Clinical findings
Patient 1
The mutation, ZNF408, p.G290S, identified in a proband, was associated with a FEVR phenotype. The family, which has one girl (the proband) and unaffected parents, was recruited into the FEVR project specifically because of the fundus appearance mimicking FEVR. The proband was a 13-day-old baby at the time of recruitment.
Patient 2
The mutation in LRP5 involved in splicing, identified in a proband was associated with a stage 4-FEVR phenotype. The family, which has two girls (the proband, and an unaffected elder sister of proband) and unaffected parents, was recruited in the FEVR project specifically because of the stage 4- FEVR. The proband was a 3-year-old girl at the time of recruitment. She had been noted as have retinal abnormalities at age 3 years. She was born full-term with a birth weight of 3.2 kg. The elder sister was examined at age 5 but she is normal.
Patient 3
The mutation in KIF11, p.1423T, identified in proband was associated with a FEVR phenotype. The family, which has two boys (the proband and an unaffected eld brother) and unaffected parents, was recruited into the FEVR project specifically because of stage-1FEVR. The proband was an 8-month-old baby at the time of recruitment. The retinal abnormalities were first noted at age 14 days. He was born full-term with a birth weight of 2.9 kg. There was no family history of health problems compatible with a diagnosis of FEVR, and the mutation was found in his unaffected father and elder brother.
Patient 4
The mutations in NR2E3, p.H361R, identified in a proband were associated with a FEVR phenotype. The family, which has one boy (the proband) and an unaffected elder sister, as well as unaffected parents, was recruited into the FEVR project specifically because of stage-4 FEVR. The proband was a 3-year-old boy at the time of recruitment.
Patient5
The mutation, KRT3, p. R271H, identified in proband was associated with a FEVR phenotype. The family, which has one boy (the proband) and unaffected parents, was recruited in the FEVR project specifically because of stage-4FEVR. The proband was a 2-month-old boy at the time of recruitment.
Patient 6
The FOXL2 mutation, p.333-337del, was identified in a proband was associated with a FEVR phenotype. The family, which has one boy (the proband) and unaffected parents, was recruited in the FEVR project specifically because of stage-4 FEVR. The proband was an 11-month-old boy at the time of recruitment.
Genetic Analysis
WES was initially performed on an affected probands (pedigree I-Ⅵ) to identify a shared disease-causing variant. The reads aligned to the human genome, mapped to the target region with a mean coverage between 99.8 and 99.9%, SNPs and indels are shown in Table 1. Between 11,631 and 11,844 were identified as SNPs from six affected individuals, whereas 833-918 indels were identified in the unaffected individuals. Analysis based on the pattern of inheritance identified common homozygous and heterozygous variants in six affected individuals (Table 1). Among the identified variants, the numbers of selected gene were displayed in Table 1 according to our mutational analysis. Further analysis based on expression profiles identified six candidate variants (Table 1). Among these, the four software packages predicted six of the missense variants to be damaging. After filtering for suspected sequencing artefacts and with a multiple allele frequency greater than 5%, the shared missense or nonsense variants identified were displayed in Table 2.
Sanger sequencing revealed the novel, homozygous missense variants identified in these probands (Fig1 family tree, and Table 3); p.G290S in the ZNF408 gene, splicing in LRP5, p.I423T in KIF11, p.H361R in NR2E3, p.R271H in KRT3, p. 333_337del inFOXL2 segregating with disease in families 1,2,3,4,5,6, respectively (Fig. 2).
In the families (pedigree I-Ⅲ), the remaining heterozygous, ZNF408, LRP5 and KIF11 genes were selected as the candidates because heterozygous mutations in these genes has previously been reported as being causative for FEVR[22]. There were no other siblings (pedigree Ⅲ) and no family history of a condition compatible with a diagnosis of FEVR. The mutation was absent father, mother, and mother in family I,Ⅱ, and Ⅲ,
respectively. In families (pedigree Ⅳ-Ⅵ), NR2E3,KRT3 and FOXL2 were selected as the candidate. The unaffected father (pedigree Ⅳ), mother (pedigree Ⅴ) and mother (pedigree Ⅵ) presented with mutations.
Sanger sequencing identified additional novel heterozygous mutations in 6 probands (Figure 1 family tree).
Gene type, amino acid changes and nucleotide changes of all mutations are shown in Table 3, and the amino acid changes in family2 (LRP5) are in an acceptor splice site.
Additionally, these variants were absent in 60 normal control individuals.