Robust fragile X (CGG)n genotype classification using a methylation specific triple PCR assay.
Explore the source record for details and available documents.
Algorithms↗
Biomedical subjects
G R Cutting
Publications and source records attributed to G R Cutting.
Cystic fibrosis transmembrane conductance regulator gene mutations: do they play a role in the aetiology of allergic bronchopulmonary aspergillosis?
BACKGROUND: Previous work suggests that cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations may be implicated in the aetiology of allergic bronchopulmonary aspergilosis (ABPA). OBJECTIVE: To compare the frequency of CF gene mutations in asthmatics with ABPA of varying severity with asthmatics who were skin prick test (SPT)-positive to Aspergillus fumigatus (Af) without evidence of ABPA and asthmatics SPT-negative to Af. METHODS: Thirty-one Caucasian patients with ABPA were identified, together with asthmatics SPT positive to Af without evidence of ABPA (n = 23) and SPT negative to Af (n = 28). Genomic DNA was tested for 16 CF mutations accounting for approximately 85% of CF alleles in Caucasian New Zealanders. RESULTS: Four (12.9%) ABPA patients were found to be carriers of a CF mutation (DeltaF508 n = 3, R117H n = 1), one (4.3%) asthmatic SPT positive to Af without ABPA (DeltaF508), and one (3.6%) asthmatic SPT negative to Af (R117H). All patients with a CF mutation had normal sweat chloride (< 40 mM). There was no significant difference between the frequency of CF mutations in the ABPA patients and asthmatics without ABPA. However, the frequency of CF mutations in the ABPA patients was significantly different (P = 0.0125) to the expected carrier rate in the general population. CONCLUSION: These results lend further support to a possible link between CF mutations and ABPA.
Adult↗
Developmentally regulated expression of GABA receptor rho1 and rho2 subunits, L7 and cone-rod homeobox (CRX) genes in mouse retina.
In this study, we compared the temporal expression pattern of four retinal genes; rho1 and rho2 that encode subunits of GABA(c) receptors, L7 that encodes Purkinje cell protein and CRX that encodes the cone-rod homeobox transcription factor. A reverse-transcription-polymerase chain reaction (RT-PCR) strategy that generated a linear correlation between the amount of retinal RNA and the amount of amplified product was used to quantify transcripts from each gene. Results with this method showed that the rho1 and L7 have similar developmental patterns. Both exhibit basal level expression before P7. From P7 to P20, the RNA levels for both genes were increased about 12-fold. After P20, the RNA levels remained unchanged. Compared to rho1 and L7, expression of rho2 began later, since the rho2 RNA could not be detected until P10. At P10, the rho2 RNA level was about 10% of its level at P35. Expression of rho2 reached its peak at a later developmental stage compared to that of rho1 and L7. The different temporal patterns were confirmed by co-amplification of rho1, rho2, and L7 in a single PCR tube. CRX RNA was detected at embryonic day 15 (E15) and increased progressively, in agreement with a prior study using in situ hybridization. These data, combined with evidence that the tissue distribution of rho1 and L7 RNA in the CNS are similar, indicates that rho1 and L7 may share common transcriptional regulatory elements. Furthermore, the difference in the timing of rho subunit expression suggests that the subunit composition of GABA(c) receptors vary during retinal development.
Aging↗
ABCD1 mutations and the X-linked adrenoleukodystrophy mutation database: role in diagnosis and clinical correlations.
X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene, which encodes a peroxisomal ABC half-transporter (ALDP) involved in the import of very long-chain fatty acids (VLCFA) into the peroxisome. The disease is characterized by a striking and unpredictable variation in phenotypic expression. Phenotypes include the rapidly progressive childhood cerebral form (CCALD), the milder adult form, adrenomyeloneuropathy (AMN), and variants without neurologic involvement. There is no apparent correlation between genotype and phenotype. In males, unambiguous diagnosis can be achieved by demonstration of elevated levels of VLCFA in plasma. In 15 to 20% of obligate heterozygotes, however, test results are false-negative. Therefore, mutation analysis is the only reliable method for the identification of heterozygotes. Since most X-ALD kindreds have a unique mutation, a great number of mutations have been identified in the ABCD1 gene in the last seven years. In order to catalog and facilitate the analysis of these mutations, we have established a mutation database for X-ALD ( http://www.x-ald.nl). In this review we report a detailed analysis of all 406 X-ALD mutations currently included in the database. Also, we present 47 novel mutations. In addition, we review the various X-ALD phenotypes, the different diagnostic tools, and the need for extended family screening for the identification of new patients.
ATP Binding Cassette Transporter, Subfamily D, Mem↗
A PDZ-binding motif is essential but not sufficient to localize the C terminus of CFTR to the apical membrane.
Localization of ion channels and transporters to the correct membrane of polarized epithelia is important for vectorial ion movement. Prior studies have shown that the cytoplasmic carboxyl terminus of the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the apical localization of this protein. Here we show that the C-terminal tail alone, or when fused to the green fluorescent protein (GFP), can localize to the apical plasma membrane, despite the absence of transmembrane domains. Co-expression of the C terminus with full-length CFTR results in redistribution of CFTR from apical to basolateral membranes, indicating that both proteins interact with the same target at the apical membrane. Amino acid substitution and deletion analysis confirms the importance of a PDZ-binding motif D-T-R-L> for apical localization. However, two other C-terminal regions, encompassing amino acids 1370-1394 and 1404-1425 of human CFTR, are also required for localizing to the apical plasma membrane. Based on these results, we propose a model of polarized distribution of CFTR, which includes a mechanism of selective retention of this protein in the apical plasma membrane and stresses the requirement for other C-terminal sequences in addition to a PDZ-binding motif.
Amino Acid Motifs↗
Mutation in the gene responsible for cystic fibrosis and predisposition to chronic rhinosinusitis in the general population.
CONTEXT: Chronic rhinosinusitis (CRS) is a common condition in the US general population, yet little is known about its underlying molecular cause. Chronic rhinosinusitis is a consistent feature of the autosomal recessive disorder cystic fibrosis (CF). OBJECTIVE: To determine whether mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, which is responsible for CF, predispose to CRS. DESIGN: Case-control study conducted from 1996 to 1999 in which the DNA of CRS patients and controls was typed for 16 mutations that account for 85% of CF alleles in the general population. Chronic rhinosinusitis patients with 1 CF mutation were evaluated for a CF diagnosis by sweat chloride testing, nasal potential difference measurement, and DNA analysis for additional mutations. SETTING: Otolaryngology-head and neck clinic of a US teaching hospital. PARTICIPANTS: One hundred forty-seven consecutive adult white patients who met stringent diagnostic criteria for CRS and 123 CRS-free white control volunteers of similar age range, geographic region, and socioeconomic status. MAIN OUTCOME MEASURES: Presence of CF mutations by DNA analysis among CRS patients vs controls. RESULTS: Eleven CRS patients were found to have a CF mutation (DeltaF508, n = 9; G542X, n = 1; and N1303K, n = 1). Diagnostic testing excluded CF in 10 of these patients and led to CF diagnosis in 1. Excluding this patient from the analyses, the proportion of CRS patients who were found to have a CF mutation (7%) was significantly higher than in the control group (n = 2 [2%]; P =.04, both having DeltaF508 mutations). Furthermore, 9 of the 10 CF carriers had the polymorphism M470V, and M470V homozygotes were overrepresented in the remaining 136 CRS patients (P =.03). CONCLUSION: These data indicate that mutations in the gene responsible for CF may be associated with the development of CRS in the general population. JAMA. 2000;284:1814-1819.
Adult↗
The PDZ-interacting domain of cystic fibrosis transmembrane conductance regulator is required for functional expression in the apical plasma membrane.
Polarization of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel to the apical plasma membrane in epithelial cells is critical for vectorial chloride transport. Previously, we reported that the C terminus of CFTR constitutes a PDZ-interacting domain that is required for CFTR polarization to the apical plasma membrane and interaction with the PDZ domain-containing protein EBP50 (NHERF). PDZ-interacting domains are typically composed of the C-terminal three to five amino acids, which in CFTR are QDTRL. Our goal was to identify the key amino acid(s) in the PDZ-interacting domain of CFTR with regard to its apical polarization, interaction with EBP50, and ability to mediate transepithelial chloride secretion. Point substitution of the C-terminal leucine (Leu at position 0) with alanine abrogated apical polarization of CFTR, interaction between CFTR and EBP50, efficient expression of CFTR in the apical membrane, and chloride secretion. Point substitution of the threonine (Thr at position -2) with alanine or valine had no effect on the apical polarization of CFTR, but reduced interaction between CFTR and EBP50, efficient expression of CFTR in the apical membrane as well as chloride secretion. By contrast, individual point substitution of the other C-terminal amino acids (Gln at position -4, Asp at position -3 and Arg at position -1) with alanine had no effect on measured parameters. We conclude that the PDZ-interacting domain, in particular the leucine (position 0) and threonine (position -2) residues, are required for the efficient, polarized expression of CFTR in the apical plasma membrane, interaction of CFTR with EBP50, and for the ability of CFTR to mediate chloride secretion. Mutations that delete the C terminus of CFTR may cause cystic fibrosis because CFTR is not polarized, complexed with EBP50, or efficiently expressed in the apical membrane of epithelial cells.
Animals↗
Effects of cystic fibrosis and congenital bilateral absence of the vas deferens-associated mutations on cystic fibrosis transmembrane conductance regulator-mediated regulation of separate channels.
The protein defective in cystic fibrosis (CF), the CF transmembrane-conductance regulator (CFTR), functions as an epithelial chloride channel and as a regulator of separate ion channels. Although the consequences that disease-causing mutations have on the chloride-channel function have been studied extensively, little is known about the effects that mutations have on the regulatory function. To address this issue, we transiently expressed CFTR-bearing mutations associated with CF or its milder phenotype, congenital bilateral absence of the vas deferens, and determined whether mutant CFTR could regulate outwardly rectifying chloride channels (ORCCs). CFTR bearing a CF-associated mutation in the first nucleotide-binding domain (NBD1), DeltaF508, functioned as a chloride channel but did not regulate ORCCs. However, CFTR bearing disease-associated mutations in other domains retained both functions, regardless of the associated phenotype. Thus, a relationship between loss of CFTR regulatory function and disease severity is evident for NBD1, a region of CFTR that appears important for regulation of separate channels.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Single-tube multiplex-PCR screen for common deletional determinants of alpha-thalassemia.
Alpha-thalassemia is very common throughout all tropical and subtropical regions of the world. In Southeast Asia and the Mediterranean regions, compound heterozygotes and homozygotes may have anemia that is mild to severe (hemoglobin [Hb] H disease) or lethal (Hb Bart's hydrops fetalis). We have developed a reliable, single-tube multiplex-polymerase chain reaction (PCR) assay for the 6 most frequently observed determinants of alpha-thalassemia. The assay allows simple, high throughput genetic screening for these common hematological disorders. (Blood. 2000;95:360-362)
Base Sequence↗
Genotype-phenotype relationships in cystic fibrosis.
The genotype-phenotype relationship in CF is complex despite its being a monogenic disorder. Factors that contribute to variability among individuals with the same genotype are an area of intense study. Nevertheless, certain conclusions can be derived from these studies. First, mutations in both CFTR alleles cause the CF phenotype. Homozygosity for delta F508 or compound heterozygosity for delta F508 and another severe mutation (e.g., G551D, W1282X) cause classic CF: obstructive pulmonary disease, exocrine pancreatic deficiency, male infertility, and elevated sweat chloride concentrations. Clinical variability is observed among patients with the classic form of CF, especially with regards to the severity of lung disease. Although understanding of the role of other genes and environment in the development of lung disease is incomplete, evidence that other factors are important raises the possibility that therapeutic intervention may be possible at several levels. Second, genotype correlates more closely with certain features of the CF phenotype than others. Mutations that allow partial function of CFTR are often associated with pancreatic sufficiency, occasionally identified with normal sweat gland function, and sporadically correlated with mild lung disease. Partially functioning mutants rarely prevent maldevelopment of the male reproductive tract; an exception is 3849 + 10 Kb C-->T. These observations suggest that certain tissues require different levels of CFTR function to avoid the pathologic manifestations typical of CF. The genetic cause of several disorders that clinically overlap CF can be attributed, in part, to mutations in CFTR. Finally, molecular analysis of disease-associated mutations identified through genotype-phenotype studies provides a mechanistic framework for genotype-based therapeutic approaches and pharmaceutical interventions.
Cystic Fibrosis↗
Mutation analysis of GABRR1 and GABRR2 in autosomal recessive retinitis pigmentosa.
Explore the source record for details and available documents.
Base Sequence↗
Simplified multiplex-PCR diagnosis of common southeast asian deletional determinants of alpha-thalassemia.
Explore the source record for details and available documents.
Asia↗
Expression of the voltage-gated chloride channel ClC-2 in rod bipolar cells of the rat retina.
Voltage-gated chloride channels (ClC) are highly conserved during evolution and appear to participate in a variety of physiological functions. Recently, ClC-2 was proposed to play a role in stabilizing the chloride equilibrium potential near or below the resting membrane potential in neurons expressing ligand-gated chloride channels. Because rod bipolar cells in mammalian retina express three forms of inhibitory ligand-gated chloride channels, we decided to study ClC-2 localization and function in the rat retina. RNA encoding ClC-1, -2, -3, -4, and -5 was detected by reverse transcription-PCR in the rat retina. ClC-2-specific antibodies identified protein on cell bodies and in synaptic layers. Double-immunofluorescence staining revealed that intense ClC-2 immunoreactivity colocalized with PKC-stained rod bipolar cells. Patch-clamp experiments performed with individual rod bipolar cells demonstrated the presence of a time-dependent, inwardly rectified current activated at hyperpolarizing membrane potentials. This current demonstrated selectivity for different anions (Cl(-) > I(-) > gluconate), was inhibited by Cd(2+), and was minimally reduced by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid. These features are consistent with currents generated by ClC-2 channels. Our data indicate that functional ClC-2 channels are present in retinal rod bipolar cells and support a role for ClC-2 in maintaining Cl(-) homeostasis in neurons with ligand-gated chloride channels.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Identification of 70 amino acids important for GABA(C) receptor rho1 subunit assembly.
gamma-Aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the mammalian central nervous system and gates at least three subclasses of receptors, termed GABA(A), GABA(B) and GABA(C). Accumulating evidence indicates that GABA(C) receptors are composed exclusively of rho subunits. The N-terminal half of the rho subunits has been shown to mediate formation of homo- and heterooligomeric GABA(C) receptors. In this study, we searched for specific sequences within the N-terminus of the rho1 subunit involved in the assembly process. Assembly sequences were localized to a 128-amino acid region by deletion of progressively larger regions of a chimeric rho1beta1 subunit previously shown to disrupt rho1 and rho2 assembly. To confirm this observation, a series of GABA(A) receptor beta subunit chimeras containing different regions of the rho1 N-terminus were tested for interference with rho1 and rho2 subunit assembly into functional GABA receptors. Transfer of 70 residues within the 128 amino acid region to the beta1 subunit created a chimera that disrupted rho1, but not rho2, assembly into functional receptors. These observations refine the location of signals involved in rho1 subunit assembly, and suggest that different signals exist for the formation of rho1 homooligomeric and rho1/rho2 heterooligomeric GABA(C) receptors.
Amino Acid Sequence↗
Accurate DNA-based diagnostic and carrier testing for X-linked adrenoleukodystrophy.
X-linked adrenoleukodystrophy is a serious and often fatal disorder, affecting the white matter of the nervous system, the adrenal cortex, and the testis. The gene mutated in X-ALD encodes a peroxisomal membrane protein, ALDP. The presence of very long chain fatty acids in plasma is highly diagnostic for affected males and carrier females, but exclusion of carrier status biochemically is unreliable. Molecular analysis of the X-ALD gene has the potential to either identify or rule out carrier status accurately, but is complicated by the existence of autosomal paralogs. We have developed and validated a robust DNA diagnostic test for this disorder involving nonnested genomic amplification of the X-ALD gene, followed by fluorescent dye-primer sequencing and analysis. This protocol provides a highly reliable means of determining carrier status in women at risk for transmitting X-ALD and is applicable to a clinical diagnostic laboratory.
ATP Binding Cassette Transporter, Subfamily D, Mem↗
Detection of a cystic fibrosis modifier locus for meconium ileus on human chromosome 19q13.
Explore the source record for details and available documents.
Child↗
GABAC receptor rho subunits are heterogeneously expressed in the human CNS and form homo- and heterooligomers with distinct physical properties.
In the central nervous system, receptors for gamma-aminobutyric acid (GABA) are responsible for inhibitory neurotransmission. Anatomical and electrophysiological studies indicate that GABAC receptors are composed of rho subunits. While the rho 1 subunit of various species forms homooligomeric receptors with GABAC-like properties, molecular cloning has identified additional rho subunits whose functional role is unclear. By RT-PCR, we demonstrated that rho 1 expression is primarily restricted to the retina, whereas the rho 2 subunit was present in all brain regions tested. Transfection of HEK-293 cells with rho 2 cDNA resulted in GABA-gated whole-cell currents that differed from those mediated by the rho 1 subunit in two respects: maximal amplitude (rho 1:rho 2 approximately 4:1) and inactivation time course (rho 1:rho 2 approximately 2:1). Cotransfection of rho 1 and rho 2 cDNA in a 1:1 ratio generated whole-cell currents with large amplitudes characteristic of rho 1 but more rapid inactivation typical for rho 2. This observation suggested formation of heterooligomeric GABAC receptors with distinct features. Therefore, we tested the assembly of rho 1 and rho 2 subunits by cotransfecting rho 2 cDNA together with a chimeric rho 1 beta 1 subunit, known to interfere with rho 1 assembly in a dominant-negative fashion. Reduction of rho 2 generated currents correlated with the ratio of chimeric to rho 2 cDNA. Secondly, we determined that the picrotoxinin sensitivity of cells transfected with various ratios of rho 1 and rho 2 cDNA differed from that expected of a pure mixture of homooligomeric receptors. The latter two observations support the idea that rho 1 and rho 2 subunits form heterooligomeric GABAC receptors in mammalian cells. Together, our results indicate that the presence of both rho subunits enables the formation of heterooligomeric receptors with physical properties distinct from homooligomers, thus increasing the diversity of GABAC receptors in the CNS.
Animals↗
Clinical and genetic studies of an autosomal dominant cone-rod dystrophy with features of Stargardt disease.
Cone-rod dystrophy (CORD) and Stargardt disease (STGD) are two hereditary retinal dystrophies with similarities to age-related macular degeneration. Cone-rod dystrophies are a group of degenerative disorders resulting in decreased visual acuity and color vision, attenuated electroretinographic (ERG) responses, and atrophic macular lesions. Autosomal dominant, autosomal recessive, and X-linked forms of cone-rod dystrophy have been reported. Stargardt disease is characterized by reduced visual acuity, atrophic macular changes, prominent 'flavimaculatus flecks' in the pigment epithelium of the posterior retina, and a virtually pathognomic 'dark choroid' pattern on fluorescein angiography. Stargardt disease is classically inherited as an autosomal recessive trait, although numerous families have been described in which features of Stargardt disease are transmitted in an autosomal dominant manner. We have identified a new kindred with autosomal dominant cone-rod dystrophy with features of Stargardt-like disease. Detailed clinical evaluation, genotype analysis, and linkage analysis were performed. Fluorescein angiography revealed a 'dark choroid' pattern in three affected subjects. Electroretinography disclosed markedly reduced scotopic and photopic responses in three affected individuals. Genetic analysis revealed linkage to known loci for cone-rod dystrophy (CORD7) and Stargardt-like disease (STGD3) on chromosome 6q14. A peak lod score of 3.3 was obtained with the marker D6S280 at straight theta =0.010. A physical map was constructed by screening a YAC library with short tandem repeat markers in the region. Screening of a candidate gene, the rho1 subunit of the GABA receptor, failed to reveal any mutations.
Adult↗