The gene LSS encodes lanosterol synthase. Previous research has shown that mutations in LSS cause congenital cataract (Chen & Liu, 2017; Zhao et al., 2015), as well as autosomal recessive hypotrichosis simplex (HS) or congenital alopecia (Besnard et al., 2019; Chen & Liu, 2017; Li et al., 2019; Romano et al., 2018). These conditions may be accompanied by a neuroectodermal phenotype, such as intellectual disability (ID), epilepsy, microcephaly, genital abnormalities in males, and other dermatological features, such as ichthyosis and erythroderma (Besnard et al., 2019). Wide intra- and interfamilial phenotypic variability is observed, with the former being less pronounced. In particular, one study reported a family in which two affected siblings presented with substantially differing degrees of ID (Besnard et al., 2019). To date, the establishment of a genotype–phenotype correlation for LSS mutations has not been possible. The present report describes four families in which LSS mutations led to HS with or without accompanying clinical features. Four of the identified mutations are novel, and one is previously known. Notably, one of the novel mutations is an apparent synonymous mutation, which leads to altered splicing, as demonstrated by cDNA sequencing. The seven-year-old daughter of first-degree cousins from Iraq (family 1) presented with a lifetime history of sparse hair, which grew to a maximum length of 1 cm (Figure 1a). Her eyelashes and eyebrows were unremarkable, and no abnormalities of the skin or nails were observed. The child also presented with a developmental speech disorder, learning difficulties, and microcephaly. Whole exome sequencing revealed the homozygous substitution c.530G > A (p.Arg177Gln) (rs142081800, gnomAD v.2.1.1 frequency = 5.66e-5) in LSS (NM_002340). Both parents were heterozygous carriers of the mutation (Figure 1a). Recent studies reported this mutation occurring in a compound heterozygous state, together with other mutations, in two further hypotrichosis patients, who presented with and without ID, respectively (Murata et al., 2021; Wada et al., 2020). Two siblings from Georgia (a boy aged 7 years and a girl aged 14 years, family 2), presented with a complete lack of scalp hair. Although both siblings had shown some degree of scalp hair growth from the age of 5 years, the hair had subsequently fallen out. Both siblings presented with sparse and very light colored eyelashes and eyebrows, and some fine hairs were evident on the back, legs, and hands. The mother described the mental development of the girl as normal. However, she reported that the boy had hearing difficulties, and experienced concentration problems at school, which has not yet been formally evaluated. Whole exome sequencing revealed that both siblings carry the mutations c.934C > T (p.Arg312Trp) (rs764497439, gnomAD v.2.1.1 frequency = 2.82e-5) and c.881G > T (p.Arg294Leu) (rs188459967, gnomAD v.2.1.1 frequency = 8.1e-6) in LSS in a compound heterozygous state (Figure 1b). The mutations co-segregate with the disease phenotype in the family. The healthy parents carry each one of the mutations in a heterozygous state. The nine-year old daughter of consanguineous parents from Syria (family 3) presented at the age of 5 years with a history of very sparse scalp hair from birth. Her younger brother was similarly affected. The parents reported that after episodes of febrile illness, a diffuse loss of scalp hair always occurred, with subsequent spontaneous regrowth. DNA was only available from the girl. Mutation analysis of LSS by Sanger sequencing revealed the homozygous missense mutation c.1702C > T (p.Arg568Trp) (rs769430360) (Figure 1c). The 3-year-old son of first-degree cousins of Afghani descent (family 4) presented with total alopecia. The parents reported that although he had had scant fluffy hair on the scalp at birth, this had fallen out completely by the age of 2 months. The child presented with missing eyebrows and body hair, and very sparse eyelashes (Figure 1d). The skin on his arms was dry, with an appearance that resembled keratosis pilaris. No other cutaneous or neurodevelopmental abnormalities were observed. Sanger sequencing revealed the synonymous variant c.393G > A (p.131Leu=) in LSS in a homozygous state (Figure 1d). Both parents were heterozygous for the mutation, which was predicted to be “disease causing” by Mutation Taster. In silico analysis for splice site effects was performed using HSF Pro (Desmet et al., 2009) and SpliceAI (Jaganathan et al., 2019). Both analyses predicted that the mutation caused the activation of a cryptic donor site. This prediction was assessed via cDNA sequencing, which involved the isolation of RNA from the boy's blood cells and reverse transcription. The results confirmed that the mutation leads to the activation of the in silico predicted cryptic donor site 2 bp adjacent to the substitution, which eventually leads to partial skipping of exon 4 and an in frame deletion of 11 amino acid residues: r.425-457del (Figure 1e). Based on the crystal structure of the human lanosterol synthase (PDB accession code 1W6K) (Thoma et al., 2004), the disease related mutations identified in this study were modeled into the structure and analyzed for their potential impact on the structural integrity of the synthase and its enzymatic function (Figure 2). This analysis showed that all mutations might have consequences on the three-dimensional structure of the protein, however, none appeared to be deleterious on the protein and its enzymatic activity. Families 1–3 each have a mutation either in homozygous (families 1 and 3) or compound heterozygous state (family 2) leading to the substitution of a surface exposed arginine. These positively charged residues may be involved in interacting with the negatively charged lipid surface and their substitution could therefore, influence the membrane association of the enzyme (Figure 2a–c). Family 2 carries an additional mutation that leads to the substitution of an arginine residue located in the close proximity of a putative membrane insertion region by a tryptophan. This substitution could change the local arrangement of the membrane insertion region and thus influence lanosterol substrate uptake (Figure 2d). Finally, the mutation that leads to the deletion of 11 amino acid residues in family 4 may have an effect on the local arrangement of the helices surrounding the lanosterol binding site and therefore, it may influence, even if not substantially, the catalytic activity of the synthase. All LSS mutations reported to date have been missense, nonsense, or splice site mutations, and no synonymous LSS mutation has yet been reported to cause HS with or without neurodevelopmental abnormalities. Based on our discovery of the pathogenic synonymous variant, which leads to aberrant splicing, we propose that the possible molecular consequences of all identified synonymous LSS mutations in patients should be carefully investigated before their pathogenicity is ruled out. With respect to genotype–phenotype correlations, it remains unclear which mutations in LSS lead to an alopecia phenotype only, and which lead to accompanying severe neurodevelopmental and dermatological phenotypes. At writing, we hypothesize that the presence and extent of such additional clinical features may be related to the degree of normal protein function. Further functional studies are warranted to understand the mechanisms underlying the phenotypic variability that arises from LSS mutations. At writing, the general (but not fixed) consensus is that prenatal diagnostics (PD) should not be offered for alopecia alone. However, given the reported interfamilial phenotypic variability (Besnard et al., 2019), the siblings of children with an alopecia only phenotype may display cognitive disability and/or a severe skin phenotype. Therefore, we must be prepared to offer PD for future pregnancies in cases where the parents are both known LSS mutation carriers or who already have an affected child. The identification of further LSS mutation carriers will facilitate our understanding of this complex and unpredictable protein. This in turn will improve predictions concerning the phenotypic outcomes of individual LSS mutations. The authors thank all family members for their participation. This study was supported by the grant I-1443-422.13/2017 (to Regina C. Betz) from the German-Israeli Foundation for Scientific Research and Development (GIF), and a grant (to Regina C. Betz and Matthias Geyer) from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), under the auspices of the Germany Excellence Strategy—EXC2151-390873048. The authors declare no conflict of interest. Nicole Cesarato, Maria Wehner, Daisy Axt, and Holger Thiele contributed to the sequencing experiments and mutation analysis; Mariam Ghughunishvili, Cristina Has, and Michael J. Lentze collected clinical data and blood samples, Axel Schmidt performed bioinformatic analysis, Matthias Geyer performed structural protein modeling and contributed to the writing of the article, F. Buket Basmanav performed data analysis, supervised the study and wrote the manuscript. Regina C. Betz collected clinical data, supervised the study and wrote the manuscript. Data are presented in the manuscript. Additional information such as experimental conditions are available upon request. Data are presented in the manuscript. Additional information such as experimental conditions are available upon request.