An Unusual Phenotype in Dutch Patients With Oculocutaneous Albinism Type 4: Evident Hypopigmentation Without Other Ocular Decits.

Purpose: To describe the phenotype of Dutch patients with oculocutaneous albinism type 4 (OCA4). Patients and Methods: We collected data on pigmentation (skin, hair, and eyes), visual acuity (VA), nystagmus, foveal hypoplasia, chiasmal misrouting, and molecular analyses of nine Dutch OCA4 patients from the Bartiméus Diagnostic Center for complex visual disorders. Results: All patients had severely reduced pigmentation of skin, hair, and eyes with iris transillumination over 360 degrees. Three unrelated OCA4 patients had normal VA, no nystagmus, no foveal hypoplasia, and no misrouting of the visual pathways. Six patients had poor visual acuity (0.6 to 1.0 logMAR), nystagmus, severe foveal hypoplasia and misrouting. We found two novel mutations in the SLC45A2 gene, c.310C>T; p.(Pro104Ser), and c.1368+3_1368+9del p.(?). Discussion: OCA4 patients of this Dutch cohort all had hypopigmentation of skin, hair, and iris translucency. However, patients were either severely affected with regard to visual acuity, foveal hypoplasia, and misrouting, or visually not affected at all. We describe for the rst time OCA4 patients with an evident lack of pigmentation, but normal visual acuity, normal foveal development and absence of misrouting. This implies that absence of melanin does not invariably lead to foveal hypoplasia and abnormal routing of the visual pathways. gene, mapped to chromosome 5p13 (OCA4; OMIM #606574). Its protein, the membrane-associated transporter (MATP), is located in melanosomes. The exact function of MATP is unknown, but it probably plays an important role in the membrane transport of melanosomes. 2 Knockdown of MATP results in a lower pH level in the melanosomes. 3 Tyrosinase activity is inhibited by an acidic environment, and consequently mutations in SLC45A2 reduce melanin synthesis by lowering or inhibiting tyrosinase The rst OCA4 patient, a Turkish patient reported in 2001 by Newton et al., showed complete lack of pigmentation of skin, hair and eyes. 4 Since then, many patients have been described with variable phenotypes, from complete absence of pigmentation to subtle hypopigmentation only. 1,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 World prevalence of OCA4 is estimated around 1:100.000, which is 3–19% of all OCA cases. 5,17,30,7,14,15,24,25,31,32 In Japan OCA4 is more common, with a frequency of 27% of all OCA cases. 33 In the Netherlands, approximately 4% of OCA is caused by mutations in SLC45A2. 1 In this study, we describe our cohort of OCA4 patients in the Netherlands, including detailed ophthalmic information. While most reports describe pigmentation levels of skin and hair, ophthalmic details are usually scarce. Some studies report absence of nystagmus in some patients. 8,9,12,14,16,17,18,19 Visual acuities in OCA4 patients in earlier reports ranged from − 0.1 to 1.5 logMAR, with most patients having poor VA. 8,17, 25,5,38,13,15,24,27 Only three patients were described with normal VA, and all three had only mild or no hypopigmentation. 8,17, Rundshagen et al. described a patient with VA of 0.2 logMAR, subtle hypopigmentation, and nystagmus. The second patient good VA was of Japanese origin. VA was 0.0 logMAR, he had brown hair and mild hypopigmentation of the skin, with the ability to tan. Other ophthalmic details were missing. An Italian patient with good VA (< 0.2 logMAR), no nystagmus, minimal iris translucency, and no fundal hypopigmentation is the only OCA4 patient decribed, to our knowledge, without foveal hypoplasia. In this patient misrouting was present, and molecular analysis revealed two mutations in the SLC45A2 gene, c.619C4G (p.L207V) and c.606G4C (p.W202C). Until now, no patients have been described with evident lack of pigmentation of skin, hair, and eyes, but with normal ocular development, -i.e. normal VA, no nystagmus, no foveal hypoplasia and normal routing of the optic nerve bers. the absence of foveal hypoplasia is occurring in less than 0.7% of albinism patients.


Introduction
Oculocutaneous albinism type four (OCA4) is one of seven known non-syndromic types of albinism with autosomal recessive inheritance. All types of oculocutaneous albinism are characterized by hypopigmentation of skin, hair, and eyes, reduced visual acuity (VA), nystagmus, foveal hypoplasia, and misrouting of the visual pathways. 1 OCA4 is caused by mutations in the SLC45A2 gene, mapped to chromosome 5p13 (OCA4; OMIM #606574). Its protein, the membrane-associated transporter (MATP), is located in melanosomes. The exact function of MATP is unknown, but it probably plays an important role in the membrane transport of melanosomes. 2 Knockdown of MATP results in a lower pH level in the melanosomes. 3 Tyrosinase activity is inhibited by an acidic environment, and consequently mutations in SLC45A2 reduce melanin synthesis by lowering or inhibiting tyrosinase The rst OCA4 patient, a Turkish patient reported in 2001 by Newton et al., showed complete lack of pigmentation of skin, hair and eyes. 4 Since then, many patients have been described with variable phenotypes, from complete absence of pigmentation to subtle hypopigmentation only. 1,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 World prevalence of OCA4 is estimated around 1:100.000, which is 3-19% of all OCA cases. 5,17,30,7,14,15,24,25,31,32 In Japan OCA4 is more common, with a frequency of 27% of all OCA cases. 33 In the Netherlands, approximately 4% of OCA is caused by mutations in SLC45A2. 1 In this study, we describe our cohort of OCA4 patients in the Netherlands, including detailed ophthalmic information.

Patients And Methods
This study was approved by the Medical Ethics Committee of the Leiden University Medical Center and adhered to the tenets of the Declaration of Helsinki.
Informed consent was obtained from all participants and/or legal guardians. An additional informed consent was obtained for the publication of the images from the patients in Fig. 1.
We identi ed nine patients from six families with two mutations in SLC45A2 from the databases of Bartiméus Diagnostic Center for complex visual disorders. Eight patients were of Dutch origin, patient 9 was of Syrian-Turkish descent. We retrospectively collected data on pigmentation levels of skin, hair, and eyes, visual acuity (VA), and other ophthalmic features of albinism, -i.e. nystagmus, foveal hypoplasia, and misrouting of the optic nerve bers. We made spectral Optical Chorence Tomography (OCT) scans to assess the amount of foveal hypoplasia. To ensure we captured the foveal region we used radial scans. We graded the foveal hypoplasia according to the scheme of Thomas et al., with grade 1 and 2 not having incursion of the inner retinal layers, and grade 3 and 4 also affecting the photoreceptor differentiation. 34 Multichannel visually evoked potentials (VEPs) were recorded to determine misrouting. We used pattern onset VEPs for the assessment of misrouting in adults, and older children, and we used ash VEP for young children, according to ISCEV standards. 35,36 We used the differential signal of the electrodes on the left hemisphere minus that of the electrodes on the right hemisphere to calculate the chiasm coeffeicient.
To conclude if misrouting was present we used the cutoff values calculated by Kruijt et al. 36 Results Patient data are shown in Table 1. P1, P2, and P3 were unrelated. They had very pale skin, were unable to tan, were very sensitive to sun exposure, had white to very light blond hair, white eyelashes, and blue irides, translucent over 360 degrees (grade 3-4 iris translucency 1 ). P1 had grade 1 fundus hypopigmentation (only hypopigmention in the (mid)periphery 1 ), but in P2, and P3 choroid vessels were easiliy visible in the posterior pole (grade 2). The main complaint of the patients was photophobia. P1 and P2 had good VA of -0.1 logMAR (1.25 Snellen). VA of P3, a four year old boy, was 0.3 logMAR (0.5 Snellen), which was within the normal range for his age. None of the three patients had nystagmus, foveal hypoplasia, or misrouting. Misrouting was absent in pattern onset as well as ash VEP in all three patients. They were diagnosed with OCA4 based on evident hypopigmentation, and two mutations in the SLC45A2 gene. P2 and P3 carried novel mutations, c.310C > T; p.(Pro104Ser), and c.1368 + 3_1368 + 9del p.(?) respectively. (Table 1) In all three patients, segregation analysis showed that the mutations were located on different alleles. Iris transillumination, fundus images, and OCT-scans of P1 and P3 are illustrated in Fig. 1. one was severely affected (not speci ed) and the other patient had mild hypopigmentation and nystagmus (further details missing). 7,24 The second mutation, c.1567G > A; p.(Ala523Thr), was described in one compound heterozygous albinism patient without phenotypic description. 37 The missense variants c.125T > C and c.310C > T found in P2 predict the amino acid substitution p.(Met42Thr) and p.(Pro104Ser) respectively, changing a highly conserved amino acid. The second mutation c.310C > T; p.(Pro104Ser) was novel. P3 had one previously reported mutation, c.1082T > C; p.(Leu361Pro), and one novel mutation, c.1368 + 3_1368 + 9del p.(?). The missense mutation c.1082T > C; p.(Leu361Pro) has been reported in a homozygous patient that was severely affected in melanin synthesis and visual function. 25 The mutation was also found in severely affected P4 and P5 from our series. The novel variant c.1368 + 3_1368 + 9del p.(?) probably results in abnormal splicing of the SLC45A2 RNA, causing a frameshift by skipping exon 6. The pathological signi cance of the novel mutations has to be further investigated.
Unravelling the genotypic pro le of all these cases does not provide a decisive clue for the variety in phenotypic presentation in this cohort. It is remarkable, that in our series of nine OCA4 patients, all patients showed obvious lack of pigmentation of skin, hair, and eyes. But, concerning visual function and ocular development, they were either on the poorer end of the spectrum for albinism, or were not affected at all.
Since the rst patient reported in 2001, many patients with OCA4 are reported. In contrast to the homogeneous hypopigmentation phenotype we found in our cohort, in the literature the degree of pigmentation seems to vary from complete lack of pigmentation to very mild hypopigmentation. 1,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 While most reports describe pigmentation levels of skin and hair, ophthalmic details are usually scarce. Some studies report absence of nystagmus in some patients. 8,9,12,14,16,17,18,19 Visual acuities in OCA4 patients in earlier reports ranged from − 0.1 to 1.5 logMAR, with most patients having poor VA. 8,17,25,5,38,13,15,24,27 Only three patients were described with normal VA, and all three had only mild or no hypopigmentation. 8,17,25 Rundshagen et al. described a patient with VA of 0.2 logMAR, subtle hypopigmentation, and nystagmus. 25 The second patient with good VA was of Japanese origin. His VA was 0.0 logMAR, he had brown hair and mild hypopigmentation of the skin, with the ability to tan. Other ophthalmic details were missing. 8 An Italian patient with good VA (< 0.2 logMAR), no nystagmus, minimal iris translucency, and no fundal hypopigmentation is the only OCA4 patient decribed, to our knowledge, without foveal hypoplasia. In this patient misrouting was present, and molecular analysis revealed two mutations in the SLC45A2 gene, c.619C4G (p.L207V) and c.606G4C (p.W202C). 17 Until now, no patients have been described with evident lack of pigmentation of skin, hair, and eyes, but with normal ocular development, -i.e. normal VA, no nystagmus, no foveal hypoplasia and normal routing of the optic nerve bers. Especially the absence of foveal hypoplasia is remarkable, occurring in less than 0.7% of albinism patients. 1 It is still unclear why mutations in genes responsible for melanin synthesis cause defects in the development of the visual system. It is assumed that lack of melanin in the retinal pigment epithelium is responsible for excessive crossing of the optic nerve bers and foveal hypoplasia. Pigment epithelium-derived factor (PEDF) is a negative regulator of angiogenesis and plays an important role in the formation of the foveal avascular zone. 39 PEDF is decreased in the absence of tyrosinase, and therefore foveal hypoplasia could be caused by reduced PEDF. 40,41 However, a role for PEDF in chiasmal misrouting has not been established.
Generally, albinism patients with more severe hypopigmentation have more severe foveal hypoplasia and worse visual acuity. 1 Patients P4-P9 from this report conform to this phenotype. In contrast, P1, P2, and P3 demonstrate that normal foveal development, and normal routing of the optic nerve bers can occur despite an evident lack of melanin. Non pathogenic variants in SLC45A5 may cause lightly pigmented skin and hair without an ocular phenotype. However, the variants found in P1, P2, and P3 were likely pathogenic, and importantly, the patients' hypopigmented phenotype was not restricted to skin and hair, as they did have grade 3-4 iris translucency.
The OCA4 phenotype of patients P1, P2, and P3 seems the very opposite of the phenotype of the FHONDA syndrome, caused by mutations in SLC38A8 42,43,44,45 . Patients with FHONDA have nystagmus, poor VA, severe foveal hypoplasia and misrouting, but no pigmentation defect. The FHONDA syndrome provided the rst convincing evidence that lack of melanin is not the only determining factor in the combined occurrence of foveal hypoplasia and misrouting.
The three OCA4 patients with normal visual development we describe in this report, are further proof that the relationship between pigmentation defect and ocular de cits in albinism is more complicated than previously thought. Further research is needed to unravel the mechanisms that cause some OCA4 patients to have a severe albinism phenotype, while others do not show any ocular de cit, apart from iris translucency.

Declarations
Data availability: All data that are not included in this published article are available from the corresponding author on request.