A genetic and clinical study of individuals with nonsyndromic retinopathy consequent upon sequence variants in HGSNAT, the gene associated with Sanfilippo C mucopolysaccharidosis

Abstract Pathogenic variants in the gene HGSNAT (heparan‐α‐glucosaminide N‐acetyltransferase) have been reported to underlie two distinct recessive conditions, depending on the specific genotype, mucopolysaccharidosis type IIIC (MPSIIIC)—a severe childhood‐onset lysosomal storage disorder, and adult‐onset nonsyndromic retinitis pigmentosa (RP). Here we describe the largest cohort to‐date of HGSNAT‐associated nonsyndromic RP patients, and describe their retinal phenotype, leukocyte enzymatic activity, and likely pathogenic genotypes. We identified biallelic HGSNAT variants in 17 individuals (15 families) as the likely cause of their RP. None showed any other symptoms of MPSIIIC. All had a mild but significant reduction of HGSNAT enzyme activity in leukocytes. The retinal condition was generally of late‐onset, showing progressive degeneration of a concentric area of paramacular retina, with preservation but reduced electroretinogram responses. Symptoms, electrophysiology, and imaging suggest the rod photoreceptor to be the cell initially compromised. HGSNAT enzymatic testing was useful in resolving diagnostic dilemmas in compatible patients. We identified seven novel sequence variants [p.(Arg239Cys); p.(Ser296Leu); p.(Phe428Cys); p.(Gly248Ala); p.(Gly418Arg), c.1543‐2A>C; c.1708delA], three of which were considered to be retina‐disease‐specific alleles. The most prevalent retina‐disease‐specific allele p.(Ala615Thr) was observed heterozygously or homozygously in 8 and 5 individuals respectively (7 and 4 families). Two siblings in one family, while identical for the HGSNAT locus, but discordant for retinal disease, suggest the influence of trans‐acting genetic or environmental modifying factors.


| INTRODUCTION
Inherited retinal dystrophy (IRD) denotes a phenotypically and genetically heterogeneous group of disorders causing retinal dysfunction with or without retinal degeneration. They are associated with over 200 genes acting in a Mendelian fashion causing retinal disease, either uniquely or in association with systemic or syndromic disease. Collectively, IRD is the most frequent cause of blindness in the working-age population, at least in England and Wales (Liew, Michaelides, & Bunce, 2014). Retinitis pigmentosa (RP), or synonymously, rod-cone dystrophy, is in turn the most prevalent form of IRD and is due to pathology primarily and initially of rod photoreceptors. Generally, patients with RP first experience loss of night vision (nyctalopia) followed by progressive loss of peripheral vision. Central vision can be preserved in some affected individuals. The age of onset and degree of sight impairment is hugely variable. Distinct genotypes in several autosomal genes, acting in a recessive Mendelian fashion (e.g., USH2A, FLVCR1, CEP290, CLN3, and MFSD8) have been shown to be associated with both syndromic or nonsyndromic forms of RP, depending on the specific component alleles. In certain genes, hypomorphic alleles have been identified that are associated with the nonsyndromic forms such as USH2A (Lenassi et al., 2015;Rivolta, Sweklo, Berson, & Dryja, 2000). Specific genotypes of HGSNAT (encoding heparanα-glucosaminide N-acetyltransferase) in which sequence variants generally cause mucopolysaccharidosis type IIIC (MPSIIIC) or Sanfilippo C syndrome (MIM 252930)-a severe childhood onset lysosomal storage disorder-have also been associated with isolated retinal disease in a few reported individuals.
There are four clinically indistinguishable (Valstar, Marchal, Grootenhuis, Colland, & Wijburg, 2011) subtypes of autosomal recessive MPSIII-A, B, C, and D-each caused by deficiency of a different enzyme involved in the stepwise degradation of heparan sulfate, a glucosaminoglycan (GAG). The membrane-bound HGSNAT (E.C. 2.3.1.3) catalyzes the transmembrane acetylation of the terminal glucosamine residue of heparan sulfate (Bame & Rome, 1986). Pathogenic variants in HGSNAT lead to the accumulation of unacetylated heparan sulfate in the lysosomes of all tissues and organs and to its excretion in the urine (Hrebícek et al., 2006).
Predominant features of MPSIIIC are progressive behavioral difficulties including hyperactivity, aggression and progressive mental deterioration leading to severe dementia. Other signs and symptoms include sleep disorders, coarse facial features, full lips, thick eyebrows, hearing impairment, vision loss (retinopathy), and seizures. The age of death for typical disease is at the end of the second or beginning of the third decade of life (Valstar, Ruijter, van Diggelen, Poorthuis, & Wijburg, 2008). In 2015, three families with specific HGSNAT genotypes were first associated with isolated late-onset slowly progressive retinal disease (Haer-Wigman et al., 2015) and this was followed by several reports, each of a few additional families Comander et al., 2017;Long et al., 2020;Van Cauwenbergh et al., 2017).
Total β-hexosaminidase was also measured in the same leucocyte homogenate as a control for sample integrity using the synthetic fluo-  Figure 1. Cystoid macular edema was a feature in some. Imaging showed loss of the ellipsoid-line and outer-nuclear layer with retained autofluoresence in the outer macular in less severely affected individuals, suggesting loss of rod photoreceptors as the primary degenerative event.
Of the nine patients who had electrodiagnostic testing, full-field ERG (Figure 2), indicated generalized photoreceptor dysfunction confined to the rod system (N = 2); mild and similar rod and cone system involvement (N = 1), rod-cone dysfunction that ranged from mild to severe (N = 5) or undetectable responses consistent with a severe loss of rod and cone function (N = 1). Of the five patients who underwent standard-field PERG testing of macular function, P50 was undetectable (N = 2), subnormal (N = 3; including one with additional delay) or normal (N = 1), but all five had a subnormal response to a large field size, in keeping with severe paracentral macular involvement. It is highlighted that macular function could not be predicted from the severity of the full-field ERG findings; two of those with relatively mild rod and cone dysfunction had undetectable PERGs, suggesting severe macular dysfunction (Figure 2a,b).

| Enzymatic analysis
Leukocyte HGSNAT activity and urinary GAG/creatinine ratio were assessed in 16 of the 20 patients (Table 1) . The PERG is recorded to an alternating chequerboard (15 × 11 ). The full-field ERGs show evidence of similar rod and cone dysfunction (a), rod > cone dysfunction (b and d) or a loss of rod function with preserved peripheral cone system function (c). PERG P50 shows reduction, in keeping with severe (a, b) or relatively mild (c, d) macular dysfunction. There was a high degree of interocular symmetry and recordings are shown from the right eye only. Patient traces are superimposed to demonstrate reproducibility. Broken lines replace blink artifacts for clarity 2.1 nmol/hr/mg protein, respectively), indicating that any HGSNAT variants were benign.
Observations of the genotypes in our cohort and those previously reported, suggest a class of allele that when paired together, or in trans with a null, give rise to the retina-only phenotype. This is analogous to the situation for genes such as USH2A (Lenassi et al., 2015;Rivolta et al., 2000) and CLN3 (Ku et al., 2017) (Comander et al., 2017), also belong to this class of alleles. In addition, the extension allele at the end of exon 18 in trans with a null, F I G U R E 4 Schematic representation of the HGSNAT gene showing the localization and distribution of variants associated with MPSIIIC and with nonsyndromic retinitis pigmentosa (RP). Exonic variants are represented below and intronic variants above. Each vertical dot represents a unique variant. Blue dots represent the 71 variants associated with MPSIIIC described to date (Martins et al., 2019). Red dots represent the 8 reported variants associated with nonsyndromic RP in HGSNAT of which 6 were novel (see text). Green dots represent the 12 variants associated with nonsyndromic retinitis pigmentosa in HGSNAT described in this study, 7 of which are novel (see Table 2) described recently in a Chinese family (Long et al., 2020) would also fit into this class of retinal disease-specific allele. Alleles found uniquely in retinal dystrophy patients, but only paired with another known allele of this class, could belong to either class and are here termed "undefined." This includes both p.(Gly418Arg) and p.
(Gly248Ala) in this study. The peptide position of these alleles is shown in Figure 4, in the context of all those previously reported in families with MPSIIIC (Martins et al., 2019) and retinal dystrophy (Table S1). There is no obvious clustering of those missense variants associated with nonsyndromic retinal disease. The phenotype presented here and by others appears distinctwith a late-onset of presentation and diagnosis, a symmetrical (both inter-and intraocular) distribution of degeneration that is pericentral.
The functional phenotype was more variable with full-field ERGs, ranging from undetectable to showing relatively mild loss of rod function (see Fig ERG1 A and D; both aged 70 years with relatively mild ERG abnormalities). The PERG indicated spared or relatively spared central macular function in the majority, consistent with a previous report that labeled the condition "pericentral RP" (Comander et al., 2017), but there were also exceptions, with severe macular dysfunction occurring in some with ERG evidence of mild peripheral retinal dysfunction. Depending upon the resources available, enzymology, rather than nucleotide sequencing, might be the most efficient test to secure a molecular diagnosis. The distinct phenotype also helps clinicians interpret variants found in panel, exome-, or genome-wide testing.
In conclusion, we have here expanded the phenotypic and genotypic spectrum of nonsyndromic HGSNAT-retinopathy, and added at least three further alleles to those that appear to confer disease that affects only the retina. The discordant siblings in our cohort suggest the action of as yet unknown trans-acting genetic and/or environmental modifiers that might determine nonpenetrance and help explain the high population prevalence of the most prevalent retinal disease specific variant.

SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of this article.