Transcript Long-Read Sequencing Unveils the Molecular Complexity of a Novel ROGDI Splicing Variant in a Tunisian Family With Kohlschütter-Tönz Syndrome

Abstract

Kohlschütter-Tönz Syndrome (KTS) is an ultra-rare autosomal recessive disorder, characterized by a clinical triad: infantile-onset epilepsy, global developmental delay, and amelogenesis imperfecta. KTS is caused by pathogenic variants in ROGDI, encoding a leucine zipper protein of unknown function. Our study characterizes a novel homozygous ROGDI variant (NM_024589.3:c.646-2A>G) identified in a Tunisian family case with KTS, renal tubular acidosis, and hyperammonemia. This variant disrupts a canonical acceptor splice site (ASS) in intron 8. Reverse-transcriptase polymerase chain reaction and targeted long-read cDNA sequencing, identified only abnormal transcripts secondary to the ROGDI ASS variant in the proband. Complex splicing events were detected including exon 9 skipping, cryptic ASS activation leading to 13-bp deletion in exon 9, and retention of intron 8 or both intron 8 and 9. These alterations were all predicted to result in nonsense mediated decay and ROGDI loss of function. By integrating complementary techniques, our study unveiled fundamental mechanisms underlying complex splice alterations, providing insights that may guide future therapeutic strategies in KTS.

Keywords: ROGDI; Kohlschütter‐Tönz Syndrome; long‐read sequencing; splicing variant.

FIGURE 1

(A) Family pedigree. (B) Brain MRI images: Diffuse cortical and subcortical atrophy, cerebellar atrophy, with passive ventricular enlargement. Red arrow: Diffuse cerebellar atrophy, (*) passive enlargement of the fourth ventricle. Red arrow head: Mega cisterna magna. (C) Sanger sequencing. (D) Sashimi plots of targeted long‐read cDNA sequencing with partial schematic representation of ROGDI. (E) Targeted‐LRS and SpliceAI predictions. (F) Schematic representation of the identified transcripts.