See related commentary on pg 1971 ORIGINAL ARTICLE

Homozygosity Mapping and Whole-Exome Sequencing to Detect SLC45A2 and G6PC3 Mutations in a Single Patient with Oculocutaneous Albinism and Neutropenia Andrew R. Cullinane1, Thierry Vilboux1, Kevin O’Brien2, James A. Curry1, Dawn M. Maynard1, Hannah Carlson-Donohoe1, Carla Ciccone1, NISC Comparative Sequencing Program3, Thomas C. Markello1, Meral Gunay-Aygun2, Marjan Huizing1 and William A. Gahl1,2

We evaluated a 32-year-old woman whose oculocutaneous albinism (OCA), bleeding diathesis, neutropenia, and history of recurrent infections prompted consideration of the diagnosis of Hermansky–Pudlak syndrome type 2. This was ruled out because of the presence of platelet d-granules and absence of AP3B1 mutations. As parental consanguinity suggested an autosomal recessive mode of inheritance, we employed homozygosity mapping, followed by whole-exome sequencing, to identify two candidate disease-causing , SLC45A2 and G6PC3. Conventional dideoxy sequencing confirmed pathogenic mutations in SLC45A2, associated with OCA type 4 (OCA-4), and G6PC3, associated with neutropenia. The substantial reduction of SLC45A2 protein in the patient’s melanocytes caused the mislocalization of tyrosinase from melanosomes to the plasma membrane and also led to the incorporation of tyrosinase into exosomes and secretion into the culture medium, explaining the hypopigmentation in OCA-4. Our patient’s G6PC3 mRNA expression level was also reduced, leading to increased apoptosis of her fibroblasts under endoplasmic reticulum stress. To our knowledge, this report describes the first North American patient with OCA-4, the first culture of human OCA-4 melanocytes, and the use of homozygosity mapping, followed by whole-exome sequencing, to identify disease-causing mutations in multiple genes in a single affected individual. Journal of Investigative Dermatology (2011) 131, 2017–2025; doi:10.1038/jid.2011.157; published online 16 June 2011

INTRODUCTION polymorphism (SNP) arrays, followed by the next-generation Until recently, molecular diagnostics for patients with rare sequencing technique of whole-exome sequencing (Ng et al., disorders relied upon sequencing of genes identified as 2009, 2010; Otto et al., 2010). This reduces the amount of candidates by virtue of the clinical phenotypes. Now, data generated and is more cost effective than whole-genome massively parallel (‘‘next generation’’) sequencing allows sequencing (Teer and Mullikin, 2010). for unfocused detection of disease-causing mutations, with- We illustrate the benefit of next-generation sequencing in out the bias introduced by phenotype. This critical advantage a patient with oculocutaneous albinism (OCA) and con- can be particularly important for patients with clinical genital neutropenia. OCA is a group of autosomal recessively manifestations that point to complex diseases. The process inherited hypopigmentary disorders that affect melanin that is generally employed involves single-nucleotide pigment synthesis or trafficking of pigmentary or organelles (King et al., 2002) in the skin, hair, and eyes 1Medical Genetics Branch, National Research Institute (Gronskov et al., 2007). Four genes are associated with OCA: (NHGRI), National Institutes of Health, Bethesda, Maryland, USA; 2Intramural TYR (OCA-1; Tomita et al., 1989), OCA2 (OCA-2; Rinchik Office of Rare Diseases Research, Office of the Director, National Institutes of et al., 1993), TYRP1 (OCA-3; Boissy et al., 1996), and Health, Bethesda, Maryland, USA and 3National Institutes of Health Intramural Sequencing Center (NISC), National Human Genome Research SLC45A2 (OCA-4; Newton et al., 2001). Mutations in these Institute, National Institutes of Health, Bethesda, Maryland, USA genes rarely cause phenotypic features other than albinism, Correspondence: Andrew R. Cullinane, Medical Genetics Branch, National but certain syndromes have OCA as an obligate component. Human Genome Research Institute (NHGRI), National Institutes of Health, Hermansky–Pudlak syndrome (HPS), e.g., is characterized by Bethesda, Maryland 20892, USA. E-mail: [email protected] OCA and a bleeding diathesis. There are 8 known human Abbreviations: ER, endoplasmic reticulum; HPS, Hermansky–Pudlak HPS genes (HPS1–8) (Oh et al., 1996; Dell’Angelica et al., syndrome; OCA, oculocutaneous albinism; SNP, single-nucleotide polymorphism 1999; Anikster et al., 2001; Suzuki et al., 2002; Li et al., Received 12 January 2011; revised 31 March 2011; accepted 5 April 2011; 2003; Zhang et al., 2003; Morgan et al., 2006), whose protein published online 16 June 2011 products are involved in the biogenesis of lysosome-related

& 2011 The Society for Investigative Dermatology www.jidonline.org 2017 AR Cullinane et al. Homozygosity Mapping and Whole-Exome Sequencing

organelles (Huizing et al., 2008). HPS commonly has other disease-associated genes, i.e., SLC45A2 causing OCA-4 and phenotypic features such as pulmonary fibrosis, which is G6PC3 causing neutropenia. Neither of these genes would associated with subtypes 1 and 4 (Oh et al., 1996; Suzuki have been pursued according to standard practice. We et al., 2002). conclude that SNP array analysis, along with whole-exome Congenital neutropenia is a heterogeneous group sequencing, can be useful in identifying disease-causing of hereditary disorders characterized by life-threatening mutations in a single affected individual, especially if the bacterial infections early in life due to a paucity of mature family is consanguineous and suitable for homozygosity neutrophils (Welte et al., 2006; Boztug et al., 2008). mapping. Mutations in several genes, including ELA2, HAX1, WAS, GFI1, and recently G6PC3 (Horwitz et al., 1999; Devriendt RESULTS et al., 2001; Person et al., 2003; Klein et al., 2007; Boztug Case report et al., 2009), cause such conditions. Neutropenia also occurs A 32-year-old Caucasian female presented to the National in patients with HPS-2 disease, due to mutations in AP3B1, Institutes of Health (NIH) Clinical Center with a history of which encodes the b-3A subunit of adaptor complex-3 inflammatory bowel disease, primary neutropenia and (Dell’Angelica et al., 1999). Most congenitally neutropenic thrombocytopenia, and a surgically repaired atrial septal patients, including those with HPS-2, respond to recombinant defect. Her medical history revealed that she was diagnosed human granulocyte colony-stimulating factor, which with OCA shortly after birth, and subsequent eye examina- increases neutrophil counts and decreases the number and tions confirmed the presence of ocular albinism (Figure 1a). severity of infections (Bonilla et al., 1989). The patient was hospitalized for E. coli sepsis in the neonatal In this article, we describe a patient of consanguineous period with a white cell count as high as 12 K ml1; her parentage who presented with findings that suggested the platelet count was 130 K ml1. In her first year of life, she possible diagnosis of HPS-2, i.e., OCA and neutropenia. suffered from recurrent ear and bladder infections. At 3 years When no mutations were found in AP3B1, we performed of age, she was hospitalized for pneumonia and was homozygosity mapping on a SNP array and whole-exome leukopenic, with a white blood cell count of 2,800 K ml1. sequencing using the patient’s genomic DNA. This allowed Thereafter, she continued to have recurrent lower urinary us to identify disease-causing mutations in two different tract infections and was placed on prophylactic antibiotics.

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Figure 1. Clinical findings and DNA analysis. (a) Fair skin and classic red hair in our patient, typical for oculocutaneous albinism type 4 patients. (b) Prominent superficial venous pattern seen in all previously described G6PC3 mutation-positive neutropenia patients. (c) Pedigree showing affected and unaffected individuals; our patient is depicted by the red box. Note the consanguineous background of the family. (d) Regions of homozygosity in the genome of the patient (shown within red boxes). (e) Chromatogram of the mutation in SLC45A2 (c.986delC, p.T329RfsX68). (f) Chromatogram of the mutation in G6PC3 (c.829C4T, p.Q277X). Control sequences are shown for comparison. Photograph published with patient’s permission.

2018 Journal of Investigative Dermatology (2011), Volume 131 AR Cullinane et al. Homozygosity Mapping and Whole-Exome Sequencing

Throughout childhood and adolescence, she had symptoms Other immunological, autoimmune, and infectious dis- of colitis, chronic abdominal pain, bloody diarrhea, recurrent ease studies were normal. She had a minimal decrease in her skin infections, easy bruising, and heavy menstrual periods. She total immunoglobulin A level, but other tests of immune was eventually diagnosed with Crohn’s disease at 16 years of competency were normal, and an assay for HIV was negative. age and failed medical therapy, resulting in a hemicolectomy at Her soluble IL-2R level was also normal. The antinuclear 17 years of age. Despite this surgery, she continued to have antigen level, extractable nuclear antigen panel, and assays symptoms of inflammatory bowel disease and has received for antibodies to platelets and neutrophils were all negative. antibiotics and immunosuppressive therapy intermittently. The patient’s parents were first cousins (Figure 1c). Because of her persistent cytopenias and history of Her brother had albinism, colitis, and a history of recurrent infections, the patient underwent a bone marrow aspiration infections. Although his medical history was similar to at 16 years of age that showed arrested neutrophil develop- the proband’s, he was never compliant with treatment and ment; granulocytic precursors were markedly increased became progressively more ill in his late 20 s and early 30 s, but matured only to the myelocyte and metamyelocyte stage. resulting in a colostomy at 32 years of age. Afterward, he The patient began receiving recombinant human granulocyte developed recurrent infections and was non-compliant with colony-stimulating factor (Neupogen, Amgen, Thousand recombinant human granulocyte colony-stimulating factor Oaks, CA), resulting in improved neutrophil counts (Supple- therapy. At 37 years of age, he was hospitalized for sepsis and mentary Table S1 online) and fewer infections, especially endocarditis, and died shortly afterward from multisystem skin abscesses. The absolute neutrophil count at 29 years of organ failure due to septic shock. age was recorded as 0; neutrophil release and chemotaxis tests were abnormal. The first report of significant thrombo- Molecular diagnostics cytopenia occurred at 29 years of age, with a platelet count of Owing to the presence of HPS-2-related symptoms of OCA, 94 K ml1. Thereafter, platelet counts ranged from 21 to bleeding and neutropenia, all coding exons and flanking 57 K ml1 and averaged 36.6 K ml1. Platelet aggregation intronic sequences of AP3B1 were sequenced using conven- studies showed an abnormal secondary aggregation tional dideoxy sequencing. No disease-causing mutations response, with no response to mini-dose ADP or adrenaline. were detected, and further analysis of mRNA and protein in However, whole-mount electron microscopy of her platelets dermal fibroblasts and melanocytes revealed no change showed the presence of dense bodies. Despite her laboratory in expression compared with control cells (data not shown). abnormalities, the patient did not experience serious bleed- As our current definition of HPS requires the absence of ing, nor did she receive platelets or DDAVP (desmopressin platelet dense bodies, and this patient had a normal acetate), even with major surgeries such as an atrial septal contingent of d-granules, we considered the possibility that defect repair at 31 years of age. this could represent a new disease. Upon admission to the NIH at 32 years of age, the proband underwent a screening examination that revealed SNP array analysis prognathism and abnormal skin findings. She had ecchy- The patient is a result of a first cousin union (Figure 1c), moses and petechiae on her extremities, an eczematous rash so autosomal recessive inheritance was suspected. Therefore, on her upper extremities, and fine telangiectasias on her a SNP–chip microarray was carried out on the patient’s arms and chest. A prominent superficial venous pattern was genomic DNA to identify regions of her genome that were present on her legs (Figure 1b). There was no evidence of skin homozygous and could harbor the disease-causing mutation. infections. Her eye exam was consistent with ocular Indeed, approximately B10% of her genome was homo- albinism, and her visual acuities were 20/160 in the right zygous including the centromeric areas of eye and 20/800 in the left eye. Her pulmonary, liver, and 4 and 5 (Figure 1d and Supplementary Figure S1 online). renal function tests were normal. These data, however, were not very informative, as the A hematological evaluation was performed to investigate regions of homozygosity were so large. the cytopenias. Her total leukocyte and neutrophil counts were low, but the remainder of her differential was normal, Whole-exome sequencing and her red blood cell and reticulocyte indices were normal We next subjected the patient’s DNA to whole-exome (Supplementary Table S2 online). A peripheral smear showed sequencing. This technique covers the 1.22% of the human neutropenia; the granulocytes contained primary granules, genome corresponding to the Consensus Conserved Domain although some were hypogranular. Rare Dohle bodies were Sequences database and greater than 1,000 non-coding also noted, and the platelets were enlarged. RNAs (Gnirke et al., 2009). After alignment of the patient’s Although the patient was receiving recombinant human DNA sequence fragments to the UCSC reference genome granulocyte colony-stimulating factor, the marrow aspirate (build 18), there were 62,235 variations and after filtering showed hypercellularity, myeloid hyperplasia with a left shift, out known SNPs using dbSNP (http://www.ncbi.nlm.nih.gov/ and increased megakaryocytes. The differential was 75% snp/), there were 13,950 variants. This number was reduced myeloid precursors, 19% erythroid precursors, 5% lympho- to 1502 variations after filtering for variants likely to affect cytes, and 1% blasts. Primary granules persisted, and some the protein sequence. After further filtering for regions of mature granulocytes had cytoplasmic vacuoles. Rare pelger- homozygosity from the SNP–chip data, the number of oid bodies were noted; there was no stainable iron. homozygous variations was reduced to 30 (Supplementary

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Table S3 online). From there, a candidate approach melanocytes, as seen in OCA-1 and OCA-3 cells (Halaban was used to identify potentially pathogenic mutations using et al., 2000; Toyofuku et al., 2001). In patient cells, tyrosinase the potential detriment (CD_Pred) score as a guide (Johnston was primarily localized to the plasma membrane as observed et al., 2010). by immunofluorescence microscopy (Figure 2d) and specifi- The most likely mutations identified were a known cally colocalized with the plasma membrane marker Nile red mutation in G6PC3 (c.829C4T, p.Q277X), which had (Figure 2f). Furthermore, after separation of plasma mem- previously been seen in a French patient (Boztug et al., brane proteins by biotinylation before cell lysis, tyrosinase 2009), and a novel mutation in SLC45A2 (c.987delA, was only found in the membrane fraction of patient cells p.A330PfsX68). Both of these mutations were in the expected (Figure 2e). Expression of tyrosinase in the patient’s melano- homozygous state. Conventional dideoxy sequencing of cytes was slightly reduced in whole-cell extracts compared G6PC3 confirmed the mutations and explained the patient’s with control cells; tyrosinase was detectable only in the neutropenia (Figure 1f), but the mutation identified by whole- growth medium of patient’s cells (Figure 2b). Furthermore, exome sequencing for SLC45A2 was incorrect. The actual isolating exosomes from the conditioned medium revealed mutation in SLC45A2 was c.986delC, p.T329RfsX68 (Figure that the secreted tyrosinase was present in exosomes 1e), a previously described mutation in five German OCA-4 (Figure 2b). patients (Rundshagen et al., 2004). An explanation for the It is possible that tyrosinase and other proteins are being discrepancy would be the misalignment of the whole-exome secreted into the medium as a result of the reduced G6PC3 reads due to presence of a known SNP (rs2287949) expression. However, Coomassie or silver staining of the gels immediately after the deletion. containing whole-cell lysates and conditioned medium revealed that there were no differences in total amounts of SLC45A2 and OCA secreted proteins between patient and control (Supplemen- To ascertain whether the mutation in SLC45A2 was likely to tary Figure S2a and b online). This suggests that the aberrant be pathogenic, expression analysis of mRNA and protein protein secretion is tyrosinase specific, and therefore likely was carried out. Quantitative real-time PCR showed due to the SLC45A2 defect. significantly reduced mRNA levels (14.1±1.24% compared with control; Po0.001; Figure 2a). SLC45A2 protein was G6PC3 and neutropenia undetectable in the patient’s melanocytes compared with The G6PC3 mutation was further investigated. Quantitative control cells by western blotting (Figure 2b). Immunofluor- real-time PCR revealed a significant reduction in the escence microscopy of normal human melanocytes revealed expression of the transcript (5.47±0.75% compared with punctate cytoplasmic staining for SLC45A2 that colocalized control; Po0.001; Figure 3a), probably due to nonsense- to PMEL17-positive stage II melanosomes (Figure 2c). As the mediated decay. As G6PC3 deficiency is related to increased patient had no detectable SLC45A2 protein expression, spontaneous apoptosis due to ER stress (Cheung et al., 2007; no signal was seen in this channel, confirming the specificity Boztug et al., 2009), we studied the rate of spontaneous of the antibody. Nevertheless, the PMEL17 localization apoptosis in our patient’s fibroblasts. The percentage of appeared comparable to that of wild-type cells, suggesting patient cells that were undergoing apoptosis was higher than that biogenesis and trafficking of PMEL17-positive organelles in control cells (23.8±2.3% for control and 27.1±1.2% for is not defective. patient; P ¼ 0.14), but this difference was not statistically In normal melanocytes, tyrosinase predominantly loca- significant (Figure 3b). However, when the cells were lizes to PMEL17-positive cytoplasmic structures, whereas subjected to ER stress using 5 mM dithiothreitol, the percen- in patient cells there was no colocalization (Figure 2d). tage of the patient’s cells undergoing apoptosis was No endoplasmic reticulum (ER) or trans-Golgi network significantly greater than for control cells (91.5±1.1% vs retention of tyrosinase was observed in the patient’s 72.6±1.3%; Po0.001).

Figure 2. Functional analysis of the oculocutaneous albinism type 4 phenotype. (a) Quantitative real-time PCR results for SLC45A2 mRNA expression in patient compared with control melanocytes. Values shown are percentage expression of SLC45A2 in patient cells compared with control cells, normalized by ACTB (error bars ¼ ±1 SEM, n ¼ 3, Po0.001). (b) Western blots of whole-cell extracts (WCEs), conditioned medium, and exosome pellets from control and patient melanocytes. There is no detectable SLC45A2 protein expression, and tyrosinase expression is reduced in patient cells compared with control cells. Tyrosinase is present in the medium from patient cells, suggesting that this protein is abnormally secreted from these cells; it is also found in the excreted exosome pellet. The media were collected from an equal number of cells, and equal volumes of concentrated proteins were loaded onto the gel. Loading was controlled by assessing b-actin expression in the whole-cell lysate of the corresponding cells. The western blot with the exosome marker TSG101 demonstrates that exosomes were isolated from the conditioned medium. (c) Control and patient cells stained for SLC45A2 and the melanosome marker PMEL17, showing colocalization (inserts) of the two proteins in control cells. Consistent with the western blot, no staining for SLC45A2 is seen in patient cells, but PMEL17 looks comparable to control cells. (d) Control and patient cells stained for tyrosinase and PMEL17 show the colocalization (inserts) of the two proteins in control cells. No colocalization is seen in patient cells, and tyrosinase appears predominantly in the plasma membrane. Nuclei are stained with 4’,6-diamidino-2-phenylindole; bar ¼ 20 mm. (e) Plasma membrane protein biotinylation assay. Only the patient’s cells showed tyrosinase on the plasma membrane. No b-actin was detected in the membrane fraction (demonstrating purity). Whole-cell lysates showed tyrosinase expression in control and patient cells (loading controlled by b-actin). (f) High-magnification images of control and patient cells stained for tyrosinase and the plasma membrane marker Nile red. Tyrosinase membrane localization in the patient is evidenced by the colocalization of tyrosinase and Nile red. Bar ¼ 5 mm.

2020 Journal of Investigative Dermatology (2011), Volume 131 AR Cullinane et al. Homozygosity Mapping and Whole-Exome Sequencing

DISCUSSION greater confidence that these mutations are causing the The application of SNP array analysis, homozygosity map- phenotypes seen in our patient. ping, and whole-exome sequencing to a patient with OCA One of the homozygous variants identified in our patient and neutropenia allowed us to diagnose two different involved SLC45A2, associated with OCA-4. Reduced mRNA disorders in a single, unique patient. This was possible, in expression, consistent with nonsense-mediated decay, com- part, because the patient was born from a consanguineous bined with absence of SLC45A2 protein detected by western mating. Furthermore, the identified mutations were present blotting and immunofluorescence in cultured melanocytes, in genes previously associated with similar phenotypes strongly suggested that the SLC45A2 mutation c.986delC is (Rundshagen et al., 2004; Boztug et al., 2009), allowing for pathogenic. This case represents the first North American

120 P<0.001 WCE Medium Exosome 100

80 Control Patient Control Patient Control Patient SLC45A2 60 Tyrosinase 40 SLC45A2 RNA TSG101 20 Percentage expression of expression Percentage β 0 -Actin Control Patient

SLC45A2 PMEL17 Merge

Control Patient Control Biotin No biotinBiotin No biotin Tyrosinase Membrane β-Actin

Tyrosinase WCE β Patient -Actin

Tyrosinase PMEL17 Merge Tyrosinase Nile red Control Control Patient Patient

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P<0.001 OCA-4 mouse model (Costin et al., 2003). However, our 120 results should be interpreted with caution, as the patient’s 100 cells have a mutation in another disease-causing gene, with unknown effects on melanocytes. 80 The second mutation in our patient involves G6PC3. Three genes are known to encode enzymes that have glucose-6- 60 activity, i.e., G6PC1, G6PC2, and G6PC3. All three proteins have the same enzymatic activity, i.e., removal

G6PC3 RNA 40 of the phosphate group from glucose-6-phosphate to produce 20 glucose. G6PC1 is expressed in the liver, kidney, and small Percentage expression of expression Percentage intestine and catalyzes the essential hydrolysis of glucose-6- 0 phosphate in the gluconeogenic and glycogenolytic path- Control Patient ways (Lei et al., 1993). G6PC2 is expressed only in pancreatic P<0.001 islet cells, where it is thought to function in glucose- 100 dependent insulin secretion by controlling free glucose levels (Martin et al., 2001; Petrolonis et al., 2004). In contrast, 80 G6PC3 is ubiquitously expressed, as evidenced by a wide range of affected tissues in patients with G6PC3 mutations. 60 For example, such patients have atrial septal defects and low platelet counts (Boztug et al., 2009), features that were also observed in our patient. 40 P=0.14 apoptosis The G6PC3 mutation c.829C4T identified in our patient was previously described in a French patient in the 20 heterozygous state (Boztug et al., 2009). However, as it is a

Percentage of cells undergoing Percentage null allele, this mutation was not functionally investigated. 0 Furthermore, expression at the protein level could not be Not treated Treated assessed because there is no commercially available antibody Control Patient against G6PC3. We did demonstrate a reduction in G6PC3 Figure 3. Functional analysis of the G6PC3 phenotype. (a) Quantitative mRNA expression in our patient’s cells (Figure 3a), consistent real-time PCR results for G6PC3 mRNA expression in patients compared with nonsense-mediated decay. This finding strongly implies with control melanocytes. Values shown are percentage expression of G6PC3 decreased protein expression as well. Functional G6PC3 is in patient cells compared with control cells, normalized by ACTB (error required to maintain neutrophil viability; absence of the bars ¼ ±1 SEM, n ¼ 3, Po0.001). (b) Flow cytometry results using the results in reduced neutrophil number and increased Annexin V apoptosis assay for control and patient cells with and without spontaneous apoptosis due to ER stress (Cheung et al., 2007; endoplasmic reticulum stress induction (error bars ¼ ±1 SEM, n ¼ 3). Without Boztug et al., 2009). We demonstrated a higher rate of 5mM dithiothreitol treatment, no significant difference can be seen; with treatment, there is a clear increase in apoptosis in patient cells. spontaneous apoptosis in our patient’s fibroblasts compared with control cells. If the same phenomenon were to occur in patient shown to have OCA-4, and to our knowledge, her neutrophils, the decreased G6PC3 protein expression could melanocytes are the only such cultures obtained from an explain the neutropenia. It could also account for the relative individual with OCA-4. paucity of mature neutrophils in the blood and bone marrow, Relatively little is known about the function of SLC45A2 in as the older cells may be more likely to undergo apoptosis human melanocytes, although mutations in the gene were than survive. identified as the cause of OCA-4 nearly a decade ago Our patient’s inflammatory bowel disease could not (Newton et al., 2001). We showed that human SLC45A2- readily be attributed to either SLC45A2 or G6PC3 mutations, deficient melanocytes exhibited a normal distribution of but may be related to another mutated gene due to PMEL17-tagged melanosomes, similar to what was observed consanguinity. in the underwhite mouse model of OCA-4 (Costin et al., Syndromic, multisystemic pigmentary disorders are 2003). We also demonstrated that, in our patient’s cells, excellent examples of diseases whose identification can be tyrosinase was mislocalized to the plasma membrane, difficult, especially when consanguinity introduces the suggesting that SLC45A2 functions in the correct trafficking possible involvement of multiple genes. One approach in of melanocyte-specific proteins to melanosomes, probably at this situation is to perform homozygosity mapping based upon the level of trans-Golgi network sorting. Moreover, the SNP array results, followed by whole-exome sequencing. presence of tyrosinase in the growth medium of the patient’s cells, and specifically in exosomes (Figure 2b), suggests that MATERIALS AND METHODS tyrosinase is also prematurely secreted out of the cell without Patient passing through melanosomes. These findings explain the The patient was enrolled in protocol 76-HG-0238 (http://www. hypopigmentation phenotype of OCA-4 and are consistent clinicaltrials.gov, NCT00369421), ‘‘Diagnosis and Treatment of with observations in melanocytes from the underwhite Inborn Errors of Metabolism’’, approved by the NHGRI Institutional

2022 Journal of Investigative Dermatology (2011), Volume 131 AR Cullinane et al. Homozygosity Mapping and Whole-Exome Sequencing

Review Board. Written, informed consent was obtained in adher- fluoride, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, ence to the Declaration of Helsinki Principles. 1mM EDTA, 1 mM EGTA, 0.27 M sucrose, 1% Triton X-100, and Complete, Mini Protease Inhibitor Cocktail (Roche Diagnostics, SNP arrays and dideoxy sequencing Indianapolis, IN). Cell lysates were centrifuged (15,000 g, for For SNP genotyping, genomic DNA was run on a Human 1M-Duo 15 minutes at 4 1C); supernatants were removed for immunoblotting DNA Analysis BeadChip and the data analyzed using the Geno- or plasma membrane protein biotinylation experiments. Twenty meStudio software (both from Illumina, San Diego, CA). For dideoxy micrograms of total protein, as determined by the Dc Protein assay sequencing, primers were designed to cover all coding exons (BioRad, Hercules, CA), were loaded onto 4–20% Tris–glycine gels. and flanking intronic regions of AP3B1, G6PC3, and SLC45A2 After blotting, the PVDF membranes (Invitrogen) were probed with (primer sequences available upon request). Direct sequencing was a rabbit polyclonal antibody against SLC45A2 (Proteintech, Chicago, carried out using the dideoxy termination method (ABI BigDye IL) or mouse monoclonal antibodies against tyrosinase (clone T311; Terminator v3.1) on an ABI 3130xl DNA sequencer (Applied Santa Cruz Biotechnology, Santa Cruz, CA) and TSG101 (clone Biosystems, Austin, TX). Results were analyzed using Sequencher 4A12; Abcam, Cambridge, MA). A mouse monoclonal antibody v4.9 software (Gene Codes, Ann Arbor, MI). All mutations were against b-actin was used as a loading control (clone AC-15; Sigma). verified bidirectionally. HRP-labeled secondary anti-mouse or anti-rabbit antibodies were used (Amersham Biosciences, Piscataway, NJ). The antigen–antibody Whole-exome sequencing and variant analysis complexes were detected with an Enhanced Chemiluminescence kit Solution hybridization exome capture was carried out using the (Amersham Biosciences). SureSelect Human All Exon System (Agilent Technologies, Santa Clara, CA), which employs biotinylated RNA baits to hybridize to Conditioned medium and exosome isolation sequences that correspond to exons. The manufacturer’s protocol To prepare serum-free conditioned medium, 3 106 cells per flask version 1.0 compatible with Illumina paired-end sequencing were washed three times and incubated for 24 hours at 37 1C with was used, with the exception that DNA fragment size and quality 15 ml serum-free culture medium. The medium was harvested and was measured using a 2% agarose gel stained with SYBR Gold centrifuged at 1,250 g for 10 minutes at 4 1C. The supernatant was (Invitrogen, Carlsbad, CA) instead of using an Agilent Bioanalyzer. ultracentrifuged at 200,000 g for 60 minutes to isolate exosomes. Flowcell preparation and 101-bp paired-end read sequencing were The exosome pellets were re-suspended in 2 Laemmli loading carried out as per protocol for the GAIIx sequencer (Illumina; buffer. The supernatant was 30 concentrated at 4 1C in Centriprep Bentley et al., 2008). A single 101-bp paired-end lane on a GAIIx YM-10 tubes (Millipore, Billerica, MA). The resulting protein flowcell was used per exome sample to generate sufficient reads to concentrates, exosome pellets, and whole-cell extracts from the align the sequence. Image analysis and base calling on all lanes of corresponding cells were subjected to western blotting as above. data were performed using Illumina Genome Analyzer Pipeline software (GAPipeline versions 1.4.0 or greater) with default Plasma membrane protein biotinylation parameters. Reads were aligned to a human reference sequence For membrane protein biotinylation, control and patient melano- (UCSC assembly hg18, NCBI build 36) using the package cytes were incubated with 500 ml of 0.25 mg ml1 EZ-Link Sulfo- called Efficient Large-scale Alignment of Nucleotide Databases NHS-SS-Biotin (Thermo Scientific, Waltham, MA) for 30 minutes at

(Illumina, San Diego, CA). Genotypes were called at all positions 4 1C. This reaction was quenched by incubating with 1 M NH4Cl for where there were high-quality sequence bases using a Bayesian 5 minutes. Protein was then extracted using the protocol above. The algorithm called the Most Probable Genotype (Teer et al., 2010). biotin-labeled proteins were separated from the total lysates using A graphical java tool VarSifter was developed by NISC to view, sort, streptavidin Dynabeads (Invitrogen) according to the manufacturer’s and filter the variants. instructions. The protein samples were loaded directly onto SDS- PAGE gels for immunoblotting analysis. Tissue culture Primary patient and control fibroblasts and melanocytes were Quantitative real-time PCR cultured from a forearm skin biopsy. Fibroblasts were grown in Total RNA was isolated using RNA-Easy Mini-Kit (Qiagen, Valencia, high-glucose (4.5 g l1) DMEM medium supplemented with 10% CA) according to the manufacturer’s protocol. RNA was treated with fetal bovine serum, 2 mML-glutamine, MEM non-essential amino a DNase kit (DNA-free) to remove all remaining DNA according to acid solution, and penicillin–streptomycin. Melanocytes were the manufacturer’s protocol (Applied Biosystems). RNA concentra- cultured in Ham’s F10 (Invitrogen, Carlsbad, CA), supplemented tion and purity were measured on the Nanodrop ND-1000 apparatus with 5% fetal calf serum (Gemini Bio-Products, West Sacramento, (Nanodrop Technologies, Wilmington, DE). First-strand comple- CA), 5 mgl1 basic fibroblast growth factor (Sigma, St Louis, MO), mentary DNA was synthesized using a high capacity RNA-to-cDNA 10 mgl1 Endothelin (Sigma), 7.5 mg l1 3-Isobutyl-1-methylxanthine kit (Applied Biosystems) according to the manufacturer’s guidelines. (Sigma), 30 mgl1 Choleratoxin (Sigma), 3.3 mgl1 Phorbol 12- Taqman gene expression master mix reagent and Assays-On- myristate 13-acetate (Sigma), and 1 ml Fungizone (Invitrogen). Demand (Applied Biosystems) were obtained for G6PC3 (assay ID Hs00292720_m1), SLC45A2 (assay ID Hs01125486_m1), and a Protein extraction and immunoblotting control gene ACTB (assay ID Hs99999903_m1). Quantitative real- Cells were grown to confluency in 25-cm2 flasks, washed twice with time PCR was performed using 100 ng complementary DNA, on ice-cold phosphate-buffered saline, and scraped into 250 ml of cell an ABI PRISM 7900 HT Sequence Detection System (Applied lysis buffer containing 50 mM Tris-HCl (at pH 7.5), 50 mM sodium Biosystems) using the comparative CT method (DDCT); this method

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measures relative gene expression (Livak and Schmittgen, 2001). The Boztug K, Welte K, Zeidler C et al. (2008) Congenital neutropenia syndromes. cycling conditions were as follows: 2 minutes at 50 1C, 10 minutes Immunol Allergy Clin North Am 28:259–75, vii-viii at 95 1C, and 40 cycles at 95 1 C for 15 seconds and 60 1C for Cheung YY, Kim SY, Yiu WH et al. (2007) Impaired neutrophil activity and 60 seconds. increased susceptibility to bacterial infection in mice lacking glucose-6- phosphatase-beta. J Clin Invest 117:784–93 Costin GE, Valencia JC, Vieira WD et al. (2003) Tyrosinase processing and Immunofluorescence microscopy intracellular trafficking is disrupted in mouse primary melanocytes Cells were grown in 4-well chamber slides and fixed using 4% carrying the underwhite (uw) mutation. A model for oculocutaneous paraformaldehyde and permeabilized using 0.1% Triton X-100. albinism (OCA) type 4. J Cell Sci 116:3203–12 Antibodies against SLC45A2 and tyrosinase were purchased for Dell’Angelica EC, Shotelersuk V, Aguilar RC et al. (1999) Altered trafficking immunoblotting. A mouse monoclonal antibody (clone HMB45) of lysosomal proteins in Hermansky-Pudlak syndrome due to mutations in the beta 3A subunit of the AP-3 adaptor. Mol Cell 3:11–21 and a rabbit polyclonal against PMEL17 were purchased from Devriendt K, Kim AS, Mathijs G et al. (2001) Constitutively activating LifeSpan BioSciences (Seattle, WA) and Neomarkers (Fremont, CA), mutation in WASP causes X-linked severe congenital neutropenia. respectively. Alexa Fluor 488 and 555 secondary antibody con- Nat Genet 27:313–7 jugates and Nile red were purchased from Invitrogen, and nuclei Gnirke A, Melnikov A, Maguire J et al. (2009) Solution hybrid selection with were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). ultra-long oligonucleotides for massively parallel targeted sequencing. Cells were imaged with a Zeiss 510 META confocal laser-scanning Nat Biotechnol 27:182–9 microscope (Carl Zeiss, Thornwood, NY), with the pinhole set to 1 Gronskov K, Ek J, Brondum-Nielsen K (2007) Oculocutaneous albinism. Orphanet J Rare Dis 2:43 airy unit. A series of optical sections were collected from the xy Halaban R, Svedine S, Cheng E et al. (2000) Endoplasmic reticulum plane and merged into maximum projection images. retention is a common defect associated with tyrosinase-negative albinism. Proc Natl Acad Sci USA 97:5889–94 Apoptosis assay Horwitz M, Benson KF, Person RE et al. (1999) Mutations in ELA2, encoding Spontaneous apoptosis was measured using the Annexin V and neutrophil elastase, define a 21-day biological clock in cyclic propidium iodide apoptosis assay (Pigault et al., 1994). The Annexin haematopoiesis. Nat Genet 23:433–6 V-FITC apoptosis detection kit was purchased from Sigma, and the Huizing M, Helip-Wooley A, Westbroek W et al. (2008) Disorders of assay carried out according to the manufacturer’s instructions with lysosome-related organelle biogenesis: clinical and molecular genetics. Annu Rev Genomics Hum Genet 9:359–86 5 105 fibroblast cells per treatment group. ER stress was induced Johnston JJ, Teer JK, Cherukuri PF et al. (2010) Massively parallel sequencing with 5 mM dithiothreitol (Sigma) for 2 hours at 37 1C. The samples of exons on the X identifies RBM10 as the gene that causes were sorted on a Becton Dickenson FACSCalibur flow cytometer a syndromic form of cleft palate. Am J Hum Genet 86:743–8 (Franklin Lakes, NJ), on which green and red fluorescence were King RA, Oetting WS, Creel D et al. (2002) Abnormalities of pigmentation. counted. Data were analyzed using FlowJo software (Tree Star, In: Emery and Rimoin’s Principles and Practice of Medical Genetics. Ashland, OR). (Rimoin DL, Connor JM, Pyeritz RE, eds). New York: Churchill Livingstone, 3731–85 Klein C, Grudzien M, Appaswamy G et al. (2007) HAX1 deficiency causes CONFLICT OF INTEREST autosomal recessive severe congenital neutropenia (Kostmann disease). The authors state no conflict of interest. Nat Genet 39:86–92 Lei KJ, Shelly LL, Pan CJ et al. (1993) Mutations in the glucose-6-phosphatase ACKNOWLEDGMENTS gene that cause glycogen storage disease type 1a. Science 262:580–3 We thank S Anderson, R Hess, R Fischer, and H Dorward for their help with the study. This study was supported by the Intramural Research Program of the Li W, Zhang Q, Oiso N et al. (2003) Hermansky-Pudlak syndrome type 7 National Human Genome Research Institute, National Institutes of Health, (HPS-7) results from mutant dysbindin, a member of the biogenesis Bethesda, MD, USA. of lysosome-related organelles complex 1 (BLOC-1). Nat Genet 35:84–9 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. SUPPLEMENTARY MATERIAL Methods 25:402–8 Supplementary material is linked to the online version of the paper at http:// Martin CC, Bischof LJ, Bergman B et al. (2001) Cloning and characterization www.nature.com/jid of the human and rat islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) genes. J Biol Chem 276:25197–207 REFERENCES Morgan NV, Pasha S, Johnson CA et al. (2006) A germline mutation in Anikster Y, Huizing M, White J et al. (2001) Mutation of a new gene causes a BLOC1S3/reduced pigmentation causes a novel variant of Hermansky- unique form of Hermansky-Pudlak syndrome in a genetic isolate of Pudlak syndrome (HPS8). Am J Hum Genet 78:160–6 central Puerto Rico. Nat Genet 28:376–80 Newton JM, Cohen-Barak O, Hagiwara N et al. (2001) Mutations in the Bentley DR, Balasubramanian S, Swerdlow HP et al. 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Person RE, Li FQ, Duan Z et al. (2003) Mutations in proto-oncogene GFI1 Teer JK, Bonnycastle LL, Chines PS et al. (2010) Systematic comparison of cause human neutropenia and target ELA2. Nat Genet 34:308–12 three genomic enrichment methods for massively parallel DNA Petrolonis AJ, Yang Q, Tummino PJ et al. (2004) Enzymatic characterization sequencing. Genome Res 20:1420–31 of the pancreatic islet-specific glucose-6-phosphatase-related protein Teer JK, Mullikin JC (2010) Exome sequencing: the sweet spot before whole (IGRP). J Biol Chem 279:13976–83 genomes. Hum Mol Genet 19:R145–51 Pigault C, Follenius-Wund A, Schmutz M et al. (1994) Formation of two- Tomita Y, Takeda A, Okinaga S et al. (1989) Human oculocutaneous albinism dimensional arrays of annexin V on phosphatidylserine-containing caused by single base insertion in the tyrosinase gene. Biochem Biophys liposomes. J Mol Biol 236:199–208 Res Commun 164:990–6 Rinchik EM, Bultman SJ, Horsthemke B et al. (1993) A gene for the mouse Toyofuku K, Wada I, Valencia JC et al. (2001) Oculocutaneous albinism types pink-eyed dilution locus and for human type II oculocutaneous albinism. 1 and 3 are ER retention diseases: mutation of tyrosinase or Tyrp1 can Nature 361:72–6 affect the processing of both mutant and wild-type proteins. FASEB J Rundshagen U, Zuhlke C, Opitz S et al. (2004) Mutations in the MATP gene 15:2149–61 in five German patients affected by oculocutaneous albinism type 4. Welte K, Zeidler C, Dale DC (2006) Severe congenital neutropenia. Hum Mutat 23:106–10 Semin Hematol 43:189–95 Suzuki T, Li W, Zhang Q et al. (2002) Hermansky-Pudlak syndrome is caused Zhang Q, Zhao B, Li W et al. (2003) Ru2 and Ru encode mouse orthologs of by mutations in HPS4, the human homolog of the mouse light-ear gene. the genes mutated in human Hermansky-Pudlak syndrome types 5 and 6. Nat Genet 30:321–4 Nat Genet 33:145–53

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