Identification of genetic suppressors for a BSCL2 lipodystrophy pathogenic variant in Caenorhabditis elegans.

Seipin (BSCL2), a conserved endoplasmic reticulum protein, plays a critical role in lipid droplet (LD) biogenesis and in regulating LD morphology, pathogenic variants of which are associated with Berardinelli-Seip congenital generalized lipodystrophy type 2 (BSCL2). To model BSCL2 disease, we generated an orthologous BSCL2 variant, seip-1(A185P), in Caenorhabditis elegans. In this study, we conducted an unbiased chemical mutagenesis screen to identify genetic suppressors that restore embryonic viability in the seip-1(A185P) mutant background. A total of five suppressor lines were isolated and recovered from the screen. The defective phenotypes of seip-1(A185P), including embryonic lethality and impaired eggshell formation, were significantly suppressed in each suppressor line. Two of the five suppressor lines also alleviated the enlarged LDs in the oocytes. We then mapped a suppressor candidate gene, lmbr-1, which is an ortholog of human limb development membrane protein 1 (LMBR1). The CRISPR/Cas9 edited lmbr-1 suppressor alleles, lmbr-1(S647F) and lmbr-1(P314L), both significantly suppressed embryonic lethality and defective eggshell formation in the seip-1(A185P) background. The newly identified suppressor lines offer valuable insights into potential genetic interactors and pathways that may regulate seipin in the lipodystrophy model.

International Cohort of Neonatal Timothy Syndrome.

INTRODUCTION: Timothy syndrome (TS) is an extremely rare, multisystem disorder classically associated with long QT, syndactyly, ventricular arrhythmias, and hypoglycaemia. A neonatal diagnosis allows maximal medical and device therapy to be implemented to avoid malignant arrhythmias and sudden cardiac death. METHODS: This was a retrospective case series study of type I TS (TS1) patients using data from the Timothy Syndrome Foundation's international registry, encompassing patients with a genetic diagnosis (CACNA1C variant G406R in exon 8A) recruited over a 28-year period. RESULTS: Forty-four cases of TS1 were included (26 male; 60%). Mean gestational age (GA) was 35.6 weeks (range 28 weeks - term), with 43% of patients born less than 37 weeks GA. In TS1 patients presenting with foetal bradycardia, mean GA was significantly lower (34.2 weeks, p < 0.05). Foetal bradycardia secondary to atrioventricular block was present in 20 patients (45%), resulting in premature delivery in 14 patients (32%). Fifteen patients (34%) were diagnosed with TS1 as neonates. Long QT at birth helped secure a diagnosis in 25 patients (57%). Syndactyly was seen in most patients (n = 40, 91%). Twenty patients died, with an average age of death of 2.3 years (range 1 month-6 years). Of the 7 patients who died before the first year of life (16%), the average age of death was 2.5 months. CONCLUSION: TS is associated with high early mortality. TS should be considered in paediatric patients presenting with long QT and syndactyly. Recognition of TS in the neonatal period allows for early intervention to prevent life-threatening arrhythmias.

A missense mutation in the C. elegans src-2 tyrosine-protein kinase reduces brood size and enhances embryonic morphogenesis defects in src-1(RNAi) conditions.

Goldenhar Syndrome is a rare congenital disorder characterized by hemifacial microsomia. Although select mutations have been mapped for this disorder, the genetic etiologies in the majority of cases remain unknown. A recent clinical report of a Goldenhar Syndrome patient identified a homozygous missense mutation in FRK , a gene associated with various types of cancer. In this work, we precisely modeled the disease-associated missense mutation in the C. elegans FRK ortholog src-2 , using CRISPR/Cas9 gene editing, and investigated the physiological role of this mutation and the src-2 gene. In addition, we generated a conserved variant in src-1 ( FYN ortholog) to assess the functional redundancy of the conserved variant. The putative pathogenic variants src-1 (Val190Ile) or src-2 (Val170Ile) caused only subtle phenotypes, suggesting that these mutations alone are not sufficient to explain the facial deformities observed in the Goldenhar Syndrome patient. However, the src-2 (Val170Ile) mutant exhibited reduced brood size and moderately enhanced embryonic developmental phenotypes, including epidermal and neuronal patterning defects, in the src-1 (RNAi) condition, indicating that the src-2 (Val170Ile) locus could play a supportive role during developmental processes. Overall, however, these studies showed that src-1 /FYN is essential for regulating embryogenesis and morphogenesis, while src-2 /FRK is largely dispensable for normal embryonic development, suggesting FYN , not FRK , is the dominant non-receptor protein kinase during embryonic development in C. elegans .

Localized TWIST1 and TWIST2 basic domain substitutions cause four distinct human diseases that can be modeled in Caenorhabditis elegans.

Twist transcription factors, members of the basic helix-loop-helix family, play crucial roles in mesoderm development in all animals. Humans have two paralogous genes, TWIST1 and TWIST2, and mutations in each gene have been identified in specific craniofacial disorders. Here, we describe a new clinical entity, Sweeney-Cox syndrome, associated with distinct de novo amino acid substitutions (p.Glu117Val and p.Glu117Gly) at a highly conserved glutamic acid residue located in the basic DNA binding domain of TWIST1, in two subjects with frontonasal dysplasia and additional malformations. Although about one hundred different TWIST1 mutations have been reported in patients with the dominant haploinsufficiency Saethre-Chotzen syndrome (typically associated with craniosynostosis), substitutions uniquely affecting the Glu117 codon were not observed previously. Recently, subjects with Barber-Say and Ablepharon-Macrostomia syndromes were found to harbor heterozygous missense substitutions in the paralogous glutamic acid residue in TWIST2 (p.Glu75Ala, p.Glu75Gln and p.Glu75Lys). To study systematically the effects of these substitutions in individual cells of the developing mesoderm, we engineered all five disease-associated alleles into the equivalent Glu29 residue encoded by hlh-8, the single Twist homolog present in Caenorhabditis elegans. This allelic series revealed that different substitutions exhibit graded severity, in terms of both gene expression and cellular phenotype, which we incorporate into a model explaining the various human disease phenotypes. The genetic analysis favors a predominantly dominant-negative mechanism for the action of amino acid substitutions at this highly conserved glutamic acid residue and illustrates the value of systematic mutagenesis of C. elegans for focused investigation of human disease processes.

emb-1 encodes the APC16 subunit of the Caenorhabditis elegans anaphase-promoting complex.

In the nematode Caenorhabditis elegans, temperature-sensitive mutants of emb-1 arrest as one-cell embryos in metaphase of meiosis I in a manner that is indistinguishable from embryos that have been depleted of known subunits of the anaphase-promoting complex or cyclosome (APC/C). Here we show that the emb-1 phenotype is enhanced in double mutant combinations with known APC/C subunits and suppressed in double mutant combinations with known APC/C suppressors. In addition to its meiotic function, emb-1 is required for mitotic proliferation of the germline. These studies reveal that emb-1 encodes K10D2.4, a homolog of the small, recently discovered APC/C subunit, APC16.

Multiple ERK substrates execute single biological processes in Caenorhabditis elegans germ-line development.

RAS-extracellular signal regulated kinase (ERK) signaling governs multiple aspects of cell fate specification, cellular transitions, and growth by regulating downstream substrates through phosphorylation. Understanding how perturbations to the ERK signaling pathway lead to developmental disorders and cancer hinges critically on identification of the substrates. Yet, only a limited number of substrates have been identified that function in vivo to execute ERK-regulated processes. The Caenorhabditis elegans germ line utilizes the well-conserved RAS-ERK signaling pathway in multiple different contexts. Here, we present an integrated functional genomic approach that identified 30 ERK substrates, each of which functions to regulate one or more of seven distinct biological processes during C. elegans germ-line development. Our results provide evidence for three themes that underlie the robustness and specificity of biological outcomes controlled by ERK signaling in C. elegans that are likely relevant to ERK signaling in other organisms: (i) multiple diverse ERK substrates function to control each individual biological process; (ii) different combinations of substrates function to control distinct biological processes; and (iii) regulatory feedback loops between ERK and its substrates help reinforce or attenuate ERK activation. Substrates identified here have conserved orthologs in humans, suggesting that insights from these studies will contribute to our understanding of human diseases involving deregulated ERK activity.