Clinical and molecular investigation in Chinese patients with glutaric aciduria type I

    Clinical and molecular investigation in Chinese patients with glutaric aciduria type I Yanghui Zhang, Haoxian Li, Ruiyu Ma, Libin Mei, Xianda Wei, Desheng Liang, Lingqian Wu PII: DOI: Reference:

S0009-8981(15)30072-3 doi: 10.1016/j.cca.2015.12.003 CCA 14194

To appear in:

Clinica Chimica Acta

Received date: Revised date: Accepted date:

30 July 2015 22 November 2015 3 December 2015

Please cite this article as: Zhang Yanghui, Li Haoxian, Ma Ruiyu, Mei Libin, Wei Xianda, Liang Desheng, Wu Lingqian, Clinical and molecular investigation in Chinese patients with glutaric aciduria type I, Clinica Chimica Acta (2015), doi: 10.1016/j.cca.2015.12.003

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ACCEPTED MANUSCRIPT Clinical and molecular investigation in Chinese patients with glutaric aciduria type I Yanghui Zhanga, Haoxian Lia,b, Ruiyu Maa, Libin Meia, Xianda Weia, Desheng Lianga,b,*,

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Lingqian Wua,b,* a

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State Key Laboratory of Medical Genetics, Central South University,110 Xiangya Road, Changsha, Hunan 410078,China b Hunan Jiahui Genetics Hospital,110 Xiangya Road, Changsha, Hunan 410078,China

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* Corresponding authors at: State Key Laboratory of Medical Genetics, Central South University, 110 Xiangya Road, Changsha, Hunan 410078, China; Tel.: +86 731 84805252; Fax: +86 731 84478152; E-mail addresses: [email protected] (D. Liang),

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[email protected] (L. Wu)

Abstract

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Glutaric aciduria type I (GA-I) is a rare autosomal recessive metabolic disorder caused by

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deficiency of Glutaryl-CoA dehydrogenase (GCDH), leading to an abnormal metabolism of lysine, hydroxylysine and tryptophan. It results in accumulations of Glutaric acid, 3- hydroxyglutaric acid

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and glutaconic acid. Clinical features include the sudden onset of encephalopathy, hypotonia and macrocephaly usually before age 18 months. Here we report five cases of GA-I confirmed with mutation analysis. GCDH gene mutations were identified in all five probands with GA-I. Three of

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them had compound heterozygous mutations and two had homozygous mutations. Mutations of two alleles (c.334G＞T and IVS11-11A＞G) were novel and both of them were confirmed to be splice site mutations by reverse transcription PCR.

Keywords Glutaric aciduria type I; GCDH; splice site mutation; newborn screening

1. Introduction Glutaric aciduria type I (OMIM #231670, GA-I) is a rare inherited metabolic disorder caused by deficiency of glutaryl-CoA dehydrogenase (GCDH). GCDH converts glutaryl-CoA to crotonyl-CoA and catalyzes the metabolism of Lysine, hydroxylysine and tryptophan. The product, citric acid, participates in the tricarboxylic acid cycle. Because of the low activity of GCDH, glutaric acid, 3- hydroxyglutaric acid and glutaconic acid accumulate and cause dysbolism and a series of nervous system impairment. In most cases, the initial episode occurs between age 3 to 18

ACCEPTED MANUSCRIPT months after a normal initial development. If untreated in time, 90% cases would have encephalopathic crisis triggered by an infectious illness or vaccination, leading to acute striatal lesion, dystonia, spastic paralysis, seizures, acidosis, hyperammonemia and dyskinesia [1]. About

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31.6% to 75% cases have macrocephaly, which may already be presented at birth [2, 3]. Urinary

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organic acid analysis by gas chromatography–mass spectrometry (GC-MS) based on increased 3-hydroxyglutaric and glutaric acids, and plasma acylcarnitine analysis by tandem mass

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spectrometry (MS-MS) can be used to screen for GA-I, while diagnosis of GA-I depends on Glutaryl-CoA dehydrogenase activity detection or genetic testing.

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GCDH gene encodes Glutaryl-CoA dehydrogenase. It localizes on human chromosome 19p13.2 and is composed of 12 exons, 11 of which are coding exons. Over 200 mutations have

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been publicly reported, most of which are missense/nonsense mutations. There is no clear evidence proving the phenotype-genotype relationship [3].

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The birth incidence of GA-1 was evaluated at 1:100,000-1:40,000 via newborn screening [4-7]. In certain ethnic groups, such as the North American Ojibway-Cree in Canada, it was proved to be

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1:300 as a widespread disease [8]. The incidence in the Southern Chinese population was

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proposed to be 1:60,000 by newborn screening [9,10].

Here we report five Chinese cases of glutaric aciduria type I who were clinically diagnosed of an accumulation of glutaric acid and 3- hydroxyglutaric acid and confirmed by Sanger sequencing.

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Three probands were detected with compound heterozygous mutations and the other two had homozygous mutants in GCDH. Among the mutations, c.334G＞T and IVS11-11A＞G were novel and both of them were considered to be splice site mutations by reverse transcription polymerase chain reaction (RT-PCR). 2. Case report Five cases were identified in Hunan Jiahui Genetics Hospital of China. They were diagnosed with GA-I based on results of urinary organic acid testing and/or plasma acylcarnitine analysis. The parents of four cases were healthy and non-consanguineous, while case 4 was from family of second cousin marriage. Due to financial disadvantage, case 1 died at 6 months old with no appropriate treatment. Cases 2 to 5 lived on a protein-restricted diet and appropriate drugs treatment after diagnosed. The clinical features of five patients were summarized in Table 1. Their clinical diagnoses of GA-I were supported by GC/MS for urinary organic acids and/or MS-MS for plasma acylcarnitine. All cases were referred to hospital for macrocephaly (cases 1, 2, 3 and 4),

ACCEPTED MANUSCRIPT dystonia (cases 1,4 and 5 ), instability of lifting up head (cases 1, 2 and 4), persistent crying(cases 1, 2 and 4), seizure (case 3) or motor regression (case 5). Their brain magnetic resonance imaging (MRI) revealed a series of nervous system impairment. This study fully complied with the Tenets

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of the Declaration of Helsinki and was approved by the Ethics Board of the State Key Laboratory

Table 1 Clinical features of 5 Chinese patients with GA-I. Gender

Age at diagnosis (months) Triggering event

Bilateral lentiform and caudate nucleus degeneration Subdural fluid accumulation Bilateral temporal arachnoid cysts

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Leukoencephalopathy

Low Response

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Persistent crying

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Macrocephaly

Dystonia

Anterior fontanel apophysis Seizure

Athetosis Emesis

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Motor regression

Glutaric aciduria (normal control 1.9) 3- hydroxyglutaric acid (normal control 0) C5DC (μmol/L) Outcome

Case 2

Case 3

Case 4

Case 5

Female 5 6 None + + + + + + + 18144.07 149.74 N.D Died

Male 8 9 None + + + + + + + ↑ ↑ 1.28 Alive

Female 4 4 None + + 1480.5 12.12 N.D Alive

Female 4 10 Fever + + + + + + + 915.34 2.7 0.57 Alive

Female 11 12 None + + + + + + + + 2484.4 46.8 N.D Alive

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Enlarged sylvian fissures

Instability of lifting up head

Case 1

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Age at onset (months)

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of Medical Genetics of China. Informed consent was obtained from all patients’ parents.

3. Molecular studies Genomic DNA and RNA were extracted from peripheral blood leucocytes using standard methods. The entire coding regions and intron–exon boundaries of GCDH were tested for mutations. Eight different mutations were identified in GCDH gene in five patients (Table 2). Two mutations (c.334G＞T and IVS11-11A＞G) have not been reported in the Human Gene Mutation Database (HGMD), Leiden Open Variation Database (LOVD), dbSNP135 or Exome Variant Server (EVS). Meanwhile, neither of them was detected in 100 unaffected Chinese individuals.

ACCEPTED MANUSCRIPT Three probands had compound heterozygous mutations and two had homozygous mutations. Case 4 had homozygous mutation c.416C＞T, which may arise from the same progenitor.

Case 2

Mutation at nucleotide level

Mutation at protein level

References

No

c.533G＞A (maternal)

p.G178E

Lin et al. [11]

IVS11-11A＞G (paternal)

p.G415fsX429

This study

p.V91_K111del

This study

p.R355C p.A298T

Goodman et al. [12]

p.G354S

Schwartz et al. [13]

No

c.334G＞T (maternal) c.1063C＞T (paternal)

Case 3

No

c.892G＞A (maternal)

Case 4

Yes

homozygous c.416C＞T (maternal/ paternal)

No

p.S139L

Goodman et al. [12]

homozygous c.1204C＞T

p.R402W

Biery et al. [14]

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Case 5

Goodman et al. [12]

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c.1060G＞A (paternal)

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Inbreeding

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Case 1

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Table 2 Mutations of GCDH gene in 5 Chinese patients with GA-I.

(maternal/ paternal)

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Using RT-PCR, c.334G＞T and IVS11-11A＞G were confirmed to be splice site mutations.

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Mutation c.334G＞T caused skipping of exon 5 and IVS11-11A＞G caused kipping of the coding

4. Discussion

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region of exon 12 and part of 3’ untranslatable region (Fig. 1).

For most patients with glutaric aciduria type I, the acute encephalopathic crisis would be triggered by an infectious illness or vaccination with fever between 6 and 18 months of age. The

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concentration of glutaric acid and 3- hydroxyglutaric acid in the brain is much higher than that in other tissues. It leads to a loss of striatal neuron at time of the acute event and an abnormal development of blood vessels or blood flow that could aggravate the striatal injury [15]. The production of cytokines and nitric oxide contributes to neuronal damage [16]. The febrile illness might be insidious and hard to define. In this study, case 4 was observed to be glassy-eyed after fever at her 4 months age, while other cases were notified no triggering event. Except case 1, four cases were placed restrictions on low-protein diet and other management. Case 2 achieved an optimal outcome with good language, motor and intelligence development, even though he had shown neurological impairment and subdural fluid accumulation which lessened at the age of 4. Case 3 walked at the age of 5 and made progress on the development, but much slower than normal peers. Cases 4 and 5 were unoptimistic with little progress. The outcome might be not noticeable due to their tender age and a short treatment time.

ACCEPTED MANUSCRIPT Glutaryl-CoA dehydrogenase is located in the mitochondrial matrix. GCDH gene encodes 438 amino acids, and 44 N-terminal amino acid residues are cut off after import into the mitochondrion. The GCDH monomer consists of three domains, two α-helical amino-terminal

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domains (residues 45-167 and residues 282-438) and a β-sheet domain in the middle (residues

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168-281) [17]. The N-terminal domain consists of six α-helices, followed by a seven-stranded pseudo-β-sheet domain, and five α-helices forms the C- terminal domain [18]. The active GCDH

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is a homotetramer and converts glutaryl-CoA to crotonyl-CoA and CO2. Flavin adenine dinucleotide (FAD) is involved in this enzymatic reaction as a coenzyme. The α-helical domain formed by the C-terminus is in the tetrameric binding surface, which constitutes the binding

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pockets of the FAD and glutaryl-CoA [19].

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Considering only of the cDNA sequence of GCDH, mutation c.334G＞T is supposed to be a nonsense mutation and results in a truncation of the peptide. However, according to the result of RT-PCR, this mutation causes exon 5 skipping and leads to an inframe deletion. In other words,

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c.334G＞T causes a mutant of p.V91_K111del and results in the translation of GCDH that lacks an α-helix(Fig. 2). This missing region lies away from the binding site of the FAD and exposes on

GCDH

would

be

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the surface of the protein. As a constituent of multimeric mitochondrial dehydrogenase complexes, bound

to

mitochondrial

proteins

directly.

Dihydrolipoamide

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S-succinyltransferase (DLST) involved in glutaryl-CoA synthesis and the β-subunit of the electron transfer flavoprotein (ETFB) serving as an electron acceptor have been designated as GCDH binding partners. Presently, the binding sites of GCDH have not been identified and are supposed

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to locate on the surface of GCDH. ETFB is assumed to bind to the N-terminal domain of GCDH [20]. The mutant of p.V91_K111del lacks an α-helix of the N-terminal domain, so the interactions between GCDH and mitochondrial proteins are likely to be affected. Furthermore, it is entirely possible to impact the whole molecular space structure of GCDH, due to the large deletion of 21 amino acid residues. IVS5+5G＞A also causes exon 5 skipping and leads to the same inframe deletion [12]. In this region, several missense mutations (p.R94L, p.R94Q and p.G101R) have been reported to be pathogenic[13,21,22], implying the indispensable role of this α-helix in GCDH protein function.

The IVS11-11A＞G mutation causes exon 12 partly skipping 232 bases, which compose the entire coding region and the next 158 bases of non-coding region of exon 12. In other words, IVS11-11A＞G causes a frameshift mutant of p.G415fsX429 and results in a lacking of the extreme α-helix of the C- terminal domain in the translation of GCDH (Fig. 2). This missing

ACCEPTED MANUSCRIPT region locates at the binding pockets of the FAD and glutaryl-CoA. This change of C-terminus affects substrate binding, resulting in an inefficient or inactive enzyme. Previously, a splicing variant of GCDH missing the 5’ region of exon 12 used to be identified as an inactive enzyme

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[23].

The pathogenicity of novel missense mutations in most Chinese cases was investigated by

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conservative comparison and protein function prediction. In this study, RT-PCR confirmed two novel mutations to be structural changing, which generate incomplete functional domains and

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obstruct the activity of glutaryl-CoA dehydrogenase.

Many Chinese cases have been reported regarding of the clinical phenotypes but only a few

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involve genetic testing. According to several reports [24-33], exon 6 and 11 are probablely hot spots in Chinese patients with GA-I. Compared with prevalent mutations in specific groups, such as c.914C＞T in Japanese [3], c.1240C＞T in European [34] and c.913G＞A in Spain [35],

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IVS11-2A＞C is common in Southern Chinese based on reports from Hong Kong and Taiwan [25, 29](unified according to NM_000159). However, this mutation is not prevalent in other Chinese

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groups. Mutations c.148T > C and c.533G＞A present a higher frequency than others, but not

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prominent.

All five patients were diagnosed on the basis of clinical characteristics and urinary organic acid analysis. No one was identified before attacked by the disease because of an absence of

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newborn screening, the criteria of which is met by GA-I. Via early diagnosis and preventive treatment, irreversible damage of the organ can be avoided by preventing neurological deterioration of the patients with GA-I. Kölker et al. [36] reported in 2004 that 51 of 65 patients identified by newborn screening kept in a good condition under appropriate management. Reported by the same researchers in 2006, patients with the phenotype of neurological deterioration received irreversible damage, as 185 of 218 patients diagnosed symptomatically had neurological complications and 49 died in childhood [37]. GA-I is not involved in the routine newborn screening program in most region of China. In this study, no patient has been identified before representation of GA-I. Therefore, the treatment of these patients is delayed. It is expected that newborn screening for GA-I would be broader conducted in China in the future to achieve a satisfactory outcome with effective treatment for the patients.

In conclusion, we have identified eight mutations in the GCDH gene in five GA-I patients.

ACCEPTED MANUSCRIPT Two of them are novel splice site mutations. Based on these results, we performed prenatal diagnosis for two families and ensured two healthy babies. Furthermore, our findings broaden the

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mutation spectrum of GCDH and extend current understanding of the effects of GCDH mutations.

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Acknowledgements

Authors thank the family members who participated in this study for their help and support.

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Contract sponsors: The National Key Basic Research Program of China (2012CB944600) and the National Key Technology R&D Program of China (2012BAI09B05).

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Fig. 1. Results of RT-PCR of two novel mutations. (A) Polyacrylamide gel electrophoresis (PAGE) of case 2 with c.334G＞T. (B) PAGE of case 1 with IVS11-11A＞G. (C) c.334G＞T caused exon 5 skipping to form a mutant of p.V91_K111del. (D) IVS11-11A＞G caused exon 12 partly skipping 232 bases to form a mutant of p.G415fsX429. (E) cDNA of GCDH. Red bands show 5′ UTR and 3′ UTR regions and blue bands show coding regions.

Fig. 2. (A) The monomer of GCDH. It consists of N-terminal α-helical domain (cyan), seven-stranded pseudo-β-sheet domain (green) and C-terminal α-helical domain (purple). (B) The homotetramer of GCDH. p.V91_K111del lacks an α-helix of N-terminal domain (red), which lies away from the binding site of the FAD and surface exposed. p.G415fsX429 lacks the extreme α-helix of the C- terminal domain (purple), which locates at the binding pockets of the FAD and glutaryl-CoA (PDB ID:1SIR).