Functional Characterization of a Novel Splice-Altering Intronic Variant in AMPD2 Associated with Pontocerebellar Hypoplasia Type 9.

backgroundA two-year-old female patient was referred to our clinic due to speech and gait disturbances, strabismus, vacant staring, truncal hypotonia, and spasticity in the extremities. The patient had a history of a complicated delivery resulting in perinatal asphyxia. Electrocardiogram (ECG), echocardiogram (ECHO) and abdominal ultrasound findings reported no abnormalities. Previously performed spinal muscular atrophy (SMA) test, chromosomal microarray and karyotype analyses yielded normal results. On clinical examination, facial dysmorphic features included: prominently low-set ears, strabismus, downslanting palpebral fissures, micrognathia, and tapering fingers.

methodsTo elucidate the patient's phenotype, trio exome sequencing was performed using DNA samples of the patient and her parents. Analysis revealed a homozygous variant in the AMPD2 gene (NM_001368809.2) c.353 + 11 C > T, with both parents identified as heterozygous carriers. The phenotype was found to be partially consistent with pontocerebellar hypoplasia type 9 (PCH9), a condition associated with the AMPD2 gene. The variant was suspected to create a novel splicing site; therefore, blood samples were collected from the patient and her parents for RNA extraction. Subsequent cDNA analyses via gel electrophoresis and Sanger sequencing confirmed the alternative splicing event. Quantitative PCR study was applied to evaluate expression.

conclusionsThis study designates the early-onset phenotype of PCH9 in a patient carrying a splice-altering variant in AMPD2. It also highlights the feasibility of functional studies in evaluating intronic variants of uncertain significance. Functional validation of such variants, which are often challenging to classify, can provide critical insights for clinical decision-making for the patient and further reproductive planning for the family.