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

Molecular Genetics and Metabolism 106 (2012) 419–423

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Microarray based mutational analysis of patients with methylmalonic acidemia: Identification of 10 novel mutations Halil Dündar a, Rıza Köksal Özgül a, b,⁎, Ayşegül Güzel-Ozantürk a, c, Ali Dursun a, Serap Sivri a, Didem Aliefendioğlu d, Turgay Coşkun a, Ayşegül Tokatlı a a

Metabolism Unit, Department of Pediatrics, Faculty of Medicine, Hacettepe University, Ankara, Turkey Institute of Child Health, Hacettepe University, Ankara, Turkey Department of Molecular Biology, Hacettepe University, Ankara, Turkey d Department of Pediatrics, Kırıkkale University, Kırıkkale, Turkey b c

a r t i c l e

i n f o

Article history: Received 18 May 2012 Received in revised form 21 May 2012 Accepted 21 May 2012 Available online 1 June 2012 Keywords: Methylmalonic acidemia Microarray sequencing Mutation MUT

a b s t r a c t 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. © 2012 Elsevier Inc. All rights reserved.

1. Introduction Methylmalonic acidemia (MMA, OMIM 251000) is an autosomal recessive inborn error of the organic acid metabolism [1–3]. MMA can result from two general classes of genetic defects; those termed cbl, which involve genes required for provision of the adenosylcobalamin cofactor and those termed mut, which involves functional defects in the gene encoding the methylmalonyl-CoA mutase apoenzyme (MCM, EC 5.4.99.2), also known as MUT. MUT, is a nuclear encoded mitochondrial enzyme in the propionate pathway in mammals that carries out the reversible conversion between L-methylmalonyl-CoA and succinyl-CoA using adenosylcobalamin (AdoCbl) as a cofactor [4], where propionylCoA produced by the catabolism is converted to succinyl-CoA before entry into the citric acid cycle. Patients with mut MMA have been divided into two subtypes, mut0, with no MCM activity in cultured fibroblasts and mut− patients with MCM residual activity stimulated by high levels of cobalamin in the culture medium [5]. Methylmalonyl-CoA, among other metabolites, accumulates in the mitochondrial matrix as a result of MCM deficiency. MethylmalonylCoA is subsequently hydrolyzed to CoA and methylmalonic acid [1,6], resulting in elevated blood and urine levels of methylmalonic ⁎ Corresponding author at: Metabolism Unit, Department of Pediatrics, Faculty of Medicine, Institute of Child Health, Hacettepe University, Sıhhiye, Ankara, Turkey. Fax:+90 312 3051863. E-mail address: [email protected] (R.K. Özgül). 1096-7192/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2012.05.014

acid. Affected individuals typically present in the first weeks or months of life with ketoacidosis, lethargy, vomiting and failure to thrive, hypotonia which may lead to early death if not treated [1,7]. Despite dietary treatment, affected patients remain vulnerable to life threatening metabolic crises and most patients have problems with growth and motor skills. Long term complications include progressive renal failure, neurological symptoms as well as cardiomyopathy [2,8]. The human MCM gene (MUT), located on chromosome 6, comprises 13 exons spanning over 35 kb. The open reading frame consists of 2.7 kb, encoding 750 amino acids [9]. The primary structure of MUT is conserved throughout evolution in organisms ranging from bacteria to humans. Human MUT (hMUT) shares 65% identity with the Propionibacterium shermanii MUT (psMUT) alpha subunit. Unlike psMUT, which is a heterodimer of one catalytic (α) and one acatalytic subunit (β) [10], hMUT is a homodimer with two catalytic (α) subunits per dimer [11]. Each hMUT monomer features a two-domain structure as observed for the α-subunit of psMUT, namely a large substrate-binding TIM barrel (N-domain) connected to a small AdoCbl-binding domain (C-domain) via a ∼100 aa inter-domain linker (Belt), with the active site situated at the N/C-domain interface [12]. In this study, custom designed microarray sequencing methodology was applied for mutation screening in a cohort of Turkish patients with mut MMA. The microarray sequencing platform was designed by our group (TR_06_01r520489, Affymetrix) for genetic screening

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H. Dündar et al. / Molecular Genetics and Metabolism 106 (2012) 419–423

Table 1 Mutation spectrum and clinical outcomes of MMA patients. Patient

Sex

Age at diagnosis

Consanguinity

Family history

Complication

1 2 3 4 5

F M F F F

6m 3d 10 d 13 m 7m

No Yes Yes No Yes

Yes Yes Yes No No

6 7 8 9

F F M F

1d 2d 1y 1m

Yes Yes Yes No

Yes Yes Yes No

RF (GFR35mil/min), Anemia MR ? Acrodermatitis metabolica GR, MR, RF (GFR 29 ml/min/) GER, Anemia MR MR ? MMR, RF

10 11 12 13 14 15 16 17 18 19

F F M M M M M M F F

1m 6d 2d 2m 6m 3d 10 m 4m 6d 12 d

Yes No Yes Yes Yes No Yes Yes Yes Yes

No Yes Yes Yes No No Yes No Yes Yes

20 21

F M

2d 9m

Yes Yes

Yes No

22 23 24

F F M

11 m 3d 4m

Yes Yes No

No Yes Yes

25 26 27 28 29 30

M M F F M F

4d 3d 1.5 m 4m 4d 8d

Yes Yes Yes Yes ? No

Yes ? Yes Yes ? No

GR Micronoduler cirrhosis, MR MR MR GR, MR DD ? ? MR, HFS, ARDS MR, VUR Grade 2, RF (GFR 49.5 ml/min) – GR, MR, Anemia, CVA (hemiparesis), RF GR MR GR, MMR, Micronoduler cirrhosis HFS, ARDS ? ? GR, MR, Anemia, RF – PH

Mutation

Domain

Outcome

p.R31*/– p.K54*/p.K54* p.Q132*/p.Q132* p.A137G/p.A137G p.L140Lfs*40/p.L140Lfs*40

Mitochondrial leader sequence N-terminal extended segment Substrate-binding TIM barrel Substrate-binding TIM barrel Substrate-binding TIM barrel

Died 16 years old Alive 11 years old Died 2 years old Died 17 m old Alive 9 years old

p.L140Lfs*40/p.L140Lfs*40 p.R152*/p.R152* p.N219Y/p.N219Y p.R228*/p.R467*

Substrate-binding TIM barrel Substrate-binding TIM barrel Substrate-binding TIM barrel Substrate-binding TIM barrel/inter-domain linker Substrate-binding TIM barrel Substrate-binding TIM barrel Substrate-binding TIM barrel Substrate-binding TIM barrel Inter-domain linker Inter-domain linker Splicing/AdoCbl binding domain AdoCbl binding domain AdoCbl binding domain AdoCbl binding domain

Alive 2 years old Alive 8 years old Lost to follow up Alive 10 years old

p.P615T/p.P615T p.P615T/p.P615T

AdoCbl binding domain AdoCbl binding domain

Alive 1 year old Alive 9.5 years old

p.P615T/p.P615T p.P615T/p.P615T p.P615L/p.P615L

AdoCbl binding domain AdoCbl binding domain AdoCbl binding domain

Alive 11 years old Alive 9 years old Alive 9 years old

AdoCbl binding domain AdoCbl binding domain AdoCbl binding domain AdoCbl binding domain AdoCbl binding domain AdoCbl binding domain/AdoCbl binding domain

Died 3.5 years old Died 9 years old Alive 4.5 years old Alive 7 years old Died 10 days old Died 20 days old

p.L305S/p.L305S p.L328F/– p.L328F/p.L328F p.T387I/p.T387I p.G454E/p.G454E p.Q514E/c.753+1T/p.P615T p.P615T/p.P615T p.P615T/p.P615T p.P615T/p.P615T

p.D625V/p.D625V p.P654Pfs*2/p.P654Pfs*2 p.L674F/p.L674F p.P706Tfs*5/p.P706Tfs*5 p.R727*/p.R727* p.P615T/p.R727*

Alive 2.5 years old Alive 7 years old Alive 3 years old Alive 5 years old Alive 1 years old Lost to follow up Lost to follow up Lost to follow up Died 8 years old Alive 10 years old

MR: mental reterdation; MMR: mental-motor reterdation; GR: growth reterdation; PH: pulmonary hypertension; RF: renal failure; GFR: glomerular filtration rate; VUR: vesicoureteral reflux; GER: gastroesophageal reflux; HFS: hemophagocytic syndrome; ARDS: acute respiratory distress syndrome; CVA: cerebrovascular accident; DD: developmental delay.

of ten different inherited metabolic diseases, including fructose 16 diphosphatese deficiency, galactosemia, glutaric aciduria type I, hereditary fructose intolerance, maple syrup urine disease, methylmalonic acidemia, phenylketonuria, tyrosinemia type 1 and Wilson's disease [13–15]. Using this microarray sequencing method, we identified 22 different mutations in MUT, of which 10 are novel. 2. Materials and methods 2.1. Patients Thirty unrelated Turkish patients diagnosed with mut MMA at Hacettepe University Department of Pediatrics, Metabolism Unit were included in the study. Written informed consent was taken from participating individuals. Institutional review ethical board approvals for the research project were obtained (Hacettepe University). Diagnosis of MMA was made by urine organic acid analysis and the blood carnitine acylcarnitine profile. 2.2. Mutation screening Genomic DNA was isolated from peripheral blood. A 50 K custom designed DNA sequencing microarray TR_06_01r520489 (Affymetrix, Santa Clara, USA) was applied for mutation screening in 30 Turkish patients diagnosed with mut MMA. The array platform was designed to detect all nucleotide substitutions except for insertion or deletion

type of nucleotide changes, with an extent of 50,000 base pairs of genomic reference sequences for selected genes. All exonic sequences and 25 base pairs of their exon–intron boundaries, 500 base pair sequences from each 5′ and 3′ untranslated regions were tiled on this array for the genes namely, ALDOB, ATP7B, BCKDHA, BCKDHB, DBT, DLD, FAH, FBP, GALT, GCDH, MUT, PAH, PCCA and PCCB [13]. Exons of MUT gene from MMA patients were amplified using MUT gene specific primers. MUT gene amplicons were generated by Polymerase Chain Reaction (PCR). PCR products were purified by MinElute 96 UF PCR Purification plate (QiaGen Hilden, Germany). DNA concentrations were measured (NanoDrop Technologies, Wilmington, DE) and quantitated amplicons were pooled. Fragmentation, labeling, and hybridization steps were carried out as recommended by the manufacturer (GeneChip CustomSeq Resequencing Array Protocol Version 5.0, Affymetrix, Santa Clara, USA). Arrays were stained and washed through GeneChip® Fluidics Station 450 (DNA ARRAY_WS4_450) and scanning of arrays were performed by GeneChip 3000 Scanner (Affymetrix, Santa Clara, USA). Processing of raw data and analysis of nucleotide sequence were performed by Affymetrix GCOS Vers. 1.4 and GSEQ 4.0 Software (Affymetrix, Santa Clara, USA). All identified nucleotide changes detected by microarray sequencing method were confirmed by direct DNA sequencing in both forward and reverse directions. Sequencing reaction was performed using BigDye Terminator Cycle Sequencing Version 3.1. ABI 3130 capillary electrophoresis system was used for automated sequencing with POP7 polymer (Applied Biosystems, Foster City, CA). Files were

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processed using sequencing analysis software. Healthy individuals (n:50) were screened by direct sequencing to further characterize novel mutations. 3. Results Clinical and laboratory data of the 30 individuals affected with mut MMA are summarized in Table 1. In brief, most of the patients had an early neonatal presentation and three of them died in neonatal period, and others presented in early childhood. The most common clinical complications were growth retardation, developmental delay/mental-motor retardation, anemia, and renal failure. Two patients (patients 11 and 24) presented with micronodular cirrhosis and hemophagocytic lymphohistiocytosis was seen in two patients (patients 18 and 25). Mutation screening study identified 57 of 60 mutant alleles in patients with mut MMA. Among identified mutant alleles, 22 different mutations were described. Seven previously reported nonsense mutations were detected, p.R31*, p.Q132* p.R152*, p.R228*, p.R467*, p.R727* [16] and p.K54* [17]. The novel mutations were: p.Q132*, c.1962_1963delTC, c.2115_2116insA, c.753+1T, p.A137G, p.T387I, p.Q514E, p.P615L, p.D625V and p.L674F. Twelve mutations, p.R31*, p.K54*, p.R152*, c.420delG, p.N219Y, p.R228*, p.L305S, p.L328F, p.G454E, p.R467*, p.P615T and p.R727*, were described previously (Table 2). The frameshift mutations include nucleotide deletions c.420delG; p.L140Lfs*40 [18] in exon 3, c.1962_1963delTC; p.P654Pfs*2 in exon 12 and single nucleotide insertion c.2115_2116insA; p.P706Tfs*5 in exon 12. Both the deletion and insertion type of mutations cause a premature termination codon, all were identified as homozygote and the patients were born to consanguineous parents. Only one mutant allele was identified in three patients; however, we suspect that the second mutation may be located in the 5′ and 3′ UTR regions or in other noncoding regions of the gene. Codon 615 was the only mutational hotspot, with p.P615T identified in 28.0% of mutated alleles. p.P615T was detected in both alleles of seven patients (patients 17, 18, 19, 20, 21, 22 and 23) and in one allele of two patients (patient 16 and patient 30). Although the mutations were distributed along the whole coding sequence of MUT, 77.1% of the mutant alleles were located in exons 3, 5, 11 and 12 in this study. No mutations were detected in exons 1, 4, 9 or 10. Four prevalent polymorphisms (p.K212K, p.A499T, p.R532H, p.I671V) were also identified in this study (Table 2). p.K212K and p.I671V polymorphisms were very common, and both polymorphisms were identified together in 11 patients. Twenty four patients were homozygous and three patients had a compound heterozygous mutations. Only one mutant allele was identified in three patients. Eighty eight percent (21/24) of the homozygous patients had documented parental consanguinity, two were nonconsanguineous, and information was not available for one patient. The rate of consanguineous marriages in our cohort was higher than the frequency of parental consanguinity reported in Turkey (http://www.aile.gov.tr/images/arastirmalar/AileYapısı.pdf. Accessed 21 Jan 2008). 4. Discussion This study expands the mutation spectrum for MUT in the Turkish population by using sequencing microarrays. The majority of mutations were found within the substrate-binding TIM barrel (Ndomain) and AdoCbl-binding domain (C-domain). p.R31* is found in the mitochondrial leader sequence and p.K54* mutation is in the Nterminal extended segment. Both these nonsense mutations produce a truncated peptide. p.G454E, p.R464* and p.Q514E mutations were identified in the inter-domain linker. The mutation p.Q132*, a novel homozygous mutation detected in this study is located within substrate-binding TIM barrel of the

421

Table 2 Identified mutations and polymorphisms in MUT Gene.

Mutation missense p.A137G # p.N219Y p.L305S p.L328F p.T387I # p.G454E p.Q514E # p.P615T p.P615L # p.D625V # p.L674F # Nonsense p.R31* p.K54* p.Q132* # p.R152* p.R228* p.R467* p.R727* Deletion p.L140Lfs*40 p.P654Pfs*2 # Insertion p.P706Tfs*5 # Splicing c.753 + 1T # Polymorphism p.K212K p.A499T p.V532H p.I671V

Nucleotide changes

Exon

Allele Frequency

c.410C>G c.655A > T c.914T>C c.982C>T c.1160C>T c.1361G>A c.1540C>G c.1843C>A c.1844C>T c.1874A > T c.2020C>T

3 3 5 5 6 7 8 11 11 11 12

0.035 0.035 0.035 0.052 0.035 0.035 0.017 0.28 0.035 0.035 0.035

c.91C>T c.160A>T c.394C>T c.454C>T c.682C>T c.1399C>T c.2179C>T

2 2 3 3 3 7 13

0.017 0.035 0.035 0.035 0.017 0.017 0.052

c.420delG c.1962_1963delTC

3 12

0.07 0.035

c.2115_2116insA

12

0.035

g.49404942G>T

Intron 3

0.017

c.636G>A c.1495G>A c.1595G>A c.2011G>A

3 8 9 12

0.35 0.05 0.11 0.38

# indicates novel mutations. Bold indicates mutation with the highest frequency.

hMUT enzyme. p.R228* was located within the substrate-binding TIM barrel, while p.R467* was located within the inter-domain linker. Patient 29 who had a homozygous p.R727* mutation in exon 13 was lost to follow up. However, pulmonary hypertension was found in patient 30, who harbored p.R727* in heterozygosity along with p.P615T. p.R727* is located within AdoCbl binding domain, causing loss of 24 amino acid residues in the C-terminus of the hMUT enzyme. Missense mutations were identified in eleven patients, four of which were found within the AdoCbl binding domain, and five were located within substrate-binding TIM barrel. Two missense mutations were located within the inter-domain linker. The recurrent missense mutation p.P615T accounted for 28% of the mutational load. The portion consisting of AdoCbl binding domain is found within the residues between 615 and 690. The loop between IIβ1 and IIα1 contains His 627, which provides the ligand to cobalt in the enzyme. His627, Asp625, and Lys621 form part of a hydrogen bonded triad of residues that is proposed to modulate the reactivity at cobalt either by acting as a proton relay [7] or by positioning the histidine ligand at an unusually long distances from the cobalt atom [19]. p.P615T and p.P615L mutations likely have an important effect on the function of this critical domain, since the proline is critical for protein folding. Five missense mutations were located within the substratebinding TIM barrel including p.A137G and p.N219Y in exon 3, p.L305S and p.L328F in exon 5 and p.T387I in exon 6. A137 is part of the Iβ2 strand and was reported to play an important role in the interaction with coenzyme A [19]. p.A137V mutation was described previously [17]. The longer side chain Val leads to a conformational change of the AdoCbl-binding domain thereby causing a loss of enzyme activity. However, the exchange of Ala with Gly would result in high flexibility around the respective Cα bonds, thereby affecting the enzyme activity. Two missense mutations were identified in the inter-domain linker including homozygous p.G454E and novel heterozygous p.Q514E mutation.

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Fig. 1. Amino acid alignment of human MCM (MUTA_HUMAN, accession no. P22033), with mouse (MUTA_MOUSE, accession no. P16332), Caenorhabditis elegans (MUTA_CAEEL, accession no. Q23381), and with the α-subunit of MCM from Propionobacterium shermanii (MUTB_PROFR, accession no. P11653) denotes novel missense mutations identified in this study; an asterisk (*) denotes amino acids identical in normal human, mouse, C. elegans MCM and P. Shermanii α-subunit sequences; and a colon (:) denotes amino acids identical in normal human and mouse MCM and either.

All of the novel missense mutations found in this study, including p.A137G, p.T387I, p.Q514E, p.P615L, p.D625V, and p.L674F, are conserved in human, mouse, Caenorhabditis elegans and Propionobacterium shermanii (Fig. 1). The preservation of these amino acid residues through evolution, along with the observation that none of these missense changes were found in healthy individuals (100 alleles) suggests that these amino acids are located in a region which is critical for the structure and function of the MCM enzyme. One mutation, located in splice site sequence (c.753 +1T) along with a heterozygous mutated allele, c.1843C>A; p.P615T could cause either alternative splicing or complete exon skipping. The AAGgtatac motif in this region of MUT is a donor site for splicing and consensus value is 75.9 (in the range of 0–100) (Human Splice Finder www.umd.be/HSF/4DACTION/input_SSF).

5. Conclusion One of the most important goals of genetic analysis is to make a correlation between genotype and phenotype in order to understand the diversity among clinical outcomes. Although a wide array of mutations were identified in our group of patients, it is difficult to correlate phenotype with genotype in a straightforward way since clinical findings in mut MMA patients vary widely. Of note, a missense mutation p.P615T accounted for 28% of all mutations, the most common mutation for Turkish patients with mut MMA. This work provides a novel microarray based sequencing approach for genetic screening and updates the spectrum of mutations with description of novel mutations for MUT in the Turkish population.

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