Seventy five karyotype-proven DS patients having age <15 years and clinical features like developmental delay, intellectual disability, Simian crease, low set ears, prominent forehead, macroglossia, and 30 age and sex-matched healthy controls were studied after appropriate informed consent (Table 1 and Table ST1). MTHFR genotype frequencies identified are as presented below.
Table 1 Total 105 participants were enrolled in the study out of which 30 are sex and age matched controls and rest 75 have DS, which were further divided into different subgroups on the basis of comorbidities
S.No
|
Types of participants enrolled in the study
|
N (Number of participants)
|
1.
|
Controls
|
30
|
2.
|
DS patients without CHD and hypothyroidism
|
15
|
3.
|
DS patients with CHD
|
27
|
4.
|
DS patients with hypothyroidism
|
16
|
5.
|
DS patients with both CHD and hypothyroidism
|
17
|
Allele and genotype frequency of C677T:
DS patients had 5.8-fold higher genotype frequency (Table 2, Fig. 3, SF1,) of MTHFR677CT (29.3%) than controls (6.6%). In Fig. SF1b, the MTHFR 677CT frequency in male DS patients (37.3%) is notably higher than that of female DS patients (12.5%), with a p-value of 0.028. Conversely, DS patients with hypothyroidism (56.3%) exhibit a significant overexpression of MTHFR 677CT when compared to DS patients with CHD (29.6%, p-value 0.084), DS patients with both hypothyroidism and CHD (17.6%, p-value 0.021), DS patients without both hypothyroidism and CHD (13.3%, p-value0.013), and controls (6.6% p-value 0.000) (Table 3 and Fig SF1c). MTHFR C677T expression is eighteen times higher in DS patients with hypothyroidism than in controls. As a result, MTHFR CC677CT is a genetic risk factor for hypothyroidism in DS patients.
Allele and genotype frequency of A1298C:
The genotype frequency of MTHFR 1298AC is 6.4 times (58.6%) higher in DS patients as compared to controls (30%) (Table 2, Figure SF2). Similarly, homozygous variation MTHFR1298CC in DS patients (20%) is also much higher as compared to controls (0%) having p-value = 0.000. Unlike MTHFR 677CT, MTHFR A1298C has no significant difference in allele frequency in between DS males and females. Similarly, in DS patients with different comorbidities we found that MTHFR 1298AC is significantly high in all DS patients {DS with CHD (p=0.002), DS with CHD and hypothyroidism(P=0.000), DS without CHD and hypothyroidism (P =0.019)} except DS with hypothyroidism (p-value=0.272) as compared to controls. DS with both hypothyroidism and CHD has significantly high expression of MTHFR 1298AC as compared to DS without both CHD and hypothyroidism (p-value=0.033), and DS with hypothyroidism (p-value=0.035). But, there is no significant difference in the expression of MTHFR 1298AC in between DS patients with CHD and DS with both CHD and hypothyroidism (P=0.653). Thus, MTHFR 1298AC genotype is associated with CHD in DS.
Except for DS without both CHD and hypothyroidism (p-value=0.155), the prevalence of genotype MTHFR 1298CC is significantly greater in all DS patients {DS with CHD (p-value=0.002), hypothyroidism (p-value=0.000), and CHD and hypothyroidism both (p-value=0.000) as compared to controls (Table 2, Figure SF2d). Similarly, MTHFR 1298CC expression is considerably higher in DS patients with both hypothyroidism and CHD than in DS patients with hypothyroidism alone (p-value=0.037) or CHD (p-value= 0.016). Thus, MTHFR 1298CC is associated with both hypothyroidism and congenital heart defect (DS) in patients.
Plasma concentrations of Homocysteine and Cysteine
Between DS patients and controls, there is no discernible change in homocysteine levels (p-value=0.99). Similarly, there was no difference (p-value=0.410) in the homocysteine levels between DS male and female patients. However, compared to controls (p-value= 0.046) and DS patients without both CHD and hypothyroidism (p-value= 0.030), the plasma of DS patients with both conditions has greater amounts of homocysteine [Fig.4 (a), ST. 2]. By multinomial logistic regression it has been found that DS with both CHD and hypothyroidism has 1.104 times, and 1.203 times more levels of homocysteine as compared to controls and DS without CHD and hypothyroidism (ST. 2). Thus, Homocysteine levels are higher in DS patients with both hypothyroidism and CHD.
The plasma levels of cysteine in DS patients are higher than in controls (p-value<0.0001), however there is no variation in cysteine levels between male and female DS patients. In comparison to controls, DS patients with CHD (p-value<0.0001), DS with hypothyroidism (p-value =0.006) and DS with both CHD and hypothyroidism (p-value=0.002) had greater levels of cysteine (Fig. 4b, ST2). Similarly, DS patients with CHD has higher levels of cysteine (p-value=0.005) than DS patients without either condition. Multinomial regression has shown that DS with CHD, DS with hypothyroidism and DS with both CHD and hypothyroidism has 1.031, 1.020, 1.024 times higher levels of cysteine as compared to controls and DS with CHD has 1.021 times higher levels of cysteine as compared DS without CHD and hypothyroidism. So, higher levels of cysteine are associated with CHD.
Relation between the levels of Homocysteine with MTHFR C677T, MTHFR A1298C and both MTHFR C677T and MTHFR A1298C
Since the MTHFR enzyme is necessary for the folate pathway and synthesis of homocysteine. Therefore, we examined the levels of homocysteine in DS patients to determine the effect of the MTHFR C677T and A1298C polymorphisms on its concentrations. In Table 5 DS patients were divided into different subgroups on the presence and absence of MTHFR variant. We discovered that DS patients with both variants there is 1.217 (Table ST3) times higher levels of homocysteine as compared to DS with only C677T variant. Similarly, there is 1.802, and 1.582 times higher levels of homocysteine in DS patients with both variants and only A1298C variant as compared to DS patients without any variant. So, the presence of compound variant and A1298C increases the plasma concentration of homocysteine in DS patients.
Table 2: Frequency of MTHFR gene variants C677T, A1298C and both C677T and A1298C in DS patients and sex and age matched healthy controls.
S.NO
|
Variants
|
Number of DS patients
|
Number of Controls
|
1.
|
No mutation
|
7
|
19
|
2.
|
MTHFR C677T
|
9
|
2
|
3.
|
MTHFR A1298C
|
46
|
9
|
4.
|
Both MTHFR C677T and MTHFR A1298C
|
13
|
0
|
Testing in mothers of DS babies
We also performed Sanger Sequencing in four mothers of DS patients the latter has homozygous MTHFR 1298CC variant and found that 2 mothers carried MTHFR 1298CC variant while the rest two had heterozygous MTHFR 1298AC.