A Novel Long-Range Deletion Spanning CDC73 and Upper-Stream Genes Discovered in a Kindred of Familial Primary Hyperparathyroidism

PurposeTo confirm the exact break-point of a novel long-range deletion discovered in one female parathyroid carcinoma (PC) patient who has a strong family history suggesting familial hyperparathyroidism, and to investigate the expression of parafibromin in the patient’s affected lesion.MethodsClinical information of one female patient as well as five of her relatives were collected. Their genomic DNA extracted from peripheral blood went through Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). After completing whole genome sequencing (WGS), clone sequencing was also performed, whose result was aligned with standard human genome database after Sanger sequencing.ResultsThe medical history of recurrent hypercalcemia after parathyroidectomy and histopathological investigation confirmed that the female patient with the diagnosis of PC. WGS displayed a novel 130kb long-range deletion spanning UCHL5 to CDC73 which was later confirmed by clone sequencing. MLPA showed similar results in four of her five relatives, suggesting these people to be carriers of the same long-range deletion, and three among them had a history of primary hyperparathyroidism (PHPT) ahead of the proband’s first visit.ConclusionWe discovered a novel 130kb long-range deletion spanning CDC73 in a family of five-person, and the existence of the deletion was related to PHPT and PC. Our discovery validated the role of CDC73 mutation in the occurrence of PHPT and PC, which provided new information to the genetic studies of PC.

Combining a COI Mini-Barcode with Next-Generation Sequencing for Animal Origin Ingredients Identification in Processed Meat Product

For revealing animal species in complex or adulterated processed meat product, we presented a method combining a novel cytochrome oxidase I (COI) mini-barcode with next-generation sequencing (NGS), which identifies various animal species (swine, bovine, Caprinae, and some of fish, shrimp, and poultry) accurately and efficiently in processed meat products. We designed a universal primer based on 140 sequences from 51 edible animal species. A mixture of 12 species raw meat samples were identified with the clone sequencing and also with a mini-barcode- (136 bp) combined NGS method, respectively. The mini-barcode of these 12 species was 100% identical to the target species sequence by Sanger sequencing. Compared to the clone sequencing method, the NGS method is superior in accuracy, sensitivity, and detection efficiency. Various edible animal species were identified in the species level both in the mixed samples and the 7 heavily processed food products. Moreover, some unlabeled species and dubious contamination were detected as well.

Impacts of Ceftriaxone Exposure During Pregnancy on Maternal Gut and Placental Microbiota and Its Influence on Maternal and Offspring Immunity in Mice (OR01-04-19)

Objectives This study aimed to investigate whether antibiotic exposure during pregnancy could alter maternal gut and placental microbiota, and consequently affect the immunity of both mother and offspring. Methods Pregnant BALB/c mice (n = 24) were gavaged with ceftriaxone from gestation day 13 to delivery. Both dams and pups were then sacrificed immediately after delivery. Spleen, placental, and fecal samples were collected from the tested dams, and blood samples were collected from both the dams and their pups. The microbiota in the feces and placenta of the dams were comprehensively analyzed using16S rRNA sequencing. Furthermore, viable bacteria in the placentas of dams were also isolated by plate cultivation then taxonomically identified in detail by clone sequencing. Serum cytokines collected from dams and pups were quantitatively profiled using Luminex. Results The maternal spleen index was significantly lower and the offspring serum interleukin-6 (IL-6) levels were significantly higher in ceftriaxone-treated mice compared with the control group. The diversity of maternal fecal microbiota was significantly lower in ceftriaxone-treated mice. The relative abundance of Bacteriodetes was significantly lower, while the relative abundance of Tenericutes was significantly higher in ceftriaxone-treated mothers. However, no significant differences in placental microbiota communities or metagenomic activity were found between the control group and the ceftriaxone-treated mice. Conclusions These results indicated that ceftriaxone exposure in pregnancy could dramatically alter maternal intestinal microbiota, which affected the immunity of the mothers and their offspring, characteristically by enhanced pro-inflammatory responses. The results from the present study also indicated that the placenta might harbor its own microbes, which may not be affected by environmental factors, such as oral administration of ceftriaxone during pregnancy. Further studies should be focused on the role of these microbes in the health of the fetus and infants. Funding Sources This work was supported by the National Natural Science Foundation of China (Grant number 81372982).

Molecular characterization of dihydroneopterin aldolase and aminodeoxychorismate synthase in common bean—genes coding for enzymes in the folate synthesis pathway

Common beans (Phaseolus vulgaris) are excellent sources of dietary folates, but different varieties contain different amounts of these compounds. Genes coding for dihydroneopterin aldolase (DHNA) and aminodeoxychorismate synthase (ADCS) of the folate synthesis pathway were characterized by PCR amplification, BAC clone sequencing, and whole genome sequencing. All DHNA and ADCS genes in the Mesoamerican cultivar OAC Rex were isolated and compared with those genes in the genome of Andean genotype G19833. Both genotypes have two functional DHNA genes and one pseudo gene. PvDHNA1 and PvDHNA2 proteins have similar secondary structures and conserved residues as DHNA homologs in Staphylococcus aureus and Arabidopsis. Sequence analysis and synteny mapping indicated that PvDHNA1 might be a duplicated and transposed copy of PvDHNA2. There is only one ADCS gene (PvADCS) identified in the bean genome and it is identical in OAC Rex and G19833. PvADCS has the conserved motifs required for catalytic activity similar to other plant ADCS homologs. DHNA and ADCS gene-specific markers were developed, mapped, and compared to their physical locations on chromosomes 1 and 7, respectively. The gene-specific markers developed in this study should be useful for detection and selection of varieties with enhanced folate contents in bean breeding programs.

Discovery of large genomic inversions using pooled clone sequencing

There are many different forms of genomic structural variation that can be broadly classified as copy number variation (CNV) and balanced rearrangements. Although many algorithms are now available in the literature that aim to characterize CNVs, discovery of balanced rearrangements (inversions and translocations) remains an open problem. This is mainly because the breakpoints of such events typically lie within segmental duplications and common repeats, which reduce the mappability of short reads. The 1000 Genomes Project spearheaded the development of several methods to identify inversions, however, they are limited to relatively short inversions, and there are currently no available algorithms to discover large inversions using high throughput sequencing technologies (HTS). Here we propose to use a sequencing method (Kitzman et al., 2011) originally developed to improve haplotype resolution to characterize large genomic inversions. This method, called pooled clone sequencing, merges the advantages of clone based sequencing approach with the speed and cost efficiency of HTS technologies. Using data generated with pooled clone sequencing method, we developed a novel algorithm, dipSeq, to discover large inversions (>500 Kbp). We show the power of dipSeq first on simulated data, and then apply it to the genome of a HapMap individual (NA12878). We were able to accurately discover all previously known and experimentally validated large inversions in the same genome. We also identified a novel inversion, and confirmed using fluorescent in situ hybridization. Availability: Implementation of the dipSeq algorithm is available at https://github.com/BilkentCompGen/dipseq