Exome sequencing revealed a novel homozygous variant in TRMT61 A in a multiplex family with atypical Cornelia de Lange Syndrome from Rwanda

Abstract

Background: In 30% of patients who exhibit the clinical profile of Cornelia de Lange Syndrome (CdLS), the genetic cause remains undetermined. This proportion tends to be higher in low-resource settings including Africa. We performed a molecular characterization of CdLS in a multiplex Rwandan family.

Methods: After a clinical evaluation of two affected siblings, DNA isolated from peripheral whole blood of the affected patients and their parents underwent Exome Sequencing (ES). Sanger sequencing validated the variant segregating with CdLS. In silico predictive tools, protein modelling, and cell-based experiments using HEK293T cells were used to investigate the pathogenicity of the variant found.

Results: We identified a family with two parents and their two offspring (male and female), who were referred for hearing impairment. The 17-year-old female presented bilateral profound hearing impairment with moderate hypertelorism, progressive visual impairment, and secondary amenorrhea. The 14-year-old male displayed intellectual disability and a bilateral profound hearing impairment with no noticeable facial dysmorphism. Following exome sequencing (ES) of DNA samples obtained from the four family members, we found that the siblings harbored a novel likely pathogenic homozygous missense variant in the TRMT61 A gene [NM_152307.3:c.665C > T p.(Ala222Val)] inherited from both heterozygous parents. In silico analysis suggested that the variant substitutes a highly conserved amino acid, and 2-D structure modelling revealed a significant decrease in the stability of the protein. Cell-based experiment in HEK293T showed that the variant significantly affected the TRMT61 A protein localization which is thought to impact the mitochondrial and cytosolic functions.

Conclusion: We reported a novel biallelic variant in TRMT61 A, [NM_152307.3:c.665C > T p.(Ala222Val)], which is associated with autosomal recessive atypical CdLS in a multiplex Rwandan family, the first report from Africa, and the second globally. The study emphasizes the need to expand the availability of ES for molecular characterization of rare diseases for the understudied genetically diverse population of Africa.

Keywords: TRMT61 A; Cornelia de Lange Syndrome; Whole exome sequencing.

Fig. 1

Pedigree, audiogram, chromatogram, amino acid conservation and protein modeling data of multiplex family RW.KG.SFS_.09. A Two-generation pedigree suggestive of an incomplete penetrance autosomal dominant mode of inheritance of the Cornelia de Lange syndrome. The black arrow indicates the proband. B Air conduction of pure tone audiometry of the affected brother and sister showing bilateral profound HI. C Photo of the affected female showing mild hypertelorism. D Chromatograms of Sanger sequencing of the missense TRMT61 A variant in the TRMT61 A gene [NM_152307.3:c.665 C > T p.(Ala222 Val)] and the reference allele (indicate the segregation on the pedigree). The position of the nucleotide change is highlighted in blue. E Evolutionary conservation of the TRMT61 A: p.(Ala222 Val) variant position (indicated by the red asterisk). F (1) Wild type, (2) Mutant, showing major structural changes (Black boxes). C/T, heterozygous mutant allele; T/T, homozygous mutant allele; Hom Mut, homozygous mutant for the variant allele; Het Ref, heterozygous reference allele

Fig. 2

Functional analysis using HEK293 T cells. A-C Confocal microscopy images at 10X showing the co-localization of the nuclear material (blue) and tGFP fused TRMT61 A protein (green) of tGFP, wild type and mutant, respectively. D Wild type TRMT61 A at 40X showing a nuclear and cytoplasmic localisation (white arrows), E Mutant TRMT61 A at 40X showing a predominant nuclear accumulation (white arrows), F-G Western blot analysis showing the decreased level of the normalised target signal in the mutant protein compared to the wild type. L: stands for the ladder, WT: wild type and MT: mutant

Fig. 3

Expression profile of TRMT61 A. A Human inner ear organoids. B-C P1 mouse