2Cornea Clinic, Aravind Eye Hospital, Madurai, Tamilnadu, India
Purpose: The main purpose of this study was to perform the genetic analysis of 55 Indian MCD families.
Methods: We have recruited 55 affected, 11 unaffected members from 55 MCD families along with 100 controls. All the study subjects underwent ocular examination before collecting the blood for the screening of CHST6 gene. Polymerase chain reaction was performed followed by bi-directional sequencing. The novel mutations were predicted by Polyphene-2, SIFT, Mutation taster and SOPMA tool.
Results: We identified 14 different mutations, 3 known SNP’s in 44 MCD patients amongst 6 were novel. Also 2 hotspot mutations were identified amongst the 14 mutations. We could not identify any mutations in the coding region of CHST6 gene in 11 MCD patients (20%).
Conclusion: Our study identified six novel mutations which will add up to the list of already known mutations identified in different ethnic populations. Our study also increases the mutational landscape of CHST6 gene. We concluded due to genetic heterogeneity there might be some other gene involved in Indian MCD patients who are negative for CHST6 mutations.
Keywords: CHST6 gene; Cornea; Heterogeneity; Mutations;
Based on the histochemical features, MCD is classified into three immunophenotypes I, II and IA [24-26]. Type I is characterized by absence or low level of sulfated KS (AgKS) in cornea as well as serum. Type II is characterized by normal or marginally reduced level of AgKS in cornea as well as serum. Type IA is characterized by low level of antigenic AgKS in serum and detectable level in keratocytes [26].
The main purpose of this study was to screen the coding region of CHST6 gene in 55 Indian families with MCD.
1. The six novel changes were segregating in the family in an autosomal recessive fashion.
2. All the six novel mutations were absent in the following databases (Ensemble, HGVS, 1000 genome project)
3. Three of the novel missense mutations were highly conserved (Val172Met, Ser248Asn, Glu274Gln) and one novel missense mutation was not conserved (Arg202His) across different orthologous species (Figure 2)
4. Mutation taster predicted all the novel mutations to be disease causing and the polyphen2, SIFT programme predicted all are damaging.
A novel homozygous missense mutation Val172Met was identified in a patient from family 5 with consanguinity, Arg202His was identified in a patient from family 42 with consanguinity, Ser248Asn was present in a patient from family 25 with consanguinity, Glu274Gln was identified in a patient from family 9 with consanguinity (Table 1). Additionally, a novel homozygous nonsense mutation Ser53X was identified in 2 patients (patient & patient’s younger brother) from family 8 without consanguinity but with MCD history (Figure 3a). One more novel homozygous nonsense mutation Ser81X was identified in a patient from family 36 without consanguinity and MCD history.
Family/ |
Mutationᵃ |
Mutation type |
Amino acid |
Age(yrs)/ |
Consanguinity |
Novel/ Previously reportedᵇ |
1 |
No mutation |
|
|
32/M |
No |
|
2 |
c.614G>A (p.R205Q) |
Homozygous missense |
Basic to polar |
35/M |
No |
India |
3 |
c.172C>T (p.Q58X) |
Homozygous nonsense |
Basic to Polar |
44/M |
Yes |
France |
4 |
No mutation |
|
|
26/M |
No |
|
5 |
c.514G>A (p.V172M) |
Homozygous missense |
Non-polar to non-polar |
44/M |
Yes |
Novel |
6 |
c.198delC (p.V66VfsX3) |
Heterozygous deletion |
Non-polar to non-polar |
18/M |
No |
India |
7 |
No mutation |
|
|
10/M |
Yes |
|
8 |
c.158C>A (p.S53X) |
Homozygous nonsense |
Polar to stop codon |
30/M |
No |
Novel |
9 |
c.820G>C (p.E274Q) |
Homozygous missense |
Acidic to polar |
60/M |
Yes |
Novel |
10 |
No mutation |
|
|
66/M |
No |
|
11 |
c.614G>A (p.R205Q) |
Heterozygous missense |
Basic to polar |
23/M |
No |
India |
12 |
c.158C>T (p.S53L) |
Homozygous missense |
Polar to basic |
32/M |
Yes |
Hotspot mutation India, America |
13 |
No mutation |
|
|
50/F |
Yes |
|
14 |
c.545delA (p.Q182RfsX198) |
Heterozygous deletion |
Polar to basic |
47/F |
Yes |
Hotspot mutation India |
15 |
c.158C>T (p.S53L) |
Homozygous missense |
Polar to non-polar |
16/F |
Yes |
Hotspot mutation |
16 |
c.158C>T (p.S53L) |
Homozygous missense |
Polar to non-polar |
32/M |
Yes |
Hotspot mutation |
17 |
c.379C>T (p.R127C) |
Homozygous missense |
Basic to polar |
33/M |
No |
Saudi Arabia, India |
18 |
No mutation |
|
|
20/M |
Yes |
|
19 |
No mutation |
|
|
38/F |
Yes |
|
20 |
c.545delA (p.Q182RfsX198) |
Homozygous deletion |
Polar to basic |
28/F |
Yes |
Hotspot mutation |
21 |
No mutation |
|
|
26/F |
No |
|
22 |
c.545delA (p.Q182RfsX198) |
Homozygous deletion |
Polar to basic |
27/F |
No |
Hotspot mutation |
23 |
No mutation |
|
|
28/F |
Yes |
|
24 |
c.545delA (p.Q182RfsX198) |
Homozygous deletion |
Polar to basic |
19/F |
Yes |
Hotspot mutation India |
25 |
c.743G>A (p.S248N) |
Homozygous missense |
Polar to Polar |
37/M |
Yes |
Novel |
26 |
c.614G>A (p.R205Q) |
Homozygous missense |
Basic to polar |
12/M |
No |
India |
27 |
c.124C>T (p.H42Y) |
Homozygous missense |
Basic to polar |
30/F |
No |
India |
28 |
c.820G>C (p.E274Q) |
Heterozygous missense |
Acidic to polar |
47/F |
Yes |
India |
29 |
c.148C>T (p.R50C) |
Homozygous missense |
Basic to polar |
40/M |
No |
India |
30 |
c.379C>T (p.R127C) |
Homozygous missense |
Basic to polar |
22/M |
No |
Saudi Arabia, India |
31 |
c.614G>A (p.R205Q) |
Homozygous missense |
Basic to polar |
52/F |
No |
India |
32 |
No mutation |
|
|
52/F |
Yes |
|
33 |
c.158C>T (p.S53L) |
Homozygous missense |
Polar to non-polar |
20/M |
No |
Hotspot mutation |
34 |
c.158C>T (p.S53L) |
Homozygous missense |
Polar to non-polar |
35/F |
Yes |
Hotspot mutation |
35 |
c.545delA (p.Q182RfsX198) |
Heterozygous deletion |
Polar to Basic |
50/M |
No |
Hotspot mutation |
36 |
c.242C>A (p.S81X) |
Homozygous nonsense |
Polar to stop codon |
33/M |
No |
Novel |
37 |
c.613C>T (p.R205W) |
Homozygous missense |
Basic to non-polar |
46/M |
No |
Korea |
38 |
c.545delA (p.Q182RfsX198) |
Homozygous deletion |
Polar to basic |
30/F |
Yes |
Hotspot mutation |
39 |
c.545delA (p.Q182RfsX198) |
Homozygous deletion |
Polar to basic |
22/M |
Yes |
Hotspot mutation |
40 |
c.158C>T (p.S53L) |
Homozygous missense |
Polar to non-polar |
30/M |
Yes |
Hotspot mutation |
41 |
c.148C>T (p.R50C) |
Homozygous missense |
Basic to polar |
25/F |
No |
India |
42 |
c.290G>A (p.R202H) |
Homozygous missense |
Basic to basic |
21/M |
Yes |
Novel |
43 |
c.545delA (p.Q182RfsX198) |
Homozygous deletion |
Polar to basic |
36/M |
No |
Hotspot mutation |
44 |
c.545delA (p.Q182RfsX198) |
Heterozygous deletion |
Polar to basic |
61/M |
No |
Hotspot mutation |
45 |
c.158C>T (p.S53L) |
Homozygous missense |
Polar to non-polar |
25/M |
Yes |
Hotspot mutation |
46 |
c.545delA (p.Q182RfsX198) |
Heterozygous deletion |
Polar to basic |
60/M |
No |
Hotspot mutation |
47 |
c.545delA (p.Q182RfsX198) |
Homozygous deletion |
Polar to basic |
27/F |
Yes |
Hotspot mutation |
48 |
c.158C>T (p.S53L) |
Homozygous missense |
Polar to non-polar |
38/F |
Yes |
Hotspot mutation |
49 |
c.158C>T (p.S53L) |
Homozygous missense |
Polar to non-polar |
31/M |
Yes |
Hotspot mutation |
50 |
c.158C>T (p.S53L) |
Homozygous missense |
Polar to non-polar |
17/F |
No |
Hotspot mutation |
51 |
c.581_586 delACCTACinsGGT |
Deletioninsertion mutation |
Non-polar to polar |
39/M |
Yes |
India |
52 |
No mutation |
|
|
36/F |
Yes |
India |
53 |
c.278G>A (p.R93H) |
Homozygous missense |
Basic to Basic |
40/M |
Yes |
India |
54 |
c.148C>T (p.R50C) |
Homozygous missense |
Basic to polar |
37/F |
Yes |
India |
55 |
c.581_586 delACCTACinsGGT |
Deletioninsertion mutation |
Non-polar to polar |
44/M |
No |
India |
A heterozygous deletion (Q182RfsX198-deletion) mutation was identified in a patient from family 44. Further, pedigree analysis revealed that the propand’s father, propand’s elder sister and propand’s elder brother had the same eye problem (No genomic DNA for analysis). But propand’s two sons are phenotypically and genotypically normal. We believe, either it could be uniparentaldisomy or the second mutation may be present in the deep intronic region or regulatory region which was not covered by Sanger sequencing.
Further, a known heterozygous single base pair deletion was also identified in one of the patient from family 6 causing a frame shift at valine-66 (Val66ValfsX3) resulting in a premature termination codon at amino acid residue 2. The second mutation in the patient could be either present in deep intronic or regulatory region of CHST6 gene which was not covered by Sanger sequencing. Furthermore, in family 3, we have identified a homozygous nonsense mutation (Glu58X) in a patient and his affected sibling that leads to the formation of truncated protein (Table 1). Interestingly, we have also identified known 6 bp deletion (homozygous state) and 3 bp Insertion (Asn194_ Arg196delinsArgCys) in two patients belonging to two unrelated families (family 51 and 55) that results in frame shifting at asparagine-194 position (Table 1).
Overall, we have sequenced 55 families, out of these families; we failed to identify any mutation in 11 MCD patients. Amongst these, 2 patients from two unrelated families had consanguinity (7,13) in their family, but had no mutations in the entire coding region of CHST6 gene.
State of |
Control |
Nonsense |
Nonsense |
Missense |
Missense |
Missense |
Missense |
Hotspot-missense |
||||||||
|
|
% |
|
% |
|
% |
|
% |
|
% |
|
% |
|
% |
|
% |
Alpha helix |
190 |
48.1 |
15 |
28.85 |
28 |
35 |
194 |
49.11 |
190 |
48.1 |
184 |
46.58 |
190 |
46.1 |
191 |
48.35 |
Extended strand |
58 |
14.68 |
14 |
26.92 |
20 |
25 |
54 |
13.67 |
58 |
14.68 |
50 |
12.66 |
58 |
14.68 |
58 |
14.68 |
β turn |
31 |
7.85 |
3 |
5.77 |
8 |
10 |
31 |
7.85 |
31 |
7.85 |
0 |
0 |
31 |
7 |
32 |
8.1 |
Random coil |
116 |
29.37 |
20 |
38.46 |
24 |
30 |
116 |
29.37 |
116 |
29.37 |
161 |
40.76 |
116 |
30.37 |
116 |
28.86 |
Instability index (II) |
US |
46.24 |
US |
58.93 |
US |
50.87 |
US |
46.24 |
US |
46.24 |
US |
45.13 |
US |
45.13 |
US |
45.1 |
The identified known mutations have been observed among patients from several populations included India Saudi Arabia, Korea, Egypt, Japan, America and France [2,3,1517,18,23]. This additionally supports our findings showed a high degree of mutational heterogeneity among the patients studied.
We identified 6 novel mutations across the CHST6 gene in 6 MCD patients as described in the result section. Of these, we have identified a novel missense mutation in patient from the family 9 that leads to the replacement of glutamic acid to glutamine (E274Q) while in an independent studies from different ethnic background (Egypt, Japanese, American) with MCD; they have identified the same missense mutation (E274K) though the glutamic acid was replaced by lysine and the same mutation was identified in a patient from family 28, but in a heterozygous state suggesting that the second mutation may be present in the deep intronic or regulatory region of CHST6 gene which was not covered by Sanger sequencing [15,18]. This mutation was highly conserved across different orthologous species (Figure 2). Polyphene 2, SIFT and Mutation taster also predicted the mutation was pathogenic in nature. In addition, we have used SOPMA tool that also predicted the un-stability (instability index 45.13) in the protein structure. Moreover, additional evidences also supports our findings that the secondary structure of protein was altered by the missense mutation. Which causes protein unstability thus leads to deficient enzyme activity [12].
Interestingly, we have also identified a novel homozygous nonsense mutation (S53X) in a patient from family 36 was highly conserved across different orthologous species. Polyphene 2, SIFT and Mutation taster also predicted the mutation was pathogenic. SOPMA also predicted that this cause changes in protein stability (instability index 58.93) that leads to the formation of unstable protein. We have identified one more novel homozygous nonsense mutation (S81X) was highly conserved with the un-stability (instability index 50.87) in the protein structure that leads to the formation of unstable protein. These nonsense mutations may lead to the absence of proteins due to nonsense-mediated decay (NMD) of the mRNA suggesting that these mutations might be expected to be associated with an early onset and/or severe form of MCD affecting both the eyes [14].
In addition, we have identified one novel homozygous missense mutation (R202H). It was not conserved across the species but Polyphene 2, SIFT and Mutation taster predicted the mutation as pathogenic in nature. Though, this particular change was not conserved. However, its mild secondary structural modifications with instability index 46.24 may be responsible for the loss of enzyme activity.
Apart from the novel mutations described above; we have identified 12 known mutations including a hotspot deletion mutation (Q182RfsX198) in the coding region of the CHST6 gene that causes frameshift changes in the upstream region of CHST6 gene due to nucleotide sequence similarity of CHST5 and CHST6 genes and the adjacent regions or it could be due to chromosomal crossover in CHST5 and CHST6 genes [18]. Previous studies have revealed that frameshift mutations of CHST6 gene may lead to severe MCD phenotypes with much deeper grey white deposits [30].
Additionally, we have also observed a hotspot missense mutation (S53L) in 10 patients from different MCD families that leads to unstable protein (instability index 45.10) this mutation was already reported in 7 patients from seven South Indian families and the same mutation was identified in an American population suggesting a hotspot mutation [2,12,19]. Previous studies suggested that this mutation might be present in the 3’-phosphate-binding domain of C-GlcNac-6-ST enzyme. This region of the CHST6 gene contains an active site which might be a mutational hotspot [31].
We failed to identify mutations in 11 patients from 11 different families out of 55 families screened. Warren, et al. also did not identify CHST6 gene mutations in 4 of 51 families screened suggesting that the mutations in these families may be present in a yet to be identified gene or may be present in a deep intronic or promoter region which can be explored by means of extensive linkage analysis [32]. And also this may be asociated with MCD type II phenotype caused by genetic abnormalities in the upstream of CHST6 gene [8].
Taken together, our results indicate the high degree of mutational heterogeneity in Indian population. The coding region of CHST6 gene significantly affected by mutations leads to unstable protein products with altered secondary structure. All the altered amino acid residues are evolutionary conserved among other mammalian species indicating severe functional loss of CHST6 gene. Functional characterization of these novel mutations identified may help to improve the understanding of the disease pathogenesis.
B. Conflict of Interest All authors of this manuscript declare that they have no conflict of interest.
C. Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the Institutional Ethical Committee of the Aravind Eye Care System, Madurai, Tamilnadu, India and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent Informed consent was obtained from all individual participants included in the study.
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