Exceptional good cognitive and phenotypic profile in a male carrying a mosaic mutation in the FMR1 gene.

Fragile X (FRAX) syndrome is a commonly inherited form of mental retardation resulting from the lack of expression of the fragile X mental retardation protein (FMRP). It is caused by a stretch of CGG repeats within the fragile X gene, which can be unstable in length as it is transmitted from generation to generation. Once the repeat exceeds a threshold length, the FMR1 gene is methylated and no protein is produced resulting in the fragile X phenotype. The consequences of FMRP absence in the mechanisms underlying mental retardation are unknown. We have identified a male patient in a classical FRAX family without the characteristic FRAX phenotype. His intelligence quotient (IQ) is borderline normal despite the presence of a mosaic pattern of a pre-mutation (25%), full mutation (60%) and a deletion (15%) in the FMR1 gene. The cognitive performance was determined at the age of 28 by the Raven test and his IQ was 81. However, FMRP expression studies in both hair roots and lymphocytes, determined at the same time as the IQ test, were within the affected male range. The percentage of conditioned responses after delay eyeblink conditioning was much higher than the average percentage measured in FRAX studies. Moreover, this patient showed no correlation between FMRP expression and phenotype and no correlation between DNA diagnostics and phenotype.

Prorenin accumulation and activation in human endothelial cells: importance of mannose 6-phosphate receptors.

ACE inhibitors improve endothelial dysfunction, possibly by blocking endothelial angiotensin production. Prorenin, through its binding and activation by endothelial mannose 6-phosphate (M6P) receptors, may contribute to this production. Here, we investigated this possibility as well as prorenin activation kinetics, the nature of the prorenin-activating enzyme, and M6P receptor-independent prorenin binding. Human umbilical vein endothelial cells (HUVECs) were incubated with wild-type prorenin, K/A-2 prorenin (in which Lys42 is mutated to Ala, thereby preventing cleavage by known proteases), M6P-free prorenin, and nonglycosylated prorenin, with or without M6P, protease inhibitors, or angiotensinogen. HUVECs bound only M6P-containing prorenin (K(d) 0.9+/-0.1 nmol/L, maximum number of binding sites [B(max)] 1010+/-50 receptors/cell). At 37 degrees C, because of M6P receptor recycling, the amount of prorenin internalized via M6P receptors was >25 times B(max). Inside the cells, wild-type and K/A-2 prorenin were proteolytically activated to renin. Renin was subsequently degraded. Protease inhibitors interfered with the latter but not with prorenin activation, thereby indicating that the activating enzyme is different from any of the known prorenin-activating enzymes. Incubation with angiotensinogen did not lead to endothelial angiotensin generation, inasmuch as HUVECs were unable to internalize angiotensinogen. Most likely, therefore, in the absence of angiotensinogen synthesis or endocytosis, M6P receptor-mediated prorenin internalization by endothelial cells represents prorenin clearance.

Cardiomyocytes bind and activate native human prorenin : role of soluble mannose 6-phosphate receptors.

Cardiomyocytes bind, internalize, and activate recombinant human prorenin through mannose 6-phosphate/insulin-like growth factor II (M6P/IGFII) receptors. To investigate whether this also applies to native human prorenin, neonatal rat myocytes were incubated for 4 hours at 37 degrees C with various prorenin-containing human body fluids. Uptake and activation by M6P/IGFII receptors were observed for plasma prorenin from subjects with renal artery stenosis and/or hypertension and for follicular fluid prorenin. The total amount of cellular renin and prorenin (expressed as percentage of the levels of renin and prorenin in the medium) after 4 hours of incubation was 4 to 10 times lower than after incubation with recombinant human prorenin. Although plasma contains alkaline phosphatases capable of inactivating the M6P label as well as soluble M6P/IGFII receptors that block prorenin binding in a competitive manner and proteins (eg, insulin, IGFII) that increase the number of cell-surface M6P/IGFII receptors, these factors were not responsible for the modest uptake of native human prorenin. Uptake did not occur during incubation of myocytes with plasma prorenin from anephric subjects or with amniotic fluid prorenin, and this was not due to the presence of excessively high levels of M6P/IGFII receptors and/or phosphatase activity in these fluids. In conclusion, myocytes are capable of binding, internalizing, and activating native human prorenin of renal and ovarian origin through M6P/IGFII receptors. Differences in prorenin glycosylation and/or phosphorylation as well as the concentration of soluble M6P/IGFII receptors and growth factors affecting cell-surface M6P/IGFII receptor density determine the amount of prorenin entering the heart and thus cardiac angiotensin II production.

High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin.

Mannose-6-phosphate (man-6-P)/insulin-like growth factor-II (man-6-P/IgF-II) receptors are involved in the activation of recombinant human prorenin by cardiomyocytes. To investigate the kinetics of this process, the nature of activation, the existence of other prorenin receptors, and binding of native prorenin, neonatal rat cardiomyocytes were incubated with recombinant, renal, or amniotic fluid prorenin with or without man-6-P. Intact and activated prorenin were measured in cell lysates with prosegment- and renin-specific antibodies, respectively. The dissociation constant (K(d)) and maximum number of binding sites (B(max)) for prorenin binding to man-6-P/IGF-II receptors were 0.6 +/- 0.1 nM and 3,840 +/- 510 receptors/myocyte, respectively. The capacity for prorenin internalization was greater than 10 times B(max). Levels of internalized intact prorenin decreased rapidly (half-life = 5 +/- 3 min) indicating proteolytic prosegment removal. Prorenin subdivision into man-6-P-free and man-6-P-containing fractions revealed that only the latter was bound. Cells also bound and activated renal but not amniotic fluid prorenin. We concluded that cardiomyocytes display high-affinity binding of renal but not extrarenal prorenin exclusively via man-6-P/IGF-II receptors. Binding precedes internalization and proteolytic activation to renin thereby supporting the concept of cardiac angiotensin formation by renal prorenin.

Functional importance of angiotensin-converting enzyme-dependent in situ angiotensin II generation in the human forearm.

To assess the importance for vasoconstriction of in situ angiotensin (Ang) II generation, as opposed to Ang II delivery via the circulation, we determined forearm vasoconstriction in response to Ang I (0.1 to 10 ng. kg(-1). min(-1)) and Ang II (0.1 to 5 ng. kg(-1). min(-1)) in 14 normotensive male volunteers (age 18 to 67 years). Changes in forearm blood flow (FBF) were registered with venous occlusion plethysmography. Arterial and venous blood samples were collected under steady-state conditions to quantify forearm fractional Ang I-to-II conversion. Ang I and II exerted the same maximal effect (mean+/-SEM 71+/-4% and 75+/-4% decrease in FBF, respectively), with similar potencies (mean EC(50) [range] 5.6 [0.30 to 12.0] nmol/L for Ang I and 3.6 [0.37 to 7.1] nmol/L for Ang II). Forearm fractional Ang I-to-II conversion was 36% (range 18% to 57%). The angiotensin-converting enzyme (ACE) inhibitor enalaprilat (80 ng. kg(-1). min(-1)) inhibited the contractile effects of Ang I and reduced fractional conversion to 1% (0.1% to 8%), thereby excluding a role for Ang I-to-II converting enzymes other than ACE (eg, chymase). The Ang II type 1 receptor antagonist losartan (3 mg. kg(-1). min(-1)) inhibited the vasoconstrictor effects of Ang II. In conclusion, the similar potencies of Ang I and II in the forearm, combined with the fact that only one third of arterially delivered Ang I is converted to Ang II, suggest that in situ-generated Ang II is more important for vasoconstriction than circulating Ang II. Local Ang II generation in the forearm depends on ACE exclusively and results in vasoconstriction via Ang II type 1 receptors.

Cultured neonatal rat cardiac myocytes and fibroblasts do not synthesize renin or angiotensinogen: evidence for stretch-induced cardiomyocyte hypertrophy independent of angiotensin II.

OBJECTIVE: The hypertrophic response of cardiomyocytes exposed to mechanical stretch is assumed to depend on the release of angiotensin (Ang) II from these cells. Here we studied the synthesis of renin-angiotensin system (RAS) components by cardiac cells under basal conditions and after stretch. METHODS: Myocytes and fibroblasts were isolated by enzymatic dissociation from hearts of 1-3-day-old Wistar rat strain pups, grown for 1 day in serum-supplemented medium and then cultured in a chemically defined, serum-free medium. Medium and cell lysate were collected 5 days later or after exposure of the cells to cyclic stretch for 24 h. Prorenin, renin and angiotensinogen were measured by enzyme-kinetic assay; Ang I and Ang II were measured by radioimmunoassay after SepPak extraction and HPLC separation. RESULTS: Prorenin, but none of the other RAS components, could be detected in the medium of both cell types. However, its levels were low and the Ang I-generating activity corresponding with these low prorenin levels could not be inhibited by the specific rat renin inhibitor CH-732, suggesting that it was most likely due to bovine and/or horse prorenin sequestered from the serum-containing medium to which the cells had been exposed prior to the serum-free period. When incubated with Ang I, both myocytes and fibroblasts generated Ang II in a captopril-inhibitable manner. Myocyte and fibroblast cell lysates did not contain prorenin, renin, angiotensinogen, Ang I or Ang II in detectable quantities. Stretch increased myocyte protein synthesis by 20%, but was not accompanied by Ang II release into the medium. CONCLUSION: Cardiac myocytes and fibroblasts do not synthesize renin, prorenin or angiotensinogen in concentrations that are detectable or, it not detectable, high enough to result in Ang II concentrations of physiological relevance. These cells do synthesize ACE, thereby allowing the synthesis of Ang II at cardiac tissue sites when renin and angiotensinogen are provided via the circulation. Ang II is not a prerequisite to observe a hypertrophic response of cardiomyocytes following stretch.

Cellular localization and tissue distribution of polycystin-1.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid-filled cysts in both kidneys, in addition to a variety of extra-renal manifestations. The PKD1 gene product, polycystin-1, encodes a novel protein with a putative role in cell-cell/cell-matrix interactions. The present study we focused on the (sub)cellular localization of polycystin-1 in cultured cells, and on its tissue distribution in various organs. In Madin Darby canine kidney (MDCK) cells, several polyclonal antibodies showed intense staining at the sites of interaction between adjacent cells, which remained after Triton extraction. Weak cytoplasmic staining was observed. No signal was detected at the free borders of cell aggregates, supporting a role for polycystin-1 in cell-cell interactions. At the tissue level, polycystin-1 expression was observed in specific cell types in tissues with known manifestations of the disease, but also in tissues of organs which have not been reported to be affected in ADPKD. Expression was frequently seen in epithelia, but also in endocrine cells (pancreatic islets, parathyroid-producing cells, clusters in the adenohypophysis, clusters in the adrenal gland, and Leydig cells in the testis). In addition, expression was observed in myocardium and more weakly in myocytes of cardiac valves, of the cerebral arteries, and of skeletal muscles.

Is there a local renin-angiotensin system in the heart?

The existence of a local renin-angiotensin system in the heart is still a controversial issue. This review discusses the evidence, obtained from studies in cardiac cells, in isolated perfused hearts and in intact animals and humans, both under normal and pathological conditions, for local production of prorenin, renin, angiotensinogen, angiotensin-converting enzyme, angiotensin I and angiotensin II at cardiac tissue sites. In addition, the role of alternative angiotensin-generating enzymes (cathepsin, chymase) and the possibility of (pro)renin uptake from the circulation is evaluated.

Mutation detection in the repeated part of the PKD1 gene.

The principle cause of one of the most prevalent genetic disorders, autosomal dominant polycystic kidney disease, involves mutations in the PKD1 gene. However, since its identification in 1994, only 27 mutations have been published. Detection of mutations has been complicated because the greater part of the gene lies within a genomic region that is reiterated several times at another locus on chromosome 16. Amplification of DNA fragments in the repeated part of the PKD1 gene will lead to coamplification of highly homologous fragments derived from this other locus. These additional fragments severely hamper point-mutation detection. None of the point mutations published to date are located in the repeated part of the PKD1 gene. However, we have reduced the problems posed by the strong homology, by using the protein-truncation test, and we have identified eight novel mutations, seven of which are located in the repeated part of the PKD1 gene.

A spectrum of mutations in the second gene for autosomal dominant polycystic kidney disease (PKD2).

Recently the second gene for autosomal dominant polycystic kidney disease (ADPKD), located on chromosome 4q21-q22, has been cloned and characterized. The gene encodes an integral membrane protein, polycystin-2, that shows amino acid similarity to the PKD1 gene product and to the family of voltage-activated calcium (and sodium) channels. We have systematically screened the gene for mutations by single-strand conformation-polymorphism analysis in 35 families with the second type of ADPKD and have identified 20 mutations. So far, most mutations found seem to be unique and occur throughout the gene, without any evidence of clustering. In addition to small deletions, insertions, and substitutions leading to premature translation stops, one amino acid substitution and five possible splice-site mutations have been found. These findings suggest that the first step toward cyst formation in PKD2 patients is the loss of one functional copy of polycystin-2.

PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein.

A second gene for autosomal dominant polycystic kidney disease was identified by positional cloning. Nonsense mutations in this gene (PKD2) segregated with the disease in three PKD2 families. The predicted 968-amino acid sequence of the PKD2 gene product has six transmembrane spans with intracellular amino- and carboxyl-termini. The PKD2 protein has amino acid similarity with PKD1, the Caenorhabditis elegans homolog of PKD1, and the family of voltage-activated calcium (and sodium) channels, and it contains a potential calcium-binding domain.

Rubinstein-Taybi syndrome caused by mutations in the transcriptional co-activator CBP.

The Rubinstein-Taybi syndrome (RTS) is a well-defined syndrome with facial abnormalities, broad thumbs, broad big toes and mental retardation as the main clinical features. Many patients with RTS have been shown to have breakpoints in, and microdeletions of, chromosome 16p13.3 (refs 4-8). Here we report that all these breakpoints are restricted to a region that contains the gene for the human CREB binding protein (CBP), a nuclear protein participating as a co-activator in cyclic-AMP-regulated gene expression. We show that RTS results not only from gross chromosomal rearrangements of chromosome 16p, but also from point mutations in the CBP gene itself. Because the patients are heterozygous for the mutations, we propose that the loss of one functional copy of the CBP gene underlies the developmental abnormalities in RTS and possibly the propensity for malignancy.

Chromosome 4 localization of a second gene for autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder. A gene defect located on the short arm of chromosome 16 is responsible for the disease in roughly 86% of affected European families. Using highly polymorphic microsatellite DNA markers, we have assigned a second gene for ADPKD to chromosome 4. In eight families with clear evidence against linkage to chromosome 16 markers, linkage analysis with the markers D4S231 and D4S423, demonstrated a multipoint lod score of 22.42.

Rubinstein-Taybi syndrome caused by submicroscopic deletions within 16p13.3.

The Rubinstein-Taybi syndrome (RTS) is a well-defined complex of congenital malformations characterized by facial abnormalities, broad thumbs and big toes, and mental retardation. The breakpoint of two distinct reciprocal translocations occurring in patients with a clinical diagnosis of RTS was located to the same interval on chromosome 16, between the cosmids N2 and RT1, in band 16p13.3. By using two-color fluorescence in situ hybridization, the signal from RT1 was found to be missing from one chromosome 16 in 6 of 24 patients with RTS. The parents of five of these patients did not show a deletion of RT1, indicating a de novo rearrangement. RTS is caused by submicroscopic interstitial deletions within 16p13.3 in approximately 25% of the patients. The detection of microdeletions will allow the objective conformation of the clinical diagnosis in new patients and provides an excellent tool for the isolation of the gene causally related to the syndrome.

Extensive cross-homology between the long and the short arm of chromosome 16 may explain leukemic inversions and translocations.

Specific rearrangements of chromosome 16 are well known in acute nonlymphocytic leukemia with abnormal eosinophils. While mapping cosmids relative to breakpoints in chromosome 16 in leukemic cells with fluorescence in situ hybridization (FISH), we have identified three areas of extensive cross-homology between 16p and 16q. Three cosmids among 99 tested showed two large signals on the short arm and one signal on the long arm of chromosome 16. A fourth cosmid showed mainly two signals on the short arm. With the 16p-specific cosmid we can demonstrate that the breakpoints of a pericentric inversion and a reciprocal (16;16) translocation, both of which are characteristic for acute leukemia, map to the most distal of two blocks on the short arm. We suggest that there may be at least two distinct repetitive elements specific for chromosome 16 interdigitated on 16p. The presence of a similar repeat in the short, as well as the long arm of the chromosome, may play a role in the origin of chromosome 16 rearrangements in acute leukemia.

Map of 16 polymorphic loci on the short arm of chromosome 16 close to the polycystic kidney disease gene (PKD1).

To define the PKD1 locus further, the gene involved in the most frequent form of adult polycystic kidney disease, probes from 16 polymorphic loci were mapped on 16p13.1-pter with the combined use of cell lines containing rearranged chromosomes and family studies. Five breakpoints in the distal part of 16p arbitrarily subdivided the loci into five groups. By analysing 58 recombination events among 259 informative meioses in 12 large families with PKD, we were able to construct a linkage map for the distal part of 16p. The order of the markers obtained with chromosomal rearrangements was confirmed by the family studies. The D16S85 locus near alpha globin, D16S21, and D16S83 map distal, or telomeric, to PKD1. The polymorphic red cell enzyme phosphoglycolate phosphatase (PGP), D16S84, D16S259, and D16S246 showed no recombination with PKD1. The remaining nine RFLPs all map proximal to the PKD1 gene. By cosmid walking, additional RFLPs were detected at the D16S21 locus. A single intrahaplotype recombination observed defines the orientation of D16S21 relative to PKD1. The new polymorphisms are valuable for presymptomatic and prenatal diagnosis of PKD1. Furthermore, our map is both a good starting point for the physical map of 16p and a useful tool for the isolation of the PKD1 gene.

Two step procedure for early diagnosis of polycystic kidney disease with polymorphic DNA markers on both sides of the gene.

Polymorphic DNA markers can now be used for presymptomatic and prenatal diagnosis of the autosomal dominant form of polycystic kidney disease (PKD). A detailed map is known for the chromosomal region around the PKD1 gene on the short arm of chromosome 16. We present here a simple, two step procedure for diagnosis of PKD1 by family studies. Using this approach, at least 92% of random subjects are informative for polymorphic DNA markers bracketing the PKD1 gene. The recombination rate between these flanking markers is on average 10%. In non-recombinants (90% of family members), the accuracy of diagnosis using DNA markers is greater than 99%. We conclude that sufficient well defined DNA markers are now available for routine diagnosis of PKD1. We recommend, however, that prenatal diagnosis of PKD by chorionic villi sampling should be attempted only after the linkage phase of the DNA markers has been established by haplotyping the index family. Since autosomal dominant PKD has been found to be genetically heterogeneous, families should be of sufficient size to rule out the rare form of PKD not caused by a mutation on the short arm of chromosome 16.