Biallelic ATOH1 Gene Variant in Siblings With Pontocerebellar Hypoplasia, Developmental Delay, and Hearing Loss

Clinical Presentation of a Family

We present a family with 2 affected siblings (the proband and his sister). The proband (Figure 1, II-1), a 14-year-old White boy, was born vaginally after an uneventful pregnancy as the first-born twin at 36 weeks of gestation. The second-born twin experienced intrauterine hypoxia; however, she showed no clinical and radiologic signs present in the proband and the affected sister. The proband's birth weight was 2,720 g (50 P), birth length 48 cm (50 P), and head circumference 35.5 cm (85 P); Apgar 8/9. At his birth, the ages of the mother and the father were 29 and 30 years, respectively. In the neonatal period, he was breastfed and had no feeding difficulties. Hypotonia was reported at age 3 months. Motor development was slow. He achieved head control at age 4 months, sat at age 3 years, and walked with a walker at age 8 years. He had severe speech delay, used one word (mum) only, and communicated nonverbally or by communication symbols. He had severe hearing loss but declined to use hearing aids. He learned to eat and dress mostly independently but was not toilet trained. His sleep pattern was normal. He never had seizures. Behavior disturbances included stereotypic hand movements, bursts of anger, and the episodes, when he gritted his teeth, looked up, and hugged his thighs with his hands. At age 14 years, his weight was 44.5 kg (25 P), height 160.5 cm (35 P), and head circumference 55.8 cm (44 P). A neurologic examination revealed horizontal nystagmus, low muscular tone, brisk reflexes, and intention tremor. He was unable to walk independently because of severe trunk ataxia. He had soft skin, hypermobility of the small joints, mild contractures of knees and elbows, and bilateral valgus feet.

His older affected sister (Figure 1, II-2) was born at full term by normal vaginal delivery. The family moved to Slovenia when she was at the age of 10 years. Birth weight was 2,750 g (5 P). Other anthropometric data at birth were not recorded. The neonatal period was uneventful. She was breastfed. At the age of 6 months, hypertonus, hyperreflexia, and repeated episodes of opisthotonus were reported. Her development was extremely delayed. She achieved short-term head control but was not able to sit or walk. She had vision and hearing impairment and reacted to auditory and tactile stimuli by smiling and making sounds. She was fed orally and had problems with constipation. She had frequent apnea attacks after the age of 20 years. At the age of 10 years, her weight was 31 kg (25–50 P), and her development was estimated to be at the level of 4 to 6 months. She had no head control, nystagmus, opisthotonus, the lower limbs in an adduction position, and athetosis of the upper limbs.

General laboratory tests including blood cell counts, liver and kidney function tests, blood glucose levels, lactate and pyruvate blood levels, and amino acid and organic acid tests revealed normal results in both siblings.

An ophthalmologic examination of the siblings at the age of 10 years revealed horizontal nystagmus, convergent strabismus, and pale optic discs. In addition, the proband had mild peripapillary chorioretinal atrophy in the right eye. The proband showed severe visual impairment (6/50), and his sister presented with blindness (6/130).

Audiogram and brain-evoked response audiometry performed in the proband showed a severe bilateral sensorineural hearing loss. His sister was clinically assessed as experiencing profound hearing loss because she only responded to loud auditory stimuli.

Several brain imaging studies were performed in both siblings; however, only CT images from the proband and MRI from his affected sister were preserved (Figure 2 and eFigure1, links.lww.com/NXG/A523). The consistent findings were posteriorly thin corpus callosum, small posterior fossa with the upward displacement of the tentorium, small cerebellum with more severely hypoplastic vermis than hemispheres, and small brainstem (eFigure 1 and eTable 1, links.lww.com/NXG/A523).

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Figure 2 CT and MR Images of the Proband II-1 and His Affected Sister II-3

(A) CT axial image of a proband II-1 demonstrating prominent hypoplasia of pons, middle cerebellar peduncles, and both cerebellar hemispheres. The vermis is absent. (B) CT sagittal image of a proband II-1 resulting in small posterior fossa and widened 4th ventricle, opened into the cerebellomedullary cistern and hypoplastic pons. Image courtesy of Allen Developing Mouse Brain Atlas. (C) MR axial image of an affected sister II-3 showing enlarged 4th ventricle with cerebellomedullary cistern, very hypoplastic pons, middle cerebellar peduncles, and cerebellar hemispheres. Please note also enlarged ocular bulbs. (D) MR sagittal image of an affected sister II-3 shows very hypoplastic pons, cystic 4th ventricle with absent vermis, and flattened posterior part of corpus callosum.

EEG performed in the proband at the age of 14 years showed slow and irregular background electrical activity with no epileptiform discharges. There was otherwise no family history of pontocerebellar dysplasia and hearing impairment, and the parents were healthy. Because the proband and his sister were likely affected by the same condition, an autosomal recessive inheritance was considered.

Genetic Investigation

Initially, we focused the analysis of ES data on the genes with established association with monogenic disorders. No known pathogenic or novel variants were identified in the set of genes associated with PCH or other neurodevelopmental disorders or hearing loss. In addition, the analysis of mitochondrial sequences, patterns of homozygosity, and copy number variants in the ES data revealed no convincing causative variants.

We therefore proceeded to the analysis of all variants identified by ES. We filtered and prioritized the ES variants as outlined in the Methods section. Performing the filtration under the recessive model, we identified a homozygous variant NM_005,172.1:c.481C>G (p.Arg161Gly) in the ATOH1 that was predicted to result in amino acid substitution of arginine to glycine at position 161 in the sequence of the protein encoded by the ATOH1. The ATOH1 gene encodes for the atonal bHLH transcription factor 1, a core transcription factor in the developing cerebellum, brainstem, and dorsal spinal cord, and in the ear. The identified missense variant results in the amino acid substitution within the evolutionarily conserved DNA-binding bHLH domain of the ATOH1 protein.

Strikingly, the variant was found in a homozygous state despite being absent from all control populations (gnomAD, GoNL, UK10K, and the in-house population). No blocks of homozygosity or structural variants were identified in the region of the ATOH1. The gnomAD currently contains 332 variants of ATOH1, of which 215 are missense variants, 113 are synonymous mutations, and only 4 variants are predicted to result in the loss of function (LoF). Among the missense and LoF variants in gnomAD, only 4 are homozygous (p.Asp138Glu, p.His237Gln, p.Ala291Thr, and p.Glu326Val), and none of them lie in the evolutionary conserved bHLH domain (p.159-211) of the ATOH1 protein. In addition, the ATOH1 displays constraint against missense variants according to the gnomAD regional constraint data in DECIPHER genome browser (missense constraint: 0.637, p-value 6.03 × 10⁻⁷).

The p.Arg161Gly variant is consistently predicted as damaging by most in silico predictors, and the affected amino acid position also shows a high degree of evolutionary conservation, with the arginine position being invariant across species. This affected amino acid position 161 lies at the beginning of the evolutionary conserved bHLH domain of the ATOH1 protein called basic region of bHLH domain, which is involved in DNA binding. At the carboxy-terminal end of this domain is the HLH region, which facilitates interactions with other transcription factors to form a heterodimer^13,14 and activate target gene transcription. Because our identified variant is in a basic region of bHLH domain, it may affect the structural composition of this protein region or influence binding affinity to DNA.

Based on the evidence presented earlier, we analyzed the segregation of this variant in the reported family. Sanger sequencing confirmed the homozygous state of the NM_005172.1:c.481C>G ATOH1 variant in the proband and affected sister. Both unaffected parents and the proband's twin were found to be heterozygous carriers of the variant, signifying that the variants segregated with the disease (Figure 1A). This variant was submitted to a ClinVar database (variation ID:873538).

We analyzed the images of the Allen mouse brain atlas¹⁵ and RNAseq data from the Allen developing human brain project¹⁶ and observed that the expression of the ATOH1 and its mouse ortholog Atoh1 is confined to developing cerebellum and primarily occurs in the prenatal stages of brain development (Figure 1B, eFigure 2, links.lww.com/NXG/A523).

Because the biallelic variants in ATOH1 have not been conclusively associated with monogenic conditions in humans (apart from a single report without details on the clinical presentation¹⁷), we attempted to use the Matchmaker Exchange networks (GeneMatcher, Phenome Central, and MyGene2) to identify additional patients with biallelic ATOH1 variants and PCH but failed to identify a suitable match.

In Silico Modeling of p.Arg161Gly in ATOH1

To determine the potential functional effect of the identified variant on the function of the ATOH1 protein, we performed in silico modeling of the p.Arg161Gly change. A bona fide homology model of the complex of the ATOH1/transcription factor E2 alpha with cognate DNA could be built using the structure of the NeuroD1/transcription factor E2 alpha/DNA complex (pdb id. 2QL2) (Figure 1C). A structural analysis of the homology model of the complex suggests that replacing R161 with G has 2 main effects:

1. Loss of a salt bridge with DNA backbone phosphate (Figure 1C), which should reduce the affinity of the protein for DNA and could also slightly change the orientation of the helix regarding the major groove of DNA, with possible implications on DNA recognition;

2. Glycine has a different conformational preference for arginine, and the helix of the bHLH motif could be more flexible or disrupted at the beginning, affecting the relative orientation of other parts of the protein.

We performed continuum electrostatic calculations to assess the effect of the variant on the free energy of binding DNA. Although the computed values depend on the assumed inner dielectric constant and the temperature, in all cases, the estimated change in the free energy of binding was unfavourable between 1.0 and 2.0 kcal/mol.

We also performed 60 ns molecular dynamics simulations in explicit water for the complex to check for structural and dynamical differences. The rather large fluctuations, possibly due to the small size of the DNA fragment, make, however, the differences observed in simulations not significant.