A small gene sequencing panel realises a high diagnostic rate in patients with congenital nystagmus following basic phenotyping

Nystagmus is a disorder of uncontrolled eye movement and can occur as an isolated trait (idiopathic INS, IINS) or as part of multisystem disorders such as albinism, significant visual disorders or neurological disease. Eighty-one unrelated patients with nystagmus underwent routine ocular phenotyping using commonly available phenotyping methods and were grouped into four sub-cohorts according to the level of phenotyping information gained and their findings. DNA was extracted and sequenced using a broad utility next generation sequencing (NGS) gene panel. A clinical subpanel of genes for nystagmus/albinism was utilised and likely causal variants were prioritised according to methods currently employed by clinical diagnostic laboratories. We determine the likely underlying genetic cause for 43.2% of participants with similar yields regardless of prior phenotyping. This study demonstrates that a diagnostic workflow combining basic ocular phenotyping and a clinically available targeted NGS panel, can provide a high diagnostic yield for patients with infantile nystagmus, enabling access to disease specific management at a young age and reducing the need for multiple costly, often invasive tests. By describing diagnostic yield for groups of patients with incomplete phenotyping data, it also permits the subsequent design of ‘real-world’ diagnostic workflows and illustrates the changing role of genetic testing in modern diagnostic workflows for heterogeneous ophthalmic disorders.

Ocular albinism (OA) is a form of albinism in which the clinical features are constrained to the eye, whilst oculocutaneous albinism (OCA) encompasses a broader phenotypic range affecting the eyes, hair and skin 4 . The ocular manifestations of OCA and OA include infantile nystagmus syndrome (INS) 5 , foveal hypoplasia, abnormal crossing pattern at the optic chiasm and iris transillumination defects 6 all of which can be subtle or incomplete 7 . Additionally, many of the ocular features seen in albinism can be seen in other disorders caused by mutations in genes that are not associated with melanin biosynthesis such as PAX6 mutations, which can cause a variety of ocular phenotypes including nystagmus and foveal hypoplasia 8 . Similarly, Chediak-Higashi syndrome and Hermansky-Pudlak syndrome, caused by the LYST and HPS genes respectively, involve many OA and OCA phenotypic traits. Despite the significant systemic health implications of these forms of syndromic albinism, most patients never undergo genetic testing.
Genes involved in the melanin biosynthesis pathway are known to cause forms of both OA and OCA. Examples include GPR143, which is causal for OA1 9 , whilst TYR, OCA2, TYRP1, SLC45A2, SLC24A5 and C10orf11 are associated with OCA subtypes 1-4 and 6-7 respectively 4 . The OCA1 gene, TYR, is known to be associated with missing heritability 10,11 . Our group and others have previously reported a compound heterozygous tri-alleleic genotype in TYR which involves both rare (AF <5%) and common (AF 28-36%) functionally damaging variants which are likely to be on trans alleles 2,12,13 . The two common TYR variants p.S192Y and p.R402Q, have previously been shown to cause a 40% reduction in tyrosinase activity and protein misfolding, respectively 14,15 .
Pigmentary abnormalities in hair and skin may be apparent during phenotyping, although this is not always the case. Consequently, a range of potential diagnoses could be made in children with nystagmus, particularly between the ages of 4-6 months 10 .
As phenotyping becomes more precise and nuanced in children with nystagmus, it is possible that candidate gene lists can become more specific 16 . For example, an abnormal electroretinogram (ERG) can be the only indication that an underlying retinal dystrophy is the cause of the nystagmus. Hence, a retinal gene panel might be the most appropriate genetic testing option. It also allows potential candidate causal variant(s) to be interpreted with greater confidence. Previous studies of next-generation sequencing in INS patients have utilised large gene panels of up to 300 genes whilst identifying candidate causal variants in a recurrent, small subset of genes [17][18][19] , or in genes for conditions which would have been identified by basic phenotyping. This suggests that pre-selecting and interpreting variants in fewer genes for phenotyped patients, may provide the most efficient workflow and highest diagnostic yield in routine clinical practice.

Aim
We describe the diagnostic yield of a clinically available 31 gene panel for nystagmus and albinism. We evaluate its clinical utility in both completely phenotyped and incompletely phenotyped patients in order to reflect the real-world limitations of phenotyping in the clinic and describe its use for patients presenting to specialist and non-specialist centres. patients. Eighty-one individuals (age range 0-18 yrs) were identified from a regional paediatric nystagmus clinic as having INS with or without clinical features suggesting albinism. All patients referred to a single, regional service were offered recruitment.

Methods
phenotyping. All patients underwent basic phenotyping as outlined in Norman et al. 2 . Briefly, this included history taking, orthoptic examination, age-appropriate visual acuity testing, anterior and posterior segment examinations and ERG. Visual evoked potential (VEP, using flash VEP for younger children and pattern onset for older patients) and optical coherence tomography (OCT) with the Leica OCT system or Spectralis OCT (Heidelberg Engineering) were performed in most patients. Eye movements were recorded in some subjects with the EYElink1000 + (SR research, Ottawa, Ontario, Canada) eye tracker. Patients with a diagnosis of a condition known to cause nystagmus (such as Down syndrome, congenital cataract or Aniridia) or where a specific diagnosis was strongly suspected (such as a cone disorder in a photophobic patient confirmed by ERG) or without an INS phenotype (such as Gaze Evoked Nystagmus, GEN due to cerebellar disease) and those who were born before 35/40 weeks gestation were excluded. For included patients, saliva samples (ORAGENE) were collected and DNA extracted using Oragene-DNA kit (OG-575) (DNA Genotek).
Probands were allocated into four phenotype subgroups; clinically IINS with complete phenotyping (group 1), clinically IINS with incomplete phenotyping (group 2), clinical phenotyping consistent with albinism with complete phenotyping (group 3), and clinical features suggestive of albinism with incomplete phenotyping (group 4) (

Variant prioritisation.
Variants were prioritised into two categories of 'assumed pathogenic' and 'assumed likely pathogenic' . ' Assumed pathogenic' was defined as a variant which had a 'pathogenic' annotation in ClinVar, 'pathogenic' annotation by InterVar or 'disease-causing mutation' (DM) in HGMD. ' Assumed likely pathogenic' was defined as a variant which was: (1) not synonymous; (2) had an allele frequency ≤5% in 1000 Genomes Project (all populations), Exome Sequencing Project 6500 (all populations) and Exome Aggregation Consortium (all populations) and; (3) had either a CADD Phred ≥15 22 or a MaxEntScan ≥|3| 28 . Variants which form part of a single likely causal genotype were identified as 'likely causal' variants whilst multiple possible causal genotypes were identified as 'reportable likely causal' . Sanger sequencing was performed to verify 'likely causal' variants which were miscalled in >10% of individuals in the cohort. A Sanger sequencing primer pair was designed using ' A Plasmid Editor (ApE)' software spanning 19 bp (forward) and 20 bp (reverse) in length.

Results
Quality control. The mean read depth across all samples was 127X (See Supplementary Table S2) with 95.8% coverage at a depth of 20X or greater across the 31 gene panel.

Assumed pathogenic variants.
A total of 46 variants across the 81 participants met the criteria for assumed likely pathogenic genetic variants. For 17 patients (21.0% of the cohort), a total of 24 variants were considered to be likely causal variants (Fig. 1). Likely causal diagnoses were identified in 7 genes (HPS5, PAX6, TYR, OCA2, CACNA1A, CACNA1F and FRMD7) from the 31 gene panel. Twenty-two heterozygous variants that were initially labelled as assumed pathogenic or assumed likely pathogenic were found in genes known to cause recessive disorders in patients without a second identified putative variant.
Assumed pathogenic and likely pathogenic variants. For the remaining 64 patients without a likely causal genotype identified, assumed pathogenic variants together with assumed likely pathogenic variants (n = 89 unique variants) were interpreted for likely causality (Fig. 2).
Individuals with likely causal variants within the PAX6 gene were attributed to two variants within the same codon, p.Q286. However, although these variants initially presented as variants of interest they had high failure rates (miscalls by GATK haplotype caller in 27.2% and 24.7% respectively of the total cohort). Subsequent verification with Sanger sequencing excluded these variants as false positives. For this reason, the two PAX6 variants of the p.Q286 codon and an assumed likely pathogenic HPS6 variant causing a p.W595G substitution (53.1% failure rate) were omitted from Fig. 2.
Nine of the 64 patients (11.1% of the total cohort) had likely causal diagnoses from assumed likely pathogenic variants with a cumulative total of 29 unique variants. Likely causal diagnoses were identified across 6 genes (TYRP1, HPS5, SACS, OCA2, CACNA1A and FRMD7) from the 31  www.nature.com/scientificreports www.nature.com/scientificreports/ variants were found in genes known to cause recessive disorders in patients without a second identified putative variant.
TYR tri-allelic genotypic cause of albinism. The TYR variant, NM_000372.4:exon4:c.G1205A:p.R402Q satisfies our assumed pathogenic criteria as it is assigned as assumed pathogenic in ClinVar despite being very common (17.7% AF in ExAC all populations). This variant is thought to form a part of a tri-allelic phenotype 2,14,15 with another common variant; NM_000372.4:exon1:c.C575A:p.S192Y and any other rare pathogenic TYR variant. Therefore, we consider it here as a unique case. Of the 55 remaining undiagnosed patients, nine were identified to have the tri-allelic genotype within TYR (Fig. 3). These nine patients originated from phenotype groups 3 (n = 6) and 4 (n = 3). Each patient had a minimum of the two common variants, NM_000372.4:exon1:c.C575A:p. S192Y (25.2% in all populations of ExAC) and NM_000372.4:exon4:c.G1205A:p.R402Q (17.7% in all populations of ExAC), and one rare assumed pathogenic variant which is deemed clinically sufficient to call as the molecular basis of albinism.
Albinism patients with partially resolved genetic aetiology. In clinical practice it is common for gene testing to yield one well described pathogenic variant in an OCA gene but the absence of a second variant meaning that a molecular diagnosis cannot be made.
There were 46 patients without an assumed pathogenic, assumed likely pathogenic or TYR tri-allelic genotype identified. Forty-two patients across groups 1 (11), 2 (10), 3 (10) and 4 (15) without genetic diagnoses were subsequently investigated for single heterozygous assumed pathogenic or assumed likely pathogenic variants in an OCA/OA genes (TYR, OCA2, TYRP1, SLC45A2, SLC24A5, C10orf11, GPR143 2 ). Strikingly, sixteen patients had a single assumed pathogenic or assumed likely pathogenic variant which are likely to contribute to an albino genotype (see Supplementary Fig. S1). This corresponds to 2/10 (20.0%), 7/10 (70.0%) and 7/15 patients (46.6%) for of the remaining unresolved cases for phenotype group 2, 3 and 4 respectively. No patients in group 1 had a single assumed pathogenic or assumed likely pathogenic variant in any albinism gene. This is insufficient for www.nature.com/scientificreports www.nature.com/scientificreports/ a clinical diagnosis but warrants further investigation as it strongly suggests other missing variants in albinism genes for these cases.
overview of diagnostic results. Table 2 summarises the number of samples harbouring likely causal variants. Overall, a clinically callable diagnostic yield of 48% was identified for the cohort as a whole. Phenotype groups 1 and 2 had similar diagnostic rates of 38% and 40% respectively. Groups 3 and 4 had a 50.0% and 57.1% diagnostic rate respectively. This shows that the additional phenotyping, beyond that of the baseline examinations prior to patient selection, did not significantly increase diagnostic yield. www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
In this study, we have utilised phenotyping methods which are currently employed in most large ophthalmology clinics worldwide. We recruited unselected, sequential patients in order to report on diagnostic yield using a UKGTN approved clinical panel based on the TruSight One 'clinical exome' panel which is being utilised in many centres as a cross-specialty, high throughput, sequencing platform. We report diagnostic yield for patients falling within the four most common clinical scenarios encountered in clinical practice; (1) complete phenotyped IINS, (2) likely IINS with incomplete phenotyping, (3) well phenotyped albinism and (4) likely albinism with incomplete phenotyping. We demonstrate a diagnostic rate across 81 patients of 43.2%, which is substantially higher than the majority of exome diagnostic analyses with the TruSight One capture 18 and reflects, in part, the necessity for basic initial phenotyping in order to exclude common, clear clinical presentations and the utility of a subpanel of genes taken from a larger 'clinical exome' panel.
Six patients had assumed pathogenic variants in genes that would not have been previously directly implicated in causing the phenotype presentation of the patient according to the available clinical information. For example, NG315 from phenotype group 1 was found to have a likely disease-causing compound heterozygous genotype in the OCA2 gene. These cases reflect the variable, often hypomorphic and overlapping phenotypes seen in children with nystagmus 4,29 , and support the argument that basic phenotyping alone prior to panel testing, may be the most efficient clinical diagnostic workflow rather than reducing the gene panel further by assuming that phenotyping has excluded or confirmed an albinism related phenotype.
The variant prioritisation category of assumed likely pathogenic identified some likely causal variants in genes for which the phenotype seen in our patients is unexpected. For example, NG381 (idiopathic nystagmus with incomplete phenotyping) was found to be homozygous for an assumed likely pathogenic splicing variant in the CACNA1F gene, which is known to cause Aland Island eye disease, cone-rod dystrophy and X-linked incomplete stationary night blindness (CSNB); all of which cause nystagmus and retinal dystrophy. It might be expected that such disorders would be identified by ERGs prior to recruitment to this study, however, this patient's ERG www.nature.com/scientificreports www.nature.com/scientificreports/ result was initially reported as normal. Interestingly, a subsequent ERG performed at an older age for this patient identified the typical features of CSNB. This case and others may support an argument that genomic testing in the future may form an earlier part of the diagnostic workflow. More detailed phenotyping, which has been outlined here and by others 30 , might then be directed towards proving or disproving diagnoses suggested by putative likely causal variants, but it is clear that clinical evaluation and baseline phenotyping will still be required regardless of the yield from genetic testing.
The high number of cases identified here with likely albinism related phenotypes, but for whom only a single albinism gene pathogenic variant was identified, mirrors that seen in clinical practice. It strongly suggests that other, as yet unknown, variants in albinism genes are contributing or that multiple variants across the melanin biosynthesis pathway may combine to contribute to cause albinism phenotypes (epistasis). Some albinism genes such as the C10orf11 gene are less well covered (70.5%) which may miss contributing variants for the albinism phenotype. The TruSight One chemistry could be backfilled to provide a more comprehensive and reliable capture of all target regions. Clearly, identification of these 'missing variants' and complex causal genotypes is likely to increase the diagnostic yield still further.
Nystagmus and albinism gene panels can vary from less than 20 to more than 300 genes 17,19,31 . Here we have used 31 genes taken as a subpanel from the commonly used TruSight One 'clinical exome' gene panel. The value of employing a large off the shelf target kit such as the TruSight One means that by revisiting the data already generated, the gene panel could be expanded retrospectively. The 'TruSight One Expanded v3.0' has also expanded on the original TruSight One capture to cover other known nystagmus-causing genes such as SLC38A8.
It is possible that a diagnostic report may initially include false positives, however, all identified variants which have been identified as 'likely causal' would be verified with Sanger sequencing before clinical reporting.

conclusions
In conclusion, the work presented here shows that for clinicians using a standard set of phenotyping criteria, the UKGTN approved 31 gene panel based on the TruSight One platform, can provide a clinically callable genetic diagnosis for 43.2% of children with INS regardless of more detailed phenotyping. This could significantly reduce the time and number of investigations that many children with nystagmus undergo and permit informed family counselling with regards to recurrence risk. This could also lead to more rapid access to tailored management, a current priority for health systems worldwide, for conditions such as spinocerebellar ataxia 6/episodic ataxia type 2 or Hermansky-Pudlak syndrome. For the future planning of diagnostic workflows and genetic testing strategies, it is important to note that the work here has shown similar diagnostic yield across all four patient groups. This suggests that clinical phenotyping (beyond the exclusion of retinal dystrophy, known underlying ophthalmic disease, prematurity or likely neurological cause) is not a key prerequisite for genetic testing, and that detailed phenotyping could be tailored subsequent to genetic testing in order to confirm or refute putative genetic diagnoses. It is also clear that diagnostic workflows for children with nystagmus should include differing genetic strategies according to the basic phenotyping results or availability of phenotyping modalities. For example, a first-line retinal gene panel for children with a retinal dystrophy identified by ERGs and a first line nystagmus/albinism gene panel for children with a clear albinism phenotype prior to ERG and VEP testing.

Data Availability
Data generated or analysed during this study are included in this published article and its Supplementary Files.