PEX1 and PEX6 encode two interacting proteins that belong to the peroxisomal import machinery and that are involved in the shuttling of PEX5, the cytosolic receptor for peroxisomal matrix proteins.32,33 Biallelic pathogenic variants in PEX1, PEX5, PEX6, or any of the other 11 PEX genes result in a PBD, which is characterized by defective peroxisome assembly due to impaired import of proteins into the peroxisomal matrix or membrane.25 Peroxisomes are found in virtually all human cells and play a crucial role in a number of metabolic pathways.34 PBDs usually have a severe progressive multi-systemic clinical presentation, including developmental delay, seizures, SNHL, retinopathy, peripheral neuropathy, leukodystrophy, and skeletal, craniofacial, and liver abnormalities.35–37 Dependent on the underlying genetic defect, however, the clinical presentation and survival of individuals with a PBD can show a wide variability ranging from the severe, early-childhood lethal Zellweger syndrome to milder phenotypes, including isolated progressive ataxia.26,27 To reflect this clinical variability, the PBDs are often referred to as Zellweger spectrum disorders. Accounting for 60% and 16%, respectively, of diagnosed cases,19 mutations in PEX1 and PEX6 represent the most common causes of PBDs. Our combined findings show that HS is caused by compound heterozygosity for a loss-of-function allele and a hypomorphic allele in PEX1 or PEX6. Alternatively, in family 1 a homozygous hypomorphic allele also results in the HS phenotype. Consequently, although PBD-affected individuals with mild or normal peroxisome functions in blood and fibroblasts and normal intellect have been described before, HS represents a discrete phenotypic entity at the mildest end of the PBD clinical spectrum. The characteristic presentation overlaps some of the clinical features observed in affected individuals with PBDs.38,39 Indeed, SNHL is a common feature of PBDs, and tooth and nail abnormalities have been described in PBD-affected individuals with prolonged survival, but always in association with additional and more severe features.40–44 Importantly, in contrast to individuals with PBDs at the severe end of the clinical spectrum, the individuals with HS showed no identifiable dysmorphic or additional neurological features. Other evidence of clinical effect due to peroxisome dysfunction was not investigated, given that there was no clinical indication. However, future assessments of individuals with HS should consider other features well described in individuals with PBDs, including brain imaging, testing for adrenal insufficiency, liver-function tests, and clinical assessment for evidence of (progressive) peripheral neuropathy. Compound heterozygosity of the hypomorphic PEX6 c.1802G>A allele has been reported previously in seven individuals with a Zellweger spectrum disorder.20,23 In all reported individuals, the p.Arg601Gln allele was in trans with a severe PEX6 allele that, when homozygous or in trans with another severe PEX6 allele, causes a severe peroxisomal phenotype (six individuals) or that is predicted to be deleterious (one individual). For three of the seven individuals, who were diagnosed at an advanced adult age, studies in fibroblasts revealed peroxisomal mosaicism at 37°C and slightly elevated C26:C22 levels (unpublished results). Other peroxisomal parameters were normal. No clinical data are available for determining whether these individuals showed a HS-like phenotype. However, these findings suggest that the PEX6 c.1802G>A allele is a risk allele for mild PBD when in trans with a severe PEX6 allele. Because the PEX6 c.1802G>A allele has a frequency of 0.41% in the European population (see ExAC Browser in the Web Resources), we expect that future WES studies will identify additional individuals who have a mild PBD due to compound heterozygosity of the PEX6 c.1802G>A allele and a severe PEX6 allele and who have not been suspected of or analyzed for a peroxisomal disorder on the basis of clinical diagnosis.