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Identification of a Novel Transcript Produced by the Gene Responsible for the Hermansky–Pudlak Syndrome in Puerto Rico

1998; Elsevier BV; Volume: 110; Issue: 5 Linguagem: Inglês

10.1046/j.1523-1747.1998.00183.x

1523-1747

Scott C. Wildenberg, James P. Fryer, John M. Gardner, Murray H. Brilliant, William S. Oetting, Richard A. King,

Herpesvirus Infections and Treatments

Hermansky–Pudlak Syndrome (HPS) is a rare, autosomal recessive disorder that is characterized by oculocutaneous albinism, a predisposition to mild bleeding caused by storage-pool deficient platelets, and a ceroid storage disorder. A gene responsible for HPS in Puerto Rico maps to chromosome 10q2 and isolation of the gene has been reported. We have now identified a variant HPS cDNA that contains the same 5′ sequence as the published HPS gene and a unique 3′ sequence. Analysis of genomic DNA suggests that the two cDNA are derived from alternative transcripts of a single gene; two polyadenylated transcripts were found in normal human melanocytes, human bone marrow cells, human melanoma cells, lymphoblastoid cell lines, and megakaryocytic leukemia cells by reverse transcriptase polymerase chain reaction and northern analysis. The splicing exhibited by this gene is identical to the splicing found to produce two alternative transcripts of the Chediak–Higashi Syndrome gene, another pigment disorder exhibiting platelet storage pool deficiency. These studies show that the HPS gene on chromosome 10 is complex and may have more than one biologically active transcript. Hermansky–Pudlak Syndrome (HPS) is a rare, autosomal recessive disorder that is characterized by oculocutaneous albinism, a predisposition to mild bleeding caused by storage-pool deficient platelets, and a ceroid storage disorder. A gene responsible for HPS in Puerto Rico maps to chromosome 10q2 and isolation of the gene has been reported. We have now identified a variant HPS cDNA that contains the same 5′ sequence as the published HPS gene and a unique 3′ sequence. Analysis of genomic DNA suggests that the two cDNA are derived from alternative transcripts of a single gene; two polyadenylated transcripts were found in normal human melanocytes, human bone marrow cells, human melanoma cells, lymphoblastoid cell lines, and megakaryocytic leukemia cells by reverse transcriptase polymerase chain reaction and northern analysis. The splicing exhibited by this gene is identical to the splicing found to produce two alternative transcripts of the Chediak–Higashi Syndrome gene, another pigment disorder exhibiting platelet storage pool deficiency. These studies show that the HPS gene on chromosome 10 is complex and may have more than one biologically active transcript. Hermansky–Pudlak Syndrome. Hermansky–Pudlak Syndrome (HPS) is a rare, autosomal recessive disorder with three distinct phenotypic features. Affected individuals have oculocutaneous albinism, a predisposition toward mild bleeding due to the lack of platelet dense bodies, and a ceroid storage disorder. Variability in the HPS phenotype suggests possible genetic heterogeneity for the disorder. This is supported by the existence of several genetically distinct mouse mutants with phenotypes similar to HPS (Novak et al., 1984Novak E.K. Hui S.W. Swank R.T. Platelet storage pool deficiency in mouse pigment mutations associated with seven distinct genetic loci.Blood. 1984; 63: 536-544Crossref PubMed Google Scholar). There is also variability among affected Puerto Rican individuals, suggesting the involvement of other genetic or environmental factors in the phenotypic expression of HPS (Summers et al., 1988Summers C.G. Knobloch W.H. Witkop C.J. King R.A. Hermansky–Pudlak syndrome: ophthalmic findings.Ophthalmol. 1988; 95: 545-554Abstract Full Text PDF PubMed Scopus (90) Google Scholar). In previous studies, we and others mapped a locus linked to HPS in a Puerto Rican population to chromosome 10q2 (Fukai et al., 1995Fukai K. Oh J. Frenk E. Almadovar C. Spritz R.A. Linkage Disequilibrium Mapping of the Gene for Hermansky–Pudlak Syndrome to Chromosome 10q23.1-q23 3 Hum Molec Genet. 1995; 4: 1665-1669Crossref PubMed Scopus (64) Google Scholar; Wildenberg et al., 1995Wildenberg S.C. Oetting W.S. Almadovar C. Krumwiede M. White J.G. King R.A. A gene causing Hermansky–Pudlak syndrome in a Puerto Rican population maps to chromosome 10q2.Am J Hum Genet. 1995; 57: 755-765PubMed Google Scholar). This region of chromosome 10q has conserved synteny with the distal arm of mouse chromosome 19 where the HPS homolog, pale ear (ep), maps (O’Brien et al., 1994O’Brien E.P. Novak E.K. Keller S.A. Poirier C. Guenet J.L. Swank R.T. Molecular map of chromosome 19 including three genes affecting bleeding time: ep, ru, and bm.Mamm Genome. 1994; 5: 356-360Crossref PubMed Scopus (27) Google Scholar; Feng et al., 1997Feng G.H. Bailin T. Oh J. Spritz R.A. Mouse pale ear (ep) is homologous to human Hermansky–Pudlak syndrome and contains a rare “AT-AC” intron.Hum Molec Genet. 1997; 5: 793-797Crossref Scopus (95) Google Scholar; Gardner et al., 1997Gardner J.M. Wildenberg S.C. Keiper N.M. et al.The mouse pale ear (ep) mutation is the homologue of human Hermansky–Pudlak Syndrome (HPS).Proc Natl Acad Sci USA. 1997; 94: 9238-9343Crossref PubMed Scopus (120) Google Scholar). Further studies allowed us to localize the gene to a small region of chromosome 10, to identify YAC clones containing this region, and to isolate a cDNA that represents the HPS gene from a human erythroleukemia cell cDNA library. The 5′ end of this cDNA is identical to the first 1145 bases of the gene previously reported to be responsible for HPS (Oh et al., 1996Oh J. Bailin T. Fukai K. et al.Positional cloning of a gene for Hermansky–Pudlak syndrome, a disorder of cytoplasmic organelles.Nat Genet. 1996; 14: 300-306Crossref PubMed Scopus (252) Google Scholar); however, the remainder of the cDNA is novel and is not included in the exon sequences of the recently published genomic sequence of the HPS gene (Bailin et al., 1997Bailin T. Oh J. Feng G.H. Fukai K. Spritz R.A. Organization and nucleotide sequence of the Hermansky-Pudlak (HPS) gene.J Invest Dermatol. 1997; 108: 923-927Abstract Full Text PDF PubMed Scopus (52) Google Scholar). Polyadenylated transcripts of the expected size for this cDNA as well as for the published cDNA have been identified in several tissues, and these transcripts appear to be alternative transcripts of the gene responsible for HPS. DNA sequencing was performed with the fmol cycle sequencing kit (Promega, Madison, WI). Primers were labeled in reactions containing 10 pmol primer, 2.5 U T4 kinase, 0.025 μCi γ-32P-dATP, and 1 × T4 PNK buffer (70 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 5 mM dithiothreitol), and incubated for 1 h at 37°C. Cycling conditions were 95°C for 1 min followed by 20 cycles of 95°C for 25 s, 58°C for 25 s, and 72°C for 20 s. Probes for Southern analysis were derived from three separate regions of the HPS gene by polymerase chain reaction (PCR) amplification of human genomic DNA or by restriction digestion of the 1.5 kb cDNA clone. Following separtation on 1% SeaKem GTG agarose (FMC BioProducts, Rockland, ME), restriction digested DNA was blotted onto a Zetabind membrane (Cuno, Meriden, CT), fixed by UV cross-linking, and prehybridized for 4 h at 55°C in 5 × sodium citrate/chloride buffer (SSC), 10 × Denhardt’s reagent, 50 mM Tris, 0.1% Na4P2O7, 1% sodium dodecyl sulfate (SDS), and 667 μg salmon sperm per ml. Hybridization was carried out in 5 × SSC, 1 × Denhardt’s reagent, 50 mM Tris, 0.1% Na4P2O7, 1% SDS, 15% dextran sulfate, 50% formamide, 100 μg salmon sperm DNA per ml, and 25 ng of α-32P-dCTP labeled probe at 42°C for 15–18 h. Post-hybridization washes were 15 min in 2 × SSC and 0.2% SDS at 42°C and 30 min at room temperature in 0.2 × SSC and 0.2% SDS. RNA was isolated from lymphoblastoid cells lines, melanoma, or megakaryocytic leukemia cells lines by a guanidinium isothiocyanate procedure (Sambrook et al., 1989Sambrook J. Fritsch E.F. Maniatis T. Extraction of RNA with guanidium thiocyanate followed by centrifugation in cesium chloride solutions.in: Ford N. Nolan C. Ferguson M. Ockler M. 2nd edn. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor1989: 7.19-7.22Google Scholar). Reverse transcription was carried out with 100 pmol oligo dT15, 200 units SuperScript II reverse transcriptase (Gibco BRL, Gaithersburg, MD), 250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2, 10 mM dithiothreitol, 30 units RNasin ribonuclease inhibitor (Promega, Madison, WI), 125 μM of each dNTP, and 1–5 μg of total RNA at 42°C for 2 h. Reverse transcription products were then amplified in reactions containing 1.5 mM MgCl2, 1×PCR buffer (50 mM KCl, 10 mM Tris-HCl pH 8.3), 20 pmol of each amplification primer, 200 μM of each dNTP, 2.5 U Ampli-Taq (Perkin Elmer, Branchburg, NJ), and 10 μl of the reverse transcription reaction. The cycling conditions consisted of 35 cycles of 95°C for 1 min, 55°C for 1 min 10 s, and 72°C for 1 min 30 s. For sequence analysis, PCR products were cloned into pCR 2.1 plasmid using the TA cloning kit (Invitrogen, Carlsbad, CA). Sequences of primers used for PCR amplifications are as follows: Primer 1: 5′-GGTGGCAGTGAGGAAGTATC-3′ Primer 2: 5′-GTCCACTGGAGCCTAAGACA-3′ Primer 3: 5′-GACTGTTAAGACCACTCCAC-3′ Primer 4: 5′-CCTTCCTTCAGCTTCTTCTC-3′ Primer 5: 5′-GAGACGGAGACAGACAGCTT-3′ Primer 6: 5′-CAGTGGAGTGGTCTTAACA-3′ Primer 7: 5′-CCGGTCATCATCTCCTCCAT-3′ Ten micrograms of total RNA from lymphocytes or 1 μg of polyadenylated RNA from melanoma or bone marrow cells (Clontech, Palo Alto, CA) was separated on 1% SeaKem GTG Agarose (FMC BioProducts, Rockland, ME) containing 1 × 2-[N-Morpholino]-ethanesulfonic acid and 5.92% formaldehyde, blotted onto a Zetabind membrane (Cuno, Meriden, CT) in 20 × SSC, and fixed to the membrane by UV cross-linking. Blots were prehybridized for 4 h at 55°C in 5 × SSC, 10 × Denhardt’s reagent, 50 mM Tris, 0.1% Na4P2O7, 1% SDS, and 40 μg salmon sperm DNA. Hybridization was carried out in 5 × SSC, 1 × Denhardt’s reagent, 50 mM Tris, 0.1% Na4P2O7, 1% SDS, 10% dextran sulfate, 50% formamide, 0.8 μg salmon sperm DNA, and 25 ng of α-32P-dCTP labeled probe at 42°C for 16–18 h. Post-hybridization washes were 15 min at room temperature in 2 × SSC, 0.2% SDS and 0.2 × SSC, 0.2% SDS at 42°C for 30 min. DNA for allele specific oligo analysis was blotted onto a Zetabind membrane (Cuno, Meriden, CT) using a 96 well Bio-Dot apparatus (Bio-Rad, Hercules, CA) and fixed by UV cross-linking. Oligonucleotide probes specific to normal or variant alleles were end-labeled with γ-32P-dATP by polynucleotide kinase (Promega, Madison, WI). Blots were prehybridized for 4 h at 55°C in 5 × SSC, 10 × Denhardt’s reagent, 50 mM Tris, 0.1% Na4P2O7, 1% SDS, and 100 μg salmon sperm DNA per ml, and hybridized with 3 M TMAC (Sigma, St. Louis, MO), 0.6% SDS, 1 mM ethylenediamine tetraacetic acid, 10 mM Na3PO4, 5 × Denhardt’s reagent, 1 mg salmon sperm DNA per ml, 26.25 pmol of unlabeled competitive primer, and 2.6 pmol of labeled primer at 55°C for 15–18 h. Post-hybridization washes were 20 min at room temperature in 3 M TMAC, 1 mM ethylenediamine tetraacetic acid, 10 mM Na3PO4, and 0.6% SDS followed by a second wash at 52°C for 20 min. Probes used were as follows: 1259C→T alteration: normal: 5′-AGTGGACCCCCGTGCCTCC-3′ mutant: 5′-AGTGGACCTCCGTGCCTCC-3′ 1328C→T alteration: normal: 5′-GCGGCTCCCCATCTTCCCT-3′ mutant: 5′-GCGGCTCCCTATCTTCCCT-3′ As previously described (Gardner et al., 1997Gardner J.M. Wildenberg S.C. Keiper N.M. et al.The mouse pale ear (ep) mutation is the homologue of human Hermansky–Pudlak Syndrome (HPS).Proc Natl Acad Sci USA. 1997; 94: 9238-9343Crossref PubMed Scopus (120) Google Scholar), the Puerto Rican HPS locus was mapped to a 1 cM region between D10S58 and D10S1433, and very close to D10S184 on chromosome 10q2, by recombination analysis and multipoint disequilibrium analysis (Terwilliger, 1995Terwilliger J.D. A powerful method for the analysis of linkage disequilibrium between trait and one or more polymorphic marker loci.Am J Hum Genet. 1995; 56: 777-787PubMed Google Scholar), and YAC containing these markers were identified (943f9 and 811d8). 1YAC clones were identified by searching the Whitehead Institute’s human genome map (http://www-genome.wi.mit.edu/cgi-bin/contig/phys_map) or by physical mapping data developed by the Genome Therapeutics Corporation (Waltham, MA). A single exon bearing fragment, 7RV-H, isolated from these YAC was used to isolate a human cDNA clone. This cDNA consisted of four EcoRI fragments whose lengths were 1.5, 1.3, 0.9, and 0.344 kb. Sequencing of these fragments revealed that the clone was a chimera consisting of two separate cDNA. The 1.3, 0.9, and 0.344 kb fragments all contained sequences identical to portions of the mRNA for human BiP protein (GenBank accession #X87949), and were not analyzed further. The sequence of the remaining 1.5 kb fragment was unique and contained partial sequence identity to the previously published HPS cDNA sequence (Oh et al., 1996Oh J. Bailin T. Fukai K. et al.Positional cloning of a gene for Hermansky–Pudlak syndrome, a disorder of cytoplasmic organelles.Nat Genet. 1996; 14: 300-306Crossref PubMed Scopus (252) Google Scholar). The complete DNA sequence of this 1.5 kb cDNA (GenBank accession #U96721) and the predicted amino acid sequence are shown in Figure 1. The gene responsible for the mouse pale ear phenotype was isolated by homology to this HPS cDNA (Gardner et al., 1997Gardner J.M. Wildenberg S.C. Keiper N.M. et al.The mouse pale ear (ep) mutation is the homologue of human Hermansky–Pudlak Syndrome (HPS).Proc Natl Acad Sci USA. 1997; 94: 9238-9343Crossref PubMed Scopus (120) Google Scholar). In a separate study, Oh et al. published the sequence of a 3.6 kb cDNA associated with HPS (Oh et al., 1996Oh J. Bailin T. Fukai K. et al.Positional cloning of a gene for Hermansky–Pudlak syndrome, a disorder of cytoplasmic organelles.Nat Genet. 1996; 14: 300-306Crossref PubMed Scopus (252) Google Scholar). Comparison of the published cDNA sequence with the sequence of the 1.5 kb cDNA suggested that both were from the same gene, but with significant differences. Bases 1–1145 of the 3.6 kb cDNA are identical to bases 17–1173 of the 1.5 kb cDNA, except for additional bases found at positions 74–76 and 119–127 in the 1.5 kb cDNA. Bases 1–16 of the 1.5 kb cDNA are absent from the 3.6 kb cDNA sequence. These additional bases are underlined in Figure 1, and their presence in the gene has been confirmed by sequencing genomic DNA. The most significant difference occurs after base 1173 of the 1.5 kb cDNA (base 1145 of the 3.6 kb cDNA). The two cDNA sequences diverge completely after this point. The 1.5 kb cDNA continues for an additional 319 bases, as shown in Figure 1, whereas the 3.6 kb cDNA continues for 2528 bases after the divergence point (Oh et al., 1996Oh J. Bailin T. Fukai K. et al.Positional cloning of a gene for Hermansky–Pudlak syndrome, a disorder of cytoplasmic organelles.Nat Genet. 1996; 14: 300-306Crossref PubMed Scopus (252) Google Scholar). The 319 bases unique to the 1.5 kb cDNA have no overlap with the 2528 bases unique to the 3.6 kb cDNA, and no homology to other cDNA could be found on a BLAST search. These bases are also excluded from the recently reported exon sequences of the HPS gene (Bailin et al., 1997Bailin T. Oh J. Feng G.H. Fukai K. Spritz R.A. Organization and nucleotide sequence of the Hermansky-Pudlak (HPS) gene.J Invest Dermatol. 1997; 108: 923-927Abstract Full Text PDF PubMed Scopus (52) Google Scholar). Sequence analysis of the region unique to the 1.5 kb cDNA in primate genomic DNA showed 98% homology between the human, gorilla, and chimpanzee sequences, and 96% homology between the human and orangutan sequences. Three probes were used to demonstrate that the two cDNA are derived from the same gene. One probe was derived from the region common to both cDNA, and the other probes were derived from the regions unique to each. For each probe, hybridization to YAC DNA (clone 811d8) and human genomic DNA identified the same specific fragments. Furthermore, PCR amplification of the common region and each unique region verified their presence in the YAC and in a 130 kb BAC clone (145 M16) reported to contain the HPS gene (Oh et al., 1996Oh J. Bailin T. Fukai K. et al.Positional cloning of a gene for Hermansky–Pudlak syndrome, a disorder of cytoplasmic organelles.Nat Genet. 1996; 14: 300-306Crossref PubMed Scopus (252) Google Scholar; Bailin et al., 1997Bailin T. Oh J. Feng G.H. Fukai K. Spritz R.A. Organization and nucleotide sequence of the Hermansky-Pudlak (HPS) gene.J Invest Dermatol. 1997; 108: 923-927Abstract Full Text PDF PubMed Scopus (52) Google Scholar) (data not shown). These results suggested that all three cDNA regions are likely to be part of a single gene. Amplification of genomic DNA further revealed that the region unique to the 1.5 kb cDNA and a portion of the common region are contained within a single exon. Human genomic DNA was amplified with primers 5 and 6 shown in Figure 2(a). The sequence of the resulting PCR product was identical to the sequence found in the 1.5 kb cDNA Figure 3. Furthermore, the sequences around the junction between the common region and the 1.5 kb cDNA unique region contained a consensus 5′ splice site. The sequence of bases 1169–1177 of the 1.5 cDNA is 5′-CAGGTGAGG-3′ as shown in Figure 3. This consensus splice site could allow splicing from the common region of the gene to the region unique to the 3.6 kb cDNA. The results of these analyses suggested a model for the genomic structure of the gene and the post-transcriptional processing that would produce the transcripts with alternative 3′ ends Figure 2a.Figure 3Sequence analysis of the HPS gene. RT-PCR products (a and c) and genomic DNA (b) were sequenced at the junction between the common region and the regions unique to the 1.5 kb cDNA (a and b) or the 3.6 kb cDNA (c). The sequence of the genomic DNA and the 1.5 kb cDNA matches the consensus 5′ splice site sequence: AG GT(A/G)AG.View Large Image Figure ViewerDownload (PPT) Messenger RNA from melanoma cells and bone marrow was hybridized to probes from the common region or from each of the unique 3′ regions. The probe from the unique region of the 1.5 kb cDNA hybridized to a major band in both tissues of ≈1.5 kb Figure 4a. The probe from the unique region of the 3.6 kb cDNA hybridized to a major band of about 3.0 kb in both tissues Figure 4a, as expected (Oh et al., 1996Oh J. Bailin T. Fukai K. et al.Positional cloning of a gene for Hermansky–Pudlak syndrome, a disorder of cytoplasmic organelles.Nat Genet. 1996; 14: 300-306Crossref PubMed Scopus (252) Google Scholar). The probe from the common region hybridized to both of these transcripts Figure 4a, and the bands identified were of approximately equal intensity as determined by densitometric analysis. The expression of the 1.5 and 3.6 kb cDNA was examined by RT-PCR in lymphoblastoid cell lines of HPS affected and unaffected individuals, normal melanocytes, and megakaryocytic leukemia cells. Total RNA was isolated, reverse transcribed with an oligo-dT primer, and the reverse transcription products were amplified with primers designed to amplify from the common region to each of the two unique 3′ regions. The 1.5 kb cDNA was amplified using primers 5 and 2 in Figure 2(a), and the 3.6 kb cDNA was amplified with primers 5 and 4. In all tissue types examined, these reactions produced fragments of the expected size Figure 5 and sequence Figure 3, indicating that both alternative transcripts are produced and polyadenylated in these cell types. To determine if a transcript containing both of the unique 3′ regions could be detected, RNA from normal melanocyte and leukemic megakaryocyte cells was reverse transcribed and amplified using primers 1 and 2 or 3 and 4 Figure 2a. These reactions did not produce any amplification products Figure 5, implying that transcripts can only contain one 3′ region or the other. Oh et al. identified a 16 base duplication mutation of the HPS gene in affected Puerto Rican individuals (Oh et al., 1996Oh J. Bailin T. Fukai K. et al.Positional cloning of a gene for Hermansky–Pudlak syndrome, a disorder of cytoplasmic organelles.Nat Genet. 1996; 14: 300-306Crossref PubMed Scopus (252) Google Scholar). This mutation is located in the unique region of the 3.6 kb cDNA. In addition to this 16 base duplication, we identified two other base changes in affected Puerto Ricans within the 3′-UTR unique to the 1.5 kb transcript: 1259C→T and 1328C→T. DNA from 81 individuals was screened for the presence of these alterations to determine their correlation with the HPS phenotype (Table 1). The 16 base duplication was analyzed by PCR amplification with the primers reported by Oh et al., 1996Oh J. Bailin T. Fukai K. et al.Positional cloning of a gene for Hermansky–Pudlak syndrome, a disorder of cytoplasmic organelles.Nat Genet. 1996; 14: 300-306Crossref PubMed Scopus (252) Google Scholar, and the alterations in the 1.5 kb cDNA unique region were analyzed by allele specific oligo hybridization. The 16 base duplication and the 1328C→T alteration segregated together completely in Puerto Rican individuals with HPS or those who were obligate heterozygotes. Neither of these alterations was found in non-Puerto Rican individuals. The 1259C→T alteration was present in DNA from many Puerto Rican and non-Puerto Rican individuals and was found in the homozygous state in five unaffected individuals.Table 1Results of HPS gene mutation analysisIndividuals examinedGenotypes16bp dupl.1259C→T1328C→TPuerto RicanHomozygous212121affectedmutantHeterozygous000Homozygous000normalPuerto RicanHomozygous000obligatemutantHeterozygotesHeterozygous121212Homozygous000normalPuerto RicanHomozygous020unaffectedmutantNon-carriersHeterozygous021Homozygous211720normalNon-Puerto RicanHomozygous030affectedmutantHeterozygous040Homozygous13613normalNon-Puerto RicanHomozygous030unaffectedmutantHeterozygous030Homozygous14813normalTotal individuals818181 Open table in a new tab We report here the identification of a novel 1.5 kb cDNA sequence associated with the HPS locus. The transcript represented by this cDNA shares 1173 bases with a previously reported cDNA and genomic sequence but diverges into a unique 319 base 3′ end. The region common to both cDNA and the two unique 3′ regions are all found within a 130 kb fragment of genomic DNA, suggesting that they are all part of a single gene, and expression studies have shown that both transcripts are produced in normal melanocytes, melanoma cells, bone marrow cells, leukemic megakaryoctes, and lymphoblastoid cell lines. Previous studies of this HPS associated gene, however, have not revealed the presence of transcripts containing the sequences unique to the 1.5 kb cDNA (Oh et al., 1996Oh J. Bailin T. Fukai K. et al.Positional cloning of a gene for Hermansky–Pudlak syndrome, a disorder of cytoplasmic organelles.Nat Genet. 1996; 14: 300-306Crossref PubMed Scopus (252) Google Scholar; Bailin et al., 1997Bailin T. Oh J. Feng G.H. Fukai K. Spritz R.A. Organization and nucleotide sequence of the Hermansky-Pudlak (HPS) gene.J Invest Dermatol. 1997; 108: 923-927Abstract Full Text PDF PubMed Scopus (52) Google Scholar). Our analysis now shows that complex post-transcriptional processing of this gene may play a role in the production of two alternative transcripts. Analysis of genomic DNA has suggested a model for the post-transcriptional processing used to produce the two alternative transcripts of this gene Figure 2a. The region unique to the 1.5 kb cDNA and a portion of the common region were found to be parts of a single exon. A consensus 5′ splice site was also found to exist in this exon at the partition between the common region and the region unique to the 1.5 kb cDNA. Because the divergence point between the two alternative cDNA lies within this exon, it appears that the transcript for the 1.5 kb cDNA is produced by polyadenylation after this exon and cleavage of the exons unique to the 3.6 kb cDNA. The 3.6 kb cDNA would then be produced by activation of the 5′ splice site within this exon to allow splicing to the sequences unique to that product. This type of RNA processing has been observed in the genes for the immunoglobulin M (IgM) heavy chain (Peterson and Perry, 1989Peterson M.L. Perry R.P. The regulated production of the mu m and mu s mRNA is dependent on the relative efficiencies of the mu s poly (A) site usage and the c mu 4-to-M1 splice.Molec Cell Bio. 1989; 9: 726-738Crossref PubMed Scopus (122) Google Scholar) and for the Chediak–Higashi Syndrome (CHS) (Barbosa et al., 1997Barbosa Mdfs Barrat F.J. Tchernev V.T. et al.Identification of mutations in two major mRNA isoforms of the Chediak–Higashi syndrome gene in human and mouse.Hum Molec Genet. 1997; 6: 1091-1098Crossref PubMed Scopus (81) Google Scholar). The IgM gene produces membrane bound (μm) or secreted (μs) protein products whose relative levels are regulated during B cell maturation. More relevant to our studies are the recent findings showing that the gene responsible for CHS produces two major transcripts using the same type of processing we have described for the HPS gene (Barbosa et al., 1997Barbosa Mdfs Barrat F.J. Tchernev V.T. et al.Identification of mutations in two major mRNA isoforms of the Chediak–Higashi syndrome gene in human and mouse.Hum Molec Genet. 1997; 6: 1091-1098Crossref PubMed Scopus (81) Google Scholar). Although disimilar in size, it is intriguing that both of these genes produce alternate transcripts in an identical manner and that HPS and CHS both exhibit oculocutaneous albinism and platelet storage pool deficiencies. The role played by the two alternative transcripts in the HPS phenotype deserves further study. It is clear that the 3.6 kb cDNA contributes to the phenotype because frame-shift mutations have been reported in the region unique to the 3.6 kb cDNA in patients from Puerto Rico, Japan, Switzerland, and Ireland (Oh et al., 1996Oh J. Bailin T. Fukai K. et al.Positional cloning of a gene for Hermansky–Pudlak syndrome, a disorder of cytoplasmic organelles.Nat Genet. 1996; 14: 300-306Crossref PubMed Scopus (252) Google Scholar). These alterations all result in aberrant translation and premature termination of the protein product. The recently cloned ep mouse mutant also contains a mutation within the region homologous to the sequences unique to the 3.6 kb cDNA (Feng et al., 1997Feng G.H. Bailin T. Oh J. Spritz R.A. Mouse pale ear (ep) is homologous to human Hermansky–Pudlak syndrome and contains a rare “AT-AC” intron.Hum Molec Genet. 1997; 5: 793-797Crossref Scopus (95) Google Scholar; Gardner et al., 1997Gardner J.M. Wildenberg S.C. Keiper N.M. et al.The mouse pale ear (ep) mutation is the homologue of human Hermansky–Pudlak Syndrome (HPS).Proc Natl Acad Sci USA. 1997; 94: 9238-9343Crossref PubMed Scopus (120) Google Scholar). The role of the 1.5 kb cDNA in HPS is, however, uncertain. We have identified two base changes in the region unique to this transcript among affected Puerto Rican individuals. One of these alterations (1259C→T) appears to be a nonpathologic base change. The other alteration (1328C→T) segregates completely with the HPS phenotype in Puerto Rican individuals and has not been observed among non-Puerto Ricans. It is not likely to be a pathogenic mutation because it is found within the 3′ untranslated region of the 1.5 kb cDNA. Its correlation with the HPS phenotype may simply be due to the fact that the Puerto Ricans we have examined all carry a single founder haplotype. The effects of this alteration have not been directly tested, however, and studies have suggested that the 3′-UTR may play a role in the proper expression of some genes (Jackson, 1993Jackson R.J. Cytoplasmic regulation of mRNA function: the importance of the 3′ untranslated region.Cell. 1993; 74: 9-14Abstract Full Text PDF PubMed Scopus (364) Google Scholar; Tsukamoto et al., 1996Tsukamoto H. Boado R.J. Pardridge W.M. Differential expression in glioblastoma multiforme and cerebral hemangioglastoma of cytoplasmic proteins that bind to different domains within the 3 prime-untranslated region of the human glucose transporter 1 (GLUT1) messenger RNA.J Clin Invest. 1996; 97: 2823-2832Crossref PubMed Scopus (37) Google Scholar). Furthermore, the sequences unique to the 1.5 kb cDNA have not yet been analyzed in non-Puerto Rican HPS individuals, so it is unknown whether there are pathologic alterations in this region among other affected individuals. It is also possible that mutations within the common region or within the region unique to the 1.5 kb cDNA lead to an as yet undetermined phenotype. The high degree of homology seen between human, gorilla, chimpanzee, and orangutan for the sequences unique to the 1.5 kb cDNA and for the consensus 5′ splice site in the exon containing these sequences, suggests that this region is required for proper function of the gene. Although a gene associated with HPS has been isolated, it appears to have a complex expression pattern. In addition to producing two transcripts with different 3′ ends, there is evidence suggesting alternative splicing within the region common to both transcripts. The biologic significance of these alternative forms remains to be seen, and knowledge gained from the study of this gene will provide further insight into the molecular systems affected by HPS. We would like to thank Dr. Harry Orr, Dr. Timothy Behrens, and Dr. Lynne Maquat for their helpful discussions and advice, and the HPS Network for their continued support. We also gratefully acknowledge Jen-I Mao from the Genome Therapeutics Corporation for providing contig and physical mapping information. This research was supported by NIH Grants #GM22167, #GM/AR56181, and #CA06927.