A screening analysis of the GJB2 c.176 del 16 mutation responsible for hereditary deafness in a Chinese family

Abstract
Objective: To determine whether a new-born child from a family carrying a deafness gene needs cochlear implantation to avoid dysphonia by screening and sequencing a deafness-related gene. Results: Both screening and sequencing results confirmed that the new born child had a normal GJB2 gene despite the fact that she has a brother suffering from hearing loss triggered by an allelic GJB2 c.176 del 16 mutation. We cloned the GJB2 genes derived from their respective blood genomic DNA into GFP fused plasmids and transfected those plasmids into the 293T cell line to test for gene function. While the mutated GJB2 gene (GJB2 c.176 del 16) of her deaf brother was found to be unable to form the gap junction structure between two adjacent cells, the baby girl’s GJB2 gene ran into no such problems. Conclusion: The screening and sequencing as well as the GJB2 gene function tests invariably showed results consistent with the ABR tested hearing phenotype, which means that the child, with a normal wild type GJB2 gene, does not need early intervention to prevent her from developing hearing loss and dysphonia at a later stage in life.

1.Introduction
Hearing loss, known as a partial or total loss of the ability to hear, can often be attributed to multiple causes such as ageing, genetics, perinatal problems and external causes like noise and illness (Deafness, 2011). In general, hereditary deafness is mostly the result of mutations in certain genes playing a decisive role in the physiological mechanism of hearing. While around 75e80% of cases of hereditary deaf- ness are known to have been caused by recessive genes, the pathogenic cause of 20e25% of them can be attributed to dominant genes. Among all such cases, 1e2% can be explained by X-linked patterns and another 1% by mito- chondrial inheritance (Rehm). Gap junction beta-2 (GJB2), whose corresponding protein is connexin 26 (Cx26), is one of the leading genetic causes of deafness. Connexins, a type of gap junction proteins, are a family of structurally related transmembrane proteins that can help form vertebrate gap junctions. Each gap junction is made up of two hemi-channels, or connexons, whose structure commonly contains six connexin molecules. Together with other proteins, they can dock with the hemi-channels of adjacent cells to form gap junctions (Jongen et al., 1991).The gap junctions formed as a result consist of cell-to-cell channels crucial to multiple physiological processes, for example, the transmission of Potassium ions in the inner ear, meaning that defects in this gene can lead to one of the most common forms of congenital deafness (Kelsell et al., 1997). In this research, we helped the parents of a 5-year-old deaf son, who recently gave birth to a new baby girl to determine whether their second child needs artificial cochlear implanta- tion to prevent her developing hearing loss by screening the genomic DNA of the entire family. Research results showed that the second new born child was fortunate enough to have a properly-functioning (wild type) GJB2 gene and run no risk of suffering from congenital hearing disabilities linked to a defective GJB2 gene.

2.Material and methods
All members of the studied family underwent consecutive auditory brainstem response (ABR) tests. Their medical his- tories showed that they had not exhibited any abnormality during pregnancy or delivery, nor had they been exposed to amino-glycosides. We used DNA Isolation kits (Tiangen, china) to isolate blood genomic DNA from the blood samples of the children and their parents. Nine hotspot mutations of deafness genes commonly found among Chinese populations were screened by adopting a method called multiplex allele specific PCR-based universal array (ASPUA) (Li et al., 2008).These mutations include the four mutations of c.35delG, c.176del16bp, c.235delC and c.299delAT in the GJB2 gene as well as c.538C > T in the GJB3 gene, c.IVS7-2A > G and c. 2168A > G in the SLC26A4 gene, and m.1555A > G and m.1494C > T in the RNR1 gene of mitochondrial DNA (mtDNA). The family was then put through bi-directional sequencing of the coding region of the GJB2 gene to exclude rare or novel pathogenic mutations. The HEK293 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The plasmids encoding WT or mutated Cx26 tagged with fluores- cent protein markers were transfected to the HEK293 cells using Shining Jet transfection reagent (Shing, China) under the manufacturer’s instructions. The intracellular localization of WT and mutant Cx26 proteins was studied through fluores- cence microscopic analysis 24 h after transfection.

3.Results
The blood genomic DNA from all members of the family were used for ASPUA molecular screening. 8 of 9 potential hotspot mutations in SLC26A4, GJB2, GJB3 and mtDNA RNR1 genes were excluded as causative factors of hearing loss except c.176 del 16, a prevalent mutation in the GJB2 gene. The existence of this mutation was subsequently reconfirmed during the sequencing process (Fig. 1). Our screening and sequencing results showed that while the heterozygous muta- tion of c.176 del 16 is found in the couple’s GJB2 genes, the deaf boy carries a homozygote mutation in GJB2; c.176 del
16. Fortunately, the new born child did not inherit any of such mutations in her allelic GJB2 genes.To see whether the GJB2 gene from the new born baby was functional, we cloned the GJB2 genes from the two children’s blood genomic DNA and constructed two respective GJB2- GFP fused plasmids. Then we transfected HEK293 cells with these two expression plasmids, which allowed the re- searchers to track the intracellular location of Cx26.
In our test, the normal Cx26-GFP protein cloned from the second child was successfully formed into some small foci localized in the cell membrane, indicative of involvement of the gap junction between two adjacent cells (Fig. 2B arrow). By contrast, the mutated Cx26 protein (GJB2 c.176 del 16) from the deaf child was defused on the full cells evenly without any specific localization on the cell membrane and no foci (gap junctions) were observed in these cells (Fig. 2A). Therefore, we reached the conclusion that the GJB2 c.176del16 mutation might potentially lead to the boy’s hear- ing loss by undermining the ability of mutant Cx26 proteins to form the gap junction structure in the cell membrane.

4. Discussion
As many studies have shown, GJB2 mutations, as one of the predominant causes of non-syndromic hearing loss, are either dominant or recessive (Han et al., 2008; Abe et al., 2000; Dai et al., 2009). In this study we first screened and sequenced the 5-year-old child diagnosed with profound hearing loss for hot-point genes (Li et al., 2008) related to the symptom. Then the same procedures were performed on the other child of the family, a healthy one born recently, in an attempt to seek out the same gene. This process was considered essential to excluding the possibilities of her developing the same hearing disabilities at a later stage in life. Luckily enough, both screening and sequencing results confirmed that the second child had a normal GJB2 gene, an indication that this new born child is unlikely to develop the same gene type of hearing disabilities as did her elder brother whose genomic type is homozygosity for the GJB2 c.176 del 16 mutation. To make a further comparison of their GJB2 genes, we cloned the sequences of their GJB2 genes derived from their respective blood genomic DNA into GFP fused-plasmids. Then 239T cells were used to measure gene function, showing that the mutated GJB2 gene (GJB2 c.176 del 16) from the first child could not form the gap junction structure between two adjacent cells, indicative of a dysfunctional GJB2 gene. This is consistent with the phenotype of the child’s deafness determined by the screening and sequencing pro- cedures. In contrast, the GJB2 gene from the second child has no problems helping form functional gap junctions needed for cell communication and is therefore considered healthy. The screening and sequencing results are both consistent with her ABR tested phenotype. Our results also echoed previous studies in that the frame shift mutation of GJB2 c.176 del 16 (Mikstiene et al., 2016; Kudo et al., 2000) is sited in the second topological domain and can erase the second transmembrane domain of Cx26. With such mutations at play, a stop codon (TGA) introduced into the GJB2 gene and helps form a truncated CX26 protein comprising 75 amino acids. This abnormal protein can seri- ously inhibit the formation of hexamers (Zhang, 2009), a process reenacted by our cell line experiments (Fig. 2).

In summary, our study helped determine the possibility of a child developing hearing loss in a family carrying a deafness-inducing gene. The methodologies adopted in this study are suitable for the identification of any hot-point or rare-point mutation in the GJB2 gene. In our case, the child was found to have a normal (wild type) GJB2 gene and we presume that no preemptive intervention was needed to prevent the devel- opment of hearing loss and language learning problems caused by mutated GJB2. However, it is C-176 impossible to exclude the existence of other causative genes in the genome of this child. So we recommend that further screening of deafness genes based on the next generation of sequencing technology should be carried out if the patients can afford it.