Variable phenotypes and outcomes associated with the MMACHC c.482G > A mutation: follow-up in a large CblC disease cohort.

BACKGROUND: The aim of this study was to characterize the variable phenotypes and outcomes associated with the methylmalonic aciduria and homocystinuria type C protein gene (MMACHC) c.482G > A mutation in 195 Chinese cases with CblC disease. METHODS: We carried out a national, retrospective multicenter study of 195 Chinese patients with CblC disease attributable to the MMACHC c.482G > A variant either in a homozygous or compound heterozygous state. The control group consisted of 200 patients diagnosed with CblC disease who did not possess the c.482G > A mutation. Clinical features, including disease onset, symptoms, biochemical metabolites, gene mutation, and follow-up outcomes were reviewed and analyzed in detail. The median follow-up period spanned 3 years and 8 months, with a range of 1 year and 2 months to 12 years and 10 months. RESULTS: Among 195 patients carrying the c.482G > A variant, 125 (64.1%) cases were diagnosed by newborn screening (NBS), 60 (30.8%) cases were detected due to disease onset, and 10 (5.1%) cases were identified from sibling diagnoses. One hundred and seventeen (93.6%) individuals who were diagnosed by NBS, and nine patients who came from sibling diagnoses remained asymptomatic in this study. From 69 symptomatic patients of the c.482G > A group, more patients presented with later onset, and the top six common clinical symptoms at disease onset were developmental delay (59.4%), lower limb weakness and poor exercise tolerance (50.7%), cognitive decline (37.7%), gait instability and abnormal posture (36.2%), seizures (26.1%), and psychiatric and behavioral disturbances (24.6%). In the 159 symptomatic patients lacking c.482G > A variants, the most frequently observed clinical manifestations at disease onset included developmental delay (81.8%), lethargy and feeding difficulty (62.9%), lower limb weakness and poor exercise tolerance (54.7%), prolonged neonatal jaundice (51.6%), vomiting (47.2%), and seizures (32.7%). Before treatment, the levels of blood propionylcarnitine, propionylcarnitine/acetylcarnitine ratio, and homocysteine in the c.482G > A group were significantly lower (P < 0.05) than those in the non-c.482G > A group, while the concentration of urinary methylmalonic acid was slightly lower (P > 0.05). The degree of decline in the above metabolites after treatment in different groups significantly differed in both plasma total homocysteine values and urinary methylmalonic acid levels (P < 0.05). In patients carrying the c.482G > A variant compared with the non-c.428G > A group, there were markedly lower rates of mortality (0.5% vs. 2.0%) and developmental delay (20.5% vs. 65.5%). When compared with individuals diagnosed due to disease onset, those identified through NBS in either group exhibited a reduced proportion of disease onset (6.7% vs. 100% in the c.482G > A group, 54.4% vs. 100% in the non-c.482G > A group), lower mortality (0.0% vs. 1.7% in the c.482G > A group, 0.0% vs. 3.6% in the non-c.482G > A group), and had a higher percentage of patients exhibiting normal psychomotor and language development (99.3% vs. 33.3% in the c.482G > A group, 58.9% vs. 10.9% in the non-c.482G > A group). CONCLUSIONS: The c.482G > A variant in MMACHC is associated with late-onset and milder phenotypes of CblC disease. Patients with this mutation tend to have a relatively better response to hydroxocobalamin, better metabolic control, and more favorable neurological outcomes. NBS and other appropriate pre-symptomatic treatments seem to be helpful in early diagnosis, resulting in favorable clinical outcomes. Video Abstract (MP4 136794 kb).

Living-donor liver transplantation for children with tyrosinemia type I.

OBJECTIVE: To evaluate the efficacy of living-donor liver transplantation (LDLT) in children with tyrosinemia type I. METHODS: Altogether 10 patients diagnosed with tyrosinemia type I underwent LDLT between June 2013 and April 2019. Cirrhosis was the indication for LDLT in all 10 patients, and hepatocellular carcinoma (HCC) was suspected in nine. Patients' outcomes, including liver function, restoration of metabolism, quality of life and physical development, were analyzed after LDLT. RESULTS: All recipients were alive with a normal liver function after a median follow-up period of 49 months. Pathological examinations detected HCC in one patient, dysplasia in five and cirrhosis in all. Nine patients were found to have elevated alpha-fetoprotein level, and their median alpha-fetoprotein level dropped from 2520 ng/mL to a normal level after LDLT, with no recurrence of HCC detected during the follow-up. Tyrosine metabolism was restored to its normal level with normalized plasma tyrosine and succinylacetone concentrations. Moreover, urinary succinylacetone excretion decreased significantly during the follow up. LDLT improved patients' renal tubular function, as evidenced by the normalized plasma phosphate concentration and improved glomerular filtration rate. Severe rickets symptoms, including spontaneous fractures and bone pain, were also ameliorated. Improved motor function was reported by all patients' parents during the follow-up. Dietary restriction was no longer required, which was associated with a favorable catch-up in growth and improved quality of life. Complete resolution of hypertrophic cardiomyopathy was observed one year after LDLT in one patient. CONCLUSION: LDLT is an effective treatment for patients with end-stage liver disease resulting from tyrosinemia type I.

Identification of predominant GNPTAB gene mutations in Eastern Chinese patients with mucolipidosis II/III and a prenatal diagnosis of mucolipidosis II.

Mucolipidosis II α/ß, mucolipidosis III α/ß, and mucolipidosis III γ are autosomal recessive disorders belonging to the family of lysosomal storage disorders caused by deficiency of the UDP-N-acetylglucosamine, a lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) localized in the Golgi apparatus, which is essential for normal processing and packaging of soluble lysosomal enzymes with initiating the first step of tagging lysosomal enzymes with mannose-6-phosphate (M6P). Mucolipidosis II and III are caused by mutations in the GNPTAB and GNPTG genes, and patients with these diseases are characterized by short stature, skeletal abnormalities, and developmental delay. In this study we report 38 patients with mucolipidosis II and III enrolled in Eastern China during the past 8 years. The diagnosis was made based on clinical characteristics and measurement of plasma lysosomal enzyme activity. Sanger sequencing of GNPTAB and/or GNPTG for all patients and real-time quantitative PCR were performed to confirm the diagnosis. In addition, 11 cases of prenatal mucolipidosis II were diagnosed based on measurement of the enzyme activity in amniotic fluid supernatant and genetic testing of cultured amniotic cells. Based on molecular genetic tests, 30 patients were diagnosed with mucolipidosis II α/ß, 6 were diagnosed with III α/ß and 2 were diagnosed with III γ. Thirty-seven different GNPTAB gene mutations were identified in 29 patients with mucolipidosis II α/ß and six patients with III α/ß. These mutations included 22 new mutations (p.W44X, p.E279X, p.W416X, p.W463X, p.Q802X, p.Q882X, p.A34P, p.R334P, p.D408N, p.D534N, p.Y997C, p.D1018V, p.L1025S, p.L1033P, c.88_89delAC, c.890_891insT, c.1150_1151insTTA, c.1523delG, c.2473_2474insA, c.2980_2983delGCCT, c.3094delA, and deletion of exon 9). Four new GNPTG gene mutations were identified (c.13delC, p.Y81X, p.G126R and c.609+1delG) in two mucolipidosis III γ patients. Among the 11 cases of prenatal diagnosis, four were mucolipidosis II fetuses, three were heterozygous, and the remaining four were normal fetuses. This study expands the mutation spectrum of the GNPTAB and GNPTG genes and contributes to specific knowledge of mucolipidosis II/III in a population from Eastern China.

Application of isoxanthopterin as a new pterin marker in the differential diagnosis of hyperphenylalaninemia.

BACKGROUND: This study aimed to explore the value of applying a new pterin marker (isoxanthopterin) to the traditional urine pterin analysis to reduce the rate of mis-diagnosis of 6-pyruvoyltetrahydropterin synthase deficiency (PTPSD) and improve the accuracy of diagnosis. METHODS: We compared the urine neopterin (N), biopterin (B), isoxanthopterin (Iso), B% and Iso% levels between patients with phenylalanine hydroxylase deficiency and those with PTPSD, and found the most specific pterin biomarkers by ROC analysis. A positive cut-off value of urine pterins was determined. The effect of combined Iso% + B + B% in reducing PTPSD mis-diagnosis was evaluated, and the different urine pterin levels in PTPSD and false PTPSD (FPTPSD) were compared. The concordance of PTPSD diagnosis by the new pterin scheme and gene mutation analysis was determined. RESULTS: (1) Urinary B, B%, Iso and Iso% were significantly lower in PTPSD than those in phenylalanine hydroxylase-deficiency group (P < 0.01); (2) Iso%, B%, and B were the most specific markers; (3) The positive cut-off values of B, B%, Iso% for PTPSD were < 0.17 mmoL/moLCr, < 5.0%, and < 9.5%, respectively; (4) urinary B + B% + Iso% scheme significantly reduced the false-positive rate of PTPSD compared to traditional ones. The Iso% levels in FPTPSD group were higher than the ones in PTPSD group; (5) an accuracy of diagnosis for PTPSD was increased by 9-19% when Iso% was introduced to urinary pterin scheme. CONCLUSIONS: Iso% is helpful to reduce the rate of misdiagnosis of PTPSD in the diagnosis by urinary pterin analysis for hyperphenylalaninemias and improve the accuracy of diagnosis. This approach is worthy of further development and increased utilization.

Eight novel MUT 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. CONCLUSIONS: 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.

Clinical features and MUT gene mutation spectrum in Chinese patients with isolated methylmalonic acidemia: identification of ten novel allelic variants.

BACKGROUND: This study aims to study MUT gene mutation spectrum in Chinese patients with isolated methylmalonic academia (MMA) and their clinical features for the potential genotype-phenotype correlation. METHODS: Forty-three patients were diagnosed with isolated MMA by elevated blood propionylcarnitine, propionylcarnitine to acetylcarnitine ratio, and urine methylmalonate without hyperhomocysteinemia. The MUT gene was amplified by polymerase chain reaction and directly sequenced. Those patients with at least one variant allele were included. The novel missense mutations were assessed by bioinformatic analysis and screened against alleles sequenced from 50 control participants. RESULTS: Among the 43 patients, 38 had typical clinical presentations, and the majority (30/38) experienced earlyonset MMA. Eight patients died and seven were lost to follow-up. Twenty patients had poor outcomes and eight showed normal development. The 43 identified MUT gene mutations had at least one variant allele, whereas 35 had two mutant alleles. Of the 33 mutations reported before, eight recurrent mutations were identified in 32 patients, and c.729_730insTT (p.D244Lfs*39) was the most common (12/78) in the mutant alleles. Of the 10 novel mutations, six were missense mutations and four were premature termination codon mutations. The six novel missense mutations seemed to be pathogenic. CONCLUSIONS: A total of 10 novel MUT mutations were detected in the Chinese population. c.729_730insTT (p.D244Lfs*39) was the most frequent mutation. A genotype-phenotype correlation could not be found, but the genotypic characterization indicated the need of genetic counseling for MMA patients and early prenatal diagnoses for high-risk families.

Eight novel mutations in the ABCD1 gene and clinical characteristics of 25 Chinese patients with X-linked adrenoleukodystrophy.

BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) is a fatal neurodegenerative disease caused by mutations in the adenosine triphosphate-binding cassette D1 (ABCD1) gene. This study aimed to retrospectively investigate the clinical characteristics of 25 patients with X-ALD including members of large pedigrees, to analyze ABCD1 gene mutations, the effect of gene novel variants on ALD protein (ALDP) structure and function, and to expand gene mutation spectrum of Chinese patients. METHODS: Twenty-five male patients diagnosed with X-ALD were enrolled in this study. The clinical characteristics of the patients were retrospectively summarized by reviewing medical records or telephone consultation. ABCD1 gene mutations were analyzed. The pathogenicity of novel missense variants was analyzed using cobalt constraint-based multiple protein alignment tool, polymorphism phenotyping, sorting intolerant from tolerant, Align-Grantham variation and Grantham deviation, and Swiss-Program Database Viewer 4.04 software, respectively. RESULTS: Childhood cerebral form ALD (CCALD) is the most common phenotype (64%) in the 25 patients with X-ALD. The progressive deterioration of neurological and cognitive functions is the main clinical feature. The demyelination of the brain white matter and elevated plasma very long chain fatty acids (VLCFAs) were found in all patients. Different phenotypes were also presented within family members of the patients. Twenty-two different mutations including 8 novel mutations in the ABCD1 gene were identified in the 25 patients. Of the mutations, 63.6% were missense mutations and 34.8% located in exon 1. The amino acid residues of three novel missense mutations in eight species were highly conserved, and were predicted to be "probably" damaging to ALDP function. The other five novel mutations were splice, nonsense, deletion or duplication mutations. CONCLUSIONS: CCALD is the most common phenotype (64%) in our patients with X-ALD. Eight novel mutations in the ABCD1 gene identified are disease-causing mutations. Brain magnetic resonance imaging and plasma VLCFA determination should be performed for the patients who present with progressive deterioration of neurological development.

QDPR gene mutation and clinical follow-up in Chinese patients with dihydropteridine reductase deficiency.

BACKGROUND: This study aimed to investigate the mutation spectrum of the QDPR gene, to determine the effect of mutations on dihydropteridine reductase (DHPR) structure/function, to discuss the potential genotypephenotype correlation, and to evaluate the clinical outcome of Chinese patients after treatment. METHODS: Nine DHPR-deficient patients were enrolled in this study and seven of them underwent neonatal screening. QDPR gene mutations were analyzed and confirmed by routine methods. The potential pathogenicity of missense variants was analyzed using Clustal X, PolyPhen program and Swiss-PDB Viewer 4.04_OSX software, respectively. The clinical outcomes of the patients were evaluated after long-term treatment. RESULTS: In 10 mutations of the 9 patients, 4 were novel mutations (G20V, V86D, G130S and A175R), 4 were reported by us previously, and 2 known mutations were identified. R221X was a hotspot mutation (27.7%) in our patients. Eight missense mutations probably had damage to protein. Six patients in this series were treated with a good control of phenylalanine level. The height and weight of the patients were normal at the age of 4 months to 7.5 years. Four patients, who underwent a neonatal screening and were treated early, showed a normal mental development. In 2 patients diagnosed late, neurological symptoms were significantly improved. CONCLUSIONS: The mutation spectrum of the QDPR gene is different in the Chinese population. Most mutations are related to severe phenotype. The determination of DHPR activity should be performed in patients with hyperphenylalaninemia. DHPR-deficient patients who were treated below the age of 2 months may have a near normal mental development.

Clinical features and mutations in seven Chinese patients with very long chain acyl-CoA dehydrogenase deficiency.

BACKGROUND: Very long chain acyl-CoA dehydrogenase deficiency (VLCADD) is an inherited metabolic disease caused by deleterious mutations in the ACADVL gene that encodes very long chain acyl-CoA dehydrogenase (VLCAD), and which can present as cardiomyopathy in neonates, as hypoketotic hypoglycemia in infancy, and as myopathy in late-onset patients. Although many ACADVL mutations have been described, no prevalent mutations in the ACADVL gene have been associated with VLCADD. Herein, we report the clinical course of the disease and explore the genetic mutation spectrum in seven Chinese patients with VLCADD. METHODS: Seven Chinese patients, from newborn to 17 years old, were included in this study. Tandem mass spectrometry was performed to screen for VLCAD deficiency. All exons and flanking introns of the ACADVL gene were analyzed using polymerase chain reaction and direct sequencing. Online analysis tools were used to predict the impact of novel mutations. RESULTS: All cases had elevated serum levels of tetradecanoylcarnitine (C14:1) which is the characteristic biomarker for VLCADD. The phenotype of VLCADD is heterogeneous. Two patients were hospitalized for hypoactivity and hypoglycemia shortly after birth. Three patients showed hepatomegaly and hypoglycemia in infancy. The other two adolescent patients showed initial manifestations of exercise intolerance or rhabdomyolysis. Three of the patients died at the age of 6-8 months. Eleven different mutations in the ACADVL gene in the 7 patients were identified, including seven reported mutations (p.S22X, p.W427X, p.A213T, p.G222R, p.R450H, c.296-297delCA, c.1605+1G>T) and four novel mutations (p.S72F, p.Q100X, p.M437T, p.D466Y). The p.R450H and p.D466Y (14.28%, 2/14 alleles) mutations were identified in two alleles respectively. CONCLUSIONS: The clinical manifestations were heterog-eneous and ACADVL gene mutations were heterozygous in the seven VLCADD Chinese patients. R450H may be a relatively common mutation in Asian populations. The genotype and phenotype had a certain correlation in our patients.

[Analysis of GALNS gene mutation in thirty-eight Chinese patients with mucopolysaccharidosis type IVA].

OBJECTIVE: Mucopolysaccharidosis (MPS) type IVA (MPS IVA) is an autosomal recessive lysosomal storage disease caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) needed to degrade glycosaminoglycanes (GAGs), accumulation of GAGs in the tissue resulting in disorder of function. So far, the small number of articles about clinical study of Chinese MPS IVA were published and only one paper about gene mutation analysis was published. This study aimed to investigate the mutation spectrum and characteristic of GALNS gene in Chinese patients with MPS IVA who were diagnosed in our hospital. METHOD: Thirty-eight patients from 36 families (male 17, female 21) were diagnosed as MPS IVA by GALNS activity determination [(0.85 ± 1.33) nmol/(17 h·mg)] and clinical symptoms during 2006-2012. The average age of diagnosis was (5.7 ± 3.6) years. Mutation analysis of GALNS gene performed performed by PCR-direct DNA sequencing for 38 patients. PCR-restriction fragment length polymorphism analysis was used for validating novel mutation, and also to assess amino acid conservation for novel missense variants in five different species. PolyPhen-2 tool was used to predict the possible impact of missense mutations on the structure and function of the human GALNS protein, etc. Analysis of GALNS activity and gene mutation in amniotic fluid were performed to provide the prenatal diagnosis for some families with MPS type IVA. RESULT: (1) Thirty-eight kinds of mutation in GALNS gene were identified in 38 patients of them, 71% were missense mutations. p. M318R was a hot-spot mutation (21%) tested. Five kinds of mutation i.e., p. P163H, p.G168L, p. A324E, p. L366P and p. F452L were only found in Chinese patients with MPS IVA. Eighteen kinds of novel mutation were detected including p. E315K, p.G304D, p.R251Q, p.Y240C, p.G161E, p.N32D, p.L390P, p. D60E, p. P420S, W403C/T404S, p.L454P, for p.W405X, p. M1I, c.409_ c.420del12, c.1176_1178del3, c.1046delG, c.1188delG and IVS9-2A>C. (2) The polymorphism of novel missense variants were ruled out by the PCR-restriction fragment length polymorphism analysis and no related mutations were found in 50 normal controls. A splice site mutation IVS9-2A>C had been validated by reverse transcription PCR direct sequencing. The amino acid of mutant position of 10 kinds of missense variants are highly conserved and only p. L454 is moderately conserved position. These missense variants were predicted to cause damage to the structure and function of human GALNS protein possibly according to the PolyPhen-2 tool, so these novel missense variants may be disease-causing mutations. (3) Prenatal diagnosis was provided for 7 families and three fetuses were diagnosed as MPS IVA. CONCLUSION: The GALNS gene mutation spectrum in Chinese patients with MPS IVA is really different from that in other countries, five kinds of mutation were only found in Chinese patients with MPS IVA. The reports of hot-spot mutation in Chinese patients were also different, and should be analyzed by more data of gene mutation analysis and epidemiological study.

[Clinical and mutational features of maternal 3-methylcrotonyl coenzyme deficiency].

OBJECTIVE: To report on 5 patients with maternal 3-methylcrotonyl coenzyme A carboxylase deficiency (MCCD) and to confirm the clinical diagnosis through mutation analysis. METHODS: Five neonates with higher blood 3-hydroxy isovalerylcarnitine (C5-OH) concentration detected upon newborn screening with tandem mass spectrometry and their mothers were recruited. Urinary organic acids were analyzed with gas chromatography mass spectrometry. Gene mutation and protein function analysis were performed by PCR direct sequencing and PolyPhen-2 software. RESULTS: Higher blood C5-OH concentrations (5.11-21.77 µmol/L) and abnormal 3-hydroxy isovalerate and 3-methylcrotonyl glycine in urine were detected in the five asymptomatic mothers, who were diagnosed as benign MCCD. Higher C5-OH concentration was also detected in their neonates by tandem mass spectrometry, which had gradually decreased to normal levels in three neonates. Four new variations, i.e., c.ins1680A(25%), c.203C > T (p.A68V), c.572T > C (p.L191P) and c.639+5G > T were detected in the MCCC1 gene, in addition with 2 mutations [c.1406G > T (p.R469L, novel variation) and c.592C > T (p.Q198X)]. The novel variations were predicted to have affected protein structure and function. CONCLUSION: For neonates with higher C5-OH concentration detected upon neonatal screening, their mothers should be also tested to rule out MCCD. Mutations in MCCC1 gene are quite common.

[Diagnosis and treatment of isolated methylmalonic acidemia].

OBJECTIVE: To explore the clinical feature, therapeutic effect and prognosis of isolated methylmalonic acidemia. METHODS: The clinical characteristics, laboratory findings, treatment and outcome of 40 patients were retrospectively analyzed. The main treatment was a low-protein diet supplemented with L-carnitine and special milk free of leucine, valine, threonine and methionine. Vitamin B12 was also given to cobalamin responders. The patients were followed up every 1-3 months. RESULTS: Mutations in the MUT gene were identified in 30 of 33 patients who had accepted DNA testing. Thirty cases were treated and followed up regularly for from 1 month to 8 years. Eight cases had died, 8 had developed normal intelligence, among whom 4 from newborn screening were asymptomatic. Psychomotor developmental delay and mental retardation were present in 14 cases. The propionylcarnitine level, ratio of propionylcarnitine/acetylcarnitine in blood, methylmalonic acid and methylcitric acid levels in urine have decreased significantly, with the median values reduced respectively from 24.15 (7.92-81.02) µmol/L, 1.08 (0.38-6.01), 705.34 (113.79-3078.60) and 7.71 (0.52-128.21) to 10.50 (3.00-30.92) µmol/L, 0.63 (0.25-2.89), 166.23 (22.40-3322.21) and 3.96 (0.94-119.13) (P < 0.05). CONCLUSION: The prognosis of isolated methylmalonic acidemia may be predicted with the enzymatic subgroup, age at onset and cobalamin responsiveness. Outcome is unfavorable in neonatal patients and those who were non-responsive to cobalamin.

[Outcomes of patients with combined methylmalonic acidemia and homocystinuria after treatment].

OBJECTIVE: Combined methylmalonic acidemia with homocystinuria is a common form of methylmalonic acidemia in China. Patients with this disease can progress to death without timely and effective treatment. This study aimed to analyze the treatment outcomes of patients with combined methylmalonic acidemia and homocystinuria. METHOD: From September 2004 to April 2012, 58 patients with combined methylmalonic acidemia and homocystinuria (34 males and 24 females) were diagnosed and treated in our hospital. Fifty cases were from clinical patients including 42 early-onset cases and 8 late-onset cases. Their age when they were diagnosed ranged from 18 days to 30.8 years. The other 8 cases were from newborn screening. All the patients were treated with vitamin B12, betaine, folic acid, vitamin B6, and L-carnitine. The physical and neuropsychological development, general laboratory tests, the levels of amino acids, acylcarnitines, and homocysteine in blood, and organic acids in urine were followed up. RESULT: The follow-up period ranged from 1 month to 7.1 years. Three cases died (all were early-onset cases). In the other patients after treatment, the symptoms such as recurrent vomiting, seizures, lethargy, and poor feeding disappeared, muscle strength and muscle tension were improved, and general biochemical abnormalities such as anemia and metabolic acidosis were corrected. Among the surviving 55 cases, 49 had neurological impairments such as developmental delay and mental retardation. The median levels of blood propionylcarnitine and its ratio with acetylcarnitine, serum homocysteine, and urine methylmalonic acid were significantly decreased (P < 0.01), from 7.73 µmol/L (ranged from 1.5 to 18.61 µmol/L), 0.74 (ranged from 0.29 to 2.06), 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 2.74 µmol/L (ranged from 0.47 to 12.09 µmol/L), 0.16 (ranged from 0.03 to 0.62), 43.8 µmol/L (ranged from 17 to 97.8 µmol/L) and 6.81 (ranged from 0 to 95.43) after treatment, respectively. CONCLUSION: Patients with combined methylmalonic acidemia and homocystinuria respond to a combined treatment consisting of supplementation of hydroxycobalamin, betaine, folic acid, vitamin B6 and L-carnitine with clinical and biochemical improvement. But the long-term outcomes are unsatisfactory, with neurological sequelae in most patients.

[Analysis of propionylcarnitine in blood and methylmalonic acid in urine of 162 patients with methylmalonic acidemia].

OBJECTIVE: To analyze the levels of methylmalonic acid and methylcitrate in urine, propionylcarnitine (C3) in plasma and C3/acetylcarnitine (C2) of patients with methylmalonic acidemia (MMA) and explore their applications in the diagnosis of MMA. METHODS: From December 2003 to March 2012, a total of 162 patients with MMA (MMA group) and 200 healthy children (control group) of Xinhua Hospital, Shanghai Jiaotong University School of Medicine were recruited. MMA patients with a definite classification were divided into 2 groups: isolate MMA group (n = 51) and MMA complicated with homocysteinemia group (n = 65). Gas chromatography-mass spectrometry was used to measure the urine levels of methylmalonic acid and methylcitrate and tandem mass spectrometry to measure the blood levels of free carnitine (C0), acylcarnitines and methionine (Met). RESULTS: In the MMA group, the median levels of methylmalonic acid (259.10 (6.73 - 6429.28)), methylcitrate (4.39 (0 - 248.96)), C3 (8.52 (1.50 - 52.11) µmol/L) and C3/C2 (0.73(0.28 - 2.89)) were all higher than the upper limit values (0.2 - 3.6, 0 - 1.1, 0.50 - 4.00 µmol/L and 0.04 - 0.25 respectively). And they were all higher than those in the control group (0 (0 - 1.87), 0.10 (0 - 1.84), 1.40 (0.53 - 3.90) µmol/L, 0.10 (0.04 - 0.23), all P < 0.01). C3/C2 increased significantly in 15 patients while the C3 level remained normal. The median level of Met was normal in the isolate MMA group. But in patients with homocysteinemia, the level of 8.71 (0.68 - 31.95) µmol/L was below the reference value (10.00 - 35.00 µmol/L) and lower than that in the isolate MMA group (15.35 (4.18 - 59.50) µmol/L) and the control group (15.59 (10.20 - 34.68) µmol/L, all P < 0.05). CONCLUSIONS: Significant increases in the urine level of methylmalonic acid and C3/C2 may be specific to MMA. Organic acid analyses of gas chromatography-mass spectrometry and acylcarnitines with tandem mass spectrometry are required for a definite diagnosis of this disorder. And repeated tests and genomic mutation analysis are necessary for patients with mildly abnormal biochemical indices.

[Analysis of clinical data and genetic mutations in three Chinese patients with tyrosinemia type I].

OBJECTIVE: To analyze clinical data and gene mutations in 3 Chinese patients with tyrosinemia type I, and to explore the correlation between genotypes and phenotypes. METHODS: Three patients suspected with tyrosinemia I were tested by tandem mass spectrometry for the level of tyrosine, phenylalanine and succinylacetone in the blood, and by gas chromatography-mass spectrometry to determine the level of succinylacetone and organic acid in their urine. With the diagnosis established, the FAH gene was analyzed with polymerase chain reaction (PCR) and direct sequencing. RESULTS: Two patients had acute onset of the disease, while another had subacute onset of the disease, with features including hepatomegaly and remarkably increased tyrosine and succinylacetone in the blood. Five mutations were detected in the FAH gene, which included c.455G>A (W152X), c.520C>T (R174X), c.974_976delCGAinsGC, c.1027 G>A (G343R) and c.1100 G>A (W367X), among which c.455G>A (W152X), c.974_976delCGAinsGC and c.1100 G>A (W367X) were not reported previously. CONCLUSION: Tyrosinemia type I may be effectively diagnosed with the level of tyrosine and succinylacetone by tandem mass spectrometry and succinylacetone in the urine by gas chromatography mass spectrometry. Detection of underlying mutations mutations will be helpful for genetic counseling and further research.

Beta-galactosidase deficiencies and novel GLB1 mutations in three Chinese patients with Morquio B disease or GM1 gangliosidosis.

BACKGROUND: This paper aims to report GLB1 activities and mutation analysis of three patients from the mainland of China, one with Morquio B disease and two with GM1 gangliosidosis. METHODS: GLB1 activity and GLB1 gene mutation were analyzed in the three patients who were clinically suspected of having Morquio B disease or GM1 gangliosidosis. Novel mutations were analyzed by aligning GLB1 homologs, 100 control chromosomes, and the PolyPhen-2 tool. RESULTS: The enzymatic activity of GLB1 was found to be 5.03, 4.20, and 4.50 nmol/h/mg in the three patients, respectively. Patient 1 was a compound heterozygote for p.[Arg148Cys] and p.[Tyr485Cys] mutations in the GLB1 gene. Patient 2 was a compound heterozygote for p.[Tyr270Phe] and p.[Leu337Pro] mutations. Patient 3 was a homozygote for p.[Asp448Val] mutation. Three mutations (p.[Tyr485Cys], p.[Tyr270Phe] and p.[Leu337Pro]) were novel variants and were predicted to damage GLB1 function. CONCLUSIONS: The enzymatic activity and related gene analysis of ß-galactosidase should be performed in clinically suspected individuals to confirm diagnosis. The three novel mutations, p.[Tyr485Cys], p.[Tyr270Phe], and p.[Leu337Pro], are thought to be disease-causing mutations.

[Primary carnitine deficiency in 17 patients: diagnosis, treatment and follow up].

OBJECTIVE: Many children were found to have low free carnitine level in blood by tandem mass spectrometry technology. In some of the cases the problems occurred secondary to malnutrition, organic acidemia and other fatty acid oxidation metabolic diseases, and some of cases had primary carnitine deficiency (PCD). In the present article, we discuss the diagnosis of PCD and evaluate the efficacy of carnitine in the treatment of PCD. METHOD: We measured the free carnitine (C0) and acylcarnitine levels in the blood of 270 000 neonates from newborns screening program and 12 000 children with suspected clinical inherited metabolic diseases by tandem mass spectrometry. The mutations of carnitine transporter protein were tested to the children with low C0 level and the diagnosis was made. The children with PCD were treated with 100 - 300 mg/kg of carnitine. RESULT: Seventeen children were diagnosed with PCD, 6 from newborn screening program and 11 from clinical patients. Mutations were found in all of them. The average C0 level [(2.9 ± 2.0) µmol/L] in patients was lower than the reference value (10 µmol/L), along with decreased level of different acylcarnitines. The clinical manifestations were diverse. For the 6 patients from newborn screening, 4 were asymptomatic, 1 showed hypoglycaemia and 1 showed movement intolerance from 2 years of age. For the 11 clinical patients, 8 showed hepatomegaly, 7 showed myasthenia, 6 showed cardiomyopathy, 1 showed chronic abdominal pain, and 1 showed restlessness and learning difficulty. Among these patients, 14 cases were treated with carnitine. Their clinical symptoms disappeared 1 to 3 months later. The C0 level in the blood rose to normal, with the average from (4.0 ± 2.7) µmol/L to (20.6 ± 8.3) µmol/L (P < 0.01). However, the level was still lower than the average level of healthy children [(27.1 ± 4.5) µmol/L, P < 0.01]. CONCLUSION: Seventeen patients were diagnosed with PCD by the test levels of free carnitine and acylcarnitines in blood with tandem mass spectrometry, and gene mutation test. Large dose of carnitine had a good effect in treatment of the PCD patients.

[GLB1 gene mutation and clinical characteristics of a patient with mucopolysaccharidosis type IVB].

OBJECTIVE: To report the results of clinical characteristics, enzyme activity determination and mutation analysis of GLB1 gene in a Chinese patient with mucopolysaccharidosis (MPS) type IVB (Morquio B disease). METHOD: A 14-year-old Chinese boy with MPS type IVB was firstly diagnosed by blood leucocytes galactosamine-6-sulfate sulfatase (GALNS) and ß-galactosidase (GLB1) determination, who was characterized by short stature, multiplex skeletal abnormalities, difficulty in walking. PCR-sequencing analysis was applied to detect the mutations in GLB1 of the patient. RESULT: The patient was characterized by dwarfism, pectus carinatum, kyphosis, normal intelligence, and no neurologic damage of spasms, linguistic capacity and so on. The patient had normal GALNS enzyme activity and very low GLB1 enzyme activity [5.03 nmol/(h·mg) vs. normal value 118 - 413 nmol/(h·mg) ] in leukocytes. A compound heterozygous missense mutations c.442C > T(p.R148C)/c.1454A > G(p.Y485C) in GLB1 gene were detected in this patient. The mutation p.Y485C is a novel variant. With the method of gene analysis of new variant, the mutation p.Y485C was considered to be a pathogenic mutation. CONCLUSION: The MPS IVB patient showed severe multiple skeletal deformities, normal intelligence, no neurologic damage and very low GLB1 enzyme activity, who carries compound heterozygous mutations p.R148C/p.Y485C. The mutation p.Y485C in GLB1 gene may be a novel pathologic mutation of MPS type IVB.