Search PubMed⌕ Search

Biomedical subjects

P C Harris

Publications and source records attributed to P C Harris.

At least 19 recordsLinked to original sources
PubMed2001

Polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease (ADPKD), is a Ca2+-permeable nonselective cation channel.

Defects in polycystin-2, a ubiquitous transmembrane glycoprotein of unknown function, is a major cause of autosomal dominant polycystic kidney disease (ADPKD), whose manifestation entails the development of fluid-filled cysts in target organs. Here, we demonstrate that polycystin-2 is present in term human syncytiotrophoblast, where it behaves as a nonselective cation channel. Lipid bilayer reconstitution of polycystin-2-positive human syncytiotrophoblast apical membranes displayed a nonselective cation channel with multiple subconductance states, and a high perm-selectivity to Ca2+. This channel was inhibited by anti-polycystin-2 antibody, Ca2+, La3+, Gd3+, and the diuretic amiloride. Channel function by polycystin-2 was confirmed by patch-clamping experiments of polycystin-2 heterologously infected Sf9 insect cells. Further, purified insect cell-derived recombinant polycystin-2 and in vitro translated human polycystin-2 had similar ion channel activity. The polycystin-2 channel may be associated with fluid accumulation and/or ion transport regulation in target epithelia, including placenta. Dysregulation of this channel provides a mechanism for the onset and progression of ADPKD.

Animals↗
PubMed2001

Treatment prospects for autosomal-dominant polycystic kidney disease.

An increased understanding of the molecular genetic and cellular pathophysiologic mechanisms responsible for the development of autosomal-dominant polycystic kidney disease (ADPKD), made possible by the advances in molecular biology and genetics of the last three decades, has laid the foundation for the development of effective therapies. As the concept that a polycystic kidney is a neoplasm in disguise is becoming increasingly accepted, the development of therapies for ADPKD may benefit greatly from the expanding body of information on cancer chemoprevention and chemosuppression. This review summarizes the observations that already have been made and discusses therapies for PKD that deserve investigation.

Anti-Inflammatory Agents↗
PubMed2000

Mutation analysis of the entire PKD1 gene: genetic and diagnostic implications.

Mutation screening of the major autosomal dominant polycystic kidney disease (ADPKD) locus, PKD1, has proved difficult because of the large transcript and complex reiterated gene region. We have developed methods, employing long polymerase chain reaction (PCR) and specific reverse transcription-PCR, to amplify all of the PKD1 coding area. The gene was screened for mutations in 131 unrelated patients with ADPKD, using the protein-truncation test and direct sequencing. Mutations were identified in 57 families, and, including 24 previously characterized changes from this cohort, a detection rate of 52.3% was achieved in 155 families. Mutations were found in all areas of the gene, from exons 1 to 46, with no clear hotspot identified. There was no significant difference in mutation frequency between the single-copy and duplicated areas, but mutations were more than twice as frequent in the 3' half of the gene, compared with the 5' half. The majority of changes were predicted to truncate the protein through nonsense mutations (32%), insertions or deletions (29.6%), or splicing changes (6.2%), although the figures were biased by the methods employed, and, in sequenced areas, approximately 50% of all mutations were missense or in-frame. Studies elsewhere have suggested that gene conversion may be a significant cause of mutation at PKD1, but only 3 of 69 different mutations matched PKD1-like HG sequence. A relatively high rate of new PKD1 mutation was calculated, 1.8x10-5 mutations per generation, consistent with the many different mutations identified (69 in 81 pedigrees) and suggesting significant selection against mutant alleles. The mutation detection rate, in this study, of >50% is comparable to that achieved for other large multiexon genes and shows the feasibility of genetic diagnosis in this disorder.

Alleles↗
PubMed2000

A human PKD1 transgene generates functional polycystin-1 in mice and is associated with a cystic phenotype.

Three founder transgenic mice were generated with a 108 kb human genomic fragment containing the entire autosomal dominant polycystic kidney disease (ADPKD) gene, PKD1, plus the tuberous sclerosis gene, TSC2. Two lines were established (TPK1 and TPK3) each with approximately 30 copies of the transgene. Both lines produced full-length PKD1 mRNA and polycystin-1 protein that was developmentally regulated, similar to the endogenous pattern, with expression during renal embryogenesis and neonatal life, markedly reduced at the conclusion of renal development. Tuberin expression was limited to the brain. Transgenic animals from both lines (and the TPK2 founder animal) often displayed a renal cystic phenotype, typically consisting of multiple microcysts, mainly of glomerular origin. Hepatic cysts and bile duct proliferation, characteristic of ADPKD, were also seen. All animals with two copies of the transgenic chromosome developed cysts and, in total, 48 of the 100 transgenic animals displayed a cystic phenotype. To test the functionality of the transgene, animals were bred with the Pkd1(del34) knockout mouse. Both transgenic lines rescued the embryonically lethal Pkd1(del34/del34) phenotype, demonstrating that human polycystin-1 can complement for loss of the endogenous protein. The rescued animals were viable into adulthood, although more than half developed hepatic cystic disease in later life, similar to the phenotype of older Pkd1(del34/+) animals. The TPK mice have defined a minimal area that appropriately expresses human PKD1. Furthermore, this model indicates that over-expression of normal PKD1 can elicit a disease phenotype, suggesting that the level of polycystin-1 expression may be relevant in the human disease.

Animals↗
PubMed2000

Screening of the PKD1 duplicated region reveals multiple single nucleotide polymorphisms and a de novo mutation in Hellenic polycystic kidney disease families.

Mutations in the PKD1 gene account for approximately 85% of cases with autosomal dominant polycystic kidney disease (ADPKD1; MIM# 601313), which is considered one of the most frequent monogenic disorders, with a frequency of approximately 1:1000. The main symptom is the formation of fluid-filled cysts in the kidneys and less often in other organs, such as the liver and pancreas. Since the cloning of the gene many mutations have been identified, although the screening is hampered by several unique features of this gene, the most significant one being that approximately 70% of the sequence at the 5'-end, is reiterated elsewhere on chromosome 16 with homology approaching 95%. Here, we used an oligonucleotide primer anchored in the unique part in exon 34, paired with a forward primer in exon 23 for specifically amplifying PKD1 sequences. We screened for mutations in samples from 32 Hellenic ADPKD families. We detected seven sequence variants, five of which most probably are single nucleotide polymorphisms (SNPs), especially useful for linkage analysis and disease association studies. One is a missense change, segregating with ADPKD in one family. The last one is a missense non-conservative change, H2921P, which appeared de novo in the proband, concurrently with the disease phenotype, and was passed on to another two generations. Two siblings who inherited the same haplotype as the proband, but not the de novo mutation, were not affected. This is only the fourth case of a molecularly documented de novo mutation in ADPKD. Somatic mosaicism in peripheral blood leukocytes of the proband was tested and excluded. Hum Mutat 16:176, 2000.

Aged↗
PubMed2000

Identification of a subtle t(16;19)(p13.3;p13.3) in an infant with multiple congenital abnormalities using a 12-colour multiplex FISH telomere assay, M-TEL.

There is increasing evidence that cytogenetically invisible chromosome rearrangements are an important cause of genetic disease. Clues to the chromosomal location of these rearrangements may be provided by a specific clinical diagnosis, which can then be investigated by targeted FISH or molecular studies. However, the phenotypic features of some microdeletion syndromes are difficult to recognise, particularly in infants. In addition, the presence of other chromosome aneuploidy may mask the typical clinical features. In the present study, the presence of tubers on cranial magnetic resonance imaging (MRI) of a 5-week-old infant prompted an investigation, by FISH, with probes from the tuberous sclerosis gene, TSC2. This and further FISH deletion mapping studies revealed a submicroscopic deletion encompassing the entire TSC2 gene and the adjacent PKD1 gene on one chromosome 16, confirming a del(16)(p13.3). Because of the large number of abnormal phenotypic features in this infant, we performed a 12-colour FISH assay (M-TEL) to screen for subtelomeric rearrangements involving the del(16p). The M-TEL assay revealed a cryptic der(16)t(16;19)(p13.3;p13.3). Further FISH with 19p and 19q subtelomeric probes demonstrated that this was derived from a balanced maternal t(16;19)(p13.3;p13.3). Importantly, 24-colour painting by multiplex FISH (M-FISH) failed to detect the translocation in either the infant or his mother. Based on our FISH mapping studies, we estimate the size of the trisomic region from 19p13.3 to be approximately 2 Mb, and the region of monosomy for 16p13.3 as 2.25 Mb. This case adds to the growing literature which indicates that many apparent chromosomal deletions are unbalanced translocations. The M-TEL assay provides a sensitive alternative to M-FISH for the detection of these subtle telomeric rearrangements.

Abnormalities, Multiple↗
PubMed2000

Rhys-Davies exsanguinator: pressure characteristics and technique for improving performance.

This paper reports the results of an investigation into the pressure exerted by the Rhys-Davies exsanguinator on the palm and dorsum of the hand. We hypothesised that, due to the shape of the hand, the palm is shielded from the full effect of the exsanguinator, but our study showed that it is compressed as well as the dorsum. However, placing a bag of fluid in the patient's palm significantly increased the pressure applied to the palm.

Adult↗
PubMed1999

Genome duplications and other features in 12 Mb of DNA sequence from human chromosome 16p and 16q.

Several publicly funded large-scale sequencing efforts have been initiated with the goal of completing the first reference human genome sequence by the year 2005. Here we present the results of analysis of 11.8 Mb of genomic sequence from chromosome 16. The apparent gene density varies throughout the region, but the number of genes predicted (84) suggests that this is a gene-poor region. This result may also suggest that the total number of human genes is likely to be at the lower end of published estimates. One of the most interesting aspects of this region of the genome is the presence of highly homologous, recently duplicated tracts of sequence distributed throughout the p-arm. Such duplications have implications for mapping and gene analysis as well as the predisposition to recurrent chromosomal structural rearrangements associated with genetic disease.

Animals↗
PubMed1999

Coordinate expression of the autosomal dominant polycystic kidney disease proteins, polycystin-2 and polycystin-1, in normal and cystic tissue.

A second gene for autosomal dominant polycystic kidney disease (ADPKD), PKD2, has been recently identified. Using antisera raised to the human PKD2 protein, polycystin-2, we describe for the first time its distribution in human fetal tissues, as well as its expression in adult kidney and polycystic PKD2 tissues. Its expression pattern is correlated with that of the PKD1 protein, polycystin-1. In normal kidney, expression of polycystin-2 strikingly parallels that of polycystin-1, with prominent expression by maturing proximal and distal tubules during development, but with a more pronounced distal pattern in adult life. In nonrenal tissues expression of both polycystin molecules is identical and especially notable in the developing epithelial structures of the pancreas, liver, lung, bowel, brain, reproductive organs, placenta, and thymus. Of interest, nonepithelial cell types such as vascular smooth muscle, skeletal muscle, myocardial cells, and neurons also express both proteins. In PKD2 cystic kidney and liver, we find polycystin-2 expression in the majority of cysts, although a significant minority are negative, a pattern mirrored by the PKD1 protein. The continued expression of polycystin-2 in PKD2 cysts is similar to that seen by polycystin-1 in PKD1 cysts, but contrasts with the reported absence of polycystin-2 expression in the renal cysts of Pkd2+/- mice. These results suggest that if a two-hit mechanism is required for cyst formation in PKD2 there is a high rate of somatic missense mutation. The coordinate presence or loss of both polycystin molecules in the same cysts supports previous experimental evidence that heterotypic interactions may stabilize these proteins.

Aged↗
PubMed1999

Polycystin-1 expression in PKD1, early-onset PKD1, and TSC2/PKD1 cystic tissue.

BACKGROUND: The mutational mechanism responsible for cyst formation in polycystic kidney disease 1 gene (PKD1) remains controversial, with data indicating a two-hit mechanism, but also evidence of polycystin-1 expression in cystic tissue. METHODS: To investigate this apparent paradox, we analyzed polycystin-1 expression in cystic renal or liver tissue from 10 patients with truncating PKD1 mutations (including one early-onset case) and 2 patients with severe disease associated with contiguous deletions of TSC2 and PKD1, using monoclonal antibodies (mAbs) to both extreme N-(7e12) and C-terminal (PKS-A) regions of the protein. Truncation of the C-terminal epitope from the putative mutant proteins in each case allowed exclusive assessment of the nontruncated protein with PKS-A. RESULTS: In adult PKD1 tissue, the majority of cysts (approximately 80%) showed polycystin-1 expression, although staining was absent in a variable but significant minority (approximately 20%), in spite of the normal expression of marker proteins. Unlike adult PKD1, however, negative cysts were rarely found in infantile PKD1 or TSC2/PKD1 deletion cases. CONCLUSIONS: If a two-hit mutational mechanism is operational, these results suggest that the majority of somatic mutations in adult PKD1 are likely to be missense changes. The low level of polycystin-1-negative cysts in the three "early-onset" cases, however, suggests that a somatic PKD1 mutation may not always be required for cyst formation.

Adult↗
PubMed1999

Autosomal dominant polycystic kidney disease: clues to pathogenesis.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutation of one of two genes: PKD1 (16p13.3) or PKD2 (4q13-23). PKD1 accounts for approximately 85% of pedigrees and is associated with significantly more severe cystic disease. The ADPKD genes encode proteins, polycystin-1 and polycystin-2, which are very different in size and structure, but which have a region of homology and may interact as part of the same complex. Polycystin-1 is a large, integral membrane protein ( approximately 460 kDa) predicted to be involved in cell-cell and/or cell-matrix interactions. Polycystin-2 ( approximately 110 kDa) is related to polycystin-1 and voltage-activated and transient receptor potential channel subunits, suggesting that the polycystins may also be associated with ion transport. A polycystin complex could regulate cellular events (that are abnormal in ADPKD) in response to specific extracellular cues, mediated by controlling cellular Ca(2+)levels and/or other signalling pathways. Recently, two further polycystin-like molecules have been identified, indicating roles for this novel protein family beyond the kidney. A wide range of different mutations to the PKD1 or PKD2 gene have been detected, most predicted to truncate and inactivate the proteins. A somatic second hit may be required for focal cyst development, although there is widespread immunohistochemical evidence of polycystin expression in cystic epithelia. Disruption of the mouse Pkd1 gene leads to death in the perinatal period with massive cystic expansion in homozygotes and age-related cyst development in heterozygotes. Normal renal development in Pkd1(del34/del34)mice up to embryonic day approximately 15.5 suggests a role for polycystin-1 in developing and maintaining the tubular architecture, consistent with the localization of the protein, rather than nephron induction. Renal cystic disease in homo- and heterozygotes of a Pkd2 mouse model with a disrupted exon 1 inserted in tandem with the normal exon (and prone to somatic recombination, which inactivates the gene) supports a role for somatic events in cystogenesis.

Genetic Heterogeneity↗
PubMed1999

Identification of a human homologue of the sea urchin receptor for egg jelly: a polycystic kidney disease-like protein.

Previous studies have shown sequence similarity between a region of the autosomal dominant polycystic kidney disease (ADPKD) protein, polycystin-1 and a sea urchin sperm glycoprotein involved in fertilization, the receptor for egg jelly (suREJ). We have analysed sequence databases for novel genes encoding PKD/REJ-like proteins and found a significant region of homology to a large open reading frame in genomic sequence from human chromosome 22. Northern analysis showed that this is a functional gene [termed the polycystic kidney disease and receptor for egg jelly related gene ( PKDREJ )], but unlike polycystin-1, has a very restricted expression pattern; the approximately 8 kb transcript was found exclusively in testis, coincident with the timing of sperm maturation. The PKDREJ transcript was cloned by screening a testis cDNA library and RT-PCR which revealed a 7660 bp mRNA terminating with a 900 bp 3'UTR and a polyA tail. Comparison with genomic sequence showed that PKDREJ is intronless; sequencing the mouse orthologue revealed a similar structure. The predicted human PKDREJ protein has 2253 amino acids (calculated molecular mass 255 kDa) and sequence similarity over approximately 2000 amino acids with polycystin-1, corresponding to the predicted membrane associated region and the area of homology ( approximately 1000 amino acids) with the suREJ protein (the REJ module). The suREJ protein binds the glycoprotein coat of the egg (egg jelly), triggering the acrosome reaction, which transforms the sperm into a fusogenic cell. The sequence similarity and expression pattern suggests that PKDREJ is a mammalian equivalent of the suREJ protein and therefore may have a central role in human fertilization.

Amino Acid Sequence↗
PubMed1998

Facilitated diagnosis of the contiguous gene syndrome: tuberous sclerosis and polycystic kidneys by means of haplotype studies.

Tuberous sclerosis (TSC) and autosomal dominant polycystic kidney disease (ADPKD) are genetically heterogeneous diseases. The major gene for ADPKD (PKD1) lies adjacent to the TSC2 gene on chromosome 16p13. Some reports in the literature referred to an unusual presentation of TSC with enlarged cystic kidneys at birth, but it was not until the localization of the TSC2 and PKD1 genes that it was possible to analyze the interaction between both genes. We describe a case of a child with TSC and enlarged cystic kidneys. The study of genetic marker segregation in the family pointed to the presence of a deletion involving the 3' region of PKD1. A further study of the region showed a deletion of 40 kb involving both PKD1 and TSC2. We suggest that an additive or synergistic effect between PKD1 and TSC2 may cause this renal phenotype. A contiguous gene syndrome involving PKD1 and TSC2 should be suspected in children with TSC and enlarged polycystic kidneys at birth. The first approach to identify a deletion of both genes could be the analysis of the segregation of PKD1 and TSC2 markers in the family.

Chromosomes, Human, Pair 16↗
PubMed1997

Cloning and characterization of a functional human homolog of Escherichia coli endonuclease III.

Repair of oxidative damage to DNA bases is essential to prevent mutations and cell death. Endonuclease III is the major DNA glycosylase activity in Escherichia coli that catalyzes the excision of pyrimidines damaged by ring opening or ring saturation, and it also possesses an associated lyase activity that incises the DNA backbone adjacent to apurinic/apyrimidinic sites. During analysis of the area adjacent to the human tuberous sclerosis gene (TSC2) in chromosome region 16p13.3, we identified a gene, OCTS3, that encodes a 1-kb transcript. Analysis of OCTS3 cDNA clones revealed an open reading frame encoding a predicted protein of 34.3 kDa that shares extensive sequence similarity with E. coli endonuclease III and a related enzyme from Schizosaccharomyces pombe, including a conserved active site region and an iron/sulfur domain. The product of the OCTS3 gene was therefore designated hNTH1 (human endonuclease III homolog 1). The hNTH1 protein was overexpressed in E. coli and purified to apparent homogeneity. The recombinant protein had spectral properties indicative of the presence of an iron/sulfur cluster, and exhibited DNA glycosylase activity on double-stranded polydeoxyribonucleotides containing urea and thymine glycol residues, as well as an apurinic/apyrimidinic lyase activity. Our data indicate that hNTH1 is a structural and functional homolog of E. coli endonuclease III, and that this class of enzymes, for repair of oxidatively damaged pyrimidines in DNA, is highly conserved in evolution from microorganisms to human cells.

Adenocarcinoma↗