Human ABL1 deficiency syndrome (HADS) is a recognizable syndrome distinct from ABL1-related congenital heart defects and skeletal malformations syndrome.

Germline gain of function variants in the oncogene ABL1 cause congenital heart defects and skeletal malformations (CHDSKM) syndrome. Whether a corresponding ABL1 deficiency disorder exists in humans remains unknown although developmental defects in mice deficient for Abl1 support this notion. Here, we describe two multiplex consanguineous families, each segregating a different homozygous likely loss of function variant in ABL1. The associated phenotype is multiple congenital malformations and distinctive facial dysmorphism that are opposite in many ways to CHDSKM. We suggest that a tight balance of ABL1 activity is required during embryonic development and that both germline gain of function and loss of function variants result in distinctively different allelic congenital malformation disorders.

Whole-exome sequencing identifies cancer-associated variants of the endo-lysosomal ion transport channels in the Saudi population.

Background: Although national efforts are underway to document the genomic variability of the Saudi population relative to other populations, such variability remains largely unexplored. Genetic variability is known to impact the fate of cells and increase or decrease the risk of a variety of complex diseases including cancer forms. Therefore, the identification of variants associated with cancer susceptibility in Saudi population may protect individuals from cancer or aid in patient-tailored therapies. The endo-lysosomal ion transport genes responsible for cationic ion homeostasis within the cell. We screened 703 single-nucleotide polymorphisms (SNPs) of the endo-lysosomal ion transporter genes in the Saudi population and identified cancer-associated variants that have been reported in other populations. Methods: Utilizing previously derived local data of Whole-Exome Sequencing (WES), we examined SNPs of TPCN1, TPCN2, P2RX4, TRPM7, TRPV4, TRPV4, and TRPV6 genes. The SNPs were identified for those genes by our in-house database. We predicted the pathogenicity of these variants using in silico tools CADD, Polyphen-2, SIFT, PrimateAI, and FATHMM-XF. Then, we validated our findings by exploring the genetics database (VarSome, dbSNP NCB, OMIM, ClinVar, Ensembl, and GWAS Catalog) to further link cancer risk. Results: The WES database yielded 703 SNPs found in TPCN2, P2RX4, TRPM7, TRPV4, and TRPV6 genes in 1,144 subjects. The number of variants that were found to be common in our population was 150 SNPs. We identified 13 coding-region non-synonymous variants of the endo-lysosomal genes that were most common with a minor allele frequency (MAF) of ≥ 1 %. Twelve of these variants are rs2376558, rs3750965, rs61746574, rs35264875, rs3829241, rs72928978, rs25644, rs8042919, rs17881456, rs4987682, rs4987667, and rs4987657 that were classified as cancer-associated genes. Conclusion: Our study highlighted cancer-associated SNPs in the endo-lysosomal genes among Saudi individuals. The allelic frequencies on polymorphic variants confer susceptibility to complex diseases that are comparable to other populations. There is currently insufficient clinical data supporting the link between these SNPs and cancer risk in the Saudi population. Our data argues for initiating future cohort studies in which individuals with the identified SNPs are monitored and assessed for their likelihood of developing malignancies and therapy outcomes.

Biallelic variation in the choline and ethanolamine transporter FLVCR1 underlies a pleiotropic disease spectrum from adult neurodegeneration to severe developmental disorders.

FLVCR1 encodes Feline leukemia virus subgroup C receptor 1 (FLVCR1), a solute carrier (SLC) transporter within the Major Facilitator Superfamily. FLVCR1 is a widely expressed transmembrane protein with plasma membrane and mitochondrial isoforms implicated in heme, choline, and ethanolamine transport. While Flvcr1 knockout mice die in utero with skeletal malformations and defective erythropoiesis reminiscent of Diamond-Blackfan anemia, rare biallelic pathogenic FLVCR1 variants are linked to childhood or adult-onset neurodegeneration of the retina, spinal cord, and peripheral nervous system. We ascertained from research and clinical exome sequencing 27 individuals from 20 unrelated families with biallelic ultra-rare missense and predicted loss-of-function (pLoF) FLVCR1 variant alleles. We characterize an expansive FLVCR1 phenotypic spectrum ranging from adult-onset retinitis pigmentosa to severe developmental disorders with microcephaly, reduced brain volume, epilepsy, spasticity, and premature death. The most severely affected individuals, including three individuals with homozygous pLoF variants, share traits with Flvcr1 knockout mice and Diamond-Blackfan anemia including macrocytic anemia and congenital skeletal malformations. Pathogenic FLVCR1 missense variants primarily lie within transmembrane domains and reduce choline and ethanolamine transport activity compared with wild-type FLVCR1 with minimal impact on FLVCR1 stability or subcellular localization. Several variants disrupt splicing in a mini-gene assay which may contribute to genotype-phenotype correlations. Taken together, these data support an allele-specific gene dosage model in which phenotypic severity reflects residual FLVCR1 activity. This study expands our understanding of Mendelian disorders of choline and ethanolamine transport and demonstrates the importance of choline and ethanolamine in neurodevelopment and neuronal homeostasis.

PMEL is mutated in oculocutaneous albinism.

Oculocutaneous albinism (OCA) is a group of Mendelian disorders characterized by hypopigmentation of skin, hair and pigmented ocular structures. While much of the genetic heterogeneity of OCA has been resolved, many patients still lack a molecular diagnosis following exome sequencing. Here, we report a consanguineous family in which the index patient presented with OCA and Hirschsprung disease but tested negative for known genetic causes of OCA. Instead, he was found to have a homozygous presumptive loss of function variant in PMEL. PMEL encodes a scaffolding protein that is essential for the normal maturation of melanosomes and normal deposition of the melanin pigment therein. Numerous PMEL vertebrate ortholog mutants have been reported and all were characterized by conspicuous pigmentary abnormalities. We suggest that the patient we report is the first human equivalent of PMEL loss of function.

A novel DPH5-related diphthamide-deficiency syndrome causing embryonic lethality or profound neurodevelopmental disorder.

PURPOSE: Diphthamide is a post-translationally modified histidine essential for messenger RNA translation and ribosomal protein synthesis. We present evidence for DPH5 as a novel cause of embryonic lethality and profound neurodevelopmental delays (NDDs). METHODS: Molecular testing was performed using exome or genome sequencing. A targeted Dph5 knockin mouse (C57BL/6Ncrl-Dph5em1Mbp/Mmucd) was created for a DPH5 p.His260Arg homozygous variant identified in 1 family. Adenosine diphosphate-ribosylation assays in DPH5-knockout human and yeast cells and in silico modeling were performed for the identified DPH5 potential pathogenic variants. RESULTS: DPH5 variants p.His260Arg (homozygous), p.Asn110Ser and p.Arg207Ter (heterozygous), and p.Asn174LysfsTer10 (homozygous) were identified in 3 unrelated families with distinct overlapping craniofacial features, profound NDDs, multisystem abnormalities, and miscarriages. Dph5 p.His260Arg homozygous knockin was embryonically lethal with only 1 subviable mouse exhibiting impaired growth, craniofacial dysmorphology, and multisystem dysfunction recapitulating the human phenotype. Adenosine diphosphate-ribosylation assays showed absent to decreased function in DPH5-knockout human and yeast cells. In silico modeling of the variants showed altered DPH5 structure and disruption of its interaction with eEF2. CONCLUSION: We provide strong clinical, biochemical, and functional evidence for DPH5 as a novel cause of embryonic lethality or profound NDDs with multisystem involvement and expand diphthamide-deficiency syndromes and ribosomopathies.

Misregulation of the expression and activity of DNA methyltransferases in cancer.

In mammals, DNA methyltransferases DNMT1 and DNMT3's (A, B and L) deposit and maintain DNA methylation in dividing and nondividing cells. Although these enzymes have an unremarkable DNA sequence specificity (CpG), their regional specificity is regulated by interactions with various protein factors, chromatin modifiers, and post-translational modifications of histones. Changes in the DNMT expression or interacting partners affect DNA methylation patterns. Consequently, the acquired gene expression may increase the proliferative potential of cells, often concomitant with loss of cell identity as found in cancer. Aberrant DNA methylation, including hypermethylation and hypomethylation at various genomic regions, therefore, is a hallmark of most cancers. Additionally, somatic mutations in DNMTs that affect catalytic activity were mapped in Acute Myeloid Leukemia cancer cells. Despite being very effective in some cancers, the clinically approved DNMT inhibitors lack specificity, which could result in a wide range of deleterious effects. Elucidating distinct molecular mechanisms of DNMTs will facilitate the discovery of alternative cancer therapeutic targets. This review is focused on: (i) the structure and characteristics of DNMTs, (ii) the prevalence of mutations and abnormal expression of DNMTs in cancer, (iii) factors that mediate their abnormal expression and (iv) the effect of anomalous DNMT-complexes in cancer.

The acute myeloid leukemia variant DNMT3A Arg882His is a DNMT3B-like enzyme.

We have previously shown that the highly prevalent acute myeloid leukemia (AML) mutation, Arg882His, in DNMT3A disrupts its cooperative mechanism and leads to reduced enzymatic activity, thus explaining the genomic hypomethylation in AML cells. However, the underlying cause of the oncogenic effect of Arg882His in DNMT3A is not fully understood. Here, we discovered that DNMT3A WT enzyme under conditions that favor non-cooperative kinetic mechanism as well as DNMT3A Arg882His variant acquire CpG flanking sequence preference akin to that of DNMT3B, which is non-cooperative. We tested if DNMT3A Arg882His could preferably methylate DNMT3B-specific target sites in vivo. Rescue experiments in Dnmt3a/3b double knockout mouse embryonic stem cells show that the corresponding Arg878His mutation in mouse DNMT3A severely impairs its ability to methylate major satellite DNA, a DNMT3A-preferred target, but has no overt effect on the ability to methylate minor satellite DNA, a DNMT3B-preferred target. We also observed a previously unappreciated CpG flanking sequence bias in major and minor satellite repeats that is consistent with DNMT3A and DNMT3B specificity suggesting that DNA methylation patterns are guided by the sequence preference of these enzymes. We speculate that aberrant methylation of DNMT3B target sites could contribute to the oncogenic potential of DNMT3A AML variant.

Oct4-Mediated Inhibition of Lsd1 Activity Promotes the Active and Primed State of Pluripotency Enhancers.

An aberrant increase in pluripotency gene (PpG) expression due to enhancer reactivation could induce stemness and enhance the tumorigenicity of cancer stem cells. Silencing of PpG enhancers (PpGe) during embryonic stem cell differentiation involves Lsd1-mediated H3K4me1 demethylation and DNA methylation. Here, we observed retention of H3K4me1 and DNA hypomethylation at PpGe associated with a partial repression of PpGs in F9 embryonal carcinoma cells (ECCs) post-differentiation. H3K4me1 demethylation in F9 ECCs could not be rescued by Lsd1 overexpression. Given our observation that H3K4me1 demethylation is accompanied by strong Oct4 repression in P19 ECCs, we tested if Oct4 interaction with Lsd1 affects its catalytic activity. Our data show a dose-dependent inhibition of Lsd1 activity by Oct4 and retention of H3K4me1 at PpGe in Oct4-overexpressing P19 ECCs. These data suggest that Lsd1-Oct4 interaction in cancer stem cells could establish a "primed" enhancer state that is susceptible to reactivation, leading to aberrant PpG expression.

The transcription factor Vezf1 represses the expression of the antiangiogenic factor Cited2 in endothelial cells.

Formation of the vasculature by angiogenesis is critical for proper development, but angiogenesis also contributes to the pathogenesis of various disorders, including cancer and cardiovascular diseases. Vascular endothelial zinc finger 1 (Vezf1), is a Krüppel-like zinc finger protein that plays a vital role in vascular development. However, the mechanism by which Vezf1 regulates this process is not fully understood. Here, we show that Vezf1-/- mouse embryonic stem cells (ESC) have significantly increased expression of a stem cell factor, Cbp/p300-interacting transactivator 2 (Cited2). Compared with WT ESCs, Vezf1-/- ESCs inefficiently differentiated into endothelial cells (ECs), which exhibited defects in the tube-formation assay. These defects were due to reduced activation of EC-specific genes concomitant with lower enrichment of histone 3 acetylation at Lys27 (H3K27) at their promoters. We hypothesized that overexpression of Cited2 in Vezf1-/- cells sequesters P300/CBP away from the promoters of proangiogenic genes and thereby contributes to defective angiogenesis in these cells. This idea was supported by the observation that shRNA-mediated depletion of Cited2 significantly reduces the angiogenic defects in the Vezf1-/- ECs. In contrast to previous studies that have focused on the role of Vezf1 as a transcriptional activator of proangiogenic genes, our findings have revealed a role for Vezf1 in modulating the expression of the antiangiogenic factor Cited2. Vezf1 previously has been characterized as an insulator protein, and our results now provide insights into the mechanism, indicating that Vezf1 can block inappropriate, nonspecific interactions of promoters with cis-located enhancers, preventing aberrant promoter activation.

Dnmt3b Methylates DNA by a Noncooperative Mechanism, and Its Activity Is Unaffected by Manipulations at the Predicted Dimer Interface.

The catalytic domains of the de novo DNA methyltransferases Dnmt3a-C and Dnmt3b-C are highly homologous. However, their unique biochemical properties could potentially contribute to differences in the substrate preferences or biological functions of these enzymes. Dnmt3a-C forms tetramers through interactions at the dimer interface, which also promote multimerization on DNA and cooperativity. Similar to the case for processive enzymes, cooperativity allows Dnmt3a-C to methylate multiple sites on the same DNA molecule; however, it is unclear whether Dnmt3b-C methylates DNA by a cooperative or processive mechanism. The importance of the tetramer structure and cooperative mechanism is emphasized by the observation that the R882H mutation in the dimer interface of DNMT3A is highly prevalent in acute myeloid leukemia and leads to a substantial loss of its activity. Under conditions that distinguish between cooperativity and processivity, we show that in contrast to that of Dnmt3a-C, the activity of Dnmt3b-C is not cooperative and confirm the processivity of Dnmt3b-C and the full length Dnmt3b enzyme. Whereas the R878H mutation (mouse homologue of R882H) led to the loss of cooperativity of Dnmt3a-C, the activity and processivity of the analogous Dnmt3b-C R829H variant were comparable to those of the wild-type enzyme. Additionally, buffer acidification that attenuates the dimer interface interactions of Dnmt3a-C had no effect on Dnmt3b-C activity. Taken together, these results demonstrate an important mechanistic difference between Dnmt3b and Dnmt3a and suggest that interactions at the dimer interface may play a limited role in regulating Dnmt3b-C activity. These new insights have potential implications for the distinct biological roles of Dnmt3a and Dnmt3b.