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Sequencing products were analyzed using the ABI Prism 3730
Capillary Sequence Detection System, and raw data were
obtained and compared to the published consensus sequences
using Sequencher 4.8 (Gene Codes Corporation, Ann Arbor, MI)
sequencing analysis software. The 342-kb deletion in the
genetic region of D13S1830 (GJB6-D13S1830) locus on chromo-
some 13q12 was analyzed by PCR and gel electrophoresis.
Detailed methodologies have previously been published.19,25
Mutation nomenclature is based on the recommendations of the
American College of Medical Genetics and the Human Genome
Variation Society.
Several in silico analysis methods for the prediction of
functional consequences of sequence variants were utilized to
provide initial classification. All sequence variants were verified
by the Human Gene Mutation Database, the National Center
for Biotechnology Information Single Nucleotide Polymorphism
database, and locus-specific mutation databases, such as
Connexin Deafness Homepage
index.php?seccion ¼ mut_db&db ¼ nonsynd&nonsynd ¼
cx26mut). For novel mutations, we used SIFT (http://blocks.
, which utilizes evolutionary infor-
mation from homologous proteins,16 and PolyPhen (http://
, which incorporates
structural information into classification rules.17 The Grantham
Scale18 was also used to evaluate the significance of amino acid
substitutions, and a determination of the likelihood of the
sequence change being pathogenic was made by the Molecular
Genetics laboratory.
Statistical Analyses
Data distributions were reported as medians with ranges
or interquartile ranges for continuous variables and frequencies
with proportions for categorical variables. Comparisons were
made among subjects who had ipsilateral hearing loss, contra-
lateral hearing loss, and bilateral hearing loss relative to the
ear with the identified EVA. Due to the non-normal distribution
of the majority of the data, nonparametric statistics were used
in the analyses. Continuous data were compared using the
Kruskal–Wallis test or the Wilcoxon rank sums test, as appro-
priate. Categorical data were compared among groups using v2
analysis or the Fisher exact test, as appropriate. Correlations
between vestibular measurements and continuous variables
such as PTA values were conducted using the Spearman rank
correlation coefficient. For assessing which factors may be inde-
pendently related (independent predictors) to having a
progressive hearing loss in an ear, multivariate analysis was
conducted using generalized estimating equations (GEE). GEE
allows for the clustering of ears in the analysis (two ears
belonging to the same individual). GEE was used for both mod-
eling of odds or risk progression as a dichotomous variable and
modeling the rate of progression (as a decibel loss per year). For
all analyses, a P value of .05 or less was considered statistically
significant. All analyses were performed using SAS for
Windows, version 9.2 (SAS Institute, Cary, NC).
RESULTS
Our database search identified 144 patients (67
males, 77 females) who met our inclusion criteria. Uni-
lateral EVA was identified in 74 (51.4%) of these
patients (Fig. 1); 42 (56.8%) had left-sided EVA, and 32
had right-sided EVA. The median age at which the hear-
ing loss was identified was 59.5 months (range, 0–324.5
months). The median follow-up time was 37.8 months
(range, 0–812.5 months), and 116 (80.5%) patients were
followed for at least 3 months. As shown in Table I,
patients with unilateral EVA were identified at a later
age than patients with bilateral EVA (70 vs. 54.5
months; P ¼ .01). They also had a shorter period of
audiometric follow-up (32.8 vs. 56.3 months; P ¼ .02)
and were more likely to have unilateral hearing loss
(46% vs. 15.7%; P < .0001).
Unilateral Versus Bilateral Hearing Loss
Forty-five patients (31.3%) with EVA had unilateral
hearing loss. In 23 (51%) of these patients, the hearing
loss was left-sided. Among the 74 patients with unilat-
eral EVA, 34 (46%) had unilateral hearing loss; in 1
patient, the unilateral loss was in the contralateral ear.
The remaining 39 patients had bilateral hearing loss.
More than 55% of patients with unilateral EVA pre-
sented with hearing loss in the contralateral ear (Fig. 1).
Among the 70 patients with bilateral EVA, only 11
(15.7%) had unilateral hearing loss. The median age at
which hearing loss was identified in all EVA patients
(n ¼ 144) was significantly higher among those with uni-
lateral hearing loss as compared to those with bilateral
loss (78.5 vs. 45.5 months; range, 16.5–158 vs. 0–324.5
months; P < .0001). This finding was consistent when
patients were stratified into a unilateral EVA group
(80.5 vs. 42.8 months; range, 16.5–158 vs. 0–324.5
Fig. 2. Schema for acquiring vestibular aqueduct measurements. (A)
Operculum measurement. (B) Midpoint measurements: half of the dis-
tance between the opercular and crus plane (A to B). OP ¼ opercu-
lum; PSSC ¼ posterior semicircular canal. [Color figure can be viewed
in the online issue, which is available at ]
Laryngoscope 123: June 2013 Greinwald et al.: Unilateral Enlarged Vestibular Aqueduct
135