Retinitis pigmentosa (RP) is a heterogeneous set of inherited retinopathies with many disease-causing genes, many known mutations, and highly varied clinical consequences. Progress in finding treatments is dependent on determining the genes and mutations causing these diseases, which includes both gene discovery and mutation screening in affected individuals and families. Despite the complexity, substantial progress has been made in finding RP genes and mutations. Depending on the type of RP, and the technology used, it is possible to detect mutations in 30–80% of cases. One of the most powerful approaches to genetic testing is high-throughput ‘deep sequencing’, that is, next-generation sequencing (NGS). NGS has identified several novel RP genes but a substantial fraction of previously unsolved cases have mutations in genes that are known causes of retinal disease but not necessarily RP. Apparent discrepancy between the molecular defect and clinical findings may warrant reevaluation of patients and families. In this review, we summarize the current approaches to gene discovery and mutation detection for RP, and indicate pitfalls and unsolved problems. Similar considerations apply to other forms of inherited retinal disease.

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Restriction fragment length polymorphisms (RFLP) analysis using the Southern blot technique can be used to recognize copy number variation of variable number of tandem repeats (VNTR) of conserved core sequences at several regions of the human genome. This new class of polymorphisms reveals a high degree of genetic variation, useful for individual identification purposes. Criticisms against forensic applications of such DNA typing data include the limitation of employing Hardy-Weinberg expectation of genotype frequencies, since several surveys indicate apparent deficiency of heterozygosity (or excess homozygosity) in comparison with Hardy-Weinberg expectations. This research postulates an alternative explanation of deficiency of apparent heterozygosity which is caused by the inability to detect extremely small-sized alleles (called 'non-detectable' alleles) due to the sensitivity of Southern gel electrophoresis. We show that the presence of 'non-detectable' alleles can produce pseudo-homozygosity and their frequencies can be predicted from the observed proportional heterozygote deficiency. Furthermore, in the covert presence of such 'non-detectable' alleles, we show that the gene-count method provides over-estimates of allele frequencies in the sample population, and hence the Hardy-Weinberg predictions of genotype frequencies avoid wrongful bias against suspects in forensic applications of DNA typing data. Applications of this theory to population data on six VNTR loci in US Caucasians and US Blacks suggest that the presence of 'non-detectable' alleles could be the major cause of apparent heterozygote deficiency, and the current approaches of predicting the population frequency of specific DNA phenotypes are practically free of the possible wrongful bias in courtroom applications of DNA typing data.

Retinitis pigmentosa (RP) is a devastating form of retinal degeneration, with significant social and professional consequences. Molecular genetic information is invaluable for an accurate clinical diagnosis of RP due to its high genetic and clinical heterogeneity. Using a gene capture panel that covers 163 of the currently known retinal disease genes, including 48 RP genes, we performed a comprehensive molecular screening in a collection of 123 RP unsettled probands from a wide variety of ethnic backgrounds, including 113 unrelated simplex and 10 autosomal recessive RP (arRP) cases. As a result, 61 mutations were identified in 45 probands, including 38 novel pathogenic alleles. Interestingly, we observed that phenotype and genotype were not in full agreement in 21 probands. Among them, eight probands were clinically reassessed, resulting in refinement of clinical diagnoses for six of these patients. Finally, recessive mutations in CLN3 were identified in five retinal degeneration patients, including four RP probands and one cone-rod dystrophy (CRD) patient, suggesting that CLN3 is a novel non-syndromic retinal disease gene. Collectively, our results underscore that, due to the high molecular and clinical heterogeneity of RP, comprehensive screening of all retinal disease genes is effective in identifying novel pathogenic mutations and provides an opportunity to discover new genotype-phenotype correlations. Information gained from this genetic screening will directly aid in patient diagnosis, prognosis, and treatment, as well as allowing appropriate family planning and counseling.

Together, the known adRP genes account for retinal disease in approximately half of the families in this survey, mostly Americans of European origin. Among the adRP genes, IMPDH1, PRPF8, PRPF31, RDS, RHO, and RP1 each accounts for more than 2% of the total; CRX, PRPF3, and RPGR each accounts for roughly 1%. Disease-causing mutations were not found in CA4, FSCN2, NRL, or RP9. Because some mutations are frequent and some regions are more likely to harbor mutations than others, more than two thirds of the detected mutations can be found by screening less than 10% of the total gene sequences. Among the remaining families, mutations may lie in regions of known genes that were not tested, mutations may not be detectable by PCR-based sequencing, or other loci may be involved.