Richard A. Spritz

Hermansky-Pudlak syndrome type 7 (HPS-7) results from mutant dysbindin, a member of the biogenesis of lysosome-related organelles complex 1 (BLOC-1)

Hermansky-Pudlak syndrome (HPS; MIM 203300) is a genetically heterogeneous disorder characterized by oculocutaneous albinism, prolonged bleeding and pulmonary fibrosis due to abnormal vesicle trafficking to lysosomes and related organelles, such as melanosomes and platelet dense granules. In mice, at least 16 loci are associated with HPS, including sandy (sdy; ref. 7). Here we show that the sdy mutant mouse expresses no dysbindin protein owing to a deletion in the gene Dtnbp1 (encoding dysbindin) and that mutation of the human ortholog DTNBP1 causes a novel form of HPS called HPS-7. Dysbindin is a ubiquitously expressed protein that binds to α- and β-dystrobrevins, components of the dystrophin-associated protein complex (DPC) in both muscle and nonmuscle cells. We also show that dysbindin is a component of the biogenesis of lysosome-related organelles complex 1 (BLOC-1; refs. 9–11), which regulates trafficking to lysosome-related organelles and includes the proteins pallidin, muted and cappuccino, which are associated with HPS in mice. These findings show that BLOC-1 is important in producing the HPS phenotype in humans, indicate that dysbindin has a role in the biogenesis of lysosome-related organelles and identify unexpected interactions between components of DPC and BLOC-1.

Mutations of PVRL1, encoding a cell-cell adhesion molecule/herpesvirus receptor, in cleft lip/palate-ectodermal dysplasia.

Cleft lip, with or without cleft palate (CL/P), is one of the most common birth defects, occurring in 0.4 to 2.0 per 1,000 infants born alive. Approximately 70% of CL/P cases are non-syndromic (MIM 119530), but CL/P also occurs in many single-gene syndromes, each affecting a protein critical for orofacial development. Here we describe positional cloning of the gene responsible for an autosomal recessive CL/P-ectodermal dysplasia (ED) syndrome (CLPED1; previously ED4; ref. 2), which we identify as PVRL1, encoding nectin-1, an immunoglobulin (Ig)-related transmembrane cell-cell adhesion molecule that is part of the NAP cell adhesion system. Nectin-1 is also the principal cell surface receptor for α-herpesviruses (HveC; ref. 7), and the high frequency of CLPED1 on Margarita Island in the Caribbean Sea might result from resistance of heterozygotes to infection by these viruses.

Mutation of PVRL1 is associated with sporadic, non-syndromic cleft lip/palate in northern Venezuela.

Non-syndromic cleft lip with or without cleft palate (CL/P, MIM 119530) is among the most common of major birth defects. Homozygosity for a nonsense mutation of PVRL1, W185X, results in an autosomal recessive CL/P syndrome on Margarita Island, CLPED1 (ref. 1). Here we demonstrate highly significant association between heterozygosity for this mutation and sporadic, non-syndromic CL/P in northern Venezuela.

Ru2 and Ru encode mouse orthologs of the genes mutated in human Hermansky-Pudlak syndrome types 5 and 6.

Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous disease involving abnormalities of melanosomes, platelet dense granules and lysosomes. Here we have used positional candidate and transgenic rescue approaches to identify the genes mutated in ruby-eye 2 and ruby-eye mice (ru2 and ru, respectively), two mimic mouse models of HPS. We also show that these genes are orthologs of the genes mutated in individuals with HPS types 5 and 6, respectively, and that their protein products directly interact. Both genes are previously unknown and are found only in higher eukaryotes, and together represent a new class of genes that have evolved in higher organisms to govern the synthesis of highly specialized lysosome-related organelles.

Hermansky-Pudlak syndrome is caused by mutations in HPS4, the human homolog of the mouse light-ear gene.

Hermansky-Pudlak syndrome (HPS) is a disorder of organelle biogenesis in which oculocutaneous albinism, bleeding and pulmonary fibrosis result from defects of melanosomes, platelet dense granules and lysosomes. HPS is common in Puerto Rico, where it is caused by mutations in the genes HPS1 and, less often, HPS3 (ref. 8). In contrast, only half of non–Puerto Rican individuals with HPS have mutations in HPS1 (ref. 9), and very few in HPS3 (ref. 10). In the mouse, more than 15 loci manifest mutant phenotypes similar to human HPS, including pale ear (ep), the mouse homolog of HPS1 (refs 13,14). Mouse ep has a phenotype identical to another mutant, light ear (le), which suggests that the human homolog of le is a possible human HPS locus. We have identified and found mutations of the human le homolog, HPS4, in a number of non–Puerto Rican individuals with HPS, establishing HPS4 as an important HPS locus in humans. In addition to their identical phenotypes, le and ep mutant mice have identical abnormalities of melanosomes, and in transfected melanoma cells the HPS4 and HPS1 proteins partially co-localize in vesicles of the cell body. In addition, the HPS1 protein is absent in tissues of le mutant mice. These results suggest that the HPS4 and HPS1 proteins may function in the same pathway of organelle biogenesis.

Novel Vitiligo Susceptibility Loci on Chromosomes 7 (AIS2) and 8 (AIS3), Confirmation of SLEV1 on Chromosome 17, and Their Roles in an Autoimmune Diathesis

To the Editor: n nGeneralized vitiligo (MIM 193200) is a common acquired disorder in which patches of white skin and overlying hair result from loss of pigment-forming melanocytes (reviewed in Bolognia et al. [1998], Kovacs [1998], and Hann and Nordlund [2000]), apparently because of a noninflammatory, T cell autoimmune response (reviewed in Ongenae et al. [2003]). Vitiligo occurs in 0.38% of whites (Howitz et al. 1977) and, in 23% of cases, is associated with other autoimmune disorders, particularly including autoimmune thyroid disease, pernicious anemia, systemic lupus erythematosus, Addison disease (Alkhateeb et al. 2003), and adult-onset insulin-dependent diabetes mellitus (our unpublished data). This complex of associated multiple autoimmune diseases most likely results from combinations of genes, some predisposing to an inherited autoimmune diathesis and others to specific forms of autoimmune disease. In the past, the association of various multiple autoimmune diseases has been termed “autoimmune polyendocrine syndrome, type 2” (APS2 [MIM 269200]), with a number of descriptive subcategories, the biological bases for which are uncertain (reviewed in Betterle et al. [2002]). n nVitiligo is a polygenic, multifactorial disorder (Majumder et al. 1988; Nath et al. 1994; Arcos-Burgos et al. 2002; Alkhateeb et al. 2003), with frequent family clustering (Mehta et al. 1973; Carnevale et al. 1980; Goudie et al. 1983; Hafez et al. 1983; Das et al. 1985; Majumder et al. 1993; Alkhateeb et al. 2003), and ∼20% of probands having at least one affected first-degree relative (Alkhateeb et al. 2003). We previously described a genome scan of 71 white multiplex families with vitiligo, and we mapped AIS1 (autoimmune susceptibility 1), a locus in chromosome segment 1p31.3-p32.2 that apparently confers susceptibility to generalized vitiligo and associated autoimmune disorders, as well as seven additional suggestive linkage signals on chromosomes 1, 7, 8, 11, 19, and 22 (Alkhateeb et al. 2002; Fain et al. 2003). n nWe have now extended this study to a total of 102 white multiplex families with vitiligo, providing substantially increased power for genomewide linkage analysis. Families were ascertained principally from the Vitiligo Society (U.K.) and the National Vitiligo Society (U.S.A.), as described elsewhere (Fain et al. 2003). Phenotypes were checked carefully by history, lesion maps, and, in most cases, physical examination and/or photographs; individuals for whom the phenotype was at all questionable were excluded. DNA was prepared from peripheral blood by standard methods, and genotyping was performed on a total of 660 individuals (300 affected with vitiligo; 192 females, 108 males) for 382 autosomal microsatellite markers from the ABI Prism 10-cM Linkage Mapping Set LMSv2-MD10, with manual checking of all genotypes by at least two people to minimize data errors, as described elsewhere (Fain et al. 2003). Multipoint nonparametric linkage analyses were performed using Allegro (Gudbjartsson et al. 2000). Heterogeneity testing between autoimmune and nonautoimmune families was performed using a predivided sample test (Morton 1956; Ott 1999). n nAs shown in table 1, analysis of the extended 102-family cohort provides continued strong support for AIS1 at 73.7 cM on chromosome 1p (LOD=5.59; P=.000000279) (all positions have been updated in accordance with the deCODE genome map [Kong et al. 2002]). In addition, two other signals that previously were only suggestive now achieve threshold criteria for significant linkage (Lander and Kruglyak 1995). These loci, now designated “AIS2” at 89.4 cM on chromosome 7 (LOD=3.73; P=.0000208) and AIS3 at 54.2 cM on chromosome 8 (LOD=3.36; P=.0000418), thus represent candidates for confirmation by analysis of a replicate family cohort. Our data also provide support for a locus at 4.3 cM on chromosome 17 (LOD=3.07; P=.0000852), most likely corresponding to SLEV1, a locus detected in multiplex families with lupus that also segregate cases of vitiligo (Nath et al. 2001). Three linkage signals that were suggestive in our previous analysis of 71 families (Fain et al. 2003)—at 12.2 cM on chromosome 1p, 4.1 cM on 11p, and 107.8 cM on 19q—fell below the threshold for suggestive linkage at the 102-family level. However, we detected two new linkage signals that met criteria for suggestive linkage (Lander and Kruglyak 1995)—at 88.1 cM on chromosome 9q (LOD=2.34; P=.000238) and at 109.4 cM on 13q (LOD=2.30; P=.000563)—that represent candidates for follow-up extension and replication linkage studies. n n n nTable 1 n nSuggestive and Significant LODs (and P Values) for 71 and 102 Multiplex White Families with Vitiligo[Note] n n n nThe 102 study families were selected solely on the basis of having multiplex cases of vitiligo. Nevertheless, half of the families segregated only vitiligo, whereas the other half also segregated various others of the vitiligo-associated autoimmune diseases (autoimmune thyroid disease, pernicious anemia, lupus, Addison disease, or adult-onset autoimmune diabetes mellitus) (Alkhateeb et al. 2002) (table 2). This provided an obvious basis for phenotypic stratification of the 102 families into autoimmunity-associated versus nonautoimmunity-associated family subgroups and analysis of the four a priori significant linkage signals in each subgroup. As shown in table 3, the AIS1, AIS2, and SLEV1 linkage signals derive principally from the autoimmunity-associated family subgroup and thus may predispose to a vitiligo-associated autoimmunity diathesis. Indeed, the AIS1 and SLEV1 LODs increased substantially on family stratification, even though the number of families was reduced by half. AIS1 was originally mapped in a large family with vitiligo, Hashimoto disease, and 21-hydroxylase autoantibody positivity (a preclinical marker for Addison disease) (Alkhateeb et al. 2002), and SLEV1 was originally mapped in families segregating lupus and vitiligo (Nath et al. 2001), so the derivation of these linkage signals from the autoimmunity-associated family subgroup is not unexpected. In contrast, the AIS3 linkage signal appears to derive principally from the nonautoimmunity-associated family subgroup. AIS3 may thus predispose to vitiligo per se, rather than to an autoimmune diathesis, although, of course, the basis of generalized vitiligo even in these families might still be autoimmune in nature. n n n nTable 2 n nVitiligo-Associated Autoimmune Diseases in the 51 Autoimmunity-Associated Families[Note] n n n n n nTable 3 n nLODs (and P Values) on Phenotypic Stratification of 102 Multiplex Families with Vitiligo into Autoimmunity-Associated and Nonautoimmunity-Associated Family Subgroups n n n nHeterogeneity testing did not quite exclude the possibility that AIS1 might also contribute to vitiligo susceptibility in the nonautoimmune families, since the total LOD score did not decrease significantly (P=.084) when the autoimmune and nonautoimmune family subgroups are combined, compared with the LOD in the autoimmune families alone. This was also the case for AIS2 and AIS3 (P=.362 and .192, respectively). These results suggest the possibility of allelic heterogeneity at these loci between these two groups of families. However, for SLEV1, there was a significant (P=.018) decrease in the LOD score when the autoimmune and nonautoimmune family subgroups were combined, a result that suggests linkage to SLEV1 in the autoimmune families and nonlinkage in the nonautoimmune families. Furthermore, although SLEV1 was originally detected in multiplex families with lupus with at least one case of vitiligo (Nath et al. 2001), there was only a single case of possible lupus among all of our 51 autoimmune families. Thus, linkage to SLEV1 in these families indicates that SLEV1 confers susceptibility to a broader range of autoimmune diseases than just lupus and vitiligo. n nOur data thus indicate that generalized vitiligo can be divided into at least two distinct phenotypic subcategories that apparently involve different loci or alleles. Vitiligo associated with a specific constellation of autoimmune diseases is linked with AIS1, AIS2, and SLEV1, whereas vitiligo unassociated with other autoimmune diseases is linked with AIS3. These findings begin to elucidate the genetic underpinnings of vitiligo and to dissect the contributions of individual loci to the vitiligo-associated autoimmune disease diathesis.

The molecular basis of oculocutaneous albinism type 1 (OCA1): sorting failure and degradation of mutant tyrosinases results in a lack of pigmentation

Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disease resulting from mutations of the tyrosinase gene (TYR). To elucidate the molecular basis of OCA1 phenotypes, we analysed the early processing and maturation of several different types of mutant tyrosinase with various degrees of structural abnormalities (i.e. two large deletion mutants, two missense mutants that completely destroy catalytic function and three missense mutants that have a temperature-sensitive phenotype). When expressed in COS7 cells, all mutant tyrosinases were sensitive to endoglycosidase H digestion, and immunostaining showed their localization in the endoplasmic reticulum (ER) and their failure to be sorted further to their target organelles. Pulse-chase experiments showed that all mutant tyrosinases were retained by calnexin in the ER and that they were degraded at similarly rapid rates, which coincided with their dissociation from calnexin. Temperature-sensitive mutant enzymes were sorted more efficiently at 31 degrees C than at 37 degrees C, and their degradation was accelerated at 37 degrees C compared with 31 degrees C. Thus in contrast to the current concept that mutant tyrosinases are transported to melanosomes but are functionally inactive there, our results suggest that mutant tyrosinases may not be transported to melanosomes in the first place. We conclude that a significant component of mutant tyrosinase malfunction in OCA1 results from their retention and degradation in the ER compartment. This quality-control process is highly sensitive to minimal changes in protein folding, and so even relatively minor mutations in peripheral sequences of the enzyme not involved with catalytic activity may result in a significant reduction of functional enzyme in melanosomes.

Deletion of the SLUG (SNAI2) gene results in human piebaldism.

Slug is a zinc‐finger neural crest transcription factor, encoded by the SLUG gene, which is critical for development of hematopoietic stem cells, germ cells, and melanoblasts in the mouse. In mouse, heterozygous and homozygous slug mutations result in anemia, infertility, white forehead blaze, and depigmentation of the ventral body, tail, and feet. This phenotype is very similar to the heterozygous W (KIT)‐mutant mouse phenotype and to human piebaldism, which is characterized by a congenital depigmented patches and poliosis (white forelock). To investigate the possibility that some cases of human piebaldism might result from abnormalities of the human SLUG (SNAI2) gene, we carried out Southern blot analysis of the SLUG gene in 17 unrelated patients with piebaldism, who lack apparent KIT mutations. Three of these patients had evident heterozygous deletions of the SLUG gene encompassing the entire coding region. Real‐time PCR confirmed the deletion in all cases. Fluoresence in situ hybridization (FISH) of genomic SLUG probes to metaphase chromosomes independently confirmed the deletion in one of the cases. These findings indicate that some cases of human piebaldism result from mutation of the SLUG gene on chromosome 8, and provide further strong evidence for the role of SLUG in the development of human melanocytes.

The rat Ruby ( R) locus is Rab38: identical mutations in Fawn-hooded and Tester-Moriyama rats derived from an ancestral Long Evans rat sub-strain.

AbstractHermansky-Pudlak syndrome (HPS) is a group of rare, recessive disorders in which oculocutaneous albinism, progressive pulmonary fibrosis, bleeding diathesis, and other abnormalities result from defective biogenesis of multiple cytoplasmic organelles. Seven different HPS genes are known in humans; in mouse, at least 16 loci are associated with HPS-like mutant phenotypes. In the rat, only two HPS models are known, Fawn-hooded (FH) and Tester Moriyama (TM), non-complementing strains in which HPS-like hypopigmentation and platelet storage pool deficiency result from a mutation of the Ruby (red eyed dilution; R) locus on Chromosome (Chr) 1. We have identified the R locus as the Rab38 gene, establishing that rat R is homologous to mouse chocolate (cht). Further, we show that FH and TM rats have identical Rab38 Met1Ile mutations, occurring on an identical Chr 1 marker allele haplotype, indicating that these two strains derive from a common ancestor. This ancestor appears to have been a sub-strain of the outbred Long Evans (LE) strain, and several modern LE sub-strains carry the Rab38 Met1Ile R mutation on the same Chr 1 marker haplotype. These findings have significant implications for the many past and ongoing studies that involve the FH and LE-derivative rat strains.nHermansky-Pudlak syndrome (HPS; MIM 203300) is a group of autosomal recessive diseases in which oculocutaneous albinism (OCA), progressive and fatal pulmonary fibrosis, and bleeding diathesis due to platelet storage pool deficiency result from defects in the biogenesis of specific cytoplasmic organelles and granules: melanosomes, lysosomes, and platelet dense granules (reviewed in Spritz 1999, 2000; Spritz et al. 2003). In humans, seven different HPS genes are known (Oh et al. 1996; Dell’Angelica et al. 1999; Anikster et al. 2001; Suzuki et al. 2002; Li et al. 2003; Zhang et al. 2003). In the mouse, at least 16 loci associated with HPS-like mutant phenotypes are known, seven of which are homologous to the human HPS loci (Swank et al. 1998; Bennett and Lamoreux 2003).

Multi-organellar disorders of pigmentation: intracellular traffic jams in mammals, flies and yeast

Several different mutant genes in humans, mice and Drosophila, most of which were identified initially on the basis of reduced pigmentation, have been associated with defects of multiple cytoplasmic organelles - melanosomes, lysosomes and granules. Recent discoveries show that several of these mutations directly affect components in the pathway of organelle-specific protein trafficking, and provide new insights into the relationships of these pathways in mammals, flies and yeast.