Seven novel mutations in mut methylmalonic aciduria

Methylmalonic aciduria (MMA) is an autosomal recessive inborn error of metabolism that results from functional defects in methylmalonyl CoA mutase (MCM), a nuclear-encoded, mitochondrial enzyme that uses the vitamin B₁₂ derivative, adenosylcobalamin (AdoCbl) as a cofactor. To date, 23 mutations have been identified at the MUT locus on the short arm of chromosome 6, causing the mut forms of MMA (mut complementation group; mut MMA, McKusick #251000). We now report seven novel mutations. Three were found in mut⁰ patients: R228Q (c759G→A) was found as a heterozygous change; G312V (c1011G→T) and 346delL (c1112delCTT) were both found as homozygous changes. Four mutations were found in mut^– patients: A191E (c648C→A) and V633G (c1974T→G) were found in the same patient; 684insL (c2128insCTC) and L685R (c2130T→G) were both found as homozygous changes. The recent modelling of the human methylmalonyl CoA mutase allowed for an interpretation of the identified mutations. Hum Mutat 11:270–274, 1998. © 1998 Wiley-Liss, Inc.     

Molecular basis of methylmalonyl-CoA mutase apoenzyme defect in 40 European patients affected by mut(o) and mut- forms of methylmalonic acidemia: identification of 29 novel mutations in the MUT gene

Methylmalonyl-CoA mutase (MCM) apoenzyme deficiency is a rare metabolic disease that may result in distinct biochemical phenotypes of methylmalonic acidemia (MMA), namely mut(o) and mut-. We analyzed a cohort of 40 MCM-deficient patients with MMA affected by either the mut(o) or the mut- form of the disease. By direct sequencing of cDNA and gDNA of the MUT gene, we detected 42 mutations, 29 of which were novel mutations. These included five frameshift mutations (insertion, deletion, or duplication of a single nucleotide), five sequence modifications in consensus splice sites, six nonsense and 12 missense mutations, and a large genomic deletion including exon 12. We explored how the 12 novel missense mutations might cause the observed phenotype by mapping them onto a three-dimensional model of the human MCM generated by homology with the P. shermanii enzyme. In this work we update the spectrum of MCM mutations (n=84), and then discuss their prevalence and distribution throughout the coding sequence in relation to the enzyme structure.     

甲基丙二酸血症患儿MUT基因突变分析

目的 检测甲基丙二酸血症(methylmalonic acidemia,MMA)患儿MUT基因突变类型及突变频率,探讨基因型与临床表型之间的关系.方法 依据串联质谱检测血酰基肉碱、气相色谱-质谱检测尿甲基丙二酸以及维生素B_(12)负荷试验等,诊断21例单纯MMA患儿;采用聚合酶链反应和直接测序法对这些患儿进行MUT基因突变检测分析.结果 在21例单纯MMA患儿中14例检测到17种MUT基因突变,其中8种为未报道突变.以c.323G>A(R108H)、c.729_730insTT(D244LfsX39)与c.1630_1631GG>TA(G544X)较为常见,突变频率分别为14.3%、10.7%及14.3%,以错义突变多见(64.7%).14例MUT突变患儿中10例为早发型,1例为迟发型,3例由新生儿出生筛查检出;11例为维生素B_(12)无效型,3例为有效型.结论 揭示了中国MMA患儿中MUT基因的部分突变谱,MUT基因突变患儿发病较早,多为维生素B_(12)无效型.     

Atypical methylmalonic aciduria: frequency of mutations in the methylmalonyl CoA epimerase gene (MCEE)

Methylmalonic aciduria is known to result from defects in the enzyme methylmalonyl CoA mutase (MCM) (mut complementation group) and from defects in the synthesis of the MCM cofactor adenosylcobalamin (cblA, cblB, cblC, cblD, and cblF groups). Two patients who excrete methylmalonic acid have recently been shown to have a homozygous nonsense mutation in the gene coding for methylmalonyl CoA epimerase (MCEE). To further understand the cause of methylmalonic acid excretion, the MCEE gene was sequenced in 229 patients with elevations of methylmalonic acid excretion for which no cause was known. Mutations in MCEE were detected in five patients: two patients homozygous for c.139C>T, p.R47X, one patient homozygous for c.178A>C, p.K60Q, and two patients heterozygous for c.427C>T, p.R143C. Fusion of fibroblast lines from two patients homozygous for c.139C>T, p.R47X did not result in correction of [(14)C]propionate incorporation toward control values while the defect in these fibroblasts was complemented by mut, cblA, and cblB fibroblasts. Infection with wild-type MCEE cDNA resulted in correction of the biochemical phenotype in cells from both patients.     

Mutation and biochemical analysis of 19 probands with mut0 and 13 with mut- methylmalonic aciduria: identification of seven novel mutations

Isolated methylmalonic acidurias (MMA-urias) comprise a group of rare autosomal recessively inherited disorders characterised by accumulation of MMA in urine and other body fluids, resulting from deficient activity of the mitochondrial enzyme methylmalonyl-CoA mutase (MCM). Isolated MMA-uria results from either MCM apoenzyme defects (mut(0) and mut(-)) or defects in synthesis of its cofactor 5-deoxyadenosylcobalamin, i.e. cblA, cblB and cblD-variant 2. To date various studies have identified 171 disease-causing mutations in the MCM gene (MUT). We report mutation analysis in 32 probands with mut MMA-uria including 13 probands with a mut(-) defect. Sixty two of 64 possible mutant alleles were identified, seven of which were novel missense alleles. We found three novel mutations (c.427C>T/p.H143Y; c.862T>C/p.S288P; c.1361G>A/p.G454E) among 19 probands with a mut(0) defect and four novel mutations (c.299A>G/p.Y100C; c.1031C>T/p.S344F; c.1097A>G/p.N366S; c.2081G>T/p.R694L) among 13 probands with a mut(-) defect. Our study provides evidence that the p.Y100C, p.R108H, p.N366S, p.V633G, p.R694W, p.R694L and p.M700K mutations are associated with a mut(-) phenotype.     

Pseudoexon exclusion by antisense therapy in methylmalonic aciduria (MMAuria)

Development of pseudoexon exclusion therapies by antisense modification of pre-mRNA splicing represents a type of personalized genetic medicine. Here we present the cellular antisense therapy and the cell-based splicing assays to investigate the effect of two novel deep intronic changes c.1957–898A>G and c.1957–920C>A identified in the methylmalonyl–coenzyme A (CoA) mutase (MUT) gene. The results show that the nucleotide change c.1957–898A>G is a pathological mutation activating pseudoexon insertion and that antisense morpholino oligonucleotide (AMO) treatment in patient fibroblasts leads to recovery of MUT activity to levels 25 to 100% of control range. On the contrary, the change c.1957–920C>A, identified in two fibroblasts cell lines in cis with c.1885A>G (p.R629G) or c.458T>A (p.D153V), appears to be a rare variant of uncertain clinical significance. The functional analysis of c.1885A>G and c.458T>A indicate that they are the disease-causing mutations in these two patients. The results presented here highlight the necessity of scanning the described intronic region for mutations in MUT-affected patients, followed by functional analyses to demonstrate the pathogenicity of the identified changes, and extend previous work of the applicability of the antisense approach in methylmalonic aciduria (MMAuria) for a novel intronic mutation. Hum Mutat 30:1–7, 2009. © 2009 Wiley-Liss, Inc.     

Microarray based mutational analysis of patients with methylmalonic acidemia: identification of 10 novel mutations

Methylmalonic acidemia is an autosomal recessive metabolic disorder affecting the propionate oxidation pathway in the catabolism of several amino acids, odd-chain fatty acids, and cholesterol. Methylmalonic acidemia is characterized by elevated levels of methylmalonic acid in the blood and urine. Mutations in the MUT gene, encoding methylmalonyl-CoA mutase carries out isomerization of L-methylmalonyl-CoA to succinyl-CoA, cause methylmalonic acidemia. In this study, 30 Turkish patients diagnosed with mut methylmalonic acidemia were screened for mutations using custom designed sequencing microarrays. The study resulted in detection of 22 different mutations, 10 of which were novel: p.Q132*, p.A137G, c.753+1T, p.T387I, p.Q514E, p.P615L, p.D625V, c.1962_1963delTC, p.L674F, and c.2115_2116insA. The most common, p.P615T, was identified in 28.0% of patients. These results suggest that microarray based sequencing is a useful tool for the detection of mutations in MUT in patients with mut methylmalonic acidemia.     

Molecular analysis of Turkish Gaucher disease patients: identification of novel mutations in glucocerebrosidase (GBA) gene

Gaucher disease (GD) is the most frequent lysosomal glycolipid storage disorder due to autosomal recessive deficiency of acid beta-glucosidase and is characterized by the accumulation of glucocerebroside. In this work we carried out molecular analysis of the glucocerebrosidase gene (GBA) in 57 unrelated patients and the alleleic frequencies of gene mutations in Turkish patients are reported. The most prevalent are L444P and N370S accounting for 42% and 30% in our patients. We identified three novel genetic alterations: two missense changes S356F, L296V that are associated with the severe phenotype of type 1 GD. 303-305delCAC was identified in a homozygous state in one patient type 1 or type 3.     

单纯型甲基丙二酸血症家系MUT基因变异的分析及产前诊断CSCD

目的对20个单纯型甲基丙二酸血症家系MUT基因的变异进行测序分析,为家系产前诊断提供依据。方法应用PCR产物直接测序法对20例单纯型甲基丙二酸血症患儿及其父母的MUT基因进行变异检测和分析,明确基因变异情况,并对9名孕妇进行产前诊断。结果20例患儿的家系共检测出19种MUT基因变异,最常见的变异为C.323G〉A（P．Arg108His）、c．1106G〉A（P．Arg369His）、C．729_730insTT（P．D244Lfs＊39）和c．1107dupT（P．T370Yfs＊22）。C．920_923delTCTT（P．F307SIs＊6）、C．419T〉C（P．Leu140Pro）和C．613G〉A（P．Glu205Lys）为未报道过的新变异。Polyphen2和Mutationtaster软件预测这3个变异均可能致病。产前诊断结果显示1例胎儿未检测到MUT基因变异,3例胎儿为MUT基因杂合变异携带者,5例胎儿为MUT基因复合杂合变异或纯合变异患儿。MUT基因正常或杂合变异携带者胎儿的家系选择继续妊娠,而MUT基因纯合变异或复合杂合变异胎儿的家系均选择终止妊娠,胎儿娩出后随访结果与产前诊断结果一致。结论MUT基因突变分析结果为家系的产前诊断提供了依据,新变异的检出丰富了MUT基因突变谱。     

Late-onset combined homocystinuria and methylmalonic aciduria (cblC) and neuropsychiatric disturbance

We report on the case of a 36-year-old Hispanic woman with a spinal cord infarct, who was subsequently diagnosed with methylmalonic aciduria and homocystinuria, cblC type (cblC). Mutation analysis revealed c.271dupA and c.482G > A mutations in the MMACHC gene. The patient had a past medical history significant for joint hypermobility, arthritis, bilateral cataracts, unilateral hearing loss, anemia, frequent urinary tract infections, and mental illness. There was no significant past history of mental retardation, failure to thrive, or seizure disorder as reported in classic cases of cblC. Prior to the thrombotic incident, the patient experienced increased paresthesia in the lower extremities, myelopathy, and impaired gait. Given her previous psychiatric history, she was misdiagnosed with malingering until hemiplegia and incontinence became apparent. The authors would like to emphasize the recognition of a neuropsychiatric presentation in late onset cblC. Ten other reported late onset cases with similar presentations are also reviewed.     

Genetic and cellular studies of oxidative stress in methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC)

Methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC) is the most frequent genetic disorder of vitamin B₁₂ metabolism. The aim of this work was to identify the mutational spectrum in a cohort of cblC-affected patients and the analysis of the cellular oxidative stress and apoptosis processes, in the presence or absence of vitamin B₁₂. The mutational spectrum includes nine previously described mutations: c.3G>A (p.M1L), c.217C>T (p.R73X), c.271dupA (p.R91KfsX14), c.331C>T (p.R111X), c.394C>T (p.R132X), c.457C>T (p.R153X), c.481C>T (p.R161X), c.565C>A (p.R189S), and c.615C>G (p.Y205X), and two novel changes, c.90G>A (p.W30X) and c.81+2T>G (IVS1+2T>G). The most frequent change was the known c.271dupA mutation, which accounts for 85% of the mutant alleles characterized in this cohort of patients. Owing to its high frequency, a real-time PCR and subsequent high-resolution melting (HRM) analysis for this mutation has been established for diagnostic purposes. All cell lines studied presented a significant increase of intracellular reactive oxygen species (ROS) content, and also a high rate of apoptosis, suggesting that elevated ROS levels might induce apoptosis in cblC patients. In addition, ROS levels decreased in hydroxocobalamin-incubated cells, indicating that cobalamin might either directly or indirectly act as a scavenger of ROS. ROS production might be considered as a phenotypic modifier in cblC patients, and cobalamin supplementation or additional antioxidant drugs might suppress apoptosis and prevent cellular damage in these patients. Hum Mutat 30:1–9, 2009. © 2009 Wiley-Liss, Inc.     

Diagnosis and treatment of methylmalonic aciduria: a case report

The methylmalonic aciduria is an organic acidemia, inherited as autosomic recessive trait, caused by a deficiency of the methylmalonyl-CoA mutase, or by defects in the biosynthesis of the cofactor adenosylcobalamin. Regarding the enzymatic defect, there are two forms: mut(o) with no detectable enzymatic activity and mut(-) with reduced activity. Its clinical presentation may vary from a severe neonatal form with acidosis and death, up to a progressive chronic form. Here we describe the case of a four year-old boy, with diagnosis of methylmalonyl-CoA mutase deficiency type mut(-) with an acute presentation. Molecular analysis of MUT gene identified two mutations c.607G>A (G203R) and c.2080C>T (R694W), later confirmed in the parents. The aim of this report is to highlight the importance of including the organic acid analysis in urine among the first line exams in acutely and severely ill children with undefined etiology. The definitive diagnosis is important because it may allow a specific treatment and a favorable evolution to prevent the secuelae.     

Molecular characterization of novel progranulin (GRN) mutations in frontotemporal dementia

Frontotemporal dementia (FTD) is a clinical term encompassing dementia characterized by the presence of two major phenotypes: 1) behavioral and personality disorder, and 2) language disorder, which includes primary progressive aphasia and semantic dementia. Recently, the gene for familial frontotemporal lobar degeneration (FTLD) with ubiquitin-positive, tau-negative inclusions (FTLD-U) linked to chromosome 17 was cloned. In the present study, 62 unrelated patients from the Washington University Alzheimer's Disease Research Center and the Midwest Consortium for FTD with clinically diagnosed FTD and/or neuropathologically characterized cases of FTLD-U with or without motor neuron disease (MND) were screened for mutations in the progranulin gene (GRN; also PGRN). We discovered two pathogenic mutations in four families: 1) a single-base substitution within the 3' splice acceptor site of intron 6/exon 7 (g.5913A>G [IVS6-2A>G]) causing skipping of exon 7 and premature termination of the coding sequence (PTC); and 2) a missense mutation in exon 1 (g.4068C>A) introducing a charged amino acid in the hydrophobic core of the signal peptide at residue 9 (p.A9D). Functional analysis in mutation carriers for the splice acceptor site mutation revealed a 50% decrease in GRN mRNA and protein levels, supporting haploinsufficiency. In contrast, there was no significant difference in the total GRN mRNA between cases and controls carrying the p.A9D mutation. Further, subcellular fractionation and confocal microscopy indicate that although the mutant protein is expressed, it is not secreted, and appears to be trapped within an intracellular compartment, possibly resulting in a functional haploinsufficiency.     

Organization of the mevalonate kinase (MVK) gene and identification of novel mutations causing mevalonic aciduria and hyperimmunoglobulinaemia D and periodic fever syndrome

Mevalonic aciduria (MA) and hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS) are two autosomal recessive inherited disorders both caused by a deficient activity of the enzyme mevalonate kinase (MK) resulting from mutations in the encoding MVK gene. Thus far, disease-causing mutations only could be detected by analysis of MVK cDNA. We now describe the genomic organization of the human MVK gene. It is 22 kb long and contains 11 exons of 46 to 837 bp and 10 introns of 379 bp to 4.2 kb. Three intron-exon boundaries were confirmed from natural splice variants, indicating the occurrence of exon skipping. Sequence analysis of 27 HIDS and MA patients confirmed all previously reported genotypes based on cDNA analysis and identified six novel nucleotide substitutions resulting in missense or nonsense mutations, providing new insights in the genotype/phenotype relation between HIDS and MA.     

Arylsulfatase A pseudodeficiency-associated mutations: Population studies and identification of a novel haplotype

Pseudodeficiency of arylsulfatase A is characterized by reduction of arylsulfatase A activity without neurodegeneration, making it an important complication when diagnosing metachromatic leukodystrophy. Two DNA substitutions are associated with arylsulfatase A pseudodeficiency. One, 1788A→G, results in the loss of an N-glycosylated asparagine in the protein, and the second, 2723A→G, removes the polyadenylation signal site of the mRNA. Previously, the polyadenylation signal site variant was observed only in the presence of the N-glycosylation site variant, although the latter has been reported to occur in the absence of the polyadenylation signal site variant. We investigated the frequencies of these alleles and their linkage disequilibrium in a number of populations and in psychiatric patients. While the N-glycosylation site variant had a high frequency in the Bantu-speaking people from Southern Africa (0.44), the San of Southern Africa (0.22), African Americans (0.37), and Cheyenne Indians (0.375), the polyadenylation signal site variant was absent in these groups. The mutated polyadenylation signal site was found only in the Caucasian groups surveyed. Two Caucasian sibs were identified with the pseudodeficiency polyadenylation signal site variant in the absence of the N-glycosylation site variant, indicating that linkage disequilibrium between the two polymorphisms is not perfect. © 1996 Wiley-Liss, Inc.     

MRX93 syndrome (BRWD3 gene): five new patients with novel mutations

Overgrowth syndromes (OGS) comprise a heterogeneous group of disorders whose main characteristic is that either the weight, height, or head circumference are above the 97th centile or 2 to 3 SD above the mean for age and sex. Additional features, such as facial dysmorphism, developmental delay or intellectual disability (ID), congenital anomalies, neurological problems and an increased risk of neoplasia are usually associated with OGS. Genetic analysis in patients with overlapping clinical features is essential, to distinguish between two or more similar conditions, and to provide appropriate genetic counseling and recommendations for follow up. In the present paper, we report five new patients (from four unrelated families) with an X-linked mental retardation syndrome with overgrowth (XMR93 syndrome), also known as XLID-BRWD3-related syndrome. The main features of these patients include ID, macrocephaly and dysmorphic facial features. XMR93 syndrome is a recently described disorder caused by mutations in the Bromodomain and WD-repeat domain-containing protein 3 (BRWD3) gene. This article underscores the importance of genetic screening by exome sequencing for patients with OGS and ID with unclear clinical diagnosis, and expands the number of reported individuals with XMR93 syndrome, highlighting the clinical features of this unusual disease.     

Molecular analysis of Turkish mucopolysaccharidosis IVA (Morquio A) patients: identification of novel mutations in the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) gene

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS; EC 3.1.6.4). The deficiency of N-acetylgalactosamine-6-sulfate sulfatase leads to lysosomal accumulation of undegraded glycosaminoglycans, keratan sulfate and chondroitin-6-sulfate. Mutation screening of the GALNS gene was performed by SSCP and direct sequence analyses using genomic DNA samples from 10 Morquio A patients. By nonradioactive SSCP screening, 6 different gene mutations and 2 polymorphisms were identified in 10 severely affected MPS IVA patients. Five of the mutations and one of the polymorphisms are novel. The vast majority of the gene alterations were found to be single nucleotide deletions (389delG, 929delG, and 763delT) or insertions (1232-1233insT). The other two mutations were one previously identified missense mutation (Q473X) and one novel nonsense (P179S) mutation. Together they account for 95% of the disease alleles of the patients investigated. Beside mutations, one previously identified E477 polymorphism and one novel W520 polymorphism were found among Turkish MPS IVA patients.