Recurrent mutation in the HMGCL gene in a family segregating HMG-CoA lyase deficiency

The gene HMGCL encodes 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase. Mutations in HMG-CoA lyase cause HMG-CoA lyase deficiency (HMGCLD), which is an autosomal recessive congenital disorder of metabolism. This study was designed to detect mutation in the 3-hydroxy-3-methylglutarylCoA lyase (HMGCL) gene in a Saudi family segregating HMG-CoA lyase deficiency (HMGCLD).Methods: Clinical and molecular genetic analysis of a Saudi family with five individuals affected with HMGCLD was performed by GC-MS, tandem MS and sequencing. This study was conducted in the Centre for Genetics and Inherited Diseases, Taibah University Almadinah Almunawwarah, Saudi Arabia from September 2015 to February 2016. Sanger sequencing of the entire coding region and the intron-exon junctions of the HMGCL gene identified a recurrent missense mutation in exon 2. This mutation (c.122G>A) causes a substitution of a highly conserved amino acid Arginine to a Glutamine residue at position 41 (p.Arg41Gln). This is the most frequent mutation found in the HMGCL gene in Saudi population and might have occurred due to a founder effect. Multiple in silico software predicted this mutation as disease-causing. Moreover, to determine the protein stability upon change in amino acid various tools including SDM, I-Mutant, mCSM and DUET were used and found that the mutation, identified in this family, is protein destabilizing. An extensive literature review was performed and all mutations reported to date in the HMGCL gene were identified and listed.


INTRODUCTION
3-Hydroxy-3-methylglutaryl-CoA (HMG CoA) lyase deficiency (HMGCLD) is a rare autosomal recessive disorder with the cardinal manifestations of metabolic acidosis without ketonuria, hypoglycemia, and a *Corresponding author.E-mail: sbasit.phd@gmail.comAuthor(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License characteristic pattern of elevated urinary organic acid metabolites, which include 3-hydroxy-3-methylglutaric, 3methylglutaric and 3-hydroxyisovaleric acids (Zapater et al., 1998).Urinary levels of 3-methylcrotonylglycine may be increased (Gibson et al., 1988).Dicarboxylic aciduria, hepatomegaly, and hyperammonemia may also be observed (Zapater et al., 1998).Presenting clinical signs include irritability, lethargy, coma, and vomiting (Gibson et al., 1988).3-Hydroxy-3-methylglutaryl coenzyme A lyase (HMGCL) catalyzes the cleavage of HMG-CoA to acetoacetic acid and acetyl-CoA, the last steps in both ketogenesis and leucine catabolism.HMGCL is located in the mitochondrial matrix as well as in the peroxisomes (Wang et al., 1996).The enzyme HMGCL is encoded by the gene HMGCL located on chromosome 1p36.11.The HMGCL gene produces two isoforms; isoform A is expressed in mitochondria and isoform B is found in peroxisomes (Menao et al., 2009).HMGCLD is rare in Europe and Japan, but a common inherited disease in Saudi Arabia and Portugal (Cardoso et al., 2004;Ozand et al., 1992;Funghini et al., 2001).HMG CoA lyase deficiency has been extensively studied and over 30 mutations in the HMGCL gene have been reported (Cardoso et al., 2004;Menao et al., 2009).In Saudi Arabia, 89% of patients have a missense mutation in exon 2 (122G>A; R41Q) (Ozand et al., 1991(Ozand et al., , 1992)).HMG-CoA lyase deficiency is treatable by diet and avoidance of prolonged fasting.Leucine is restricted and supplementary glucose is given to prevent hypoglycemia.Without treatment, death occur early (Duran et al., 1979;Gibson et al., 1988).
In this study, we performed molecular genetic analysis of a family segregating HMGCLD.The study included two siblings manifesting the disease.A recurrent missense mutation in the HMGCL gene was identified.We also employed in silico analysis to demonstrate that this mutation is protein destabilizing.

Collection of samples and extraction of nucleic acid
A large family with a total of five individuals affected with HMGCLD was identified in Madinah Maternity and Children Hospital (MMCH) in September 2015.Clinical examinations were performed in the MMCH, and all genetic analyses were conducted in the Center for Genetics and Inherited Diseases (CGID), Taibah University during September 2015 to February 2016.Prior to the start the research, ethical approval was obtained from the Ethical Review Committee of MMCH.Five individuals were included; two affected (IV: 1, IV: 3), one normal (IV: 2) and two carriers (III: 1, III: 2).Peripheral blood samples were collected from all 5 family members in EDTAcontaining tubes.A Qiagen Mini Genomic DNA Extraction Kit (Skelton House, Lloyd Street North Manchester, UK) was used to isolate genomic DNA following the manufacturer's instructions.

Polymerase chain reaction (PCR) for coding exons amplification
Primers flanking all coding exons and intron-exon junctions were designed using primer 3 software.PCR was performed to amplify nine coding exons of HMGCL in a final volume of 25 µl containing 12.5 µl of GoTaq® Green Master Mix, 50 ng of genomic DNA, 10 pmol of each forward and reverse primer, and 7.5 µl of dH2O.Thermal-cycling conditions consisted of 3 stages; initial denaturation at 94°C for 3 min, 33 cycle of 94°C for 30 s, 58-62°C for 30 s, 72°C for 1 min, and a final extension at 72°C for 10 min.PCR products were electrophoresed on 2% of agarose gels.

DNA sequencing
PCR cleanup was done to remove unconsumed nucleotides and remaining primers with exoSAP-IT reagent (Central Expressway Santa Clara, CA 95051, USA

Clinical description of the family
Two individuals (IV: 1, IV: 3) with HMGCLD, an unaffected sibling (IV: 2) and both parents (III: 1; III: 2) from a consanguineous Saudi family were included in this study after informed written consent (Figure 1).Hypoglycemic seizures, metabolic acidosis and hepatomegaly were hall marks.Both patients underwent urine organic acid profiling by gas chromatography/mass spectroscopy (GC/MS) and acylcarnitine profiling using tandem mass spectrometry (MS/MS).Urine organic acid profiles showed elevated levels of 3-hydroxyisovaleric, highly elevated levels of 3-methylglutaric and 2 isomers of 3-methylglutaconic and 3-hydroxy-3-methylglutaric acid.Quantitative blood acylcarnitine profiling revealed elevated levels of hydroxyl-C5-carnitine (2.66 uM) and free-CO-carnitine (93 uM).The data confirmed the diagnosis of HMGCLD in both patients.

Genetic analysis
The family was tested to detect the known inheritable genetic defects causing 3-hydroxymethyl-3methylglutaryl-CoA (HMG CoA) lyase deficiency.In screening the HMGCL gene, we detected one pathogenic mutation in the DNA of 2 HMGCLD patients.Sequence analysis showed that both affected patients were homozygous for a missense mutation resulting in a G to A transition in position 122 (c.122G>A) in exon 2 of the gene (Figure 2).The mutation resulted in a substitution of an arginine for a glutamine amino acid residue at position 41 (p.Arg41Gln).As anticipated, both parents were found to be heterozygous for the allele (Figure 2).In addition, the screening of HMGCL in both patients revealed 2 previously described homozygous SNPs (rs719400 (T>C)

In silico analysis
The mutation identified in this study changes a conserved amino acid Arginine to Glutamine.In silico analysis predicted that this mutation is probably pathogenic.I-Mutant software (used for prediction of protein stability upon single point mutation) predicted the mutant protein as less stable or with decreased stability (Capriotti et al., 2005).Moreover, DUET (predicting effects of mutations on protein stability via an integrated computational approach), SDM (predicting effects of mutations on protein stability and malfunction) and mCSM (predicting the effect of mutation in protein using graph based signatures) were used for prediction of effect of mutation on the protein and found that this mutation is indeed destabilizing (Pires et al., 2014a, b;Worth et al., 2011).

DISCUSSION
HMG CoA lyase deficiency is an early onset disease and is inherited in an autosomal recessive manner.Metabolic acidosis and hyperammoniemia are prominent clinical manifestation of the illness.Patients show symptoms of the disorders in infancy.Clinically, patients show different acute episodes including vomiting, hypotonia, lethargy, diarrhea, cyanosis, dehydration, hypothermia, hepatomegalia and macrocephalia (Gibson et al., 1988a, b).Some patients may present non-common signs as hepatomegalia, macrocephalia, dilated cardiomyopathy with arrhythmia, and delayed development (Pié et al., 2007).About 20% of HMG-CoA lyase deficiency cases progress and cause permanent neurological damage and death (Gibson et al., 1988a).
In the first year of an infant's life, a restricted low-fat diet is important to avoid metabolic stress and to maintain the patient's general health.Low intake of fat and protein improves a patient's condition by decreasing the formation of acetoacetate in the body (Dasouki et al., 1987).This allows the patient's body to better control gluconeogenesis by lowering the need for the acetyl-CoA that hinders the activity of pyruvate carboxylase (Dasouki et al., 1987).Patients who are presented in hospital with acute episodes are given glucose and bicarbonate to control hypoglycemia and acidosis, respectively.The severity of illness decreases with age, and adults generally are free of symptoms (Pié et al., 2007).
Missense mutations in protein-coding regions in the HMGCL gene are the most frequent genomic alteration causing the disorders.Nonsense mutations and in-frame indels are less frequent.To date, 22 missense and 06 nonsense mutations have been reported and found to be distributed along the entire coding part the gene (Table 1).In addition, there are six mutations in the intergenic regions affecting the splicing sites.
Our mutational analysis found a missense mutation in exon 2 (c.122G>A; p.Arg41Gln) in both affected individuals.Arg41Gln mutation, in comparison to all known mutations, is found to have the most sever effect on the catalytic activity of the enzyme (Fu et al., 2006).Arg41 is a critical residue due to its involvement in the stabilization of enolized form of acetyl-CoA product (Figure 3).Arg41 is found on the active site of the enzyme and acts as salt bridge to the C-carboxyl group of hydroxyglutaric acid (Fu et al., 2006).Arg41 position explains the extreme reduction of catalytic activity of the enzyme by2.7×105-fold in Vmax compared to the normal enzyme (Tuinstra et al., 2004).
Arg 41 Identification of a missense mutation (c.122G>A; p.Arg41Gln) in our patients agrees with the results presented in previous studies where this mutation was found to be present almost exclusively in patients from Saudi Arabia.Eighty-nine percent of HMGCLD cases in Saudi Arabia carry this pathogenic mutation (Al-Sayed et al., 2006).This observation might be explained by a founder effect.This hypothesis is strengthened based on the observation that the same mutation occurs in Turkish and Italian patients who were originally from the Arabian Peninsula suggesting that the c.122G>A mutation first appeared in this region.Screening for this mutation can improve genetic counseling and prenatal diagnosis of HMGCLD in Saudi Arabia.

Figure 1 .
Figure 1.Pedigree of the family segregating HMGCLD.Open symbols represent unaffected subjects and filled symbols represent affected subjects.Double lines indicate consanguineous marriages.

Figure 2 .
Figure 2. Sequence analysis of the missense mutation identified in a family segregating HMGCLD.The upper panel (A) represents the nucleotide sequences of the affected individuals, and the lower panel (B) represents the nucleotide sequence of the heterozygous carriers.Arrow in panel A indicates the position of the nucleotide change.

Figure 3 .
Figure 3. Three dimensional structure of the HMGCL protein.Arrow points toward the position of the mutated arginine residue.
). Cycle-sequencing reactions for the nine protein-coding exons and their splice junctions were performed

Table 1 .
List of all known mutations reported to date in the HMGCL gene.