Variants in myelin regulatory factor (MYRF) cause autosomal dominant and syndromic nanophthalmos in humans and retinal degeneration in mice
[['Uncorrected refractive error is the leading cause of moderate to severe visual impairment in the world [1] .', 'Nanophthalmos comprises a spectrum of conditions characterized by small , structurally normal eyes and resultant high hyperopia or farsightedness .', 'It is estimated to affect ~1% of the English population and constitutes a significant visual burden in these people [2] .', 'Nanophthalmos can predispose individuals to vision-threatening angle closure glaucoma , increase the risk of cataract surgery complications , and is associated with amblyopia , exudative retinal and choroidal detachments , and retinal degeneration [3 , 4] .', 'Hyperopia is a salient feature of specific molecular subtypes of inherited retinal degenerations [5–11] .', 'The heritability of hyperopia is estimated through twin studies to be between 70 and 90 percent , suggesting that hyperopia is strongly influenced by genetic factors [12 , 13] .', 'To date , five loci and four genes have been found in association with nanophthalmos , including sporadic , autosomal dominant and recessive forms [4] .', 'Although large-scale genetic epidemiology studies remain to be done , known genes likely explain only a small fraction of disease burden .', 'For example , in Chinese populations , variants in two of the most commonly mutated genes in isolated recessive nanophthalmos , MFRP and PRSS56 , account for less than 6% and 8% of cases , respectively [14 , 15] .', 'Thus far , genes identified in Mendelian cases of nanophthalmos have different roles in the retina ( PRSS56 , CRB1 ) [16–18] and the retinal pigment epithelium ( BEST1/VMD2 , MFRP ) [19 , 20] .', 'The precise mechanisms by which they cause defects in ocular growth are largely unknown .', 'Species differences in ocular dimensions between mice and humans have made studying disorders of eye size more challenging [21] .', 'Pathogenic variants in nanophthalmos genes are frequently associated with retinal degeneration [22–25] , and this is often the most prominent feature in animal models [17 , 20 , 26] .', 'Myelin regulatory factor ( MYRF ) is a membrane-associated homo-trimeric protein that self-cleaves to release an N-terminal domain that activates transcription [27–29] .', 'Myrf has a significant role in regulating the formation and maintenance of myelination in the central nervous system ( CNS ) both during development and in adulthood [30–32] .', 'Recent studies in human disease suggest that deleterious de novo variants in MYRF are associated with congenital diaphragmatic hernia , cardiac anomalies including Scimitar syndrome , urogenital anomalies , and an encephalopathy syndrome [33–36] , consistent with expression in a range of tissues .', 'However , the role of MYRF in ocular development has not yet been explored , and ocular phenotypes in syndromic children have not yet been evaluated .', 'We previously described the first large family with autosomal dominant nanophthalmos ( NNO1 ) and mapped the locus to a 14 . 7 centimorgan ( cM ) region on chromosome 11 [37] .', 'Here , we show that an inherited deleterious splice site mutation in MYRF that segregates with nanophthalmos in this family .', 'We further demonstrate a frameshift MYRF variant in a child with syndromic features and an overlapping ocular phenotype .', 'Additionally , we show that Myrf is expressed in ocular tissues and plays an important role in the development of the retina and RPE , and that it genetically and physically interacts with Tmem98 , another gene implicated in nanophthalmos .', 'Thus , we have identified a new pathway in eye growth and development and expanded the spectrum of phenotypes caused by MYRF mutations .'], ['We first described the NNO1 family in 1998 [37] .', 'Its phenotype is characterized by high hyperopia ( +7 . 25 to +13 . 0 D ) and short axial length ( 17 . 55 to 19 . 28 mm ) , with a high incidence of angle-closure glaucoma and vision loss [37] .', 'To refine the clinical features of the affected individuals , we conducted deep ocular phenotyping in this Caucasian family to ascertain additional details of ocular structure and function .', 'Individual IV-22 ( Fig 1 ) , a 31-year-old woman , had best-corrected Snellen visual acuity of 20/30 OD and 20/20 OS .', 'Before bilateral cataract surgery for narrow angles , manifest refraction showed significant hyperopia: +9 . 50 sphere OD and +10 . 25 sphere OS .', 'Anterior segment exam after cataract surgery showed peripherally shallow angles despite central deepening; intraocular pressure was within normal limits .', 'Peripheral anterior synechiae concerning for plateau iris syndrome were confirmed by ultrasound biomicroscopy ( Fig 1A ) .', 'Fundus exam was notable for crowded optic discs ( OD > OS ) with small optic cups and vascular tortuosity ( Fig 1B and 1C ) .', 'There was a small area of non-specific stippled hyperautofluorescence in the inferior periphery of the left eye , with an otherwise normal appearing ultra-widefield short-wavelength fundus autofluorescence ( Fig 1D and 1E ) .', 'Spectral domain optical coherence tomography ( SD-OCT ) revealed undulations of the outer retina/RPE consistent with choroidal folds in the right eye ( Fig 1F ) and a normal foveal contour and structure in the left eye ( Fig 1G ) .', 'Electroretinography showed mildly reduced amplitudes but preserved implicit times in all waveforms under both scotopic and photopic conditions .', 'Electro-oculogram Arden ratios were normal ( 2 . 10 and 2 . 18 in the right and left eye , respectively ) .', 'These results were consistent with a diagnosis of nanophthalmos with no evidence of narrow angle glaucoma .', 'There was no evidence of retinal or RPE disease with the exception of the small area of peripheral pigment mottling in the left eye and choroidal folds in the right eye .', 'We conducted a medical record review of available family members .', 'This revealed dextrocardia , a rare cardiac anomaly [38] , in 4 affected and no unaffected individuals ( Fig 2A ) .', 'One of these dextrocardia patients ( IV-19 ) was also noted to have hypoplastic right lung and pulmonary artery stenosis .', 'We originally mapped the NNO1 locus to a 14 . 7 cM region on chromosome 11 between microsatellite markers D11S905 and D11S987 , with a maximum multipoint LOD score of 6 . 31 at recombination fraction θ = 0 [37] .', 'As a first step in identifying the underlying gene for NNO1 , we screened blood samples from individuals in the family with additional microsatellite markers and reconstructed haplotypes to narrow the non-recombinant interval ( Fig 2 ) .', 'This analysis refined the locus to a 3 . 5 MB region between D11S4191 and D11S1883 , which contains 135 genes ( Fig 2 ) .', 'The locus included the VMD2/BEST1 gene previously implicated in nanophthalmos that encodes a calcium-activated chloride channel associated with nanophthalmos and four types of retinal dystrophy [19 , 24 , 39–45] .', 'Sanger sequencing of all of the coding elements of BEST1 , including 50–100 bp of flanking intronic sequences , revealed no pathogenic variants ( S1 Fig ) .', 'Long range PCR was used to exclude exonic deletions of BEST1 ( S1 Fig ) .', 'Sequencing of these fragments , with identification of SNPs in the fragments , confirmed that both copies of BEST1 were being amplified using this method .', 'Molecular analysis was then extended to include introns and untranslated regions ( UTR1/2 ) of BEST1 .', 'This identified a range of common variants and known SNPs , but no ultra-rare variants that were considered either to be novel or potentially pathogenic .', 'Furthermore , observation of normal Arden ratios on the EOG in the nanophthalmos individual suggest an alternative to BEST1 as the cause of disease , as individuals carrying pathogenic mutations in BEST1-associated nanophthalmos have an abnormal EOG Arden ratio [24] .', 'Given the large number of candidate genes within the interval , we employed a new approach to identify candidate variants , combining pooled whole exome sequencing together with our linkage data .', 'We separately pooled DNA samples of 26 affected individuals and 13 unaffected individuals for whole exome sequencing and subtractive filtering ( S2 Fig ) .', 'The unaffected pool was selected to maximize the representation of the non-disease haplotypes found in affected individuals .', 'We expected the difference in minor allele fraction in the affected vs . unaffected pool ( ΔMAF ) to be between 0 . 4 and 0 . 6 for variants within the linkage interval , but we used a more conservative cutoff of 0 . 1 to improve the sensitivity of detecting variants .', 'We performed whole exome sequencing as described in the methods and identified 1144 variants within the linkage interval .', 'After filtering for rare nonsynonymous coding exonic , splice junction , or microRNA variants with ΔMAF>0 . 10 , three candidate variants remained ( S1 Table ) .', 'No coding or non-coding variants were found in BEST1 .', 'As expected for variants on the disease haplotype located within the linkage interval , all of these variants segregated perfectly with the disease phenotype ( Fig 3 , S4 Fig ) .', 'We excluded two of these candidate variants , in ZP1 and PPP1R32 , on the basis of the presence of homozygotes within the Genome Aggregation Database ( gnomAD ) [46] and allele frequency ( 0 . 2% ) being higher than the rate of severe nanophthalmos in the general population ( S1 Table ) .', 'Furthermore , ZP1 has undetectable expression in human ocular tissues by RT-PCR and expression sequence tags ( ESTs ) in the Unigene database ( National Center for Biotechnology Information ) , and PPP1R32 loss-of-function mice had no clear ocular phenotype in the International Mouse Phenotyping Consortium Database [47] .', 'The remaining candidate variant was a conserved splice acceptor mutation in the myelin regulatory factor gene ( NM_001127392 . 2; MYRF c . 3376-1G>A ) .', 'This variant was absent in 246126 alleles in gnomAD , highly conserved in all vertebrate species , and in the top 0 . 1% of damaging variants by CADD score [48] .', 'Evaluation of evolutionary constraint showed that MYRF was significantly constrained against missense and loss-of-function ( LoF ) variation ( pLI = 1 . 00 ) , while PPR1R32 was not ( S3 Fig ) .', 'Taken together , these findings suggested that MYRF c . 3376-1G>A was the most promising candidate mutation .', 'To validate MYRF as a nanophthalmos gene , we identified an additional , unrelated syndromic patient with a deleterious variant in MYRF and evaluated him for ocular phenotypes .', 'The 8-year-old male subject was initially seen in the comprehensive ophthalmology clinic at age 4 .', 'His visual acuity was 20/30 in each eye at that time with a +9 . 00 sphere cycloplegic refraction and an otherwise unremarkable ocular exam .', 'A focused follow-up exam at age 8 revealed +7 . 00 sphere manifest refraction and B-scan ultrasonography demonstrated normal ocular structures in the setting of short eyes ( 17 mm axial length in each eye , Fig 1J ) .', 'Fundus exam appeared normal .', 'SD-OCT showed a flattened foveal contour with a visible inner nuclear layer ( INL ) at the foveal center and mild increase in outer nuclear layer ( ONL ) thickness and outer segment lengthening , in line with grade I foveal hypoplasia based on published criteria [49] ( Fig 1H and 1I ) .', 'These findings supported a diagnosis of nanophthalmos .', 'Additionally , this child had systemic findings of mitral valve prolapse , unilateral cryptorchidism , and micropenis , consistent with MYRF spectrum disorders [33 , 34 , 36] .', 'Clinical exome sequencing revealed a c . 769dupC ( p . S264QfsX74 ) variant in MYRF .', 'Chromosomal microarray was normal .', 'The patient’s mother was a mosaic carrier of this variant , was asymptomatic , and had a normal ocular exam .', 'This variant was absent from the gnomAD database and expected to be deleterious as the RNA species would be expected to undergo nonsense-mediated decay given the early truncation of the protein prior to the DNA-binding , transmembrane , and the intramolecular chaperone assembly ( ICA ) domains .', 'To evaluate the prevalence of MYRF mutations in high hyperopia and nanophthalmos , we systematically screened 60 independent cases of high hyperopia and nanophthalmos .', 'These included both sporadic and inherited cases , where nanophthalmos was defined as axial length <21 mm in the smaller eye and high hyperopia was defined as cycloplegic or manifest refraction with spherical equivalent >+5 . 50 D . We identified several rare variants , but none are likely to be causative based on higher allele frequencies in gnomAD controls than the expected prevalence of nanophthalmos in the population ( S2 Table ) .', 'These results lend further support that loss-of-function variants in MYRF lead to nanophthalmos , which can present in syndromic or predominantly isolated forms .', 'To determine whether other rare variants in MYRF were associated with nanophthalmos or high hyperopia , we identified individuals in The Genomic Ascertainment Cohort ( TGAC ) , whose exome data were available from the ClinSeq project [50] .', 'We selected three individuals with variants in MYRF ( S3 Table ) that were predicted to be deleterious by a high CADD score and very rare ( <0 . 1% ) allele frequency for ocular phenotyping .', 'None of the carriers of these variants had high hyperopia or nanophthalmos , as determined by our clinical criteria ( see methods ) .', 'No loss-of-function variants were identified in the TGAC cohort , and it is not unusual for in silico damaging variants of unknown significance to be tolerated [51] .', 'MYRF is a 27-exon gene that encodes a homo-trimeric membrane protein that autoproteolytically cleaves and translocates to the nucleus to regulate transcription [27–30 , 52] .', 'The MYRF c . 3376-1G>A variant is predicted to disrupt a conserved splice accepter upstream of the last exon ( Fig 4 ) , and this residue is highly conserved in all vertebrate species .', 'Using the Alternative Splice Site Predictor tool ( ASSP ) [53] , we predicted that the G>A change would result in loss of the splice acceptor site for the last exon , and formation of a new splice acceptor site 1 basepair ( bp ) downstream of the original site .', 'To test this hypothesis , we first designed a mini-gene assay to test the effect of the loss of the canonical splice acceptor site in MYRF .', 'We cloned the human wild-type and mutant genomic sequence from MYRF exon 26 through the polyadenylation site in exon 27 into the CS2 expression vector [54] , which uses a CMV promoter to ubiquitously drive gene expression .', 'Subsequently , we transfected each of these constructs into HEK293T cells , isolated RNA , and then reverse-transcribed and sequenced the resulting cDNA .', 'The wild-type construct showed consistent splicing from exon 26 to exon 27 of MYRF ( Fig 4A ) .', 'The MYRF c . 3376-1G>A mutation , however , abolished this splice acceptor site and led to use of a splice site 1 bp downstream as predicted .', 'To further confirm that the aberrant MYRF RNA is generated in vivo , we collected blood from individual IV-22 and isolated RNA to look at the relative abundance of RNA species in this individual .', 'Given the low level of MYRF expression in blood , we cloned reverse-transcribed and PCR amplified sequences spanning exons 26 to 27 to better quantify the relative abundance of spliced transcripts in vivo .', 'The mutant splice product was generated in 3/7 clones , consistent with the predicted 50% from monoallelic expression ( Fig 4B ) .', 'These results are consistent with the suggestion that the MYRF c . 3376-1G>A variant generates a stable mRNA product , and that the relative expression of wild-type and mutant RNA species is roughly equivalent .', 'The splice alteration is predicted to lead to a 1bp frameshift in the coding sequence of the C-terminus resulting in replacement of the final 26 amino acids of the protein with a different 30 amino acids , and disruption of a putative glycosylation site ( Fig 4C ) .', 'To define the role of MYRF in the pathogenesis of nanophthalmos , we first systematically evaluated RNA expression in human ocular and adnexal tissues .', 'We identified highest levels of MYRF expression in the RPE/choroid and in the optic nerve , with low levels in other ocular tissues relative to extraocular muscle ( Fig 5 ) .', 'These results are consistent with the known role of MYRF in myelination of the optic nerve and relative expression data based on RNAseq experiments in human ocular tissues [55] .', 'A similar pattern of expression was observed in mouse ocular tissues with a 99 . 5±1 . 8 fold greater expression in RPE as compared to retina by qRT-PCR .', 'Immunofluorescence staining of mouse retinal sections for MYRF using the validated N-terminal antibody [28 , 56] showed consistent expression in the RPE in human and mouse tissue; however , it is likely that nonspecific cross-reactivity to retinal tissue complicates the interpretation of these results ( S5 Fig ) .', 'To understand the pathophysiology of MYRF mutations causing eye disease , we generated a loss-of-function mouse model in the early eye field .', 'Specifically , we used a Cre driver that begins expression in the early developing retina and RPE at embryonic day ( E ) 10 . 5 ( RxCre ) [57] to delete exon 8 of Myrf ( Myrffl ) , leading to a non-functional protein in these cells [30] .', 'Gross examination of eyes and RPE flatmounts from RxCre;Myrffl/fl mice at postnatal day ( P ) 22 revealed patchy loss of RPE pigmentation in all mutant mouse eyes compared to heterozygous ( RxCre;Myrf+/fl ) and wild-type littermates ( Fig 5B and 5C ) .', 'Gross analysis of axial length in enucleated eyes , measured as central cornea to optic nerve distance , did not reveal any substantial or statistically significant differences in eye size among genotypes at P22 or gross differences at other time points ( S6 Fig ) : 2 . 93±0 . 07 mm for control ( n = 7 animals , 14 eyes ) , 2 . 95±0 . 09 mm RxCre;Myrf+/fl ( n = 3 animals , 6 eyes ) , and 2 . 95±0 . 07 mm RxCre;Myrffl/fl mice ( n = 3 animals , 6 eyes ) .', 'To detect more subtle biometric changes , we conducted anterior chamber and vitreous chamber analysis using SD-OCT in a separate cohort of animals .', 'Measurement of anterior chamber depth did not reveal any statistically significant difference among the genotypes ( S6 Fig ) : 0 . 31±0 . 02 mm for control ( n = 8 eyes from 4 mice ) , 0 . 30±0 . 02 mm for RxCre;Myrf+/fl ( n = 5 eyes from 3 mice ) , 0 . 31±0 . 03 mm for RxCre;Myrffl/fl ( n = 11 eyes from 6 mice ) , one-way ANOVA F = 0 . 29 , p = 0 . 79 ( S6H Fig ) .', 'Likewise , there was a minimal statistical or biological differences on the vitreous chamber depth: 0 . 69±0 . 02 for control ( n = 8 eyes from 4 mice ) , 0 . 67±0 . 006 mm for RxCre;Myrf+/fl ( n = 6 eyes from 3 mice ) , 0 . 67±0 . 02 mm for RxCre;Myrffl/fl ( n = 12 eyes from 6 mice ) , one-way ANOVA F = 2 . 058 , p = 0 . 15 ( S6G Fig ) .', 'Given that Myrf is absent in early development in RxCre;Myrffl/fl mice , we evaluated the histologic appearance at various developmental time points .', 'We noted that patchy loss of RPE pigmentation is present as early as E15 . 5 ( Fig 5D , S7 Fig ) .', 'Histologic analysis confirmed segmental losses of RPE granular pigment and thinning of the choroid with otherwise maintained cells ( Fig 5E–5G ) .', 'To investigate whether the patchy appearance of pigmentation was due to mosaic deletion from the RxCre driver , we crossed the RxCre mice with the Rosa26floxYFP reporter line [58] .', 'Our results revealed that there was constitutional expression of YFP throughout RPE and weaker , but uniform expression in the retina ( S8 Fig ) .', 'In RxCre;Myrffl/fl;R26floxYFP mice , RPE cells were likewise uniformly YFP+ , suggesting that variability in pigmentation is not due to selective loss of MYRF in these areas .', 'Additionally , we generated Myrf+/-and RxCre;Myrffl/- mice to determine whether Cre deletion efficiency plays a role in the severity and uniformity of the RPE phenotype .', 'RxCre;Myrffl/- showed comparable and patchy RPE pigmentation loss and similar retinal phenotype to RxCre;Myrffl/fl ( S9 Fig , Fig 6C ) .', 'Taken together , these results suggest that the variability and changes in pigment deposition through development were less likely to be due to differential deletion of the Myrf floxed allele or issues with Cre recombination efficiency .', 'Given the disparate ocular anatomy in the mouse versus human eye and the difficulty in observing small differences in mouse eye size , we next evaluated the effects of Myrf deletion on the structure of the outer retina .', 'Histologic analysis of RxCre;Myrffl/fl revealed shortened photoreceptor inner and outer segments ( IS and OS ) and thinning of the ONL ( 65% of wild-type , p = 0 . 001 ) especially overlying depigmented areas ( Fig 6 ) .', 'Histologic analysis at post-natal day ( P ) 3 did not reveal any differences in retinal structure ( Fig 6A ) .', 'RxCre;Myrffl/fl mice eyes had shortened outer segments by P22 ( Fig 6B and 6C ) .', 'To quantify the loss of photoreceptors , we identified the fraction of cone and rod photoreceptors by immunostaining against rhodopsin ( rhodopsin ) or cone-arrestin ( cones ) ( Fig 6D and 6E ) .', 'Our results showed a statistically significant loss of IS/OS area compared to total retinal area ( 12±2% vs . 21±3% , p = 0 . 001 , S4 Table , Fig 6F ) in RxCre;Myrffl/fl mice compared to controls and preferential loss of cone photoreceptors ( 2 . 3±0 . 4% vs . 3 . 5±0 . 7% of total retina cells , p = 0 . 004 ) .', 'Furthermore , we see a greater effect on IS/OS area than on ONL area ( 20±2% vs . 24±3% in RxCre;Myrffl/fl vs . control , p = 0 . 01 , S4 Table ) We did not see any significant differences in any of these measures between control and RxCre;Myrf+/fl eyes .', 'We also evaluated measurements of the retinal thickness , and the number of cells in each cell layer .', 'These analyses did not show any significant differences in the number or fraction of cells in the retinal ganglion cell layer ( S4 Table ) .', 'A small , but significant difference was noted in overall retinal area in the RxCre;Myrffl/fl vs . control mice ( S4 Table ) .', 'This was consistent with measurements of retinal thickness obtained by SD-OCT at P22 in a separate cohort of mice ( S6F Fig ) : 0 . 232±0 . 006 mm for control ( n = 8 eyes from 4 mice ) ; 0 . 220 ±0 . 003 mm for RxCre;Myrf+/fl ( n = 6 eyes from 3 mice ) , p = 0 . 0003; 0 . 211±0 . 005 mm for RxCre;Myrffl/fl ( n = 12 eyes from 6 mice ) , p = 1 . 6x10-6 vs . control and p = 0 . 00013 vs . RxCre;Myrf+/fl .', 'To further define the retinal phenotype of RxCre;Myrffl/fl mice and confirm the retinal degeneration phenotype , we next pursued in vivo structural and functional testing using a separate cohort from our histologic analysis .', 'Using 10-month-old RxCre;Myrffl/fl and age-matched heterozygous and wild-type controls , we evaluated the appearance of the retina by fundus photography and SD-OCT .', 'Photography showed patchy areas of RPE and retinal atrophy , with variable pigment deposition in RxCre;Myrffl/fl , but not control mice ( Fig 7A ) .', 'RxCre;Myrf+/fl mice had a few smaller , but similar looking lesions .', 'On SD-OCT , there was evidence of outer retinal disruption in RxCre;Myrffl/fl with severe loss of the ONL and RPE in affected areas in the retinal periphery and milder changes centrally ( Fig 7B and 7C ) .', 'RxCre;Myrf+/fl eyes were grossly normal appearing on SD-OCT ( Fig 7B and 7C ) .', 'To evaluate the functional consequences of these effects , we conducted electroretinograms ( ERGs ) on these mice .', 'We saw significantly diminished scotopic and photopic responses in RxCre;Myrffl/fl eyes compared to control eyes at all stimulus intensities ( Fig 7D–7I , S5 Table ) .', 'In RxCre;Myrffl/fl , scotopic a-wave ERG amplitudes were reduced by 45–50% and B-wave amplitudes are reduced 35–45% , while photopic B-wave amplitudes were reduced 50–60% ( Fig 7G–7I ) .', 'Photopic flicker peak amplitudes were likewise reduced by 43% .', 'Taken together , our findings strongly support that RxCre;Myrffl/fl eyes undergo retinal degeneration , with both rod and cone pathways affected .', 'It remains to be determined whether the RxCre;Myrf+/fl mice have a more subtle , late onset retinal degeneration , but we did not observe any histologic changes in the early postnatal period or ERG or SD-OCT findings in 10-month-old mice ( Figs 6 and 7 ) .', 'Given that mouse MYRF functions as a transcription factor [28 , 29 , 59] and is present in fetal RPE , we hypothesized that it might regulate transcription of other RPE-expressed genes associated with nanophthalmos in humans .', 'To investigate this , we evaluated expression of Best1 , Mfrp , Myrf , and Tmem98 by qRT-PCR in developing ( P7 ) and mature eyes ( P22 ) from RxCre;Myrffl/fl , RxCre;Myrf+/fl , and wild-type littermate controls .', 'As expected , Myrf RNA levels were nearly absent in RxCre;Myrffl/fl mutants and reduced in RxCre;Myrf+/fl mice ( Fig 8A ) .', 'Consistent with our previous findings that MYRF has a binding site just upstream of the transcriptional start site of the Tmem98 gene in oligodendrocytes [28] , we noted that Tmem98 mRNA levels were significantly reduced in eyes from RxCre;Myrffl/fl mice as compared to heterozygous and wild-type controls ( Fig 8A ) .', 'There was no difference in mRNA level between the genotypes for Mfrp or Best1 .', 'Given the reduction in Tmem98 mRNA in mice lacking Myrf , we evaluated the effect at the protein level by immunostaining in sections and RPE flatmounts at different developmental stages .', 'Our results demonstrated a profound reduction in TMEM98 immunostaining with a small fraction of cells with diffuse cellular localization pattern , possibly mosaicism-related ( Fig 8 and S10 Fig ) .', 'These findings are seen early in development ( E15 . 5 , Fig 8C ) and persist through later developmental stages ( Fig 8D and 8E ) .', 'In contrast , we saw no distinguishable difference in staining for MFRP ( S11 Fig ) .', 'As feedback loops are a common regulatory mechanism in development [60] , we hypothesized that TMEM98 may function in a feedback loop with MYRF .', 'To evaluate this possibility , we conducted co-immunoprecipitation experiments with HA-tagged TMEM98 and myc-tagged MYRF ( Fig 9 ) in HEK-293T cells to look for a direct interaction between these proteins .', 'We were able to pull down HA-TMEM98 with the full-length myc-MYRF and vice versa , suggesting that these two proteins interact directly ( Fig 9B ) .', 'We observed that TMEM98 preferentially interacts with the un-cleaved form of MYRF; no band for the MYRF N-terminal cleavage product was observed with the HA-TMEM98 immunoprecipitation , suggesting that TMEM98 does not interact with the N-terminal cleavage product that comprises the functional transcription factor .', 'TMEM98 also stabilized the un-cleaved form of MYRF in a dose-dependent manner ( Fig 9C ) .', 'To further define the interaction site of these two proteins , we generated a truncating mutant containing the first 846 amino acids of MYRF ( deleting much of the ER-lumenal component of the protein ) , and a mutant consisting of only the C-terminal cleavage product .', 'Both of these truncated proteins retained their ability to pull down TMEM98 ( Fig 9 ) , suggesting that the region in MYRF that is critical for this interaction is downstream of the cleavage site in the intramolecular chaperone or transmembrane domains , between amino acids 586 and 846 .'], ['Since the discovery of the membrane-associated Myrf transcription factor [28–30] , much has been learned about its role in human disease , and its structure and function .', 'De novo variants have been implicated in a new syndrome involving congenital diaphragmatic hernia , cardiac defects , and urogenital anomalies [33 , 34 , 36] , and in an encephalopathy syndrome [35] .', 'The mutational spectrum of these variants predominantly includes truncating mutations that would be predicted to undergo nonsense-mediated decay or missense mutations within the DNA binding and intramolecular chaperone auto-processing ( ICA ) domains [28 , 29 , 59] .', 'Both of the functional domains are critical to processing and protein function , and are conserved in lower order species including the slime mold Dictyostelium [59 , 65] .', 'The splice site variant in the NNO1 family is vertically transmitted , affects the C-terminus of the protein , and results in a predominantly ocular phenotype .', 'Although significant advances have been made in elucidating the role of the N-terminal domains of Myrf , including the transcriptional targets and the cleavage process [28 , 29 , 56 , 59 , 66] , the role of the C-terminus remains unclear .', 'Interestingly , much of the C-terminus of MYRF is absent in lower eukaryotes [65] , suggesting that this domain likely developed disparate functions in higher eukaryotes .', 'Our large family with nanophthalmos represents an expansion in phenotype of that seen in the newly described MYRF-associated syndrome [33 , 34 , 36] .', 'Patients in our large family have a severe ocular phenotype , but few have systemic features .', 'Four affected individuals have reported dextrocardia , a very uncommon finding in the general population [38] .', 'Scimitar syndrome , an anomalous venous return syndrome associated with MYRF mutations [33] , may be misdiagnosed as isolated dextrocardia [67] .', 'As such , dextrocardia in these individuals may represent a milder form of the cardiac anomalies present in syndromic patients carrying MYRF loss of function variants .', 'The differences in pathogenesis of MYRF variants and in ocular and systemic phenotypes could be explained in several ways .', 'First , MYRF may be a dosage sensitive transcription factor with ocular tissues being more sensitive than cardiac and urogenital tissues .', 'In this model , the C-terminal alleles would be hypomorphic causing mild disruption in protein function , while early truncating or non-functional alleles would lead to haploinsufficiency in all affected tissues .', 'The variable penetrance of the dextrocardia phenotype may be consistent with this model .', 'However , data from animal model studies favors an alternative model , as Myrf heterozygous mice ( Myrf+/- and Myrffl/- ) are viable and have normal Mendelian segregation ratios ( 40 Myrf+/- vs . 38 Myrf+/+ pups , χ2 p = 0 . 82 ) .', 'This would be contrary to what would be expected with the severe human phenotype [33 , 34 , 36] .', 'Furthermore , RxCre;Myrf+/fl and Myrf+/- mice have a minimal ocular phenotype ( Figs 5–7 , S9 Fig ) .', 'Alternatively , MYRF variants may lead to dominant negative effects in protein function , through their incorporation into trimers .', 'This effect may alter protein-protein interactions , cleavage , or ability to activate transcription .', 'Differential effects may be observed in different tissues , such that the eye is more sensitive to changes in the C-terminus of the protein .', 'Future work will be necessary to determine which of these mechanisms explains the ocular and systemic phenotypes in these patients .', 'Our results support the need for comprehensive ophthalmic evaluation and potential intervention in patients with MYRF mutations , even if they are presumed asymptomatic , as subnormal vision may be overlooked in severe systemic disorders .', 'Uncorrected high hyperopia can be associated with strabismus and amblyopia in early childhood , which may cause permanent vision loss [68] .', 'Proper early spectacle correction along with surveillance and treatment of ocular motility abnormalities may help to improve final visual outcomes [69 , 70] and consequently quality of life .', 'Nanophthalmos may also predispose to angle closure glaucoma early in life [71 , 72] .', 'The risks of this visually disastrous complication may be mitigated with early intervention by peripheral iridotomy or lens extraction , and avoidance of triggering medications [73] .', 'On the other end of the syndromic spectrum , cardiac evaluation in NNO1 family members and other cases with MYRF mutations may also be warranted , as Scimitar syndrome and other anomalous pulmonary venous return phenotypes are often asymptomatic , but can be associated with dyspnea , fatigue , and recurrent respiratory infections [74] .', 'High hyperopia is strongly heritable and known genes likely account for a small fraction of explained cases [12–15] .', 'Our analysis of unrelated high hyperopia and nanoph
['Introduction', 'Results', 'Discussion', 'Materials and methods']
['Nanophthalmos is a rare , potentially devastating eye condition characterized by small eyes with relatively normal anatomy , a high hyperopic refractive error , and frequent association with angle closure glaucoma and vision loss .', 'The condition constitutes the extreme of hyperopia or farsightedness , a common refractive error that is associated with strabismus and amblyopia in children .', 'NNO1 was the first mapped nanophthalmos locus .', 'We used combined pooled exome sequencing and strong linkage data in the large family used to map this locus to identify a canonical splice site alteration upstream of the last exon of the gene encoding myelin regulatory factor ( MYRF c . 3376-1G>A ) , a membrane bound transcription factor that undergoes autoproteolytic cleavage for nuclear localization .', 'This variant produced a stable RNA transcript , leading to a frameshift mutation p . Gly1126Valfs*31 in the C-terminus of the protein .', 'In addition , we identified an early truncating MYRF frameshift mutation , c . 769dupC ( p . S264QfsX74 ) , in a patient with extreme axial hyperopia and syndromic features .', 'Myrf conditional knockout mice ( CKO ) developed depigmentation of the retinal pigment epithelium ( RPE ) and retinal degeneration supporting a role of this gene in retinal and RPE development .', 'Furthermore , we demonstrated the reduced expression of Tmem98 , another known nanophthalmos gene , in Myrf CKO mice , and the physical interaction of MYRF with TMEM98 .', 'Our study establishes MYRF as a nanophthalmos gene and uncovers a new pathway for eye growth and development .']
['Hyperopia or farsightedness is a common condition that can cause visual impairment especially in children .', 'The extreme of this condition is called nanophthalmos , a small crowded eye in which inappropriate drainage of aqueous humor from the eye can lead to glaucoma and vision loss .', 'We previously described a large family with inherited nanophthalmos , but the genetic defect that segregated in this family was unknown .', 'Here , we have used a new approach combining linkage analysis and pooled sequencing to identify the genetic cause in this family .', 'We identified a splice site mutation that causes the myelin regulatory factor ( MYRF ) gene to produce an aberrant protein .', 'Additionally , a child with syndromic manifestations and a deleterious MYRF variant shared the same eye condition .', 'Using a mouse model in which MYRF is absent from eye tissue during early development , we established a role for this transcription factor in the development of the retinal pigment epithelium and retina .', 'We showed that MYRF interacts with and regulates expression of another membrane protein , TMEM98 , which has been implicated in nanophthalmos .', 'Our study establishes MYRF as a new disease gene for nanophthalmos and a regulator of eye development .']
['medicine and health sciences', 'ocular anatomy', 'social sciences', 'neuroscience', 'genetic mapping', 'animal models', 'retinal disorders', 'model organisms', 'animal anatomy', 'experimental organism systems', 'molecular biology techniques', 'eyes', 'zoology', 'research and analysis methods', 'artificial gene amplification and extension', 'animal studies', 'animal cells', 'mouse models', 'sensory receptors', 'head', 'molecular biology', 'animal ocular anatomy', 'signal transduction', 'haplotypes', 'cellular neuroscience', 'retina', 'psychology', 'anatomy', 'cell biology', 'polymerase chain reaction', 'heredity', 'ophthalmology', 'genetics', 'neurons', 'biology and life sciences', 'ocular system', 'cellular types', 'afferent neurons', 'photoreceptors', 'sensory perception', 'retinal degeneration']