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1.  Exon skipping through the creation of a putative exonic splicing silencer as a consequence of the cystic fibrosis mutation R553X 
Journal of Medical Genetics  2007;44(5):341-346.
Nonsense mutations that occur more than 50 bases upstream of terminal spliced junctions are generally thought to lead to degradation of the corresponding transcripts by the process of nonsense‐mediated mRNA decay. It has also been proposed that some nonsense mutations may affect splicing by the process of nonsense‐associated altered splicing (NAS), or by the disruption of a splicing regulatory element. In this study, the effect of the R553X mutation on the splicing of exon 11 of the cystic fibrosis transmembrane conductance regulator gene was investigated. Evidence that R553X causes exon 11 to skip through the creation of a putative exonic splicing silencer (ESS) was provided. The putative ESS appears to be active when located immediately upstream of a 5′ splice site. These findings argue against the possibility that R553X‐associated exon 11 skipping is caused by NAS. The study further suggests that aminoglycoside antibiotic treatment would not be effective for patients with the R553X mutation, owing to the skipping of exon 11, and further emphasises the need for detailed mechanistic characterisation of the consequences of nonsense disease mutations.
PMCID: PMC2597982  PMID: 17475917
2.  Modifier gene study of meconium ileus in cystic fibrosis: statistical considerations and gene mapping results 
Human genetics  2009;126(6):763-778.
Cystic fibrosis (CF) is a monogenic disease due to mutations in the CFTR gene. Yet, variability in CF disease presentation is presumed to be affected by modifier genes, such as those recently demonstrated for the pulmonary aspect. Here, we conduct a modifier gene study for meconium ileus (MI), an intestinal obstruction that occurs in 16–20% of CF newborns, providing linkage and association results from large family and case–control samples. Linkage analysis of modifier traits is different than linkage analysis of primary traits on which a sample was ascertained. Here, we articulate a source of confounding unique to modifier gene studies and provide an example of how one might overcome the confounding in the context of linkage studies. Our linkage analysis provided evidence of a MI locus on chromosome 12p13.3, which was segregating in up to 80% of MI families with at least one affected offspring (HLOD = 2.9). Fine mapping of the 12p13.3 region in a large case–control sample of pancreatic insufficient Canadian CF patients with and without MI pointed to the involvement of ADIPOR2 in MI (p = 0.002). This marker was substantially out of Hardy–Weinberg equilibrium in the cases only, and provided evidence of a cohort effect. The association with rs9300298 in the ADIPOR2 gene at the 12p13.3 locus was replicated in an independent sample of CF families. A protective locus, using the phenotype of no-MI, mapped to 4q13.3 (HLOD = 3.19), with substantial heterogeneity. A candidate gene in the region, SLC4A4, provided preliminary evidence of association (p = 0.002), warranting further follow-up studies. Our linkage approach was used to direct our fine-mapping studies, which uncovered two potential modifier genes worthy of follow-up.
PMCID: PMC2888886  PMID: 19662435
3.  Refinement of the Genomic Structure of STX1A and Mutation Analysis in Nondeletion Williams Syndrome Patients 
Williams syndrome (WS) is a contiguous gene deletion disorder in which the commonly deleted region contains at least 17 genes. One of these genes, Syntaxin 1A (STX1A), codes for a protein that is highly expressed in the nervous system and is essential for the docking of synaptic vesicles with the presynaptic plasma membrane. In this study, we refine the complete genomic structure of the human STX1A gene by direct sequencing and primer walking of bacterial artificial chromosome (BAC) clones and show that STX1A contains at least 10 exons and 9 introns. The length of exons range from 27 bp to 138 bp and all splice sites conform to the GT-AG rule. Investigation of the STX1A gene sequence in five WS patients without detectable deletions did not identify any point mutations. Although the regulatory elements that control STX1A transcription were not examined, these results do not support a role for STX1A in the WS phenotype.
PMCID: PMC2893211  PMID: 11977160 CAMSID: cams402
Williams syndrome; STX1A; genomic structure; point mutation
4.  A 1.5 million–base pair inversion polymorphism in families with Williams-Beuren syndrome 
Nature genetics  2001;29(3):321-325.
Williams-Beuren syndrome (WBS) is most often caused by hemizygous deletion of a 1.5-Mb interval encompassing at least 17 genes at 7q11.23 (refs. 1, 2). As with many other haploinsufficiency diseases, the mechanism underlying the WBS deletion is thought to be unequal meiotic recombination, probably mediated by the highly homologous DNA that flanks the commonly deleted region3. Here, we report the use of interphase fluorescence in situ hybridization (FISH) and pulsed-field gel electrophoresis (PFGE) to identify a genomic polymorphism in families with WBS, consisting of an inversion of the WBS region. We have observed that the inversion is hemizygous in 3 of 11 (27%) atypical affected individuals who show a subset of the WBS phenotypic spectrum but do not carry the typical WBS microdeletion. Two of these individuals also have a parent who carries the inversion. In addition, in 4 of 12 (33%) families with a proband carrying the WBS deletion, we observed the inversion exclusively in the parent transmitting the disease-related chromosome. These results suggest the presence of a newly identified genomic variant within the population that may be associated with the disease. It may result in predisposition to primarily WBS-causing microdeletions, but may also cause translocations and inversions.
PMCID: PMC2889916  PMID: 11685205 CAMSID: cams401
5.  Human Chromosome 7: DNA Sequence and Biology 
Scherer, Stephen W. | Cheung, Joseph | MacDonald, Jeffrey R. | Osborne, Lucy R. | Nakabayashi, Kazuhiko | Herbrick, Jo-Anne | Carson, Andrew R. | Parker-Katiraee, Layla | Skaug, Jennifer | Khaja, Razi | Zhang, Junjun | Hudek, Alexander K. | Li, Martin | Haddad, May | Duggan, Gavin E. | Fernandez, Bridget A. | Kanematsu, Emiko | Gentles, Simone | Christopoulos, Constantine C. | Choufani, Sanaa | Kwasnicka, Dorota | Zheng, Xiangqun H. | Lai, Zhongwu | Nusskern, Deborah | Zhang, Qing | Gu, Zhiping | Lu, Fu | Zeesman, Susan | Nowaczyk, Malgorzata J. | Teshima, Ikuko | Chitayat, David | Shuman, Cheryl | Weksberg, Rosanna | Zackai, Elaine H. | Grebe, Theresa A. | Cox, Sarah R. | Kirkpatrick, Susan J. | Rahman, Nazneen | Friedman, Jan M. | Heng, Henry H. Q. | Pelicci, Pier Giuseppe | Lo-Coco, Francesco | Belloni, Elena | Shaffer, Lisa G. | Pober, Barbara | Morton, Cynthia C. | Gusella, James F. | Bruns, Gail A. P. | Korf, Bruce R. | Quade, Bradley J. | Ligon, Azra H. | Ferguson, Heather | Higgins, Anne W. | Leach, Natalia T. | Herrick, Steven R. | Lemyre, Emmanuelle | Farra, Chantal G. | Kim, Hyung-Goo | Summers, Anne M. | Gripp, Karen W. | Roberts, Wendy | Szatmari, Peter | Winsor, Elizabeth J. T. | Grzeschik, Karl-Heinz | Teebi, Ahmed | Minassian, Berge A. | Kere, Juha | Armengol, Lluis | Pujana, Miguel Angel | Estivill, Xavier | Wilson, Michael D. | Koop, Ben F. | Tosi, Sabrina | Moore, Gudrun E. | Boright, Andrew P. | Zlotorynski, Eitan | Kerem, Batsheva | Kroisel, Peter M. | Petek, Erwin | Oscier, David G. | Mould, Sarah J. | Döhner, Hartmut | Döhner, Konstanze | Rommens, Johanna M. | Vincent, John B. | Venter, J. Craig | Li, Peter W. | Mural, Richard J. | Adams, Mark D. | Tsui, Lap-Chee
Science (New York, N.Y.)  2003;300(5620):767-772.
DNA sequence and annotation of the entire human chromosome 7, encompassing nearly 158 million nucleotides of DNA and 1917 gene structures, are presented. To generate a higher order description, additional structural features such as imprinted genes, fragile sites, and segmental duplications were integrated at the level of the DNA sequence with medical genetic data, including 440 chromosome rearrangement breakpoints associated with disease. This approach enabled the discovery of candidate genes for developmental diseases including autism.
PMCID: PMC2882961  PMID: 12690205 CAMSID: cams403
6.  Complex two-gene modulation of lung disease severity in children with cystic fibrosis 
The Journal of Clinical Investigation  2008;118(3):1040-1049.
Although cystic fibrosis (CF) is a monogenic disease, its clinical manifestations are influenced in a complex manner. Severity of lung disease, the main cause of mortality among CF patients, is likely modulated by several genes. The mannose-binding lectin 2 (MBL2) gene encodes an innate immune response protein and has been implicated as a pulmonary modifier in CF. However, reports have been conflicting, and interactions with other modifiers have not been investigated. We therefore evaluated the association of MBL2 with CF pulmonary phenotype in a cohort of 1,019 Canadian pediatric CF patients. MBL2 genotypes were combined into low-, intermediate-, and high-expression groups based on MBL2 levels in plasma. Analysis of age at first infection with Pseudomonas aeruginosa demonstrated that MBL2 deficiency was significantly associated with earlier onset of infection. This MBL2 effect was amplified in patients with high-producing genotypes of transforming growth factor beta 1 (TGFB1). Similarly, MBL2 deficiency was associated with more rapid decline of pulmonary function, most significantly in those carrying the high-producing TGFB1 genotype. These findings provide evidence of gene-gene interaction in the pathogenesis of CF lung disease, whereby high TGF-β1 production enhances the modulatory effect of MBL2 on the age of first bacterial infection and the rate of decline of pulmonary function.
PMCID: PMC2248329  PMID: 18292811
7.  Mutations in the Cystic Fibrosis Transmembrane Regulator Gene and In Vivo Transepithelial Potentials 
Aim: To examine the relationship between cystic fibrosis transmembrane regulator gene mutations (CFTR) and in vivo transepithelial potentials.
Methods: We prospectively evaluated 162 men including 31 healthy subjects, 21 obligate heterozygotes, 60 with congenital bilateral absence of the vas deferens (CBAVD) and 50 with CF by extensive CFTR genotyping, sweat chloride and nasal potential difference testing.
Results: Six (10%) men with CBAVD carried no CFTR mutations, 18 (30%) carried one mutation, including the 5T variant, and 36 (60%) carried mutations on both alleles, for a significantly higher rate carrying one or more mutations than healthy controls (90% versus 19%, p < 0.001). There was an overlapping spectrum of ion channel measurements among the men with CBAVD, ranging from values in the control and obligate heterozygote range at one extreme, to values in the CF range at the other. All pancreatic-sufficient patients with CF and 34 of 36 patients with CBAVD with mutations on both alleles carried at least one mild mutation. However, the distribution of mild mutations in the two groups differed greatly. Genotyping, sweat chloride and nasal potential difference (alone or in combination) excluded CF in all CBAVD men with no mutations. CF was confirmed in 56% and 67% of CBAVD men carrying 1 and 2 CFTR mutations, respectively.
Conclusion: Abnormalities of CFTR transepithelial function correlate with the number and severity of CFTR gene mutations.
PMCID: PMC2648063  PMID: 16840743
CFTR mutations; congenital bilateral absence of the vas deferens; cystic fibrosis; nasal potential difference; sweat chloride
8.  Slc25a13-Knockout Mice Harbor Metabolic Deficits but Fail To Display Hallmarks of Adult-Onset Type II Citrullinemia 
Molecular and Cellular Biology  2004;24(2):527-536.
Adult-onset type II citrullinemia (CTLN2) is an autosomal recessive disease caused by mutations in SLC25A13, the gene encoding the mitochondrial aspartate/glutamate carrier citrin. The absence of citrin leads to a liver-specific, quantitative decrease of argininosuccinate synthetase (ASS), causing hyperammonemia and citrullinemia. To investigate the physiological role of citrin and the development of CTLN2, an Slc25a13-knockout (also known as Ctrn-deficient) mouse model was created. The resulting Ctrn−/− mice were devoid of Slc25a13 mRNA and citrin protein. Liver mitochondrial assays revealed markedly decreased activities in aspartate transport and the malate-aspartate shuttle. Liver perfusion also demonstrated deficits in ureogenesis from ammonia, gluconeogenesis from lactate, and an increase in the lactate-to-pyruvate ratio within hepatocytes. Surprisingly, Ctrn−/− mice up to 1 year of age failed to show CTLN2-like symptoms due to normal hepatic ASS activity. Serological measures of glucose, amino acid, and ammonia metabolism also showed no significant alterations. Nitrogen-loading treatments produced only minor changes in the hepatic ammonia and amino acid levels. These results suggest that citrin deficiency alone may not be sufficient to produce a CTLN2-like phenotype in mice. These observations are compatible, however, with the variable age of onset, incomplete penetrance, and strong ethnic bias seen in CTLN2 where additional environmental and/or genetic triggers are now suspected.
PMCID: PMC343808  PMID: 14701727
9.  Genome-wide detection of segmental duplications and potential assembly errors in the human genome sequence 
Genome Biology  2003;4(4):R25.
Rapid computational heuristics have been developed based on BLAST analysis to detect segmental duplications, as well as regions containing potential sequence misassignments in the human genome assemblies. Most of the sequences in the human genome that have undergone recent segmental duplications have now been identified.
Previous studies have suggested that recent segmental duplications, which are often involved in chromosome rearrangements underlying genomic disease, account for some 5% of the human genome. We have developed rapid computational heuristics based on BLAST analysis to detect segmental duplications, as well as regions containing potential sequence misassignments in the human genome assemblies.
Our analysis of the June 2002 public human genome assembly revealed that 107.4 of 3,043.1 megabases (Mb) (3.53%) of sequence contained segmental duplications, each with size equal or more than 5 kb and 90% identity. We have also detected that 38.9 Mb (1.28%) of sequence within this assembly is likely to be involved in sequence misassignment errors. Furthermore, we have identified a significant subset (199,965 of 2,327,473 or 8.6%) of single-nucleotide polymorphisms (SNPs) in the public databases that are not true SNPs but are potential paralogous sequence variants.
Using two distinct computational approaches, we have identified most of the sequences in the human genome that have undergone recent segmental duplications. Near-identical segmental duplications present a major challenge to the completion of the human genome sequence. Potential sequence misassignments detected in this study would require additional efforts to resolve.
PMCID: PMC154576  PMID: 12702206
10.  Comparative analysis of the gene-dense ACHE/TFR2 region on human chromosome 7q22 with the orthologous region on mouse chromosome 5 
Nucleic Acids Research  2001;29(6):1352-1365.
Chromosome 7q22 has been the focus of many cytogenetic and molecular studies aimed at delineating regions commonly deleted in myeloid leukemias and myelodysplastic syndromes. We have compared a gene-dense, GC-rich sub-region of 7q22 with the orthologous region on mouse chromosome 5. A physical map of 640 kb of genomic DNA from mouse chromosome 5 was derived from a series of overlapping bacterial artificial chromosomes. A 296 kb segment from the physical map, spanning Ache to Tfr2, was compared with 267 kb of human sequence. We identified a conserved linkage of 12 genes including an open reading frame flanked by Ache and Asr2, a novel cation-chloride cotransporter interacting protein Cip1, Ephb4, Zan and Perq1. While some of these genes have been previously described, in each case we present new data derived from our comparative sequence analysis. Adjacent unfinished sequence data from the mouse contains an orthologous block of 10 additional genes including three novel cDNA sequences that we subsequently mapped to human 7q22. Methods for displaying comparative genomic information, including unfinished sequence data, are becoming increasingly important. We supplement our printed comparative analysis with a new, Web-based program called Laj (local alignments with java). Laj provides interactive access to archived pairwise sequence alignments via the WWW. It displays synchronized views of a dot-plot, a percent identity plot, a nucleotide-level local alignment and a variety of relevant annotations. Our mouse–human comparison can be viewed at Laj is available at, along with online documentation and additional examples of annotated genomic regions.
PMCID: PMC29746  PMID: 11239002
11.  Replication Delay along FRA7H, a Common Fragile Site on Human Chromosome 7, Leads to Chromosomal Instability 
Molecular and Cellular Biology  2000;20(12):4420-4427.
Common fragile sites are specific chromosomal loci that show gaps, breaks, or rearrangements in metaphase chromosomes under conditions that interfere with DNA replication. The mechanism underlying the chromosomal instability at fragile sites was hypothesized to associate with late replication time. Here, we aimed to investigate the replication pattern of the common fragile site FRA7H, encompassing 160 kb on the long arm of human chromosome 7. Using in situ hybridization on interphase nuclei, we revealed that the replication of this region is initiated relatively early, before 30% of S phase is completed. However, a high fraction (∼35%) of S-phase nuclei showed allelic asynchrony, indicating that the replication of FRA7H is accomplished at different times in S phase. This allelic asynchrony is not the result of a specific replication time of each FRA7H allele. Analysis of the replication pattern of adjacent clones along FRA7H by using cell population and two-color fluorescent in situ hybridization analyses showed significant differences in the replication of adjacent clones, under normal growth condition and upon aphidicolin treatment. This pattern significantly differed from that of two nonfragile regions which showed a coordinated replication under both conditions. These results indicate that aphidicolin is enhancing an already existing difference in the replication time along the FRA7H region. Based on our replication analysis of FRA7H and on previous analysis of the common fragile site FRA3B, we suggest that delayed replication is underlying the fragility at aphidicolin-induced common fragile sites.
PMCID: PMC85809  PMID: 10825205
12.  Permeability of Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels to Polyatomic Anions  
The Journal of General Physiology  1997;110(4):355-364.
Permeability of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel to polyatomic anions of known dimensions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. Biionic reversal potentials measured with external polyatomic anions gave the permeability ratio (PX/PCl) sequence NO3− > Cl− > HCO3− > formate > acetate. The same selectivity sequence but somewhat higher permeability ratios were obtained when anions were tested from the cytoplasmic side. Pyruvate, propanoate, methane sulfonate, ethane sulfonate, and gluconate were not measurably permeant (PX/PCl < 0.06) from either side of the membrane. The relationship between permeability ratios from the outside and ionic diameters suggests a minimum functional pore diameter of ∼5.3 Å. Permeability ratios also followed a lyotropic sequence, suggesting that permeability is dependent on ionic hydration energies. Site-directed mutagenesis of two adjacent threonines in TM6 to smaller, less polar alanines led to a significant (24%) increase in single channel conductance and elevated permeability to several large anions, suggesting that these residues do not strongly bind permeating anions, but may contribute to the narrowest part of the pore.
PMCID: PMC2229373  PMID: 9379168
pore size; channel selectivity; anion permeation; lyotropic sequence; cystic fibrosis

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