A case of siblings with juvenile retinitis pigmentosa associated with NEK1 gene variants

ABSTRACT Background Axial spondylometaphyseal dysplasia(axial SMD) is associated with early-onset retinal dystrophy and various skeletal dysplasias of varying severity. NEK1 is the causative gene for short rib polydactyly syndrome and axial SMD. Here, we report a case of siblings with juvenile retinitis pigmentosa (RP) and NEK1 variants not associated with systemic disorders. Materials and Methods The patients were a 7-year-old-girl and a 9-year-old boy with RP, who were followed for 9 years. Whole exome sequencing (WES) was performed on the siblings and their parents, who were not consanguineous. Results The corrected visual acuity of the girl and the boy at first visit was binocular 20/63 and 20/100 OD and 20/63 OS, respectively. The siblings had narrowing of retinal blood vessels and retinal pigment epithelium atrophy in the fundus and showed an extinguished pattern in electroretinogram. On optical coherence tomography, there was a mottled ellipsoid band with progressive loss in the outer macular, the edges of which corresponded to the ring of hyperautofluorescence on fundus autofluorescence imaging. The siblings showed progressive visual field constriction. Radiological examination did not reveal any skeletal abnormalities. We identified two rare heterozygous NEK1 variants in the patients: c.240 G>A; p.(M80I) and c.634_639dup;p.(V212_L213dup). Heterozygous variants were recognized in the father and mother, respectively. According to the guidelines of the American College of Medical Genetics and Genomics, both variants were classified as likely pathogenic. Conclusion This is the first report of RP patients with NEK1 variants not associated with skeletal abnormalities.


Introduction
Axial spondylometaphyseal dysplasia (axial SMD, MIM#602271) is a genetic disorder of the bone characterized by progressive postnatal growth failure with shortening of the limbs due to the metaphyseal dysplasia of the tubular bones and deformed, hypoplastic thorax (1)(2)(3).Recent molecular genetic studies have revealed that C21ORFf2 and NEK1 are the causative genes of axial SMD (4,5).Axial SMD is frequently associated with retinal dystrophy (1)(2)(3)6), and cases of cone-rod dystrophy or early-onset retinal dystrophy without axial SMD caused by variants in the C21ORF2 gene were previously reported (7)(8)(9).The NEK1 gene has been pointed out as the causative gene for short rib polydactyly (SRPS) and axial SMD (5,10).SRPS is associated with severe skeletal dysplasia and visceral dysfunction, which often results in perinatal mortality (11,12).It has been reported that axial SMD caused by NEK1 gene variants is also associated with severe retinal dystrophy (5).Here, we report a case of siblings of juvenile-onset RP with variants in the NEK1 gene not associated with systemic abnormalities.

Ethics statements
This study was approved by the Institutional Review Board for Human Genetic and Genome Research at Hamamatsu University School of Medicine (permit no.14-040).All study procedures adhered to the tenets of the Declaration of Helsinki.Written informed consent was obtained from the patients and their parents after all the study procedures were explained in detail.

Patients
A 7-year-old girl (proband) and her 9-year-old brother were introduced to the Hamamatsu University Hospital.The parents were not consanguineous.The patients were followed up for 9 years.We underwent ophthalmic examinations, including BCVA, using a Landolt C chart, refraction measurement, and kinetic visual fields on the Goldmann perimeter.In addition, slit-lamp biomicroscopy, ophthalmoscopy after pupillary dilation, fundus photography, fundus autofluorescence imaging (FAF, Heidelberg Engineering, Heidelberg, Germany), and optical coherence tomography (OCT, Carl Zeiss Meditec AG, Dublin, CA, USA and Heidelberg Engineering) were performed.

Genetic analysis
Genomic DNA was extracted using the QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.WES was performed on four family members (I-1, I-2, II-1, and II-2).Exome data processing, variant calling, and variant annotation were performed as previously described (13) using human GRCh38 as the reference genome.To identify the disease-causing variants, we focused on nonsynonymous variants and splice-site variants, which are within 10 bp of the exon-intron boundaries (±10 bp), and excluded synonymous and non-coding exonic variants from the analysis.We treated common genetic variants (allele frequency >0.01 for recessive variants and >0.001 for dominant variants) in any of the ethnic subgroups found in the following single nucleotide polymorphism databases and in-house exome data (n = 218) as putative non-pathogenic sequence alterations: Genome Aggregation Database (https://gnomad.broadinstitute.org/),Human Genetic Variation Database (http://www.genome.med.kyoto-u.ac.jp/SnpDB/), and Integrative Japanese Genome Variation Database (4.7KJPN; https ://ijgvd.megabank.tohoku.ac.jp/).
Potential pathogenic variants detected by WES were validated using Sanger sequencing according to the standard protocol (14).Sanger sequencing segregation analyses were performed on four family members to investigate the cosegregation of the potentially pathogenic variants.The following primer sets for NEK1 were used: exon 4 of NEK1: forward primer 5ʹ-GCAGCTGCTTTTTAGTAAGCTTTT-3ʹ, and reverse primer 5ʹ-TGGCAGCTAATTAGAGGTAAATA TGAG-3ʹ.exon 9 of NEK1: forward primer 5ʹ-GGCTGATATTAATTTACAGATAACCAA-3ʹ, and reverse primer 5ʹ-AGATCCAGATGTGTTCTCCTCAAC-3ʹ.The NCBI Reference Sequence of NEK1 (NM_012224.4) was used.

Clinical characteristics
The girl's corrected visual acuity at the first visit was right 20/ 100 OD and 20/63 OS.Refraction was −3.75-0.50x5 OD, and −4.50-0.50x5 OS.The boy's corrected visual acuity at the first visit was 20/63 OU.Refraction was −0.50-1.50x40 OD, and −1.00 OS.The ocular fundus on both of them showed narrowing of retinal blood vessels, tilted optic discs, and extensive retinal pigment epithelium atrophy (Figure 1).In the male sibling, there are peripheral white dots and occasional hyperpigmented spots.The brother had a few pigmentation spots since he was 12 years old, but the 15-year-old sister had no pigmentation spots.The siblings showed progressive visual field constriction (Figure 2), and their corrected visual acuity in the last assessment was 20/200 OU.On OCT, there was a mottled ellipsoid band with progressive loss in the outer macular, the edges of which corresponded to the ring-shaped hyperfluorescence on FAF (Figure 3).Throughout the followup period, the siblings had a similar clinical course based on fundus findings, visual fields, OCT, and FAF.Electroretinograms from the International Society for Clinical Electrophysiology of Vision (ISCEV) protocol (15) showed an extinguished pattern (Suppl 1).The patients were diagnosed with RP.They had no physical problems diagnosed by pediatricians.The heights of the 7-year-old girl and the 9-yearold boy were 118 cm and 125 cm, respectively, which did not deviate from the normal range of the Japanese population.Although the siblings are a little short in height, no abnormalities in height development have been observed until now.A skeletal survey did not reveal any skeletal abnormalities (Suppl 2, 3).

Genetic studies
To elucidate the pathogenesis, we performed WES on four family members.Given that our patients were two siblings from the same healthy parents, autosomal recessive inheritance was presumed.WES data of the two affected children (II-1 and II-2) and parents (I-1 and I-2) were analyzed based on the autosomal recessive model.Consequently, we identified two rare heterozygous NEK1 variants in both children: c.240 G>A; p.(M80I) and c.634_639dup;p.(V212_L213dup).Recently, NEK1 was identified as the causative gene for axial SMD, which is characterized by dysplasia of the axial skeleton and retinal dystrophy (5).The NEK1 variants were validated by Sanger sequencing and co-segregated (Figure 4).Neither variant was found in any database described in the methods, indicating their extreme rarity.In addition, the variants were located in the well-conserved serine/threonine kinase catalytic domain of NEK1.The p.(M80I) variant was predicted to be pathogenic by four different computational programs (SIFT, PolyPhen2, Mutation Taster, and CADD).On the other hand, p.(V212_L213dup) was analyzed using the PSI-blast-based secondary structure prediction (PSIPRED) program (accessed 6 September 2022.http://bioinf.cs.ucl.ac.uk/psipred/) to determine its effect on the secondary structure of NEK1.Specifically, an alpha helix consisting of ten contiguous amino acids (208-MKNLVLKIIS-217) in the wild-type NEK1 was changed to an   alpha helix consisting of eight amino acids (211-LVLVLKII -218) in the mutant NEK1.This suggests that p. (V212_L213dup) is deleterious to the helical structure of the protein.According to the guidelines of the American College of Medical Genetics and Genomics ( 16), both variants were classified as likely pathogenic.Another rare compound heterozygous variant was identified (c.1111 G>A:p.(V371M) and c.7488 G>C:p.(E2496D) in PIEZO1).Variants in PIEZO1 are known to cause two distinct disorders: dehydrated hereditary stomatocytosis with autosomal dominant inheritance and lymphatic malformation with autosomal recessive inheritance.However, our patients' phenotypes are different from the two disorders.

Discussion
NEK1 gene variants have been reported to exhibit various skeletal abnormalities and retinal dystrophy (5,10).This is the first report of retinal dystrophy alone caused by NEK1 gene abnormalities without skeletal abnormalities.Patients with axial SMD may present postnatal failure to thrive, proximal limb shortening and short stature in early childhood, and short trunk in late childhood (1)(2)(3).Axial SMD is characterized by disability and later metaphyseal dysplasia, which may increase susceptibility to respiratory tract infections.C21ORF2 (603191 21q22) and NEK1 (604588 4q33) have been reported as causative genes for axial SMD (4,5).RP and cone-rod dystrophy are retinal dystrophies associated with axial SMD.There were a few reports of isolated retinal dystrophy, including conerod retinal dystrophy or early-onset retinal dystrophy due to C21ORF2 gene variants (7)(8)(9).The patients of the present report show a new phenotype with juvenile RP caused by NEK1 gene variants without general abnormalities.
In the siblings, the electroretinogram showed extinguished rod and cone responses in the ISCEV protocol (15).The retina of the proband showed mottled retinal pigment epithelium changes without retinal pigment, although a few pigments were observed at the mid-peripheral fundus in the 12-yearold brother.FAF showed ring-shaped hyperfluorescence, which is often observed in RP.These findings are consistent with those of patients with juvenile RP.
We followed them due to visual impairment from childhood.Although they had impaired vision, they completed high school education.The visual fields were also concentrically constricted, leaving only 10° in the center of the 17-year-old brother.Wang et al (4).reported the case of an 11-year-old boy with NEK1 variants and skeletal dysplasia showing widespread retinal degeneration and an extremely diminished mixed rodcone response on full-field electroretinography.Therefore, the prognosis of visual function was poor.
The siblings had no systemic abnormalities.We have previously encountered a few cases of syndromic RP, including Usher syndrome due to USH2A gene variants and Bardet-Biedl syndrome due to BBS10 gene variants (17,18).In these syndromic RP cases, appropriate medical interviews are required for the diagnosis.Although a genetic diagnosis might be easier, we believe that appropriate genetic counseling and informed consent are necessary.On the other hand, there are rare cases of retinal dystrophy alone due to a genetic abnormality reported as a systemic disease.The accumulation of this type of cases is important for ophthalmologists.
C21ORF2, which is reported to be a causative gene for syndromic ciliopathies such as axial SMD and nonsyndromic retinal dystrophies, including cone-rod dystrophy, is localized in the connecting cilium of photoreceptor cells (5,7,19).These findings indicated that the variants of C21ORF2 may cause several types of retinal dystrophy in a syndromic and non-syndromic manner.On the other hand, NEK1 interacts with C21ORF2 and retinal ciliopathy protein SPATA7, which are part of the same protein complex.Further, the partial rescue of ciliopathy features in NEK1 null zebrafish by human C21ORF2 has revealed the functional interaction between NEK1 and C21ORF2 (20), suggesting that NEK1 may contribute not only to syndromic ciliopathies but also to nonsyndromic retinal dystrophies.
It is unclear why the lesion is only retinal dystrophy, although the variants fall in the catalytic domain, which may be expected to cause severe or lethal systemic disease.To our knowledge, the in-frame deletion/duplications have never been reported in NEK1, and we first reported the in-frame duplication of two amino acids.Although it is difficult to predict functional alteration caused by the duplication, it might be possible that this novel variant is associated with the retinal only phenotype in the patient.

Figure 1 .
Figure 1.Fundus photograph of siblings.a, Right eye; b, Left eye of a girl (15 years).c, right eye; d, left eye of a boy (17 years).In both cases, the ocular fundus shows narrowing of the retinal blood vessels and retinal pigment epithelium atrophy.A few pigments were observed in the right eye of the boy.

Figure 2 .
Figure 2. Visual field of siblings.a, Left eye; b, Right eye of a girl (9 years).c, left eye; d, right eye of a boy (10 years).e, left eye; f, right eye of the boy (17 years).The siblings showed progressive visual field constriction.

Figure 3 .
Figure 3. Optical coherence tomography (OCT) and fundus autofluorescence (FAF) of the siblings.a, OCT of the girl (right eyes, 11 y and 15 y).b, FAF of the girl (right eye, 14 years) c, OCT of the boy (right eyes, 13 years and 17 years).FAF of the boy (right eye, 16 years) OCT revealed an unclear ellipsoid zone.FAF examination showed ring-shaped hyperfluorescence in the macula.Both eyes showed similar images symmetrically.

Figure 4 .
Figure 4. Two rare variants of the NEK1 gene.a, family tree.Unaffected members are shown as unfilled circles (females) or squares (males), whereas affected members are shown as filled circles or squares.The female proband (II-2) is indicated by an arrow, and + refers to the normal allele.b, Two rare heterozygous NEK1 variants in the affected siblings: c.240G>A;p.(M80I)and c.634_639dup;p.(V212_L213dup).Heterozygous variants were recognized in both the father and mother.