Smith-Lemli-Opitz Syndrome

Richard I. Kelley, M.D., Ph.D.

 

Introduction

Smith-Lemli-Opitz syndrome (SLOS) is a well-known malformation syndrome with principal characteristics of psychomotor and growth retardation, cleft palate, hypospadias, postaxial polydactyly, and a distinctive craniofacial appearance consisting of microcephaly, ptosis, inner epicanthal folds, anteverted nares, and micrognathia. The first three cases of SLOS, all males with microcephaly and hypogenitalism, were described in 1964 (Smith et al. 1964) in three families with surnames beginning with R, S, and H, hence the designation by the original authors as the "RSH" syndrome. This report was soon followed by the description of many new cases and the delineation of additional characteristics including Hirschsprung's disease; sexual ambiguity or partial sex reversal in 46,XY males; abnormal cerebral gyri and cerebellar hypoplasia; and structural defects of the heart, lungs, liver, and kidneys (Fine et al. 1968; Finley et al. 1969; Opitz et al. 1969; Kaufman et al. 1974; Cherstvoy et al. 1975; Gold and Pfaffenbach 1975; Johnson 1975). For the more severely affected children, life-span was often limited by lethal internal malformations, but many less severely involved patients have survived to older childhood and adulthood (Opitz et al. 1969). The occurrence of the syndrome in siblings of both sexes and birth to consanguineous parents indicated that SLOS is inherited as an autosomal recessive Mendelian disorder (Opitz et al. 1969).

In the first 20 years following the initial description of SLOS, a wide spectrum of abnormalities was documented in more than 120 cases (Opitz et al. 1969; Cherstvoy et al. 1975; Johnson 1975; Jeanty et al. 1977). As a result, classical SLOS came to be recognized as a relatively common disorder among children with congenital malformation syndromes. In 1987, Curry and colleagues (Curry et al. 1987) collected 19 new cases of a severe, congenitally lethal, apparently autosomal recessive disorder with many characteristics of SLOS syndrome and summarized the findings in 19 similarly affected, previously published cases. The diagnosis of "SLOS type II" as possibly distinct from classical SLOS was proposed for these severely affected patients. However, not all dysmorphologists have considered SLOS II to be genetically distinct from the classical syndrome, a view that has also been supported by quantitative analysis of the distribution of malformations (Bialer et al. 1987).

Despite the frequency of SLOS and the many different clinical, laboratory, and pathological studies published about SLOS since 1964, the genetic cause of SLOS remained elusive for many years. However, by the mid-1980s, a number of abnormalities of steroid metabolism in SLOS had been reported. In part, the search for defects of steroid metabolism had been prompted by the sexual ambiguity common in the more severely affected male patients and by the finding of enlarged, lipid-depleted adrenal glands in some patients (McKeever and Young 1990). The steroid abnormalities included aberrant patterns of steroid sulfates in plasma and urine (Chasalow et al. 1985), low basal and stimulated levels of testosterone, low maternal estriol levels in mid and late gestation (McKeever and Young 1990), and hypocholesterolemia (Curry et al. 1987). Although these abnormalities collectively pointed to a fundamental disturbance of sterol or cholesterol metabolism, the primary defect remained unknown until Natowicz and Evans (Natowicz and Evans 1994) discovered that a patient with hypocholesterolemia and a relatively severe form of SLOS had essentially undetectable levels of normal urinary bile salts. This observation led to an analysis of bile acids and plasma sterols in the patient by Tint and colleagues and their discovery (Irons et al. 1993) that, in addition to a low plasma level of cholesterol, the patient had a greater than 1000-fold increase in the level of 7&endash;dehydrocholesterol (cholesta-5,7-dien-3ß-ol; "7DHC'), the immediate precursor of cholesterol in the Kandutsch-Russell (1960) pathway for biosynthesis of cholesterol. The same biochemical abnormality was later found in additional patients with a clinical diagnosis of SLOS (Tint et al. 1994). Although 7DHC represented the major sterol in plasma of the patients, large amounts of two other diene sterols - isodehydrocholesterol (cholesta-6,8-dien-3ß-ol) and 8&endash;dehydrocholesterol (cholesta-5,8(9)-dien-3ß-ol), both presumably related to 7DHC by isomerization - were also found, forming a distinctive pattern of plasma diene sterols. Similar sterol abnormalities were present in all tissues as well. Subsequent to the initial reports of Tint et al (Irons et al. 1993; Tint et al. 1994), the same diene sterol pattern has been found in almost all patients with both classical SLOS and the more severe SLOS type II, as well as in patients with variant syndromes that could not be assigned the diagnosis of SLOS on clinical grounds alone (Cunniff et al. 1996). Based on these biochemical abnormalities, Tint et al (Tint et al. 1994) have speculated that Smith-Lemli-Opitz syndrome is caused by a primary deficiency of 3ß-hydroxysteroid-D7-reductase (7DHC-reductase), a microsomal enzyme that converts 7&endash;dehydrocholesterol to cholesterol. Preliminary studies of 7DHC-reductase activity in cultured skin fibroblasts from patients with SLOS have shown reduced levels of the enzyme (Shefer et al. 1995). However, whether the primary genetic defect lies in the 7DHC reductase enzyme protein or in an associated factor or transport system remains to be determined, perhaps ultimately by molecular as well as biochemical analysis. The reports of two patients with SLOS and de novo balanced chromosomal translocations with a common breakpoint at 7q32.1 (Curry et al. 1987; Wallace et al. 1994) suggest that at least one genetic locus for SLOS resides at that position.

The discovery of a fundamental defect of sterol biosynthesis in SLOS syndrome has provided clinicians with not only a sensitive and accurate laboratory method for postnatal and antenatal diagnosis, but also a rationale for treatment and for elucidation of the biochemistry and physiology of the profound abnormalities of tissue morphogenesis characteristic of SLOS. In addition, the association of defective sterol biosynthesis with a multiple congenital anomaly syndrome has major implications for clinical and biochemical genetics as a whole, as discussed later.

Diagnosis of Smith-Lemli-Opitz Syndrome

A. Clinical Characteristics

Prior to the recognition of abnormal cholesterol biosynthesis in SLOS, the diagnosis was made strictly on clinical grounds. The clinical findings most characteristic of SLOS are microcephaly, blepharoptosis, cleft palate, cataracts, and postaxial (ulnar/fibular side) polydactyly (Opitz et al. 1969; Johnson 1975; Jeanty et al. 1977). Although each of these malformations can be found in many other genetic syndromes, the overall facial appearance of most children with SLOS is quite distinctive and the diagnosis easily confirmed by the recognition of other physical abnormalities listed in table 1.

 

Table 1 - Clinical Characteristics of Smith-Lemli-Opitz Syndrome

More than 50% of patients

10 to 50% of patients

Mental retardation (100%)

Cataracts

Microcephaly (>90%)

Postaxial polydactyly

Hypotonia

Cardiac defect

Epicanthal folds

Sexual ambiguity

Blepharoptosis

Prenatal growth retardation

Low set, abnormal pinnae

Renal cystic dysplasia

Cleft palate

Cholestatic liver disease

Broad maxillary alveolar ridges

Adrenal hyperplasia

Small tongue

Abnormal pulmonary lobation

Micrognathia

Hirschsprung's disease

Hypospadias*

Equinovarus deformity

Cryptorchidism*

Pyloric stenosis

Syndactyly of toes 2/3 (95%)

Postnatal growth retardation

* greater than 50% of 46,XY patients

 

With the availability of a laboratory test for SLOS, it is now possible to be more definitive about the diagnosis of classical SLOS and to separate out children with SLOS-like syndromes who do not have the biochemical markers now considered requisite for SLOS. In testimony to both the distinctiveness of the syndrome and the value of clinical dysmorphology, very few patients given the diagnosis of SLOS by experienced geneticists have not had the characteristic diene sterol abnormalities now associated with SLOS. Interestingly, one of the most sensitive clinical markers for SLOS with 7DHC-emia is syndactyly of toes 2/3 which, although a characteristic of a number of genetic syndromes, occurs in more than 98% of patients with increased levels of 7DHC (Cunniff et al. 1996). Other especially important characteristics found in a high proportion of patients are broad alveolar ridges, micrognathia, cleft palate, and, in males, hypospadias or more severe genital malformations. Many patients with SLOS are unusually irritable as infants and as older children and suffer from severe colic and a wide variety of functional gastrointestinal problems. Despite the severe deficiency of cholesterol in the brain (Tint et al. 1995), some affected children who survive are capable of remarkably good intellectual function, with developmental quotients in the moderately or even mildly retarded range. Although the majority of patients function in the moderate to severely retarded range, they nonetheless can be very interactive children with substantial receptive language and communication abilities.

A phenomenon that has occurred with other well-established clinical syndromes when a definitive biochemical marker is discovered is expansion of the clinical phenotype. This has now also occurred with SLOS in some quite remarkable ways. For example, a number of children whose only clinical abnormalities are developmental delay and 2/3 toe syndactyly have now been found to have diagnostically increased levels of the three diene sterols increased in classical SLOS (Cunniff et al. 1996). These less severely affected children will often have only mildly reduced or even normal plasma cholesterol levels, as described below. At the other clinical extreme, neonates who die at birth from multiple severe malformations but who have uncertain clinical diagnoses have been found by sterol analysis of plasma or autopsy tissues to have SLOS (Kelley 1995; Cunniff et al. 1996). Frank holoprosencephaly has also been confirmed biochemically as a presentation of SLOS (Muenke et al. 1994). Furthermore, because the biochemical defect of SLOS is expressed in amniotic fluid early in pregnancy (Abuelo et al. 1995; Rossiter et al. 1995), a number of fetuses with multiple severe malformations have been shown biochemically to have SLOS by sterol analysis of amniotic fluid, cultured amniocytes, or fetal tissues (R. Kelley, unpublished observations). Among these have been a number of fetuses noted by sonography to have hydrops, severe nuchal edema, or renal agenesis and oligohydramnios, all clinical indications for amniocentesis and more detailed fetal assessment.

 

Pathology of Smith-Lemli-Opitz Syndrome

1. General. Unlike most disorders of intermediary metabolism, where affected children are clinically normal at birth, Smith-Lemli-Opitz syndrome is characterized by major abnormalities of development before birth. These disturbances of morphogenesis affect all germ layers and must begin as early as six weeks gestation, when the heart chambers and cerebral ventricles are forming. There are still major effects on morphogenesis evident at 12 weeks gestation when the terminal digits are developing. Thirty years before the discovery of abnormal cholesterol metabolism in SLOS, investigators studying cholesterol biosynthesis found that fetal rats exposed to an inhibitors of 7DHC-reductase developed a variety of malformations. These included brain malformations such as pituitary agenesis and holoprosencephaly (Barbu et al. 1984; Repetto et al. 1990) as well as malformations of the lungs, kidneys, and limbs (Roux and Aubry 1966), all of which are affected in SLOS. Because similar defects were found when pregnant rats were exposed to inhibitors of other enzymes of cholesterol biosynthesis, and because the malformations could be largely prevented by feeding the pregnant rats a high cholesterol diet (Barbu et al. 1988), the deficiency of cholesterol rather than the accumulation of sterol intermediates was suspected to be the proximate cause of the malformations.

2. Nervous System. Studies of the CNS pathology of SLOS are few, but the pattern of malformations is itself instructive. For example, the characteristic midfacial abnormalities of SLOS suggest disturbances in the development of the forebrain or neural crest, a major embryological element of the midface. The intestinal aganglionosis of Hirschsprung's disease, a common finding in severe SLOS may also be caused by abnormal development of the neural crest. Furthermore, cardiovascular malformations in SLOS are predominantly defects of the endocardial cushions, which also are important derivatives of the neural crest (Lin et al. 1995). The central nervous system pathology varies from micrencephaly with no identifiable structural abnormalities to frank holoprosencephaly (Muenke et al. 1994). In the one detailed review of the histopathology of the CNS (Cherstvoy et al. 1984), abnormalities noted in more than half of the patients were inferior olivary and fascia dentate dysplasia, abnormalities of neuronal distribution and maturation in the 1st, 3rd, and 5th cortical layers, and ectopic neurons and Purkinje cells in the white matter. The majority of children with SLOS have mild microcephaly, typically 2 to 3 SD below normal, and frontal lobe hypoplasia. The degree of mental retardation varies from profound to mild. In the more severely affected children there is a wide range of cerebral and cerebellar defects, including white matter hypoplasia, enlarged ventricles, partial or complete agenesis of the corpus callosum, dysplastic cerebral gyri, cerebral heterotopias, microphthalmia, and cerebellar hypoplasia (Cherstvoy et al. 1975; Cherstvoy et al. 1984). Other less frequently occurring abnormalities that reflect abnormal CNS or sensory development are mild to moderate sensorineural hearing loss and, occasionally, seizure disorders.

3. Heart. A recent study of cardiac defects in SLOS (Lin et al. 1995) found the three most common lesions to be secundum atrial septal defect, endocardial cushion defects, and anomalous pulmonary venous return. Patent ductus arteriosus is also relatively common. In contrast, conotruncal defects, which predominate in a number of genetic syndromes, are relatively rare. Although almost all categories of cardiac defects have been reported at least occasionally in SLOS, the proportion of historical cases with heart defects that had abnormal cholesterol metabolism cannot be known. Early death in SLOS is usually caused by either severe cardiac defects or pulmonary hypoplasia.

4. Gastrointestinal Tract. Gastrointestinal abnormalities in SLOS are very common causes of morbidity and mortality. Except the most mildly affected children, pyloric stenosis is common and may present as usual in the first few weeks of life or much later in some. Dysmotility also seems to be a common problem, but frank abnormalities of the small and large intestine, including malrotations and intestinal aganglionosis (Hirschsprung's disease) are not uncommon. In the more severely affected infants, involvement of the liver and gall bladder, including intra- and extrahepatic biliary atresia, is not uncommon (Curry et al. 1987). Progressive cholestatic liver disease, possibly caused by the presence of abnormal bile acids or the deficiency of normal bile or bile acids, is a rare but usually lethal complication (Curry et al. 1987); R. Kelley, unpublished). However, there is hope that early diagnosis and treatment with cholesterol and bile acid supplements will prevent fatal liver disease.

5. Kidney and Adrenal Abnormalities. The most common malformations of the urinary tract are renal hypoplasia and microcystic kidney disease (Donnai et al. 1986; Curry et al. 1987). Although unilateral or bilateral renal agenesis has not been reported very frequently, neonates or fetuses with SLOS and renal agenesis, whose facial characteristics would be distorted by the Potter oligohydramnios sequence of fetal deformations, could easily have been missed prior to the recognition of the cholesterol abnormalities. As noted above, adrenal hyperplasia with lipid depletion is a common finding and is associated with low maternal estriol levels during pregnancy (McKeever and Young 1990) as well as generally low levels of a number of adrenal steroids postnatally. However, interestingly, there have been no reported cases of SLOS with neonatal adrenal insufficiency characteristic of classical forms of congenital adrenal hyperplasia, such as 21&endash;hydroxylase deficiency. This may be because even the most severely affected SLOS patients synthesize small amounts of cholesterol from which steroid hormones can be made by otherwise normal pathways of steroidogenesis.

6. Pulmonary Malformations. Pulmonary insufficiency is a relatively common cause of death among infants with severe forms of SLOS (Rutledge et al. 1984; Donnai et al. 1986). Pulmonary hypertension and very small lung volumes are commonly described premortem. At autopsy, abnormal or absent pulmonary lobation and associated pulmonary vessel malformations as well as a decreased number of alveoli are typically found. Of note, pulmonary hypoplasia and lobation abnormalities have figured prominently in several "new" lethal syndromes reported in the last 15 years (Kohler 1983; Rutledge et al. 1984; Donnai et al. 1986; Verloes et al. 1991). In retrospect, some of these patients almost certainly had SLOS, because similarly affected neonates and fetuses have more recently been shown to have the characteristic sterol abnormality of SLOS.

7. Genital Malformations. Genital malformations are very important for the clinical recognition of SLOS and the reason for the well-known male predominance among reported cases of SLOS. Hypospadias - from simple penile to third degree - cryptorchidism, and bifid scrotum are the most common genital malformations. However severely ambiguous genitalia and even completely normal-appearing female genitalia are not uncommon in 46,XY males with severe SLOS. Although the clinically apparent "sex-reversal" in SLOS is never complete, ovarian tissue, blind vaginal pouches, and other Müllerian remnants are often found (Bialer et al. 1987). With the knowledge of deficient cholesterol biosynthesis in SLOS, the cause of the genital malformations has been assumed to be deficient fetal synthesis of androgenic steroids. However, because pituitary agenesis is relatively common in the rat biochemical model for SLOS, deficient hypothalamic-pituitary function may also play a role in the genital maldevelopment in some patients. Moreover, deficient steroidogenesis cannot explain the persistence of Müllerian structures in some of the severely affected males. In the few patients who have been studied in detail endocrinologically, levels of testosterone are low, and even in those whose testosterone is low-normal, FSH may be high. In one adult with classical SLOS, a testicular biopsy showed essentially absent spermatogenesis and a marked deficiency of spermatogonia (Hoefnagel et al. 1969). A marked deficiency of Leydig cells was also noted, which may explain the persistence of Müllerian duct derivatives in some.

8. Skeletal Abnormalities. The majority of infants with SLOS have normal to slightly below normal birth weights and lengths, but mesomelic dwarfing has been reported in more severely affected children and fetuses. Malformations of the feet and hands are well known and constitute some of the most important diagnostic characteristics of SLOS. Polydactyly is characteristically postaxial and may range from a minimus digit to a complete extra finger. Severe bilateral upper limb deficiency has also been reported (Singer et al. 1989) in an infant later found to have the biochemical abnormalities of classical SLOS. Other important skeletal findings include short first metacarpals, syndactyly of toes 2/3, a preponderance of whorl digital patterns, and, in more severely affected children, equinovarus deformity and polysyndactyly of the feet. At birth, a large proportion of infants are also noted to have hip contractures.

9. Oral Malformations. The most distinctive oral malformation in SLOS is broadening and irregularity of the alveolar ridges. In older children, a grossly disordered dental pattern is also typical of the syndrome. Among the more severely affected children, microglossia and gingival cysts are common and apparently redundant sublingual tissue especially distinctive (Curry et al. 1987). Midline clefts are another important hallmark of SLOS occurring in at least 50% of patients and ranging from a bifid uvula or the U-shaped cleft soft palate of the Pierre Robin sequence to a submucus or complete cleft of the hard palate. Unilateral or bilateral cleft lip is not characteristic of SLOS, but midline clefts occur in the rare SLOS patient manifesting a complete or partial holoprosencephaly sequence (Muenke et al. 1994); Kelley, unpublished).

 

Biochemical Basis of Smith-Lemli-Opitz Syndrome

A. Normal Sterol Biosynthesis

Sterols are essential cellular components in both the plant and animal kingdoms, and are synthesized by a complex series of steps beginning with 3-hydroxy-3-methylglutaryl(HMG)-CoA and ending with the neutral 30&endash;carbon sterol, lanosterol (4,4,14-trimethylcholesta-8(9),24-dien-3ß-ol), the precursor of all other sterols. From lanosterol, cholesterol is synthesized by a complex series of mostly microsomal oxidations, reductions and demethylations. The first reaction in the cholesterol biosynthetic pathway, the reduction of HMG-CoA to mevalonic acid is catalyzed by HMG-CoA reductase and is generally accepted as the rate limiting step in the synthesis of cholesterol (Brown and Goldstein 1980). However, because of the great many biologically important compounds whose synthesis also begins with HMG-CoA reductase - dolichols, coenzyme Q, heme, and protein isoprenoid groups - the entire pathway is under complex, multilevel regulation to assure that, for example, dietary excess of cholesterol does not lead to excessive down-regulation of the synthesis of other critical metabolites in the pathway. The individual enzymes, cofactors, carrier proteins, and intracellular transport steps involved in the conversion of lanosterol to cholesterol are still incompletely characterized. Although the synthesis of cholesterol occurs principally in the microsomes, recent evidence suggests that much of the same cholesterol biosynthetic pathway exists in the peroxisome and that certain essential enzymes, such as mevalonate kinase, may exist solely within the peroxisome (Stamellos et al. 1992).

In one proposed pathway for synthesis of cholesterol in higher vertebrates, the Kandutsch-Russell pathway (Kandutsch and Russell 1960), the 7,8 unsaturated bond in 7DHC is reduced by an NADPH-dependent microsomal reductase to form cholesterol. However, not all cholesterol need be synthesized by this specific route, wherein the 24,25 unsaturated bond is reduced before the final series of steps for placement of the single 5,6 double bond in cholesterol. In the brain, for example, the relative abundance of desmosterol (cholesta-5,24-dien-3ß-ol) suggests that, under some circumstances, reduction of the 24,25 double bond may be the last step in the synthesis of cholesterol. Evidence that the Kandutsch-Russell pathway is the principal pathway for most human sterol synthesis has been mostly indirect and based on observations such as the finding that, among the various precursors of cholesterol, the level of lathosterol correlates well with the intrinsic rate of cholesterol biosynthesis (Kempen et al. 1988). 7DHC itself has been reported as a minor constituent of plasma and solid tissues. In 1991, Axelson reported that 7DHC and two other diene sterols, isodehydrocholesterol (cholesta-6,8-dien-3ß-ol) and 8-dehydrocholesterol (cholesta-5,8(9)-dien-3ß-ol) exist in normal plasma in relatively constant proportions, possibly because of a chemical equilibrium among these three diene sterols (Axelson 1991). 7DHC was found to be increased up to three-fold in conditions associated with increased loss of bile acids, such as ileal resection. 7DHC also holds special biochemical interest as a precursor of vitamin D3 via photic conversion to pre-vitamin D3 in skin. The ability of 7DHC to enter other pathways in normal individuals, such as adrenal steroid and bile acid biosynthesis is not certain, but evidence indicates that at least trace amounts of 7DHC may indeed be metabolized in these pathways (Chasalow et al. 1985; Natowicz and Evans 1994).

Of special importance to understanding the prenatal and postnatal physiology of SLOS are data that indicate that, in contrast to many other metabolites, most cholesterol for fetal growth is not obtained from the mother via the placenta but must be synthesized in situ by the fetus (Carr and Simpson 1982; Bellknap and Dietschy 1988). That very little if any cholesterol in the fetus is supplied by the maternal circulation is perhaps best illustrated by the finding that severely affected newborns with SLOS may have plasma cholesterol levels as low as 1 mg/dl at birth, barely 2% of the normal newborn cholesterol level (Cunniff et al. 1996). Similarly, deficiencies of cholesterol are found throughout the body of newborns with SLOS, with the most severe deficiency occurring in the brain.

Cholesterol synthesized prenatally has many different fates. Whereas most cholesterol becomes a relatively passive constituent of cell and organellar membranes and is therefore essential for normal prenatal growth, substantial amounts of cholesterol are also delivered to pathways for bile acid and steroid hormone synthesis. Prenatal deficiency of steroid hormone biosynthesis is well known and a common feature of the congenital adrenal hyperplasias, in which male genital malformations similar to those of SLOS occur. The consequences of defects of bile acid synthesis in syndromes other than SLOS can be largely postnatal, as in cerebrotendinous xanthomatosis (Björkhem and Muri-Boberg 1995), as well as prenatal, as in several different primary defects of bile acid biosynthesis associated with severe congenital cholestatic liver disease (Björkhem and Muri-Boberg 1995). Liver disease from biliary stasis has also occurred in a few of the more severely affected SLOS patients. An especially important fate of fetal cholesterol is the synthesis of estriol by combined action of the fetal adrenals and the placenta. In retrospect, the observation that mothers of SLOS children had low estriol levels during pregnancy (Donnai et al. 1987) was an important clue to the nature of the primary biochemical defect in SLOS.

 

B. Sterol Metabolism in Smith-Lemli-Opitz Syndrome

Because cholesterol is a constituent of all tissues and is present (in association with proteins) in all body fluids, the biochemical diagnosis of SLOS can now be made by sterol analysis of almost any patient specimen other than urine, which itself can be used for presumptive diagnosis of SLOS via bile acid analysis (Kelley 1995). The standard method for diagnosis of SLOS is quantification of plasma sterols by gas chromatography or gas chromatography/mass spectrometry, which allows separation of cholesterol from the diagnostic diene sterols (Tint et al. 1994; Kelley 1995). Although most SLOS patients have low total plasma cholesterol levels, screening for SLOS by routine laboratory measurement of cholesterol alone is not reliable. This is because many of the more mildly affected patients will have low normal cholesterol levels, even at birth, and because the presence of substantial amounts of 7DHC in plasma often leads to overestimation of cholesterol by some colorimetric assays. Caution must also be exercised in excluding the diagnosis of SLOS in an older child on biochemical criteria alone. Whereas the typical level of 7DHC at the time of diagnosis is between 100 and 200 µg/ml (normal: less than 0.25 µg/ml), levels as low as 1.5 µg/ml - too low to be detected by gas chromatography alone - have been found in children with SLOS. Conversely, because the carrier frequency of SLOS may be as high as 2% (Opitz et al. 1996), the chance of finding a mildly increased level of 7DHC because the patient is coincidentally a heterozygote for SLOS is not insignificant. In such individuals, measurement of 7DHC in cells cultured in the absence of cholesterol offers a suitable method to assess the significance of equivocal plasma levels of 7DHC. Another important factor to consider when testing patients for SLOS is that 7DHC is a comparatively unstable, oxygen-sensitive diene sterol. As a result, small but nonetheless diagnostic elevations of 7DHC may be lost during shipment or storage of samples.

The measurement of cholesterol and its precursor sterols in cultured cells is important for diagnosing SLOS in deceased patients for whom only cultured cells are available for testing (Kelley 1995). However, lymphoblasts and fibroblasts must be cultured in cholesterol-depleted medium for several days (lymphoblasts) or weeks (fibroblasts) to be certain of eliciting the diagnostic sterol abnormality. Interestingly, there is relatively little difference in the severity of the biochemical defect, as expressed in cultured cells, between clinically mild and severe cases of SLOS (Cunniff et al. 1996). Preliminary studies of 7DHC-reductase have shown markedly reduced activities in cultured skin fibroblasts from all degrees of severity of SLOS, but with statistically slightly greater activity in cells of SLOS type I patients versus type II patients (Shefer et al. 1995). However, as yet, there are no data establishing that a deficiency of 7DHC-reductase is the primary genetic defect causing SLOS.

Because cholesterol is present in all tissues, patient samples other than plasma or cultured cells, such as frozen tissues or even newborn screening (Guthrie) cards stored in state newborn screening laboratories, can be used for diagnosis. Formalin-preserved tissues can sometimes yield diagnostic elevations of 7DHC or 8DHC when all traces of formalin are removed, but this is a less reliable method of diagnosis. In other cases where testable samples are not available, measurement of 7DHC in parents' plasma or, better, cultured lymphoblasts or fibroblasts, can yield mild but diagnostic elevations of 7DHC. In fact, cultured cells from parents when grown in the absence of cholesterol will have up to 20-fold elevations in the ratio of 7DHC to cholesterol compared with control cells (R. Kelley, unpublished data). However, cells from a few obligate heterozygotes for SLOS have demonstrated only slight elevations in 7DHC that cannot be distinguished reliably from normal.

 

C. Prenatal Diagnosis

Prenatal diagnosis of SLOS has now been accomplished in many pregnancies both retrospectively and prospectively. As reported by several authors (Donnai et al. 1986; Abuelo et al. 1995; Rossiter et al. 1995) one of the early signs of an affected fetus is an abnormally low level of estriol, a fetal sterol product, in maternal serum and amniotic fluid. Other components of the commonly used, mid-gestational "triple screen," chorionic gonadotropin and alpha-fetoprotein, are also depressed in some affected pregnancies. A number of affected fetuses have also been identified by the discovery of suggestive fetal abnormalities, such as nuchal edema, polydactyly, cystic kidneys, ambiguous genitalia, or a 46,XY karyotype in a phenotypically female fetus. The level of 7DHC in the amniotic fluid of affected pregnancies is typically more than 1000-fold elevated. There are also often marked increases in the amniotic fluid level of lathosterol, the immediate precursor of 7DHC in more severely affected pregnancies. In some fluids from affected pregnancies, the level of cholesterol is abnormally low whereas in others the level is surprisingly normal. Cultured amniocytes can also yield diagnostically increased levels of 7DHC; however, both normal and mutant amniocytes grow poorly in the absence of cholesterol. As a result, achieving sufficient growth of amniocytes without complete suppression of the biochemical defect by cholesterol in the culture medium may sometimes be difficult. Direct analysis of mid-gestation placental villi and cultured villus cells has also yielded diagnostic elevations of 7DHC (R. Kelley, unpublished), but the applicability of chorionic villus sampling (CVS) in the first trimester has not yet been established as a certain method of prenatal diagnosis.

 

D. Biochemical-Clinical Correlations

Like most Mendelian syndromes, SLOS demonstrates much phenotypic variability between individual sibships and less so within sibships. Overall, the range of clinical severity in SLOS has been so great that separate disorders - types I and II SLOS - have been proposed to describe, respectively, mild and severe forms of SLOS. However, early experience with biochemical testing for SLOS has shown that the majority of patients with strong clinical criteria for SLOS have abnormal diene sterol patterns that differ only in magnitude. Using the clinical severity scale developed by Bialer, et al (1987), relatively little correlation between the plasma level of 7DHC and the clinical severity score is found. In contrast, there is a strong inverse correlation between severity and the plasma cholesterol level. Moreover, the most severely affected newborns have been those with plasma levels of cholesterol less than 10 mg/dl and levels of 7DHC less than 60 µg/dl, lower than that of most patients with comparatively mild forms of SLOS. Although clinical severity does not correlate well with the level of 7DHC, there is nonetheless a strong inverse correlation between cholesterol and 7DHC among patients beyond infancy. This apparent reciprocal relationship between cholesterol and 7DHC is also evident in children with SLOS following dietary supplementation with cholesterol. In these children, the level of 7DHC slowly falls over many months of therapy as the level of cholesterol rises. The apparent ability of dietary cholesterol to suppress diagnostic increases in the level of 7DHC emphasizes the importance of assessment of cholesterol metabolism in cultured cells for patients with clinical characteristics of SLOS but normal plasma sterol levels.

 

Biochemical Pathology and Teratology

Almost 30 years before the description of the cholesterol synthetic defect in SLOS, Roux and Aubry (1966) studied the teratological effects of an inhibitor of 7DHC reductase (AY&endash;9944) on the development of fetal rats. The malformations they described included pituitary agenesis, neural tube defects, holoprosencephaly and its secondary craniofacial malformations, and maxillary and mandibular hypoplasia. All of these occur in SLOS. The observed plasma sterol abnormalities in the treated rats were also similar to those eventually described in SLOS. After one week of treatment with AY&endash;9944, the plasma cholesterol levels fell to 50% of baseline and the level of 7DHC rose to the same level as cholesterol (Barbu et al. 1984). Similar findings were reported by Xu et a.l (1995) using another inhibitor of 7DHC-reductase, BM 15.766. The only difference in the sterol patterns was that 8&endash;dehydrocholesterol was not increased in the AY-9944-treated rats to the degree it is in SLOS plasma, which may reflect a slow rate of isomerization of 7DHC to 8DHC. Of considerable interest was that feeding the rats before pregnancy with large amounts of cholesterol largely prevented malformations in the fetuses exposed to AY-9944 (Barbu et al. 1984). This observation suggests that the teratogenic action of AY&endash;9944 may not be mediated directly by the drug itself or increased levels of 7DHC, but, instead, by the deficiency of cholesterol. This possibility is also supported by the strong inverse correlation between clinical severity of patients with SLOS and their plasma cholesterol levels. Rat embryos are believed to obtain almost all of their needed cholesterol from the mother until the 13th day of gestation, which is beyond the critical exposure time for the teratogenic effect of AY-9944 (Barbu et al. 1984). The situation in the human fetus may be more complex. Although there is evidence that in humans very little cholesterol is transported from mother to fetus (Carr and Simpson 1982), there may be an influence of the maternal cholesterol level during the earliest weeks of embryogenesis when the feto-placental unit is forming, a period that overlaps with the developmental period of many of the cerebral and cardiac malformations characteristic of SLOS.

 

Genetics of Smith-Lemli-Opitz Syndrome

Smith-Lemli-Opitz syndrome of all degrees of severity appears to be inherited as an autosomal recessive Mendelian disorder. Although earlier epidemiologic studies estimated the incidence of SLOS to be 1 in 20,000 births (Lowry and Yong 1980), more recent studies in a completely ascertained population in Czechoslovakia suggest an incidence closer to 1 in 10,000 births for definite cases and as high as 1 in 4000 births when probable cases are included (Opitz et al. 1996). The fact that the frequency of consanguinity among SLOS parents appears to be relatively low and that families with two affected sibships appear to be common also suggests that the gene for SLOS is relatively common (Cunniff et al. 1996). Many of the diagnostically less certain cases in the past have been females, in whom SLOS is more difficult to diagnose because of the absence of external genital malformations. The easier recognition of SLOS when genital malformations are present is most likely why in historical series of SLOS the male to female ratio is often greater than 2 (Johnson 1975; Jeanty et al. 1977). More recent experience with SLOS identified by biochemical testing also suggests considerable under-ascertainment of SLOS by physical criteria alone, because when SLOS is diagnosed biochemically following ascertainment by a non-genital malformation, the sex ratio is much closer to 1 (Cunniff et al. 1996). Underascertainment of severely affected SLOS cases also occurs. It has long been known that among siblings of probands with clinically diagnosed SLOS the segregation ratio is significantly less than the expected 0.25 for an autosomal recessive disorder (Johnson 1975). Some have speculated that the lower than expected segregation ratio is caused by undiagnosed fetal losses. Indeed, with the availability of prenatal testing by measurement of 7DHC in amniotic fluid, some multiply malformed fetuses that would have gone undiagnosed in the past are now being identified as SLOS. Because of the severity of visceral malformations in these fetuses, other SLOS pregnancies could spontaneously terminate even earlier in gestation, leading to a lower than predicted frequency of SLOS in siblings. On the other hand, a comparison of the frequencies of various malformations between clinically ascertained and biochemically ascertained cases (Table 2) indicates that the historical, clinically-defined series probably included two or more genetic disorders.

 

Table 2 - Smith-Lemli-Opitz syndrome - Percent Anomalies

Malformation

Johnson - 1975

Bialer et al. - 1987

This series

N = 55

N = 121

N = 77

Heart

"occasional"

NR

38

Renal cysts/agenesis

NR

NR

13

Bowel

22

26

43

Cataract

"occasional"

10

18

Cleft palate

40

34

52

2/3 Toe syndactyly

73

NR

99

This is especially evident in the frequency of toe syndactyly, which was present in 98% of biochemically defined cases of SLOS (Cunniff et al. 1996), but only 75% of the cases collected from the literature by Johnson (1975). Another result of biochemical screening for SLOS is that other syndromes not previously linked with SLOS have been found to have the same sterol abnormality. Because frank holoprosencephaly with midline cleft lip has occurred in one patient with the classical biochemical pattern of SLOS (Muenke et al. 1994), patients with clinically similar autosomal recessive forms of holoprosencephaly, such as pseudotrisomy 13 syndrome (holoprosencephaly-polydactyly syndrome; (Verloes et al. 1991) may also be found to have the biochemical profile of SLOS or another fundamental abnormality of cholesterol biosynthesis.

Although SLOS has often been divided into type I for the classical syndrome and type II for the more severe syndrome delineated by Curry et al. (1987), there has always been doubt that these two extremes of severity represent different genetic entities rather than variability within a single genetic disorder (Bialer et al. 1987; Penchaszadeh 1987). As noted above, both type I and type II SLOS have essentially the same abnormality of sterol metabolism except that the more severely affected patients with 7DHC-emia have, on average, lower plasma cholesterol levels. Patients with cholesterol levels below 10 mg/dl are almost always classified clinically as type II. So far, none of the many other theoretically possible defects of sterol metabolism distal to lanosterol have been described among either typical or atypical cases of SLOS. Until direct molecular genetic tests are developed, complementation analysis may be the only way to determine if different genetic forms of SLOS exist. Based on the description of two patients with biochemically confirmed SLOS and de novo chromosomal translocations with common breakpoints at 7q32.1, work has begun on identifying a possible SLOS gene at this chromosomal location (Wallace et al. 1994; Alley et al. 1995). At the time of this writing, the candidate region on 7q had been narrowed to approximately 1000 kilobases (Alley et al. 1995). Evidence cited for a gene for SLOS at 7q34 is less convincing (Berry et al. 1989) but still important to note. There are as yet no data published suggesting an increased incidence of SLOS in any particular ethnic group. However, among patients identified by biochemical testing, African-American and Asian children appear to be under represented ((Cunniff et al. 1996); R. Kelley, unpublished).

 

Differential Diagnosis

The clinical diagnosis of classic SLOS is a relatively simple matter for an experienced clinical geneticist, and biochemical confirmation is straightforward. Biochemical testing for diagnosis of SLOS should be entertained in any infant or fetus with polydactyly, 2/3 toe syndactyly, cataracts, cleft palate, ambiguous genitalia, or apparent sex reversal in a 46,XY patient. However, recognition of SLOS is more difficult at the two extremes of severity. Among the least affected patients, mild developmental delay and 2/3 toe syndactyly may be the only abnormalities found (Stephan et al. 1995; Cunniff et al. 1996). An abnormal level of 7DHC has also been a relatively unexpected finding in children from cleft palate or cataract clinics referred to geneticists for evaluation of developmental delay or growth retardation. A diagnosis of SLOS is also easily missed because of either the lack of classical external malformations that suggest SLOS or because a malformation such as holoprosencephaly has disrupted the diagnostically important facial characteristics. Furthermore, the extreme hypotonia and liver disease of some severely affected infants has led to the suspicion of Zellweger (cerebrohepatorenal) syndrome or related disorders of peroxisomal metabolism or biogenesis. Other diagnoses that have been made or entertained in some SLOS patients include Meckel syndrome (Lowry 1983), autosomal recessive holoprosencephaly (Muenke et al. 1994), pseudotrisomy 13 (holoprosencephaly-polydactyly syndrome) (Verloes et al. 1991), Gardner-Silengo-Wachtel syndrome (Greenberg et al. 1987), Campomelic dwarfism (Curry et al. 1987), hydrolethalus syndrome (Salonen 1990), Pallister-Hall hamartoblastoma syndrome (Donnai et al. 1987), and other severe phenotypes (Rutledge et al. 1984). Although some of these conditions, such as Meckel syndrome and campomelic dwarfism, are easily distinguished from SLOS, further study may show that other syndromes overlap biochemically with SLOS. For example, the only other known defect of cholesterol biosynthesis, mevalonic aciduria, is characterized by failure to thrive, hypocholesterolemia, cerebellar hypoplasia, and midfacial malformations (Hoffmann et al. 1986).

 

Treatment

For the first thirty years following its original description, SLOS could only be treated supportively. The most commonly management problems were gastrointestinal disorders, especially non-specific dysmotility, pyloric stenosis, and Hirschsprung disease. Equally common were frequent respiratory tract infections, complications of cardiac disease, and otherwise non-specific failure-to-thrive. As reported in a number of patient series. mortality in the first few years often was between 25 and 50%. With the recognition and treatment of the severe deficiency of cholesterol biosynthesis in SLOS, the medical and developmental outcome of patients may change substantially. The earliest results of dietary therapy, although only anecdotal, have been quite encouraging (Irons et al. 1994) especially with regard to growth and behavior.

The estimated daily synthetic need for cholesterol during infancy is 30 - 40 mg/kg/d (Acosta 1994; Cruz et al. 1994). Although natural breast milk has a high concentration of cholesterol, up to 120 mg/liter, even this amount supplies only about half the minimum daily requirement, or even less when one considers the large deficit of cholesterol that an SLOS infant has at birth. Moreover, the deficiency of cholesterol synthesis could easily be made worse with diarrhea and loss of gram amounts of cholesterol-derived bile acids present in the gut and enterohepatic circulation of an infant, even the SLOS infant who has a reduced pool of bile acids.

Initial treatment plans for SLOS (Irons et al. 1994) provided 50 mg/kg/d cholesterol either in a natural form (eggs, cream, liver, meats) or in the form of purified food-grade cholesterol, with or without supplements of bile acids: cholic acid, chenodeoxycholic acid, or ursodeoxycholic acid. For substantially growth retarded children, treatment with cholesterol supplements has sometimes been followed by striking increases in the rate of growth - up to 250 g per week - for several months until more appropriate weight and height percentiles are reached (R. Kelley, unpublished observations). Such rapid growth has occurred with combined cholesterol and bile acid supplementation as well as with cholesterol supplementation alone. Parents of SLOS patients also reported marked reduction in irritability, a well-known characteristic of SLOS children. Following treatment with cholesterol supplements, plasma cholesterol levels of most SLOS children rise slowly, but in some the cholesterol levels do not increase substantially until after the initial, rapid catch-up growth, as if most of the supplementary cholesterol is being preferentially incorporated directly into the tissues. Bile acids, in particular ursodeoxycholic acid, may be specifically beneficial in those few SLOS patients who have persistent liver disease. However, because bile acids may down-regulate tissue levels of LDL receptors, the more rapid rise in plasma cholesterol levels found in bile-acid supplemented patients could reflect impaired tissue uptake rather than enhanced intestinal absorption of cholesterol.

The clinical problems of SLOS that arise from congenital malformations are many and frequently require surgical treatment and intensive supportive medical care. In addition to Hirschsprung disease, which may affect up to 30% of patients with severe SLOS, there is a high incidence of other structural and functional gastrointestinal problems, including microgastria, pyloric stenosis, gastroesophageal reflux, severe colic, and dysfunctional swallowing. A large proportion of even the less severely affected patients require gavage or gastrostomy feeding for many months or indefinitely. Funduplications are frequently done and frequently unsuccessful in treating gastroesophageal reflux. Although many patients have almost a complete deficiency of normal bile acids, fat malabsorption and deficiencies of fat soluble vitamins appear to be uncommon. A number of the more severely affected patients have developed progressive cholestatic liver disease from which they have eventually succumbed. Cholestatic liver disease has occurred in the absence of peripheral hyperalimentation but can become rapidly worse with hyperalimentation. Treatment with ursodeoxycholic acid in one patient led to rapid reversal of the hyperbilirubinemia (R. Kelley, unpublished).

 

Smith-Lemli-Opitz Syndrome: A Paradigm for

Inborn Errors of Metabolite Biosynthesis

Despite the many advances in diagnostic molecular and biochemical genetics, the ability of the clinician to identify the primary cause of a patient's congenital malformations or developmental problems is still limited. Even in the best institutions, less than half of patients with developmental disabilities can be given a specific genetic diagnosis. For genetic developmental syndromes that are either more common or occur with increased frequency in genetic isolates, molecular genetics will eventually uncover the primary genetic lesion. But for syndromes that are rare or highly variable, a biochemical approach to diagnosis remains important if not essential. The great expansion of the SLOS clinical phenotype, the development of prenatal diagnosis and, most importantly, the possibility of meaningful therapy for SLOS patients were all spawned within a few months of the astute observations of several biochemical geneticists studying a single patient.

Although the concept of a "metabolic malformation syndrome" has precedents in both peroxisomal disorders and the congenital adrenal hyperplasias, SLOS is the best example yet of a biochemical disease that affects the synthesis of a critical metabolite for which the fetus is responsible for all synthesis. Because cholesterol is not transported to any significant degree from the mother to the fetus, patients with SLOS suffer a variety of systemic and cerebral malformations, more or less in proportion to the severity of the deficiency of cholesterol biosynthesis. This relationship of clinical severity to fetal metabolite deficiency is supported both by the recent clinical biochemical studies and by the earlier animal experiments with chemical inhibitors of 7DHC-reductase. Clearly, SLOS is only one of many theoretically possible inborn errors of metabolite biosynthesis. However, the vast majority of known inborn errors of metabolism - aminoacidopathies, organic acidopathies, lysosomal storage disease, etc. - are those affecting metabolite catabolism, not synthesis. Of the more than 30 steps in the biosynthesis of cholesterol, only two - mevalonate kinase deficiency and SLOS - have been described as genetic disorders. There must be at least an equal number of theoretically possible genetic diseases caused by deficient biosynthesis of other isoprenoid compounds, such as dolichols and coenzyme Q. In large part, the failure to identify these many genetic defects of metabolite biosynthesis is caused by the lack of appropriate screening techniques for neutral and complex lipids and other water-insoluble compounds. These will need to be developed before many more syndromes of metabolite biosynthesis, like SLOS, will be discovered.

 

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July, 1996