Int J Clin Exp Pathol 2015;8(8):9337-9341 www.ijcep.com /ISSN:1936-2625/IJCEP0010308 Original Article Genetic analysis of four cases of methylmalonic aciduria and homocystinuria, cblC type# Jun Wang1, Erzhen Li1, Liwen Wang1, Zhilong Wang2, Shenghai Yang1, Qiao Zhou2, Qian Chen1 1Department of Neurology, Capital Institute of Pediatrics, Beijing, PR China; 2Clinical Laboratory of Zhongke, Beijing, PR China Received May 17, 2015; Accepted June 27, 2015; Epub August 1, 2015; Published August 15, 2015 Abstract: Methylmalonic aciduria and homocystinuria, cblC type, is the most common disorder of intracellular vita- min B12 (cobalamin, cbl) metabolism, which results in impaired biosynthesis of methylcobalamin and adenosylco- balamin. The gene MMACHC responsible for the cblC type had been identified, which enables molecular diagnos- tics. Here, we report four cblC type cases, which were identified by the typical manifestations, and a new approach of next-generation sequencing platform in pediatrics for genetic diseases, further confirmed by Sanger sequencing of the whole MMACHC gene. The article will replenish the mutational information of related genes to the cblC type, which makes for detecting of cblC disease through the newborn screening. Keywords: Methylmalonic aciduria and homocystinuria, MMACHC, mutation, vitamin B12 (cobalamin, Cb1) Introduction disease through expanded newborn screening, and increases the possibility to improve the out- Combined methylmalonic aciduria and homo- come in patients with cblC disease. To date, the cystinuria, cblC type (MIM# 277400), is autoso- most prevalent mutation was c271dupA which mal recessive mode of inheritance. It is claimed account for the mutant alleles characterized in to be the most common inborn error of intracel- a crowd of cblC patients from around the world lular cobalamin metabolism, caused by muta- [1, 5, 6]. Here we report four cases with five tions in the MMACHC gene located in chromo- mutations, including one novel change, and one some region 1p34.1 with five exons [1]. case without any mutation in MMACHC gene. Individuals with cblC deficiency do not synthe- The full identification of mutations in the size AdoCbl and MeCbl, cofactors for the meth- MMACHC gene is benefit for the detection of ylmalonyl-CoA mutase and methionine syn- cblC disease. thase enzymes, and display methylmalonic aciduria and homocystinuria. The patients with Case reports cblC disease display a wide spectrum of clinical manifestations including feeding difficulties, Case 1 failure to thrive, hematologic, neurologic, meta- bolic (acidosis), ophthalmologic and dermato- A 4-month-old girl was admitted to hospital for logic abnormalities. The cblC disease can light cognitive impairment and not be amused develop severe complications despite treat- and vertical head instability. The patient pre- ment [2, 3]. The pathophysiology of cblC is not sented aware consciousness, normal myody- fully understood, but three important factors namia but higher muscle tone by clinical exami- contribute to the disease-related complica- nation. Pregnancy history described that it is tions. They are increased homocysteine con- the first child and first production. The new born centrations, impaired methyl group metabo- has not fetal distress and asphyxia history. lism, and oxidative stress [3, 4]; Characterization Related biochemical studies revealed that of the variation in the MMACHC gene can facili- Lactic acid 5.4 mmol/L↑ (reference 4 mmol/L), tate the prenatal and early diagnosis of cblC blood ammonia 50 µmol/L (reference 20-60 Genetic analysis of four cases of methylmalonic aciduria and homocystinuria Table 1. 48 genes related to organic acid Case 3 metabolic disease MUT MMAA MMAB MCEE A 9-month-old girl was admitted to hospital. She can’t sit alone, pronounce summation tone MMACHC MMADHC LMBRD1 PCCA and has poor eyesight. Cranial MRI enhance- PCCB MTHFR MTRR CBS ment scanning was cerebral dysplasia. EEG MTR PAH PTS GCH1 showed hypsarrhythmia. Blood screening test QDPR PCBD1 GLDC GCSH showed Lactic acid 5.4 mmol/L↑. Plasma homo- MAT1A HAL IVD HGD cysteine was 174.22 µmol/L. Urine screening PRODH ALDH4A1 MCCC1 MCCC2 test showed. Methylmalonic aciduria (MMA HIBCH HMGCL AUH L2HGDH 5066 times increased), and after 4 days of DNAJC19 MVK BTD HLCS injection Cobamamide, MMA 526.5 times dec- D2HGDH L2HGDH GCDH BCAT2 reased. ETFA FAH TAT HPD Case 4 ETFB ETFDH HMGCL UGT1A1 A 9-month and 19-days-old boy presented sit- ting instability, intermittent lethargy, somno- μmol/L). Cranial MRI enhancement scanning lence, and vomited for 9 months. The EGG displays the result of hydrocephalus. EEG video showed that slow background activity showed atypical hypsarrhythmia. Plasma amino with DQ41. The patient presented aware con- acids analysis by tandem mass spectrometry sciousness, weak myodynamia and muscle (MS/MS) showed higher homocysteine, citrul- tone of four limbs by clinical examination. ine/arginine, propionyl-L-carnitine, C3; propio- Plasma amino acid test showed higher C3, C3/ nyl-L-carnitine/free carnitine, C3/CO; propionyl- C16, and homocysteine of 27.13 μmol/L. L-carnitine/acetyl-carnitine, C3/C2; and octa- Methylmalonic aciduria increased to 15601.8 noyl-canitine/kawi-acyl-carnitine, C8/C12. Be- times of the normal level by urine organic acid sides, organic acid test of urine by MS/MS test. showed an excessive urinary excretion of meth- ylmalonic acid 18076 times of average level Genomic DNA analysis which supported methylmalonic aciduria (refer- ence range < 2 mmol/mol). Besides, plasma Since the publication of the first graft of the homocysteine was 260.64 µmol/L, higher than human genome sequence, the field of genom- the normal level (reference range 1-20 µmol/L ics has been changed dramatically; and with in blood). Combined the two characteristic bio- the development of high-throughput methods chemical parameters and clinical manifesta- that could be used to interrogate the wealth of tions, the patient was diagnosed as methylma- data available in the human genome. In the lonic aciduria and homocystinuria. present study, we used 48 genes related to organic acid metabolic disease (Table 1), Case 2 including: MUT, MMAA (cblA), MMAB (cblB), An 11-year-old boy was admitted to hospital MMACHC (cblC), MMADHC (cblD), LMBRD1 because of “intermittent headache, spasm, (cblF) and other genes. This technology com- somnolence, paralysis of facial features”. The bined the designed gene capture probe and patient is with aware consciousness. The skull customer DNA library. The gene fragments CT demonstrated that bilateral lateral ventricle were hybridized to the probe, and adsorbed to triangle choroid plexus slightly punctuated with the beads through biotin and streptavidin-bio- high density of shadow. Cranial MRI enhance- tin; and then the nonspecific binding DNA frag- ment scanning was normal. EEG showed slow ments were washed out. Finally the captured background activity. The results of plasma total target DNA fragment was identified by new-gen- homocysteine and urine organic acid analysis eration sequencing platform in pediatrics, are homocysteine of 396.84 µmol/L and meth- which can quickly distinguishe the targeted ylmalonic acidria of 2058 times of average gene (MMACHC) and determine its mutation level respectively, both elevated far more than locus. In order to avoid the false positive errors. the normal level. Plasma amino acids analysis The pathogenic mutation was further confirmed showed higher Hcy, C3/CO, C3/C2, C8/C2 and by Sanger direct sequencing the whole target C8/C12, similar to the status of case 1. gene. 9338 Int J Clin Exp Pathol 2015;8(8):9337-9341 Genetic analysis of four cases of methylmalonic aciduria and homocystinuria clearly. In these cases, low homocysteine level after treat- ment of B12 was observed which indicates that the patient is responded to B12, a charac- teristic of the cblC disease type of combined methylmalonic aci- duria and homocystinuria. However, the fourth case sho- wed no response to treatment of vitamin B12. His mum did not showed lack of Vitamin B12 dur- ing pregnancy. All gDNA samples of these four cases were checked for molecular diag- nostics. In the first case, two mutations c.609G>A p.W203Term (Figure 1A) and c.440_441del (Figure 1B) were identified in the MMACHC gene; the deletion mutation (c.440_441del) sited on the binding domain of cyano- cobalamine has not been report- Figure 1. MMACHC gene mutations of case 1. (point mutation is indenti- ed previously for resulting in fied by arrow and deletions and insertions are marked by black frame). cblC disease. In the second case, two mutations in gene MMACHC were identified, one Discussion insertion mutation c.567_568insT p.Ile190fs (Figure 2A) in exon 4 lead to the frame shift and Individuals with cblC disease caused by muta- early termination of its allele. The other muta- tions in MMACHC gene have aroused atten- tion is c.482G>A p.R161Q (Figure 2B) which tions from all over the world. While most muta- has been reported in previous cases disease tions are private, clear ethnic observations [1, 7]. In third case, one pair of homozygosis have been made for more common mutations. mutation was indentified, c.609G>A p. Clinical heterogeneity in cblC disease has been W203Term (Figure 3). Similar to the finding was clearly started by Morel et al [6]. The mutations reported by Lerner-Ellis et al. It is indicated that of MMACHC gene can lead to onset of cblC dis- this mutation c.609G>A p.W203Term is not ease at different age. ethnic. In the fourth case, we did not found any mutation of his MMACHC gene, but this patient Herein, we have applied a facility to distin- displays clinical manifestations of cblC type. guished mutated genes, which can carefully Therefore, we speculate that the mutation sequence the MMACHC gene in affected indi- might occur in the regulatory regions of viduals and has a greater than 95% chance of MMACHC gene. In order to illustrate the real definitively confirming the diagnosis of a sus- reason, we should evaluate the expression pected case of cblC disease.