Hermansky-Pudlak syndrome type 3 in Ashkenazi Jews and other non-Puerto Rican patients with hypopigmentation and platelet storage-pool deficiency

Abstract

Hermansky-Pudlak syndrome (HPS), consisting of oculocutaneous albinism and a bleeding diathesis due to the absence of platelet dense granules, displays extensive locus heterogeneity. HPS1 mutations cause HPS-1 disease, and ADTB3A mutations cause HPS-2 disease, which is known to involve abnormal intracellular vesicle formation. A third HPS-causing gene, HPS3, was recently identified on the basis of homozygosity mapping of a genetic isolate of HPS in central Puerto Rico. We now describe the clinical and molecular characteristics of eight patients with HPS-3 who are of non-Puerto Rican heritage. Five are Ashkenazi Jews; three of these are homozygous for a 1303+1G-->A splice-site mutation that causes skipping of exon 5, deleting an RsaI restriction site and decreasing the amounts of mRNA found on northern blotting. The other two are heterozygous for the 1303+1G-->A mutation and for either an 1831+2T-->G or a 2621-2A-->G splicing mutation. Of 235 anonymous Ashkenazi Jewish DNA samples, one was heterozygous for the 1303+1G-->A mutation. One seven-year-old boy of German/Swiss extraction was compound heterozygous for a 2729+1G-->C mutation, causing skipping of exon 14, and resulting in a C1329T missense (R396W), with decreased mRNA production. A 15-year-old Irish/English boy was heterozygous for an 89-bp insertion between exons 16 and 17 resulting from abnormal splicing; his fibroblast HPS3 mRNA is normal in amount but is increased in size. A 12-year-old girl of Puerto Rican and Italian background has the 3,904-bp founder deletion from central Puerto Rico on one allele. All eight patients have mild symptoms of HPS; two Jewish patients had received the diagnosis of ocular, rather than oculocutaneous, albinism. These findings expand the molecular diagnosis of HPS, provide a screening method for a mutation common among Jews, and suggest that other patients with mild hypopigmentation and decreased vision should be examined for HPS.

Figure 1

Whole-mount electron micrographs of platelet dense bodies. A, Normal platelets showing two to six dense bodies per platelet. B, Platelets of patient 91, which are devoid of dense bodies. C, Platelets of patient 100, which are totally lacking dense granules.

Figure 2

Northern blots demonstrating HPS3 expression. A, Multiple-tissue northern blots hybridized with an HPS3 cDNA (upper panel) and β-actin (lower panel) probe. The tissue/cell source of RNA is indicated above each lane. p = peripheral; PML = promyelocytic leukemia; CML = chronic myelogenous leukemia; LL = lymphoblastic leukemia; Adeno Ca = colorectal adenocarcinoma. B, Northern blots of mRNA isolated from fibroblasts cultured from patients with HPS who did not have HPS-1 or HPS-2 disease. Patient numbers correspond to a master list of NICHD patients with HPS.

Figure 3

Multiplex PCR amplification identifying the 3,904-bp founder deletion of central Puerto Rico. Patient 90 and her father (F) have the deletion in the heterozygous state, but the mother (M) lacks the deletion. M = 100-bp DNA ladder; N1 and N2 = normal control DNA; HPS-3 = DNA from a central Puerto Rican patient with HPS-3 who was homozygous for the deletion.

Figure 4

Mutation analysis in patient 91 and his family. A, PCR amplification of a 1,058-bp HPS3 cDNA fragment showing heterozygous skipping of exon 14 (with an additional 950-bp band) in patient 91, his two full sisters, and his mother. B, PCR amplification of exon 6 HPS3 genomic DNA (233-bp), followed by restriction enzyme digestion with DraIII. The paternal C1329T mutation introduces a DraIII recognition site, resulting in 102-bp and 131-bp bands after digestion. Patient 91, his father, and his affected sister carry the C1329T mutation.

Figure 5

Demonstration of a new splice site in patient 34. A, PCR amplification of a 956-bp cDNA fragment in normal cDNA (N1 and N2). Amplification reveals an 89-bp insertion. B, Sequencing indicates a heterozygous G44101A mutation in intron 16. C, Schematic drawing of the 89-bp insertion the patient’s cDNA. Exon 16A is expressed in HPS3 cDNA in a very rare alternative splice form in normal fibroblasts; however, the G44101C mutation introduces a new consensus splice site that results in insertion of 89-bp of exon 16A in the patient’s cDNA.

Figure 6

Ashkenazi Jewish mutation in HPS3. A, PCR amplification of a 908-bp HPS3 cDNA fragment, showing homozygous and heterozygous skipping of exon 5 (714-bp band). B, PCR amplification of exon 5 genomic DNA (328-bp) followed by RsaI restiction enzyme digestion. The 1303+1G→A splice-site mutation deletes an RsaI restriction site. Normal genomic DNA is cut into 251-bp, 42-bp, and 35-bp bands; homozygously mutated genomic DNA is cut into 251-bp and 77-bp bands. Patients 44, 89, and 100 exhibit homozygous 1303+1G→A mutations. Patients 26 and 86 carry the mutation in the heterozygous state. Patients 34, 90, and 91 do not have this mutation.

Figure 7

Hypopigmentation of hair, iris, and retina in patients with HPS-3. A, Hair pigmentation in patients of increasing age (see table 1). Color ranges from tan (patient 34) to dark brown (patient 90). Patient 100 tints her hair lighter, but her eyebrows are natural in color. B, Increasing severity of iris transillumination (top panel) and retinal hypopigmentation (bottom panel) in four patients with HPS-3. The picture on the left shows the retina of an unaffected control subject. Iris transillumination consists of light transmitted through the iris as a result of reduced pigment in that tissue. The normal iris remains dark when light is shone through the pupil. In the HPS-3 retinas, patchy hypopigmentation results from a reduction of pigment epithelium compared with normal retina (left). The degree of fundus hypopigmentation corresponds somewhat with the extent of iris transillumination.