Spectrum of mutations in patients with organic acidurias from Ukraine

O. I. Barvinska, N. V. Olkhovych, T. O. Shkurko © 2018 O. I. Barvinska et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 616.575-056.7-07


Introduction
Organic acidurias are a subgroup of inborn errors of metabolism which includes more than 33 inherited disorders by SSIEM classification 2011 [1] and are characterized by the cumulative prevalence of 1:7962 live birth [2].The most prevalent disorders in this group are isolated methylmalonic aciduria (MIM 251000, deficiency of methylmalonyl-CoA mutase (EC 5.4.99.2)), isovaleric aciduria (MIM 243500, deficiency of isovaleryl-CoA dehydrogenase (EC1.3.99.10)), propionic aciduria (MIM 606054, deficiency of propionyl-CoA carboxylase (EC 6.4.1.3)),and glutaric aciduria type 1 (MIM 231670, deficiency of glutaryl-CoA-dehydrogenase (EC 1.3.99.7)).Organic acidurias are caused by the deficiency of enzymes and transporters which take part in metabolism of amino acids, carbohydrates Biomedicine ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell. 2018. Vol. 34.N 2. P 107-116 doi: http://dx.doi.org/10.7124/bc.000975or lipid intermediates [3].Such alterations of metabolism lead to some common clinical manifestations such as developmental delay, seizures, lethargy, coma, vomiting, failure to thrive, hepatomegaly, respiratory distress and some unique ones such as dystonic-dyskinetic disorder, macrocephaly in case of GA I, sweaty odor of feet in case of IVA [3][4][5].The most common biochemical features are hyperammonemia, metabolic acidosis, ketosis and hypoglycemia.Without treatment these disorders could result in coma or death very quickly.Therefore, early diagnosis is crucial for decreasing a level of disability, mortality and morbidity of patients.In all developed countries the majority of organic acidurias are detected within the newborn screening by tandem mass spectrometry of acylcarnitines in dry blood spots [6].The diagnosis is confirmed by gas chromatography, mass spectrometry or molecular genetic analysis.Glutaric aciduria I (GA I) and isovaleric aciduria (IVA) are monogenic autosomal recessive inherited disorders, caused by mutations in genes GCDH (mapped to 19p13.2, includes 12 exons) and IVD (mapped to 15q.14-15, includes 12 exons) respectively [4,5].Noteworthy, methylmalonic aciduria (MMA) is a big heterogenous group of inherited disorders which are remarkable for elevation of methylmalonic acid in biological fluids.In terms of biochemical features these disorders are divided into two groups -isolated MMA and combined MMA.Isolated MMA is a clinical condition which could be caused by mutations in such genes as MUT, MMAA, MMAB and MCEE, while combined MMA manifests in complex with homocystinuria/homocysteinemia and is caused by mutations in the genes MMACHC, MMADHC, LMBRD1, ABCB4, HCFC1 [7].The isolated MMA is a more prevalent disorder than the combined one and 60 % of mutations are detected in the MUT gene (mapped to 6p.12.3, consists of 13 exon s) [8].The current mutation database HGMD Professional 2017.4 describes 361 mutations in the MUT gene, 87 rearrangements in the IVD gene, and 208 the in GCDH gene.The absence of highly predominant mutations in the genes MUT, IVD and GCDH and the need to define and distinguish different types of MMA stipulate the use of sequencing as a common method of confirming MMA, IVA and GA I.

Aim
To investigate a disease-causing mutations spectrum in patients with organic acidurias from Ukraine.

Materials and Methods
This study included 8 patients (4 males and 4 females) aged from 10 days to 3 years.These individuals were selected for organic aciduria diagnosis confirmation by selective screening of inborn errors of fatty acid and amino acid metabolism (2011-2016).All selected patients with suspected isolated methylmalonic aciduria, glutaric aciduria type I or isovaleric aciduria had elevations of typical acylcarnitines in dry blood spots and organic acids in urine.All parents gave their written informed consent on these investigations.The study was approved by the Committee of Bioethics of NSCH "OKhMATDYT".
Dry blood spots were collected on Whatman TM N 903 specimen collection papers and dried according to all requirements.
Genomic DNA was extracted from dry blood spots with NEOGENE reagents kit (Ukraine) according to the manufacturer's protocol.
The exons and flanking intronic regions of the MUT, IVD and GCDH genes were amplified by the polymerase chain reaction (PCR) from genomic DNA in SimpliAmp™ Thermal Cycler (Thermo Fisher Scientific) with the amplification kit GenPak®PCRCore NEOGENE (Ukraine) and primers developed with Primer3 software.The primers are presented in Table 1.
The visualization of PCR products was performed with capillary electrophoresis on MCE-202 MultiNA Shimadzu (Japan) using DNA 500 Shimadzu kit (Japan), 1000 Shimadzu kit (Japan) and molecular weight marker 25 bp DNA Ladder Invitrogen by Life Technologies (USA), and dye SYBR® Gold Molecular probes by Life Technologies (USA).
The PCR products were sequenced directly using forward and reverse primers on genetic analyzer ABI 3130 by Applied Biosystems (USA).The preparation of samples to sequenc- The assessment of pathogenicity of novel missense mutations was verified with Mutation Taster, Polyphen2, Provean software.Nucleotide sequence changes which result in frameshift were considered pathogenic.The prediction of the effect of novel missense mutations on the structure of protein was made with DeepView4.1 software, the modelling involved the use of a crystallographic structure of human methylmalonyl-CoA mutase PDB ID: 2XIJ previously reported by Froese et al. [9] and human isovaleryl-CoA dehydrogenase PDB ID: 1IVH reported by Tifanny et al. [10].The analysis of mutations was performed using the normal human MUT (NM_000255.3),IVD (NM_002225.3),GCDH (NM_000159.3)sequences as a reference.The nomenclature of mutations was based on coding sequences according to the standards of the Human Genome Variation Society [11].

Results and Discussion
The sequencing of exons and intron flanking regions of the MUT, GCDH, IVD genes was performed to confirm the diagnosis of isolated methylmalonic aciduria in 4 patients, glutaric aciduria type I in 2 patients and isovaleric aciduria in 2 patients with a specific profile of elevated acylcarnitines in dry blood spots and organic acids in urine.
According to the gene MUT sequence, two different missense mutations in this gene were identified in 2 patients with suspected isolated type of MMA.Patient 1 carried mutation in 3 exon p. N219Y, c.655A > T in homozygous state (Table 2).This rearrangement has already been described in the HGMD database and is considered to be a frequent mutation, as its presence in 25 alleles out of 302 in Caucasian population was previously published [8].
Patient 2 had a missense mutation in exon 5 of the MUT gene p.R326G, c.976A > G in heterozygous state which has not been added to HGMD database yet (Fig. 1A) (Table 2).The analysis of this novel missense mutation with Mutation Taster, PolyPhen2 and Provean Predictor software showed that it had a pathogenic effect (Table 3).The 3D model of methylmalonyl-CoA mutase (MCM) revealed that the replacement of positively charged large amino acid Arg with a small neutral amino acid Gly in the 326 th position causes breaking of 4 hydrogen bonds, which Arg326 creates with Thr92, Phe91, Gly380 and Ala330 in the N-terminal domain (Fig. 1B).Consequently, such alterations result in the tertiary conformation changes of methylmalonyl-CoA mutase that likely affect the protein function.
The mutation in the second allele of Patient 2 was not detected in any exon from 2 to 13 and its intronic flanking regions.The only additional findings in Patient 2 were three polymorphisms p. K212K, p. R532H, p. I671V in homozygous state.No mutations were found in exons 2-13 with flanking regions of introns of the MUT gene in two other patients with suspected MMA.In these two patients it is necessary to search for mutations in regulatory regions or introns of the MUT gene or other genes MMAA, MMAB, MCEE which are responsible for other types of isolated MMA [7].
The sequence of the GCDH gene, performed in order to confirm the diagnosis of glutaric aciduria type I in Patient 3, detected missense rearrangement p. R383C, c.1147 C > T in homozygous state (Table 2).This mutation is considered to be rare, for example, in Spanish population it was present in one allele out of 70 and in 3 alleles out of 38 in Japanese population [5,17].The diagnosis of glutaric aciduria type I was also confirmed in Patient 4 with the identification of two known missense mutations p. G390R, c.1168G > C -a rare variant in Caucasian population, and p. A421V, c. 1262 C > T -prevalent in Old Order Amish population [14,15,18].
The sequencing of the IVD gene in Patient 5 with suspected isovaleric aciduria revealed a known mutation p. R53P, c.158 G > C in heterozygous state (Table 2).Previously this mutation was reported in two alleles out of 16 in the USA studies and in 5 alleles out of 46 in Turkish population [16,19].A novel duplication of 7 nucleotides -c.49dupTGTGGCG (Table 2) was detected in the second allele of Patient 5 (Fig. 2A).This mutation causes a frameshift in exon 1, changing amino acid sequence and resulting in crucial alteration of tertiary structure of isovaleryl-CoA dehydrogenase with the loss of all cofactor and substrate binding sites.Such a truncated and changed protein could not perform its functions.The analysis of the IVD gene of Patient 6 revealed missense rearrangement p. S133C,   c.398 C > G in homozygous state (Table 2) (Fig. 2B).This mutation has not been described in mutation database HGMD yet.The analysis of this rearrangement by Mutation Taster, Polyphen2, Provean software predicted it as the disease-causing one (Table 3).This mutation causes a change of one polar amino acid Serine to another polar amino acid Cysteine in the N-terminal domain of isovaleryl Co-A dehydrogenase (IVD).Such replacement causes breaking of one hydrogen bond which Ser133 creates with Ala129 and lengthens the other which Ser133 creates with Ser256 (Fig. 2C).Also, other hydrogen bonds with Tyr134, Ala136, His137, Val130 remain unchanged.
Taking into consideration the fact that this amino acid residue is close to FAD-binding site, such alterations could affect cofactor binding and functional activity of protein.
To sum up, the sequencing of the MUT, IVD and GCDH genes allowed identifying a spectrum of mutations in Ukrainian patients with organic acidurias which is represented by known but rare variants (p.R53P in the IVD gene and p. R383C, p. G390R in the GCDH gene) or novel mutations (p.R326G in the MUT gene, p. S133C and c.49dupTGTGGCG in the IVD gene).The only exceptions were missense mutation p. N219Y in the MUT gene and p. A421V in the GCDH gene which are considered to be common variants for Caucasians and Amish population respectively [7,18].

Conclusions
The sequence analysis of the MUT gene confirmed the diagnosis of classic isolated methylmalonic aciduria in two patients out of 4 suspected ones.In these patients two missense mutations were detected, including one com-monly known mutation p. N219Y (2/8 alleles) and the other undisclosed one -p.R326G (1/8 alleles).According to the results of the GCDH gene sequence, three missense rearrangements p. R383C (2/4 alleles), p. G390R (1/4 alleles), p. A421V (1/4 alleles) were detected in patients with suspected glutaric aciduria type I and the previous diagnosis was confirmed.Two missense mutations -recurrent p. R53P (1/4 alleles) and novel p. S133C (2/4 alleles), and one novel duplication c.49dupTGTGGCG (1/4 alleles) in the IVD gene were revealed in patients with a probable diagnosis of isovaleric aciduria.All new missense mutations were analyzed with Mutation Taster, Polyphen2, Provean Predictor software and were predi cted as deleterious ones.Threedimensional modelling of novel missense mutations also revealed alterations in the structure of protein which could affect its functions.In perspective, these data could be used for the development of a molecular genetic diagnostic algorithm for the patients with isolated MMA, GA I and IVA.

Fig. 1 .Fig. 2 .
Fig. 1.A -Image of MUT gene sequence of Patient 2 with rearrangement c.976A > G in heterozygous state.B -Fragments of three-dimensional models of human wild-type MCM (green amino acid residue) and MCM with mutation p. R326G (red amino acid residue).

Table 1 . Primers for exons and flanking intronic regions of genes
GCDH, IVD and MUT