Downloaded from http://bjo.bmj.com/ on November 15, 2016 - Published by group.bmj.com Clinical science The ophthalmic presentation of Hermansky–Pudlak syndrome 6 Sarah Hull,1,2 Gavin Arno,1,2 Graham E Holder,1,2 Vincent Plagnol,3 Keith Gomez,4 Ri Liesner,5,6 Andrew R Webster,1,2 Anthony T Moore1,2,7

1University College London ABSTRACT identified, which all encode protein complexes Institute of Ophthalmology, Background Hermansky–Pudlak syndrome (HPS) may involved in the biogenesis of lysosome-related orga- London, UK 2 fi present to the ophthalmologist with signs suggestive of nelles (BLOC), including melanosomes in melano- Moor elds Eye Hospital, 6 London, UK oculocutaneous albinism. Consideration of HPS as a cytes and delta granules in platelets. HPS6 is a 3University College London differential diagnosis is important due to its potential subunit of the BLOC 2 complex in addition to Genetics Institute, London, UK 4 systemic complications. HPS6 is a rarely reported HPS3 and HPS5. HPS6 is an integral part of the Haematology Department, subtype. retrograde motor complex for lysosomal transport Royal Free London NHS 7 Foundation Trust, London, UK Methods Three patients from two families underwent from cell membrane to the perinuclear region. To 5Haematology Department, clinical examination, imaging and targeted systemic date, six families have been reported with Great Ormond Street Hospital investigations. Electrophysiology with visual-evoked HPS6-related disease characterised by OCA with – for Children NHS Trust, potentials (VEPs) was performed in both children of bleeding diatheses.8 10 London, UK 6Haematology Department, family 1. Whole exome sequencing (WES) was In this report, the initial isolated ophthalmic University College London performed on the proband of family 1. Bidirectional presentation of HPS6-related disease in three Hospitals, London, UK Sanger sequencing of the single exon and intron–exon patients is described and the utility of exome 7 San Francisco Medical Centre, boundaries of HPS6 was performed on all affected sequencing is demonstrated. University of California, patients and segregation confirmed in available relatives. San Francisco, California, USA Results Two siblings presented in infancy with METHODS Correspondence to nystagmus and reduced vision. They were initially The study protocol adhered to the tenets of the Professor Anthony T Moore, diagnosed with isolated foveal hypoplasia with no Declaration of Helsinki and received approval from Inherited Eye Diseases, UCL aberrant chiasmal misrouting on VEPs. WES performed the local ethics committee. Written, informed Institute of Ophthalmology, fi 11-43 Bath St., London in the proband when 10 years of age identi ed a novel consent was obtained from all participants prior to EC1V 9EL, UK; homozygous missense variant in HPS6 and further their inclusion in this study with parental written [email protected] questioning elicited a history of nose bleeds and mild consent provided on behalf of the children involved bruising. Segregation supported causality of this variant in this study. All underwent full clinical examin- Received 4 November 2015 in the affected younger sibling. In the third unrelated ation and imaging including anterior segment and Accepted 2 January 2016 Published Online First patient, an initial diagnosis of ocular albinism was made colour fundus photography (Topcon Great Britain, 28 January 2016 at 3 months with HPS only diagnosed at 26 years. Berkshire, UK), fundus autofluorescence imaging Biallelic, truncating mutations in HPS6 were identified by and spectral domain optical coherence tomography candidate Sanger sequencing and included a novel (OCT, Spectralis, Heidelberg Engineering, variant. Abnormal platelet function consistent with HPS Heidelberg, Germany). Electroretinography was was confirmed in all patients. performed using eyelid electrodes for the two chil- Conclusions The diagnosis of HPS in all patients was dren of family 1 according to previously described delayed due to a mild systemic phenotype. Next- protocols.11 12 In addition, both children under- generation sequencing can aid diagnosis of syndromic went VEP recording to evaluate chiasmal routing as conditions with important consequences for preventing previously described.13 morbidity. All patients underwent haematological assess- ment and investigation with platelet function tests. Electron microscopy of platelets from patient 3 was INTRODUCTION additionally performed. Oculocutaneous albinism (OCA) is a recessive dis- The proband of family 1 underwent whole order of melanogenesis presenting in infancy with exome sequencing (WES, AROS Applied nystagmus.1 It is characterised by iris transillumin- Biotechnology, Aarhus, Denmark) using a solution- ation, foveal hypoplasia, chiasmal misrouting on phase Agilent SureSelect 38 Mb exome capture visual-evoked potentials (VEPs) and variably reduced (SureSelect Human All Exon Kit; Agilent pigmentation of the fundus, hair and skin. OCA is a Technologies, Santa Clara, California, USA) and the feature of certain syndromes including Hermansky– Illumina HiSeq2000 sequencer (Illumina, San Pudlak syndrome (HPS, MIM#203300) associated Diego, California, USA). Reads were aligned to the with bleeding diathesis, pulmonary fibrosis and hg19 human reference sequence using Novoalign granulomatous colitis and Chediak–Higashi syn- (Novocraft, Selangor, Malaysia) V.2.05. The drome (MIM#214500) with immunodeficiency and ANNOVAR tool (Openbioinformatics.org) was neurological involvement.23 used to annotate single-nucleotide polymorphisms To cite: Hull S, Arno G, HPS is an inherited disorder of lysosomal organ- (SNPs) and small insertions/deletions. Holder GE, et al. Br J elle biogenesis most prevalent in Puerto Rico at a A likely pathogenic variant in HPS6 was further Ophthalmol rate of 1 in 1800 due to founder mutations in investigated in the proband, affected brother and – 2016;100:1521 1524. HPS1 and HPS3.45Nine HPS genes have been parents with bidirectional Sanger sequencing of the

Hull S, et al. Br J Ophthalmol 2016;100:1521–1524. doi:10.1136/bjophthalmol-2015-308067 1521 Downloaded from http://bjo.bmj.com/ on November 15, 2016 - Published by group.bmj.com Clinical science single exon of HPS6 and the exon–intron boundaries. DNA was keeping with her parents. Her electroretinogram (ERG) and amplified using specifically designed primers by PCR and the VEPs performed at age 1 and repeated age 5 were normal; there resulting fragments were sequenced using standard protocols was no evidence of retinal dysfunction nor of chiasmal misrout- (details upon request). ing. Reduced foveal reflexes were noted and OCT scan at age 5 In the third patient, candidate gene investigation with Sanger demonstrated foveal hypoplasia (figure 1). Her younger brother sequencing of HPS6 was performed based on the clinical pheno- was first evaluated at 5 months and was also diagnosed with iso- type. In addition, given a potential history of parental consan- lated foveal hypoplasia. His fundi and his hair were noted to be guinity, autozygosity mapping using a SNP microarray blonder than his sister. Full-field ERG at 8 months was normal, containing 730 525 SNPs (OmniExpress, Illumina, San Diego, and there was no chiasmal misrouting on VEPs. California, USA) was performed including all nine HPS loci. WES in patient 1 identified a novel, homozygous missense Mutation nomenclature was assigned in accordance with variant in HPS6, c.779G>A; p.Gly260Glu, predicted to be GenBank Accession number NM_024747.5 with nucleotide damaging in silico (SIFT score 0.03, Polyphen2 score 1.00) and position 1 corresponding to the A of the ATG initiation codon. segregating with disease within the family (figure 2). Subsequent Variants were identified as novel if not previously reported in re-evaluation elicited a history of mild bruising at the time of the literature and if absent from dbSNP available at http://www. immunisations for both children and for patient 1, nose bleeds ncbi.nlm.nih.gov/projects/SNP/, NHLBI GO Exome Sequencing every 1–2 weeks from the age of 6 years. Haematological inves- Project, Seattle, WA available at http://evs.gs.washington.edu/ tigation was consistent with a platelet storage pool disorder and EVS/ and 1000 genomes project available at http://www. HPS was confirmed (table 1). 1000genomes.org/ and the Exome Aggregation Consortium Patient 3, the second child of parents of Russian–Palestinian (ExAC) Cambridge, Massachusetts, USA (http://exac. origin, presented at 3 months of age with reduced vision, nys- broadinstitute.org) all accessed on 27 October 2015. The likely tagmus, iris transillumination and blonde fundi. In addition, pathogenicity of novel missense variants was assessed using the right Axenfeld anomaly and left posterior embryotoxin were predictive algorithms of ‘Sorting Intolerant From Tolerant’ noted (figure 1). An initial diagnosis of mild ocular albinism was (SIFT) available at http://sift.jcvi.org and Polymorphism made, amended to oculocutaneous albinism at 9 years due to Phenotyping V.2 (PolyPhen-2) available at http://genetics.bwh. likely inheritance pattern and the observation of mildly reduced harvard.edu/pph2. cutaneous/hair pigmentation.14 There were no complications from hernia surgery at 6 years but at 10 years there was pro- RESULTS longed bleeding for 3–4 h following a dental extraction. Two siblings, from distantly consanguineous parents of Punjabi Haematological investigations at 26 years identified a platelet Afghan descent, presented in infancy with reduced vision, fine storage pool disorder consistent with HPS (table 1). Due to the horizontal nystagmus but no iris transillumination on undilated mild systemic presentation, candidate gene sequencing of HPS6 examination. Patient 1 was first reviewed at 1 year of age. The was performed and identified biallelic premature termination fundi were not blonde and cutaneous/hair pigmentation was in codons (PTC), c.902dupT; p.Thr303Hisfs*64 (novel) and

Figure 1 Ophthalmic imaging in Hermansky–Pudlak type 6. (a) Anterior segment photograph photography, (b) colour fundus photograph, (c) fundus autofluorescence imaging, (d) optical coherence tomography (OCT) of the macula. Patient 1, left eye; (a) dark brown iris; (b–d) all demonstrate the absence of a fovea. Patient 2, right eye; (a) dark brown iris; (b) blonder fundus than sister; (b–d) demonstrate no fovea. Patient 3; (a.i) right Axenfeld anomaly, (a.ii) right transillumination defect, (a.iii) left posterior embryotoxin, (a.iv) left transillumination defect; (b) blonde peripheral right fundus; (b–d) right eye imaging, lack of fovea.

1522 Hull S, et al. Br J Ophthalmol 2016;100:1521–1524. doi:10.1136/bjophthalmol-2015-308067 Downloaded from http://bjo.bmj.com/ on November 15, 2016 - Published by group.bmj.com Clinical science

Figure 2 Molecular results. Pedigrees for the two families and chromatograms from Sanger sequencing of HPS6. Family 1 chromatograms demonstrate heterozygous mutation in carrier parent and homozygous mutation in affected patient. Family 2, compound heterozygous duplications are shown to be biallelic from a single Sanger sequencing experiment with out-of-phase chromatogram from initial duplication subsequently becoming in phase from later duplication on other allele.

c.1083dupC; p.Gly362Argfs*5 (1 in 120 260 on ExAC), with onset of pulmonary fibrosis is not until the fourth decade.18 19 predicted truncation of each allele at the same codon. With the limited number of patients with HPS6 reported to Autozygosity mapping identified no regions of homozygosity date, there is the theoretical possibility that these complications over any of the nine HPS loci. could still be associated. Patients 1 and 2 have an unusual presentation with a lack DISCUSSION of iris transillumination and no chiasmal misrouting on VEPs, This report describes the phenotype in three patients with HPS6 which led to an initial diagnosis of isolated foveal hypoplasia. and identifies two novel mutations. The systemic phenotype in A lack of iris transillumination has not been previously all three patients is particularly mild. reported in HPS although the degree of iris defects can range HPS6 was first reported in a single patient with OCA, bleed- from mild spoke like defects to complete transillumin- ing diathesis but no pulmonary and gastrointestinal involve- ation.20 21 Abnormal chiasmal misrouting is regarded as a car- ment.8 A further report identified mutations in HPS6 as a cause dinal feature of OCA and the degree of misrouting has been of OCA with mild bleeding diathesis in an extended Israeli shown to negatively correlate with the loss of pigmenta- Bedouin pedigree, all affected members having classical features tion.13 22 VEP characterisation in HPS is limited; a report of of OCA including iris transillumination.9 A detailed series of 11 patients with molecularly uncharacterised HPS identified four patients with mutations in HPS6 demonstrated variable aberrant chiasmal misrouting in all patients.23 The lack of degrees of albinism diagnosed in infancy; all had iris transillu- misrouting in the siblings in this report may reflect the mild- mination and bleeding diatheses.10 There have been no reported ness of their hypopigmentation. Further investigation of VEPs complications of pulmonary fibrosis or granulomatous colitis. in other patients with HPS in particular the BLOC2 subtypes This is similar to the present series of patients and to other may demonstrate similar findings. Visual acuities were signifi- BLOC2 subtypes, HPS3 and HPS5, which also manifest with cantly reduced, likely related to foveal hypoplasia and nystag- – milder systemic disease than the other forms of HPS.15 17 mus. The vision levels found were similar to those reported Although colitis may present within the first decade of life, the in other patients with HPS6.10

Table 1 Key molecular, visual and haematological findings Visual function Haematology investigations Pt number, age last review Visual acuity log MAR Platelet ATP:ADP ratio Platelet function (family number) (Snellen) Refraction (0.9–2.3) tests Electron microscopy

Pt 1 R 0.48 (6/19) R +7.00/ 13.9 Normal aggregation Not done −2.00×180 Significantly reduced 12 years (GC 18806) L 0.40 (6/15) L +6.00/ ATP release −1.00×180 Pt 2 R 0.76 (6/38) R +5.50/ 31.4 Reduced aggregation Not done −3.00×180 Significantly reduced 4 years (GC 18806) L 0.78 (6/38) L +5.50/ ATP release −3.00×180 Pt 3 R 0.82 (6/38) R −1/−1.25×180 10.8 Reduced aggregation Reduced platelet dense granules 29 years (GC 15023) L 0.94 (6/48) L −4/−3×180 Absent ATP release EM, electron microscopy.

Hull S, et al. Br J Ophthalmol 2016;100:1521–1524. doi:10.1136/bjophthalmol-2015-308067 1523 Downloaded from http://bjo.bmj.com/ on November 15, 2016 - Published by group.bmj.com Clinical science

Patient 3 in this series has a similar ocular phenotype to previ- 3 Desai N, Weisfeld-Adams JD, Brodie SE, et al. Optic neuropathy in late-onset ously reported patients with HPS6 with iris transillumination, a neurodegenerative Chédiak–Higashi syndrome. Br J Ophthalmol 2015. 4 Oh J, Bailin T, Fukai K, et al. Positional cloning of a gene for blonde fundus and foveal hypoplasia. In addition, she has fea- Hermansky–Pudlak syndrome, a disorder of cytoplasmic organelles. Nat Genet tures of anterior segment dysgenesis with Axenfeld anomaly and 1996;14:300–6. posterior embryotoxin, reported in up to one third of patients 5 Anikster Y, Huizing M, White J, et al. Mutation of a new gene causes a unique with HPS.20 Interestingly, she underwent abdominal surgery at form of Hermansky–Pudlak syndrome in a genetic isolate of central Puerto Rico. – 6 years without bleeding complications but had prolonged Nat Genet 2001;28:376 80. 6 Cullinane AR, Curry JA, Carmona-Rivera C, et al. A BLOC-1 mutation screen reveals bleeding post dental extraction when 10 years of age. that PLDN is mutated in Hermansky–Pudlak Syndrome type 9. Am J Hum Genet The unbiased platform of WES identified a novel missense 2011;88:778–87. variant in HPS6 in family 1, which is predicted to be damaging 7 Li K, Yang L, Zhang C, et al. HPS6 interacts with dynactin p150Glued to mediate in silico. This allowed further clinical assessment and targeted retrograde trafficking and maturation of lysosomes. J Cell Sci 2014;127:4574–88. 8 Zhang Q, Zhao B, Li W, et al. Ru2 and Ru encode mouse orthologs of the genes investigation of platelet function. Only one of nine previously – 8–10 mutated in human Hermansky Pudlak syndrome types 5 and 6. Nat Genet reported pathogenic variants was missense (p.Thr272Ile). 2003;33:145–53. This was identified in a conjunction with a PTC in a patient 9 Schreyer-Shafir N, Huizing M, Anikster Y, et al. A new genetic isolate with a unique diagnosed in early childhood with HPS and typical phenotypic phenotype of syndromic oculocutaneous albinism: clinical, molecular, and cellular features.10 Biallelic missense variants may manifest a milder characteristics. Hum Mutat 2006;27:1158. 10 Huizing M, Pederson B, Hess RA, et al. Clinical and cellular characterisation of phenotype if the protein function is less functionally impaired Hermansky–Pudlak syndrome type 6. J Med Genet 2009;46:803–10. than truncating variants. However, a large series of patients 11 Holder G, Robson A. Paediatric electrophysiology: a practical approach. In: Pediatric would be needed to substantiate this potential correlation. The ophthalmology, neuro-ophthalmology, genetics. Lorenz B, Moore A, eds. Berlin: PTCs identified in patient 3 are predicted to produce a trun- Springer, 2006, pp 133–55. 12 McCulloch DL, Marmor MF, Brigell MG, et al. ISCEV Standard for full-field clinical cated protein and not undergo nonsense mediated decay as – 24 electroretinography (2015 update). Doc Ophthalmol 2015;130:1 12. there is only a single exon of HPS6. This has been substan- 13 Dorey SE, Neveu MM, Burton LC, et al. The clinical features of albinism and their tiated by mRNA investigation in the previously reported correlation with visual evoked potentials. Br J Ophthalmol 2003;87:767–72. Bedouin family.9 Both variants would truncate the protein at the 14 Charles SJ, Green JS, Grant JW, et al. Clinical features of affected males with X linked ocular albinism. Br J Ophthalmol 1993;77:222–7. same codon of 367, which is the same predicted truncation – 9 15 Huizing M, Anikster Y, Fitzpatrick DL, et al. Hermansky Pudlak syndrome type 3 in codon of a previously reported variant, p.Leu356Argfs*11. Ashkenazi Jews and other non-Puerto Rican patients with hypopigmentation and This study demonstrates that HPS6 can be associated with platelet storage-pool deficiency. Am J Hum Genet 2001;69:1022–32. mild and late systemic manifestations and a diagnosis of HPS 16 Huizing M, Hess R, Dorward H, et al. Cellular, molecular and clinical should therefore be considered in any patient presenting with characterization of patients with Hermansky–Pudlak syndrome type 5. Traffic 2004;5:711–22. foveal hypoplasia, ocular albinism or OCA. An accurate diagno- 17 Tsilou ET, Rubin BI, Reed GF, et al. Milder ocular findings in Hermansky–Pudlak sis will ensure appropriate precautions during surgical or dental syndrome type 3 compared with Hermansky–Pudlak syndrome type 1. procedures.25 Ophthalmology 2004;111:1599–603. 18 Gahl WA, Brantly M, Kaiser-Kupfer MI, et al. 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1524 Hull S, et al. Br J Ophthalmol 2016;100:1521–1524. doi:10.1136/bjophthalmol-2015-308067 Downloaded from http://bjo.bmj.com/ on November 15, 2016 - Published by group.bmj.com

The ophthalmic presentation of Hermansky− Pudlak syndrome 6

Sarah Hull, Gavin Arno, Graham E Holder, Vincent Plagnol, Keith Gomez, Ri Liesner, Andrew R Webster and Anthony T Moore

Br J Ophthalmol 2016 100: 1521-1524 originally published online January 28, 2016 doi: 10.1136/bjophthalmol-2015-308067

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