Molecular genetic characterization of cblC defects in 126 pedigrees and prenatal genetic diagnosis of pedigrees with combined methylmalonic aciduria and homocystinuria

Methylmalonic acidaemia or aciduria (MMA), which is the most common inborn error of organic acid metabolism [1], is inherited as an autosomal recessive disease caused by deficiency in methylmalonyl coenzyme A mutase (MCM) or intracellular cobalamin (cbl) metabolism [2]. Based on the patients’ biochemical features, MMA can be classified as isolated MMA or combined methylmalonic aciduria and homocystinuria, and the latter consists of five subtypes, including cblC, cblD, cblF, cblJ and cblX deficiencies [3, 4]. These defects result in methylcobalamin and adenosylcobalamin dysfunction, leading to the subsequent accumulation of methylmalonic acid and homocysteine in the blood and urine, which is a biochemical hallmark of this disorder [5, 6]. In China, the most commonly affected patients have combined methylmalonic acidaemia and homocystinuria, accounting for approximately 80% of all MMA cases. Among the subtypes, the cblC defect (MIM 277400) is the most common [7] and is caused by mutations in the MMACHC gene, which is located in chromosome region 1p34.1; the gene contains five exons and encodes a 282 amino acid protein [8].

An increasing number of cblC patients have benefited from an early diagnosis and better outcomes through expanded newborn screening, particularly gas chromatography-mass spectrometry (GC-MS) [9]. Despite the reduced mortality and morbidity in MMA children, their quality of life remains very low, and the families of these patients suffer a significant economic burden. The most effective measure to reduce the social and family pressure is to avoid the birth of children with MMA by prenatal diagnosis. Measuring the activity of methylmalonyl CoA mutase in amniotic fluid, amniotic fluid cells and chorionic cells and quantifying cbl metabolites in amniotic fluid cells have been employed in prenatal diagnosis [10, 11]. Combined with these biochemical analyses, genetic analyses are also currently used. Other studies report that prenatal diagnosis is performed through simple genetic diagnosis. Here, our study employs a simple genetic diagnosis.

This study was approved by the Scientific and Experimental Research Medical Ethics Committee of the First Affiliated Hospital of Zhengzhou University. All analysed samples were obtained after signed informed consent was provided.

MMA is the most prevalent organic aciduria involving intermediate metabolism due to enzymatic block. Based on the age at the presentation of the initial symptoms and diagnosis (from newborn to older than 20 years), patients are divided into the following two categories: early-onset and late-onset cblC deficiency. The latter is a rare disorder, and diagnosis is often delayed. Most reported patients with the cblC type present in the first year and typically in the neonatal period with lethargy, vomiting, hypotonia, respiratory distress and developmental delay, which is referred to as early-onset cblC [13, 14]. The other type is the late-onset cblC form, and patients with cblC exhibit ataxia, dementia, psychosis and other neurologic abnormalities generally after 4 years of age [15, 16].

Currently, newborns are screened by MS and GC-MS, and abnormalities in early-onset and late-onset children can be easily observed in urine and blood. Early diagnosis and treatment are beneficial in children with cblC defects, resulting in improved quality of life and reduced suffering from pain.

Treatment options like betaine, hydroxocobalamin, methylfolate, vitamin B6 and L-carnitine are applied to these patients clinically, the combined treatment can effectively improve the clinical manifestations in acute phase and alleviate biochemical abnormalities in hematuria. The median levels of serum homocysteine, and urine methylmalonic acid were significantly decreased (p < 0.01), from 97.3 μmol/L (ranged from 25.1 to 250 μmol/L) and 168.55 (ranged from 3.66 to 1032.82) before treatment to 43.8 μmol/L (ranged from 17 to 97.8 μmol/L) and 6.81 (ranged from 0 to 95.43) after treatment, respectively [17]. For those from a rural area they suffer more pain due to undertreatment, or late initiation of optimal treatment in China. Even with enough combined treatment, along with much lower levels of serum homocysteine and urine methylmalonic acid than before, their long-term outcomes are not encouraging, along with severe neurological sequelae in most patients.

To date, more than 90 mutations in this gene, including missense mutations, nonsense mutations, small indels, and splicing region mutations, have been reported in the literature [18]. Given the high allelic heterogeneity in MMA, the spectrum of variants differs in various populations worldwide. Thus, mutational analysis of MMA patients is crucial in different populations. Specifically, c.271dupA (21%), c.394C > T (21%) and c.609G > A (11%) are the most frequent variants in Europe based on a study conducted in 2014 [19]. Other studies have reported that the c.394C > T variant is the most common allele in Native American and Middle Eastern MMA patients [20, 21]. However, numerous children with the cblC type have been diagnosed as homozygous for the c.609G > A variant, and more importantly, these children were East Asians [12, 20]. Another Chinese study reported that 55.4% (51/92) of all variants were c.609G > A [22]. Similarly, in this study, c.609G > A accounted for 48.41% of the variants. Among the 32 homozygous variants, 29 variants were homozygous for c.609G > A. Thus, we further confirm that c.609G > A is a very common mutation in China. Following the most common c.609G > A variant is c.658_660delAAG, which accounted for 13.49% in this study and is also a very common mutation in Chinese patients [8].

Of the 31 different variants identified in this study, 2 variants were novel. One variant, i.e., the nonsense variant c.637G>T (p.Glu213Ter), results in a truncated protein at residue 213. The other variant, i.e., small deletion c.463_465delGGG (p.Gly155del), causes the deletion of a glycine at residue 155. The c.463G > C (Gly155Arg) and c.464G > A (Gly155Glu) variants have been previously reported to be pathogenic [23]. In addition, the highly conserved results of the PROVEAN and PolyPhen analyses of glycine 155 support the pathogenicity of the deletion. However, further research should be performed to verify our hypothesis.

Although early detection and treatment can improve the outcome in cblC patients, most probands die at a young age. Thus, preimplantation genetic diagnosis or screening (PGD/PGS) or prenatal diagnosis are the best currently available prevention methods. MMA can be diagnosed as early as the first trimester by chronic villus sampling, which is beneficial for families with probands.

However, undeniably, this study also has some limitations in terms of prenatal diagnosis. First, in families with deceased probands, we first detected the MMACHC gene variants in the parents. If both parents harboured a heterozygous variant of the MMACHC gene, we deduced that the proband genotype harboured compound heterozygous or homozygous variants. Direct testing of the probands was not performed. Second, traditional methods used for prenatal diagnosis include biochemical detections, such as measuring the activity of methylmalonyl CoA mutase in the amniotic fluid, amniotic fluid cells and chorionic cells and quantifying cbl metabolites in amniotic fluid cells. Our study exclusively performed a simple genetic diagnosis to these pedigrees with explicit homozygous or compound heterozygous mutations without traditional biochemical measurements. However, for those pedigrees with only one or no definite deleterious alleles, or some variants of unknown significance, we suggest them to other Hospital to perform prenatal diagnosis with biochemical testing, so here we do not collect them into our study, and we are unable to help them. Finally, pregnant women bearing MMA foetuses typically choose to terminate the pregnancy, which also represents a type of injury to the pregnant women. Overall, preimplantation genetic diagnosis can be useful for avoiding miscarriages or the induction of labour in early pregnancy and is a future direction in the development of MMA prenatal diagnosis.