Eight novel <Emphasis Type="Italic">MUT</Emphasis> loss-of-function missense mutations in Chinese patients with isolated methylmalonic academia

Background:Isolated methylmalonic acidemia is a rare autosomal recessive metabolic disorder mostly caused by mutations in the methylmalonyl coenzyme A mutase (MCM) gene (MUT).This study aimed to verify whether missense mutations in MUT in Chinese patients affect the stability and enzymatic activity of MCM.Methods:Eight Chinese patients were identified with novel mutations.Plasmids carrying the wild-type and mutated MUT cDNA were constructed and transfected into HEK293T cells for functional analyses.The expression and activity of MCM were determined by western blot and ultra-performance liquid chromatography,respectively.Results:All patients had high levels of blood propionylcarnitine and urinary methylmalonyl acid.By the end of the study,two patients were lost to follow-up,three died,and three survived with mental retardation.Compared to the wild-type protein,the expression levels of all missense mutations of in vitro MCM protein were decreased (P＜0.05) except those for I597R,and the MCM activity of the mutations was reduced in a permissive assay.Conclusion:The missense mutations L140P,A141T,G161V,W309G,I505T,Q514K,I597R and G723D affected the stability and enzymatic activity of MCM,indicating that they had a disease-causing capacity.     

Stabilization of blood methylmalonic acid level in methylmalonic acidemia after liver transplantation

Chen PW, Hwu WL, Ho MC, Lee NC, Chien YH, Ni YH, Lee PH. Stabilization of blood methylmalonic acid level in methylmalonic acidemia after liver transplantation.
Pediatr Transplantation 2010: 14: 337–341. © 2009 John Wiley & Sons A/S. Abstract: Methylmalonic acidemia with complete mutase deficiency (mut⁰ type) is an inborn error of metabolism with high mortality and morbidity. LT has been suggested to be a solution to this disease, but elevation of urinary and blood MMA was still observed after LT. In this study, we measured dry blood spot MMA and its precursor propionyl‐carnitine (C3‐carnitine) for mut⁰ patients. The results revealed that when C3‐carnitine rose during metabolic stress, MMA rose exponentially (up to 1000 μmol/L) in patients who did not undergo LT. In patients who underwent LT, MMA rose to 100–200 μmol/L when C3‐carnitine reached 10–20 μmol/L. However, when C3‐carnitine rose further to 40–50 μmol/L, MMA levels just stayed put. Therefore, LT stabilized blood MMA level, though there might be a threshold for blood MMA clearance by the donor liver. This finding should be critical to understand the long‐term outcome for LT in methylmalonic acidemia.     

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??Objective??To investigate the changes and diagnostic value of urinary hydrogen sulfide??H2S?? in the patients with methylmalonic acidemia??MMA??. Methods??The urine samples from 20 cases??13 males and 7 females??patient group?? of MMA were collected when the patients visited Peking University First Hospital from September 2016 to November 2017. The normal urine samples were collected from 20 healthy children??11 males and 9 females??control group?? who had normal development and metabolic condition. H2S in the urine of normal children and MMA children was determined by free radical detector. The differences of urinary H2S concentration between the two groups were analyzed. The ROC curve of urine samples was evaluated. Results??The urinary H2S concentration of the 20 MMA cases was higher than that in the control group??64.9±52.0?? μmol/L vs.??43.9±40.0?? μmol/L. The difference was significant statistically??Z??-3.435??P??0.001??. The area under the curve??AUC?? was 0.818 in the ROC curve analysis of MMA with urinary H2S concentration and 47.72 μmmol/L in urine H2S as a cut-off value. The sensitivity and specificity were 95% and 60% respectively. Conclusion??The concentration of urinary H2S is increased in the patients with MMA. Urinary H2S concentration might be a biochemical marker for the early diagnosis MMA in neonatal screening. It is helpful to evaluate the treatment patients and follow-up of MMA.     

甲基丙二酸血症诊治研究进展

甲基丙二酸血症是一种常见的有机酸血症,属于常染色体隐性遗传病,临床表现无特异性,以反复呕吐及嗜睡、惊厥等神经系统症状为主.诊断依靠串联质谱检测血中的酰基肉碱和气相色谱-质谱检测尿甲基丙二酸.对伴有同型半胱氨酸血症患儿,治疗以维生素B12、甜菜碱和左旋肉碱为主;对不伴有同型半胱氨酸血症患儿以限制天然蛋白质摄入,给予去除异亮氨酸、缬氨酸、甲硫氨酸和苏氨酸的特殊奶粉及左旋肉碱治疗为主.维生素B12治疗有效型预后较好,治疗无效型预后较差.     

Renal cell carcinoma harboring somatic TSC2 mutations in a child with methylmalonic acidemia

Pediatric renal cell carcinoma (RCC) is a rare cancer that can be associated with inherited diseases including tuberous sclerosis complex (TSC) caused by germline mutations in TSC1 or TSC2. Somatic mutations in TSC1 and TSC2 have also been reported in adult RCC, which predict response to mTOR inhibitors. Here, we present the first case of RCC in a child with methylmalonic acidemia (MMA). Clinical whole exome sequencing of blood and tumor samples confirmed the diagnosis of MMA and revealed two somatic inactivating mutations in TSC2, suggesting the potential consideration of an mTOR inhibitor in the event of tumor recurrence.     

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??Abstract??Objective??To summarize the clinical features and prognosis of epilepsy in children with MMA. Methods??From Jan. 1997 to Dec. 2009 in Department of Pediatrics??Peking University First Hospital??the medical records of hospitalized MMA patients complicated with epilepsy were retrospectively reviewed. The clinical manifestations??laboratory examination results??and treatment modalities were analyzed. Results??From 63 pediatric inpatients diagnosed with MMA??27 children ??42.9%?? complicated with epilepsy were enrolled in this study. These 27 patients were also accompanied with other neurological manifestations?? including mental retardation or regression ??n = 22????lethargy ??n = 10????increased muscle tone ??n = 8????muscle hypotonia ??n = 8????recurrent vomiting ??n = 4????tremor ??n = 2????ataxia ??n = 2????and abnormal posture ??n = 1??.The onset age of seizure ranged from 8 days to 11 years. The seizure types included partial seizure??n = 21????generalized tonic-clonic seizure ??n = 5????tonic seizure ??n = 3????myoclonic seizure ??n = 3????and epileptic spasm ??n = 2??. Five patients had 2 or 3 seizure types. Nine patients ??33.3%?? had a history of status epilepticus. EEG showed slow background activity in 17 patients??focal or multifocal discharges in 16 patients??generalized discharges in 4 patients??hypsarrathmia in 2 patients??and suppression-burst pattern in 1 patient. Cranial MRI showed cerebral atrophy ??n = 14????white matter changes ??n = 12????agenesis of corpus callosum ??n = 2????abnormal signal in basal ganglia ??n = 2????and cerebellar atrophy ??n = 1??. Twenty-one patients were MMA combined with homocysteinemia.Seventeen patients were confirmed with cobalamin C disease and one with partial mutase deficiency ??mut-??. Vitamin B12-responsive patients had a better outcome compared with vitamin B12-unresponsive patients. Conclusion??Epilepsy is a common manifestation of patients with MMA.Partial seizures is more common than other seizure types.Urine organic acid analysis should be performed for children with unknown cause of epilepsy combined with other neurological manifestations?? so as to promptly identify the etiology and improve the prognosis.     

Juvenile gout in methylmalonic acidemia

Methylmalonic acidemia (MMA) is an inborn error of metabolism caused by either deficiency of the enzyme methylmalonyl‐CoA mutase or a defect in adenosyl‐cobalamin synthesis. Chronic kidney disease is its common complication and, in combination with persistent acidosis, leads to hyperuricemia. Symptomatic hyperuricemia or gout, however, has not been reported in MMA. We herein report two pediatric cases of MMA caused by MMAB mutations (cblB defect) with renal tubular acidosis, chronic kidney disease, hyperuricemia, and gout. The clinical findings of gout in these cases included recurrent first metatarsophalangeal arthritis and/or tophi. The patients responded to treatment with colchicine and allopurinol.     

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??Objective To analyze the clinical features?? mutation types?? treatment and outcomes of methylmalonic acidemia combined with homocysteinemia??MMA-HC?? in children??in order to improve the clinical understanding of this disease.
Methods The clinical data??mutation types??treatment and outcomes of 11 children with MMA-HC were retrospectively analyzed to find the relationships of clinical phenotypes and genotypes with the prognosis of the early-onset??≤1 year?? and late-onset ????1 year?? patients. Results In the 9 early-onset children with more serious clinical manifestations??the major gene mutation type was c.609G??A and C.658_660delAAG??the mutation types of 2 cases of late-onset children were c.482G??A/c.609 G??A and c.394C??T/c.80A??G compound heterozygous mutations. The children with C.609G??A/c.658_660delAAG compound heterozygous mutation in early-onset patients had poorer clinical manifestations and prognosis. In contrast??the other early-onset children were relatively good. After treatment??the prognosis of late-onset children were usually good. Conclusion Combined-type MMA children have various clinical manifestations and individual differences. Seizures??lethargy??feeding difficulty and no increase of weight are the common symptoms in early-onset children??while in late-onset patients there is occult onset??chronic mental disorders??mental retardation and weakness of two legs. Early treatment with hydroxycobalamin can significantly improve the clinical symptoms in the children??but the prognosis still depends on the clinical phenotype and genotype.     

儿童危重型甲基丙二酸血症临床特点及基因分析

目的分析危重型甲基丙二酸血症的临床及基因学特点。方法回顾分析2015年9月至2018年12月34例危重型甲基丙二酸血症患儿的临床资料,其中11例行基因检测。结果 34例患儿中男18例、女16例,中位发病年龄为11.5个月。所有患儿均存在2个及以上脏器功能障碍或衰竭,除常见血液系统、肝、肾、心、肺血管及神经系统损害外,11例合并纤维蛋白原水平降低,3例合并第四脑室中、侧孔先天性闭锁脑发育畸形(Dandy-Walker畸形)。11例行基因检测的患儿中,9例为MMACHC基因突变,c.609GA位点突变为合并型甲基丙二酸血症热点突变,其中2例行基因检测的脑发育畸形患儿均存在c.609GA位点突变;另2例为单纯型甲基丙二酸血症,均为甲基丙二酰辅酶A变位酶(MUT)基因突变所致。结论危重型甲基丙二酸血症患儿神经损害最常见,部分可合并脑发育畸形,且可能与c.609GA基因位点突变有关;可合并纤维蛋白原水平降低。     

Dietary therapy in two patients with vitamin B12-unresponsive methylmalonic acidemia

The biochemical and therapeutic responses to dietary therapy were studied in a 25-month-old girl and a 1-month-old girl with methylmalonic acidemia (MMA-emia), which was unresponsive to vitamin B₁₂.The minimum daily intake of protein which patients could tolerate and display a good development was between 1.0 and 1.2 g per kg body weight. Supplementation with amino acid mixture devoid of toxic amino acids was required to prevent protein malnutrition when daily protein intake was restricted to 0.6 g per kg body weight. Caloric intake should be sufficient, not only to promote growth but also to prevent a rise in MMA level, especially when a patient has ketoacidosis.It was found that MMA excretion per mg creatinine in random urine specimens correlated significantly with serum MMA and twenty four-hour output of MMA per kg body weight. Therefore measurement of MMA in a single urine specimen is useful for evaluating the in vivo accumulation of MMA.     

利用CRISPR/Cas9构建甲基丙二酸血症cblC型W203X突变小鼠模型

目的 利用CRISPR/Cas9技术构建与人甲基丙二酸血症cblC型W203X突变型一致的小鼠模型。方法 通过BLAST比对人和小鼠cblC基因和蛋白序列的保守性,应用CRISPR/Cas9技术进行小鼠受精卵显微注射,获得杂合子F1代小鼠,F1代小鼠通过杂交获得W203X纯合突变型小鼠,并对纯合突变型、同窝杂合型及野生型3种类型小鼠进行血代谢产物丙酰肉碱检测。结果 人和小鼠甲基丙二酸血症cblC型的致病基因MMACHC的核苷酸和氨基酸序列高度保守。通过CRISPR/Cas9技术成功获得W203X纯合突变型小鼠,该小鼠模型在生后24 h丙酰肉碱明显升高（P < 0.001）。结论 利用CRISPR/Cas9技术成功构建了与人甲基丙二酸血症cblC型W203X突变型一致的小鼠模型。     

基因捕获联合高通量测序技术在甲基丙二酸血症诊断中的应用

目的 探讨新一代基因测序平台(HiSeq2000)在甲基丙二酸血症(MMA)诊断中的价值.方法 1.采集已经临床确诊的9例MMA患儿外周血并提取DNA,将设计好的基因捕获探针与患儿DNA文库混合,然后利用基因捕获联合高通量测序分析技术对有机酸代谢相关的48个基因全部外显子区域进行测序.2.获得原始数据,去除接头和过滤低质量数据,对数据进行SNP、InDel等分析,并采用Sanger测序方法对异常位点进行测序验证.3.气相色谱-质谱联用分析技术对尿液标本的有机酸测定及其他辅助检查.结果 1.突变基因:7例存在MMACHC基因突变,7例患儿中共检测出7种突变,包括c.482G＞A、c.567_568insT、c.609G＞A、c.440_ 441del、c.80A＞G、c.315C＞G、c.90G＞A,其中c.440_441 del为未见报道突变;1例存在甲基丙二酰辅酶A变位酶(MUT)基因突变,均为内含子异常,分别是c.754-1G＞C、c.1677-1G＞A,其中c.754-1G＞C为未见报道突变;1例未检测到突变.2.临床症状:9例患儿均存在智力运动发育迟缓,伴抽搐3例,反复顽固性代谢性酸中毒、头痛及面瘫、反复性溶血各1例.脑电图异常9例,头颅磁共振异常8例,尿液中甲基丙二酸水平均升高(273.4 ～146 022.8倍),血同型半胱氨酸水平增高8例(27.13～ 396.84 μmol/L,正常＜20 μmol/L).3.Sanger测序验证:均符合新一代测序平台结果,无假阳性存在.结论 1.进一步证明c.609G＞A突变位点为中国MMA合并同型半胱氨酸血症患儿的热点突变位点.MMA基因突变类型较多,与临床症状无明显相关性.2.推测MMACHC基因c.440_441 del突变及MUT基因c.754-1G＞C突变为新发突变.3.基因捕获联合高通量基因测序技术一次性捕获突变基因数量大,获取疾病的遗传信息量广,具有高通量、高灵敏度、高效等特点,适合于MMA患儿检测,也可为其他儿科临床常见遗传性疾病的检测提供参考.     

甲基丙二酸血症伴同型半胱氨酸血症患儿基因突变分析

目的 检测甲基丙二酸血症伴同型半胱氨酸血症患儿的MMACHC基因突变类型及突变频率.方法 依据串联质谱检测血中的酰基肉碱、气相色谱-质谱检测尿甲基丙二酸、血清同型半胱氨酸测定及维生素B12负荷试验等,对28例甲基丙二酸血症伴同型半胱氨酸血症患儿进行诊断;应用聚合酶链反应(PCR)对这些患儿及其部分直系亲属、健康对照组的MMACHC基因外显子进行扩增,通过DNA直接测序进行基因突变分析.结果 在28例患儿中27例检测到突变,共10种,其中2例仪检测到1个杂合突变,3例仅检测到多态性.突变集中在外显子3和4上(91.3%),以609G>A(W203X)最常见,突变频率为53.6%,其中10例为纯合突变,10例为杂合突变,其次为658_660delAAG(K220del),突变频率为8.9%,均为杂合突变.另外检测到6种未报道突变,分别为1A>G、365A>T、658_del660AAG、301-3_327del 30、567_568insT和625_626insT.3种基因多态性分别为-302T>G(rs3748643)、-234A>G(rs3728644)和321G>A(rs2275276).结论 揭示了中国甲基丙二酸血症伴同型半胱氨酸血症患儿的部分基因突变谱,其中609G>A(W203X)可能为热点突变.     

Phenotypic and mutation spectrums of Thai patients with isovaleric acidemia

Background: Isovaleric acidemia (IVA) is an autosomal recessive disorder caused by deficiency of isovaleryl‐CoA dehydrogenase (IVD). Clinical features include vomiting, lethargy, metabolic acidosis, and “sweaty feet” odor. The pathognomonic metabolite, isovalerylglycine, is detected on urine organic acid analysis. Clinical diagnosis of IVA can be confirmed on mutation analysis of the IVD gene. Methods: The cases of five unrelated Thai patients with IVA, identified on urine organic acid analysis, are described. Mutation analysis of the IVD gene was performed using polymerase chain reaction sequencing of the entire coding regions. Results: Four out of the five IVA patients had an acute neonatal form. The hematologic abnormalities were common and thus could be presenting symptoms in the absence of metabolic acidosis. As for the neurological outcome, only one patient had normal intelligence. Mutation analysis of the IVD gene identified the mutations c.457–3_2CA>GG, c.1199A>G (p.Y371C), c.281C>G (p.A65G), c.358G>A (p.G91R), and c.827T>C (p.L247P). The poor outcome in most patients might be explained by the delayed diagnosis and initial unavailability of the metabolic formulas and medications in Thailand. The c.457–3_2CA>GG mutation was identified in all of the present patients. This suggests that it is the most common mutation in the Thai population. Therefore, it could be a founder mutation in Thai subjects. One of the present Thai IVA patients also had the p.Y371C mutation, which is common in Han Chinese subjects. In addition, two novel mutations, p.A65G and p.L247P, were identified. Conclusion: The present study provides additional knowledge on the genotype–phenotype of IVA, suggesting that IVD mutations in Asian populations are distinct from these in Western populations.     

甲基丙二酸尿症合并同型半胱氨酸血症致多系统损害

目的 甲基丙二酸尿症合并同型半胱氨酸血症是甲基丙二酸尿症中的特殊类型,对5例患儿进行回顾性分析,研究本症的临床表现、生化特点、诊断与治疗方法。方法 应用气相色谱-质谱联用分析法（gas chromatography-mass spectrometry,GC-MS）进行尿有机酸分析,采用荧光偏振免疫测定法检测血浆同型半胱氨酸。确诊后给予钴胺素、左旋肉碱、甜菜碱等药物治疗,予以长期随访。结果 5例患儿（男2例,女3例）起病年龄3个月～13岁。4例表现为进行性智力运动障碍伴蛋白尿、血尿,1例因肌肉酸痛就诊,伴抽搐2例,伴周围神经损害2例,肝功异常2例。5例患儿尿甲基丙二酸为29．4～805．9mg／g肌酐（正常对照为0．2～3．6mg／g肌酐）,血浆中同型半胱氨酸42．5～215．22μmol／L（正常对照5～15μmol／L）,均明显增高,血浆游离肉碱浓度下降。经维生素B12左旋肉碱治疗后,患儿尿甲基丙二酸浓度明显下降,经甜菜碱治疗后血浆同型半胱氨酸逐渐降低。结论 甲基丙二酸尿症并同型半胱氨酸血症可导致多系统损害,临床表现多样;早期诊断、早期治疗是改善预后的关键;对不明原因的脑病、周围神经病、肾损害等多脏器损害患者,应尽早进行尿有机酸分析等特殊检查,对于甲基丙二酸尿症患者应进行血浆同型半胱氨酸测定。     

Evaluation and management of patients with propionic acidemia undergoing liver transplantation: a comprehensive review

Propionic acidemia is a rare metabolic disorder that often results in episodic hyperammonemia, basal ganglia infarction, mental retardation, and cardiomyopathy. OLT has been used as a treatment for propionic acidemia, but its benefit in patients with this disease is unclear. The current study was undertaken to clarify the role of OLT in the management of this disease. The medical literature, a national registry of US OLT recipients, and a single institution liver transplant experience were reviewed for cases of OLT for propionic acidemia. Accumulated cases demonstrate that OLT has resulted in clear evidence of clinical improvement in several patients, often obviating the need for dietary restriction or other forms of medical management. OLT appears to halt the decline in neurocognitive function often associated with propionic acidemia. In total, 12 patients with propionic acidemia have undergone a total of 14 OLTs. A quantitative analysis of outcomes shows an overall patient survival rate of 72.2% at one year after OLT. In conclusion, OLT should be considered a treatment option for patients with propionic acidemia who continue to experience episodes of hyperammonemia in spite of maximal medical therapy. Early OLT may limit the development of mental retardation and/or cardiomyopathy.