Whole Exome Sequencing Identifies a Novel Homozygous Missense Mutation in the CSB Protein-Encoding ERCC6 Gene in a Taiwanese Boy with Cockayne Syndrome

Background: Cockayne syndrome (CS) is a rare form of dwarfism that is characterized by progressive premature aging. CS is typically caused by mutations in the excision repair cross-complementing protein group 6 (ERCC6) gene that encodes the CS group B (CSB) protein. Using whole exome sequencing, we recently identified a novel homozygous missense mutation (Leu536Trp) in CSB in a Taiwanese boy with CS. Since the current database (Varsome) interprets this variant as likely pathogenic, we utilized a bioinformatic tool to investigate the impact of Leu536Trp as well as two other variants (Arg453Ter, Asp532Gly) in similar articles on the CSB protein structure stability. Methods: We used iterative threading assembly refinement (I-TASSER) to generate a predictive 3D structure of CSB. We calculated the change of mutation energy after residues substitution on the protein stability using I-TASSER as well as the artificial intelligence program Alphafold. Results: The Asp532Gly variant destabilized both modeled structures, while the Leu536Trp variant showed no effect on I-TASSER’s model but destabilized the Alphafold’s modeled structure. Conclusions: We propose here the first case of CS associated with a novel homozygous missense mutation (Leu536Trp) in CSB. Furthermore, we suggest that the Asp532Gly and Leu536Trp variants are both pathogenic after bioinformatic analysis of protein stability.

Autosomal Recessive Congenital Dyserythropoietic Anemia Type III Is Caused By Mutations in the Centralspindlin RACGAP1 Component

An autosomal dominant form of congenital dyserythropoietic anemia type III (CDA III) is caused by a missense mutation in the KIF23 gene whose protein product, mitotic kinesin-like protein (MKLP1), is part of the centralspindlin complex involved in cytokinesis. Several case reports suggested the existence of an autosomal recessive inheritance form of CDA III so far not genetically characterized. By means of whole exome sequencing in a Spanish CDA III family with healthy parents, we identified in the male proband a novel homozygous missense mutation p.Pro432Ser in the RACGAP1 gene, which encodes for the RACGAP1 protein (Rac GTPase-activating protein 1, also known as MgcRacGAP or CYK-4), the partner of MKLP1 in the centralspindlin complex. A second CDA III Spanish patient has a different rare and novel homozygous missense mutation, p.Thr220Ala, in the RACGAP1 gene. Both patients presented with macrocytic anemia, aberrant multinucleated erythroblasts in the bone marrow typically seen in CDA III cases, no iron overload and skull defects secondary to severe anemia. Silencing of RACGAP1 using siRNA in HeLa cells mimics the cytokinesis defect observed in the bone marrow of our patients. Both mutations disrupt normal cytokinesis and alter the GTPase balance in patients' cells. We conclude that the autosomal recessive form of CDA type III is caused by mutations in the RACGAP1 gene, encoding for RACGAP1 protein, which is the partner of MKLP1 in the centralspindlin complex critical for cytokinesis and now both proteins are associated with CDA type III. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

A Novel Homozygous Missense Mutation of WEE2 Causes Female Infertility Characterized by Fertilization Failure

Purpose: To investigate the genetic cause of infertility in a female patient due to repeated fertilization failure.Methods: Whole exome sequencing was performed to obtain the candidate mutation. Sanger sequencing was used to identify the mutation of the proband and other family members. SIFT, Polyphen-2, and Mutation Taster were used to predict the pathogenicity of mutations. The online software Arpeggio and the mCSM online service were used to analyze the effect of the mutation on protein structure and stability. Results: We identified a novel homozygous missense mutation c.T1199A:p.L400Q (Leu400Gln) in WEE2 gene in a female proband with infertility caused by fertilization failure. Conclusions: We discovered a novel homozygous missense mutation c.T1199A:p.L400Q (Leu400Gln) of the WEE2 gene in an infertile female whose oocytes had undergone complete fertilization failure, either after ICSI or RICSI. Our findings extend the mutant spectrum of WEE2 , a genetic cause for fertilization failure, and provide a theoretical basis for clinical diagnosis of the pathogenic causes of infertility.