Clinical characteristics of Guillain-Barré syndrome in patients with primary Sjögren's syndrome.

To investigate the clinical characteristics of Guillain-Barré syndrome (GBS) in patients with primary Sjögren's syndrome (SS). Records of patients with positive anti-SSA antibodies hospitalized in the Beijing Tiantan Hospital between December 2011 and May 2020 were retrieved. Patients who fulfilled the criteria for diagnosis of GBS and primary SS were included, and their clinical data were analyzed. Among the 785 patients with positive anti-SSA, 52 patients were identified in this study. They were 27 males and 25 females with median age of 59 years old. Besides anti-SSA antibodies, multiple autoantibodies were detected in these patients including antinuclear antibody, anti-Ro52, anti-mitochondrial M2, anti-thyroid peroxidase and anti-thyroglobulin autoantibodies. Preceding infection was reported in 42 patients. Hyporeflexia/areflexia and limbs weakness were the most common manifestation and 35 patients presented cranial nerve injuries. GBS disability score of 3, 4 and 5 was scaled in 28 (53.8%), 15 (28.8%) and 3 (5.8%) patients respectively. Forty-six patients received intravenous immunoglobulin (IVIG) monotherapy, 5 patients were treated by IVIG plus glucocorticoids, and 51 patients improved during hospitalization. The frequency of male gender among the patients with both GBS and primary SS suggests an independent onset of GBS and the co-existence of these autoimmune diseases in patients with multiple autoantibodies. Majority of patients with GBS and primary SS experience benign disease course.

Correction to: Genetic polymorphisms and mutation rates of 16 X-STRs in a Han Chinese population of Beijing and application examples in second-degree kinship cases.

The above article was published online with incorrect author name.

Genetic polymorphisms and mutation rates of 16 X-STRs in a Han Chinese population of Beijing and application examples in second-degree kinship cases.

As a supplementary tool in forensic cases, X chromosomal short tandem repeats (X-STRs) might bridge large pedigree gaps and bring inspiration to forensic practices for the special mode of inheritance. To standardize the application of X-STRs, the DNA Commission of the International Society for Forensic Genetics (ISFG) presented recommendations concentrating on biostatistical evaluations. Following this guideline, in this study, 1247 (655 females and 592 males) unrelated individuals and 770 families originating from a Han Chinese population of Beijing were investigated with 16 X-STRs. The combined PDF and PDM were 0.999999999999994 and 0.999999997, respectively. The combined MECKrüger, MECKishida, MECDesmarais, and MECDesmarais duo were 0.999972736708864, 0.999999975670766, 0.999999975720931, and 0.999993489709197, respectively. In addition, a population comparison demonstrated that genetic heterogeneity widely exists between the Han population of Beijing and other populations, especially southern Han Chinese, European, and West African populations. Additionally, the overall mutation rates of the paternal and maternal germlines of the 16 X-STRs were 0.0021 and 0.0003, respectively. Among them, HPRTB showed the highest paternal mutation rate of 0.0094. Finally, based on these forensic parameters, the likelihood ratios of four second-degree kinship cases were evaluated. Comparing with autosomal STR, X-STR showed significant advantages for hypothesis exclusion. Our study indicated that the 16 X-STR loci are highly polymorphic in the Han population of Beijing and could be a satisfactory complimentary tool for forensic applications.

MicroRNA profile analysis for discrimination of monozygotic twins using massively parallel sequencing and real-time PCR.

In general, it is extremely problematic to discriminate between monozygotic twins (MZTs), who share the same genomic DNA sequence, using traditional DNA-based identification methods such as short tandem repeat profiling. MicroRNAs (miRNAs) have shown potential in forensic applications owing to their low molecular weight, abundant and tissue-specific expression. In this study, we utilized massively parallel sequencing technology to perform genome-wide profiling of miRNAs in the blood from four pairs of healthy MZTs. On average, 158 miRNAs were detected in each individual and 14% of which were differentially expressed within each pair of MZTs. The miRNAs with the most significant differences in expression between the twins were confirmed using real-time polymerase chain reaction. Our results demonstrated that miRNAs have potential for use as molecular markers in MZTs discrimination.

Development of a massively parallel sequencing assay for investigating sequence polymorphisms of 15 short tandem repeats in a Chinese Northern Han population.

Massively parallel sequencing (MPS) has been used in forensic genetics in recent years owing to several advantages, e.g. MPS can provide precise descriptions of the repeat allele structure and variation in the repeat-flanking regions, increasing the discriminating power among loci and individuals. However, it cannot be fully utilized unless sufficient population data are available for all loci. Thus, there is a pressing need to perform population studies providing a basis for the introduction of MPS into forensic practice. Here, we constructed a multiplex PCR system with fusion primers for one-directional PCR for MPS of 15 commonly used forensic autosomal STRs and amelogenin. Samples from 554 unrelated Chinese Northern Han individuals were typed using this MPS assay. In total, 313 alleles obtained by MPS for all 15 STRs were observed, and the corresponding allele frequencies ranged between 0.0009 and 0.5162. Of all 15 loci, the number of alleles identified for 12 loci increased compared to capillary electrophoresis approaches, and for the following six loci more than double the number of alleles was found: D2S1338, D5S818, D21S11, D13S317, vWA, and D3S1358. Forensic parameters were calculated based on length and sequence-based alleles. D21S11 showed the highest heterozygosity (0.8791), discrimination power (0.9865), and paternity exclusion probability in trios (0.7529). The cumulative match probability for MPS was approximately 2.3157 × 10-20 .

Haplotypic polymorphisms and mutation rate estimates of 22 Y-chromosome STRs in the Northern Chinese Han father-son pairs.

Y chromosome Short tandem repeats (Y-STRs) analysis has been widely used in forensic identification, kinship testing, and population evolution. An accurate understanding of haplotype and mutation rate will benefit these applications. In this work, we analyzed 1123 male samples from Northern Chinese Han population which including 578 DNA-confirmed father-son pairs at 22 Y-STRs loci. A total of 537 haplotypes were observed and the overall haplotype diversity was calculated as 1.0000 ± 0.0001. Except that only two haplotypes were observed twice, all the rest of the 535 were unique. Furthermore, totally 47 mutations were observed during 13,872 paternal meiosis. The mutation rate for each locus estimates ranged from 0.0 to 15.6 × 10-3 with an average mutation rate 3.4 × 10-3 (95% CI 2.5-4.5 × 10-3). Among the 22 loci, DYS449, DYS389 II and DYS458 are the most prone to mutations. This study adds to the growing data on Y-STR haplotype diversity and mutation rates and could be very useful for population and forensic genetics.

A 472-SNP panel for pairwise kinship testing of second-degree relatives.

Kinship testing based on genetic markers, as forensic short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), has valuable practical applications. Paternity and first-degree relationship can be accurately identified by current commonly-used forensic STRs and reported SNP markers. However, second-degree and more distant relationships remain challenging. Although ∼105-106 SNPs can be used to estimate relatedness of higher degrees, genome-wide genotyping and analysis may be impractical for forensic use. With rapid growth of human genome data sets, it is worthwhile to explore additional markers, especially SNPs, for kinship analysis. Here, we reported an autosomal SNP panel consisted of 342 SNP selected from >84 million SNPs and 131 SNPs from previous systems. We genotyped these SNPs in 136 Chinese individuals by multiplex amplicon Massively Parallel Sequencing, and performed pairwise gender-independent kinship testing. The specificity and sensitivity of these SNPs to distinguish second-degree relatives and the unrelated was 99.9% and 100%, respectively, compared with 53.7% and 99.9% of 19 commonly-used forensic STRs. Moreover, the specificity increased to 100% by the combined use of these STRs and SNPs. The 472-SNP panel could also greatly facilitate the discrimination among different relationships. We estimated that the power of ∼6.45 SNPs were equivalent to one forensic STR in the scenario of 2nd-degree relative pedigree. Altogether, we proposed a panel of 472 SNP markers for kinship analysis, which could be important supplementary of current forensic STRs to solve the problem of second-degree relative testing.

Inconsistent genotyping call at DYS389 locus and implications for interpretation.

The male-specific Y chromosome short tandem repeat (STR) locus is used widely in forensic case, which are useful molecular tool to providing the biological evidence for male/female mixture and paternal lineage cases. The Y-STR analysis has been greatly facilitated by advent of commercial multiplex kit. However, even with well-designed robust multiplex kit, abnormal genotyping profile may be observed when encountering with mutations, such as deletion/duplication within the target region or mutation at the primer binding site. In this study, a single-allele shift by five nucleotides for the DYS389I marker between the AmpFlSTR® Yfiler® and Yfiler® Plus PCR amplification kits while the same allele count for DYS389II was observed in eight unrelated Chinese male individuals. After further investigations by re-amplified with three additional multiplex kits, sanger, and next-generation sequencing, the discordance was finally proven caused by existing rare mutation in those sample, which contained two adjacent SNPs only one base apart in the sequence. This paper describes the molecular basis of the discordance at DYS389I genotyping between different commercial multiplex kits and could provide available information for enhancing of interpretation of abnormal Y-STR genotyping in forensic practice.

Tri-allelic patterns of STRs and partially homologous non-sister chromatid crossover observed in a parentage test.

A maternity testing case is reported, in which the child showed tri-allelic patterns in two short tandem repeat (STR) loci. The genotypes of Penta D of the mother and the child were 9,13 and 9,10,13, respectively. Those of D21S11 were 32.2,35 and 29,35, respectively, but intensity ratio of alleles 29 and 35 of the child was 1:2. These results suggested the copy number variations (CNVs) or trisomy of chromosome 21. By further examination using STR-based chromosome aneuploidy detection kit, three alleles were detected in D21S1411, LFG21 and Penta D, and 2 alleles with intensity ratio of 1:2 were observed in D21S2502, D21S1435, D21S11 and D21S1246. Karyotype and whole-genome SNP array analyses showed that the child had a free trisomy 21. In addition, partially homologous non-sister chromatid crossover occurred at the region 19181770-39499178 on the long arm of chromosome 21.

Genetic variability and forensic efficiency of 39 microsatellite loci in the Li ethnic group from Hainan Island in the South China Sea.

BACKGROUND: Investigation of allele and genotype frequencies of microsatellite loci in various populations is an essential pre-requisite in forensic application. AIM: The present study obtained population genetic data and forensic parameters of 39 autosomal Short Tandem Repeats (STRs) loci from a Chinese Li ethnic group and estimated the genetic relationships between Li and other reference populations. SUBJECTS AND METHODS: Thirty-nine STR loci, which include D19S433, D5S818, D21S11, D18S51, D6S1043, D3S1358, D13S317, D7S820, D16S539, CSF1PO, Penta D, D2S441, vWA, D8S1179, TPOX, Penta E, TH01, D12S391, D2S1338, FGA, D6S477, D18S535, D19S253, D15S659, D11S2368, D20S470, D1S1656, D22-GATA198B05, D8S1132, D4S2366, D21S1270, D13S325, D9S925, D3S3045, D14S608, D10S1435, D7S3048, D17S1290 and D5S2500, were amplified in two multiplex DNA-STR fluorescence detection systems for 189 unrelated healthy individuals of the Chinese Li ethnic group. The allele frequency distribution and several parameters commonly used in forensic science were statistically analysed. RESULTS: A total of 378 alleles were observed with corresponding allelic frequencies ranging from 0.0026-0.5899. The power of discrimination and power of exclusion ranged from 0.7569-0.9672 and 0.2513-0.7355, respectively. The power of exclusion (PE) ranged from 0.2580-0.7943 for trio paternity cases and 0.1693-0.5940 for duo paternity cases. The polymorphism information content (PIC) ranged from 0.5001-0.8611. The cumulative match probability across these 39 loci was 2.4242 × 10-38. CONCLUSION: The results indicate that 39 STR loci are polymorphic among the Li ethnic group in Hainan Island in the South China Sea. This set of polymorphic STR loci provide highly polymorphic information and forensic efficiency for forensic individual identification and paternity testing, as well as basic population data for population genetics and anthropological research.

Whole genome nucleosome sequencing identifies novel types of forensic markers in degraded DNA samples.

In the case of mass disasters, missing persons and forensic caseworks, highly degraded biological samples are often encountered. It can be a challenge to analyze and interpret the DNA profiles from these samples. Here we provide a new strategy to solve the problem by taking advantage of the intrinsic structural properties of DNA. We have assessed the in vivo positions of more than 35 million putative nucleosome cores in human leukocytes using high-throughput whole genome sequencing, and identified 2,462 single nucleotide variations (SNVs), 128 insertion-deletion polymorphisms (indels). After comparing the sequence reads with 44 STR loci commonly used in forensics, five STRs (TH01, TPOX, D18S51, DYS391, and D10S1248)were matched. We compared these "nucleosome protected STRs" (NPSTRs) with five other non-NPSTRs using mini-STR primer design, real-time PCR, and capillary gel electrophoresis on artificially degraded DNA. Moreover, genotyping performance of the five NPSTRs and five non-NPSTRs was also tested with real casework samples. All results show that loci located in nucleosomes are more likely to be successfully genotyped in degraded samples. In conclusion, after further strict validation, these markers could be incorporated into future forensic and paleontology identification kits, resulting in higher discriminatory power for certain degraded sample types.

MAGE-A1 promotes melanoma proliferation and migration through C-JUN activation.

MAGE-A1 belongs to the chromosome X-clustered genes of cancer-testis antigen family and is normally expressed in the human germ line but is also overexpressed in various tumors. Previous studies of MAGE-A1 in melanoma mainly focused on methylation changes or its role in immunotherapy, however, its biological functions in melanoma have remained unknown. In order to determine the role of MAGE-A1 in melanoma growth and metastasis, we manipulated melanoma cell lines with overexpression and knockdown of MAGE-A1. Integration of cell proliferation assays, transwell migration and invasion assays, and RNA-Seq analysis revealed that up-regulation of MAGE-A1 dramatically promoted proliferation, migration, and invasion of human melanoma cell lines in vitro, while down-regulation of MAGE-A1 inhibited those characteristics associated with tumor cells. Furthermore, transcriptome sequencing revealed that MAGE-A1 exerts its tumor promoting activity by activating p-C-JUN directly or through ERK-MAPK signaling pathways. Based on our findings, we propose that MAGE-A1 may be a potential therapeutic target for melanoma patients.

Development of a rapid 21-plex autosomal STR typing system for forensic applications.

DNA-STR genotyping technology has been widely used in forensic investigations. Even with such success, there is a great need to reduce the analysis time. In this study, we established a new rapid 21-plex STR typing system, including 13 CODIS loci, Penta D, Penta E, D12S391, D2S1338, D6S1043, D19S433, D2S441 and Amelogenin loci. This system could shorten the amplification time to a minimum of 90 min and does not require DNA extraction from the samples. Validation of the typing system complied with the Scientific Working Group on DNA Analysis Methods (SWGDAM) and the Chinese National Standard (GA/T815-2009) guidelines. The results demonstrated that this 21-plex STR typing system was a valuable tool for rapid criminal investigation.

A novel strategy for forensic age prediction by DNA methylation and support vector regression model.

High deviations resulting from prediction model, gender and population difference have limited age estimation application of DNA methylation markers. Here we identified 2,957 novel age-associated DNA methylation sites (P < 0.01 and R(2) > 0.5) in blood of eight pairs of Chinese Han female monozygotic twins. Among them, nine novel sites (false discovery rate < 0.01), along with three other reported sites, were further validated in 49 unrelated female volunteers with ages of 20-80 years by Sequenom Massarray. A total of 95 CpGs were covered in the PCR products and 11 of them were built the age prediction models. After comparing four different models including, multivariate linear regression, multivariate nonlinear regression, back propagation neural network and support vector regression, SVR was identified as the most robust model with the least mean absolute deviation from real chronological age (2.8 years) and an average accuracy of 4.7 years predicted by only six loci from the 11 loci, as well as an less cross-validated error compared with linear regression model. Our novel strategy provides an accurate measurement that is highly useful in estimating the individual age in forensic practice as well as in tracking the aging process in other related applications.

[Molecular networks and mechanisms of epithelial-mesenchymal transition regulated by miRNAs in the malignant melanoma cell line].

Melanoma is a malignant cutaneous cancer of high metastasis and lethal rates. Epithelial-mesenchymal transition (EMT) plays an important role in the embryonic developmental process that is often activated during tumorigenesis and metastasis. In this study, we integrated of mRNA and miRNA transcriptome sequencing data of melanocyte and melanoma cell lines to identify genes involved in the process of tumor EMT in the first place, and uncovered 11 miRNAs including miR-130a-3p, miR-130b-3p, miR-125a-5p, miR-30a-3p, miR-195-5p, miR-345-5p, miR-509-3-5p, miR-374a-5p, miR-509-5p, miR-148a-3p and miR-330-3p, negatively related with EMT genes using the Mirsystem software. Bioinformatics analysis with target genes of these miRNAs revealed two networks closely related with cellular development and cell-to-cell interactions, as well as multiple signaling pathways participating in EMT. Validation of the 11 miRNAs with molecular biology experiments demonstrated that four miRNAs regulated oncogenes in melanomas, including miR-195-5p, miR-130a-3p, miR-509-5p, and miR-509-3-5p. Our study integrates two kinds of omics data to screen for EMT-related miRNAs, providing a new research idea in the precision genomics of cancer research.

[Genetic variability and phylogenetic analysis of 39 short tandem repeat loci in Beijing Han population].

In this study, we studied the genetic polymorphisms of short tandem repeat (STR) loci from 13 CODIS and 26 non-CODIS system in Beijing Han population for the first time, and established a database of 39 STR loci whose forensic parameters were further evaluated. Our results demonstrated no significant deviation from the Hardy-Weinberg equilibrium of 39 STR loci and no pairwise linkage disequilibrium between them. The power of discriminations, expected heterozygosity, polymorphic information content, and power of exclusion of 39 STR loci ranged from 0.7740-0.9818, 0.6000-0.9350, 0.5317-0.9047 and 0.2909-0.8673. The cumulated discrimination power and cumulative probability of exclusion were 0.999999999999999999999999999999999999999964971 and 0.999999999973878, respectively. Moreover, the genetic distance was calculated based on allele frequency and phylogenetic tree was built using STR loci data from Beijing Han and other 11 Chinese ethnic groups.This study provides important basic data for Chinese forensic DNA database and population genetics database, and has important significance in carrying out forensic individual identification, paternity testing, and population genetic study.

Concurrent copy number variations on chromosome 8 and 22 combined with mutation at FGA locus revealed in a parentage testing case.

Copy number variations (CNVs) are one of the major sources of human genetic diversity and are associated with rare genomic disorders as well as complex traits and diseases. A copy number variation was observed at the D8S1179 locus during routine STR based parentage testing, in which the child exhibited three alleles, "13, 15, 16", with the putative father a homozygous "15" and the mother homozygous "13". In addition, in the same testing case, there was a one-step mutation at the STR locus FGA, in which the putative father was a "22, 24", the mother was a "22, 25", and the child was a "22, 23". After further investigations by re-amplified with different primer sets, clone-based sequencing, karyotype analysis and whole-genome SNP analysis, the results showed that the child had the CNVs at chromosome 8q24.3 and 22q11.21. In conclusion, for parentage testing cases encountered with tri-allele patterns, more testings, such as cloning sequencing, karyotyping, or even whole genome analysis, as well as more appropriate statistical estimations might be conducted to further confirm or exclude the relationship.

Insulin-like growth factor binding protein 5 (IGFBP5) functions as a tumor suppressor in human melanoma cells.

The insulin-like growth factor binding protein 5 (IGFBP5), which is often dysregulated in human cancers, plays a crucial role in carcinogenesis and cancer development. However, the function and underlying mechanism of IGFBP5 in tumor growth and metastasis has been elusive, particularly in malignant human melanoma. Here, we reported that IGFBP5 acts as an important tumor suppressor in melanoma tumorigenicity and metastasis by a series of experiments including transwell assay, xenograft model, in vivo tumor metastasis experiment, and RNA-Seq. Overexpression of IGFBP5 in A375, a typical human melanoma cell line, inhibited cell malignant behaviors significantly, including in vitro proliferation, anchorage-independent growth, migration and invasion, as well as in vivo tumor growth and pulmonary metastasis. In addition, overexpression of IGFBP5 suppressed epithelial-mesenchymal transition (EMT), and decreased the expression of E-cadherin and the key stem cell markers NANOG, SOX2, OCT4, KLF4, and CD133. Furthermore, IGFBP5 exerts its inhibitory activities by reducing the phosphorylation of IGF1R, ERK1/2, and p38-MAPK kinases and abating the expression of HIF1α and its target genes, VEGF and MMP9. All these findings were confirmed by IGFBP5 knockdown in human melanoma cell line A2058. Taken together, these results shed light on the mechanism of IGFBP5 as a potential tumor-suppressor in melanoma progression, indicating that IGFBP5 might be a novel therapeutic target for human melanoma.