Short Tandem Repeat Assay Research Articles

Development and validation of a novel forensic STR multiplex assay for blue (Anthropoides paradiseus), wattled (Bugeranus carunculatus), and grey-crowned crane (Balearica regulorum)

The blue crane (Anthropoides paradiseus), wattled crane (Bugeranus carunculatus), and grey-crowned crane (Balearica regulorum) are species of concern as their populations are declining and they face several threats including habitat loss, disturbance and illegal trade. In South Africa, these species are bred in captivity for trade purposes which is permitted and regulated globally under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Legal sustainable trade through captive breeding of endangered wildlife species such as cranes has been promoted to counteract the illegal trade of individuals from the wild. Captive breeding independent of wild populations may reduce the harvest pressures on wild bird populations which in turn benefit the recovery of exploited species. This approach is considered to be controversial by some individuals. Although captive breeding of endangered species, for both population sustainability and commercial purposes, is promoted to aid in conserving species, concerns have been raised with regards to breeding facilities being used for laundering of animals. To monitor the legal trade of cranes in South Africa a short tandem repeat (STR) assay following recommendations of the International Society for Forensic Genetics (ISFG) was developed and validated. An STR assay comprising of four multiplexes that include 16 STR markers and two gender determination markers was proven to be highly informative with average polymorphic information content (PIC) values of 0.806, 0.646 and 0.725 for A. paradiseus, B. regulorum and B. carunculatus respectively. In addition, the assay showed sufficient discriminatory power for parentage assignment of closely related individuals in all three species (A. paradiseus: PI = 1.7×10−24, PIsibs = 4.7×10−08, and B. carunculatus: PI = 1.4×10−19, PIsibs = 2.9×10−07 and B. regulorum: PI = 1.7×10−12, PIsibs = 5.0×10−05). Analysis of 251 samples suggested that the validated multiplex assay ensures reliability, reproducibility, and repeatability for applications in forensic case work where illegal trade of offspring is suspected through verifying parentage of captive birds in breeding facilities.

Change in Visual Acuity of Patients With Fuchs Endothelial Corneal Dystrophy Over 1 Year.

To determine whether the clinical and paraclinical course of Fuchs endothelial corneal dystrophy (FECD) over 1 year is related to the extent of triplet repetition in the transcription factor 4 (TCF4) gene. A prospective study with a 1-year follow-up was conducted. A total of 104 patients (160 eyes) with FECD and an equivalent number of age- and sex-matched control subjects without FECD were included. At inclusion, the corneas were graded using the modified Krachmer grade (KG) and patients were genotyped for the number of trinucleotide repeats (TNRs) in the TCF4 gene by the short tandem repeat assay. Visual acuity, Scheimpflug tomographic features, and the Visual Function and Corneal Health Status using a visual disability instrument were measured on 2 visits at 1-year intervals. KGs ranged from 1 to 6, and 46% of eyes had grades 1 to 4. 71% of the patients harbored TNR expansion (>40) versus 13% in control subjects ( P < 0.001). Severity at inclusion was higher in the presence of TNR expansion when considering eyes independently (mean grade ±SD, 4.08 ± 1.42) without TNR expansion and 4.66 ± 1.27 with TNR expansion ( P = 0.024). In 1 year, the ETDRS score significantly decreased by -2.97 (95% confidence interval -4.69 to -1.26, P = 0.001) and the ETDRS score with glare by -4.25 (95% confidence interval -6.22 to -2.27, P < 10 -5 ). There was no relationship between the rate of decline and TNR expansion or KG. Central corneal thickness and Visual Function and Corneal Health Status scores did not significantly vary. It is possible to measure a subtle progression of FECD over a period as short as 1 year. We did not find a relationship between the presence of TNR expansion and the speed of deterioration over 1 year. This work should facilitate the design of future clinical trials on FECD.

Development and validation of a novel 30-plex STR assay for canine individual identification and parentage testing

Domestic dogs are helpers in outdoor human work and companions for families; thus, individual canine identification and parentage testing are crucial in certain fields, including forensics and breeding programs. In this study, a six-dye fluorescent labeling multiplex amplification system containing 29 canine short tandem repeats (STRs) and the sex-determining marker DAmel was developed. The system was called the Tronfo Canine 30-plex STR Kit and was further validated according to the Scientific Working Group on DNA Analysis Methods and the Organization of Scientific Area Committees for Wildlife Forensics guidelines, including tests for PCR conditions, precision, species specificity, sensitivity, stability, repeatability and reproducibility, a population study, and a study of 16 paternity test cases. The results indicated that the novel canine STR assay was accurate, specific, reproducible, stable, and robust. Complete profiles were obtained with 31.25 pg of canine DNA. Additionally, 500 unrelated canine individuals were investigated using this novel system, and the combined power of discrimination and exclusion values were 0.999999999999999999 and 0.999996451039850, respectively. These results suggest that the Tronfo Canine 30-plex STR Kit is highly polymorphic, informative, and suitable for individual canine identification and parentage testing.

Development and Application of a Short Tandem Repeat Multiplex Typing Assay for Candida tropicalis.

Candida tropicalis is a clinically important yeast that causes candidemia in humans with a high mortality rate. The yeast primarily infects immunocompromised patients, and causes outbreaks in health care facilities. Antifungal resistant isolates have been reported. We developed a short tandem repeat (STR) typing scheme for C. tropicalis to enable fast, cost-effective, and high-resolution genotyping. For the development of the typing scheme, 6 novel STR markers were selected, combined into 2 multiplex PCRs. In total, 117 C. tropicalis isolates were typed, resulting in the identification of 104 different genotypes. Subsequently, the outcome of STR typing of 10 isolates was compared to single nucleotide polymorphism (SNP) calling from whole-genome sequencing (WGS). Isolates with more than 111 SNPs were differentiated by the typing assay. Two isolates, which were identical according to SNP analysis, were separated by STR typing in 1 marker. To test specificity, the STR typing was applied to 15 related yeast species, and we found no amplification of these targets. For reproducibility testing, 2 isolates were independently typed five times, which showed identical results in each experiment. In summary, we developed a reliable and multiplex STR genotyping for C. tropicalis, which was found to correlate well to SNP calling by WGS. WGS analysis from and extensive collection of isolates is required to establish the precise resolution of this STR assay. IMPORTANCE Candida tropicalis frequently causes candidemia in immunocompromised patients. C. tropicalis infections have a high mortality rate, and the yeast is able to cause outbreaks in health care facilities. Further, antifungal resistant isolates are on the rise. Genotyping is necessary to investigate potential outbreaks. Here, we developed and applied a STR genotyping scheme in order to rapidly genotype isolates with a high-resolution. WGS SNP outcomes were highly comparable with STR typing results. Altogether, we developed a rapid, high-resolution, and specific STR genotyping scheme for C. tropicalis.

Optimization and Validation of Candida auris Short Tandem Repeat Analysis.

ABSTRACTCandida auris is an easily transmissible yeast with resistance to different antifungal compounds. Outbreaks of C. auris are mostly observed in intensive care units. To take adequate measures during an outbreak, it is essential to understand the transmission route, which requires isolate genotyping. In 2019, a short tandem repeat (STR) genotyping analysis was developed for C. auris. To determine the discriminatory power of this method, we performed STR analysis of 171 isolates with known whole-genome sequencing (WGS) data using Illumina reads, and we compared their resolutions. We found that STR analysis separated the 171 isolates into four clades (clades I to IV), as was also seen with WGS analysis. Then, to improve the separation of isolates in clade IV, the STR assay was optimized by the addition of 2 STR markers. With this improved STR assay, a total of 32 different genotypes were identified, while all isolates with differences of >50 single-nucleotide polymorphisms (SNPs) were separated by at least 1 STR marker. Altogether, we optimized and validated the C. auris STR panel for clades I to IV and established its discriminatory power, compared to WGS SNP analysis using Illumina reads.IMPORTANCE The emerging fungal pathogen Candida auris poses a threat to public health, mainly causing outbreaks in intensive care units. Genotyping is essential for investigating potential outbreaks and preventing further spread. Previously, we developed a STR genotyping scheme for rapid and high-resolution genotyping, and WGS SNP outcomes for some isolates were compared to STR data. Here, we compared WGS SNP and STR outcomes for a larger sample cohort. Also, we optimized the resolution of this typing scheme with the addition of 2 STR markers. Altogether, we validated and optimized this rapid, reliable, and high-resolution typing scheme for C. auris.

P293 Development of a multiplex PCR short tandem repeat typing scheme for Candida tropicalis

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM Candida tropicalis is a clinically relevant yeast that causes candidemia in humans with a high mortality rate. The yeast primarily infects immunocompromised patients and causes outbreaks in health care facilities. Antifungal-resistant isolates have been reported. Here, we report a short tandem repeat (STR) typing scheme, for C. tropicalis to enable fast, cost-effective, and high-resolution genotyping. This novel typing approach was applied to 117 clinical isolates. For the development of the typing scheme six novel STR markers were selected, combined into two multiplex PCRs, and used to type 117 C. tropicalis isolates, resulting in the identification of 104 different genotypes. The outcome of STR typing of 10 isolates was then compared to single nucleotide polymorphism (SNP) calling from whole genome sequencing (WGS). Isolates with >111 SNPs were also differentiated by the STR assay. Two isolates that were identical according to SNP analysis were separated by STR typing in one marker. To test specificity, STR typing was applied to 15 related yeast species and we found no amplification of these targets. For reproducibility testing, two isolates were typed independently 5-times, which showed identical results in each experiment. In summary, we developed a reliable, rapid, and high-resolution STR genotyping for C. tropicalis, which was found to correlate well to SNP calling by WGS.

Analysis of loss of heterozygosity at HLA loci in a patient with leukemia

To detect loss of heterozygosity (LOH) at human leukocyte antigen (HLA) loci in a Chinese patient with leukemia after haploidentical hematopoietic stem cell transplantation. HLA genotyping was carried out on peripheral blood, hair follicle and buccal swab samples derived from the patient after the transplantation as well as peripheral blood samples from his parents by using PCR-sequence specific oligonucleotide probe method and PCR-sequence based typing method. Short tandem repeat (STR) loci were detected by using a 23 site STR assay kit and a self-developed 6 STR loci assay for the HLA regions. After the transplantation, the HLA genotype of the peripheral blood sample of the patient was identical to his father. The patient was HLA-A*02:01,24:02, C*03:03,03:04, B*13:01,15:01, DRB1*08:03,12:02, DQB1*03:01,06:01 for his hair follicle specimen. However, homozygosity of the HLA loci was found in his buccal swab sample. Only the HLA-A*24:02-C*03:03-B*15:01-DRB1*08:03-DQB1*06:01 haplotype from his father's was present, while the HLA-A*02:01-C*03:04-B*13:01-DRB1*12:02-DQB1*03:01 haplotype from his mother was lost. After the transplantation, the alleles of the 23 STR sites in the patient's peripheral blood sample were consistent to his father, with no allelic loss detected in his buccal swab sample. However, at least 4 STR loci in the HLA region were lost in his buccal swab sample. LOH at the HLA loci has been detected in the buccal swab sample of a patient with leukemia who received haploidentical hematopoietic stem cell transplantation.

Identification of an SRY-negative 46,XX infertility male with a heterozygous deletion downstream of SOX3 gene

BackgroundA male individual with a karyotype of 46,XX is very rare. We explored the genetic aetiology of an infertility male with a kayrotype of 46,XX and SRY negative.MethodsThe peripheral blood sample was collected from the patient and subjected to a few genetic testing, including chromosomal karyotyping, azoospermia factor (AZF) deletion, short tandem repeat (STR) analysis for AMELX, AMELY and SRY, fluorescence in situ hybridization (FISH) with specific probes for CSP 18/CSP X/CSP Y/SRY, chromosomal microarray analysis (CMA) for genomic copy number variations(CNVs), whole-genome analysis(WGA) for genomic SNV&InDel mutation, and X chromosome inactivation (XCI) analysis.ResultsThe patient had a karyotype of 46,XX. AZF analysis showed that he missed the AZF region (including a, b and c) and SRY gene. STR assay revealed he possessed the AMELX in the X chromosome, but he had no the AMELY and SRY in the Y chromosome. FISH analysis with CSP X/CSP Y/SRY showed only two X centromeric signals, but none Y chromosome and SRY. The above results of the karyotype, FISH and STR analysis did not suggest a Y chromosome chimerism existed in the patient's peripheral blood. The result of the CMA indicated a heterozygous deletion with an approximate size of 867 kb in Xq27.1 (hg19: chrX: 138,612,879–139,480,163 bp), located at 104 kb downstream of SOX3 gene, including F9, CXorf66, MCF2 and ATP11C. WGA also displayed the above deletion fragment but did not present known pathogenic or likely pathogenic SNV&InDel mutation responsible for sex determination and development. XCI assay showed that he had about 75% of the X chromosome inactivated.ConclusionsAlthough the pathogenicity of 46,XX male patients with SRY negative remains unclear, SOX3 expression of the acquired function may be associated with partial testis differentiation of these patients. Therefore, the CNVs analysis of the SOX3 gene and its regulatory region should be performed routinely for these patients.

Should Patients with Kearns-Sayre Syndrome and Corneal Endothelial Failure Be Genotyped for a TCF4 Trinucleotide Repeat, Commonly Associated with Fuchs Endothelial Corneal Dystrophy?

The aim of this study was to describe the ocular phenotype in a case with Kearns-Sayre syndrome (KSS) spectrum and to determine if corneal endothelial cell dysfunction could be attributed to other known distinct genetic causes. Herein, genomic DNA was extracted from blood and exome sequencing was performed. Non-coding gene regions implicated in corneal endothelial dystrophies were screened by Sanger sequencing. In addition, a repeat expansion situated within an intron of TCF4 (termed CTG18.1) was genotyped using the short tandem repeat assay. The diagnosis of KSS spectrum was based on the presence of ptosis, chronic progressive external ophthalmoplegia, pigmentary retinopathy, hearing loss, and muscle weakness, which were further supported by the detection of ~6.5 kb mtDNA deletion. At the age of 33 years, the proband’s best corrected visual acuity was reduced to 0.04 in the right eye and 0.2 in the left eye. Rare ocular findings included marked corneal oedema with central corneal thickness of 824 and 844 µm in the right and left eye, respectively. No pathogenic variants in the genes, which are associated with corneal endothelial dystrophies, were identified. Furthermore, the CTG18.1 genotype was 12/33, which exceeds a previously determined critical threshold for toxic RNA foci appearance in corneal endothelial cells.

O-090 Correcting a PLCζ mutation in the human germ line to overcome hereditary infertility

Abstract Study question Can clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing result in the correction of a single base pair substitution that causes male infertility? Summary answer CRISPR/Cas9 administration during intracytoplasmic sperm injection (ICSI) leads to correction attempts of mutant phospholipase C zeta (PLCζ), howeverc loss-of-heterozygosity (LOH). What is known already Failed fertilization after ICSI can be caused by mutations in the sperm-related oocyte factor PLCζ which can be overcome by assisted oocyte activation (AOA). In this way, children may inherit the infertility-causing mutation. Mutation transmission can be overcome through CRISPR/Cas9 delivery during ICSI. In previous studies using CRISPR/Cas9 in the human germline for mutation correction, loss-of-heterozygosity (LOH, loss of the allele of one of the parents) was observed. Two different explanations were given, namely partial or complete paternal chromosomal loss or the correction of the mutation by using the maternal wild-type allele instead of the exogeneous supplied repair template. Study design, size, duration We injected a gRNA-Cas9 protein complex to target the PLCζ mutant allele, a repair template harboring the desired nucleotide substitution and an additional synonymous variant to track template usage, together with patient’s sperm. To overcome fertilization failure, AOA was applied during ICSI. After a culture period of maximal 6 days the embryos were collected. At day 3, some embryos were dissociated in individual blastomeres. The extracted DNA was analyzed through different genetic sequencing techniques. Participants/materials, setting, methods Donated sperm of a patient experiencing complete fertilization failure after routine ICSI, harboring a heterozygous base pair substitution in PLCZ1 (c.136-1G&amp;gt;C), was utilized. Sperm was injected in donated in vitro matured oocytes or in vivo matured oocytes containing clusters of smooth endoplasmic reticulum. Next-generation sequencing was used to assess correction potential. Short tandem repeat (STR) and single nucleotide polymorphism (SNP) assays were used to determine whether the sperm contained the mutation and to evaluate LOH. Main results and the role of chance CRISPR/Cas9 injections had no significant impact (p &amp;gt; 0.05) on embryonic development. Due to the heterozygous nature of the mutation, 47% (27/58) of the embryos originated from mutated sperm injection. The CRISPR components showed a high specificity with absence of insertions/deletions in 97% of the embryos originating from wild-type sperm (n = 31). Embryos originating from mutant sperm (n = 27) fall into three categories:(1) 22% showed the untargeted mutant allele, (2) 52% showed additional mutagenesis and (3) 26% showed the wild-type allele, which could be explained by correction. Mosaicism, defined as various editing events, was present in 17% (1), 21% (2) and 71% (3) of the embryos. The low occurrence of the synonymous variant, incorporated in the repair template, suggests that the template is not used during correction attempts. In only 29% (2/7) and 14% (1/7) of the ‘corrected embryos’, respectively long (&amp;gt;18Mb) or medium width LOH (4Mb) was observed through STR analysis. SNP analysis in closer proximity showed in 71% (5/7) of the embryos LOH, even in the absence of LOH through STR, suggesting also the occurrence of short width LOH. These results will be studied in more detail before definitive conclusions can be made. Chromosomal LOH will be studied by ddRADseq. Limitations, reasons for caution The occurrence of mosaicism and LOH might complicate the use of traditional CRISPR/Cas9 in human embryos and should be studied in detail to draw definite conclusions on its potential future use. To this end, genomic data have been produced from both individual blastomeres and whole-embryos which will be further analyzed. Wider implications of the findings Our findings demonstrate caution to use CRISPR/Cas9 to correct mutations in the germ line. They seem to contradict other reports that show predominant lack of mosaicism and presence of long width LOH. A deeper evaluation will be undertaken to define the length and type of LOH in this study. Trial registration number Not Applicable

Comparison of single nucleotide polymorphisms and short tandem repeats as markers for differentiating between donors and recipients in solid organ transplantation.

BackgroundTo analyze transplant rejection and to distinguish between donor and recipient, it is necessary to select a marker from single nucleotide polymorphism (SNP), short tandem repeat (STR), and human leukocyte antigen (HLA) testing. SNPs are bi-allelic and the polymerase chain reaction method used for SNP testing has the advantage of lower cost than sequencing methods. In this study, we aimed to distinguish donors from recipients using a combination of existing commercialized STRs and the SNPs identified.MethodsAll selected SNPs complied with the following criterion known and validated minor allele frequency (MAF) ≥43% in Korean and reported ethnicities from global populations (HapMap, 1000 Genomes, and the Korean Reference Genome project). The STR assays were performed for 16 tetranucleotide repeat loci.ResultsDNA from the 52 donor/recipient pairs were tested for informative markers. The median age of the recipients was 47 years. MAF in the 52 pairs was 1.0%–76.0%. The probability of informative genotypes (I) was 0.001–0.124. The summation of I was 0.680. In the 52 donor recipient pairs, the selected SNPs showed a 0.031 average probability of being informative. The probability of identity in our study was 0.122–0.348. SNP panel configuration distinguished 100% of 52 donors/recipient pairs.ConclusionsDonors and recipients were distinguished by STR and 22 SNPs with MAF identified from SNP databases. Seventeen SNPs were able to distinguish between donors and recipients (I value=0.039).

Authentication, characterization and contamination detection of cell lines, xenografts and organoids by barcode deep NGS sequencing.

Misidentification and contamination of biobank samples (e.g. cell lines) have plagued biomedical research. Short tandem repeat (STR) and single-nucleotide polymorphism assays are widely used to authenticate biosamples and detect contamination, but with insufficient sensitivity at 5–10% and 3–5%, respectively. Here, we describe a deep NGS-based method with significantly higher sensitivity (≤1%). It can be used to authenticate human and mouse cell lines, xenografts and organoids. It can also reliably identify and quantify contamination of human cell line samples, contaminated with only small amount of other cell samples; detect and quantify species-specific components in human–mouse mixed samples (e.g. xenografts) with 0.1% sensitivity; detect mycoplasma contamination; and infer population structure and gender of human samples. By adopting DNA barcoding technology, we are able to profile 100–200 samples in a single run at per-sample cost comparable to conventional STR assays, providing a truly high-throughput and low-cost assay for building and maintaining high-quality biobanks.

Association of rs613872 and Trinucleotide Repeat Expansion in the TCF4 Gene of German Patients With Fuchs Endothelial Corneal Dystrophy.

To investigate single nucleotide polymorphisms (SNPs) and trinucleotide repeat (TNR) expansion in the transcription factor 4 (TCF4) gene in a large cohort of German patients with Fuchs endothelial corneal dystrophy (FECD). Genomic DNA was obtained from 398 patients with FECD and from 58 non-FECD controls. Thirty-seven previously reported SNPs were evaluated by genotyping. The 398 FECD samples were analyzed for TNR expansions by short tandem repeat assays and Southern blotting. The possible associations between the TNR length and clinical parameters (age, sex, visual acuity, and central corneal thickness) were analyzed in 132 patients. The SNPs in COL8A2, TCF8, LOXHD1, and AGBL1 showed no heterogeneity in 36 cases, although SLCA411 showed 3 nonsense mutations. SNPs were detected for TCF4 (rs613872, rs2123392, rs17089887, rs1452787, and rs1348047), but only rs613872 showed a significant association with FECD (P = 9.93 × 10). Overall, 315/398 (79%) patients harbored TNR lengths >50, whereas no non-FECD controls harbored TNR lengths >50. The TCF4 SNP rs613872 genotype was TT: 39 (67%), TG: 18 (31%), and GG: 1 (2%) in non-FECD controls; TT: 39 (47%), TG: 38 (46%), and GG: 6 (7%) in FECD cases harboring TNR <50; and TT: 23 (8%), TG: 224 (79%), and GG: 38 (13%) in FECD cases harboring TNR >50 (P = 2.93 × 10). No significant association was detected between the TNR length and clinical parameters. Our large German cohort demonstrated a significant association between the risk allele G in rs613872 and FECD, irrespective of TNR expansion, although this risk allele was more frequent in FECD cases with TNR expansion than without.

Effect of Trinucleotide Repeat Expansion on the Expression of TCF4 mRNA in Fuchs' Endothelial Corneal Dystrophy.

PurposeCTG trinucleotide repeat (TNR) expansion is frequently found in transcription factor 4 (TCF4) in Fuchs' endothelial corneal dystrophy (FECD), though the effect of TNR expansion on FECD pathophysiology remains unclear. The purpose of this study was to evaluate the effect of TNR expansion on TCF4 expression in corneal endothelium of patients with FECD.MethodsPeripheral blood DNA and Descemet membrane with corneal endothelium were obtained from 203 German patients with FECD. The CTG TNR repeat length in TCF4 was determined by short tandem repeat (STR) assays and Southern blotting using genomic DNA. Genotyping of rs613872 in TCF4 was performed by PCR. TCF4 mRNA levels in corneal endothelium were evaluated by quantitative PCR using three different probes. Control corneal endothelial samples were obtained from 35 non-FECD subjects.ResultsThe STR assay and Southern blotting showed that 162 of the 203 patients with FECD (80%) harbored CTG trinucleotide repeat lengths larger than 50. Quantitative PCR using all three probes demonstrated that TCF4 mRNA is significantly upregulated in the corneal endothelium of patients with FECD, regardless of the presence of TNR expansion. However, the length of the TNR tended to show a positive correlation with TCF4 expression level. No correlation was shown between the genotype of TCF4 SNP, rs613872, and the level of TCF4 expression.ConclusionsOur findings showed that TCF4 mRNA is upregulated in the corneal endothelium of patients with FECD. Further studies on the effects of TCF4 upregulation on corneal endothelial cell function will aid in understanding the pathophysiology of FECD.

Repurposing the Cord Blood Bank for Haplobanking of HLA-Homozygous iPSCs and Their Usefulness to Multiple Populations.

Although autologous induced pluripotent stem cells (iPSCs) can potentially be useful for treating patients without immune rejection, in reality it will be extremely expensive and labor-intensive to make iPSCs to realize personalized medicine. An alternative approach is to make use of human leukocyte antigen (HLA) haplotype homozygous donors to provide HLA matched iPSC products to significant numbers of patients. To establish a haplobank of iPSCs, we repurposed the cord blood bank by screening ∼4,200 high resolution HLA typed cord blood samples, and selected those homozygous for the 10 most frequent HLA-A,-B,-DRB1 haplotypes in the Korean population. Following the generation of 10 iPSC lines, we conducted a comprehensive characterization, including morphology, expression of pluripotent markers and cell surface antigens, three-germ layer formation, vector clearance, mycoplasma/microbiological/viral contamination, endotoxin, and short tandem repeat (STR) assays. Various genomic analyses using microarray and comparative genomic hybridization (aCGH)-based single nucleotide polymorphism (SNP) and copy number variation (CNV) were also conducted. These 10 HLA-homozygous iPSC lines match 41.07% of the Korean population. Comparative analysis of HLA population data shows that they are also of use in other Asian populations, such as Japan, with some limited utility in ethnically diverse populations, such as the UK. Taken together, the generation of the 10 most frequent Korean HLA-homozygous iPSC lines serves as a useful pointer for the development of optimal methods for iPSC generation and quality control and indicates the benefits and limitations of collaborative HLA driven selection of donors for future stocking of worldwide iPSC haplobanks. Stem Cells 2018;36:1552-1566.

Blood chimerism in twins

Chimerism is a phenomenon in which an individual has cells with different genetic content from different zygotes. In dizygotic twins (DTs), chimerism is believed to occur through placental anastomoses that enable the bidirectional exchange of hematopoietic stem cells. Little is still known about chimerism frequency in twins, but several studies have shown a relation between chimerism and some conditions such as autism, Alzheimer's disease, and a group of autoimmune diseases such as Sjögren syndrome, systemic lupus erythematosus, and systemic sclerosis. In addition to chimerism of ABO blood groups being possibly mistaken for ABO subgroups, these autoimmune diseases may affect other serologic immunohematologic tests. This study aimed to determine the frequency of chimerism in DTs through ABO and D testing using the tube method, column agglutination, and short tandem repeat (STR) assays. Among the 103 subjects assessed for this study, 24 subjects (12 pairs) were excluded because STR assays showed they were monozygotic; of the remaining, 70 subjects (35 pairs) were DTs and 9 subjects came from gestations of trizygotic triplets. No ABO or D chimerism was detected in any subject through serologic assays, and STR assays did not detect any blood chimerism. Although there was no evidence of chimerism found in this study, we emphasize the importance of observing the family background of individuals with suspected ABO subgroup in complex immunohematologic studies because ABO antigen-antibody reactions are similar in both circumstances, and chimerism can be overlooked. Moreover, the use of the STR analysis method in chimerism studies can be important to help differentiate chimerism and ABO subgroups.

Using long ssDNA polynucleotides to amplify STRs loci in degraded DNA samples.

Obtaining informative short tandem repeat (STR) profiles from degraded DNA samples is a challenging task usually undermined by locus or allele dropouts and peak-high imbalances observed in capillary electrophoresis (CE) electropherograms, especially for those markers with large amplicon sizes. We hereby show that the current STR assays may be greatly improved for the detection of genetic markers in degraded DNA samples by using long single stranded DNA polynucleotides (ssDNA polynucleotides) as surrogates for PCR primers. These long primers allow a closer annealing to the repeat sequences, thereby reducing the length of the template required for the amplification in fragmented DNA samples, while at the same time rendering amplicons of larger sizes suitable for multiplex assays. We also demonstrate that the annealing of long ssDNA polynucleotides does not need to be fully complementary in the 5’ region of the primers, thus allowing for the design of practically any long primer sequence for developing new multiplex assays. Furthermore, genotyping of intact DNA samples could also benefit from utilizing long primers since their close annealing to the target STR sequences may overcome wrong profiling generated by insertions/deletions present between the STR region and the annealing site of the primers. Additionally, long ssDNA polynucleotides might be utilized in multiplex PCR assays for other types of degraded or fragmented DNA, e.g. circulating, cell-free DNA (ccfDNA).

OR33 The importance of detection of donor-chimerism and anti-donor or anti-recipeint HLA antibodies after liver-contained intestinal transplantation

Acute graft versus host disease (aGvHD) is a devastating complication of transplantation, with the highest incidence after liver-contained intestinal transplantation (LIT). Its prompt diagnosis with analysis of involved cell components is critical for treatment plan. Here we present two aGvHD cases after LIT, diagnosed with donor-chimerism by short tandem repeat assay along with detection of de novo anti-recipient or donor specific HLA antibodies (dRSA or dDSA). Case 1 : A 2-year girl presented with skin rash, and fever 2 weeks after LIT for trichohepatoenteric syndrome. On post operative day (POD) 15, donor-derived CD8 cell was detected with 60%, and peaked on POD 20 (74% CD8, 91% CD19) (Fig. 1A). Importantly, multiple dRSAs were also detected (Fig. 1B). The diagnosis of combined cellular and humoral aGvHD elicited anti-B cell therapy with bortezomib/rituximab. Subsequently, all dRSAs but DQ8 declined to Case 2: A 41-year man developed acute cellular rejection a week after LIT for metastatic carcinoid tumors. Rejection was resolved with thymoglobuline treatment, but DQ2 dDSA persisted on POD 31-100. Along with skin rash, donor-chimerism was detected on POD 62 (45% CD8, 53% CD19), and persisted for the next 40 days with absence of dRSA. Accordingly, cellular aGvHD was treated with photopheresis and steroid, with significant reduction of both DQ2 dDSA and donor-chimerism (Fig. 1C and D). After resolution of aGvHD, he developed intra-abdominal sepsis from surgical complication and died on POD 200. Simultaneous occurrence of aGvHD and circulating dDSA reflects lymphocyte reactivity from both recipient and donor. Even though both patients succumbed due to complicated courses, donor-chimerism and HLA antibody detection provided critical information of donor-derived T and B cell involvement that guided treatment plan for dRSA, dDSA, and aGvHD.

P162 Use of an automated chimerism calculator to increase speed, precision, and statistical analysis of results

Aim Chimerism testing is a valuable tool for determining the proportion of donor vs. recipient DNA in a sample. The commonly used polymerase chain reaction amplifications of short tandem repeats assay (Promega, Madison, WI) produces results for 21 loci, but only 9 loci are used in manual calculations. We sought to improve the tedious and time consuming process of manually calculating a patient’s percent chimerism by developing an automated chimerism calculator for rapid and accurate chimerism calculations based off of all 21 loci. Methods The calculator was developed using Microsoft Excel Macros and is able to report the percent donor chimerism +/− the margin of error. To determine whether precision is improved by using 21 loci, the results of a 21 loci analysis and a 9 loci analysis were compared in 111 patient samples. Additionally, each of the 21 loci was evaluated for its average deviation from the mean. Finally, 298 patient samples were analyzed by the program in order to determine if precision is equal at all chimerism percentages. Results When the same loci are analyzed, the chimerism calculator results are identical to the manual calculations, but completed in 10% of the time. The analysis of patient samples using 21 and 9 loci yielded, on average, chimerism percentages within 2.0% of each other. The average margin of error using 9 loci for analysis is 2.41% compared to 0.8% using all 21 loci. Among the 21 loci, the average deviation from the mean was found to range from 8.9% and 5.9%. Finally, we found that the average margin of error for patients between 90 and 100 percent donor chimerism is 0.19% while patients between 30 and 40 percent donor chimerism have 10.1% margin of error. Conclusions The chimerism calculator allows for routine use of all 21 STR loci with significantly reduced analysis time, 30% less margin of error, and an informative statistical analysis. In various laboratory settings, similar methods of computer assisted chimerism analysis can be easily implemented.

TGC repeat expansion in the TCF4 gene increases the risk of Fuchs' endothelial corneal dystrophy in Australian cases.

Fuchs’ endothelial corneal dystrophy (FECD) is a progressive, vision impairing disease. Common single nucleotide polymorphisms (SNPs) and a trinucleotide repeat polymorphism, thymine-guanine-cytosine (TGC), in the TCF4 gene have been associated with the risk of FECD in some populations. We previously reported association of SNPs in TCF4 with FECD risk in the Australian population. The aim of this study was to determine whether TGC repeat polymorphism in TCF4 is associated with FECD in the Australian population. In 189 unrelated Australian cases with advanced late-onset FECD and 183 matched controls, the TGC repeat polymorphism located in intron 3 of TCF4 was genotyped using a short tandem repeat (STR) assay. The repeat length was verified by direct sequencing in selected homozygous carriers. We found significant association between the expanded TGC repeat (≥ 40 repeats) in TCF4 and advanced FECD (P = 2.58 × 10−22; OR = 15.66 (95% CI: 7.79–31.49)). Genotypic analysis showed that 51% of cases (97) compared to 5% of controls (9) were heterozygous or homozygous for the expanded repeat allele. Furthermore, the repeat expansion showed stronger association than the most significantly associated SNP, rs613872, in TCF4, with the disease in the Australian cohort. This and haplotype analysis of both the polymorphisms suggest that considering both the polymorphisms together rather than either of the two alone would better predict susceptibility to FECD in the Australian population. This is the first study to report association of the TGC trinucleotide repeat expansion in TCF4 with advanced FECD in the Australian population.