The Relationship Between HIF1α and Clock Gene Expression in Patients with Obstructive Sleep Apnea

Figures & data

Table 1 Primer Sequence Details of the Analyzed Gene (F: Forward Primer, R: Reverse Primer)

Gene	Sequence
Human_Gapdh _F	TCGGAGTCAACGGATTTGGT
Human_Gapdh _R	TTCCCGTTCTCAGCCTTGAC
Human_Timeless _F	AGGAGCGGAGTGCAGATTTT
Human_Timeless _R	TCGAGAATGCCTGTTGCCTC
Human_Clock _F	AAGTTAGGGCTGAAAGACGACGA
Human_Clock _R	GAACTCCGAGAAGAGGCAGAAG
Human_Bmal1 _F	AGGATGGCTGTTCAGCACATGA
Human_Bmal1 _R	CAAAAATCCATCTGCTGCCCTG
Human_Per1_F	CTGAGGAGGCCGAGAGGAAAGAA
Human_Per1_R	AGGAGGAGGAGGCACATTTAC
Human_ Per 2_F	AAGCAGGTGAAAGCCAATGAAGA
Human_ Per 2 _R	CCACCGCAAACATATCGGCATT
Human_ Per 3_F	TGCAGGGCATCCTCCCTTTGA
Human_ Per 3_R	TCCGGCTCCAGGGATTCACAA
Human_ Cry1_F	CTGCGTCTACATCCTGGACC
Human_ Cry1_R	GAAGCAAAAATCGCCACCTGT
Human_ Cry2_F	TCCCTAGCATGTCAGCCCGTT
Human_ Cry2_R	AGGATTTGAGGCACTGTTCCGA
Human_ Ck1δ_F	CTCACAGAATAGCATTCCTTTCG
Human_ Ck1δ_R	TGCACGACAGACTGAAGACC
Human_ Rorα_F	TCGCAGCGATGAAAGCTCAAAT
Human_ Rorα_R	GTGGCATTGCTTTGCTGACT
Human_ NR1D1_F	ACAGCTGACACCACCCAGATC
Human_ NR1D1_R	CATGGGCATAGGTGAAGATTTCT
Human_ NPAS2_F	ATCCTGCAGGCCAATATCCG
Human_ NPAS2_R	CTGCTGCAGGAACATCTGGA
Human_ HIF1α_F	GCCGCTGGAGACACAATCAT
Human_ HIF1α_R	GAAGTGGCTTTGGCGTTTCA
Human_ HIF1β_F	GACTACTGCCAACCCCGAAA
Human_ HIF1β_R	CTCATCATCCGACCTGGCAA

Table 2 General Characteristics of the Study Participants

	Control (AHI<15 Events/h, n=22)	OSA (AHI≥15 Events/h, n=21)	P value	no NH (T90<0, n=22)	NH (T90>0, n=21)	P value
Age (years)	39.0±7.8	38.9±8.3	0.969	37.5±6.9	40.4±8.8	0.238
BMI (kg/m2)	25.1±3.1	28.1±2.4	0.001	25.0±3.1	28.2±2.3	<0.001
Current smokers(n)	3	5	0.457	4	4	1.000
Hypertension (n)	5	9	0.203	4	10	0.055
AHI (events/h)	1.6 (0.6, 6.9)	42.4 (21.1, 63.1)	<0.001	1.6 (0.6, 8.6)	40.4 (18.2, 63.2)	<0.001
ODI (events/h)	1.4(0.3,4.9)	38.6(19.1,60.1)	<0.001	1.4(0.3,4.9)	38.6(17.0,60.1)	<0.001
Mean SpO2(%)	97.3 (95.6, 98.1)	95.8 (94.0, 97.4)	0.029	97.7 (95.8, 98.4)	95.1 (94.0, 96.7)	<0.001
LSpO2(%)	91.6±4.0	81.7±6.7	<0.001	92.4±2.6	80.8±5.8	<0.001
T90(%)	0(0, 0)	0.6(0.2,1.6)	<0.001	0(0, 0)	0.6(0.3, 1.6)	<0.001
ALT(U/L)	25(19, 35.5)	46(27.5, 56)	0.017	25(19, 33.2)	44(27.5, 62.5)	0.011
Cr (umol/L)	80.1±8.2	75.4±11.5	0.128	80.3±7.8	75.2±11.7	0.099
Glucose (mmol/L)	4.9±0.6	5.2±0.6	0.082	4.8±0.6	5.3±0.6	0.008
HBA1C(%)	5.2(5.0, 5.5)	5.5(5.3, 5.6)	0.025	5.2(5.0, 5.5)	5.5(5.4, 5.6)	0.006
TG (mmol/L)	1.1(0.8, 1.8)	2.4(1.5, 4.2)	0.004	1.1(0.8, 1.8)	2.4(1.5, 4.2)	0.004
TC (mmol/L)	4.7±0.7	4.8±0.8	0.722	4.7±0.7	4.8±0.8	0.965
LDL (mmol/L)	3.2±0.7	2.9±0.8	0.357	3.2±0.7	2.9±0.8	0.200
HDL (mmol/L)	1.2±0.3	1.0±0.2	0.026	1.2±0.3	1.0±0.2	0.005
UA (umol/L)	391.6±74.8	425.4±93.5	0.197	410.1±95.8	406.0±74.8	0.874
hsCRP (mg/L)	0.8(0.6, 1.1)	2.0(0.8, 2.6)	0.002	0.9(0.6, 1.8)	1.2(0.8, 2.4)	0.194

Notes: Data are presented as mean values and SD, counts, or medians and interquartile ranges, as appropriate. Differences tested by t-test for each OSA subset against Controls.

Abbreviations: AHI, Apnea-hypopnea index; ALT, Alanine aminotransferase; BMI, Body mass index; Cr, Creatinine; HBA1C, hemoglobin A1C; HDL, high-density lipoprotein; hsCRP, high-sensitivity C-reactive protein; LDL, low-density lipoprotein; LSpO₂, Lowest pulse arterial oxygen saturation; T90 (%), percent of time spent with SpO₂ below 90%; TC, Total cholesterol; TG, Triglyceride; UA, Uric acid.

Figure 1 The percentage difference of morning-evening variation in clock gene expression for subjects with and without OSA. (A) expression of HIF1α increased by 23% in subjects with OSA when compared with those without OSA (P = 0.008) (B and C) Relative gene expression of Timeless (R = −0.223, P = 0.038) and Cry2 (R= −0.348, P = 0.012) in subjects with OSA decreased with increasing AHI. Data represent means ± SEM. **P < 0.01.

Figure 2 The percentage difference of morning-evening variation in clock gene expression for subjects with and without NH. (A-D) The percentage difference of Bmal1, Rorα, and HIF1α were up-regulated by 16% (P = 0.006), 14% (P = 0.027), and 25% (P = 0.005), respectively, in subjects with nocturnal hypoxemia. (E) The protein concentration of HIF1α was increased in patients with NH after one-night of hypoxia but did not reach a significant statistical difference. Data represent means ± SEM. * P < 0.05, ** P < 0.01.

Figure 3 Correlations between morning-evening variation of HIF-1α and circadian genes mRNA levels (Bmal1, Cry1, and CK1δ), PSG parameters (AHI, ODI LSpO₂, and Mean SpO₂). (A–C) Correlation between morning-evening variation for HIF1α and Bmal1, Cry1, and CK1δ mRNA level (R = 0.638, P < 0.001; R = 0.327, P = 0.032; R = 0.332, P = 0.030, respectively). (D–G) Correlation between morning-evening variation for HIF1α mRNA level and PSG parameters (AHI, ODI, LSpO₂, and Mean SpO₂) (R = 0.234, P = 0.131; R = 0.277, P = 0.072; R = −0.464, P = 0.009; R = −0.500, P = 0.003, respectively).