Clinical summary
An unrelated natural couple who gave birth to two affected siblings continuously (Fig. 1a) came to the outpatient for genetic counseling. The proband is a 4 years old boy with nystagmus and delayed intelligence development, and he has a 10 years old brother shares similar features (Fig.1b, c). Axial brain magnetic resonance imaging (MRI) was performed for him and hypoplastic cerebellar vermis, abnormal fourth ventricle, elongated superior cerebellar peduncles, and so-called “molar tooth sign” (MTS) were found (Fig.1d, e). Thus, we initially diagnosed this patient with JS depending on the specific clinical characteristics. There was no phenotypic abnormality discovered in their parents.
Molecular genetic analysis
To elucidate the underlying genetic cause of JS, we performed WES on this family. A heterozygous nonsense mutation (c.5953G>T [p.E1985*]) in CEP290 gene, a known causative gene of JS, was identified in the proband, his affected brother and his mother but not the father. Furthermore, there was no report about this variation in any database, including ExAC browser, 1000 genome project, or genomAD. In addition, this mutation site is 100% highly conserved in many species (Fig. 2a). However, the nature of JS associated with CEP290 is an autosomal recessive inheritance, so we considered there should be another mutation of CEP290. We further analyzed the CEP290 variants in the WES data, and strikingly, a suspected 65.97-kb deletion in 12q21.32 (88523465-88589431) was found in the two affected individuals and their father but absent in the mother (Fig. 2b), which covered exon 1 to 10 of CEP290. Furthermore, this CEP290 deletion was confirmed by chromosome CGH analysis. Intriguingly, a heterozygous 29.8-kb deletion in 12q21.32 (88525732-88823847) was detected out, which gives rise to exon 1 to exon 6 deletion of CEP290 (Fig. 2c). In view of the coding sequence (CDS) region of CEP290 started in exon 2, deletions found through these two methods both cause a complete translation deficiency. These findings concluded that the biallelic loss-of-function of CEP290 variations were the genetic cause of JS in this family.
The identification and negative effect of CEP290 mutations in the JS family
To understand the putative contribution of the point mutation (c.5953G>T [p.E1985*]) to the proband's phenotype, this mutation was validated by Sanger sequencing in this family, and was observed in the proband, his affected brother, and their mother, meanwhile which was absent in their father (Fig. 3a). This variation resulted in a premature stop codon in transcribed mRNA (NM_025114.3, exon 43) and thus a truncated CEP290 protein, which leads to the missing of CC (coiled-coils) domain XIII, P-loop (ATP/GTP-binding site motif A) domain, KID (RepA/Rep+ protein kinase interaction domain) IV- VI(15) which results in the loss of function of CEP290 protein (Fig. 3b).
To verify the CNV, quantitative PCR was performed to analysis genome relative copy number of exon 1, exon 2, exon 6, exon 7, exon 10 and exon 11 of CEP290 in the family. Results showed that compared to the mother, the father and the siblings only had half-fold copy for exon 1, exon 2, exon 6, exon 7 and exon 10, meanwhile exon 11 were equivalent (Fig. 3c). Furthermore, leukocyte mRNA levels of CEP290 also revealed significant decrease in the two siblings compared to their parents by RT-PCR (Fig. 3d). Thus, the novel biallelic loss-of-function variants in CEP290 could reduce CEP290 expression seriously and further caused JS in these two siblings.