Учебники / Genetic Hearing Loss Willems 2004

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Figure 1 Pedigree of the DFNA17 family with progressive high-frequency hearing impairment associated with cochleosaccular degeneration.

Figure 2 Pure tone thresholds for the right ear averaged for 500, 1000, 2000, and 3000 Hz. Test results show a mild hearing impairment in all subjects at age 13 with progression of the loss with aging.

Figure 3 Variability in hearing loss. Shown is the hearing impairment observed for di erent a ected individuals between the ages of 18 and 20 years. The proband, III-2, shows the most significant hearing impairment when compared with his three children and his nephew.

number of a ected individuals, it was not possible to determine the rate of progression of hearing impairment. In some members of the family with significant hearing impairment, the word identification scores were good considering the degree of hearing loss. The preservation of clarity with a significant sensorineural hearing impairment is reminiscent of Schucknecht’s strial presbyacusis.

Distortion product otoacoustic emissions (DPOAE) were also performed on the a ected and una ected members of the family. Presence or absence of otoacoustic emissions reflected the level of hearing loss of the individual being tested. OAE were present in individuals with mild hearing loss and absent in members with profound impairment. Therefore, OAE cannot be used for early identification of individuals harboring the mutated gene prior to the development of hearing impairment. Similar to OAE, auditory brainstem responses (ABR) reflected the severity of hearing impairment. ABR were normal in individuals with mild hearing impairment and absent in deaf individuals.

III.TEMPORAL BONE FINDINGS

The temporal bone of the proband, a 61-year-old profoundly deaf man, demonstrated classic findings associated with Scheibe, or cochleosaccular, degeneration: degeneration of the stria vascularis, the organ of Corti, and the saccular sensory epithelium. There was collapse of Reissner’s membrane onto the spiral ligament and area of organ of Corti. Stria vascularis was either absent or replaced by basophilic granules. The population of spiral ganglion neurons was reduced to one third of normal levels. Consistent with cochleosaccular degeneration, the utricle and the ampullae of the semicircular canals appeared to be completely normal.

IV. MYH9, A CONVENTIONAL NONMUSCLE MYOSIN

The DFNA17 locus was mapped to a relatively large genetic region of 17–23 cM on chromosome 22q12.2–q13.3, typical for the size of the family studied

(2). The low resolution of the linked region precluded use of the positional cloning approach to identify the disease gene. The candidate gene approach, default alternative to positional cloning, was thus pursued to identify the mutant gene within the DFNA17 locus. Analysis of the chromosome 22gene map identified 163 candidate genes within the region spanning the DFNA17 locus (6). One of the candidate genes encoding the nonmuscle myosin heavy chain A (MYH9), was selected for further analysis. The selection of MYH9, a member of the class II or conventional myosin, was based upon identification of mutations in several other myosin heavy-chain genes that are pathogenically linked to HHI (7).

The class II myosins are broadly expressed in skeletal, cardiac, and smooth muscles as well as nonmuscle tissues and consist of a pair of heavy chains, a pair of light chains, and a pair of regulatory light chains (8). The N-terminal motor domain is the most highly conserved region of the myosin heavy chain and contains the ATPand actin-binding sites. The apparent molecular weight of the class II myosin heavy chain is 200 kDa. The myosin that mediates skeletal muscle contraction, also known as the sarcomeric myosin, represents the most well-characterized representative of the class II myosin family. Cardiac and smooth muscle cells also express isoforms of class II myosin, distinct from the sarcomeric myosin that mediates contraction in these muscle cells.

Characterization of myosins in nonmuscle cells has identified two distinct isoforms of myosin II: nonmuscle myosin-IIA (MYH9) (9,10) and nonmuscle myosin-IIB (MYH10) (10). MYH9 and MYH10 have been mapped to 22q11.2 and 17q13, respectively, and exhibit 85% identity in their motor

domains. Several skeletal-muscle heavy-chain genes have also been localized to the region containing MYH10. Most cells express relatively equal amounts of each of these myosins, with a few exceptions; platelets express MYH9 only (11,12) while neuronal tissues predominantly express MYH10. MYH9 and MYH10 demonstrate overlapping but distinct intracellular locations when coexpressed within the same cell type (13,14). In vitro motility studies of these two isoforms has shown that MYH9 is several-fold faster than MYH10 in its rate of ATP hydrolysis and movement of actin filaments (15). The di erences in their localization and their in vitro characteristics suggest di ering in vivo functions.

Sequence analysis of MYH9 in the DFNA17 family identified a G–A transition at nucleotide 2114 that cosegregated with the inherited autosomal dominant hearing impairment (3). This missense mutation changes codon

Figure 4 DFNA17 mutation R705H resides in a highly conserved and functionally important myosin heavy-chain motor domain.

705 from an invariant arginine (R) to histidine (H), R705H. The identified mutation R705H within MYH9 occurs in a highly conserved and functionally critical region within the carboxy-terminal half of the myosin heavychain motor domain that forms the globular head in the hexameric myosin molecule (Fig. 4). Within the motor domain, R705 resides in a highly conserved linker region spanning 16 amino acids that contains two free thiol groups, C704 (SH1) and C694 (SH2). Crystal structure of the globular head of the skeletal muscle myosin has shown that the two thiols are part of two short a-helices joined through a kink at the conserved G696 residue (16,17). The SH1 and SH2 helices are believed to play a key role in the conformational changes that occur in the myosin head during force generation coupled to ATP hydrolysis. X-ray crystallographic studies suggest that during the power stroke, the light-chain-binding domain (LCBD) swings relative to the catalytic/ATP-binding domain. The pivot point of this swinging motion is considered to be in the vicinity of the SH1-SH2 helix (18). Not surprisingly, in vitro and in vivo modifications of the SH1 helix including cross-linking of SH1-SH2 groups or alteration/substitution of SH1/SH2 have been shown to disrupt the mechanical function of the myosin motor domain (19,20). Thus, studies investigating myosin structure and function have demonstrated that the functional integrity of myosin is critically dependent upon its flexibility at the SH1–SH2 helix. The strict conservation of this linker region among myosin II subtypes within and between species further underscores its functional importance. The R705H mutation in the DFNA17 family may cause an altered conformation of the SH1 helix that a ects its flexibility and movement, thus disrupting the mechanical function of the motor domain.

V.MYH9 EXPRESSION IN THE MAMMALIAN COCHLEA

Localization of MYH9 expression in situ in the mammalian cochlea has particular importance for understanding and interpreting the histopathology associated with its dysfunction. Within the rat cochlea, MYH9 expression was localized in three distinct tissue types: the organ of Corti, the subcentral region of the spiral ligament, and Reissner’s membrane (Fig. 5) (3). Within the organ of Corti, the MYH9 expression was identified throughout the outer hair cells including the cuticular plate. The CSD histopathology of the a ected DFNA17 proband demonstrated a collapsed Reissner’s membrane, a two-cell-layer-wide membranous barrier that forms the upper boundary of the cochlear duct. MYH9 dysfunction in the Reissner’s membrane may contribute to its compromised cytoarchitecture, thus disturbing cochlear fluid homeostasis and a ecting cochlear function. The cochlear tissue con-

Figure 5 (A) Within the rat cochlea, MYH9 expression was localized in three distinct tissue types: the organ of Corti, the subcentral region of the spiral ligament, and Reissner’s membrane. (B) Control cochlear section with competing peptide shows no staining.

spicuous by its absence of MYH9 expression is the stria vascularis, the lateral wall of the cochlear duct, and whose dysfunction is generally considered to be the cause of CSD. The absence of MYH9 expression is the stria vascularis suggests that this tissue type is not directly responsible for the underlying pathophysiology of CSD.

MYH9 is also expressed within the spiral ligament consisting of connective tissue made up of fibrocytes. These fibrocytes of the spiral ligament are classified according to the distinct isoforms of the Na+, K+- ATPase that they express as well as by their cytoskeletal components. The subcentric region of the spiral ligament, immnoreactive to the MYH9