**6. Next-generation sequencing technology in dissecting the background of hearing loss**

Syndromic X-linked HL is represented by the **Alport** (OMIM#301050) and **Mohr-Tranebjaerg** (MTS, OMIM#304700) **syndromes**. The Alport syndrome is characterized by glomerulonephropathy leading to progressive renal failure, varying severity of progressive sensorineural HL (occurs typically after 10 years of age and affects mainly high frequencies), and variable ocular anomalies. In 85% of patients with Alport syndrome, dominant pathogenic variants in the *COL*4*A*5 gene on the X chromosome are found. Approximately 15% of patients develop Alport syndrome due to recessive variants in the *COL*4*A*4 or *COL*4*A*3 genes, cases with auto-

In the Mohr-Tranebjaerg syndrome, also known as progressive deafness syndrome with blindness, dystonia, fractures and mental deficiency, progressive pre- or postlingual sensorineural deafness occurs in the early childhood and is a presenting symptom. MTS is caused by

Mutations in the **mitochondrial genome** can lead to HL that is an isolated feature or part of genetic syndrome, i.e., mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), myoclonic epilepsy with ragged-red fibers (MERFF) or Kearns-Sayre syndrome (KSS), a mitochondrial myopathy, characterized by ptosis, ophthalmoplegia, muscle weakness, cerebellar ataxia, diabetes mellitus, and/or endocrinopathies [33]. Aminoglycoside ototoxicity has been associated with pathogenic variants in *MT-RNR1* [34]. Pathogenic variant m.3243A > G in the *MT-TL1* gene, which is causative of MELAS, was found in patients with diabetes mellitus and HL or HL exclusively [35]. The data raise questions on mitochondrial

Regarding the heterogeneity of genetically related HL, the precise evaluation of its cause is a challenging task. Apart from some, rather rare, unquestionable phenotypic manifestations, many of the cases of possibly genetically related HL (syndromic and nonsyndromic) are hard to distinguish. It should be noted that there is a group of useful, assistive algorithms, which

The Face2Gene (http://suite.face2gene.com/) is a collection of phenotyping applications, which enable accurate and comprehensive assessment of a patient based on characteristic dysmorphic facial features. As the variations in the face and skull are very common and are present in about 40% of genetic disorders, they are very useful in identification of the disorder. Briefly, the picture of patient face is uploaded to an application, than the quantification of the similarity is calculated with the usage of the algorithm database. The result is the list of syndromes with analogous morphology. The algorithm Face2Gene is accessible free of charge to all healthcare

The AudioGene (http://audiogene.eng.uiowa.edu/) predicts the genes underling autosomal dominant nonsyndromic hearing loss (ADNSHL) based on the audiometric data. This algorithm also takes into account the age of the patient, which is essential in the ADNSHL study

are helpful for both: establishing the diagnosis and targeting a diagnostic approach.

somal dominant inheritance, have also been occasionally reported.

variant penetrance, tissue specificity, and heteroplasmy level.

recessive variants of the *TIMM*8*A* gene.

38 An Excursus into Hearing Loss

**5. Assistive algorithms**

specialists.

In almost all genetically related diseases (included hearing impairment), there is a need for tailored diagnostic strategies in searching for their molecular background. The milestone in this research area was the development of the method for the DNA sequencing by Frederick Sanger in 1975 [39]. Whereas the direct sequencing (called also Sanger sequencing—tribute to its inventor) allows to debunk the molecular cause of disease in a limited number of genes, e.g., when HL background analyses were limited to the *GJB*2 gene, there was a significant gap in our knowledge and diagnostic capabilities. This gap has been filled out by introducing NGS technology, also called massive parallel sequencing (MPS) or high-throughput DNA sequencing. The NGS developed over the last decade has revolutionized the genetic research and diagnostic practice, mainly because of reducing costs and time of DNA sequencing. This technology gives a unique opportunity to analyze in one experiment the sequence of more than 1 million base pairs, thus sequencing of the whole genome or thousands of genes simultaneously in a few days has become possible [40]. The most commonly used forms of NGS in searching for pathogenic variants underling HL are whole exome sequencing (WES) and multigene panels.

An undisputable advantage of the WES is the possibility of simultaneous sequencing of the whole coding DNA sequence (protein coding part within all human genes ~20,000) regardless of the disease studied. This makes WES a universal test for almost all known genetic diseases. The multigene panel sequencing is a more economical solution, especially dedicated for the diagnostic purpose. It should be noted that with the panel sequencing, only already known genes associated with a given disease are analyzed. Although the targeted NGS has many advantages, whole-genome sequencing (WGS) is still the state of art approach with its high cost representing a main drawback. The application of high-throughput DNA sequencing methods generates a vast amount of information, which accelerated the discovery of new, causative genes for many diseases. Also in the field of genetically related HL, with the NGS technology, novel genes have been discovered for all modes of inheritance described above. The important, newly discovered genes causative of recessive HL are: *ADCY*1*, BDP*1*, SYNE*4*, ELMOD*3*, CABP*2*, GRXCR*2*, OTOGL, TPRN*, and *TSPEAR*. For dominant HL, the *CEACAM*16, *P*2*RX*2, and *OSBPL*2 genes were revealed, also gene for the DFNX4 locus (i.e., *SMPX*) was identified. Another remarkable achievement obtained by the NGS technology is the identification of genes incorrectly classified as pathogenic, the examples of such events are: *MYO*1*A* and *RAB*40*AL* [41–43].

**Nonsyndromic deafness**—deafness not associated with pathological symptoms/signs from

Genetic Basis of Hearing Loss

41

http://dx.doi.org/10.5772/intechopen.73557

**Partial deafness**—hearing loss assessed by an audiometric test as a normal or little elevated hearing threshold within low frequencies and significantly raised hearing threshold in high

**Sex-linked inheritance**—type of the Mendelian inheritance of a trait in which the defective

**Syndromic deafness**—deafness as a part of a syndrome i.e., associated with pathological

**Whole exome sequencing** (WES)—approach based on NGS technology, allowing to sequence

**Whole genome sequencing** (WGS)—approach based on NGS technology allowing to analyze

Department of Genetics, Institute of Physiology and Pathology of Hearing, Warsaw, Poland

[1] Ołdak M. Chapter 8—Next generation sequencing in vision and hearing impairment. Clinical Applications for Next-Generation Sequencing. Boston: Academic Press; 2016.

[2] Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular

[3] Oldak M, Ozieblo D, Pollak A, Stepniak I, Lazniewski M, Lechowicz U, et al. Novel neuro-audiological findings and further evidence for TWNK involvement in Perrault

**Post-lingual hearing loss**—hearing loss with late onset (after speech development). **Prelingual hearing loss**—hearing loss with early onset (before speech development).

other systems.

frequencies.

gene is located on X or Y chromosome.

symptoms/signs from other organs.

all protein-coding regions (exons).

the whole genome DNA sequence.

Agnieszka Pollak\* and Monika Ołdak

\*Address all correspondence to: a.pollak@ifps.org.pl

Pathology. Genetics in Medicine. 2015;**17**(5):405-424

syndrome. Journal of Translational Medicine. 2017;**15**(1):25

**Author details**

**References**

p. 153-170

For the clinical diagnostic purpose, there are many commercial tests based on NGS, which differ in technologies and numbers of genes included. Heretofore, at least 20 commercially available tests, based on the NGS technology, focused on genetically related HL may be applied [1]. Due to the constant reduction of costs and availability, diagnostic approach based on the NGS technology in the nearest future will become a standard, which will significantly improve the level of patient care.

#### **Index of technical terms**

**Autosomal dominant inheritance** (AD)—type of Mendelian inheritance of a trait in which a defective copy of a gene (localized on autosome) dominates over the normal one. For the symptoms to occur, presence of only one defective copy is sufficient.

**Autosomal recessive inheritance** (AR)—type of Mendelian inheritance of a trait in which two copies of defective gene (localized on autosomes) are required in order for the disease to develop.

*DFNB***1 locus**—most common locus causative for nonsyndromic hearing loss, containing *GJB*2 and *GJB*6 genes.

**Direct sequencing**—a technology allowing to determine the sequence of nucleotides in DNA invented in 1977 by Frederic Sanger and Alan R. Coulson, based on the chain-dideoxy terminator method, also called Sanger sequencing.

**Genetic pedigree**—illustration of genetic relationship of a family, including information about health history of the family members.

**Heteroplasmy**—coexistence of more than one mtDNA type within an individual.

**Homoplasmy**—presence of a uniform type of mtDNA within an individual.

**Mendelian inheritance**—type of transmission of genes according to Gregor Mendel's set of laws, also called classical inheritance. Mendelian inheritance comprises of autosomal dominant, autosomal recessive, and X-linked type of inheritance.

**Mitochondrial DNA** (mtDNA)—small circular genome localized in the mitochondria.

**Mitochondrial inheritance**—non-Mendelian type of inheritance, occurring when a defective gene is located within the mitochondrial genome, inheritance of a trait encoded by this gene takes place exclusively from mother to offspring.

**Next generation sequencing** (NGS)—also known as high-throughput sequencing, a technology allowing to establish the sequence of DNA larger than 1 million base pairs in a single experiment.

**Nonsyndromic deafness**—deafness not associated with pathological symptoms/signs from other systems.

**Partial deafness**—hearing loss assessed by an audiometric test as a normal or little elevated hearing threshold within low frequencies and significantly raised hearing threshold in high frequencies.

**Post-lingual hearing loss**—hearing loss with late onset (after speech development).

**Prelingual hearing loss**—hearing loss with early onset (before speech development).

**Sex-linked inheritance**—type of the Mendelian inheritance of a trait in which the defective gene is located on X or Y chromosome.

**Syndromic deafness**—deafness as a part of a syndrome i.e., associated with pathological symptoms/signs from other organs.

**Whole exome sequencing** (WES)—approach based on NGS technology, allowing to sequence all protein-coding regions (exons).

**Whole genome sequencing** (WGS)—approach based on NGS technology allowing to analyze the whole genome DNA sequence.
