Novel myosin mutations for hereditary hearing loss revealed by targeted genomic capture and massively parallel sequencing

Abstract

Hereditary hearing loss is genetically heterogeneous, with a large number of genes and mutations contributing to this sensory, often monogenic, disease. This number, as well as large size, precludes comprehensive genetic diagnosis of all known deafness genes. A combination of targeted genomic capture and massively parallel sequencing (MPS), also referred to as next-generation sequencing, was applied to determine the deafness-causing genes in hearing-impaired individuals from Israeli Jewish and Palestinian Arab families. Among the mutations detected, we identified nine novel mutations in the genes encoding myosin VI, myosin VIIA and myosin XVA, doubling the number of myosin mutations in the Middle East. Myosin VI mutations were identified in this population for the first time. Modeling of the mutations provided predicted mechanisms for the damage they inflict in the molecular motors, leading to impaired function and thus deafness. The myosin mutations span all regions of these molecular motors, leading to a wide range of hearing phenotypes, reinforcing the key role of this family of proteins in auditory function. This study demonstrates that multiple mutations responsible for hearing loss can be identified in a relatively straightforward manner by targeted-gene MPS technology and concludes that this is the optimal genetic diagnostic approach for identification of mutations responsible for hearing loss.

Figure 1

Analysis of myosin VI mutations. (a) Pedigrees and segregation analysis of Family AV. (b) Partial sequences of MYO6 exon 10 from a normal hearing and affected proband AV-III-1 demonstrating the c.897G>T splicing mutation. (c) cDNA was amplified from blood from the patients of Family AV, using primers from exon 9–12 to generate a 545-bp fragment. Exon 10 was lost in the resultant cDNA of patient AV-III-1, confirmed by sequencing. (d) Pedigrees and segregation analysis of Family QS009. (e) Partial sequences of MYO6 from an affected proband demonstrating the c.2777T>A, p.Leu926Gln, as shown by a chromatogram. (f) Schematic representation of myosin VI shows the location of the potential splicing mutation c.897G>T in the ATPase activity domain in the motor and p.Leu926Gln, located in a potential proximal dimerization region in the tail of the myosin VI protein.

Figure 2

Analysis of myosin VIIA mutations. (a) Pedigrees and segregation analysis of Family D79. (b) Partial sequences of MYO7A from a normal hearing and affected proband D79-II-1 demonstrating the c.29T>C and c.1969C>T mutations. (c) Pedigrees and segregation analysis of Family D75. (d) Partial sequences of MYO7A from an affected proband demonstrating the c.620A>G mutation. (e) Pedigrees and segregation analysis of Family QS025. (f) Chromatogram of the c.6211C>T mutation. (g) Pedigrees and segregation analysis of families QS004 and QS013. (h) Chromatogram of the c.4153-2A>G mutation. (i) Myosin VIIA mutations p.Asn207Ser and p.Arg657Trp are superimposed on a structural model of the myosin VIIa motor domain, based on the structure of myosin V bound to ATP. Note in black the location of the p.Asn207Ser mutation that may result in poor nucleotide binding and hydrolysis. The p.Arg657Trp mutation is likely to impair the communication between the active site and the lever arm as it is located at the interface between two connectors of the motor relay (yellow) and the SH1 helix (red) that controls the rotation of the lever arm. (j) Schematic representation of the myosin VIIA protein shows the location of p.Val10Ala, p.Arg657Trp, p.Asn207Ser, p.Gln2071* and c.4153-2A>G.

Figure 3

Analysis of myosin XVA mutations. (a) Pedigree and segregation analysis of Family BA. (b) Partial sequence of MYO15A from an affected proband BA-II-1 demonstrating the compound heterozygote mutation c.1223C>T, p.Ala408Val, which appeared with the known p.Glu1414Lys mutation. (c) Pedigree and segregation analysis of Family 1234. (d) Partial sequences of MYO15A from an affected proband 1234-II-1 demonstrating the mutation c.8467G>A, p.Asp2823Asn. (e) Schematic representation of the myosin XVA protein shows the location of the p.Ala408Val, p.Glu1414Lys and p.Asp2823Asn mutations.