Smith–Lemli–Opitz syndrome is an inborn error of cholesterol synthesis. It is an autosomal recessive, multiple malformation syndrome caused by a mutation in the enzyme 7-Dehydrocholesterol reductase encoded by the DHCR7 gene. It causes a broad spectrum of effects, ranging from mild intellectual disability and behavioural problems to lethal malformations.
Signs and symptoms
SLOS can present itself differently in different cases, depending on the severity of the mutation and other factors. Originally, SLOS patients were classified into two categories (classic and severe) based on physical and mental characteristics, alongside other clinical features. Since the discovery of the specific biochemical defect responsible for SLOS, patients are given a severity score based on their levels of cerebral, ocular, oral, and genital defects. It is then used to classify patients as having mild, classical, or severe SLOS.
Physical characteristics
The most common facial features of SLOS include microcephaly, bitemporal narrowing (reduced distance between temples), ptosis, a short and upturned nose, micrognathia, epicanthal folds, and capillary hemangioma of the nose. Other physical characteristics include: low-set and posteriorly rotated ears high-arched, narrow, hard palate cleft lip/palate agenesis or hypoplasia of the corpus callosum cerebellar hypoplasia increased ventricular size decreased frontal lobe size polydactyly of hands or feet short, proximally placed thumb other finger malformations syndactyly of second and third toes ambiguous or female-like male genitalia congenital heart defects renal, pulmonary, liver and eye abnormalities
Behavioural characteristics
Certain behaviours and attributes are commonly seen among patients with SLOS. They may have low normal intelligence, and react negatively or with hypersensitivity to different sensory stimuli. This is particularly true for certain auditory and visual stimuli. Many patients show aggressiveness and self-injurious behaviours, and sleep disturbances are common. Specific behaviours resembling those of people with autism are often present as well as hyperactivity, which provides genetic and biological insights into autism spectrum disorders. The autistic behaviours most characteristic of SLOS patients are opisthokinesis (an upper body movement), stretching of the upper body, and hand flicking. Autism is typically diagnosed separately from SLOS using the DSM-V, and approximately 50–75% of SLOS patients meet the criteria for autism.
Other behaviours associated with SLOS can be linked directly to physical abnormalities. For example, infants often show feeding problems or feeding intolerance, and patients may require increased caloric intake due to accelerated metabolism. Recurrent infections, including ear infections and pneumonia, are also common.
Biochemical phenotype
Given that SLOS is caused by a mutation in an enzyme involved in cholesterol synthesis, the resulting biochemical characteristics may be predictable. Most patients have lowered plasma cholesterol levels (hypocholesterolemia). However, approximately 10% may show normal cholesterol levels, and decreased concentrations of cholesterol are not solely indicative of SLOS. Increased levels of cholesterol precursors are also common in SLOS. In particular, elevated levels of 7-dehydrocholesterol are fairly specific to SLOS.
Genetics
DHCR7
The gene encoding DHCR7 (labeled as DHCR7) was cloned in 1998, and has been mapped to chromosome 11q12–13. It is 14100 base pairs of DNA in length, and contains nine exons, the corresponding mRNA is 2786 base pairs in length (the remaining DNA sequence is intronic). The structure of the DHCR7 rat gene is very similar to the structure of the human gene.
The highest levels of DHCR7 expression have been detected in the adrenal gland, the testis, the liver and in brain tissue. Its expression is induced by decreased sterol concentrations via sterol regulatory binding proteins (SREBP). There is also evidence that its activity may be regulated by tissue specific transcription, and alternative splicing.
As outlined above, the enzyme DHCR7 catalyzes the reduction of 7DHC to cholesterol, as well as the reduction of 7-dehydrodesmosterol to desmosterol. It requires NADPH as a cofactor for this reduction, and may involve the activity of cytochrome-P450 oxidoreductase. It is also thought to contain iron. DHCR7 is an integral membrane protein of the endoplasmic reticulum, and computer models have predicted up to nine transmembrane domains. DHCR7 is most efficient at reducing 7DHC, but it is known to reduce the carbon 7 double bond of other sterols, indicating a range of substrate specificity. The human version of this enzyme is predicted to have a molecular weight of 54,489 kDa, and an isoelectric point of 9.05.
The amino acid sequence that encodes DHCR7 is predicted to contain 475 amino acids, as well as several protein motifs. It contains multiple sterol reductase motifs, as would be expected given its function. It contains a potential sterol-sensing domain (SSD), whose function is unknown but thought to be necessary for binding sterol substrates. It also includes multiple sites of phosphorylation, including potential protein kinase C and tyrosine kinase sites (regulatory enzymes responsible for phosphorylation). The exact function of phosphorylating DHCR7 is yet unknown, but it is thought to be involved in the regulation of its activity.
Mutations and incidence
SLOS is an autosomal recessive disorder. More than 130 different types of mutations have been identified. Missense mutations (single nucleotide change resulting in a code for a different amino acid) are the most common, accounting for 87.6% of the SLOS spectrum. These typically reduce the function of the enzyme but may not inhibit it completely. Much depends on the nature of the mutation (i.e. which amino acid is replaced and where). Null mutations are much less common, these mutations produce either a completely dysfunctional enzyme, or no enzyme at all. Thus, missense mutations may be more common overall because they are less lethal than nonsense mutations; nonsense mutations may simply result in spontaneous abortion.
The IVS8-1G>C is the most frequently reported mutation in DHCR7. This disrupts the joining of exons eight and nine, and results in the insertion of 134 nucleotides into the DHCR7 transcript. This is a nonsense mutation, thus patients that are homozygous for this allele are severely affected. It is thought that this mutation first occurred in the British Isles, and it has a carrier (those that are heterozygous for the allele but not affected) frequency of 1.09% for Caucasians of European heritage. The frequency of mutations differs for various ethnicities, depending on the origin of the mutation. In all Caucasian populations, this particular mutation has an estimated carrier frequency of 3%.
The next most common mutation is 278C>T, and results in a threonine at the amino acid position 93. It is a missense mutation and tends to be associated with less severe symptoms. This mutation is the most common one seen in patients of Italian, Cuban, and Mediterranean descent.
The third most common mutation is 452G>A. This nonsense mutation causes protein termination, such that the enzyme DHCR7 would not be formed. It is thought to have arisen in Southern Poland and is most common in Northern Europe.
Other mutations are less common, although appear to target certain protein domains more so than others. For example, the sterol reductase motifs are common sites of mutation. Overall, there is an estimated carrier frequency (for any DHCR7 mutation causing SLOS) of 3–4% in Caucasian populations (it is less frequent among Asian and African populations). This number indicates a hypothetical birth incidence between 1/2500 and 1/4500. However, the measured incidence is between 1/10,000 to 1/60,000 (it differs depending on heritage and descent). This is much lower than expected. This indicates that many cases of SLOS are undetected, and is likely due to either spontaneous abortion caused by severe mutations (miscarriage), or mild cases that are undiagnosed. Females lack the characteristic genital malformations that affected males have, and thus are less likely to be correctly diagnosed.
Cholesterol metabolism and function
Metabolism
Cholesterol can be obtained through the diet, but it can also be formed by metabolism in the body. Cholesterol metabolism primarily takes place in the liver, with significant amounts in the intestine as well. It should also be noted that cholesterol cannot pass the blood–brain barrier, thus within the brain, biosynthesis is the only source of cholesterol.
In humans, cholesterol synthesis begins with the mevalonate pathway (see diagram), leading to the synthesis of farnesyl pyrophosphate (FPP). This pathway uses two acetyl-CoA and two NADPH to make mevalonate, which is metabolized to isopentenyl pyrophosphate (IPP) using three ATP. From there, three IPP are needed to make one FPP. The combination of two FPP leads to the formation of squalene; this represents the first committed step towards cholesterol biosynthesis. Squalene leads to the creation of lanosterol, from which there are multiple pathways that lead to cholesterol biosynthesis. The rate limiting step of cholesterol synthesis is the conversion of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to mevalonate, this is an early step in the mevalonate pathway catalyzed by HMG-CoA reductase.
Through a complicated series of reactions, lanosterol leads to the formation of zymosterol. As shown in a diagram to the right, it is at this point that the pathway diverges. In humans, the main pathway leading to cholesterol is known as the Kandutsch–Russell pathway. Zymosterol is metabolized to 5α-cholesta-7,24-dien-3β-ol, then to lathosterol, and then to 7-dehydrocholesterol, or 7-DHC. 7-DHC is the immediate precursor to cholesterol, and the enzyme DHCR7 is responsible for converting 7-DHC to cholesterol. DHCR7 reduces the double bond on carbon 7 of 7-DHC, leading to the unesterified product. Mutations in this enzyme are responsible for the wide range of defects present in SLOS. In another pathway leading to cholesterol synthesis, DHCR7 is required for the reduction of 7-Dehydrodesmosterol to desmosterol.
Regulation
Regulation of cholesterol synthesis is complex and occurs primarily through the enzyme HMG-CoA reductase (catalyst of the rate-limiting step). It involves a feedback loop that is sensitive to cellular levels of cholesterol. The four main steps of regulation are: The synthesis of the enzyme HMG-CoA reductase is controlled by sterol regulatory element binding protein (SREBP). This is a transcription factor that is inactive when cholesterol levels are high, and active when cholesterol levels are low. When cholesterol levels fall, SREBP is released from the nuclear membrane or endoplasmic reticulum, it then migrates to the nucleus and causes the transcription of the HMG-CoA reductase gene. The translation (creating the enzyme from the mRNA transcript) of HMG-CoA reductase is inhibited by derivatives of mevalonate and by dietary cholesterol. The degradation of HMG-CoA reductase is tightly controlled. The part of the enzyme that is bound to the endoplasmic reticulum senses signals, such as increased cholesterol levels, that lead to its degradation or proteolysis. When HMG-CoA reductase is phosphorylated, its activity decreases. This means cholesterol synthesis is reduced when cell energy (ATP) levels are low.
Function