Investigation of 7-dehydrocholesterol reductase pathway to elucidate off-target prenatal effects of pharmaceuticals: a systematic review

Abstract

Mendelian diseases contain important biological information regarding developmental effects of gene mutations that can guide drug discovery and toxicity efforts. In this review, we focus on Smith-Lemli-Opitz syndrome (SLOS), a rare Mendelian disease characterized by compound heterozygous mutations in 7-dehydrocholesterol reductase (DHCR7) resulting in severe fetal deformities. We present a compilation of SLOS-inducing DHCR7 mutations and the geographic distribution of those mutations in healthy and diseased populations. We observed that several mutations thought to be disease causing occur in healthy populations, indicating an incomplete understanding of the condition and highlighting new research opportunities. We describe the functional environment around DHCR7, including pharmacological DHCR7 inhibitors and cholesterol and vitamin D synthesis. Using PubMed, we investigated the fetal outcomes following prenatal exposure to DHCR7 modulators. First-trimester exposure to DHCR7 inhibitors resulted in outcomes similar to those of known teratogens (50 vs 48% born-healthy). DHCR7 activity should be considered during drug development and prenatal toxicity assessment.

Figure 1

Full-term SLOS patients are typically compound heterozygous for two distinct mutations in DHCR7 (a), whereas homozygous null individuals are detected less frequently due to prenatal lethality (b) depicts the autosomal inheritance of SLOS in children and how compound heterozygosity is responsible for the disease phenotype. Many SLOS genetic studies focus on compound heterozygous patients (a) because most homozygous phenotypes result in prenatal fatalities, reducing the detection rate (b). Both W151X and IVS8-1G>C are null mutations in DHCR7 meaning that they reduce DHCR7 expression to almost 0% in the homozygous state. Therefore, if an individual is homozygous for either of these mutations or heterozygous for the combo then little to no DHCR7 expression would result. On the other hand, T93M is a non-null mutation in DHCR7 that reduces DHCR7 expression by 5% when compared to normal. Therefore, a compound heterozygous patient with one IVS8-1G>C null mutation and one T93M mutation would have around 45% functional DHCR7 and SLOS would result, but prenatal fatality would be averted (a). DHCR7, 7-dehydrocholesterol reductase; SLOS, Smith–Lemli–Opitz syndrome.

Figure 2

Certain exons and functional regions are enriched for SLOS-inducing mutations in DHCR7 and mutation spectrum varies by region and ethnicity. The overall exon distribution of alleles is shown in (a) for all 1024 alleles. Notice that the intronic null mutation (renders exon 9 non-existent) is the most common followed by mutations in exon 9. Exons 4 and 6 also feature prominently in SLOS-inducing mutations. (b) depicts the proportion of distinct DHCR7 mutations (left) and overall mutations (right) that occur in the trans-membrane region of the protein. The 3-dimensional protein structure for DHCR7 has yet to be published. However, three different models have been described indicating transmembrane domains including the Human Protein Reference Database, Waterham and Wanders and Fitzky et al. Note that the Waterham model results in the largest proportion (62.109%) of SLOS-inducing mutations being flagged as occurring in the transmembrane domain. (c) The allele frequency distribution for patients with ethnicity or country of origin information (170 patients had this information available). Some ethnicities only had one patient including, Ashkenazi, Turkish and Spanish/African (a patient with mixed ancestry). (d) The exon locations for each compound heterozygote pair (each patient has 2 mutations in DHCR7). Note that different ethnicities or countries of origin have different mutation patterns. For example Asian patients frequently have one mutation in exon 8 and one mutation in exon 9; whereas patients from Europe (unspecified lower left-hand corner of (d) or Northern Europe tend to have one mutation in exon 6 and other in exon 9. Patients from Southern Europe frequently had two different mutations in exon 9 or one mutation in exon 4 and one in exon 9. Ethnicity groupings: Europe (European but not otherwise specified and N-S Europeans); N. Europe (Dutch, Hungarian, Polish, German, Austrian, United Kingdom, Irish); S. Europe (French, Italian, Spanish, Portuguese, Greek); S. America (Brazil, United States of America-Hispanic); Asia (Korean, Japanese, Japanese-Dutch). DHCR7, 7-dehydrocholesterol reductase; SLOS, Smith–Lemli–Opitz syndrome.

Figure 3

Literature-derived pathway illustrates how 7-dehydrocholesterol reductase effects vitamin D production by removing 7-dehydrocholesterol and the effects of drugs on this pathway. Drugs that enhance DHCR7 function result in reduction in vitamin D production. This occurs because DHCR7 causes more 7-dehydrocholesterol to be converted to cholesterol. This indirectly inhibits vitamin D production by reducing the amount of 7-dehydrocholesterol that can be converted into cholecalciferol in the skin. Homozygous null mutations in DHCR7 completely inhibit functionality and result in patients with SLOS with heterozygous patients showing increased protection against rickets and osteomalacia. The dashed line indicates a feedback inhibition loop. 1 (ref. 53), 2 (ref. 45), 3 (ref. 94), 4 (ref. 7), 5 (ref. 57), 6 (ref. 58), 7 (ref. 59), 8 (ref. 54), 9 (ref. 28), 10 (ref. 50). DHCR7, 7-dehydrocholesterol reductase; SLOS, Smith–Lemli–Opitz syndrome.

Figure 4

DHCR7 SLOS-inducing mutation frequency in SLOS patients vs frequency from ExAC population. Some mutations occurred more frequently in the SLOS population given their frequency in the healthy population (for example, T93M, V326L, R404C, R352W). A possible explanation for this could be that these mutations occur in certain ethnic populations and those populations are under-represented in the ExAC population. DHCR7, 7-dehydrocholesterol reductase; SLOS, Smith–Lemli–Opitz syndrome.

Figure 5

Fetal outcomes of prenatal exposure to DHCR7 modulators compared to CDC prevalence and a known teratogenic drug (i.e., isotretinoin or accutane) and a known pregnancy safe drug (that is, levothyroxine). (a) contains aggregated results across all trimesters for regarding pregnancy outcomes. (b) Only born-healthy and born with fetal malformations, defects or congenital anomalies. Therefore, we excluded spontaneous abortions, elective terminations, stillbirths, ectopic pregnancies and neonatal deaths. The CDC provides data on live-born babies, elective terminations and fetal losses. We report fetal losses as spontaneous abortions in (a). We also split the number of live-born babies into born with defects and born-healthy using the statistic that 3% of live-born babies has a congenital anomaly. Notice that in (a) the number of fetal malformations/anomalies is higher among DHCR7 modulators including those that increase expression of DHCR7 (5.5%). However, drugs that inhibit DHCR7 have an increase in fetal malformations (10.9%). None of these levels comes close to the known teratogen, isotretinoin or accutane, with 47.7% born with malformations (out of total born). Many patients that are pregnant elect to terminate their pregnancy (a), which may be due to detect anomalies, however data on malformations among aborted fetuses is typically not available. We took all reported results where first-trimester exposure occurred (even if exposure persisted throughout the pregnancy) or where the exposure was listed as ‘early pregnancy' as this appeared to indicate first-trimester exposure and these are shown in (c). Many known first-trimester accutane or isotretinoin resulted in elective terminations or induced abortions. Therefore, we included elective terminations and spontaneous abortions in (d) along with live births (healthy or malformed).