American Journal of Molecular Biology, 2013, 3, 229-234 AJMB Published Online October 2013 (
Confirmation of diagnosis in Romanian children with
DFNB1 related hearing loss
Cristina Dragomir1*, Adriana Stan1, Dragos Tiberiu Stefanescu1, Lorand Savu1,
Codrut Sarafoleanu2, Adrian Toma3, Emilia Severin2
1Genetic Lab, 43B Ghencea Avenue, Bucharest, Romania
2Carol Davila, University of Medicine and Pharmacy, 37 Dionisie Lupu St, Bucharest, Romania
3Panait Sirbu Hospital, Calea Giulesti, Bucharest, Romania
Received 26 May 2013; revised 20 June 2013; accepted 12 July 2013
Copyright © 2013 Cristina Dragomir et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
DFNB1 locus has been linked to a nonsyndromic “in-
visible disability” called cong enital sensorineural hea-
ring loss and deafness. Mutations of GJB2 and GJB6
genes are associated with deafness at the DFNB1 lo-
cus. The diagnosis of DFNB1 is made with molecular
genetic testing. DNA-based testing can be used both
prenatally and postnatally. Purpose: To get evidence
for implementation of newborn hearing screen ing pro-
grams at national level; to use the molecular testing of
children at risk for confirmation of diagnosis and
early intervention. OAEs and ABR were performed
for 4303 newborns. Audiologic evaluation of 38 chil-
dren suspected of having hearing loss was performed
too. Physical examinations and family history were
used to get information about congenital deafness.
DNA from blood samples was isolated, and two PCR
multiplex assays were developed to detect DFNB1
mutations. Only 23 newborns were screened positive.
Newborns were referred to audiologic evaluation,
genetic counseling and testing for the etiologic diag-
nosis. Physical examination revealed no other abnor-
mal findings. GJB2 mutations were detected in 36.03%
of patients, and all of them have 35delG mutation.
None of them was found to have GJB6 mutations.
Our results suggested that molecular testing was an
accurate method of early determining cause of con-
genital hearing loss and helped us to exclude GJB6
gene from the routine hearing screening protocol.
Keywords: DFNB1; Molecular Genetic Testing; GJB2;
Hearing loss (HL) affects both children and adults. Age-
related HL is more common than HL in children but “the
earlier hearing loss occurs in a child’s life, the more se-
rious the effects on the child’s development. Similarly,
the earlier, the problem is identified and intervention
begun, the less serious the ultimate impact” [1]. In this
respect, early diagnosis, early management of hearing
aids, and an early start on special education programs
and using assistive, adaptive and rehabilitative devices
could help improvement of a child’s hearing.
Genetic HL has diverse etiologies and many genes are
known to be involved in hearing process [2-4]. Among
them, mutations of GJB2 and GJB6 genes at DFNB1
(OMIM 220290) locus are estimated to play an important
role to congenital hearing loss [5-8]. Nowadays, the ge-
netic analysis for hearing loss is used for early diagnosis
followed by early intervention programs (invasive inter-
ventions, such as, otologic surgery and cochlear implan-
tation or non-invasive interventions, such as rehabilita-
tion), and serves as an explanation for etiology at any age,
and for reproductive alternatives in families with affected
The aims of the study were to get evidence for imple-
mentation of newborn hearing screening programs in all
neonatal care settings and to use the molecular genetic
testing of children at risk (suspected on basis of signs,
symptoms or family history) for confirmation of diagno-
sis and early intervention.
With these aims, we proposed a two-stage study de-
sign: stage I—to estimate the carrier frequency of the
three mutations namely 35 delG, del(GJB6-D13S1830)
and del(GJB6-D13S1854) using amniotic fluid from an
unselected group of 350 pregnant women [9] and stage
II—to detect mutations of the GJB2 and GJB6 genes in a
*Corresponding author.
C. Dragomir et al. / American Journal of Molecular Biology 3 (2013) 229-234
selected group of 61 neonates and children from all over
the Romanian zones for which early diagnosis is neces-
sary and available.
2.1. Neonates
Otoacoustic emissions (OAEs) and auditory brainstem
evoked response audiometry (ABR) were performed for
4303 babies born in “Panait Sirbu” Hospital during 2009.
Physical examinations and family history were used to
get information about hereditary/syndromic hearing loss.
All newborns were tested in newborn setting using Echo-
screen (Fischer-Zoth).
2.2. Infants
Pediatricians from several regions of Romania referred
38 unrelated children (18 males and 20 females) with
congenital hearing loss to our ORL/Audiology Clinic.
All children were Caucasians and ranging in age from 1
to 7 years. Audiologic evaluation, physical examinations,
medical and family history and CT examination of the
temporal bone were used to get information about con-
genital deafness.
2.3. DNA Extraction
Standard protocols were used to obtain blood samples
from patients and controls for the mutation analysis.
DNA from blood samples was isolated and two PCR
multiplex assays were developed to detect DFNB1 muta-
2.4. ARMs Multiplex PCRs and Multiplex PCR
DNA was collected from 61 unrelated patients and their
parents (child-parents trio). We performed the DNA ex-
traction from peripheral blood cells collected in EDTA
vacutainers using the QIAamp DNA Blood Mini Kit
(QIAGEN) according with the QIAamp® DNA Mini and
Blood Mini Handbook. We proceed from 200 µl whole
blood sample and we obtained aproximatively 100 ng
DNA/µl. We used 400 ng DNA for the detection of 35
delG GJB2 gene mutation and respectively 800 ng DNA
for the detection of the two mutations of the GJB6 gene
(GJB6-D13S1830 and GJB6-D13S1854 deletions).
We developed two PCR multiplex assays to detect the
35 delG GJB2 mutation using either the normal or the
mutant primer along with the common primer proposed
by Smith and co-workers [7] and the β-actin primers. The
sequences of forward and revers β-actin primers were
β-actin was used as internal control to monitor the
PCR efficiency of two reactions.
Both reactions were performed in a final volume of 25
µl using 400 ng DNA, 1x PCR buffer, 5 pmol each pri-
mer, 1.5 mM MgCl2, 200 nM each dNTPs and 1.5 U
Power QTaq DNA polymerase (Genomics BioSci&Tech).
PCR conditions were as follow: initial denaturation at 95
˚C for 5 minutes followed by 30 cycles of denaturation at
95˚C for 40 sec, annealing at 60˚C for 30 sec, extension
at 72˚C and a final extension step at 72˚C for 7 min. The
expected size of the amplified product was 202 bp for
GJB2 and 294 bp for the β-actin amplicons.
To detect the del(GJB6-D13S1830) and del(GJB6-
D13S1854) mutations we used the primers and PCR con-
ditions as described by Del Castillo et al., 2005. 800 ng
of genomic DNA was amplified for 25 cycles in a 25 µl
PCR reaction mixture containing 1x PCR buffer, 1.5 mM
MgCl2, 200 µM each dNTP, 10 pmol each primer and 1.5
U of Power QTaq DNA polymerase (Genomics BioSci &
Tech). The method described by Del Castillo et al., 2005,
allows the amplification of DNA fragments in a single
step. The DNA fragments contain the breakpoint junction
of each deletion [for del(GJB6-D13S1854—564 bp and
for del(GJB6-D13S1830)—460 bp], as well as a frag-
ment with GJB6 exon 1 (333 bp) which is used as an in-
ternal control of PCR multiplex efficiency. This method
also allows the detection of both heterozygous and ho-
mozygous forms of the two deletions. The exon 1 is not
amplified when the deletions are present in either homo-
zygous or compound heterozygous forms [8]. Positive
controls were kindly offered by Dr. Ignacio Del Castillo
from the Unidad de Genetica Molecular, Hospital Ramon
y Cajal, Madrid, Spain.
The PCR amplification reactions were run on a Ge-
neAmp PCR System 9700 thermal cycler (Applied Bio-
The PCR products were subjected to capillary elec-
trophoresis using the QIAxcel Instrument (Qiagen). The
QIAxcel system performs fully automated separation of
DNA fragments according to their molecular weight and
use capillary electrophoresis to enable high resolution
and high sensitivity separation of DNA samples [10].
The DNA-based testing can be used for diagnostic
testing, determination of carrier status and prenatal di-
agnosis of hearing loss.
Written informed consent was obtained from all par-
ents of our minor patients and provided to the Ethics
Committee of the Carol Davila University of Medicine
and Pharmacy—Bucharest, who approved the conduct of
this study in accordance with the Helsinki Declaration.
3.1. Neonates
An unselected population of 4303 neonates was screened
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C. Dragomir et al. / American Journal of Molecular Biology 3 (2013) 229-234
Copyright © 2013 SciRes.
by 48 h of age, before discharge, of which 4280 babies
had a pass response in both ears at either the initial or
follow-up screen.
Only 23 neonates with a positive identification within
newborn hearing screening program were referred to
audiologic evaluation to confirm if the HL is present.
Physical examination revealed no other findings associ-
ated with a syndrome. Four newborns with hearing par-
ents were found to have different types of HL: two pre-
sented sensorineural hearing loss, one had unilateral
hearing loss and one had auditory neuropathy. They were
referred to genetic counseling and testing for the etio-
logic diagnosis.
Incidence of congenital HL among neonates was 0.93
per 1000 live births (four children were clinically diag-
nosed with HL, out of a total neonate population of
3.2. Infants
Clinical signs could direct the molecular diagnosis to
specific genes. So, all of 38 children with congenital HL
expressed DFNB1 phenotype associated with senso-
rineural, prelingual, stable (until now), bilateral, all fre-
quencies affected, and moderately severe hearing im-
pairment. All parents had normal hearing and no con-
sanguine family was identified. Seven children had a
history of HL in a close (second or third degree relatives)
or distant family member. No sex predilection was no-
ticed. No other physical anomalies were detected.
3.3. ARMS Multiplex PCRs and Multiplex PCR
GJB2 mutations were detected in 36.03% of the patients
(n = 22, 4 neonates and 18 children, out of a total of 61),
all of them having 35 delG mutation. Homozygous status
for 35 delG mutation was found in 20 patients and 2 pa-
tients were heterozygous for this mutation. Figure 2
shows a representative example of 35 delG GJB2 muta-
tion detection. The PCR reaction with the normal and
common pair of primers tests for the presence of normal
allele, whereas the PCR reaction with the mutant and
common pairs of primers detects the presence of mutant
Two of the 61 homozygous samples and the two ho-
mozygous samples were included in the sample set pre-
sented in Figure 1.
Beside the screening for the 35delG mutation we also
tested for the del(GJB6-D13S1830) and del(GJB6-
D13S1854) mutations. These multiplex PCR tests are
shown in Figure 2, for the same samples presented in
Figure 1.
The band corresponding to exon 1 of GJB6 gene is
presented in all samples indicating that the samples were
neither homozygous nor compound heterozygous for the
two above mentioned mutations. The fact that no bands
for either of the two mutations are present indicates that
the samples are not heterozygous either.
Figure 1. Results of DFNB1 genetic testing.
C. Dragomir et al. / American Journal of Molecular Biology 3 (2013) 229-234
Figure 2. Detection of 35 delG mutation in the GJB2 gene. Panel (a): PCR amplification of genomic DNA
using normal and common primers for GJB2 and β-actin primers. Panel (b): PCR amplification of genomic
DNA using mutant and common primers for GJB2 and β-actin primers. M represents the pUC18/HaeIII
marker, Lane 1—homozygous 35delG positive control, Lane 2—heterozygous 35delG positive control,
Lane 3—wild-type control, Lane 11—NTC (no template control), Lanes 4, 5—wild-type DNA samples,
Lanes 6, 7, 8—35delG homozygous DNA samples, Lanes 9, 10—heterozygous DNA samples.
None of 61 investigated children was found to have
del(GJB6-D13S1830) and del(GJB6-D13S1854) muta-
Until now, many studies investigating populations from
Europe, Asia, Australia or North America, have shown
that DFNB1 accounts for approximately 50% of con-
genital, severe-to-profound, and autosomal recessive non-
syndromic hearing loss [11-21]. GJB2 encodes connexin
26, and GJB6 encodes connexin 30. These are the only
two genes known to be associated with deafness at the
DFNB1 lo cus. Approximately 98% of individuals with
DFNB1 have two identifiable GJB2 mutations (i.e. , they
are homozygotes or compound heterozygotes). More
than half of all persons of northern European ancestry
with two identifiable GJB2 mutations are homozygous
for the c.35 delG point mutation [7]. Approximately 2%
of individuals with DFNB1 have one identifiable GJB2
mutation and one of two large deletions that include a
portion of GJB6. Clinical signs direct the molecular di-
agnosis to specific genes.
Considering the data from the literature and the facts
that Romanians are of Caucasian origin and settled in
South Eastern Europe, we decided to start the investiga-
tions of DFNB1 related HL. In this respect, our results
show the presence and the higher frequency of 35 delG
GJB2 mutation among persons of South Eastern Europe
and confirm, once more, the European origin of the mu-
tation. In European countries, the prevalence of the 35
delG mutation, in normal-hearing individuals, has been
estimated to range from 2 to 4% [22]. Absence of GJB6
mutations could be explained by the small size sample
investigated or the frequency of the mutations is not sig-
nificant for Romanian population comparable to the data
from Italy, Belgium, Turkey and Slovakia [8,23-26].
HL is the most common birth defect and the most
prevalent sensorineural disorder in developed countries
[27]. One of every 500 newborns has bilateral permanent
sensorineural hearing loss 40 dB; by adolescence, the
prevalence increases to 3.5 per 1000 [28]. The results of
the study are of medical interest in audiological practice
or family planning but also in the characterization of epide-
miological data. The incidence of congenital HL is lower
among Romanian neonates than in many European coun-
tries but undoubtedly the number of neonates is still sig-
nificant. In Romania an estimated 200,000 babies are
born in Romania each year. This means that around 200
babies with congenital HL should be born each year.
Based on our results, newborn hearing screening test
detects neonates at-risk and combines with molecular
genetic testing, which could early clarify the status of the
patient. As early as possible after that, the treatment
should start: first, to fit with hearing aids and enrollment
in appropriate educational programs and second, to pre-
vent the consequences. So, we suggest that newborn
hearing screening test could be offered as a routine test
during postpartum hospitalization because it is available
and cost-effective. Except few hospitals, the test is of-
fered by request and usually the parents notice the
Copyright © 2013 SciRes. OPEN ACCESS
C. Dragomir et al. / American Journal of Molecular Biology 3 (2013) 229-234 233
handicap of their child too late for intervention. We con-
sider molecular diagnosis as part of evaluation strategy
because it confirms the diagnosis in a proband, which
could clarify the carrier status of the parents and allows
the identification of recurrence risk in future pregnancies.
Molecular diagnosis is a complement to clinical exami-
nation and not an alternative. The test is available and
cost-effective. Non-invasive methods can be used to col-
lect DNA from high-risk neonates and children. More-
over, in Romania rehabilitation and support services are
available too.
Also, our results give an overview of the main causes
of congenital HL, exclude the GJB6 gene from hearing
screening protocols and offer a new strategy for preven-
tion of hearing loss in Romania.
We thank all families for their participation in this study.
[1] ASHA Web Site (2005) Audiology information series:
Effects of hearing loss on development.
[2] ACMG (2002) Genetics evaluation guidelines for the
etiologic diagnosis of congenital hearing loss. Genetic
Evaluation of Congenital Hearing Loss Expert Panel:
ACMG statement. Genetics in Medicine, 4, 162-171.
[3] Morton, N.E. (1991) Genetic epidemiology of hearing
impairment. Annals of the New York Academy of Sciences,
630, 16-31.
[4] Van Camp, G., Willems, P.J. and Smith, R.J. (1997)
Nonsyndromic hearing impairment: Unparalleled hetero-
geneity. The American Journal of Human Genetics, 60,
[5] Zelante, L., Gasparini, P., Estivill, X., Melchionda, S.,
D’Agruma, L., Govea, N., Mila, M., Monica, M.D., Lutfi,
J., Shohat, M., Manfield, E., Delgroso, K., Rappaport, E.,
Surrey, S. and Fortina, P. (1997) Connexin 26 mutations
associated with the most common form of non-syndromic
neurosensory autosomal recessive deafness (DFNB1) in
Mediterraneans. Human Molecular Genetics, 6, 1605-1609.
[6] Morell, R.J., Kim, H.J., Hood, L.J., Goforth, L., Friderici,
K., Fisher, R., Van Camp, G., Berlin, C.I., Oddoux, C.,
Ostrer, H., Keats, B., Friedman, T.B. (1998) Mutations in
the connexin 26 gene (GJB2) among Ashkenazi Jews
with nonsyndromic recessive deafness. The New England
Journal of Medicine, 339, 1500-1505.
[7] Scott, D.A., Kraft, M.L., Carmi, R., Ramesh, A., Elbe-
dour, K., Yairi, Y., Srikumari Srisailapathy, C.R., Rosen-
gren, S.S., Markham, A.F., Mueller, R.F., Lench, N.J.,
Van Camp, G., Smith, R.J.H. and Sheffield, V.C. (1998)
Identification of mutation in the connexin 26 gene that
cause autosomal recessive nonsyndromic hearing loss.
Human Mutation, 11, 387-394.<38
[8] Del Castillo, I., Villamar, M., Moreno-Pelayo, M.A., Del
Castillo, F.J., Alvarez, A., Telleria, D. and Menendez, I.,
Moreno, F. (2002) A deletion involving the connexin 30
gene in nonsyndromic hearing impairment. The New
England Journal of Medicine, 346, 243-249.
[9] Dragomir C., Stan A., Stefanescu T.D., Savu L. and Se-
verin E. (2011) Prenatal Screening for the 35delG GJB2,
Del (GJB6-D13S1830), and Del (GJB6-D13S1854) Mu-
tations in Romanian Population. Genetic Testing and Mo-
lecular Biomarkers, 15, 749-753.
[10] Wang, X.W., Rinehart, T.A., Wadl, P.A., Spiers, J.M.,
Hadziabdic, D., Windham1 M.T. and Robert Trigiano N.
(2009) A new electrophoresis technique to separate mic-
rosatellite alleles, African Journal of Biotechnology, 8,
[11] Denoyelle, F., Weil, D., Maw, M.A., Wilcox, S.A., Lench,
N.J., Allen-Powell, D.R., Osborn, A.H., Dahl, H.H.,
Middleton, A., Houseman, M.J., Dode, C., Marlin, S.,
Boulila-ElGaied, A., Grati, M., Ayadi, H., BenArab, S.,
Bitoun, P., Lina-Granade, G., Godet, J., Mustapha, M.,
Loiselet, J., El-Zir, E., Aubois, A., Joannard, A., Petit, C.,
et al. (1997) Prelingual deafness: High prevalence of a
30delG mutation in the connexin 26 gene. Human Mo-
lecular Genetics, 6, 2173-2177.
[12] Estivill, X., Fortina, P., Surrey, S., Rabionet, R., Mel-
chionda, S., D’Agruma, L., Mansfield, E., Rappaport, E.,
Govea, N., Mila, M., Zelante, L. and Gasparini, P. (1998)
Connexin-26 mutations in sporadic and inherited senso-
rineural deafness. Lancet, 351, 394-398.
[13] Green, G.E., Scott, D.A., McDonald, J.M., Woodworth,
G.G., Sheffield, V.C. and Smith, R.J.H. (1999) Carrier
rates in the mid-western United States for GJB2 muta-
tions causing inherited deafness. JAMA, 281, 2211-2116.
[14] Kudo, T., Ikeda, K., Kure, S., Matsubara, Y., Oshima, T.,
Watanabe, K., Kawase, T., Narisawa, K. and Takasaka, T.
(2000) Novel mutations in the connexin 26 gene (GJB2)
responsible for childhood deafness in the Japanese popu-
lation. American Journal of Medical Genetics, 90, 141-
[15] Anichkina, A., Kulenich, T., Zinchenko, S., Shagina, I.,
Polyakov, A., Ginter, E., Evgrafov, O., Viktorova, T. and
Khusnitdonova, E. (2001) On the origin and frequency of
the 35delG allele in GJB2-linked deafness in Europe.
European Journal of Human Genetics, 9, 151.
[16] Lucotte, G. and Mercier, G. (2001) Meta-analysis of
GJB2 mutation 35delG frequencies in Europe. Genet Test,
5, 149-152.
Copyright © 2013 SciRes. OPEN ACCESS
C. Dragomir et al. / American Journal of Molecular Biology 3 (2013) 229-234
Copyright © 2013 SciRes.
[17] Hwa, H.L., Ko, T.M., Hsu, C.J., Huang, C.H., Chiang,
Y.L., Oong, J.L., Chen, C.C. and Hsu, C.K. (2003) Mu-
tation spectrum of the connexin 26 (GJB2) gene in
Taiwanese patients with prelingual deafness. Genetics in
Medicine, 5, 161-165.
[18] Rothrock, C.R., Murgia, A., Sartorato, E.L., Leonardi, E.,
Wei, S., Lebeis, S.L., Yu, L.E., Elfenbein, J.L., Fisher,
R.A. and Friderici, K.H. (2003) Connexin 26 35delG
does not represent a mutational hotspot. Human Genetics,
113, 18-23.
[19] Najmabadi, H., Nishimura, C., Kahrizi, K., Riazalhos-
seini, Y., Malekpour, M., Daneshi, A., Farhadi, M., Moh-
seni, M., Mahdieh, N., Ebrahimi, A., Bazazzadegan, N.,
Naghavi, A., Avenarius, M., Arzhangi, S. and Smith, R.J.
(2005) GJB2 mutations: Passage through Iran. American
Journal of Medical Genetics Part A, 133, 132-137.
[20] Lazar, C., Popp, R., Trifa, A., Mocanu, C., Al-Khzouz, C.,
Tomescu, E., Figan, I. and Grigorescu-Sido, P. (2010)
Prevalence of the c.35delG and p.W24X mutations in the
GJB2 gene in patients with nonsyndromic hearing loss
from North-West Romania. International Journal of Pe-
diatric Otorhinolaryngology, 74, 351-355.
[21] Mahdieh N., Rabbani B., Wiley S., Akbari M.T. and Zei-
nali S. (2010) Genetic causes of nonsyndromic hearing
loss in Iran in comparison with other populations. Journal
of Human Genetics, 55, 639-648.
[22] Gasparini, P., Rabionet, R., Barbujani, G., Melchionda, S.,
Petersen, M. and Brondum-Nielsen, K. (2000) High car-
rier frequency of the 35delG deafness mutation in Euro-
pean populations. Genetic Analysis Consortium of GJB2
35delG. European Journal of Human Genetics, 8, 19-23.
[23] Del Castillo, I., Moreno-Pelayo, M.A., Del Castillo, F.J.,
Brownstein, Z., Marlin, S., Adina, Q., Cockburn, D.J.,
Pandya, A., Siemering, K.R., Chamberlin, G.P., Balana,
E., Wuyts, W., Maciel-Guerra, A.T., Alvarez, A., Villa-
mar, M., Shohat, M., Abelivich, D., Dahl, H.H.M., Esti-
vill, X., Gasparini, P., Hutchin, T., Nance, W.E., Sarto-
rato, E.L., Smith, R.J.H., Van Camp, G., Avraham, K.B.,
Petit, C. and Moreno, F. (2003) Prevalence and evolu-
tionary origins of the del (GJB6-D13S1830) mutations in
the DFNB1 locus in hearing impaired subjects: A mul-
ticenter study. The American Journal of Human Genetics,
73, 1452-1458.
[24] Del Castillo, F.J., Rodrigues-Ballesteros, M., Alvarez, A.,
Hutchin, T., Leonardi, E., De Oliveira, C.A., Azaiez, H.,
Brownstein, Z., Avenarius, M.R., Marlin, S., Pandya, A.,
Shahin, H., Siemering, K.R., Weil, D., Wuyts, W., Ag-
uirre, L.A., Martin, Y., Moreno-Pelayo, M.A., Villamar,
M., Avraham, K.B., Dahl, H.H.M., Kanaan, M., Nance,
W.E., Petit, C., Smith, R.J.H., Van Camp, G., Sartorato,
E.L., Murgia, A., Moreno, F. and Del Castillo, I. (2005)
A novel deletion involving the connexin-30 gene, del
(GJB6-d13s1854), found in trans with mutations in the
GJB2 gene (connexin-26) in subjects with DFNB1 non-
syndromic hearing impairment. Journal of Medical Gene-
tics, 42, 588-594.
[25] Sirmaci, A., Akcayoz-Duman, D. and Tekin, M. (2006).
The c.IVS1+1G>A mutation in the GJB2 gene is preva-
lent and large deletions involving the GJB6 gene are not
present in Turkish population. Journal of Genetics, 85,
[26] Minarik, G., Tretiarova D., Szemes T. and Kadasi L.
(2012) Prevalence of DFNB1 mutations in Slovak popu-
lation with non-syndromic hearing loss. International
Journal of Pediatric Otorhinolaryngology, 76, 400-403.
[27] Hilgert, N., Smith, R.J. and Van Camp, G. (2009)
Forty-six genes causing nonsyndromic hearing impair-
ment: Which ones should be analyzed in DNA diagnos-
tics. Mutation Research, 681, 189-96.
[28] Morton, C.C. and Nance, W.E. (2006) Newborn hearing
screening—A silent revolution. The New England Jour-
nal of Medicine, 354, 2151-2164.