Reduced cone photoreceptor function and subtle systemic manifestations in two siblings with loss of SCLT1

ABSTRACT

Background

The sodium channel and clathrin linker 1 gene (SCLT1) has been involved in the pathogenesis of various ciliopathy disorders such as Bardet-Biedl syndrome, orofaciodigital syndrome type IX, and Senior-Løken syndrome. Detailed exams are warranted to outline all clinical features. Here, we present a family with a milder phenotype of SCLT1-related disease.

Material and Methods

Comprehensive eye examination including fundus images, OCT, color vision, visual fields and electroretinography were performed. Affected individuals were assessed by a pediatrician and a medical geneticist for systemic features of ciliopathy. Investigations included echocardiography, abdominal ultrasonography, blood work-up for diabetes, liver and kidney function. Genetic testing included NGS retinal dystrophy panel, segregation analysis and transcriptome sequencing.

Results

Two male children, age 10 and 8 years, were affected with attention deficit hyperactivity disorder (ADHD), obesity and mild photophobia. The ophthalmic exam revealed reduced best-corrected visual acuity (BCVA), strabismus, hyperopia, astigmatism and moderate red-green defects. Milder changes suggesting photoreceptors disease were found on retinal imaging. Electroretinogram confirmed cone photoreceptors dysfunction. Genetic testing revealed a homozygous likely pathogenic, splice-site variant in SCLT1 gene NM_144643.3: c.1439 + 1del in the proband and in the affected brother. The unaffected parents were heterozygous for the SCLT1 variant. Transcriptome sequencing showed retention of intron 16 in the proband.

Conclusions

In this report, we highlight the importance of further extensive diagnostics in patients with unexplained reduced vision, strabismus, refractive errors and ADHD spectrum disorders. SCLT1-related retinal degeneration is very rare and isolated reduced function of cone photoreceptors has not previously been observed.

KEYWORDS:

• SCLT1
• retinal degeneration
• cone dysfunction
• ciliopathy
• Bardet-Biedl syndrome

1. Introduction

The sodium channel and clathrin linker 1 gene (SCLT1), located on human chromosome 4q28.2, has been reported in the pathogenesis of ciliopathy disorders such as Bardet-Biedl syndrome (BBS) (OMIM # 209900) (1–4), orofaciodigital syndrome type 9 (5) (OMIM # 258865) and Senior-Løken syndrome (OMIM # 266900) (6).

SCLT1 encodes a linker protein associated with the sodium voltage-gated channel alpha subunit 10 and clathrin, which is a core protein involved in ciliogenesis (7). Specifically, SCLT1 is one of the components of the distal appendages (DAPs) of centrioles (7), that anchor the cilia to the plasma membrane. Other DAP components have also been linked to ciliopathy diseases, and similarly to SCLT1, the affected individuals may show different clinical features. For instance, impaired function of CEP164 has been shown to cause nephronophthisis with or without retinal degeneration (OMIM # 614845) (8), but also was reported in BBS (9). Further, alterations in CEP83, which were first described to cause nephronophthisis and intellectual disability (OMIM # 615862) (10), later were shown to cause isolated retinitis pigmentosa (RP) (11).

Ciliopathies are mostly autosomal recessive conditions caused by pathogenic variants in a wide range of genes coding for proteins responsible for ciliogenesis, cilia structure and/or function (12–14). These disorders can be both syndromic and non-syndromic, in which retinal degeneration (RD) is a cardinal clinical feature (15,16). The spectrum of disorders ranges from isolated RD, such as Leber congenital amaurosis due to pathogenic variants in CEP290 (17–19), to complex syndromes with multiple organ dysfunction such as BBS characterised by a combination of RD, obesity, polydactyly, hypogonadism, kidney disease and learning difficulties (20–25). Other examples of syndromic ciliopathies are Alström syndrome (OMIM #203800) (26–30), Senior-Løken syndrome (31–34) and orofaciodigital syndrome (35,36) among others. Furthermore, ciliopathies with milder clinical features have been also recently reported (37–39).

To date, only few patients with SCLT1-related retinal degeneration of RP type have been reported (1,2,6). Here, we report two siblings affected with ADHD, obesity, refractive error, esotropia and unexplained reduced vision, and homozygous splice variants in SCLT1.

2. Material and methods

2.1. Clinical assessments

The study adhered to the tenets of the Declaration of Helsinki. Legal guardians have given written consent to participate in this study. The proband (III-1) and his sibling (III-2) underwent comprehensive eye examinations (St. Erik Eye Hospital, Stockholm, Sweden), pediatric assessment (community-based pediatrician), and genetic testing (Department of Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden). The parents (II-2 and II-1) underwent eye exams and genetic testing. Ophthalmological evaluations included: best corrected visual acuity (BCVA), color vision test (HRR), cycloplegic refraction, ophthalmoscopy, Goldman visual fields, fundus photography (Optos Nikon, Tokyo, Japan), optical coherence tomography (Topcon OCT), full field electroretinogram (ERG, Diagnosys, LLC, Lowell, MA, USA) according to the ISCEV standards (40). Pediatric evaluation included comprehensive clinical exam and ancillary tests, such as echocardiography, ultrasonography of abdomen, blood morphology and blood tests to assess liver function (ASAT, ALAT, lipid profile), kidney function (creatinine, electrolytes), glucose and HbA1c. The children were referred for psychological assessment of cognitive function.

2.2. Genetic analysis

The proband (patients III-1) was analysed at the Department of Clinical Genetics, Karolinska University Hospital with massive parallel sequencing of a retinal dystrophy panel of 285 genes (Table S1). Testing of patient III-2 as well as the healthy parents (II-1 and II-2) was performed with polymerase-chain reaction (PCR) and Sanger sequencing of the SCLT1 (NM_144643.3) variant identified. Primers are available on request.

Effects on RNA were evaluated by whole transcriptome sequencing on RNA isolated from blood of the proband according to standard protocols. Briefly, total RNA was isolated and library preparation was done by the Illumina mRNA stranded method with sequencing on Nova Seq S4. The resulting data was aligned to reference genome using the Spliced Transcripts Alignment to a Reference (STAR). The resulting alignment were visualized in Integrated Genomics Viewer (IGV) to detect aberrant splicing.

3. Results

3.1. Patient III-1 (proband)

The male child of Middle East origin from consanguineous parents, who were second cousins, (Figure 1) presented at one and a half years of age at the eye clinic (St. Erik Eye Hospital) due to left esotropia. Cycloplegic refraction revealed significant hyperopia and fundus examination was normal. Between 6- and 8-years of age, vision loss and abnormal foveal reflex were observed (Table 1, Figure 2a). Nystagmus was not present. Extensive diagnostics revealed mild increased autofluorescence in the posterior pole, and abnormalities in the outer nuclear layer corresponding to photoreceptors (Figure 2a). The patient had moderate colour vision defects. Full field ERG showed normal function of rod photoreceptors and reduced function of cone photoreceptors (Figure 2a, Table S2). Systemic manifestations included well-controlled obesity and ADHD-spectrum disorders. Dysmorphic features or polydactyly were not present. Despite mild phenotype (reduced function of cone photoreceptors, obesity and ADHD), ciliopathy was suggested as a possible disease causing mechanism. The proband was referred for comprehensive pediatric evaluation regarding ciliopathy (Table 1). A psychological assessment of cognitive function was normal. Genetic testing with a retinal dystrophy gene panel (Table S1) revealed a homozygous splice variant in SCLT1 gene NM_144643.3: c.1439 + 1del, which is novel (Figure 1). Segregation analysis in the affected younger brother showed the same genotype, and the unaffected parents were heterozygous carriers. The proband was assessed clinically and obtained genetic counselling by a medical geneticist at the Karolinska University Hospital.

Figure 1. Pedigree and segregation analysis. Proband (III-1) and affected younger brother (III-2) are homozygous for splice-site variant in SCLT1 gene NM_144643.3: c.1439 + 1del while parents (II-1 and II-2) are heterozygous. The arrow depicts proband, horizontal lines—parents examined clinically. Black squares: affected male individuals. Double horizontal lines: consanguinity.

Figure 2a. Ophthalmological phenotype (Patient III-1). (a): fundus images and (b): OCT shows increased autofluorescence signal in the posterior pole and disruptions in the outer nuclear layer corresponding to photoreceptors; (c): Goldman visual fields (tested to II4E isopter) present well- preserved fields; (d): upper panel—full filed ERG shows reduced function of cone photoreceptors with preserved function of rod photoreceptors, lower panel—age-adjusted normative data.

Table 1. Clinical features.

Ocular features	III-1 (age 10 years)	III-2 (age 8 years)
BCVA (logMAR)	0.1 OD/0.2 OS (4 year of age) 0.2 OD/0.4 OS (6 year of age) 0.4 OD/0.5 OS (8–10 year of age)	0.3 OD/OS (5–8 year of age)
Cycloplegic refraction	+8.0 OD/OS (1.5 year of age) +7.5–2.0 × 130 OD/+7.5–2.0 × 30 OS (6–10 year of age)	+4.0–1.0 × 90 OD/OS (5 year of age) +5.0–2.0 × 90 OD/OS (8 year of age)
Eye alignment	Left esotropia	Esoforia
Color vision (HRR)	Moderate red-green defect	Moderate red-green defect
Fundus	Abnormal foveal reflex	Abnormal foveal reflex
FAF	Decreased central hypo-FAF, surrounded by diffuse increase of FAF	Decreased central hypo-FAF, surrounded by diffuse increase of FAF
OCT macula	Retinal thinning, CST 168 µm, thickening of ELM, abnormal ONL	Retinal thinning, CST 167 µm, thickening of ELM, abnormal ONL
Goldman Visual fields	Well-preserved	Not performed
ERG	Cone dysfunction	Cone dysfunction
Systemic findings:
ADHD/autism	Yes	Yes
Polydactyly	No	No
Obesity	Yes (well-controlled)	Yes (well-controlled)
Diabetes	No	No
Echocardiography	Normal	Normal
Ultrasonography abdomen	Normal (liver, kidneys)	Normal (liver, kidneys)
ASAT, ALAT	Normal	Normal
Cholesterol, TG, LDL, HDL	Normal	Normal
Creatinine	Normal	Normal

Legend: III-1: proband; III-2: affected younger brother; BCVA: best corrected visual acuity (logMAR); OD: right eye; OS: left eye; HRR: Hardy Rand and Rittler pseudoisochromatic test; FAF: fundus autofluorescence; CST: central subfield thickness; ELM: external limiting membrane; ONL: outer nuclear layer; ERG: electroretinogram; ADHD: Attention deficit hyperactivity disorder; ASAT: aspartate aminotransferase; ALAT: alanine aminotransferase; TG: triglycerides; LDL: low-density lipoprotein; HDL: high-density lipoprotein.

The homozygous splice-site variant in SCLT1 gene (c.1439 + 1del) (Figure 1) was novel (not present in ClinVar (41) or gnomAD (42)) and affected the 5’ canonical splice site of exon 16 at the first position of intron 16. Splicing predictions in Alamut Visual Plus v1.2 (Sophia genetics, Boston, USA) predicted a loss of donor site with score 100%. Transcriptome sequencing of the proband verified the presence of aberrant splicing, with retention of intron 16 sequences (Figure 3). Translation of this aberrant transcript is predicted to result in a protein with a pre-mature stop codon 29 amino acids downstream of p.D480.

Figure 3. DNA and RNA sequencing results. A) Electropherogram from genomic DNA sequence in parent II-1 (top) and proband III-1 (bottom). Arrow indicates heterozygote position in parent, and deletion in proband. B) Sashimi-plot from transcriptome sequencing showing region Exon15 to exon 17 in SCLT1-gene. Top trace in red represents proband III-1, and traces in blue and green, are normal controls with wild-type genotype.

3.2. Patient III-2

The proband’s younger brother was referred to the eye clinic at the age of 5 years due to reduced vision. His eye exam revealed esophoria, and cycloplegic refraction showed moderate hyperopia and astigmatism (Table 1). His vision remained stable over the course of 3 years. Comprehensive eye exam including structural and functional retinal parameters showed similar results to his brother (Patient III-1), however Patient III-2 was affected to a lesser extent (Figure 2b, Table S2). Similarly to his brother, this patient also underwent an extensive pediatric evaluation regarding ciliopathy, which was unremarkable except for ADHD and well-controlled obesity as seen in the proband. DNA sequencing revealed the same homozygous SCLT1 splice variant as observed in the proband (Figure 1).

Figure 2b. Ophthalmological phenotype (Patient III-2). (a): fundus images and (b): OCT shows increased autofluorescence signal in the posterior pole and disruptions in the outer nuclear layer corresponding to photoreceptors; (c): upper panel—full filed ERG presents reduced function of cone photoreceptors with preserved function of rod photoreceptors, lower panel—age-adjusted normative data.

3.3. Patient II-1 and II-2

The 39-year-old father (II-1) and 38-year-old mother (II-2) did not report any clinical symptoms, and their eye exams were normal. Segregation analysis confirmed that both parents were heterozygous carriers of pathogenic splice-site variant in SCLT1 (Figure 1).

4. Discussion

We report two siblings from a consanguineous family affected with SCLT1-related disease presenting with refractive errors, strabismus, reduced function of cone photoreceptors, ADHD and obesity. Compared to previous reports, the affected individuals in the family presented here have milder systemic and ocular phenotype.

4.1. The SCLT1-related condition is a very rare form of ciliopathy and presents with heterogenous phenotype

Animal studies showed that Sclt1-/- mice exhibit typical ciliopathy phenotypes, including cystic kidney, cleft palate and polydactyly (43).

The first published case with pathogenic variants in SCLT1 gene was a severely affected male child diagnosed with orofaciodigital syndrome type 9 (OMIM #258865) (5) who passed away at the age of 3 months due to a cardiorespiratory arrest. Since then, six individuals from five families with pathogenic SCLT1 variants have been reported. The clinical picture was variable with all individuals displaying retinal symptoms and many systemic features, such as liver and kidney disease, cognitive impairment, hypogonadism, obesity and polydactyly (Table 2) (1,2,4,6). In contrast, the two siblings presented here had mild systemic features, such as ADHD and obesity (Table 1).

Table 2. Clinical features of individuals with pathogenic variants in SCLT1.

Publication	Clinical diagnosis	Gender	Age of last exam	SCLT1 variant NM_144643.4	Ocular symptoms	Systemic manifestations
Adly et al., 2014	OFD	M	3 months	Homozygote c.290 + 2T>C	bilateral coloboma	severe midline cleft lip, microcephaly, agenesis of corpus callosum, pachygyria, abnormal genitalia, congenital heart disease
Katagiri et al., 2018	SLS	F	6 years	Compound heterozygote c.1218 + 4dup c.1631A>G	retinal degeneration with non-recordable electroretinogram (at the age of 6 years)	nephronophtisis, intellectual disability and obesity
Shamseldin et al., 2020	BBS	F		Homozygote c.290 + 2T>C	rod-cone dystrophy	pituitary hypoplasia, obesity, and polydactyly
Morisada N et al., 2020	BBS	F		Compound heterozygote c.1218 G > A c.1631A > G	RP	intellectual disability, autism, motor developmental delay, renal cysts
Morisada N et al., 2020	BBS	M	3 years	Compound heterozygote c.1218 G > A c.1631A > G	RP	hepatic fibrosis, kidney failure, micropenis, mild cognitive impairment, short stature, obesity
Horiuchi et al., 2020	BBS		22 years	No genetic data reported	RP	hepatic fibrosis, renal failure, obesity, mental retardation and hypogonadism
Current study	SCLT1-related disease	M	8 years	c.1439 + 1del, p? hom	Reduced cone photoreceptors function	ADHD, obesity
Current study	SCLT1-related disease	M	10 years	c.1439 + 1del, p? hom	Reduced cone photoreceptors function	ADHD, obesity

Legend: OFD: orofaciodigital syndrome; SLS: Senior-Løken syndrome; BBS: Bardet-Biedl syndrome; F: female; M: male; hom: homozygous; RP: retinitis pigmentosa; ADHD: Attention deficit hyperactivity disorder.

4.2. SCLT1-related retinal degeneration spans from generalised, advanced retinal degeneration of RP type to milder condition affecting only cone photoreceptors

All previously reported patients with SCLT1-related retinal disease had advanced retinal degeneration (1,2,4,6) at an early age with non-recordable electroretinogram (2,6), whereas the two individuals presented here had milder ocular phenotype (Table 1 , Figure 2a,b). Both children had subtle fundus changes, which could had been missed with ophthalmoscopic exam. Electroretinogram revealed normal function of rod photoreceptors and reduced function of cone photoreceptors, as seen in cone dystrophies. Importantly, the visual functions remained relatively stable and well-preserved until the most recent visit at the age of 8 and 10 years, respectively. This is the first report of milder clinical phenotype caused by pathogenic variants in SCLT1 gene, thereby providing evidence of both systemic and ocular heterogeneity.

4.3. Pathogenic variants in SCLT1 gene may affect splicing

A few case reports of individuals with BBS/ciliopathy disorder and loss of function genetic variants in SCLT1 have been reported of which three are splice variants with the loss of exons 5, 14 and 17 respectively (1,2,5,6). All variants resulted in severely affected individuals with a range of systemic manifestations typical for ciliopathies (Table 2). In contrast, our patients had mild clinical phenotype due to a novel splice-site variant (NM_144643.3: c.1439 + 1del) in SCLT1 gene (Figure 1) in a homozygous state leading to retention of intron 16. Splice-site variants have been reported as common disease causing variations, with estimated frequency as high as 5.9–8.5% of all currently known pathogenic variants (44). Also, splice-site variants have been detected in a wide range of genes causative for RD (45) and they may lead to modified explanations of disease mechanisms. The variant identified in this family, c.1439 + 1del, was predicted to cause a loss of donor-site and was shown by RNA sequencing to cause a retention of intron 16. Control samples did not show any inclusion of sequences from intron 16 (n = 10). The resulting transcript would include a pre-mature stop codon 29 amino acids downstream, which could putatively lead to an alternative truncated protein, although other mechanisms cannot be ruled out.

In this report, we highlight the importance of extensive diagnostics in patients with unexplained reduced vision, refractive errors, strabismus, obesity and autism/ADHD spectrum disorders. We and other authors (16,37,46) showed that ciliopathy diseases may present with mild systemic and ocular phenotypes, and thereby can be underdiagnosed, especially in young children. SCLT1-related retinal degeneration is an ultra-rare condition, and isolated cone photoreceptors dysfunction has not been previously observed. Regular follow-up of our patients is important to determine natural history of SCLT1-related retinal degeneration and to clarify if this is a case of cone dystrophy or an early stage of disease leading to cone-rod dystrophy. Also, in line with previous studies (47,48), we would like to emphasize the need of extensive clinical examinations together with genetic diagnostics in inherited retinal conditions, which enables identification of systemic associations early on. These actions have not only a role in preventive management strategies for the disease but also an individual’s general health, well-being and family planning (49,50).

Acknowledgments

We greatly thank all the family members and patients who participated in this study.

Disclosure statement

AL has received honoraria from Illumina, MKGP from Novartis (unrelated to this study).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/13816810.2023.2215332.

Additional information

Funding

MKGP is supported by research grants from Karolinska Institutet Research Foundation [K828262023]; Carmen och Bertil Regners Stiftelse for Research in Eye Diseases [K828262033]; Kronprinsessan Margaretas Arbetsnämnd för synskadade KMA [K828262013]. AL is supported by grants from the Swedish Research Council (2019-02078), the Swedish Rare Diseases Research foundation (Sällsyntafonden) and the Swedish Brain Foundation (FO2020-0351). AM is supported by grants from Her Majesty the Quenn Silvia’s Jubilee Fund. Several of the authors of this publication are members of the European Reference Network on Rare Congenital Malformations and Rare Intellectual Disability ERN-ITHACA [EU Framework Partnership Agreement ID: 3HP-HP-FPA ERN-01-2016/739516], MKGP is currently active in a management team of ERN-EYE (European Reference Network dedicated to Rare Eye Diseases).