jMorp: Japanese Multi-Omics Reference Panel update report 2023.

Modern medicine is increasingly focused on personalized medicine, and multi-omics data is crucial in understanding biological phenomena and disease mechanisms. Each ethnic group has its unique genetic background with specific genomic variations influencing disease risk and drug response. Therefore, multi-omics data from specific ethnic populations are essential for the effective implementation of personalized medicine. Various prospective cohort studies, such as the UK Biobank, All of Us and Lifelines, have been conducted worldwide. The Tohoku Medical Megabank project was initiated after the Great East Japan Earthquake in 2011. It collects biological specimens and conducts genome and omics analyses to build a basis for personalized medicine. Summary statistical data from these analyses are available in the jMorp web database (https://jmorp.megabank.tohoku.ac.jp), which provides a multidimensional approach to the diversity of the Japanese population. jMorp was launched in 2015 as a public database for plasma metabolome and proteome analyses and has been continuously updated. The current update will significantly expand the scale of the data (metabolome, genome, transcriptome, and metagenome). In addition, the user interface and backend server implementations were rewritten to improve the connectivity between the items stored in jMorp. This paper provides an overview of the new version of the jMorp.

Analysis of randomly mutated AlSRKb genes reveals that most loss-of-function mutations cause defects in plasma membrane localization.

Only very limited information is available on why some nonsynonymous variants severely alter gene function while others have no effect. To identify the characteristic features of mutations that strongly influence gene function, this study focused on SRK which encodes a highly polymorphic receptor kinase expressed in stigma papillary cells that underlies a female determinant of self-incompatibility in Brassicaceae. A set of 300 Arabidopsis thaliana transformants expressing mutated SRKb from A. lyrata was constructed using error-prone PCR and the genotype and self-incompatibility phenotype of each transformant were determined. Almost all the transformants showing the self-incompatibility defect contained mutations in AlSRKb that altered localization to the plasma membrane. The observed mutations occurred in amino acid residues that were highly conserved across S haplotypes and whose predicted locations were in the interior of the protein. Our findings suggested that mutations causing the self-incompatibility defect were more likely to result from changes to AlSRKb biosynthesis than from loss of AlSRKb function. In addition, we examined whether the RandomForest and Extreme Gradient Boosting methods could predict the self-incompatibility phenotypes of SRK mutants.

Medication use before and during pregnancy in Japan: the Tohoku Medical Megabank Project Birth and Three-Generation Cohort Study.

PURPOSE: To elucidate the status of medication use among pregnant women in Japan, by means of a multigenerational genome and birth cohort study: the Tohoku Medical Megabank Project Birth and Three-Generation Cohort Study (TMM BirThree Cohort Study). METHODS: Questionnaires were distributed to pregnant women participating in the TMM BirThree Cohort Study (from July 2013 to March 2017) around 12 weeks (early pregnancy) and 26 weeks (middle pregnancy). We analysed medication use over three periods: (1) 12 months prior to pregnancy diagnosis, (2) the period between pregnancy diagnosis and around week 12 of pregnancy, and (3) post around week 12 of pregnancy. RESULTS: In total, 19,297 women were included in the analysis. The proportion of pregnant women using medications was 49.0% prior to pregnancy diagnosis, 52.1% from diagnosis to week 12, and 58.4% post week 12 of pregnancy. The most frequently prescribed medications were loxoprofen sodium hydrate (5.5%) prior to pregnancy diagnosis, magnesium oxide (5.9%) from diagnosis to week 12, and ritodrine hydrochloride (10.5%) post week 12 of pregnancy. The number of women who used suspected teratogenic medications during early pregnancy was 96 prior to pregnancy diagnosis, 48 from diagnosis to week 12, and 54 post week 12 of pregnancy. CONCLUSION: We found that ~ 50% of the pregnant women used medications before and during pregnancy and some took potential teratogenic medications during pregnancy. In birth genomic cohort study, it is expected that investigations into the safety and effectiveness of medications used during pregnancy will advance.

A novel variant in the transmembrane 4 domain of ANO3 identified in a two-year-old girl with developmental delay and tremor.

ANO3 encodes Anoctamin-3, also known as TMEM16C, a calcium-activated chloride channel. Heterozygous variants of ANO3 can cause dystonia 24, an adult-onset focal dystonia. Some pediatric cases have been reported, but most patients were intellectually normal with some exceptions. Here, we report a two-year-old girl who showed mild to moderate developmental delay, tremor, and ataxic gait, but no obvious dystonia. Trio exome sequencing identified a heterozygous de novo missense variant NM_031418.4:c.1809T>G, p.(Asn603Lys) in the ANO3 gene. Three cases with ANO3 variants and intellectual disability have been reported, including the present case. These variants were predicted to face in the same direction on the same alpha-helix (the transmembrane 4 domain), suggesting an association between these variants and childhood-onset movement disorder with intellectual disability. In pediatric cases with developmental delay and movement disorders such as tremor and ataxia, specific variants in the transmembrane 4 domain of ANO3 may be a cause, even in the absence of dystonia.

Novel POLE mutations identified in patients with IMAGE-I syndrome cause aberrant subcellular localisation and protein degradation in the nucleus.

BACKGROUND: DNA replisome is a molecular complex that plays indispensable roles in normal DNA replication. IMAGE-I syndrome is a DNA replisome-associated genetic disease caused by biallelic mutations in the gene encoding DNA polymerase epsilon catalytic subunit 1 (POLE). However, the underlying molecular mechanisms remain largely unresolved. METHODS: The clinical manifestations in two patients with IMAGE-I syndrome were characterised. Whole-exome sequencing was performed and altered mRNA splicing and protein levels of POLE were determined. Subcellular localisation, cell cycle analysis and DNA replication stress were assessed using fibroblasts and peripheral blood from the patients and transfected cell lines to determine the functional significance of POLE mutations. RESULTS: Both patients presented with growth retardation, adrenal insufficiency, immunodeficiency and complicated diffuse large B-cell lymphoma. We identified three novel POLE mutations: namely, a deep intronic mutation, c.1226+234G>A, common in both patients, and missense (c.2593T>G) and in-frame deletion (c.711_713del) mutations in each patient. The unique deep intronic mutation produced aberrantly spliced mRNAs. All mutants showed significantly reduced, but not null, protein levels. Notably, the mutants showed severely diminished nuclear localisation, which was rescued by proteasome inhibitor treatment. Functional analysis revealed impairment of cell cycle progression and increase in the expression of phospho-H2A histone family member X in both patients. CONCLUSION: These findings provide new insights regarding the mechanism via which POLE mutants are highly susceptible to proteasome-dependent degradation in the nucleus, resulting in impaired DNA replication and cell cycle progression, a characteristic of DNA replisome-associated diseases.

jMorp updates in 2020: large enhancement of multi-omics data resources on the general Japanese population.

In the Tohoku Medical Megabank project, genome and omics analyses of participants in two cohort studies were performed. A part of the data is available at the Japanese Multi Omics Reference Panel (jMorp; https://jmorp.megabank.tohoku.ac.jp) as a web-based database, as reported in our previous manuscript published in Nucleic Acid Research in 2018. At that time, jMorp mainly consisted of metabolome data; however, now genome, methylome, and transcriptome data have been integrated in addition to the enhancement of the number of samples for the metabolome data. For genomic data, jMorp provides a Japanese reference sequence obtained using de novo assembly of sequences from three Japanese individuals and allele frequencies obtained using whole-genome sequencing of 8,380 Japanese individuals. In addition, the omics data include methylome and transcriptome data from ∼300 samples and distribution of concentrations of more than 755 metabolites obtained using high-throughput nuclear magnetic resonance and high-sensitivity mass spectrometry. In summary, jMorp now provides four different kinds of omics data (genome, methylome, transcriptome, and metabolome), with a user-friendly web interface. This will be a useful scientific data resource on the general population for the discovery of disease biomarkers and personalized disease prevention and early diagnosis.

Metabolic and pathologic profiles of human LSS deficiency recapitulated in mice.

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.

Germline-Activating RRAS2 Mutations Cause Noonan Syndrome.

Noonan syndrome (NS) is characterized by distinctive craniofacial appearance, short stature, and congenital heart disease. Approximately 80% of individuals with NS harbor mutations in genes whose products are involved in the RAS/mitogen-activating protein kinase (MAPK) pathway. However, the underlying genetic causes in nearly 20% of individuals with NS phenotype remain unexplained. Here, we report four de novo RRAS2 variants in three individuals with NS. RRAS2 is a member of the RAS subfamily and is ubiquitously expressed. Three variants, c.70_78dup (p.Gly24_Gly26dup), c.216A>T (p.Gln72His), and c.215A>T (p.Gln72Leu), have been found in cancers; our functional analyses showed that these three changes induced elevated association of RAF1 and that they activated ERK1/2 and ELK1. Notably, prominent activation of ERK1/2 and ELK1 by p.Gln72Leu associates with the severe phenotype of the individual harboring this change. To examine variant pathogenicity in vivo, we generated zebrafish models. Larvae overexpressing c.70_78dup (p.Gly24_Gly26dup) or c.216A>T (p.Gln72His) variants, but not wild-type RRAS2 RNAs, showed craniofacial defects and macrocephaly. The same dose injection of mRNA encoding c.215A>T (p.Gln72Leu) caused severe developmental impairments and low dose overexpression of this variant induced craniofacial defects. In contrast, the RRAS2 c.224T>G (p.Phe75Cys) change, located on the same allele with p.Gln72His in an individual with NS, resulted in no aberrant in vitro or in vivo phenotypes by itself. Together, our findings suggest that activating RRAS2 mutations can cause NS and expand the involvement of RRAS2 proto-oncogene to rare germline disorders.

Heterozygous calcyclin-binding protein/Siah1-interacting protein (CACYBP/SIP) gene pathogenic variant linked to a dominant family with paucity of interlobular bile duct.

Paucity of interlobular bile ducts (PILBD) is a heterogeneous disorder classified into two categories, syndromic and non-syndromic bile duct paucity. Syndromic PILBD is characterized by the presence of clinical manifestations of Alagille syndrome. Non-syndromic PILBD is caused by multiple diseases, such as metabolic and genetic disorders, infectious diseases, and inflammatory and immune disorders. We evaluated a family with a dominantly inherited PILBD, who presented with cholestasis at 1-2 months of age but spontaneously improved by 1 year of age. Next-generation sequencing analysis revealed a heterozygous CACYBP/SIP p.E177Q pathogenic variant. Calcyclin-binding protein and Siah1 interacting protein (CACYBP/SIP) form a ubiquitin ligase complex and induce proteasomal degradation of non-phosphorylated ß-catenin. Immunohistochemical analysis revealed a slight decrease in CACYBP and ß-catenin levels in the liver of patients in early infancy, which almost normalized by 13 months of age. The CACYBP/SIP p.E177Q pathogenic variant may form a more active or stable ubiquitin ligase complex that enhances the degradation of ß-catenin and delays the maturation of intrahepatic bile ducts. Our findings indicate that accurate regulation of the ß-catenin concentration is essential for the development of intrahepatic bile ducts and CACYBP/SIP pathogenic variant is a novel cause of PILDB.

Alternative microexon splicing by RBFOX2 and PTBP1 is associated with metastasis in colorectal cancer.

The splicing of microexons (very small exons) is frequently dysregulated in the brain of individuals with autism spectrum disorder. However, little is known of the patterns, regulatory mechanisms and roles of microexon splicing in cancer. We here examined the transcriptome-wide profile of microexon splicing in matched colorectal cancer (CRC) and normal tissue specimens. Out of 1492 microexons comprising 3 to 15 nucleotides, 21 (1%) manifested differential splicing between CRC and normal tissue. The 21 genes harboring the differentially spliced microexons were enriched in gene ontology terms related to cell adhesion and migration. RNA interference-mediated knockdown experiments identified two splicing factors, RBFOX2 and PTBP1, as regulators of microexon splicing in CRC cells. RBFOX2 and PTBP1 were found to directly bind to microexon-containing pre-mRNAs and to control their splicing in such cells. Differential microexon splicing was shown to be due, at least in part, to altered expression of RBFOX2 and PTBP1 in CRC tissue compared to matched normal tissue. Finally, we found that changes in the pattern of microexon splicing were associated with CRC metastasis. Our data thus suggest that altered expression of RBFOX2 and PTBP1 might influence CRC metastasis through the regulation of microexon splicing.

Variants That Affect Function of Calcium Channel TRPV6 Are Associated With Early-Onset Chronic Pancreatitis.

BACKGROUND & AIMS: Changes in pancreatic calcium levels affect secretion and might be involved in development of chronic pancreatitis (CP). We investigated the association of CP with the transient receptor potential cation channel subfamily V member 6 gene (TRPV6), which encodes a Ca2+-selective ion channel, in an international cohort of patients and in mice. METHODS: We performed whole-exome DNA sequencing from a patient with idiopathic CP and from his parents, who did not have CP. We validated our findings by sequencing DNA from 300 patients with CP (not associated with alcohol consumption) and 1070 persons from the general population in Japan (control individuals). In replication studies, we sequenced DNA from patients with early-onset CP (20 years or younger) not associated with alcohol consumption from France (n = 470) and Germany (n = 410). We expressed TRPV6 variants in HEK293 cells and measured their activity using Ca2+ imaging assays. CP was induced by repeated injections of cerulein in TRPV6mut/mut mice. RESULTS: We identified the variants c.629C>T (p.A210V) and c.970G>A (p.D324N) in TRPV6 in the index patient. Variants that affected function of the TRPV6 product were found in 13 of 300 patients (4.3%) and 1 of 1070 control individuals (0.1%) from Japan (odds ratio [OR], 48.4; 95% confidence interval [CI], 6.3-371.7; P = 2.4 × 10-8). Twelve of 124 patients (9.7%) with early-onset CP had such variants. In the replication set from Europe, 18 patients with CP (2.0%) carried variants that affected the function of the TRPV6 product compared with 0 control individuals (P = 6.2 × 10-8). Variants that did not affect the function of the TRPV6 product (p.I223T and p.D324N) were overrepresented in Japanese patients vs control individuals (OR, 10.9; 95% CI, 4.5-25.9; P = 7.4 × 10-9 for p.I223T and P = .01 for p.D324N), whereas the p.L299Q was overrepresented in European patients vs control individuals (OR, 3.0; 95% CI, 1.9-4.8; P = 1.2 × 10-5). TRPV6mut/mut mice given cerulein developed more severe pancreatitis than control mice, as shown by increased levels of pancreatic enzymes, histologic alterations, and pancreatic fibrosis. CONCLUSIONS: We found that patients with early-onset CP not associated with alcohol consumption carry variants in TRPV6 that affect the function of its product, perhaps by altering Ca2+ balance in pancreatic cells. TRPV6 regulates Ca2+ homeostasis and pancreatic inflammation.

Wnt5a in cancer-associated fibroblasts promotes colorectal cancer progression.

Cancer-associated fibroblasts (CAFs) are a major component of the tumor microenvironment and have been shown to promote cancer aggressiveness. In our previous study, analysis of expression profiles obtained from paired CAFs and normal fibroblasts from colorectal cancer (CRC) tissue revealed that gene sets related to the Wnt signaling pathway were highly enriched in colorectal CAFs. Furthermore, among the components of the ß-catenin-independent Wnt pathway, Wnt5a was highly expressed in CAFs. Since Wnt5a is considered to be a regulator of CRC progression in CAFs, we performed immunohistochemical analysis on Wnt5a in 171 patients who underwent surgery for CRC. Positive staining for Wnt5a was often found in cancer stroma, particularly in fibromatous areas, although the immunoreactivity for Wnt5a was weak in cancer cells. Wnt5a status in CAFs was significantly associated with tumor size, depth of invasion, lymphatic and vascular invasion, lymph node metastasis, TNM stage, and recurrence. Subsequent in vitro analyses using human recombinant Wnt5a protein revealed that cancer cell proliferation and migration were significantly increased by stimulation with Wnt5a. Our findings suggest that Wnt5a-derived CAFs play a crucial role in CRC progression and have potential as a target of anti-cancer therapies.

A novel deletion in the C-terminal region of HSPB8 in a family with rimmed vacuolar myopathy.

Heat shock protein family B member 8, encoded by HSPB8, is an essential component of the chaperone-assisted selective autophagy complex, which maintains muscle function by degrading damaged proteins in the cells. Mutations in HSPB8 have been reported to cause Charcot-Marie-Tooth type 2L, distal hereditary motor neuropathy IIa, and rimmed vacuolar myopathies (RVM). In this study, we identified a novel heterozygous frameshift variant c.525_529del in HSPB8 in a large Japanese family with RVM, using whole exome sequencing. Three affected individuals had severe respiratory failure, which has not been addressed by previous studies. Muscle atrophy in the paraspinal muscles was also a clinical feature of the individuals affected with RVM in this study. The frameshift mutation was located in the last coding exon, and the mutated protein was predicted to harbor an isoleucine-leucine-valine (ILV) sequence, which corresponds to the IXI/V (isoleucine, X amino acids, and isoleucine or valine) motif. The IXI/V motif is essential for assembly into larger oligomers in other small heat shock proteins and all frameshift mutants of HSPB8 were predicted to share the ILV sequence in the C-terminal extension. The in silico prediction tools showed low protein solubility and increased aggregation propensity for the region around the ILV sequence. The IXI/V motif might be associated with the pathogenesis of HSPB8-related RVM.

Biallelic variants/mutations of IL1RAP in patients with steroid-sensitive nephrotic syndrome.

Nephrotic syndrome (NS) is a renal disease characterized by severe proteinuria and hypoproteinemia. Although several single-gene mutations have been associated with steroid-resistant NS, causative genes for steroid-sensitive NS (SSNS) have not been clarified. While seeking to identify causative genes associated with SSNS by whole-exome sequencing, we found compound heterozygous variants/mutations (c.524T>C; p.I175T and c.662G>A; p.R221H) of the interleukin-1 receptor accessory protein (IL1RAP) gene in two siblings with SSNS. The siblings' parents are healthy, and each parent carries a different heterozygous IL1RAP variant/mutation. Since IL1RAP is a critical subunit of the functional interleukin-1 receptor (IL-1R), we investigated the effect of these variants on IL-1R subunit function. When stimulated with IL-1ß, peripheral blood mononuclear cells from the siblings with SSNS produced markedly lower levels of cytokines compared with cells from healthy family members. Moreover, IL-1R with a variant IL1RAP subunit, reconstituted on a hematopoietic cell line, had impaired binding ability and low reactivity to IL-1ß. Thus, the amino acid substitutions in IL1RAP found in these NS patients are dysfunctional variants/mutations. Furthermore, in the kidney of Il1rap-/- mice, the number of myeloid-derived suppressor cells, which require IL-1ß for their differentiation, was markedly reduced although these mice did not show significantly increased proteinuria in acute nephrotic injury with lipopolysaccharide treatment. Together, these results identify two IL1RAP variants/mutations in humans for the first time and suggest that IL1RAP might be a causative gene for familial NS.

Identification and Validation of Combination Plasma Biomarker of Afamin, Fibronectin and Sex Hormone-Binding Globulin to Predict Pre-eclampsia.

The purpose of the present study was to identify a plasma protein biomarker able to predict pre-eclampsia (PE). Comprehensive quantitative proteomics using mass spectrometry with sequential window acquisition of all theoretical fragment ion spectra (SWATH-MS) was applied to plasma samples of 7 PE and 14 healthy pregnant women (for PE subjects, plasma samples were taken before onset of PE), and 11 proteins were selected as candidates potentially able to differentiate the two groups. Plasmas collected at gestational weeks 14-24 from 36 PE and 120 healthy pregnant women (for PE subjects, plasma samples were taken before onset of PE) were used to conduct selected reaction monitoring quantification analysis, optimize protein combinations and conduct internal validation, which consisted of 30 iterations of 10-fold cross-validation using multivariate logistic regression and receiver operating characteristic (ROC) analysis. The combination of afamin, fibronectin, and sex-hormone-binding globulin was selected as the best candidate. The 3-protein combination predictive model (predictive equation and cut-off value) generated using the internal validation subjects was successfully validated in another group of validation subjects (36 PE and 54 healthy (for PE subjects, plasma samples were taken before onset of PE)) and showed good predictive performance, with the area under the curve (AUC) 0.835 and odds ratio 13.43. In conclusion, we newly identified a 3-protein combination biomarker and established a predictive equation and cut-off value that can predict the onset of PE based on analysis of plasma samples collected during gestational weeks 14-24.

Correction: Biallelic GALM pathogenic variants cause a novel type of galactosemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Recurrent de novo MAPK8IP3 variants cause neurological phenotypes.

c-Jun-amino-terminal kinase-interacting protein 3 (JIP3), encoded by MAPK8IP3, is an adaptor protein of the kinesin-1 complex and essential for axonal transport in neurons. However, an association between MAPK8IP3 variants and human disease has not been established. We identified 5 individuals from four families with recurrent de novo variants c.1732C>T (p.Arg578Cys) and c.3436C>T (p.Arg1146Cys) in MAPK8IP3. The core phenotype includes spastic diplegia, intellectual disability, cerebral atrophy, and corpus callosum hypoplasia. Zebrafish embryos overexpressing human mutant JIP3 showed axon varicosities of the posterior lateral line nerve, suggesting an adverse effect on the developing axons. Our results suggest that MAPK8IP3 variants cause a neurodevelopmental disease. ANN NEUROL 2019;85:927-933.

jMorp: Japanese Multi Omics Reference Panel.

We developed jMorp, a new database containing metabolome and proteome data for plasma obtained from >5000 healthy Japanese volunteers from the Tohoku Medical Megabank Cohort Study, which is available at https://jmorp.megabank.tohoku.ac.jp. Metabolome data were measured by proton nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS), while proteome data were obtained by nanoLC-MS. We released the concentration distributions of 37 metabolites identified by NMR, distributions of peak intensities of 257 characterized metabolites by LC-MS, and observed frequencies of 256 abundant proteins. Additionally, correlation networks for the metabolites can be observed using an interactive network viewer. Compared with some existing databases, jMorp has some unique features: (i) Metabolome data were obtained using a single protocol in a single institute, ensuring that measurement biases were significantly minimized; (ii) The database contains large-scale data for healthy volunteers with various health records and genome data and (iii) Correlations between metabolites can be easily observed using the graphical viewer. Metabolites data are becoming important intermediate markers for evaluating the health states of humans, and thus jMorp is an outstanding resource for a wide range of researchers, particularly those in the fields of medical science, applied molecular biology, and biochemistry.

Omics research project on prospective cohort studies from the Tohoku Medical Megabank Project.

Population-based prospective cohort studies are indispensable for modern medical research as they provide important knowledge on the influences of many kinds of genetic and environmental factors on the cause of disease. Although traditional cohort studies are mainly conducted using questionnaires and physical examinations, modern cohort studies incorporate omics and genomic approaches to obtain comprehensive physical information, including genetic information. Here, we report the design and midterm results of multi-omics analysis on population-based prospective cohort studies from the Tohoku Medical Megabank (TMM) Project. We have incorporated genomic and metabolomic studies in the TMM cohort study as both metabolome and genome analyses are suitable for high-throughput analysis of large-scale cohort samples. Moreover, an association study between the metabolome and genome show that metabolites are an important intermediate phenotype connecting genetic and lifestyle factors to physical and pathologic phenotypes. We apply our metabolome and genome analyses to large-scale cohort samples in the following studies.

Biallelic GALM pathogenic variants cause a novel type of galactosemia.

PURPOSE: Galactosemia is caused by metabolic disturbances at various stages of galactose metabolism, including deficiencies in enzymes involved in the Leloir pathway (GALT, GALK1, and GALE). Nevertheless, the etiology of galactosemia has not been identified in a subset of patients. This study aimed to explore the causes of unexplained galactosemia. METHODS: Trio-based exome sequencing and/or Sanger sequencing was performed in eight patients with unexplained congenital galactosemia. In vitro enzymatic assays and immunoblot assays were performed to confirm the pathogenicity of the variants. RESULTS: The highest blood galactose levels observed in each patient were 17.3-41.9 mg/dl. Bilateral cataracts were observed in two patients. In all eight patients, we identified biallelic variants (p.Arg82*, p.Ile99Leufs*46, p.Gly142Arg, p.Arg267Gly, and p.Trp311*) in the GALM encoding galactose mutarotase, which catalyzes epimerization between ß- and α-D-galactose in the first step of the Leloir pathway. GALM enzyme activities were undetectable in lymphoblastoid cell lines established from two patients. Immunoblot analysis showed the absence of the GALM protein in the patients' peripheral blood mononuclear cells. In vitro GALM expression and protein stability assays revealed altered stabilities of the variant GALM proteins. CONCLUSION: Biallelic GALM pathogenic variants cause galactosemia, suggesting the existence of type IV galactosemia.