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The pathogenicity of SLC38A8 in five families with foveal hypoplasia and congenital nystagmus
Experimental Eye Research 193 (2020) 107958
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The pathogenicity of SLC38A8 in five families with foveal hypoplasia and congenital nystagmus
T
Chen Weinera,b,∗, Idan Hechtb,c, Ygal Rotenstreichb,d, Sharon Guttmanb,c, Lior Orb,c, Yair Moradb,c, Guy Shapirab, Noam Shomronb,e,f, Eran Prasa,b,c a
Matlow's Ophthalmo-genetic Laboratory, Department of Ophthalmology, Shamir Medical Center (formerly Assaf Harofeh Medical Center), Zerifin, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel c Department of Ophthalmology, Shamir Medical Center, (formerly Assaf Harofeh Medical Center), Zerifin, Israel d Electrophysiology Clinic and Retinal Research Laboratory, Goldschleger Eye Institute, Sheba Medical Center, Israel e Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel f Edmond J. Safra Center of Bioinformatics, Tel Aviv University, Tel Aviv, Israel b
A R T I C LE I N FO
A B S T R A C T
Keywords: Foveal hypoplasia FVH2 SLC38A8 FHONDA syndrome Karaite jews
Purpose: A recently described subtype of foveal hypoplasia with congenital nystagmus and optic-nerve-decussation defects was found to be associated with mutations in the SLC38A8 gene. The aim of this study is to advance the clinical and molecular knowledge of SLC38A8 gene mutations. Methods: Five Israeli families with congenital foveal hypoplasia were studied, two of Karait Jewish origins and three of Indian Jewish origins. Subjects underwent a comprehensive ophthalmic examination including retinal photography and ocular coherence tomography. Molecular analysis including whole exome sequencing and screening of the SLC38A8 gene for specific disease-causing variants was performed. Results: Eight affected individuals were identified, all had congenital nystagmus and all but one had hypoplastic foveal pits. Anterior segment dysgenesis was observed in only one patient, one had evidence of developmental delay and another displayed early age-related macular degeneration (AMD). Molecular analysis revealed a recently described homozygous mutation, c.95T > G; p.Ile32Ser, in two families of Jewish Indian descent, and the same mutation in two families of Karaite Jewish descent. In a patient with only one pathogenic mutation (c.95T > G; p.Ile32Ser), a possible partial clinical expression of the disorder was seen. One patient of Jewish Indian descent was found to be compound heterozygous for c.95T > G; p.Ile32Ser and a novel mutation c.490_491delCT; p.L164Vfs*41. Conclusions: In five unrelated families with congenital nystagmus and foveal hypoplasia, mutations in the SLC38A8 gene were identified. Possible partial expression in a heterozygous patient was observed and novel potential disease-related phenotypes were identified including early-onset AMD and developmental delay. A novel mutation was also identified and a similar mutation in both Indian and Karaite Jewish ethnicities could be suggestive for common ancestry.
1. Introduction Foveal hypoplasia is a congenital disorder defined as the lack of foveal depression with continuity of all retinal layers in the foveal area (Al-Araimi et al., 2013). It is frequently found in relation to different forms of albinism but has also been described in other eye conditions, and as an isolated finding (Oliver et al., 1987; Campbell et al., 2019). Several gene mutations were found to be associated with foveal hypoplasia (Oliver et al., 1987; Perez et al., 2014). Many are also associated with oculocutaneous or ocular albinism such as X-linked ocular
∗
albinism type I (MIM 300500; GPR143 mutations, MIM 300808), autosomal recessive oculocutaneous albinism type IA (MIM, 203100), oculocutaneous albinism type IB (MIM 606952; TYR mutations, MIM 606933), and X-linked Aland Island disease (MIM 300600; CACNA1F mutations, MIM 300110). Foveal hypoplasia has also been described in cases of Stickler syndrome (COL2A1 mutations, MIM 108300) (Matsushita et al., 2017). Foveal hypoplasia as an isolated finding can also occur and has been described in PAX6 oculopathy (MIM 607108) and X-linked GPR143 (p.S89F mutation) (Azuma et al., 1996). A recently described subtype of foveal hypoplasia with congenital
Corresponding author. Department of Ophthalmology, Shamir Medical Center, Be'er Ya'akov, 70300, Israel. E-mail address: [email protected] (C. Weiner).
https://doi.org/10.1016/j.exer.2020.107958 Received 29 August 2019; Received in revised form 16 January 2020; Accepted 3 February 2020 Available online 04 February 2020 0014-4835/ © 2020 Elsevier Ltd. All rights reserved.
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All patients underwent a comprehensive ophthalmic examination by an experienced ophthalmologist (E.P) including slit-lamp bio-microscopy, retinal photography and Spectral Domain Ocular Coherence Tomography (SD-OCT, Spectralis, Heidelberg Engineering, Germany). Visual evoked potentials (VEP) were obtained from selected patients to document a decussation defect if present and was performed according to the protocol for ocular albinism (Dorey et al., 2003). Molecular analysis was performed at the Matlow's OphthalmoGenetic Laboratory, Shamir Medical Center, Israel. Initially, Whole Exome Sequencing (WES) was performed on DNA samples from all subjects of Karaite Jewish origin (families #1 and #2, 10 subjects). On samples from families #3 and #4 (Indian Jewish origin, 4 subjects) we performed direct sequencing of the founder mutation c.95T > G; p.Ile32Ser (ClinVar ID: VCV000125442; dbSNP ID: rs587777253) by Sanger sequencing. Finally, on samples from family #5 (Indian Jewish origin, 2 subjects) we started with the founder mutation by direct sequencing followed by complete gene sequencing, also using Sanger sequencing. Linkage and haplotype analysis was performed using Single Nucleotide Polymorphism (SNPs) extracted from the WES raw data, and in patients who did not undergo WES, we profiled their haplotype using PCR and Sanger sequencing. WES was performed by a commercial company, as previously described by us (Pras et al., 2015). The R package CODEX2 was used to call Copy Number Variants (Jiang et al., 2018).
nystagmus and without albinism (MIM 615585) was associated with biallelic mutations in Solute Carrier Family 38 Member 8 (SLC38A8, NG_034136) (Perez et al., 2014; Toral et al., 2017; Poulter et al., 2013). This recessively inherited disorder has been termed FHONDA syndrome, FVH2, or foveal hypoplasia-2 by some (Perez et al., 2014; Toral et al., 2017; Poulter et al., 2013; Shields et al., 2015). The SLC38 family are protein-coding genes, also known as sodiumcoupled neutral amino acid transporters (SNATs). The SLC38A8 gene product, SNAT8, expressed mostly in neurons. This protein functions as a sodium-coupled amino acid transporter with high preference for glutamate as a substrate (Hagglund et al., 2015). Studies have shown tissue specificity in the brain, in a subset of glial cells, and throughout the neuronal retina with higher expression in the inner and outer plexiform layer and the photoreceptor layer (Poulter et al., 2013; Hagglund et al., 2015; Pochini et al., 2014). To date, about 11 disease-predicted-mutations have been reported in multiple ethnicities, including three families of Indian Jews. The c.95T > G; p.Ile32Ser founder mutation in Indian Jews was first identified by Perez et al., in 2014 (Perez et al., 2014). Foveal hypoplasia and nystagmus were the dominant phenotype present in all affected individuals, followed by optic-nerve-decussation defects and less common were anterior segment abnormalities, microphthalmia, coloboma, and foveal thickness abnormalities (Perez et al., 2014; Toral et al., 2017; Poulter et al., 2013; Shields et al., 2015). In this study, we describe the clinical and molecular characteristics of five Israeli families with congenital nystagmus and foveal hypoplasia carrying mutations in the SLC38A8 gene.
3. Results 3.1. Clinical findings
2. Materials and methods Five non-related Jewish families, with a total of eight affected individuals, were identified, their pedigrees along with detailed clinical and molecular findings are displayed in Fig. 1 and Table 1. All affected individuals had congenital nystagmus and all but one (NY0035 Family #1) had hypoplastic foveal pits showing lack of foveal depression, persisting nerve fiber layer across the fovea, and multiple inner retinal layers which are normally absent at the center of the fovea, present across the fovea (Fig. 2). Visual acuities of affected individuals ranged between 6/18 and 6/
The study and informed consent procedures were approved by the Institutional Review Board of Shamir Medical Center and followed the tenets of the Declaration of Helsinki. Informed written consent was obtained from all the participants involved in the study. Eight affected individuals of five Israeli families with congenital foveal hypoplasia and nystagmus were included in the study. Two families of Karait Jewish and three families of Indian Jewish “Bene Israel” origin (Mumbai region) as illustrated in Fig. 1.
Fig. 1. Pedigrees of families reported in this study. HMZ – Homozygous; HTZ – Heterozygous; WT – Wild Type. 2
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60 and none had strabismus. Visual evoked potentials testing of two subjects from family #1 (NY0035 and NY0034) showed normal decussation in subject NY0035 (Karaite heterozygous patient). However subject NY0034 (Son of NY0035) showed excessive crossing of optic nerve fibers at the chiasm from the right eye to the left brain and from the left eye to the right brain (Fig. 3). None of the patients exhibited abnormal cutaneous or ocular pigmentation and no iris transillumination defects were seen. Although anterior segment anomalies are considered part of the disorder and have been reported in 47–100% of cases (Al-Araimi et al., 2013; Oliver et al., 1987; Perez et al., 2014; Poulter et al., 2013), only one patient out of eight affected participants showed anterior segment anomalies, in the form of mild posterior embryotoxon (NY0036, Family #2, Fig. 4). Interestingly, all homozygous patients had foveal hypoplasia, while the Karaite heterozygous patient (NY0035) had a normal-appearing fovea. SD-OCT images of patient NY0035 showed preserved foveal pits and preserved macular morphology in both eyes, including a sloping foveal depression, thickening of the photoreceptor nuclear layer at the fovea and displacement of the inner retinal layers (further detailed in Fig. 2). Further evaluation of this subject revealed better visual acuities (20/25 in the right eye and 20/50 in the left; attributed to mild amblyopia) as opposed to participants with hypoplastic foveal pits whose visual acuities ranged from 20/60 to 20/200. On VEP testing, normal decussation at the chiasm was recorded in this patient, as opposed to her son (NY0034) in which smaller amplitude responses with an excessive crossing of optic nerve fibers (Fig. 3). Taken together, the clinical picture of patient NY0035 reveals a milder form of the disorder compared to other patients either from the same or different families, raising the question of whether she presents a phenocopy; e.g. congenital nystagmus from another unknown cause or in fact a partial expression of the same syndrome. Additionally, findings not previously reported in this syndrome include one patient (NY0050, Family #3) with evidence of developmental delay presenting as lack of verbal and continence skills by the age of three years, and another patient (NY0048, Family #4) displaying early age-related macular degeneration (AMD), as depicted in Fig. 5.
Indian Jews Father 33 M RP0058 F5
BCVA, Best corrected visual acuity. CN, Congenital nystagmus. ASD, Anterior Segment Dysgenesis. DD, Developmental Delay. CUSM, Central, Unsteady, Maintained. CF, Counting fingers. AMD, Age-related macular degeneration.
Normal None – Not affected
Hypoplasia CN CUSM
1|2 2|1 1|2 RP0057 F5
F
2
Proband
Indian Jews
c.95T > G p.Ile32Ser c.490_491delCT p.L164Vfs*41 c.490_491delCT p.L164Vfs*41
Affected
Hypoplasia CN + Early AMD 20/150//20/200 2|2 NY0048 F4
F
74
Proband
Indian Jews
c.95T > G p.Ile32Ser
Affected
Unknown Unknown CN CN + DD 2|2 2|2 NY0049 NY0050 F3 F3
M M
1 3
Proband Brother
Indian Jews Indian Jews
c.95T > G p.Ile32Ser c.95T > G p.Ile32Ser
Affected Affected
– CUSM
Hypoplasia Normal Normal Normal CN with ASD None None None G G G G
p.Ile32Ser p.Ile32Ser p.Ile32Ser p.Ile32Ser
2 1 1 1
| | | |
2 2 2 2 > > > > c.95T c.95T c.95T c.95T Karaite Karaite Karaite Karaite Proband Brother Brother Mother 50 55 59 79 NY0036 NY0041 NY0042 NY0040 F2 F2 F2 F2
M M M F
| | | | | | p.Ile32Ser p.Ile32Ser p.Ile32Ser p.Ile32Ser p.Ile32Ser p.Ile32Ser G G G G G G Karaite Karaite Karaite Karaite Karaite Karaite Proband Spouse Daughter Daughter Son Son 37 39 6 10 15 7 NY0035 NY0038 NY0033 NY0039 NY0034 NY0037 F1 F1 F1 F1 F1 F1
F M F F M M
Origin Relation to proband Age (years) Gender Patient ID Family ID
Table 1 Clinical and molecular details.
Affected Not affected Not affected Not affected
– – – 20/300//CF
Normal Normal Hypoplasia Normal Hypoplasia Normal CN None CN None CN None 20/25//20/50 20/20//20/20 20/60//20/60 – 20/60//20/60 – Affected Not affected Affected Not affected Affected Not affected 1 1 2 1 2 1 > > > > > > c.95T c.95T c.95T c.95T c.95T c.95T
2 2 2 2 2 2
Zygosity Variant
Affected
BCVA (Right//Left)
Nystagmus Phenotype
Fovea
C. Weiner, et al.
3.2. Molecular analysis DNA sequencing identified bi-allelic SLC38A8 mutations in all seven patients with foveal hypoplasia; of them, six were homozygous for c.95T > G; p.Ile32Ser and one (RP0057 Family #5) was a compoundheterozygous for the same mutation and for a novel mutation c.490_491delCT; p.L164Vfs*41 (ClinVar ID: SCV000987705, Fig. 6 and Table 1). Surprisingly, the more frequent mutation c.95T > G; p.Ile32Ser that has been previously described in Indian Jews (Perez et al., 2014), and was also found in three Indian Jewish families from the present cohort, was also found in the Karaite Jewish patients, an undescribed yet ethnicity for this mutation (Family #1 and Family #2). Another interesting finding regards patient NY0035, manifesting a milder phenotype, in which both WES and Sanger sequencing of the gene coding exons, splice site regions, and untranslated region did not identify a second disease-related mutation (aside c.95T > G; p.Ile32Ser). This might suggest that partial expression of the syndrome is evident in this heterozygous patient. Attempting to address this hypothesis we have re-analyzed NY0035 WES data, and ruled out carriage for any mutation in known congenital nystagmus related genes (e.g. CACNAF1, CEP290, CNGA3, CNGB3, CRB1, CRX, FRMD7, GPR143, GUCY143, NMNAT1, PAX6, RPGRIP1, WDR19, SLC45A2, TYR, TYRP1, and OCA1-4). Using the WES data, we constructed the haplotypes of Family #1 and #2 (Supplemental Fig. 1), showing that NY0035 (F1–I:2) from Family #1 shares the same haplotype as the healthy individual NY0040 (F2–I:2) from Family #2 with an exception for a single synonymous variant c.609G > A; p.V203V. Furthermore, an unaffected child of NY0035 (F1–I:2) (NY0037 F1-II:3) inherited the paternal 3
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Fig. 2. SD-OCT images of patient NY0036 from family #2, and patient NY0035 from family #1, both of Karait Jewish descent. (A) SD-OCT image of patient NY0036 from family #2, aged 50. Notice the absence of foveal pit and the high reflectivity across the fovea, suggesting a persisting nerve fiber layer. Multiple inner retinal layers, normally absent at the center of the fovea, persist across the fovea. The external limiting membrane, photoreceptor inner segment layer, and photoreceptor outer segment layer appear normal. (B) Despite evident nystagmus, optical coherence tomography images of patient NY0035 showed preserved foveal pits and preserved macular morphology in both eyes. Normal morphology can be seen including a sloping foveal depression, thickening of the hyporeflective photoreceptor nuclear layer at the fovea and displacement of the nerve fiber layer, ganglion cell layer, inner plexiform layer, inner nuclear layer, and outer plexiform layer. c entral macular thickness was 217 μm and 219 μm in the right and left eyes respectively.
coastline of India) (Pras et al., 2015; Jiang et al., 2018). The majority immigrated to Israel and ever since have kept somewhat isolated and are therefore a relatively inbred small community. Based on the different heritage and origins of the Israeli Indian and Karaite Jews, it is somewhat surprising that similar disease phenotypes with identical homozygous mutations appear in both populations. It is possible that this is a result of a coincidental mutation in the same location (hot spot), however, given the results of the haplotype analysis the possibility of the existence of a founder mutation, suggestive of common ancestry is more plausible. Despite this, historical records do not reveal emigration between Mesopotamia and the Konkan area and no other genetic disorders typically common to these two populations were identified by the authors (Astren, 2004; Parfitt, 2002). One of our patients (NY0035), was found to be heterozygous for c.95T > G; p.Ile32Ser and presented with a milder phenotype. She had congenital nystagmus yet no foveal hypoplasia and normal visual evoked potentials results. One explanation may be the existence of a genocopy, an additional mutation in a different gene with possible parallel effects, or by the presence of a low penetrance mutation in noncoding regions. This last explanation, although not ruled out, seems unlikely given the haplotype analysis which showed equivalent haplotypes in unaffected members (NY0037 and NY0040), nor we found CNVs suggestive of chromosomal aberration in the gene of interest region. Another interesting possibility is that despite the known recessive mode of inheritance, the disorder may be expressed in individuals carrying only one mutated allele. Partial expression in a heterozygous patient could explain not only the fact the individual was affected with only one allele mutated but also the milder, partial clinical presentation (Table 1). Partial (or incomplete) disease expression in heterozygous patients has been described in other syndromes such as familial
mutated allele (NY0038 F1 I:1) and the maternal alternative allele colored purple, making the possibility that both subjects (NY0035 and NY0037) carry disease-associated alleles on both chromosomes unlikely. Finally, we performed copy number variation analysis to rule out the possibility of a large chromosomal duplication or deletion in this locus. In both comparative and non-comparative analysis, no copy number variations were found that could suggest a chromosomal aberration. Haplotype analysis revealed that both Indian Jews and Karaite patients who carry the pathogenic mutation p.I32S share a common haplotype spanning about 0.15 Mb (Supplemental Fig. 1). The centromeric side is flanked by a recombination evident in Family-5 II:1 at rs77876966 (positioned on chr16:84,016,604), and the telomeric side of the haplotype is bordered by a recombination in Family-4 II:1, at rs2288019 (Chr16:84,176,028). This may indicate that the Karaite Jews and Indian Jews share a common ancestry. 4. Discussion The ocular findings seen in our study have been previously described in Israeli Jews from Indian heritage but are described here for the first time in those of Karaite Jewish origins. These two populations, while both of Jewish religion has been historically isolated from one another. Karaite Jews, practice Karaism, which is a variant of postbiblical Judaism (Polliack, 2003). Their origins have been traced to the 8th and 9th century Mesopotamia (present-day Iraq) (Astren, 2004). Today the total number of Karaites is quite small (up to 35,000 worldwide), with the majority being concentrated in Israel (Stanford Libraries, 2018). On the other hand, the Jewish Indian community, also called “Bene Israel”, originate from the Konkan area (the western 4
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Fig. 3. Visual evoked potentials result of two subjects from family #1. Visual evoked potentials result of two subjects from family #1 (NY0035 and NY0034). Subject NY0035 (the proband, Karaite heterozygous patient) showed normal decussation. Subject NY0034 (Son of the proband) showed smaller amplitudes responses with the excessive crossing of optic nerve fibers at the chiasm from the right eye to the left brain and from the left eye to the right brain.
et al., 2001). All previously described patients with this syndrome had hypoplastic fovea and congenital nystagmus, with one possibly explaining the other (Al-Araimi et al., 2013; Perez et al., 2014; Toral et al., 2017; Poulter et al., 2013). This patient (NY0035) intriguingly has congenital nystagmus with normal foveal pits and visual acuity, suggesting that nystagmus may not originate from inadequate image formation. Previous descriptions of the syndrome typically included anterior segment dysgenesis, however, this finding has been shown to be a variable component of the phenotype. Only three out of eleven families reported by Poulter et al. showed anterior segment dysgenesis and none among nine patients of Jewish Indian descent reported by Perez et al. (Perez et al., 2014; Poulter et al., 2013). Our results reinforce these findings as only one patient out of eight had anterior segment dysgenesis. It is possible that the specific mutation seen in the majority of the patients described by Peretz et al. and us (c.95T > G; p.Ile32Ser) is associated to a lesser degree with anterior segment dysgenesis (Perez et al., 2014). Moreover, whether anterior segment dysgenesis is part of the syndrome or not remains unclear. On the other hand, this specific mutation might predispose to other related phenotypes. Patient (NY0050, Family #3) had evidence of severe developmental delay presenting as lack of verbal and continence skills by the age of three years. Perez et al. have also noted three patients with mild developmental delay, also homozygous to the same mutation (c.95T > G; p.Ile32Ser) (Perez et al., 2014). These functional deficits could be related to the presumed neurological function of the SLC38A8 gene and specifically to the (c.95T > G; p.Ile32Ser) mutation (Hagglund et al., 2015). Although many aspects of the gene and protein functionality are still unclear, it is believed to function as glutamine transporter, found in high concentrations in the brain extracellular fluid (Poulter et al., 2013; Hagglund et al., 2015; Pochini et al., 2014). It is, therefore, possible
Fig. 4. Image of patient NY0036, the proband of family #2. Notice the posterior embryotoxon marked by the arrow (image quality is limited by nystagmus).
Mediterranean fever, cystinuria, sickle cell trait, and Tay-Sachs disease, where a heterozygous mutation of a traditionally recessive disease, manifests a partial phenotype (Livneh et al., 2001; Procopio et al., 2018; Myerowitz, 1997; Tsaras et al., 2009). In cystinuria, for example, a kidney stone disease traditionally described as autosomal recessive, bi-allelic carriers develop kidney stones in 85% of cases, yet 13% of heterozygous carriers will also develop kidney stones (Pras et al., 1998). In familial Mediterranean fever, another traditionally autosomal recessive disease, heterozygous carriers usually remain unaffected, yet the disease may be precipitated by co-existing Behcet's disease (Livneh 5
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Fig. 5. Fundoscopy images of patient NY0048, Family #4. Fundoscopy and SD-OCT images of subject NY0048, the proband of family #4, showing evidence of early-onset AMD, first documented at the age of 60 years.
(WGS) was not performed but Whole Exome Sequencing was done in only selected patients. Third, the clinical evaluation did not include visual evoked potentials in all cases. Lastly, not all subjects were available for clinical examination or consented to molecular analysis. To conclude, in five families with foveal hypoplasia and nystagmus carrying SLC38A8 gene mutations, similar biallelic mutations were identified in two distinct populations of Indian and Karaite Jewish origins. A possible partial expression in a heterozygous patient is seen and new potential disease-related phenotypes, along with a novel mutation of the SLC38A8 gene are described. These results could help improve our understanding of the relationship between the SLC38A8 gene with foveal hypoplasia and other ocular and systemic manifestations with potential insights into future genetic counseling.
Fig. 6. Sanger sequence chromatogram of c.490_491delCT; p.L164Vfs*41. Reference sequence of SLC38A8 (ENSE00001104170) is represented at the top. Middle chromatogram is the wild type (wt) sequence. At the bottom, is the patient (Family #5 RP0057) sequence, demonstrating double base deletion resulting in a complete CDS frame-shift followed by stop codon 41 amino acids downstream.
Funding Funding of this study was supported by the Claire and Amedee Maratier Institute for the Study of Blindness and Visual Disorders (TAU#: 32003197000); The Ernest And Nusia Gothelf research funds (TAU#: 32003125000), Sackler Faculty of Medicine, TelAviv University, Tel-Aviv, Israel. This work was also supported in part by grant no. 7205 from the Chief Scientist Office of the Ministry of Health, Israel; ‘Lirot’ Association and the Consortium for Mapping Retinal Degeneration Disorders in Israel, and by Grants BR-GE-02140639-TECH and BR-GE-0518-0734-TECH from the Foundation Fighting Blindness, Grant 3-12583 from the Israeli Ministry of Health. This work was performed in partial fulfillment of the requirements for a Ph.D. degree by Chen Weiner at the Sackler Faculty of Medicine, Tel Aviv University, Israel.
that other extraocular manifestations exist affecting verbal and developmental aspects. Another interesting observation is the evidence of early-onset AMD seen in patient NY0048 (Family #4, Fig. 5), which was first documented at the age of 60 years. AMD is typically seen above the age of 75 years and the prevalence at the age of 60 years has been estimated to be only between 0.1% and 0.4% (Lambert et al., 2016). This finding is not only linked to the same tissue shown to be affected by the SLC38A8 gene, but the majority of previously reported cases were young and so this phenotype could have been missed. This study has several limitations. First, it includes only five families, all Jewish origins and so clinical and molecular findings may differ from other populations. Second, Whole Genome Sequencing 6
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Declaration of competing interest
H., 2017. Foveal hypoplasia in patients with stickler syndrome. Ophthalmology 124 (6), 896–902. Myerowitz, R., 1997. Tay-Sachs disease-causing mutations and neutral polymorphisms in the Hex A gene. Hum. Mutat. 9 (3), 195–208. Oliver, M.D., Dotan, S.A., Chemke, J., Abraham, F.A., 1987. Isolated foveal hypoplasia. Br. J. Ophthalmol. 71 (12), 926–930. Parfitt, T., 2002. The Lost Tribes of Israel : the History of a Myth. Weidenfeld & Nicolson, London. Perez, Y., Gradstein, L., Flusser, H., Markus, B., Cohen, I., Langer, Y., Marcus, M., Lifshitz, T., Kadir, R., Birk, O.S., 2014. Isolated foveal hypoplasia with secondary nystagmus and low vision is associated with a homozygous SLC38A8 mutation. Eur. J. Hum. Genet. 22 (5), 703–706. Pochini, L., Scalise, M., Galluccio, M., Indiveri, C., 2014. Membrane transporters for the special amino acid glutamine: structure/function relationships and relevance to human health. Front. Chem. 2, 61. Polliack, M., 2003. Karaite Judaism : a Guide to its History and Literary Sources. Brill, Leiden ; Boston. Poulter, J.A., Al-Araimi, M., Conte, I., van Genderen, M.M., Sheridan, E., Carr, I.M., Parry, D.A., Shires, M., Carrella, S., Bradbury, J., et al., 2013. Recessive mutations in SLC38A8 cause foveal hypoplasia and optic nerve misrouting without albinism. Am. J. Hum. Genet. 93 (6), 1143–1150. Pras, E., Kochba, I., Lubetzky, A., Pras, M., Sidi, Y., Kastner, D.L., 1998. Biochemical and clinical studies in Libyan Jewish cystinuria patients and their relatives. Am. J. Med. Genet. 80 (2), 173–176. Pras, E., Kristal, D., Shoshany, N., Volodarsky, D., Vulih, I., Celniker, G., Isakov, O., Shomron, N., Pras, E., 2015. Rare genetic variants in Tunisian Jewish patients suffering from age-related macular degeneration. J. Med. Genet. 52 (7), 484–492. Procopio, V., Manti, S., Bianco, G., Conti, G., Romeo, A., Maimone, F., Arrigo, T., Cutrupi, M.C., Salpietro, C., Cuppari, C., 2018. Genotype-phenotype correlation in FMF patients: a "non classic" recessive autosomal or "atypical" dominant autosomal inheritance? Gene 641, 279–286. Shields, R.A., Cavuoto, K.M., McKeown, C.A., Chang, T.C., 2015. Unilateral foveal hypoplasia in a child with bilateral anterior segment dysgenesis. Clin. Case Rep. 3 (7), 676–678. Stanford Libraries University of stanford. [cited 2018 15/12]. Available from: https:// library.stanford.edu/spc/exhibitspublications/past-exhibits/karaite-jews-egyptisrael-and-san-francisco-bay-area. Toral, M.A., Velez, G., Boudreault, K., Schaefer, K.A., Xu, Y., Saffra, N., Bassuk, A.G., Tsang, S.H., Mahajan, V.B., 2017. Structural modeling of a novel SLC38A8 mutation that causes foveal hypoplasia. Mol. Genet. Genom. Med. 5 (3), 202–209. Tsaras, G., Owusu-Ansah, A., Boateng, F.O., Amoateng-Adjepong, Y., 2009. Complications associated with sickle cell trait: a brief narrative Review. Am. J. Med. 122 (6), 507–512.
The authors declare that they have no conflicts of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.exer.2020.107958. References Al-Araimi, M., Pal, B., Poulter, J.A., van Genderen, M.M., Carr, I., Cudrnak, T., Brown, L., Sheridan, E., Mohamed, M.D., Bradbury, J., et al., 2013. A new recessively inherited disorder composed of foveal hypoplasia, optic nerve decussation defects and anterior segment dysgenesis maps to chromosome 16q23.3-24.1. Mol. Vis. 19, 2165–2172. Astren, F., 2004. Karaite Judaism and Historical Understanding. University of South Carolina Press, Columbia. Azuma, N., Nishina, S., Yanagisawa, H., Okuyama, T., Yamada, M., 1996. PAX6 missense mutation in isolated foveal hypoplasia. Nat. Genet. 13 (2), 141–142. Campbell, P., Ellingford, J.M., Parry, N.R.A., Fletcher, T., Ramsden, S.C., Gale, T., Hall, G., Smith, K., Kasperaviciute, D., Thomas, E., et al., 2019. Clinical and genetic variability in children with partial albinism. Sci. Rep. 9 (1). Dorey, S.E., Neveu, M.M., Burton, L.C., Sloper, J.J., Holder, G.E., 2003. The clinical features of albinism and their correlation with visual evoked potentials. Br. J. Ophthalmol. 87 (6), 767–772. Hagglund, M.G.A., Hellsten, S.V., Bagchi, S., Philippot, G., Lofqvist, E., Nilsson, V.C.O., Almkvist, I., Karlsson, E., Sreedharan, S., Tafreshiha, A., et al., 2015. Transport of Lglutamine, L-alanine, L-arginine and L-histidine by the neuron-specific Slc38a8 (SNAT8) in CNS. J. Mol. Biol. 427 (6 Pt B), 1495–1512. Jiang, Y., Wang, R., Urrutia, E., Anastopoulos, I.N., Nathanson, K.L., Zhang, N.R., 2018. CODEX2: full-spectrum copy number variation detection by high-throughput DNA sequencing. Genome Biol. 19 (1). Lambert, N.G., ElShelmani, H., Singh, M.K., Mansergh, F.C., Wride, M.A., Padilla, M., Keegan, D., Hogg, R.E., Ambati, B.K., 2016. Risk factors and biomarkers of age-related macular degeneration. Prog. Retin. Eye Res. 54, 64–102. Livneh, A., Aksentijevich, I., Langevitz, P., Torosyan, Y., G-Shoham, N., Shinar, Y., Pras, E., Zaks, N., Padeh, S., Kastner, D.L., et al., 2001. A single mutated MEFV allele in Israeli patients suffering from familial Mediterranean fever and Behcet's disease (FMF-BD). Eur. J. Hum. Genet. 9 (3), 191–196. Matsushita, I., Nagata, T., Hayashi, T., Kimoto, K., Kubota, T., Ohji, M., Kusaka, S., Kondo,
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The pathogenicity of SLC38A8 in five families with foveal hypoplasia and congenital nystagmus
T
Chen Weinera,b,∗, Idan Hechtb,c, Ygal Rotenstreichb,d, Sharon Guttmanb,c, Lior Orb,c, Yair Moradb,c, Guy Shapirab, Noam Shomronb,e,f, Eran Prasa,b,c a
Matlow's Ophthalmo-genetic Laboratory, Department of Ophthalmology, Shamir Medical Center (formerly Assaf Harofeh Medical Center), Zerifin, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel c Department of Ophthalmology, Shamir Medical Center, (formerly Assaf Harofeh Medical Center), Zerifin, Israel d Electrophysiology Clinic and Retinal Research Laboratory, Goldschleger Eye Institute, Sheba Medical Center, Israel e Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel f Edmond J. Safra Center of Bioinformatics, Tel Aviv University, Tel Aviv, Israel b
A R T I C LE I N FO
A B S T R A C T
Keywords: Foveal hypoplasia FVH2 SLC38A8 FHONDA syndrome Karaite jews
Purpose: A recently described subtype of foveal hypoplasia with congenital nystagmus and optic-nerve-decussation defects was found to be associated with mutations in the SLC38A8 gene. The aim of this study is to advance the clinical and molecular knowledge of SLC38A8 gene mutations. Methods: Five Israeli families with congenital foveal hypoplasia were studied, two of Karait Jewish origins and three of Indian Jewish origins. Subjects underwent a comprehensive ophthalmic examination including retinal photography and ocular coherence tomography. Molecular analysis including whole exome sequencing and screening of the SLC38A8 gene for specific disease-causing variants was performed. Results: Eight affected individuals were identified, all had congenital nystagmus and all but one had hypoplastic foveal pits. Anterior segment dysgenesis was observed in only one patient, one had evidence of developmental delay and another displayed early age-related macular degeneration (AMD). Molecular analysis revealed a recently described homozygous mutation, c.95T > G; p.Ile32Ser, in two families of Jewish Indian descent, and the same mutation in two families of Karaite Jewish descent. In a patient with only one pathogenic mutation (c.95T > G; p.Ile32Ser), a possible partial clinical expression of the disorder was seen. One patient of Jewish Indian descent was found to be compound heterozygous for c.95T > G; p.Ile32Ser and a novel mutation c.490_491delCT; p.L164Vfs*41. Conclusions: In five unrelated families with congenital nystagmus and foveal hypoplasia, mutations in the SLC38A8 gene were identified. Possible partial expression in a heterozygous patient was observed and novel potential disease-related phenotypes were identified including early-onset AMD and developmental delay. A novel mutation was also identified and a similar mutation in both Indian and Karaite Jewish ethnicities could be suggestive for common ancestry.
1. Introduction Foveal hypoplasia is a congenital disorder defined as the lack of foveal depression with continuity of all retinal layers in the foveal area (Al-Araimi et al., 2013). It is frequently found in relation to different forms of albinism but has also been described in other eye conditions, and as an isolated finding (Oliver et al., 1987; Campbell et al., 2019). Several gene mutations were found to be associated with foveal hypoplasia (Oliver et al., 1987; Perez et al., 2014). Many are also associated with oculocutaneous or ocular albinism such as X-linked ocular
∗
albinism type I (MIM 300500; GPR143 mutations, MIM 300808), autosomal recessive oculocutaneous albinism type IA (MIM, 203100), oculocutaneous albinism type IB (MIM 606952; TYR mutations, MIM 606933), and X-linked Aland Island disease (MIM 300600; CACNA1F mutations, MIM 300110). Foveal hypoplasia has also been described in cases of Stickler syndrome (COL2A1 mutations, MIM 108300) (Matsushita et al., 2017). Foveal hypoplasia as an isolated finding can also occur and has been described in PAX6 oculopathy (MIM 607108) and X-linked GPR143 (p.S89F mutation) (Azuma et al., 1996). A recently described subtype of foveal hypoplasia with congenital
Corresponding author. Department of Ophthalmology, Shamir Medical Center, Be'er Ya'akov, 70300, Israel. E-mail address: [email protected] (C. Weiner).
https://doi.org/10.1016/j.exer.2020.107958 Received 29 August 2019; Received in revised form 16 January 2020; Accepted 3 February 2020 Available online 04 February 2020 0014-4835/ © 2020 Elsevier Ltd. All rights reserved.
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All patients underwent a comprehensive ophthalmic examination by an experienced ophthalmologist (E.P) including slit-lamp bio-microscopy, retinal photography and Spectral Domain Ocular Coherence Tomography (SD-OCT, Spectralis, Heidelberg Engineering, Germany). Visual evoked potentials (VEP) were obtained from selected patients to document a decussation defect if present and was performed according to the protocol for ocular albinism (Dorey et al., 2003). Molecular analysis was performed at the Matlow's OphthalmoGenetic Laboratory, Shamir Medical Center, Israel. Initially, Whole Exome Sequencing (WES) was performed on DNA samples from all subjects of Karaite Jewish origin (families #1 and #2, 10 subjects). On samples from families #3 and #4 (Indian Jewish origin, 4 subjects) we performed direct sequencing of the founder mutation c.95T > G; p.Ile32Ser (ClinVar ID: VCV000125442; dbSNP ID: rs587777253) by Sanger sequencing. Finally, on samples from family #5 (Indian Jewish origin, 2 subjects) we started with the founder mutation by direct sequencing followed by complete gene sequencing, also using Sanger sequencing. Linkage and haplotype analysis was performed using Single Nucleotide Polymorphism (SNPs) extracted from the WES raw data, and in patients who did not undergo WES, we profiled their haplotype using PCR and Sanger sequencing. WES was performed by a commercial company, as previously described by us (Pras et al., 2015). The R package CODEX2 was used to call Copy Number Variants (Jiang et al., 2018).
nystagmus and without albinism (MIM 615585) was associated with biallelic mutations in Solute Carrier Family 38 Member 8 (SLC38A8, NG_034136) (Perez et al., 2014; Toral et al., 2017; Poulter et al., 2013). This recessively inherited disorder has been termed FHONDA syndrome, FVH2, or foveal hypoplasia-2 by some (Perez et al., 2014; Toral et al., 2017; Poulter et al., 2013; Shields et al., 2015). The SLC38 family are protein-coding genes, also known as sodiumcoupled neutral amino acid transporters (SNATs). The SLC38A8 gene product, SNAT8, expressed mostly in neurons. This protein functions as a sodium-coupled amino acid transporter with high preference for glutamate as a substrate (Hagglund et al., 2015). Studies have shown tissue specificity in the brain, in a subset of glial cells, and throughout the neuronal retina with higher expression in the inner and outer plexiform layer and the photoreceptor layer (Poulter et al., 2013; Hagglund et al., 2015; Pochini et al., 2014). To date, about 11 disease-predicted-mutations have been reported in multiple ethnicities, including three families of Indian Jews. The c.95T > G; p.Ile32Ser founder mutation in Indian Jews was first identified by Perez et al., in 2014 (Perez et al., 2014). Foveal hypoplasia and nystagmus were the dominant phenotype present in all affected individuals, followed by optic-nerve-decussation defects and less common were anterior segment abnormalities, microphthalmia, coloboma, and foveal thickness abnormalities (Perez et al., 2014; Toral et al., 2017; Poulter et al., 2013; Shields et al., 2015). In this study, we describe the clinical and molecular characteristics of five Israeli families with congenital nystagmus and foveal hypoplasia carrying mutations in the SLC38A8 gene.
3. Results 3.1. Clinical findings
2. Materials and methods Five non-related Jewish families, with a total of eight affected individuals, were identified, their pedigrees along with detailed clinical and molecular findings are displayed in Fig. 1 and Table 1. All affected individuals had congenital nystagmus and all but one (NY0035 Family #1) had hypoplastic foveal pits showing lack of foveal depression, persisting nerve fiber layer across the fovea, and multiple inner retinal layers which are normally absent at the center of the fovea, present across the fovea (Fig. 2). Visual acuities of affected individuals ranged between 6/18 and 6/
The study and informed consent procedures were approved by the Institutional Review Board of Shamir Medical Center and followed the tenets of the Declaration of Helsinki. Informed written consent was obtained from all the participants involved in the study. Eight affected individuals of five Israeli families with congenital foveal hypoplasia and nystagmus were included in the study. Two families of Karait Jewish and three families of Indian Jewish “Bene Israel” origin (Mumbai region) as illustrated in Fig. 1.
Fig. 1. Pedigrees of families reported in this study. HMZ – Homozygous; HTZ – Heterozygous; WT – Wild Type. 2
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60 and none had strabismus. Visual evoked potentials testing of two subjects from family #1 (NY0035 and NY0034) showed normal decussation in subject NY0035 (Karaite heterozygous patient). However subject NY0034 (Son of NY0035) showed excessive crossing of optic nerve fibers at the chiasm from the right eye to the left brain and from the left eye to the right brain (Fig. 3). None of the patients exhibited abnormal cutaneous or ocular pigmentation and no iris transillumination defects were seen. Although anterior segment anomalies are considered part of the disorder and have been reported in 47–100% of cases (Al-Araimi et al., 2013; Oliver et al., 1987; Perez et al., 2014; Poulter et al., 2013), only one patient out of eight affected participants showed anterior segment anomalies, in the form of mild posterior embryotoxon (NY0036, Family #2, Fig. 4). Interestingly, all homozygous patients had foveal hypoplasia, while the Karaite heterozygous patient (NY0035) had a normal-appearing fovea. SD-OCT images of patient NY0035 showed preserved foveal pits and preserved macular morphology in both eyes, including a sloping foveal depression, thickening of the photoreceptor nuclear layer at the fovea and displacement of the inner retinal layers (further detailed in Fig. 2). Further evaluation of this subject revealed better visual acuities (20/25 in the right eye and 20/50 in the left; attributed to mild amblyopia) as opposed to participants with hypoplastic foveal pits whose visual acuities ranged from 20/60 to 20/200. On VEP testing, normal decussation at the chiasm was recorded in this patient, as opposed to her son (NY0034) in which smaller amplitude responses with an excessive crossing of optic nerve fibers (Fig. 3). Taken together, the clinical picture of patient NY0035 reveals a milder form of the disorder compared to other patients either from the same or different families, raising the question of whether she presents a phenocopy; e.g. congenital nystagmus from another unknown cause or in fact a partial expression of the same syndrome. Additionally, findings not previously reported in this syndrome include one patient (NY0050, Family #3) with evidence of developmental delay presenting as lack of verbal and continence skills by the age of three years, and another patient (NY0048, Family #4) displaying early age-related macular degeneration (AMD), as depicted in Fig. 5.
Indian Jews Father 33 M RP0058 F5
BCVA, Best corrected visual acuity. CN, Congenital nystagmus. ASD, Anterior Segment Dysgenesis. DD, Developmental Delay. CUSM, Central, Unsteady, Maintained. CF, Counting fingers. AMD, Age-related macular degeneration.
Normal None – Not affected
Hypoplasia CN CUSM
1|2 2|1 1|2 RP0057 F5
F
2
Proband
Indian Jews
c.95T > G p.Ile32Ser c.490_491delCT p.L164Vfs*41 c.490_491delCT p.L164Vfs*41
Affected
Hypoplasia CN + Early AMD 20/150//20/200 2|2 NY0048 F4
F
74
Proband
Indian Jews
c.95T > G p.Ile32Ser
Affected
Unknown Unknown CN CN + DD 2|2 2|2 NY0049 NY0050 F3 F3
M M
1 3
Proband Brother
Indian Jews Indian Jews
c.95T > G p.Ile32Ser c.95T > G p.Ile32Ser
Affected Affected
– CUSM
Hypoplasia Normal Normal Normal CN with ASD None None None G G G G
p.Ile32Ser p.Ile32Ser p.Ile32Ser p.Ile32Ser
2 1 1 1
| | | |
2 2 2 2 > > > > c.95T c.95T c.95T c.95T Karaite Karaite Karaite Karaite Proband Brother Brother Mother 50 55 59 79 NY0036 NY0041 NY0042 NY0040 F2 F2 F2 F2
M M M F
| | | | | | p.Ile32Ser p.Ile32Ser p.Ile32Ser p.Ile32Ser p.Ile32Ser p.Ile32Ser G G G G G G Karaite Karaite Karaite Karaite Karaite Karaite Proband Spouse Daughter Daughter Son Son 37 39 6 10 15 7 NY0035 NY0038 NY0033 NY0039 NY0034 NY0037 F1 F1 F1 F1 F1 F1
F M F F M M
Origin Relation to proband Age (years) Gender Patient ID Family ID
Table 1 Clinical and molecular details.
Affected Not affected Not affected Not affected
– – – 20/300//CF
Normal Normal Hypoplasia Normal Hypoplasia Normal CN None CN None CN None 20/25//20/50 20/20//20/20 20/60//20/60 – 20/60//20/60 – Affected Not affected Affected Not affected Affected Not affected 1 1 2 1 2 1 > > > > > > c.95T c.95T c.95T c.95T c.95T c.95T
2 2 2 2 2 2
Zygosity Variant
Affected
BCVA (Right//Left)
Nystagmus Phenotype
Fovea
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3.2. Molecular analysis DNA sequencing identified bi-allelic SLC38A8 mutations in all seven patients with foveal hypoplasia; of them, six were homozygous for c.95T > G; p.Ile32Ser and one (RP0057 Family #5) was a compoundheterozygous for the same mutation and for a novel mutation c.490_491delCT; p.L164Vfs*41 (ClinVar ID: SCV000987705, Fig. 6 and Table 1). Surprisingly, the more frequent mutation c.95T > G; p.Ile32Ser that has been previously described in Indian Jews (Perez et al., 2014), and was also found in three Indian Jewish families from the present cohort, was also found in the Karaite Jewish patients, an undescribed yet ethnicity for this mutation (Family #1 and Family #2). Another interesting finding regards patient NY0035, manifesting a milder phenotype, in which both WES and Sanger sequencing of the gene coding exons, splice site regions, and untranslated region did not identify a second disease-related mutation (aside c.95T > G; p.Ile32Ser). This might suggest that partial expression of the syndrome is evident in this heterozygous patient. Attempting to address this hypothesis we have re-analyzed NY0035 WES data, and ruled out carriage for any mutation in known congenital nystagmus related genes (e.g. CACNAF1, CEP290, CNGA3, CNGB3, CRB1, CRX, FRMD7, GPR143, GUCY143, NMNAT1, PAX6, RPGRIP1, WDR19, SLC45A2, TYR, TYRP1, and OCA1-4). Using the WES data, we constructed the haplotypes of Family #1 and #2 (Supplemental Fig. 1), showing that NY0035 (F1–I:2) from Family #1 shares the same haplotype as the healthy individual NY0040 (F2–I:2) from Family #2 with an exception for a single synonymous variant c.609G > A; p.V203V. Furthermore, an unaffected child of NY0035 (F1–I:2) (NY0037 F1-II:3) inherited the paternal 3
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Fig. 2. SD-OCT images of patient NY0036 from family #2, and patient NY0035 from family #1, both of Karait Jewish descent. (A) SD-OCT image of patient NY0036 from family #2, aged 50. Notice the absence of foveal pit and the high reflectivity across the fovea, suggesting a persisting nerve fiber layer. Multiple inner retinal layers, normally absent at the center of the fovea, persist across the fovea. The external limiting membrane, photoreceptor inner segment layer, and photoreceptor outer segment layer appear normal. (B) Despite evident nystagmus, optical coherence tomography images of patient NY0035 showed preserved foveal pits and preserved macular morphology in both eyes. Normal morphology can be seen including a sloping foveal depression, thickening of the hyporeflective photoreceptor nuclear layer at the fovea and displacement of the nerve fiber layer, ganglion cell layer, inner plexiform layer, inner nuclear layer, and outer plexiform layer. c entral macular thickness was 217 μm and 219 μm in the right and left eyes respectively.
coastline of India) (Pras et al., 2015; Jiang et al., 2018). The majority immigrated to Israel and ever since have kept somewhat isolated and are therefore a relatively inbred small community. Based on the different heritage and origins of the Israeli Indian and Karaite Jews, it is somewhat surprising that similar disease phenotypes with identical homozygous mutations appear in both populations. It is possible that this is a result of a coincidental mutation in the same location (hot spot), however, given the results of the haplotype analysis the possibility of the existence of a founder mutation, suggestive of common ancestry is more plausible. Despite this, historical records do not reveal emigration between Mesopotamia and the Konkan area and no other genetic disorders typically common to these two populations were identified by the authors (Astren, 2004; Parfitt, 2002). One of our patients (NY0035), was found to be heterozygous for c.95T > G; p.Ile32Ser and presented with a milder phenotype. She had congenital nystagmus yet no foveal hypoplasia and normal visual evoked potentials results. One explanation may be the existence of a genocopy, an additional mutation in a different gene with possible parallel effects, or by the presence of a low penetrance mutation in noncoding regions. This last explanation, although not ruled out, seems unlikely given the haplotype analysis which showed equivalent haplotypes in unaffected members (NY0037 and NY0040), nor we found CNVs suggestive of chromosomal aberration in the gene of interest region. Another interesting possibility is that despite the known recessive mode of inheritance, the disorder may be expressed in individuals carrying only one mutated allele. Partial expression in a heterozygous patient could explain not only the fact the individual was affected with only one allele mutated but also the milder, partial clinical presentation (Table 1). Partial (or incomplete) disease expression in heterozygous patients has been described in other syndromes such as familial
mutated allele (NY0038 F1 I:1) and the maternal alternative allele colored purple, making the possibility that both subjects (NY0035 and NY0037) carry disease-associated alleles on both chromosomes unlikely. Finally, we performed copy number variation analysis to rule out the possibility of a large chromosomal duplication or deletion in this locus. In both comparative and non-comparative analysis, no copy number variations were found that could suggest a chromosomal aberration. Haplotype analysis revealed that both Indian Jews and Karaite patients who carry the pathogenic mutation p.I32S share a common haplotype spanning about 0.15 Mb (Supplemental Fig. 1). The centromeric side is flanked by a recombination evident in Family-5 II:1 at rs77876966 (positioned on chr16:84,016,604), and the telomeric side of the haplotype is bordered by a recombination in Family-4 II:1, at rs2288019 (Chr16:84,176,028). This may indicate that the Karaite Jews and Indian Jews share a common ancestry. 4. Discussion The ocular findings seen in our study have been previously described in Israeli Jews from Indian heritage but are described here for the first time in those of Karaite Jewish origins. These two populations, while both of Jewish religion has been historically isolated from one another. Karaite Jews, practice Karaism, which is a variant of postbiblical Judaism (Polliack, 2003). Their origins have been traced to the 8th and 9th century Mesopotamia (present-day Iraq) (Astren, 2004). Today the total number of Karaites is quite small (up to 35,000 worldwide), with the majority being concentrated in Israel (Stanford Libraries, 2018). On the other hand, the Jewish Indian community, also called “Bene Israel”, originate from the Konkan area (the western 4
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Fig. 3. Visual evoked potentials result of two subjects from family #1. Visual evoked potentials result of two subjects from family #1 (NY0035 and NY0034). Subject NY0035 (the proband, Karaite heterozygous patient) showed normal decussation. Subject NY0034 (Son of the proband) showed smaller amplitudes responses with the excessive crossing of optic nerve fibers at the chiasm from the right eye to the left brain and from the left eye to the right brain.
et al., 2001). All previously described patients with this syndrome had hypoplastic fovea and congenital nystagmus, with one possibly explaining the other (Al-Araimi et al., 2013; Perez et al., 2014; Toral et al., 2017; Poulter et al., 2013). This patient (NY0035) intriguingly has congenital nystagmus with normal foveal pits and visual acuity, suggesting that nystagmus may not originate from inadequate image formation. Previous descriptions of the syndrome typically included anterior segment dysgenesis, however, this finding has been shown to be a variable component of the phenotype. Only three out of eleven families reported by Poulter et al. showed anterior segment dysgenesis and none among nine patients of Jewish Indian descent reported by Perez et al. (Perez et al., 2014; Poulter et al., 2013). Our results reinforce these findings as only one patient out of eight had anterior segment dysgenesis. It is possible that the specific mutation seen in the majority of the patients described by Peretz et al. and us (c.95T > G; p.Ile32Ser) is associated to a lesser degree with anterior segment dysgenesis (Perez et al., 2014). Moreover, whether anterior segment dysgenesis is part of the syndrome or not remains unclear. On the other hand, this specific mutation might predispose to other related phenotypes. Patient (NY0050, Family #3) had evidence of severe developmental delay presenting as lack of verbal and continence skills by the age of three years. Perez et al. have also noted three patients with mild developmental delay, also homozygous to the same mutation (c.95T > G; p.Ile32Ser) (Perez et al., 2014). These functional deficits could be related to the presumed neurological function of the SLC38A8 gene and specifically to the (c.95T > G; p.Ile32Ser) mutation (Hagglund et al., 2015). Although many aspects of the gene and protein functionality are still unclear, it is believed to function as glutamine transporter, found in high concentrations in the brain extracellular fluid (Poulter et al., 2013; Hagglund et al., 2015; Pochini et al., 2014). It is, therefore, possible
Fig. 4. Image of patient NY0036, the proband of family #2. Notice the posterior embryotoxon marked by the arrow (image quality is limited by nystagmus).
Mediterranean fever, cystinuria, sickle cell trait, and Tay-Sachs disease, where a heterozygous mutation of a traditionally recessive disease, manifests a partial phenotype (Livneh et al., 2001; Procopio et al., 2018; Myerowitz, 1997; Tsaras et al., 2009). In cystinuria, for example, a kidney stone disease traditionally described as autosomal recessive, bi-allelic carriers develop kidney stones in 85% of cases, yet 13% of heterozygous carriers will also develop kidney stones (Pras et al., 1998). In familial Mediterranean fever, another traditionally autosomal recessive disease, heterozygous carriers usually remain unaffected, yet the disease may be precipitated by co-existing Behcet's disease (Livneh 5
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Fig. 5. Fundoscopy images of patient NY0048, Family #4. Fundoscopy and SD-OCT images of subject NY0048, the proband of family #4, showing evidence of early-onset AMD, first documented at the age of 60 years.
(WGS) was not performed but Whole Exome Sequencing was done in only selected patients. Third, the clinical evaluation did not include visual evoked potentials in all cases. Lastly, not all subjects were available for clinical examination or consented to molecular analysis. To conclude, in five families with foveal hypoplasia and nystagmus carrying SLC38A8 gene mutations, similar biallelic mutations were identified in two distinct populations of Indian and Karaite Jewish origins. A possible partial expression in a heterozygous patient is seen and new potential disease-related phenotypes, along with a novel mutation of the SLC38A8 gene are described. These results could help improve our understanding of the relationship between the SLC38A8 gene with foveal hypoplasia and other ocular and systemic manifestations with potential insights into future genetic counseling.
Fig. 6. Sanger sequence chromatogram of c.490_491delCT; p.L164Vfs*41. Reference sequence of SLC38A8 (ENSE00001104170) is represented at the top. Middle chromatogram is the wild type (wt) sequence. At the bottom, is the patient (Family #5 RP0057) sequence, demonstrating double base deletion resulting in a complete CDS frame-shift followed by stop codon 41 amino acids downstream.
Funding Funding of this study was supported by the Claire and Amedee Maratier Institute for the Study of Blindness and Visual Disorders (TAU#: 32003197000); The Ernest And Nusia Gothelf research funds (TAU#: 32003125000), Sackler Faculty of Medicine, TelAviv University, Tel-Aviv, Israel. This work was also supported in part by grant no. 7205 from the Chief Scientist Office of the Ministry of Health, Israel; ‘Lirot’ Association and the Consortium for Mapping Retinal Degeneration Disorders in Israel, and by Grants BR-GE-02140639-TECH and BR-GE-0518-0734-TECH from the Foundation Fighting Blindness, Grant 3-12583 from the Israeli Ministry of Health. This work was performed in partial fulfillment of the requirements for a Ph.D. degree by Chen Weiner at the Sackler Faculty of Medicine, Tel Aviv University, Israel.
that other extraocular manifestations exist affecting verbal and developmental aspects. Another interesting observation is the evidence of early-onset AMD seen in patient NY0048 (Family #4, Fig. 5), which was first documented at the age of 60 years. AMD is typically seen above the age of 75 years and the prevalence at the age of 60 years has been estimated to be only between 0.1% and 0.4% (Lambert et al., 2016). This finding is not only linked to the same tissue shown to be affected by the SLC38A8 gene, but the majority of previously reported cases were young and so this phenotype could have been missed. This study has several limitations. First, it includes only five families, all Jewish origins and so clinical and molecular findings may differ from other populations. Second, Whole Genome Sequencing 6
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Declaration of competing interest
H., 2017. Foveal hypoplasia in patients with stickler syndrome. Ophthalmology 124 (6), 896–902. Myerowitz, R., 1997. Tay-Sachs disease-causing mutations and neutral polymorphisms in the Hex A gene. Hum. Mutat. 9 (3), 195–208. Oliver, M.D., Dotan, S.A., Chemke, J., Abraham, F.A., 1987. Isolated foveal hypoplasia. Br. J. Ophthalmol. 71 (12), 926–930. Parfitt, T., 2002. The Lost Tribes of Israel : the History of a Myth. Weidenfeld & Nicolson, London. Perez, Y., Gradstein, L., Flusser, H., Markus, B., Cohen, I., Langer, Y., Marcus, M., Lifshitz, T., Kadir, R., Birk, O.S., 2014. Isolated foveal hypoplasia with secondary nystagmus and low vision is associated with a homozygous SLC38A8 mutation. Eur. J. Hum. Genet. 22 (5), 703–706. Pochini, L., Scalise, M., Galluccio, M., Indiveri, C., 2014. Membrane transporters for the special amino acid glutamine: structure/function relationships and relevance to human health. Front. Chem. 2, 61. Polliack, M., 2003. Karaite Judaism : a Guide to its History and Literary Sources. Brill, Leiden ; Boston. Poulter, J.A., Al-Araimi, M., Conte, I., van Genderen, M.M., Sheridan, E., Carr, I.M., Parry, D.A., Shires, M., Carrella, S., Bradbury, J., et al., 2013. Recessive mutations in SLC38A8 cause foveal hypoplasia and optic nerve misrouting without albinism. Am. J. Hum. Genet. 93 (6), 1143–1150. Pras, E., Kochba, I., Lubetzky, A., Pras, M., Sidi, Y., Kastner, D.L., 1998. Biochemical and clinical studies in Libyan Jewish cystinuria patients and their relatives. Am. J. Med. Genet. 80 (2), 173–176. Pras, E., Kristal, D., Shoshany, N., Volodarsky, D., Vulih, I., Celniker, G., Isakov, O., Shomron, N., Pras, E., 2015. Rare genetic variants in Tunisian Jewish patients suffering from age-related macular degeneration. J. Med. Genet. 52 (7), 484–492. Procopio, V., Manti, S., Bianco, G., Conti, G., Romeo, A., Maimone, F., Arrigo, T., Cutrupi, M.C., Salpietro, C., Cuppari, C., 2018. Genotype-phenotype correlation in FMF patients: a "non classic" recessive autosomal or "atypical" dominant autosomal inheritance? Gene 641, 279–286. Shields, R.A., Cavuoto, K.M., McKeown, C.A., Chang, T.C., 2015. Unilateral foveal hypoplasia in a child with bilateral anterior segment dysgenesis. Clin. Case Rep. 3 (7), 676–678. Stanford Libraries University of stanford. [cited 2018 15/12]. Available from: https:// library.stanford.edu/spc/exhibitspublications/past-exhibits/karaite-jews-egyptisrael-and-san-francisco-bay-area. Toral, M.A., Velez, G., Boudreault, K., Schaefer, K.A., Xu, Y., Saffra, N., Bassuk, A.G., Tsang, S.H., Mahajan, V.B., 2017. Structural modeling of a novel SLC38A8 mutation that causes foveal hypoplasia. Mol. Genet. Genom. Med. 5 (3), 202–209. Tsaras, G., Owusu-Ansah, A., Boateng, F.O., Amoateng-Adjepong, Y., 2009. Complications associated with sickle cell trait: a brief narrative Review. Am. J. Med. 122 (6), 507–512.
The authors declare that they have no conflicts of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.exer.2020.107958. References Al-Araimi, M., Pal, B., Poulter, J.A., van Genderen, M.M., Carr, I., Cudrnak, T., Brown, L., Sheridan, E., Mohamed, M.D., Bradbury, J., et al., 2013. A new recessively inherited disorder composed of foveal hypoplasia, optic nerve decussation defects and anterior segment dysgenesis maps to chromosome 16q23.3-24.1. Mol. Vis. 19, 2165–2172. Astren, F., 2004. Karaite Judaism and Historical Understanding. University of South Carolina Press, Columbia. Azuma, N., Nishina, S., Yanagisawa, H., Okuyama, T., Yamada, M., 1996. PAX6 missense mutation in isolated foveal hypoplasia. Nat. Genet. 13 (2), 141–142. Campbell, P., Ellingford, J.M., Parry, N.R.A., Fletcher, T., Ramsden, S.C., Gale, T., Hall, G., Smith, K., Kasperaviciute, D., Thomas, E., et al., 2019. Clinical and genetic variability in children with partial albinism. Sci. Rep. 9 (1). Dorey, S.E., Neveu, M.M., Burton, L.C., Sloper, J.J., Holder, G.E., 2003. The clinical features of albinism and their correlation with visual evoked potentials. Br. J. Ophthalmol. 87 (6), 767–772. Hagglund, M.G.A., Hellsten, S.V., Bagchi, S., Philippot, G., Lofqvist, E., Nilsson, V.C.O., Almkvist, I., Karlsson, E., Sreedharan, S., Tafreshiha, A., et al., 2015. Transport of Lglutamine, L-alanine, L-arginine and L-histidine by the neuron-specific Slc38a8 (SNAT8) in CNS. J. Mol. Biol. 427 (6 Pt B), 1495–1512. Jiang, Y., Wang, R., Urrutia, E., Anastopoulos, I.N., Nathanson, K.L., Zhang, N.R., 2018. CODEX2: full-spectrum copy number variation detection by high-throughput DNA sequencing. Genome Biol. 19 (1). Lambert, N.G., ElShelmani, H., Singh, M.K., Mansergh, F.C., Wride, M.A., Padilla, M., Keegan, D., Hogg, R.E., Ambati, B.K., 2016. Risk factors and biomarkers of age-related macular degeneration. Prog. Retin. Eye Res. 54, 64–102. Livneh, A., Aksentijevich, I., Langevitz, P., Torosyan, Y., G-Shoham, N., Shinar, Y., Pras, E., Zaks, N., Padeh, S., Kastner, D.L., et al., 2001. A single mutated MEFV allele in Israeli patients suffering from familial Mediterranean fever and Behcet's disease (FMF-BD). Eur. J. Hum. Genet. 9 (3), 191–196. Matsushita, I., Nagata, T., Hayashi, T., Kimoto, K., Kubota, T., Ohji, M., Kusaka, S., Kondo,
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