The epitranscriptome toolbox.

In the last decade, the notion that mRNA modifications are involved in regulation of gene expression was demonstrated in thousands of studies. To date, new technologies and methods allow accurate identification, transcriptome-wide mapping, and functional characterization of a growing number of RNA modifications, providing important insights into the biology of these marks. Most of the methods and approaches were developed for studying m6A, the most prevalent internal mRNA modification. However, unique properties of other RNA modifications stimulated the development of additional approaches. In this technical primer, we will discuss the available tools and approaches for detecting and studying different RNA modifications.

N4-acetylation of Cytidine in mRNA by NAT10 Regulates Stability and Translation.

Arango et al. expand the repertoire of epitranscriptomic modifications by identifying N4-acetylcytidine in mRNA catalyzed by the known dual acetyltransferase NAT10. It occurs mainly in the coding sequence, likely in wobble positions of select codons, where it promotes stability and translation, possibly by safeguarding cognate codon-anticodon interaction.

The m6A epitranscriptome: transcriptome plasticity in brain development and function.

The field of epitranscriptomics examines the recently deciphered form of gene expression regulation that is mediated by type- and site-specific RNA modifications. Similarly to the role played by epigenetic mechanisms - which operate via DNA and histone modifications - epitranscriptomic modifications are involved in the control of the delicate gene expression patterns that are needed for the development and activity of the nervous system and are essential for basic and higher brain functions. Here we describe the mechanisms that are involved in the writing, erasing and reading of N6-methyladenosine, the most prevalent internal mRNA modification, and the emerging roles played by N6-methyladenosine in the nervous system.

Loud and Clear Epitranscriptomic m1A Signals: Now in Single-Base Resolution.

In this issue of Molecular Cell, Li et al. (2017) map N1-methyladenosine (m1A) with base precision, parsing the methylome into subsets differing in location, sequence-structure features, and catalyzing enzymes, thereby aiding functional investigation.

Whole-exome sequencing reveals a monogenic cause in 56% of individuals with laterality disorders and associated congenital heart defects.

BACKGROUND: The molecular basis of heterotaxy and congenital heart malformations associated with disruption of left-right asymmetry is broad and heterogenous, with over 25 genes implicated in its pathogenesis thus far. OBJECTIVE: We sought to elucidate the molecular basis of laterality disorders and associated congenital heart defects in a cohort of 30 unrelated probands of Arab-Muslim descent, using next-generation sequencing techniques. METHODS: Detailed clinical phenotyping followed by whole-exome sequencing (WES) was pursued for each of the probands and their parents (when available). Sanger sequencing was used for segregation analysis of disease-causing mutations in the families. RESULTS: Using WES, we reached a molecular diagnosis for 17 of the 30 probands (56.7%). Genes known to be associated with heterotaxy and/or primary ciliary dyskinesia, in which homozygous pathogenic or likely pathogenic variants were detected, included CFAP53 (CCDC11), CFAP298 (C21orf59), CFAP300, LRRC6, GDF1, DNAAF1, DNAH5, CCDC39, CCDC40, PKD1L1 and TTC25. Additionally, we detected a homozygous disease causing mutation in DAND5, as a novel recessive monogenic cause for heterotaxy in humans. Three additional probands were found to harbour variants of uncertain significance. These included variants in DNAH6, HYDIN, CELSR1 and CFAP46. CONCLUSIONS: Our findings contribute to the current knowledge regarding monogenic causes of heterotaxy and its associated congenital heart defects and underscore the role of next-generation sequencing techniques in the diagnostic workup of such patients, and especially among consanguineous families.

RNA modifications: what have we learned and where are we headed?

Proper control of the transcriptome is key for diverse aspects of gene expression, cellular functions and development, and its disruption can result in disease. A rapidly accumulating wealth of studies are identifying and functionally characterizing diverse types of RNA base modifications in protein-coding and non-coding RNAs, which have energized the emerging field of 'epitranscriptomics'. In this Viewpoint article, five experts discuss our latest understanding of RNA modifications, including recommendations for best practices and visions for the future.

A multidisciplinary nephrogenetic referral clinic for children and adults-diagnostic achievements and insights.

BACKGROUND: Genetic kidney diseases contribute a significant portion of kidney diseases in children and young adults. Nephrogenetics is a rapidly evolving subspecialty; however, in the clinical setting, increased use of genetic testing poses implementation challenges. Consequently, we established a national nephrogenetics clinic to apply a multidisciplinary model. METHODS: Patients were referred from different pediatric or adult nephrology units across the country if their primary nephrologist suspected an undiagnosed genetic kidney disease. We determined the diagnostic rate and observed the effect of diagnosis on medical care. We also discuss the requirements of a nephrogenetics clinic in terms of logistics, recommended indications for referral, and building a multidisciplinary team. RESULTS: Over 24 months, genetic evaluation was completed for a total of 74 unrelated probands, with an age range of 10 days to 72 years. The most common phenotypes included congenital anomalies of the kidneys and urinary tract, nephrotic syndrome or unexplained proteinuria, nephrocalcinosis/nephrolithiasis, tubulopathies, and unexplained kidney failure. Over 80% of patients were referred due to clinical suspicion of an undetermined underlying genetic diagnosis. A molecular diagnosis was reached in 42/74 probands, yielding a diagnostic rate of 57%. Of these, over 71% of diagnoses were made via next generation sequencing (gene panel or exome sequencing). CONCLUSIONS: We identified a substantial fraction of genetic kidney etiologies among previously undiagnosed individuals which influenced subsequent clinical management. Our results support that nephrogenetics, a rapidly evolving field, may benefit from well-defined multidisciplinary co-management administered by a designated team of nephrologist, geneticist, and bioinformatician. A higher resolution version of the Graphical abstract is available as Supplementary information.

The dynamic N(1)-methyladenosine methylome in eukaryotic messenger RNA.

Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N(6)-methyladenosine (m(6)A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N(1)-methyladenosine (m(1)A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m(1)A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m(1)A in promoting translation of methylated mRNA.

Atypical immune phenotype in severe combined immunodeficiency patients with novel mutations in IL2RG and JAK3.

Mutations in the common gamma chain of the interleukin 2 receptor (IL2RG) or the associated downstream signaling enzyme Janus kinase 3 (JAK3) genes are typically characterized by a T cell-negative, B cell-positive, natural killer (NK) cell-negative (T-B+NK-) severe combined immunodeficiency (SCID) immune phenotype. We report clinical course, immunological, genetic and proteomic work-up of two patients with different novel mutations in the IL-2-JAK3 pathway with a rare atypical presentation of T-B+NK- SCID. Lymphocyte subpopulation revealed significant T cells lymphopenia, normal B cells, and NK cells counts (T-B+NK+SCID). Despite the presence of B cells, IgG levels were low and IgA and IgM levels were undetectable. T-cell proliferation in response to mitogens in patient 1 was very low and T-cell receptor V-beta chain repertoire in patient 2 was polyclonal. Whole-exome sequencing revealed novel mutations in both patients (patient 1-c.923delC frame-shift mutation in the IL2RG gene, patient 2-c.G172A a homozygous missense mutation in the JAK3 gene). Bioinformatic analysis of the JAK3 mutation indicated deleterious effect and 3D protein modeling located the mutation to a surface exposed alpha-helix structure. Our findings help to link between genotype and phenotype, which is a key factor for the diagnosis and treatment of SCID patients.

Netrin-G2 dysfunction causes a Rett-like phenotype with areflexia.

We describe the underlying genetic cause of a novel Rett-like phenotype accompanied by areflexia in three methyl-CpG-binding protein 2-negative individuals from two unrelated families. Discovery analysis was performed using whole-exome sequencing followed by Sanger sequencing for validation and segregation. Functional studies using short-hairpin RNA for targeted gene knockdown were implemented by the transfection of mouse cultured primary hippocampal neurons and in vivo by in utero electroporation. All patients shared a common homozygous frameshift mutation (chr9:135073515, c.376dupT, p.(Ser126PhefsTer241)) in netrin-G2 (NTNG2, NM_032536.3) with predicted nonsense-mediated decay. The mutation fully segregated with the disease in both families. The knockdown of either NTNG2 or the related netrin-G family member NTNG1 resulted in severe neurodevelopmental defects of neuronal morphology and migration. While NTNG1 has previously been linked to a Rett syndrome (RTT)-like phenotype, this is the first description of a RTT-like phenotype caused by NTNG2 mutation. Netrin-G proteins have been shown to be required for proper axonal guidance during early brain development and involved in N-methyl- d-aspartate-mediated synaptic transmission. Our results demonstrating that knockdown of murine NTNG2 causes severe impairments of neuronal morphology and cortical migration are consistent with those of RTT animal models and the shared neurodevelopmental phenotypes between the individuals described here and typical RTT patients.

Whole exome sequencing (WES) approach for diagnosing primary immunodeficiencies (PIDs) in a highly consanguineous community.

Primary immunodeficiencies (PIDs) are a heterogeneous group of monogenic inborn errors of immunity. The genetic causes of these diseases can be identified using whole exome sequencing (WES). Here, DNA samples from 106 patients with a clinical suspicion of PID were subjected to WES in order to test the diagnostic yield of this test in a highly consanguineous community. A likely genetic diagnosis was achieved in 70% of patients. Several factors were considered to possibly influence the diagnostic rate of WES among our cohort including early age, presence of consanguinity, family history suggestive of PID, the number of family members who underwent WES and the clinical phenotype of the patient. The highest diagnostic rate was in patients with combined immunodeficiency or with a syndrome. Notably, WES findings altered the clinical management in 39% (41/106) of patients in our cohort. Our findings support the use of WES as an important diagnostic tool in patients with suspected PID, especially in highly consanguineous communities.

Nm-seq maps 2'-O-methylation sites in human mRNA with base precision.

The ribose of RNA nucleotides can be 2'-O-methylated (Nm). Despite advances in high-throughput detection, the inert chemical nature of Nm still limits sensitivity and precludes mapping in mRNA. We leveraged the differential reactivity of 2'-O-methylated and 2'-hydroxylated nucleosides to periodate oxidation to develop Nm-seq, a sensitive method for transcriptome-wide mapping of Nm with base precision. Nm-seq uncovered thousands of Nm sites in human mRNA with features suggesting functional roles.

A founder truncating variant in GDF1 causes autosomal-recessive right isomerism and associated congenital heart defects in multiplex Arab kindreds.

The genetic basis of congenital heart malformations associated with disruption of left-right (L-R) asymmetry is broad and heterogenous, with variants in over 25 genes implicated thus far. Of these, deleterious variants in the Growth/Differentiation Factor 1 (GDF1) gene have been shown to cause heterotaxy with varied complex heart malformations of left-right patterning, in 23 individuals reported to date, either in monoallelic or biallelic state. We report three unrelated individuals exhibiting right isomerism with congenital heart defects, each originating from a consanguineous kindred of Arab-Muslim descent. Using whole exome sequencing, a shared novel homozygous truncating c.608G > A (p.W203*) variant in the GDF1 gene was revealed as the molecular basis of their disease. Subsequently, targeted sequencing of this variant showed full segregation with the disease in these families, with a total of over 15 reportedly affected individuals, enabling genetic counseling, prenatal diagnosis, and planning of future pregnancies. Our findings further confirm the association of biallelic GDF1 variants, heterotaxy and congenital heart defects of left-right patterning, and expand the previously described phenotypic spectrum and mutational profile. Moreover, we suggest targeted screening for the p.W203* variant in relevant clinical circumstances.

Gene expression regulation mediated through reversible m6A RNA methylation.

Cellular RNAs carry diverse chemical modifications that used to be regarded as static and having minor roles in 'fine-tuning' structural and functional properties of RNAs. In this Review, we focus on reversible methylation through the most prevalent mammalian mRNA internal modification, N(6)-methyladenosine (m(6)A). Recent studies have discovered protein 'writers', 'erasers' and 'readers' of this RNA chemical mark, as well as its dynamic deposition on mRNA and other types of nuclear RNA. These findings strongly indicate dynamic regulatory roles that are analogous to the well-known reversible epigenetic modifications of DNA and histone proteins. This reversible RNA methylation adds a new dimension to the developing picture of post-transcriptional regulation of gene expression.

Congenital neutropenia with variable clinical presentation in novel mutation of the SRP54 gene.

BACKGROUND: The SRP54 (signal recognition protein 54) is a conserved component of the ribonucleoprotein complex that mediates cotranslational targeting and translocation of proteins to the endoplasmic reticulum. In 2017, mutations in the gene have been described as a cause of congenital neutropenia with or without pancreatic insufficiency, and since then, only limited cases were added to the literature. METHODS: Two patients with neutropenia underwent hematological, immunological, and genetic work-up, including lymphocyte phenotyping, immunoglobulins, and complement levels, antineutrophil and antinuclear antibodies, bone marrow FISH panel for myelodysplastic syndrome, whole-exome sequencing, and in silico proteomic analysis. RESULTS: Clinical findings in the two families revealed a wide spectrum of immunological and clinical manifestations, ranging from mild asymptomatic neutropenia during febrile illnesses to severe neutropenia and life-threatening infection requiring leg amputation. Immunological and hematological work-up showed isolated neutropenia with normal lymphocyte subpopulations, immunoglobulin and complement levels, and negative autoimmune tests. Bone marrow aspirations showed variability ranging from normal myelopoiesis to myeloid maturation arrest at the promyelocytic stage, with normal FISH panel for myelodysplastic syndrome. Genetic analysis identified a novel, de novo, in-frame deletion in the SRP54 gene, c.342-344delAAC, p.T115del. In silico proteomic analysis suggested impaired SRP54 protein function due to reduced GTP activity and stability. CONCLUSIONS: We describe congenital neutropenia with variable clinical presentation in novel mutation of the SRP54 gene.

The Duffy antigen receptor for chemokines, ACKR1,- 'Jeanne DARC' of benign neutropenia.

Benign neutropenia, observed in different ethnic groups, is the most common form of neutropenia worldwide. A specific single nucleotide polymorphism, rs2814778, located at the promoter of the ACKR1 (previously termed DARC) gene, which disrupts a binding site for the GATA1 erythroid transcription factor, resulting in a ACKR1-null phenotype, was found to serve as a predictor of low white blood cell and neutrophil counts in African-Americans and Yemenite Jews. Individuals with benign neutropenia due to the ACKR1-null allele have been found to have an increased susceptibility to human immunodeficiency virus infection and, on the other hand, a protective effect against malaria. The associated protective effect may explain the spread of the ACKR1-null allele by natural selection. The reviewed relationships between ACKR1 polymorphism and various pathological states may have important clinical implications to individuals with and without benign neutropenia. Potential mechanisms for ACKR1 (previously termed DARC) modulation during neutrophil recruitment to inflammation, and chemokine bioavailability in the circulation and in local tissue are reviewed and discussed.

Disruption of Thrombocyte and T Lymphocyte Development by a Mutation in ARPC1B.

Regulation of the actin cytoskeleton is crucial for normal development and function of the immune system, as evidenced by the severe immune abnormalities exhibited by patients bearing inactivating mutations in the Wiskott-Aldrich syndrome protein (WASP), a key regulator of actin dynamics. WASP exerts its effects on actin dynamics through a multisubunit complex termed Arp2/3. Despite the critical role played by Arp2/3 as an effector of WASP-mediated control over actin polymerization, mutations in protein components of the Arp2/3 complex had not previously been identified as a cause of immunodeficiency. Here, we describe two brothers with hematopoietic and immunologic symptoms reminiscent of Wiskott-Aldrich syndrome (WAS). However, these patients lacked mutations in any of the genes previously associated with WAS. Whole-exome sequencing revealed a homozygous 2 bp deletion, n.c.G623DEL-TC (p.V208VfsX20), in Arp2/3 complex component ARPC1B that causes a frame shift resulting in premature termination. Modeling of the disease in zebrafish revealed that ARPC1B plays a critical role in supporting T cell and thrombocyte development. Moreover, the defects in development caused by ARPC1B loss could be rescued by the intact human ARPC1B ortholog, but not by the p.V208VfsX20 variant identified in the patients. Moreover, we found that the expression of ARPC1B is restricted to hematopoietic cells, potentially explaining why a mutation in ARPC1B has now been observed as a cause of WAS, whereas mutations in other, more widely expressed, components of the Arp2/3 complex have not been observed.

Two phases of mitogenic signaling unveil roles for p53 and EGR1 in elimination of inconsistent growth signals.

Normal cells require continuous exposure to growth factors in order to cross a restriction point and commit to cell-cycle progression. This can be replaced by two short, appropriately spaced pulses of growth factors, where the first pulse primes a process, which is completed by the second pulse, and enables restriction point crossing. Through integration of comprehensive proteomic and transcriptomic analyses of each pulse, we identified three processes that regulate restriction point crossing: (1) The first pulse induces essential metabolic enzymes and activates p53-dependent restraining processes. (2) The second pulse eliminates, via the PI3K/AKT pathway, the suppressive action of p53, as well as (3) sets an ERK-EGR1 threshold mechanism, which digitizes graded external signals into an all-or-none decision obligatory for S phase entry. Together, our findings uncover two gating mechanisms, which ensure that cells ignore fortuitous growth factors and undergo proliferation only in response to consistent mitogenic signals.

Somatic NRAS mutation in patient with generalized lymphatic anomaly.

Generalized lymphatic anomaly (GLA or lymphangiomatosis) is a rare disease characterized by a diffuse proliferation of lymphatic vessels in skin and internal organs. It often leads to progressive respiratory failure and death, but its etiology is unknown. Here, we isolated lymphangiomatosis endothelial cells from GLA tissue. These cells were characterized by high proliferation and survival rates, but displayed impaired capacities for migration and tube formation. We employed whole exome sequencing to search for disease-causing genes and identified a somatic mutation in NRAS. We used mouse and zebrafish model systems to initially evaluate the role of this mutation in the development of the lymphatic system, and we studied the effect of drugs blocking the downstream effectors, mTOR and ERK, on this disease.