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Objectives: Ameloblastoma (AM) has been known as a benign but locally invasive tumour with high recurrence rates. Invasive behaviour of the AM results in destruction of the adjacent jawbone and the non‐detectable remnants during surgery, interrupting the complete elimination of cancer cells. Methods: To explore novel targets for the tumour cell invasion, a transcriptomic analysis between AM and odontogenic keratocyst were performed through next‐generation sequencing in detail. Results: Enrichment of CACNA1C gene (encoding Cav1.2) in AM, a subunit of the L‐type voltage‐gated calcium channel (VGCC) was observed for the first time. The expression and channel activity of Cav1.2 was confirmed by immunostaining and calcium imaging in the patient samples or primary cells. Verapamil, L‐type VGCC blocker revealed suppression of the Ca2+‐induced cell aggregation and collective invasion of AM cells in vitro. Furthermore, the effect of verapamil in suppressing AM invasion into the adjacent bone was confirmed through orthotopic xenograft model specifically. Conclusion: Taken together, Cav1.2 maybe considered to be a therapeutic candidate to decrease the collective migration and invasion of AM. [ABSTRACT FROM AUTHOR]

X-linked microhaplotypes (X-MHs) have the potential to be a valuable supplementary tool in complex kinship identification or the resolution of DNA mixtures, because they bring together the distinctive genetic pattern of X chromosomal markers and the benefits of microhaplotypes (MHs). In this study, we used the 1000 Genome database to screen and select 63 X-MHs; 18 MHs were filtered out though a batch sequencing assessment of the DNA samples collected from 112 unrelated Chinese Han individuals. The resulting 45-plex panel performed well in comprehensive assessments including repeatability, sensitivity, species specificity, resistance to PCR inhibitors or degradation, mutation rate, and accuracy in detecting DNA mixture samples. The minimum amount of DNA template that can be tested with this panel is 0.5 ng. Additionally, the alleles of the minor contributor can be accurately detected when the mixture rate is larger than 1:9 in female-male mixture or 1:19 in male-male mixture. Then, we calculated population parameters on each MH based on the allele frequency data obtained from the sequence results of the aforementioned 112 unrelated samples. Combining these parameters on each MH, it can be calculated that TDP m , TDP f , CPET, CPED FM , CPED FF and CNCEP 3 of the 45-plex system were 1–8.99×10−13, 1–1.62×10−19, 0.9999999995, 0.9999981, 0.9955, 0.9999971 and 0.99940, respectively, indicating that the panel is capable in personal identification and parentage testing. To reveal the unique advantage of X-MHs in the analyses of complex kinship and male DNA mixture, further assessments were made. For complex kinship identification, 22 types of individual pairs with different second-degree kinship were simulated and different types of likelihood ratios (LR) were calculated for each. The results revealed that the panel can achieve accuracy of approximately 70 %∼80 % when dividing each of the three types of second-degree kinships into three or four groups. Theoretically, such sub-division cannot be done by using independent autosomal markers. For male DNA mixture analysis without suspects, the maximum likelihood ratio strategy was derived and employed in the estimation of the number of male contributors (NOMC). Simulations were conducted to verify the efficacy of the 45-plex panel in the field and to compare it with autosomal markers by assuming the 45 MHs as autosomal ones. The results showed that X-MHs can achieve higher accuracy in the estimation of NOMC than autosomal ones when the mixed males were unrelated. The results highlighted the unique value of X-linked MHs in complex kinship and male mixture analyses. • A novel X-linked microhaplotype (MH) panel containing 45 MHs was developed. • Such panel performed well in multiple evaluations of sequencing quality. • Likelihood ratios to distinguish different 2nd-degree kinships with X-MH markers. • X-based maximum LR strategy to estimate number of male contributors in a mixture. • Simulation proved X-MH markers' advance in kinship and male mixture analyses. [ABSTRACT FROM AUTHOR]

High-throughput next-generation sequencing (NGS) is a feasible technique to detect considerably more markers and simultaneously obtain length and sequence information in a single reaction. In this study, we developed an NGS panel including 42 commonly used autosomal short tandem repeats (STRs) and amelogenin on the Illumina MiSeq FGx™. Sequencing accuracy was validated by the consistency of 2800M Control DNA detected using the ForenSeq™ DNA Signature Prep Kit and Sanger sequencing. Nomenclature incompatibility was found between NGS-STR and CE-STR typing at 9 loci (D3S3045, D6S477, D7S3048, D9S925, D14S608, D17S1290, D18S535, D21S1270, GATA198B05), despite the correct sequence. The difference was caused by the two different methods of identifying motif sequence and a one-to-one correspondence can be found. We evaluated the panel by investigating consistency, sequencing sensitivity and the effectiveness of the 2nd-degree relationship identification. Herein, we present sequencing results from 58 unrelated individuals of the Hebei Han population. The total discrimination power (TDP) and cumulative probability of exclusion for trio paternity testing (CPEtrio) of the 42 NGS-STR panels reached 1–2.84 × 10−57 and 1–9.87 × 10−21, respectively. By family simulation and likelihood ratio (LR) calculation, this panel was shown to have effectiveness for the 2nd-degree kinship identification similar to the ForenSeq™ DNA Signature Prep Kit and certain advantages compared with it due to the relatively small number of loci. As expected, it provides new data for the development of NGS-STR typing technology. [ABSTRACT FROM AUTHOR]

With growing cancer morbidity, forensics cases in which archived tumour tissues can be used as biological samples are increasing, and an effective method to identify the body source of tumour tissues is needed. Single nucleotide polymorphisms (SNPs) may be a promising biomarker to identify the source of tumour tissues because of their low mutation rate and small amplicon size. Next-generation sequencing techniques offers the ability to detect hundreds of SNPs in a single run. The Precision ID Identity Panel (Thermo Fisher Scientific, Waltham, MA, USA) detects 90 autosomal SNPs for individual identification and 34 lineage-informative SNPs on Y chromosome using the Ion PGM system (Thermo Fisher Scientific). In this study, we evaluated performance of the panel for individual identification of tumour tissues. One hundred and fifty pairs of tumour tissues and corresponding normal tissues were analysed. Loss of heterozygosity was detected only in tumour tissues. The identity-by-state (IBS) scoring system was adopted to identify the body source of tumour tissues. The IBS score, as well as the number of loci with 2 alleles (A2), 1 allele (A1) and 0 alleles (A0) shared, were analysed within each tumour-normal pair, unrelated individual pairs, parent–offspring pairs and full-sibling pairs. According to the probability distribution, threshold of A2 in the range of 69 to 89 could achieve accuracy > 99% in identifying the source of tumour tissues. Thus, we developed a new strategy (process and criteria) to identify the source of tumour tissues that could be used in practice. [ABSTRACT FROM AUTHOR]

The scope of forensic kinship analysis is being extended to more distant or complex relationships. However, current methods and standards in this field do not meet the needs of casework. The next-generation sequencing (NGS) technology may hold an advantage in this field to traditional methods due to its strong power to get much more genetic information. To evaluate the effectiveness of NGS to identify the 2nd-degree kinship pairs, DNA samples of 227 individuals from 49 Hebei Han pedigrees were tested by Goldeneye™ 20A kit using capillary electrophoresis (CE) to confirm the relationships within each pedigree, and those of 111 individuals within 97 confirmed grandparent-grandchild or avuncular pairs were analyzed by ForenSeq™ DNA Signature Prep Kit using MiSeq® FGx™ DNA sequencing platform. We calculated the likelihood ratio (LR) based on ITO method and the identical by state (IBS) score of 97 kinship pairs and compared with those of 97 unrelated pairs. According to the results summarized and analyzed by Fisher discriminant analysis and leave-one-out cross-validation (LOOCV) method, ITO method showed higher accuracy than IBS method, even with less information. Therefore, we proposed a recommendation of the thresholds for pairwise 2nd-degree kinship identification for Hebei Han population based on ITO method. When using ITO method based on 94 SNPs and the length information of 27 autosomal STRs, cumulative likelihood ratio (CLR) > 1 and CLR < 0.1 are recommended as the thresholds of confirming and excluding, respectively. The accuracy applying such thresholds is greater than 95%, indicating the promising application value of NGS in this field and providing a direction for further kinship identification strategy selection. Further studies are needed to get the population genetic data of loci contained in the kit based on all sequence information including flanking regions to make full use of the NGS data to improve the accuracy of kinship analysis. [ABSTRACT FROM AUTHOR]

Background: Retinitis pigmentosa (RP) is a group of inherited retinal diseases that result in severe progressive visual impairment. Aims: The purpose of this article was to apply targeted next-generation sequencing (NGS) to identify the causative mutation in a Chinese RP family. Methods: Blood samples were collected from a Chinese proband diagnosed with RP and her family members. A total of 163 genes that have been previously found to be involved in inherited retinal diseases were selected for NGS. Rigorous NGS data analysis; Sanger sequencing validation; and segregation analysis were applied to evaluate a novel frameshift mutation. Results: Sequence analysis revealed that the proband and her affected sister both carried a novel homozygous frameshift mutation in MERTK (p.I103Nfs*4). Other family members carrying a heterozygous mutation were unaffected. This mutation was found to cosegregate with the disease phenotype in this family. This mutation was not found in 1,000 control individuals. Conclusions: The targeted NGS strategy employed provides an efficient tool for RP pathogenic gene detection. This study identified a new autosomal recessive mutation in the RP-related gene MERTK, which expands the spectrum of RP disease-causing mutations. [ABSTRACT FROM AUTHOR]

Background: Retinitis pigmentosa (RP) is a group of rare inherited retinal dystrophies that result in a progressive loss of vision. Molecular diagnosis of RP is difficult due to its phenotypic and genetic heterogeneities. Aims: To investigate causative genetic mutations in a collection of RP cases: one Indian and two Chinese families with autosomal-recessive RP and two sporadic patients with RP. Materials and Methods: A total of 163 genes, which have previously been found to be involved in inherited retinal disorders, were selected for targeted next-generation sequencing (NGS). Stringent NGS data analyses followed by confirmation using Sanger sequencing and segregation analyses were applied to evaluate all identified pathogenic mutations. Results: Four novel frameshift mutations and two compound heterozygous mutations were identified in RP1. In addition, all mutations were found to co-segregate with the disease in the three familial cases; none of the mutations were detected in control samples. Conclusion: This study expands the mutational spectrums of RP1 for RP. [ABSTRACT FROM AUTHOR]