Sebastián Menao

Ten novel HMGCL mutations in 24 patients of different origin with 3-hydroxy-3-methyl-glutaric aciduria.

3‐Hydroxy‐3‐methylglutaric aciduria is a rare autosomal recessive genetic disorder that affects ketogenesis and L‐leucine catabolism. The clinical acute symptoms include vomiting, convulsions, metabolic acidosis, hypoketotic hypoglycaemia and lethargy. To date, 33 mutations in 100 patients have been reported in the HMGCL gene. In this study 10 new mutations in 24 patients are described. They include: 5 missense mutations: c.109G>A, c.425C>T, c.521G>A, c.575T>C and c.598A>T, 2 nonsense mutations: c.242G>A and c.559G>T, one small deletion: c.853delC, and 2 mutations in intron regions: c.497+4A>G and c.750+1G>A. Two prevalent mutations were detected, 109G>T (E37X) in 38% of disease alleles analyzed and c.504_505delCT in 10% of them. Although patients are mainly of European origin (71%) and mostly Spanish (54%), the group is ethnically diverse and includes, for the first time, patients from Pakistan, Palestine and Ecuador. We also present a simple, efficient method to express the enzyme and we analyze the possible functional effects of missense mutations. The finding that all identified missense mutations cause a >95% decrease in the enzyme activity, indicates that the disease appears only in very severe genotypes.”

Characterization of splice variants of the genes encoding human mitochondrial HMG-CoA lyase and HMG-CoA synthase, the main enzymes of the ketogenesis pathway

The genes HMGCS2 and HMGCL encode the two main enzymes for ketone-body synthesis, mitochondrial HMG-CoA synthase and HMG-CoA lyase. Here, we identify and describe possible splice variants of these genes in human tissues. We detected an alternative transcript of HMGCS2 carrying a deletion of exon 4, and two alternative transcripts of HMGCL with deletions of exons 5 and 6, and exons 5, 6 and 7, respectively. All splice variants maintained the reading frame. However, Western blot studies and overexpression measurements in eukaryotic or prokaryotic cell models did not reveal HL or mHS protein variants. Both genes showed a similar distribution of the inactive variants in different tissues. Surprisingly, the highest percentages were found in tissues where almost no ketone bodies are synthesized: heart, skeletal muscle and brain. Our results suggest that alternative splicing might coordinately block the two main enzymes of ketogenesis in specific human tissues.

New case of mitochondrial HMG-CoA synthase deficiency: Functional analysis of eight mutations

Mitochondrial HMG-CoA synthase deficiency is a rare inherited metabolic disorder that affects ketone-body synthesis. Acute episodes include vomiting, lethargy, hepatomegaly, hypoglycaemia, dicarboxylic aciduria, and in severe cases, coma. This deficiency may have been under-diagnosed owing to the absence of specific clinical and biochemical markers, limitations in liver biopsy and the lack of an effective method of expression and enzyme assay for verifying the mutations found. To date, eight patients have been reported with nine allelic variants of the HMGCS2 gene. We present a new method of enzyme expression and a modification of the activity assay that allows, for first time, the functional study of missense mutations found in patients with this deficiency. Four of the missense mutations (p.V54M, p.R188H, p.G212R and p.G388R) did not produce proteins that could have been detected in soluble form by western blot; three produced a total loss of activity (p.Y167C, p.M307T and p.R500H) and one, variant p.F174L, gave an enzyme with a catalytic efficiency of 11.5%. This indicates that the deficiency may occur with partial loss of activity of enzyme. In addition, we describe a new patient with this deficiency, in which we detected the missense allelic variant, c.1162G>A (p.G388R) and the nonsense variant c.1270C>T (p.R424X).

Characterization of a novel HMG-CoA lyase enzyme with a dual location in endoplasmic reticulum and cytosol

A novel lyase activity enzyme is characterized for the first time: HMG-CoA lyase-like1 (er-cHL), which is a close homolog of mitochondrial HMG-CoA lyase (mHL). Initial data show that there are nine mature transcripts for the novel gene HMGCLL1, although none of them has all its exons. The most abundant transcript is called “variant b,” and it lacks exons 2 and 3. Moreover, a three-dimensional model of the novel enzyme is proposed. Colocalization studies show a dual location of the er-cHL in the endoplasmic reticulum (ER) and cytosol, but not in mitochondria or peroxisomes. Furthermore, the dissociation experiment suggests that it is a nonendoplasmic reticulum integral membrane protein. The kinetic parameters of er-cHL indicate that it has a lower Vmax and a higher substrate affinity than mHL. Protein expression and lyase activity were found in several tissues, and were particularly strong in lung and kidney. The occurrence of er-cHL in brain is surprising, as mHL has not been found there. Although mHL activity is clearly associated with energy metabolism, the results suggest that er-cHL is more closely related to another metabolic function, mostly at the pulmonary and brain level.