Association of selected genetic variants in CBS and MTHFR genes in a cohort of children with homocystinuria in Sri Lanka

Background Homocystinuria is an inherited, inborn error of homocysteine metabolism, which leads to the abnormal accumulation of homocysteine and its metabolites in blood and urine, resulting in various complications. Variants in the cystathionine β-synthase (CBS) and methylenetetrahydrofolate reductase (MTHFR) genes interrupt the formation of the corresponding enzymes and prevent homocysteine from being metabolised; hence, the homocysteine levels in plasma increase than the optimum levels. Materials and methods In the current study, eight clinically confirmed children with homocystinuria were detected to study the chosen variants in the CBS gene (c.833 T>C and c.19del) and in the MTHFR gene (c.665 C>T, c.1286 A>C) using SNaPshot mini-sequencing and direct sequencing. Results After screening eight patients, none had the c.833T>C, but four patients were in the homozygous state for the c.19del variant in the CBS gene. Furthermore, seven were heterozygous for c.1286A>C, while one patient was heterozygous for c.665C>T in the MTHFR gene. Conclusion According to the results, c.19del is common in the studied cohort of Sri Lankan children, while c.833T>C is absent, whereas c.1286A>C was more frequent than c.665C>T. To our knowledge, the current study was the first report to discuss the genetic impact of homocystinuria in Sri Lanka; further comprehensive studies are necessary with a larger sample size to establish the association of these variants with the disease in Sri Lanka, which can be beneficial in enhanced patient care and for prospective studies.


Background
Homocystinuria is an inborn metabolism error and an autosomal recessive inherited disorder in the transsulfuration or methylation pathway, which leads to high plasma/ urinary levels of homocysteine and methionine and low level of cystathionine and cysteine. A genetic defect in one of the enzymes (CBS-cystathionine β-synthase, MTHFR-methylenetetrahydrofolate reductase, MTR-5-methyltetrahydrofolate-homocysteine methyltransferase, MTRR -5-methyltetrahydrofolate-homocysteine methyltransferase reductase) in homocysteine metabolism may lead to metabolic disruption. In addition, nutritional deficiency of one or more of the cofactors (vitamins B2, B6, B12, and folate) in the homocysteine metabolism is also causing metabolic disruption. As a result, homocysteine levels are increased (hyperhomocysteinemia) [1], and it can be confirmed by measuring plasma total homocysteine levels in the blood. Samarasinghe et al. Journal of Genetic Engineering and Biotechnology (2022) 20:164 The most common form of homocystinuria is caused by a deficiency in the production of cystathionine β-synthase (CBS) enzyme (OMIM no. 236200). This enzyme catalyses the first committed step of transsulfuration by conjugating homocysteine and serine to form cystathionine and then subsequently converting into cysteine and -ketoglutarate [2]. The human CBS gene contains 17 exons and is located on chromosome 21q22.3 [3], encoding 552 amino acid length CBS enzyme, which assembles into a homo-tetrameric protein with 63 kDa subunits [4]. Variants in the CBS gene interrupt the CBS enzyme production, preventing homocysteine from being further metabolised. Thus, homocysteine levels increase in blood, and excess is excreted in the urine. According to the Human Gene Mutation Database, in 2022, nearly 200 different genetic variants are reported in the CBS gene [5].
Furthermore, the variants in the MTHFR gene could interfere with the methylenetetrahydrofolate reductase enzyme (MTHFR) production (OMIM no. 236250), disrupting the conversion of folic acid into 5-methyltetrahydrofolate, which in turn is used as the methyl donor in the remethylation pathway when converting homocysteine into methionine, leading to the interruption of methionine formation from homocysteine. However, homocysteine forms methionine in an uninterrupted remethylation pathway by adding a methyl group from 5-methyltetrahydrofolate, which is formed when folic acid is converted into 5-methyltetrahydrofolate by the MTHFR enzyme. The human MTHFR gene contains 12 exons, and it is located on chromosome 1p36.3. The gene encodes MTHFR protein, a homodimer consisting of an N-terminal catalytic domain in each subunit, which binds NADPH (nicotinamide adenine dinucleotide phosphate) and FAD (flavin adenine dinucleotide) and C-terminal regulatory domains. The catalytic domain of the protein carries out the entire enzyme reaction [6].
Severe hyperhomocysteinemia has a worldwide incidence of 1:344,000. The most common symptoms associated with this condition are ocular abnormalities such as ectopia lentis (dislocation of the eye lens), myopia and glaucoma, and skeletal abnormalities with marfanoid features such as long limbs, high-arched palate, knock knees, and sunken or funnel chest. Infants who develop homocystinuria may fail in growth and fail to gain weight at the expected rate leading to developmental delays. Furthermore, severe complications include myocardial infarction, neurodegenerative diseases, pregnancy complications [7], mental retardation, osteoporosis, and vascular complications (thromboembolism) are shown in patients that cause morbidity and mortality in untreated patients with higher homocysteine levels [2].
According to the Human Gene Mutation Database, more than 200 pathogenic mutations in the CBS gene and more than 40 in the MTHFR gene have been identified worldwide related to homocystinuria. Yet, there are only a few reports from Asian populations [5]. However, several studies have been conducted in Sri Lanka to study the allelic variants of these genes about diseases such as cardiovascular diseases; the knowledge regarding homocystinuria is very low. Thus, it is important to find out the variations of the genes associated with the disease in the Sri Lankan population, which can be used to develop novel therapeutics and enhance patient care.

Methods
The current study was designed to detect the selected variants in CBS and MTHFR genes associated with homocystinuria in a cohort of children in Sri Lanka. Two variants from each gene, c.833 T>C/exon 8 (rs5742905) and c.19del/exon 1 (rs748695461) in the CBS gene and c.665C>T/exon 5 (rs1801133) and c.1286A>C/exon 8 (rs1801131) in the MTHFR gene, were selected based on the literature as they were common in other populations. The current study tested eight clinically and biochemically confirmed children with homocystinuria.

Patient recruitment and sample processing
Ethical approval for the study was obtained from the Ethics Review Committee of the Faculty of Medicine, University of Colombo, Sri Lanka (EC-19-092), prior to sampling. Furthermore, additional ethical approval (LRH/DA/05/2019) was granted by the Ethics Committee of the Lady Ridgeway Hospital for Children (LRH), Colombo, Sri Lanka, as patient recruitment was conducted at the hospital.
Sample collection was carried out on eight clinically and biochemically confirmed children diagnosed between 5 and 10 years of age with homocystinuria. They are currently being followed up at their respective clinics at LRH. Genomic DNA was extracted using the QIAamp DNA Blood Mini Kit (cat. no. 51104, QIAGEN, Hilden, Germany), followed by polymerase chain reaction (PCR) for each selected variant. Sociodemographic and clinical data were obtained from the patients by filling a data collection sheet, medical records based on patient guardian interviews and patient records, and pathology reports. As per the hospital records, homocysteine and methionine levels of the patient samples were detected using high-performance liquid chromatography, and the vitamin B12 levels of patient samples were detected using gas chromatography-mass spectrometry.

Results
Results of the SNP mini-sequencing were analysed by using Gene Marker software V2.6.4, while sequencing results were analysed using Mutation Surveyor ® v4.0.9 by aligning with the Human CBS (GenBank accession number-NG_008938) and MTHFR (GenBank accession number-NG_013351.1) reference sequences at the National Centre for Biotechnology Information (NCBI). ClinVar was used to classify the variants based on the clinical significance (https:// www. ncbi. nlm. nih. gov/ clinv ar/). Protein structure prediction for the identified variants was built using SWISS-MODEL (https:// swiss model. expasy. org/) and used P42898 (MTHFR_HUMAN) and P35520 (CBS_HUMAN) for comparison. TM align software (https:// zhang lab. ccmb. med. umich. edu/ TM-align/) was used for protein structure comparison and for constructing superimposed images. Table 1 summarises the status of four variants in CBS and MTHFR genes with the patients' general information, clinicopathological characteristics, and biochemical parameters of eight patients studied in the current research work.

c.833T>C in CBS
According to the results obtained via the GeneMarker software, all the subjected samples have one peak at the expected position of the SNP, which indicated the presence of only one nucleotide, wild-type threonine. The electropherograms of all the samples indicated the presence of homozygous wild-type condition for c.833T>C (Fig. 1a). This missense variant occurred due to the substitution of thymine by cytosine at 833rd position in exon 8 of the CBS gene, altering isoleucine to threonine at the 278th position in the protein (p.Ile278Thr). According to ClinVar, this variant is classified as a pathogenic variant. However, the result was further checked and confirmed for the zygosity by carrying out the Sanger sequencing for one of the representative samples.

c.1286A>C in MTHFR
According to the results, seven samples showed two peaks at the expected position of the SNP, indicating the presence of adenine and cytosine nucleotides (Fig. 1b).
Only one peak was observed in one sample, indicating the nucleotide adenine. As a result, it was concluded that seven samples had the heterozygous condition, while one had the homozygous wild-type condition for the SNP c.1286A>C. In this missense variant, adenine is substituted by cytosine at the 1286th position in exon 8, altering glutamate to alanine at the 429th position (p.Glu429Ala) in the MTHFR enzyme. This genetic change is classified as "likely pathogenic. " Direct sequencing further confirmed these results using one representative heterozygous sample.

Results of sequencing c.665C>T in MTHFR
Direct sequencing was carried out for all the samples to check the SNP c.665C>T. Then, the resultant sequence was aligned with the reference sequence of the MTHFR gene using Mutation Surveyor ® V4.0.9. According to the results, except for one sample, all the other samples did not show overlapped peaks at the expected position, which confirmed the homozygous wild-type condition for the SNP c.665C>T. In this missense variant, a cytosine at the 655th position of exon 5 of MTHFR is substituted by thymine; altering alanine to valine in the enzyme at the 222nd position (p.Ala222Val) is classified as a pathogenic variant with clinical interpretation as drug responsive. The resultant sample with the heterozygous condition is illustrated in Fig. 2.

c.19del in the CBS
Direct sequencing was carried out in all samples. Then, the resultant sequences were aligned with the reference sequence of the CBS gene using Mutation Surveyor ®  Fig. 3.

Discussion
This study was mainly focused on detecting the selected variants of CBS and MTHFR genes and their association with homocystinuria. A cohort of children who are clinically confirmed to have homocystinuria were the study participants being followed up by a consultant chemical pathologist at the Lady Ridgeway Hospital for Children, Colombo.
In the current study, eight individuals were screened for the c.833T>C variant in CBS, and among these individuals, none of them showed the variant, and all those individuals were homozygous wild type. Hence, the assumption can be made that there is a likelihood that this mutation is not common in Sri Lankan population. When considering the world context, c.833T>C (I278T) was found in patients with homocystinuria of different ethnic backgrounds with a prevalence of 1:344,000 [8] and was reported to be associated with pyridoxine responsiveness [9]. The presence of the CBS c.833T>C (p.I278T) mutation was tested in 500 newborns in the Danish population and appeared to be having heterozygous state in 1.4%, corresponding to the prevalence of predicted homozygosity of 1:20,400 [10]. In addition, after screening 1133 newborns in Norway, a heterozygous state of c.833T>C was detected with a frequency rate of 0.62%, while the predicted frequency of homozygosity was 1:104,000 [11]. According to the study conducted among Czech and Slovak Republics in 2001, it was found that 5 out of 1284 screened newborns are heterozygous for c.833T>C, with a frequency rate of 0.389%, while predicting the frequency of homozygosity was 1:264,000 [12]. It was detected heterozygote frequencies of 1.5% for c.833T>C after screening 200 unrelated healthy adults in Germany and predicted homozygote frequency of 1:17,800 [13], while in the Netherlands, it was 0.4% of heterozygosity frequency and 1:250,000 of predicted homozygote frequency [14]. A total of 304,086 newborns were screened in Kuwait between January 2015 and December 2020, and only six newborn children were identified with homocystinuria with a prevalence of 1:50,000, and no c.833T>C mutation was detected [15]. The highest prevalence was reported in Qatar at 1:1800 as per a study done on 126 Qatari patients between 2016 and 2017 by Al-Dewik et al. [16]. According to Shawky et al., the prevalence of homocystinuria in the Northeast region of Cairo, Egypt, is 1.96% [17]. Studies among the Asian population related to homocystinuria are less, and few studies were conducted among Korean, Chinese, Pakistani, and Indian nationalities. Mutation analysis was done on six individuals in the Korean population [18], and research conducted among individuals of a Chinese family [19] did not identify the c.833T>C mutation. In 2011, c.833T>C and c.1006C>T in the CBS gene were detected in Hong Kong patients with homocystinuria. However, those are the only reported mutations in the CBS gene in the Chinese population [20] and were also the first time that c.833T>C was reported in the Asian population. Kaur R. et al. conducted a study on 16 northern Indian children in 2020 who did not identify with the c.833T>C mutation [21]. In 2021, Wasim M. et al. conducted a mutation analysis on 429 patients with an intellectual disability from northern areas of Punjab, Pakistan. Only nine patients were identified with homocystinuria, and none had c.833T>C mutation [22]. A study on three patients in a Pakistani consanguineous family with symptoms in 2021 confirmed homocystinuria was not detected with c.833T>C mutation [23]. Although the prevalence of c.833T>C is likely less among the Asian population, but it is the most prevalent mutation worldwide in the CBS gene related to homocystinuria, thus, it is important to find out the presence and prevalence of this mutation in Sri Lanka.
Among the 485 SNPs recorded in the NCBI SNP database, at least 40 variants in the MTHFR gene have been identified in people with homocystinuria. Out of them, c.665C>T and c.1286A>C are the two major variants in the MTHFR gene responsible for increasing homocysteine levels in the blood [24]. According to previous studies, the presence of c.665C>T variant in either a homozygous or a compound heterozygous form with c.1286A>C in the MTHFR gene leads to the severe reduction of the MTHFR enzyme levels, causing an elevated level of homocysteine in the blood [25]. In the current study, none of the patients shows the coexistence of c.665C>T and c.1286A>C in the MTHFR gene. For c.665C>T, the enzyme activity of heterozygous and homozygous mutant individuals is 67% and 25%, respectively, compared to the wild-type ones. However, c.1286A>C, the enzyme activity of heterozygous and homozygous mutant individuals, is 83% and 61%, respectively, which of the wild-type subjects [25]. A study done on the geographic and ethnic distribution of the c.665C>T variant in newborns showed that homozygous mutant type (TT genotype) was prevalent in Northern China (20%), Southern Italy (26%), and Mexico (32%) where the frequency of the T allele in newborns of Asian origin was low [26]. Alternatively, a study done in Sri Lanka by Amarakoon et al., to check the association of the variants with hyperhomocysteinemia (high homocysteine levels), showed that 89.9% of individuals were homozygous wild type, while there were individuals with heterozygous and homozygous mutants for c.665C>T with the prevalence rate of 3.8% and 6.3%, respectively. In addition, 41.8% of individuals were homozygous wild type, and the rest were heterozygous and homozygous mutants for the c.1286A>C in the MTHFR gene with rates of 32.7% and 25.5%, respectively [27]. According to another study done in Sri Lanka by Perera et al., MTHFR variants for ischemic heart disease (IHD) showed prevalence rates of 72.8%, 24.7%, and 2.5% for CC (homozygous wild type), CT (heterozygous mutant), and TT (homozygous mutant) genotypes in c.665C>T, respectively, showing CC genotype was the predominant genotype among IHD patients and controls. In contrast, the TT genotype was detected only among very few IHD patients. Prevalence rates of c.1286A>C were 50% for the AA (homozygous wild type), 37.3% for the AC (heterozygous), and 12.7% for CC (homozygous mutant) genotypes for the whole study population, where the AA genotype was the predominant genotype among controls, and AC genotype was the predominant genotype among IHD patients [28]. According to the analysis of the current study on MTHFR gene variants, seven samples showed heterozygous mutant status for c.1286A>C, while one patient had the heterozygous mutant for c.665C>T condition. Hence, the frequency of c.1286A>C was 87.5%, and c.665C>T was 12.5%, indicating that the c.1286A>C SNP was much more likely to be prominent than the SNP c.665C>T among patients with homocystinuria in the studied cohort. Thus, the presence of c.665C>T variant in the MTHFR gene dissociates the active dimer into monomers, losing the MTHFR enzyme's FAD-binding capacity, which leads to hypomethylation and increases the homocysteine levels [29]. According to the direct sequencing results, the presence of c.19del variant in the CBS gene was shown in four samples with the homozygous state, while the other four samples did not show this variant. Thus, the frequency for this variant is 50% in the studied cohort, showing a significant appearance of the variant in patients with homocystinuria in Sri Lanka. According to the ExAC browser, c.19del was previously reported in a heterozygous state in two South Asian alleles, which was not found to be related to homocystinuria. In a study done by Kaur R. et al., in 2020, out of sixteen children, four patients were confirmed to have c.19del in North Indian children with homocystinuria [21]. However, these frequencies will not reflect the actual frequency distributions of these variants in Sri Lanka, as the number of samples studied in this preliminary research is insufficient to conclude a result. Thus, a randomly selected larger cohort needs to be studied in the future to determine the prevalence of the above variants in children with homocystinuria in Sri Lanka.
According to the overall result shown in Table 1, indicating the fact that all the children have symptoms more or less similar associated with homocystinuria, where four children (S003, S004, S005, and S006) had comparatively severe symptoms than the other patients. Patients S003 and S004 are children of a non-consanguineous marriage, who were presented with severe clinical symptoms such as ectopia lentis, myopia, glaucoma, visual impairment, learning difficulties, marfonoid features, and personality changes, having elevated plasma total homocysteine and methionine levels, and were detected for having c.19del variant in homozygous condition. Since this is a recessive inherited disorder, where both patients in this family are homozygous for the condition, their parents should be heterozygous, asymptomatic, and carriers. According to the data obtained from the patients, they have a suspected family history of this disease where their paternal grandfather had similar eye problems. This paternal pedigree likely shares this variant over a few generations as heterozygous carriers. Since parents are not related, analysing the pedigrees of families (paternal and maternal), if possible, is required to conclude the inheritance and origin of this mutation.
Another two siblings, S005 and S006, were diagnosed with homocystinuria based on the detection of elevated blood homocysteine and severe clinical symptoms such as ectopia lentis, myopia, visual impairment, learning difficulties, and marfonoid features. They are children of consanguineous parents and had no evidence of family history for this condition. S005 and S006 are siblings who are homozygous for c.19del in the CBS gene.
Due to the recessive inheritance of the disease and parents being related, the genetic variant is expected to be shared and formed over a few generations. As these two children have two different SNP conditions for the gene, MTHFR pedigree analysis is necessary to confirm the inheritance. However, patient S005 has the heterozygous condition for SNP c.665C>T, and S006 has the heterozygous condition for SNP c.1286A>C. According to the gender difference, different effects can occur in homocysteine levels in normal and heterozygous states. Females are found to have lower homocysteine levels than males. This difference is mainly due to the protective effect of oestrogen and other physiological factors in women. The severity of symptoms and plasma homocysteine levels are more prominent in the homozygous TT genotype in c.665C>T [30]. However, no such difference was seen between the heterozygous A>C patients and heterozygous C>T when comparing the symptoms in the studied cohort. Hence, the effect of the variant might be almost the same according to the results obtained. However, the sample size is comparatively small to conclude the difference between symptoms and genetic conditions.
All samples with c.19del in the CBS gene were from the southern province in Sri Lanka. Except for one, all samples from the southern province shared heterozygosity for the c.1286A>C variant in the MTHFR gene. Only one has the heterozygous condition for c.665C>T, while others have the homozygous wild type in the southern province. They might be likely to share a common genetic basis. So, studying these families' pedigrees would significantly impact the origin and inheritance of these mutations.
The primary purpose of treatment for homocystinuria is to reduce plasma homocysteine levels, normalise methionine levels, and alleviate a patient's clinical symptoms. Administration of betaine of 2-9 g/d in children to facilitate an alternate pathway for methylation reactions, then hydroxycobalamin of 1-2 g/d (vit. B12) as a cofactor for methionine synthase, folate of 400 mg/d to increase residual MTHFR function and pyridoxine (vit. B6) to maximise the transsulfuration pathway as a cofactor for CBS, and riboflavin (vit. B2) for the flavin component of the MTHFR enzyme are some treatment options for homocystinuria. There is only limited success with methionine supplements and MTHFR enzyme replacements [31]. According to murine studies, it was identified that the prophylactic treatment with high-dose folate and betaine in pregnant and lactating women benefits the high-risk families [32]. As per Table 2, the onset homocysteine levels of the patients have been considerably higher than the optimum range (4.6-8.1 μmol/L). However, after the treatment of betaine and pyridoxine, On the contrary, most of the patients have noticeably higher methionine levels than the normal range (6-60 μmol/L) initially, and in some patients, their methionine levels have further increased after treatment of betaine which was given to reduce the homocysteine levels and to aid the methylation reaction. Vitamin B12 levels of a normal person vary from 140-650 pmol/L. Furthermore, all the patients had vitamin B12 within the above range when this study was conducted. Vitamin B12 acts as a cofactor for the enzyme MTHFR, and if this vitamin is deficient, it affects the activity of the enzyme interfering with the metabolic pathway and causing a high amount of homocysteine in blood or urine. Vitamin B12 levels of these patients were within the normal range at the time of the study, indicating that the disease could not be due to the vitamin B12 deficiency but to the mutations of genes that aid the methylation reaction.

Conclusion
This is the first study to deliver a genetic characterisation of homocystinuria in Sri Lanka to the best of our knowledge. When comparing the results of this study with the other studies done worldwide, the results merely gave a pattern among the Sri Lankan population. As mentioned, the variant conditions did not follow a constant distribution but can be varied depending on different sociodemographic factors. Hence, further studies are needed with a larger cohort to confirm these conditions associated with the disease. Furthermore, it is important to study the correlation of both of these variants for the occurrence of the disease and its symptoms. Further studies are needed with a larger cohort of patients to assess the effect of both of these variants.