MMA,which refers to a cohort of organic aciduria caused by deficiency in methylmalonyl coenzyme A mutase (MCM) or intracellular cobalamin (cbl) metabolism, is the most common form of organic aciduria in the mainland of China. Recently, thanks to newborn screening by MS-MS or GC-MS, an increasing number of MMA patients could get early diagnosis and effective treatment. However, the long-term outcomes are not encouraging, along with severe neurological and renal complications. Therefore it is very important to prevent the recurrence in families with MMA child birth history. This study reports prenatal molecular genetic diagnosis for 130 pedigrees with clear gene variants as early as the first trimester by chorionic villus sampling, which is the best currently available prevention method.
Among the 244 pedigrees, 168 (68.9%) of which were combined MMA, the remaining 76 (31.1%) were isolated MMA, showing that MMA combined with hyperhomocystinuria is the major biochemical phenotype, and their proportion approaches what have been reported in other literatures [18, 32]. Most of the patients who were clinically diagnosed other than identified via newborn screening present in the first year and typically in the neonatal period.
For the isolated MMA, severe metabolic crises were very common, which is rarely seen in cblC patiens. Neuropsychiatric damages like seizure and hypotonia are frequently found in these patients, which could also occur in cblC patients. other neural complications like feeding problems, delayed development and intellectual disability could be seen both in isolated and combined MMA. Renal insufficiency is frequently reported in mutase-deficient methylmalonic academia, and they are susceptible to metabolic strokes. While pulmonary arterial hypertensions and hemolytic uremic syndrome could occur in cblC patients. Less common features of combined MMA include eye problems and megaloblastic anemia, but rare in isolated patients.
One patient with mitochondrial DNA depletion sysdrome (MDS) caused by SUCLG1 gene variants had lower blood methylmalonic acid than others patients, but he got severe neurological damages. One pedigree with diseased proband was clinically diagnosed MMA with high methylmalonic acid in the urine, the parents both harboured the c.981-2A > TA heterozygous variant in LMBRD1 gene(CblF).
In a total of 244 families, only 20 children were discovered by newborn screening. The remaining were diagnosed based on the probands' clinical manifestations, and the results of MS-MS and (or) GC-MS. They all suffer much pain because of MMA and usually lost optimal treatment opportunities, until they were clearly diagnosed and got prompt treatment. However, the prognosis of those 20 patients detected by neonatal screening is very good, which indicated that neonatal screening is a key strategy of early diagnosis and effective treatment. Cobalamin and levocarnitine were all used to treat MMA. The therapy of isolated MMA relies on stopping protein intake, whereas this is contra-indicated in cblC patients, who have low methionine levels.
In our research, there are 244 probands who received sequencing analysis, and we found different pathogenic variants in 4 genes including MMACHC, MUT, LMBRD1 and SUCLG1, among which MMACHC and MUT gene variants are the most prevalent, verifying the complexity of genotypes in Chinese MMA patients. For 8 probands with negative gene variants and 6 probands with only one heterozygous gene variant, they were all detected by Next Generation Sequencing, which may be due to variations outside the scope of current techniques or caused by other genes which is outside the range of genes in our NGS inherited metabolic disorders panel..
Among the MMA patients with genetic diagnosis, 158 cases were MMACHC gene mutations, which confirmed that cblC type is the main type of MMA with homocysteinemia in China. Of the 76 isolated MMAs that received genetic analysis, 70 (92.1%) were MUT gene variants, confirming that the mutase deficiency caused by MUT gene deficiency is the main type of simple MMA in China. At the same time, MMA caused by 1 cases of LMBRD1, 1 case of SUCLG1 gene defect were found, suggesting that cblF type MMA is a rare type in China, which are different from other countries. Other genes defect like SUCLG1 and SUCLA2, which could cause mitochondrial DNA depletion sysdrome (MDS) accompanied by the phenotype of methylmalonic acid in urine and blood, should also come into our consideration in clinical practice.
To date, it has been reported more than 90 variants in MMACHC gene and 250 variants in MUT gene in the literature, respectively, including missense mutation, nonsense mutation, small insertions and deletions, splicing region mutation et al [33, 34]. In view of the high allelic heterogeneity in MMA, the spectrums of variants in MMACHC and MUT gene are different in various populations around the world, suggesting that mutation analysis of MMA patients is significant in China. Our research shows that c.609G > A (48.28%), c.658_660delAAG (13.17%) and c.567_568insT (7.84%) are the most frequent variants for cblC defect patients. And for MUT type patients, c.729_730insTT (16.78%), c.1106G > A (7.69%), c.914T > C (6.99%) and c.323G > A (6.29%) are more frequent than other variants.
It has been illustrated that for cblC type patients, c.271dupA were the most common allele in different European countries from their study [35, 36]. Other literature had indicated that c.394C > T variant is the most frequent variants in Native American and Middle Eastern MMA patients [36, 37]. For East Asians, a great number of cblC defect patients were homozygous in c.609G > A variant . Lots of Chinese study shows that c.609G > A was the most common mutation in Chinese cblC defect patients [38, 39].
Similarily, in our study, among 38 homozygous variants, 34 of them were homozygous for c.609G > A, which accounts for 48.28%. Therefore, we can further confirm that the c.609G > A variant is the most common mutation in Chinese population. It was followed by c.658_660delAAG and c.567_568insT variants, which accounted for 13.17% and 7.84%, respectively, and these two variants are very common mutation in Chinese patients.
For MUT type patients, considering the wide spectrum of variants, their distribution is heterogeneous from exon 1 to exon 13. Thus, mutation analysis is crucial in different populations. The c.278G > A and c.329A > G are the most frequent variants in Saudi Arabian patients based on a study conducted in 2014 . The c.322C > T mutation present in 41% of black patients  and 60% of Hispanic patients . The mutation c.731A > T was the first frequent mutation reported in Caucasian population , c.425G > T in Japanese patients , and c.1280G > A and c.1630_1631delGGinsTA are the frequent mutations in Asian patients . A Chinese research in 2012 shows that c.729_730insTT and c.1280G > A were the most frequent variants in the mut-type MMA patients . Another Chinese article revealed that c.323G > A, c.729_730insTT and c.1630_1631delGGinsTA were more frequent in their research in 2009 . Thus we could get a conclusion that maybe c.729_730insTT is a hotspot mutation in Chinese Patients .
In our patients, the c.729_730insTT variant, detected in 16.78% of the MUT cases, was the very common variant. The other frequently observed variants were c.1106G > A, c.914T > C and c.323G > A, with each variant accounting for 7.69%, 6.99%, 6.29%, respectively. This reflects that c.729_730insTT and c.323G > A are frequent variants in Chinese population.
A total of 34 variants were identified among our 158 cblC defect pedigrees, including 2 novel variants, namely the c.89G > A and c.415_416delinsTA variant, each of them are nonsense variant, yielding a truncated protein at residue 30 and 139, respectively, resulting in incomplete protein activity. The the softwares’ prediction are all deleterious, however, further research should be performed to confirm their pathogenecity..
In the 60 different variants we have identified among 70 MUT type patients, 41 of them were previously reported and 16 of these variants were novel, including one 5’UTR variant (c.-39-2A > G), 1 splicing mutations (c.1560 + 2T > C), 1 small deletion (c.1233_1235delCAT), 3 deletions of single nucleotide (c.1102delG, c.1645delC and c.1787delA), 3 insertions of single nucleotide (c.445_446insA, c.1107dupT and c.1581_1582insA ), 1 nonsense (c.7A > T) and 6 missense mutations (c.422C > A, c.652C > G, c.893T > A, c.1330A > T c.1673C > T, c.1678T > C), most of which have been found only once and are restricted to one single family.
Methylmalonyl-CoA mutase (MCM) is encoded by MUT gene encoding 750 amino acids, containing 2 important main functional domains:1)an N-terminal (β/α)8 barrel domain (residues 86–423) that accommodates the substrate and 2) a C-terminal (α/β)5 cofactor (adenosylcobalamin)-binding domain (residues 578–750). The two main domains are connected by a long linker region (residues 424–577) that could enclose the (β/α)8 barrel domain [9, 48].
In our study, 6 novel frameshift variants and one nonsense variants were detected, leading to introduction of a subsequent premature termination codon and production of truncated protein. The novel splicing variant is predicted to affect splicing, and the stability of mRNA, and 5’UTR variant would affect the level of translation  .One deletion of 3 nucleotides causes Isoleucine deletion at redisue 410, which could affect the binding of substrate. The remaining 9 novel missense variants were identified in 9 different patients. Seven of them are located in the N-terminal (β/α)8 barrel domain, while 2 others (c.1673C > T and c.1678T > C) are located in the linker domain. The former would influence the binding of substrate, and the latter may affect the binding of substrates, enzymes and cobalamin. The novel missense mutations, being likely disease-causing, in 9 different pedigrees all occurred in segments highly conserved across the tested species, suggesting that these residues are crucial for stability and function of MCM.
One patient had the c.826-2A > G variant in SUCLG1 gene in a homozygous state, which had been reported to be harmful . Another pedigree with diseased proband, the parents both harboured the c.981-2A > TA heterozygous variant in LMBRD1 gene. Some Database indicated that this variant is binign (Illumina Clinical Services Laboratory), another one literature showed that its’ pathogenicity is uncertain . However, the Mutationtaster predicted that it’s disease-causing, the specific function needs further experimental study.
For all the novel variants, Mutation taster, Polymorphism phenotyping (PolyPhen) and SIFT were calculated, and the results show that they are all disease causing. However, further research should be done to verify our speculation of the pathogenicity.
For all genes, sequence analysis is performed first, followed by deletion or duplication analysis if only one pathogenic or no variant has been detected. Here in our study real-time quantitative PCR (Q-PCR) technology was used to test deletions/duplications in the MMACHC/MUT genes. After Q-PCR, only 2 pedigrees have found exon 13 deletion in MUT gene . The exon 13 deletion, was confirmed by microarray-based comparative genomic hybridization .
According to the current station, early detection and treatment are critical strategies to improve the outcome of MMA patients, however, varying degrees of psychoneurotic sequel would lower the quality of their life. Thus preimplantation genetic diagnosis or screening (PGD/PGS) are the optimal effective prevention ways at present, which would be a prevailing trend to help those affected with MMA in the development of MMA prenatal diagnosis, although it costs a lot.