Eman R. Youness .Sherien M. El-Daly .Hanaa Reyad Abdallah .Hala T. El-Bassyouni .Hisham Megahed .Azzah A. Khedr .Marwa Elhady .Walaa Alsharany Abuelhamd

Down syndrome, Turner syndrome, and Klinefelter syndrome are the most common chromosomal disorders worldwide. Down syndrome is caused by an extra copy of the 21st chromosome and has an incidence of 1 in 800 to 1000 live births. Turner syndrome results from X-chromosome monosomy and has an incidence of 1 in 2000 of live-born female infants. Klinefelter syndrome is caused by an extra X chromosome in male infants and has an incidence of 1 in 600 live-born male infants. All of these syndromes have greater liabilities for metabolic and cardiovascular comorbidities [ 1]. A complementary health care system is required for early detection of comorbidities among these children to improve their quality of life [ 2– 5]. Several non-traditional modifiable risk factors have been identified including homocysteinemia [ 6]. Homocysteine is a sulfhydryl-containing amino acid produced as an intermediate product during the metabolism of methionine and cysteine. Homocysteine is considered as a key element of the methylation cycle [ 7]. Homocysteinemia is caused by either a genetic disorder or other pathogenic disorders, including chromosomal and environmental factors [ 8].

There is emerging evidence linking elevated circulating homocysteine level with dyslipidemia and cardiovascular morbidities including atherosclerosis, ischemic heart disease, thromboembolism, and cerebrovascular accident. Homocysteinemia is considered as an independent risk factor for cardiovascular mortality [ 9, 10]. Elevated homocysteine induces vascular endothelium injury, smooth muscle cell proliferation and inflammation leading to the formation of atheromatous plaques, which can result in ischemic insults. Additionally, homocysteine decreases the serum level of high-density lipoprotein (HDL) by suppressing apo-A lipoprotein synthesis and by increasing HDL clearance. Furthermore, homocysteine increases low-density lipoprotein (LDL), cholesterol peroxidation and thrombosis activation [ 11]. However, data regarding the relationship between circulating homocysteine level and dyslipidemia are limited, especially in children with chromosomal disorders.

We conducted this cross-sectional case–control study to explore the relation between serum homocysteine level, lipid profile and body mass index in children with non-mosaic numerical chromosomal disorders including Klinefelter syndrome, Down syndrome, and Turner syndrome for early detection of cardiovascular co-morbidities.

We included 60 children with documented diagnosis of non-mosaic numerical chromosomal disorders (aneuploidy); 18 children with Klinefelter syndrome (XXY), 22 as Down syndrome (trisomy 21) and 20 as Turner syndrome (monosomy X). Thirty-seven healthy normal children (body mass index between 5 and 85th percentiles) were included as the control group. The patients were recruited from the Clinical Genetic Department, National Research Center, Egypt. Healthy controls were selected from Al-Zahraa hospital, Al-Azhar University, Egypt. Both groups were matched for age. Written consent for publication of the case details together with imaging or videos has been obtained from participants (or their parent or legal guardian in the case of children under 16). The study protocol was approved by the Ethics Committee of Faculty of Medicine for Girls, Al-Azhar University (RHDIRP2018122001-Study Approval Number 2019/12323).

All included children were subjected to a detailed history taking with special emphasis on dietetic history, physical activity, and lifestyle. Complete general and systematic examination and anthropometric measurements including weight and height were done using standardized equipment. Body weight (kg) was assessed while the child was wearing light clothes, height (cm) was measured while the child was bare foot with knees stretched [ 12]. All parameters were recorded to the nearest 0.1 value. All measurements were plotted on age- and sex-specific growth charts [ 13].

Body mass index (BMI) was calculated as body weight in kilograms divided by square of height in meters. Obesity was defined as BMI ≥ 95th percentile, whereas overweight was defined as BMI ≥ 85th but less that 95th percentile. Children were considered of normal BMI if their BMI ranged between the 5th and 85th percentiles for age- and sex-specific percentiles [ 14]. Children who had any acute medical illness, hepatic, or renal impairment or those who received drugs affecting lipid profile as corticosteroids were excluded.

Peripheral blood samples were collected from both groups and were subjected to conventional G- banding technique on blood lymphocytes [ 15]. Blood samples were collected after a 12-hour overnight fasting and were centrifuged to collect serum; then stored at ＜ 80 °C until analyzed. An Olympus AU400 automatic analyzer (Olympus Corporation, Tokyo, Japan) was used to measure serum total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG) and fasting blood glucose (FBG); fasting blood insulin (FBI) was measured with commercial kits (Roche Diagnostics, Indianapolis, IN, USA). Low-density lipoprotein-cholesterol (LDL-C) was calculated using Friedewald equation: LDL-C = TC – HDL-C–(TG/5) [ 16, 17].

Homocysteine serum level was determined using reversed phase high-performance liquid chromatography according to the method described by Melnyk et al. [ 16]. Homocysteine was separated using high-performance liquid chromatography, Agilent 1100 series with a reversed-phase C18 column (3 μm bead size; 3.9 × 150 mm). The mobile phase formed of 40 mmol/L sodium phosphate monobasic, monohydrate: 8 mmol/L heptane sulfonic acid and 18% (v/v) methanol adjusted to pH 3.1 which was filtered through a 0.45-μm membrane filter. The isocratic elution had done by flow rate of 1.0 mL/min at 40 °C. The detector wavelength was at 260 nm.

Data analysis was performed using the statistical package for social sciences (SPSS, USA). Quantitative data were expressed as mean ± standard deviation. Differences between groups were analyzed using independent student t test, ANOVA test and post hoc analysis. Pearson correlation coefficient was used to assess correlations between investigated parameters. Regression analysis was also done.P values ＜ 0.05 were considered significant.

The results are elucidated in Fig.1. All patients were subjected to cytogenetic analysis. Chromosomes were arranged in karyograms (Fig.2 a–c). All samples had nonmosaic aneuploidy in all cells as follows: 18 Klinefelter syndrome (XXY), 22 Down syndrome (trisomy 21) and 20 Turner syndrome (monosomy X). Serum homocysteine levels were significantly higher in children with chromosomal disorders than in healthy controls. Furthermore, BMI, total cholesterol and LDL level were significantly higher in children with chromosomal disorders (Tables 1 and 2). Serum homocysteine level had a significant positive correlation with low-density lipoprotein in all groups of children with chromosomal disorders, whereas homocysteine level had a significant positive correlation with total cholesterol level only in children with Down syndrome. Homocysteine level had a significant positive correlation with BMI in children with Down and Turner syndromes (Table 3). Despite of the significant positive correlations between homocysteine serum levels and body mass index in children with chromosomal disorders, there was no significant difference in homocysteine level between children with and without obesity. Binary logistic regression analysis demonstrated a significant association between homocysteinemia and LDL level in children with chromosomal disorders (Table 4).

Table 1 Clinical data and lipid profile of the studied groups

Table 2 Correlation between lipid profile and homocysteine among the studied groups

Table 3 Comparison between chromosomal disorders children with and without obesity as regarding homocysteine level

Table 4 Logistic regression analysis for the association of low-density lipoprotein (LDL) and BMI in children with chromosomal disorders

Subjects with chromosomal disorders are at higher risk for cardiometabolic comorbidities that develop earlier than in the general population. Homocysteinemia has emerged as an independent risk factor for cardiovascular diseases. Reports showed that about 40% of patients with cerebrovascular disorders have homocysteinemia. There are controversial reports regarding the relationship between homocysteine and lipid profile under several normal and pathological conditions [ 18]. However, there are no sufficient data exploring the prevalence of homocysteinemia in children with chromosomal disorders and its relation to dyslipidemia. Controversy exists in the identification of the cutoff level of homocysteinemia in children and adolescents that varied according to age and ethnicity between 8.3 and 13.75 nmol/L. In the present study, we relied on a cutoff level ＞ 95th percentile for age and sex to identify homocysteinemia [ 19, 20].

Our study demonstrated higher homocysteine serum level in children with Klinefelter, Turner and Down syndromes than in healthy children with positive correlation between homocysteine level and BMI. These findings could be explained by overweight and obesity among children with chromosomal disorders especially those with Down syndrome. These findings are in accordance with Kumar et al. [ 21], who reported that none of normal weight children had homocysteinemia while 37.5% of overweight children and 36.5% of obese children had homocysteinemia. However, homocysteinemia in obese children with chromosomal disorders exceeded that observed in healthy children.

Further analysis of our results demonstrated that homocysteine level was significantly higher in normal weight children with chromosomal disorders compared with normal weight healthy children suggesting the presence of other causes for homocysteinemia rather than increased BMI. This result indicates that numerical chromosomal aberrations play a role in the development of homocysteinemia.

Schulze et al. [ 22] suggested that homocysteinemia in children with numerical chromosomal aberrations could be attributed to genetic polymorphisms, physical activity patterns, adiposity, and nutritional deficiency of folic acid and vitamin B12. In children with Turner syndrome, previous reports demonstrated that homocysteinemia may be related to female sex hormones deficiency. Estrogen defi-ciency caused by monosomy X chromosome may represent a possible cause of homocysteinemia in children with Turner syndrome [ 23]. Homocysteinemia in children with Klinefelter syndrome may be related to increased muscle mass. Furthermore, previous evidence showed increased homocysteine level after treatment with testosterone in patients with Klinefelter syndrome [ 24].

Our results demonstrated higher levels of total cholesterol and LDL in children with homocysteinemia. Regression analysis revealed a significant association between homocysteinemia and LDL level. These findings are in accordance with previous studies that have shown a strong association between homocysteinemia and dyslipidemia. This could be attributed to the effect of homocysteine, which promotes the formation and secretion of cholesterol by the hepatocytes and may contribute to the direct correlation between cholesterol and homocysteine level [ 25]. Experimental studies demonstrated that homocysteine thiolactone triggers the aggregation of LDL leading to the formation of foam cells in cultured human macrophages [ 26].

Dong et al., [ 27] reported a significant positive correlation between homocysteine and LDL level in patients with hypothyroidism, which is commonly associated with both Down syndrome and Turner syndrome. Furthermore, reduction of homocysteine level was associated with decreased LDL.

Our findings were in agreement with those of Yakub et al. [ 28] who reported that homocysteinemia was correlated with hypertension, dyslipidemia, and obesity. Experimental studies have demonstrated that hypomethylation due to homocysteinemia may be attributed to visceral fat accumulation. Furthermore, homocysteinemia may alter the activity of some inhibitory enzymes which are involved in HDL metabolism [ 29]. On the other hand, previous studies reported an association between homocysteinemia and the metabolic syndrome. Additionally, it was reported that homocysteinemia has a thrombogenic and atherogenic effect that is attributed to premature atherosclerosis leading to several cardiometabolic events including stroke and ischemic heart disease [ 30].

In conclusion, our findings suggested a strong association between homocysteinemia and dyslipidemia in children with non-mosaic numerical chromosomal disorders who are known to have high risks of cardiovascular morbidities. Furthermore, BMI was positively correlated with homocysteinemia in children with numerical chromosomal aberrations but not in children with normal karyotypes.

Acknowledgements Authors thank El- Azhar university and National Research Centre for their help and collaboration.

Author contributions Conceptualization: ER, HT and HM, Data curation: ME, SM, AA, HR and WA, formal analysis: ME and HR, Investigation: AA and HR, Writing–review & editing: ER, ME and HT.

Funding No financial benefits have been received or will be received from any party related directly or indirectly to the subject of this article.Data availability All data generated or analyzed during this study are included in this published article (and its supplementary information files).

Declarations

Ethical approval Written consent for publication of the case details together with imaging or videos have been obtained from participants (or their parent or legal guardian in the case of children under 16.

Conflict of interest No financial or non-financial benefits have been received or will be received from any party related directly or indirectly to the subject of this article.