Demographic and clinical characteristics of the patients:
Among all PCR positive patients 77: 53.8% were serious cases followed by non-serious 66: 46.2%. Males were 80: 55.9% with the age of presentation of more than 50 years (106, 74.1%). Most of the patients had diabetes mellitus (71, 49.4%) and hypertension (83, 58%). Few of them also had other known diseases of like cardiovascular disease (22, 15.4%), respiratory disease (7, 4.9%) and others (Cardiovascular includes the ischemic heart disease, coronary artery disease and valvular disease, it does not include the arterial diseases and hypertension while endocrine diseases do not include the diabetes mellitus). Fever (90, 62.9%) was the most frequent symptom encountered in all patients with the dyspnea (87, 60.8%) being the second common symptom. Almost all inflammatory markers were raised in every patient and procalcitonin (139, 97.2%) being the most frequently raised marker.
On comparing the demographic and clinical characteristics with the severity of the COVID-19 disease the significant statistical association of fever (p value, 0.0002), cough (p value, 0.004), dyspnea (p value, < 0.001), loss of taste (p value, 0.043), de-dimer (p value, < 0.001), LDH (p value, < 0.001), CRP (p value, < 0.001) and outcome (p value, < 0.001 was found). Fever, cough and dyspnea are the symptoms that were frequently present in every case of severity from mild to critical while the loss of taste and smell were more related to mild and moderate diseases. Table: 1
Table 1
Statistical association of demographic and clinical characteristics with severity of COVID-19
Characteristics
|
n = 143
|
Severity
|
P valuea
|
Serious
77 (53.8)
|
Non-Serious
66 (46.2)
|
Age (Years)
<=50
> 50
|
37 (25.9)
106 (74.1)
|
18 (12.6)
59 (41.3)
|
19 (13.3)
47 (32.9)
|
0.566
|
Gender
Male
Female
|
80 (55.9)
63 (44.1)
|
44 (30.8)
33 (23.1)
|
36 (25.2)
30 (21)
|
0.866
|
Ethnicity
Sindhi
Urdu
Punjabi
Pathan
|
35 (24.5)
82 (57.3)
18 (12.6)
8 (5.6)
|
15 (10.5)
45 (31.5)
14 (9.8)
3 (2.1)
|
20 (14)
37 (25.9)
4 (2.8)
5 (3.5)
|
0.081
|
Hypertension
Yes
No
|
83 (58)
60 (42)
|
46 (32.2)
31 (21.7)
|
37 (25.9)
29 (20.3)
|
0.735
|
Diabetes Mellitus
Yes
No
|
71 (49.7)
72 (50.3)
|
42 (29.4)
35 (24.5)
|
29 (20.3)
37 (25.9)
|
0.242
|
Fever
Yes
No
|
90 (62.9)
53 (37.1)
|
59 (41.3)
18 (12.6)
|
31 (21.7)
35 (24.5)
|
0.001*
|
Cough
Yes
No
|
58 (40.6)
85 (59.4)
|
40 (28)
37 (25.9)
|
18 (12.6)
48 (33.6)
|
0.004*
|
Dyspnea
Yes
No
|
87 (60.8)
56 (39.2)
|
64 (44.8)
13 (9.1)
|
23 (16.1)
43 (30.1)
|
0.001*
|
Arthralgia
Yes
No
|
18 (12.6)
125 (87.4)
|
9 (6.3)
68 (47.6)
|
9 (6.3)
57 (39.9)
|
0.803
|
Ageusia
Yes
No
|
4 (2.8)
139 (97.2)
|
0 (0)
77 (53.8)
|
4 (2.8)
62 (43.4)
|
0.043*
|
Anosmia
Yes
No
|
6 (4.2)
137 (95.8)
|
2 (1.4)
75 (52.4)
|
4 (2.8)
62 (43.3)
|
0.415
|
TLC
Raised
Normal
Below Normal
|
120 (83.9)
21 (14.7)
2 (1.4)
|
62 (43.3)
15 (10.5)
0 (0)
|
58 (40.6)
6 (4.2)
2 (1.4)
|
0.075
|
De-Dimer
Raised
Normal
|
105 (73.4)
38 (26.6)
|
66 (46.2)
11 (7.7)
|
39 (27.3)
27 (18.9)
|
< 0.001*
|
Ferritin
Raised
Normal
|
100 (69.9)
43 (30.1)
|
56 (39.2)
21 (14.7)
|
44 (30.8)
22 (15.4)
|
0.468
|
LDH
Raised
Normal
|
127 (88.8)
16 (11.2)
|
76 (53.1)
1 (0.7)
|
51 (35.7)
15 (10.5)
|
< 0.001*
|
CRP
Raised
Normal
|
92 (64.3)
51 (35.7)
|
62 (43.3)
15 (10.5)
|
30 (21)
36 (25.2
|
< 0.001*
|
Pro-calcitonin
Raised
Normal
|
139 (97.2)
4 (2.8)
|
74 (51.7)
1 (0.7)
|
65 (45.5)
3 (2.1)
|
0.642
|
Outcome
Discharged
Deaths
|
110 (76.9)
33 (23.1)
|
48 (33.6)
29 (20.3)
|
62 (43.4)
4 (2.8)
|
< 0.001*
|
Categorical data represented as n and frequency in percentages (%), *significant p value (P < 0.05), aChi square test/Fischer’s Exact Test, TLC: Total Leukocyte Count, LDH: Lactate Dehydrogenase, CRP: C-Reactive Protein.
Association of ACE2 variants and the severity of the disease.
In current survey we found 29 ACE2 variants in the selected range of base pairs (position: 15592100 to 15592699) from which 7 variants have no change in the sequence while 22 ACE2 variants had altered genotypes. Among these 15 were the intronic variants, 4 were splice donor region variants, 2 were missense and remaining one was synonymous variant. Two of the variants were found to have insertion/deletion and one had insertion. These polymorphisms were analyzed using only a dominant model due to the low number of minor homozygotes (MAF < 0.1, Table: 2). Given these associations were used in further analyses and the linkage disequilibrium existent between gene polymorphisms (Figure: 1 and 2), we performed an exploratory selection of associations using a method to control the expected proportion of false positives (False Discovery Rate [FDR]) instead the Bonferroni correction, which is more stringent. Therefore, associations with FDR < 0.1 were used in haplotype analyses. First, haplotype blocks were generated by the algorithm of four-gamete rules observed at frequency > 0.01. For each block, we tested if the observed frequencies of haplotypes were deviated from expected under linkage equilibrium. Finally, we assessed the association between haplotypes and phenotypes by means of a permutation procedure. (Supplementary Table: 1)
Table 2
Minor allele frequency (MAF) and results of exact test to assess deviations from Hardy-Weinberg equilibrium (HWE).
ACE2
|
Major Allele
|
Minor Allele
|
Class
|
Consequences
|
MAF
|
HWE
|
rs768883316
|
T
|
A
|
SNP
|
Intronic Variant
|
0.055
|
2.6928E-6
|
rs560997634
|
A
|
C
|
SNP
|
Intronic Variant
|
0.006
|
1.00
|
rs201159862
|
T
|
A
|
SNP
|
Intronic Variant
|
0.024
|
0.0206
|
rs751170930
|
C
|
G
|
Insertion
|
Splice donor region variant
|
0.012
|
0.0206
|
rs1569241829
|
C
|
G
|
SNP
|
Splice donor 5th base variant
|
0.012
|
0.0206
|
rs2285666
|
T
|
G
|
SNP
|
Splice donor region variant
|
0.497
|
8.2762E-15
|
rs756737634
|
T
|
G
|
Indel
|
Splice donor region variant
|
0.018
|
0.0206
|
rs146991645
|
T
|
G
|
SNP
|
Synonymous variant
|
0.018
|
0.0206
|
rs1601703288
|
T
|
A
|
SNP
|
Missense variant
|
0.018
|
1.00
|
rs1391451327
|
A
|
T
|
SNP
|
Missense variant
|
0.055
|
3.5006E-5
|
rs1927830489
|
G
|
T
|
SNP
|
Intronic Variant
|
0.006
|
1.00
|
rs1927831624
|
T
|
C
|
SNP
|
Intronic Variant
|
0.006
|
1.00
|
rs764947941
|
A
|
T
|
SNP
|
Intronic Variant
|
0.012
|
0.0206
|
rs752242172
|
G
|
T
|
SNP
|
Intronic Variant
|
0.006
|
1.00
|
rs73195521
|
G
|
T
|
SNP
|
Intronic Variant
|
0.006
|
0.0206
|
rs781378335
|
T
|
C
|
SNP
|
Intronic Variant
|
0.018
|
0.0206
|
rs756597390
|
A
|
C
|
SNP
|
Intronic Variant
|
0.012
|
0.0206
|
rs780478736
|
G
|
A
|
SNP
|
Intronic Variant
|
0.345
|
2.8697E-14
|
rs148006212
|
T
|
G
|
SNP
|
Intronic Variant
|
0.012
|
0.0206
|
rs768583671
|
A
|
C
|
Indel
|
Intronic variant
|
0.006
|
1.00
|
Most prevalent variant seen was rs2285666 for which 49.2% CC, 45.2% TT, 4.8%, CT heterozygocity and 0.8% AA genotypic carrier were found in COVID-19 positive patients. The second frequent SNP noted was rs1391451327 having the 93.5% AA, 5.7% TT and 0.8% GG genotypic carriers followed by rs781378335 with the genotypic frequency of 97.6% TT and 2.4% CC. Though the SNPs have insignificant association with the severity of the disease but the changes were seen in the more severe and critical patients admitted in the ICU. So these variants may have increased susceptibility to acquire the severe or critical COVID-19 infection. Few of the genotypic variants of ACE2 were also associated with the mild and moderate cases. Table: 3 However the variants with multiple genotypes were also not found to be associated with the severity of COVID-19 in the analysis of the dominant model. Table: 4.
Table 3
Genetic association study of ACE2 variants with severity of COVID-19
ACE2 Single nucleotide variant
|
All, n = 124
|
Severity
|
P
|
OR (CI 95%)
|
FDRb
|
Serious
n = 68
|
Non-Serious
n = 56
|
rs180878567
Reference (AA)
Altered (TT)
Altered (GG)
|
121 (0.976)
2 (0.016)
1 (0.008)
|
68 (1.00)
0 (0)
0 (0)
|
53 (0.946)
2 (0.036)
1 (0.018)
|
0.999
|
NA
|
1.998
|
rs751227277
Reference (GG)
Altered (TT)
|
123 (0.992)
1 (0.008)
|
67 (0.985)
1 (0.015)
|
56 (1.00)
0 (0)
|
1.000
|
NA
|
1.571
|
rs1391451327
Reference (AA)
Altered (GG)
Altered (TT)
|
116 (0.935)
1 (0.008)
7 (0.057)
|
61 (0.897)
1 (0.015)
6 (0.088)
|
55 (0.982)
0 (0)
1 (0.018)
|
0.096
|
0.538 (0.260–1.116)
|
2.112
|
rs1469916451
Reference (TT)
Altered (CC)
|
122 (0.984)
2 (0.016)
|
67 (0.985)
1 (0.015)
|
55 (0.982)
1 (0.018)
|
0.890
|
0.906 (0.224–3.664)
|
2.797
|
rs1601703288
Reference (TT)
Altered (AA)
|
121 (0.976)
3 (0.024)
|
67 (0.985)
1 (0.015)
|
54 (0.964)
2 (0.036)
|
0.463
|
0.739 (0.329–1.659)
|
5.093
|
rs201159862
Reference (TT)
Altered (AA)
|
121 (0.976)
3 (0.024)
|
67 (0.985)
1 (0.015)
|
54 (0.964)
2 (0.036)
|
0.463
|
0.739 (0.329–1.659)
|
3.395
|
rs751170930
Reference (CC)
Altered (GG)
|
123 (0.992)
1 (0.008)
|
67 (0.985)
1 (0.015)
|
56 (1.00)
0 (0)
|
1.000
|
NA
|
1.466
|
rs756737634
Reference (TT)
Altered (GG)
|
122 (0.984)
2 (0.016)
|
67 (0.985)
1 (0.015)
|
55 (0.982)
1 (0.018)
|
0.890
|
0.821 (0.50-13.427)
|
2.447
|
rs1927831624
Reference (TT)
Altered (CC)
|
123 (0.992)
1 (0.008)
|
67 (0.985)
1 (0.015)
|
56 (1.00)
0 (0)
|
1.000
|
NA
|
1.375
|
rs764947941
Reference (AA)
Altered (TT)
|
123 (0.992)
1 (0.008)
|
67 (0.985)
1 (0.015)
|
56 (1.00)
0 (0)
|
1.000
|
NA
|
1.294
|
rs73195521
Reference (TT)
Altered (GG)
|
123 (0.992)
1 (0.008)
|
67 (0.985)
1 (0.015)
|
56 (1.00)
0 (0)
|
1.000
|
NA
|
1.222
|
rs752242172
Reference (GG)
Altered (TT)
|
122 (0.984)
2 (0.016)
|
67 (0.985)
1 (0.015)
|
55 (0.982)
1 (0.018)
|
1.000
|
NA
|
1.157
|
rs781378335
Reference (TT)
Altered (CC)
|
121 (0.976)
3 (0.024)
|
68 (1.00)
0 (0)
|
53 (0.946)
3 (0.054)
|
0.999
|
NA
|
1.831
|
rs756597390
Reference (AA)
Altered (CC)
Altered (TT)
|
122 (0.984)
1 (0.008)
1 (0.008)
|
67 (0.985)
0 (0)
1 (0.015)
|
55 (0.982)
1 (0.018)
0 (0)
|
0.622
|
0.686 (0.154–3.062)
|
2.736
|
rs148006212
Reference (TT)
Altered (GG)
|
123 (0.992)
1 (0.008)
|
67 (0.985)
1 (0.015)
|
56 (1.00)
0 (0)
|
1.000
|
NA
|
1.100
|
rs780478736
Reference (GG)
Altered (AA)
|
121 (0.976)
3 (0.024)
|
67 (0.985)
1 (0.015)
|
54 (0.964)
2 (0.036)
|
0.463
|
0.635 (0.189–2.136)
|
2.546
|
rs768583671
Reference (AA)
Altered (CC)
Altered (GG)
Altered (TT)
|
121 (0.976)
1 (0.008)
1 (0.008)
1 (0.008)
|
65 (0.955)
1 (0.015)
1 (0.015)
1 (0.015)
|
56 (1.00)
0 (0)
0 (0)
0 (0)
|
0.999
|
NA
|
1.1.690
|
rs2285666
Reference (CC)
Altered (AA)
Altered (CT)
Altered (TT)
|
61 (0.492)
1 (0.008)
6 (0.048)
56 (0.452)
|
32 (0.48)
1 (0.015)
2 (0.029)
33 (0.485)
|
29 (0.51)
0 (0)
4 (0.07)
23 (0.42)
|
0.966
|
0.993 (0.716–1.377)
|
2.125
|
rs1569241829
Reference (CC)
Altered (GG)
|
123 (0.992)
1 (0.008)
|
67 (0.985)
1 (0.015)
|
56 (1.00)
0 (0)
|
1.000
|
NA
|
1.047
|
rs768883316
Reference (TT)
Altered (CC)
Altered (AA)
|
117 (0.944)
2 (0.016)
5 (0.04)
|
64 (0.942)
0 (0)
4 (0.058)
|
53 (0.946)
2 (0.036)
1 (0.018)
|
0.655
|
1.140 (0.642–2.023)
|
2.401
|
rs560997634
Reference (AA)
Altered (CC)
|
122 (0.984)
2 (0.016)
|
67 (0.985)
1 (0.015)
|
55 (0.982)
1 (0018)
|
0.890
|
1.218 (0.074–19.92)
|
2.175
|
rs1927830489
Reference (GG)
Altered (TT)
|
123 (0.992)
1 (0.008)
|
68 (1.00)
0 (0)
|
55 (0.0982)
1 (0.018)
|
1.000
|
NA
|
1.00
|
bBenjamini-Hockberg method. CI, confidence Interval; FDR, false discovery rate; NA, does not apply; OR, odds ratio.
Table 4
Dominant model analyses for ACE2 genetic variants with multiple genotypes
ACE2 Single nucleotide variant
|
Genotype
|
Severity
|
P
|
OR (CI 95%)
|
FDRb
|
Serious
n = 68
|
Non-Serious
n = 56
|
rs1391451327
|
AA
GG + TT
|
61 (0.897)
7 (0.103)
|
55 (0.982)
1 (0.018)
|
0.090
|
0.158 (0.19–1.329)
|
0.54
|
rs2285666
|
CC
AA + TT + CT
|
32 (0.47)
36 (0.53)
|
29 (0.517)
27 (0.483)
|
0.600
|
0.828 (0.408–1.680)
|
1.80
|
rs768883316
|
TT
CC + AA
|
64 0.941)
4 (0.059)
|
53 (0.946)
3 (0.054)
|
0.900
|
0.906 (0.194–4.227)
|
1.35
|
rs180878567
|
AA
TT + GG
|
68 (1.00)
0 (0)
|
53 (0.946)
3 (0.054)
|
0.999
|
NA
|
1.19
|
rs756597390
|
AA
CC + TT
|
67 (0.985)
1 (0.015)
|
55 (0.982)
1 (0.018)
|
0.890
|
1.218 (0.074–19.92)
|
1.78
|
rs768583671
|
AA
CC + GG + TT
|
65 (0.955)
3 (0.045)
|
56 (1.00)
0 (0)
|
0.999
|
NA
|
0.999
|
bBenjamini-Hockberg method. CI, confidence Interval; FDR, false discovery rate; NA, does not apply; OR, odds ratio.
Furthermore, we also sought out to find the association of all ACE2 variants with the different ethnic groups, age groups, comorbids and gender found in current study. Among all 22 ACE2 variants none of them showed any statistical significance after applying the regression model with the ethnicity while only rs2285666 had significant statistical link with the gender (p value 0.034) and rs768883316 had significant statistical association with the age groups (p value 0.026). Table: 5 and 6.
Table 5
Genotype distribution in COVID-19 severity groups disaggregated by gender.
ACE2 Single nucleotide variant
|
Age Group
|
Severity
|
Genotype frequency
|
P
|
OR (CI 95%)
|
CC
|
AA
|
TT
|
rs768883316
|
< =50 Years (n = 29)
> 50 Years (n = 95)
|
Serious (n = 15)
Non serious (n = 14)
Serious (n = 53)
Non serious (n = 42)
|
0 (0)
0 (0)
0 (0)
2 (0.048)
|
4 (0.266)
0 (0)
0 (0)
1 (0.024)
|
11 (0.734)
14 (1.00)
53 (1.00)
39 (0.928)
|
0.026
|
1.953 (1.085–3.514)
|
CI, confidence Interval; NA, does not apply; OR, odds ratio.
Table 6
Genotype distribution in COVID-19 severity groups disaggregated by age groups.
ACE2 Single nucleotide variant
|
Gender
|
Severity
|
Genotype frequency
|
P
|
OR (CI 95%)
|
CC
|
AA
|
CT
|
TT
|
rs2285666
|
Male (n = 69)
Female (n = 55)
|
Serious (n = 38)
Non serious (n = 31)
Serious (n = 30)
Non serious (n = 25)
|
21 (0.553)
19 (0.613)
11 (0.367)
10 (0.4)
|
0 (0)
0 (0)
1 (0.034)
0 (0)
|
0 (0)
1 (0.032)
2 (0.066)
3 (0.12)
|
17 (0.447)
11 (0.355)
16 (0.533)
12 (0.48)
|
0.034
|
1.438 (1.028–2.011)
|
CI, confidence Interval; FDR, false discovery rate; NA, does not apply; OR, odds ratio.
Haplotypes ATC of three polymorphisms (rs560997634, rs201159862 and rs751170930) commonly found in 120 (69.77%) patients including the 54; 43.54%, serious and 66; 53.22% non-serious and TTTGTAGTTAGTA haplotype consisting of 13 polymorphisms (rs756737634, rs146991645, rs1601703288, rs1927830489, rs1927831624, rs764947941, rs752242172, rs73195521, rs781378335, rs756597390, rs780478736, rs148006212, rs768583671) in 112 (90.32%) with serious 48; 38.7%, non-serious 64; 51.61% had significance statistical association with the severity having p = value 0.029 and 0.001 respectively. Table: 7.
Table 7
Frequency of ACE2 Haplotypes distribution in COVID-19 patients
Haplotype
|
Frequency
|
Severity
|
OR (95%: CI)
|
p-value
|
ATC
|
120 (69.77%)
|
Non-Serious (54; 43.54%)
Serious (66; 53.22%)
|
3.27 (1.10–9.70)
|
0.029*
|
TTTGTAGTTAGTA
|
112 (90.3%)
|
Non-Serious (48; 38.70%)
Serious (64; 51.61%)
|
5.44 (2.12–13.93)
|
< 0.001*
|
OR, odds ratio; CI, confidence interval; *P < 0.05 was considered statistically significant