Identification of novel mutations by targeted exome sequencing and the genotype-phenotype assessment of patients with achromatopsia

Achromatopsia (ACHM) is an early-onset and mostly stationary retinal dystrophy characterized
by amblyopia (severely reduced visual acuity), pendular nystagmus, photophobia, and
reduced or complete loss of color discrimination 1]. Currently, the diagnosis of ACHM is based on medical history, typical clinical manifestations,
color discrimination problems in color vision testing, absent or reduced photopic
(cone) responses and normal scotopic (rod) responses in electroretinograms (ERGs),
and the presence of a normal fundus or only minor changes upon fundus photography
2].

To date, five causative genes (CNGA3, CNGB3, GNAT2, PDE6C and PDE6H) have been identified in ACHM patients 1], 3]–6]. These genes encode crucial components of the cone phototransduction cascade. Mutations
in CNGA3 and CNGB3, which encode the ?-subunit and ?-subunit of the CNG, account for 25 % and 40–50 %
of affected individuals in multiple ethnic groups, respectively 3], 7]. Mutations in CNGA3 are considered the most common cause of ACHM and cone-rod dystrophies (CORDs) in
Chinese, in which only cone photoreceptors are usually affected, although CNGA3 mutations have been reported in a patient with CORDs and Leber congenital amaurosis
(LCA) 8], 9]. However, approximately 20–30 % of ACHM cases appear to lack pathogenic mutations,
which is likely due to the limitations in the number of screened regions 1], 3]–5], 10].

Traditional techniques using Sanger sequencing for molecular diagnosis have several
limitations, including being time-intensive and inconvenient for large scale analysis.
Next-generation sequencing (NGS) has been shown to identify variants rapidly and systematically
on an extremely large scale, which has greatly accelerated the development of gene
discovery and molecular diagnosis 11]. NGS is increasingly being used for discovery of causative genes of Mendelian diseases
and for genetic diagnosis 12], 13]. Targeted exome sequencing (TES) is an efficient method of NGS based on custom designed
capture panels. TES can be used to identify disease-causing genes and to screen for
mutations in hundreds of loci in genetically heterogeneous diseases. TES is less costly
than whole genome sequencing (WGS) for mapped chromosomal regions, as well as for
whole exome sequencing (WES). Thus, TES has several advantages compared to other approaches
for reducing costs while enriching for discovery of highly penetrant variants 14], and for identifying pathogenic mutations with respect to both efficiency and accuracy.
To date, some studies performed WES and detected causative mutations of ACHM 15], 16], but these lacked the TES approach to aid in molecular diagnosis of ACHM. Considering
the enormous genotypic and phenotypic heterogeneity of inherited retinal dystrophies,
there remains great potential to discover novel mutations or genes. In our study,
we performed TES to screen 201 disease-causing genes of inherited retinal dystrophies
in two families with ACHM, and discovered novel disease causative mutations, which
broaden the spectrum of ACHM in Chinese.