Genetic mapping and pedigree analysis of non-syndromic congenital deafness in Surabaya, Indonesia

Introduction

Hearing loss is a health problem affecting about 6-8% of the population in developing countries and is partly a congenital disability.¹ Congenital hearing loss (CHL) is one of the most common disorders in humans, with an incidence of 1-2 out of 1000 newborns.² Congenital hearing loss can be caused by genetic or environmental factors or the interaction of these two. Genetic factors play about 50-75% as the cause of hearing loss. 30% of congenital hearing loss is syndromic (SHL) with abnormalities in other organ systems, and 70% is non-syndromic. Approximately 600 SHL-related syndromes have been identified, including Usher, Pandred, Stickler, Branchio-to-renal, Down's syndromes, et cetera.² Based on data from the hereditary hearing loss homepage, 132 mutations genes associated with sensorineural hearing loss (SNHL): 51 autosomal dominant (DFNA), 77 autosomal recessive (DFNB), and 5 X-chromosome (DFNX).^2,3

Hereditary hearing loss is classified into syndromic and non-syndromic types. The syndromic type accounts for 30% of genetic deafness.^4,5 According to World Health Organization (WHO) data, there are 466 million people in the world experiencing a hearing loss (6.1% of the total population), of which 34 million are found in children and 432 million in adults.⁶ In 2018, 2.6% of the Indonesian population experienced hearing loss. The population with hearing loss in Indonesia is distributed in several age groups, most of which are above 75 years, with a prevalence of 36.6%.⁷ Congenital hearing loss related to genetic factors is found in two forms, namely: syndromic hearing loss and non-syndromic hearing loss.³ Non-syndromic hearing loss is a hearing loss that is not associated with other physical disorders. Non-syndromic hearing loss can be inherited in autosomal recessive, autosomal dominant, X-chromosome-associated, and mitochondrial inheritance. Non-syndromic hearing loss causes prelingual hearing loss in children with sensorineural types ranging from severe hearing loss to total deafness.^4,5

More than 60 genes and some proteins are involved in the pathogenesis of SNHL. To date, mutations in four connexin genes, including Gap Junction Beta 2 (GJB2)/Connexin 26 (Cx26), GJB3 (Cx31), GJB4 (Cx30.3), and GJB6 (Cx30), have been associated with SNHL.⁴ Research in Surabaya in 2019 reported that changes or variations in the nucleotide G to A at sequence 8473 in the GJB2 gene, changing the encoded amino acid (valine to leucine) were found in patients with Hereditary Hearing Loss in Deaf School type B Surabaya. This was assumed to be related to the occurrence of congenital deafness.⁶

Genetically inherited hearing loss affects various molecular processes, including gene mutations that interfere with the function of transcription factors, potassium and chloride channels, connexins, and stereocilia. With genetic testing, the identification of mutations can produce an accurate prognosis for deafness. Genetic information can help predict whether the hearing loss will persist or worsen, and determine the type of damage in the hearing system. Furthermore, knowing the degree of damage to the inner ear could help to determine whether the patients need hearing aid or cochlear implant surgery to improve their hearing.^7,8

WHO held a multi-nation consultative meeting in Colombo in 2021 and recommended every stakeholder in various countries, especially in the Southeast Asian region to focus on preventive and early detection efforts related to ear health. This meeting was the basis for the Sound Hearing 2030 program to improve the quality of life in the Asian region by developing a right to better hearing program. With this program, it is expected that the prevalence of hearing loss will be reduced approximately 90% by 2030.⁹

Non-syndromic hearing loss is a problem because there is lack of knowledge of the cause. In contrast, in the syndromic type, the pathophysiology of typical symptoms could be explored. This study aimed to find out the causes of non-syndromic type congenital deafness through a genetic approach; to detect the presence or absence of genetic mutations that caused deafness; to identify specific mutations, and to ascertain how the pattern of inheritance of these mutations.

Methods

Hybridization

The hybridization kit used in the study was Hearing Loss Susceptibility GenoArray Diagnostic Kit (ref HB_HLS GA) by Hybribio limited, Sheung Wan, Hong Kong) to analyze genetic mutation. Thirteen mutations in four genes (GJB2, GJB3, SLC26A4 and 12S rRNA) are evaluated simultaneously. By using polymerase chain reaction (PCR) to amplify extracted DNA from PB (Peripheral Blood), amplified DNA amplicons are then hybridized with specific probes located inside the “HybriMem box” under hybribio patented “flow-through hybridization” technology followed by colorimetric result obtained using enzyme immunoassay method without using primer as in DNA sequencing (Figure 1).

Figure 1. Hearing Loss Susceptibility GenoArray Diagnostic Kit.

(A) HybriMem Box. The wells detect specific gene mutation. (B) DNA HybriMax HHM-3 device, containing 15 HybriMem Box.

The hybridization reagents consist of hybridization solution, blocking solution, enzyme conjugate, solution A, washing solution, NBT/BCIP, hybridmem box. The composition of hybridization solution were 120ml 2X SSC and 0.1% SDS. The composition of blocking solution was 30ml TBS with <0.1% detergent. The composition of enzyme conjugate were 15ml <0.0003% Streptavidin-Alkaline, Phosphatase Conjugate, and Stabilizer. The composition of solution A was 100ml Tris-HCl buffer with 0.05% sodium azide. The composition of washing solution were 96ml 0.5X SSC and <0.1% SDS Solution. The composition of NBT/BCIP was 15ml 1-5% (w/w) NBT/BCIP. The composition of hybrimem box was 30 Nylon Membrane coated with specific DNA probes.

There were 3 steps in hybridization process. First step was running at 45°C from (number 1-11), second step (number 12-20) at 25°C, and third step (number 21-28) at 36°C. The steps were:

• 1. Set HybriMax temperature at 45°C. (Step 1-11 running at 45°C)

• 2. When the temperature of HybriMax reaches 45°C, add 0.8ml of 45°C pre-warmed Hybridization Solution into the sample well where “HybriMem” is located for 2 min.

• 3. Pump away all the solvent, then pump off.

• 4. Add 0.8ml of 45°C pre-warmed Hybridization Solution into the same wells.

• 5. Add group 1 and group 2 denatured DNA amplified samples from PCR tubes into one well, pipetting 2-3 times to mix the solution carefully.

• 6. Close the lid.

• 7. Incubate for 20 min. (Hybridization)

• 8. Pump away all the solvent, then pump off.

• 9. Add 0.8ml of 45°C washing Solution. (Washing Step)

• 10. Repeat Steps 8-9 four times more.

• 11. Pump off.

• 12. Set HybriMax temperature at 25°C.

• 13. When the temperature of HybriMax reaches around 30°C, add 0.5ml of Blocking Solution into the well. (Temperature is going down from 45°C to 25°C)

• 14. Pump away all the solvent, then pump it off.

• 15. Again, add 0.5ml of Blocking Solution and Incubate for 5 min; pump away all the solvent, then pump off.

• 16. When the temperature of HybriMax reaches 25°C, add 0.5ml of Enzyme Conjugate and Incubate for 5 min.

• 17. Pump away all the solvent then pump off.

• 18. Add 0.8ml of Solution A. (Washing Step)

• 19. Repeat Step 17-18 four times more.

• 20. Pump off.

• 21. Set HybriMax temperature at 36°C.

• 22. When the temperature of HybriMax reaches 36°C, add 0.5ml of NBT/BCIP Solution (brown bottle) and Incubate for 5 min. Close the lid. (Coloring)

• 23. Pump away all the solvent.

• 24. Keep the pump on.

• 25. Add 0.8ml of Hybridization Solution. (Washing Step)

• 26. Repeat Step 25 three times more.

• 27. Add 1.0ml of Distilled Water. (Rinsing Step)

• 28. Pump off

• 29. Remove the fixing cover and all accessories. Using forceps to take out the membranes and dry them on absorbent paper.

• 30. Interpret the results by color visualization observed on membrane.

Results

The characteristics of 49 subjects were as follows: the percentage of male and female participants was 51% and 49%. There were no subject with age$\le$10 years, nor >30 years, the highest number of subjects was in age range 11-20 years (91.84%), with the highest education level was senior high school 28 subjects (57.14%) (Table 1).¹⁸

The results of the audiogram examination, showed the type of deafness was sensorineural in all subjects, with the highest number was profound hearing loss (>90dB) in 46 subjects (93.88%), and the least number was severe hearing loss (71-90dB) in right ears 3 subjects, and left ears 3 subjects (6.12%) (Table 2)

The results of the hybridization examination in Table 3 showed that genetic mutations occurred in 6 subjects (12.25%) with criteria for genetic mutations of the GJB2 gene 1 subject (2.04%), PDS gene 1 subject (2.04%), mtDNA gene 1 subject (2.04%), and in 3 subjects (6.13%) unknown (genetic mutation was found, but the type was unknown).¹⁹ The pattern of inheritance of the genetic mutation occurred in autosomal recessive 1 subject (2.04%), sex-linked 1 subject (2.04%), and mitochondrial 1 subject (2.04%).

Discussion

Non-syndromic congenital deafness has 70% of the features that often appears in congenital hearing loss. The most common forms of inheritance in NSHL are autosomal recessive 77%, followed by autosomal dominant 22%, X-linked 1%, and mitochondrial <1%. ⁸ Genetic mutations that cause NSHL vary between different populations and ethnicities. The most common mutation is GJB 2 which is encoded by the protein connexin 26. The population in Saudi Arabia has higher OTOF gene mutations than the GJB2 gene. Mutations of GJB2, GJB3, SLC26A4, and mitochondrial 12SRNA were common causes of NSHL found in China.^7,10

A similar study in Surabaya, Indonesia, showed that there were more females than males with hearing loss, different in Iraq where there were more males than females. Gender has no significant relationship with the occurrence of deafness.¹¹

The results of the audiogram examination, showed that the type of deafness was sensorineural in all subjects, with the highest number was profound hearing loss (>90dB) in 46 subjects (93.88%). In a research profile of 430 patients with congenital deafness in America, the highest degree of the hearing was very severe (>90dB) in 133 samples, with no lateralization (symmetrical) in 311 samples (Raymond, 2019).¹² Hearing loss may manifest in the ears as bilateral or unilateral and symmetrical or asymmetrical. Genetic deafness was mostly found as bilateral (44%), asymmetrical (22%) and unilateral (2%). There was a negative correlation between age and deafness after environmental factors were excluded in 1119 patients; congenital onset occurred in 45%, childhood 30%, and adulthood 28%. A study of 200 samples in the Netherlands stated that deafness could be found 50% as congenital, 38% in the first decade, and 20% in the second decade of life.²

The results of the hybridization examination in Table 1 showed that genetic mutations occurred in 6 subjects (12.25%), with criteria for genetic mutations of the GJB2 gene 1 subject (2.04%), PDS gene 1 subject (2.04%), mtDNA gene 1 subject (2.04%), and 3 subjects (6.13%), unknown (genetic mutation was found but the type was not known). Research in Bosnia and Herzegovina described that in the results of exome sequencing of patients with non-syndromic deafness, 68% showed negative results, 18% positive mutations, while in syndromic deafness it was more genetic mutations were found.¹³ Following this study, genetic examinations for non-syndromic deafness did not always reveal mutations. In this study, due to limitations in the examination, only a few genes could be detected.

The pattern of inheritance of genetic mutations in this study occurred in autosomal recessive one subject (2.04%), sex-linked one subject (2.04%), and mitochondrial one subject (2.04%). Non-syndromic hearing loss (NSHL) was inherited in an autosomal recessive manner (75-80%), autosomal dominant (20-25%), and 1-2% X-chromosome and mitochondria-linked. After aging, the prevalence of autosomal dominant inheritance and mitochondrial inheritance increases while autosomal recessive inheritance decreases.^7,14 Autosomal dominant is described as a child whose mother has a no mutations gene and a father who mutates the dominant gene, can inherit a 50% chance of being deaf. Only one copy of the inherited mutation gene can cause deafness in a child. So in every pregnancy, there is a 50% chance that the child will be deaf. Autosomal recessive is when the chromosomes of the father and mother have a recessive mutation in the same gene and are inherited in 50% of their offspring, but it takes two mutated genes to produce a deafness phenotype. In X-linked inheritance, boys have a higher chance of deafness. Mitochondrial inheritance is only inherited by eggs from the mother, and all offspring will be deaf.¹⁵

The results of hybridization of research and family samples obtained one sample of IVS-M/IVS-N mutation; one sample of 299M/299N mutation; one sample of the 1555M mutation; one sample of mutations 235M/235N, 7445M/7445N, and IVS-M/IVS-N; one sample of mutations 235M/235N, 7445M/7445N, 538M/538N, and IVS-M/IVS-N. The interpretation of the results of this hybridization was, if an IVS-M/IVS-N mutation was found, it was defined as a SLC26A4-IVS(7)2 mutation; mutation 299M/299N defined as GJB2-299; the 1555M mutation was defined as mtDNA1555; mutation 235M/235N defined as heterozygous GJB2-235 mutation; mutations 7445M/7445N defined as mtDNA7445 mutations, mutations 538M/538N defined as mutations GJB3-538. The family of patients with mtDNA 1555 (1555M homozygous) had a deaf father and mother, but only the mother had the 1555M mitochondrial mutation. The result of inheritance in their offspring was deafness in all children. This was related to the reference where mitochondrial inheritance can only be inherited by egg cells from the mother, which caused all of the offspring to be deaf.^15,16

The hybridization kit used by researchers had limitations in assessing the genetic mutations that occured. The Hearing Loss Susceptibility GenoArray Diagnostic Kit is designed for rapid screening with accurate results through dot mutations known to be associated with hereditary hearing loss. Thirteen mutations in four genes (GJB2, GJB3, SLC26A4 and 12S rRNA) were evaluated simultaneously. In this study, Knowledge of mutations could help identify hearing loss at birth and could advise avoiding taking certain types of antibiotics that are associated with deafness in children who carry the gene mutation.¹⁷ Researchers took hybribio as a genetic mutation screening method because the GJB2, GJB3, mitochondrial and SLC26A4 genes are genetic mutations that often appear in Asian populations.¹⁷

In the pedigree pattern, the SLC26A4 family (IVS-M/IVS-N) had its own pattern, where the grandmother had 4 genetic mutations (235M/235N, 7445M/7445N, 538M/538N, IVS-M/IVS-N), the mother had 3 genetic mutations (235M/235N, 7445M/7445N, IVS-M/IVS-N), and the child had only 1 genetic mutation. This was interesting because the inherited mutation was only 1 gene while the other genes were missing (235M/235N, 7445M/7445N, 538M/538N). The most common genes in cases of autosomal recessive hearing loss in order of highest frequency are the GJB2, SLC26A4, and MYO15A genes.⁷

In the GJB2 family pattern (299M/299N heterozygous) a father who carried the genetic mutation but was not deaf with a mother not found the mutation gene, had deaf and normal sons. This pattern was an autosomal recessive genetic mutation. Under the reference that in cases of non-syndromic hearing loss, the most common mutation occurred in GJB2.⁷

Conclusion

Non-syndromic congenital deafness in Surabaya was caused by genetic mutations GJB2, SLC26A4 and mtDNA 1555, which were passed down through families with diverse inheritance patterns. Genetic mutation is one of the causes of non-syndromic congenital deafness. Hybridization examination is a limited screening method to determine promptly the presence of genetic mutations that caused deafness in GJB2, GJB3, mtDNA, and SLC26A4 mutations that often occur in Asia. Genetic counselling is needed to predict the risk of deafness that will be passed down, prepare genetic screening methods, and be able to determine the prognosis of the therapy that will be carried out. Further examination with next generation sequencing method on all samples is needed to find more genetic mutations that cause non-syndromic deafness.