Download Arg 41 - Saudi Medical Journal

Recurrent mutation in the HMGCL gene in a family segregating HMG-CoA lyase deficiency
Essa Alharby1, Omhani Malibari2, Hamad S. Al-Otaibi3, Muhammad Saud3, Ghadeer Al-Harbi1,
Mohammad I. Samman1,3, Sulman Basit1
1. Center for Genetics and Inherited Diseases, Taibah University Almadinah Almunawwarah
2. Department of Metabolic Diseases, King Abdulla Medical City-Madinah Maternity and
Children Hospital, Almadinah Almunawwarah, Saudi Arabia
3. College of Applied Medical Science, Taibah University Almadinah Almunawwarah
Essa Alharby; [email protected], 00966560980011
Omhani Malibari; [email protected], 00966505536181
Hamad S. Al-Otaibi; [email protected],
Muhammad Saud; [email protected]
Ghadeer Al-Harbi; [email protected], 00966505664906
Mohammad I. Samman; [email protected], 00966504127745
Sulman Basit; [email protected], 00966535370209
Corresponding Author: Sulman Basit, PhD
Center for Genetics and Inherited Diseases, Taibah University Almadinah Almunawwarah
Email: [email protected]
Cell: 00966535370209
ABSTRACT
Objective: 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-methylglutaryl-CoA lyase (HMGCL) gene in a 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 respectively. Study was
conducted in Center for Genetics and Inherited Diseases, Taibah University during September
2015 to February 2016.
Results: Sanger sequencing of entire coding and intron-exon junctions of the HMGCL gene in five
members of the family 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).
Conclusion: This is the most frequent mutation found in the HMGCL gene in Saudi population
and might have occurred due to founder effect. Multiple in silico software predicted this mutation
as a 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. Moreover, extensive literature review was
performed and all mutations reported to date in the HMGCL gene were identified and enlisted.
Key words: HMGCL gene, HMG-CoA lyase deficiency, Homozygous mutation, Protein stability
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 characteristic pattern of elevated urinary organic acid metabolites, which
include 3-hydroxy-3-methylglutaric, 3-methylglutaric and 3-hydroxyisovaleric acids. Urinary
levels of 3-methylcrotonylglycine may be increased. Dicarboxylic aciduria, hepatomegaly, and
hyperammonemia may also be observed. 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 step
of 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. HMGCL gene produces two different isoforms; isoform A is
expressed in Mitochondria and isoform B is found in peroxisomes (Menao et al., 2009).
HMGCLD is a rare metabolic disorder 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 on HMGCL gene have
been reported (Cardoso et al., 2004; Menao et al., 2009). In Saudi Arabia, 89% of patients have a
missense mutation on exon 2 (122G>A) R41Q (Ozand et al., 1991, 1992). HMG-CoA lyase
deficiency is treatable by diet and avoidance of prolonged fasting. Leucine is restricted and
supplementary glucose given to prevent hypoglycemia. Without treatment, death occurs early
(Duran et al., 1979; Gibson et al., 1988).
We performed molecular genetic analysis of a family segregating HMGCLD with two siblings
manifesting the disease and identified a recurrent missense mutation in the HMGCL gene. We used
in silico analysis to prove that this mutation is indeed protein destabilizing.
MATERIAL AND METHODS
Collection of Samples and Extraction of nucleic acid
It is a cross-sectional study where a large family with total five individuals affected with
HMGCLD was ascertained from Madinah Maternity and Children Hospital (MMCH) in
September 2015. Clinical examination was performed in MMCH and all genetic work was carried
out in Center for Genetics and Inherited Diseases (CGID) during September 2015 to February
2016. Prior to start the research work, ethical approval was obtained from Ethical Review
Committee of MMCH. Five individuals including two affected (IV:1, IV:3), a normal (IV:2) and
two carriers (III:1, III:2) were available for this study. Thus, peripheral blood samples were
collected from 5 members of a family in an EDTA-containing tubes. Qiagen Mini Genomic DNA
Extraction Kit was used to isolate Genomic DNA following manufacturer's instructions.
Polymerase chain reaction (PCR) for coding exons amplification
Primers flanking all coding exons and intron-exon junctions were designed. PCR was performed
to amplify nine coding exons of HMGCL gene in a final volume of 25 ul containing 12.5 ul of
GoTaq® Green Master Mix, 50 ng of genomic DNA, 10 pmol of each forward and reverse primer,
and 7.5 ul of dH2O. Thermal cycling conditions consisted of 3 stages; where first stage used for
initial denaturation at 94°C for 3 min and second stage consist of 33 cycle of 94°C for 30sec, 5862 °C for 30 sec, 72 °C for 1 min and last stage for final extension at 72 °C for 10 min. PCR
products were electrophoresed on 2% of agarose gel for evaluating the efficiency of PCR.
DNA Sequencing
PCR cleanup was done to remove unconsumed nucleotides and remaining primers with exoSAPIT reagent. Cycle-sequencing reactions for the nine protein-coding exons and their splice junctions
in two directions were done by following the instructions of the BigDye Terminator v3.1
Cycle Sequencing Kit. Final cleanup of all nine reactions was performed with BigDye
XTerminator® Purification Kit prior to sequencing.
I: 1
II: 1
II: 2
III: 1
IV: 1
I: 2
IV: 2
II: 3
III: 3
III: 2
IV: 3
II: 4
IV: 4
IV: 5
III: 4
IV: 6
IV: 7
Figure 1: Pedigree of the family segregating HMGCLD. Open symbols represent unaffected subjects and filled
symbols affected persons. Double lines indicate consanguineous marriages.
RESULTS
Clinical Description of the family
Two individuals (IV:1, IV:3) with HMGCLD, an unaffected sib (IV:2) and both parents (III:1,
III:2) from a consanguineous Saudi family was 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 profile showed elevated 3-hydroxyisovaleric, highly elevated 3-methylglutaric and 2 isomers
of 3-methylglutaconic and 3-hydroxy-3-methylglutaric acid. Quantitative blood acylcarnitine
profile showed elevated hydroxyl-C5-carnitine (2.66 uM) and an elevated free-CO-carnitine (93
uM). This confirms the diagnosis of HMGCLD in both patients.
Genetic Analysis
The family was tested to detect the inheritable defects causing 3-hydroxymethyl-3-methylglutarylCoA (HMG CoA) lyase deficiency. Screening HMGCL gene, we found one pathogenic mutation
in the genomic DNA of 2 HMGCLD patients. Sequence analysis showed that both affected share
a homozygous allele of the mutation. It is a missense mutation involving G to A transition in
position 122 (c.122G>A) in exon 2 of the gene (Figure 2). The mutation causes a substitution of
an Arginine to a Glutamine amino acid residue at position 41 (p.Arg41Gln). As anticipated, botyh
parents were found heterozygous for the allele (Figure 2). In addition, screening of HMGCL gene
in patients revealed 2 previously described SNPs in homozygous manner; rs719400 (T>C) in exon
7 and rs2076344 (T>C) in the intergenic region downstream of exon 3.
C
Arg
41
Figure 2. Sequence analysis of the missense mutation identified in a family segregating HMGCLD. The upper panel
(A) represents the nucleotide sequences in the affected individuals, and the lower panel (B) in the heterozygous
carriers. Arrow in panel A indicate the position of the nucleotide change. Three dimensional structure of the HMGCL
protein (C). Arrow points toward the position of the mutated residue Arginine.
DISCUSSION
3-hydroxymethyl-3-methylglutaryl-CoA (HMG CoA) lyase deficiency is an early onset disease
and inherit 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, 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 infant’s life, a restricted low-fat diet is important to avoid metabolic stress and
maintain patient’s general health condition. Low intake of fat and protein improves patient’s
condition by decreasing the formation of acetoacetate in the body. This will allow patient’s body
to have control over gluconeogenesis by lowering the need of acetyl-CoA that hinders the activity
of pyruvate carboxylase (Dasouki et al., 1987). Patients who are presented in hospital with acute
episodes are giving glucose and bicarbonate to control hypoglycemia and acidosis, respectively.
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, in-frame indels, and nonsense mutations are
less frequent. To date, 28 missense and nonsense mutations have been reported and found to be
distributed along the entire coding part the gene (Table1). In addition, there are six mutations in
the intergenic regions affecting the splicing sites.
Mutation
Missense
Nonsense
Missense
Nonsense
Missense
Missense
Missense
Missense
Missense
Missense
Nonsense
Nonsense
Missense
Missense
Missense
Nonsense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Missense
Nonsense
Missense
Deletion
Deletion
Deletion
Deletion
Deletion
Deletion
Deletion
Deletion
Deletion
Insertion
Intronic
Intronic
Intronic
Intronic
Intronic
Intronic
Exon
Ex2
Ex2
Ex2
Ex2
Ex2
Ex2
Ex2
Ex2
Ex3
Ex3
Ex3
Ex4
Ex5
Ex5
Ex5
Ex6
Ex7
Ex7
Ex7
Ex7
Ex7
Ex7
Ex8
Ex8
Ex8
Ex8
Ex9
Ex4
Ex1
Ex2
Ex6
Ex7
Ex8
E3,4,5,6
Ex9
Ex 2,3,4,5,6
Ex5
Ex2
Int3
Int6
Int5
Int7
Int6
Int8
cDNA Position
c.109G>A
c.109G>T
c.122G>A
c.122C>T
c.124C>G
c.125G>A
c.126G>T
c.144G>T
c.208G>C
c.225C>G
c.242G>A
c.286C>T
c.425C>T
c.494G>A
c.521G>A
c.559G>T
c.575T>C
c.598A>T
c.602C>A
c.608G>A
c.610G>A
c.698A>G
c.788T>C
c.796T>C
c.820G>A
c.835G>A
c.922C>T
c.434A>T
c.27delG
c.202_207delCT
c.504_505delCT
c. 564_750del
c.853delC
c.145_561del
c. 914-915del TT
c. 61–561del
c. 374-375delTC
c. 137_138insA
IVS3 + 1G > A
IVS6-1G > A
IVS5+4A>G
IVS7+1G>A
IVS6+1G>A
IVS8+1G>C
Codon Change
p. Glu37Lys
p. Glu37X
p. Arg41Gln
p. Arg41X
p. Asp42His
p. Asp42Gly
p. Asp42Glu
p. Lys48Asn
p. Val70Leu
p. Ser75Arg
p. Trp81X
p. Gln96X
p. Ser142Phe
p. Arg165Gln
p. Cys174Tyr
p. Glu187X
p. Phe192Ser
p. Ile200Phe
p. Ser201Tyr
p. Gly203Glu
p. Asp204Asn
p. His233Arg
p. Leu263Pro
p. Cys266Arg
p. Gly274Arg
p. Glu279Lys
p. Gln308X
p. Gln 148-Glu 187 del
p. Pro9fsX
p. Ser69CysfsX
p. Val168fsX
p. Val188-Gln250del
p. L285X
p. Asn49-Glu187del
p. Phe305TyrfsX
p. Val21-Glu187del
p. V125fs
p. Asn46_Glu47insX
Splice donor site alteration
Splice Acceptor site alteration
Splice donor site alteration
Splice donor site alteration
Splice donor site alteration
Splice donor site alteration
Effect
AAS
PTC
AAS
PTC
AAS
AAS
AAS
AAS
AAS
AAS
PTC
PTC
AAS
AAS
AAS
PTC
AAS
AAS
AAS
AAS
AAS
AAS
AAS
AAS
AAS
AAS
PTC
Aberrant splicing
PTC
PTC
PTC
PTC
PTC
PTC
PTC
PTC
PTC
PTC
Skipping Exon3
Skipping Exon7
Skipping exon 5
Retention of intron 8
Retention of intron 7
Skipping Exon 8
Reference
2
34
17
17
17
17
19
21
20
17
9
2
22
2
2
2
2
20
23
20
24
25
26
27
8
8
8
28
29
30
8
2
31
17
31
26
32
8
22
2
2
1
33
Table 1: List of all known mutations reported to date in the HMGCL gene
Our mutational analysis found a missense mutation in exon 2 (c.122G>A; p.Arg41Gln) in both
affected individuals. This result resembles previous studies where this mutation is almost
exclusively present in patients from Saudi Arabia. 89% of HMGCLD cases in Saudi Arabia carry
this pathogenic mutation (Al-Sayed et al., 2006). This observation might be explained by founder
effect. This hypothesis is strengthened with the observation of same mutation in Turkish and Italian
patients who were originally from the Arabian Peninsula which suggests that 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.
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