Disorders of sterol synthesis: Beyond Smith–Lemli–Opitz syndrome^†

INTRODUCTION

Since the recognition that Smith–Lemli–Opitz syndrome (SLOS) is a disorder of cholesterol biosynthesis, human disorders associated with additional enzymes involved in the conversion of lanosterol to cholesterol have been identified; for a recent review see Porter and Herman [2011]. In Table I, we list all of the known enzymes involved in this process along with the associated human genes and disorders. To date, human disorders have been identified for all but two of the enzymes, CYP51, a lanosterol demethylase, and HSD17β7, a ketosteroid reductase that is part of a C4 demethylase complex (see below).

Two of the disorders, desmosterolosis and lathosterolosis, are extremely rare autosomal recessive malformation syndromes, one of which, lathosterolosis, resembles SLOS. Two X-linked, male-lethal disorders, X-linked dominant chondrodysplasia punctata (CDPX2) and CHILD syndrome, present with primarily skin and skeletal findings and are associated with heterozygous mutations in the genes EBP and NSDHL that encode a sterol isomerase and C4-methyl sterol dehydrogenase, respectively.

Two X-linked, male-lethal disorders, X-linked dominant chondrodysplasia punctata (CDPX2) and CHILD syndrome, present with primarily skin and skeletal findings and are associated with heterozygous mutations in the genes EBP and NSDHL that encode a sterol isomerase and C4-methyl sterol dehydrogenase, respectively.

However, recently, distinct syndromes with significant neurologic phenotypes have been described in surviving males with hypomorphic mutations in these two X-linked genes. The most recently described disorder, SC4MOL deficiency, is autosomal recessive and is associated with severe skin manifestations, immune dysfunction, and mild intellectual disability. SC4MOL is part of the demethylase complex that also includes NSDHL and HSD17β7. Finally, Hydrops-Ectopic Calcification-Moth-eaten (HEM) or Greenberg dysplasia is an autosomal recessive perinatal lethal disorder that results from mutations in the gene encoding the Lamin B receptor (LBR) [Waterham et al., 2003; Wassif et al., 2007]. Whether the pathology in HEM dysplasia results from disruption of structural functions of the protein in the nuclear membrane or from its sterol C14-reductase enzymatic activity remains controversial [Clayton et al., 2010].

An additional human disorder that has been included with disorders of post-squalene cholesterol biosynthesis in the past is Antley–Bixler syndrome. This disorder is associated with craniosynostosis, choanal atresia, skeletal defects, and ambiguous genitalia and results from a deficiency of lanosterol C14-demethylase (CYP51) enzymatic activity. However, the mutant gene encodes a P450 oxidoreductase (POR protein) that serves as an electron donor to many cytoplasmic (Type II) P450 enzymes, including CYP51. These cases of Antley–Bixler are now considered the severe end of a spectrum of POR deficiency which is primarily a group of disorders of sexual development and steroidogenesis. For recent reviews, see Arlt [2007], Cragun and Hopkin [2009], Porter and Herman [2011]. Antley–Bixler syndrome will not be discussed here.

As noted above, no patients have been identified with deficiency of HSD17β7 sterol reductase. Based on the phenotype of a recent hypomorphic targeted mouse allele, one might expect a lethal phenotype with skeletal and CNS defects [Stottmann et al., 2011].

Comprehensive reviews describing the disorders of post-squalene cholesterol biosynthesis have recently been published [Horvat et al., 2011; Porter and Herman, 2011]. Thus, we will focus here on the clinical aspects of the eight disorders mentioned above and compare and contrast the findings with those found in SLOS. We will also briefly discuss possible mechanisms of disease pathogenesis that rely heavily on mouse models for the disorders.

DESMOSTEROLOSIS

Desmosterolosis results from deficiency of 3β-hydroxysteroid-Δ24-reductase (DHCR24), an enzyme that reduces the C24 double bond in the aliphatic side chain of desmosterol. Until recently, only two patients with this disorder had been described in the medical literature; their phenotypes were very different. The first reported case was a 46, XX female born at 34 weeks gestation who died at 1 hr of age due to poor respiratory effort and lung hypoplasia. She had macrocephaly, cleft palate, total anomalous pulmonary venous drainage, ambiguous genitalia with clitoromegaly, short limbs with generalized osteosclerosis, and CNS anomalies, including a hypoplasic corpus callosum, immature gyri, and enlarged ventricles [FitzPatrick et al., 1998]. Her facial features were reminiscent of SLOS, and markedly increased levels of desmosterol were detected in tissue samples from the infant. A second patient was subsequently reported who exhibited a milder phenotype that included microcephaly, a small area of cutis aplasia of the scalp, dysmorphic facies, submucous cleft of the palate, persistent patent ductus arteriosus, bilateral clubfeet, mild contractures of the hands, and complete agenesis of the corpus callosum. At 3 years of age, the child had severe intellectual disability and poor weight gain and linear growth (see Fig. 1) [Andersson et al., 2002]. Elevated desmosterol levels were detected in plasma from this patient, as well as in lymphoblasts cultured in lipid-depleted medium. Subsequently, Waterham et al. [2001] identified recessive mutations in the DHCR24 sterol-Δ²⁴-reductase gene in both patients. DNA constructs containing each mutation were also prepared and tested in a heterologous yeast expression assay for their ability to convert desmosterol to cholesterol. The second patient had significant residual activity, consistent with his milder clinical course.

Recently, seven additional patients with desmosterolosis have been identified, including four surviving affected cousins from a large consanguineous Bedouin family [Zolotushko et al., 2011]; an isolated case diagnosed at 5 days of age [Fig. 2; Schaaf et al., 2011]; and two unpublished cases known to one of the authors [Kelley and Kratz, unpublished work]. Six of these additional patients were living at the time they were identified or reported; they ranged in age from 4 months to 15 years. One of the new unpublished cases died in the perinatal period, similar to the original case of FitzPatrick et al. [1998]. All of these patients had elevated levels of desmosterol in plasma, tissue, and/or cultured cells and six had molecular confirmation of the diagnosis, with the identification of homozygous or compound heterozygous mutations in DHCR24.

As shown in Table II, all known cases of desmosterolosis have had significant CNS involvement, including structural malformations and intellectual disability of varying degree in long-term survivors. Agenesis of the corpus callosum is the most consistent CNS anomaly and was present in all reported patients (9/9), while most also had dilated ventricles (8/9). These findings are consistent with the accumulation of desmosterol in the developing CNS relative to the amount present in other tissues, although its exact functions in the brain are not known [Tint et al., 2006; Porter and Herman, 2011]. Most of the patients were dysmorphic, with some features similar to those seen in SLOS (see Table II). A variety of mild to severe skeletal anomalies were also commonly described, including contractures or arthrogryposis (6/9). Other clinical features were highly variable, including the presence of microcephaly (5/9) or macrocephaly (2/9). It should be noted that the four cousins from the consanguineous family all had a very similar and severe phenotype with microcephaly, seizures (3/4), complete or partial agenesis of the corpus callosum and reduced white matter on MRI, severe intellectual disability, and severe failure to thrive [Zolotushko et al., 2011].

A targeted mouse model for desmosterolosis has been generated. However, the phenotype appears to depend on the genetic background. Wechsler et al. [2003] reported that null mice were viable, albeit with some prenatal losses and postnatal growth retardation and infertility, while Mirza et al. [2006] reported that the mutation was perinatal lethal with abnormal development of the skin producing a restrictive dermopathy. In contrast, treatment of pregnant rats with pharmacologic inhibitors of sterol-Δ²⁴-reductase is teratogenic and produces CNS, GU, and skeletal anomalies, as well as cataracts [Roux, 1964; Gofflot et al., 2003; Cenedella, 2009]. The absence of major malformations in the targeted alleles likely reflects significant maternal cholesterol transfer to the rodent fetus through midgestation [Woollett, 2005], while pharmacologic agents lower maternal cholesterol, reducing placental transfer.

LATHOSTEROLOSIS

Lathosterolosis results from deficiency of sterol-C5-desaturase (SC5D), an enzyme that converts lathosterol to 7-dehydrocholesterol, the immediate precursor of cholesterol. Two unrelated patients with lathosterolosis were described by Brunetti-Pierri et al. [2002, Patient 1] and Krakowiak et al. [2003, Patient 2]. A clinical update of the first case, as well as a brief description of an affected sibling terminated at 21 weeks, was subsequently reported [Rossi et al., 2007]. Both liveborn infants exhibited an “SLOS-like” phenotype (see Table II). The first patient, a female, had microcephaly and dysmorphic facies with a receding forehead, bilateral epicanthal folds, short nose with anteverted nares, prominent upper lip, high palate, and micrognathia (Fig. 3). Skeletal findings included postaxial polydactyly of the left foot with mild 2–4 toe syndactyly and more significant syndactyly of the 5th and extra 6th digits. She had a horseshoe kidney, progressive cholestatic liver disease, and truncal hypotonia. Brain imaging was normal and no ichthyosis, chondrodysplasia punctata, or genital abnormalities were detected. Severe intellectual disability became apparent as she grew older, and her facies came to resemble more those found in SLOS. When examined again at age 6–7 years (Fig. 3C), she had severe microcephaly, mild conductive hearing loss, and bilateral cataracts requiring surgery. She also developed pathologic fractures secondary to severe osteoporosis. Her liver disease progressed to failure, with continued cholestasis, persistently elevated serum transaminases, and hepatic fibrosis and portal hypertension [Rossi et al., 2005, 2007]. Liver involvement has been reported in up to 5% of patients with SLOS, usually in more severely affected infants, and often carries a poor prognosis [Rossi et al., 2005]. A previous, karyotypically normal (46,XX) pregnancy was terminated at 21 weeks for multiple anomalies that included microcephaly, a lumbosacral meningocele with Type II Arnold–Chiari malformation, four limb hexadactyly, and clubfeet. Extramedullary hematopoiesis with hemosiderin deposition within periportal hepatocytes was present in the liver at autopsy. No foamy storage material or lamellar inclusions were noted in any of the tissues examined (see below).

The second unrelated patient has been described in brief [Parnes et al., 1990; Krakowiak et al., 2003]. He presented with symmetric IUGR with microcephaly, hypotonia, congenital cataracts and microcorneae, penoscrotal hypospadias and undescended testes, postaxial polydactyly of the feet, and bilateral 2,3-toe syndactyly. Craniofacial abnormalities included a prominent metopic suture, ptosis and downslanting palpebral fissures, short prominent nose with short philtrum, downturned mouth, high palate with prominent alveolar ridges, and micrognathia. The neck was short with redundant skin. He exhibited failure to thrive and developed myoclonic jerks at 2 months of age and progressive hepatoplenomegaly, corneal clouding, and gingival hypertrophy. He was suspected to have a storage disease and somatic, but not CNS, lysosomal accumulations of mucopolysaccharides and lipids were noted at autopsy when he died at 18 weeks of age. Subsequent examination by electron microscopy of cultured skin fibroblasts from the first patient revealed the presence of lamellar inclusions which increased upon culture in lipid-depleted media and appeared to be degraded within cellular lysosomes [Rossi et al., 2007]. Similar inclusions were found in cultured fibroblasts of Patient 2, as well as in cells from sterol-C5-reductase (Sc5d) deficient mice [Krakowiak et al., 2003].

Plasma cholesterol levels were normal in the first patient, while both liveborn patients had abnormal sterol profiles with accumulation of lathosterol in plasma (Patient 1) or in cultured fibroblasts (Patient 2). Using sequence from a human SC5DL cDNA isolated by homology to the yeast enzyme, Patient 1 and the aborted fetus were found to be compound heterozygotes for the missense mutations R29Q and G211D, while Patient 2 was homozygous for the missense mutation Y46S. Detection of residual conversion of labeled mevalonate to cholesterol in cultured cells from both patients confirmed that these mutations act as hypomorphs, at least in vitro [Brunetti-Pierri et al., 2002; Krakowiak et al., 2003].

Krakowiak et al. [2003] generated a mouse model for lathosterolosis by targeted disruption of the murine Sc5d gene in ES cells. Homozygotes are stillborn, edematous, and growth retarded. Eighty-eight percent of homozygotes examined had a cleft palate, and they had plugged or absent nostrils. They exhibited a variety of skeletal defects including shortened limbs, a kinked tail, postaxial polydactyly of the forelimbs, duplication of the distal phalanx of the 4th digit, and soft tissue syndactyly.

Krakowiak et al. generated a mouse model for lathosterolosis by targeted disruption of the murine Sc5d gene in ES cells. Homozygotes are stillborn, edematous, and growth retarded. Eighty-eight percent of homozygotes examined had a cleft palate, and they had plugged or absent nostrils. They exhibited a variety of skeletal defects including shortened limbs, a kinked tail, postaxial polydactyly of the forelimbs, duplication of the distal phalanx of the 4th digit, and soft tissue syndactyly.

They also had hypomineralization of the bones and an enlarged liver. Tissues and serum from affected pups demonstrated markedly elevated levels of lathosterol and decreased cholesterol. These mice were generated, in part, to examine whether the phenotypic features of SLOS are caused by a lack of cholesterol or by an accumulation of 7-dehydrocholesterol (7DHC). Both compounds would be expected to be low in a Sc5d^−/− mouse. The authors concluded that common malformations identified in the Sc5d−/− and Slos−/− mice likely result from decreased cholesterol rather than accumulation of sterol intermediates, such as 7DHC and/or lathosterol, and may involve impaired hedgehog signaling [Krakowiak et al., 2003]. Unique features in lathosterolosis, such as the progressive liver disease and intracellular storage defects, may indeed result from accumulation of lathosterol, although additional studies will be necessary to prove this hypothesis.

Despite increased awareness of and biochemical screening for disorders of cholesterol synthesis in infants with multiple malformations, no additional cases of lathosterolosis have been identified. That recessive null alleles for SC5D would be lethal is supported by the finding that mutations in the known human cases probably function as hypomorphs, as well as by the perinatal lethality of Sc5d−/− mice.

X-LINKED DOMINANT CHONDRODYSPLASIA PUNCTATA

X-linked dominant chondrodysplasia punctata (CDPX2, Conradi–Hünermann syndrome) is a rare, X-linked, often male-lethal disorder that is associated with skin, skeletal, and ophthalmologic anomalies in affected heterozygous females (for recent reviews see Dempsey et al. [2011] and Porter and Herman [2011]). The incidence of CDPX2 has been estimated as ≤1/400,000 live births, based on rates of biochemical diagnoses in one large laboratory compared to those diagnosed with SLOS, with an incidence of 1 in 80,000 [Kelley and Hennekam, 2000; Craig et al., 2006]. However, this number may be an underestimate based on underdiagnosis of very mild cases, as well as possible misdiagnoses.

The major clinical findings in heterozygous females with CDPX2 are typically present at birth and involve the skin and skeleton. There is rhizomelic shortening of the limbs that is often asymmetric. Radiographs in infancy demonstrate stippling of the epiphyses (chondrodysplasia punctata) due to abnormal deposition of calcium (see Fig. 4). Epiphyseal stippling can be found in several genetic disorders, including other types of chondrodysplasia punctata, generalized peroxisomal disorders, and trisomy 18 and 21, as well as in acquired disorders, such as warfarin embryopathy and maternal SLE [Irving et al., 2008; Wainwright and Beighton, 2010]. However, the stippling found in CDPX2 is often more widespread, with involvement of the vertebrae and tracheal cartilage, in addition to the long bones. Once normal epiphyseal ossification occurs, stippling can no longer be detected. Short stature and scoliosis are common in CDPX2 and may be congenital. Clubfoot and joint contractures have also been reported, and vertebral anomalies may contribute to scoliosis.

There is a characteristic craniofacial appearance in CDPX2 females that includes frontal bossing, a flat nasal bridge, and midface hypoplasia. Postaxial polydactyly occurs in 5–10% of reported cases and appears to occur most often in this form of inherited CDP [Herman, 2000; Herman et al., 2002; Hennekam et al., 2010]. Adult height is often reduced, averaging 60–63 in. in mildly affected females [Has et al., 2000].

Skin findings at birth typically include a scaling, erythematous eruption with a linear or patchy distribution that follows lines of X-inactivation, also called the lines of Blaschko (see Fig. 5) [Happle, 1993; Traupe, 1999]. Occasionally, a diffuse erythroderma is present [Kalter et al., 1989]. The initial eruption usually fades over the first few months of life and may be replaced with linear or whorled pigmentary abnormalities (hyper or hypopigmentation) and/or atrophic patches that may involve hair follicles (follicular atrophoderma). The nails are occasionally involved, but the teeth are normal. Patches of scarring alopecia are common, and the hair has been described as coarse and lusterless.

Histologic examination of ichthyotic regions of skin in infancy demonstrates hyperkeratosis, acanthosis, and parakeratosis, with the presence of calcium in the stratum corneum and follicular plugging. These latter features are not found in involved skin in CHILD syndrome (see below) or in other types of inherited ichthyoses [Sybert, 1997; Hoang et al., 2004]. Inflammatory infiltrates of neutrophils or lymphocytes may also be found in areas of involved skin.

Cataracts are found in approximately 65% of affected females. They are often congenital and may be bilateral, unilateral, or sectorial [Happle, 1981; Hennekam et al., 2010]. Microphthalmia and microcornea have also been reported. Sensorineural hearing loss occurs in approximately 10% of patients, and there are single reports of affected females with a tethered cord, Dandy–Walker malformation, and cervical myelopathy. Intelligence is usually normal. Other reported visceral anomalies include congenital heart disease and hydronephrosis or other developmental renal anomalies. Newly diagnosed patients should receive a renal ultrasound and an echocardiogram if there is a murmur present, as well as a careful ophthalmologic exam, assessment of hearing, and orthopedics evaluation.

The phenotype in affected females is extremely variable. At the severe end, one can see early fetal loss and stillbirths, with severe skeletal and internal anomalies [Rakheja et al., 2007]. In these cases, a skeletal dysplasia, with asymmetric shortening of the long bones detected on prenatal ultrasound may suggest CDPX2, among other diagnoses [Pryde et al., 1993]. The identification of the genetic basis for CDPX2 and the ability to screen for mutations in mildly affected individuals has led to an expanded phenotypic range for the disorder. Some gene carriers may demonstrate only short stature, and completely asymptomatic carrier mothers of infants with classic CDPX2 have been identified [Has et al., 2000; Herman et al., 2002]. Gonadal mosaicism has also been described [Has et al., 2000], which is important for genetic counseling regarding recurrence risks. Finally, several males with typical features of CDPX2 have been reported with a 47,XXY karyotype or somatic mosaicism as a mechanism [Sutphen et al., 1995; Has et al., 2000; Aughton et al., 2003; Arnold et al., 2012]. Males with 46, XY karyotypes and non-mosaic hypomorphic mutations in the EBP gene have a distinct phenotype that is discussed below (see the Hemizygous Male EBP Deficiency Section).

In 1999, Kelley et al. [1999] searched for sterol abnormalities in several patients with various types of chondrodysplasia punctata. They demonstrated abnormal sterol profiles in tissue samples from several females with CDPX2, with increased levels of 8-dehydrocholesterol (8DHC) and cholesta-8(9)-en 3β-ol [8(9)chl].

In 1999, Kelley et al. searched for sterol abnormalities in several patients with various types of chondrodysplasia punctata. They demonstrated abnormal sterol profiles in tissue samples from several females with CDPX2, with increased levels of 8-dehydrocholesterol (8DHC) and cholesta-8(9)-en 3β-ol [8(9)chl].

The pattern of sterols present suggested a block at the level of 3β-hydroxysteroid-Δ⁸–Δ⁷-sterol isomerase, an enzyme encoded by the X-linked emopamil binding protein (EBP) gene. The accumulation of 8DHC is presumed to result from the action of lathosterol 5-desaturase (SC5D) on the increased levels of 8(9)chl. The detection of specific elevations of 8DHC and 8(9)chl in plasma in a female with clinical features of CDPX2 has a high correlation with detection of an EBP mutation. In one series, heterozygous EBP mutations were identified in 20/22 females (91%) with the characteristic abnormal sterol profile [Herman et al., 2002]. Similar results have been found by others [Has et al., 2002; Whittock et al., 2003]. Thus, plasma sterol analysis can be a useful biochemical screening test for CDPX2, particularly in atypical cases. It should be noted, however, that the levels of these sterol intermediates are typically 1–10% of total plasma sterols, much lower than the levels of intermediates found in SLOS, and plasma total cholesterol levels do not differ from those found in the general population.

The gene defect underlying CDPX2 was identified in 1999 by two groups based on the pattern of sterol abnormalities in human patients [Braverman et al., 1999] and by homology with the X-linked mouse mutant tattered [Derry et al., 1999]. The EBP gene was originally defined as a sigma type receptor for a variety of drugs, including tamoxifen [Hanner et al., 1995; Silve et al., 1996]. It was subsequently shown to have Δ⁸–Δ⁷-sterol isomerase activity and can complement Saccharomyces cerevisiae that lack the yeast ortholog (ERG2) [Braverman et al., 1999].

The EBP gene maps to Xp11.22, spans ∼7.0 kb, and has five exons, four of which are coding. At least 65 different EBP mutations have been reported in females with CDPX2 according to the Human Gene Mutation Database (HMGD) at www.hgmd.cf.ac.uk [Stenson et al., 2009] including missense, nonsense, frameshift, and splicing mutations, as well as small insertions and deletions. Canueto et al. [2012] provide a nice summary and schematic representation of different human EBP mutations identified as of 2011. There are several recurrent mutations, primarily at CpG dinucleotides representing mutation “hot spots” [Herman et al., 2002]. There are no significant genotype/phenotype correlations, with wide phenotypic variation within single families. These findings are likely due to the fact that random X-inactivation is the major determinant of clinical severity in affected tissues in individual female patients.

Finally, there are several females with focal dermal hypoplasia caused by submicroscopic deletions of the X-linked PORCN gene that also include the adjacent EBP locus [Grzeschik et al., 2007; Bornholdt et al., 2009]. None of the females exhibited any features of CDPX2. All of the patients had extreme skewing of X-inactivation in favor of the normal X chromosome (>95%), likely accounting for their survival and lack of additional phenotypes.

HEMIZYGOUS MALE EBP DEFICIENCY

Although mutations in EBP causing CDPX2 were once assumed to be uniformly lethal in males early in gestation, sterol-Δ⁸–Δ⁷-isomerase deficiency has been found in a small number of males based on an abnormal sterol profile and/or hemizygosity for a damaging EBP mutation, see Figures 6 and 7 [Milunsky et al., 2003; Kelley et al., 2005; Furtado et al., 2010; Arnold et al., 2012; and Kelley and Kratz, unpublished data]. As stated above, males with a 47,XXY karyotype or mosaic for an EBP mutation exhibit most of the classic clinical findings of CDPX2 found in heterozygous females, including bone shortening and/or asymmetry, epiphyseal stippling, scoliosis, ichthyosis, alopecia, and mild facial dysmorphism. In contrast, males hemizygous for non-mosaic mutations in EBP have very different features, including distinct facial dysmorphisms; cardiac defects (5/8); genital abnormalities, such as cryptorchidism (9/10) and/or hypospadias (4/8); and developmental brain abnormalities, including Dandy–Walker anomalies (7/8) and agenesis of the corpus callosum (6/8) (see Table II). Skeletal defects may include 2,3 toe syndactyly, polydactyly, and epiphyseal stippling. Collodion baby and/or diffuse congenital ichthyosis have been noted in most of the patients (8/10), although skin and skeletal anomalies may be totally absent. At least eight of the males are deceased (between 1 day of age and 4.5 years). Those surviving beyond infancy have had significant intellectual disability.

Increased levels of 8(9)-cholestenol and 8-dehydrocholesterol have been observed in all EBP-deficient males where sterol analysis was performed [Milunsky et al., 2003; Furtado et al., 2010; Kelley and Kratz, unpublished data]. In three families where mutation analysis of EBP was performed, affected males carried a missense mutation (L91P in one family and W47C in two unrelated families) inherited from a clinically unaffected mother [Milunsky et al., 2003; Furtado et al., 2010]. It has been presumed that the mutations function as hypomorphs, which is supported by the lack of any clinical symptoms in the carrier females. However, functional studies of the mutations to prove this hypothesis have not been performed.

CHILD SYNDROME

CHILD syndrome (congenital hemidysplasia with ichthyosiform erythroderma and limb defects) is an extremely rare, X-linked male-lethal disorder, first described under this acronym by Happle et al. [1980]. Fewer than 100 cases have been reported in the medical literature. The majority of cases have been sporadic, although rare mother to daughter transmission has been described. For recent reviews, see Happle [2010] and Porter and Herman [2011].

The hallmarks of CHILD syndrome are the presence of unilateral ichthyosiform skin lesions and ipsilateral limb reduction defects [Happle et al., 1980, 1995; Bornholdt et al., 2005]. The skin lesion(s), which may be extensive, typically affect one side of the body, with a sharp line of demarcation at the midline.

The hallmarks of CHILD syndrome are the presence of unilateral ichthyosiform skin lesions and ipsilateral limb reduction defects. The skin lesion(s), which may be extensive, typically affect one side of the body, with a sharp line of demarcation at the midline.

Happle et al. [1995, 2010] have argued that the characteristic CHILD syndrome skin lesion is a distinct type of inflammatory nevus consisting of an erythematous base and yellow, waxy scales, although some regions may be more “warty” or verrucous in appearance. The lesions are present at birth or within the first few months of life. Lesions may occur on the contralateral side, and bilateral, more symmetric involvement has been described [Fink-Puches et al., 1997; Konig et al., 2002]. Some of the lesions may follow the lines of Blaschko [Happle, 1993; Happle, 2010], although most do not. Unlike CDPX2, the lesions often persist throughout life, although there may be some resolution over time, and new lesions may continue to appear. Lesions are often observed in skin folds, a finding called ptychotropism [Happle, 1990]. The face is usually spared, but the scalp may be involved. Alopecia can occur, usually on the more involved side, and nails are often dystrophic. Involvement of the right side of the body is more common than the left.

All of the disorders of post-squalene cholesterol biosynthesis are associated with skeletal defects. However, aplasia of an entire limb, severe phocomelia, or significant limb hypoplasia on the side of skin involvement, is unique to human CHILD syndrome. X-rays in infancy may demonstrate epiphyseal stippling of the involved limb(s), similar to what is found in CDPX2. Milder defects, including distal digit shortening, syndactyly and/or polydactyly, oligo- or hypodactyly, clefting of the hand or foot, hypoplastic or hemivertebrae, and scoliosis have also been reported [Peter and Meinecke, 1993; Murata et al., 2003; Bornholdt et al., 2005; Bittar et al., 2006; Happle, 2010]. Following the discovery of the NSDHL gene and the ability to perform molecular diagnosis, milder cases with minimal to no skin and/or skeletal involvement have been identified [Murata et al., 2003; Bittar et al., 2006].

Visceral involvement is fairly common, and occurs more frequently in cases with extensive skin and skeletal involvement or in those with left-sided predominance. Deafness was reported in 3/22 cases in one series (14%; 2 sensorineural and 1 unilateral, type unspecified) [Bornholdt et al., 2005]. Mild cognitive problems have been reported in a few surviving females, although intelligence is usually normal. Documented CNS malformations are present in only a small number of cases (≤10%) [Bornholdt et al., 2005; Hennekam, 2005; Schmidt-Sidor et al., 2008]. The most common abnormalities are hypoplasia of the involved side of the brain and/or cranial nerve involvement.

A variety of congenital heart defects were noted in 10–20% of cases prior to gene identification. However, in the cases reported since 2000 with NSDHL mutations, a single instance of hypoplastic left heart syndrome was noted in a newborn with CNS malformations who died [Schmidt-Sidor et al., 2008]. Three of 23 (13%) patients reported by Bornholdt et al. [2005] had unilateral renal agenesis. Lung hypoplasia with a small chest wall leading to respiratory distress has also been reported [Hummel et al., 2003; Schmidt-Sidor et al., 2008]. Single cases with classic features of CHILD syndrome and optic atrophy [Knape et al., 2010] or congenital hip dislocation and thrombocytosis [Chander et al., 2010] have recently been described, although no molecular studies confirming the presence of NSDHL mutations were reported. A squamous cell carcinoma arising within the CHILD nevus has been reported in a 33-year-old female [Jacyk and La Cock, 2006].

Recently, several females with CHILD syndrome have had novel surgical or medical treatments of their skin lesions: In two patients, the CHILD lesion received dermabrasion and then the area was covered with split skin grafts taken from an unaffected region of the same patient [Konig et al., 2010]. Paller et al. [2011] used a topical lotion containing a statin and cholesterol to treat two patients with CHILD syndrome. Both had improvement in their skin disease; cholesterol treatment alone had no effect. The authors suggest that both deficiency of cholesterol and accumulation of toxic metabolites above the enzymatic block contribute to the pathogenesis of the disorder, at least in skin.

CHILD syndrome results primarily from mutations in the X-linked NSDHL (NADH steroid dehydrogenase-like) gene that is involved in the demethylation of C4-methyl groups from the sterol intermediate lanosterol [Konig et al., 2000]. The first mutations in the Nsdhl gene were reported in mice with the X-linked, male lethal bare patches (Bpa) and striated (Str) mutations [Liu et al., 1999]. The function of NSDHL as a C-4 sterol dehydrogenase was substantiated by the accumulation of 4α-monomethyl and 4,4′-dimethyl sterol intermediates in tissue samples and cultured skin fibroblasts from heterozygous Bpa/Str mutant females. In addition, the mouse NSDHL protein can rescue the conditional lethality of S. cerevisiae that lack the orthologous ERG26 protein involved in the synthesis of ergosterol [Lucas et al., 2003].

At least 23 distinct NSDHL mutations have been reported in unrelated CHILD syndrome patients according to the HMGD database [Stenson et al., 2009]. The missense mutation A105V has been reported in five unrelated cases and involves a CpG dinucleotide that can be a hotspot for mutations. Four of the reported cases are familial, including one family with five mildly affected females in three generations [Bittar et al., 2006]. A complete NSDHL gene deletion in an affected female, as well as a microdeletion encompassing the promoter and exon 1 of NSDHL and the entire centromeric CETN2 gene, have been reported [Bornholdt et al., 2005; Raychaudhury et al., 2012]. All of the mutation-positive affected cases are female with the exception of one male with the nonsense mutation R88X, a 46,XY blood karyotype, and classic CHILD syndrome with extensive unilateral skin involvement and lower limb hypoplasia [Happle et al., 1996]. The authors postulated that an early somatic mutation accounted for his postnatal survival. Indeed, in DNA prepared from cultured skin fibroblasts from the unaffected side, only the normal allele was detected [Bornholdt et al., 2005]. As with CDPX2, there are no clear genotype/phenotype correlations in affected females, probably because the pattern of X-inactivation in affected tissues influences the phenotype as much or more than the mutation itself.

Grange et al. [2000] reported a female with clinical features of CHILD syndrome and a nonsense mutation R110X in the EBP sterol-Δ⁸–Δ⁷-isomerase gene, that is associated with CDPX2. The patient exhibited primarily unilateral ichthyosiform skin lesions with ipsilateral limb hypoplasia and patchy alopecia. Subsequently, a second female with typical clinical features of CHILD syndrome and an EBP mutation was identified [Kelley and Herman, 2001 and D.K. Grange, A. Metzenberg, G.E. Herman, and R.I. Kelley, unpublished results]. There has been some disagreement about the clinical diagnosis of the first case [Grange and Kelley, 2000; Happle et al., 2000] and whether only females with NSDHL mutations should be considered to have CHILD syndrome. However, given the recent description of males with hypomorphic NSDHL mutations and a distinct neurologic phenotype called CK syndrome (see below), it appears reasonable to use the designation of CHILD syndrome based on the clinical features present, as suggested by Grange and Kelley [2000].

Although CHILD syndrome results from a defect in cholesterol biosynthesis, cholesterol levels and sterol profiles in plasma from affected females are normal. The normal plasma sterol profile has been postulated to result from toxicity of the 4α-methyl sterols and loss of cells expressing the affected X chromosome. Elevated 4α-methyl sterols and 4α-carboxymethylcholesta-8(9)-en-3β-ol are found in affected skin and in cultured fibroblasts or lymphoblasts grown in lipid-depleted medium [see Table III and Hummel et al., 2003]. Because plasma sterol studies are normal, molecular diagnosis should be employed first in any patient suspected to have CHILD syndrome, and, if negative, sterol analysis of affected skin or cultured cells performed.

The unilateral distribution of anomalies and skin lesions in CHILD syndrome does not follow the pattern of X-inactivation and remains unexplained. The existence of familial cases with an NSDHL mutation excludes somatic mosaicism as a unifying mechanism [Bornholdt et al., 2005; Bittar et al., 2006]. Happle has proposed that disruption of a clone of early midline “organizer” cells expressing a mutant NSDHL allele could affect X-inactivation and the lateralization process itself [Happle, 1993; Konig et al., 2000]. While some asymmetry may be noted in affected heterozygous Bpa and Str female mice, limb reduction anomalies and diffuse unilateral skin lesions have never been observed [Phillips, 1963; Phillips et al., 1973; Cunningham et al., 2009; and G.E. Herman, unpublished results]. Understanding the basis for the unilateral findings in human CHILD syndrome may, thus, be extremely difficult to model experimentally.

CK SYNDROME

Recently, two families with males with X-linked intellectual disability and hemizygous NSDHL mutations have been described [du Souich et al., 2009; Tarpey et al., 2009; McLarren et al., 2010]. The disorder has been given the eponym of CK syndrome, after the first patient described [see Fig. 8; du Souich et al., 2009]. All of the 13 affected males identified to date demonstrate mild to severe intellectual disability and microcephaly. Most had hypotonia and little to no speech development. All developed seizures during infancy which range from brief absence episodes to prolonged generalized tonic-tonic seizures [du Souich et al., 2009; Du Souich et al., 2011b]. Three males (two from one family, one from the other) who had brain MRIs had evidence of cerebral cortical malformations most consistent with polymicrogyria and/or pachygyria. Similar facial dysmorphisms were observed in all affected males and included a long thin face, particularly among adults, almond-shaped eyes with epicanthal folds, upslanting palpebral fissures, posteriorly rotated ears, high nasal bridge, and a high palate with dental crowding (see Fig. 9). While retro- or micrognathia was present in childhood, the jaw became more prominent with age. Mild skeletal findings include an asthenic build with hyperextensible joints, long fingers and toes, and spinal abnormalities such as scoliosis, kyphosis, and/or lordosis. Significant behavioral problems occurred in most of the affected males and included ADHD, irritability, and aggression. While some of the males demonstrated autistic behaviors, they did not fulfill diagnostic criteria for an autism spectrum disorder based on psychological testing. Ophthalmologic anomalies, such as strabismus and optic atrophy, were also identified in some patients. There were no features of CHILD syndrome present and other visceral malformations have not been described. Congenital aortic stenosis seen in the proband from Family 1 may be coincidental since it has not been reported in any of the other affected males.

A comparison of the clinical features found in CK syndrome with other selected X-linked syndromes with IDD is provided by du Souich et al. [2009]. Although males with CK syndrome have microcephaly and significant intellectual disability, their physical features, in general, are very different from those found in SLOS, desmosterolosis, and hemizygous male EBP deficiency, which are also disorders with significant neurologic manifestations (see Table II).

All of the affected CK males tested had normal sterol profiles, total cholesterol levels, steroid hormone levels, and lipoprotein profiles in plasma. However, when mutant lymphoblasts were cultured in lipid-depleted media, accumulations of 4-methyl sterols, similar to those seen in CHILD syndrome, were found [McLarren et al., 2010].

Carrier females are not dysmorphic and have normal intelligence. However, they often demonstrate abnormal and antisocial behavior and have problems with their working memory on psychological testing [Du Souich et al., 2011a; 2012]. No skewing of X-inactivation was found in blood cells from several carrier females tested.

Linkage analysis and gene sequencing led to the identification of a 3 base pair deletion of a single amino acid in exon 7 (p.K232del) of NSDHL in the first family [McLarren et al., 2010], while, in the second family, X chromosome exome sequencing revealed an insertion and frameshift mutation near the C-terminus of NSDHL that disrupted the ER localization signal of the protein [Tarpey et al., 2009]. Functional studies in vitro demonstrated that both mutations act as hypomorphs [McLarren et al., 2010]. The authors speculate that the neurodevelopmental phenotypes and psychopathology in affected males and carrier females, respectively, result from accumulation of methylsterols in the brain rather than from cholesterol deficiency, as in SLOS.

SC4MOL DEFICIENCY

He et al. [2011] reported a female with autosomal recessive sterol C4 methyl oxidase (SC4MOL) deficiency. Based on homology with yeast, the enzyme is expected to catalyze successive oxidations of 4α-dimethyl and monomethylsterols to 4α-carboxymethylsterols, which then undergo decarboxylation catalyzed by NSDHL.

The phenotype in the first SC4MOL deficient patient included severe psoriasiform dermatitis, arthralgias, immune dysfunction, congenital cataracts, failure to thrive, short stature, microcephaly, and intellectual disability. Skin involvement was first noted on her abdomen at age 2 years and progressed throughout her body by age 6 years. Total serum cholesterol was low at 85 mg/dl (normal 140–176 mg/dl) with markedly elevated levels of 4,4′-dimethyl and 4α-monomethylsterols present by gas chromatography/mass spectrometry (see Table III). Unlike the sterol profiles found in CHILD or CK syndrome, the sterol intermediates were easily detected in plasma, as well as in cultured skin fibroblasts grown in complete or lipid-depleted media. The authors also found increased proliferation of affected skin fibroblasts grown in lipid-depleted media that they attributed to the meiosis-activating effects of the sterol metabolites that accumulate [Byskov et al., 2002; Rozman et al., 2002]. They postulated that the hyperproliferative skin phenotype in disorders of the C-4 demethylase complex results from elevations of these intermediates.

The patient had a dramatic response to treatment with an oral statin plus cholesterol and bile acid supplementation, with improved growth and weight gain. Her arthralgias also resolved. Topical application of a statin plus cholesterol produced marked improvement in her skin disease [Vockley et al., 2011].

Subsequently, three additional patients with SCMOL deficiency have been reported, in brief [He et al., 2012]. Two of these cases also had ichthyosiform dermatitis, while the third demonstrated dry skin and hair loss. All of them exhibited small size, microcephaly, congenital cataracts, and intellectual disability, as found in the first reported patient. The first patient is a compound heterozygote for two missense variants (H173Q, Y244C) in the SC4MOL gene, while mutations in the other patients have not been reported.

One of the new cases was examined for possible immune dysfunction and had findings similar to those in the first reported patient: Both demonstrated altered immunocyte profiles with increased expression of CD25 and toll-like receptor (TLR) 2 in granulocytes. Similar proinflammatory markers and neutrophil infiltration are seen in idiopathic psoriasis, and skin flakes from several patients with psoriasis have been shown to accumulate methylsterols [Grange et al., 2001]. Meiosis-activating sterols serve as ligands for liver X receptors (LXRs), which can help regulate immune function, and the authors postulate that this interaction is the link between the enzymatic block and altered immune function [He et al., 2011]. Mild immune dysfunction has been reported in some SLOS patients; however, comprehensive immune profiling and functional analyses have, in general, not been performed for any of the other disorders.

HEM DYSPLASIA

Hydrops-ectopic calcification-moth-eaten (HEM) or Greenberg dysplasia was first described in 1988 in a stillborn infant and subsequent affected fetus from a consanguineous mating [Greenberg et al., 1988]. Since then, additional cases have been reported [Chitayat et al., 1993; Horn et al., 2000; Trajkovski et al., 2002; Offiah et al., 2003; Oosterwijk et al., 2003; Konstantinidou et al., 2008] and summarized in Clayton et al. [2010]. The disorder appears to be uniformly lethal prenatally.

Hydrops-ectopic calcification-moth-eaten (HEM) or Greenberg dysplasia was first described in 1988 in a stillborn infant and subsequent affected fetus from a consanguineous mating. Since then, additional cases have been reported and summarized in Clayton et al. The disorder appears to be uniformly lethal prenatally.

Cardinal features include non-immune hydrops fetalis and a severe short-limbed dwarfism with disorganized cartilaginous and bony architecture. Radiographic findings include a moth-eaten appearance to the markedly shortened long bones, and a small thorax, as well as the presence of ectopic calcifications and platyspondyly. Postaxial polydactyly has been noted in several cases. Common additional findings outside the skeleton include lung hypoplasia and extramedullary hematopoiesis with an enlarged spleen and/or liver, while omphalocele, intestinal malrotation, and hypolobated lungs have occasionally been reported in Horn et al. [2000].

Based on the presence of severely disorganized chondrogenesis and abnormal calcification that resembles that found in CDPX2, Kelley and Wilcox examined sterols extracted from cartilage from several fetuses with HEM and identified mildly elevated levels of cholesta-8,14-dien-3β-ol and cholesta-8,14,24-trien-3β-ol, suggesting a block at the level of the 3β-hydroxysteroid-Δ¹⁴-reductase (sterol C14-reductase) [Kelley et al., 1999]. Subsequently, mutations were identified in the gene encoding the lamin B receptor in four fetuses with HEM dysplasia [Waterham et al., 2003; Konstantinidou et al., 2008; Clayton et al., 2010]. The lamin B receptor is a protein of the nuclear membrane with a C-terminal transmembrane domain that has sterol C14 reductase activity [Silve et al., 1998]. The mutations found in HEM dysplasia include two point mutations within the sterol reductase domain, as well as two frameshift mutations that produce truncated proteins (see schematic in Fig. 1B of Clayton et al. [2010]).

Controversy regarding whether HEM dysplasia is a disorder of sterol biosynthesis has arisen for several reasons. First, there are two human sterol-Δ¹⁴-reductase enzymes based on homology with the yeast ERG24 gene. The first, TM7SF2, encodes a ubiquitously expressed ER transmembrane protein that is linked to the DHCR7 locus on chromosome 11q [Holmer et al., 1998]. However, mutations have not been detected in TM7SF2 in human HEM dysplasia patients [Kelley and Herman, 2001; Waterham et al., 2003]. Furthermore, while a human LBR expression construct was able to rescue the ergosterol biosynthesis defect of ERG24, a similar TM7SF2 construct did not, suggesting that LBR is the major human sterol-Δ¹⁴-reductase [Silve et al., 1998; Prakash and Kasbekar, 2002].

Second, heterozygous mutations in LBR are associated with the Pelger–Huet anomaly (PHA), a benign autosomal dominant abnormality of leukocyte development that results in hypolobulation of neutrophils, as well as abnormal chromatin structure and nuclear shape [Hoffmann et al., 2002]. Several PHA homozygotes have also been described. Their neutrophils have a single ovoid nucleus with coarse and clumped chromatin [Hoffmann et al., 2007]. In one series of homozygous PHA patients, additional variable features include some intellectual disability (3/11), macrocephaly (2/11), seizures (2/11), and mild skeletal anomalies, such as short stature (2/11), postaxial polydactyly (1/11), or shortened metacarpals (1/11) [Oosterwijk et al., 2003]. However, none of these individuals had findings reminiscent of HEM dysplasia.

Third, phenotypes resulting from mutations in the lamin B receptor in other mammals are highly variable. The ichthyosis mouse (ic) also results from mutations in Lbr [Shultz et al., 2003]. Heterozygous ic mice have white blood cell abnormalities very similar to those found in PHA, while homozygotes for a null allele whose phenotype was analyzed in detail, ic^J, also exhibited sparse hair or complete alopecia, growth retardation, variable syndactyly, and ichthyosis, particularly on the tail. Based on the frequencies of affected ic^J/ic^J pups, there was approximately 50% pre- or perinatal lethality for the mutation. In contrast, homozygous Lbr deficiency in the rabbit has a severe skeletal phenotype resembling HEM dysplasia (reviewed in Oosterwijk et al., [2003] and Shultz et al. [2003]).

Wassif et al. [2007] have suggested that HEM dysplasia is a laminopathy rather than a defect in cholesterol biosynthesis based on phenotypes identified in mice carrying different combinations of the ichthyosis (ic^J) Lbr null mutation and a targeted Tm7sf2−/− (also called Dhcr14−/−) null allele. Dhcr14 null mice had no observable phenotype and did not accumulate C14 sterols, consistent with the work of Bennati et al. [2008] who also found normal cholesterol synthesis in Tm7sf2 knockout mice. However, Clayton et al. [2010] have argued that human PHA results from disruption of the structural and nuclear envelope functions of LBR, while HEM dysplasia results from sterol C14-reductase deficiency. This hypothesis is based, in part, on their recent studies demonstrating that 2 missense mutations observed in HEM dysplasia fetuses disrupt enzyme activity, but not nuclear morphology and do not completely rescue reductase deficient yeast mutants. They also demonstrated that wild type LBR protein colocalizes with the ER membrane protein calnexin, as well as being present in nuclear membranes. In the one fetus studied, 8,14-cholestadien-3β-ol was significantly increased in muscle tissue [Offiah et al., 2003]. The authors suggest that differences in the role of sterol C14 reductases and LBR among mammalian species may account for the discrepant findings between their work and findings in the mouse. They cite the example of differences in phenotypes with NSDHL deficiency in females with CHILD syndrome versus bare patches mice as a known example of such species differences. Given the rarity of HEM dysplasia and its uniform prenatal lethality, with the potential for misdiagnosis or failure to retrieve tissue for molecular and biochemical studies, it may take considerable time and effort to resolve this issue.

CONCLUSIONS

We have presented updated descriptions of 8 disorders involving 6 enzymes of post-squalene cholesterologenesis, including recently published data and some contributed unpublished results. Together, this information begins to present a clearer picture of the phenotypes for these rare, but important, developmental genetic conditions. As noted previously, all of the disorders are associated with major somatic and/or CNS malformations and dysmorphic facies, suggesting that perturbations of the pathway have potent teratogenic effects.

While these disorders may share some features with each other and with SLOS, as demonstrated in Table II, there are many distinctions as well. For hemizygous male EBP deficiency, a specific syndromic phenotype is emerging with characteristic facies and frequent corpus callosal and Dandy–Walker malformations, cataracts, congenital heart defects, genito-urinary abnormalities (GU), and minor skeletal anomalies, and diffuse congenital ichthyosis. There is high postnatal mortality from cardiac disease, respiratory insufficiency and/or infections. Some of the clinical features are also found in heterozygous females with EBP mutations and CDPX2, albeit often with an asymmetric or patchy distribution. Although there are only two families reported to date with CK syndrome, their features are very different from those seen in the other disorders, with a long thin face, thin build (as adults), and abnormal cortical development with intellectual disability and seizures (but not agenesis of the corpus callosum or Dandy–Walker malformation). Somatic major malformations are rare in CK syndrome. There is virtually no overlap with CHILD syndrome seen in heterozygous females with NSDHL mutations. For desmosterolosis, with nine affected individuals from six families, the clinical phenotype still remains highly variable, perhaps due, in part, to the fact that there are multiple substrates for the deficient enzyme within the pathway. The most unifying feature in this disorder appears to be the presence of complete or partial agenesis of the corpus callosum. For lathosterolosis, there remain only the originally reported two liveborn cases. Both patients were dysmorphic with microcephaly and congenital or postnatal cataracts. The phenotypic spectrum of this disorder remains incomplete, although liver involvement with features of a storage disease appears to be unique. Finally, a phenotype is beginning to emerge for SC4MOL deficiency, with four known cases with at least some phenotypic information. Prominent skin hyperproliferation and immune defects in this disorder have been attributed to specific effects of the intermediates that accumulate and are known to induce meiosis in germ cells.

For most of these disorders, plasma sterol analysis is an extremely useful, cost effective screening test. The overall breadth and diversity of clinical features warrants early screening in infants with any combination of the following: dysmorphic features, hypotonia, one or more congenital anomalies, ichythosis, or minor skeletal anomalies. A sterol biosynthetic disorder should also be considered in older children with intellectual disability and CNS malformations or dysmorphic features with or without other malformations. It is clear that the absence of skeletal, growth, or skin findings or features suggestive of SLOS does not exclude a diagnosis of some of these disorders. However, a normal sterol profile also does not exclude certain disorders of cholesterol biosynthesis, particularly a defect in NSDHL (CHILD and CK syndromes) or mildly affected females with CDPX2.

The pathogenesis of non-SLOS sterol disorders remains unclear. One can postulate that these abnormal phenotypes result from cholesterol deficiency during early embryonic development, accumulation of toxic sterol intermediates proximal to each enzymatic block, abnormal feedback regulation, generation of abnormal bioactive oxysterols, and/or abnormal signaling by hedgehog proteins that normally contain bound cholesterol. It is very likely that multiple mechanisms occurring at different stages of development and/or in different tissues are involved, and data are beginning to accumulate to support some of these mechanisms.

Cholesterol is an essential component of cell membranes and a major determinant of plasma membrane fluidity. The cholesterol biosynthetic pathway is intimately tied to numerous important cellular functions, including signaling in lipid rafts and the formation of isoprenoids, steroid hormones, bile acids, vitamin D, and oxysterols. Thus, it is likely that at least some of the teratogenic effects in these disorders result from a lack of cholesterol for incorporation into membranes, particularly in the brain where all cholesterol must be synthesized in situ once the blood–brain barrier forms. Methylated sterol intermediates, in particular, are unable to fulfill the role of cholesterol within membranes and result in dramatic changes in fluidity within the lipid bilayers [Bloch, 1983; Xu et al., 2001; Wang et al., 2004]. Studies of S5cd−/− mice support the hypothesis that at least some of the malformations in these disorders result from decreased cholesterol rather than accumulation of sterol intermediates [Krakowiak et al., 2003]. However, unique features of each disorder would argue against the lack of cholesterol as the only mechanism. Response to treatment of skin lesions in CHILD syndrome and SC4MOL deficiency with a combination of statins and cholesterol, but not cholesterol alone, argues for a pathogenic mechanism involving both product depletion and substrate accumulation.

It remains attractive to postulate important roles for hedgehog proteins in disease pathogenesis. One or more of the hedgehog proteins are involved in many of the developmental processes that are perturbed in these disorders, including establishing a proper midline and left–right axis, CNS development, limb patterning, chondrocyte differentiation, and hair follicle development, among others. We [Jiang and Herman, 2006] and others [Stottmann et al., 2011] have recently demonstrated defective hedgehog signaling in vivo in recipient cells using two mouse models of cholesterol synthesis disorders, NSDHL deficiency and HSD17β7 deficiency, respectively.

Many unanswered questions remain concerning the clinical aspects of these disorders as well: Why are there different types of CNS malformations associated with different disorders, and what can this tell us about cholesterol and lipid metabolism in normal CNS development? Why does the brain appear to be spared in HEM dysplasia? What is the mechanism for unilateral skin lesions and limb reduction defects in human CHILD syndrome? Why are the clinical features of SC4MOL deficiency and CHILD or CK syndrome so different given that they act at the same step in the pathway? The availability of additional animal models, including conditional and inducible conditional mouse alleles in which postnatal development can be examined, will be extremely important to answer some of these questions. However, careful clinical phenotyping, as well as biochemical and molecular studies of additional patients, is critical to improve the diagnosis of these disorders, as well as our understanding of disease pathogenesis, particularly where phenotypes in model organisms differ from those in humans. Determining the mechanisms associated with these diseases, particularly the CNS malformations, intellectual disability, and behavior problems, may lead to improved patient care and may also advance our understanding of more common neurodevelopmental disorders, such as autism.