Minimal Efficacy of Nitisinone Treatment in a Novel Mouse Model of Oculocutaneous Albinism, Type 3.

Purpose: Oral nitisinone has been shown to increase fur and ocular pigmentation in a mouse model of oculocutaneous albinism (OCA) due to hypomorphic mutations in tyrosinase (TYR), OCA1B. This study determines if nitisinone can improve ocular and/or fur pigmentation in a mouse model of OCA type 3 (OCA3), caused by mutation of the tyrosinase-related protein 1 (Tyrp1) gene. Methods: Mice homozygous for a null allele in the Tyrp1 gene (C57BL/6J-Tyrp1 b-J/J) were treated with 8 mg/kg nitisinone or vehicle every other day by oral gavage. Changes in fur and ocular melanin pigmentation were monitored. Mature ocular melanosome number and size were quantified in pigmented ocular structures by electron microscopy. Results: C57BL/6J-Tyrp1 b-J/J mice carry a novel c.403T>A; 404delG mutation in Tyrp1, predicted to result in premature truncation of the TYRP1 protein. Nitisinone treatment resulted in an approximately 7-fold increase in plasma tyrosine concentrations without overt toxicity. After 1 month of treatment, no change in the color of fur or pigmented ocular structures was observed. The distribution of melanosome cross-sectional area was unchanged in ocular tissues. There was no significant difference in the number of pigmented melanosomes in the RPE/choroid of nitisinone-treated and control groups. However, there was a significant difference in the number of pigmented melanosomes in the iris. Conclusions: Treatment of a mouse model of OCA3 with oral nitisinone did not have a favorable clinical effect on melanin production and minimally affected the number of pigmented melanosomes in the iris stroma. As such, treatment of OCA3 patients with nitisinone is unlikely to be therapeutic.

Associations of OCA2-HERC2 SNPs and haplotypes with human pigmentation characteristics in the Brazilian population.

Panels composed of Single Nucleotide Polymorphisms (SNPs) in genes related to pigmentation, when associated with different phenotypes, may assist in predicting the physical appearance of an individual, being very useful in forensic caseworks. We evaluated the association of seven OCA2-HERC2 SNPs and haplotypes with pigmentation characteristics (eye, skin, hair and freckles) in the highly admixed and phenotypically heterogeneous Brazilian population. All the seven SNPs evaluated presented one allele associated with phenotypes from at least two pigmentation features and the alternative allele associated with the opposite phenotypes from the same trait. The genotypic associations followed the same pattern for all seven SNPs. Nine haplotypes were observed in our sample and eight were associated with at least two pigmentation traits. Such SNPs and haplotypes could be deemed as good predictors for the presence of freckles and for skin, eye and hair pigmentation in the Brazilian population.

PARTIAL OCULOCUTANEOUS ALBINISM AND IMMUNODEFICIENCY SYNDROMES: TEN YEARS EXPERIENCE FROM A SINGLE CENTER IN TURKEY.

BACKGROUND AND AIM: Partial oculocutaneous albinism and immunodeficiency (OCA-ID) diseases are autosomal recessive syndromes characterized by partial hypopigmentation and recurrent infections. Moreover, some OCA-ID syndromes confer susceptibility to develop a life-threatening hyperinflammatory condition called hemophagocytic lymphohistiocytosis (HLH). We investigated the genetic, clinical and immunological characteristics of 20 OCA patients. MATERIAL AND METHODS: Herein, we present the clinical and immunological characteristics of 20 OCA patients who referred to the Department of Pediatric Immunology, Erciyes University Medical Faculty in Kayseri, Turkey between 2004 and 2014. RESULTS: Of the 20 OCA patients, 7 fulfilled diagnostic criteria for HLH, 9 showed defective functions of CD8 T cells and natural killer cells, and 8 received a definitive molecular diagnosis. Among the patients, we also report a patient diagnosed with two different genetic defects, in TYR and JAK3 genes, causing, respectively, OCA and ID. CONCLUSION: Our results illustrate the variability of clinical presentations and disease severity in OCA-ID patients, with consequent challenges in diagnosing and treating these patients.

Super-resolution microscopy as a potential approach to diagnosis of platelet granule disorders.

BACKGROUND: Many platelet functions are dependent on bioactive molecules released from their granules. Deficiencies of these granules in number, shape or content are associated with bleeding. The small size of these granules is such that imaging them for diagnosis has traditionally required electron microscopy. However, recently developed super-resolution microscopes provide sufficient spatial resolution to effectively image platelet granules. When combined with automated image analysis, these methods provide a quantitative, unbiased, rapidly acquired dataset that can readily and reliably reveal differences in platelet granules between individuals. OBJECTIVE: To demonstrate the ability of structured illumination microscopy (SIM) to efficiently differentiate between healthy volunteers and three patients with Hermansky-Pudlak syndrome. METHODS: Blood samples were taken from three patients with Hermansky-Pudlak syndrome and seven controls. Patients 1-3 have gene defects in HPS1, HPS6 and HPS5, respectively; all controls were healthy volunteers. Platelet-rich plasma was isolated from blood and the platelets fixed, stained for CD63 and processed for analysis by immunofluorescence microscopy, using a custom-built SIM microscope. RESULTS: SIM can successfully resolve CD63-positive structures in fixed platelets. A determination of the number of CD63-positive structures per platelet allowed us to conclude that each patient was significantly different from all of the controls with 99% confidence. CONCLUSIONS: A super-resolution imaging approach is effective and rapid in objectively differentiating between patients with a platelet bleeding disorder and healthy volunteers. CD63 is a useful marker for predicting Hermansky-Pudlak syndrome and could be used in the diagnosis of patients suspected of other platelet granule disorders.

A new genetic variant of mdx mice: study of the phenotype.

Genetic selection in a colony of mdx mice (suffering from X-chromosome-linked muscular dystrophy) resulted in generation of their new genetic variant. In this new variant, the genetic, biochemical, and histological markers of muscular dystrophy are combined with signs of oculocutaneous albinism (skin and fur depigmentation), transillumination of the iris, sharply reduced pigmentation of the retinal epithelium, and increase of the eyeball refraction). Two sensorimotor tests (negative geotaxis and wire back down hanging) detected other phenotypical characteristics of albino mdx mice carrying, in addition to the mutation in the dystrophin gene exon 23 (intrinsic of the "classical" black mdx mice), an extra mutation responsible for pigmentation disorders. Slow geotaxis, despite longer wire back down hanging capacity, was regarded as aggravation of the neurological dysfunction in albino mdx mice in comparison with black mdx mice.

Abnormal expression and subcellular distribution of subunit proteins of the AP-3 adaptor complex lead to platelet storage pool deficiency in the pearl mouse.

The pearl mouse is a model for Hermansky Pudlak Syndrome (HPS), whose symptoms include hypopigmentation, lysosomal abnormalities, and prolonged bleeding due to platelet storage pool deficiency (SPD). The gene for pearl has recently been identified as the beta3A subunit of the AP-3 adaptor complex. The objective of these experiments was to determine if the expression and subcellular distribution of the AP-3 complex were altered in pearl platelets and other tissues. The beta3A subunit was undetectable in all pearl cells and tissues. Also, expression of other subunit proteins of the AP-3 complex was decreased. The subcellular distribution of the remaining AP-3 subunits in platelets, macrophages, and a melanocyte-derived cell line of pearl mice was changed from the normal punctate, probably endosomal, pattern to a diffuse cytoplasmic pattern. Ultrastructural abnormalities in mutant lysosomes were likewise apparent in mutant kidney and a cultured mutant cell line. Genetically distinct mouse HPS models had normal expression of AP-3 subunits. These and related experiments strongly suggest that the AP-3 complex regulates the biogenesis/function of organelles of platelets and other cells and that abrogation of expression of the AP-3 complex leads to platelet SPD.

Platelet von Willebrand factor in Hermansky-Pudlak syndrome.

The Hermansky-Pudlak Syndrome (HPS) is an autosomal recessive inherited disorder characterized by oculocutaneous albinism, tissue accumulation of ceroid pigment, and a mild to moderate bleeding diathesis attributed to storage-pool deficient (SPD) platlets. Patients have platelet aggregation and release abnormalities. In addition, low levels of plasma von Willebrand factor (vWF) antigen in some HPS patients have been associated with a greater bleeding tendency than would be predicted from either condition alone. Other HPS patients have severe bleeding despite normal levels of plasma vWF, suggesting that at least one additional factor is responsible for their bleeding diathesis. Because platelet vWF levels have been well correlated with clinical bleeding times in patients with von Willebrand's disease, we have measured the platelet vWF activity and antigen levels in 30 HPS patients and have attempted to correlate their clinical bleeding with these values. The platelet vWF activity levels in patients was significantly lower than that of normal subjects (P < 0.0001). The patients as a group also had slightly lower values of plasma vWF activity when compared with normals (P-0.03). In 11 of the HPS patients, the multimeric structure of plasma vWF showed a decrease in the high molecular weight multimers and an increase in the low molecular weight multimers. In correlating the platelet and plasma vWF values with the bleeding histories, we were not able to show a predictable relationship in the majority of the patients.

The lysosomal granule membrane protein, LAMP-2, is also present in platelet dense granule membranes.

Lysosomal Associated Membrane Protein-2 (LAMP-2) is an inherent component of lysosomal granule membranes in diverse cell types, including platelets. We examined platelets for evidence of LAMP-2 in dense granule membranes as CD63 has previously been shown to be present in both lysosomal and dense granule membranes. Immunological techniques were used to examine the localization of LAMP-2 in control platelets and those from an individual with Hermansky-Pudlak syndrome (HPS), a condition characterised by platelet dense granule deficiency. Immunoblotting studies demonstrated that LAMP-2 was enriched in a dense granule preparation. Flow cytometry of thrombin-stimulated control platelets was consistent with biphasic surface expression of LAMP-2. The early expression was accompanied by dense granule, but minimal lysosomal granule, release. The late expression was accompanied by additional lysosomal granule release only. Thrombin stimulation of HPS platelets showed only late, lysosome-associated LAMP-2 expression. Immunoelectron microscopy indicated the presence of LAMP-2 in the membranes of serotonin-containing granules as identified by an anti-serotonin polyclonal antibody. These data indicate that LAMP-2 is present in the membranes of platelet dense granules in addition to lysosomal granules, and has a similar distribution to CD63.

Oculocutaneous albinism and bruising in two sisters--probable Hermansky-Pudlak syndrome.

Cases of Hermansky-Pudlak syndrome may present to the dermatologist, ophthalmologist or haematologist. Classically the diagnosis rests on the triad of oculocutaneous albinism, a bruising tendency and the presence of characteristic pigmented-containing cells in the bone marrow. Specific tests of platelet function can, however, suggest the diagnosis without recourse to invasive techniques. We describe the disease in two young sisters.

Studies on the platelet surface-associated von Willebrand factor.

The contribution of platelets to the prevention and control of bleeding depends not only on the plasma and vessel wall von Willebrand (vW) factor, but also on the vW factor originating from platelets. Although platelet vW factor is mainly stored in the alpha-granules, small amounts of vW factor have been detected on the platelet surface under conditions without direct stimulation. This communication focuses on the small amount of vW factor associated with the surface of platelets. EDTA-washed platelets in a calcium-free medium containing prostaglandin E1 (PGE1) were exposed to anti-vW factor F(ab')2 fragments. The platelet-associated anti-vW factor antibodies were separated from the free fragments by gel chromatography. Normal platelets were compared to platelets from patients with Bernard Soulier syndrome, Glanzmann's thrombasthenia, and Hermansky-Pudlak syndrome. These experiments showed that, in the absence of extracellular calcium, vW factor can be detected on the surface of washed platelets independent of glycoproteins Ib and IIb-IIIa, and despite markedly reduced intracellular calcium content. This particular platelet surface expression of vW factor is probably the result of disturbing the platelet during the in vitro manipulation. von Willebrand factor is an extremely important element in the multiple molecular interactions required to stop and anchor the platelet in areas of high flow rates. Mechanical perturbation of platelets leading to surface expression of the vW factor helps increase its availability for such critical interactions.

Synergistic effect of storage pool deficient platelets and low plasma von Willebrand factor on the severity of the hemorrhagic diathesis in Hermansky-Pudlak syndrome.

A study of 565 Puerto Rican patients with storage pool deficient (SPD) Hermansky-Pudlak syndrome (HPS) demonstrated that most HPS patients had minor bleeding episodes while others had repeated, severe hemorrhagic episodes requiring transfusion. The severity of bleeding in these latter patients could not be attributed to their SPD alone. As swine with SPD platelets and low von Willebrand factor antigen (vWF:Ag) have more severe hemorrhages than pigs with either defect alone, 146 albino patients and 46 normally pigmented patients were examined for their level of vWF:Ag. The risk of SPD HPS patients having severe, repeated bleeding episodes increased when vWF:Ag fell below 70 U/dL. Family studies indicated that low vWF:Ag levels were more frequently associated with O blood group than from a gene suppressing production or release of vWF1. HPS patients should be tested for vWF:Ag levels.

The protein CD63 is in platelet dense granules, is deficient in a patient with Hermansky-Pudlak syndrome, and appears identical to granulophysin.

The levels and expression of the proteins CD63 and granulophysin in platelets from control and from a Hermansky-Pudlak syndrome subject (a condition characterized by dense granule and lysosomal deficiencies and the accumulation of ceroid-like material in reticuloendothelial cells) were examined. Immunofluorescence studies indicated that anti-CD63 and anti-granulophysin antibodies recognized similar numbers of granules; coapplication of antibodies did not identify more granules than the individual antibodies. Significantly fewer granules were recognized in Hermansky-Pudlak syndrome platelets than in control using either antibody. Immunoblotting studies demonstrated that anti-CD63 and anti-granulophysin antibodies apparently recognize the same protein, which was deficient in Hermansky-Pudlak platelets. Analysis by fluorescence-activated cell sorter (FACS) showed biphasic expression of CD63 and granulophysin after thrombin stimulation of control but not Hermansky-Pudlak platelets. Anti-CD63 effectively blocked detection of the protein by anti-granulophysin using immunofluorescence, ELISA, immunoblotting, and FACS analysis. Amino-terminal sequencing over the first 37 amino acids revealed that granulophysin was homologous to CD63, melanoma antigen ME491, and pltgp40. These results suggest that granulophysin and CD63 are possibly identical proteins. This is the first report of a protein present in platelet dense granules, lysosomes, and melanocytes, but deficient in a patient with Hermansky-Pudlak syndrome.

Human platelets deficient in dense granules contain normal amounts of pp60c-src.

We have determined that pp60c-src, a protein tyrosine kinase abundant in normal platelets, is present at comparable levels in platelets that are deficient in dense granules (Hermansky-Pudlak syndrome). Relative quantitation of pp60c-src was performed by immunoblot analysis after protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our data suggest that human platelet dense granules, unlike chromaffin cell secretory granules, are not the major intracellular site of localization of pp60c-src.

Identification of a platelet dense granule membrane protein that is deficient in a patient with the Hermansky-Pudlak syndrome.

Monoclonal antibodies were raised after injecting mice with isolated human dense granules. Several of these monoclonals were found to recognize a 40-Kd dense granule membrane protein. Western blot and immunofluorescent analysis confirmed the dense-granule specificity. After thrombin activation, the protein was found in patches on the external platelet membrane. By Western blot and slot blot analysis, the protein was found to be markedly deficient in a patient with the Hermansky-Pudlak syndrome. Studies of neutrophils and endothelial cells show the presence of immunologically related granule-membrane protein(s). Western blots using four anti-synaptophysin antibodies and three antibodies to the platelet 40-Kd protein suggest that the protein may share some homology with, but is not identical to, the synaptosomal membrane protein synaptophysin.

In quest of the tyrosinase-positive oculocutaneous albinism gene.

The gene which causes tyrosinase-positive oculocutaneous albinism (ty-pos OCA) is not known. Forty-seven Bantu-speaking Negroid families with ty-pos OCA were studied in an attempt to find linkage to the gene. Fifteen 'classical' and seven DNA polymorphisms were used in the search for linkage. Close linkage was excluded for the Rh, Gc and beta-globin loci. There is no suggestion of linkage to MNS, ABO, PGM1, 6PGD, ACP1, GPX1, GLO1, GPT1, PEP A, Tf, alpha 1-AT, Hp, DQA, DXA and three arbitrary restriction fragment length polymorphisms (RFLPs). There is a slightly positive lod score for pAW101 (D14S1) (0.591 for theta = 0.2). An 'interesting' lod score was obtained with Bf and a haplotype generated by the markers DQA and DXA (1.575 for theta = 0.1 and 0.979 for theta = 0.2, respectively). Further testing of markers on chromosome 6p are indicated. Although ty-pos OCA in Southern Africa is likely to be a homogeneous disorder, genetic heterogeneity cannot be excluded as differences due to the presence/absence of ephelides within families have been observed. To date 57% of the genome has been excluded from linkage with ty-pos OCA.