Further evidence of exclusion of linkage between type II autosomal dominant retinitis pigmentosa (ADRP) and D3S47 on 3q

Linkage of the anonymous DNA marker D3S47 (CRI-C17) and autosomal dominant retinitis pigmentosa (ADRP) was tested in a large, extended family with type II (late onset) ADRP. D3S47 has been shown previously to be tightly linked to the RP locus in one family with type I (early onset) ADRP (McWilliams et al., 1989, Genomics 5: 619-622). Linkage between ADRP type II and D3S47 has recently been excluded in a single family (Ingelhearn et al., 1990, Genomics 6: 168-173). Results of our linkage analysis clearly establish that type II ADRP in our family is unlinked to D3S47. These findings support the hypothesis that type II ADRP is genetically distinct from type I ADRP.     

No evidence of linkage between the locus for autosomal dominant retinitis pigmentosa and D3S47 (C17) in three Australian families

Summary A linkage analysis has been performed on three Australian families segregating for autosomal dominant retinitis pigmentosa (ADRP). No evidence of linkage has been found in any of the pedigrees studied between the locus D3S47 and the gene for ADRP. The D3S47 locus was found to show very close linkage with the ADRP gene in a large Irish pedigree. Our study together with a similar report on a British family indicates that there is genetic heterogeneity in this disease.     

Linkage analysis of human chromosome 4: exclusion of autosomal dominant retinitis pigmentosa (ADRP) and detection of new linkage groups

As part of our ongoing linkage studies of degenerative retinal diseases, we tested seven DNA markers and two classical genetic markers from chromosome 4 in two extended families with autosomal dominant retinitis pigmentosa (ADRP). Our goals were (1) to detect or exclude linkage of ADRP to markers spanning most of chromosome 4 and (2) to contribute useful new information regarding the linkage map of this chromosome. Our results exclude linkage of ADRP from more than 82% of chromosome 4. We detected four new linkage relationships: loose linkage of K082 (D4S10) and G1E5 (D4S21) at a distance of 21 cM; loose linkage of 4F2 (D4S18) and GC protein at a distance of 19 cM; tight linkage (i.e., no recombinants) between B3D (D4S44), B5A (D4S40), and the MNS blood group; and tight linkage between 4F2 and GDS5 (D4S23). These data, combined with previously reported data, exclude ADRP from approximately 35% of the human genome.     

No evidence for linkage between late onset autosomal dominant retinitis pigmentosa and chromosome 3 locus D3S47 (C17): evidence for genetic heterogeneity

Retinitis pigmentosa is an inherited form of blindness caused by progressive retinal degeneration. P. McWilliam et al. (1989, Genomics 5: 619-622) demonstrated close genetic linkage between autosomal dominant retinitis pigmentosa (ADRP) and locus D3S47 (C17) in a single early onset pedigree. The marker C17 maps to the long arm of chromosome 3. Clinically, the disease phenotype has been subdivided into at least two forms on the basis of age of onset, as well as electrodiagnostic criteria. We demonstrate that C17 is unlinked in a late onset pedigree, indicating that the phenotypic variation seen reflects underlying genetic heterogeneity.     

Autosomal dominant retinitis pigmentosa: Localization of a disease gene (RP6) to the short arm of chromosome 6

DNA from members of an Irish pedigree presenting with late onset autosomal dominant retinitis pigmentosa (ADRP) have been typed with a series of genetic markers from chromosome 6p. Positive two-point lod scores have been obtained with five markers (D6S89: theta = 0.10, Z = 3.338; D6S109: theta = 0.10, Z = 3.932; D6S105: theta = 0.00, Z = 6.081; HLA-DRA: theta = 0.00, Z = 4.364; and RDS: theta = 0.00, Z = 5.376). In a series of overlapping multipoint analyses a lod score of 6.6 was obtained, maximizing at HLA-DRA and hence localizing the ADRP gene (RP5) segregating in this pedigree to 6p. These data provide direct evidence for an additional autosomal dominant RP locus and strongly implicate the human equivalent of the mouse retinal degeneration slow (rds) gene, peripherin-rds, as a candidate for autosomal dominant retinitis pigmentosa.     

A 3-bp deletion in the rhodopsin gene in a family with autosomal dominant retinitis pigmentosa.

Autosomal dominant retinitis pigmentosa (ADRP) has recently been linked to locus D3S47 (probe C17), with no recombination, in a single large Irish family. Other ADRP pedigrees have shown linkage at zero recombination, linkage with recombination, and no linkage, demonstrating genetic heterogeneity. The gene encoding rhodopsin, the rod photoreceptor pigment, is closely linked to locus D3S47 on chromosome 3q. A point mutation changing a conserved proline to histidine in the 23d codon of the gene has been demonstrated in affected members of one ADRP family and in 17 of 148 unrelated ADRP patients. We have sequenced the rhodopsin gene in a C17-linked ADRP family and have identified in the 4th exon and in-frame 3-bp deletion which deletes one of the two isoleucine monomers at codons 255 and 256. This mutation was not found in 30 other unrelated ADRP families. The deletion has arisen in the sequence TCATCATCAT, deleting one of a run of three x 3-bp repeats. The mechanism by which this occurred may be similar to that which creates length variation in so-called mini- and microsatellites. Thus ADRP is an extremely heterogeneous disorder which can result from a range of defects in rhodopsin and which can have a locus or loci elsewhere in the genome.     

Linkage to D3S47 (C17) in one large autosomal dominant retinitis pigmentosa family and exclusion in another: confirmation of genetic heterogeneity.

Recently Dryja and his co-workers observed a mutation in the 23d codon of the rhodopsin gene in a proportion of autosomal dominant retinitis pigmentosa (ADRP) patients. Linkage analysis with a rhodopsin-linked probe C17 (D3S47) was carried out in two large British ADRP families, one with diffuse-type (D-type) RP and the other with regional-type (R-type) RP. Significantly positive lod scores (lod score maximum [Zmax] = +5.58 at recombination fraction [theta] = .0) were obtained between C17 and our D-type ADRP family showing complete penetrance. Sequence and oligonucleotide analysis has, however, shown that no point mutation at the 23d codon exists in affected individuals in our complete-penetrance pedigree, indicating that another rhodopsin mutation is probably responsible for ADRP in this family. Significantly negative lod scores (Z less than -2 at theta = .045) were, however, obtained between C17 and our R-type family which showed incomplete penetrance. Previous results presented by this laboratory also showed no linkage between C17 and another large British R-type ADRP family with incomplete penetrance. This confirms genetic heterogeneity. Some types of ADRP are being caused by different mutations in the rhodopsin locus (3q21-24) or another tightly linked gene in this region, while other types of ADRP are the result of mutations elsewhere in the genome.     

Linkage mapping of autosomal dominant retinitis pigmentosa (RP1) to the pericentric region of human chromosome 8.

Linkage mapping in a large, seven-generation family with type 2 autosomal dominant retinitis pigmentosa (ADRP) demonstrates linkage between the disease locus (RP1) and DNA markers on the short arm of human chromosome 8. Five markers were most informative for mapping ADRP in this family using two-point linkage analysis. The markers, their maximum lod scores, and recombination distances were ANK1 (ankyrin)--2.0 at 16%; D8S5 (TL11)--5.3 at 17%; D8S87 [a(CA)n repeat]--7.2 at 14%; LPL (lipoprotein lipase)--1.5 at 26%; and PLAT (plasminigen activator, tissue)--10.6 at 7%. Multipoint linkage analysis, using a simplified pedigree structure for the family (which contains 192 individuals and two inbreeding loops), gave a maximum lod score of 12.2 for RP1 at a distance 8.1 cM proximal to PLAT in the pericentric region of the chromosome. Based on linkage data from the CEPH (Paris) reference families and physical mapping information from a somatic cell hybrid panel of chromosome 8 fragments, the most likely order for four of these five loci and the diseases locus is 8pter-LPL-D8S5-D8S87-PLAT-RP1. (The precise location of ANK1 relative to PLAT in this map is not established). The most likely location for RP1 is in the pericentric region of the chromosome. Recently, several families with ADRP with tight linkage to the rhodopsin locus at 3q21-q24 were reported and a number of specific rhodopsin mutations in families with ADRP have since been reported. In other ADRP families, including the one in this study, linkage to rhodopsin has been excluded. Thus mutations at two different loci, at least, have been shown to cause ADRP. There is no remarkable clinical disparity in the expression of disease caused by these different loci.     

Inhibition of ADRP prevents diet-induced insulin resistance

Diets with high fat content induce steatosis, insulin resistance, and type 2 diabetes. The lipid droplet protein adipose differentiation-related protein (ADRP) mediates hepatic steatosis, but whether this affects insulin action in the liver or peripheral organs in diet-induced obesity is uncertain. We fed C57BL/6J mice a high-fat diet and simultaneously treated them with an antisense oligonucleotide (ASO) against ADRP for 4 wk. Glucose homeostasis was assessed with clamp and tracer techniques. ADRP ASO decreased the levels of triglycerides and diacylglycerol in the liver, but fatty acids, long-chain fatty acyl CoAs, ceramides, and cholesterol were unchanged. Insulin action in the liver was enhanced after ADRP ASO treatment, whereas muscle and adipose tissue were not affected. ADRP ASO increased the phosphorylation of insulin receptor substrate (IRS)1, IRS2, and Akt, and decreased gluconeogenic enzymes and PKCepsilon, consistent with its insulin-sensitizing action. These results demonstrate an important role for ADRP in the pathogenesis of diet-induced insulin resistance.     

Gene mapping and mutation screening in candidate genes in a Chinese family of autosomal dominant retinitis pigmentosa

We wanted to find the gene defect in a Chinese pedigree with autosomal dominant form of retinitis pigmentosa (ADRP). A small Chinese family with retinitis pigmentosa was collected. The genetic analysis of the family suggested an autosomal dominant pattern. Microsatellite (STR) markers tightly linked to candidate genes for ADRP were selected for linkage analysis. We got a maximum LOD score of 0.87 between markers D19S210 and D19S418. Precursor mRNA-processing factor (PRPF) 31, 3, 8, rhodopsin (RHO), peripherin 2 (PRPH2 or RDS), rod outer segment protein 1 (ROM1), neural retina leucine zipper (NRL), cone-rod homeobox-containing (CRX), inosine-5-prime-monophosphate dehydrogenase, type I (IMPDH1) and retinitis pigmentosa 1 (RPI) were amplified by polymerase chain reaction (PCR) and screened by direct sequencing. One new sequence variation was found. It was the missence mutation c.148G > C (D50H) occurred in exon 1 of RDS gene which existed in all the effected individuals and one unaffected family member. The DNA sequence variation didn't cosegregate with the RP disease. We considered this transition was one new polymorphism which we speculate involved in the pathogenesis of ADRP and increased the risk of ADRP. Further study should be conducted to confirm the causative gene of this family.     

Regulation of ADRP expression by long-chain polyunsaturated fatty acids in BeWo cells, a human placental choriocarcinoma cell line

Transplacental transfer of maternal fatty acids is critical for fetal growth and development. In the placenta, a preferential uptake of fatty acids toward long-chain polyunsaturated fatty acids (LCPUFAs) has been demonstrated. Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that has been ascribed a role in cellular fatty acid uptake and storage. However, its role in placenta is not known. We demonstrate that ADRP mRNA and protein are regulated by fatty acids in a human placental choriocarcinoma cell line (BeWo) and in primary human trophoblasts. LCPUFAs of the n-3 and n-6 series [arachidonic acid (20:4n-6), docosahexaenoic acid (22:6n-3), and eicosapentaenoic acid (20:5n-3)] were more efficient than shorter fatty acids at stimulating ADRP mRNA expression. The fatty acid-mediated increase in ADRP mRNA expression was not related to the differentiation state of the cells. Synthetic peroxisome proliferator-activated receptor and retinoic X receptor agonists increased ADRP mRNA level but had no effect on ADRP protein level in undifferentiated BeWo cells. Furthermore, we show that incubation of BeWo cells with LCPUFAs, but not synthetic agonists, increased the cellular content of radiolabeled oleic acid, coinciding with the increase in ADRP mRNA and protein level. These studies provide new information on the regulation of ADRP in placental trophoblasts and suggest that LCPUFA-dependent regulation of ADRP could be involved in the metabolism of lipids in the placenta.     

Recombination between rhodopsin and locus D3S47 (C17) in rhodopsin retinitis pigmentosa families

Autosomal dominant retinitis pigmentosa (adRP) has shown linkage to the chromosome 3q marker C17 (D3S47) in two large adRP pedigrees known as TCDM1 and adRP3. On the basis of this evidence the rhodopsin gene, which also maps to 3q, was screened for mutations which segregated with the disease in adRP patients, and several have now been identified. However, we report that, as yet, no rhodopsin mutation has been found in the families first linked to C17. Since no highly informative marker system is available in the rhodopsin gene, it has not been possible to measure the genetic distance between rhodopsin and D3S47 accurately. We now present a linkage analysis between D3S47 and the rhodopsin locus (RHO) in five proven rhodopsin-retinitis pigmentosa (rhodopsin-RP) families, using the causative mutations as highly informative polymorphic markers. The distance, between RHO and D3S47, obtained by this analysis is theta = .12, with a lod score of 4.5. This contrast with peak lod scores between D3S47 and adRP of 6.1 at theta = .05 and 16.5 at theta = 0 in families adRP3 and TCDM1, respectively. These data would be consistent with the hypothesis that TCDM1 and ADRP3 represent a second adRP locus on chromosome 3q, closer to D3S47 than is the rhodopsin locus. This result shows that care must be taken when interpreting adRP exclusion data generated with probe C17 and that it is probably not a suitable marker for predictive genetic testing in all chromosome 3q-linked adRP families.     

ADRP/adipophilin is degraded through the proteasome-dependent pathway during regression of lipid-storing cells

Adipose differentiation-related protein (ADRP) is a major protein associated with lipid droplets in various types of cells, including macrophage-derived foam cells and liver cells. However, the role of ADRP in the processes of formation and regression of these cells is not understood. When J774 murine macrophages were incubated with either VLDL or oleic acid, their content of both ADRP and triacylglycerol (TG) increased 3- to 4-fold. Induction of ADRP during TG accumulation was also observed in oleic acid-treated HuH-7 human liver cells. Addition of triacsin C, a potent inhibitor of acyl-CoA synthase, for 6 h decreased the amount of TG in VLDL-induced foam cells and oleic acid-treated liver cells; it decreased the amount of ADRP protein in parallel, indicating the amount of ADRP reduced during regression of the lipid-storing cells. Addition of a proteasome inhibitor during triacsin C treatment abolished the ADRP decrease and accumulated polyubiquitinated ADRP. In addition, the proteasome inhibitor reversed not only the degradation of ADRP but also TG reduction by triacsin C. These results suggest that cellular amounts of ADRP and TG regulate each other and that the ubiquitin-proteasome system is involved in degradation of ADRP during regression of lipid-storing cells.     

ADRP is dissociated from lipid droplets by ARF1-dependent mechanism

Adipocyte differentiation-related protein (ADRP) is a member of PAT proteins existing in lipid droplets (LDs). By yeast two-hybrid screening, we identified ADP-ribosylation factor 1 (ARF1) as a binding partner of ADRP. The interaction of ADRP and ARF1 was verified by GST pull-down and co-immunoprecipitation experiments. Interestingly, ADRP precipitated the GDP-bound ARF1 preferentially to the GTP-bound ARF1. Consistent with this, either brefeldin A (BFA), a fungal metabolite to inhibit ARF-GEF, or a dominant-negative mutant of ARF1 caused dissociation of ADRP from LD. On the other hand, overexpression of wild-type ARF1 did not promote the ADRP dissociation or new LD formation. By using deletion mutants, a central domain of ADRP, which is dispensable for LD binding, was shown to bind to ARF1. The present study showed that the GDP-bound ARF1 induces dissociation of ADRP from the LD surface, and that LD is a target of BFA action.     

Autosomal dominant retinitis pigmentosa: linkage to rhodopsin and evidence for genetic heterogeneity

Retinitis pigmentosa (RP) is the most prevalent human retinopathy of genetic origin. Chromosomal locations for X-linked RP and autosomal dominant RP genes have recently been established. Multipoint analyses with ADRP and seven markers on the long arm of chromosome 3 demonstrate that the gene for rhodopsin, the pigment of the rod photoreceptors, cosegregates with the disease locus with a maximum lod score of approximately 19, implicating rhodopsin as a causative gene. Recent studies have indicated the presence of a point mutation at codon 23 in exon 1 of rhodopsin which results in the substitution of histidine for the highly conserved amino acid proline, suggesting that this mutation is a cause of rhodopsin-linked ADRP. This mutation is not present in the Irish pedigree in which ADRP has been mapped close to rhodopsin. Another mutation in the rhodopsin gene or in a gene closely linked to rhodopsin may be involved. Moreover, the gene in a second ADRP pedigree, with Type II late onset ADRP, does not segregate with chromosome 3q markers, indicating that nonallelic as well as perhaps allelic genetic heterogeneity exists in the autosomal dominant form of this disease.     

Gene mapping and mutation screeneng in candidate genes in a Chinese family of autosomal dominant retinitis pigmentosa

We wanted to find the gene defect in a Chinese pedigree with autosomal dominant form of retinitis pigmentosa (ADRP). A small Chinese family with retinitis pigmentosa was collected. The genetic analysis of the family suggested an autosomal dominant pattern. Microsatellite (STR) markers tightly linked to candidate genes for ADRP were selected for linkage analysis. We got a maximum LOD score of 0.87 between markers D19S210 and D19S418. Precursor mRNA-processing factor (PRPF) 31, 3, 8, rhodopsin (RHO), peripherin 2 (PRPH2 or RDS), rod outer segment protein 1 (ROM1), neural retina leucine zipper (NRL), cone-rod homeobox-containing (CRX), inosine-5-prime-monophosphate dehydrogenase, type I (IMPDH1) and retinitis pigmentosa 1 (RP1) were amplified by polymerase chain reaction (PCR) and screened by direct sequencing. One new sequence variation was found. It was the missence mutation c.148G > C (D50H) occurred in exon 1 of RDS gene which existed in all the effected individuals and one unaffected family member. The DNA sequence variation didn’t cosegregate with the RP disease. We considered this transition was one new polymorphism which we speculate involved in the pathogenesis of ADRP and increased the risk of ADRP. Further study should be conducted to confirm the causative gene of this family.     

Recruitment of TIP47 to lipid droplets is controlled by the putative hydrophobic cleft

Adipose differentiation-related protein (ADRP) and TIP47 show sequence similarity, particularly in their N-terminal PAT-1 domain. Under standard culture conditions, ADRP existed in most lipid droplets (LDs), whereas TIP47 was observed only in some LDs and recruited to LDs on treatment with fatty acids. By analyzing deletion mutants, we found that the C-terminal half of TIP47, or more specifically the putative hydrophobic cleft [S.J. Hickenbottom, A.R. Kimmel, C. Londos, J.H. Hurley, Structure of a lipid droplet protein; the PAT family member TIP47, Structure (Camb) 12 (2004) 1199-1207.], was involved in LD targeting and responsiveness to fatty acids. The result contrasted with that observed for ADRP and implied a distinct LD-targeting mechanism for TIP47. Consistent with this, overexpression of Rab18 decreased ADRP, but not TIP47, from LDs, and TIP47 did not displace pre-existing ADRP from LDs. But ADRP may be a factor to control the TIP47 behavior, because TIP47 in LDs increased upon down-regulation of ADRP. The results suggested that the putative hydrophobic cleft is critical for the unique characteristics of TIP47.     

Mutational analysis of the rhodopsin gene in Chinese ADRP families by conformation sensitive gel electrophoresis

Retinitis pigmentosa is a very heterogeneous group of retinal degenerations, with multiple genes identified in each mode of inheritance. For autosomal dominant retinitis pigmentosa (ADRP), the most common gene is the rhodopsin (RHO) gene, mutations in which contribute to about 25% of ADRP in Caucasian population. To investigate the frequency and pattern of RHO point mutations in Chinese patients with ADRP, we have screened the five coding exons and splice sites of the RHO gene in 50 unrelated probands from Chinese ADRP families and 100 normal controls to identify disease-associated mutations, using conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing. Two RHO mutations, Pro347Leu and Pro327 (1-bp del), were identified each in one family, thus the frequency of RHO mutations among ADRP families in this study is less than 14% (2/50=4%, 95% confidence interval: 1-14%), lower than that in Europe and North America, which may reflect an ethnic difference between Chinese and Caucasian populations. Loss of all phosphorylation sites at the C-terminus and a highly conserved sequence QVS(A)PA may occur because of Pro327(1-bp del). CSGE was found to be a sensitive, simple and practical method for the screening of a large number of samples under highly reproducible conditions, and could be utilized in routine molecular diagnostic laboratories.     

ADRP stimulates lipid accumulation and lipid droplet formation in murine fibroblasts

Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that is expressed early during adipose differentiation. The present study was undertaken to reveal the role of ADRP in adipose differentiation. In murine fibroblasts infected with green fluorescent protein (GFP)-ADRP fusion protein expression adenovirus vector, confocal microscopic analysis showed the number and size of lipid droplets apparently increased comparing with those of control cells. Overexpressed GFP-ADRP were mainly located at the surface of lipid droplets and appeared to be "ring-shaped." Triacylglycerol content was also significantly (P < 0.001) increased in GFP-ADRP-overexpressed cells compared with control cells. ADRP-induced lipid accumulation did not depend on adipocyte-specific gene induction, such as peroxisome proliferator-activated receptor-gamma, lipoprotein lipase, or other lipogenic genes, including acyl-CoA synthetase, fatty acid-binding protein, and fatty acid transporter. In conclusion, ADRP stimulated lipid accumulation and lipid droplet formation without induction of other adipocyte-specific genes or other lipogenic genes in murine fibroblasts. The detailed molecular mechanisms of ADRP on lipid accumulation remain to be elucidated.