OBJECTIVES: Mutations in the SLC26A4 gene (7q22.3-7q31.1) are considered one of 
the most common causes of genetic hearing loss. There are two clinical forms 
related to these mutations: syndromic and non-syndromic deafness. The first one 
is named Pendred Syndrome (PS) when deafness is associated with thyroid goiter; 
the second is called DFNB4, when no other symptoms are present. Both are 
transmitted as an autosomal recessive trait, but simple heterozygotes can 
develop both forms of deafness. Actually it is thought that Pendred Syndrome 
occurs when both alleles of SLC26A4 gene are mutated; DFNB4 seems due to 
monoallelic mutations. PS and DFNB4 can be associated with inner ear 
malformations. In most of the cases (around 80%), these consist in Enlarged 
Vestibular Aqueduct (EVA). EVA can also be present without SLC26A4 mutations. 
Understanding the role of new SLC26A4 variants should facilitate clinical 
assessment, as well as diagnostic and therapeutic approaches. This investigation 
aims to detect and report genetic causes of two unrelated Italian boys with 
hearing loss.
METHODS: Patients and family members underwent clinical, audiological and 
genetic evaluations. To identify genetic mutations, DNA sequencing of SLC26A4 
gene (including all 21 exons, exon-intron boundaries and promoter region) was 
carried out.
RESULTS: Both probands were affected by congenital, progressive and fluctuating 
mixed hearing loss. Temporal bone imaging revealed a bilateral EVA with no other 
abnormalities in both cases. Probands were heterozygotes for previously 
undescribed mutations in the SLC26A4 gene: R409H/IVS2+1delG (proband 1) and 
L236P/K590X (proband 2). No other mutations were detected in GJB2, GJB6 genes or 
mitochondrial DNA (mit-DNA).
CONCLUSIONS: The IVS2+1delG and K590X mutations have not yet been described in 
literature but there is some evidence to suggest that they have a pathological 
role. The results underlined the importance of considering the complete DNA 
sequencing of the SLC26A4 gene for differential molecular diagnosis of deafness, 
especially in those patients affected by congenital, progressive and fluctuating 
mixed hearing loss with bilateral EVA.