Clinical presentation, molecular analysis and follow-up of patients with mut methylmalonic acidemia in Shandong province, China

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Pediatrics and Neonatology xxx (xxxx) xxx

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Original Article

Clinical presentation, molecular analysis and follow-up of patients with mut methylmalonic acidemia in Shandong province, China Bingjuan Han a,1, Wenying Nie a,1, Meng Sun a, Yingxia Liu a,b,*, Zhiyang Cao c,** a

Jinan Maternal and Child Care Hospital, Jinan 250001, Shandong Province, China Department of Immunology, Nanjing Medical University, Nanjing 211166, Jiangsu Province, China c Nanjing Jiangning Hospital, Affiliated to Nanjing Medical University, Nanjing 211100, Jiangsu Province, China b

Received Feb 26, 2019; received in revised form Jun 6, 2019; accepted Jul 24, 2019

Available online - - -

Keywords follow-up; isolated methylmalonic acidemia; methylmalonyl-CoA mutase; MUT

Background: The mut methylmalonic acidemia (MMA) caused by the deficiency of methylmalonyl-CoA mutase (MCM) activity, which results from defects in the MUT gene. The aim of this study was to summarize the clinical and biochemical data, spectrum of mutations, treatment regime and follow-up of patients with mut MMA from Jan 2013 to Dec 2017 in Shandong province, China. Methods: Twenty patients were diagnosed with isolated mut MMA by elevated C3, C3/C2, and urine methylmalonic acid levels without hyperhomocysteinemia. The MUT gene was amplified and sequenced. Most patients received treatment with specific medical nutrition and oral L-carnitine after diagnosis. Metabolic parameters, clinical presentation and mental development were followed up. Results: Among 20 patients with mut MMA, 14 had clinical presentations, and 12 presented in the neonatal period. Three patients died of metabolic crises triggered by infection. Twenty-three different mutations were detected, and four mutations (c.613G > A, c.446A > G, c.920923delTCTT and c.1359delT) were novel. Most patients received timely treatment and had favorable metabolic responses, with reductions in C3, C3/C2 and urine MMA. We obtained 16 records of DQ/IQ assessments. Six patients exhibited normal development, but ten patients suffered from neurological symptoms of varying degrees and had low DQ/IQ scores.

Abbreviations: MMA, Methylmalonic acidemia; MCM, methymalonyl-CoA mutase; AdoCbl, adenosylcobalamin; C3, propionylcarnitine; C2, acetylcarnitine; BCAA, branched-chain amino acid; DQ, developmental quotient; IQ, intelligence quotient; ALT, aspartate transaminase; AST, alanine transaminase. * Corresponding author. No. 101, Longmian Avenue, Jiangning District, Nanjing, Jiangsu Province, China. Fax: þ86 25 86868499. ** Corresponding author. No. 168, Gushan Road, Jiangning District, Nanjing, Jiangsu Province, China. Fax: þ86 25 52281256. E-mail addresses: [email protected] (Y. Liu), [email protected] (Z. Cao). 1 Contributed equally to this work. https://doi.org/10.1016/j.pedneo.2019.07.004 1875-9572/Copyright ª 2019, Taiwan Pediatric Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BYNC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article as: Han B et al., Clinical presentation, molecular analysis and follow-up of patients with mut methylmalonic acidemia in Shandong province, China, Pediatrics and Neonatology, https://doi.org/10.1016/j.pedneo.2019.07.004

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B. Han et al Conclusion: Our study contributes toward expanding the knowledge of the genetic basis of mut MMA. The c.914T > C was the most frequent mutation, and four novel mutations were detected. Patients diagnosed by newborn screening and treated at the presymptomatic stage may have better outcomes. However, these limited data do not allow any definitive statements on possible genotypeephenotype correlations that can influence the outcomes of mut MMA. Nonetheless, it is necessary for high-risk families to have early prenatal diagnoses. Copyright ª 2019, Taiwan Pediatric Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/).

1. Introduction Methylmalonic acidemia (MMA) consists of a group of autosomal recessive disorders affecting catabolic pathways of isoleucine, valine, methionine, threonine, thymine, and odd-carbon-number fatty acids, and MMA is characterized by the accumulation of methylmalonyl-CoA and methylmalonic acid in body fluids.1 MMA can result from two general classes of genetic defects: those termed mut, which involve defects in methylmalonyl-CoA mutase (MCM), and those termed cbl, which involve defects in the synthesis of adenosylcobalamin (AdoCbl) as a cofactor.1 Isolated MMA is characterized by MMA without hyperhomocysteinemia and is caused by defects in MCM or by defects in its coenzyme, AdoCbl.2 Defects in AdoCbl leading to isolated MMA can be categorized into three groups: cblA, cblB, and cblD-variant 2. While mutations in the MUT gene are responsible for MCM deficiency, mutations in three genes (MMAA, MMAB, MMADHC ) are responsible for cblA, cblB, and cblD-variant 2 disease, respectively.3 MCM deficiency leads to the impaired metabolism of amino acids valine, isoleucine, methionine and threonine, as well as that of odd-chain fatty acids and the side chain of cholesterol. Defects in MCM can be divided into subgroups: mut0 with complete loss of MCM activity and mut with partial MCM activity.4 Mut0 manifests in patients as early as the neonatal period, with poor feeding, recurrent vomiting, lethargy, hypotonia, respiratory distress, progressive alterations in consciousness, and lifethreatening metabolic ketoacidosis. Without appropriate treatment, the progression of symptoms may result in deep coma, neurological damage (especially involving the basal ganglia) and, in some cases, death. Mut patients have a milder phenotype and present within 1e2 years of life. With appropriate therapy, most patients survive the acute metabolic crisis but still suffer from long-term complications, such as chronic renal failure, neurodevelopmental delay and neurological complications.5 The management of isolated MMA remains particularly challenging and controversial. The estimated incidence of MMA ranges from 1:48,000 to 1:250,000 worldwide.6 The MUT gene has been mapped to chromosome region 6p12.3, which consists of 13 exons spanning over 35 kb and encodes 750 amino acids.7 To date, 246 mutations have been identified in the MUT gene in various populations (Human Gene Database, http://www. hgmd.cf.ac.uk/ac/). Here, the aim of this study was to summarize the clinical and biochemical data, spectrum of

mutations, treatment regime and follow-up of patients with mut MMA from Jan 2013 to Dec 2017 in Shandong province, China.

2. Patients and methods 2.1. Patients and diagnosis Twenty patients were enrolled from Jinan Maternal and Child Care Hospital, Shandong province, between Jan 2013 and Dec 2017. The levels of propionylcarnitine (C3) and C3/ acetylcarnitine (C2) in the blood were measured with tandem mass spectrometry.8 To further confirm the diagnosis, the levels of organic acids in urine were measured with gas chromatography-mass spectrometry in suspected patients. In addition, the concentration of total plasma homocysteine was quantified. All tests were performed as routine clinical and biochemical investigations and in accordance with the ethical principles of the Declaration of Helsinki. Informed consent was obtained from the guardians of all patients for inclusion into the study. This study was approved by the ethical committee of Jinan Maternal and Child Care Hospital.

2.2. DNA sequence analysis Genomic DNA was extracted from the peripheral blood leukocytes of patients using a blood genomic DNA extraction kit. The entire coding sequence and flanking intronic regions of MUT were amplified by polymerase chain reaction. The primers used in this study are shown in Supplementary Table 1.9 Purified PCR products were sequenced. Mutation analysis was performed using the normal MUT genomic DNA (NM_000255.3) sequence as a reference.

2.3. Treatment and follow-up Metabolic treatments, including a high-calorie diet, reduced natural protein intake, medical foods, supplementation with special formulas (isoleucine-, methionine-, threonine- and valine-free formula), and oral L-carnitine, were given to most patients. To observe the effect of hydroxocobalamin (OHCbl), these patients were given a loading test of OHCbl (1 mg/day  3 days, by intramuscular injection). Responsiveness to intramuscular OHCbl was

Please cite this article as: Han B et al., Clinical presentation, molecular analysis and follow-up of patients with mut methylmalonic acidemia in Shandong province, China, Pediatrics and Neonatology, https://doi.org/10.1016/j.pedneo.2019.07.004

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Clinical Presentation and Molecular Analysis of patients with mut MMA defined as a decrease in urinary methylmalonate excretion of more than 30% or to an undetectable level. Changes in the levels of C3, the C3/C2 ratio, and urine MMA as well as mental development were monitored during treatment. The developmental quotient (DQ) was evaluated using the Revised Gesell Developmental Evaluation for children (<4 years), which provides a developmental profile in five domains, namely, adaptive, gross motor, fine motor, language, and personalesocial domains. A child was considered to have a developmental concern if his or her DQ in any specific domain was  75.10,11 The intelligence quotient (IQ) was assessed by the Wechsler Intelligence Scale for Children (WISC) for children (>4 years).12 Low intelligence was defined as an IQ score < 80.

2.4. In silico analysis MutationTaster was used to predict the significance of the novel mutations (missense, insertion and deletion mutations) (www.mutationtaster.org/).

3. Results Twenty patients representing nineteen families (cases 13 and 14 are siblings) were included in this study. The parents of all patients were healthy nonconsanguineous couples. The biochemical data are summarized in Table 1. All patients presented with elevated C3 (>5 mmol/L) and C3/C2 ratio (>0.2) in the blood. In addition, the patients demonstrated increased urine MMA (>3.6 mg/g Cr) (Table 1) and normal total plasma homocysteine levels (data not shown). Therefore, the patients were diagnosed with isolated MMA. Seven patients showed acute metabolic decompensation at first onset. They had elevated ammonia as well as a low pH and HCO3-. Isolated MMA can be classified as mut, cblA, cblB, or cblD-variant 2. Sequencing of the MUT gene is used to confirm the diagnosis of mut MMA. MUT mutations were found in the twenty patients. As shown in Table 1, 23 different mutations were identified. Thus, the diagnosis of mut MMA was confirmed in the patients. The most frequent mutation was c.914T > C (6/20). Four of these mutations had not been reported, including two missense mutations (c.613G > A and c.446A > G) and two deletion mutations (c.920-923delTCTT and c.1359delT). Moreover, in silico analysis was performed using a bioinformatics tool (MutationTaster). Bioinformatic analysis revealed that the novel mutations were predicted to be disease-causing mutations. Thirteen patients were diagnosed during the neonatal period, 10 by newborn screening (cases 1, 2, 3, 8, 9, 14, 15, 17, 19, and 20). Seven patients were diagnosed after the neonatal period between 2 months and 5 years of age. Four patients (cases 2, 3, 13, and 17) were identified by newborn screening before clinical symptoms were observed. Six patients exhibited symptoms when the screening results were returned. Case 13 was asymptomatic and diagnosed due to her younger brother (case 14, diagnosed by newborn screening) at the age of 5 years. The nine remaining patients were ascertained clinically. Most patients experienced their first metabolic crisis at ages between 2 days and 2 years and 8 months. The early-onset patients

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presented with clinical symptoms within 1 month after birth. Twelve patients experienced early-onset MMA. Three patients (cases 4, 8, and 15) died during acute metabolic crisis triggered by infection at the ages of 30 days, 2 months, and 1 year, respectively. Most late-onset patients had their first metabolic crises triggered by infections or vaccination. The initial symptoms frequently included recurrent vomiting, poor feeding, hypotonia, seizures, lethargy, failure to thrive, mental retardation and developmental delay. After diagnosis, most patients received treatment with specific medical nutrition (‘low-protein/high-energy’ diet combined with special formulas containing minimal to no valine, isoleucine, methionine or threonine) and oral Lcarnitine to prevent secondary carnitine deficiency as quickly as possible. Cases 13 and 14 received oral L-carnitine supplementation without a protein-restricted diet. In this study, all patients were unresponsive to OHCbl. Most patients who received treatment had a favorable metabolic response with reductions in C3, the C3/C2 ratio and urine MMA (Table 2). However, the metabolic parameters did not reach the levels of those of healthy people. No patient had undergone kidney or liver transplantation by the end of this study. Although the biochemical responses of the patients were favorable, their clinical outcomes were variable. The longterm outcomes were unsatisfactory despite conventional treatment and improvements in biochemical abnormalities. Three patients died of metabolic crisis. The parents of case 7 did not agree to have the assessment of DQ. The records of the DQ/IQ assessment were available for 16 patients (Table 3). The ages at the last visit ranged from 7 months to 8 years. Among four patients who were diagnosed by newborn screening and treated before onset, three patients (cases 2, 14, and 17) remained asymptomatic without acute decompensation episode, and case 3 exhibited normal development without neurological impairment. Four early-onset patients (cases 1, 9, 19, and 20), who had exhibited clinical symptoms when the screening results were returned, suffered from neurological symptoms of various degrees and had low DQ/IQ scores. The first metabolic crisis of case 10 was due to influenza vaccination, and the patient was then diagnosed as mut MMA at the age of 2 years and 8 months. This patient had normal DQ. Interestingly, case 13, who was diagnosed at the age of 5 years due to her affected younger brother and who did not receive any treatment before diagnosis, remained asymptomatic at the age of 8 years. Other surviving patients who were ascertained clinically presented with poor growth and neurological complications, such as mental retardation and motor dysfunction to varying degrees. None of the surviving patients had renal or other organ complications by the end of this study.

4. Discussion MMA is the most common form of organic acidemia in China.13 The mut MMA is responsible for a large number of cases of isolated MMA. In this study, we summarized the clinical course, mutational spectrum, treatment and follow-up data of twenty patients with mut MMA from

Please cite this article as: Han B et al., Clinical presentation, molecular analysis and follow-up of patients with mut methylmalonic acidemia in Shandong province, China, Pediatrics and Neonatology, https://doi.org/10.1016/j.pedneo.2019.07.004

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Biochemical data and genotypic features of twenty patients with mut MMA.

Case Gender Newborn Age of Age at C3a, mmol/L C3/C2 ratiob Urine Ammoniad,mmol/L pHe number screening onset diagnosis MMAc, mg/g Cr

HCO3-f ALTg ASTh MUT genotype

F M F

Yes Yes Yes

2d e 20d

3d 3d 10d

11.4 6.76 27.72

1.44 0.62 0.86

332.65 282.19 291.66

197 NA 52

7.36 21.1 NA NA 7.35 22.6

12 26 21

30 32 28

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

F F F M M M M F M F M F M F M F M

No No No No Yes Yes No No No No Yes Yes No Yes No Yes Yes

2d 6d 6m 10d 3d 2d 2y8m 10d 1y e e 4d 10m e 2d 3d 3d

10d 11m 6m 3m 10d 10d 2y8m 20d 1y2m 5y 5d 8d 1y5m 4d 4d 5d 6d

33.01 84.42 65.44 6.01 84.24 18.46 6.38 58.88 29.15 7.32 5.47 41.89 66.09 29.37 33.26 42.46 21.58

1.93 0.86 1.26 0.23 2.79 0.8 0.37 2.74 1.28 0.51 0.21 1.05 1.04 0.76 0.84 1.21 0.35

862.12 1046.33 897.21 43.77 1046.33 433 20.31 1188.29 154.07 45.43 44.64 956.53 616.59 329.2 286.55 443.05 126.8

307 205 167 161 328 202 60 178 210 NA NA 321 193 NA 103 188 145

7.12 7.30 7.29 7.37 7.06 7.19 7.35 7.16 7.35 NA NA 7.09 7.35 NA 7.35 7.38 7.36

38 48 35 39 36 29 22 17 56 23 19 35 58 28 32 35 28

46 57 32 47 41 39 29 32 67 29 29 47 72 32 37 38 34

13.6 18.7 20.1 22.9 7.8 16.4 23.0 15.8 19.2 NA NA 10.5 20.8 NA 21.1 20.2 20.9

c.914T > C/c.1106G > A c.1663G > A/c.914T > C c.1677-1G > A c.299A > G c.914T > C/c.729-730insTT c.920-923delTCTT/c.1741C > T c.1677-1G > A/c.914T > C c.1663G > A/c.613G > A c.322C > T/c.944dupT c.1106G > A/c.1359delT c.1106G > A/c.446A > G c.729-730insTT/c.729-730insTT c.914T > C/c.970G > A c.2168G > A/c.920-923delTCTT c.2168G > A/c.920-923delTCTT c.1777G > T/c.729-730insTT c.424A > G/c.636G > A c.323G > A/c.914T > C c.103C > T/c.322C > T c.1399C > T/c.755dupA c.1106G > A/c.441T > A

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1 2 3

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“-”: having no onset yet; NA: not assayed. a Normal range of C3 0.2e4.5 mmol/L. b Normal C3/C2 ratio <0.2. c Normal range of urine MMA 0.2e3.6 mg/g Cr. d Normal range of ammonia 10e47 mmol/L. e Normal range of pH 7.35e7.45. f Normal range of HCO3-22e27 mmol/L. g Normal range of ALT 0e41 U/L. h Normal range of AST 0e42 U/L.

B. Han et al

Please cite this article as: Han B et al., Clinical presentation, molecular analysis and follow-up of patients with mut methylmalonic acidemia in Shandong province, China, Pediatrics and Neonatology, https://doi.org/10.1016/j.pedneo.2019.07.004

Table 1

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Clinical Presentation and Molecular Analysis of patients with mut MMA Table 2 Biochemical data in patients with mut MMA after treatment. Case number

C3a, mmol/L

C3/C2 ratiob

Urine MMAc, mg/gCr

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

3.83 3.18 23.93 53.49 56.42 34.42 2.57 63.16 14.3 4.85 20.73 9.54 5.62 4.49 25.07 49.34 12.47 17.36 11.31 18.66

0.76 0.17 0.72 1.42 0.62 0.97 0.1 0.61 0.52 0.22 0.82 0.37 0.21 0.14 0.82 1.17 0.85 0.75 1.18 0.28

74.9 28.44 73 502.5 323.33 619.59 10.77 419.3 282.83 5.78 100.9 37.1 23.88 15.16 738.66 105.9 109.67 33.52 202.3 104.58

a b c

Normal range of C3 0.2e4.5 mmol/L. Normal C3/C2 ration <0.2. Normal range of urine MMA 0.2e3.6 mg/gCr.

Shandong province, China. A definitive laboratory diagnosis is crucial for confirming MMA since its clinical presentation is nonspecific and mimics common conditions such as other inborn errors of metabolism. These patients had typical findings of isolated MMA. Isolated MMA can be classified as mut, cblA, cblB, or cblD-variant 2. To identify the

Table 3

Results of DQ/IQ tests at last visit.

Case number

Newborn screening

Age of onset

Age at diagnosis

Age at test

1 2 3 5 6 9 10 11 12 13 14 16 17 18 19 20

Yes Yes Yes No No Yes No No No No Yes No Yes No Yes Yes

2d e 20d 6d 6m 2d 2y8m 10d 1y e e 10m e 2d 3d 3d

3d 3d 10d 11m 6m 10d 2y8m 20d 1y2m 5y 5d 1y5m 4d 4d 5d 6d

2y10m 13.5m 3.5y 5y 3y2m 8.5m 3y5m 2y8m 2y10m 8y 3y 5y 1y 13m 1y7m 7m

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classification, mutation analyses were performed. Here, twenty-three different mutations of the MUT gene were identified. Therefore, the patients were diagnosed with mut MMA. The most frequent mutation was c.914T > C (6/ 20). The finding of our study appears to differ from that of a previous report. Liu et al. reported that c.729_730insTT is the most frequent mutation in northern Chinese patients and that c.1280G > A is the most frequent mutation in southern Chinese patients.14 In Japanese patients, the relatively frequent mutations are c.385þ5G > A, c.1106G > A, c.1481T > A, c.2179C > T and c.349G > T.15,16 The c.278G > A mutation is the most prevalent mutation in Saudi patients.17 Furthermore, to confirm whether the mutations were novel, the sequencing data were compared with those from the Human Gene Mutation Database and ExAC database. Among 23 mutations, four mutations (c.613G > A, c.446A > G, c.920-923delTCTT and c.1359delT) were not found in these databases. Two deletion mutations led to a shift in the reading frame, creating new stop codons and truncated proteins. Missense mutations caused amino acid changes. In silico analysis revealed that the four novel mutations were predicted to be diseasecausing mutations using MutationTaster. These mutations may exert their harmful effects by affecting protein structure and function. Most patients were treated in a timely manner with specific medical nutrition and oral L-carnitine after diagnosis. The goals of treatment are to achieve normal development and to prevent episodes of metabolic decompensation. The mainstay of nutrition therapy is low protein intake, limiting but ensuring essential requirements of isoleucine, valine, methionine, and threonine to reduce elevated concentrations of metabolites.18 L-Carnitine is given to compensate for secondary carnitine deficiency caused by urinary loss of carnitine bound to organic acids. In addition, L-carnitine enhances propionyl group elimination, regenerates CoA and transforms toxic CoA esters into

DQ

Full-Scale IQ

Gross motor

Fine motor

Adaptive

Language

Personalsocial

54 133 86

54 89 81

72 100 81

42 89 85

63 78 79

30 71 81 55 18

31 60 82 46 6

35 71 85 65 23

24 71 90 74 20

35 77 79 74 22

96

97

95

100

90

77 65 45 63

85 69 40 78

95 65 42 78

92 77 40 85

94 69 45 78

35

97 40

“-”: having no onset yet; d: day; m: month; y: year.

Please cite this article as: Han B et al., Clinical presentation, molecular analysis and follow-up of patients with mut methylmalonic acidemia in Shandong province, China, Pediatrics and Neonatology, https://doi.org/10.1016/j.pedneo.2019.07.004

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6 less toxic carnitine esters that can be eliminated in urine.19 Furthermore, L-carnitine exhibits antioxidant activity by reducing free radicals and enhancing enzymatic activity involved in the defense against reactive species.20 No risks related to high levels of free or total carnitine have been reported, so L-carnitine therapy is considered safe.18 Most patients had a favorable metabolic response with reduction of C3, the C3/C2 ratio and urine MMA. However, their clinical outcomes were variable. Among 20 patients, 14 had clinical presentations of varying degrees, and 12 presented during the neonatal period. Surprisingly, among four patients who were diagnosed by newborn screening and treated before onset, three remained asymptomatic, and one exhibited normal development without neurological impairment. Thus, patients diagnosed by newborn screening and treated at the presymptomatic stage may have better outcomes. In this study, we did not assay the activity of MCM due to a lack of technical capability. Moreover, it was difficult to assess the precise correlation between clinical features and genotype because of the high frequency of compound heterozygotes. According to previous reports, four mutations (c.322C > T, c.729-730insTT, c. 914T > C and c.1677-1G > C) are associated with the mut0 subtype,9,21,22 two mutations (c.299A > G and c.323G > A) are associated with the mut subtype,21,22 and the c.1106G > A mutation is associated with both the mut0 and mut subtypes.9,22,23 Case 11 was homozygous for c.729-730insTT, experienced an acute decompensation episode at the age of 3 days and presented with failure to thrive and mild mental retardation at the age of 6 years. Among six patients with compound heterozygotes for c.914T > C, the patients carrying mut and mut0 mutations in the compound heterozygous state seemed to have relatively good clinical conditions. The mut mutation may play a dominant role when it is associated with the mut0 mutation in the compound heterozygous state, which may partly explain the late-onset symptoms and normal DQs of cases 3 and 17 with the mut mutation (c.299A > G, c.323G > A) and the mut0 mutation (c.914T > C) compared to the severe mental retardation and developmental delay of case 6 with two mut0 mutations (c.914T > C and c.16771G > A). Furthermore, the death of case 4 with two mut0 mutations (c.914T > C and 729-730insTT) also supports this notion. Our findings are in agreement with the findings by Aquaviva et al.23 The patients with two protein-truncating mutations are predicted to have a severe phenotype.9,24 In our study, the three patients (cases 5, 15 and 19) carrying two proteintruncating mutations presented in the first month of life and had severe phenotypes. Furthermore, case 15 died of metabolic crisis at the age of 1 year. Therefore, our results are in agreement with this notion of severity. Four novel mutations were identified in our study. The c.920923delTCTT (Y316Lfs*11) mutation was predicted to produce a truncated protein and be disease-causing using a bioinformatics tool (MutationTaster). Three patients (cases 5, 13, and 14) carried this mutation in compound heterozygous state. Cases 13 and 14 are siblings. Case 13 was diagnosed at the age of 5 years due to her affected younger brother who was diagnosed by newborn screening. Before diagnosis, case 13 did not receive any treatment. Interestingly, Cases 13 and 14 were and had normal DQ/IQ, whereas

B. Han et al case 5 presented with severe mental retardation, hypotonia and anemia. Another mutation in cases 13 and 14 was c.2168G > A (G723D). The residual activity of the c.2168G > A mutation was 44.1% compared to that of wildtype MCM,25 which partly explained the good outcomes of cases 13 and 14. However, Han et al. reported that a patient carrying the c.2168G > A mutation in compound heterozygous state presented with failure to thrive, seizures, hypotonia and mental retardation.25 Moreover, the c.2168G > A mutation was predicted to be disease-causing using MutationTaster. As demonstrated, in silico prediction tools are unable to resolve this uncertainty in all cases. Other modifying genes may affect the clinical phenotype. Furthermore, other factors, such as the environment, ethnicity, and individual genetic variations, can also be involved. In conclusion, 23 different mutations in the MUT gene were detected in 20 patients with mut MMA, and these mutations included four novel mutations. This work is sure to contribute to expanding the knowledge of the genetic basis of mut MMA. However, these limited data do not allow definitive statements on the genotypeephenotype correlations that could influence the outcomes of mut MMA. Nonetheless, it is necessary for high-risk families to have early prenatal diagnosis. To study the mutation spectrum of disease-causing genes associated with mut MMA in the Chinese population, a large-scale investigation is necessary.

Conflicts of interest The authors have no conflicts of interest relevant to this article.

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Please cite this article as: Han B et al., Clinical presentation, molecular analysis and follow-up of patients with mut methylmalonic acidemia in Shandong province, China, Pediatrics and Neonatology, https://doi.org/10.1016/j.pedneo.2019.07.004

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Clinical Presentation and Molecular Analysis of patients with mut MMA

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Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.pedneo.2019.07.004.

Please cite this article as: Han B et al., Clinical presentation, molecular analysis and follow-up of patients with mut methylmalonic acidemia in Shandong province, China, Pediatrics and Neonatology, https://doi.org/10.1016/j.pedneo.2019.07.004