Fibroblast Growth Factor 6.

Fibroblast Growth Factor 6 (FGF6), also referred to as HST2 or HBGF6, is a member of the Fibroblast Growth Factor (FGF), the Heparin Binding Growth Factor (HBGF) and the Heparin Binding Secretory Transforming Gene (HST) families. The genomic and protein structure of FGF6 is highly conserved among varied species, as is its expression in muscle and muscle progenitor cells. Like other members of the FGF family, FGF6 regulates cell proliferation, differentiation, and migration. Specifically, it plays key roles in myogenesis and muscular regeneration, angiogenesis, along with iron transport and lipid metabolism. Similar to others from the FGF family, FGF6 also possesses oncogenic transforming activity, and as such is implicated in a variety of cancers.

Pluripotency of a founding field: rebranding developmental biology.

The field of developmental biology has declined in prominence in recent decades, with off-shoots from the field becoming more fashionable and highly funded. This has created inequity in discovery and opportunity, partly due to the perception that the field is antiquated or not cutting edge. A 'think tank' of scientists from multiple developmental biology-related disciplines came together to define specific challenges in the field that may have inhibited innovation, and to provide tangible solutions to some of the issues facing developmental biology. The community suggestions include a call to the community to help 'rebrand' the field, alongside proposals for additional funding apparatuses, frameworks for interdisciplinary innovative collaborations, pedagogical access, improved science communication, increased diversity and inclusion, and equity of resources to provide maximal impact to the community.

Sf3b4 regulates chromatin remodeler splicing and Hox expression.

SF3B proteins form a heptameric complex in the U2 small nuclear ribonucleoprotein, essential for pre-mRNA splicing. Heterozygous pathogenic variants in human SF3B4 are associated with head, face, limb, and vertebrae defects. Using the CRISPR/Cas9 system, we generated mice with constitutive heterozygous deletion of Sf3b4 and showed that mutant embryos have abnormal vertebral development. Vertebrae abnormalities were accompanied by changes in levels and expression pattern of Hox genes in the somites. RNA sequencing analysis of whole embryos and somites of Sf3b4 mutant and control litter mates revealed increased expression of other Sf3b4 genes. However, the mutants exhibited few differentially expressed genes and a large number of transcripts with differential splicing events (DSE), predominantly increased exon skipping and intron retention. Transcripts with increased DSE included several genes involved in chromatin remodeling that are known to regulate Hox expression. Our study confirms that Sf3b4 is required for normal vertebrae development and shows, for the first time, that like Sf3b1, Sf3b4 also regulates Hox expression. We propose that abnormal splicing of chromatin remodelers is primarily responsible for vertebral defects found in Sf3b4 heterozygous mutant embryos.

Single substitution in H3.3G34 alters DNMT3A recruitment to cause progressive neurodegeneration.

Germline histone H3.3 amino acid substitutions, including H3.3G34R/V, cause severe neurodevelopmental syndromes. To understand how these mutations impact brain development, we generated H3.3G34R/V/W knock-in mice and identified strikingly distinct developmental defects for each mutation. H3.3G34R-mutants exhibited progressive microcephaly and neurodegeneration, with abnormal accumulation of disease-associated microglia and concurrent neuronal depletion. G34R severely decreased H3K36me2 on the mutant H3.3 tail, impairing recruitment of DNA methyltransferase DNMT3A and its redistribution on chromatin. These changes were concurrent with sustained expression of complement and other innate immune genes possibly through loss of non-CG (CH) methylation and silencing of neuronal gene promoters through aberrant CG methylation. Complement expression in G34R brains may lead to neuroinflammation possibly accounting for progressive neurodegeneration. Our study reveals that H3.3G34-substitutions have differential impact on the epigenome, which underlie the diverse phenotypes observed, and uncovers potential roles for H3K36me2 and DNMT3A-dependent CH-methylation in modulating synaptic pruning and neuroinflammation in post-natal brains.

Craniofacial Defects in Embryos with Homozygous Deletion of Eftud2 in Their Neural Crest Cells Are Not Rescued by Trp53 Deletion.

Embryos with homozygous mutation of Eftud2 in their neural crest cells (Eftud2ncc-/-) have brain and craniofacial malformations, hyperactivation of the P53-pathway and die before birth. Treatment of Eftud2ncc-/- embryos with pifithrin-α, a P53-inhibitor, partly improved brain and craniofacial development. To uncover if craniofacial malformations and death were indeed due to P53 hyperactivation we generated embryos with homozygous loss of function mutations in both Eftud2 and Trp53 in the neural crest cells. We evaluated the molecular mechanism underlying craniofacial development in pifithrin-α-treated embryos and in Eftud2; Trp53 double homozygous (Eftud2ncc-/-; Trp53ncc-/-) mutant embryos. Eftud2ncc-/- embryos that were treated with pifithrin-α or homozygous mutant for Trp53 in their neural crest cells showed reduced apoptosis in their neural tube and reduced P53-target activity. Furthermore, although the number of SOX10 positive cranial neural crest cells was increased in embryonic day (E) 9.0 Eftud2ncc-/-; Trp53ncc-/- embryos compared to Eftud2ncc-/- mutants, brain and craniofacial development, and survival were not improved in double mutant embryos. Furthermore, mis-splicing of both P53-regulated transcripts, Mdm2 and Foxm1, and a P53-independent transcript, Synj2bp, was increased in the head of Eftud2ncc-/-; Trp53ncc-/- embryos. While levels of Zmat3, a P53- regulated splicing factor, was similar to those of wild-type. Altogether, our data indicate that both P53-regulated and P53-independent pathways contribute to craniofacial malformations and death of Eftud2ncc-/- embryos.

Snrpb is required in murine neural crest cells for proper splicing and craniofacial morphogenesis.

Heterozygous mutations in SNRPB, an essential core component of the five small ribonucleoprotein particles of the spliceosome, are responsible for cerebrocostomandibular syndrome (CCMS). We show that Snrpb heterozygous mouse embryos arrest shortly after implantation. Additionally, heterozygous deletion of Snrpb in the developing brain and neural crest cells models craniofacial malformations found in CCMS, and results in death shortly after birth. RNAseq analysis of mutant heads prior to morphological defects revealed increased exon skipping and intron retention in association with increased 5' splice site strength. We found increased exon skipping in negative regulators of the P53 pathway, along with increased levels of nuclear P53 and P53 target genes. However, removing Trp53 in Snrpb heterozygous mutant neural crest cells did not completely rescue craniofacial development. We also found a small but significant increase in exon skipping of several transcripts required for head and midface development, including Smad2 and Rere. Furthermore, mutant embryos exhibited ectopic or missing expression of Fgf8 and Shh, which are required to coordinate face and brain development. Thus, we propose that mis-splicing of transcripts that regulate P53 activity and craniofacial-specific genes contributes to craniofacial malformations. This article has an associated First Person interview with the first author of the paper.

TMED2 binding restricts SMO to the ER and Golgi compartments.

Hedgehog (HH) signaling is important for embryonic pattering and stem cell differentiation. The G protein-coupled receptor (GPCR) Smoothened (SMO) is the key HH signal transducer modulating both transcription-dependent and transcription-independent responses. We show that SMO protects naive mouse embryonic stem cells (ESCs) from dissociation-induced cell death. We exploited this SMO dependency to perform a genetic screen in haploid ESCs where we identify the Golgi proteins TMED2 and TMED10 as factors for SMO regulation. Super-resolution microscopy shows that SMO is normally retained in the endoplasmic reticulum (ER) and Golgi compartments, and we demonstrate that TMED2 binds to SMO, preventing localization to the plasma membrane. Mutation of TMED2 allows SMO accumulation at the plasma membrane, recapitulating early events after HH stimulation. We demonstrate the physiologic relevance of this interaction in neural differentiation, where TMED2 functions to repress HH signal strength. Identification of TMED2 as a binder and upstream regulator of SMO opens the way for unraveling the events in the ER-Golgi leading to HH signaling activation.

The imperative for scientific societies to change the face of academia: Recommendations for immediate action.

As organizations that facilitate collaboration and communication, scientific societies have an opportunity, and a responsibility, to drive inclusion, diversity, equity, and accessibility in science in academia. The American Association for Anatomy (AAA), with its expressed and practiced culture of engagement, can serve as a model of best practice for other professional associations working to become more inclusive of individuals from historically underrepresented groups. In this publication, we acknowledge anatomy's exclusionary past, describe the present face of science in academia, and provide recommendations for societies, including the AAA, to accelerate change in academia. We are advocating for scientific societies to investigate inequities and revise practices for inclusivity; develop and empower underrepresented minority leadership; and commit resources in a sustained manner as an investment in underrepresented scientists who bring diverse perspectives and lived experiences to science in academia.

Mutation in Eftud2 causes craniofacial defects in mice via mis-splicing of Mdm2 and increased P53.

EFTUD2 is mutated in patients with mandibulofacial dysostosis with microcephaly (MFDM). We generated a mutant mouse line with conditional mutation in Eftud2 and used Wnt1-Cre2 to delete it in neural crest cells. Homozygous deletion of Eftud2 causes brain and craniofacial malformations, affecting the same precursors as in MFDM patients. RNAseq analysis of embryonic heads revealed a significant increase in exon skipping and increased levels of an alternatively spliced Mdm2 transcript lacking exon 3. Exon skipping in Mdm2 was also increased in O9-1 mouse neural crest cells after siRNA knock-down of Eftud2 and in MFDM patient cells. Moreover, we found increased nuclear P53, higher expression of P53-target genes and increased cell death. Finally, overactivation of the P53 pathway in Eftud2 knockdown cells was attenuated by overexpression of non-spliced Mdm2, and craniofacial development was improved when Eftud2-mutant embryos were treated with Pifithrin-α, an inhibitor of P53. Thus, our work indicates that the P53-pathway can be targeted to prevent craniofacial abnormalities and shows a previously unknown role for alternative splicing of Mdm2 in the etiology of MFDM.

Spliceosomopathies and neurocristopathies: Two sides of the same coin?

Mutations in core components of the spliceosome are responsible for a group of syndromes collectively known as spliceosomopathies. Patients exhibit microcephaly, micrognathia, malar hypoplasia, external ear anomalies, eye anomalies, psychomotor delay, intellectual disability, limb, and heart defects. Craniofacial malformations in these patients are predominantly found in neural crest cells-derived structures of the face and head. Mutations in eight genes SNRPB, RNU4ATAC, SF3B4, PUF60, EFTUD2, TXNL4, EIF4A3, and CWC27 are associated with craniofacial spliceosomopathies. In this review, we provide a brief description of the normal development of the head and the face and an overview of mutations identified in genes associated with craniofacial spliceosomopathies. We also describe a model to explain how and when these mutations are most likely to impact neural crest cells. We speculate that mutations in a subset of core splicing factors lead to disrupted splicing in neural crest cells because these cells have increased sensitivity to inefficient splicing. Hence, disruption in splicing likely activates a cellular stress response that includes increased skipping of regulatory exons in genes such as MDM2 and MDM4, key regulators of P53. This would result in P53-associated death of neural crest cells and consequently craniofacial malformations associated with spliceosomopathies.

Transmembrane emp24 domain proteins in development and disease.

Regulated transport through the secretory pathway is essential for embryonic development and homeostasis. Disruptions in this process impact cell fate, differentiation and survival, often resulting in abnormalities in morphogenesis and in disease. Several congenital malformations are caused by mutations in genes coding for proteins that regulate cargo protein transport in the secretory pathway. The severity of mutant phenotypes and the unclear aetiology of transport protein-associated pathologies have motivated research on the regulation and mechanisms through which these proteins contribute to morphogenesis. This review focuses on the role of the p24/transmembrane emp24 domain (TMED) family of cargo receptors in development and disease.

Snap29 mutant mice recapitulate neurological and ophthalmological abnormalities associated with 22q11 and CEDNIK syndrome.

Synaptosomal-associated protein 29 (SNAP29) encodes a member of the SNARE family of proteins implicated in numerous intracellular protein trafficking pathways. SNAP29 maps to the 22q11.2 region and is deleted in 90% of patients with 22q11.2 deletion syndrome (22q11.2DS). Moreover, bi-allelic SNAP29 mutations in patients are responsible for CEDNIK (cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma) syndrome. A mouse model that recapitulates abnormalities found in these syndromes is essential for uncovering the cellular basis of these disorders. In this study, we report that mice with a loss of function mutation of Snap29 on a mixed CD1;FvB genetic background recapitulate skin abnormalities associated with CEDNIK, and also phenocopy neurological and ophthalmological abnormalities found in CEDNIK and a subset of 22q11.2DS patients. Our work also reveals an unanticipated requirement for Snap29 in male fertility and supports contribution of hemizygosity for SNAP29 to the phenotypic spectrum of abnormalities found in 22q11.2DS patients.

Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation.

BACKGROUND: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method). RESULTS: We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. CONCLUSION: We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

Loss of function mutation of Eftud2, the gene responsible for mandibulofacial dysostosis with microcephaly (MFDM), leads to pre-implantation arrest in mouse.

Mutations in EFTUD2 are responsible for the autosomal dominant syndrome named MFDM (mandibulofacial dysostosis with microcephaly). However, it is not clear how reduced levels of EFTUD2 cause abnormalities associated with this syndrome. To determine if the mouse can serve as a model for uncovering the etiology of abnormalities found in MFDM patients, we used in situ hybridization to characterize expression of Eftud2 during mouse development, and used CRISPR/Cas9 to generate a mutant mouse line with deletion of exon 2 of the mouse gene. We found that Eftud2 was expressed throughout embryonic development, though its expression was enriched in the developing head and craniofacial regions. Additionally, Eftud2 heterozygous mutant embryos had reduced EFTUD2 mRNA and protein levels. Moreover, Eftud2 heterozygous embryos were born at the expected Mendelian frequency, and were viable and fertile despite being developmentally delayed. In contrast, Eftud2 homozygous mutant embryos were not found post-implantation but were present at the expected Mendelian frequency at embryonic day (E) 3.5. Furthermore, only wild-type and heterozygous E3.5 embryos survived ex vivo culture. Our data indicate that Eftud2 expression is enriched in the precusor of structures affected in MFDM patients and show that heterozygous mice carrying deletion of exon 2 do not model MFDM. In addition, we uncovered a requirement for normal levels of Eftud2 for survival of pre-implantation zygotes.

Control of anterior GRadient 2 (AGR2) dimerization links endoplasmic reticulum proteostasis to inflammation.

Anterior gradient 2 (AGR2) is a dimeric protein disulfide isomerase family member involved in the regulation of protein quality control in the endoplasmic reticulum (ER). Mouse AGR2 deletion increases intestinal inflammation and promotes the development of inflammatory bowel disease (IBD). Although these biological effects are well established, the underlying molecular mechanisms of AGR2 function toward inflammation remain poorly defined. Here, using a protein-protein interaction screen to identify cellular regulators of AGR2 dimerization, we unveiled specific enhancers, including TMED2, and inhibitors of AGR2 dimerization, that control AGR2 functions. We demonstrate that modulation of AGR2 dimer formation, whether enhancing or inhibiting the process, yields pro-inflammatory phenotypes, through either autophagy-dependent processes or secretion of AGR2, respectively. We also demonstrate that in IBD and specifically in Crohn's disease, the levels of AGR2 dimerization modulators are selectively deregulated, and this correlates with severity of disease. Our study demonstrates that AGR2 dimers act as sensors of ER homeostasis which are disrupted upon ER stress and promote the secretion of AGR2 monomers. The latter might represent systemic alarm signals for pro-inflammatory responses.

TMED2/emp24 is required in both the chorion and the allantois for placental labyrinth layer development.

TMED2, a member of the transmembrane emp24 domain (TMED) family, is required for transport of cargo proteins between the ER and Golgi. TMED2 is also important for normal morphogenesis of mouse embryos and their associated placenta, and in fact Tmed2 homozygous mutant embryos arrest at mid-gestation due to a failure of placental labyrinth layer formation. Differentiation of the placental labyrinth layer depends on chorioallantoic attachment (contact between the chorion and allantois), and branching morphogenesis (mingling of cells from these two tissues). Since Tmed2 mRNA was found in both the chorion and allantois, and 50% of Tmed2 homozygous mutant embryos failed to undergo chorioallantoic attachment, the tissue-specific requirement of Tmed2 during placental labyrinth layer formation remained a mystery. Herein, we report differential localization of TMED2 protein in the chorion and allantois, abnormal ER retention of Fibronectin in Tmed2 homozygous mutant allantoises and cell-autonomous requirement for Tmed2 in the chorion for chorioallantoic attachment and fusion. Using an ex vivo model of explanted chorions and allantoises, we showed that chorioallantoic attachment failed to occur in 50% of samples when homozygous mutant chorions were recombined with wild type allantoises. Furthermore, though expression of genes associated with trophoblast differentiation was maintained in Tmed2 mutant chorions with chorioallantoic attachment, expression of these genes was attenuated. In addition, Tmed2 homozygous mutant allantoises could undergo branching morphogenesis, however the region of mixing between mutant and wild type cells was reduced, and expression of genes associated with trophoblast differentiation was also attenuated. Our data also suggest that Fibronectin is a cargo protein of TMED2 and indicates that Tmed2 is required cell-autonomously and non-autonomously in the chorion and the allantois for placental labyrinth layer formation.

Low Dietary Folate Interacts with MTHFD1 Synthetase Deficiency in Mice, a Model for the R653Q Variant, to Increase Incidence of Developmental Delays and Defects.

Background: Suboptimal folate intake, a risk factor for birth defects, is common even in areas with folate fortification. A polymorphism in methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), R653Q (MTHFD1 c.1958 G > A), has also been associated with increased birth defect risk, likely through reduced purine synthesis. Objective: We aimed to determine if the interaction of MTHFD1 synthetase deficiency and low folate intake increases developmental abnormalities in a mouse model for MTHFD1 R653Q. Methods: Female Mthfd1S+/+ and Mthfd1S+/- mice were fed control or low-folate diets (2 and 0.3 mg folic acid/kg diet, respectively) before mating and during pregnancy. Embryos and placentas were examined for anomalies at embryonic day 10.5. Maternal 1-carbon metabolites were measured in plasma and liver. Results: Delays and defects doubled in litters of Mthfd1S+/- females fed low-folate diets compared to wild-type females fed either diet, or Mthfd1S+/- females fed control diets [P values (defects): diet 0.003, maternal genotype 0.012, diet × maternal genotype 0.014]. These adverse outcomes were associated with placental dysmorphology. Intrauterine growth restriction was increased by embryonic Mthfd1S+/- genotype, folate deficiency, and interaction of maternal Mthfd1S+/- genotype with folate deficiency (P values: embryonic genotype 0.045, diet 0.0081, diet × maternal genotype 0.0019). Despite a 50% increase in methylenetetrahydrofolate reductase expression in low-folate maternal liver (P diet = 0.0007), methyltetrahydrofolate concentration decreased 70% (P diet <0.0001) and homocysteine concentration doubled in plasma (P diet = 0.0001); S-adenosylmethionine decreased 40% and S-adenosylhomocysteine increased 20% in low-folate maternal liver (P diet = 0.002 and 0.0002, respectively). Conclusions: MTHFD1 synthetase-deficient mice are more sensitive to low folate intake than wild-type mice during pregnancy. Reduced purine synthesis due to synthetase deficiency and altered methylation potential due to low folate may increase pregnancy complications. Further studies and individualized intake recommendations may be required for women homozygous for the MTHFD1 R653Q variant.

Non-alcoholic fatty liver disease in mice with heterozygous mutation in TMED2.

The transmembrane emp24 domain/p24 (TMED) family are essential components of the vesicular transport machinery. Members of the TMED family serve as cargo receptors implicated in selection and packaging of endoplasmic reticulum (ER) luminal proteins into coatomer (COP) II coated vesicles for anterograde transport to the Golgi. Deletion or mutations of Tmed genes in yeast and Drosophila results in ER-stress and activation of the unfolded protein response (UPR). The UPR leads to expression of genes and proteins important for expanding the folding capacity of the ER, degrading misfolded proteins, and reducing the load of new proteins entering the ER. The UPR is activated in non-alcoholic fatty liver disease (NAFLD) in human and mouse and may contribute to the development and the progression of NAFLD. Tmed2, the sole member of the vertebrate Tmed ß subfamily, exhibits tissue and temporal specific patterns of expression in embryos and developing placenta but is ubiquitously expressed in all adult organs. We previously identified a single point mutation, the 99J mutation, in the signal sequence of Tmed2 in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Histological and molecular analysis of livers from heterozygous mice carrying the 99J mutation, Tmed299J/+, revealed a requirement for TMED2 in liver health. We show that Tmed299J/+ mice had decreased levels of TMED2 and TMED10, dilated endoplasmic reticulum membrane, and increased phosphorylation of eIF2α, indicating ER-stress and activation of the UPR. Increased expression of Srebp1a and 2 at the newborn stage and increased incidence of NAFLD were also found in Tmed299J/+ mice. Our data establishes Tmed299J/+ mice as a novel mouse model for NAFLD and supports a role for TMED2 in liver health.

Moderate folic acid supplementation and MTHFD1-synthetase deficiency in mice, a model for the R653Q variant, result in embryonic defects and abnormal placental development.

BACKGROUND: Moderately high folic acid intake in pregnant women has led to concerns about deleterious effects on the mother and fetus. Common polymorphisms in folate genes, such as methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase (MTHFD1) R653Q, may modulate the effects of elevated folic acid intake. OBJECTIVES: We investigated the effects of moderate folic acid supplementation on reproductive outcomes and assessed the potential interaction of the supplemented diet with MTHFD1-synthetase (Mthfd1S) deficiency in mice, which is a model for the R653Q variant. DESIGN: Female Mthfd1S+/+ and Mthfd1S+/- mice were fed a folic acid-supplemented diet (FASD) (5-fold higher than recommended) or control diets before mating and during pregnancy. Embryos and placentas were assessed for developmental defects at embryonic day 10.5 (E10.5). Maternal folate and choline metabolites and gene expression in folate-related pathways were examined. RESULTS: The combination of FASD and maternal MTHFD1-synthetase deficiency led to a greater incidence of defects in E10.5 embryos (diet × maternal genotype, P = 0.0016; diet × embryonic genotype, P = 0.054). The methylenetetrahydrofolate reductase (MTHFR) protein and methylation potential [ratio of S-adenosylmethionine (major methyl donor):S-adenosylhomocysteine) were reduced in maternal liver. Although 5-methyltetrahydrofolate (methylTHF) was higher in maternal circulation, the methylation potential was lower in embryos. The presence of developmental delays and defects in Mthfd1S+/- embryos was associated with placental defects (P = 0.003). The labyrinth layer failed to form properly in the majority of abnormal placentas, which compromised the integration of the maternal and fetal circulation and presumably the transfer of methylTHF and other nutrients. CONCLUSIONS: Moderately higher folate intake and MTHFD1-synthetase deficiency in pregnant mice result in a lower methylation potential in maternal liver and embryos and a greater incidence of defects in embryos. Although maternal circulating methylTHF was higher, it may not have reached the embryos because of abnormal placental development; abnormal placentas were observed predominantly in abnormally developed embryos. These findings have implications for women with high folate intakes, particularly if they are polymorphic for MTHFD1 R653Q.