Evaluation of homocysteine levels in individuals having nonsyndromic cleft lip with or without palate.

CONTEXT: Nonsyndromic cleft lip with or without palate (NSCL ± P) is a genetic predisposition involving defects in shape and makeup of the lip and palate. Elevation of homocysteine (Hcy) levels is seen in medical complications such as developmental anomalies causing neural tube defects, congenital vascular diseases, neurodegenerative and psychiatric conditions. Evaluation of serum Hcy levels forms an important feature to look further into molecular aspects. AIMS: The aim of this study was to evaluate the Hcy levels in NSCL ± P cases by comparing with control cases having no orofacial deformities. SETTINGS AND
DESIGN: This study was performed with a biochemical assay in a research laboratory.
MATERIALS AND METHODS: A cross-sectional prevalence study was done to compare the concentrations of Hcy between 25 NSCL ± P patients and 15 healthy controls. Blood samples were collected from both the patients and controls and assessed for serum Hcy level using competent chemiluminescent immunoassay technique. STATISTICAL ANALYSIS USED: Student's t-test was used for statistical analysis.
RESULTS: The average Hcy concentration was 9.5 μmol/L in control group. There was an increase in Hcy concentration among the NSCL ± P cases with an average value of 18.4 μmol/L. The results were found to be statistically significant using Student's t-test.
CONCLUSIONS: The results of this study indicate that Hcy concentration has a significant elevation in NSCL ± P patients when compared with that of control cases.

INTRODUCTION

Nonsyndromic cleft lip with or without palate (NSCL ± P) is a genetic predisposition prevalent in the Indian population.[1] It involves defects in the shape and makeup of lip and palate characterized by closure defects of lip, alveolus and/or palate. There have been numerous experimental evidence suggesting the etiology for the disease which gives an approximate frequency of disease incidence in around 1/1000 infants with ethnic and geographic variations.[2] Orofacial clefts are one of the most challenging congenital malformations exhibiting varying phenotype in terms of location of cleft, presence or absence of cleft palate and incomplete fusion of orofacial complex.[3] NSCL ± P has a multifactorial etiology including abnormal homocysteine (Hcy) level and anomalous developmental end points which is not well understood.[4] The other etiological factors include heredity, consanguinity, maternal environment and demographic factors, and aspects such as intrauterine posture, drugs, vitamins, alcohol consumption, smoking, infections and diet.[5] Hence, complexity of the disease gives a scope for research in terms of generating experimental evidence regarding the genetic, environmental and other factors that play a role in causing orofacial defects. Fetal environment can be seen as one of the potential causes of this particular developmental anomaly. Pregnant mothers exposed to nicotine products either by smoking or by indirect ingestion were found to have orofacial malformations in their offspring.[6]

The risk of NSCL ± P is expected to be influenced by the patterns of single nucleotide polymorphisms (SNPs), and the resulting variations can have functional consequences ranging from severe to none. As an example, the C677T mutation is considered a risk factor of neural tube defects as it lowered the plasma level of folate.[7] Previous studies in NSCL ± P confirmed the role of SNPs in the form of methylene-tetra-hydrofolate reductase (MTHFR) C677T genotype in the mothers who conferred an increased risk of NSCL ± P in their offspring.[8] However, there are also reports emphasizing no significant difference in C677T and A1298C polymorphisms between cleft lip/palate (CL/P) patients, their mothers and controls.[13] These contradicting conclusions at the level of genetic and phenotypic expressions suggest a need for enough evidence to establish exact mechanisms that lead to these developmental anomalies.

One of the main suggested factors responsible for the formation of orofacial clefts is elevated Hcy levels resulting from the errors in Hcy metabolic pathway. Hcy is a sulfur-containing amino acid present in the body which does not take part in the formation of proteins.[9] It is formed as an intermediate in the methionine pathway.[10] Hcy, under normal conditions, is converted to either methionine by remethylation or to cystathionine through trans-sulfuration pathway [Figure 1].[11] The remethylation reaction catalyzed by the enzyme methionine synthase (MS) requires folate and vitamin B12 (or betaine in another type of reaction) as cofactors whereas the trans-sulfuration reaction is catalyzed by cystathionine-β-synthase (CBS) using pyridoxal-5’- phosphate (coenzyme form of vitamin B6) as a cofactor. S-adenosylmethionine allosterically inhibits MTHFR and also activates CBS, thus coordinating the two pathways.[11]

Figure 1

Overview of homocysteine metabolic pathway

MTHFR gene regulates the concentration of Hcy in blood by causing demethylation of 5-methyltetrahydrofolate to tetrahydrofolate and hence forming methionine from Hcy in the process.[12] Any error in the molecular composition of MTHFR gene sequence or gene regulatory mechanism might result in the accumulation of Hcy in blood which might have a role in the formation of orofacial clefts.

In addition to orofacial cleft formation, disturbed Hcy and folate metabolism is also implicated in many different congenital birth defects such as congenital heart disease, cleft lip, late pregnancy complications, different kinds of neurodegenerative and psychiatric diseases, osteoporosis and cancer.[13]

Incidence of NSCL ± P was reported more in males who had orofacial deformities even at relatively lower level of Hcy as compared with females, for whom higher levels of Hcy were required for the deformities to occur. In other words, the incidence of NSCL ± P was more prevalent in males than in females.[1]

This study was conducted with the aim of investigating serum Hcy as a risk factor for NSCL ± P. An ideal threshold level of Hcy needs to be established to determine the nutritional deficiencies related to orofacial clefting. Through this study, we confirm the association of elevated levels of Hcy with cleft lip and palate formation with results showing a significant difference in the mean values of Hcy concentration between controls and patients.

MATERIALS AND METHODS

The study participants were enrolled from the pediatric and general ward of the Hospital. A total of 40 participants were considered, of which 15 were grouped as controls and 25 were grouped as patients. Participants in the control group were the ones not affected with any kind of orofacial deformities. All the cases were evaluated for individual organ system before the surgical procedure as per the protocol. There were no associated syndromes or defects in patients as verified by both case record details, physical and other systemic diagnostic examinations. The systemic evaluation was done for cardiovascular system, central nervous system, respiratory system, Ear Nose and Throat and skin conditions. Condition/diseases where Hcy levels were elevated such as congenital vascular diseases, neurodegenerative and psychiatric conditions were excluded in both patient group and control group. This record was obtained from presurgical reports before cheiloplasty surgical procedure. The patient group comprised participants having NSCL ± P with varying degrees of penetrance and phenotypic expressions. The age of participants ranged between 6 and 20 years. Ethical clearance was obtained for the study from the Institutional Ethical Clearance Committee.

Blood samples were collected separately from both control and patient groups and assessed for the concentration of Hcy from serum using competent chemiluminescent immunoassay technology. This is a one-step immunoassay for quantitative determination of total L-Hcy in human serum or plasma. Bound or dimerized Hcy (oxidized form) is reduced by dithiothreitol to free Hcy, which is then converted to S-adenosylhomocysteine (SAH) by the action of the recombinant enzyme SAH hydrolase. The presence of SAH is measured as relative light units (RLUs). There is an inverse relationship between the amount of Hcy present in the patients’ sample and the amount of RLUs detected by the system.[14]

Statistical analysis

Student's t-test was used to analyze the values of Hcy concentrations obtained in blood samples of both control and patient groups.

RESULTS

Mean Hcy level in control group samples was 9.5 ± 1.99 μmol/L and 18.4 ± 6.76 μmol/L in patient group samples [Figure 2]. P value obtained by Student's t-test was 6.84 × 10-6, which is lesser than 0.05. This confirms the significance of the results obtained. Control group comprising individuals without CL/P had relatively moderate level of Hcy in their serum samples [Figure 3] whereas patient group with NSCL ± P had elevated level of Hcy [Figure 4].

Figure 2

Comparing homocysteine levels in control and patient groups

Figure 3

Homocysteine concentration of participants in control group

Figure 4

Homocysteine concentration of participants in patient group

DISCUSSION

The average Hcy level among healthy Indian population is reported to be 12.5 μmol/L. In this study, the average Hcy level was recorded as 9.5 ± 1.99 μmol/L (mean ± standard deviation) in control group and 18.4 ± 6.76 μmol/L in patient group. While the former had Hcy levels at relatively healthier range, the latter exhibited elevated levels of Hcy, indicating a possibility of the accumulation of Hcy in blood. Hence, it can be implied that there is a clear increase in the concentration of Hcy in cases having NSCL ± P.

Several intervention and case–control studies have proposed that maternal preconceptional use of multivitamins containing folic acid protects against NSCL ± P occurrence and recurrence.[1516] Folate is a one-carbon donor which is involved in the biosynthesis of purines, pyrimidines and Hcy remethylation, thereby producing methyl groups for methylation of DNA, proteins and lipids. Therefore, folate is important for the expression of several genes essential for cellular multiplication and differentiation during embryogenesis.[17] Imbalance in interconversion mechanism of Hcy and methionine could result in the accumulation of Hcy and lack of free methyl groups that are crucial in post-transcriptional and translational regulation of several genes.

Some have elevated Hcy levels caused by the deficiency of B vitamins and folate in their diets. High Hcy levels are also seen in people with kidney disease, low levels of thyroid hormones, psoriasis and with certain medications (such as antiepileptic drugs and methotrexate). It has been recognized that some people have a common genetic variant (called MTHFR) that impairs their ability to process folate. This defective gene leads to the elevated levels of Hcy in some people who inherit MTHFR variants from both parents.[1819]

The results can also be correlated to the investigations about the compromised supply of myo-inositol, zinc and excessive exposure to glucose which could have a role in the developmental pathogenesis of NSCL ± P. Zinc finger proteins are important in controlling genes in embryonic development. An inadequate zinc status has been postulated to be teratogenic in both animal and human studies. Besides that zinc is involved in the conversion of 5-methyl tetrahydrofolate into tetrahydrofolate by the zinc-dependent MS enzyme.[20212223] Both MS and MTHFR enzyme functions in tandem to regulate the amount of Hcy in blood. It was reported that zinc concentration in serum was low in the mothers of neural tube-affected offspring. It should be also noted that NSCL ± P and neural tube defects both arise from neural crest cells.[24] It has been shown that Hcy enters the fetus through amniotic fluid and induces apoptosis of the palatal mesenchyme that prevents fusion of the palate.[25] Hence, there is a close relationship between zinc concentration and accumulation of Hcy in the blood. Reduction of zinc and folate in the metabolic system can be directly correlated with the accumulation of Hcy in the blood which might act as an important biochemical feature of developmental anomalies in the orofacial region. However, no significant history of maternal folate and zinc status of the investigated cleft children was available for the study.

Determining maternal folate status forms an important parameter in Hcy metabolism since folate-deficient mothers develop a risk of CL/P occurrence or recurrence in their children.[717]

Therefore, biochemical determination of Hcy compound could act as an indicator to certain molecular mechanism in Hcy metabolic pathway, wherein genes such as MTHFR and MS could play a major role in the development of craniofacial anomalies and act as molecular markers of the disease incidence.

In control samples, there was elevated serum Hcy level in about 2–3 cases. However, it is not of significance since the rise in value was quite negligible, and ranges characterized for normal, abnormal and moderately elevated Hcy levels are very large.

CONCLUSION

The genetic basis of NSCL ± P is to be investigated with all possible molecular techniques in the larger Indian cohort. Our study concluded that total serum Hcy concentrations can be a marker for the cleft lip patients. Direct investigation of MTHFR gene polymorphism and Hcy analysis are further being carried out using Sanger sequencing to identify gene variants. Maternal and offspring zinc and folate concentration can also be evaluated as it can influence Hcy accumulation. These studies would establish a link between a group of possible candidate genes and serum concentrations of Hcy, zinc, folate, etc., in the Indian cohort.

Financial support and sponsorship

This study is financially supported by the VGST, Government of Karnataka, under the grant no: VGST/SMYSR/GRD-393/2014-15.

Conflicts of interest

There are no conflicts of interest.