**2. Disorders of the pre-squalene cholesterol pathway**

#### **2.1 Mevalonate kinase deficiency**

Mevalonate kinase phosphorylates mevalonate, the product of the reduction of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA), which is important in cholesterol biosynthesis and for farnesylation and isoprenylation of proteins [13]. Mevalonate kinase deficiency (MKD) is a recessively inherited autoinflammatory disorder in the isoprenoid biosynthetic pathway with a spectrum of manifestations, including the well-defined allelic clinical phenotypes of HIDS and MA [14], both of which were identified in the mid-1980s [15, 16].

Mevalonate kinase is essential for the biosynthesis of non-sterol isoprenoids, which mediate protein prenylation. MKD is caused by mutations in the *MKD* gene which encodes mevalonate kinase, with the degree of residual enzyme activity largely determining disease severity. MKD leads to perturbations in the mevalonate pathway of cholesterol synthesis with episodes of hyperinflammation [17]. MKD is now viewed as a phenotypic continuum based on the degree of enzyme deficiency, with MA the most severe phenotype and HIDS the mild end of the spectrum [18].

MKD is characterised by autoinflammatory flares with fever, abdominal pain, mucoid and cutaneous lesions and arthralgias [14]. The more severely affected patients with MA classically have developmental delay, dysmorphism, psychomotor retardation, hepatosplenomegaly and ocular abnormalities [14]. During attacks, patients with MKD have increased levels of acute-phase proteins including C-reactive protein and cytokines such as TNF-α, IL-6 and interferon-γ [19, 20]. The MA phenotype characteristically presents in the first few months of life, with antenatal presentations linked with a high rate of stillbirth [21]. Commonly reported dysmorphic features include frontal bossing, hypertelorism, long eyelashes and triangular-shaped facies, as well as failure to thrive, developmental delay, ataxia, seizures, myopathies and autoinflammatory attacks [21, 22]. MA is a multisystem phenotype with gastrointestinal manifestations including cholestasis and liver dysfunction [23], and ocular findings including recurrent conjunctivitis, cataracts and uveitis [24].

The HIDS phenotype typically presents with recurrent (four-to-six weekly) self-limited bouts of multisystem inflammation characterised by fever, abdominal pain, adenopathy, rash and arthralgia [14]. As these are common symptoms of many childhood infectious illnesses, the diagnosis of HIDS is often delayed for many years. HIDS episodes usually last 3–7 days, occurring in a cyclical fashion or induced by a provocative physiological stress such as illness, injury or vaccination. Acute abdominal pain may be the most marked and debilitating feature of systemic inflammation and can mimic a 'surgical acute abdomen' [24]. A long-term followup study of 103 HIDS patients revealed that the frequency of the attacks decreases

**135**

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

ated with MA [25].

cell transplantation [14].

**2.2 Squalene synthase deficiency**

over time, but 50% of patients greater than 20 years of age still experience six or

Although the precise pathogenesis of MKD remains unclear, increasing evidence suggests that deficiency in protein prenylation leads to innate immune activation and systemic hyperinflammation, which has assisted in the development of cytokine-directed biologic therapy. Corticosteroids induce a complete response in 24% of HIDS patients [30]. Biologics targeting IL-1, including anakinra and canakinumab, and TNF-α blocking agents such as etanercept and adalimumab, have been used with varying success [30]. Some cases that have failed to respond to anakinra have demonstrated a successful reduction in symptoms with tocilizumab, a monoclonal antibody targeted against the IL-6 receptor [31]. One patient with MKD, treated with alendronate for steroid-induced osteoporosis, subsequently achieved complete remission [32]. Alendronate inhibits farnesol-pyrophosphate synthase. For refractory cases of MA phenotype, the last consideration for therapy includes liver transplantation or haematopoietic stem

Blockade of the mevalonate pathway with the HMG-CoA reductase inhibitors reduces both mevalonic acid levels and residual isoprenoid production and but can trigger disease flares [22]. The inflammatory hyper-responsiveness in MKD appears to be due to lack of isoprenoid products and not accumulation of mevalonic acid. This appears to be due to the need for geranylgeranylation rather than other mevalonate pathway products, such as cholesterol biosynthesis, in mediating the hypersecretion of IL-1β [27]. The use of statins in this disease process has therefore largely been abandoned [30]. *Mvk*+/<sup>−</sup> mice do have some features of immune dysfunction, including increased serum IgD and TNF-α levels, as well as increased expression of

Squalene synthase deficiency (SQSD) is a recently identified pre-squalene defect

to have been characterised. In 2018, three patients were reported with this novel cholesterol biosynthesis defect [4]. Salient clinical features include facial dysmorphism, dry skin with photosensitivity, generalised tonic-clonic seizures, structural brain malformations, cortical visual impairment, profound global developmental

activation markers on T-lymphocytes and macrophages [33].

delay and genital malformations in the two males [4].

The epidemiology of MKD is largely unknown. At least 300 people are documented worldwide, the majority with HIDS, although this is likely to be underdiagnosed as recurrent fevers in childhood are a common occurrence. The highest documented prevalence is in the Netherlands, with an estimated 1:200,000 affected nationwide, consequent to a high carrier rate which is estimated at 1:65 [24, 25]. Elevations in IgD in MKD are inconsistent and can be normal in up to 20% of cases [24]. Serum amyloidosis is a long-term sequela of prolonged inflammatory activation, with elevations in serum amyloid A noted in approximately 3% of HIDS patients [24]. Urinary excretion of mevalonic acid can persist in MA and maybe present in some HIDS patients during febrile attacks [21]. The diagnosis is confirmed by identifying pathogenic mutations in the *MVK* gene; currently more than 120 sequence variants in this gene have been reported in association with MKD [26], most of which are missense mutations that impair mevalonate kinase stability [27]. Some genotype-phenotype correlations exist: *MVK* variants located in the core of the protein (affecting folding and stability) are highly associated with the more severe MA phenotype [25, 28, 29]. In contrast, other variants such as the C-terminal V377I substitution typically manifest as the HIDS phenotype and are rarely associ-

more attacks per year, impacting on the quality of life [24].

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

*Apolipoproteins, Triglycerides and Cholesterol*

**2.1 Mevalonate kinase deficiency**

which were identified in the mid-1980s [15, 16].

Modulating flux through the cholesterol biosynthesis pathway has been of interest for many years as a pharmacological treatment option for hypercholesterolemia. The statin family of drugs inhibit HMG-CoA reductase, the rate limiting step in the pre-squalene pathway, and similar efforts have focused on inhibitors of squalene synthase as this enzyme is the first committed step in cholesterol biosynthesis. Animal and human models of squalene synthase inhibitors generated a complex array of farnesol-derived metabolites [10–12], the recognition of which was instrumental in describing SQSD, a newly described pre-squalene cholesterol biosynthesis defect [4]. That pathogenesis of the cholesterol biosynthesis defects is complex,

Mevalonate kinase phosphorylates mevalonate, the product of the reduction of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA), which is important in cholesterol biosynthesis and for farnesylation and isoprenylation of proteins [13]. Mevalonate kinase deficiency (MKD) is a recessively inherited autoinflammatory disorder in the isoprenoid biosynthetic pathway with a spectrum of manifestations, including the well-defined allelic clinical phenotypes of HIDS and MA [14], both of

Mevalonate kinase is essential for the biosynthesis of non-sterol isoprenoids, which mediate protein prenylation. MKD is caused by mutations in the *MKD* gene which encodes mevalonate kinase, with the degree of residual enzyme activity largely determining disease severity. MKD leads to perturbations in the mevalonate pathway of cholesterol synthesis with episodes of hyperinflammation [17]. MKD is now viewed as a phenotypic continuum based on the degree of enzyme deficiency, with MA the most severe phenotype and HIDS the mild end of the spectrum [18]. MKD is characterised by autoinflammatory flares with fever, abdominal pain, mucoid and cutaneous lesions and arthralgias [14]. The more severely affected patients with MA classically have developmental delay, dysmorphism, psychomotor retardation, hepatosplenomegaly and ocular abnormalities [14]. During attacks, patients with MKD have increased levels of acute-phase proteins including C-reactive protein and cytokines such as TNF-α, IL-6 and interferon-γ [19, 20]. The MA phenotype characteristically presents in the first few months of life, with antenatal presentations linked with a high rate of stillbirth [21]. Commonly reported dysmorphic features include frontal bossing, hypertelorism, long eyelashes and triangular-shaped facies, as well as failure to thrive, developmental delay, ataxia, seizures, myopathies and autoinflammatory attacks [21, 22]. MA is a multisystem phenotype with gastrointestinal manifestations including cholestasis and liver dysfunction [23], and ocular findings including recurrent conjunctivitis, cataracts

The HIDS phenotype typically presents with recurrent (four-to-six weekly) self-limited bouts of multisystem inflammation characterised by fever, abdominal pain, adenopathy, rash and arthralgia [14]. As these are common symptoms of many childhood infectious illnesses, the diagnosis of HIDS is often delayed for many years. HIDS episodes usually last 3–7 days, occurring in a cyclical fashion or induced by a provocative physiological stress such as illness, injury or vaccination. Acute abdominal pain may be the most marked and debilitating feature of systemic inflammation and can mimic a 'surgical acute abdomen' [24]. A long-term followup study of 103 HIDS patients revealed that the frequency of the attacks decreases

reflective of the multisystem nature of the clinical phenotypes.

**2. Disorders of the pre-squalene cholesterol pathway**

**134**

and uveitis [24].

over time, but 50% of patients greater than 20 years of age still experience six or more attacks per year, impacting on the quality of life [24].

The epidemiology of MKD is largely unknown. At least 300 people are documented worldwide, the majority with HIDS, although this is likely to be underdiagnosed as recurrent fevers in childhood are a common occurrence. The highest documented prevalence is in the Netherlands, with an estimated 1:200,000 affected nationwide, consequent to a high carrier rate which is estimated at 1:65 [24, 25].

Elevations in IgD in MKD are inconsistent and can be normal in up to 20% of cases [24]. Serum amyloidosis is a long-term sequela of prolonged inflammatory activation, with elevations in serum amyloid A noted in approximately 3% of HIDS patients [24]. Urinary excretion of mevalonic acid can persist in MA and maybe present in some HIDS patients during febrile attacks [21]. The diagnosis is confirmed by identifying pathogenic mutations in the *MVK* gene; currently more than 120 sequence variants in this gene have been reported in association with MKD [26], most of which are missense mutations that impair mevalonate kinase stability [27]. Some genotype-phenotype correlations exist: *MVK* variants located in the core of the protein (affecting folding and stability) are highly associated with the more severe MA phenotype [25, 28, 29]. In contrast, other variants such as the C-terminal V377I substitution typically manifest as the HIDS phenotype and are rarely associated with MA [25].

Although the precise pathogenesis of MKD remains unclear, increasing evidence suggests that deficiency in protein prenylation leads to innate immune activation and systemic hyperinflammation, which has assisted in the development of cytokine-directed biologic therapy. Corticosteroids induce a complete response in 24% of HIDS patients [30]. Biologics targeting IL-1, including anakinra and canakinumab, and TNF-α blocking agents such as etanercept and adalimumab, have been used with varying success [30]. Some cases that have failed to respond to anakinra have demonstrated a successful reduction in symptoms with tocilizumab, a monoclonal antibody targeted against the IL-6 receptor [31]. One patient with MKD, treated with alendronate for steroid-induced osteoporosis, subsequently achieved complete remission [32]. Alendronate inhibits farnesol-pyrophosphate synthase. For refractory cases of MA phenotype, the last consideration for therapy includes liver transplantation or haematopoietic stem cell transplantation [14].

Blockade of the mevalonate pathway with the HMG-CoA reductase inhibitors reduces both mevalonic acid levels and residual isoprenoid production and but can trigger disease flares [22]. The inflammatory hyper-responsiveness in MKD appears to be due to lack of isoprenoid products and not accumulation of mevalonic acid. This appears to be due to the need for geranylgeranylation rather than other mevalonate pathway products, such as cholesterol biosynthesis, in mediating the hypersecretion of IL-1β [27]. The use of statins in this disease process has therefore largely been abandoned [30]. *Mvk*+/<sup>−</sup> mice do have some features of immune dysfunction, including increased serum IgD and TNF-α levels, as well as increased expression of activation markers on T-lymphocytes and macrophages [33].

#### **2.2 Squalene synthase deficiency**

Squalene synthase deficiency (SQSD) is a recently identified pre-squalene defect to have been characterised. In 2018, three patients were reported with this novel cholesterol biosynthesis defect [4]. Salient clinical features include facial dysmorphism, dry skin with photosensitivity, generalised tonic-clonic seizures, structural brain malformations, cortical visual impairment, profound global developmental delay and genital malformations in the two males [4].

Gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy profiles yielded a consistent and complex pattern of abnormal metabolites including accumulation of methylsuccinic acid, mevalonate lactone, mesaconic acid, 3-methyladipic acid, saturated and unsaturated branchedchain dicarboxylic acids and glucuronides derived from farnesol [4]. A similar metabolite profile has previously been observed in the urine of animal models and humans treated with pharmacological inhibitors of squalene synthase, as well as in animals loaded with farnesol [10–12]. This urine metabolic profile is specific to and thus diagnostic of SQSD. Plasma total farnesol levels (the sum of free farnesol and farnesyl-pyrophosphate) in affected individuals were, however, significantly increased (1.5–3.9 mmol/L; reference <0.12) while plasma squalene levels were reduced or normal (0.17–0.93 mmol/L, reference 0.36–1.04).

A range of pathogenic *FDFT1* molecular variants have been described in the three SQSD patients identified thus far (a sibship and an unrelated patient) [4]. The sibship was compound heterozygous for a maternally-inherited 120 kb deletion, resulting in loss of exons 6–10 of *FDFT1* and the entire coding sequence of the neighbouring *CTSB* gene (encoding cathepsin B (OMIM 116810)); and a paternally inherited variant c.88024\_88023delinsAG, which created a novel splice acceptor site. The unrelated patient was homozygous for a novel 16-bp intronic deletion. Functional characterisation of the variants demonstrated a partial splicing defect and altered promoter and/or enhancer activity, reflecting essential mechanisms for regulating cholesterol biosynthesis and/or uptake in steady state [4].

*Fdft1*-null mice demonstrate embryonic lethality at day 12.5 in conjunction with growth restriction and neurodevelopmental disorders [34]. The fact that the *FDFT1* variants in the human SQSD cases are compatible with life may be explained by the fact that all individuals have some form of residual FDFT1 activity, either resulting from the diminished levels of correctly-spliced enzyme or by functional compensation for disrupted regulation [4].

#### **2.3 Lanosterol synthase deficiency**

In the cholesterol biosynthesis pathway, lanosterol synthase leads to the cyclisation of (S)-2,3-oxidosqualene into lanosterol. Pathogenic mutations in the *LSS* gene have recently been reported in a spectrum of clinical phenotypes including congenital cataracts in three families [35], hypotrichosis simplex (HS) in three families [36] and a more severe neuroectodermal syndrome formerly named alopecia with mental retardation (APMR) syndrome in six unrelated families [37]. HS (OMIM 618275) is a rare form of hereditary alopecia characterised by childhood onset of diffuse and progressive scalp and body hair loss [36]. APMR syndrome (OMIM 203650) is a rare disorder with autosomal recessive transmission. A recent report identified 11 individuals from seven unrelated families affected with alopecia, male genital abnormalities, variable MRI abnormalities and neurological symptoms [37]. In this cohort, total alopecia was universal with other common dermatological manifestations including ichthyosis and erythroderma. Neurological manifestations included significant developmental delay, microcephaly, epilepsy and hypomyelination [37].

Sterol profiling in lanosterol synthase deficiency cases has not identified any specific abnormalities, thus supporting the previously proposed hypothesis of an alternative cholesterol pathway [36]. *LSS* variants identified to date include truncating, missense and splicing variants. *LSS* has also been associated with congenital cataracts in rat [38]. Mice homozygous for the Lsstm1b(KOMP)Wtsi allele demonstrate variable lethality, from embryonic day 9.5 to postnatal prior to weaning [39].

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

cholesterol biosynthesis [48, 52].

**3. Disorders of the post-squalene cholesterol pathway**

**3.1 Hydrops-ectopic calcification-moth-eaten skeletal dysplasia**

Most proximal in the post-squalene pathway is hydrops-ectopic calcification-moth-

eaten (HEM) skeletal dysplasia, or Greenberg dysplasia. This very rare and severe autosomal recessive disorder was first described in 1988 [40] with only 11 examples identified in the literature to date. All but one of these have been lethal *in utero*, with the remaining case dying at 2 days of age [41]. HEM skeletal dysplasia is characterised by significant non-immune hydrops fetalis, erroneous chondro-osseous calcification of vertebrae, ribs, pelvis, larynx and trachea as well as a diagnostic mottled 'moth-eaten' appearance of long bones on radiography [42–44]. Further skeletal abnormalities can include rhizomelic and mesomelic shortening of the limbs, platyspondyly, decreased skull ossification and distal dysmorphisms such as absent phalanges or postaxial polydactyly [42–45]. Non-skeletal congenital malformations include pulmonary hypoplasia, intestinal malrotation, cystic hygroma and excessive extramedullary haematopoiesis [45, 46]. Histology shows significant bone and cartilage disorganisation [43, 45]. HEM skeletal dysplasia was first suggested as an inborn error of cholesterol

biosynthesis by Kelley et al. [47] with identification of increased levels of 4,4-dimethylcholesta-8 [9],14-dien-3β-ol and 4,4-dimethylcholesta-8(9),14,24 trien-3β-ol in cultured fibroblasts, indicating a deficiency of sterol ∆14-reductase. This enzyme converts these sterols to 4,4-dimethylcholesta-8(9)-en-3β-ol and 4,4-dimethylcholesta-8(9),24-dien-3β-ol, respectively. This point on the cholesterol biosynthesis pathway is unique with sterol ∆14-reductase activity by both the lamin B receptor (LBR) and a second enzyme DHCR14 (TM7FS2), although functional redundancy is disputed [48, 49]. It was originally thought that the more prominent role in sterol biosynthesis was that of DHCR14 compared to the lamin B receptor. However, it has more recently been demonstrated that it is a deficiency in the lamin B receptor due to mutations in *LBR* at 1q42.12 that is causative for HEM skeletal dysplasia [50, 51] and that it is the LBR, not DHCR14, that is required for

The involvement of *LBR* has raised contention as to whether HEM skeletal dysplasia should be classified as a laminopathy rather than as an error of cholesterol synthesis [49]; however, it is appropriate to recognise that mutations in *LBR* can cause different disorders in different contexts. The type of mutation (missense, nonsense or splice-site), the functional location of each mutation in the *LBR* gene and the residual protein activity affect the clinical outcome of this disorder [53, 54]. The LBR protein has both a nuclear domain involved in anchoring chromatin to the nuclear membrane, and a transmembrane domain with sterol ∆14-reductase activity critical for cholesterol synthesis [48], the latter primarily where mutations causing HEM dysplasia are located [50]. Some mutations found in *LBR* in HEM dysplasia patients have been identified in the heterozygous state in the relatively benign autosomal dominant condition of Pelger-Huët anomaly in which granulocytes have bilobed nuclei but patients are otherwise clinically normal. These two conditions may represent different allele patterns of the same disorder for some mutations [53, 55]. The less common homozygous Pelger-Huët is clinically more severe with round or ovoid granulocyte nuclei and some cases with mild skeletal abnormalities [56, 57]. This highlights the role of the lamin B receptor sterol reductase function as essential in prenatal development but also the phenotypic continuum that can occur for various allele combinations of the *LBR* gene. Species variation with respect to the role of the LBR can make mouse model outcomes difficult to elucidate. Studies of mutations in both *LBR* and *DHCR14/ TM7FS2* have been investigated in ichthyosis (*ic*) mice with contrasting conclusions, including those highlighted above and as reviewed by Herman and Kratz [58].

*Apolipoproteins, Triglycerides and Cholesterol*

Gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy profiles yielded a consistent and complex pattern of abnormal metabolites including accumulation of methylsuccinic acid, mevalonate lactone, mesaconic acid, 3-methyladipic acid, saturated and unsaturated branchedchain dicarboxylic acids and glucuronides derived from farnesol [4]. A similar metabolite profile has previously been observed in the urine of animal models and humans treated with pharmacological inhibitors of squalene synthase, as well as in animals loaded with farnesol [10–12]. This urine metabolic profile is specific to and thus diagnostic of SQSD. Plasma total farnesol levels (the sum of free farnesol and farnesyl-pyrophosphate) in affected individuals were, however, significantly increased (1.5–3.9 mmol/L; reference <0.12) while plasma squalene levels were

A range of pathogenic *FDFT1* molecular variants have been described in the three SQSD patients identified thus far (a sibship and an unrelated patient) [4]. The sibship was compound heterozygous for a maternally-inherited 120 kb deletion, resulting in loss of exons 6–10 of *FDFT1* and the entire coding sequence of the neighbouring *CTSB* gene (encoding cathepsin B (OMIM 116810)); and a paternally inherited variant c.88024\_88023delinsAG, which created a novel splice acceptor site. The unrelated patient was homozygous for a novel 16-bp intronic deletion. Functional characterisation of the variants demonstrated a partial splicing defect and altered promoter and/or enhancer activity, reflecting essential mechanisms for regulating cholesterol biosynthesis and/or uptake in

*Fdft1*-null mice demonstrate embryonic lethality at day 12.5 in conjunction with growth restriction and neurodevelopmental disorders [34]. The fact that the *FDFT1* variants in the human SQSD cases are compatible with life may be explained by the fact that all individuals have some form of residual FDFT1 activity, either resulting from the diminished levels of correctly-spliced enzyme or by functional compensa-

In the cholesterol biosynthesis pathway, lanosterol synthase leads to the cyclisation of (S)-2,3-oxidosqualene into lanosterol. Pathogenic mutations in the *LSS* gene have recently been reported in a spectrum of clinical phenotypes including congenital cataracts in three families [35], hypotrichosis simplex (HS) in three families [36] and a more severe neuroectodermal syndrome formerly named alopecia with mental retardation (APMR) syndrome in six unrelated families [37]. HS (OMIM 618275) is a rare form of hereditary alopecia characterised by childhood onset of diffuse and progressive scalp and body hair loss [36]. APMR syndrome (OMIM 203650) is a rare disorder with autosomal recessive transmission. A recent report identified 11 individuals from seven unrelated families affected with alopecia, male genital abnormalities, variable MRI abnormalities and neurological symptoms [37]. In this cohort, total alopecia was universal with other common dermatological manifestations including ichthyosis and erythroderma. Neurological manifestations included significant developmental delay, microcephaly, epilepsy and hypomyelin-

Sterol profiling in lanosterol synthase deficiency cases has not identified any specific abnormalities, thus supporting the previously proposed hypothesis of an alternative cholesterol pathway [36]. *LSS* variants identified to date include truncating, missense and splicing variants. *LSS* has also been associated with congenital cataracts in rat [38]. Mice homozygous for the Lsstm1b(KOMP)Wtsi allele demonstrate variable lethality, from embryonic day 9.5 to postnatal prior to weaning [39].

reduced or normal (0.17–0.93 mmol/L, reference 0.36–1.04).

**136**

ation [37].

steady state [4].

tion for disrupted regulation [4].

**2.3 Lanosterol synthase deficiency**
