Clinical features
The proband was a 31-year-old Chinese Han male patient who was admitted with the chief complaint of “poor vision for 29 years, walking imbalance for 2 years.” He was born to normal non-consanguineous parents and his parents denied any family history of genetic disorders. His critical developing points are as follow: head raising at 3 months, sitting at 6 months, standing at 14 months, and walking at 24 months. At the age of 2 years, he was demonstrated difficult to track and find toys. At the age of 5 years old, he was diagnosed with macular degeneration because of night blindness and findings of fundoscopy at an ophthalmic clinic. Concurrently, his naked eye vision was 0.1 OD and 0.09 OS, and the corrected visual acuity was about 0.5 in both eyes. Subsequently, he was treated with vitamin B12, B1, and B6 during the following 24 years; his visual acuity remained stable but the visual field continued to become narrower. He also experienced intention tremors and walking imbalance in both extremities from the age of 29 years. He couldn’t ride a bicycle because of the narrowed visual field but he can walk down the stairs without holding rails. There were no other symptoms of sensory neuropathy and vestibular dysfunction. Nonetheless, he exhibited normal intelligence and is currently working as a bank staff. He had no Polydactyly.
Physical examination on admission: The patient was conscious, fluent in language, normal in intelligence, tubular visual field, decreased smooth pursuit, and slight nystagmus on bilateral gaze. Although the muscle strength and tension were normal, he has slight intention tremor on finger—nose pointing, unstable at toe -heel walking and straight walking. Minimal-Mental Status Examination (MMSE) score was 30/30, and Montreal Cognitive Assessment (MoCA) score was 29/30 (which was normal). Blood tests, including complete blood count, liver and kidney function, electrolytes, creatine kinase, and lactic acid, were normal. Other examinations, including electrocardiogram, electroencephalogram, abdominal and kidney ultrasound, were normal.
Ophthalmology examinations showed tubular visual field, retina pigmentation on Fundus photograph, retinal nerve fiber layer thinning on optical coherence tomography (OCT) (Fig. 1A–D), which were signs of retinal dystrophy. Brain magnetic resonance imaging (MRI) showed a molar tooth sign (MTS) in the midbrain and cauda cerebelli hypoplasia (Fig. 1E). Based on the clinical characteristics of poor vision, night blindness, tubular visual field, intention tremor and imbalance, combined with retina pigmentation and molar tooth sign on brain MRI, the proband was clinically diagnosed as JS.
Molecular analyses
Total genomic DNA was extracted from the blood of proband and his parents using a DNEasy Blood and Tissue Kit (Qiagen, Hilden, Germany). The genomic DNA of the proband was enriched for coding exons using Agilent SureSelect Exon capture Kit and sequenced on Illumina HiSeq X Ten platform. The WES data were analyzed for single-nucleotide variants and insertion–deletion polymorphisms using Genome Analysis Toolkit (GATK). The pathogenicity of the detected mutations was analyzed using Mutation Taster (http://www.mutationtaster.org/), SIFT (http://sift.jcvi.org/) and PolyPhen-2 (http://genetics.bwh.harvard. edu/pph2/). The results revealed two compound heterozygous variants at NM_017651.4: c.2105C>T (p.T702M) (Fig. 2A) and c.1330A>T (p.I444F) (Fig. 2A) in the AHI1 gene. The c.2105C>T variant was inherited from the unaffected mother, and the c.1330A>T variant was inherited from the unaffected father (Fig. 1A). According to the American College of Medical Genetics and Genomics (ACMG) guidelines [10], both the c.2105C>T and the c.1330A>T mutations were predicted to be of uncertain significance. However, c.2105C>T has been previously reported as pathogenic (rs756276537) in the compound heterozygous pattern with c.903_910insAp.T304fs*309 [11] or alongside another pathogenic variant (p.E281*) [12]. On the other hand, c.1330A>T mutation has not been reported in the popular public databases, but its pathogenicity could be predicted by Mutation Taster, SIFT and PolyPhen-2. The CADD phred score was 28.2.
Protein structure prediction
3D structure homology modeling of Jouberin protein encoded by the wild-type AHI1 gene (NM_017651.4) and mutated AHI1 gene was predicted using I-TASSER (https://zhanglab.ccmb.med.umich.edu/I-TASSER-MR/), and the architecture was visualized using PyMOL (PyMOL Molecular Graphics System, Version 1.5, Schrödinger, LLC). Homozygous mutation of c.2105C>T (p.T702M) could cause the replacement of a hydroxylic polarly amino acid threonine by a sulfuric non-polarly methionine. The difference of biochemical property of the amino acids would influence protein-folding, which resulted in local 3D structure from β-sheet to D-loop (Fig. 2B), and also decreased the protein stability (ΔΔG of − 0.82 kcal/mol) based on the result of I-Mutant (http://gpcr2.biocomp.unibo.it/cgi/predictors/I-Mutant3.0/I-Mutant3.0.cgi). For the mutation c.1330A>T (p.I444F), even amino acid Ile and Phe are both nonpolar and neutral charged, they still show difference fragment structure with aliphatic and aromatic, respectively. When insight into protein structure, it would change the 3D structure from α-helix to D-loop (Fig. 2B), and decreases protein stability (ΔΔG of − 1.08 kcal/mol).