Brian W. Rigatti

X-linked cone-rod dystrophy (locus COD1): identification of mutations in RPGR exon ORF15.

X-linked cone-rod dystrophy (COD1) is a retinal disease that primarily affects the cone photoreceptors; the disease was originally mapped to a limited region of Xp11.4. We evaluated the three families from our original study with new markers and clinically reassessed all key recombinants; we determined that the critical intervals in families 2 and 3 overlapped the RP3 locus and that a status change (from affected to probably unaffected) of a key recombinant individual in family 1 also reassigned the disease locus to include RP3 as well. Mutation analysis of the entire RPGR coding region identified two different 2-nucleotide (nt) deletions in ORF15, in family 2 (delAG) and in families 1 and 3 (delGG), both of which result in a frameshift leading to altered amino acid structure and early termination. In addition, an independent individual with X-linked cone-rod dystrophy demonstrated a 1-nt insertion (insA) in ORF15. The presence of three distinct mutations associated with the same disease phenotype provides strong evidence that mutations in RPGR exon ORF15 are responsible for COD1. Genetic heterogeneity was observed in three other families, including the identification of an in-frame 12-nt deletion polymorphism in ORF15 that did not segregate with the disease in one of these families.

Identification of Sites in Domain I of Perlecan That Regulate Heparan Sulfate Synthesis

Perlecan is primarily a heparan sulfate containing proteoglycan found in all basement membranes. Rotary shadowed images of perlecan show it to contain three glycosaminoglycan (GAG) side chains extending from one end of its core protein. Domain I is at the N terminus of perlecan and contains three closely spaced Ser-Gly-Asp sequences that may serve in GAG attachment. We evaluated the serines in these three sequences for GAG attachment by preparing a cDNA construct encoding for the N-terminal half (domains I, II, and III) of perlecan and then a series of constructs containing deletions and mutations within domain I of the domain I/II/III construct, expressing these constructs in COS-7 cells, and then analyzing the recombinant product for GAG side chains and GAG type. The results showed that all three serine residues in the Ser-Gly-Asp sequences in domain I can accept both chondroitin and heparan sulfate side chains but that a cluster of acidic residues N-terminal to these sequences is the primary determinant responsible for targeting these sites for heparan sulfate. Furthermore, there are two elements that can enhance heparan sulfate synthesis at a targeted site: 1) the presence of a the SEA module in the C-terminal region of domain I and 2) the presence of multiple acceptors in close proximity. These results indicate that the proportion of heparan and chondroitin sulfate at any one site in domain I of perlecan is regulated by multiple factors.

Nuclear retinoic acid receptors in the lacrimal gland

The lacrimal gland secretes and metabolizes retinoids and responds to retinoic acid in culture. Like other retinoid responsive organs it is expected to express the nuclear retinoid receptors. The goal of this study was to identify the retinoic acid receptors (RAR) in the lacrimal glands of rats, rabbits, and humans. Total RNA was prepared from whole lacrimal glands and rat lacrimal gland acinar cells grown in culture. RNA was subjected to Northern blot analysis and probed for the RAR alpha, RAR beta, and RAR gamma mRNAs. Nuclear extracts of rat and rabbit lacrimal glands were incubated with 3H-all-trans retinoic acid and analyzed by gel filtration chromatography. Western blots of the nuclear extracts were probed using monoclonal antibodies to RAR alpha and RAR beta. Rat lacrimal gland expresses RAR alpha mRNA with two transcripts (3.8 and 3.0 kb), a single RAR beta mRNA transcript (3.3 kb), and a single RAR gamma mRNA transcript (3.3 kb). Cultured rat lacrimal acinar cells also expressed the mRNA for all three RAR subtypes. Rabbit lacrimal glands express mRNAs for RAR alpha (3.7 and 2.9 kb) and RAR beta (3.2 kb) but RAR gamma mRNA is not detectable. Human lacrimal glands also express mRNA for RAR alpha (3.5 and 2.3 kb), RAR beta (3.4 kb) and RAR gamma (3.0 kb). Lacrimal gland nuclear extracts contain proteins in the 50 kDa range that specifically bind retinoic acid with Kd = 1.25 nM in rat lacrimal gland and 0.3 nM in rabbit. The monoclonal antibodies identified RAR alpha and RAR beta in both rat and rabbit lacrimal glands. The results of this study support a role for retinoids in maintaining the structure and function of the lacrimal gland. The presence of RARs suggests potential interactions of these receptors with other members of their superfamily, including androgen and thyroid receptors, which also may be involved in lacrimal function.

Simultaneous effects of the platelet 5-HT2 and alpha2-adrenergic receptor populations on phosphoinositide hydrolysis

The interaction of multiple receptor populations on a common second messenger system is a critical aspect of cell function and may be involved in pathology. We studied the interactions of the 5-HT2, alpha 2-adrenergic and prostaglandin (PGI2) receptors on phosphoinositide (PI) turnover in human platelets. Serotonin and epinephrine (EPI) stimulated PI hydrolysis in a dose-dependent manner. The PI turnover response to serotonin was mediated by the 5-HT2 receptor. The PI response to EPI was mediated by alpha 2-adrenergic receptors. An additive PI turnover response was generated by the combination of 5-HT and EPI. The sum of the maximal responses to 5-HT (72.5 +/- 4.9%) and EPI (56.0 +/- 4.2%) approximated the maximal response (129.3 +/- 9.5) to the combination. Prostacyclin (PGI2) at 1 microgram/mL reduced PI turnover by 21.8 +/- 1.1%. The PI response to 5-HT and EPI was not significantly altered once the reduction in the baseline PI turnover by PGI2 is taken into account. Similarly, PGI2 did not reduce PI hydrolysis stimulated by a combination of 5-HT (0.2 mM) and EPI (0.1 mM) once the decrease in baseline was taken into account (p greater than 0.20). The summation of serotonin stimulation of PI turnover by a combination of both epinephrine and serotonin was blocked by either yohimbine or ketanserin. These studies indicate: (1) the pool of phospholipases appears to exceed the maximal capacity of the individual alpha 2-adrenergic and 5-HT2 receptor populations to activate this second messenger system. (2) inhibition of serotonin or epinephrine-stimulated PI turnover by prostacyclin is due to a lowering of basal PI turnover. Future studies should examine other cell systems to assess the generalizability of these findings regarding the differences in effects on a second messenger system when activated by one receptor population as opposed to two different receptor types.

Refinement of the physical location and the genomic characterization of the CRSP2 (EXLM1) gene on Xp11.4.

In the course of our search for the gene responsible for X-linked cone-rod dystrophy (COD1), we constructed a physical map and contig (encompassing the region between DXS556 and DXS228), and identified sequences showing homologies to the expressed sequence tags (ESTs) that matched CRSP2 (EXLM1) transcript. We confirmed the expression of the CRSP2 gene in the retina and refined its exact genomic location between DXS1368 and DXS993. We demonstrated that the entire transcript is encoded within 31 exons. Primers were designed for mutation analysis of the exons by direct sequencing of PCR products from genomic DNA, and revealed no mutations in COD1 families. We subsequently excluded CRSP2 as a candidate for COD1 by demonstrating the causative mutations in the RPGR. However, due to its expression in different tissues and its contribution to transcriptional regulation, CRSP2 may be a candidate for other diseases that map to this region of the X chromosome.