Genomic epidemiology reveals the dominance of Hennepin County in the transmission of SARS-CoV-2 in Minnesota from 2020 to 2022.

IMPORTANCE: We analyzed over 22,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes of patient samples tested at Mayo Clinic Laboratories during a 2-year period in the COVID-19 pandemic, which included Alpha, Delta, and Omicron variants of concern to examine the roles and relationships of Minnesota virus transmission. We found that Hennepin County, the most populous county, drove the transmission of SARS-CoV-2 viruses in the state after including the formation of earlier clades including 20A, 20C, and 20G, as well as variants of concern Alpha and Delta. We also found that Hennepin County was the source for most of the county-to-county introductions after an initial predicted introduction with the virus in early 2020 from an international source, while other counties acted as transmission "sinks." In addition, major policies, such as the end of the lockdown period in 2020 or the end of all restrictions in 2021, did not appear to have an impact on virus diversity across individual counties.

εγ-Thalassemia, a New Hemoglobinopathy Category.

BACKGROUND: Large ß-globin gene cluster deletions (hereditary persistence of fetal hemoglobin [Hb] or ß-, δß-, γδß-, and ϵγδß-thalassemia), are associated with widely disparate phenotypes, including variable degrees of microcytic anemia and Hb F levels. When present, increased Hb A2 is used as a surrogate marker for ß-thalassemia. Notably, ϵγδß-thalassemias lack the essential regulatory locus control region (LCR) and cause severe transient perinatal anemia but normal newborn screen (NBS) results and Hb A2 levels. Herein, we report a novel deletion of the ϵ, Aγ, Gγ, and ψß loci with intact LCR, δ-, and ß-regions in 2 women and newborn twins. METHODS: Capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), gap-polymerase chain reaction (gap-PCR), and long-read sequencing (LRS) were performed. RESULTS: NBS showed an Hb A > Hb F pattern for both twins. At 20 months, Hb A2 was increased similarly to that in the mother and an unrelated woman. Unexplained microcytosis was absent and the twins lacked severe neonatal anemia. MLPA, LRS, and gap-PCR confirmed a 32 599 base pair deletion of ϵ (HBE1) through ψß (HBBP1) loci. CONCLUSIONS: This deletion represents a hemoglobinopathy category with a distinct phenotype that has not been previously described, an ϵγ-thalassemia. Both the NBS Hb A > F pattern and the subsequent increased Hb A2 without microcytosis are unusual. A similar deletion should be considered when this pattern is encountered and appropriate test methods selected for detection. Knowledge of the clinical impact of this new category will improve genetic counselling, with distinction from the severe transient anemia associated with ϵγδß-thalassemia.

Genomic epidemiology reveals the dominance of Hennepin County in transmission of SARS-CoV-2 in Minnesota from 2020-2022.

SARS-CoV-2 has had an unprecedented impact on human health and highlights the need for genomic epidemiology studies to increase our understanding of virus evolution and spread, and to inform policy decisions. We sequenced viral genomes from over 22,000 patient samples tested at Mayo Clinic Laboratories between 2020-2022 and use Bayesian phylodynamics to describe county and regional spread in Minnesota. The earliest introduction into Minnesota was to Hennepin County from a domestic source around January 22, 2020; six weeks before the first confirmed case in the state. This led to the virus spreading to Northern Minnesota, and eventually, the rest of the state. International introductions were most abundant in Hennepin (home to the Minneapolis/St. Paul International (MSP) airport) totaling 45 (out of 107) over the two-year period. Southern Minnesota counties were most common for domestic introductions with 19 (out of 64), potentially driven by bordering states such as Iowa and Wisconsin as well as Illinois which is nearby. Hennepin also was, by far, the most dominant source of in-state transmissions to other Minnesota locations (n=772) over the two-year period. We also analyzed the diversity of the location source of SARS-CoV-2 viruses in each county and noted the timing of state-wide policies as well as trends in clinical cases. Neither the number of clinical cases or major policy decisions, such as the end of the lockdown period in 2020 or the end of all restrictions in 2021, appeared to have impact on virus diversity across each individual county.

Comparison of TCF4 repeat expansion length in corneal endothelium and leukocytes of patients with Fuchs endothelial corneal dystrophy.

Expansion of CTG trinucleotide repeats (TNR) in the transcription factor 4 (TCF4) gene is highly associated with Fuchs Endothelial Corneal Dystrophy (FECD). Due to limitations in the availability of DNA from diseased corneal endothelium, sizing of CTG repeats in FECD patients has typically been determined using DNA samples isolated from peripheral blood leukocytes. However, it is non-feasible to extract enough DNA from surgically isolated FECD corneal endothelial tissue to determine repeat length based on current technology. To circumvent this issue, total RNA was isolated from FECD corneal endothelium and sequenced using long-read sequencing. Southern blotting of DNA samples isolated from primary cultures of corneal endothelium from these same affected individuals was also assessed. Both long read sequencing and Southern blot analysis showed significantly longer CTG TNR expansion (>1000 repeats) in the corneal endothelium from FECD patients than those characterized in leukocytes from the same individuals (<90 repeats). Our findings suggest that the TCF4 CTG repeat expansions in the FECD corneal endothelium are much longer than those found in leukocytes.

Improved Characterization of Complex ß-Globin Gene Cluster Structural Variants Using Long-Read Sequencing.

Complex insertion-deletion (indel) events in the globin genes manifest in widely variable clinical phenotypes. Many are incompletely characterized because of a historic lack of efficient methods. A more complete assessment enables improved prediction of clinical impact, which guides emerging therapeutic choices. Current methods have limited capacity for breakpoint assignment and accurate assessment of mutation extent, especially in cases containing duplications or multiple deletions and insertions. Technology, such as long-read sequencing, holds promise for significant impact in the characterization of indel events because of read lengths that span large regions, resulting in improved resolution. Four known complex ß-globin gene cluster indel types were assessed using single-molecule, real-time sequencing technology and showed high correlation with previous reports, including the Caribbean locus control deletion (g.5,305,478_5,310,336del), a large ß-gene duplication containing the Hb S mutation (g.4,640,335_5,290,171dup with g.5,248,232T>A, c.20A>T; variant allele fraction, 64%), and two nested variants (double deletions with intervening inversion): the Indian Gγ(Aγδß)0-thalassemia (g.5,246,804-5,254,275del, g.5,254,276_5,269,600inv, and g.5,269,601_5,270,442del) and the Turkish/Macedonian (δß)0 thalassemia (g.5,235,064_5,236,652del, g.5,236,653_5,244,280inv, and g.5,244,281_5,255,766del). Our data confirm long-read sequencing as an efficient and accurate method to identify these clinically significant complex events. Limitations include high-complexity sample preparation requirements, which hinder routine use in clinical laboratories. Continued improvements in sample and data workflow processes are needed to accommodate volumes in a tertiary clinical laboratory.

Characterization of a dual media system for culturing primary normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells.

Primary cultures of human corneal endothelial cells (HCECs) are an important model system for studying the pathophysiology of corneal endothelium. The purpose of this study was to identify and validate an optimal primary culture model of normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells by comparing cell morphology and marker expression under different media conditions to in vivo donor tissues. Primary and immortalized HCECs, isolated from normal and FECD donors, were cultured in proliferation media (Joyce, M4, Bartakova) alone or sequentially with maturation media (F99, Stabilization 1, M5). CD56, CD73 and CD166 expressions were quantified in confluent and matured cell lines by flow cytometry. HCECs that were allowed to proliferate in Joyce's medium followed by maturation in low-mitogen containing media yielded cells with similar morphology to corneal endothelial tissues. Elevated expression of CD56 and CD166 and low expression of CD73 correlated with regular, hexagonal-like HCEC morphology. CD56:CD73 > 2.5 was most consistent with normal HCEC morphology and mimicked corneal endothelial tissue. Immortalization of normal HCECs by hTERT transduction showed morphology and CD56:CD73 ratios similar to parental cell lines. HCECs established from FECD donors showed reduced CD56:CD73 ratios compared to normal HCECs which coincided with reduced uniformity and regularity of cell monolayers. Overall, a dual media system with Joyce's medium for proliferation and a low-mitogen media for maturation, provided normal cultures with regular, hexagonal-like cell morphologies consistent with corneal endothelial cells in vivo. CD56:CD73 expression ratio >2.5 was predictive of in vivo-like cellular morphology.

Long-read targeted sequencing uncovers clinicopathological associations for C9orf72-linked diseases.

To examine the length of a hexanucleotide expansion in C9orf72, which represents the most frequent genetic cause of frontotemporal lobar degeneration and motor neuron disease, we employed a targeted amplification-free long-read sequencing technology: No-Amp sequencing. In our cross-sectional study, we assessed cerebellar tissue from 28 well-characterized C9orf72 expansion carriers. We obtained 3507 on-target circular consensus sequencing reads, of which 814 bridged the C9orf72 repeat expansion (23%). Importantly, we observed a significant correlation between expansion sizes obtained using No-Amp sequencing and Southern blotting (P = 5.0 × 10-4). Interestingly, we also detected a significant survival advantage for individuals with smaller expansions (P = 0.004). Additionally, we uncovered that smaller expansions were significantly associated with higher levels of C9orf72 transcripts containing intron 1b (P = 0.003), poly(GP) proteins (P = 1.3 × 10- 5), and poly(GA) proteins (P = 0.005). Thorough examination of the composition of the expansion revealed that its GC content was extremely high (median: 100%) and that it was mainly composed of GGGGCC repeats (median: 96%), suggesting that expanded C9orf72 repeats are quite pure. Taken together, our findings demonstrate that No-Amp sequencing is a powerful tool that enables the discovery of relevant clinicopathological associations, highlighting the important role played by the cerebellar size of the expanded repeat in C9orf72-linked diseases.

Relationship of Body Mass Index With Fuchs Endothelial Corneal Dystrophy Severity and TCF4 CTG18.1 Trinucleotide Repeat Expansion.

PURPOSE: To investigate the association of body mass index (BMI) with Fuchs endothelial corneal dystrophy (FECD) severity and TCF4 CTG18.1 expansion. METHODS: A total of 343 patients with FECD were enrolled from the Mayo Clinic. FECD severity was graded by slit-lamp biomicroscopy. BMI values were obtained from the electronic medical records. DNA extracted from leukocytes was analyzed for CTG18.1 expansion length, with ≥40 repeats considered expanded. Wilcoxon signed-rank tests were used to compare FECD grade and CTG18.1 expansion length in patients by BMI (<25, ≥25 to <30, and ≥30 kg/m2). FECD grade was regressed on age, sex, BMI, and CTG18.1 expansion and, separately, BMI on CTG18.1 expansion. Models were investigated for effect modification by age and sex with an interaction term of P < 0.05 considered statistically significant. RESULTS: When examining the association between BMI and FECD, there was a significant interaction between BMI and sex (P for interaction = 0.004). When controlling for age and CTG18.1 expansion, a positive association was observed between BMI and FECD grade in women, but not in men. In addition, BMI was not associated with CTG18.1 expansion when controlling for age and sex. CONCLUSIONS: BMI was positively associated with FECD severity among women but not men. There was no significant association between BMI and CTG18.1 expansion. These findings suggest that increased BMI is potentially a modifiable risk factor for FECD disease progression among women.

Disease Expression and Familial Transmission of Fuchs Endothelial Corneal Dystrophy With and Without CTG18.1 Expansion.

Purpose: To characterize inheritance, penetrance, and trinucleotide repeat expansion stability in Fuchs endothelial corneal dystrophy (FECD). Methods: One thousand unrelated and related subjects with and without FECD were prospectively recruited. CTG18.1 repeat length (CTG18.1L) was determined via short tandem repeat assay and Southern blotting of leukocyte DNA. Multivariable logistic regression and generalized estimating equation models were employed. Results: There were 546 unrelated FECD cases (67.6% female; 70 ± 10 years) and 235 controls (63.8% female; 73 ± 8 years; all ≥ 50 years). CTG18.1 expansion (CTG18.1exp+) was observed in 424 (77.7%) cases and 18 (7.7%) controls (P = 2.48 × 10-44). CTG18.1 expansion was associated with FECD severity (P = 5.62 × 10-7). The family arm of the study included 331 members from 112 FECD-affected families; 87 families were CTG18.1exp+. Autosomal dominant inheritance with variable expression of FECD was observed, regardless of expansion status. FECD penetrance of CTG18.1 expansion increased with age, ranging from 44.4% in the youngest (19-46 years) to 86.2% in the oldest (64-91 years) age quartiles. Among 62 parent-offspring transmissions of CTG18.1exp+, 48 (77.4%) had a change in CTG18.1L ≤ 10 repeats, and eight (12.9%) were ≥50 repeats, including five large expansions (∼1000-2000 repeats) that contracted. Among 44 offspring who did not inherit the CTG18.1exp+ allele, eight (18.2%) exhibited FECD. Conclusions: CTG18.1 expansion was highly associated with FECD but demonstrated incomplete penetrance. CTG18.1L instability occurred in a minority of parent-offspring transmissions, with large expansions exhibiting contraction. The observation of FECD without CTG18.1 expansion among family members in CTG18.1exp+ families highlights the complexity of the relationship between the FECD phenotype and CTG18.1 expansion.

Identification of Possible Risk Variants of Familial Strabismus Using Exome Sequencing Analysis.

PURPOSE: To investigate candidate genes associated with familial strabismus and propose a theory of their interaction in familial strabismus associated with early neurodevelopment. METHODS: Eighteen families, including 53 patients diagnosed with strabismus and 34 unaffected family members, were analyzed. All patients with strabismus and available unaffected family members were evaluated using whole exome sequencing. The primary outcome was to identify rare occurring variants among affected individuals and investigate the evidence of their genetic heterogeneity. These results were compared with exome sequencing analysis to build a comprehensive genetic profile of the study families. RESULTS: We observed 60 variants from 58 genes in 53 patients diagnosed with strabismus. We prioritized the most credible risk variants, which showed clear segregation in family members affected by strabismus. As a result, we found risk variants in four genes (FAT3, KCNH2, CELSR1, and TTYH1) in five families, suggesting their role in development of familial strabismus. In other families, there were several rare genetic variants in affected cases, but we did not find clear segregation pattern across family members. CONCLUSION: Genomic sequencing holds great promise in elucidating the genetic causes of strabismus; further research with larger cohorts or other related approaches are warranted.

TCF4-mediated Fuchs endothelial corneal dystrophy: Insights into a common trinucleotide repeat-associated disease.

Fuchs endothelial corneal dystrophy (FECD) is a common cause for heritable visual loss in the elderly. Since the first description of an association between FECD and common polymorphisms situated within the transcription factor 4 (TCF4) gene, genetic and molecular studies have implicated an intronic CTG trinucleotide repeat (CTG18.1) expansion as a causal variant in the majority of FECD patients. To date, several non-mutually exclusive mechanisms have been proposed that drive and/or exacerbate the onset of disease. These mechanisms include (i) TCF4 dysregulation; (ii) toxic gain-of-function from TCF4 repeat-containing RNA; (iii) toxic gain-of-function from repeat-associated non-AUG dependent (RAN) translation; and (iv) somatic instability of CTG18.1. However, the relative contribution of these proposed mechanisms in disease pathogenesis is currently unknown. In this review, we summarise research implicating the repeat expansion in disease pathogenesis, define the phenotype-genotype correlations between FECD and CTG18.1 expansion, and provide an update on research tools that are available to study FECD as a trinucleotide repeat expansion disease. Furthermore, ongoing international research efforts to develop novel CTG18.1 expansion-mediated FECD therapeutics are highlighted and we provide a forward-thinking perspective on key unanswered questions that remain in the field.

Transplant chimerism in porcine structural vascularized bone allotransplants.

BACKGROUND: Bone allotransplant viability can be maintained long-term by implanting arteriovenous (AV) bundles and creating an autogenous neoangiogenic circulation. Only short-term immunosuppression is required. This study investigates the origin of viable osteocytes observed in areas of active bone remodeling in orthotopically transplanted tibiae in a Yucatan mini-pig model. METHODS: Segmental tibial defects created in female Yucatan minipigs (N = 14) were reconstructed with a matched vascularized composite allotransplant from a male donor. The circulation was microsurgically restored, with simultaneous autogenous AV-bundle implantation in group 1 (N = 7). A ligated AV-bundle was implanted as a no-angiogenesis control in group 2 (N = 7). After 20-weeks, repopulation of the allotransplant was assessed by real-time qPCR measurement of relative copy numbers of a Y chromosome-specific gene (SRY) and an autosomal housekeeping gene, ribosomal protein L4 (RPL4). A lower SRY/RPL4 ratio demonstrates replacement of male allogeneic cells with female, autogenous cells in the sample. Genomic DNA was extracted from cross-sections of the allotransplant, liver and spleen. Additionally, areas of new bone formation within the allotransplant were sampled by laser capture microdissection. A comparison was made between groups as well as male control samples. RNA was extracted from bone as well, as a measure of metabolically active cells. RESULTS: Laser-captured areas of new bone formation in animals with both normal and ligated AV-bundles were found to have significantly lower relative copy numbers of SRY (p = 0.03) than control specimens from male bone, indicating replacement by female (autogenous) bone-forming cells. Analysis of an entire segment of the allotransplant from Group 1 was similarly reduced (p = 0.04), unlike that from Group 2. RNA expression of SRY was observed in both groups. No chimerism could be found in non-bone tissues (liver and spleen). CONCLUSION: We observed a significant level of transplant chimerism in areas of new bone formation sampled by laser capture microdissection. The migration of autogenous cells including osteocytes was seen in both groups. Survival of some allogeneic (male) cells was also demonstrable. No microchimerism was found in liver and spleen.

Correction to: Recommendations for performance optimizations when using GATK3.8 and GATK4.

Following publication of the original article [1], the author explained that Table 2 is displayed incorrectly. The correct Table 2 is given below. The original article has been corrected.

Recommendations for performance optimizations when using GATK3.8 and GATK4.

BACKGROUND: Use of the Genome Analysis Toolkit (GATK) continues to be the standard practice in genomic variant calling in both research and the clinic. Recently the toolkit has been rapidly evolving. Significant computational performance improvements have been introduced in GATK3.8 through collaboration with Intel in 2017. The first release of GATK4 in early 2018 revealed rewrites in the code base, as the stepping stone toward a Spark implementation. As the software continues to be a moving target for optimal deployment in highly productive environments, we present a detailed analysis of these improvements, to help the community stay abreast with changes in performance. RESULTS: We re-evaluated multiple options, such as threading, parallel garbage collection, I/O options and data-level parallelization. Additionally, we considered the trade-offs of using GATK3.8 and GATK4. We found optimized parameter values that reduce the time of executing the best practices variant calling procedure by 29.3% for GATK3.8 and 16.9% for GATK4. Further speedups can be accomplished by splitting data for parallel analysis, resulting in run time of only a few hours on whole human genome sequenced to the depth of 20X, for both versions of GATK. Nonetheless, GATK4 is already much more cost-effective than GATK3.8. Thanks to significant rewrites of the algorithms, the same analysis can be run largely in a single-threaded fashion, allowing users to process multiple samples on the same CPU. CONCLUSIONS: In time-sensitive situations, when a patient has a critical or rapidly developing condition, it is useful to minimize the time to process a single sample. In such cases we recommend using GATK3.8 by splitting the sample into chunks and computing across multiple nodes. The resultant walltime will be nnn.4 hours at the cost of $41.60 on 4 c5.18xlarge instances of Amazon Cloud. For cost-effectiveness of routine analyses or for large population studies, it is useful to maximize the number of samples processed per unit time. Thus we recommend GATK4, running multiple samples on one node. The total walltime will be ∼34.1 hours on 40 samples, with 1.18 samples processed per hour at the cost of $2.60 per sample on c5.18xlarge instance of Amazon Cloud.

Sentieon DNASeq Variant Calling Workflow Demonstrates Strong Computational Performance and Accuracy.

As reliable, efficient genome sequencing becomes ubiquitous, the need for similarly reliable and efficient variant calling becomes increasingly important. The Genome Analysis Toolkit (GATK), maintained by the Broad Institute, is currently the widely accepted standard for variant calling software. However, alternative solutions may provide faster variant calling without sacrificing accuracy. One such alternative is Sentieon DNASeq, a toolkit analogous to GATK but built on a highly optimized backend. We conducted an independent evaluation of the DNASeq single-sample variant calling pipeline in comparison to that of GATK. Our results support the near-identical accuracy of the two software packages, showcase optimal scalability and great speed from Sentieon, and describe computational performance considerations for the deployment of DNASeq.

Gene Expression and Missplicing in the Corneal Endothelium of Patients With a TCF4 Trinucleotide Repeat Expansion Without Fuchs' Endothelial Corneal Dystrophy.

Purpose: CTG trinucleotide repeat (TNR) expansion in an intron of the TCF4 gene is the most common genetic variant associated with Fuchs' endothelial corneal dystrophy (FECD). Although several mechanisms have been implicated in the disease process, their exact pathophysiologic importance is unclear. To understand events leading from TCF4 TNR expansion to disease phenotype, we characterized splicing, gene expression, and exon sequence changes in a rare cohort of patients with TNR expansions but no phenotypic FECD (RE+/FECD-). Methods: Corneal endothelium and blood were collected from patients undergoing endothelial keratoplasty for non-FECD corneal edema. Total RNA was isolated from corneal endothelial tissue (n = 3) and used for RNASeq. Gene splicing and expression was assessed by Mixture of Isoforms (MISO) and MAP-RSeq software. Genomic DNA was isolated from blood mononuclear cells and used for whole genome exome sequencing. Base calling was performed using Illumina's Real-Time Analysis. Results: Three genes (MBNL1, KIF13A, AKAP13) that were previously identified as misspliced in patients with a CTG TNR expansion and FECD disease (RE+/FECD+) were found normally spliced in RE+/FECD- samples. Gene expression differences in pathways associated with the innate immune response, cell signaling (e.g., TGFß, WNT), and cell senescence markers were also identified between RE+/FECD- and RE+/FECD+ groups. No consistent genetic variants were identified in RE+/FECD- patient exomes. Conclusions: Identification of novel splicing patterns and differential gene expression in RE+/FECD- samples provides new insights and more relevant gene targets that may be protective against FECD disease in vulnerable patients with TCF4 CTG TNR expansions.

Amplification-free long-read sequencing of TCF4 expanded trinucleotide repeats in Fuchs Endothelial Corneal Dystrophy.

Amplification of a CAG trinucleotide motif (CTG18.1) within the TCF4 gene has been strongly associated with Fuchs Endothelial Corneal Dystrophy (FECD). Nevertheless, a small minority of clinically unaffected elderly patients who have expanded CTG18.1 sequences have been identified. To test the hypothesis that the CAG expansions in these patients are protected from FECD because they have interruptions within the CAG repeats, we utilized a combination of an amplification-free, long-read sequencing method and a new target-enrichment sequence analysis tool developed by Pacific Biosciences to interrogate the sequence structure of expanded repeats. The sequencing was successful in identifying a previously described interruption within an unexpanded allele and provided sequence data on expanded alleles greater than 2000 bases in length. The data revealed considerable heterogeneity in the size distribution of expanded repeats within each patient. Detailed analysis of the long sequence reads did not reveal any instances of interruptions to the expanded CAG repeats, but did reveal novel variants within the AGG repeats that flank the CAG repeats in two of the five samples from clinically unaffected patients with expansions. This first examination of the sequence structure of CAG repeats in CTG18.1 suggests that factors other than interruptions to the repeat structure account for the absence of disease in some elderly patients with repeat expansions in the TCF4 gene.

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

PURPOSE: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Purpose: CTG 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. Methods: Peripheral 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. Results: The 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. Conclusions: Our 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.

CDKN2A Germline Rare Coding Variants and Risk of Pancreatic Cancer in Minority Populations.

Background: Pathogenic germline mutations in the CDKN2A tumor suppressor gene are rare and associated with highly penetrant familial melanoma and pancreatic cancer in non-Hispanic whites (NHW). To date, the prevalence and impact of CDKN2A rare coding variants (RCV) in racial minority groups remain poorly characterized. We examined the role of CDKN2A RCVs on the risk of pancreatic cancer among minority subjects.Methods: We sequenced CDKN2A in 220 African American (AA) pancreatic cancer cases, 900 noncancer AA controls, and 183 Nigerian controls. RCV frequencies were determined for each group and compared with that of 1,537 NHW patients with pancreatic cancer. Odds ratios (OR) and 95% confidence intervals (CI) were calculated for both a case-case comparison of RCV frequencies in AAs versus NHWs, and case-control comparison between AA cases versus noncancer AA controls plus Nigerian controls. Smaller sets of Hispanic and Native American cases and controls also were sequenced.Results: One novel missense RCV and one novel frameshift RCV were found among AA patients: 400G>A and 258_278del. RCV carrier status was associated with increased risk of pancreatic cancer among AA cases (11/220; OR, 3.3; 95% CI, 1.5-7.1; P = 0.004) compared with AA and Nigerian controls (17/1,083). Further, AA cases had higher frequency of RCVs: 5.0% (OR, 13.4; 95% CI, 4.9-36.7; P < 0.001) compared with NHW cases (0.4%).Conclusions: CDKN2A RCVs are more common in AA than in NHW patients with pancreatic cancer and associated with moderately increased pancreatic cancer risk among AAs.Impact: RCVs in CDKN2A are frequent in AAs and are associated with risk for pancreatic cancer. Cancer Epidemiol Biomarkers Prev; 27(11); 1364-70. ©2018 AACR.