Clinical report - mother
A 36-year-old woman who visited our clinic was the first child of parents who had a non-consanguineous marriage. Her family history was positive for epilepsy, and she had deafness since childhood. Her hearing loss started when she was 5 years old with occasional dizziness, frequent falls, loss of balance, otalgia, and frequent episodes of acute otitis media, which more frequently affected the right ear. After a year, at the age of 6 years, she presented with profound hearing loss and underwent a hearing device implantation. However, she showed no improvement and experienced loss in sound discrimination until the age of 25 years. Thus, she received supportive therapy with bilabial pronunciation during 6–7 years of age to preserve spoken language. She used to have frequent upper tract respiratory infections and asthma. She had never presented with nyctalopia or visual acuity decline (shown in Fig. 2). Ophthalmological studies showed that all anterior segment findings were within normal limits, and she had normal pupils with no afferent pupillary defect. Fundus examination revealed normal findings. Audiometry findings revealed a profound hearing loss in both the ears with responses at 67.5 dB and 63.75 dB for right and left ears, respectively. Visual field examination showed pattern II with bilateral inferotemporal loss, more markedly in the right eye. The patient has two sons, aged 15 and 11 years, respectively. The oldest son presented multiple seizures episodes, whose description is provided in the subsequent section. After obtaining consent from the patient and her family members to participate in the study, peripheral blood sample was collected for whole-exome sequencing (WES) and Sanger sequencing.
Clinical report - child
The 15-year-old boy was the first child of non-consanguineous parents. His family history was positive for epilepsy. In addition, his mother had neurosensorial progressive hearing loss since 6 years of age with audiometry findings revealing bilateral hearing loss with responses at 67 and 85 dB in right and left ear, respectively, with 100% discrimination and refractive disorder. The patient’s younger brother aged 12 years is undergoing schooling and shows no cognitive impairment. The patient was born at term by vaginal delivery after an uneventful pregnancy with a birth weight of 2,700 g (body length and head circumference at birth were not reported). At 30 months of life, the patient started to have clusters of tonic spasms at night, on average 10 episodes each night, with gaze deviation on some occasions and without sphincter relaxation, which were more severe at the beginning of the sleep cycle and persisted in following days. At the age of 3 years, the patient developed cardiorespiratory arrest, which required an extended stay in the intensive care unit (ICU) which was followed by global developmental. The developmental milestones and the ages at which the patient achieved them were as follows: crawling at 11 months, walking at 18 months, and poor speech at 2 years. During the stay in the ICU, the patient was diagnosed to have clusters of tonic spasms and seizures. Subsequently, he was treated with valproic acid and clonazepam until he reached the age of 5 years. However, epilepsy remained substantially drug-resistant. During this time, the patient recovered standing and walking and started saying a few words. At the age of 7 years, he developed epileptic encephalopathy requiring ICU and adjustment of the epileptic drugs with levetiracetam, clobazam, and topiramate, which resulted in partial control of the epileptogenic activity. In the following years, seizures occurred several times a week on some occasions associated with fever requiring the adjustment of anticonvulsant therapy. Additionally, he required ICU hospitalization owing to infectious disease.
The patient was additionally assessed by the genetics department for infantile epileptic encephalopathy, swallowing disorder, cognitive deficits, and psychomotor developmental delay with speech delay. Physical examination revealed the following: at 14 years, anthropometric parameters such as head circumference was 49 cm (p < 1; z-score: −4.71), weight was 35.1 kg (p = 1; z-score: −3.26), and length was 143.5 cm (p < 1; z-score: −3.63). Dysmorphisms included microcephaly, long face, bilateral epicanthal folds, overfolded helix, high set nasal root implantation and prominent nasal bridge, retrognathia, several hyperpigmented lesions in the chest, and four café-au-lait macules (shown in Fig. 2). The neurological examination revealed that the patient had a childish attitude, poor speech with prominent restrictive vocabulary (12 to 15 words), mainly guttural sounds, and echolalic syllables. He could nominate some words after his parents assigned them. He communicated with his mother by gestures and followed simple commands. On few occasions, he had an aggressive conduct. At the last observation, the patient was 15 years old and occasionally presents generalized tonic-clonic seizures. The child was treated with levetiracetam every 8 hours. The last severe seizure episode was in January 2021, and he has not presented any new sleep spasm episodes in the previous years. The patient presented an auditive evoke potential score of 30 dB with v wave, representing a normal register.
He had previously undergone normal brain magnetic resonance imaging. In addition, previous assessments for plasma amino acids and acyl carnitines, array Comparative Genomic Hybridization (CGH), and electroencephalography were all normal.
WES was conducted as trio-WES, wherein both parents and their affected child underwent sequencing simultaneously. The exome was captured using a SureSelectXT Human All Exon V5 capture kit (Agilent) of 51 Mb, and sequencing was performed using an Illumina Hiseq 2000 sequencing system (Illumina, San Diego, CA). In addition, the mother underwent WES to identify any germline mutations that could explain her Usher syndrome. During the sequencing, paired readings of 101 nucleotides in length were obtained. Subsequently, the different variants were analyzed with a focus on genes related to the patient’s phenotype. Only variants in the coding region and those within ± 20 bp of flanking intron regions with minor allele frequency <1% were evaluated and compared using datasets obtained from the 1000 Genomes Project Consortium, dbSNP, Exome Variant Server, and Exome Aggregation Consortium databases. Parental carrier status of the clinically relevant variants was confirmed by Sanger sequencing.
Sanger sequencing was performed to verify and validate variants within the family. Genomic DNA was extracted and amplified by polymerase chain reaction (PCR) using oligonucleotide primers designed to amplify exons 19 and 73 of ADGRV1 (GeneBank NG_007083) and exon 3 of KCNC2 (GeneBank NC_000012.12). Primer sequences and PCR conditions will be provided upon request. PCR products were sequenced using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and were analyzed using the ABI Prism 3500 Genetic Analyzer (Applied Biosystems).
Protein structure modeling and stability analysis
To identify the potentially pathogenic genetic variants in mother and her child, the amino acid sequence of human ADGRV1 (residues 1108–1208 and 2814–3048) and KCNC2 (residues 453–502) were obtained from the Uniprot entries Q8WXG9 and Q96PR1, respectively. Three models were generated ab initio using I-Tasser [Roy et al., 2010; Yang et al., 2014], Robetta [Kim et al., 2004], and SwissModel [Bienert et al., 2017; Waterhouse et al., 2018]. These models were validated using the structure validation algorithms Verify3D [Eisenberg et al., 1997], ProSA-web [Wiederstein and Sippl, 2007], and ERRAT [Colovos and Yeates, 1993]. Then, the best in silico models were chosen to predict the change in protein stability following single point mutation using MAESTRO web [Laimer et al., 2015], Rhapsody [Ponzoni et al., 2020], I-Mutant [Capriotti et al., 2006], DynaMut2 [Rodrigues et al., 2021], PremPS [Chen et al., 2020], CUPSAT [Parthiban et al., 2006], and SDM and mCSM-membrane (only for KCNC2 model) [Pires et al., 2020].
The pathogenic effects of three missense mutations were analyzed using 10 in silico prediction tools: Polyphen-2 [Ramensky et al., 2002], DANN [Quang et al., 2015], EIGEN [Dong et al., 2015; Ionita-Laza et al., 2016], FATHMM-MLK [Shihab et al., 2013], MutationTaster [Schwarz et al., 2014], BayesDel noAF [Feng, 2017], LRT [Chun and Fay, 2009], MetaLR [Dong et al., 2015], SIFT [Kumar et al., 2009], and SIFT4G [Sim et al., 2012].