Genetic diagnostics in pediatric hearing loss
Hearing impairment in newborn children is one of the most frequent forms of sensorineural disorders, affecting 1 in 1000 infants. In half of the cases the hearing loss has a genetic basis, and over 70 genes have been identified so far, making hearing loss genetically exceptionally heterogeneous. Early detection in newborns, in combination with a genetic diagnosis is critical for the selection of a proper intervention and the development of speech, language and communication skills.
by Dr Isabelle Schrauwen
Hearing impairment in infants can be due to environmental influences such as cytomegalovirus infection, but in industrialized countries, however, most cases of early-onset hearing impairment have a genetic basis. Genetic hearing loss is non-syndromic in 70% of cases, whereas other symptoms (apart from hearing loss) are noticeable in 30% of cases (syndromic hearing loss). Autosomal recessive non-syndromic hearing loss (ARNSHL) is most common (80%) and is typically prelingual in onset, and autosomal dominant non-syndromic hearing loss (ADNSHL), X-linked and mitochondrial hearing loss are less frequent (20 and <1% respectively). To date, over 70 genes have been found to be implicated in non-syndromic hearing loss (NSHL), of which 40 are autosomal recessive. The most frequent causes of ARNSHL in most populations are mutations in GJB2, with a frequency ranging from 10 to 50% of all ARNSHL cases.
The implementation of newborn hearing screening in many countries has lead to an early detection of hearing loss and deafness in infants. This, together with improved genetic diagnostics and neuroimaging, has lead to a better understanding and better intervention of hearing loss overall .
The importance of a genetic diagnosis in pediatric hearing impairment
Clinical tests are not always sufficient for an accurate diagnosis and genetic diagnostics can provide answers that clinical tests cannot. Identification of the genetic cause can help predict the progression of the hearing loss and also direct the choice of the most appropriate treatment or method of communication. In addition, some apparent forms of non-syndromic hearing loss can be diagnosed to be syndromic as they give other symptoms at a later age (such as goitre in Pendred syndrome or retinitis pigmentosa in Usher syndrome). For Usher syndrome, preventative measures can be taken including sunlight protection and vitamin therapy to minimize the rate of progression of retinitis pigmentosa . Furthermore, autosomal recessive mutations in GJB2 often cause a stable form of hearing loss and patients usually have good prospects with a cochlear implant. Knowing the gene responsible can also be very important to the parents, reducing their feelings of guilt and predicting the likelihood of subsequent children having hearing loss.
In addition, more extensive screening will also be very useful in providing a more accurate picture of the prevalence of different types of deafness affecting people across the world. Finally, advances in molecular and cellular therapies for hearing loss are also gene-specific , and identification of the genetic cause is key.
Until recently, routine molecular diagnostics for hearing impairment consisted of the gene-specific sequencing of certain deafness genes, mainly with Sanger sequencing. GJB2 testing is offered most frequently in routine diagnostics, as it is responsible for a large number of ARNSHL cases. When there is evidence of progression of the hearing loss, or the presence of a goitre, an enlarged vestibular aqueduct (EVA), or Mondini dysplasia, SLC26A4 will be analysed, and when a specific phenotype is seen, other genes might also be analysed (OTOF, TECTA, COCH, WFS1, or a mitochondrial mutation). The selection criteria are typically: (1) high frequency cause of deafness (i.e. GJB2); (2) association with another recognizable feature (i.e. SLC26A4 and EVA); or (3) a recognizable
audioprofile (i.e. WFS1) .
Syndromic forms of deafness usually only have one or a few candidate genes responsible for each syndrome. However, for non-syndromic deafness, it is very difficult, and often impossible, to determine candidate genes because of the large number of causative genes leading to a relatively indistinguishable phenotype. GJB2 sequencing will identify 10–50% of ARNSHL cases, but the remaining cases of hearing loss display a high degree of genetic heterogeneity and unless a specific audioprofile is present it is hard to diagnose these with a gene-specific test. Traditionally, with gene-specific tests, it has therefore been difficult to establish a genetic diagnosis due to extreme genetic heterogeneity and a lack of phenotypic variability.
The analysis of multiple mutations in several genes in parallel was made possible by the development of single nucleotide extension microarrays . These microarrays detect a specific mutation by hybridizing primers to patient DNA, followed by a single base extension. This technology therefore only detects known mutations, and a panel of 198 mutations in 8 genes [GJB2, GJB6, GJB3, GJA1, SLC26A4, SLC26A5 and the mitochondrial genes encoding 12S rRNA and tRNA-Ser(UCN)] underlying sensorineural (mostly non-syndromic) hearing loss has been developed . Although new mutations cannot be picked up, this technique can provide some additional diagnostic value in GJB2 negative cases.
An Affymetrix resequencing microarray capable of resequencing 13 genes mutated in NSHL was also developed (GJB2, GJB6, CDH23, KCNE1, KCNQ1, MYO7A, OTOF, PDS, MYO6, SLC26A5, TMIE, TMPRSS3, USH1C) , but the number of genes here is also limited and specific kinds of mutations such as insertion/deletion (indel) mutations cannot be detected accurately.
Custom gene enrichment with next-generation sequencing
The need for new and better diagnostic methods for extremely heterogeneous diseases has been filled by the availability of next-generation sequencing, which has made it possible to sequence a large number of genes at the same time. This has lead to an immense growth of custom hearing-loss gene panels. Several labs have adopted this approach in-house already [7–9], and several labs offer this test for ARNSHL, ADNSHL, some cases of syndromic hearing loss, or all of the above.
The most commonly available systems for massive parallel sequencing are: Illumina, 454, or SOLiD. The Illumina platform is the most widely used platform to date and relies on cyclic reversible termination technology. Before massive parallel sequencing, DNA will be enriched for a custom selection of hearing-loss genes. In a diagnostic setting, sensitivity and specificity are important, and different enrichment methods perform differently in these criteria. Capture enrichment methods have been used more often and are easy to use, but PCR-based methods seem to have a better performance. A portion of targeted bases in repetitive regions cannot be captured, whereas PCR is able to enrich 100% of the desired target area. This is crucial to the sensitivity of detecting variants.
Although PCR-based techniques are usually more labour-intensive, microdroplet PCR methods have improved this greatly . By using barcoding, custom hearing-loss panels are now offered for a competitive price in several labs across the world, and depending on the genes included in the panel, will offer a genetic diagnosis in the majority of cases.
Exome sequencing is also emerging as a diagnostic tool for many diseases and has decreased in price significantly in recent years. Exome sequencing targets every coding exon in the genome for enrichment prior to next-generation sequencing. Though current exome kits provide insufficient target enrichment in a diagnostic setting for deafness , as the regions of interest might not been completely covered and coverage depth may not be high enough for a diagnostic setting. Exome sequencing has therefore a decreased sensitivity to detect mutations in known genes compared to the custom panels available, but does allow the identification of new genes. In addition, given the amount of data that arises from exome sequencing, identification of the causative mutation among the list of variants will be more challenging. Although over 70 genes have already been discovered, there are still many more to be found, and the identification of new genes will greatly improve our understanding of deafness. Since its introduction, exome sequencing has lead to a fast rise in the identification of hearing-loss-related genes.
Future techniques and conclusions
Other technologies, such as Ion torrent, Pacific Biosystems, and specifically the emerging Oxford Nanopore technique, might offer very cost-effective sequencing methods for the future of molecular diagnostics in many diseases. Furthermore, genome sequencing might be shown useful in the diagnosis of hearing loss if the price of sequencing keeps dropping.
In conclusion, a genetic test ideally has to be sensitive, specific, accurate and low in cost. Gene-specific analysis of GJB2 will detect a 10–40% of ARNSHL cases, and custom gene panels with next-generation sequencing will provide a diagnosis in the majority of genetic hearing-loss cases. It is anticipated that within the coming years genetic testing will be routinely implemented in pediatric hearing loss, leading to better intervention and choice of treatment.
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Isabelle Schrauwen PhD 1,2
1 Department of Medical Genetics, University of Antwerp, Antwerp, Belgium
2 The Translational Genomics Research Institute (TGen), Phoenix, AZ, USA