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

Abstract

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.

Figure 1

Seventeen of 81 patients with assumed pathogenic variants were determined to harbour likely causal genotypes. Samples, blue background indicates male whilst pink indicates female, orange cells indicate heterozygous variants and red cells indicate homozygous and hemizygous variants. Samples are ordered by phenotypic group, and cells with a ‘C’ denote assignment of a likely causal variant. The TYR variant, NM_000372:exon4:c.G1205A:p.R402Q (bold) would fail the MAF filter detailed above (17.7% AF in ExAC all populations) for putative variants but is highlighted here as it is listed as ‘pathogenic’ in ClinVar and is of relevance to subsequent work in this publication. Chrom, chromosome; Position, location of 5′ base of variant in hg38; Ref, reference allele; Alt, alternative allele; Variant type, consequence of the variant (s = synonymous, ns = nonsynonymous, sp = splicing, sg = stopgain); Gene.refGene, gene symbol; Omim Inheritance, inheritance as listed on OMIM for the gene in OCA/nystagmus; Amino acid, amio acid change; avsnp144, dbSNP144 rsID; ExAC ALL, Alternate allele frequency from ExAC database (all populations); CADD Phred, Combined Annotation Dependent Depletion score (Phred scale); MaxEntScan diff, the difference in score between MaxEntScan reference allele and alternative allele; ClinSig, clinical significance (clinvar) annotated ‘p’ if ‘pathogenic’; InterVar, annotated as ‘p’ if identified as ‘pathogenic’ by InterVar; HGMD 2016 CLASS, annotated as DM for disease-causing mutation; Variant category, 1 = assumed pathogenic, 2 = assumed likely pathogenic.

Figure 2

Nine patients with assumed likely pathogenic variants determined to be likely causal. For 64 patients investigated for likely causal genotypes by investigating variants which were assumed likely pathogenic or as a combination of assumed pathogenic and assumed likely pathogenic, 13 patients were determined to have likely causal genotypes. Samples, blue background indicates male whilst pink indicates female, orange cells indicate heterozygous variants and red indicates homozygous variants. Samples are ordered by phenotypic group, and cells with a ‘C’ denotes a likely causal variant,‘R’ denotes a reportable variant, ‘-’ denotes variant miscall. Chrom, chromosome; Position, location of 5′ base of variant in hg38; Ref, reference allele; Alt, alternative allele; Variant type, consequence of the variant (s = synonymous, ns = nonsynonymous, sp = splicing, sg = stopgain); Gene.refGene, gene symbol; Omim Inheritance, inheritance as listed on OMIM for the gene in OCA/nystagmus; Amino acid, amio acid change; avsnp144, dbSNP144 rsID; ExAC ALL, Alternate allele frequency from ExAC database (all populations); CADD Phred, Combined Annotation Dependent Depletion score (Phred scale); MaxEntScan diff, the difference in score between MaxEntScan reference allele and alternative allele; ClinSig, clinical significance (clinvar) annotated ‘p’ if ‘pathogenic’; InterVar, annotated as ‘p’ if identified as ‘pathogenic’ by InterVar; HGMD 2016 CLASS, annotated as DM for disease-causing mutation; Variant category, 1 = assumed pathogenic, 2 = assumed likely pathogenic.

Figure 3

Nine samples were identified to have a likely causal tri-allelic genotype for albinism within TYR. For 55 patients which did not have likely causal genotypes with assumed pathogenic or assumed likely pathogenic variants, nine were identified to have tri-allelic causal genotypes in TYR. All TYR variants identified as assumed pathogenic or assumed likely pathogenic with the addition of S192Y and R402Q are listed. Position, location of 5′ base of variant in hg38; Alt, alternative allele; Variant type, consequence of the variant (s = synonymous, ns = nonsynonymous, sp = splicing, sg = stopgain); AAchange; avsnp144, dbSNP144 rsID; ExAC ALL, Alternate allele frequency from ExAC database (all populations); CADD Phred, Combined Annotation Dependent Depletion score (Phred scale); MaxEntScan diff, the difference in score between MaxEntScan reference allele and alternative allele; ClinSig, clinical significance (clinvar); InterVar, pathogenicity category according to InterVar interpretation; HGMD 2016 class, HGMD annotation for pathogenicity; Samples, orange indicates heterozygous variants, red indicates homozygous variants. Samples are ordered by phenotypic group, ‘c’ was used to indicate a likely causal variant, and grey highlights the common variants involved in the tri-allelic genotype outlined by Norman et al. (S192Y and R402Q).