Novel biallelic LSS variants in autosomal recessive hypotrichosis simplex: insights from a multi-omics approach.

Biallelic pathogenic variants in the lanosterol synthase gene LSS involved in the cholesterol biosynthesis cause alopecia with intellectual disability, a rare recessive neuroectodermal syndrome

Mitochondrial integrity is fundamental to cellular function, upheld by a network of proteases that regulate proteostasis and mitochondrial dynamics. Among these proteases, AFG3L2 is critical due to its roles in maintaining mitochondrial homeostasis, regulating mitochondrial protein quality, and facilitating mitochondrial biogenesis. Mutations in AFG3L2 are implicated in a spectrum of diseases, including spinocerebellar ataxia type 28 (SCA28) and spastic ataxia 5 (SPAX5), as well as other systemic conditions. This study employs a multi-omics approach to investigate the biochemical impact of AFG3L2 mutations in immortalized lymphoblastoid cell lines derived from a patient with biallelic variants leading to spastic ataxia (SPAX5). Our proteomic analysis revealed AFG3L2 impairment, with significant dysregulation of proteins critical for mitochondrial function, cytoskeletal integrity, and cellular metabolism. Specifically, disruptions were observed in mitochondrial dynamics and calcium homeostasis, alongside downregulation of key proteins like COX11, a copper chaperone for complex IV assembly, and NFU1, an iron-sulfur cluster protein linked to spastic paraparesis and infection-related worsening. Lipidomic analysis highlighted substantial alterations in lipid composition, with significant decreases in sphingomyelins, phosphatidylethanolamine, and phosphatidylcholine, reflecting disruptions in lipid metabolism and membrane integrity. Metabolomic profiling did not reveal any significant findings. Our comprehensive investigation into loss of functional AFG3L2 elucidates a pathophysiology extending beyond mitochondrial proteostasis, implicating a wide array of cellular processes. The findings reveal substantial cellular disturbances at multiple levels, contributing to neurodegeneration through disrupted mitochondrial respiratory chain, calcium homeostasis, cytoskeletal integrity, and altered lipid homeostasis. This study underscores the complexity of SPAX5 pathophysiology and the importance of multi-omics approaches in developing effective strategies to address the impact of loss of functional AFG3L2. Our data also highlight the value of immortalized lymphoblastoid cells as a tool for pre-clinical testing and research, offering a detailed biochemical fingerprint that enhances our understanding of SPAX5 and identifies potential areas for further investigation.

Multi-omics approach characterises CRLS1 deficiency, a novel mitochondrial disorder

Congenital cataract is the most common cause of lifelong visual loss in children worldwide, which has significant genotypic and phenotypic heterogeneity. The LSS gene encodes lanosterol synthase (LSS), which acts on the cholesterol biosynthesis pathway by converting (S)-2,3-oxidosqualene to lanosterol. The biallelic pathogenic variants in the LSS gene were found in congenital cataract, Alopecia-intellectual disability syndrome, hypotrichosis simplex, and mutilating palmoplantar keratoderma. In this study, we reported the first congenital nuclear cataract combined with hypotrichosis in a 12-year-old boy with biallelic LSS variants (c.1025T>G; p.I342S and c.1531_1532insT; p.L511Ffs*17) by exome sequencing. Reviewing all reported patients with LSS variants indicated that p.W629 might be a hotspot for hypospadias and p.I342S was associated with congenital cataract. Patients with one or two truncation variants tend to have multisystem symptoms compared with those with two missense variants. These findings deepen the understanding of LSS variants and contribute to the genetic counseling of affected families.

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Autosomal recessive hypotrichosis (LAH3) is a rare hair disorder characterized by sparse hair on scalp and the rest of the body of affected individuals. Recently mutations in a G protein-coupled receptor gene, P2RY5, located at LAH3 locus, have been reported in several families with autosomal recessive hypotrichosis simplex and woolly hair. For the present study, 22 Pakistani families with autosomal recessive hypotrichosis were enrolled. Genotyping using microsatellite markers linked to three autosomal recessive forms of hypotrichosis (LAH1, LAH2, LAH3) showed the linkage of 2 families to the LAH2 locus and 14 to the LAH3 locus. The remaining 6 families were not linked to any of the three loci. Families linked to LAH3 locus were further subjected to screening of the P2RY5 gene with direct DNA sequencing. Three previously reported variants, c.69insCATG (p.24insHfs52), c.188A > T (p.D63V) and c.565G > A (p.E189K) were observed in eight families. Four novel nonsynonymous sequence variants, c.8G > C (p.S3T), c.36insA (p.D13RfsX16), c.160insA (p.N54TfsX58) and c.436G > A (p.G146R) were found to segregate within six families.

Hypomyelinating leukodystrophies (HLDs) are rare genetic neurodevelopmental disorders characterized by defective myelin formation. The genetic cause of these disorders has been ascribed to mutations in genes encoding myelin protein components, such as proteolipid protein 1 (PLP1) and myelin basic protein (MBP), or in genes encoding for transcription and translation-related proteins. Particularly, biallelic pathogenic variants in POLR3A, POLR3B, POLR3K, POLR3D, POLR1C lead to the insurgence of RNA Polymerase III (Pol III)-related HLDs (POLR3-HLDs). The molecular mechanisms linking Pol III dysfunction to hypomyelination remain largely elusive, though the main hypothesis is that impaired Pol III activity likely disrupts gene expression and cellular homeostasis processes critical for myelin development and lipid metabolism. In this study, we analyzed a family trio consisting of unaffected carrier parents and a proband affected by POLR3A-related HLD, carrying compound heterozygous variants (p.Phe601Tyr and p.Gly1358Arg). We investigated the structural and functional consequences of two POLR3A variants using protein modeling, functional assays and multi-omics profiling in subject-specific primary fibroblasts. Structural analysis revealed alterations in DNA-binding regions and a likely impact on protein stability, whilst functional assays showed an impairment in cellular proliferation. Lipidomic and transcriptomic profiling revealed that p. Gly1358Arg mutation predominantly affects lipidomic metabolism, while p. Phe601Tyr was associated with a widespread transcriptional dysregulation. Both mutations ultimately caused a significant reduction in lipid droplets in the proband's cells. These results demonstrate mutation-specific pathogenetic mechanisms in POLR3A-HLD and underline the utility of integrative multi-omics approaches in elucidating the molecular basis of rare neurodevelopmental disorders.

Four hypotrichosis families with mutations in the gene LSS presenting with and without neurodevelopmental phenotypes.

Mutations in lipase H cause autosomal recessive hypotrichosis simplex with woolly hair

Autosomal recessive hypotrichosis simplex (ARHS) presents with progressive hair loss mainly affecting the scalp area. In a small number of families, the condition has been associated with mutations in three distinct genes: DSG4, LIPH and LPAR6. To identify the molecular basis of ARHS in a consanguineous family of Turkish extraction. We used a combination of microsatellite marker screening and direct sequencing. We identified a novel missense mutation (c.C587T) in the human LPAR6 gene, resulting in the amino acid substitution p.P196L. The mutation affects a highly conserved amino acid residue, and is predicted to disrupt signalling through the P2Y5 receptor. This study provides further evidence supporting a role for the lysophosphatidyl signalling pathway in hair growth and differentiation. In addition, this paper reports, for the first time to our knowledge, the use of homozygosity mapping as a premutation screening tool in the diagnosis of a group of inherited hair disorders.

Hereditary alopecia/hypotrichosis is a large group of rare genetic disorders characterized by hair loss. Many causative genes and their corresponding chromosomal locations have been identified for several types of hereditary alopecia/hypotrichosis in recent years, including Marie-Unna hereditary hypotrichosis （U2HR, 8p21.3）, autosomal dominant hypotrichosis simplex （APCDD1, 18p11.22; RPL21, 13q12）, autosomal recessive hypotrichosis simplex （DSG4, 18q12.1; DSC3, 18q21.1; LIPH, 3q26-27; P2RY5, 13q13-14; 10q11.23-22.3; ？p21.3-p22.3）, autosomal recessive woolly hair/hypotrichosis （LIPH, 3q26-27; P2RYS, 13q13- 14）, autosomal dominant woolly hair/hypotrichosis （KRT74, 12q12-14） and ichthyosis follicularis-alopecia- photophobia syndrome （MBTPS2, Xp22）. These genes play critical roles in the regulation of hair follicle morphogenesis and growth cycles, and their pathogenic mutations may lead to abnormality in hair follicle morphogenesis and development, thus result in alopecia/hypotrichosis. Key words: Alopecia; Heredity; Molecular biology; Genes; Mutation

Hypotrichosis simplex comprises a group of non-syndromic human alopecias. Diffuse loss of hair typically starts in early childhood and progresses throughout adolescence. We and others have previously reported mutations in the P2RY5 gene and the LIPH gene as being causal factors of autosomal recessive hypotrichosis simplex with or without woolly hair. In the present study, we analyzed one Turkish family and two non-related girls of Indian ethnicity affected with hypotrichosis and woolly hair for mutations in these genes. We identified as yet unreported mutations in the P2RY5 gene: a 1-base pair deletion (c.472delC) and a 4-base pair duplication (c.64_67dupTGCA), both of which lead to frameshifts resulting in truncated proteins. Our study increases the spectrum of known P2RY5 mutations and highlights the importance of this receptor in human hair growth and texture.

Pathogenic biallelic variants in LSS are associated with three Mendelian rare disease traits including congenital cataract type 44, autosomal recessive hypotrichosis type 14, and alopecia-intellectual disability syndrome type 4 (APMR4). We performed trio research exome sequencing on a family with a four-year-old male with global developmental delay, epilepsy and striking alopecia, and identified novel compound heterozygous LSS splice site (c.14+2T>C) and missense (c.1357 G>A; p.V453L) variant alleles. Rare features associated with APMR4 such as cryptorchidism, micropenis, mild cortical brain atrophy and thin corpus callosum were detected. Previously unreported APMR4 findings including cerebellar involvement in the form of unsteady ataxic gait, small vermis with prominent folia, were noted. A review of all reported variants to date in 29 families with LSS-related phenotypes showed an emerging genotype-phenotype correlation. Our report potentially expands LSS-related phenotypic spectrum and highlights the importance of performing brain imaging in LSS-related conditions.

Autosomal recessive hypotrichosis simplex (ARHS) manifests with paucity of hair appearing during early childhood. We assessed four affected families. We initially genotyped three of these families for a panel of microsatellite markers spanning all ARHS-associated loci and obtained data suggesting linkage to 3q27, encompassing LIPH, which had previously been shown to be associated with ARHS. Accordingly, a homozygous duplication mutation in exon 2 of this gene (c.280_369dup; p.Gly94_Lys123dup) was found to segregate with the disease in all the families. Through the identification of the first duplication mutation in the human LIPH gene, we provide further evidence supporting a role for the phospholipase signalling pathway in hair growth and differentiation.

A recent genome‐wide association study showed eight regions for alopecia areata (AA), including CTLA4 , IL‐2/IL‐22 , HLA‐class II , UL16‐binding protein‐3 and ‐6 , syntaxin 17 , IL‐2RA , peroxiredoxin 5 and Eos . Candidate genes associated with androgenetic alopecia are divided into androgen‐related and ‐unrelated genes. The former includes 5 α‐ reductase isozymes , androgen receptor and ectodysplasin A2 receptor , which is located close to androgen receptor (AR) gene, and possible AR coregulator such as histone deacetylase 9 . The latter contains PAX1 and FOXA2 . Regarding congenital hypotrichosis, mutations in EDA‐A1 , EDAR and EDA‐RADD , p63 and P‐cadherin are pointed out for ectodermal dysplasia, SPINK5 for Netherton syndrome, desmoglein 4, lipase H (LIPH) and LPAR6 (P2RY5) for localised autosomal recessive hypotrichosis, lipase H (LIPH) and LPAR6 (P2RY5) for autosomal recessive woolly hair, keratin 74 for autosomal dominant woolly hair, corneodesmosin and APCDD for hypotrichosis simplex and hairless for Marie‐Unna hypotrichosis. Key Concepts: Recent studies using mainly genome‐wide association analysis revealed novel candidate genes associated with alopecia, providing informative explanation for the pathogenesis. Genome‐wide association studies suggested the importance of innate and acquired immunity in alopecia areata pathogenesis and possibility of androgen‐independent pathogenic pathway in androgenetic alopecia. Novel genes associated with congenital hypotrichosis have been found, providing new clues to search not only the pathogenesis but also molecular mechanism of normal hair growth.