and LvRab7 were 1383, 873, and 767 nucleotides in length and they encoded proteins of 211, 212, and 205 amino acids, respectively. Using qRT-PCR, the mRNA expression levels of the three proteins were determined in the hepatopancreas of L. vannamei at different stages after infectious hypodermal and hematopoietic necrosis virus and white spot syndrome virus challenge. The results indicated that the mRNA expression levels of LvRab5B, LvRab6A, and LvRab7 were all significantly up-regulated after virus injection, suggesting that these genes may play essential roles in the immune response to viral infection in shrimp.

Identification of highly expressed host microRNAs that respond to white spot syndrome virus infection in the Pacific white shrimp Litopenaeus vannamei (Penaeidae).

MicroRNAs (miRNAs) are known to play an important role in regulating both adaptive and innate immunity. Pacific white shrimp (Litopenaeus vannamei) is the most widely farmed crustacean species in the world. However, little is known about the role miRNAs play in shrimp immunity. To understand the impact of viral infection on miRNA expression in shrimp, we used high-throughput sequencing technology to sequence two small RNA libraries prepared from L. vannamei under normal and white spot syndrome virus (WSSV) challenged conditions. Approximately 19,312,189 and 39,763,551 raw reads corresponding to 17,414,787 and 28,633,379 high-quality mappable reads were obtained from the two libraries, respectively. Twelve conserved miRNAs and one novel miRNA that were highly expressed (>100 RPM) in L. vannamei were identified. Of the identified miRNAs, 8 were differentially expressed in response to the virus infection, of which 1 was upregulated and 7 were downregulated. The prediction of miRNA targets showed that the target genes of the differentially expressed miRNAs were related to immunity, apoptosis, and development functions. Our study provides the first characterization of L. vannamei miRNAs in response to WSSV infection, which will help to reveal the roles of miRNAs in the antiviral mechanisms of shrimp.

High-resolution melting curve analysis of the ADSL and LPL genes and their correlation with meat quality and blood parameters in chickens.

Adenylosuccinate lyase (ADSL) and lipoprotein lipase (LPL) are key enzymes in the metabolism of inosine monophosphate (IMP) and fat mass, which are important factors in meat quality evaluation. In this study, we selected 50 hens from the ISA B-line layers and Guangxi Yellow chickens, slaughtered the chickens at 120 days old, and analyzed polymorphisms in the ADSL and LPL genes using the high-resolution melting curve method. Blood lipid parameters, intramuscular fat (IMF), and IMP content were higher (P < 0.05) in Guangxi Yellow chickens than in ISA B-line layers, while LPL activity was lower (P < 0.05). In exon 2 of the ADSL gene, a C3484T mutation was identified. In both breeds, the CC genotype showed the highest IMP, and IMP was the lowest in the TT genotype. In the 5ꞌ regulatory region of the LPL gene, a C293T mutation was identified. In both breeds, the CC genotype showed the lowest LPL and IMF, while IMF was the highest in the TT genotype. The percentages of individuals with the TT type in the ADSL gene, which was associated with the lowest IMP, were 16.0 and 52.0% in Guangxi chickens and ISA layers, respectively. The percentages of individuals with the CC type of the LPL gene, which was associated with the lowest LPL and IMF, were 28.0 and 44.0%, respectively. The ADSL and LPL gene mutations are correlated with differences in meat quality in different chicken breeds, and high-resolution melting curve is an effective prediction technology for these mutations.

Developmental changes in the expression of the GLUT2 and GLUT4 genes in the longissimus dorsi muscle of Yorkshire and Tibetan pigs.

Glucose transporter proteins 2 and 4 (GLUT2 and GLUT4) play important roles in glucose transport and energy metabolism. Changes in the levels of GLUT2 and GLUT4 mRNA were measured in longissimus dorsi muscle from the lean Yorkshire and fat Tibetan pig breeds at six different time points (1, 2, 3, 4, 5, and 6 months) with quantitative real-time polymerase chain reactions. The results showed that GLUT2 and GLUT4 mRNA were abundantly expressed in the longissimus dorsi muscle and that the developmental expression patterns were similar in both breeds. Tibetan pigs exhibited higher intramuscular fat and GLUT2 mRNA levels, while Yorkshire pigs exhibited a higher myofiber cross-sectional area (CSA) and GLUT4 mRNA levels. Furthermore, the changes in the GLUT4 mRNA levels were strongly and positively correlated with the CSA over a period of six months. These results exhibit time- and breed-specific expression patterns of GLUT2 and GLUT4, which highlight their potential as candidate genes for assessing adipose deposition and muscle development in pigs. These differences in the expression of GLUT family genes may also have indications for meat quality.

Cytogenetic and molecular identification of a wheat-Leymus mollis alien multiple substitution line from octoploid Tritileymus x Triticum durum.

Leymus mollis (Trin.) Pilger (NsNsXmXm, 2n = 28), a wild relative of common wheat, possesses many traits that are potentially valuable for wheat improvement. In order to exploit and utilize the useful genes of L. mollis, we developed a multiple alien substitution line, 10DM50, from the progenies of octoploid Tritileymus M842-16 x Triticum durum cv. D4286. Genomic in situ hybridization analysis of mitosis and meiosis (metaphase I), using labeled total DNA of Psathyrostachys huashanica as probe, showed that the substitution line 10DM50 was a cytogenetically stable alien substitution line with 36 chromosomes from wheat and three pairs of Ns genome chromosomes from L. mollis. Simple sequence repeat analysis showed that the chromosomes 3D, 6D, and 7D were absent in 10DM50. Expressed sequence tag-sequence tagged sites analysis showed that new chromatin from 3Ns, 6Ns, and 7Ns of L. mollis were detected in 10DM50. We deduced that the substitution line 10DM50 was a multiple alien substitution line with the 3D, 6D, and 7D chromosomes replaced by 3Ns, 6Ns, and 7Ns from L. mollis. 10DM50 showed high resistance to leaf rust and significantly improved spike length, spikes per plant, and kernels per spike, which are correlated with higher wheat yield. These results suggest that line 10DM50 could be used as intermediate material for transferring desirable traits from L. mollis into common wheat in breeding programs.

Dephosphorylation of NSSR1 regulates alternative splicing of the GluR-B minigene.

Neural salient serine/arginine-rich protein 1 (NSSR1, alternatively SRp38) is an important splicing factor that can repress pre-mRNA alternative splicing in cells during heat shock and mitosis. We show here that NSSR1 protein is dephosphorylated when cells are heat shocked or incubated with kinase inhibitor K252a. Both heat shock and K252a treatment increase the truncated splicing isoform of the GluR-B minigene pre-mRNA. We also investigated the roles of the RRM motif and three RS domains of NSSR1 in in vivo pre-mRNA splicing. The results show that deletion of the RRM motif did not affect GluR-B minigene pre-mRNA splicing, but deletion of any one of the three RS domains increases the truncated splicing isoform of the GluR-B minigene. We further show that an SRSRSK sequence in the RS3 domain may play an important role in the function of NSSR1 in pre-mRNA splicing.

Cloning, characterization, and expression of the macrophage migration inhibitory factor gene from the Pacific white shrimp Litopenaeus vannamei (Penaeidae).

The macrophage migration inhibitory factor (MIF) is an important proinflammatory cytokine that mediates both innate and adaptive immune responses. In this study, we identified a homolog of MIF in the Pacific white shrimp Litopenaeus vannamei. The MIF cDNA contained a 363-bp open reading frame encoding a 120-amino acid protein with a calculated molecular mass of 13.442 kDa and a theoretical isoelectric point of 6.57. The L. vannamei MIF shared high amino acid identity with MIFs of other invertebrates. Tissue distribution analysis by quantitative real-time polymerase chain reaction (qRT-PCR) revealed that the L. vannamei MIF was abundantly expressed in the blood, heart, and hepatopancreas, was moderately expressed in the gill, and was weakly expressed in the muscle and intestine. Furthermore, in order to gain a basic understanding of the role of MIF in the shrimp immune response against viral infection, its mRNA expression was determined in the hepatopancreas of L. vannamei at different stages after white spot syndrome virus (WSSV) challenge using qRT-PCR. The result indicated that the expression of MIF was significantly upregulated after WSSV injection, suggesting that MIF may be involved in the response to viral infection in shrimp.

A novel SCAR marker for detecting Psathyrostachys huashanica Keng chromatin introduced in wheat.

In this study, we cloned and sequenced a 938-base pair polymorphic band, pHs27, in the tightly linked random amplified polymorphic DNA marker OPU10 and converted it into a sequence-characterized amplified region (SCAR) marker referred to as RHS141, which was specific for the Ns genome of Psathyrostachys huashanica. A GenBank basic local alignment search tool search showed that the sequence of pHs27 had no primary sequence homology with known sequences, and Southern blotting confirmed this result. This SCAR marker was used to detect Ns genome chromatin in wheat, and it was successfully amplified in P. huashanica itself, a complete set of wheat-P. huashanica disomic addition lines (1Ns-7Ns), and undetermined homoeologous group addition lines. This SCAR marker will be a powerful tool for the marker-assisted selection of P. huashanica chromosome(s) in a wheat background, and it should also allow wheat breeders to screen for the excellent traits found in P. huashanica chromatin.

IL-10 promoter SNPs and susceptibility to leprosy in ethnic groups from southwest China.

The purpose of this study was to determine whether interleukin-10 (IL-10) promoter polymorphisms are associated with leprosy or their subtypes in ethnic groups from southwest China. Genotyping using TaqMan® SNP Genotyping Master Mix and ABI 7500 real-time PCR system was performed for IL-10 T3575A, G2849A, C2763A, A1082G, C819T, and C592A in 189 healthy controls (40 ± 18 years) and 193 patients (46 ± 18 years) with leprosy [multibacillary, N = 131; paucibacillary (PB), N = 62]. The allelic frequencies of -2763C (97.9 vs 94.0%, P = 0.0074) and -1082A (92.8 vs 88.6%, P = 0.0452) in leprosy patients were significantly higher than in control subjects. The genetic frequency of -2763CC and -1082AA was not only significantly higher among leprosy patients than among control subjects [odds ratio (OR) = 3.33, 95% confidence interval (95%CI) = 1.39-7.99, P = 0.0071 and OR = 1.76, 95%CI = 1.02-3.03, P = 0.0420, respectively] but also significantly higher among PB patients than among control subjects (OR = 2.46, 95%CI = 1.22-4.96, P = 0.0115 and OR = 5.58, 95%CI = 2.06-15.12, P = 0.0007, respectively). The frequency of IL-10 haplotype 3575A/2849G/2763A/1082G/819C/592C was significantly higher among leprosy patients (OR = 5.57, 95%CI = 1.13-27.52, P = 0.0351) and PB patients (OR = 10.5, 95%CI = 1.36- 81.05, P = 0.0241) than among control subjects. IL-10 promoter -2763C/CC,-1082A/AA and haplotype 3575A/2849G/2763A/1082 G/819C/592C are associated with susceptibility to leprosy and the PB subtype in southwest China.

Cloning, partial sequence, and single-nucleotide polymorphism of the ryanodine receptor gene of the Pacific white shrimp Litopenaeus vannamei (Penaeidae).

Ryanodine receptor/calcium release channel is a large protein that plays an essential role in muscle contraction; mutations in the ryanodine receptor gene affect sensitivity to stress. As a first step towards investigating the relationship between the ryanodine receptor and shrimp cramped muscle syndrome, we cloned, partially sequenced, and examined single-nucleotide polymorphisms (SNPs) of the ryanodine receptor gene of the Pacific white shrimp (Litopenaeus vannamei). The nucleotide sequence of a 15.06-kb L. vannamei genomic DNA segment containing a partial ryanodine receptor gene sequence was determined (deposited in GenBank nucleotide database: HM367069). Direct sequencing of PCR-amplified ryanodine receptor exons with their intron-flanking regions in 10 cramped muscle syndrome shrimp and 10 healthy shrimp, revealed seven SNPs. Five of them (1713A/G, 1749T/C, 1755T/C, 3965G/A, and 8737C/T) are located in exons; however, they appear to be neutral (synonymous), since they do not alter the encoded amino acid. The other SNPs (1553C/T and 13337A/G) are in introns. The SNPs identified in the ryanodine receptor gene could be useful for association studies aimed at determining the physiological role of the ryanodine receptor in cramped muscle syndrome of shrimp.