**4. Cholesterol biosynthesis genes in other Mendelian diseases**

Inherited defects in genes encoding cholesterol biosynthetic enzymes or regulators of cholesterol homeostasis create severe clinical phenotypes as discussed above and highlighted in **Table 1**. The central nervous system is highly susceptible to perturbations in cholesterol biosynthesis, with manifestations including structural brain malformations, defects in myelin structures and, in some cases, profound developmental delay. While the cholesterol biosynthesis defects are genetically distinct individual disorders, their characterisation has demonstrated interrelation between human disease processes. This underscores the importance of cholesterol in normal cellular function and opens the possibility of novel therapies for Mendelian disorders associated with cholesterol synthesis, transport and regulation. Lessons learnt from abrogation of the cholesterol biosynthesis pathway, either by deliberate pharmacological manipulation or via inherited Mendelian diseases, serve to provide vital information amongst a raft of seemingly unrelated human disease such as inflammatory bowel disease (IBD), the cholesterol trafficking disorders Niemann-Pick disease type C (NPC, OMIM 257220) and Tangier disease (TD, OMIM 205400) and neurodegenerative diseases such as Alzheimer's disease (OMIM 104300).

NPC is an autosomal recessive lysosomal storage disorder of cholesterol trafficking due to mutations in the *NPC1* and *NPC2* genes [152]. *NPC1* encodes a 13-transmembrane-spanning protein in late endosomes/lysosomes, while *NPC2* encodes a soluble lysosomal cholesterol-binding protein [153]. This is a devastating disease characterised by a relentless neurodegenerative disease course that is usually fatal in the second decade of life, although a subset of patients will die in infancy consequent to hepatic or pulmonary failure [152]. Free cholesterol is

**143**

SLOS and TD.

*Human Cholesterol Biosynthesis Defects DOI: http://dx.doi.org/10.5772/intechopen.87150*

storage metabolite in NPC [154].

for NPC were negative [160].

stored in the late endosome/lysosome with minimal escape of cholesterol from the acidic compartment to the endoplasmic reticulum. NPC leads to a block in trafficking/fusion essential for the functioning of the endosomal/lysosomal system, causing the secondary storage of cholesterol, glycosphingolipids and sphingomyelin [154]. It is likely that cholesterol accumulation is a secondary

TD has been reported in approximately 100 patients and is caused by mutations in the gene encoding ABCA1 [155, 156]. Patients have minimal circulating HDL and accumulate cholesterol, leading to the formation of foam cells and the development of cardiovascular disease, orange-coloured tonsils, enlarged spleen, liver and lymph nodes and peripheral neuropathy. The membrane-associated protein ABCA1 regulates cellular cholesterol and phospholipid homeostasis by functioning as a cholesterol efflux pump [157]. Tangier disease patients have structurally abnormal late endocytic vesicles, which are also observed in the cells of patients with NPC disease [158]. There exists a link between ABCA1 expression and function with the NPC pathway [158, 159]. NPC disease is characterised at the cellular level by storage of glycosphingolipids, fatty acids, cholesterol, sphingomyelin and sphingosine. NPC cells also have low levels of calcium in the late endosome/lysosome. These cellular hallmarks were also identified in TD patients, suggesting that the loss of function of ABCA1 inhibits the NPC pathway through an unknown mechanism. A recent serendipitous clinical observation has provided a further link between TD and the NPC pathway: an adult patient thought to have NPC1 was treated with miglustat and demonstrated measurable clinical improvements in neurological and haematological parameters. TD was ultimately diagnosed when the molecular investigations

SLOS cellular pathophysiology should theoretically be correctable with cholesterol replacement therapy, as this should bypass the enzymatic defect in the conversion of 7DHC to cholesterol. However, when SLOS patient fibroblasts are cultured in a lipid-depleted medium to induce *de novo* cholesterol synthesis, cells exhibit a significant cholesterol trafficking defect leading to the accumulation of unesterified cholesterol in the late endosome/lysosome, which mimics the fate of LDL-derived cholesterol in NPC cells [161]. This proposes a possible mechanistic convergence between these very different inborn errors of metabolism. 7DHC could be interfering with the function of NPC1 and NPC2 by inhibition, akin to the U18666A drug that induces NPC cellular phenotypes [162]. In SLOS patient fibroblasts, accumulation of 7DHC led to the accumulation of metabolic indicators of NPC, that is, the lysosomal storage of cholesterol, sphingomyelin and multiple glycosphingolipids [163]. Elevated sphingosine levels in SLOS patient cerebrospinal fluid have been described. This serendipitous discovery of a link between the NPC pathway, two cholesterol trafficking disorders and the prototypic cholesterol biosynthesis defect SLOS will prove important in delineating the pathogenesis of these diseases and the development of novel therapies. Miglustat is an iminosugar drug that inhibits glucosylceramide synthase, the enzyme that catalyses the first step in glycosphingolipid biosynthesis, and it is in use as a substrate reduction therapy for a number of lysosomal storage defects including NPC [164]. The finding that SLOS and TD involve secondary inhibition of the NPC pathway suggests that miglustat could be a novel therapy for

The recently described SQSD exhibits a characteristic sterol pattern dominated by farnesol-derived dicarboxylic acids secondary to accumulation of farnesol-PP proximal to the enzymatic block. The role of these metabolites in the pathogenesis of this rare disease remains to be determined but is of interest as farnesol and its products exhibit a wide variety of biological activities including

#### *Human Cholesterol Biosynthesis Defects DOI: http://dx.doi.org/10.5772/intechopen.87150*

*Apolipoproteins, Triglycerides and Cholesterol*

Desmosterolosis (OMIM 602398) is currently the final inborn error of cholesterol biosynthesis and is caused by defective enzymatic function of 3-hydroxysterol-delta 24-reductase (DHCR24). This reaction causes the reduction of the C-24 bond in the aliphatic side chain of cholesterol [140]. Reduction of the C-24 bond catalysed by DHCR24 can occur at different times in the cholesterol synthetic pathway: this step occurs early in the Kandutsch-Russel cholesterol synthetic pathway [6] but is the

While first described in 1998, the molecular mechanisms of desmosterolosis were not characterised until 2001 [140, 141]. To date, only nine cases have been reported and clinical features include SLOS-like dysmorphism, thick alveolar ridges, gingival nodules, cleft palate, short limbs, severe congenital heart defect, atherosclerosis, arthrogryposis, ambiguous genitalia, microcephaly, agenesis of the corpus callosum, global developmental delay and intellectual impairment [141–147]. The diagnosis of desmosterolosis is made by demonstrating elevated levels of desmosterol by GC-MS analysis, with serum cholesterol levels usually normal [141, 142]. Reported *DHCR24* pathogenic mutations thus far have all been

A targeted mouse model for desmosterolosis has been generated, and *Dhcr24*<sup>−</sup>/<sup>−</sup>

Inherited defects in genes encoding cholesterol biosynthetic enzymes or regulators of cholesterol homeostasis create severe clinical phenotypes as discussed above and highlighted in **Table 1**. The central nervous system is highly susceptible to perturbations in cholesterol biosynthesis, with manifestations including structural brain malformations, defects in myelin structures and, in some cases, profound developmental delay. While the cholesterol biosynthesis defects are genetically distinct individual disorders, their characterisation has demonstrated interrelation between human disease processes. This underscores the importance of cholesterol in normal cellular function and opens the possibility of novel therapies for Mendelian disorders associated with cholesterol synthesis, transport and regulation. Lessons learnt from abrogation of the cholesterol biosynthesis pathway, either by deliberate pharmacological manipulation or via inherited Mendelian diseases, serve to provide vital information amongst a raft of seemingly unrelated human disease such as inflammatory bowel disease (IBD), the cholesterol trafficking disorders Niemann-Pick disease type C (NPC, OMIM 257220) and Tangier disease (TD, OMIM 205400) and neurodegenerative diseases such as Alzheimer's disease

NPC is an autosomal recessive lysosomal storage disorder of cholesterol trafficking due to mutations in the *NPC1* and *NPC2* genes [152]. *NPC1* encodes a 13-transmembrane-spanning protein in late endosomes/lysosomes, while *NPC2* encodes a soluble lysosomal cholesterol-binding protein [153]. This is a devastating disease characterised by a relentless neurodegenerative disease course that is usually fatal in the second decade of life, although a subset of patients will die in infancy consequent to hepatic or pulmonary failure [152]. Free cholesterol is

mice are viable with some postnatal growth retardation and infertility [148]. Pharmacological inhibitors of DHCR24 have been developed for studies in rat models [135, 149, 150]. Treatment of pregnant rats with these inhibitors of sterol-D24-reductase is teratogenic and produces cataracts, CNS abnormalities, genitouri-

**4. Cholesterol biosynthesis genes in other Mendelian diseases**

penultimate step in the Bloch pathway of cholesterol synthesis [5].

**3.6 Desmosterolosis**

missense mutations.

nary and skeletal anomalies [149–151].

**142**

(OMIM 104300).

stored in the late endosome/lysosome with minimal escape of cholesterol from the acidic compartment to the endoplasmic reticulum. NPC leads to a block in trafficking/fusion essential for the functioning of the endosomal/lysosomal system, causing the secondary storage of cholesterol, glycosphingolipids and sphingomyelin [154]. It is likely that cholesterol accumulation is a secondary storage metabolite in NPC [154].

TD has been reported in approximately 100 patients and is caused by mutations in the gene encoding ABCA1 [155, 156]. Patients have minimal circulating HDL and accumulate cholesterol, leading to the formation of foam cells and the development of cardiovascular disease, orange-coloured tonsils, enlarged spleen, liver and lymph nodes and peripheral neuropathy. The membrane-associated protein ABCA1 regulates cellular cholesterol and phospholipid homeostasis by functioning as a cholesterol efflux pump [157]. Tangier disease patients have structurally abnormal late endocytic vesicles, which are also observed in the cells of patients with NPC disease [158]. There exists a link between ABCA1 expression and function with the NPC pathway [158, 159]. NPC disease is characterised at the cellular level by storage of glycosphingolipids, fatty acids, cholesterol, sphingomyelin and sphingosine. NPC cells also have low levels of calcium in the late endosome/lysosome. These cellular hallmarks were also identified in TD patients, suggesting that the loss of function of ABCA1 inhibits the NPC pathway through an unknown mechanism. A recent serendipitous clinical observation has provided a further link between TD and the NPC pathway: an adult patient thought to have NPC1 was treated with miglustat and demonstrated measurable clinical improvements in neurological and haematological parameters. TD was ultimately diagnosed when the molecular investigations for NPC were negative [160].

SLOS cellular pathophysiology should theoretically be correctable with cholesterol replacement therapy, as this should bypass the enzymatic defect in the conversion of 7DHC to cholesterol. However, when SLOS patient fibroblasts are cultured in a lipid-depleted medium to induce *de novo* cholesterol synthesis, cells exhibit a significant cholesterol trafficking defect leading to the accumulation of unesterified cholesterol in the late endosome/lysosome, which mimics the fate of LDL-derived cholesterol in NPC cells [161]. This proposes a possible mechanistic convergence between these very different inborn errors of metabolism. 7DHC could be interfering with the function of NPC1 and NPC2 by inhibition, akin to the U18666A drug that induces NPC cellular phenotypes [162]. In SLOS patient fibroblasts, accumulation of 7DHC led to the accumulation of metabolic indicators of NPC, that is, the lysosomal storage of cholesterol, sphingomyelin and multiple glycosphingolipids [163]. Elevated sphingosine levels in SLOS patient cerebrospinal fluid have been described. This serendipitous discovery of a link between the NPC pathway, two cholesterol trafficking disorders and the prototypic cholesterol biosynthesis defect SLOS will prove important in delineating the pathogenesis of these diseases and the development of novel therapies. Miglustat is an iminosugar drug that inhibits glucosylceramide synthase, the enzyme that catalyses the first step in glycosphingolipid biosynthesis, and it is in use as a substrate reduction therapy for a number of lysosomal storage defects including NPC [164]. The finding that SLOS and TD involve secondary inhibition of the NPC pathway suggests that miglustat could be a novel therapy for SLOS and TD.

The recently described SQSD exhibits a characteristic sterol pattern dominated by farnesol-derived dicarboxylic acids secondary to accumulation of farnesol-PP proximal to the enzymatic block. The role of these metabolites in the pathogenesis of this rare disease remains to be determined but is of interest as farnesol and its products exhibit a wide variety of biological activities including

cell growth inhibition, induction of apoptosis and regulation of bile acid secretion [165]. Evidence is emerging that dysregulation of the mevalonate pathway may be involved in the progression of neurodegeneration in disorders such as Alzheimer's disease [166].

Inflammatory bowel disease (IBD) comprises a spectrum of phenotypes from Crohn's disease to ulcerative colitis. IBD usually occurs in young adults; however, onset in infancy and childhood are described. IBD occurs both in isolation and in monogenic syndromes with early-onset autoinflammation including the *NOD2*, *ATG16L1*, *IL23R*, *IL10R*, *IL10* and *XIAP* genes which have previously been correlated with IBD both in multifactorial and in Mendelian models [167]. MVK mutations may perhaps then synergistically augment the risk of developing IBD, especially as severe neonatal onset colitis responsive to anakinra has been reported as a feature of MVK deficiency [14, 168, 169].

Recent studies have implicated the accumulation of pre-cholesterol sterols and the replacement of cholesterol with some of these sterols in lipid rafts as playing a key role in the underlying pathophysiology of cholesterol synthesis defects [170]. The meiosis-activating sterols were the first group of cholesterol biogenesis intermediates that were found to have important extrahepatic functions in mammals. Mutations in sterol-C4-methyl oxidase-like gene (*SC4MOL*) are causative for a rare autosomal recessive syndrome associated with psoriasiform dermatitis, arthralgias, congenital cataracts, microcephaly and developmental delay [171, 172]. This gene encodes a sterol-C4-methyl oxidase (SMO) which catalyses demethylation of C4-methylsterols in the cholesterol synthesis pathway [172]. C4-methylsterols are meiosis-activating sterols, and further work is required to understand the role of these novel biomolecules in the pathogenesis of the cholesterol biosynthesis defects.
