REEP4 variant analysis in blepharospasm and other neurological disorders

Introduction: In preceding work, a deleterious REEP4 variant [GRCh38/hg38, NC_000008.11:g.22140245G>A, NM_025232.4:c.109C>T, p.Arg37Trp] was found to co-segregate with blepharospasm (BSP) in a large African-American pedigree. Other REEP4 variants have been reported in genetic screening studies of dystonia. The REEP4 paralogs, REEP1 and REEP2, are associated with spastic paraplegia. The causal contributions of REEP4 variants to dystonia and other neurological disorders remains indecisive. Methods: Sanger sequencing was used to screen subjects (N = 307) with BSP and BSP-plus dystonia affecting additional anatomical segments (BSP+) phenotypes for variants in REEP4. In silico tools were used to examine the deleteriousness of reported (ClinVar) and previously published REEP4 variants. Results: No highly deleterious variant was identified in coding or contiguous splice site regions of REEP4 in our cohort of 307 subjects. In silico analysis identified numerous deleterious REEP4 variants in published screening studies of dystonia and several highly deleterious single nucleotide REEP4 variants in ClinVar. Conclusion: Highly deleterious REEP4 variants are rare in BSP and BSP+ phenotypes.

Even though late-onset isolated dystonias including BSP and BSP+ have a notable heritable component, large pedigrees adequately powered for linkage analysis are rare.Although rare cases of isolated BSP have been linked to THAP1 mutations [16], the genetic underpinnings of this important focal dystonia remained largely unknown until identification of a deleterious co-segregating REEP4 variant [GRCh38/hg38, NC_000008.11:g.22140245G>A, NM_025232.4:c.109C>T,p.Arg37Trp] in seven subjects with BSP or BSP+ from a 3-generation African-American BSP/BSP+ pedigree [17].Follow-up screenings of large cohorts of patients with BSP and other forms of dystonia for REEP4 variants is a logical and necessary next step in the study of BSP [18,19].Additional REEP4 variants have been identified in single cases of BSP, but to our knowledge, no other highly deleterious and co-segregating variants have been reported in other multiplex pedigrees.
Although not yet reported in SGP31 and SPG72, dystonia is a phenotypic feature of many SPGs [23].In aggregate, existing genetic, cellular, and clinical data suggest that REEP4 is a candidate gene for dystonia and other neurological disorders.

Materials and methods
The DNA samples used in this study were collected by the Dystonia Coalition (DC) and acquired from the Coriell Institute for Medical Research (Camden, New Jersey, United State).The DC is part of the Rare Diseases Clinical Research Network, which is funded by the National Institutes of Health and led by the National Center for Advancing Translational Sciences (NCATS).The DC is funded under a grant (U54NS116025) as a collaboration between NCATS and the National Institute of Neurological Disorders and Stroke.DNA analyses were approved by the University of Memphis Institutional Review Board.The cohort reported herein consisted of 307 subjects with BSP (N = 200) or BSP+ (N = 107) phenotypes.BSP+ phenotypes included subjects with BSP along with various combinations of lower facial, oromandibular and cervical dystonia.There were 224 females and 83 males with ages of acquisition ranging from 19 to 87 years.The median age at acquisition was 63 years with a mean ± standard deviation of 63.1 ± 11.0 years.Self-declared races included 259 whites, 1 Native American, 1 Pacific Islander, 11 Asians, 20 African Americans, 5 multi-racial, and 10 of other, not reported, or unknown race.Genome assembly GRCh38.p14served as the reference for primer design and variant annotation.Primers were designed to cover the coding regions of REEP4 along with exon-intron boundaries (Supplementary Table S1).Sequencing also extended into proximal intergenic regions 5′ and 3′ to REEP4.Unidirectional Sanger sequencing was completed in the entire cohort of 307 subjects with BSP and BSP+ phenotypes.Bidirectional Sanger sequencing was used to confirm all identified variants.
CADD-Phred-scores [27,28], MetaLR [29], and REVEL [30] were used to access variant deleteriousness.A CADD-Phred score of 10 indicates that the variant is among the 10% most deleterious in the genome, a score of 20 indicates that the variant is among the 1% most deleterious variant in the genome, and so on.Pathogenicity classification followed the standards set by the American College of Medical Genetics and Genomics [31], considering factors such as population data, variant databases, co-segregation, disease databases, and location of the variant within established functional domains of the encoded protein.
On 28 July 2023, ClinVar reported clinical significance for 93 variants (1 likely benign, 22 of uncertain significance, 4 likely pathogenic, and 66 pathogenic).Of the 66 pathogenic variants, 57 were duplications and 7 were deletions, all large structural variants affecting more than one gene.Of the 16 single nucleotide variants (SNVs), 1 was likely benign and 15 were of uncertain significance (Table 2).All SNVs were associated with an "inborn genetic disease" and deposited by a single submitter.Thirteen of these SNVs are highly deleterious to protein function with CADD scores >20 (Table 2).The variants are distributed across the encoded REEP4 from amino acid (aa) residues 16 to 226 (REEP4 Isoform 1 = 257 aa).
Three REEP4 screening studies have been published to date.Hammer and colleagues [18] examined 132 patients (116 white) diagnosed with BSP or BSP+ phenotypes.A second study included 78 Han Chinese [19] and a third study from Hungary screened 47 nonrelated patients with BSP and 74 patients with cervical dystonia [32].A total of 70 patients harbored a REEP4 variant.A total of 19 variants were reported in the literature.The most common (N =27) was the synonymous variant (p.Ile43Ile) in Exon 3. Seven published variants included in Table 2 have CADD-Phred scores >20.0 but 3/7 have population prevalence rates above 0.01% (>1/10,000).We classified 10 of the 19 published variants as benign due to CADD-Phred scores <10 and high population prevalence rates.The p.Arg37Trp variant [17] was classified as likely pathogenic due to positive co-segregation in a large pedigree, CADD-Phred score >30, high REVEL and MetaLR scores, and low population prevalence.The other 8 variants were classified as "uncertain significance."A total of 11 unique unflagged pLoF REEP4 variants is reported in 12 individuals within the gnomAD v3.1.2database.Nine of these variants are in coding regions of REEP4 (7 frameshift, 2 stop gained), and two are splice acceptors.CADD-Phred scores for these pLoF variants range from 25.7 to 45.0 with a mean of 31.9.The gnomAD v3.1.2database does not provide Loss Intolerance probability (pLI).For the gnomAD v2.1.1 database, the pLI score for REEP4 is 0.03.

Discussion
Without comprehensive co-segregation analyses in pedigrees with dystonia and trio analyses in early-onset neurological disorders, it is not possible to convincingly ascribe pathogenicity to published and reported REEP4 variants.Moreover, the study of dystonia genetics is compromised by subtle phenotypes, incomplete penetrance, possible pleiotropy, and the largely unexplored possibilities of recessive and oligogenic inheritance patterns.Assuming penetrance of 20% for a BSP-associated gene and BSP population prevalence of 50-100 cases per million [34], a possibly pathogenic variant should be seen in no more than 20/100,000 alleles.The variants tabulated herein that exceeded that threshold are unlikely to be causal in monogenic fashion.
In addition to lack of co-segregation analyses (e.g., phenotyping and genotyping all available family members) there are other obvious limitations to our screening study.First, Sanger sequencing will often miss exonic deletions and large structural variants.Second, our study was limited to BSP as driven by our previous identification of REEP4 p.Arg37Trp in an African-American family.It is possible that REEP4 plays a more important role in other forms of focal dystonia or spastic paraplegia.In this regard, a novel variant in ATP5MC3 co-segregated with both dystonia and spastic paraplegia in a large multiplex pedigree [35].Third, we have not characterized the biological effects of individual variants using cellular, invertebrate, or vertebrate model systems.In silico assessments of deleteriousness are informative but, in isolation, cannot establish causality.
The REEP family of REEP1-6 can be divided into subfamilies REEP1-4 and REEP5-6.The REEP1-4 subfamily can be separated into REEP1-2 and REEP3-4 groups based on structural/functional similarities [36].While REEP1 and REEP2 are linked to HSP, REEP6 is associated with retinitis pigmentosa 77 [37].REEP3 and REEP5 have not yet been associated with specific neuro-ophthalmological or general medical disorders.REEP4 protein is expressed in regions of the brain (basal ganglia and cerebellum) that play a role in the pathophysiology of dystonia 2 , and very few pLoF variants are included in the gnomAD v3.1.2database.This information suggests a possible role for REEP4 in neuro-ophthalmological disorders.In this regard, the gnomAD v2.1.1 pLI score for single nucleotide variants must be recognized but interpreted with caution [38].
Several non-coding intronic and nearby intergenic REEP4 variants were identified in our screening study but all of these were present at higher frequency in the gnomAD v.3.1.2database.These differences in allele frequency are likely due to population stratification since the majority of Coriell DC samples were likely collected from only a few high enrolling sites whereas the gnomAD v.3.1.2data was derived from more geographically diverse populations.One or more of these variants in isolation or a combination of variants (haplotype) could, in theory, play a role in REEP4 expression.For instance, the intergenic variant NC_000008.11:g.22137633C>G is located within a regulatory region (OREG0018606) [39], binding site for transcription factors TFAP2C (OREG1194750), FOXP1 (OREG1608060), and CTCF (OREG1385204), and distal enhancer-like signature (dELS, EH38E2616119) [40].
In conclusion, more work is required to establish a convincing role for REEP4 in dystonia and other neurological disorders.Of note, REEP4 may make a more significant contribution to African American dystonia, whereas our work herein and other published screening studies of REEP4 were focused on white Americans, Chinese, and Hungarians.Future   NA, not applicable.