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Biomedical subjects

J A Phillips

Publications and source records attributed to J A Phillips.

At least 55 records · Page 3Linked to original sources
PubMed1998

Characterization of an intron splice enhancer that regulates alternative splicing of human GH pre-mRNA.

Splicing of pre-mRNA transcripts is regulated by consensus sequences at intron (intervening sequence, IVS) boundaries and the branch site. In vitro studies have shown that the small introns of some genes also require intron splice enhancers (ISE) to modulate splice site selection. An autosomal dominant form of isolated GH deficiency (IGHD-II) is caused by mutations in IVS3 of the GH-1 gene that cause exon 3 (E3) skipping, resulting in truncated hGH products that prevent secretion of normal hGH. Interestingly, some of these IGHD-II mutations perturb an ISE that is buried in IVS3. We localized this ISE by quantitating the effects of deletions within IVS3 on E3 skipping. The importance of individual nucleotides to ISE function was determined by analyzing the effects of point mutants and additional deletions. Our results show that (i) an ISE with a G2X1-4G3motif resides in IVS3 of GH-1; (ii) both runs of Gs are required for ISE function; (iii) a single copy of the ISE regulates E3 skipping and (iv) ISE function can be modified by an adjacent AC element. Our findings reveal a new mechanism by which mutations can cause inherited human endocrine disorders and suggest that (i) ISEs may regulate splicing of transcripts of other genes and (ii) mutations of these ISEs or of the trans -acting factors that bind them may cause other genetic disorders.

Alternative Splicing↗
PubMed1998

The PROP1 2-base pair deletion is a common cause of combined pituitary hormone deficiency.

Combined pituitary hormone deficiency (CPHD) has an incidence of approximately 1 in 8000 births. Although the proportion of familial CPHD cases is unknown, about 10% have an affected first degree relative. We have recently reported three mutations in the PROP1 gene that cause CPHD in human subjects. We report here the frequency of one of these mutations, a 301-302delAG deletion in exon 2 of PROP1, in 10 independently ascertained CPHD kindreds and 21 sporadic cases of CPHD from 8 different countries. Our results show that 55% (11 of 20) of PROP1 alleles have the 301-302delAG deletion in familial CPHD cases. Interestingly, although only 12% (5 of 42) of the PROP1 alleles of our 21 sporadic cases were 301-302delAG, the frequency of this allele (in 20 of 21 of the sporadic subjects given TRH stimulation tests) was 50% (3 of 6) and 0% (0 of 34) in the CPHD cases with pituitary and hypothalamic defects, respectively. Using whole genome radiation hybrid analysis, we localized the PROP1 gene to the distal end of chromosome 5q and identified a tightly linked polymorphic marker, D5S408, which can be used in segregation studies. Analysis of this marker in affected subjects with the 301-302delAG deletion suggests that rather than being inherited from a common founder, the 301-302delAG may be a recurring mutation.

Alleles↗
PubMed1998

Clinical and molecular characterization of Brazilian patients with growth hormone gene deletions.

Genomic DNA from 23 patients with isolated growth hormone (GH) deficiency (12 males and 11 females: heights -4.9 +/- 1.4 SDS) was screened for GH gene deletions by restriction endonuclease analysis of polymerase chain reaction amplification products. Three unrelated patients had typical features of severe GH deficiency and deletions (6.7 kb in two and 7.6 kb in one) of the GH gene. The two patients with 6.7-kb deletions developed growth-attenuating anti-GH antibodies whereas the patient with the 7.6-kb deletion continued to grow with GH replacement therapy. Our finding that 3/23 (approximately 13%) Brazilian subjects had GH gene deletions agrees with previous studies of severe isolated GH deficiency subjects in other populations. Two of three subjects (67%) with deletions developed blocking antibodies despite administration of exogenous GH at low doses. Interestingly, only 1/10 of cases with affected relatives or parental consanguinity had GH-1 gene deletions.

Brazil↗
PubMed1998

Growth disorders caused by genetic defects in the growth hormone pathway.

The growth hormone (GH) pathway is composed of a series of interdependent genes whose products are required for normal growth (Fig 1). The GH pathway genes include ligands (GH and insulin-like growth factor 1 [lGF-1]), transcription factors (prophet of pit 1, or prop 1 and pit 1), agonists and antagonists (growth hormone-releasing hormone [GHRH] and somatostatin), and receptors (GHRH receptor [GHRHR] and the GH receptor [GHR]). These genes are expressed in different organs and tissues, including the hypothalamus, pituitary, liver, and bone. Effective and regulated expression of the growth hormone pathway is essential for growth in stature as well as homeostasis of carbohydrate, protein, and fat metabolism.

Growth Disorders↗
PubMed1997

DiGeorge anomaly and chromosome 10p deletions: one or two loci?

We report on a patient with DiGeorge syndrome (DGS) phenotype or anomaly and an unbalanced translocation [45,XY,-10,-22,+der(10),t(10;22)(p13;q11)] resulting in monosomy of 10p13-pter and 22q11-pter. Because both regions involved in this rearrangement have been implicated in DGS, we performed a molecular cytogenetic analysis of both loci in this patient. Results indicate that the chromosome 22 DGS locus is intact but that the terminal deletion of the short arm of chromosome 10 is adjacent to or partially overlapping with the recently defined consensus deleted region observed in DGS patients with 10p deletions. We conclude that the DGS anomaly in our patient is likely to be due to haploinsufficiency of genes located on chromosome 10p. Most, if not all, of the region included in the previously described 10p smallest region of deletion overlap is not deleted in our patient. Therefore, this deletion breakpoint either narrows the previously proposed 10p region or defines a second region within 10p critical for the DGS anomaly.

Adult↗
PubMed1997

CD4+ T cells from IRF-1-deficient mice exhibit altered patterns of cytokine expression and cell subset homeostasis.

Interferon regulatory factor-1 (IRF-1) is a member of a family of transcription factors that regulate an array of genes involved in cell growth, differentiation, and death. Analysis of cytokine expression by stimulated CD4+ cells from IRF-1(-/-) and IRF-1(+/+) mice revealed that IRF-1 deficiency resulted in an elevated production of Th2-related cytokines and a compensatory decrease in the expression of naive cell- and Th1-related cytokines. The altered cytokine profiles of IRF-1(-/-) cells could be explained, in part, by a shift in the representation of subsets of CD4+ cells; IRF-1(-/-) mice exhibited a decreased percentage of naive cells (a major source of IL-2) but increased numbers of memory or effector cells (the source of Th2-related cytokines). We analyzed purified, phenotypically matched memory/effector cells from IRF-1(-/-) and IRF-1(+/+) mice and found that the increased Th2:Th1 cytokine ratio was still evident in the IRF-1(-/-) group, thus suggesting that IRF-1 is involved in the polarization of the cytokine repertoire in CD4+ cells. Our data indicate that IRF-1 plays an important role in the maintenance of CD4+ cell subset homeostasis and in the expression of cytokines by naive and memory/effector cells.

Animals↗
PubMed1997

Pharmacokinetics, metabolism, and elimination of a 20-mer phosphorothioate oligodeoxynucleotide (CGP 69846A) after intravenous and subcutaneous administration.

The pharmacokinetics, tissue distribution and metabolism of CGP 69846A, a 20-mer phosphorothioate oligodeoxynucleotide targeted against the 3'-untranslated region of human c-raf-1 kinase mRNA, were investigated in vivo in rats after intravenous and subcutaneous administration. Intravenous disposition studies with [3H]CGP 69846A were supported with analysis by capillary gel electrophoresis and electrospray mass spectrometry. In combination, these techniques provide a detailed account of the pharmacokinetic and metabolic profile for this compound. The elimination of CGP 69846A after a single intravenous dose was studied over extended periods in mice using whole-body autoradiography and capillary gel electrophoresis. Subcutaneous administration to rats resulted in a significant bioavailability with peak plasma levels 4.5-fold lower than after intravenous dosing. This dose route resulted in low interanimal variability and only slightly greater metabolism of the oligonucleotide compared to the intravenous administration.

Animals↗
PubMed1997

The human HNP36 gene is localized to chromosome 11q13 and produces alternative transcripts that are not mutated in multiple endocrine neoplasia, type 1 (MEN I) syndrome.

Multiple endocrine neoplasia, type 1 (MEN I), is an autosomal dominant syndrome of selected endocrine neoplasms whose causative gene, a suspected tumor suppressor, has been localized to chromosome 11q13, but has not been identified. Recently, the HNP36 cDNA was identified as a novel growth factor responsive gene of undetermined biological function that is expressed in the pituitary and parathyroid glands. In studies seeking the function of the HNP36 gene product, the gene was localized by fluorescence in situ hybridization within the 11q13 segment. Further analysis of radiation-reduced hybrid DNAs and chromosome 11-specific YAC clones established that the HNP36 gene is within 80 kb of D11S913, a marker tightly linked to the MEN1 gene. Consequently, the HNP36 gene was studied as a candidate for the MEN1 gene. The human HNP36 gene was cloned and determined to consist of 12 exons. Expression of the HNP36 gene from pituitary and parathyroid tissue and four patient tumors or lymphoblasts was confirmed by RT-PCR amplification of the coding sequences, and HNP36 transcripts were analyzed for mutations. All tissues expressed three HNP36 gene transcripts that result from alternative splicing and appear to encode related, but distinct, proteins. However, DNA sequence determination of the RT-PCR products from MEN I-associated tumors found no deletions and identified a single nucleotide difference that may be a polymorphism. Thus, mutations in the coding segments of the HNP36 gene are not the cause of the MEN I syndrome. Nevertheless, the assignment of the HNP36 gene to 11q13 and identification of new potential gene products provides a novel growth-regulated genetic candidate for other disorders whose genes map to this locus.

Alternative Splicing↗
PubMed1997

Skeletal muscle histology and biochemistry of an elite sprinter, the African cheetah.

To establish a skeletal muscle profile for elite sprinters, we obtained muscle biopsy samples from the vastus lateralis, gastrocnemius and soleus of African cheetahs (Acinonyx jubatus). Muscle ultrastructure was characterized by the fiber type composition and mitochondrial volume density of each sample. Maximum enzyme activity, myoglobin content and mixed fiber metabolite content were used to assess the major biochemical pathways. The results demonstrate a preponderance of fast-twitch fibers in the locomotor muscles of cheetahs; 83% of the total number of fibers examined in the vastus lateralis and nearly 61% of the gastrocnemius were comprised of fast-twitch fibers. The total mitochondrial volume density of the limb muscles ranged from 2.0 to 3.9% for two wild cheetahs. Enzyme activities reflected the sprinting capability of the cheetah. Maximum activities for pyruvate kinase and lactate dehydrogenase in the vastus lateralis were 1519.00 +/- 203.60 and 1929.25 +/- 482.35 mumol min-1.g wet wt-1, respectively, and indicated a high capacity for glycolysis. This study demonstrates that the locomotor muscles of cheetahs are poised for anaerobically based exercise. Fiber type composition, mitochondrial content and glycolytic enzyme capacities in the locomotor muscles of these sprinting cats are at the extreme range of values for other sprinters bred or trained for this activity including greyhounds, thoroughbred horses and elite human athletes.

Acinonyx↗
PubMed1997

Specific dynamic action of a large carnivorous lizard, Varanus albigularis.

Varanus albigularis inhabits grasslands of southern and eastern Africa and experiences months of fasting during the dry season (May-December) followed by voracious feeding during the wet season (January-April). Previous studies have found that sit-and-wait foraging snakes, which also experience long intervals between large meals, exhibit unprecedented increases in post-feeding metabolism, which reflects the added cost of up-regulating a previously quiescent gut and digesting a large meal. Hence we measured pre- and post-prandial oxygen consumption rates (VO2) of adult V. albigularis in order to observe whether they exhibit similarly large metabolic responses to digestion as sit-and-wait foraging snakes. Following the consumption of meals consisting of ground turkey and snails, hard-boiled eggs, or juvenile rats, lizards rapidly increased their VO2 to peak within 24-27 hr at 7-10 times pre-feeding values (mean = 0.035 mL O2.g-1.h-1). During the 60-90 hr of significantly elevated VO2, the extra oxygen consumed (the specific dynamic action) represented an energy expenditure of 830-1260 kJ. For meals that were fully digested, specific dynamic action equalled 24% of ingested energy. The magnitudes of V. albigularis post-prandial metabolic responses are similar to those previously observed for sit-and-wait foraging snakes. Like sit-and-wait foraging snakes, V. albigularis may also down-regulate intestinal performance during their months of fasting (suggested by their relatively low standard metabolic rate) and then up-regulate their gut (bearing its high energetic cost) upon feeding.

Animals↗
PubMed1997

Genomic organization of a human cystine transporter gene (SLC3A1) and identification of novel mutations causing cystinuria.

Cystinuria is a common inherited aminoaciduria that leads to recurrent cystine nephrolithiasis. Mutations in a gene encoding a renal amino acid transporter (SLC3A1) have been identified in patients with cystinuria establishing one molecular cause for the disease. To facilitate systematic screening of this gene for mutations, we have delineated the complete genomic organization of the SLC3A1 coding region using polymerase chain reaction strategies. The complete coding region of the gene is contained within a single yeast artificial chromosome clone and consists of 10 exons and 9 introns. Oligonucleotide primers capable of amplifying selected exons have been made and used in mutational analysis of DNA from 24 cystinuria probands. We illustrate the usefulness of this approach by identifying two novel SLC3A1 mutations. One novel mutation causes replacement of a highly conserved arginine residue (arginine-452) with tryptophan in the cytoplasmic loop between the putative third and fourth membrane spanning segments. A second previously unreported mutation results in replacement of a highly conserved tyrosine (tyrosine-461) residue with histidine in the same region of the protein. In addition, we detected three previously reported SLC3A1 mutations, R270X, 1500 +1/G to T, and M467T, the latter being present in approximately 20% of cystinuria chromosomes examined. Our findings provide a foundation for the development of more accessible diagnostic screening assays for detecting SLC3A1 mutations using patient genomic DNA, and also contribute to the emerging spectrum of cystinuria genotypes.

Amino Acid Sequence↗
PubMed1997

Localization of the gene for familial primary pulmonary hypertension to chromosome 2q31-32.

Primary pulmonary hypertension (PPH), an often fatal disease, is characterized by elevated pulmonary artery pressures in the absence of a secondary cause. Endovascular occlusion in the smallest pulmonary arteries occurs by proliferation of cells and matrix, with thrombus and vasospasm. Diagnosis is often delayed because the initial symptoms of fatigue and dyspnea on exertion are nonspecific and definitive diagnosis requires invasive procedures. The average life expectancy after diagnosis is two to three years with death usually due to progressive right heart failure. The aetiology of the disease is unknown. Although most cases appear to be sporadic, approximately 6% of cases recorded in the NIH Primary Pulmonary Hypertension Registry are inherited in an autosomal dominant manner with reduced penetrance. Following a genome-wide search using a set of highly polymorphic short tandem repeat (STR) markers and 19 affected individuals from six families, initial evidence for linkage was obtained with two chromosome 2q markers. We subsequently genotyped patients and all available family members for 19 additional markers spanning approximately 40 centiMorgans (cM) on the long arm of chromosome 2. We obtained a maximum two-point lod score of 6.97 at theta = 0 with the marker D2S389; multipoint linkage analysis yielded a maximum lod score of 7.86 with the marker D2S311. Haplotype analysis established a minimum candidate interval of approximately 25 cM.

Centromere↗
PubMed1997

A novel mechanism of aberrant pre-mRNA splicing in humans.

Eukaryotic pre-mRNA splicing is regulated by consensus sequences at the intron boundaries and branch site. Recently, Sirand-Pugnet et al. reported the importance of an additional intronic sequence, an (A/U)GGG repeat in chicken beta-tropomyosin that is a binding site for a protein required for spliceosome assembly. Interestingly, we have detected mutations in IVS3 of the human growth hormone (GH) gene that affect a putative, homologous consensus sequence and which also perturb splicing. In a series of dominant-negative GH mutations that cause exon skipping, we found two mutations that do not occur within the 5' and 3' splice sites, or branch consensus sites. The first mutation is a G-->A transition of the 28th base (+28G-->A) of and the second deletes 18 bp (del+28-45) of IVS3 of the human GH gene. These mutations segregated with autosomal dominant GH deficiency in both kindreds and no other allelic GH gene changes were detected. RT-PCR amplification of transcripts from expression vectors containing the +28G-->A or del+28-45 alleles yielded products showing a >10-fold preferred use of alternative splicing, similar to findings previously reported for IVS3 donor site mutations. Both mutations are located 28 bp downstream from the 5' splice site and examination of the sequences perturbed revealed an intronic XGGG repeat similar to the repeat found to regulate mRNA splicing in chicken beta-tropomyosin. Interestingly, the XGGG repeats involved in our mutations exhibit homologous spacing to those in a so-called 'winner' RNA sequence. Binding of A1 heterogeneous nuclear ribonucleoprotein (hnRNP) by 'winner' sequences in pre-mRNA transcripts is thought to play an important role in pre-mRNA packaging and transport as well as 5' splice site selection in pre-mRNAs that contain multiple 5' splice sites. Our findings suggest that (i) XGGG repeats may regulate alternative splicing in the human GH gene and (ii) mutations of these repeats cause GH deficiency by perturbing alternative splicing. Mutations of homologous intron sequences may underlie other human diseases.

Alleles↗
PubMed1997

Heterogeneity in clinical manifestation of autosomal dominant neurohypophyseal diabetes insipidus caused by a mutation encoding Ala-1-->Val in the signal peptide of the arginine vasopressin/neurophysin II/copeptin precursor.

Autosomal dominant neurohypophyseal diabetes insipidus (ADNDI) is a familial form of diabetes insipidus due to progressive vasopressin deficiency with onset typically at 1-6 yr of age. Affected individuals demonstrate specific degeneration of the vasopressinergic magnocellular neurons in the hypothalamic supraoptic and paraventricular nuclei and loss of the posterior pituitary bright spot on magnetic resonance imaging. The genetic locus of ADNDI is the arginine vasopressin-neurophysin II (AVP-NPII) gene. Mutations that cause ADNDI have been found to occur both within the signal peptide of the prepro-AVP-NPII precursor and within the coding sequence for neurophysin II, but not within the coding sequence for AVP itself. We evaluated the AVP-NPII genes in two independent families with ADNDI and identified a mutation (C280-->T) in the coding sequence for the signal peptide of the prepro-AVP-NPII precursor in both families. This mutation encodes an Ala-->Val substitution at the C-terminus of the signal peptide (-1 amino acid). This mutation predicts the complete inability of signal peptidase to cleave the signal peptide from the preproprecursor and supports the hypothesis that the progressive neural degeneration that underlies ADNDI is caused by accumulation of malprocessed precursor. However, considerable heterogeneity in the age of onset (1-28 yr of age) and the severity of diabetes insipidus among affected members of these two families suggests that additional factors modulate the rate and extent of progression of the neurodegeneration that results from this one specific ADNDI mutation.

Alanine↗
PubMed1997

Detection of growth hormone gene defects by dideoxy fingerprinting (ddF).

We carried out screening for mutations in the GH-1 gene in 29 sporadic Japanese subjects with severe Isolated Growth Hormone Deficiency (IGHD) by dideoxy fingerprinting (ddF). Three of 29 (approximately 10%) were heterozygous for each of the following GH-1 gene mutations including: 1) an G-->A transition in the third codon of the GH-1 signal peptide of exon 1 resulting in a Threonine to Alanine substitution, 2) a G-->A transition in the first base of the donor splice site of IVS 3 (+1G-->A) and 3) a G-->A transition in the 183rd codon of the GH-1 mature peptide of exon 5 resulting in an Arginine to Histidine substitution. One of three was heterozygous for both mutations of 1) and 2). The IVS 3 (+1G-->A) mutation has been previously reported in affected individuals from three unrelated families with IGHD type II (autosomal dominant form). This mutation destroys the GH IVS 3 donor splice site, causing skipping of exon 3 and loss of the codons for amino acids 32-71 of the mature GH peptide. Our findings indicate that 1) ddF screening of genomic DNAs provides a practical tool to detect GH gene mutations and 2) some sporadic cases of IGHD may be caused by GH gene alternations.

Autoradiography↗
PubMed1997

Allelic variations in the human growth hormone-1 gene promoter of growth hormone-deficient patients and normal controls.

OBJECTIVE: Isolated growth hormone deficiency (IGHD) type IB is suggested to be more probably due to alterations in the genes directly involved in the hypothalamo-pituitary axis and/or in the specific transcriptional regulation (cis-trans coupling) of the hGH-1 gene than to alterations in the gene itself. In this study we analyzed the hGH-1 gene promoter region for structural alterations and allelic variations. METHODS: The hGH-1 gene promoter region was analyzed by PCR, cycle sequencing and direct-blotting electrophoresis in a total of 212 individuals including 113 patients with IGHD type IB, 21 unaffected family members and 78 normal controls. RESULTS: Twenty-two sequence variation sites were identified. Of these, 14% were located around the region of -1075bp, 77% between -550bp and the translational start site (+1bp) and 9% within the first intron. Only one variation site affected a characterized cis-acting element, namely that of NF-1. Importantly, all the variations found in patients were also observed in non-affected family members as well as in normal unrelated controls. CONCLUSIONS: These findings imply that it is not a single variation within the GH-1 gene promoter, and therefore in the cis-acting elements, which causes IGHD. However, we can not exclude the possibility that combinations of variations might perturb expression. Furthermore, these data illustrate the normal heterogeneity of the GH-1 gene promoter region, a fact that has to be borne in mind whenever transcriptional studies are performed.

Alleles↗
PubMed1997

Cystic fibrosis in the Brazilian population: DF508 mutation and KM-19/XV-2C haplotype distribution.

We have used PCR amplification of DNA obtained from Guthrie cards to identify the DF508 mutation and correlate it with the allele frequencies at two polymorphic loci (XV-2C and KM-19) closely linked to the cystic fibrosis gene. The DNA came from 193 white Brazilian families affected by cystic fibrosis and living in five different states of Brazil. The distribution of the haplotypes derived from the DF508 and non-DF508 XV-2C/KM-19 genotypes indicates that 88% of the DF508 alleles are linked to haplotype B and suggests that high heterogeneity exists among the non-DF508 cystic fibrosis alleles occurring in different states. Our data can be used to compare linkage disequilibrium between Brazilians and other heterogeneous populations where the DF508 mutation frequency is low and where many different rare mutations account for the remaining recessive cystic fibrosis alleles.

Adolescent↗