Novel pathogenic variants of SLC38A8 gene and literature review

Purpose This study aimed to analyze the clinical and genetic characteristics of 6 Chinese Han patients with foveal hypoplasia (FH) caused by the variants of solute carrier family 38 member 8 (SLC38A8) gene, and to describe the genotype and phenotype of SLC38A8 gene variants from previous literature. Methods All subjects underwent comprehensive ophthalmic examinations including slit lamp microscope, fundoscopy, and retinoscopy refraction. Optical coherence tomography (OCT) was performed to evaluate the structural grade of foveal hypoplasia. Pathogenic variants of SLC38A8 gene were identi�ed using panel-based next-generation sequencing, direct Sanger sequencing, and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) techniques. Further, all previously reported cases of SLC38A8 variants were re-analyzed together with the novel ones identi�ed in this study. Results Nystagmus and FH were present in 6 patients with variants of SLC38A8 gene, accompanied by a normal anterior segment. Grade 4 FH was identi�ed in 4 patients who could cooperate with the OCT scan. A total of 12 variants of SLC38A8 gene were identi�ed, including 9 novel variants. The missense variants were predicted to be pathogenic by the online programs. Systematical analysis revealed that half of the variants (30/60) were missense, and the majority of which (23/30) were distributed in the transmembrane (TM) domains. Grade 4 FH was detected in the majority of patients (66%, 23/35), and anterior segment dysgenesis (ASD)was found in 16.5% of patients (15/91). There was no statistical difference in the clinical features between the subgroups of patients with 0, 1 and 2 missense variants. Conclusion Severe arrest of foveal development was identi�ed in patients with variants of SLC38A8. The novel identi�ed variants may expand the spectrum of pathogenic variants of SLC38A8. This study summarized the phenotypic and genotypic characteristics of SLC38A8variants, which would help the FH patients with early diagnosis.


Introduction
The solute carrier family 38 member 8 (SLC38A8) gene is located on the chromosome 16q23.3,and it is composed of 11 exons, encoding a protein of putative sodium-dependent amino-acid/proton antiporter.The SLC38A8 protein consists of 11 transmembrane domains, and it is a member of the SLC38 sodium-coupled neutral amino acid transporter family proteins.It is mainly expressed in the central nervous system and retina, which plays an important role in foveal development.Variations of SLC38A8 gene may result in foveal hypoplasia (FH) with or without optic nerve misrouting and/or anterior segment dysgenesis (ASD) [1].
To date, 51 variants of SLC38A8 gene have been reported to be associated with FH (http://www.hgmd.cf.ac.uk/ ac/index.php).The present study aimed to describe the clinical characteristics of 6 Chinese patients with FH due to pathogenic variants of SLC38A8 gene, and to analyze these variants together with those previously reported in the literature in an attempt to nd the spectrum of SLC38A8 variation, and to summarize the clinical and genetic characteristics.

Methods
Patients: A total of 6 Han Chinese patients (P01~P06) with SLC38A8 variants were involved in this study.All patients underwent careful evaluation of their eyes, including their best corrected visual acuity (BCVA), color vision, the anterior segment, and fundus of each eye.Cycloplegic refraction retinoscopy was examined by certi cated optometrists and measured in diopters (D).Fundus photography was carried out using Canon CX-1 (Canon, Tokyo, Japan) or Retcam III (RetCam III Imaging System; Clarity Medical Systems Inc., Pleasanton, CA, USA).Optical coherence tomography (OCT) (Heidelberg Engineering, Munich, Germany) was conducted to evaluate the retinal structure.FH is classi ed into grade 1~4 according to the Leicester Grading System [2].Full-eld electroretinography (ERG) was carried out using the RETI-port-scan 21 system (Roland Consult GmbH, Brandenburg, Germany) according to the standards of the International Society for Clinical Electrophysiology of Vision (http://www.iscev.org).Genetic testing was performed on all patients.The study was approved by the Ethics Committee of Beijing Children's Hospital (Beijing, China), and it was conducted in accordance with the Declaration of Helsinki.Informed consent was obtained from all the parents or guardians for the study, in accordance with the guidelines on sample collection for human genetic diseases issued by the Ministry of Public Health of China.
Genetic testing: In the present study, 2 milliliters peripheral blood was collected from each participant.Genomic DNA was extracted using the QIAamp Blood Midi kit (QIAGEN, Heidelberg, Germany) according to the manufacture's standard protocols.The panel-based next-generation sequencing was carried out by Mygenomic Biochemical Co., Ltd.(Beijing, China).The constructed DNA library was sequenced on an Illumina HiSeq X Ten platform (Illumina Inc., San Diego, CA, USA), and the captured raw data were mapped to the Genome Reference Consortium Human genome build 38 (GRCh38).
The effects of missense variants on protein stability were predicted by DynaMut2 program (http://biosig.unimelb.edu.au/dynamut2).Splice-site variations were analyzed by the SpliceAI program [3].Assessment of pathogenicity was based on the ACMG/ AMP guidelines(American College of Medical Genetics and Genomics/Association for Molecular Pathology) [4] Mutations were named following the nomenclature recommended by the Human Genomic Variation Society (HGVS) [5].
Quantitative reverse transcription-polymerase chain reaction (RT-qPCR): In the present study, RT-qPCR was employed to explore a large deletion of exons1-6 in SLC38A8 gene.Ampli cation was performed in a 20-μL volume of PCR buffer, containing 5 ng of DNA, 10 µM of each primer, and 10 µL of ChamQ Universal SYBR qPCR Master Mix (#Q711; Vazyme Co., Ltd., China).RT-qPCR was performed by an ABI 7500 Real-Time PCR system (Applied Biosystems, Waltham, MA, USA).The products were calculated using the comparative Ct method (ΔΔCt), with an equation of 2 -ΔΔCt , and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference gene.The primers used for RT-qPCR are listed in Supplementary Table 1.
Literature review: Literature search was carried out using Google Scholar, PubMed, OMIM and Web of Science databases to retrieve articles related to mutations of SLC38A8 gene.
Statistical analysis: Statistical analysis was performed using SPSS software 22.0 (IBM, Armonk).Medians and ranges were used for continuous variables.Frequencies and percentages were used for categorical variables.Kruskal-Wallis test was used to compare skewed distributed variables between groups.Fisher's test was used to compare the categorical variables in different groups.If the P value <0.05, the difference was considered to be statistically signi cant.

Results
Clinical data: In the present study, 4 boys and 2 girls aged 1-8 years old were included.They were referred to Beijing Children's Hospital for nystagmus that was developed in the rst 3~6 months after birth.The color of their skin, hair, and iris was normal.Five patients (P01~P05) underwent refractive examination.Besides, P01 and P04 patients had compound hyperopic astigmatism, and P02 and P03 patients had mixed astigmatism; P05 patient had a high myopia of more than -6.0D.Four patients (P01~P04) contributed actively to color vision tests and visual acuity, and showed normal color vision and reduced visual acuity that ranged from 0.52 to 0.69 logMAR.Furthermore, P01, P02, and P04 patients had strabismus, with exotropia in P02 and P04 patients and esotropia in P01 patient.Detailed data are listed in Table 1.FH was found in all patients (Fig. 1), accompanied by a normal anterior segment.Tessellated fundus was found in 4 patients (P01~03, and P05), and their choroidal vessels were visible in the mid-periphery of the retina.The structure of the retina was evaluated using OCT in P01, P02, P04, and P05 patients, and FH grade 4 was found based on the diagnostic criteria of the Leicester Grading System (Fig. 2a), with the absence of the extrusion of plexiform layers, foveal pit, outer nuclear layer (ONL) widening, and outer segment (OS) lengthening.ERG recordings were performed on P01 patient, which showed normal a-and b-wave under the scotopic and photopic conditions (Fig. 2b), indicating that both rod and cone photoreceptor cells had normal function.
Variant analysis: As an autosomal recessive disorder, 6 compound heterozygous variants were detected in the patients (Fig. 3).There were 12 distinct variants, of which 9 variants were novel and 3 variants were previously reported (Table 2).In addition,  The missense variants were predicted to be pathogenic by the online programs (Table 2).Multiple sequence alignments of protein showed that all wildtype amino acids were highly conserved in the different species from Danio to humans (Fig. 4a~e).Gibbs free energy gap difference (ΔΔG) between the mutant (ΔGm) and wild type (ΔGw) protein indicated negative changes and predicted to be destabilizing (Fig. 4f).
The nonsense variant of c.1025C>A (p.Ser342*) may produce an abnormal mRNA with a premature terminate codon (PTC) which may be degraded due to the nonsense-mediated decay (NMD) mechanism.While, the variant of c.1306T>C (p.Ter436ArgextTer34) may lead to an extension of mRNA and produce an abnormal protein which can either be degraded or gain an abnormal function because of a large protein.
The large deletion in P05 patient included the entire exons 1 to 6, with the start codon located within it.RT-qPCR indicated the loss of heterozygosity of exon1 and exon6 in patient and her mother (Fig. 3e).The removal of the start codon may result in the absence of translation or translation starting at a cryptic site downstream, leading to a truncated protein without the N-terminal region.
Abbreviations: DC, Disease causing; D, Damaging; PD, Probably damaging; LP, Likely pathogenic; VUS, variant of uncertain signi cance Spectrum analysis and clinical review of SLC38A8 variants: A total of 60 pathogenic variants of SLC38A8 were detected in 70 families with FH in previous reports and the present study (Supplementary Table 2).Half of families (35/70) carried a compound heterozygous variant, while the others carried a homozygous variant.Fig. 5a schematically shows SLC38A8 protein and locations of variants of amino acids.Half of variants (30/60) were missense, followed by nonsense (8/60) and frameshift (8/60), deletion (8/60), splicing (5/60), and C-termination extension (1/60) (Fig. 5b).It is noteworthy that the majority of missense variants (23/30) were distributed in the transmembrane domains (TMs) of the encoded protein, of which 9 variants were located in the TM6.The nonsense variants were mainly distributed in the domains between the transmembrane regions.The p.Glu233Lys variant was recurrent in different patients, suggesting that it could be a mutational hotspot.
In order to further analyze the correlation between the genotype and the phenotype, we divided the 98 patients into three groups according to the number of missense variants (Table 3).Fifty-seven patients had 2 missense variants, 20 patients with 1 missense variant, and 21 patients had no missense variant.In these three groups, there was no statistical difference in the prevalence of ASD, strabismus, chiasmal misrouting or the FH of grade4.Patients without a missense variant showed a better visual acuity compared to patients with 1 or 2 missense variants.However, this difference was not statistically signi cant.

Discussion
In the present study, the clinical and genetic characteristics of 6 Chinese patients with FH caused by variants of SLC38A8 were described.We identi ed 12variants of SLC38A8 gene in these patients, including 9 novel variants.In addition, we reviewed the mutation spectrum of SLC38A8 gene, and systematically analyzed the phenotypes and genotypes based on the data presented in this study and previous literature.
As one member of the SLC38 sodium-coupled neutral amino acid transporter (SNAT) family, SLC38A8 was expressed in the neural retina [1], and SLC38A8 expression level was present in the cone-enriched organoid differentiation expression data[8], suggesting that SLC38A8 may play an important role in foveal development.SLC38A8 has a high preference for transporting glutamate and gamma aminobutyric acid (GABA) [9], while the GABA/GABAergic dysregulation exhibited to have a signi cant effect on axonal projection or orientation selectivity in retina [10,11].However, the molecular mechanisms are still unclear.Biallelic variants of SLC38A8 gene may cause autosomal recessive FH with or without ASD.To our knowledge, only 5 patients with variants of SLC38A8 gene have yet been reported in Chinese population [12].
In the present study, all the 6 patients had compound heterozygous variants.A total of 12 variants of SLC38A8 gene were identi ed, of which 9 were novel, which signi cantly extended the genotypic heterogeneity.Importantly, 6 missense sites were localized in the TM regions, and other two sites were very near to the TM regions (extracellular domain between the TM4 and TM5).Moreover, analysis of previous studies revealed that the most missense variants were also localized in TM regions, and TM6 was the hotspot of missense variants, in which 8 missense variants were reported [1,6,[12][13][14].The locations of these missense variants may be critical for the function of the protein.These variants were predicted to be pathogenic by potentially altering the connectivity with other amino acids, may resulting in disruption of glutamine transport.The nonsense variants were mainly distributed in the domains between the transmembrane regions.The novel nonsense, splicing, stop-loss, and large deletion identi ed in 3 patients were all predicted to be pathological.However, the exact mechanism of the pathogenicity of these variants needs additional functional studies.
Variants of SLC38A8 gene were reported to be responsible for the FHONDA syndrome [1], consisting of FH, optic nerve decussation defects, and ASD [15].However, in the present study, not all the patients ful ll the above clinical phenotypes.Six patients in the present study exhibited phenotypic homogeneity, including congenital nystagmus, FH, and reduced BCVA.Severe FH (grade 4) and poor cone PRS (absence of widening of ONL and lengthening of OS) were identi ed in 4 patients, indicating more severe arrest of foveal development and worse visual prognosis in patients with variants of SLC38A8 gene.A previous study indicated that the extent of cone PRS was associated with BCVA [16].These results had signi cant diagnostic and prognostic value and may facilitate prioritization of genetic testing of SLC38A8 in patients with poor cone PRS and low BCVA [17].
FH accompanied by misrouting optic nerve was mainly considered as the feature of albinism, which may be attributed to the defect of melanin biosynthesis.Nevertheless, in variants of SLC38A8, FH and optic nerve decussation defect may occur independently without abnormal melanin synthesis.In the most of previous studies, the skin, hair, iris, and retina of patients had no sign of hypopigmentation [14,18,19], indicating that SLC38A8 may have no effect on melanin biosynthesis.Furthermore, Poulter et al. demonstrated that pigmentation was unaffected in the eye of embryonic Medaka sh using morpholino-mediated ablation of Slc38a8 [1], thus, variants of SLC38A8 gene and albinism might cause different diseases.
However, in the present study, tessellated fundus was identi ed in 4 patients and the choroidal vessels could be found in midperiphery.Besides, P02 patient was clinically initially diagnosed with ocular albinism in the clinic, due to the grade 4 FH and hypopigmentation in the mid-peripheral fundus.
Tessellated fundus was also described in variants of SLC38A8 in another Chinese study [12].Moreover, SLC38A8 genewas hypothesized to function in the downstream of GPR143 gene, as patients consistently exhibited a severe albinism-like retinal phenotype [20].Importantly, ASD was also found in patients with ocular albinism [21,22], and Michaelides et al. speculated that FHONDA syndrome may be a type of mild albinism [23].It is noteworthy that variants of GPR143 gene in Chinese population can present untypical signs of hypopigmentation in iris and retina.Therefore, the detailed ocular examination, inheritance patterns, and molecular con rmation may be advantageous for a more accurate diagnosis for patients with FH.
The clinical analysis of all patients revealed that ASD was identi ed in 16.5% of patients with the variantsof SLC38A8 gene.SLC38A8 transcript was also shown differentially expressed in the corneal endothelium and stem cells derived lens, indicating that SLC38A8 gene may regulate the development of anterior segment [24].However, ASD was not identi ed in 6 patients in the present study.Moreover, the prevalence of embryotoxon/Axenfeld's anomaly in the normal population was 7-32% [25,26], suggesting that ASD was not a frequent abnormality in this disorder.Hence, the relationship between the incidence of ASD and variants of SLC38A8 gene remains elusive.In addition, the present study investigated the genotype-phenotype correlation with regard to the prevalence of ASD, strabismus, chiasmal misrouting and the grade 4 FH between the subgroups of patients with 0, 1 and 2 missense variants.The results demonstrated that the correlation between the genotype and the phenotype remains unclear, needing more research.

Conclusion
The present study described the clinical characteristics of FH patients associated with variants of SLC38A8 gene and expanded the mutational spectrum of this disorder in Chinese population.It would be necessary to screen SLC38A8 gene in cases with isolated FH, due to the similar phenotype to variants of GPR143 gene, and the results suggested a common mechanism in the macular development of these variants.These results assist scholars to explore the relationship between the FH and SLC38A8 gene.
One limitation of the present study was that the pathogenicity of new variants was predicted by bioinformatic tools, and functional studies were not been performed.

Declarations Statements and Declarations
Funding No funds, grants or other support was received.

Table 2
Analysis of identi ed SLC38A8 variations in six Chinese patients

Table 3
Clinical features of 98 patients in different groups according to the number of missense variants