BACKGROUND: Hemophilia A (HEMA) is an X-linked bleeding disorder caused by reduced/absent coagulation factor VIII expression, as a result of pathogenic variants in the F8 gene. Preimplantation prevention of HEMA should ideally include direct pathogenic F8 variant detection, complemented by linkage analysis of flanking markers to identify the high-risk F8 allele. Linkage analysis is particularly indispensable when the pathogenic variant cannot be detected directly or identified. This study evaluated the suitability of a panel of F8 intragenic and extragenic short tandem repeat markers for standalone linkage-based preimplantation genetic testing for monogenic disorder (PGT-M) of the Inv22 pathogenic variant, an almost 600 kb paracentric inversion responsible for almost half of all severe HEMA globally, for which direct detection is challenging.
METHODS: Thirteen markers spanning 1 Mb and encompassing both F8 and the Inv22 inversion interval were genotyped in 153 unrelated females of Viet Kinh ethnicity.
RESULTS: All individuals were heterozygous for ≥ 1 marker, ~ 90% were heterozygous for ≥ 1 of the five F8 intragenic markers, and almost 98% were heterozygous for ≥ 1 upstream (telomeric) and ≥ 1 downstream (centromeric) markers. A prospective PGT-M couple at risk of transmitting F8 Inv22 were fully informative at four marker loci (2 intra-inversion, 1 centromeric, 1 telomeric) and partially informative at another five (2 intra-inversion, 3 centromeric), allowing robust phasing of low- and high-risk haplotypes. In vitro fertilization produced three embryos, all of which clearly inherited the low-risk maternal allele, enabling reliable unaffected diagnoses. A single embryo transfer produced a clinical pregnancy, which was confirmed as unaffected by amniocentesis and long-range PCR, and a healthy baby girl was delivered at term.
CONCLUSIONS: Robust and reliable PGT-M of HEMA, including the common F8 Inv22 pathogenic variant, can be achieved with sufficient informative intragenic and flanking markers.

The SeqStudio™ for human identification (HID) is a new benchtop capillary electrophoresis (CE) platform recently developed by Applied Biosystems for genotyping and sequencing short tandem repeat (STR) fragments. Compared to the previous series of CE systems developed by this maker, it is more compact and easier to use. Moreover, by allowing the detection of 4 to 8 fluorescent dyes, it seems to be fully compatible with the different kits of autosomal and gonosomal STR markers usually used in forensic genetics, which are available in trade and supplied by various manufacturers. However, being a new CE model, before its routine use in forensic genetics applications, it should undergo appropriate analytical validation studies in its own laboratories to understand its potential and limitations. A series of experiments on DNA samples coming from cell line controls, using the GlobalFiler™ IQC Amplification Kit, were carried out to meet this purpose. The SeqStudio™ Genetic Analyzer for HID\'s findings on genotyping reproducibility (precision and accuracy of sizing), sensitivity, signal variability between dyes (intra- and inter-color channel balance), and stutter ratios are reported. These findings confirm the validity of this new CE system and its capability to generate reliable results.

In recent years, influenced by the legalization of Cannabis sativa in some countries and regions, the number of people who smoke or abuse C. sativa has continuously grown, cases of transnational C. sativa trafficking have also been increasing. Therefore, fast and accurate identification and source tracking of C. sativa have become urgent social needs. In this study, we developed a new 19-plex short tandem repeats (STRs) typing system for C. sativa, which includes 15 autosomal STRs (D02-CANN1, C11-CANN1, 4910, B01-CANN1, E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159, nH09, CS1, ANUCS 305, 3735, and ANUCS 302 and 9043), two X-chromosome STRs (ANUCS 501 and 1528), one sex-determining marker (DM016, on Y-chromosome), and a quality control marker (DM029, on autosome). The whole polymerase chain reaction (PCR) process could finish within 1 h, making the system suitable for fast detection. The PCR products were detected and separated with an Applied Biosystems 3500XL Genetic Analyser. Developmental validation studies indicated that the 19-plex typing system was accurate, reliable and sensitive, which could also deconvolute mixed C. sativa samples. Specifically, the sensitivity study showed that a full genotyping profile was obtainable with as low as 125 pg of C. sativa DNA. The species specificity study demonstrated that this multiplex has no cross-reactivity with common non C. sativa DNA. In the population study, a total of 162 alleles at 15 autosomal STRs and 14 alleles at two X-chromosome STRs were detected among 85 samples. The efficiency parameters, including the total discrimination power (TDP) and the combined power of exclusion (CPE) of the system, were calculated to exceed 0.999 999 999 999 988 and 0.998 455 889 684 078, respectively, further proving that the system could meet the needs of individual identification. To the extent of the known studies, this is the first study that included the C. sativa sex-determining marker. In conclusion, the developed new 19-plex STR typing system can successfully achieve the purposes of species identification, gender determination, and individual identification, which could be a powerful tool in tracing trade routes of particular drug syndicates or dealers or in linking certain C. sativa to a crime scene.

Rare adrenal choriocarcinoma should be identified as gestational or nongestational choriocarcinoma because of their different treatment and prognosis. A 29-year-old parous women underwent curettage and right-oviduct resection successively due to irregular vaginal bleeding and positive human chorionic gonadotropin (HCG). Postoperative pathological examinations revealed no intrauterine and extrauterine pregnancy. After that, HCG continued to rise. A 7.6×10.3×11.0 cm mass was present in the left adrenal gland with an uneven inner density and a complete capsule by computed tomography (CT). A biopsy was performed on the mass, which showed us choriocarcinoma. Seven cycles of chemotherapy made her complete response and under supervision. Recurrent diagnosis was done after 3 months. The tumor specimen, the patient\'s blood, and her husband\'s blood were drawn for short tandem repeat (STR) analysis using polymerase chain reaction amplification kit. The genotype of the tumor cells was both maternal and patrilineal, which led to the diagnosis of adrenal gestational choriocarcinoma. The patient was scheduled for adrenalectomy and various chemotherapeutic interventions before and after operation. She achieved complete response and was being followed up again. STR analysis first aids in precise classification of this rare adrenal choriocarcinoma. We encourage using the method to analyze choriocarcinoma outside reproductive organs.

Several strongly conserved DNA sequence patterns in and between introns and intergenic regions (IIRs) consisting of short tandem repeats (STRs) with repeat lengths <3 bp have already been described in the kingdom of Animalia. In this work, we expanded the search and analysis of conserved DNA sequence patterns to a wider range of eukaryotic genomes. Our aims were to confirm the conservation of these patterns, to support the hypothesis on their functional constraints and/or the identification of unknown patterns. We pairwise compared genomic DNA sequences of genes, exons, CDS, introns and intergenic regions of 34 Embryophyta (land plants), 30 Protista and 29 Fungi using established k-mer-based (alignment-free) comparison methods. Additionally, the results were compared with values derived for Animalia in former studies. We confirmed strong correlations between the sequence structures of IIRs spanning over the entire domain of Eukaryotes. We found that the high correlations within introns, intergenic regions and between the two are a result of conserved abundancies of STRs with repeat units ≤2 bp (e.g., (AT)n). For some sequence patterns and their inverse complementary sequences, we found a violation of equal distribution on complementary DNA strands in a subset of genomes. Looking at mismatches within the identified STR patterns, we found specific preferences for certain nucleotides stable over all four phylogenetic kingdoms. We conclude that all of these conserved patterns between IIRs indicate a shared function of these sequence structures related to STRs.

Massively parallel sequencing (MPS), or next generation sequencing (NGS), is a promising methodology for the detection of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) in forensic genetics. Here, the prototype SifaMPS Panel is designed to simultaneously target 87 STRs and 294 SNPs with forensic interest in a single multiplex in conjunction with the TruSeq™ Custom Amplicon workflow and MiSeq FGx™ System. Two in-house python scripts are adopted for the fastq-to-genotype interpretation of MPS data concerning STR and SNP, respectively. In the present study, by sequencing 50 Chinese Hans and many other DNA samples involved in validation studies, system parameters including the depth of coverage (DoC), heterozygote balance (Hb) and sequence coverage ratios (SCRs), as well as different forensic parameters of STRs and SNPs in a population study, were calculated to evaluate the overall performance of this new panel and its practicality in forensic application. In general, except for two STRs (DYS505 and DYS449) and one SNP (rs4288409) that performed poorly, the other 85 STRs and 293 SNPs in our panel had good performance that could strengthen efficiency for human identification and paternity testing. In addition, discordant STR genotype results between those generated from capillary electrophoresis (CE) and from the MPS platform were clearly illustrated, and these results could be a useful reference for applying these particular non-CODIS STRs in forensic practice.

A growing number of studies have shown immunotherapy to be a promising treatment strategy for several types of cancer. Short tandem repeats (STRs) have been proven to be alternative markers for the evaluation of hypermutability in gastrointestinal (GI) cancers. However, the status of STRs and microsatellite instability (MSI) in other tumors have not yet been investigated. To further compare STR and MSI alterations in different tumors, a total of 407 paired DNAs were analyzed from the following eight tumor types: breast cancer (BC), hepatocellular cancer (HCC), pancreatic cancer (PC), colorectal cancer (CRC), gastric cancer (GC), lung cancer (LC), esophageal cancer (EC), and renal cell cancer (RCC). The STR alteration frequencies varied in different tumors as expected. Interestingly, none of the patients possessed MSI-low (MSI-L) or MSI-high (MSI-H), except for the GI patients. The highest STR alteration was detected in EC (77.78%), followed by CRC (69.77%), HCC (63.33%), GC (54.55%), LC (48.00%), RCC (40.91%), BC (36.11%), and PC (25.71%). The potential cutoff for hypermutability was predicted using the published objective response rate (ORR), and the cutoff of LC and HCC was the same as that of GI cancers (26.32%). The cutoffs of 31.58% and 10.53% should be selected for BC and RCC, respectively. In summary, we compared MSI and STR status in eight tumor types, and predicted the potential threshold for hypermutability of BC, HCC, CRC, GC, LC, EC, and RCC.

Laboratory investigations were conducted to evaluate the effect of ultraviolet radiation components and solar radiation exposure as a function of time on the degradation of whole human blood DNA from the standpoint of forensic analysis. Ten μL of whole human male blood samples were exposed to UV-A, UV-B, UV-C, and solar radiation at 20 min intervals up to 120 min. Allele frequencies of 16 short tandem repeat (STR) markers were monitored by employing current forensic typing DNA techniques. The STR markers were grouped into high, medium, and low molecular weight categories. Results revealed that even 20 min exposure to 4.89 eV UV-C photons (ʎ = 254 nm) with radiation intensity of 1200 μW/cm2 would degrade whole human male blood DNA samples significantly, making them unfit for human identification due to the breakdown of high molecular weight STRs. Exposure of blood samples to 4.11 eV UV-B photons (ʎ = 302 nm) with radiation intensity of 900 μW/cm2 resulted in complete degradation of high molecular weight STRs after 60 min. Partial breakdown of medium and low molecular weight STRs started after 80 min exposure. The degradation index (DI) values appear to show that the degradation of the DNA template molecule was relatively less in the low molecular weight DNA fragments as compared with high molecular weight DNA fragments. This finding indicates that genetic profiles obtained from whole human male blood exposed to this radiation for 60 min will give inconclusive results. Samples exposed up to 120 min to 3.40 eV UV-A photons (ʎ = 365 nm) and 3.10-3.94 eV photons of solar radiation did not appear to produce appreciable degradation in any of three molecular weight STRs in the whole human blood DNA samples.

The X-chromosome has been suggested to play a role in prostate cancer (PrCa) since epidemiological studies have provided evidence for an X-linked mode of inheritance for PrCa based on the higher relative risk among men who report an affected brother(s) as compared to those reporting an affected father. The aim of this study was to examine the potential association between the forensic STR markers located at four regions Xp22.31, Xq11.2-12, Xq26.2, and Xq28 and the risk of BPH and PrCa to confirm the impact of ChrX in the PrCa incidence. This may be helpful in the incorporation of STRs genetic variation in the early detection of men population at risk of developing PrCa.
DNA samples from 92 patients and 156 healthy controls collected from two medical centers in Riyadh, Saudi Arabia were analyzed for four regions located at X-chromosome using the Investigator® Argus X-12 QS Kit.
The results demonstrated that microvariant alleles of (DXS7132, DXS10146, HPRTB, DXS10134, and DXS10135) are overrepresented in the BPH group (p < 0.00001). Allele 28 of DXS10135 and allele 15 of DXS7423 could have a protective effect, OR 0.229 (95%CI, 0.066-0.79); and OR 0.439 (95%CI, 0.208-0.925). On the other hand, patients carrying allele 23 of DXS10079 and allele 26 of DXS10148 presented an increased risk to PrCa OR 4.714 (95%CI, 3.604-6.166).
The results are in concordance with the involvement of the X chromosome in PrCa and BPH development. STR allele studies may add further information from the definition of a genetic profile of PrCa resistance or susceptibility. As TBL1, AR, LDOC1, and RPL10 genes are located at regions Xp22.31, Xq11.2-12, Xq26.2, and Xq28, respectively, these genes could play an essential role in PrCa or BPH.

DNA profiling that relies on sets of highly polymorphic autosomal STR markers is widely used in the forensic field for human identification and paternity testing. However, the number of markers that are included in the STR kits that are currently available is insufficient to conclusively prove or disprove a relationship between individuals, especially when complex family scenarios are suspected or indirect analyses are required. In these cases, it becomes necessary to increase the number of loci under analysis to reach an adequate likelihood ratio (LR). In this study, we discovered 18 new autosomal non-CODIS STR loci (D1S1616, D1S1608, D2S437, D3S2457, D4S2406, D4S3249, D5S2843, D5S2501, D6S1010, D8S1039, D12S1301, D14S586, D15S815, SHGC-145653, CHLC.GATA14D12, D1S1603, HUMUT7148, and CHLC.GATA84D07) by web scanning and experimental screening. On the basis of this discovery, we developed a novel multiplex typing system named the \"SiFaSTR 21plex_NCII Typing System\" comprising 1) the 18 non-CODIS autosomal STRs mentioned above, 2) a CODIS locus of D2S1338, and 3) Amelogenin and DYS391. A forensic developmental validation, including sensitivity, species specificity, concordance, reproducibility, sample suitability, testing stability, and mixture testing, was performed following SWGDAM. The results of the validation studies indicated that this system is accurate, reliable and suitable for human DNA profiling. The sensitivity study of the system demonstrated that a full profile was obtainable with DNA as low as 125 pg. Species specificity was proven by the lack of cross-reactivity with a series of common animal species. The stability study demonstrated that 1 ng of control DNA could be fully genotyped with concentrations of haematin ≤ 150 μM, indigotin ≤ 5000 ng/μl, urea ≤ 16000 ng/μl, nigrosine ≤ 100 ng/μl and humic acid ≤ 20 ng/μl. In the mixture test, all of the minor alleles could be called at mixed ratios of 1:1, 1:3 and 3:1. We also investigated the allelic frequencies and forensic parameters of the included markers in 259 Chinese Han individuals. The forensic efficiency parameters, including the total power of discrimination (TDP) and the combined exclusion power in duos (CPEduos) and in trios (CPEtrios) of the system were calculated to be greater than 0.9999999, 0.9997347 and 0.9999997, respectively. This result proved that the system is suitable for human identification and paternity testing. The 18 newly discovered non-CODIS STRs and the developed system will be a valuable supplementary tool for the forensic community and will help solve complex paternity cases, evolutionary studies and population investigations.