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Diogo Hipólito-Fernandes,1 Maria Elisa Luís,1 Pedro Gil,1 Vitor Maduro,1 João Feijão,1 Tun Kuan Yeo,2 Oleksiy Voytsekhivskyy,3 Nuno Alves1 1Department of Ophthalmology, Centro Hospitalar Universitário De Lisboa Central, Lisbon 1169-050, Portugal; 2Department of Ophthalmology, Tan Tock Seng Hospital, National Healthcare Group Eye Institute, Singapore; 3Department of Ophthalmology, Kyiv Clinical Ophthalmology Hospital Eye Microsurgery Center, Medical City, Kyiv 03680, UkraineCorrespondence: Diogo Hipólito-FernandesDepartment of Ophthalmology, Centro Hospitalar Universitário De Lisboa Central, Alameda De Santo António Dos Capuchos, Lisbon 1169-050, PortugalTel +351 21 313 6300Email cdiogo777@gmail.comPurpose: To compare the accuracy of a newly developed intraocular lens (IOL) power formula (VRF-G) with twelve existing formulas (Barret Universal II, EVO 2.0, Haigis, Hill-RBF 2.0, Hoffer Q, Holladay 1, Kane, Næeser 2, PEARL-DGS, SRK/T, T2 and VRF).Methods: Retrospective case series including 828 patients having uncomplicated cataract surgery with the implantation of a single IOL model (SN60WF). Using optimised constants, refraction prediction error of each formula was calculated for each eye. Subgroup analysis was performed based on the axial length (short ≤ 22.0mm; medium > 22.0mm to < 26.0mm; long ≥ 26.0mm). Main outcomes included mean prediction error (ME) mean (MAE) and median absolute error (MedAE), in diopters (D), and the percentage of eyes within ± 0.25D, ± 0.50D, ± 0.75D and ± 1.00D.Results: Formulas absolute errors were statistically different among them (p< 0.001), with Kane having the lowest MAE of all formulas, followed by EVO 2.0 and VRF-G, which had the lowest MedAE. The Kane formula had the highest percentage of eyes within ± 0.25D (47.0%) and ± 1.00D (97.7%) and the VRF-G formula had the highest percentage of eyes within ± 0.50D (79.5%). For all AL subgroups, Kane, EVO 2.0 and VRF-G formulas had the most accurate performances (lowest MAE).Conclusion: New generation formulas may help us in achieving better refractive results, lowering the variance in accuracy in extreme eyes – Kane, EVO 2.0 and VRF-G formulas are promising candidates to fulfil that goal.Keywords: intraocular lens power calculation formulas, biometry, cataract, phacoemulsification, formulas accuracy

Author(s): J. W. Thomas [1, 11]; J. W. Touchman [1, 2, 11]; R. W. Blakesley [1, 2]; G. G. Bouffard [1, 2]; S. M. Beckstrom-Sternberg [1, 2]; E. H. Margulies [...]

Background/aims: To investigate the influence of anterior chamber depth (ACD) and lens thickness (LT) on 9 intraocular lens (IOL) power calculation formulas accuracy, in patients with normal axial lengths., Methods: Retrospective case series, including patients having uncomplicated cataract surgery with insertion of a single IOL model, divided into three groups according to preoperative ACD. Each group was further subdivided into three subgroups, according to the LT. Using optimised constants, refraction prediction error was calculated for Barrett Universal II, Emmetropia Verifying Optical (EVO) V.2.0, Haigis, Hill-RBF V.2.0, Hoffer Q, Holladay 1, Kane, PEARL-DGS and SRK/T formulas. Mean prediction error, mean and median absolute error (MedAE) and the percentage of eyes within ±0.25D, ±0.50D and ±1.00D were also calculated., Results: The study included 695 eyes from 695 patients. For ACD ≤3.0 mm and ≥3.5 mm, mean prediction error of SRK/T, Hoffer Q and Holladay 1 was significantly different from 0 (p<0.05). PEARL-DGS, Kane, EVO V.2.0 and Barrett Universal II were more accurate than the Hoffer Q in ACD ≤3.0 mm (p<0.05). Kane, PEARL-DGS, EVO V.2.0 and Barrett Universal II revealed the lowest variance of mean and MedAE by ACD and LT subgroup. Haigis and Hill-RBF V.2.0 were significantly influenced by LT, independently of the ACD, with a myopic shift with thin lenses and a hyperopic shift with thick lenses (p<0.05)., Conclusion: New generation formulas, particularly Kane, PEARL-DGS and EVO V.2.0, seem to be more reliable and stable even in eyes with extreme ACD-LT combinations., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Our study aimed to assess and compare the accuracy of 8 intraocular lens (IOL) power calculation formulas (Barrett Universal II, EVO 2.0, Haigis, Hoffer Q, Holladay 1, Kane and PEARL-DGS) in patients submitted to combined phacovitrectomy for vitreomacular (VM) interface disorders., Methods: Retrospective chart review study including axial-length matched patients submitted to phacoemulsification alone (Group 1) and combined phacovitrectomy (Group 2). Using optimized constants in both groups, refraction prediction error of each formula was calculated for each eye. The optimised constants from Group 1 were also applied to patients of Group 2 - Group 3. Outcome measures included the mean prediction error (ME) and its standard deviation (SD), mean (MAE) and median (MedAE) absolute errors, in diopters (D), and the percentage of eyes within ± 0.25D, ± 0.50D and ± 1.00D., Results: A total of 220 eyes were included (Group 1: 100; Group 2: 120). In Group 1, the difference in formulas absolute error was significative (p = 0.005). The Kane Formula had the lowest MAE (0.306) and MedAE (0.264). In Group 2, Kane had the overall best performance, followed by PEARL-DGS, EVO 2.0 and Barrett Universal II. The ME of all formulas in both Groups 1 and 2 were 0.000 (p = 0.934; p = 0.971, respectively). In Group 3, a statistically significant myopic shift was observed for each formula (p < 0.001)., Conclusion: Surgeons must be careful regarding IOL power selection in phacovitrectomy considering the systematic myopic shift evidenced-constant optimization may help eliminating such error. Moreover, newly introduced formulas and calculation methods may help us achieving increasingly better refractive outcomes both in cataract surgery alone and phacovitrectomy., (© 2021. The Author(s).)

Purpose: To describe the surgical approach and long-term outcomes of Descemet-stripping automated endothelial keratoplasty (DSAEK) for endothelial failure secondary to phakic intraocular lenses (pIOL)., Methods: Retrospective, interventional case series of 18 eyes of 13 patients who developed endothelial failure secondary to pIOL implant. Patients were submitted to pIOL explant and DSAEK for visual rehabilitation, with or without phacoemulsification plus posterior-chamber intraocular lens implant. The minimum follow-up time was 3 years and the main outcome measures were best-corrected visual acuity (BCVA), endothelial cell density (ECD) and percentage of endothelial cell loss (ECL), intraoperative and postoperative complications., Results: Patients' mean age was 47.44 (7.86) years with a mean follow-up time of 55.61 (14.13) months. Sixteen eyes (88.9%) had an angle-supported pIOL. The mean pIOL implant-to explant time was 8.17 (2.73) years. BCVA improved from 1.00 (0.36) logMAR preoperatively to 0.29 (0.36) logMAR at 1 year of follow-up ( p  < 0.001), remaining stable over the entire follow-up period ( p  > 0.05). At the last visit, all corneas were clear, with a mean ECD of 724.74 (325.57) cells/mm 2 . During the follow-up, two eyes (11.1%) required a re-DSAEK due to early graft failure (<3 months)., Conclusion: Our outcomes reveal that an individualized and careful approach may result in a stable visual rehabilitation in young patients with corneal decompensation following pIOL implant.

After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist 1,2 . Here we present a human genome assembly that surpasses the continuity of GRCh38 2 , along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome 3 , we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.

Anterior lenticonus is a characteristic ocular feature of Alport syndrome, leading to progressive vision deterioration. Surgical lens removal may be an option in such cases, and the role of femtosecond laser-assisted cataract surgery (FLACS) has been recently described. Herein, we report the third described case, to our knowledge, of bilateral anterior lenticonus surgically approached through FLACS. A 25-year-old man with X-linked Alport syndrome complained of bilateral progressive vision loss. Ophthalmological evaluation revealed a corrected distance visual acuity of 20/63 in both eyes and bilateral anterior lenticonus associated with anterior polar cataract. FLACS was performed, followed by IOL placement on the capsular bag, without any intraoperative complications. One month postoperatively, uncorrected distance visual acuity was 20/20 in both eyes. Considering these results and the information published so far, this technology might be a good option for these patients.

Traditionally, the use of genomic information for personalized medical decisions relies on prior discovery and validation of genotype-phenotype associations. This approach constrains care for patients presenting with undescribed problems. The National Institutes of Health (NIH) Undiagnosed Diseases Program (UDP) hypothesized that defining disease as maladaptation to an ecological niche allows delineation of a logical framework to diagnose and evaluate such patients. Herein, we present the philosophical bases, methodologies, and processes implemented by the NIH UDP. The NIH UDP incorporated use of the Human Phenotype Ontology, developed a genomic alignment strategy cognizant of parental genotypes, pursued agnostic biochemical analyses, implemented functional validation, and established virtual villages of global experts. This systematic approach provided a foundation for the diagnostic or non-diagnostic answers provided to patients and serves as a paradigm for scalable translational research.

ATP6V1H is a component of a large protein complex with vacuolar ATPase (V-ATPase) activity. We identified two generations of individuals in which short stature and osteoporosis co-segregated with a mutation in ATP6V1H. Since V-ATPases are highly conserved between human and zebrafish, we generated loss-of-function mutants in atp6v1h in zebrafish through CRISPR/Cas9-mediated gene knockout. Homozygous mutant atp6v1h zebrafish exhibited a severe reduction in the number of mature calcified bone cells and a dramatic increase in the expression of mmp9 and mmp13. Heterozygous adults showed curved vertebra that lack calcified centrum structure and reduced bone mass and density. Treatment of mutant embryos with small molecule inhibitors of MMP9 and MMP13 significantly restored bone mass in the atp6v1h mutants. These studies have uncovered a new, ATP6V1H-mediated pathway that regulates bone formation, and defines a new mechanism of disease that leads to bone loss. We propose that MMP9/MMP13 could be therapeutic targets for patients with this rare genetic disease., Competing Interests: The authors have declared that no competing interests exist.

Failure to thrive arises as a complication of a heterogeneous group of disorders. We describe two female siblings with spastic paraplegia and global developmental delay but also, atypically for the HSPs, poor weight gain classified as failure to thrive. After extensive clinical and biochemical investigations failed to identify the etiology, we used exome sequencing to identify biallelic UNC80 mutations (NM&#95;032504.1:c.[3983-3&#95;3994delinsA];[2431C>T]. The paternally inherited NM&#95;032504.1:c.3983-3&#95;3994delinsA is predicted to encode p.Ser1328Argfs*19 and the maternally inherited NM&#95;032504.1:c.2431C>T is predicted to encode p.Arg811*. No UNC80 mRNA was detectable in patient cultured skin fibroblasts, suggesting UNC80 loss of function by nonsense mediated mRNA decay. Further supporting the UNC80 mutations as causative of these siblings' disorder, biallelic mutations in UNC80 have recently been described among individuals with an overlapping phenotype. This report expands the disease spectrum associated with UNC80 mutations. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The National Institutes of Health Undiagnosed Diseases Program (NIH UDP) applies translational research systematically to diagnose patients with undiagnosed diseases. The challenge is to implement an information system enabling scalable translational research. The authors hypothesized that similar complex problems are resolvable through process management and the distributed cognition of communities. The team, therefore, built the NIH UDP integrated collaboration system (UDPICS) to form virtual collaborative multidisciplinary research networks or communities. UDPICS supports these communities through integrated process management, ontology-based phenotyping, biospecimen management, cloud-based genomic analysis, and an electronic laboratory notebook. UDPICS provided a mechanism for efficient, transparent, and scalable translational research and thereby addressed many of the complex and diverse research and logistical problems of the NIH UDP. Full definition of the strengths and deficiencies of UDPICS will require formal qualitative and quantitative usability and process improvement measurement.

Background: Mutations of TCF4, which encodes a basic helix-loop-helix transcription factor, cause Pitt-Hopkins syndrome (PTHS) via multiple genetic mechanisms. TCF4 is a complex locus expressing multiple transcripts by alternative splicing and use of multiple promoters. To address the relationship between mutation of these transcripts and phenotype, we report a three-generation family segregating mild intellectual disability with a chromosomal translocation disrupting TCF4., Results: Using whole genome sequencing, we detected a complex unbalanced karyotype disrupting TCF4 (46,XY,del(14)(q23.3q23.3)del(18)(q21.2q21.2)del(18)(q21.2q21.2)inv(18)(q21.2q21.2)t(14;18)(q23.3;q21.2)(14pter®14q23.3::18q21.2®18q21.2::18q21.1®18qter;18pter®18q21.2::14q23.3®14qter). Subsequent transcriptome sequencing, qRT-PCR and nCounter analyses revealed that cultured skin fibroblasts and peripheral blood had normal expression of genes along chromosomes 14 or 18 and no marked changes in expression of genes other than TCF4. Affected individuals had 12-33 fold higher mRNA levels of TCF4 than did unaffected controls or individuals with PTHS. Although the derivative chromosome generated a PLEKHG3-TCF4 fusion transcript, the increased levels of TCF4 mRNA arose from transcript variants originating distal to the translocation breakpoint, not from the fusion transcript., Conclusions: Although validation in additional patients is required, our findings suggest that the dysmorphic features and severe intellectual disability characteristic of PTHS are partially rescued by overexpression of those short TCF4 transcripts encoding a nuclear localization signal, a transcription activation domain, and the basic helix-loop-helix domain.

Background: Whole-exome sequencing (WES) is rapidly evolving into a tool of choice for rapid, and inexpensive identification of molecular genetic lesions within targeted regions of the human genome. While biases in WES coverage of nucleotides in targeted regions are recognized, it is not well understood how repetition of WES improves the interpretation of sequencing results in a clinical diagnostic setting., Method: To address this, we compared independently generated exome-capture of six individuals from three-generations sequenced in triplicate. This generated between 48x-86x mean target depth of high-quality mapped bases (>Q20) for each technical replicate library. Cumulatively, we achieved 179 - 208x average target coverage for each individual in the pedigree. Using this experimental design, we evaluated stochastics in WES interpretation, genotyping sensitivity, and accuracy to detect de novo variants., Results: In this study, we show that repetition of WES improved the interpretation of the capture target regions after aggregating the data (93.5 - 93.9 %). Compared to 81.2 - 89.6 % (50.2-55.4 Mb of 61.7 M) coverage of targeted bases at ≥20x in the individual technical replicates, the aggregated data covered 93.5 - 93.9 % of targeted bases (57.7 - 58.0 of 61.7 M) at ≥20x threshold, suggesting a 4.3 - 12.7 % improvement in coverage. Each individual's aggregate dataset recovered 3.4 - 6.4 million bases within variable targeted regions. We uncovered technical variability (2-5 %) inherent to WES technique. We also show improved interpretation in assessing clinically important regions that lack interpretation under current conditions, affecting 12-16 of the 56 genes recommended for secondary analysis by American College of Medical Genetics (ACMG). We demonstrate that comparing technical replicate WES datasets and their derived aggregate data can effectively address overall WES genotyping discrepancies., Conclusion: We describe a method to evaluate the reproducibility and stochastics in exome library preparation, and delineate the advantages of aggregating the data derived from technical replicates. The implications of this study are directly applicable to improved experimental design and provide an opportunity to rapidly, efficiently, and accurately arrive at reliable candidate nucleotide variants.

Acute lymphoblastic leukemia is a malignant hematopoietic neoplasia, which is rare in adults. Although ocular fundus alterations may be commonly observed in the course of the disease, such alterations are rarely the presenting signs of the disease. Here we describe the case of a patient with painless and progressive loss of visual acuity (right eye, 2/10; left eye, 3/10) developing over two weeks, accompanied by fever and cervical lymphadenopathy. Fundus examination showed bilateral macular serous detachment, which was confirmed by optical coherence tomography. Fluorescein angiography revealed hyperfluorescent pinpoints in the posterior poles. The limits of the macular detachment were revealed in the late phase of the angiogram. The results of blood count analysis triggered a thorough, systematic patient examination. The diagnosis of acute lymphoblastic leukemia B (CD10+) was established, and intensive systemic chemotherapy was immediately initiated. One year after the diagnosis, the patient remains in complete remission without any ophthalmologic alterations.

Ablepharon macrostomia syndrome (AMS) and Barber-Say syndrome (BSS) are rare congenital ectodermal dysplasias characterized by similar clinical features. To establish the genetic basis of AMS and BSS, we performed extensive clinical phenotyping, whole exome and candidate gene sequencing, and functional validations. We identified a recurrent de novo mutation in TWIST2 in seven independent AMS-affected families, as well as another recurrent de novo mutation affecting the same amino acid in ten independent BSS-affected families. Moreover, a genotype-phenotype correlation was observed, because the two syndromes differed based solely upon the nature of the substituting amino acid: a lysine at TWIST2 residue 75 resulted in AMS, whereas a glutamine or alanine yielded BSS. TWIST2 encodes a basic helix-loop-helix transcription factor that regulates the development of mesenchymal tissues. All identified mutations fell in the basic domain of TWIST2 and altered the DNA-binding pattern of Flag-TWIST2 in HeLa cells. Comparison of wild-type and mutant TWIST2 expressed in zebrafish identified abnormal developmental phenotypes and widespread transcriptome changes. Our results suggest that autosomal-dominant TWIST2 mutations cause AMS or BSS by inducing protean effects on the transcription factor's DNA binding., (Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Intellectual disability (ID) is a heterogeneous condition arising from a variety of environmental and genetic factors. Among these causes are defects in transcriptional regulators. Herein, we report on two brothers in a nonconsanguineous family with novel compound heterozygous, disease-segregating mutations (NM&#95;015979.3: [3656A > G];[4006C > T], NP&#95;057063.2: [H1219R];[R1336X]) in MED23. This gene encodes a subunit of the Mediator complex that modulates the expression of RNA polymerase II-dependent genes. These brothers, who had profound ID, spasticity, congenital heart disease, brain abnormalities, and atypical electroencephalography, represent the first case of MED23-associated ID in a non-consanguineous family. They also expand upon the clinical features previously reported for mutations in this gene., (© 2015 Wiley Periodicals, Inc.)

Background: Snyder-Robinson Syndrome (SRS) is an X-linked intellectual disability disorder also characterized by osteoporosis, scoliosis, and dysmorphic facial features. It is caused by mutations in SMS, a ubiquitously expressed gene encoding the polyamine biosynthetic enzyme spermine synthase. We hypothesized that the tissue specificity of SRS arises from differential sensitivity to spermidine toxicity or spermine deficiency., Methods: We performed detailed clinical, endocrine, histopathologic, and morphometric studies on two affected brothers with a spermine synthase loss of function mutation (NM&#95;004595.4:c.443A > G, p.Gln148Arg). We also measured spermine and spermidine levels in cultured human bone marrow stromal cells (hBMSCs) and fibroblasts using the Biochrom 30 polyamine protocol and assessed the osteogenic potential of hBMSCs., Results: In addition to the known tissue-specific features of SRS, the propositi manifested retinal pigmentary changes, recurrent episodes of hyper- and hypoglycemia, nephrocalcinosis, renal cysts, and frequent respiratory infections. Bone histopathology and morphometry identified a profound depletion of osteoblasts and osteoclasts, absence of a trabecular meshwork, a low bone volume and a thin cortex. Comparison of cultured fibroblasts from affected and unaffected individuals showed relatively small changes in polyamine content, whereas comparison of cultured osteoblasts identified marked differences in spermidine and spermine content. Osteogenic differentiation of the SRS-derived hBMSCs identified a severe deficiency of calcium phosphate mineralization., Conclusions: Our findings support the hypothesis that cell specific alterations in polyamine metabolism contribute to the tissue specificity of SRS features, and that the low bone density arises from a failure of mineralization.

Background: Myelin protein zero (MPZ) is a critical structural component of myelin in the peripheral nervous system. The MPZ gene is regulated, in part, by the transcription factors SOX10 and EGR2. Mutations in MPZ, SOX10, and EGR2 have been implicated in demyelinating peripheral neuropathies, suggesting that components of this transcriptional network are candidates for harboring disease-causing mutations (or otherwise functional variants) that affect MPZ expression., Methodology: We utilized a combination of multi-species sequence comparisons, transcription factor-binding site predictions, targeted human DNA re-sequencing, and in vitro and in vivo enhancer assays to study human non-coding MPZ variants., Principal Findings: Our efforts revealed a variant within the first intron of MPZ that resides within a previously described SOX10 binding site is associated with decreased enhancer activity, and alters binding of nuclear proteins. Additionally, the genomic segment harboring this variant directs tissue-relevant reporter gene expression in zebrafish., Conclusions: This is the first reported MPZ variant within a cis-acting transcriptional regulatory element. While we were unable to implicate this variant in disease onset, our data suggests that similar non-coding sequences should be screened for mutations in patients with neurological disease. Furthermore, our multi-faceted approach for examining the functional significance of non-coding variants can be readily generalized to study other loci important for myelin structure and function.

The comparative mapping and sequencing of vertebrate genomes is now a key priority for the Human Genome Project. In addition to finishing the human genome sequence and generating a 'working draft' of the mouse genome sequence, significant attention is rapidly turning to the analysis of other model organisms, such as the laboratory rat (Rattus norvegicus). As a complement to genome-wide mapping and sequencing efforts, it is often important to generate detailed maps and sequence data for specific regions of interest. Using an adaptation of our previously described approach for constructing mouse comparative and physical maps, we have established a general strategy for targeted mapping of the rat genome. Specifically, we constructed a framework comparative map of human Chromosome (Chr) 7 and the orthologous regions of the rat genome, as well as two large (>1-Mb) P1-derived artificial chromosome (PAC)-based physical maps. Generation of these physical maps involved the use of mouse-derived probes that cross-hybridized with rat PAC clones. The first PAC map encompasses the cystic fibrosis transmembrane conductance regulator gene (Cftr), while the second map allows a three-species comparison of a genomic region containing intra- and inter-chromosomal evolutionary rearrangements. The studies reported here further demonstrate that cross-species hybridization between related animals, such as rat and mouse, can be readily used for the targeted construction of clone-based physical maps, thereby accelerating the analysis of biologically interesting regions of vertebrate genomes.

The human genome is by far the largest genome to be sequenced, and its size and complexity present many challenges for sequence assembly. The International Human Genome Sequencing Consortium constructed a map of the whole genome to enable the selection of clones for sequencing and for the accurate assembly of the genome sequence. Here we report the construction of the whole-genome bacterial artificial chromosome (BAC) map and its integration with previous landmark maps and information from mapping efforts focused on specific chromosomal regions. We also describe the integration of sequence data with the map.

The identification of the cystic fibrosis transmembrane conductance regulator gene (CFTR) in 1989 represents a landmark accomplishment in human genetics. Since that time, there have been numerous advances in elucidating the function of the encoded protein and the physiological basis of cystic fibrosis. However, numerous areas of cystic fibrosis biology require additional investigation, some of which would be facilitated by information about the long-range sequence context of the CFTR gene. For example, the latter might provide clues about the sequence elements responsible for the temporal and spatial regulation of CFTR expression. We thus sought to establish the sequence of the chromosomal segments encompassing the human CFTR and mouse Cftr genes, with the hope of identifying conserved regions of biologic interest by sequence comparison. Bacterial clone-based physical maps of the relevant human and mouse genomic regions were constructed, and minimally overlapping sets of clones were selected and sequenced, eventually yielding approximately 1.6 Mb and approximately 358 kb of contiguous human and mouse sequence, respectively. These efforts have produced the complete sequence of the approximately 189-kb and approximately 152-kb segments containing the human CFTR and mouse Cftr genes, respectively, as well as significant amounts of flanking DNA. Analyses of the resulting data provide insights about the organization of the CFTR/Cftr genes and potential sequence elements regulating their expression. Furthermore, the generated sequence reveals the precise architecture of genes residing near CFTR/Cftr, including one known gene (WNT2/Wnt2) and two previously unknown genes that immediately flank CFTR/Cftr.