Background
Methods
Recruitment of participants and clinical data collection
Type of Mating | No. of hearing impaired mates | No. of hearing partners | Other hearing impaired members in the family | Other hearing members in the family | Total |
---|---|---|---|---|---|
Deaf marrying deaf (DXD) | 120 | 0 | 63 | 154 | 337 |
Deaf marrying normal hearing (DXN) | 46 | 46 | 50 | 142 | 284 |
TOTAL | 166 | 46 | 113 | 296 | 621 |
GJB2 and GJB6 mutation analysis
In silico analysis of novel variants
Homology modeling
Control study
Results
Type of marriage based on consanguinity | PARENTAL CONSANGUINITY | In DXD mating | ||
---|---|---|---|---|
In husbands’ parents (%) | In wives’ parents (%) | Combined (%) | ||
Consanguineous | 24 (40%) | 30 (50%) | 54 (45%) | 2 (3.33%) |
Non Consanguineous | 36 (60%) | 30 (50%) | 66 (55%) | 58 (96.67%) |
TOTAL | 60 | 60 | 120 | 60 |
Type of marriage based on consanguinity | PARENTAL CONSANGUINITY | In DXN mating | ||
---|---|---|---|---|
In husbands’ parents (%) | In wives’ parents (%) | Combined (%) | ||
Consanguineous | 17 (36.96%) | 13 (28.26%) | 30 (32.61%) | 18 (39.13%) |
Non Consanguineous | 29 (63.04%) | 33 (71.74%) | 62 (67.39%) | 28 (60.87%) |
TOTAL | 46 | 46 | 92 | 46 |
Outcome of GJB2 mutation screening
S. No. | GJB2 VARIANTS | DOMAIN/ LOCATION | No. of alleles in DXD mates (n=236) | Frequency in DXD mating (%) | No of alleles in affected partners of DXN mating (n=92) | Frequency in HI partners of DXN mating (%) | No of alleles in normal partners of DXN mating (n=86) | Frequency in normal hearing partners of DXN (%) | No. of alleles in normal hearing control (n=330) | Frequency in Normal hearing controls (%) | |
---|---|---|---|---|---|---|---|---|---|---|---|
CODON | PROTEIN | ||||||||||
1 | c.71 G>A | p.W24X | TM1 | 60 | 25.42 | 28 | 30.43 | 6 | 6.98 | 6 | 1.82 |
2 | c.79 G>A | p.V27I | TM1 | 2 | 0.85 | 0 | 0 | 0 | 0 | 0 | 0 |
3 | c.104 T>G | p.I35S | TM1 | 2 | 0.85 | 0 | 0 | 0 | 0 | 0 | 0 |
4 | c. 109 G>A | p.V37I | TM1 | 1 | 0.42 | 0 | 0 | 0 | 0 | 0 | 0 |
5 | c. 126 G>T | p.E42D | EC1 | 3 | 1.27 | 0 | 0 | 0 | 0 | 0 | 0 |
6 | c.135 A>G | p.G45G | EC1 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0.3 |
7 | c.165 C>A | p.T55T | EC1 | 1 | 0.42 | 0 | 0 | 0 | 0 | 3 | 0.91 |
8 | c. 185 A>G | p.N62S | EC1 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0.3 |
9 | c. 224 G>A | p. R75Q | TM2 | 1 | 0.42 | 0 | 0 | 0 | 0 | 0 | 0 |
10 | c.231 G>A | p.W77X | TM2 | 5 | 2.12 | 1 | 1.09 | 0 | 0 | 0 | 0 |
11 | c.240 G>A | p.Q80Q | TM2 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0.61 |
12 | c.257C>T | p.T86M | TM2 | 0 | 0 | 2 | 2.17 | 1 | 1.16 | 0 | 0 |
13 | c.262 G>A | p.A88A | TM2 | 0 | 0 | 2 | 2.17 | 1 | 1.16 | 0 | 0 |
14 | c.341 A>G | p.E114G | IC2 | 2 | 0.85 | 0 | 0 | 0 | 0 | 0 | 0 |
15 | c. 370 C>T | p.Q124X | IC2 | 3 | 1.27 | 2 | 2.17 | 1 | 1.16 | 0 | 0 |
16 | c.380 G>A | p.R127H | IC2 | 25 | 10.59 | 4 | 4.35 | 6 | 6.98 | 57 | 17.27 |
17 | c.439 G>A | p.E147K | TM3 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0.3 |
18 | c.457 G>A | p.V153I | TM3 | 14 | 5.93 | 1 | 1.09 | 3 | 3.49 | 6 | 1.82 |
19 | c.493 C>T | p.R165W | EC2 | 1 | 0.42 | 0 | 0 | 1 | 1.16 | 3 | 0.91 |
20 | c. 551G>A | p.R184Q | EC2 | 1 | 0.42 | 0 | 0 | 0 | 0 | 0 | 0 |
21 | c.585 G>A | p.M195I | TM4 | 0 | 0 | 1 | 1.09 | 0 | 0 | 1 | 0.3 |
22 | c.675 A>T | p.P225P | IC3 | 0 | 0 | 1 | 1.09 | 0 | 0 | 1 | 0.3 |
23 | c.IVS 1+1 G>A (-3172 G>A) | Intronic Splice site region | 4 | 1.7 | 0 | 0 | 0 | 0 | 0 | 0 | |
TOTAL | 125 | 52.96 | 42 | 45.65 | 19 | 22.09 | 82 | 24.85 |
Outcome of GJB6 mutation screening
Large deletions- 309 kb deletion (GJB6-D13S1830) & 232 kb deletion (GJB6-D13S1854) in GJB6 gene
Point mutations in GJB6 gene
S. No. | GJB6 Variants | Domain/ Location | Effect | Alleles in DXD (n=170)* | Alleles in DXN (n=64)* | Overall Allelic Frequency (%) | |
---|---|---|---|---|---|---|---|
Codon | Protein | ||||||
1 | c.311 G>A | p.R104H | IC2 | Missense mutation; Transition; NOVEL; Possibly pathogenic | 2 (1.18%) | 0 | 0.85% |
2 | c.170 A>G | p.Q57R | EC1 | Missense mutation; Transition; NOVEL; Possibly pathogenic | 1 (0.59%) | 0 | 0.43% |
3 | c.301 G>C | p.E101Q | IC2 | Missense mutation; Transversion; NOVEL; Possibly pathogenic | 0 | 1 (1.56%) | 0.43% |
Genotypes of GJB2 & GJB6 mutations
S. No. | GJB2 and GJB6 genotypes | HI Husband (n=58)* | Frequency % | HI Wife (n=60) | Frequency % | Total (n=118) | Combined Frequency (%) |
---|---|---|---|---|---|---|---|
I | GJB2-Biallelic & Triallelic | ||||||
1 | W24X/W24X | 13 | 22.41 | 13 | 21.67 | 26 | 22.03 |
2 | V153I/V153I | 1 | 1.72 | 2 | 3.33 | 3 | 2.54 |
3 | W77X/W77X | 1 | 1.72 | 1 | 1.67 | 2 | 1.7 |
4 | R127H/R127H | 1 | 1.72 | 1 | 1.67 | 2 | 1.7 |
5 | R127H/V153I | 1 | 1.72 | 1 | 1.67 | 2 | 1.7 |
6 | V27I/E114G | 1 | 1.72 | 1 | 1.67 | 2 | 1.7 |
7 | E42D/E42D | 0 | 0 | 1 | 1.67 | 1 | 0.85 |
8 | W77X/Q124X | 0 | 0 | 1 | 1.67 | 1 | 0.85 |
9 | W24X/I35S | 0 | 0 | 1 | 1.67 | 1 | 0.85 |
10 | Q124X/IVS1+1G>A | 0 | 0 | 1 | 1.67 | 1 | 0.85 |
11 | R75Q#/V153I | 0 | 0 | 1 | 1.67 | 1 | 0.85 |
12 | V37I/V153I | 1 | 1.72 | 0 | 0 | 1 | 0.85 |
13 | V153I/R165W | 0 | 0 | 1 | 1.67 | 1 | 0.85 |
14 | R184Q#/Q124X/IVS1+1G>A | 1 | 1.72 | 0 | 0 | 1 | 0.85 |
15 | W24X/T55T/R127H | 1 | 1.72 | 0 | 0 | 1 | 0.85 |
II | GJB2-Monoallelic | ||||||
1 | R127H/+ | 9 | 15.52 | 9 | 15 | 18 | 15.25 |
2 | W24X/+ | 4 | 6.9 | 2 | 3.33 | 6 | 5.08 |
3 | V153I/+ | 2 | 3.45 | 1 | 1.67 | 3 | 2.54 |
4 | IVS1+1G>A/+ | 0 | 2 | 1.67 | 2 | 1.7 | |
5 | I35S/+ | 0 | 1 | 1.67 | 1 | 0.85 | |
III | GJB6-Monoallelic | ||||||
1 | R104H/+ | 1 | 1.72 | 0 | 0 | 1 | 0.85 |
2 | Q57R/+ | 1 | 1.72 | 0 | 0 | 1 | 0.85 |
IV | GJB2/GJB6-Digenic | ||||||
1 | E42D/+; R104H/+ | 1 | 1.72 | 0 | 0 | 1 | 0.85 |
S. No. | Genotypes | Affected Partner (n= 46) | Frequency (%) | Normal hearing partner (n=43)* | Frequency (%) |
---|---|---|---|---|---|
I | GJB2-Biallelic | ||||
1 | W24X/W24X | 11 | 23.91 | 0 | 0 |
2 | Q124X/Q124X | 1 | 2.17 | 0 | 0 |
3 | T86M/T86M | 1 | 2.17 | 0 | 0 |
4 | W24X/W77X | 1 | 2.17 | 0 | 0 |
5 | W24X/A88A | 1 | 2.17 | 0 | 0 |
6 | R153I/R165W | 0 | 1 | 2.33 | |
6 | M195I/P225P | 1 | 2.17 | 0 | 0 |
II | GJB2-Monoallelic | ||||
1 | W24X/+ | 4 | 8.7 | 6 | 13.95 |
T86M/+ | 0 | 0 | 1 | 2.33 | |
Q124X/+ | 0 | 0 | 1 | 2.33 | |
2 | R127H/+ | 4 | 8.7 | 6 | 13.95 |
3 | V153I/+ | 1 | 2.17 | 2 | 4.65 |
4 | A88A/+ | 1 | 2.17 | 1 | 2.33 |
III | GJB6-Monoallelic | ||||
1 | E101Q/+ | 1 | 2.17 | 0 | 0 |
S.No. | GJB2 genotype | Normal hearing control population (n=165) | Frequency % |
---|---|---|---|
1 | W24X/+ | 5 | 3.02 |
2 | N62S/+ | 1 | 0.61 |
3 | E147K/T55T | 1 | 0.61 |
4 | W24X/M195I/P225P | 1 | 0.61 |
1 | R127H/R127H | 5 | 3.02 |
2 | R127H/R165W | 1 | 0.61 |
3 | R127H/T55T | 1 | 0.61 |
4 | V153I/R165W | 2 | 1.21 |
5 | Q80Q/R127H | 1 | 0.61 |
6 | R127H/+ | 44 | 26.67 |
7 | V153I/+ | 4 | 2.41 |
8 | Q80Q/+ | 1 | 0.61 |
9 | T55T/+ | 1 | 0.61 |
10 | G45G/+ | 1 | 0.61 |
69 | 41.82 |
Novel variants in GJB2 and GJB6 genes
p.Q57R variant (in GJB6 gene)
p.E101Q variant (in GJB6gene)
In silico analysis of novel variants observed in GJB2 and GJB6 genes
Mutation | SIFT | PolyPhen-2 | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
HumDiv | HumVar | |||||||||
Score | Median Sequence conservation | Sequences represented at position | Comment | Score | Sensitivity, Specificity | Comment | Score | Sensitivity, Specificity | Comment | |
p.E42D (GJB2) | 0.57 | 3.05 | 42 | Tolerated | 0.038 | 0.94, 0.82 | Benign | 0.052 | 0.93, 0.63 | Benign |
p.Q57R (GJB6) | 0.00 | 3.09 | 35 | Affect protein function | 1.000 | 0.00, 1.00 | Probably damaging | 1.000 | 0.00, 1.00 | Probably damaging |
p.E101Q (GJB6) | 0.48 | 3.10 | 29 | Tolerated | 0.183 | 0.92, 0.87 | Benign | 0.114 | 0.90, 0.69 | Benign |
p.R104H (GJB6) | 0.01 | 3.09 | 35 | Affect protein function | 0.990 | 0.41, 0.98 | Probably damaging | 0.749 | 0.77, 0.86 | Possibly damaging |
GJB2
|
GJB6
| |||||
---|---|---|---|---|---|---|
Property | Native | p.E42D | Native | p.Q57R | p.E101Q | p.R104H |
Number of amino acids | 226 | 226 | 261 | 261 | 261 | 261 |
Molecular weight | 26215 | 26201 | 30387.4 | 30415.5 | 30386.4 | 30368.4 |
Theoretical pI (Isoelectric point) | 9.11 | 9.11 | 8.81 | 8.92 | 8.92 | 8.68 |
Total number of negatively charged residues (Asp + Glu) | 18 | 18 | 23 | 23 | 22 | 23 |
Total number of positively charged residues (Arg + Lys) | 27 | 27 | 29 | 30 | 29 | 28 |
Total number of atoms | 3721 | 3718 | 4274 | 4280 | 4275 | 4268 |
Ext. coefficient assuming all pairs of Cys residues form cystines | 52410 | 52410 | 52410 | 52410 | 52410 | 52410 |
Ext. coefficient assuming all Cys residues are reduced | 51910 | 51910 | 51910 | 51910 | 51910 | 51910 |
Estimated half life (mammalian reticulocytes, in vitro) | 30 hrs | 30 hrs | 30 hrs | 30 hrs | 30 hrs | 30 hrs |
Instability index | 42.8 | 42.8 | 43.11 | 42.60 | 43.11 | 44.01 |
Aliphatic index | 98.67 | 98.67 | 91.07 | 91.07 | 91.07 | 91.07 |
Grand average of hydropathicity (GRAVY) | 0.288 | 0.288 | 0.055 | 0.051 | 0.055 | 0.06 |
Genotype-phenotype correlation of GJB2/GJB6 mutations in the incidence of hearing loss in DXD families
BOTH PARTNERS HAVING GJB2 | ONLY ONE PARTNER WITH GJB2 | ONLY ONE PARTNER WITH GJB6 | ONLY ONE PARTNER WITH GJB2/GJB6 | |||||||
---|---|---|---|---|---|---|---|---|---|---|
S. No. | SUBGROUPS IN DXD MATING BASED ON PHENOTYPE OF OFFSPRING | BOTH HOMOZYGOUS OR COMPOUND HETEROZYGOUS | BOTH HETEROZYGOUS | ONE HOMOZYGOUS, ONE HETEROZYGOUS | HOMOZYGOUS | HETEROZYGOUS | ONE HETEROZYGOUS | DIGENIC | NON-GJB2 & NON-GJB6 | TOTAL (%) |
1 | Non-complementary with all deaf offspring | 8 | 0 | 0 | 1 | 1 | 1 | 0 | 6* | 17 (28.33%) |
2 | Complementary with all hearing offspring | 0 | 1 | 1 | 11 | 6 | 0 | 1 | 10# | 30 (50%) |
3 | Mixed offspring (hearing and hearing impaired) | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 1 | 2 (3.33%) |
4 | NO OFFSPRING | 0 | 0 | 1 | 4 | 0 | 1 | 0 | 5 | 11 (18.33%) |
5 | TOTAL | 8 | 1 | 2 | 16 | 8 | 2 | 1 | 22 | 60 |
Group I
Group II
Group III
Group IV
Role of GJB2/GJB6 mutations in the incidence of hearing loss in DXN families
Group-I families: Consanguineously mating DXN families with and without GJB2/GJB6 mutation affliction
Group II families: Non-consanguineously mating DXN families with and without GJB2/GJB6 mutation affliction
Statistical analysis for significance
S. No. | Group | GJB2 Positive Alleles | GJB2 Negative Alleles | Total Alleles | Percentage (%) | |
---|---|---|---|---|---|---|
1 | DXD-Both Affected Partners | Observed (O) | 125 | 111 | 236 | 52.96 |
Expected (E) | 85.01 | 150.99 | ||||
2 | DXN- Affected Partners | Observed (O) | 42 | 50 | 92 | 45.65 |
Expected (E) | 33.14 | 58.86 | ||||
3 | DXN-Normal Hearing Partners | Observed (O) | 19 | 67 | 86 | 22.09 |
Expected (E) | 30.98 | 55.02 | ||||
4 | Control | Observed (O) | 82 | 248 | 330 | 24.85 |
Expected (E) | 124.88 | 205.12 | ||||
TOTAL | 268 | 476 | 744 | |||
Chi-Square Value ( Σ(O-E)2/E) | 58.21 (P< 0.001) |