Database resources of the National Center for Biotechnology Information.

The National Center for Biotechnology Information (NCBI) provides a large suite of online resources for biological information and data, including the GenBank® nucleic acid sequence database and the PubMed database of citations and abstracts published in life science journals. The Entrez system provides search and retrieval operations for most of these data from 38 distinct databases. The E-utilities serve as the programming interface for the Entrez system. Augmenting many of the web applications are custom implementations of the BLAST program optimized to search specialized data sets. New resources released in the past year include PubMed Labs and a new sequence database search. Resources that were updated in the past year include PubMed, PMC, Bookshelf, genome data viewer, Assembly, prokaryotic genomes, Genome, BioProject, dbSNP, dbVar, BLAST databases, igBLAST, iCn3D and PubChem. All of these resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov.

DbVar and DGVa: public archives for genomic structural variation.

Much has changed in the last two years at DGVa (http://www.ebi.ac.uk/dgva) and dbVar (http://www.ncbi.nlm.nih.gov/dbvar). We are now processing direct submissions rather than only curating data from the literature and our joint study catalog includes data from over 100 studies in 11 organisms. Studies from human dominate with data from control and case populations, tumor samples as well as three large curated studies derived from multiple sources. During the processing of these data, we have made improvements to our data model, submission process and data representation. Additionally, we have made significant improvements in providing access to these data via web and FTP interfaces.

CFTR transcription defects in pancreatic sufficient cystic fibrosis patients with only one mutation in the coding region of CFTR.

BACKGROUND: Patients with cystic fibrosis (CF) manifest a multisystem disease due to deleterious mutations in each gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). However, the role of dysfunctional CFTR is uncertain in individuals with mild forms of CF (ie, pancreatic sufficiency) and mutation in only one CFTR gene. METHODS: Eleven pancreatic sufficient (PS) CF patients with only one CFTR mutation identified after mutation screening (three patients), mutation scanning (four patients) or DNA sequencing (four patients) were studied. Bi-directional sequencing of the coding region of CFTR was performed in patients who had mutation screening or scanning. If a second CFTR mutation was not identified, CFTR mRNA transcripts from nasal epithelial cells were analysed to determine if any PS-CF patients harboured a second CFTR mutation that altered RNA expression. RESULTS: Sequencing of the coding regions of CFTR identified a second deleterious mutation in five of the seven patients who previously had mutation screening or mutation scanning. Five of the remaining six patients with only one deleterious mutation identified in the coding region of one CFTR gene had a pathologic reduction in the amount of RNA transcribed from their other CFTR gene (8.4-16% of wild type). CONCLUSIONS: These results show that sequencing of the coding region of CFTR followed by analysis of CFTR transcription could be a useful diagnostic approach to confirm that patients with mild forms of CF harbour deleterious alterations in both CFTR genes.

The third international hackathon for applying insights into large-scale genomic composition to use cases in a wide range of organisms.

In October 2021, 59 scientists from 14 countries and 13 U.S. states collaborated virtually in the Third Annual Baylor College of Medicine & DNANexus Structural Variation hackathon. The goal of the hackathon was to advance research on structural variants (SVs) by prototyping and iterating on open-source software. This led to nine hackathon projects focused on diverse genomics research interests, including various SV discovery and genotyping methods, SV sequence reconstruction, and clinically relevant structural variation, including SARS-CoV-2 variants. Repositories for the projects that participated in the hackathon are available at https://github.com/collaborativebioinformatics.

Acquired pseudoxanthoma elasticum presenting after liver transplantation.

BACKGROUND: Pseudoxanthoma elasticum (PXE) is thought to be a metabolic disorder resulting from mutations in the gene encoding the cellular transporter, ABCC6, which is primarily expressed in liver and kidney. We encountered 3 patients who developed clinical and histopathological evidence of PXE after liver transplantation, suggesting that PXE could have been acquired from the transplanted organ. OBJECTIVE: We sought to delineate the clinical features and screen each patient and samples of donor liver for mutations in the ABCC6 gene. METHODS: Each patient underwent full clinical examination, skin biopsy, and ophthalmologic examination, and whole genome sequencing using standard techniques. Fixed samples of donor liver tissue were available for mutation analysis in two patients and of donor kidney tissue in one. RESULTS: All 3 patients had unequivocal clinical and histopathologic evidence of PXE. No patient (or family member available for screening) had evidence of mutations in ABCC6. Neither liver specimen nor the single available kidney specimen showed evidence of mutations in ABCC6. LIMITATIONS: Liver tissue was not available from one patient and DNA was of poor quality in another, resulting in limited screening. Genetic testing does not detect ABCC6 mutations in 10% of patients with confirmed PXE. CONCLUSION: Although we were unable to demonstrate ABCC6 mutations in limited screening of fixed donor livers, the absence of any PXE mutations in the affected patients, the timing of onset of PXE, and the known acquisition of other metabolic disorders and coagulopathies from donor livers suggest that PXE was likely acquired via liver transplantation.

The incidence of duplicate genetic testing.

PURPOSE: Duplicate genetic testing (DGT) should give the same results as the initial genetic test. Therefore, DGT is indicated only in the rare instances where the initial results require confirmation. The objective of this study was to determine the incidence of DGT by reviewing TPMT, HFE, and CYP450 2D6 polymorphism testing performed in our institution's laboratories in 2006. A secondary objective was to determine the savings in charges that resulted from a system in place to limit HFE DGT. METHODS: A retrospective records review at an academic medical center. RESULTS: The percentage of patients having the same genetic test more than once in 2006 was 3.3% (253/7710) for TPMT, 0.3% for HFE (24/7851), and 0.9% (4/433) for CYP450 2D6 testing. Retail laboratory charges for the DGT identified in 2006 were $76,728. To estimate the incidence of DGT over a longer period of time than 2006, an all-time records review was performed on a subset of internal patients and found the all-time incidence of DGT for TPMT, HFE, and CYP450 2D6 testing to be 6.9%, 1.9%, and 0.9%, respectively. No case of DGT with an appropriate indication for duplicate testing was found. A system in place to decrease HFE DGT is estimated to have saved $77,479 in charges for 2006 (95% CI, $35,512-184,015). CONCLUSIONS: Indicated DGT is rare and decreasing DGT could result in significant savings. Institutions should consider implementing a systems-based process to limit DGT.

Medical errors related to inappropriate genetic testing in liver transplant patients.

Genetic testing of genes that encode proteins expressed by liver hepatocytes (clotting factors, alpha 1-antitrypsin, cytochrome P450 enzymes) is common in clinical practice. These tests use DNA extracted from peripheral blood lymphocytes (PBL) and are based on the assumption that PBL DNA can be used as a surrogate for hepatocyte DNA. However, in individuals who have undergone liver transplantation, hepatocyte DNA is that of the donor while PBL DNA remains that of the recipient. It follows that in liver transplant patients, genetic testing of the recipient's PBL DNA does not provide accurate results for proteins expressed by donor hepatocytes. Therefore, genetic testing of clotting factors, alpha 1-1-antitrypsin, cytochrome P450 enzymes, and other proteins expressed by hepatocytes is unreliable and inappropriate in liver transplant patients (inappropriate genetic testing). A review of the records of 215 consecutive liver transplant patients at our institution identified: one medical error and one near-miss medical error related to inappropriate genetic testing, 14 cases of inappropriate genetic testing, and 21 unnecessary duplicate genetic testing requests. We recommend laboratories performing genetic testing create systems to prevent inappropriate and duplicate genetic testing and that physicians be cognizant of the appropriate indications for genetic testing in liver transplant patients.

Quantitative methods for the analysis of CFTR transcripts/splicing variants.

In cystic fibrosis (CF), transcript analysis and quantification are important for diagnosis, prognosis and also as surrogate markers for some therapies including gene therapy. Classical RNA-based methods require significant expression levels in target samples for appropriate analysis, thus PCR-based methods are evolving towards reliable quantification. Various protocols for the quantitative analysis of CFTR transcripts (including those resulting from splicing variants) are described and discussed here.

Functional analysis of synonymous substitutions predicted to affect splicing of the CFTR gene.

BACKGROUND: Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Over 1800 CFTR mutations have been reported, and about 12% of mutations are believed to impair pre-mRNA splicing. Given that several synthetic, non-splice-junction synonymous substitutions have been reported to alter splicing in CFTR, we predicted that naturally occurring synonymous substitutions may be erroneously classified as functionally neutral. METHODS: Computational tools were used to predict the effect of synonymous substitutions on CFTR pre-mRNA splicing. The functional consequences of selected substitutions were evaluated using a minigene splicing assay. RESULTS: Two synonymous mutations were shown to have a dramatic effect on CFTR pre-mRNA splicing, and consequently could alter protein integrity and phenotypic outcome. CONCLUSIONS: Traditional methods of mutation analysis overlook splicing defects that occur at internal positions in coding exons, especially synonymous substitutions. We show that bioinformatics tools and minigene splicing assays are a potent combination to prioritize and identify mutations that cause aberrant CFTR pre-mRNA splicing.

A variable dinucleotide repeat in the CFTR gene contributes to phenotype diversity by forming RNA secondary structures that alter splicing.

Dinucleotide repeats are ubiquitous features of eukaryotic genomes that are not generally considered to have functional roles in gene expression. However, the highly variable nature of dinucleotide repeats makes them particularly interesting candidates for modifiers of RNA splicing when they are found near splicing signals. An example of a variable dinucleotide repeat that affects splicing is a TG repeat located in the splice acceptor of exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Higher repeat numbers result in reduced exon 9 splicing efficiency and, in some instances, the reduction in full-length transcript is sufficient to cause male infertility due to congenital bilateral absence of the vas deferens or nonclassic cystic fibrosis. Using a CFTR minigene system, we studied TG tract variation and observed the same correlation between dinucleotide repeat number and exon 9 splicing efficiency seen in vivo. Replacement of the TG dinucleotide tract in the minigene with random sequence abolished splicing of exon 9. Replacements of the TG tract with sequences that can self-base-pair suggested that the formation of an RNA secondary structure was associated with efficient splicing. However, splicing efficiency was inversely correlated with the predicted thermodynamic stability of such structures, demonstrating that intermediate stability was optimal. Finally, substitution with TA repeats of differing length confirmed that stability of the RNA secondary structure, not sequence content, correlated with splicing efficiency. Taken together, these data indicate that dinucleotide repeats can form secondary structures that have variable effects on RNA splicing efficiency and clinical phenotype.

Atypical 5' splice sites cause CFTR exon 9 to be vulnerable to skipping.

The molecular basis of the skipping of constitutive exons in many messenger RNAs is not fully understood. A well-studied example is exon 9 of the human cystic fibrosis transmembrane conductance regulator gene (CFTR), in which an abbreviated polypyrimidine tract between the branch point A and the 3' splice site is associated with increased exon skipping and disease. However, many exons, both in CFTR and in other genes and have short polypyrimidine tracts in their 3' splice sites, yet they are not skipped. Inspection of the 5' splice sites immediately up- and downstream of exon 9 revealed deviations from consensus sequence, so we hypothesized that this exon may be inherently vulnerable to skipping. To test this idea, we constructed a CFTR minigene and replicated exon 9 skipping associated with the length of the polypyrimidine tract upstream of exon 9. We then mutated the flanking 5' splice sites and determined the effect on exon skipping. Conversion of the upstream 5' splice site to consensus by replacing a pyrimidine at position +3 with a purine resulted in increased exon skipping. In contrast, conversion of the downstream 5' splice site to consensus by insertion of an adenine at position +4 resulted in a substantial reduction in exon 9 skipping, regardless of whether the upstream 5' splice site was consensus or not. These results suggested that the native downstream 5' splice site plays an important role in CFTR exon 9 skipping, a hypothesis that was supported by data from sheep and mouse genomes. Although CFTR exon 9 in sheep is preceded by a long polypyrimidine tract (Y(14)), it skips exon 9 in vivo and has a nonconsensus downstream 5' splice site identical to that in humans. On the other hand, CFTR exon 9 in mice is preceded by a short polypyrimidine tract (Y(5)) but is not skipped in vivo. Its downstream 5' splice site differs from that in humans by a 2-nt insertion, which, when introduced into the human CFTR minigene, abolished exon 9 skipping. Taken together, these observations place renewed emphasis on deviations at 5' splice sites in nucleotides other than the invariant GT, particularly when such changes are found in conjunction with other altered splicing sequences, such as a shortened polypyrimidine tract. Thus, careful inspection of entire 5' splice sites may identify constitutive exons that are vulnerable to skipping.

Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign.

An abbreviated tract of five thymidines (5T) in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is found in approximately 10% of individuals in the general population. When found in trans with a severe CFTR mutation, 5T can result in male infertility, nonclassic cystic fibrosis, or a normal phenotype. To test whether the number of TG repeats adjacent to 5T influences disease penetrance, we determined TG repeat number in 98 patients with male infertility due to congenital absence of the vas deferens, 9 patients with nonclassic CF, and 27 unaffected individuals (fertile men). Each of the individuals in this study had a severe CFTR mutation on one CFTR gene and 5T on the other. Of the unaffected individuals, 78% (21 of 27) had 5T adjacent to 11 TG repeats, compared with 9% (10 of 107) of affected individuals. Conversely, 91% (97 of 107) of affected individuals had 12 or 13 TG repeats, versus only 22% (6 of 27) of unaffected individuals (P<.00001). Those individuals with 5T adjacent to either 12 or 13 TG repeats were substantially more likely to exhibit an abnormal phenotype than those with 5T adjacent to 11 TG repeats (odds ratio 34.0, 95% CI 11.1-103.7, P<.00001). Thus, determination of TG repeat number will allow for more accurate prediction of benign versus pathogenic 5T alleles.