**Mitochondrial HMG–CoA Synthase Deficiency**

María Arnedo, Mónica Ramos, Beatriz Puisac, Mª Concepción Gil-Rodríguez, Esperanza Teresa, Ángeles Pié, Gloria Bueno, Feliciano J. Ramos, Paulino Gómez-Puertas and Juan Pié *Unit of Clinical Genetics and Functional Genomics, School of Medicine, University of Zaragoza Spain* 

### **1. Introduction**

188 Advances in the Study of Genetic Disorders

Wanders, RJ., Schutgens, RB., & Zoeters, BH. (1988b). Prenatal diagnosis of 3-hydroxy-3-

Wang, SP., Robert, MF., Gibson, KM., Wanders, RJ., & Mitchell, GA. (1996). 3-Hydroxy-3-

Wang, SP., Marth, JD., Oligny, LL., Vachon, M., Robert, MF., Ashmarina, L., & Mitchell, GA.

Watson, MS., Mann, MY., Lloyd-Puryear, MA., & Rinaldo, M. (2006). Newborn screening:

Wilson, WG., Cass, MB., Søvik, O., Gibson, KM., & Sweetman, L. (1984). A child with acute

Wysocki, SJ., & Hahnel, R. (1976a). 3-Hydroxy-3-methylglutaric aciduria: deficiency of 3-

Wysocki, SJ., & Hahnel. R. (1976b). 3-Hydroxy-3-methylglutaric aciduria: 3-hydroxy-3-

Wysocki, SJ., & Hähnel, R. (1986). 3-Hydroxy-3-methylglutaryl-coenzyme a lyase deficiency:

Yalçinkaya, C., Dinçer, A., Gündüz, E., Fiçicioğlu, C., Koçer, N., & Aydin, A. (2007). MRI

Yýlmaz, Y., Ozdemir, N., Ekinci, G., Baykal, T., & Kocaman, C. (2006). Corticospinal tract

Zapater, N., Pié, J., Lloberas, J., Rolland, MO., Leroux, B., Vidailhet, M., Divry, P., Hegardt,

Zoghbi, HY., Spence, JE., Beaudet, AL., O'Brien, WE., Goodman, CJ., & Gibson, KM. (1986).

*Neurology*, Vol. 35, No. 2, (August 2006), pp. 139-141, ISSN 0887-8994 Zafeiriou, DI., Vargiami, E., Mayapetek, E., Augoustidou-Savvopoulou, P., & Mitchell, GA.

Vol. 358, No. 2, (October 1998), pp. 197-203, ISSN 0003-9861

(September 1986), pp. 367-369, ISSN 0364-5134.

Vol. 33, No. 1, (April 1996), pp. 99-104, ISSN: 0888-7543

1998) pp. 2057-2062, ISSN 0964-6906

(September 1976), pp.349-351, ISSN 0009-8981

2007), pp. 375-380, ISSN 0887-8994

2007), pp. 47-50, ISSN 0887-8994

No. 2, (December 1976), pp. 373-375, ISSN 0009-8981

252S, ISSN 1098-3600

289-291, ISSN 0340-6199

ISSN 0141-8955

0141-8955

methylglutaric aciduria via enzyme activity measurements in chorionic villi, chorionic villous fibroblasts or amniocytes using a simple spectrophotometric method. *Journal of Inherited Metabolic Disease*, Vol. 11, No. 4, (1988), pp. 430, ISSN

methylglutaryl CoA lyase (HL): mouse and human HL gene (*HMGCL*) cloning and detection of large gene deletions in two unrelated HL-deficient patients. *Genomics,* 

(1998). 3-Hydroxy-3-methylglutaryl-CoA lyase (HL): gene targeting causes prenatal lethality in HL-deficient mice. *Human Molecular Genetics*, Vol. 7, No.13, (December

toward a uniform screening panel and system. *Genetics in Medicine*, 8 Suppl 1: 1S-

pancreatitis and recurrent hypoglycemia due to 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. *European Journal of Pediatrics*, Vol. 142, No. 4, (September 1984), pp.

hydroxy-3-methylglutaryl coenzyme A lyase. *Clinica Chimica Acta,* Vol. 71, No. 2,

methylglutaryl-coenzyme A lyase levels in leucocytes. *Clinica Chimica Acta,* Vol. 73,

a review. *Journal of Inherited Metabolic Disease*, Vol. 9, No. 3, (1986), pp. 225-233,

and MRS in HMG-CoA lyase deficiency. *Pediatrics Neurology*, Vol. 20, No. 5, (May

involvement in a patient with 3-HMG coenzyme A lyase deficiency. *Pediatrics* 

(2007). 3-Hydroxy-3-methylglutaryl coenzyme a lyase deficiency with reversible white matter changes after treatment. *Pediatrics Neurology*, Vol. 37, No.1, (July

FG., & Casals, N. (2001). Two missense point mutations in different alleles in the 3 hydroxy-3-methylglutaryl coenzyme A lyase gene produce 3-hydroxy-3 methylglutaric aciduria in a French patient. *Archives of Biochemistry and Biophysics,*

Atypical presentation and neuropathological studies in 3-hydroxy-3 methylglutaryl-CoA lyase deficiency. *Annals of Neurology*, Vol. 20, No. 3, The mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase deficiency (MIM 600234) is an autosomic recessive inborn error of metabolism, hard to characterize and probably underdiagnosed (Thompson et al., 1997). The enzyme failure is caused by mutations in the gene *HMGCS2,* located in chromosome 1. The illness was first diagnosed in 1997 (Thompson et al., 1997), in a six-year old boy, who presented a semicomatose state after three days with gastroenteritis and diet (Bouchard et al., 2001). Up to date, only eight patients have been reported with an estimated incidence of <1/1,000,000, although it could be higher because some patients probably have been misdiagnosed as Reye syndrome.

The mitochondrial HMG-CoA synthase enzyme (mHS, EC 4.1.3.5) has a main role in the synthesis of the ketone bodies and in the HMG-CoA formation. Ketone bodies act as an alternative glucose fuel in a number of tissues as heart, muscle and brain, and have a critical role during metabolic stress and starving, situations where the symptoms of the disease appear.

#### **2. Ketogenesis**

The ketogenesis is a metabolic process that takes place inside the mitochondria. The acetyl-CoA, originated during the fatty acids β-oxidation, is converted to acetoacetate, βhidroxybutyrate and acetone. These metabolites are known as ketone bodies and act as a glucose alternative fuel in many tissues as heart, skeletal muscle and brain. In brain, ketone bodies play an essential role in situations of hypoglycemia (Edmond, 1992; Zammit & Moir, 1994). Besides their alternative fuel role, ketone bodies are lipogenic precursors too. They play a role during the myelinating process in the neonates' brain (Nehlig & Pereira de Vasconcelos, 1993), and in a minor degree in the mammary gland during suckling (Zammit, 1981). Moreover, they seem to have some functions in metabolism regulation (Robinson & Williamson, 1980).

The main tissue where ketogenesis takes place is the liver (Zammit & Moir, 1994). However, it has been also found in kidney, adipose tissue (Thumelin et al., 1993), and intestine of

Mitochondrial HMG–CoA Synthase Deficiency 191


There is an alternative ketogenic pathway different from the previously described, which takes place with substrates of aminoacids catabolism. Both pathways converge in the

The mHS catalyzes the second reaction of the main pathway of the synthesis of ketone

The mHS enzyme catalyzes the condensation of acetyl-CoA and acetoacetyl-CoA into HMG-CoA. The HMG-CoA, produced inside the mitochondria, drives towards the formation of ketone bodies. The mHS catalyzed reaction follows a Bi Bi Ping-Pong substitution mechanism (Cleland, 1963; Miziorko et al., 1975). This reaction has three steps (Miziorko et



High concentrations of acetoacetyl-CoA inhibit the first reaction step, because it competes with acetyl-CoA for the active site of the enzyme (Page & Tubbs, 1978). Magnesium can act

Human mHS has 508 amino acids and its structure is a homodimer formed by two identical monomers linked by a salt bridge (Figure 3). At the N-terminal end is the leader peptide,

In 1985, the catalytic sequence of the enzyme was identified in a purified protein from chicken liver (Miziorko & Behnke, 1985). This sequence has 21 aa and a 100% homology with HMG-CoA synthases from other mammals. The catalytic aminoacid Cys129, whose human homolog is the Cys166, is located inside this region (Misra et al., 1995). The protein analysis shows three regions rich in proline, glutamine, serine and threonine, which could be PEST sequences (Pro-Glu-Ser-Thr) (Rogers et al., 1986). These regions are characterized by a quick turnover of proteins (Boukaftane et al., 1994; Ayté et al.,

Recently, the human enzyme has been crystallized and its alpha/beta structure, with 17 beta sheets and 19 alpha helices, has been well characterized. The tunnel of substrate entry and

give HMG-CoA, which will be attached to the enzyme by a covalent bond.


with 37 aa, which drives the protein to the mitochondria.

bodies and it is considered as the key enzyme of pathway regulation.

decarboxylation of acetoacetate.

mitochondrial HL enzyme.

**3.1 Enzymatic reaction** 

al., 1977) (Figure 2):

(Miziorko et al., 1977).

as an inhibitor of the reaction too.

**3.2 Protein structure** 

1990).

**3. mHS enzyme** 

suckling rats (Hahn & Taller, 1987) and, in a minor degree, in cortical astrocytes of newborn rats (Cullingford et al., 1998; Blázquez et al., 1998).

Ketone bodies can be elevated in physiological situations as: fasting, prolonged exercise, high-fat diet or pregnancy (Felig & Lynch, 1970), in the neonate (Hawkins et al., 1971); and in pathologic situations as: diabetes (Bates et al., 1968), obesity and disease of the glucose or glycogen metabolism (Mitchell et al., 1997).

Fig. 1. Ketone bodies synthesis pathway

The main pathway of ketone bodies from acetyl-CoA includes the following reactions (Figure 1):


There is an alternative ketogenic pathway different from the previously described, which takes place with substrates of aminoacids catabolism. Both pathways converge in the mitochondrial HL enzyme.

### **3. mHS enzyme**

190 Advances in the Study of Genetic Disorders

suckling rats (Hahn & Taller, 1987) and, in a minor degree, in cortical astrocytes of newborn

Ketone bodies can be elevated in physiological situations as: fasting, prolonged exercise, high-fat diet or pregnancy (Felig & Lynch, 1970), in the neonate (Hawkins et al., 1971); and in pathologic situations as: diabetes (Bates et al., 1968), obesity and disease of the glucose or

The main pathway of ketone bodies from acetyl-CoA includes the following reactions



reaction is catalyzed by the acetoacetyl-CoA thiolase enzyme.

rats (Cullingford et al., 1998; Blázquez et al., 1998).

glycogen metabolism (Mitchell et al., 1997).

Fig. 1. Ketone bodies synthesis pathway

responsible of the disease.

(Figure 1):

The mHS catalyzes the second reaction of the main pathway of the synthesis of ketone bodies and it is considered as the key enzyme of pathway regulation.

### **3.1 Enzymatic reaction**

The mHS enzyme catalyzes the condensation of acetyl-CoA and acetoacetyl-CoA into HMG-CoA. The HMG-CoA, produced inside the mitochondria, drives towards the formation of ketone bodies. The mHS catalyzed reaction follows a Bi Bi Ping-Pong substitution mechanism (Cleland, 1963; Miziorko et al., 1975). This reaction has three steps (Miziorko et al., 1977) (Figure 2):


High concentrations of acetoacetyl-CoA inhibit the first reaction step, because it competes with acetyl-CoA for the active site of the enzyme (Page & Tubbs, 1978). Magnesium can act as an inhibitor of the reaction too.

#### **3.2 Protein structure**

Human mHS has 508 amino acids and its structure is a homodimer formed by two identical monomers linked by a salt bridge (Figure 3). At the N-terminal end is the leader peptide, with 37 aa, which drives the protein to the mitochondria.

In 1985, the catalytic sequence of the enzyme was identified in a purified protein from chicken liver (Miziorko & Behnke, 1985). This sequence has 21 aa and a 100% homology with HMG-CoA synthases from other mammals. The catalytic aminoacid Cys129, whose human homolog is the Cys166, is located inside this region (Misra et al., 1995). The protein analysis shows three regions rich in proline, glutamine, serine and threonine, which could be PEST sequences (Pro-Glu-Ser-Thr) (Rogers et al., 1986). These regions are characterized by a quick turnover of proteins (Boukaftane et al., 1994; Ayté et al., 1990).

Recently, the human enzyme has been crystallized and its alpha/beta structure, with 17 beta sheets and 19 alpha helices, has been well characterized. The tunnel of substrate entry and

Mitochondrial HMG–CoA Synthase Deficiency 193

Fig. 3. Structural location of missense mutations in human mHS, represented as blue spheres

Studies of gene expression in human tissues in basal conditions showed a tissue-specific expression. High mRNA levels are detected in the liver, the organ with the highest ketogenic capacity. Expression is also high in colon, which is related to the fermentative processes that take place in it (Mascaró et al., 1995). A minor level of mRNA expression has been observed in heart, skeletal muscle, gonads, kidney and pancreas (Mascaró et al., 1995; Royo et al., 1993). Studies in rats have found mRNA expression in cortical astrocytes from newborns (Cullingford et al., 1998; Blazquez et al., 1998), and in the intestine of suckling

Changes in the level of gene expression have been reported during development. In suckling rats, the highest levels of expression were detected at the third day of life, decreasing progressively until weaning, when mRNA levels are similar to those of adult

Production of ketone bodies in hepatic mitochondria is a complex and highly regulated process (Guzmán & Geelen, 1993; Zammit & Moir, 1994). Their synthesis increases in starving situations and in high fat intake, while it decreases after feeding or insulin administration and in the suckling-weaning transition (Williamson & Whitelaw, 1978;

animals (Bekesi & Williamson, 1990; Thumelin et al., 1993; Serra et al., 1993).

**3.3 Enzyme expression** 

well fed rats (Serra et al., 1993).

McGarry & Foster, 1980; Robinson et al., 1980).

**3.4 Enzyme regulation** 

product exit is located outside of the molecule, at the opposite side of the interaction between the two monomers (Shafqat et al., 2010).

Fig. 2. A. Acetyl-enzyme intermediate formation. B. HMG-CoA formation

product exit is located outside of the molecule, at the opposite side of the interaction

Fig. 2. A. Acetyl-enzyme intermediate formation. B. HMG-CoA formation

between the two monomers (Shafqat et al., 2010).

A

B

Fig. 3. Structural location of missense mutations in human mHS, represented as blue spheres

#### **3.3 Enzyme expression**

Studies of gene expression in human tissues in basal conditions showed a tissue-specific expression. High mRNA levels are detected in the liver, the organ with the highest ketogenic capacity. Expression is also high in colon, which is related to the fermentative processes that take place in it (Mascaró et al., 1995). A minor level of mRNA expression has been observed in heart, skeletal muscle, gonads, kidney and pancreas (Mascaró et al., 1995; Royo et al., 1993). Studies in rats have found mRNA expression in cortical astrocytes from newborns (Cullingford et al., 1998; Blazquez et al., 1998), and in the intestine of suckling animals (Bekesi & Williamson, 1990; Thumelin et al., 1993; Serra et al., 1993).

Changes in the level of gene expression have been reported during development. In suckling rats, the highest levels of expression were detected at the third day of life, decreasing progressively until weaning, when mRNA levels are similar to those of adult well fed rats (Serra et al., 1993).

#### **3.4 Enzyme regulation**

Production of ketone bodies in hepatic mitochondria is a complex and highly regulated process (Guzmán & Geelen, 1993; Zammit & Moir, 1994). Their synthesis increases in starving situations and in high fat intake, while it decreases after feeding or insulin administration and in the suckling-weaning transition (Williamson & Whitelaw, 1978; McGarry & Foster, 1980; Robinson et al., 1980).

Mitochondrial HMG–CoA Synthase Deficiency 195

(fever, stress, exercise). In normal conditions, metabolic pathways that provide alternative glucose sources, as the synthesis of ketone bodies, are activated. A defect of ketogenesis can

In all the cases reported, clinical symptoms have appeared in childhood, specially during the first year of life; however, two of the 8 cases were diagnosed at the age of four and six

Clinical features of mHS deficiency are unspecific (Table 1), which makes it hard to diagnose. Initial symptoms include vomiting and lethargy that can progress to coma. In most cases these symptoms are accompanied with hepatomegaly (Thompson et al., 1997) and in some cases with respiratory disease and encephalopathy. These symptoms quickly improve after glucose administration. Up to date, all the reported cases have had a favorable outcome, despite severe acute episodes. All the reported patients with mHS deficiency are

Hypoglycemia is the main biochemical anomaly detected during an acute episode. Increase of the levels of plasmatic free fatty acids and urine dicarboxylic acids is also found. Plasma levels of acylcarnitines, lactate and ammonium are within normal limits (Morris et al., 1998).

This disease, as opposite to the HL deficiency, does not show a characteristic organic acids pattern in urine. The mHS substrate does not accumulate because it can be metabolized to

An enzymatic assay in liver biopsy is needed to confirm the mHS deficiency, because liver is the tissue with the highest enzyme expression. This method has several limitations, being the main one that in liver homogenate we cannot distinguish between mHS and cHS enzyme activity. This problem impairs the interpretation of the results and explains that the assay has only been carried out in two patients (Thompson et al., 1997; Morris et al., 1998). The possibility to use other tissues or alternative cells as lymphocytes or fibroblasts has been proposed, but the low expression levels in these tissues did not allow investigators to obtain

Molecular analysis is the method of choice to confirm the clinical diagnosis. It is done from genomic DNA of the patient, by PCR amplification and sequencing of the *HMGCS2* gene.

The HL is the immediately posterior to mHS enzyme in the pathway of the ketone bodies synthesis. HL deficiency (3-hydroxy-3-methylglutaric aciduria) has very similar clinical manifestations to mHS deficiency, although there are significant differences that the

trigger a coma in the individual with low glucose availability.

In some cases, metabolic acidosis has been reported (Table 1).

**4.1 Clinical features** 

years, respectively.

still alive.

**4.2 Diagnosis** 

**4.2.1 Biochemical data** 

**4.2.2 Enzyme activity** 

**4.2.3 Molecular diagnosis** 

acetyl-CoA during the β-oxidation.

valid measurable levels (Thompson et al., 1997).

**4.2.4 Differential diagnosis with HMG-CoA lyase deficiency** 

clinicians should know in order to differentiate them.

Initially, the acyl-CoA input to the mitochondria was considered the principal check-point of ketogenesis (Guzman & Geelen, 1993; McGarry & Brown, 1997). However, later studies showed that the mHS enzyme can regulate ketogenesis (Williamson et al., 1968). Therefore, metabolic conditions that imply an increase of ketone bodies are linked to an increase of the levels of *HMGCS2* gene expression (Casals et al., 1992) and of the mHS enzyme (Serrá et al., 1993). Meanwhile, conditions that cause a decrease of the synthesis of ketone bodies are related to a decrease in the mRNA levels of the *HMGCS2* gene (Casals et al., 1992) and of the mHS enzyme (Serrá et al., 1993).

Currently, two check-points are recognized in the control of the synthesis of ketone bodies, one at the same step that of the Carnitine Palmitoyl Transferase 1 reaction, which provides the substrate acetyl-CoA, and another in the ketogenic pathway, at the step of the mHS enzyme (Williamson et al., 1968; Dashti & Ontko, 1979). Regulation of mHS is the most important step and depends on two mechanisms: a long term regulation of the transcription of the *HMGCS2* gene (Casals et al., 1992; Hegardt, 1999), and a short term regulation of the protein. This one depends on succinyl and desuccinyl enzymes reactions (Lowe & Tubbs, 1985; Quant et al., 1990). All of these mechanisms are influenced by nutritional and hormonal factors.

#### **3.5 Isoenzymes**

Two HMG-CoA synthase isoenzymes which catalyze the same reaction are known, however, they are located in different structures inside the cell. The mHS (Clinkenbeard et al., 1975) is involved in the synthesis of ketone bodies in the mitochondria, while the cytosolic HMG-CoA synthase (cHS) is critical for the synthesis of cholesterol (Clinkenbeard et al., 1975; Reed et al., 1975).

Expression pattern of cHS is different from the one described for mHS. The cytosolic enzyme is expressed in most of the tissues, and its gene is considered a house-keeping gene. High mRNA levels are observed in liver followed by heart, placenta and pancreas (Mascaró et al., 1995). Their transcriptional regulation is different from the mHS and it is negatively regulated by cholesterol and other isoprenoids (Hua et al., 1993).

The activity measures of the HMG-CoA synthase in a liver crude extract is divided between a 20-40% for the cHS and a 60-80% for the mHS (Clinkenbeard et al., 1975). Surprisingly, there is not reported any case with cHS deficiency.

### **4. mHS deficiency**

Deficiency of mHS is an inborn error of metabolism that affects the synthesis of ketone bodies. It is a very rare autosomal recessive disorder reported in 1997 (Thompson et al., 1997), in a six year old boy. The patient had hypoglycemia, hypoketonemia and semicomatous state, after three days with gastroenteritis and dieting. Symptoms quickly reversed after intravenous administration of glucose. Liver biopsy showed a decrease in the activity of mHS. Molecular study of the gene confirmed the diagnosis (Bouchard et al., 2001). Generally, the disease shows unspecific clinical symptoms and metabolites excretion profile, sometimes attributed to a fatty acid β-oxidation enzyme defect and that makes it to be underdiagnosed. Some authors pointed out that it can be misdiagnosed as Reye syndrome and be associated with the sudden infant death syndrome (Thompson et al., 1997). Symptoms usually appear after situations of starving and/or high energy expenditure (fever, stress, exercise). In normal conditions, metabolic pathways that provide alternative glucose sources, as the synthesis of ketone bodies, are activated. A defect of ketogenesis can trigger a coma in the individual with low glucose availability.

#### **4.1 Clinical features**

194 Advances in the Study of Genetic Disorders

Initially, the acyl-CoA input to the mitochondria was considered the principal check-point of ketogenesis (Guzman & Geelen, 1993; McGarry & Brown, 1997). However, later studies showed that the mHS enzyme can regulate ketogenesis (Williamson et al., 1968). Therefore, metabolic conditions that imply an increase of ketone bodies are linked to an increase of the levels of *HMGCS2* gene expression (Casals et al., 1992) and of the mHS enzyme (Serrá et al., 1993). Meanwhile, conditions that cause a decrease of the synthesis of ketone bodies are related to a decrease in the mRNA levels of the *HMGCS2* gene (Casals et al., 1992) and of the

Currently, two check-points are recognized in the control of the synthesis of ketone bodies, one at the same step that of the Carnitine Palmitoyl Transferase 1 reaction, which provides the substrate acetyl-CoA, and another in the ketogenic pathway, at the step of the mHS enzyme (Williamson et al., 1968; Dashti & Ontko, 1979). Regulation of mHS is the most important step and depends on two mechanisms: a long term regulation of the transcription of the *HMGCS2* gene (Casals et al., 1992; Hegardt, 1999), and a short term regulation of the protein. This one depends on succinyl and desuccinyl enzymes reactions (Lowe & Tubbs, 1985; Quant et al., 1990). All of these mechanisms are influenced by nutritional and

Two HMG-CoA synthase isoenzymes which catalyze the same reaction are known, however, they are located in different structures inside the cell. The mHS (Clinkenbeard et al., 1975) is involved in the synthesis of ketone bodies in the mitochondria, while the cytosolic HMG-CoA synthase (cHS) is critical for the synthesis of cholesterol (Clinkenbeard

Expression pattern of cHS is different from the one described for mHS. The cytosolic enzyme is expressed in most of the tissues, and its gene is considered a house-keeping gene. High mRNA levels are observed in liver followed by heart, placenta and pancreas (Mascaró et al., 1995). Their transcriptional regulation is different from the mHS and it is negatively

The activity measures of the HMG-CoA synthase in a liver crude extract is divided between a 20-40% for the cHS and a 60-80% for the mHS (Clinkenbeard et al., 1975). Surprisingly,

Deficiency of mHS is an inborn error of metabolism that affects the synthesis of ketone bodies. It is a very rare autosomal recessive disorder reported in 1997 (Thompson et al., 1997), in a six year old boy. The patient had hypoglycemia, hypoketonemia and semicomatous state, after three days with gastroenteritis and dieting. Symptoms quickly reversed after intravenous administration of glucose. Liver biopsy showed a decrease in the activity of mHS. Molecular study of the gene confirmed the diagnosis (Bouchard et al., 2001). Generally, the disease shows unspecific clinical symptoms and metabolites excretion profile, sometimes attributed to a fatty acid β-oxidation enzyme defect and that makes it to be underdiagnosed. Some authors pointed out that it can be misdiagnosed as Reye syndrome and be associated with the sudden infant death syndrome (Thompson et al., 1997). Symptoms usually appear after situations of starving and/or high energy expenditure

regulated by cholesterol and other isoprenoids (Hua et al., 1993).

there is not reported any case with cHS deficiency.

mHS enzyme (Serrá et al., 1993).

hormonal factors.

**3.5 Isoenzymes** 

et al., 1975; Reed et al., 1975).

**4. mHS deficiency** 

In all the cases reported, clinical symptoms have appeared in childhood, specially during the first year of life; however, two of the 8 cases were diagnosed at the age of four and six years, respectively.

Clinical features of mHS deficiency are unspecific (Table 1), which makes it hard to diagnose. Initial symptoms include vomiting and lethargy that can progress to coma. In most cases these symptoms are accompanied with hepatomegaly (Thompson et al., 1997) and in some cases with respiratory disease and encephalopathy. These symptoms quickly improve after glucose administration. Up to date, all the reported cases have had a favorable outcome, despite severe acute episodes. All the reported patients with mHS deficiency are still alive.

### **4.2 Diagnosis**

### **4.2.1 Biochemical data**

Hypoglycemia is the main biochemical anomaly detected during an acute episode. Increase of the levels of plasmatic free fatty acids and urine dicarboxylic acids is also found. Plasma levels of acylcarnitines, lactate and ammonium are within normal limits (Morris et al., 1998). In some cases, metabolic acidosis has been reported (Table 1).

This disease, as opposite to the HL deficiency, does not show a characteristic organic acids pattern in urine. The mHS substrate does not accumulate because it can be metabolized to acetyl-CoA during the β-oxidation.

#### **4.2.2 Enzyme activity**

An enzymatic assay in liver biopsy is needed to confirm the mHS deficiency, because liver is the tissue with the highest enzyme expression. This method has several limitations, being the main one that in liver homogenate we cannot distinguish between mHS and cHS enzyme activity. This problem impairs the interpretation of the results and explains that the assay has only been carried out in two patients (Thompson et al., 1997; Morris et al., 1998). The possibility to use other tissues or alternative cells as lymphocytes or fibroblasts has been proposed, but the low expression levels in these tissues did not allow investigators to obtain valid measurable levels (Thompson et al., 1997).

#### **4.2.3 Molecular diagnosis**

Molecular analysis is the method of choice to confirm the clinical diagnosis. It is done from genomic DNA of the patient, by PCR amplification and sequencing of the *HMGCS2* gene.

#### **4.2.4 Differential diagnosis with HMG-CoA lyase deficiency**

The HL is the immediately posterior to mHS enzyme in the pathway of the ketone bodies synthesis. HL deficiency (3-hydroxy-3-methylglutaric aciduria) has very similar clinical manifestations to mHS deficiency, although there are significant differences that the clinicians should know in order to differentiate them.

Mitochondrial HMG–CoA Synthase Deficiency 197

acid, the 3-methylglutaconic acid and the 3-hydroxy-3-methylglutaric acid. Neither of these metabolites is increased in mHS deficiency. Clinical and biochemical data of both

Treatment is symptomatic during acute episodes and consists of intravenous administration of glucose to correct hypoglycemia. Long-term maintenance therapy includes low-fat diet and avoidance of fasting periods of more than 12 hours. It is also important to avoid situations of metabolic stress, mainly produced by recurrent illness that can be prevented by

Although mHS is codified by a gene (Ayte et al., 1990) different from the cHS gene (Gil et al., 1986), their high homology suggests a common origin about 500 millions years ago,

Human *HMGCS2* gene is located in chromosome 1 (1p12-p13), between the markers WI-7519 and D1S514. It has 10 exons and 9 introns, with a total size of 21,708 bp (Figure 4). Exons length oscillates between 107 and 846 bp, and the intron's size varies between 0.4 and

So far, no splice variants of the gene have been experimentally confirmed, although the current Expressed Sequence Tag database includes a variant with a deletion of exon 4.

Up to date, 8 patients have been diagnosed by molecular analysis. Among them nine allelic variants have been identified: seven missense variants, one nonsense and one intronic mutation (Figures 3 and 4). One of these variants, the Y167C, affects to a closed Cys166 amino acid, which is considered the catabolic site of the enzyme (Hegard, 1999). The nonsense variant R424X (Bouchard et al., 2001; Morris et al., 1998), produced a truncated protein of 424 amino acids that probably cannot be incorporated into the active protein dimer. The intronic mutation is located in the first nucleotide of the intron 5 (c.1016+1G>A). In general, this type of mutations produces the deletion of the affected exon (exon 5 in this case), although in this disease the mechanism has not been confirmed (Zschocke et al., 2002). In order to prove the effect of the mutation in the enzyme activity, protein over-expression has been tried with recombinant DNA techniques although with no success (Bouchard et al.,

Fig. 4. Scheme of the mutations located in the human gene *HMGCS2*

deficiencies are included in Table 1.

extra caloric intake (Morris et al., 1998).

when vertebrates appeared (Boukaftane et al., 1994).

4.1 kDa. cDNA has a total size of 2,082 bp.

**4.3 Treatment** 

**5.** *HMGCS2* **gene** 

**5.1 Mutation update** 

2001).


+Always; -Never; +/- Sometimes.

#### Table 1. Signs and Biochemical data of mHS and HL deficiency

Patients with HL deficiency may present acute crisis as well, during the neonatal or early infancy periods. Both diseases appear when exogenous supply of glucose fails (fasting periods), or when excessive glucose consumption exists (stress, fever or exercise). In the majority of patients with mHS deficiency, the acute crisis begins with hepatomegaly and superficial coma. However, in HL deficiency, or other β-oxidation enzymes, symptoms of the deficiency usually involve several tissues (Thompson et al., 1997). In the latter, most of the patients show muscular weakness and myopathy (Ribes et al., 2003). In the HL deficiency symptoms as dehydration, hypotonia, hypothermia, tachypnea, lethargy, coma and even death, have been reported (Wysocki & Hahnel, 1986; Menao et al., 2009). Up to date, no patient with mHS deficiency has died due to this disease.

Complications of 3-hydroxy-3-methylglutaric aciduria include, macrocephaly (Stacey et al., 1985), development delay and dilated cardiomyopathy with arrhythmia (Gibson et al., 1994). Microcephaly has been reported in one patient (Lisson et al., 1981). Up to date, no complications have been reported in patients with mHS deficiency.

The most important difference between both diseases is found in the urine profile of the organic acids, which is characteristic only in HL deficiency. Metabolites that accumulate in urine are the 3-hydroxy-isovaleric acid, the 3-methylcrotonyl glycine, the 3-methylglutaric acid, the 3-methylglutaconic acid and the 3-hydroxy-3-methylglutaric acid. Neither of these metabolites is increased in mHS deficiency. Clinical and biochemical data of both deficiencies are included in Table 1.

#### **4.3 Treatment**

196 Advances in the Study of Genetic Disorders

Encephalopathy +/- +/- Hypotonia - +/- Lethargy +/- +/- Abnormal Breathing +/- +/- Coma +/- +/- Hepatomegaly + +/- Normal development + +/- Death - +/-

Hypoglycaemia + + Hypoketonemia + + Hyperammonemia - +/- Transaminase +/- +/- Metabolic acidosis +/- + High urine dicarboxylic acid + -

Normal level of carnitine + +/-

Patients with HL deficiency may present acute crisis as well, during the neonatal or early infancy periods. Both diseases appear when exogenous supply of glucose fails (fasting periods), or when excessive glucose consumption exists (stress, fever or exercise). In the majority of patients with mHS deficiency, the acute crisis begins with hepatomegaly and superficial coma. However, in HL deficiency, or other β-oxidation enzymes, symptoms of the deficiency usually involve several tissues (Thompson et al., 1997). In the latter, most of the patients show muscular weakness and myopathy (Ribes et al., 2003). In the HL deficiency symptoms as dehydration, hypotonia, hypothermia, tachypnea, lethargy, coma and even death, have been reported (Wysocki & Hahnel, 1986; Menao et al., 2009). Up to

Complications of 3-hydroxy-3-methylglutaric aciduria include, macrocephaly (Stacey et al., 1985), development delay and dilated cardiomyopathy with arrhythmia (Gibson et al., 1994). Microcephaly has been reported in one patient (Lisson et al., 1981). Up to date, no

The most important difference between both diseases is found in the urine profile of the organic acids, which is characteristic only in HL deficiency. Metabolites that accumulate in urine are the 3-hydroxy-isovaleric acid, the 3-methylcrotonyl glycine, the 3-methylglutaric


**SIGNS** 

**BIOCHEMICAL DATA** 

High 3-hydroxy-3-methylglutaric, 3 hydroxyisovaleric, 3-methylglutaconic

Table 1. Signs and Biochemical data of mHS and HL deficiency

date, no patient with mHS deficiency has died due to this disease.

complications have been reported in patients with mHS deficiency.

and 3-methylglutaric acids

+Always; -Never; +/- Sometimes.

**Signs and laboratory data mHS deficiency HL deficiency** 

Treatment is symptomatic during acute episodes and consists of intravenous administration of glucose to correct hypoglycemia. Long-term maintenance therapy includes low-fat diet and avoidance of fasting periods of more than 12 hours. It is also important to avoid situations of metabolic stress, mainly produced by recurrent illness that can be prevented by extra caloric intake (Morris et al., 1998).

### **5.** *HMGCS2* **gene**

Although mHS is codified by a gene (Ayte et al., 1990) different from the cHS gene (Gil et al., 1986), their high homology suggests a common origin about 500 millions years ago, when vertebrates appeared (Boukaftane et al., 1994).

Human *HMGCS2* gene is located in chromosome 1 (1p12-p13), between the markers WI-7519 and D1S514. It has 10 exons and 9 introns, with a total size of 21,708 bp (Figure 4). Exons length oscillates between 107 and 846 bp, and the intron's size varies between 0.4 and 4.1 kDa. cDNA has a total size of 2,082 bp.

So far, no splice variants of the gene have been experimentally confirmed, although the current Expressed Sequence Tag database includes a variant with a deletion of exon 4.

#### **5.1 Mutation update**

Up to date, 8 patients have been diagnosed by molecular analysis. Among them nine allelic variants have been identified: seven missense variants, one nonsense and one intronic mutation (Figures 3 and 4). One of these variants, the Y167C, affects to a closed Cys166 amino acid, which is considered the catabolic site of the enzyme (Hegard, 1999). The nonsense variant R424X (Bouchard et al., 2001; Morris et al., 1998), produced a truncated protein of 424 amino acids that probably cannot be incorporated into the active protein dimer. The intronic mutation is located in the first nucleotide of the intron 5 (c.1016+1G>A). In general, this type of mutations produces the deletion of the affected exon (exon 5 in this case), although in this disease the mechanism has not been confirmed (Zschocke et al., 2002).

In order to prove the effect of the mutation in the enzyme activity, protein over-expression has been tried with recombinant DNA techniques although with no success (Bouchard et al., 2001).

Fig. 4. Scheme of the mutations located in the human gene *HMGCS2*


Table 2. Clinical findings and biochemical data from patients with mHS deficiency

Mitochondrial HMG–CoA Synthase Deficiency 199

Another approach consisted in studying, with an indirect method, the mutation effect in MEV-1 cell cultures (Aledo et al., 2001). These studies were based in the cell mevalonate auxotrophy correction when they are transfected with cDNA of the *HMGCS2* gene. The expression of the mHS gene gives to MEV-1 cells the capacity to synthesize HMG-CoA inside the mitochondria, which is transformed in mevalonate and cholesterol (Ortiz et al., 1994). As expected, the mutated mHS cDNA transfection does not correct the MEV-1 cells

Recently, new mutations in mHS deficiency patients have been reported, but no clinical information was given. There are 8 missense mutations and one nonsense (Shafqat et al.,

In Table 2 an updated list of mutations reported in the *HMGCS2* gene is displayed, together

With the current knowledge available, it is difficult to establish strong genotype-phenotype correlations, among other reasons, for instance, because we ignore relevant information such as the levels of enzyme activity in affected patients. The seriousness of the disease may be more related to the agent that triggered hypoglycemia and to the time without treatment

Although mHS deficiency is an extremely rare disease, it is likely underdiagnosed and its prevalence is higher than estimated. Pediatricians may suspect this disorder in infants with vomiting, mild hepatomegaly, hypoglycemia, metabolic acidosis, increased levels of plasmatic free fatty acids and dicarboxylic aciduria, specially if symptoms appeared after a situation of metabolic stress, usually due to dieting in a gastroenteritis or during an infection process. Currently, the only reliable diagnostic –confirmatory- test is the molecular analysis of the gene, since measurements of the levels of enzymatic activity are masked by the

This study was supported by Spanish grants from: Diputación General de Aragón (Ref.# Grupo Consolidado B20) and University of Zaragoza (Ref.# PIF-UZ\_2009-BIO-02). We also

Aledo, R., Zschocke, J., Pie, J., Mir, C., Fiesel, S., Mayatepek, E., Hoffmann, GF., Casals, N., &

*Human Genetics,* Vol. 1, No. 109, (July 2001), pp. 19-23, ISSN 11479731 Ayte, J., Gil-Gomez, G., Haro, D., Marrero, PF., & Hegardt, FG. (1990). Rat mitochondrial

*America,* Vol. 10, No. 87, (May 1990), pp. 3874-3878, ISSN 1971108

Hegardt, FG. (2001). Genetic basis of mitochondrial HMG-CoA synthase deficiency.

and cytosolic 3-hydroxy-3-methylglutaryl-CoA synthases are encoded by two different genes. *Proceedings of the National Academy of Sciences of the United States of* 

thank to "Biomol-Informatics SL -www.bioinfo.es-" for bioinformatic support.

auxotrophy, which proves the deficiency of HMG-CoA synthesis.

2010).

with the patients´ clinical data.

than with the mutation itself.

activity of isoenzyme cHS.

**8. Acknowledgment** 

**9. References** 

**7. Conclusion** 

**6. Genotype-phenotype correlations** 

on Inborn Errors of Metabolism, San Diego CA and by Shafqat et al., 2010. Mutations I56N, T233A, K243E and R84X were reported by Shafqat et al., 2010. No clinical data were available from the patients who carried these mutations.

Reference Bouchard 2001; Thompson 1997 Bouchard 2001; Morris 1998 Aledo 2001 Zschocke 2002 Wolf 2003 Wolf 2003 Aledo 2006 Aledo 2006 Mutations G169D, T233A, K243E, L266S, I407T, R505Q, R84X and exon 1 deletion were reported by Pitt et al., at the 11th International Congress

G212R/IVS5+1G>A

9 months

Patient 4

Table 2. Clinical findings and biochemical data from patients with mHS deficiency

V54M/Y167C

4 years and

Patient 5

6 months

Genotype

Disease

Patient

debut

F174L/F174L

6 years

Patient 1

R424X/unknow

1 year and 4

Patient 2

months

G212R/R500H

11 months

Patient 3

n

n

V54M/Y167C

1 year and 7

Patient 6

months

Vomiting, hepatomegaly,

M307T/R188H

7 months

Patient 7

hipoglycemia hipoketonemia,

encephalopathy.

Vomiting, lethargy

M307T/R188H

1 year

Patient 8

Acylcarnitine Normal Normal Normal Normal Normal Normal Normal Normal Organic acids Normal Dicarboxylic aciduria Dicarboxylic aciduria Dicarboxylic aciduria Dicarboxylic aciduria Dicarboxylic aciduria Dicarboxylic aciduria Normal Symptoms and signs Gastroenteritis, hypoglycemia, hipoketonemia, superficial coma Diarrhea, vomiting, hepatomegaly, hypoglycemia, hipoketonemia, coma Gastroenteritis, vomiting, hepatomegaly, hypoglycemia, hipoketonemia, coma Gastroenteritis, diarrhea, vomiting, hepatomegaly, hypoglycemia, hipoketonemia, coma Gastroenteritis, diarrhea, vomiting, hypoglycemia, hipoketonemia, coma Hepatomegaly, normal hepatic functio

Another approach consisted in studying, with an indirect method, the mutation effect in MEV-1 cell cultures (Aledo et al., 2001). These studies were based in the cell mevalonate auxotrophy correction when they are transfected with cDNA of the *HMGCS2* gene. The expression of the mHS gene gives to MEV-1 cells the capacity to synthesize HMG-CoA inside the mitochondria, which is transformed in mevalonate and cholesterol (Ortiz et al., 1994). As expected, the mutated mHS cDNA transfection does not correct the MEV-1 cells auxotrophy, which proves the deficiency of HMG-CoA synthesis.

Recently, new mutations in mHS deficiency patients have been reported, but no clinical information was given. There are 8 missense mutations and one nonsense (Shafqat et al., 2010).

In Table 2 an updated list of mutations reported in the *HMGCS2* gene is displayed, together with the patients´ clinical data.

### **6. Genotype-phenotype correlations**

With the current knowledge available, it is difficult to establish strong genotype-phenotype correlations, among other reasons, for instance, because we ignore relevant information such as the levels of enzyme activity in affected patients. The seriousness of the disease may be more related to the agent that triggered hypoglycemia and to the time without treatment than with the mutation itself.

### **7. Conclusion**

Although mHS deficiency is an extremely rare disease, it is likely underdiagnosed and its prevalence is higher than estimated. Pediatricians may suspect this disorder in infants with vomiting, mild hepatomegaly, hypoglycemia, metabolic acidosis, increased levels of plasmatic free fatty acids and dicarboxylic aciduria, specially if symptoms appeared after a situation of metabolic stress, usually due to dieting in a gastroenteritis or during an infection process. Currently, the only reliable diagnostic –confirmatory- test is the molecular analysis of the gene, since measurements of the levels of enzymatic activity are masked by the activity of isoenzyme cHS.

### **8. Acknowledgment**

This study was supported by Spanish grants from: Diputación General de Aragón (Ref.# Grupo Consolidado B20) and University of Zaragoza (Ref.# PIF-UZ\_2009-BIO-02). We also thank to "Biomol-Informatics SL -www.bioinfo.es-" for bioinformatic support.

### **9. References**


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**10** 

*1,3Romania 2USA* 

**Alström Syndrome** 

*2The Jackson Laboratory, Bar Harbor, ME,* 

*1"Ovidius" University, Faculty of Medicine, Constanta,* 

*3"Ovidius" University, Faculty of Pharmacy, Constanta* 

Cristina Maria Mihai1, Jan D. Marshall2 and Ramona Mihaela Stoicescu3

Recent advancements in genetic research that have elucidated the function of some of the rare disease-causing genes have suggested that a large number of genetic disorders with widely divergent phenotypes, that were not previously identified as related, may be, in fact, highly related in cellular function or common pathways. A classic example of this is the recent category of disorders called ciliopathies. Cilia and flagella are ancient, evolutionarily conserved organelles that project from cell surfaces to perform diverse biological roles, including whole-cell locomotion; movement of fluid; chemo-, mechano-, and photosensation; and sexual reproduction. Over the past ten years, several studies demonstrated the connections between cilia, basal bodies and human diseases with a wide phenotypic spectrum, including randomization of body symmetry, obesity, cystic kidney diseases and retinal degeneration. Defects in ciliary structure or function can lead to a broader set of developmental and adult phenotypes, with mutations in ciliary proteins now associated with nephronophthisis, Joubert Syndrome, Meckel-Gruber Syndrome, Bardet-Biedl Syndrome, and Alström Syndrome (ALMS), [Badano et al., 2006]. Further study of these diverse ciliopathies could lead to an understanding of the phenotypic patterns that could potentially have predictive and therapeutic value. Alström Syndrome (ALMS; MIM #203800), first described by Carl-Henry Alström, in 1959 [Alström et al., 1959], is a rare condition that affects many body systems. ALMS is characterized by a constelation of serious or life-threatening medical problems including sensory deficits, obesity, type 2 diabetes mellitus, and multiple organ failure. The signs and symptoms of ALMS vary in severity, and not all affected individuals have all of the characteristic features of the disorder, making the diagnosis more difficult. Additionally, many of the signs and symptoms of this condition begin in infancy or early childhood, although some appear later in life. The major phenotypes usually observed in children with ALMS include cone–rod retinal dystrophy beginning in infancy and leading to juvenile blindness, sensorineural hearing impairment, insulin resistance, and obesity, and congestive heart failure (CHF) due to dilated cardiomyopathy (DCM). As patients reach adolescence, more of the major phenotypes develop, including type 2 diabetes mellitus, hypertriglyceridemia, hypothyroidism, and short adult stature. Males and females have hypogonadism and are infertile. Pulmonary, hepatic, and renal phenotypes are progressive [Marshall et al. 1997,

**1. Introduction** 

esters in livers of fed or starved pregnant, lactating and weaned rats. *Biochemical Journal*, Vol. 198, No. 1 (July 1981), pp. 75-83 ISSN 7326003

