inherited retinal dystrophies ( irds ) , characterized by retinal involvement as the main clinical presentation , comprise many overlapping conditions , such as retinitis pigmentosa ( rp ) , leber congenital amaurosis ( lca ) , and stargardt disease .
the disease incidence for rp is 1/35004000 people in the usa and europe ; for stargardt disease , it is 1/800010,000 individuals and for lca it is 23/100,000 newborns . in most cases ,
however , additional tissues / organs can be involved ( syndromic conditions ) , and it can be difficult to assign a precise clinical diagnosis of non - syndromic versus syndromic conditions . for some syndromic conditions , such as juvenile batten disease , retinal dystrophy is the earliest clinical sign , and variable phenotypes are not uncommon .
in addition , the presence of some clinical signs can be co - incidental , such as the presence of both rp and hearing loss in patients not affected with usher syndrome , which may reflect the relatively high incidence of these diseases. even for non - syndromic irds , assigning a definitive clinical diagnosis can be difficult .
clinical presentations can be overlapping and/or progressive , and it may take several decades for the full disease spectrum to manifest . moreover , intrafamilial variability is not uncommon , to the extent that family members with the same causative mutation(s ) can show different phenotypes .
mutations in particular genes can be causative for both non - syndromic and syndromic conditions . for instance , cep290 mutations have been identified in patients with lca ( non - syndromic ) , bardet - biedl ( bbs ) , joubert , meckel , and senior - lken   syndromes , while ush2a mutations can manifest as either usher syndrome type ii or non - syndromic rp .
the large number of genes identified in irds ( locus heterogeneity ; > 200 genes )   has complicated molecular diagnosis , and thus , the clinical utility of target sequencing only the more common rp genes is limited .
in fact , the overall mutation detection rate for many ird - associated genes is very low , except for conditions such as stargardt disease and achromatopsia where disease - specific panels are successfully utilized due to the limited number of genes involved .
furthermore , even though certain genes implicated in irds are associated with specific inheritance modes ( autosomal recessive , autosomal dominant , or x - linked ) , the difficulty in reliably establishing an inheritance mode for certain patients makes targeted gene sequencing less effective in these instances .
although over 4000 mutations have been characterized in 200 ird - associated genes , in general , their prevalence is low .
the existence of multigenic inheritance patterns and genetic modifiers further obscure molecular diagnoses , with evidence for phenotypic variability due to complex genetic architectures emerging for irds . in the case of usher syndrome
, pdzd7 is implicated as a retinal disease modifier , and a contributor to digenic disease in combination with mutations in ush2a and gpr98 
  and also dfnb31 
 .
the interaction of the bbsome components ( complex of 7 bbs proteins ) with cep290 in cep290-related ciliopathies amply illustrates the complexity of multigenic traits , and partly explains both inter / intrafamilial phenotypic variability and phenotypic overlap among distinct syndromes .
locus and allelic heterogeneities therefore limit the effectiveness of targeted strategies , as many pathogenic variants go undetected . taken together with multigenic inheritance patterns and genetic modifiers , sequencing all known genes implicated in irds simultaneously seems the best current approach for the definitive molecular diagnosis of irds , and next - generation sequencing ( ngs ) has revolutionized this field
. this review will compare ngs with other molecular diagnostic strategies for irds with a particular focus on the challenges ahead .
before the arrival of ngs , when sanger sequencing was routinely performed , common approaches to molecular diagnosis included : targeted sequencing of selected genes ; screening known mutations in a large number of ird - associated genes by array ; and screening mutations by pcr and single strand conformation polymorphism .
targeted ngs of ird - associated genes was established in 2012 [ 1417 ] , and in just 3 years ngs has become the preferred testing method for ird patients .
while the unparalleled specificity of pcr and the  to - see - is - to - believe method of sanger sequencing have long been the benchmark for molecular diagnosis of irds , the arrival of ngs has dramatically changed this field .
the illumina ngs sequencing platform is becoming the de facto method for laboratories working on rare genetic diseases , with target enrichment one of the most critical steps .
the introduction of hybridization - based enrichment methods , such as agilent sureselect reagents , has markedly simplified enrichment procedures .
hybridization baits from any number of genes can now be ordered from several vendors . however ,
the gold standard for target enrichment in the era of sanger sequencing has been pcr - based , and it offers unmatched target specificity . however , three major factors limit its role in this ngs 
gold rush : primer design has mostly been a slow , labor - intensive process .
the rule of thumb for automation , especially with respect to complicated biological systems , is that it can never be perfect .
this is true for pcr primer design and for the designing of baits for the capture method . making redundant primers when targeting large numbers of genes can significantly increase costs .
the presence of snps in the primer regions may increase the chance of allele dropout .
manual pcr set - up is laborious and error - prone , and primer maintenance and storage requires additional effort . when large numbers of amplicons are sequenced , the task becomes increasingly labor intensive and less manageable .
the arrival of a liquid - phase , hybridization - based method , and other modified methods , have eliminated the preceding first and third factors .
the single tube method can streamline enrichment , and is especially desirable for clinical laboratories .
however , users should be aware of its limitations ; non - specific enrichment and enrichment of homologues and pseudogenes can not be avoided , and the on - target sequencing rate is lower than the more specific pcr - based method .
both non - specific and false - positive variants are then filtered out during data analysis .
this approach of indiscriminate sequencing followed by variant filtering is at the core of hybridization - based enrichment methods .
however , to increase coverage of target regions to , for example , 95% , the sequence depth is usually lowered to , approximately , 20. based on our experience , when the sequence depth is lowered , additional false - positive variants appear . to eliminate these
we believe this is one of the most common causes of false negativity . however , there is currently no consensus as to what the best sequence depth rate should be . to add complexity
, illumina offers several different ngs sequencers ( miseq , nextseq , and hiseq ) .
the balance of cost / run time with test specificity / sensitivity is a matter of judgment for each laboratory .
about 5 years ago , wes was heralded as the  panel killer and promoted as the one - stop testing platform for rare genetic diseases .
this expectation seemingly stemmed from widespread frustrations associated with testing rare genetic diseases , given that clinical diagnoses are not infrequently incorrect [ 2022 ] . for
a patient with differential diagnoses , running entire panels for multiple diseases can be cost - prohibitive .
therefore , the arrival of a simple , comprehensive testing step ( wes ) was particularly attractive .
the uneven performance of wes , however , has limited its clinical utility , and it was clearly an intermediary before whole genome sequencing ( wgs ) .
from a cost perspective , wes was favorable , but this is diminishing as wgs is becoming cheaper . at this time , limiting analysis to less than whole exomes , such as medically relevant exomes , is popular for some clinical testing .
the current consensus about wes is that it can be an alternative approach after exhausting the more specific panel testing .
the rapidly declining cost for wgs may see it replace wes as an alternative approach in the foreseeable future .
the first public record of us$1000 genomes was in december 2001 ; when it will finally become a reality in clinical practice is purely speculative at this time .
following the history of wes in the clinical market , it is safe to say that clinical sequencing is at least several years behind research sequencing .
it is difficult to argue that wgs is a viable or sustainable business model currently for the clinical market , even with the introduction of hiseq x 10 from illumina .
the true cost from a stand - alone and sustainable , commercial molecular diagnostic laboratory is much higher than the advertised cost of sequencing a whole genome today .
junk dna , which constitutes > 90% of our genome , has major biological functions . for conditions such as irds ,
clinical presentations are relatively specific and the total number of genes involved is currently approximately 230 . sequencing all known genes ( at least the coding regions ) implicated in irds simultaneously will certainly reach the us$ 1000 milestone in the near future .
ultimately , sequencing the whole genome and extracting specific data for analysis will make more economic sense for irds .
. however , due to the large number of ird - associated genes , sequencing all known genes simultaneously will require thousands of pcrs per patient , and is thus unsuitable in the clinical setting .
multiplex pcr can potentially reduce the workload : however , designing a multiplex pcr platform on this scale is difficult to achieve as pcrs compete less favorably in a multiplex environment .
recently , a lab - on - a - chip ( smartchip ) platform was developed to run up to 5184 simplex pcrs .
the pcr primer set is printed in each individual well , and duplicated to avoid allele dropout .
smartchips are manufactured and packed individually and stored at room temperature , simplifying pcr set - up to the addition of reagents onto the chips , with no requirement for primer storage or maintenance .
for example , our rd smartpanel ( version 7 ) covers 233 genes with 4658 simplex pcrs duplicated on two smartchips . on average , six low coverage regions ( < 100 sequencing depth ) occur , which are compensated for by manual pcr and sanger sequencing .
our stargardt / macular dystrophy panel covers 10 genes with 551 simplex pcrs duplicated on one smartchip .
entire coding regions from all 10 genes and the reported deep intronic mutations in abca4 are 100% covered ( > 100 sequencing depth ) . this new approach offers unprecedented advantages over traditional pcr set - up , multiplex pcr set - up , and capture / hybridization - based methods . a simple , specific , comprehensive , reproducible , and reliable platform is important to improve mutation detection rates .
therefore , smartpanels are a fast , reliable , and less expensive first - line screening tool
. there are three major limitations for smartpanel analysis : insertion / deletion mutations can not be reliably detected by pcr enrichment unless deletions are homozygous ; mutations outside coding / target regions are not sequenced unless specific primer sets are designed to amplify mutation - specific regions ( e.g. , abca4 deep intronic mutations ) ; and the extremely repetitive region in rpgr orf15 can not be sequenced by specific pcr amplification of < 500 bp amplicons .
array cgh analysis for the detection of insertion / deletion mutations and specific methods to sequence rpgr orf15 are therefore still required for comprehensive testing .
until recently , the clinical utility of molecular diagnoses was limited owing to factors including : the absence of impending therapeutic options ; the time and cost of testing methods ; and the use of hypothesis - driven approaches , for which 
success was impeded by the limited number of known ird - associated genes , the variable confidence in diagnoses and/or inheritance patterns and the difficulty in interpreting variant pathogenicity .
irds are particularly amenable to gene therapy , and today many clinical trials are in progress , for which knowledge of the causative gene is required .
this is also true for family planning options , such as pre - implantation genetic diagnosis .
moreover , identifying the disease mechanism in early stage disease , and in syndromic cases where ird is the first clinical sign , can reduce medical costs and patient anxiety by avoiding unnecessary testing , resulting in better patient management .
the molecular diagnosis of ird patients therefore represents an excellent model for cost savings , and will undoubtedly become increasingly popular . in the past few years , the generation of fast , powerful , and cost - effective gene discovery and mutation detection methods , in addition to the development of variant pathogenicity assessment tools , has markedly increased the clinical utility of molecular diagnoses . with > 230 ird - associated genes identified , the non - hypothesis - driven approach of testing all known ird - associated genes by targeted - capture ngs is now the method of choice , often revealing unsuspected disease mechanisms .
correspondingly , the simultaneous sequencing of a large number of genes has resulted in increased detection of variants of unknown significance , which require interpretation for clinical purposes .
the development of databases such as clinvar   and wes variant allele frequency by exac browser   have gradually improved variant interpretation .
similarly , programs such as sift , polyphen-2 , and nnsplice   are now widely used to predict the influence of a variant on protein localization , structure , and/or function . however , in silico predictions are not always consistent with functional studies and , despite recent advances , pathogenicity assessment remains challenging , particularly for hypomorphic , synonymous and non - coding variants .
ultimately , better tools are required , as well as improved knowledge of gene regions and , in the case of wes and wgs , genes of unknown significance . despite the potential benefits ,
several ethical issues remain . the generation of new guidelines and legislation has not kept pace with the rapid advances in mutation detection discovery .
for example , there are currently no restrictions on testing asymptomatic minors for later onset conditions . in this context ,
incidental findings are also of considerable concern , particularly for wes and wgs , and increasingly for specific panel testing with the progressive addition of syndromic genes . at the laboratory level , while there are arguments against strict regulation in some contexts ( such as for rare genetic conditions where not enough controls exist to validate tests ) , a general consensus and additional guidelines are required
. the recent release of reference dna by national institute of standards & technology ( nist ) is a good starting point , enabling clinical laboratories to sequence the same reference dna for comparison .
however , as each clinical testing laboratory still sets its own criteria , including for coverage , cutoff threshold , variant filtering , and variant calling , more work is clearly required to maximize the potential of molecular diagnoses for greater clinical utility .
overall , the progress in molecular diagnosis of irds is unprecedented , with the arrival of ngs opening a new frontier .
wes , wgs , and specific panel sequencing each have their own pros and cons ( table 1 ) , and there is still no single platform offering the ideal solution today , especially when cost is factored in .
insurance coverage is not available in many cases , mainly because the clinical utility is yet to be proven . even with this limitation , an exciting trend is emerging .
cln3 , the gene implicated in juvenile batten disease , is also mutated in some patients with non - syndromic ird .
similarly , bbs1 , implicated in bardet  biedl syndrome , is also mutated in some patients with non - syndromic ird .
it is probably true that most of the common ird - associated genes are already identified , and some patients may in fact have mutations in recognized syndromic genes , which have never been analyzed in non - syndromic patients .
alternatively , mutations can also be present outside typically sequenced regions , such as the deep intronic abca4 mutations , or can not be identified through a pcr - based approach , such as insertion / deletion mutations .
moreover , unconventional mutation mechanisms relating to genetic modifiers , and multigenic and multiallelic inheritance will need to be more systematically studied .
nonetheless , even with the current mutation detection rate of between 50 and 60% , this is already a very significant improvement from only a few years ago . 

ngs : next - generation sequencing ; sscp : single strand conformation polymorphism ; wes : whole exome sequencing ; wgs : whole genome sequencing .
any attempt to predict the status 5 years from now will most likely be somewhat off the mark .
it is likely that in 5 years , if not sooner , wgs will be where wes stands today . in our opinion , wes served as an intermediary between the unleashing of sequencing power and the full implementation of wgs , and we predict it will most likely lose its appeal as a diagnostic platform .
pcr - based enrichment may still have a role to play , but the extraction of target genes will likely happen after sequencing the whole genome ( selective analysis of a set of genes ) , unless there is a significant pricing advantage for pcr enrichment .
technological advancements will most likely be the catalyst for lowering costs and improving accessibility of testing .
large - scale genotype  phenotype correlation studies will be required to unravel complex genetic mechanisms for some ird patients .
the concept of a single - gene disease will be challenged , and many genetic modifiers will be identified . as a result
, the value of molecular diagnosis as a first - line diagnostic tool may be truly appreciated , especially in less developed countries with limited retina specialists . even in developed countries ,
the many benefits of molecular diagnosis are likely to elevate it to a standard medical diagnostic procedure for irds .
 next - generation sequencing , also known as massively paralleled sequencing , offers an unprecedented opportunity to study disease mechanisms of inherited retinal dystrophies : a dramatic change from a few years ago . 
 the specific involvement of the retina and the manageable number of genes to sequence make inherited retinal dystrophies an attractive model to study genotype  phenotype correlations . 
 costs are reducing rapidly and the current overall mutation detection rate of approximately 60% offers real potential for personalized medicine and treatments . 
 this report addresses the challenges ahead , which include : better understanding of the mutation mechanisms of syndromic genes in apparent non - syndromic patients ; finding mutations in patients who have tested negative or inconclusive ; better variant calling , especially for intronic and synonymous variants ; more precise genotype  
 phenotype correlations and making genetic testing more broadly accessible . 
The diagnostic challenges of inherited retinal dystrophies
Sanger sequencing versus NGS
Whole exome sequencing
Panel sequencing still makes sense today but whole genome sequencing is coming
Smartpanel  a high throughput & fast enrichment method based on simplex PCR
The clinical utility of molecular diagnoses for IRD patients is becoming clearer
Expert commentary
Five-year view