Next-generation sequencing to solve complex inherited retinal dystrophy: A case series of multiple genes contributing to disease in extended families

Abstract

Purpose: With recent availability of next-generation sequencing (NGS), it is becoming more common to pursue disease-targeted panel testing rather than traditional sequential gene-by-gene dideoxy sequencing. In this report, we describe using NGS to identify multiple disease-causing mutations that contribute concurrently or independently to retinal dystrophy in three relatively small families.

Methods: Family members underwent comprehensive visual function evaluations, and genetic counseling including a detailed family history. A preliminary genetic inheritance pattern was assigned and updated as additional family members were tested. Family 1 (FAM1) and Family 2 (FAM2) were clinically diagnosed with retinitis pigmentosa (RP) and had a suspected autosomal dominant pedigree with non-penetrance (n.p.). Family 3 (FAM3) consisted of a large family with a diagnosis of RP and an overall dominant pedigree, but the proband had phenotypically cone-rod dystrophy. Initial genetic analysis was performed on one family member with traditional Sanger single gene sequencing and/or panel-based testing, and ultimately, retinal gene-targeted NGS was required to identify the underlying cause of disease for individuals within the three families. Results obtained in these families necessitated further genetic and clinical testing of additional family members to determine the complex genetic and phenotypic etiology of each family.

Results: Genetic testing of FAM1 (n = 4 affected; 1 n.p.) identified a dominant mutation in RP1 (p.Arg677Ter) that was present for two of the four affected individuals but absent in the proband and the presumed non-penetrant individual. Retinal gene-targeted NGS in the fourth affected family member revealed compound heterozygous mutations in USH2A (p. Cys419Phe, p.Glu767Serfs*21). Genetic testing of FAM2 (n = 3 affected; 1 n.p.) identified three retinal dystrophy genes (PRPH2, PRPF8, and USH2A) with disease-causing mutations in varying combinations among the affected family members. Genetic testing of FAM3 (n = 7 affected) identified a mutation in PRPH2 (p.Pro216Leu) tracking with disease in six of the seven affected individuals. Additional retinal gene-targeted NGS testing determined that the proband also harbored a multiple exon deletion in the CRX gene likely accounting for her cone-rod phenotype; her son harbored only the mutation in CRX, not the familial mutation in PRPH2.

Conclusions: Multiple genes contributing to the retinal dystrophy genotypes within a family were discovered using retinal gene-targeted NGS. Families with noted examples of phenotypic variation or apparent non-penetrant individuals may offer a clue to suspect complex inheritance. Furthermore, this finding underscores that caution should be taken when attributing a single gene disease-causing mutation (or inheritance pattern) to a family as a whole. Identification of a disease-causing mutation in a proband, even with a clear inheritance pattern in hand, may not be sufficient for targeted, known mutation analysis in other family members.

Figure 1

Pedigrees of families clinically diagnosed with autosomal dominant retinitis pigmentosa (adRP) with suspected non-penetrance (FAM1, FAM2) and dominant pedigree (FAM3). Filled symbols indicate diagnosis of RP. Individuals for whom DNA samples were available are indicated with identification (ID) numbers; probands are marked with arrows. ‘-’ indicates the presence of a mutation, and ‘+’’ indicates the presence of a wild-type allele, detailed in Table 1.

Figure 2

ffERG panels for FAM1, FAM2, and FAM3. A: FAM1 unaffected full-field electroretinography (ffERG) cone and rod responses for patient #5908, heterozygous carrier of a mutation in USH2A. Non-detectable ffERG rod response and minimally reduced from normal cone response for patient #2334, compound heterozygous for two mutations in USH2A. B: Three members of FAM2 with reduced-to-minimal rod and cone ERG function. Patient #8438 was found to have two compound heterozygous mutations in USH2A, and patient #10228 had mutations in two autosomal dominant retinitis pigmentosa (adRP) genes, PRPH2 and PRPF8, as well as a single, heterozygous mutation in USH2A. Patient #10534 had a mutation in one adRP gene, PRPF8, as well as a single, heterozygous mutation in USH2A. C: Four members of FAM3 with varying degrees of ffERG dysfunction. Proband #5250 shows moderately reduced rod and cone responses and was found to harbor mutations in two autosomal dominant genes, PRPH2 and CRX. The proband’s son, #10396, had minor ffERG changes at age 8 years and was found to carry the mutation in CRX. Two additional family members, #6275 and #6121, carry only a single mutation in PRPH2 and show reduced-to-minimal ffERG responses.

Figure 3

Fundus and SD-OCT images from individuals in three families diagnosed with inherited retinal disease. Representative central views of the left eye and the corresponding spectral domain optical coherence tomography (SD-OCT) image are shown.