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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., DeJesus-Hernandez et al. employ an innovative long-read sequencing method to examine the length of the expanded C9orf72 repeat that represents the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. Smaller expansion size confers a survival benefit for patients.

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 (TCF4 CTG repeat expansions in the FECD corneal endothelium are much longer than those found in leukocytes.

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. \ud\udMethods: 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. \ud\udResults: 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. \ud\udConclusions: 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.

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 (

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.

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.

We report a female infant identified by newborn screening for severe combined immunodeficiencies (NBS SCID) with T cell lymphopenia (TCL). The patient had persistently elevated alpha-fetoprotein (AFP) with IgA deficiency, and elevated IgM. Gene sequencing for a SCID panel was uninformative. We sought to determine the cause of the immunodeficiency in this infant. We performed whole-exome sequencing (WES) on the patient and parents to identify a genetic diagnosis. Based on the WES result, we developed a novel flow cytometric panel for rapid assessment of DNA repair defects using blood samples. We also performed whole transcriptome sequencing (WTS) on fibroblast RNA from the patient and father for abnormal transcript analysis. WES revealed a pathogenic paternally inherited indel in ATM. We used the flow panel to assess several proteins in the DNA repair pathway in lymphocyte subsets. The patient had absent phosphorylation of ATM, resulting in absent or aberrant phosphorylation of downstream proteins, including γH2AX. However, ataxia-telangiectasia (AT) is an autosomal recessive condition, and the abnormal functional data did not correspond with a single ATM variant. WTS revealed in-frame reciprocal fusion transcripts involving ATM and SLC35F2 indicating a chromosome 11 inversion within 11q22.3, of maternal origin. Inversion breakpoints were identified within ATM intron 16 and SLC35F2 intron 7. We identified a novel ATM-breaking chromosome 11 inversion in trans with a pathogenic indel (compound heterozygote) resulting in non-functional ATM protein, consistent with a diagnosis of AT. Utilization of several molecular and functional assays allowed successful resolution of this case.

Purpose To identify RNA missplicing events in human corneal endothelial tissue isolated from Fuchs' endothelial corneal dystrophy (FECD). Methods Total RNA was isolated and sequenced from corneal endothelial tissue obtained during keratoplasty from 12 patients with FECD and 4 patients undergoing keratoplasty or enucleation for other indications. The length of the trinucleotide repeat (TNR) CTG in the transcription factor 4 (TCF4) gene was determined using leukocyte-derived DNA analyzed by a combination of Southern blotting and Genescan analysis. Commercial statistical software was used to quantify expression of alternatively spliced genes. Validation of selected alternative splicing events was performed by using RT-PCR. Gene sets identified were analyzed for overrepresentation using Web-based analysis system. Results Corneal endothelial tissue from FECD patients containing a CTG TNR expansion sequence in the TCF4 gene revealed widespread changes in mRNA splicing, including a novel splicing event involving FGFR2. Differential splicing of NUMA1, PPFIBP1, MBNL1, and MBNL2 transcripts were identified in all FECD samples containing a TNR expansion. The differentially spliced genes were enriched for products that localize to the cell cortex and bind cytoskeletal and cell adhesion proteins. Conclusions Corneal endothelium from FECD patients harbors a unique signature of mis-splicing events due to CTG TNR expansion in the TCF4 gene, consistent with the hypothesis that RNA toxicity contributes to the pathogenesis of FECD. Changes to the endothelial barrier function, a known event in the development of FECD, was identified as a key biological process influenced by the missplicing events.

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 (STR) assays and Southern blotting. The possible associations between TNR length and clinical parameters (age, sex, visual acuity, and central corneal thickness) were analyzed in 132 patients. RESULT: 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(−12)). Overall, 315/398 (79%) patients harbored TNR lengths >50, while no non-FECD control 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 (P=2.93×10(−25)). No significant association was detected between clinical parameters and TNR length. 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.

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.

Introduction: Globin gene sequencing has long been impeded by repetitive regions, homology and structural variation. Currently, long-read sequencing technology has been limited by high cost and error rate. These challenges can be mitigated by sample multiplexing of targeted capture sequencing and the use of consensus reads, respectively. This ongoing study analyzes the utility of long range sequencing technology in identifying historically problematic beta globin cluster mutations. Materials and methods: Long range sequencing was performed on complex beta globin gene cluster cases utilizing the Sequel platform (Pacific Biosciences, Menlo Park, California, United States). Sample input was 1 microgram of DNA extracted from whole blood. Circular Consensus Sequence (CCS) FASTQ sequences files were mapped to reference genome (GRCh37/hg19) using NGMLR, followed by annotation of variants using a custom bioinformatics workflow. Comparison to multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing, array CGH, short-range NGS (300 bp), hemoglobin protein, and clinical data was performed. Results: CCS read length varied from 1 to 10 kb, average 3.5. Analysis of the beta globin cluster region showed impressive superiority and comprehensiveness to Sanger, MLPA, array CGH and short range sequencing technology: 1) single nucleotide variants (SNVs) were identified to a sensitivity and specificity of 99.5%; 2) large structural variants (SVs) such as large deletions, duplications, insertions, crossovers and fusions spanning many kilobases were characterized in the heterozygous, homozygous and compound heterozygous state to a precise genomic coordinate (table 1); 3) phasing SNVs identified Hb S haplotypes (Central African region (CAR), Benin, Senegal, Arab-Indian and Cameroon). Conclusion: 1) Long range sequencing holds significant promise in genotyping hemoglobin disorders. This method improves efficiency with potential for a single test offering with increased resolution, particularly in large deletion analysis. Complex cases greatly benefited from improved range and resolution. 2) The decreased requirement of blood volume for extensive testing is of particular interest for the pediatric population. 3) Genotyping of Hb S patients detected by positive newborn screening will help predict need for prophylaxis or therapy by assessing variant zygosity, phasing and haplotypes. In conclusion, we believe that long range sequencing has immense potential to contribute extensively to thalassemia and hemoglobinopathy diagnostics and is far superior to the currently available technologies for beta globin cluster analysis. Disclosures Jen: Celgene: Employment.

Purpose Hereditary hyperferritinemia cataract syndrome (HHCS), an autosomal-dominant disorder characterized by hyperferritinemia and bilateral cataracts, is caused by mutations in the iron-responsive element of the ferritin light chain ( FTL ) gene. The purpose of this study is to describe the genotypic and phenotypic manifestations of HHCS observed in 2 large sets of unrelated American families. Methods Forty-five patients were recruited from 2 unrelated families. Each underwent ophthalmological and general physical evaluation as well as laboratory testing of serum ferritin, iron, transferrin saturation, and total iron binding capacity. Serum DNA was evaluated for mutations by DNA amplification and sequencing of the FTL gene. Results Numerous cortical and nuclear white opacities in a stellate pattern occurred in 22 affected individuals and were the only clinical manifestation of HHCS. Of the 22, 16 (73%) demonstrated >1.00 D of astigmatism. Genetic analysis revealed mutation G32A in Pedigree 1 and mutation G32T in Pedigree 2, both heterozygous and located in the iron-responsive element of the ferritin light chain mRNA. Serum ferritin levels of affected subjects ranged from 555 to 2,453 μg/L (normal range, 24–336 μg/L male, 11–307 μg/L female), with greater ferritin levels and more severe cataracts associated with mutation G32A. Conclusions Most clinical and genetic findings from these families are consistent with previous reports of HHCS. Astigmatism, previously not associated with HHCS, was present in the majority. Ferritin levels and age of cataract surgery varied among subjects with both FTL gene mutations, suggesting that phenotypic variability is modulated by other genetic or environmental factors.

Fuchs Endothelial Corneal Dystrophy (FECD) is a late onset, autosomal dominant eye disease that can lead to loss of vision. Expansion of a CTG trinucleotide repeat in the third intron of the transcription factor 4 (TCF4) gene is highly associated with FECD. However, only about 75% of FECD patients in the northern European population possess an expansion of this repeat. The remaining FECD cases appear to be associated with variants in other genes. To better understand the pathophysiology of this disease, we compared gene expression profiles of corneal endothelium from FECD patients with an expanded trinucleotide repeat (RE+) to those that do not have a repeat expansion (RE-). Comparative analysis of these two cohorts showed widespread RNA mis-splicing in RE+, but not in RE- samples. Quantitatively, we identified 39 genes in which expression was significantly different between RE+ and RE- samples. Examination of the mutation profiles in the RE- samples did not find any mutations in genes previously associated with FECD, but did reveal one sample with a rare variant of laminin subunit gamma 1 (LAMC1) and three samples with rare variants in the gene coding for the mitochondrial protein peripheral-type benzodiazepine receptor-associated protein 1 (TSPOAP1).

Purpose RNA toxicity from CTG trinucleotide repeat (TNR) expansion within noncoding DNA of the transcription factor 4 (TCF4) and DM1 protein kinase (DMPK) genes has been described in Fuchs' endothelial corneal dystrophy (FECD) and myotonic dystrophy, type 1 (DM1), respectively. We prospectively evaluated DM1 patients and their families for phenotypic FECD and report the analysis of CTG expansion in the TCF4 gene and DMPK expression in corneal endothelium. Methods FECD grade was evaluated by slit lamp biomicroscopy in 26 participants from 14 families with DM1. CTG TNR length in TCF4 and DMPK was determined by a combination of Gene Scan and Southern blotting of peripheral blood leukocyte DNA. Results FECD grade was 2 or higher in 5 (36%) of 14 probands, significantly greater than the general population (5%) (P < 0.001). FECD segregated with DM1; six of eight members of the largest family had both FECD and DM1, while the other two family members had neither disease. All DNA samples from 24 subjects, including four FECD-affected probands, were bi-allelic for nonexpanded TNR length in TCF4 (

Mechanisms underlying successful composite tissue transplantation must include an analysis of transplant chimerism, which is little studied, particularly in calcified tissue. We have developed a new method enabling determination of lineage of selected cells in our model of vascularized bone allotransplantation. Vascularized femoral allotransplantation was performed from female Dark Agouti (DA) donor rats to male Piebald Virol Glaxo (PVG) recipients, representing a major histocompatibility mismatch. Four groups differed in use of immunosuppression (+/-2 weeks Tacrolimus) and surgical revascularization, by implantation of either a patent or a ligated saphenous arteriovenous (AV) bundle. Results were assessed at 18 weeks. Bone blood flow was measured by the hydrogen washout technique and transverse specimens were prepared for histology. Real-time PCR was performed on DNA from laser capture microdissected cortical bone regions to determine the extent of chimerism. To do so, we analyzed the relative expression ratio of the sex-determining region Y (Sry) gene, specific only for recipient male rat DNA, to the cyclophilin housekeeper gene. Substantial transplant chimerism was seen in cortical bone of all groups (range 77-97%). Rats without immunosuppression and with a patent AV bundle revealed significantly higher chimerism than those with immunosuppression and a ligated AV bundle, which maintained transplant cell viability. We describe a new method to study the extent of chimerism in rat vascularized bone allotransplants, including a sex-mismatched transplantation model, laser capture microdissection of selected bone regions, and calculation of the relative expression ratio.

Background: Expansion of intronic (CTG·CAG)n repeats in TCF4 is found in most Fuchs endothelial corneal dystrophy (FECD) patients. Results: RNA foci co-localizing with the splicing factor MBNL1 are found in FECD cells, and changes in mRNA splicing occur. Conclusion: Trinucleotide repeat expansion in FECD is associated with RNA focus formation and missplicing. Significance: RNA toxicity occurs in a disease affecting millions of patients., Fuchs endothelial corneal dystrophy (FECD) is an inherited degenerative disease that affects the internal endothelial cell monolayer of the cornea and can result in corneal edema and vision loss in severe cases. FECD affects ∼5% of middle-aged Caucasians in the United States and accounts for >14,000 corneal transplantations annually. Among the several genes and loci associated with FECD, the strongest association is with an intronic (CTG·CAG)n trinucleotide repeat expansion in the TCF4 gene, which is found in the majority of affected patients. Corneal endothelial cells from FECD patients harbor a poly(CUG)n RNA that can be visualized as RNA foci containing this condensed RNA and associated proteins. Similar to myotonic dystrophy type 1, the poly(CUG)n RNA co-localizes with and sequesters the mRNA-splicing factor MBNL1, leading to missplicing of essential MBNL1-regulated mRNAs. Such foci and missplicing are not observed in similar cells from FECD patients who lack the repeat expansion. RNA-Seq splicing data from the corneal endothelia of FECD patients and controls reveal hundreds of differential alternative splicing events. These include events previously characterized in the context of myotonic dystrophy type 1 and epithelial-to-mesenchymal transition, as well as splicing changes in genes related to proposed mechanisms of FECD pathogenesis. We report the first instance of RNA toxicity and missplicing in a common non-neurological/neuromuscular disease associated with a repeat expansion. The FECD patient population with this (CTG·CAG)n trinucleotide repeat expansion exceeds that of the combined number of patients in all other microsatellite expansion disorders.

The single nucleotide variant (SNV), rs613872, in the transcription factor 4 (TCF4) gene was previously found to be strongly associated (P = 6 × 10(-26)) with Fuchs' endothelial corneal dystrophy (FECD). Subsequently, an intronic expansion of the repeating trinucleotides, TGC, was found to be even more predictive of disease. We performed comprehensive sequencing of the TCF4 gene region in order to identify the best marker for FECD within TCF4 and to identify other novel variants that may be associated with FECD.Leukocyte DNA was isolated from 68 subjects with FECD and 16 unaffected individuals. A custom capture panel was used to isolate the region surrounding the two previously validated markers of FECD. Sequencing of the TCF4 coding region, introns and flanking sequence, spanning 465 kb was performed at1000× average coverage using the Illumina HiSequation 2000.TGC expansion (50 repeats) was present in 46 (68%) FECD-affected subjects and one (6%) normal subject. A total of 1866 variants, including 1540 SNVs, were identified. Only two previously reported SNVs resided in the TCF4 coding region, neither of which segregated with disease. No variant, including TGC expansion, correlated perfectly with disease status. Trinucleotide repeat expansion was a better predictor of disease than any other variant.Complete sequencing of the TCF4 genomic region revealed no single causative variant for FECD. The intronic trinucleotide repeat expansion within TCF4 continues to be more strongly associated with FECD than any other genetic variant.

Fuchs endothelial corneal dystrophy (FECD) is a common, familial disease of the corneal endothelium and is the leading indication for corneal transplantation. Variation in the transcription factor 4 (TCF4) gene has been identified as a major contributor to the disease. We tested for an association between an intronic TGC trinucleotide repeat in TCF4 and FECD by determining repeat length in 66 affected participants with severe FECD and 63 participants with normal corneas in a 3-stage discovery/replication/validation study. PCR primers flanking the TGC repeat were used to amplify leukocyte-derived genomic DNA. Repeat length was determined by direct sequencing, short tandem repeat (STR) assay and Southern blotting. Genomic Southern blots were used to evaluate samples for which only a single allele was identified by STR analysis. Compiling data for 3 arms of the study, a TGC repeat length >50 was present in 79% of FECD cases and in 3% of normal controls cases (p50 TGC repeats, 13 (20%) had 50 repeats, 60 (95%) had 50 TGC repeats identifying FECD in this patient cohort was 79% and 96%, respectively Expanded TGC repeat was more specific for FECD cases than the previously identified, highly associated, single nucleotide polymorphism, rs613872 (specificity = 79%). The TGC trinucleotide repeat expansion in TCF4 is strongly associated with FECD, and a repeat length >50 is highly specific for the disease This association suggests that trinucleotide expansion may play a pathogenic role in the majority of FECD cases and is a predictor of disease risk.

Mechanisms underlying successful composite tissue transplantation must include an analysis of transplant chimerism, which is little studied, particularly in calcified tissue. We have developed a new method enabling determination of lineage of selected cells in our model of vascularized bone allotransplantation. Vascularized femoral allotransplantation was performed from female Dark Agouti (DA) donor rats to male Piebald Virol Glaxo (PVG) recipients, representing a major histocompatibility mismatch. Four groups differed in use of immunosuppression (+/-2 weeks Tacrolimus) and surgical revascularization, by implantation of either a patent or a ligated saphenous arteriovenous (AV) bundle. Results were assessed at 18 weeks. Bone blood flow was measured by the hydrogen washout technique and transverse specimens were prepared for histology. Real-time PCR was performed on DNA from laser capture microdissected cortical bone regions to determine the extent of chimerism. To do so, we analyzed the relative expression ratio of the sex-determining region Y (Sry) gene, specific only for recipient male rat DNA, to the cyclophilin housekeeper gene. Substantial transplant chimerism was seen in cortical bone of all groups (range 77-97%). Rats without immunosuppression and with a patent AV bundle revealed significantly higher chimerism than those with immunosuppression and a ligated AV bundle, which maintained transplant cell viability. We describe a new method to study the extent of chimerism in rat vascularized bone allotransplants, including a sex-mismatched transplantation model, laser capture microdissection of selected bone regions, and calculation of the relative expression ratio.

PURPOSE The purpose of this study was to evaluate the association between the intronic expansion of a trinucleotide repeat (TNR) in the TCF4 gene and Fuchs' endothelial corneal dystrophy (FECD) in a Japanese population. METHODS Forty-seven Japanese FECD patients and 96 age-matched controls were recruited. FECD patients and controls were examined by slit-lamp and noncontact specular microscopy. The repeat length was determined by direct sequencing and short tandem repeat assay of PCR-amplified DNA and Southern blotting of unamplified DNA. RESULTS A TNR expansion, defined as >50 CTG repeats in the TCF4 gene was identified in 12 of 47 FECD cases (26%) and 0 of 96 controls (0%; P < 0.001). Sensitivity and specificity in this study were 26% and 100%, respectively. The clinical characteristics of FECD patients with TNR expansion were not distinct from those without TNR expansion. CONCLUSIONS These findings show for the first time in a Japanese population the association of the TNR expansion in TCF4 with FECD. In contrast to Caucasian cohorts in whom the TNR expansion is present in most patients with FECD, a CTG expansion is present in a minority of Japanese subjects, indicating other genetic variants as common causes of phenotypically identical disease in this population.