Integrated Proteomic and N-Glycoproteomic Profiling of Placental Tissues of Patients with Preeclampsia.

Background: Preeclampsia (PE) is a multi-system disorder of pregnancy that poses a serious threat to maternal and perinatal health worldwide. This study aims to evaluate the global alterations of protein expression and N-glycosylations that are crucial for PE pathogenesis. Here, tandem mass tag labeling combined with LC-MS/MS was employed to determine the global expression of all proteins and intact glycopeptide in placentas from three healthy pregnant women, three patients with early-onset severe PE, and three patients with late-onset severe PE. Results: A total of 2260 proteins were quantified across 9 placental tissues, of which 37 and 23 were differentially expressed in the early-onset and late-onset PE groups, compared to the controls. A total of 789 glycopeptides were accurately quantified, which were derived from 204 glycosylated sites in 159 glycoproteins and were modified by 59 N-Linked glycans. A total of 123 differently expressed glycopeptides, which were from 47 glycoproteins were identified among three groups. Through a combined analysis of proteomic and glycoproteomic data, it was found that the changes in 10 glycoproteins were caused by the difference in glycosylation level but not in the protein abundance level. Conclusion: This is the first study to conduct an integrated proteomic and glycoproteomic characterization of placental tissues of PE patients. Our findings suggest that glycosylation modification may affect the molecular function of proteins through changes in the glycosylation structure or the occupancy of glycosylation, which will provide new insights to help elucidating the pathogenic mechanism of PE.

Advances of multiplex ligation-dependent probe amplification technology in molecular diagnostics.

Multiplex ligation-dependent probe amplification (MLPA) is a multiplex copy number analysis tool which is routinely used to detect large mutations in genetic diseases. With continuous modifications, MLPA has been extended for the detection of DNA methylation variation, single nucleotide polymorphisms, chromosome abnormalities and other forms of genomic variation. The combination with other techniques has even enlarged the application of MLPA in molecular diagnostics of various human diseases. In this review, the principle of MLPA-based techniques as well as their main and latest applications in clinical detection are described. It is believed that with improved automation, increased multiplexing, lower cost and the combination with other technologies, MLPA will play an increasingly important role in molecular diagnosis of human disease.

A novel single-tube multiplex real-time PCR assay for genotyping of thiopurine intolerance-causing variant NUDT15 c.415C>T.

Thiopurines are commonly used in the treatment of acute lymphoblastic leukaemia and autoimmune conditions, can be limited by myelosuppression. The NUDT15 c.415C>T variant is strongly associated with thiopurine-induced myelosuppression, especially in Asians. The purpose of this study was to develop a fast and reliable genotyping method for NUDT15 c.415C>T and investigate the polymorphic distribution among different races in China. A single-tube multiplex real-time PCR assay for NUDT15 c.415C>T genotyping was established using allele-specific TaqMan probes. In 229 samples, the genotyping results obtained through the established method were completely concordant with those obtained by Sanger sequencing. The distributions of NUDT15 c.415C>T among 173 Han Chinese, 48 Miaos, 40 Kazakhs, and 40 Kirghiz were different, with allelic frequencies of 0.06, 0.02, 0.07, and 0, respectively. This method will provide a powerful tool for the implementation of the genotyping-based personalized prescription of thiopurines in clinical settings.

Screening and Functional Analysis of TPO Gene Mutations in a Cohort of Chinese Patients With Congenital Hypothyroidism.

Backgrounds: As a crucial enzyme in thyroid hormone synthesis, the genetic defective thyroid peroxidase (TPO) was one of the main genetic factors leading to congenital hypothyroidism (CH). Methods: Mutations in the TPO gene were screened and identified in 219 patients with CH from northwest China by using high-throughput sequencing and bioinformatics analysis. The biological function of detected variants was studied by in vitro experiments and homology modeling. Results: Nineteen rare variants, including seven novel ones, were detected in 17 of 219 patients (7.8%). Most cases were detected with one single heterozygous variant, and only two patients were detected with multiple variants, i.e., compounds for (1) IVS7-1G>A, p.Ala443Val, and p.Arg769Trp and (2) p.Asn592Ser and p.Asn798Lys. The biological function of the four missense mutations (i.e., p.Ala443Val, p.Arg769Trp, p.Asn592Ser, and p.Asn798Lys) they carried were further studied. Experimental data showed that these four mutations did not affect the protein expression level of the TPO gene but remarkably reduced the peroxidase activity toward guaiacol oxidation, retaining 8-32% of activity of the wild-type protein. The comparison of the predicted 3-D structures of wild-type and mutant TPO proteins showed that these four amino acid substitutions changed the non-covalent interactions of studied residues that might alter the structure and function of the TPO protein. Conclusion: This study was the first to analyze the TPO mutation spectrum of patients with CH in northwest China. Our data indicated that the TPO mutation was not a common reason to cause CH in China. The functional data may help to clarify the structure-function relationship of the TPO protein and provide further evidence for the elucidation of the genetic etiology of CH.