Molecular and genetic characterization of a large Brazilian cohort presenting hearing loss.

Hearing loss is one of the most common sensory defects, affecting 5.5% of the worldwide population and significantly impacting health and social life. It is mainly attributed to genetic causes, but their relative contribution reflects the geographical region's socio-economic development. Extreme genetic heterogeneity with hundreds of deafness genes involved poses challenges for molecular diagnosis. Here we report the investigation of 542 hearing-impaired subjects from all Brazilian regions to search for genetic causes. Biallelic GJB2/GJB6 causative variants were identified in 12.9% (the lowest frequency was found in the Northern region, 7.7%), 0.4% carried GJB2 dominant variants, and 0.6% had the m.1555A > G variant (one aminoglycoside-related). In addition, other genetic screenings, employed in selected probands according to clinical presentation and presumptive inheritance patterns, identified causative variants in 2.4%. Ear malformations and auditory neuropathy were diagnosed in 10.8% and 3.5% of probands, respectively. In 3.8% of prelingual/perilingual cases, Waardenburg syndrome was clinically diagnosed, and in 71.4%, these diagnoses were confirmed with pathogenic variants revealed; seven out of them were novel, including one CNV. All these genetic screening strategies revealed causative variants in 16.2% of the cases. Based on causative variants in the molecular diagnosis and genealogy analyses, a probable genetic etiology was found in ~ 50% of the cases. The present study highlights the relevance of GJB2/GJB6 as a cause of hearing loss in all Brazilian regions and the importance of screening unselected samples for estimating frequencies. Moreover, when a comprehensive screening is not available, molecular diagnosis can be enhanced by selecting probands for specific screenings.

Genetics of Hearing Impairment in North-Eastern Romania-A Cost-Effective Improved Diagnosis and Literature Review.

BACKGROUND: We have investigated the main genetic causes for non-syndromic hearing impairment (NSHI) in the hearing impairment individuals from the North-Eastern Romania and proposed a cost-effective diagnosis protocol. METHODS: MLPA followed by Sanger Sequencing were used for all 291 patients included in this study. RESULTS: MLPA revealed abnormal results in 141 cases (48.45%): 57 (40.5%) were c.35delG homozygous, 26 (18.44%) were c.35delG heterozygous, 14 (9.93%) were compound heterozygous and 16 (11.35%) had other types of variants. The entire coding region of GJB2 was sequenced and out of 150 patients with normal results at MLPA, 29.33% had abnormal results: variants in heterozygous state: c.71G>A (28%), c.457G>A (20%), c.269T>C (12%), c.109G>A (12%), c.100A>T (12%), c.551G>C (8%). Out of 26 patients with c.35delG in heterozygous state, 38.46% were in fact compound heterozygous. CONCLUSIONS: We identified two variants: c.109G>A and c.100A>T that have not been reported in any study from Romania. MLPA is an inexpensive, rapid and reliable technique that could be a cost-effective diagnosis method, useful for patients with hearing impairment. It can be adaptable for the mutation spectrum in every population and followed by Sanger sequencing can provide a genetic diagnosis for patients with different degrees of hearing impairment.

Enhancing Genetic Medicine: Rapid and Cost-Effective Molecular Diagnosis for a GJB2 Founder Mutation for Hearing Impairment in Ghana.

In Ghana, gap-junction protein ß 2 (GJB2) variants account for about 25.9% of familial hearing impairment (HI) cases. The GJB2-p.Arg143Trp (NM_004004.6:c.427C>T/OMIM: 121011.0009/rs80338948) variant remains the most frequent variant associated with congenital HI in Ghana, but has not yet been investigated in clinical practice. We therefore sought to design a rapid and cost-effective test to detect this variant. We sampled 20 hearing-impaired and 10 normal hearing family members from 8 families segregating autosomal recessive non syndromic HI. In addition, a total of 111 unrelated isolated individuals with HI were selected, as well as 50 normal hearing control participants. A restriction fragment length polymorphism (RFLP) test was designed, using the restriction enzyme NciI optimized and validated with Sanger sequencing, for rapid genotyping of the common GJB2-p.Arg143Trp variant. All hearing-impaired participants from 7/8 families were homozygous positive for the GJB2-p.Arg143Trp mutation using the NciI-RFLP test, which was confirmed with Sanger sequencing. The investigation of 111 individuals with isolated non-syndromic HI that were previously Sanger sequenced found that the sensitivity of the GJB2-p.Arg143Trp NciI-RFLP testing was 100%. All the 50 control subjects with normal hearing were found to be negative for the variant. Although the test is extremely valuable, it is not 100% specific because it cannot differentiate between other mutations at the recognition site of the restriction enzyme. The GJB2-p.Arg143Trp NciI-RFLP-based diagnostic test had a high sensitivity for genotyping the most common GJB2 pathogenic and founder variant (p.Arg143Trp) within the Ghanaian populations. We recommend the adoption and implementation of this test for hearing impairment genetic clinical investigations to complement the newborn hearing screening program in Ghana. The present study is a practical case scenario of enhancing genetic medicine in Africa.

Expanded carrier screening for preconception reproductive risk assessment: Prevalence of carrier status in a Mexican population.

OBJECTIVE: Genetic carrier screening has the potential to identify couples at risk of having a child affected with an autosomal recessive or X-linked disorder. However, the current prevalence of carrier status for these conditions in developing countries is not well defined. This study assesses the prevalence of carrier status of selected genetic conditions utilizing an expanded, pan-ethnic genetic carrier screening panel (ECS) in a large population of Mexican patients. METHODS: Retrospective chart review of all patients tested with a single ECS panel at an international infertility center from 2012 to 2018 were included, and the prevalence of positive carrier status in a Mexican population was evaluated. RESULTS: Eight hundred five individuals were analyzed with ECS testing for 283 genetic conditions. Three hundred fifty-two carriers (43.7%) were identified with 503 pathogenic variants in 145 different genes. Seventeen of the 391 participating couples (4.34%) were identified as being at-risk couples. The most prevalent alleles found were associated with alpha thalassemia, cystic fibrosis, GJB2 nonsyndromic hearing loss, biotinidase deficiency, and familial Mediterranean fever. CONCLUSION: Based on the prevalence and severity of Mendelian disorders, we recommend that couples who wish to conceive regardless of their ethnicity background explore carrier screening and genetic counseling prior to reproductive medical treatment.

Public Health Burden of Hearing Impairment and the Promise of Genomics and Environmental Research: A Case Study in Ghana, Africa.

Hearing impairment (HI) is one of the most disabling conditions of major global health burden that contributes adversely to the social and economic development of a country, if not managed properly. A proper assessment of the nationwide burden and etiology of HI is instrumental in the prevention, treatment, and management of the condition. This article sought to perform an expert review of HI in Ghana to determine the present knowledge of its burden and possible causes of the condition. A literature search was conducted in PubMed using the following keywords: "hearing loss" OR "hearing impairment" OR "deafness" AND "Ghana." The literature was scanned until July 20, 2017, with specific inclusion of targeted landmark and background articles on HI. From the search, 18 of out 5869 articles were selected and considered for the review. The results of the search indicated that there were no extensive studies to determine the national burden of HI in Ghana. However, the few studies assessed suggested that the disease is either acquired or inherited. The burden of acquired HI was higher in adults than children, women than men, and people working in a noisy environment. Regarding the genetic cause, specific founder mutations in GJB2 gene (R143W, L79P, V178A, R184Q, A197S, I203K, and L214P) was the only identified genetic cause of HI in Ghana, but the other HI genes were not investigated. There has been some modest effort to study HI in Ghana, but comprehensive studies on the genetic and environmental etiologies (using the "multi-OMICS" approaches), classification, and burden of HI on Ghana are needed.

Barriers to early cochlear implantation.

OBJECTIVE: Identify variables associated with paediatric access to cochlear implants (CIs). DESIGN: Part 1. Trends over time for age at CI surgery (N = 802) and age at hearing aid (HA) fitting (n = 487) were examined with regard to periods before, during, and after newborn hearing screening (NHS). Part 2. Demographic factors were explored for 417 children implanted under 3 years of age. Part 3. Pre-implant steps for the first 20 children to receive CIs under 12 months were examined. RESULTS: Part 1. Age at HA fitting and CI surgery reduced over time, and were associated with NHS implementation. Part 2. For children implanted under 3 years, earlier age at HA fitting and higher family socio-economic status were associated with earlier CI. Progressive hearing loss was associated with later CIs. Children with a Connexin 26 diagnosis received CIs earlier than children with a premature / low birth weight history. Part 3. The longest pre-CI steps were Step 1: Birth to diagnosis/identification of hearing loss (mean 16.43 weeks), and Step 11: MRI scans to implant surgery (mean 15.05 weeks) for the first 20 infants with CIs under 12 months. CONCLUSION: NHS implementation was associated with reductions in age at device intervention in this cohort.

Reproductive management through integration of PGD and MPS-based noninvasive prenatal screening/diagnosis for a family with GJB2-associated hearing impairment.

A couple with a proband child of GJB2 (encoding the gap junction protein connexin 26)-associated hearing impairment and a previous pregnancy miscarriage sought for a reproductive solution to bear a healthy child. Our study aimed to develop a customized preconception-to-neonate care trajectory to fulfill this clinical demand by integrating preimplantation genetic diagnosis (PGD), noninvasive prenatal testing (NIPT), and noninvasive prenatal diagnosis (NIPD) into the strategy. Auditory and genetic diagnosis of the proband child was carried out to identify the disease causative mutations. The couple then received in-vitro-fertilization treatment, and eight embryos were obtained for day 5 biopsy. PGD was performed by short-tandem-repeat linkage analysis and Sanger sequencing of GJB2 gene. Transfer of a GJB2c.235delC heterozygous embryo resulted in a singleton pregnancy. At the 13th week of gestation, genomic DNA (gDNA) from the trio family and cell-free DNA (cfDNA) from maternal plasma were obtained for assessment of fetal chromosomal aneuploidy and GJB2 mutations. NIPT and NIPD showed the absence of chromosomal aneuploidy and GJB2-associated disease in the fetus, which was later confirmed by invasive procedures and postnatal genetic/auditory diagnosis. This strategy successfully prevented the transmission of hearing impairment in the newborn, thus providing a valuable experience in reproductive management of similar cases and potentially other monogenic disorders.

Screening of genetic alterations related to non-syndromic hearing loss using MassARRAY iPLEX® technology.

BACKGROUND: Recent advances in molecular genetics have enabled to determine the genetic causes of non-syndromic hearing loss, and more than 100 genes have been related to the phenotype. Due to this extraordinary genetic heterogeneity, a large percentage of patients remain without any molecular diagnosis. This condition imply the need for new methodological strategies in order to detect a greater number of mutations in multiple genes. In this work, we optimized and tested a panel of 86 mutations in 17 different genes screened using a high-throughput genotyping technology to determine the molecular etiology of hearing loss. METHODS: The technology used in this work was the MassARRAY iPLEX® platform. This technology uses silicon chips and DNA amplification products for accurate genotyping by mass spectrometry of previous reported mutations. The generated results were validated using conventional techniques, as direct sequencing, multiplex PCR and RFLP-PCR. RESULTS: An initial genotyping of control subjects, showed failures in 20 % of the selected alterations. To optimize these results, the failed tests were re-designed and new primers were synthesized. Then, the specificity and sensitivity of the panel demonstrated values above 97 %. Additionally, a group of 180 individuals with NSHL without a molecular diagnosis was screened to test the diagnostic value of our panel, and mutations were identified in 30 % of the cases. In 20 % of the individuals, it was possible to explain the etiology of the HL. Mutations in GJB2 gene were the most prevalent, followed by other mutations in in SLC26A4, CDH23, MT-RNR1, MYO15A, and OTOF genes. CONCLUSIONS: The MassARRAY technology has the potential for high-throughput identification of genetic variations. However, we demonstrated that optimization is required to increase the genotyping success and accuracy. The developed panel proved to be efficient and cost-effective, being suitable for applications involving the molecular diagnosis of hearing loss.

[Assessment of the curative effective of cochlear implantation in childer with GJB2-associated NSSNHL].

OBJECTIVE: To analyze the curative effect of CI in children with GJB2-associated NSSNHL. METHOD: The evaluations of curative effect with CI include auditory threshold, IT-MAIS/MAIS, CAP, SIR. MESP. The outcomes of 40 cases with GJB2-associated NSSNHI, were compared 80 patients with negative results of screening of gene mutation (control group). RESULT: In comparison with control group the auditory threshold in children with GJB2-associated NSSNIL is better, however had no significant difference in other tests (P > 0.05). CONCLUSION: CI could he performed on children with GJB2-associated NSSNHL. Postoperative outcomes of hearing and speech were satisfied.

A filter paper-based microdevice for low-cost, rapid, and automated DNA extraction and amplification from diverse sample types.

A plastic microfluidic device that integrates a filter disc as a DNA capture phase was successfully developed for low-cost, rapid and automated DNA extraction and PCR amplification from various raw samples. The microdevice was constructed by sandwiching a piece of Fusion 5 filter, as well as a PDMS (polydimethylsiloxane) membrane, between two PMMA (poly(methyl methacrylate)) layers. An automated DNA extraction from 1 µL of human whole blood can be finished on the chip in 7 minutes by sequentially aspirating NaOH, HCl, and water through the filter. The filter disc containing extracted DNA was then taken out directly for PCR. On-chip DNA purification from 0.25-1 µL of human whole blood yielded 8.1-21.8 ng of DNA, higher than those obtained using QIAamp® DNA Micro kits. To realize DNA extraction from raw samples, an additional sample loading chamber containing a filter net with an 80 µm mesh size was designed in front of the extraction chamber to accommodate sample materials. Real-world samples, including whole blood, dried blood stains on Whatman® 903 paper, dried blood stains on FTA™ cards, buccal swabs, saliva, and cigarette butts, can all be processed in the system in 8 minutes. In addition, multiplex amplification of 15 STR (short tandem repeat) loci and Sanger-based DNA sequencing of the 520 bp GJB2 gene were accomplished from the filters that contained extracted DNA from blood. To further prove the feasibility of integrating this extraction method with downstream analyses, "in situ" PCR amplifications were successfully performed in the DNA extraction chamber following DNA purification from blood and blood stains without DNA elution. Using a modified protocol to bond the PDMS and PMMA, our plastic PDMS devices withstood the PCR process without any leakage. This study represents a significant step towards the practical application of on-chip DNA extraction methods, as well as the development of fully integrated genetic analytical systems.

Noninvasive prenatal testing for autosomal recessive conditions by maternal plasma sequencing in a case of congenital deafness.

PURPOSE: The goals of our study were to develop a noninvasive prenatal test for autosomal recessive monogenic conditions and to prove its overall feasibility and potential for clinical integration. METHODS: We recruited a pregnant woman and her spouse, who had a proband child suffering from congenital deafness, and obtained the target-region sequencing data from a semicustom array that used genomic and maternal plasma DNA from three generations of this family. A haplotype-assisted strategy was developed to detect whether the fetus inherited the pathogenic mutations in the causative gene, GJB2. The parental haplotype was constructed using a trio strategy through two different processes, namely, the grandparent-assisted haplotype phasing process and the proband-assisted haplotype phasing process. The fetal haplotype was deduced afterward based on both the maternal plasma sequencing data and the parental haplotype. RESULTS: The accuracy levels of paternal and maternal haplotypes obtained by grandparent-assisted haplotype phasing were 99.01 and 97.36%, respectively, and the proband-assisted haplotype phasing process yielded slightly lower accuracies of 98.73 and 96.79%, respectively. Fetal inheritance of the pathogenic gene was deduced correctly in both processes. CONCLUSION: Our study indicates that the strategy of haplotype-based noninvasive prenatal testing for monogenic conditions has potential applications in clinical practice.

Barriers to early pediatric cochlear implantation.

OBJECTIVES: Universal newborn hearing screening has significantly improved the ability to identify patients with congenital sensorineural hearing loss (SNHL), which results in earlier treatment and better hearing and development outcomes. It is recommended that patients born with SNHL who meet criteria receive cochlear implants (CIs) by a target age of 12 months, however many children are being implanted at an older age. This study aims to describe populations of pre-lingual patients with SNHL that are at risk for delayed implantation and to identify and analyze barriers that cause this delay. METHODS: Charts of patients receiving a CI between January 2008 and June 2012 at a tertiary care cochlear implant center were reviewed retrospectively. We looked at patient demographics, age at hearing loss diagnosis, age at implantation, and etiology of hearing loss. Barriers to implantation were identified through surveys completed by team members. RESULTS: Fifty-seven CI recipients were identified of which 42 were in patients with pre-lingual SNHL. SNHL etiology included: cochlear dysplasia (18%), GJB2/GJB6 (17%), acquired (10%) extreme prematurity (9%), and idiopathic (46%). The median age of SNHL diagnosis for pre-lingual patients was 15 months. Compared to private insurance, public insurance status was associated with SNHL diagnosis at a significantly later median age (20.0 vs. 4.0 months, p=0.024), and with a significantly longer median interval from diagnosis to implantation (25.5 vs. 11.0 months, p=0.029). While cochlear implant team members identified delayed insurance approval and medical comorbidities as reasons for delayed implantation, the most significant factor identified was parental, with delayed/missed appointments or reluctance for evaluations or surgery. CONCLUSION: 52% of patients with pre-lingual SNHL that met criteria for CI were implanted more than 12 months after diagnosis. Having public or no insurance was significantly associated with delayed implantation. Parental barriers were most common factors cited for delays in implantation. Overcoming these delays necessitates appropriate identification of at risk patients and creating a system to educate families and chaperone them through the process.

Diagnostic application of targeted resequencing for familial nonsyndromic hearing loss.

Identification of causative genes for hereditary nonsyndromic hearing loss (NSHL) is important to decide treatment modalities and to counsel the patients. Due to the genetic heterogeneity in sensorineural genetic disorders, the high-throughput method can be adapted for the efficient diagnosis. To this end, we designed a new diagnostic pipeline to screen all the reported candidate genes for NSHL. For validation of the diagnostic pipeline, we focused upon familial NSHL cases that are most likely to be genetic, rather than to be infectious or environmental. Among the 32 familial NSHL cases, we were able to make a molecular genetic diagnosis from 12 probands (37.5%) in the first stage by their clinical features, characteristic inheritance pattern and further candidate gene sequencing of GJB2, SLC26A4, POU3F4 or mitochondrial DNA. Next we applied targeted resequencing on 80 NSHL genes in the remaining 20 probands. Each proband carried 4.8 variants that were not synonymous and had the occurring frequency of less than three among the 20 probands. These variants were then filtered out with the inheritance pattern of the family, allele frequency in normal hearing 80 control subjects, clinical features. Finally NSHL-causing candidate mutations were identified in 13(65%) of the 20 probands of multiplex families, bringing the total solve rate (or detection rate) in our familial cases to be 78.1% (25/32) Damaging mutations discovered by the targeted resequencing were distributed in nine genes such as WFS1, COCH, EYA4, MYO6, GJB3, COL11A2, OTOF, STRC and MYO3A, most of which were private. Despite the advent of whole genome and whole exome sequencing, we propose targeted resequencing and filtering strategy as a screening and diagnostic tool at least for familial NSHL to find mutations based upon its efficacy and cost-effectiveness.

Digenic inheritance in autosomal recessive non-syndromic hearing loss cases carrying GJB2 heterozygote mutations: assessment of GJB4, GJA1, and GJC3.

OBJECTIVE: Autosomal recessive non-syndromic hearing loss (ARNSHL) can be caused by many genes. However, mutations in the GJB2 gene, which encodes the gap-junction (GJ) protein connexin (Cx) 26, constitute a considerable proportion differing among population. Between 10 and 42 percent of patients with recessive GJB2 mutations carry only one mutant allele. Mutations in GJB4, GJA1, and GJC3 encoding Cx30.3, Cx43, and Cx29, respectively, can lead to HL. Combination of different connexins in heteromeric and heterotypic GJ assemblies is possible. This study aims to determine whether variations in any of the genes GJB4, GJA1 or GJC3 can be the second mutant allele causing the disease in the digenic mode of inheritance in the studied GJB2 heterozygous cases. METHODS: We examined 34 unrelated GJB2 heterozygous ARNSHL subjects from different geographic and ethnic areas in Iran, using polymerase chain reaction (PCR) followed by direct DNA sequencing to identify any sequence variations in these genes. Restriction fragment length polymorphism (RFLP) assays were performed on 400 normal hearing individuals. RESULTS: Sequence analysis of GJB4 showed five heterozygous variations including c.451C>A, c.219C>T, c.507C>G, c.155_158delTCTG and c.542C>T, with only the latter variation not being detected in any of control samples. There were three heterozygous variations including c.758C>T, c.717G>A and c.3*dupA in GJA1 in four cases. We found no variations in GJC3 gene sequence. CONCLUSION: Our data suggest that GJB4 c.542C>T variant and less likely some variations of GJB4 and GJA1, but not possibly GJC3, can be assigned to ARNSHL in GJB2 heterozygous mutation carriers providing clues of the digenic pattern.

Molecular dynamics simulations of the Cx26 hemichannel: evaluation of structural models with Brownian dynamics.

The recently published crystal structure of the Cx26 gap junction channel provides a unique opportunity for elucidation of the structure of the conductive connexin pore and the molecular determinants of its ion permeation properties (conductance, current-voltage [I-V] relations, and charge selectivity). However, the crystal structure was incomplete, most notably lacking the coordinates of the N-terminal methionine residue, which resides within the pore, and also lacking two cytosolic domains. To allow computational studies for comparison with the known channel properties, we completed the structure. Grand canonical Monte Carlo Brownian dynamics (GCMC/BD) simulations of the completed and the published Cx26 hemichannel crystal structure indicate that the pore is too narrow to permit significant ion flux. The GCMC/BD simulations predict marked inward current rectification and almost perfect anion selectivity, both inconsistent with known channel properties. The completed structure was refined by all-atom molecular dynamics (MD) simulations (220 ns total) in an explicit solvent and POPC membrane system. These MD simulations produced an equilibrated structure with a larger minimal pore diameter, which decreased the height of the permeation barrier formed by the N terminus. GCMC/BD simulations of the MD-equilibrated structure yielded more appropriate single-channel conductance and less anion/cation selectivity. However, the simulations much more closely matched experimentally determined I-V relations when the charge effects of specific co- and posttranslational modifications of Cx26 previously identified by mass spectrometry were incorporated. We conclude that the average equilibrated structure obtained after MD simulations more closely represents the open Cx26 hemichannel structure than does the crystal structure, and that co- and posttranslational modifications of Cx26 hemichannels are likely to play an important physiological role by defining the conductance and ion selectivity of Cx26 channels. Furthermore, the simulations and data suggest that experimentally observed heterogeneity in Cx26 I-V relations can be accounted for by variation in co- and posttranslational modifications.

Genotyping with a 198 mutation arrayed primer extension array for hereditary hearing loss: assessment of its diagnostic value for medical practice.

Molecular diagnostic testing of individuals with congenital sensorineural hearing loss typically begins with DNA sequencing of the GJB2 gene. If the cause of the hearing loss is not identified in GJB2, additional testing can be ordered. However, the step-wise analysis of several genes often results in a protracted diagnostic process. The more comprehensive Hereditary Hearing Loss Arrayed Primer Extension microarray enables analysis of 198 mutations across eight genes (GJB2, GJB6, GJB3, GJA1, SLC26A4, SLC26A5, MTRNR1 and MTTS1) in a single test. To evaluate the added diagnostic value of this microarray for our ethnically diverse patient population, we tested 144 individuals with congenital sensorineural hearing loss who were negative for biallelic GJB2 or GJB6 mutations. The array successfully detected all GJB2 changes previously identified in the study group, confirming excellent assay performance. Additional mutations were identified in the SLC26A4, SLC26A5 and MTRNR1 genes of 12/144 individuals (8.3%), four of whom (2.8%) had genotypes consistent with pathogenicity. These results suggest that the current format of this microarray falls short of adding diagnostic value beyond the customary testing of GJB2, perhaps reflecting the array's limitations on the number of mutations included for each gene, but more likely resulting from unknown genetic contributors to this phenotype. We conclude that mutations in other hearing loss associated genes should be incorporated in the array as knowledge of the etiology of hearing loss evolves. Such future modification of the flexible configuration of the Hereditary Hearing Loss Arrayed Primer Extension microarray would improve its impact as a diagnostic tool.

Easy, rapid, and cost-effective methods for identifying carriers of recurrent GJB2 mutations causing nonsyndromic hearing impairment in the Greek population.

A variety of techniques have been developed for screening the GJB2 gene for known and unknown mutations, especially the most common mutation in the Caucasian population, the c.35delG. Other mutations that have been so far characterized in the GJB2 gene seem to have different geographical distributions, and therefore there is an interest in identifying recurrent mutations specific for each population and developing easy and rapid screening techniques. Here we present easy screening protocols for already identified recurrent mutations in the Greek population. Developing easy, rapid, and cost-effective screening methods will facilitate the detection of GJB2 recurrent mutation carriers, at large, in the Greek population.

Application of SNaPshot multiplex assays for simultaneous multigene mutation screening in patients with idiopathic sensorineural hearing impairment.

OBJECTIVES/HYPOTHESIS: To develop a cost-effective and robust genetic diagnostic tool for patients with idiopathic nonsyndromic sensorineural hearing impairment. STUDY DESIGN: Development of a diagnostic tool and validation in a prospective cohort. METHODS: Twenty common sequence variants in GJB2, SLC26A4, and the mitochondrial 12S rRNA gene were selected based on our previous epidemiological study. These variants were analyzed using the SNaPshot technique. The efficacies of the SNaPshot multiplex assays were determined by using a prospective cohort composed of 214 unrelated Taiwanese patients with idiopathic sensorineural hearing impairment. The results of the assays were compared to the results obtained by direct sequencing. RESULTS: We developed a diagnostic technique consisting of two consecutive panels of SNaPshot multiplex assays, with each panel screening 10 common sequence variants. Theoretically, this design can detect more than 98% of the known deafness-associated sequence variants in Taiwanese individuals. A total of 126 (58.9%) patients were diagnosed as having at least one sequence variant using the SNaPshot multiplex assays. In total, the SNaPshot assays yielded an accuracy of more than 99%. CONCLUSIONS: The strengths of SNaPshot multiplex assays include high accuracy, high sensitivity, high flexibility (the examination panel can be easily expanded for additional mutations), low cost (less than US $10 per patient), and easy implementation for any institute with a DNA sequencer. Although only 20 to 30 mutations can be examined in two to three runs of the SNaPshot assay, this technology may be suitable for first-pass screening of deafness-associated mutations in populations with a relatively homogeneous ethnic background.

Impact of the national hearing screening programme in China.

China has a large population with different levels of medical care among the eastern, central and western areas. The national universal newborn hearing screening (UNHS) programme was initiated in 1999 and then progressively implemented nationwide. A "National UNHS Experts Group" was set up, formulating the national UNHS administration rules and technological specifications. 3 March had been named as national "ear-care day" since 2000 and such social activities help make deafness prevention work more widely accepted. UNHS in China presently has 3 phases due to disparities in economic development. 1) Implementation in stages: in economically under-developed areas. 2) Implementation completed: in the coastal cities. 3) Beyond basic UNHS: i) Development of a completed UNHS system including follow-up and quality control based on the neonatal disease screening system, ii) Exploration of a new public health care programme: simultaneous screening of newborn hearing and ocular disease, iii) Carrying out of a multi-centre study on high-risk factors and GJB2 gene mutations in newborn with non-syndromic hearing impairment. The incidence of newborn bilateral hearing loss was 2.22 per 1000, and 2.74 per 1000 for unilateral hearing loss. Though UNHS have been carried out widely in the eastern parts of China, there are difficulties for its implementation in the western regions. Economic development and technical expertise are the main restricting factors.

A new, easy, and rapid high-throughput detection method for the common GJB2 (CX26), 35delG mutation.

GJB2 (Gap Junction protein beta type 2; Connexin 26, CX26) is known for its contribution to nonsyndromic recessive deafness (NSRD). One particular mutation, 35delG, a deletion of one guanine from a stretch of six leading to a frame shift early in the gene, has a high prevalence in populations from European descent. 35delG testing therefore has become a standard test in genetic diagnostic laboratories. Most of the currently available methods for the detection of 35delG are relatively time consuming, and not suited for high-throughput diagnostic testing. Within this paper we present a real-time PCR genotyping assay based on melting curve analysis, requiring only a single preparation step before the actual analysis. The assay was optimized on a panel of 48 samples with known 35delG genotypes and subsequently tested using a large Belgian population (N = 460) with unknown 35delG status. For the latter set of samples, real-time PCR results were validated with SNAPShot, an assay used in our laboratory for diagnostic purposes. The real-time PCR genotyping method has proven to be highly reliable, rapid, cost-effective, and suitable for high-throughput screening. We believe that this genetic test for 35delG will find widespread applications in the DNA diagnostic field.