Mucopolysaccharidosis type VII (Sly syndrome) presenting as neonatal cholestasis with hepatosplenomegaly.
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Cholestasis↗
Biomedical subjects
L A Clarke
Publications and source records attributed to L A Clarke.
PCR amplification introduces errors into mononucleotide and dinucleotide repeat sequences.
The polymerase chain reaction (PCR) is used universally for accurate exponential amplification of DNA. We describe a high error rate at mononucleotide and dinucleotide repeat sequence motifs. Subcloning of PCR products allowed sequence analysis of individual DNA molecules from the product pool and revealed that: (1) monothymidine repeats longer than 11 bp are amplified with decreasing accuracy, (2) repeats generally contract during PCR because of the loss of repeat units, (3) Taq and proofreading polymerase Pfu generate similar errors at mononucleotide and dinucleotide repeats, and (4) unlike the parent PCR product pool, individual clones containing a single repeat length produce no "shadow bands". These data demonstrate that routine PCR amplification alters mononucleotide and dinucleotide repeat lengths. Such sequences are common components of genetic markers, disease genes, and intronic splicing motifs, and the amplification errors described here can be mistaken for polymorphisms or mutations.
Dinucleotide Repeats↗
Working strokes by single molecules of the kinesin-related microtubule motor ncd.
The ncd protein is a dimeric, ATP-powered motor that belongs to the kinesin family of microtubule motor proteins. Here we resolve single mechanochemical cycles of recombinant, dimeric, full-length ncd, using optical-tweezers-based instrumentation and a three-bead, suspended-microtubule assay. Under conditions of limiting ATP, isolated and transient microtubule-binding events exhibit exponentially distributed and ATP-concentration-dependent lifetimes. These events do not involve consecutive steps along the microtubule, quantitatively confirming that ncd is non-processive. At low loads, a single motor molecule produces ATP-triggered working strokes of about 9 nm, which occur at the ends of binding events.
Adenosine Triphosphate↗
Cell type specificity in alternative splicing of the human mismatch repair gene hMSH2.
Human non-polyposis colorectal cancer is caused by germline mutations in the DNA mismatch repair genes hMSH2 and hMHL1. Several alternatively spliced mRNA species of these genes are present in peripheral blood lymphocytes of normal individuals, which can confound RT-PCR based techniques of mutation detection. Using RT-PCR, we compared the pattern of alternative splicing in whole peripheral blood lymphocytes (PBLs), separated T and B cells, lymphoblastoid cell lines (LCLs) from the same individuals, and a variety of tissues. Alternatively spliced forms of hMLH1 lacking exons 9/10, 10/11 and 9/10/11 were found to have similar patterns of expression in T cells, B cells, and LCLs. By contrast, a subset of hMSH2 transcripts, some of which were produced by utilisation of novel splicing motifs, were generally expressed in T but not in B cells. LCLs derived from the same blood samples showed no expression of any hMSH2 splicing variants. The hMSH2 delta ex13 transcript, while absent from LCLs, was expressed in whole PBLs and both T and B cell fractions. This transcript was furthermore largely undetectable in tissues other than mononuclear blood cells. These data provide evidence for tissue specificity in the regulation of alternative splicing in hMSH2. In particular we show that LCLs generally do not express alternatively spliced forms of hMSH2 mRNA and are thus suited for RT-PCR based mutation screening in that gene.
Alternative Splicing↗
Tumor-absorbed-dose estimates versus response in tositumomab therapy of previously untreated patients with follicular non-Hodgkin's lymphoma: preliminary report.
I-131-radiolabeled tositumomab (Anti-B1 Antibody), in conjunction with unlabeled tositumomab, was employed in a phase II clinical trial for the therapy of 76 previously-untreated follicular-non-Hodgkin's-lymphoma patients at the University of Michigan Cancer Center. For all patients, conjugate-view images were obtained at six to eight time points on seven consecutive days after a tracer infusion of the antibody. A SPECT image set was obtained on day two or three after the therapy infusion for 57 of the patients. Of these, 55 are suitable for dosimetric evaluation. To date, we have completed analysis and response characterization of 20 patients from the subset of 55. All 20 patients had either a complete response (CR) or a partial response (PR). Conjugate-views provided a time-activity curve for a composite of nearby, individual tumors. These tumors were unresolved in the anterior-posterior projection. Pre-therapy CT provided volume estimates. Therapy radiation dose was computed for the composite tumor by standard MIRD methods. Intra-therapy SPECT allowed the calculation of a separate dose estimate for each individual tumor associated with the composite tumor. Average dose estimates for each patient were also calculated. The 30 individual tumors in PR patients had a mean radiation dose of (369 +/- 54) cGy, while the 56 individual tumors in CR patients had a mean radiation dose of (720 +/- 80) cGy. According to a mixed ANOVA analysis, there was a trend toward a significant difference between the radiation dose absorbed by individual tumors for PR patients and that for CR patients. When the radiation dose depended on only the patient response, the p value was 0.04. When the radiation dose depended on the pre-therapy volume of the individual tumor as well as on the patient response, the p value was 0.06. Since the patient response was complete in 75% of the patients, the analysis of the total cohort of 55 evaluable patients is needed to have a larger number of PR patients to better test the trend toward a significant difference. A pseudo-prediction analysis for patient-level dose and response was also carried out. The positive predictive value and the negative predictive value were 73% and 80%, respectively when a patient's average radiation dose was used. The predictive values were 73% and 60%, respectively, when the patient's average base-10 logarithm of radiation dose was used. A complete overlap for the dose range of CR patients compared to that for PR patients precluded higher predictive values. In conclusion, there was a trend toward a significant difference in the radiation dose between CR and PR patients, but it was only moderately predictive of response.
Antibodies, Monoclonal↗
Pathological exon skipping in an HNPCC proband with MLH1 splice acceptor site mutation.
One of the most commonly mutated mismatch repair genes in human nonpolyposis colorectal cancer (HNPCC) is MLH1. We identified a splice site mutation in MLH1 in a colorectal cancer proband (T-to-A at position -11 of intron 1 splice acceptor) and investigated its functional consequences by RT-PCR, using lymphocyte mRNA from the proband, two noncarrying siblings, and one unrelated individual. Subcloning of PCR products followed by sequencing of individual clones revealed increased transcript heterogeneity in the mutation carrier, attributable to the presence of a variety of mRNA forms lacking exon 2, or combinations of exons 2, 4, 6, 9, and 10. The full-length transcript subcloned from the mutation carrier was detected with a much reduced frequency, suggesting that only the wild-type allele produced functional MLH1 mRNA. The three noncarriers expressed some previously described transcripts and several novel variants, but none that lacked exon 2. The results are consistent with the hypothesis that this splice site mutation causes skipping of MLH1 exon 2 in a large proportion of mRNA transcripts derived from the mutated allele. Such an observation strengthens the case for identifying the mutation as pathogenic in this HNPCC family, which is of interest given the rarity of exon skipping defects resulting from splice acceptor site mutations outside the invariant AG dinucleotide.
Adaptor Proteins, Signal Transducing↗
Recombinant proteins for genetic disease.
The era of molecular biology has led to the development of powerful tools capable of generating therapeutics for genetic disorders. Although there is much current emphasis placed on the development of 'gene therapy' for human disease, developments in the production and availability of recombinant proteins are likely to have a more substantial impact on genetic disease in the short term. The clinical evaluation of recombinant or purified proteins serves as an initial 'proof of principle' of gene-based therapies and thus should expedite advances in this area. Current examples include the use of bovine adenosine deaminase for a form of severe combined immune deficiency (SCID) (Hilman BC, Sorensen RU. Management options: SCIDS with adenosine deaminase deficiency. Ann Allergy 72: 1994: 395-403) and glucocerebrosidase for Gaucher disease (Niederau C, vom Dahl S, Haussinger D. First long-term results of imiglucerase therapy of type 1 Gaucher disease. Eur J Med Res 1998: 3: 25-30). The success of these two enzyme replacement regimes in human clinical trials has been a main impetus for the development of gene-based therapies for these disorders. The 'molecularization of medicine' has led to a more thorough understanding of the molecular basis of disease and disease pathogenesis. The availability of recombinant proteins and the development of appropriate delivery systems will result in more widespread use of these agents. Protein engineering will generate agents with novel functions as is already witnessed with the generation of fusion proteins. This review will highlight advances in the use of recombinant proteins for genetic disease and future potential uses of recombinant proteins.
Animals↗
Murine MPS I: insights into the pathogenesis of Hurler syndrome.
Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disease resulting from deficiency of the lysosomal enzyme alpha-L-iduronidase. A murine model which shows complete deficiency in alpha-L-iduronidase activity has been developed and shows phenotypic features similar to severe MPS I in humans. Here we report on the long-term clinical, biochemical, and pathological course of MPS I in mice with emphasis on the skeletal and central nervous system (CNS) manifestations. Affected mice show a progressive clinical course with the development of coarse features, altered growth characteristics and a shortened life span. Progressive lysosomal accumulation is seen in all tissues. Skeletal manifestations represent the earliest clinical finding in MPS I mice with histologic analysis of growth plate and cortical bone revealing evidence that significant early pathology is present. Analysis of the CNS has revealed the novel finding of progressive neuronal loss within the cerebellum. In addition, brain tissue from MPS I mice shows increased levels of GM2 and GM3 gangliosides. This murine model clearly shows phenotypic and pathologic features which mimic those seen in severe human MPS I and should be an invaluable tool for the study of the pathogenesis of generalized storage disorders.
Animals↗
Is prolactin a gonadotrophic hormone in red deer (Cervus elaphus)? Pattern of expression of the prolactin receptor gene in the testis and epididymis.
This study investigated the pattern and site of expression of the prolactin receptor gene in the testis and epididymis of red deer collected during the breeding season (n=3). Ribonuclease protection assays using 50 microg total RNA and a 300 bp [32P]-labelled antisense cRNA probe, generated from the extracellular domain of the red deer prolactin receptor, confirmed the expression of the receptor in both the testis and epididymis; a higher level of prolactin receptor mRNA was detected in the epididymis compared with the testis (170.4+/-1.5 x 10(3) and 26.3+/-2.7 x 10(3) arbitrary units respectively; P<0.05). In situ hybridisation using 300 bp [33P]-labelled sense and antisense cRNA probes generated from the extracellular domain of the receptor localised the expression sites to the seminiferous tubules and interstitial compartments of the testis and the epithelial layer of the epididymal duct. Quantification of grain numbers demonstrated a higher level of expression of the receptor in the epididymis compared with the interstitial and seminiferous tubule compartments of the testis (18.1+/-4.4 x 10(2), 10.1+/-2.0 x 10(2) and 8.3+/-0.8 x 10(2) grains/microm2 respectively; P<0.05). However, no differences were detected in the level of expression of the receptor between the interstitial and seminiferous tubule compartments of the testis. Immunocytochemistry using an anti-prolactin receptor antibody, raised against a peptide sequence from the extracellular domain of the rat prolactin receptor, localised expression of the receptor gene to the Leydig cells, pachytene spermatocytes, round spermatids and elongating spermatids. In the epididymis, the receptor was localised to the epithelial layer within the epididymal ducts. Expression of the prolactin receptor gene in the red deer testis and epididymis suggests a role for the hormone in steroidogenesis and spermatogenesis.
Animals↗
Alternative splicing of the prolactin receptor gene generates a 1.7 kb RNA transcript that is linked to prolactin function in the red deer testis.
A cDNA encoding a putative non-membrane bound prolactin receptor was amplified by RT-PCR from red deer (Cervus elaphus) testis. Sequence analysis suggests that the testicular cDNA is generated by alternative splicing resulting in the deletion of exons 7 and 8, which code for: (a) the final 53 aa of the extracellular domain of the receptor including the fifth conserved cysteine residue and the WS x WS motif, (b) the entire transmembrane domain, (c) the first three cytoplasmic amino acid residues, and (d) two nucleotides of the fourth cytoplasmic amino acid codon. The resultant RNA would encode a putative protein of 174 aa due to a single bp frame shift and a premature stop codon. Northern blot analysis confirmed that the PCR-amplified cDNA is encoded by a specific 1.7 kb RNA transcript whereas the membrane bound receptor is encoded by transcripts of 3.5 and 2.5 kb. HPLC studies using media from 293 cells transfected with the 1.7 kb cDNA failed to detect any specific binding for prolactin. These data suggest that: (a) the deletion in the 1.7 kb transcript alters the structure of the prolactin binding domain in the putative protein encoded by the 1.7 kb transcript, and (b) alternative splicing of the prolactin receptor gene toward the 1.7 kb transcript is a means of down-regulating the expression of the full length prolactin receptor and hence may modify the role of prolactin in the testis of seasonally breeding mammals such as red deer. The sequence reported in this paper has been deposited in the Genbank/EMBL data base with accession number Y14753.
Alternative Splicing↗
Murine mucopolysaccharidosis type I: targeted disruption of the murine alpha-L-iduronidase gene.
Mucopolysaccharidosis type I (MPS I) is considered to represent the prototypical mucopolysaccharide storage disorder. Although a spectrum of severity is seen within the MPS I subgroup, Hurler syndrome represents the most severe and frequent manifestation of MPS I. We describe here the generation of a murine model for Hurler syndrome by targeted disruption of the murine Idua gene. Homozygous Idua -/- mice have no detectable alpha-L-iduronidase enzyme activity and show increased urinary glycosaminoglycan levels. Although normal appearing at birth, Idua -/- mice develop a flattened facial profile and thickening of the digits discernible by 3 weeks of age. No obvious growth deficiency nor mortality is seen within the first 20 weeks of life. Radiographs reveal anterior flaring of the ribs and thickening of the facial bones as early as 4 weeks of age with more extensive dysostosis detectable by 15 weeks of age. At 4 weeks of age, lysosomal storage is noted primarily within reticuloendothelial cells with abundant lysosomes noted in Kupffer cells, splenic sinusoidal lining cells, and glial cells. More widespread lysosomal storage is noted by 8 weeks of age in hepatocytes, chondrocytes, neurons, as well as renal tubular cells. Thus, targeted disruption of the murine Idua locus has produced a murine strain representative of the severe form of MPS I. This model should permit detailed evaluation of the pathophysiology of lysosomal storage disorders and provide a small animal model for the testing and development of enzyme replacement and gene therapy regimes.
Abnormalities, Multiple↗
Expression and localization of prolactin receptor messenger ribonucleic acid in red deer ovary during the estrous cycle and pregnancy.
In this study, expression of the prolactin receptor (PRL-R) gene in the ovaries of cycling and pregnant red deer (Cervus elaphus) hinds was investigated. A 1.9-kilobase (kb) cDNA encoding the cervine long-form PRL-R was amplified by reverse transcriptase polymerase chain reaction from corpus luteum (CL) and liver poly(A)+ RNA. Northern hybridization revealed a major mRNA transcript of 3.5 kb in both tissues. PRL-R mRNA transcripts were localized by in situ hybridization in 15-micron frozen sections of red deer ovaries, collected during the estrous cycle and early pregnancy, with homologous 45-mer [35S]dATP-labeled sense and antisense oligonucleotide probes. Specific hybridization was assessed by measurement of autoradiograph optical density (OD) in CL, follicles and stroma. PRL-R mRNA expression was higher (p < 0.001) in the CL (OD = 22.2 +/- 3.77, n = 11 CL) than in follicles (OD = 2.8 +/- 0.10, n = 224 follicles) and was undetectable in the stroma (OD < 1, limit of detection). No differences in abundance of PRL-R mRNA were observed between follicles divided on the basis of size, health vs. atresia, or stage of estrous cycle or pregnancy, or between CL from pregnant and nonpregnant hinds. In the follicles, PRL-R mRNA was localized to the theca layer. These results suggest a direct role for PRL in red deer ovarian physiology during the estrous cycle and pregnancy.
Animals↗
A unique point mutation in the fibroblast growth factor receptor 3 gene (FGFR3) defines a new craniosynostosis syndrome.
The underlying basis of many forms of syndromic craniosynostosis has been defined on a molecular level. However, many patients with familial or sporadic craniosynostosis do not have the classical findings of those craniosynostosis syndromes. Here we present 61 individuals from 20 unrelated families where coronal synostosis is due to an amino acid substitution (Pro250Arg) that results from a single point mutation in the fibroblast growth factor receptor 3 gene on chromosome 4p. In this instance, a new clinical syndrome is being defined on the basis of the molecular finding. In addition to the skull findings, some patients had abnormalities on radiographs of hands and feet, including thimble-like middle phalanges, coned epiphyses, and carpal and tarsal fusions. Brachydactyly was seen in some cases; none had clinically significant syndactyly or deviation of the great toe. Sensorineural hearing loss was present in some, and developmental delay was seen in a minority. While the radiological findings of hands and feet can be very helpful in diagnosing this syndrome, it is not in all cases clearly distinguishable on a clinical basis from other craniosynostosis syndromes. Therefore, this mutation should be tested for in patients with coronal synostosis.
Adult↗
Cloning, sequencing and functional analysis of a truncated cDNA encoding red deer prolactin receptor: an alternative tyrosine residue mediates beta-casein promoter activation.
This study reports the isolation and in vitro characterisation of a truncated cDNA encoding the red deer long form prolactin receptor. The cDNA sequence predicts a protein of 557 amino acids which differs from the rat sequence by a 3' truncation of the cytoplasmic domain located 34 residues before the stop codon. The deer sequence shares the regions of homology which are important for maintenance of structural and functional integrity, high affinity binding and signal transduction. However, the truncated deer receptor lacks the most C-terminal tyrosine residue in the intracellular domain which is believed to be essential for activation of the beta-casein promoter. Transfection studies of the cervine cDNA into human 293 fibroblast cells confirmed the expression of a receptor that has high affinity binding to ovine prolactin (Ka = 0.65 x 10(9)M(-1) and Bmax = 548.6 fmol/mg protein). Co-transfection of CHO cells with expression vector encoding the cervine prolactin receptor cDNA along with a fusion gene containing the promoter region of beta-casein followed by beta-luciferase coding sequence led to 8.13 +/- 0.13-fold induction of luciferase enzyme activity in the presence of 400 ng/ml ovine prolactin. This was comparable to fold induction observed with the wild type long form rat prolactin receptor (6.37 +/- 0.48); macaque growth hormone receptor was without effect. Western blot analysis demonstrated tyrosine phosphorylation of the cervine receptor and the associated kinase Jak2 following stimulation with prolactin. This confirms that the cervine cDNA although truncated is fully functional and that Jak2 and an alternative tyrosine residue in the intracellular domain are involved in the signalling pathway leading to activation of the beta-casein promoter. Northern blot analysis provides evidence that the prolactin receptor in the liver is encoded by transcripts of approximately 2.5 and 3.5 kb. Comparison of Northern blots of different deer species suggests that the receptor is conserved amongst the Cervidae. Northern blot analysis of red deer testis suggests that this species expresses a second form of the receptor, encoded by a transcript of 1.7 kb, which may correspond to a smaller receptor form or a binding protein.
Amino Acid Sequence↗
Identical mutations in three different fibroblast growth factor receptor genes in autosomal dominant craniosynostosis syndromes.
Pfeiffer syndrome (PS; McKusick MIM 101,600) is an autosomal dominant craniosynostosis syndrome with characteristic craniofacial anomalies and broad thumbs and big toes. We have previously demonstrated genetic heterogeneity in PS and mapped a gene to chromosome 8 (ref. 3) and a second to chromosome 10 (ref. 4). The gene on chromosome 8 is the fibroblast growth factor receptor 1 (FGFR1) with a common mutation (C755G) predicting a Pro252Arg substitution. The gene on chromosome 10 is FGFR2 with several different mutations causing sporadic and familial PS (Table 1). We report a recurrent single point mutation in the FGFR3 gene, located on chromosome 4p, in ten unrelated families with craniosynostosis syndromes. This mutation (C749G) predicts a Pro250Arg amino acid substitution in the extracellular domain of the FGFR3 protein. Interestingly, this common mutation occurs precisely at the analogous position within the FGFR3 protein as the mutations in FGFR1 (Pro252Arg) and FGFR2 (Pro253Arg) previously reported in Pfeiffer and Apert syndromes, respectively.
Acrocephalosyndactylia↗
Molecular genetics of mucopolysaccharidosis type I: diagnostic, clinical, and biological implications.
Mucopolysaccharidosis type I (MPS-I) is an autosomal recessive disease caused by mutations in the alpha-L-iduronidase (IDUA) gene. These mutations lead to a deficiency of the glycosidase alpha-L-iduronidase (IDUA), which is required for the degradation of heparan sulphate and dermatan sulphate and thus the storage of these glycosaminoglycans in the lysosome. There is a wide range of clinical phenotypes in MPS-I (eponyms: Hurler syndrome, severe; Hurler/Scheie syndrome, intermediate; Scheie syndrome, mild), which makes prediction of disease severity and genetic counselling difficult. However, since cloning of the IDUA gene, mutation analysis has provided some molecular explanations for the range of MPS-I phenotypes, in turn facilitating the selection and evaluation of patients undergoing experimental treatment protocols such as bone marrow transplantation. A total of 46 mutations now have been defined for MPS-I consisting of 8 nonsense mutations, 21 missense mutations, 3 splice site mutations, and 14 minor deletions and/or insertions. Furthermore, 30 polymorphisms or nonpathogenic sequence variants have been defined, including 7 amino acid substitutions. Among patients of European origin, there are two major MPS-I mutations and a number of less frequent mutations. It is possible to follow mutation analysis of 292 patients, which can be divided into eight main patient groups of different ethnic and/or geographic origin with significant variation in mutant allele frequencies. A complex picture of molecular heterogeneity is emerging, building a valuable database for genotype/phenotype correlation. Mutation analysis is also providing some of the first clues into the structure and function of IDUA.
Codon, Nonsense↗
Analysis of desmethylmethsuximide using high-performance liquid chromatography.
Analysis of desmethylmethsuximide by high-performance liquid chromatography (HPLC) is described. After adding an internal standard (IS), 200 microliters of plasma was buffered to pH 4.5 and extracted with dichloroethane. The organic solvent was then evaporated to dryness and the residue reconstituted in 100 microliters of mobile phase prior to injecting a 20 microliters aliquot onto a Hypersil 5 MOS column, which was eluted with acetonitrile/acetate buffer (pH 5.5) 36:64 vol/vol. Constituents were separated in approximately 8 min. Using this method, down to 1.0 mg/L of desmethylmethsuximide in plasma can be accurately determined. The method is suitable for therapeutic monitoring of desmethylmethsuximide in patient samples.
Anticonvulsants↗
Expression of the prolactin receptor gene during the breeding and non-breeding seasons in red deer (Cervus elaphus): evidence for the expression of two forms in the testis.
The red deer is a seasonally breeding mammal with a circannual cycle of prolactin secretion which reaches its peak during the non-breeding season. This study investigated expression of the prolactin receptor gene in red deer tissues collected in the breeding and non-breeding seasons. A 562 bp fragment of the extracellular domain of the red deer prolactin receptor cDNA was amplified from red deer liver poly(A)+ RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) using primers designed from the human sequence. Northern blots were prepared using 10-20 micrograms poly(A)+ RNA. The blots were hybridized to the 562 bp cDNA labelled by random priming with alpha 32P-dCTP. A main transcript of 3.5 kb was expressed in liver, heart, kidney and testis throughout the year and in epididymis during the breeding season only. In the testis an additional major transcript of 1.7 kb was present during the breeding and non-breeding seasons. Competitive binding assays using 125I-ovine prolactin (125I-oPRL) were performed on microsomal membrane fractions prepared from liver. Scatchard analyses confirmed the presence of a single class of lactogen-binding receptor with a mean Ka of 0.87 +/- 0.12 x 10(9) M-1 and a Bmax of 73.6 +/- 9.8 fmol/mg protein (n = 5). Cross-linking of 125I-oPRL to liver microsomes with 0.5 mM disuccinimidyl suberate followed by SDS-PAGE revealed a major band of molecular mass 56 kDa which was displaced by ovine prolactin, suggesting a specific lactogen-binding entity of 33 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗