Functional Study of Al Kaissi Syndrome Caused by a Prenatal CDK10 Compound Heterozygous Mutation

To editor: Al Kaissi syndrome is an autosomal recessive developmental disorder caused by mutations in the cyclin-dependent kinase 10 (CDK10) gene located on chromosome 16q24 (online mendelian inheritance in man (OMIM): 617964). Its main clinical features include developmental delay, spinal and facial deformity, and delayed psychomotor development, usually accompanied by moderate to severe intellectual disability. CDK10 is a CDC2-related kinase involved in diverse cellular activities, including cell proliferation, transcription, and cell cycle regulation. Additionally, CDK10 plays a role in the modulation of cancer cell proliferation, motility, and chemosensitivity. The disease is relatively rare, with only 13 families reported globally, and only one in China.1–2 The subject was a fetus at 20 weeks and 3 days of gestation, ultrasound performed at 20 weeks and 2 days of gestation demonstrated the onset of hydrocephalus. Whole-exome sequencing (WES), copy number variation (CNV) detection, and pathogenicity assessment of variant sites were performed on peripheral blood of the parents and skin tissue obtained after pregnancy termination. Quantitative Polymerase Chain Reaction (qPCR) and western blotting (WB) were combined with minigene technology to comprehensively elucidate the pathogenic mechanism underlying CDK10 gene mutations at the transcriptional and protein levels (Supplementary Fig. 1, Appendix 1, https://links.lww.com/MFM/A119). The clinical manifestations of the fetus and its siblings are shown in Supplementary Table 1 and Supplementary Fig. 2, https://links.lww.com/MFM/A119. CNV-seq analysis of the subject’s samples revealed no chromosomal aneuploidies or pathogenic CNVs exceeding 100 kb (Supplementary Fig. 3A, https://links.lww.com/MFM/A119). Initial analysis of the WES results3–6 identified no pathogenic single-nucleotide variants or insertions/deletions associated with the phenotype. However, two loci of interest were noted (Supplementary Fig. 3B, https://links.lww.com/MFM/A119). The father carried a c.613C > T (p.R205*) variant in exon 7 of CDK10. The mother was a heterozygous carrier of a c.933-2A > G variant in intron 11 of the same gene. Analysis of the fetal skin tissue sample showed that the fetus had inherited both mutations, establishing a compound heterozygous genotype. While the clinical pathogenicity of these loci could not be established based on the available information, their association with the phenotype in this case warranted further investigation. To further strengthen the evidence linking this variant to the clinical phenotype in the present case, comprehensive phenotyping and familial segregation analysis are warranted. We therefore performed genetic testing on the proband’s parents to trace the origin of the variant, followed by first-generation validation to assess its segregation within the family (see Supplementary Fig. 3C and 3D, https://links.lww.com/MFM/A119 for the supplementary genetic testing report). The results were as follows: The older sister carried the c.613C > T variant in exon 7 of CDK10 but was wild-type at the c.933-2A > G site in intron 11. The older brother was wild-type at both the c.613C > T site in exon 7 and the c.933-2A > G site in intron 11 of CDK10. Mutations in the CDK10 gene (OMIM: 603464) cause the autosomal recessive disorder Al Kaissi syndrome (OMIM: 617694). The clinical manifestations observed in these subjects closely align with phenotypic data documented in public databases. According to the American College of Medical Genetics and Genomics guidelines and the ClinGen Sequence Variant Interpretation Expert Group’s recommendations, the c.613C > T and c.933-2A > G mutations in CDK10 are classified as disease-causing, providing a reliable basis for our research.7 Variant 1: CDK10: NM_052988.5: exon9:c.613C > T:p.R205*: (1) PVS1 (very strong): Introduces a premature stop codon, leading to loss of protein function. (2) PM2 (moderate): Extremely rare in population databases (gnomAD frequency = 0.000123). (3) PM3 (moderate): In compound heterozygosity with c.933-2A > G. Variant 2: CDK10: NM_052988.5: intron11:c.933-2A > G: (1) PVS1 (very strong): Affects a canonical splice acceptor site (−2 position), disrupting mRNA splicing. (2) PM2 (moderate): Absent from multiple public population databases. (3) PM3 (moderate): In compound heterozygosity with c.613C > T. For the functional verification of the c.933-2A > G variant in CDK10, we tested whether the mutation affected mRNA splicing in the fetus’s mother, using samples from normal subjects and the fetus’s sister as controls. Agarose gel electrophoresis of the RT-PCR products yielded a single band of the expected size (370 bp) for the normal subjects and the sister, which were confirmed as wild type via direct Sanger sequencing. In contrast, the mother’s sample generated multiple bands. Following amplicon purification and TA cloning, Sanger sequencing identified three distinct splicing outcomes (Supplementary Fig. 4A, https://links.lww.com/MFM/A119): (1) normal splicing, (2) retention of 14 bp from the right side of intron 11, and (3) complete intron 11 retention. Analysis of sequencing results suggested that because the sister and the healthy controls lacked the mutation, their samples generated one normally spliced product. However, the mother’s c.933-2A > G heterozygous mutation resulted in the production of standard and abnormally spliced transcripts (outcomes 2 and 3) (Supplementary Fig. 4B and Supplementary Table 2, https://links.lww.com/MFM/A119). These findings indicated that the c.933-2A > G mutation affects mRNA splicing, leading to altered protein expression (Supplementary Fig. 4C, Supplementary Table 3, https://links.lww.com/MFM/A119). For the c.613C > T: p.R205* mutation, we examined CDK10 transcript levels using the pCMV-3XFlag-Neo (EGFP) expression vector. qPCR results revealed that the levels of mutant mRNA were considerably lower than those of wild-type mRNA across three independent replicates (Supplementary Fig. 5A, https://links.lww.com/MFM/A119). WB analysis of the pCMV-3XFlag-Neo (EGFP)-CDK10 series identified single protein bands at the expected sizes of 42 kDa (wild type) and 25 kDa (mutant). Compared to the wild type, the expression of the mutant protein was significantly down regulated (Supplementary Fig. 5B and 5C, https://links.lww.com/MFM/A119). In summary, the c.613C > T: p.R205* mutation in CDK10 is a nonsense mutation that yields a truncated protein. Our in vitro assay using the pCMV-3XFlag-Neo (EGFP) expression system confirmed that this mutation results in the down regulation of CDK10 mRNA expression and a significant reduction in CDK10 protein levels. Finally, we performed bioinformatic analysis on the identified variants. For the c.933-2A > G splice-site variant in intron 11 (Class I mutation region), predictions from three tools (Rare Disease Data Center, Varseak, Splicing Predictor by Artificial Intelligence) indicated a 90% probability of splicing disruption (Supplementary Fig. 6, Appendix 1 and 2, https://links.lww.com/MFM/A119). For the c.613C > T (p.R205*) nonsense variant in conserved exon 9 (Class III region), splicing predictors did not identify a considerable impact. However, mutation taster classified it as pathogenic, likely causing nonsense-mediated mRNA decay (NMD), and PolyPhen-2 predicted it to be “probably damaging” (Supplementary Fig. 7, Appendix 2, https://links.lww.com/MFM/A119). Congenital disabilities comprise structural, functional, or metabolic abnormalities present at birth. Given its large population base, hundreds of thousands of such cases are documented annually in China, which imposes significant economic and psychological burdens on families and the healthcare system, and represents a notable public health challenge.8 Many studies have shown that congenital disabilities due to mutations in CDK10 seriously compromise neonatal survival. In this study, fetal ultrasound revealed widening of the lateral ventricles, irregular spinal alignment, and hydrocephalus, which strongly suggested an underlying genetic disorder. In this study, we analyzed two mutation sites (c.613C > T and c.933-2A > G) in the CDK10 gene employing a dual-track molecular validation strategy. By combining qPCR and WB with minigene technology, we characterized the pathogenic mechanism of these variants at transcriptional and protein levels (Supplementary Fig. 8, https://links.lww.com/MFM/A119). We found that the c.613C > T nonsense mutation resulted in diminished CDK10 mRNA levels and the production of a truncated protein,.we speculate that this nonsense mutation may reduce the stability of the mutant transcript by potentially triggering the NMD pathway. For the c.933-2A > G splicing site mutation, using the minigene system, we clarified the molecular mechanism leading to abnormal splicing. In this work, we also obtained complete family samples (the patient and both parents), thereby enabling the verification of the genetic origins of the mutations, which is of substantial importance in rare disease research. Professional genetic counseling was subsequently provided to the family. Lastly, the newly identified CDK10 gene mutation site differs considerably from those previously reported abroad and the exon deletion mutations documented in China. This finding expands the mutation spectrum of the CDK10 gene and provides novel molecular markers for clinical diagnosis. It is important to note the limitations of our study. First, due to the family’s refusal to consent to an autopsy, we were unable to provide results from fetal autopsy, radiography, or computed tomography scans. Consequently, we could not confirm the presence of vertebral abnormalities characteristic of Al Kaissi syndrome (such as butterfly vertebrae or hemivertebrae) in the fetus. This also prevented the establishment of a definitive prenatal radiological diagnosis of the syndrome, which limits our understanding of the full phenotypic spectrum of the syndrome during the fetal stage. Despite this limitation, we believe our case offers valuable and novel clinical insights. The presentation of severe hydrocephalus leading to termination of pregnancy was not part of the classical description of Al Kaissi syndrome. Accordingly, our report potentially expands the known phenotypic spectrum of this rare syndrome to include severe central nervous system anomalies. This association underscores the diagnostic challenges in prenatal medicine and the importance of considering rare genetic syndromes even when common findings such as hydrocephalus are present. Secondly, the technical methods used have certain limitations. WB can only detect changes in protein expression levels, not alterations in the enzymatic activity or subcellular localization of mutant proteins. This could lead to the underestimation of the pathogenicity of mutant proteins, particularly their potential dominant adverse effects. Furthermore, owing to the difficulty in repeatedly obtaining patient tissue samples, we used a minigene system for splicing analysis. Although the results were stable and reliable, the system could not fully replicate the in vivo splicing regulatory environment. This study provides essential references for the molecular diagnosis of CDK10-related diseases and the genetic counseling of affected patients. Future research employing patient-derived cell models or gene editing technologies will be instrumental in further elucidating the pathogenic mechanisms underlying these mutations. Such advancements are expected to expand our understanding of the molecular basis of rare genetic syndromes and lead to refined targeted diagnostic strategies. Funding None. Conflicts of Interest The authors declare no conflicts of interest. Data Availability The datasets analyzed during the current study are available from the corresponding author on reasonable request.