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. 2020 Feb 26;16(2):e1008628. doi: 10.1371/journal.pgen.1008628

Metabolic and pathologic profiles of human LSS deficiency recapitulated in mice

Yoichi Wada 1, Atsuo Kikuchi 1,*, Akimune Kaga 2, Naoki Shimizu 3, Junya Ito 3, Ryo Onuma 3, Fumiyoshi Fujishima 4, Eriko Totsune 1, Ryo Sato 1, Tetsuya Niihori 5, Matsuyuki Shirota 6, Ryo Funayama 7, Kota Sato 8,9, Toru Nakazawa 8,9,10,11,12, Keiko Nakayama 7, Yoko Aoki 5, Setsuya Aiba 13, Kiyotaka Nakagawa 3, Shigeo Kure 1
Editor: Luke Engelking14
PMCID: PMC7062289  PMID: 32101538

Abstract

Skin lesions, cataracts, and congenital anomalies have been frequently associated with inherited deficiencies in enzymes that synthesize cholesterol. Lanosterol synthase (LSS) converts (S)-2,3-epoxysqualene to lanosterol in the cholesterol biosynthesis pathway. Biallelic mutations in LSS have been reported in families with congenital cataracts and, very recently, have been reported in cases of hypotrichosis. However, it remains to be clarified whether these phenotypes are caused by LSS enzymatic deficiencies in each tissue, and disruption of LSS enzymatic activity in vivo has not yet been validated. We identified two patients with novel biallelic LSS mutations who exhibited congenital hypotrichosis and midline anomalies but did not have cataracts. We showed that the blockade of the LSS enzyme reaction occurred in the patients by measuring the (S)-2,3-epoxysqualene/lanosterol ratio in the forehead sebum, which would be a good biomarker for the diagnosis of LSS deficiency. Epidermis-specific Lss knockout mice showed neonatal lethality due to dehydration, indicating that LSS could be involved in skin barrier integrity. Tamoxifen-induced knockout of Lss in the epidermis caused hypotrichosis in adult mice. Lens-specific Lss knockout mice had cataracts. These results confirmed that LSS deficiency causes hypotrichosis and cataracts due to loss-of-function mutations in LSS in each tissue. These mouse models will lead to the elucidation of the pathophysiological mechanisms associated with disrupted LSS and to the development of therapeutic treatments for LSS deficiency.

Author summary

Skin lesions, cataracts, and congenital anomalies have been frequently associated with inherited deficiencies of cholesterol synthetic enzymes. LSS encodes lanosterol synthase, an enzyme in the cholesterol biosynthesis pathway, and biallelic mutations in LSS have been reported in families with congenital cataracts and hypotrichosis. However, it remains to be clarified whether these phenotypes are caused by LSS enzymatic deficiency in each tissue, and disruption of LSS enzymatic activity in vivo has not yet been validated. We showed that LSS metabolic inhibition in patients with biallelic LSS mutations and congenital hypotrichosis in vivo by measuring metabolites of the LSS enzyme in the forehead sebum, which would be good biomarkers for the diagnosis of LSS deficiency. We recapitulated hypotrichosis and cataracts by creating tissue-specific Lss knockout mice. Our mouse studies confirmed that LSS deficiency causes hypotrichosis and cataracts due to loss-of-function mutations in LSS in each tissue. These mice will lead to the elucidation of the pathophysiological mechanisms associated with disrupted LSS and to the development of therapeutic treatments for LSS deficiency.

Introduction

Cholesterol is essential for the regulation of biological functions in animals. In cell membranes, cholesterol enhances fluidity, reduces permeability and is a constituent of lipid rafts [1]. Moreover, cholesterol or its intermediates are precursors of vitamin D, steroid hormones, and bile acids. Cholesterol itself alters the activity of proteins such as sonic hedgehog (Shh) [2][3] which is involved in embryogenesis and tissue growth. In the cholesterol synthesis pathway, beginning with acetyl-CoA, lanosterol synthase (LSS) converts (S)-2,3-epoxysqualene to lanosterol [4] (S1 Fig). Biallelic mutations in LSS were first reported in families with congenital cataracts [5,6]. In addition, very recently, hypotrichosis has been reported to be an associated phenotype with biallelic LSS mutations [7,8]. Out of 24 patients, all had either cataracts (5/24) or hypotrichosis (20/24), and one had both [5–8]. Thus, cataracts and hypotrichosis are core symptoms in patients with biallelic LSS mutations. However, the pathophysiology in patients largely remains to be clarified. First, it is not proven that biallelic LSS mutations cause LSS deficiency because the metabolite profiles in the previously reported patients have not been compatible with LSS enzymatic dysfunctions. The cholesterol or the intermediate levels in serum or plasma did not significantly change between the patients and controls [7,8]. Second, animal models that reproduce the LSS-deficient state remain to be developed. Although the Shumiya cataract rat has biallelic Lss mutations [9], this rat has additional mutations in Fdft1, which encodes an upstream enzyme in the cholesterol synthetic pathway. Moreover, the metabolic profiles of the lens were inconsistent with LSS enzymatic inhibition in the Shumiya cataract rat. There is no report of model mice that reproduce cataract or hypotrichosis due to the embryonic lethality of the Lss null mice [10]. Collectively, it has not been established whether biallelic LSS mutations cause LSS enzymatic deficiency and the local symptoms in each tissue or if they lead to systemic effects on abnormal metabolites, and this is due to the lack of an appropriate model animal and the difficulty in measuring metabolites of the LSS enzyme.

Here, we report two siblings with novel biallelic LSS mutations who presented congenital hypotrichosis, midline anomalies, such as cleft palate and agenesis of the corpus callosum, and no cataracts. The aims of this study were to confirm an LSS enzymatic disruption, that is, the presence of LSS deficiency, in patients with biallelic LSS mutation and to create Lss-deficient mice to elucidate whether LSS enzymatic depletion in each tissue is responsible for the symptoms in that tissue.

Results

Clinical information of patients with congenital hypotrichosis and anomalies

We studied two sibling patients with congenital hypotrichosis and several anomalies (Fig 1a). The parents and the first child had no medical problems, including congenital anomalies. The first patient (patient II-2) was the second boy in a nonconsanguineous Japanese family (Fig 1b). This patient was delivered at 36 weeks of gestation due to low amniotic fluid and fetal growth restriction (FGR). His birth weight was 1,848 g (-2.26 SD), and height was 42.0 cm (-2.03 SD). At birth, mild ichthyosis was observed on his chest, abdomen, back, and upper extremities (S2 Fig). This ichthyosis disappeared by the age of 2 months. His body hair never grew despite treatment, such as topical vitamin D and steroid therapy. He had intermittent exotropia, hypospadias and partial agenesis of the corpus callosum (S3 Fig). At the most recent follow-up (18 years old), his height was 157.1 cm (-2.35 SD), and his body weight was 66.1 kg (0.54 SD), showing significant growth retardation.

Fig 1. Identification of biallelic LSS mutations in patients with hypotrichosis; the LSS mutations of the patients were hypomorphic and led to decreased enzymatic activity in vivo, and analysis of skin pathology revealed acanthosis and hypoplastic hair follicles.

Fig 1

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a, Pedigree of the family. b, Photographs of patients II-2 and II-3. c-f, Histopathology of skin from patients II-2 and II-3. The epidermises exhibited psoriasiform acanthosis (arrow) and spongiosis (arrowhead). Scale bars = 600 μm (c, e), 60 μm (d, f). g, Immunoblot analysis of LSS expression in PBMCs from patients and controls. h, Overexpressed FLAG-LSS protein was detected with an anti-FLAG antibody. β-Actin was used as a loading control. Representative results from three independent experiments are shown. WT, wild type. i, j, LC-MS/MS analysis of (S)-2,3-epoxysqualene to lanosterol (i) and squalene to lanosterol (j) ratios in forehead sebum from the patients (n = 2), unaffected family members (n = 3), and controls (n = 37). P-values were calculated using Tukey-Kramer tests.

The second patient (patient II-3) was the third boy in the family (Fig 1b). He was delivered at 32 weeks of gestation to FGR. His birth weight was 1,180 g (-2.86 SD), and height was 37.0 cm (-2.36 SD). As with the first patient, this patient exhibited treatment-resistant hypotrichosis and had a cleft palate and umbilical herniation. MR images of his head demonstrated a normal corpus callosum (S3 Fig). At the most recent follow-up (16 years old), his height was 147.8 cm (-3.69 SD), and his body weight was 71.0 kg (1.09 SD), showing growth retardation similar to patient II-2.

Their motor and mental development of the patients are normal, and they do not present with cataracts. Histological analysis showed hypoplastic hair follicles, psoriasiform acanthosis, spongiosis, and hyperplastic sebaceous glands (Fig 1c–1f). The hair follicles were hypoplastic, similar to what is observed with lanugo. Ultrastructural examination by transmission electron microscopy (TEM) revealed that the number of hair matrix cells or melanin granules was decreased, and that the inner root sheath was thin (S4 Fig). The basement layer cells tightly adhered to each other, and the desmosomes developed normally.

LSS was identified as a single candidate gene by whole-exome sequencing

We performed family-based exome sequencing, for which we included the affected patients, an unaffected sibling and the unaffected parents. The mean depth of each exome was approximately 120. We identified only one candidate gene, LSS (NM_002340.5), for an autosomal recessive model in both patients (S1 Table). No rare de novo variants or rare X-linked variants shared by the patients were identified. Sanger sequencing validated compound heterozygous mutations in LSS, namely, c.[530G>A];[701_716del] (p.[Arg177Gln];[Arg234Profs*2]), both of which were absent or extremely rare in ExAC, gnomAD, and the Human Genetic Variation Database (HGVD). The missense variant, p.Arg177Gln, was also predicted to be deleterious or pathogenic in silico (combined annotation-dependent depletion score, SIFT, Polyphen-2, and MutationTaster) (S2 Table), and it affects evolutionarily conserved residues (S5 Fig).

In vitro and in vivo assays revealed that the LSS mutations were hypomorphic and led to decreased enzymatic activity

An immunoblot analysis showed decreased expression of LSS protein in peripheral blood mononuclear cells (PBMCs) in the patients compared to the controls (Fig 1g). The c.701_716del (p.Arg234Profs*2) transcript was detected at a much lower level than the wild-type allele in the Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCLs) from the patients. Suppression of nonsense-mediated mRNA decay (NMD) by cycloheximide treatment increased the levels of the frameshifted transcript, indicating that the frameshifted allele resulted in an unstable transcript that was subjected to NMD (S6 Fig). To test the stability of the mutant LSS proteins, we overexpressed wild-type or mutant FLAG-tagged LSS in HeLa cells. Exogenous expression of both LSS mutants was markedly reduced (Fig 1h). The result of in vitro LSS expression suggested that the mutant protein stability was affected, although caution must be taken because overexpression studies sometimes do not reflect the protein stability of the mutants under physiological conditions. Next, we measured the levels of intermediate metabolites of cholesterol synthesis in human specimens to show LSS enzymatic inhibition in vivo. In plasma, we detected only (S)-2,3-epoxysqualene but not lanosterol by LC-MS/MS analysis (S7 Fig). By contrast, in sebum from the skin, namely, the affected organ in the patients, we successfully detected squalene, (S)-2,3-epoxysqualene, and lanosterol by LC-MS/MS analysis. The ratios of (S)-2,3-epoxysqualene to lanosterol, that is, the ratios of the substrate to the product of LSS catalysis, in the sebum from the affected patients were significantly elevated compared to those of the unaffected family members and healthy volunteers (Fig 1i). The ratios of the squalene to lanosterol were not different between the patients and the unaffected family members (Fig 1j). Cholesterol could not be detected in the sebum of all of them, and the serum levels were not significantly different among the groups (S3 Table).

Lss epidermis-specific knockout mice were neonatal lethal because of skin barrier dysfunction that led to severe dehydration

To confirm that loss-of-function of the cutaneous LSS protein causes hypotrichosis, we created epidermis-specific Lss knockout mice; Lss constitutive knockout mice were previously reported to be embryonic lethal, as mentioned above. Lss flox/flox;Keratin14-Cre/+ (Lssf/f-K14) mice were obtained from breeding Lss wild/flox;K14-Cre/+ male mice and Lss flox/flox (Lssf/f) female mice (S8 Fig). The Lssf/f-K14 mice did not have macroscopic whiskers (Fig 2a). Genotype distribution of the Lssf/f-K14 mice and the litters at 18.5 days post coitum were observed to conform to a Mendelian distribution (based on 51 litters, S4 Table). Quantitative PCR (qPCR) analysis showed that Lss mRNA levels in the Lssf/f-K14 mice were significantly lower than those in the Lssf/f littermates in the epidermis but not significantly different in the liver (Fig 2b). Lipid measurements showed that (S)-2,3-epoxysqualene was present in the skin epidermis of the Lssf/f-K14 mice but not in the skin of the Lssf/f littermates (Fig 2c). Lanosterol and squalene were undetectable in the epidermis of both the Lssf/f-K14 and Lssf/f mice. Cholesterol was not significantly different between the epidermis of Lssf/f-K14 and Lssf/f mice (S9 Fig).

Fig 2. Epidermis-specific Lss knockout neonatal mice exhibited no vibrissae and died of severe dehydration due to skin barrier dysfunction.

Fig 2

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a, Facial appearance of a neonatal Lssf/f or Lssf/f-K14 mouse. The magnified images show the presence of whiskers in an Lssf/f mouse but not in an Lssf/f-K14 mouse. b, Quantitative PCR analysis of Lss in the epidermis (left) and liver (right) of newborn Lssf/f-K14 or Lssf/f mice. Relative amounts of mRNA were determined using the comparative Ct method. Each bar represents the mean ± SEM (n = 6 per group). P-values were calculated using Student’s t-tests. NS, not significant. c, Lipid analysis of newborn Lssf/f-K14 (n = 7) or Lssf/f (n = 8) skin for (S)-2,3-epoxysqualene. All bars represent the means. P-values were calculated using Student’s t-tests. d, Percent body weight based on the birth weights over time. The body weight loss of Lssf/f -K14 mice (n = 5), which was over 10% until 6 hours after birth, was significantly higher than the loss observed in Lssf/f (n = 11) or Lsswild/f-K14 (n = 9) mice. No significant reduction in weight loss between Lssf/f (n = 11) and Lsswild/f-K14 (n = 9) mice was observed. Each bar represents the mean ± SD. *P < 0.0001, P-values were calculated using Student’s t-tests. e, Dye penetration assay on a newborn Lssf/f-K14 or Lssf/f mouse. The experiments were repeated three times. f, Measurement of TEWL in newborn Lssf/f-K14 (n = 5) or Lssf/f (n = 7) mice. P-values were calculated using Student’s t-tests. g, TEM of the epidermis of newborn Lssf/f-K14 or Lssf/f mice. Desmosomes were hypoplastic in the Lssf/f-K14 mice compared to those observed in Lssf/f littermates. Scale bars = 100 nm. Data are from at least two independent experiments. P-values were calculated using Student’s t-tests.

Their hair growth could not be investigated because all of the Lssf/f-K14 mice died within a few hours after birth. The body weight loss exhibited by these mice was significantly greater than that of Lsswild/f-K14 and Lssf/f mice, and it was greater than 10% until 6 hours after birth (Fig 2d); thus, severe dehydration immediately after birth was suspected as the cause of death. A dye penetration assay using toluidine blue showed that the Lssf/f-K14 mice exhibited higher levels of staining than the Lssf/f littermates (Fig 2e), which indicated outside-in skin barrier dysfunction in the Lssf/f-K14 mice. Transepidermal water loss (TEWL) was significantly higher in the Lssf/f-K14 mice than it was in the Lssf/f littermates, which indicated inside-out skin barrier impairment in the Lssf/f-K14 mice (Fig 2f). A light microscopic study did not show apparent differences between the Lssf/f-K14 and Lssf/f mice (S10a Fig). Expression of the LSS protein was decreased in the epidermal basement membrane of the Lssf/f-K14 mice compared to what was observed in the Lssf/f littermates by immunohistochemistry (S10b Fig). The number of follicles and the thickness of the epidermis were not significantly different between the Lss/f-K14 and Lssf/f mice. TEM revealed hypomorphic changes in the desmosomes of the Lssf/f-K14 mice (Fig 2g).

Epidermal Lss ablation led to transient epilation in tamoxifen-inducible knockout mice

Next, we utilized a tamoxifen-inducible Cre/loxP system and generated Lss flox/flox;Keratin14-Cre-ERT/+ (Lssf/f-K14ERT) mice to avoid neonatal death. Epilation in the Lssf/f-K14ERT mice occurred over 3 weeks after intraperitoneal injections of tamoxifen. In the following 1 to 2 weeks, the hair loss area peaked (Fig 3a and 3b). The fur gradually regrew, but it was not completely restored and remained sparse (Fig 3b). The surfaces of the ears or tails were reddish and rough (Fig 3b). Tamoxifen-treated Lssf/f or untreated Lssf/f-K14ERT mice did not exhibit any phenotype (Fig 3a). The ratios of (S)-2,3-epoxysqualene to lanosterol were significantly higher in the sebum of tamoxifen-treated Lssf/f-K14ERT mice compared to the ratios in the tamoxifen-treated Lssf/f or untreated Lssf/f-K14ERT mice (Fig 3c). Histological analysis of the Lssf/f-K14ERT mice revealed hypoplastic hair follicles, hyperplastic sebaceous glands, and thin root sheaths, which was consistent with the pathological results obtained from the patients (Fig 3d). The epidermises exhibited hyperplasia, parakeratosis, hyperkeratosis and liquefaction degeneration. Immunohistochemical analysis showed that the LSS protein levels decreased in the sebaceous gland and epidermal basal layer cells after tamoxifen treatment (Fig 3e). In contrast, the keratinocytes in the surface layer exhibited positive immunostaining for LSS. As the proportion of immunostained cells in the sebaceous glands decreased, the proliferation of the nearby epidermal cells increased (S11 Fig).

Fig 3. Inducible epidermis-specific deletion of Lss caused alopecia that was spontaneously and partially reversed in the mice.

Fig 3

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a, Gross appearance of the Lssf/f-K14ERT or Lssf/f mice pre- and postadministration of intraperitoneal tamoxifen. Lssf/f-K14ERT mice 4 weeks after tamoxifen treatment, which affected baldness from the head to the back. The experiments were repeated at least three times. b, The peak of hair loss in Lssf/f-K14ERT mice was 5 weeks after the tamoxifen treatment (top). The hair had spontaneously regrown 7 weeks after the treatment (bottom). W, weeks. c, Skin sebum analysis of Lssf/f-K14ERT (n = 15) or Lssf/f (n = 13) mice pre- and postadministration of intraperitoneal tamoxifen or vehicle. At 6 weeks of age, (S)-2,3-epoxysqualene was not detected in either Lssf/f-K14ERT (n = 3) or Lssf/f (n = 3) mice. After 4 weeks of treatment, the ratios of (S)-2,3-epoxysqualene to lanosterol in the sebum of tamoxifen-treated Lssf/f-K14ERT mice (n = 6) were significantly elevated compared to those of vehicle-treated Lssf/f (n = 4), tamoxifen-treated Lssf/f (n = 6), or vehicle-treated Lssf/f-K14ERT (n = 6) mice. P-values were calculated using Tukey-Kramer tests. NS, not significant. d, H&E staining and e, LSS immunohistochemistry of skin from Lssf/f-K14ERT or Lssf/f mice 4 weeks after tamoxifen treatment. The epidermis of Lssf/f-K14ERT mice showed severe acanthosis compared to Lssf/f mice. LSS protein expression was decreased in the epidermal basement membrane (open arrowhead) and sebaceous cells (closed arrowhead) of the Lssf/f-K14 mice compared to the expression in Lssf/f mice. Scale bars = 100 nm.

In addition, we topically administered 4-hydroxytamoxifen, which is an activated form of tamoxifen in vivo, on the skin of Lssf/f-K14ERT mice or littermates. Consistent with the results of the intraperitoneal injections with tamoxifen, the 4-hydroxytamoxifen-treated areas exhibited transient epilation in Lssf/f-K14ERT mice (S12 Fig). Lssf/f (n = 2) and Lsswild/f-K14ERT (n = 2) mice, which were the littermates of the treated Lssf/f-K14ERT mice, did not show hair loss following topical administrations of 4-hydroxytamoxifen.

Lens-specific Lss knockout mice had congenital cataracts

We created Lss flox/flox;Pax6-Cre/+ (Lssf/f-Pax6) mice in which the Pax6 promoter was selected to create lens-specific knockout model mice [11,12]. The Lssf/f-Pax6 mice exhibited microphthalmia and small cloudy lenses that were not observed in the controls (Fig 4a–4d). Pathological analysis of the Lssf/f-Pax6 mice indicated lens swelling, degeneration, and liquefaction (Fig 4e–4h), which implied cataract changes. Immunohistochemical analysis demonstrated that the LSS protein levels were lower in the Lssf/f-Pax6 mice than they were in the Lssf/f mice (Fig 4i–4l). The inner retina, which is the inner nuclear layer and ganglion cell layer, was thinner in Lssf/f-Pax6 mice than it was in Lssf/f mice (S13 Fig). In particular, there were fewer retinal ganglion cells in Lssf/f-Pax6 mice than there were in Lssf/f mice. (S)-2,3-epoxysqualene, lanosterol, squalene, and cholesterol were not detected in the lenses of Lssf/f or Lssf/f-Pax6 mice.

Fig 4. Lens-specific deletion of Lss caused cataracts in mice.

Fig 4

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a-d, Images of Lssf/f-PAX6 or Lssf/f mice (14 weeks of age) and their lenses. e-l, H&E staining and immunohistochemistry of the lenses of Lssf/f-PAX6 or Lssf/f mice. These experiments were repeated at least three times. Scale bars = 500 μm (b), 50 μm (c).

Discussion

Here, we describe two sibling patients with biallelic LSS loss-of-function mutations that led to compromised LSS protein expression and an increased ratio of substrate to product in the patients’ sebum, indicating LSS enzymatic inhibition in vivo. The LSS enzyme disruption was also reproducible in the skin sebum of inducible epidermis-specific Lss knockout mice. Previous studies could not demonstrate LSS dysfunction by analysis of blood samples [7,8]. Measurement of cholesterol intermediates from skin, an affected organ, but not blood, allowed us to validate the LSS enzymatic block. The ratio of (S)-2,3-epoxysqualene to lanosterol in the sebum would be a good biomarker for the diagnosis of LSS deficiency. Hypotrichosis and cataracts were reproduced by creating tissue-specific Lss knockout mice. Our results confirmed that hypotrichosis and cataracts, the core symptoms in patients with biallelic LSS mutations, are caused by LSS enzymatic deficiency in each tissue, not by circulating abnormal metabolites in the blood.

We recapitulated hypotrichosis and cataracts by creating tissue-specific Lss knockout mice, and these outcomes are caused by knocking out Lss in the respective organs. Both of these symptoms are also associated with other inherited disorders of cholesterol biosynthesis. Hypotrichosis is observed in other conditions associated with deficiencies of cholesterol synthesis enzymes, including Greenberg dysplasia, SC4MOL deficiency, X-linked dominant chondrodysplasia punctata-2 (CDPX2), and congenital hemidysplasia with ichthyosiform erythroderma and limb defects (CHILD) syndrome [13–17]. Cataracts are also known to develop in patients with deficiencies of cholesterol synthesis enzymes, such as SC4MOL deficiency, CDPX2, lathosterolosis and Smith-Lemli-Opitz syndrome (SLOS) [16,18–20]. However, given that most mice that have had cholesterol synthesis genes knocked out are embryonic lethal [21], conventional knockout mouse studies are not feasible for recapitulation of the symptoms in patients with these deficiencies. Moreover, it is not understood whether symptoms of patients with abnormal cholesterol metabolism are local events or are secondary effects caused by enzyme deficits. Our study validated that local abnormalities in cholesterol metabolism can cause symptoms in each tissue using conditional knockout mouse models; the results are similar to those from a previous mouse study of Insig-1/2, which is involved in a feedback system of cholesterol synthesis [22].

Tamoxifen-induced Lss knockout in the mouse epidermis led to epilation. The hyperkeratosis observed in the epidermis was consistent with the ichthyosis observed in the patient during the neonatal period (S2 Fig) and with the histological analysis of the patient’s skin (Fig 1c and 1d). Recent studies also demonstrated similar dermatological findings, including ichthyosis, in other patients with biallelic LSS mutations [8]. As with hypotrichosis, hyperkeratosis also occurs in other disorders of cholesterol biosynthesis [13–17]. Interestingly, the epilation of the tamoxifen-treated mice was transient. A possible explanation for the hair regrowth is that the surrounding cells might supply LSS-deficient cells with deficient products or consume excess substrates. Consistent with this hypothesis, a severe phenotype was observed in parts of the body with few subcutaneous tissues, such as the ears (Fig 3b) and tail. Contrary to the tamoxifen-inducible system, the phenotypes of LSS deficiency were not fully reproduced in the Lssf/f-K14 mice due to neonatal lethality. The mice failed to develop normal skin, which especially affected the skin barrier function. The failure to develop a skin barrier is consistent with the mouse knockout of DHCR24, which catalyzes cholesterol intermediates [23]. Of note, in our experiments, only one mouse grew to the adult stage (S14 Fig). The mouse grew normally and could not be differentiated from the other littermates until weaning but gradually exhibited baldness and affected skin scales after approximately 6 weeks of age. Unlike the tamoxifen-treated Lssf/f-K14ERT mice, alopecia was not recovered in this mouse until the mouse died of unknown causes at 10 weeks of age. Considering that the hair follicles of epidermis-specific knockout mice developed normally (S11 Fig), cell-autonomous cholesterol biosynthesis by LSS enzyme might be crucial not for morphogenesis and primary hair development but for secondary hair germ in hair development. By preventing water evaporation from the skin, Lssf/f-K14 mice might avoid neonatal lethality. If the Lssf/f-K14 mice could survive and reach adulthood, these mice would be excellent animal models for clarifying the pathophysiology related to disrupted Lss and investigating treatments.

The pathogenesis of LSS deficiency is unknown, but the mechanism is likely attributed to aberrant SHH or WNT signaling. Although the association of LSS mutations and holoprosencephaly was not proven by pedigree analysis in a previous study [24], some patients with LSS mutations, including our patients, have midline anomalies or growth failure [8], which are observed in diseases of cholesterol metabolism or SHH signaling [25,26]. SLOS, which impairs cholesterol synthesis due to DHCR7 deficiency and often produces similar symptoms, has been proposed to be involved in the SHH pathway [20,27]. WNT signaling, which interacts with SHH-related molecules during embryogenesis in several tissues, is also required for hair follicle generation [28]. Thus, we surveyed whether LSS deficiency caused Shh or Wnt dysregulation. However, there were no significant differences in Shh or Wnt transcriptional responses against the exogenous Shh or Wnt protein between Lss-depleted and nondepleted MEFs (S15 Fig). These results might imply that the underlying mechanism is not simply caused by the upregulation or downregulation of these signaling pathways because spatial and gradient SHH or WNT signaling seems to be key in skin formation [29]. Moreover, the reason why each patient with biallelic LSS mutations shows different combinations of phenotypes, such as hypotrichosis and/or cataracts, is also unknown [5–8]. Further experimental studies are required to understand the pathogenesis and mechanism of the phenotypic variation in LSS deficiency.

In conclusion, our study verified that biallelic LSS mutations caused LSS enzymatic dysfunction in skin through analysis of sebum. Our mouse studies underscored that hypotrichosis and cataracts are caused by LSS enzymatic dysfunctions in each tissue. Local treatment in LSS deficiency might alleviate symptoms; patients with CHILD syndrome, a deficiency of downstream intermediates of LSS, was successfully treated by topical application of statin or cholesterol plus statin [15,30]. The mouse model of LSS deficiency might serve as a pathophysiological research model and aid in the development of novel treatments.

Materials and methods

Sequencing analysis

Whole-exome sequencing was performed as described elsewhere [31] but with minor modifications. Briefly, genomic DNA was purified using a SureSelectXT Human All Exon V5 (50 Mb) Kit (Agilent Technologies, Santa Clara, CA, USA) and was sequenced on a HiSeq2000 (Illumina, San Diego, CA, USA) with 101-base-pair paired-end reads. The reads were mapped to the human genome build UCSC hg19 using Novoalign v. 2.08.05 (http://www.novocraft.com), and single-nucleotide variants (SNVs) and insertions and/or deletions (indels) were identified using the Genome Analysis Toolkit (GATK) v. 1.6–13. After quality filtering, variants were annotated using ANNOVAR. Nonsynonymous SNVs, splice-site variants and indels were retained. We excluded variants present at <1% minor allele frequency (MAF) in the 1000 Genomes database, the ExAC database version 0.3 (all and East Asian populations) and the HGVD. Sanger sequencing was performed to validate the variants identified by whole-exome sequencing.

Plasmids

Wild-type, R234fs, and R177Q human LSS cDNAs (NM_002340.5, carrying SNP rs2254422 and rs2254524) were prepared by RT-PCR with RNA from the blood of the patients and their family members. cDNAs were subcloned into pCMV6-AN-DDK (PS100014, OriGene, Rockville, MD, USA). The final constructs were verified by sequencing.

Cell culture and transfection

HeLa cells (RCB0007, provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing low levels of glucose, which was supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin (100 U/ml and 100 μg/ml, respectively); cells were maintained at 37°C in a 5% CO2 incubator. The pCMV6-FLAG-LSS (wild-type, R234fs, and R177Q) plasmid was transfected into HeLa cells using Lipofectamine LTX (15338100, Thermo Fisher Scientific Inc., MA, USA). Twenty-four hours after transfection, cell lysates were harvested and subjected to immunoblot analysis.

Western blot analysis

Proteins extracted from PBMCs or HeLa cells expressing FLAG-LSS were subjected to immunoblot analysis. Mouse anti-FLAG (F3165, Sigma-Aldrich, Merck Millipore), rabbit anti-LSS (13715-1-AP, Proteintech, Rosemont, IL, USA), HRP-conjugated anti-mouse IgG (NA9310V, GE Healthcare UK Ltd., Buckinghamshire, England), HRP-conjugated anti-rabbit IgG (NA9340V, GE Healthcare UK Ltd.), and peroxidase-conjugated anti-β-actin (017–24573, Wako, Osaka, Japan) antibodies were used.

Sample collection and preparation for LC-MS/MS analysis

Human sebum was collected using acetone-soaked cotton pads as described previously [32], and it was extracted with acetone. Mouse sebum was collected similarly and was syringe filtered (GL Chromatodisc 25N, GL Sciences, Tokyo, Japan). Mouse skin was sampled as described previously [33]. Mouse skin homogenates were prepared using 0.9% KCl containing 1 mM ethylenediaminetetraacetic acid. Total lipids from the above samples and human serum were extracted by the Folch method as described previously [34,35] but with modifications. All samples were dissolved in methyl tert-butyl ether (MTBE)/methanol (2:1) prior to analysis.

LC-MS/MS analysis

Samples were analyzed with a QTRAP 4000 mass spectrometer equipped with an Exion HPLC system (SCIEX, Tokyo, Japan). Samples were eluted from a C30 column (YMC Carotenoid 250×4.6 mm, YMC, Kyoto, Japan) at a flow rate of 1.0 mL/min with a binary gradient consisting of solvent A (methanol/MTBE/water, 83:15:2) and solvent B (methanol/MTBE/water, 8:90:2). The gradient profile was as follows: 0–10 min, 0–50% B, linear; 10–20 min, 100% B; 20–25 min, 0% B. The column temperature was maintained at 40°C. Compounds were detected with atmospheric-pressure chemical ionization in positive ion mode, and they were identified through comparison of peaks between the samples and commercial standards. Compounds were quantified with external calibration curves using the corresponding standards. Further MS conditions are described in S5 Table.

Mice

Cryopreserved sperm of Lss tm1a knockout mice were purchased from the European Mouse Mutant Archive. Heterozygous Lss tm1a mice were generated at the Institute for Animal Experimentation, Tohoku University Graduate School of Medicine, and then they were crossed with Flpe mice (RBRC01834, RIKEN BRC, Japan) [36] to obtain Lss wild/flox mice that had a conditional allele (S8 Fig). Lss wild/flox mice were bred with K14-Cre transgenic mice (stock no. 004782; The Jackson Laboratory, USA) to generate epidermis-specific knockout mice. Lssf/f-K14ERT or Lssf/f-Pax6 mice were similarly created using K14-CreERT transgenic mice (stock no. 005107; The Jackson Laboratory, USA) or PAX6-Cre transgenic mice (stock no. 024688; The Jackson Laboratory, USA), respectively. All mice in these studies were on a C57BL/6 background except the K14-CreERT mice, which were a strain of ICR mice backcrossed with C57BL/6 mice for at least 5 generations. Day 0.5 of pregnancy (E0.5) was defined as the morning on the day after overnight mating. Mouse tail genomic DNA was extracted with a Maxwell 16 Mouse Tail DNA Purification Kit (Promega, USA). Mouse genotypes were determined by probe-based PCR analysis. The PCR primers (Nihon Gene Research Laboratories, Miyagi, Japan) and probes (Integrated DNA Technologies, Coralville, IA, USA) are described in S6 Table. Tamoxifen (T5648, Sigma-Aldrich, USA) was dissolved in corn oil, and 1 mg was injected intraperitoneally daily for 5 consecutive days. 4-Hydroxytamoxifen (H954725, Toronto Research Chemicals, Toronto, Canada) was dissolved in 100% acetone at 4 mg/dl, and 0.4 mg was topically applied daily for 5 consecutive days.

qPCR analysis

The mouse epidermises were isolated after allowing the skin to float on trypsin for 24 hours. Epidermis and liver were homogenized using a pellet pestle cordless motor (Kimble, Vineland, NJ, USA). RNA was extracted with RNeasy (QIAGEN, Hilden, Germany), and reverse transcription was performed with a PrimeScript RT Reagent Kit with gDNA Eraser (TakaraBio, Shiga, Japan). cDNA was amplified with TaKaRa Ex Taq (RR001A, TaKaRa Bio Inc., Otsu, Japan) and PrimeTime qPCR probe assays (Integrated DNA Technologies, Coralville, IA, USA). qPCR primers and probes for Lss (Mm.PT.58.41802559) and Gapdh (Mm.PT.39a.1) were used. qPCR reactions were performed using StepOnePlus (Sigma-Aldrich, St. Louis, MO, USA) with standard conditions.

Histology and immunohistochemistry

Skin samples were fixed in formalin, embedded, and cut into 5-μm sections. HE staining was performed using standard procedures. For immunostaining, slides were incubated with an anti-LSS antibody according to the manufacturer’s instructions (13715-1-AP, Proteintech, Rosemont, IL, USA). Images were captured with a BIOREVO BZ-9000 microscope (Keyence, Osaka, Japan).

Electron microscopy

Mouse skin samples were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer overnight at 4°C. After being cut into small pieces, the specimen was postfixed in 1% osmium tetroxide for 90 min on ice, rinsed in distilled water, successively dehydrated in 50, 60, 70, 80, 90, and 95% ethanol for 10 min each (except for 70% ethanol, which was used for 25 min). Then, specimens were incubated in for 20 minutes 100% ethanol (3 times), treated with propylene oxide for 10 min (2 times), and finally, embedded in epoxy resin. Ultrathin (80 nm) sections were prepared on an ultramicrotome (EM UC-7, Leica, Heerbrugg, Switzerland) with a diamond knife, and then slides were stained with 2% uranyl acetate and 1% lead citrate and were viewed using an electron microscope (H-7600, Hitachi, Tokyo, Japan).

Skin barrier function assays

A dye penetration assay was performed as described previously [37]. Briefly, newborn mice were rinsed in PBS and successively dehydrated in 25, 50, 75, and 100% methanol for 1 min each. Mice were then rehydrated with PBS, stained with 0.05% toluidine blue in PBS for 10 min, destained with PBS for 20 min, and photographed. All steps were performed at room temperature. TEWL was recorded using AquaFlux (Biox, London, UK). Measurements were performed after calibrating the device at room temperature.

Statistical analysis

Statistical analysis was performed with JMP Pro 13.0. P-values <0.05 were considered statistically significant.

Ethics statement

Approval for human subject research was obtained from the ethics committee of Tohoku University Graduate School of Medicine (approval number: 2015-1-694). Written informed consent was obtained from all participants in the study. Approval for animal experiments was provided by the Animal Care Facility of the Tohoku University Graduate School of Medicine (approval number: 2016MdA-371, 2016MdA-372).

Supporting information

S1 Fig. The metabolic pathway of cholesterol biosynthesis.

Cholesterol is synthesized from acetyl CoA via the Bloch or Kandutsch-Russell pathway. In this synthesis pathway, LSS converts (S)-2,3-epoxysqualene to lanosterol. The abbreviations are as follows: FDFT1, farnesyldiphosphate farnesyltransferase 1; ff-MAS, follicular fluid meiosis-activating sterol; t-MAS, testis-meiosis activating sterol; LBR, lamin B receptor; SC4MOL, sterol C4-methyloxidase-like; NSDHL, NAD(P)H steroid dehydrogenase-like protein; HSD17B7, 17-beta-hydroxysteroid dehydrogenase; EBP, emopamil-binding protein; SC5DL, sterol C5-desaturase; DHCR7, 7-dehydrocholesterol reductase; and DHCR24, 24-dehydrocholesterol reductase.

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S2 Fig. Patient II-2 at 1 month of age.

The patient exhibited ichthyosis-like scales on the skin of his chest, abdomen and upper extremities but not on his face and head.

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S3 Fig. Sagittal T1-weighted head MRI images of patients II-2 and II-3.

Sagittal T1-weighted head MRI shows partial agenesis of the corpus callosum (from the posterior midbody to splenium) in patient II-2, while the MRI shows a normal corpus callosum in patient II-3.

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S4 Fig. Transmission electron microscopy images of the skin of patient II-3.

(a) Hair matrix of the patient. The number of melanin granules was small. The inner root sheath was thin. (b) The basement layer cells were tightly adhered to each other. (c) The desmosome was well developed. Scale bars = 200 μm (a), 50 μm (b), and 2.5 μm (c).

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S5 Fig. Conservation of a missense variant in LSS.

The R177Q residue is conserved in organisms from humans to zebrafish. LSS homologs were aligned with Clustal Omega.

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S6 Fig. The frameshift variant c.701_716del in LSS leads to NMD.

EBV-LCLs from patient II-2 and II-3 were treated with 100 μg/mL cycloheximide (037–20991, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 4 hours to suppress NMD. Total RNA was extracted with an RNeasy Kit (Qiagen Inc., Valencia, CA, USA). RT-PCR was performed using a PrimeScript™ II High Fidelity RT-PCR Kit (TaKaRa, Shiga, Japan) to amplify the cDNA surrounding the c.701_716del mutation, which was followed by Sanger sequencing.

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S7 Fig. Mass spectra was collected by LC-MS/MS for the plasma from the patients or family members.

Blue arrows indicate peaks of 2,3-oxidosqualene. Lanosterol could not be detected by this analysis.

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S8 Fig. Diagrams of the knockout allele.

The tm1a allele was a knockout first allele, and it was converted to a conditional allele, tm1c (flox), by FLP-mediated recombination. The tm1c (flox) allele was converted to a deletion allele by Cre-mediated recombination.

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S9 Fig. Cholesterol quantification in newborn Lssf/f-K14 or Lssf/f skin.

Mouse skin was gravimetrically weighed, and homogenates were prepared using 0.9% KCl containing 1 mM ethylenediaminetetraacetic acid. Total lipids were extracted from the homogenates using the Folch method [34, 35], and the extract was dissolved in 100 μL of 2-propanol. Human sebum was collected using acetone-soaked cotton pads [32], and it was dissolved in 200 μL of 2-propanol. The total cholesterol content in each sample was measured enzymatically with a commercial kit (Cholesterol E; Wako Pure Chemical Industries, Ltd., Osaka, Japan) according to the manufacturer’s protocol. Each bar represents the mean ± SEM (n = 5 per group). P-values were calculated using Student’s t-tests. NS, not significant.

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S10 Fig. Light microscopic images of Lssf/f-K14 and Lssf/f mouse skin.

(a) Histological analysis did not show macroscopic differences in epidermal structure between the Lssf/f-K14 and Lssf/f mice. (b) Immunohistochemistry showed that expression of the LSS protein was decreased in the epidermal basement membrane (open arrowhead) and hair follicles (closed arrowhead) of the Lssf/f-K14 mice compared to the Lssf/f littermates. Scale bars = 100 μm.

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S11 Fig. Immunohistochemical analysis of skin from Lssf/f-K14ERT mice with alopecia.

Keratosis is mild in epidermal areas near immunostained sebaceous cells (a, b). In contrast, severe keratosis is found in epidermal areas lacking immunostained sebaceous cells (c). Scale bars = 100 μm.

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S12 Fig. Topical tamoxifen-treated Lssf/f-K14ERT mice.

Images of Lssf/f-K14ERT mice arranged chronologically after topical application of 4-hydroxytamoxifen, which was applied within the black rectangle. Depilation gradually occurred after 3 weeks of 4-hydroxytamoxifen treatment. The depilation area was greatest after 4 weeks. Similar to the effects of intraperitoneal injection of tamoxifen, the hair gradually regrew. Gray hairs were mixed among the hairs in the recovered area.

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S13 Fig. Microscopic analysis of the retina in Lssf/f-Pax6 and Lssf/f mice.

The inner retina, which is the inner nuclear layer and ganglion cell layer, was thin in Lssf/f-Pax6 mice compared to Lssf/f mice. There were fewer retinal ganglion cells in Lssf/f-Pax6 mice than there were in Lssf/f mice. In contrast, there was no obvious difference in the thickness of the outer nuclear layers between Lssf/f-Pax6 and Lssf/f mice. These pictures were captured using an Olympus BX53 microscope (Olympus, Tokyo, Japan). A 10x objective lens was used. Scale bars = 50 μm.

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S14 Fig. The surviving Lssf/f-K14 mouse.

Sequential images of an adult Lssf/f-K14 mouse (a-d). At weaning, the mouse could not be differentiated from the other littermates. The mouse gradually lost hair after 6 weeks of age (a). Desquamation started at 9 weeks of age (c). A partial skin defect on the head with oozing was observed. The mouse died due to unknown causes at 10 weeks of age (d). Analysis of histological specimens from the dead mouse revealed severe hyperkeratosis, dyskeratosis, and hypertrophic sebaceous cells (e). Scale bars = 100 μm.

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S15 Fig. Transcriptional responses against the exogenous Shh or Wnt protein between Lss-depleted and nondepleted MEFs.

We measured the transcriptional responses of MEFs against the exogenous Shh or Wnt protein as previously described [27, 38] with minor modifications. Embryos (E13.5–14.5) were harvested by aseptic techniques from pregnant Lssf/f mice that had been mated with Lssf/f male mice. The heads, extremities, and tails were cut off, and the livers were removed. The remaining body parts were minced by scissors and transferred to a 50 mL tube with PBS. The tube was centrifuged at 200 g for 5 min. The supernatant was aspirated, and the pellet was resuspended with 1 mL 0.25% trypsin-EDTA per embryo. The suspension was incubated for 15 min in a 37°C water bath with shaking. Fetal bovine serum was added (the same volume as trypsin) to the suspension. After letting the suspension sit for several minutes and allowing the embryo fragments sank to the bottom of the tube, the serous part was removed first and filtered, and then the remaining thick part was passed through a 100 μm filter. The filtered solution was centrifuged at 200 g for 5 min. The supernatant was aspirated, the pellet was resuspended in DMEM, and the suspension was transferred to a dish. MEFs were plated at 5 × 104 cells in 250 μL of DMEM per 48-well plate and transduced with AAV1 or a mock control (105548-AAV1 or 105537-AAV1, respectively, Addgene, Cambridge, MA, USA) at a multiplicity of infection of 5 ×105. The next day, 250 μL of DMEM was added to each well. The following day, 2 μg/mL Shh (464-SH-025/CF, R&D systems, Minneapolis, MN, USA) or 100 ng/mL Wnt‐3a (1324-WN, R&D systems, Minneapolis, MN, USA) were added to the medium. Three days after transduction, total RNA was extracted with RNeasy (QIAGEN, Hilden, Germany), and reverse transcription was performed with a PrimeScript RT Reagent Kit with gDNA Eraser (TakaraBio, Shiga, Japan). cDNA was amplified with TaKaRa Ex Taq (RR001A, TaKaRa Bio Inc., Otsu, Japan) and PrimeTime qPCR probe assays (Integrated DNA Technologies, Coralville, IA, USA). qPCR primers and probes for Gli1 (Mm.PT.58.11933824), Axin2 (Mm.PT.58.8726473), Lss (Mm.PT.58.41802559), and Gapdh (Mm.PT.39a.1) were used. qPCR reactions were cycled using StepOnePlus (Sigma-Aldrich, St. Louis, MO, USA) with standard conditions.

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S1 Table. Variant filtering strategy.

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S2 Table. Mutations in LSS.

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S3 Table. Laboratory results of the patients with LSS biallelic mutations and unaffected family members in this study.

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S4 Table. Genotype distribution of neonatal mice born from Lsswild/flox-Keratin14 male and Lssflox/flox female mice.

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S5 Table. MS parameters used for LC-MS/MS analysis.

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S6 Table. Primers and probes.

The forward primers of Lss tm1a, Lss tm1c, and Lss tm1d were the same. The existence of the Pax6-Cre allele was confirmed using generic Cre primers and a probe.

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Acknowledgments

The authors would like to thank the patients, families and healthy volunteers who participated in this study. We thank Yoko Chiba, Kumi Ito, Miyuki Tsuda, Mami Kikuchi, Makiko Nakagawa, Yoko Tateda, and Kiyotaka Kuroda for providing technical assistance.

We also acknowledge the technical assistance of the Biomedical Research Core of the Tohoku University Graduate School of Medicine and the Biomedical Research Unit of Tohoku University Hospital. We deeply appreciate Keisuke Nishio and Prof. Ichiro Miyoshi for recovering cryopreserved mouse sperm. We thank Naomi Sato for expert pathology-related advice.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This research was supported by the Japan Agency for Medical Research and Development (AMED) through grant number JP17ek0109151 (Initiative on Rare and Undiagnosed Diseases [IRUD] to S.K.) and JSPS KAKENHI grant number JP17K10044 (to At.K. and K.N.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Gregory S Barsh, Luke Engelking

23 Sep 2019

Dear Dr Kikuchi,

Thank you very much for submitting your Research Article entitled 'Metabolic and pathologic profiles of human LSS deficiency recapitulated in mice' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review again a much-revised version. We cannot, of course, promise publication at that time.

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Gregory Barsh

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All three reviewers were enthusiastic about the major findings of this manuscript. However, a number of issues were identified that will require revision and/or clarification.  These issues will need to be addressed in a point-by-point rebuttal.  Lastly, the following issues in particular may require additional experimental data:

- Photomicrographs of the hair and ocular structures in addition to the lens

- Measurements of sterol intermediates

- Gene and or protein expression data exploring which signaling pathways, such as Wnt or Hh-related pathways, are impacted by LSS deficiency

- Inclusion of the suggested controls for the transfection experiments in hela cells

- Methods used for lanosterol and 2,3-epoxysqualene quantification

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors submit an interesting study whereby they describe two new patients with novel biallelic mutations in lanosterol synthetase (LSS) who exhibit hair and midline structural abnormalities and a concomitant increase in the substrate:product (S-2,3-epoxysqualene:lanosterol) ratio secondary to an enzymatic block. Additionally, the authors generate tissue-specific LSS-deficient mouse lines with hair/skin and lens abnormalities; the skin from these tissue-specific LSS-deficient mice demonstrate increased S-2,3-epoxysqualene:lanosterol ratios which may alter normal skin homeostasis and hair follicle cycling. The authors provide extensive data throughout the manuscript; however, there are some concerns:

1) In Figure 1g, the authors illustrate the S-2,3-epoxysqualene:lanosterol ratio from the sebum of the affected patients, their unaffected family members, and controls. Given that cholesterol is essential in the formation of an intact skin barrier, it would be beneficial to show the values of all sterol intermediates and cholesterol in the sebum in these groups since one can speculate that these skin and hair abnormalities may be due to either a deficiency of cholesterol, a buildup of toxic intermediates, or both.

2) In addition to the high magnification photomicrograph in Figure 1e, a lower magnification photomicrograph of the hair follicles is needed to assess the extent of hair follicle pathology.

3) As stated above in Concern 1, values for the remainder of the sterol intermediates and cholesterol would be useful in explaining the pathophysiology seen in the LSSf/f-K14 mice illustrated in Figure 2.

4) Have the authors considered treating the 4-week-old tamoxifen-treated LSSf/f-K14ERT mice that show hair loss and skin abnormalities with statins and/or cholesterol + statins? As the authors suggest in the Discussion, this therapy may reduce these intermediates and reverse the pathology seen in these mice, particularly since the hair follicle cycles continuously throughout the majority of a mammal’s lifetime.

5) Figure 4 depicts the lens pathology seen in mice with LSS deficiency restricted to the lens. From the photomicrographs shown in Figure 4b, there appears to be pathology within other ocular/intraocular structures, such as within the cornea and retina, than in just the lens. Given Pax6-Cre is expressed during early eye development, loss of LSS within other ocular/intraocular tissues may not be surprising. It would be interesting to determine if the LSSf/f-Pax6 mice have retinal or other ocular/intraocular abnormalities besides those of the lens. Lastly, the authors do not state the age of these mice and do not show the LC-MS/MS data of lens or other ocular structures.

6) Additional points include:

-Line 135: Is the variant supposed to be p.Arg177Gln instead of p.Arg177Glu?

-Line 226: Should it be “increased ratio of substrate to product” instead of decreased?

-The flow of the overall manuscript is rather choppy at times and could use proofreading by a native English speaker for better flow and readability.

Overall, the manuscript is interesting and not only provides more clarification of the tissue-specific pathophysiology seen in these malformation syndromes but also hints at diagnostic assays/therapeutic interventions for LSS deficiency.

Reviewer #2: In the manuscript entitled "Metabolic and pathologic profiles of human LSS deficiency recapitulated in mice ", the authors demonstrate disruption of lanosterol synthetase in an inducible mouse model of LSS deficiency results in biochemical deficits and tissue specific changes present within affected human subjects. The work presented within the manuscript is important to the fields of cholesterol biosynthesis defects, rare disease research, and epidermal function. While the authors nicely demonstrate the identification of defined mutations within a small patient cohort of LSS deficiency and recapitulate some of the tissue deficits within a newly derived mouse model, the data presented are not likely of broad enough scientific significance for acceptance in PLOS Genetics. A significant advancement forward for LSS deficiency, such as identification of causative pathways downstream of LSS mutation or amelioration of pathological findings, would greatly enhance the significance of the manuscript.

Concerns related to the manuscript are detailed below.

1. While the methodology utilized within the manuscript is solid, the data presented, including generation of an inducible Lss mouse model, is largely descriptive. While the inducible Lss mouse is an exciting development for elucidating the pathological mechanisms subsequent to Lss disruption, the addition of mechanistic data is really needed. Are there specific signaling pathways subsequent to Lss deletion that are pathogenic?

2. It’s not clear the significance of the experiments detailing FLAG-tagged LSS mutations and changes in protein expression of specific mutants, particularly in HeLa cells that are demonstrated to already express LSS protein. How a transient transfection of a plasmid containing a mutant cDNA for LSS leads to decreased expression of native LSS is confusing. Further, why specific mutations affect this versus others is similarly confusing. Controls for this experiment for expression levels of the plasmids in an LSS null cell line would be critical.

3. The clinical findings from the two subjects identified are similar but not identical to the K14 mouse model used. The presence of cleft palate, midline anomalies, and corpus collosum agenesis in particular would not be affected by K14-Cre mediated deletion of Lss. Are these deficits present in the newly created Lss mutant mouse if a constitutive or embryonic Cre-driver used?

4. The quantitation and detection of sterols is not clearly defined. I believe there are typos within the methods, including what temperature was utilized for compound ionization. Further, how the compounds of interest were identified (i.e. through comparison to a known standard?) was performed. The number of samples analyzed as biological vs. technical replicates is also not clear. Further, the explanation regarding detection of lanosterol and 2,3-epoxysqualene in plasma vs. sebum of patients is unclear. It’s not clear why lanosterol would be detected.

5. For the early lethality of the Lss mouse presumably associated with dehydration, it would be of interest to know if lethality of the mouse could be prevented by administration of fluids. Further, if this is the presumed cause of death and the identified patients clearly have epidermal loss of Lss, it’s unclear why their lifespans are extended in comparison to the mouse.

Reviewer #3: This manuscript by Wada et al. is a carefully crafted insight into LSS deficiency. The authors identified two sibling patients with hypotrichosis and other abnormalities, and performed exome sequencing leading to the identification of LSS mutations as the likely cause. Consistent with this, they found an increase in the substrate of LSS ((S)-2,2-epoxysqualene) in sebum from the patients – previous efforts had not been able to confirm a lack of LSS activity. They then made skin and lens-specific Lss-deficient mice to show that LSS deficiency results in similar skin problems and cataracts, as indicated in humans. This work is the first to confirm that a lack of LSS activity in the affected tissues leads to the specific phenotypes observed in patients with LSS deficiency. This is significant and has important implications for the treatment of this disease, as well as broader implications for other cholesterol synthesis enzyme defects associated with diseases. I have only minor comments, which I list below.

Minor comments:

1. Page 5, line 64: please rephrase “Cholesterol is an essential nutrient” as this is not strictly correct.

2. Supp Fig 1: Only LBR is listed – please also include DHCR14 (these enzymes catalyse the same step). Zymosterol is listed twice – it should be zymostenol in the Kandutsch-Russell pathway. Dehydrodesmosterol should be 7-dehydrodesmosterol. Dihydrolanosterol is, I think, more commonly referred to as 24,25-dihydrolanosterol.

3. Page 5, line 73-74: The sentence about the 24 patients is confusing – suggest rephrasing to “Out of 24 patients, all had either cataracts (5/24) or hypotrichosis (20/24), and one had both”.

4. Page 5, line 86: Should this be a citation to the publication by Dickinson et al 2016 Nature as indicated on the website?

5. Page 9, line 151-152: The sentence about W581R and G588S is confusing – consider revising or removing.

6. Fig 2c: The authors note that lanosterol was undetectable in the skin of these mice, but in humans, and the tamoxifen-inducible knockout mice, they were able to detect lanosterol and present a ratio. Could the authors speculate on why lanosterol was not detectable? Further, was there any difference in the levels of cholesterol itself?

7. Fig 2b, S9 Fig: Can these be presented with individual data points instead, as in other graphs?

8. Fig 2d: Was the decrease in body weight of Lssf/f-K14 mice significantly different from the others? This should be indicated in the text/figure.

9. The figure legends include some results which may be better placed in the main text – please consider revising.

10. Fig 4c: The authors indicate that lanosterol levels are not significantly different – could the authors comment on why they are unchanged? Are total cholesterol levels changed?

11. S7 Fig: What is the large peak? Is it cholesterol?

12. S12 Fig: What happens if control mice are treated with a topical application of 4-hydroxytamoxifen?

13. Some of the supplementary material could be included in the main figures e.g. S5, S7, S9 – I will leave this for the authors to consider.

Typographical/visual comments:

1. Page 5, line 66: cholesterol and intermediates are “precursors” to vitamin D, etc, rather than “components” of. Please rephrase.

2. Page 8, line 135: The mutation is mislabelled as Glu – should be Gln.

3. Page 13, line 226: Should this be an increased ratio rather than decreased?

4. In some places, LSS is referred to as lanosterol synthase, but in others it is lanosterol synthetase – please be consistent.

5. Page 8, line 145: Please define EB-LCLs (and PBMCs page 18, line 342 and fig 1 legend).

6. Fig 3b: Please label what the top and bottom pictures represent.

7. Fig 4a,b,c: For clarity, please consider labelling mouse/lens/H&E/immunohistochemistry.

8. S4 Fig: Please indicate which patient.

9. Fig 1f: Please align labels over bands.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Laura Sharpe

Decision Letter 1

Gregory S Barsh, Luke Engelking

29 Dec 2019

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Dear Dr Kikuchi,

Thank you very much for submitting your Research Article entitled 'Metabolic and pathologic profiles of human LSS deficiency recapitulated in mice' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some aspects of the manuscript that should be improved.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

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Please let us know if you have any questions while making these revisions.

Yours sincerely,

Luke Engelking

Guest Editor

PLOS Genetics

Gregory Barsh

Editor-in-Chief

PLOS Genetics

Editor comments to author:

Thank you for your revised manuscript, which is now acceptable for publication pending the clarification of the textual description of Fig. S13, as suggested by reviewer 2.

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors have extensively addressed all reviewers' concerns and have added new data to support their findings. Overall, this study is an interesting one; and the data presented here will act as a springboard for further investigations, particularly mechanistic ones, into disorders due to cholesterol biosynthesis defects.

Reviewer #2: In the manuscript entitled "Metabolic and pathologic profiles of human LSS deficiency recapitulated in mice ", the authors demonstrate disruption of lanosterol synthetase in an inducible mouse model of LSS deficiency results in biochemical deficits and tissue specific changes present within affected human subjects. The work presented within the manuscript is important to the fields of cholesterol biosynthesis defects, rare disease research, and epidermal function. The authors nicely demonstrate the identification of defined mutations within a small patient cohort of LSS deficiency and recapitulate some of the tissue specific deficits within a newly derived Lss epidermal-specific knockout mouse.

From a positive perspective, the authors attempted to answer the issues raised by reviewers after the initial submission. The manuscript is improved from the first submission and the authors have added some additional supplementary data, attempted to answer biochemical and signaling questions posed by reviewers, and clarified the text. However, as noted in my initial review, I still believe the work as presented is lacking a significant advancement forward for LSS deficiency. Though the study has demonstrated loss of LSS function within the epidermis results in neonatal lethality and lens specific Lss knockout induces tissue phenotypes, there is still a lack of data demonstrating the mechanisms downstream of Lss loss leading to lethality or tissue phenotypes.

- Biochemically, there appears to be no loss of epidermal cholesterol (Fig S9) which would mechanistically explain the barrier deficiency presented in Figure 2. It’s possible 2,3-epoxysqualene accumulation at picomolar/mg levels as demonstrated in Figure 2 could exert oxidative stress on the epidermis, inducing apoptosis or lipid peroxidation. However, this is not explored. While I am distinctly aware of the difficulty with these types of analyses in limited tissue samples, these are critical data.

- While the authors did test LSS effects on SHH and Wnt signaling in MEF isolations, no deficits were identified. It’s not clear if this lack of effect is due to a true maintenance of signaling in the absence of LSS activity or limitations due to the assay. Possible explanations that come to mind include cell type of choice representing mesodermal vs ectodermal lineage of the epidermis, method of LSS deletion via AAV1 (though the qPCR analysis demonstrates fairly efficient knockdown of Lss), or limitations of the qPCR assay used. The methodology suggests MEFs were isolated with FBS, AAV1 transduction performed, and RNA isolated 3 days post-transduction. Was the biochemical defect confirmed at this time point? Further, since both SHH and Wnt signaling utilize morphogen gradients through ligand secretion by one cell type and generally ligand-responsive signaling in a separate cell, I believe IHC analyses specific to the epidermis for SHH and/or Wnt responsiveness would be a better assay of choice. However, based on a lack of cholesterol loss within the epidermis (Fig S9), it's not clear if either of these pathways would be substantially disrupted in this tissue.

In my view, any of these three outcomes would represent a significant advancement for Lss deficiency:

1) identification of causative pathways downstream of LSS loss

2) determining the biochemical changes responsible for the pathological findings

3) correction (or prevention) of pathological findings.

Any of these would be acceptable. But as the work currently stands and in comparison to previously published studies on LSS, the major finding of epidermal or lens-specific loss of Lss with confusing biochemistry (no cholesterol loss) and no well-defined mechanism is troublesome.

Minor issues to clarify related to newly presented data

S13 - “There were fewer retinal ganglion cells in Lssf/f-Pax6 mice than there were in Lssf/f mice. In contrast, the outer nuclear layers were not found to be different between Lssf/f-Pax6 and Lssf/f mice.”

” – this is not actually quantified or explained well. What does ‘different’ mean?

Reviewer #3: The authors have done their best to address comments by the reviewers. The measurement of additional sterols would indeed be interesting, but is quite difficult and so it is understandable that this has not been as extensive as hoped.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Decision Letter 2

Gregory S Barsh, Luke Engelking

26 Jan 2020

Dear Dr Kikuchi,

We are pleased to inform you that your manuscript entitled "Metabolic and pathologic profiles of human LSS deficiency recapitulated in mice" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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Comments from the reviewers (if applicable):

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Acceptance letter

Gregory S Barsh, Luke Engelking

21 Feb 2020

PGENETICS-D-19-01355R2

Metabolic and pathologic profiles of human LSS deficiency recapitulated in mice

Dear Dr Kikuchi,

We are pleased to inform you that your manuscript entitled "Metabolic and pathologic profiles of human LSS deficiency recapitulated in mice" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

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Thank you again for supporting PLOS Genetics and open-access publishing. We are looking forward to publishing your work!

With kind regards,

Kaitlin Butler

PLOS Genetics

On behalf of:

The PLOS Genetics Team

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. The metabolic pathway of cholesterol biosynthesis.

    Cholesterol is synthesized from acetyl CoA via the Bloch or Kandutsch-Russell pathway. In this synthesis pathway, LSS converts (S)-2,3-epoxysqualene to lanosterol. The abbreviations are as follows: FDFT1, farnesyldiphosphate farnesyltransferase 1; ff-MAS, follicular fluid meiosis-activating sterol; t-MAS, testis-meiosis activating sterol; LBR, lamin B receptor; SC4MOL, sterol C4-methyloxidase-like; NSDHL, NAD(P)H steroid dehydrogenase-like protein; HSD17B7, 17-beta-hydroxysteroid dehydrogenase; EBP, emopamil-binding protein; SC5DL, sterol C5-desaturase; DHCR7, 7-dehydrocholesterol reductase; and DHCR24, 24-dehydrocholesterol reductase.

    (TIF)

    Click here for additional data file. (892.6KB, tif)
    S2 Fig. Patient II-2 at 1 month of age.

    The patient exhibited ichthyosis-like scales on the skin of his chest, abdomen and upper extremities but not on his face and head.

    (TIF)

    Click here for additional data file. (9.3MB, tif)
    S3 Fig. Sagittal T1-weighted head MRI images of patients II-2 and II-3.

    Sagittal T1-weighted head MRI shows partial agenesis of the corpus callosum (from the posterior midbody to splenium) in patient II-2, while the MRI shows a normal corpus callosum in patient II-3.

    (TIF)

    Click here for additional data file. (975.9KB, tif)
    S4 Fig. Transmission electron microscopy images of the skin of patient II-3.

    (a) Hair matrix of the patient. The number of melanin granules was small. The inner root sheath was thin. (b) The basement layer cells were tightly adhered to each other. (c) The desmosome was well developed. Scale bars = 200 μm (a), 50 μm (b), and 2.5 μm (c).

    (TIF)

    Click here for additional data file. (3.8MB, tif)
    S5 Fig. Conservation of a missense variant in LSS.

    The R177Q residue is conserved in organisms from humans to zebrafish. LSS homologs were aligned with Clustal Omega.

    (TIF)

    Click here for additional data file. (452.4KB, tif)
    S6 Fig. The frameshift variant c.701_716del in LSS leads to NMD.

    EBV-LCLs from patient II-2 and II-3 were treated with 100 μg/mL cycloheximide (037–20991, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 4 hours to suppress NMD. Total RNA was extracted with an RNeasy Kit (Qiagen Inc., Valencia, CA, USA). RT-PCR was performed using a PrimeScript™ II High Fidelity RT-PCR Kit (TaKaRa, Shiga, Japan) to amplify the cDNA surrounding the c.701_716del mutation, which was followed by Sanger sequencing.

    (TIF)

    Click here for additional data file. (2.4MB, tif)
    S7 Fig. Mass spectra was collected by LC-MS/MS for the plasma from the patients or family members.

    Blue arrows indicate peaks of 2,3-oxidosqualene. Lanosterol could not be detected by this analysis.

    (TIF)

    Click here for additional data file. (1.5MB, tif)
    S8 Fig. Diagrams of the knockout allele.

    The tm1a allele was a knockout first allele, and it was converted to a conditional allele, tm1c (flox), by FLP-mediated recombination. The tm1c (flox) allele was converted to a deletion allele by Cre-mediated recombination.

    (TIF)

    Click here for additional data file. (617.2KB, tif)
    S9 Fig. Cholesterol quantification in newborn Lssf/f-K14 or Lssf/f skin.

    Mouse skin was gravimetrically weighed, and homogenates were prepared using 0.9% KCl containing 1 mM ethylenediaminetetraacetic acid. Total lipids were extracted from the homogenates using the Folch method [34, 35], and the extract was dissolved in 100 μL of 2-propanol. Human sebum was collected using acetone-soaked cotton pads [32], and it was dissolved in 200 μL of 2-propanol. The total cholesterol content in each sample was measured enzymatically with a commercial kit (Cholesterol E; Wako Pure Chemical Industries, Ltd., Osaka, Japan) according to the manufacturer’s protocol. Each bar represents the mean ± SEM (n = 5 per group). P-values were calculated using Student’s t-tests. NS, not significant.

    (TIF)

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    S10 Fig. Light microscopic images of Lssf/f-K14 and Lssf/f mouse skin.

    (a) Histological analysis did not show macroscopic differences in epidermal structure between the Lssf/f-K14 and Lssf/f mice. (b) Immunohistochemistry showed that expression of the LSS protein was decreased in the epidermal basement membrane (open arrowhead) and hair follicles (closed arrowhead) of the Lssf/f-K14 mice compared to the Lssf/f littermates. Scale bars = 100 μm.

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    S11 Fig. Immunohistochemical analysis of skin from Lssf/f-K14ERT mice with alopecia.

    Keratosis is mild in epidermal areas near immunostained sebaceous cells (a, b). In contrast, severe keratosis is found in epidermal areas lacking immunostained sebaceous cells (c). Scale bars = 100 μm.

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    S12 Fig. Topical tamoxifen-treated Lssf/f-K14ERT mice.

    Images of Lssf/f-K14ERT mice arranged chronologically after topical application of 4-hydroxytamoxifen, which was applied within the black rectangle. Depilation gradually occurred after 3 weeks of 4-hydroxytamoxifen treatment. The depilation area was greatest after 4 weeks. Similar to the effects of intraperitoneal injection of tamoxifen, the hair gradually regrew. Gray hairs were mixed among the hairs in the recovered area.

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    S13 Fig. Microscopic analysis of the retina in Lssf/f-Pax6 and Lssf/f mice.

    The inner retina, which is the inner nuclear layer and ganglion cell layer, was thin in Lssf/f-Pax6 mice compared to Lssf/f mice. There were fewer retinal ganglion cells in Lssf/f-Pax6 mice than there were in Lssf/f mice. In contrast, there was no obvious difference in the thickness of the outer nuclear layers between Lssf/f-Pax6 and Lssf/f mice. These pictures were captured using an Olympus BX53 microscope (Olympus, Tokyo, Japan). A 10x objective lens was used. Scale bars = 50 μm.

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    S14 Fig. The surviving Lssf/f-K14 mouse.

    Sequential images of an adult Lssf/f-K14 mouse (a-d). At weaning, the mouse could not be differentiated from the other littermates. The mouse gradually lost hair after 6 weeks of age (a). Desquamation started at 9 weeks of age (c). A partial skin defect on the head with oozing was observed. The mouse died due to unknown causes at 10 weeks of age (d). Analysis of histological specimens from the dead mouse revealed severe hyperkeratosis, dyskeratosis, and hypertrophic sebaceous cells (e). Scale bars = 100 μm.

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    S15 Fig. Transcriptional responses against the exogenous Shh or Wnt protein between Lss-depleted and nondepleted MEFs.

    We measured the transcriptional responses of MEFs against the exogenous Shh or Wnt protein as previously described [27, 38] with minor modifications. Embryos (E13.5–14.5) were harvested by aseptic techniques from pregnant Lssf/f mice that had been mated with Lssf/f male mice. The heads, extremities, and tails were cut off, and the livers were removed. The remaining body parts were minced by scissors and transferred to a 50 mL tube with PBS. The tube was centrifuged at 200 g for 5 min. The supernatant was aspirated, and the pellet was resuspended with 1 mL 0.25% trypsin-EDTA per embryo. The suspension was incubated for 15 min in a 37°C water bath with shaking. Fetal bovine serum was added (the same volume as trypsin) to the suspension. After letting the suspension sit for several minutes and allowing the embryo fragments sank to the bottom of the tube, the serous part was removed first and filtered, and then the remaining thick part was passed through a 100 μm filter. The filtered solution was centrifuged at 200 g for 5 min. The supernatant was aspirated, the pellet was resuspended in DMEM, and the suspension was transferred to a dish. MEFs were plated at 5 × 104 cells in 250 μL of DMEM per 48-well plate and transduced with AAV1 or a mock control (105548-AAV1 or 105537-AAV1, respectively, Addgene, Cambridge, MA, USA) at a multiplicity of infection of 5 ×105. The next day, 250 μL of DMEM was added to each well. The following day, 2 μg/mL Shh (464-SH-025/CF, R&D systems, Minneapolis, MN, USA) or 100 ng/mL Wnt‐3a (1324-WN, R&D systems, Minneapolis, MN, USA) were added to the medium. Three days after transduction, total RNA was extracted with RNeasy (QIAGEN, Hilden, Germany), and reverse transcription was performed with a PrimeScript RT Reagent Kit with gDNA Eraser (TakaraBio, Shiga, Japan). cDNA was amplified with TaKaRa Ex Taq (RR001A, TaKaRa Bio Inc., Otsu, Japan) and PrimeTime qPCR probe assays (Integrated DNA Technologies, Coralville, IA, USA). qPCR primers and probes for Gli1 (Mm.PT.58.11933824), Axin2 (Mm.PT.58.8726473), Lss (Mm.PT.58.41802559), and Gapdh (Mm.PT.39a.1) were used. qPCR reactions were cycled using StepOnePlus (Sigma-Aldrich, St. Louis, MO, USA) with standard conditions.

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    S1 Table. Variant filtering strategy.

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    S2 Table. Mutations in LSS.

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    S3 Table. Laboratory results of the patients with LSS biallelic mutations and unaffected family members in this study.

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    S4 Table. Genotype distribution of neonatal mice born from Lsswild/flox-Keratin14 male and Lssflox/flox female mice.

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    S5 Table. MS parameters used for LC-MS/MS analysis.

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    S6 Table. Primers and probes.

    The forward primers of Lss tm1a, Lss tm1c, and Lss tm1d were the same. The existence of the Pax6-Cre allele was confirmed using generic Cre primers and a probe.

    (DOCX)

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    Submitted filename: Response_to_Reviewers_LSS_PG_20200122_ak_ATSKSJ-6.docx

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    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

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