The patient is a 65-year-old woman who has been complaining of progressive difficulty in walking since childhood (approximately 8–10 years). She reported leg stiffness leading to frequent falls. Additionally, she showed a head tremor, presenting as a yes-yes movement (onset at 50 years), and a disabling depressive mood disorder for many years. The neurological examination revealed a spastic gait, difficulties in standing on the toes and heels, brisk deep tendon reflexes at upper limbs, clonic patellar reflexes, brisk ankle reflexes, as well as bilateral Babinski and Hoffmann sign and brisk jaw jerk, severe lower limb spasticity, normal muscle strength at four limbs, impaired perception of pain and vibration, yes-yes head tremor, upper limb postural and kinetic tremor. The patient refused a full neuropsychological assessment, but major cognitive impairment was not clinically evident.

Walking difficulties due to leg stiffness were reported in the father (deceased due to complications of Chronic Obstructive Pulmonary Disorder), two paternal uncles (deceased for unknown reasons), and the patient’s sister (deceased from colon cancer) who could not be examined directly. However, their clinical records were consistent with spastic paraplegia. (shown in Fig. 1A).

A Pedigree of the family. Squares indicate males and circles indicate females. The family tree shows the affected member (fully black circle), members with gait impairment who did not undergo genetic/clinical analysis (half black circles and squares), and asymptomatic members (blank). Circles and squares with the bar represent deceased subjects The circle with the arrow indicates the proband. Roman numerals indicate the different generations. B In silico evaluations of the variant identifified (on the right) compared with the normal protein (on the left). The position of the substitution D417N is indicated with an arrow

Over time, several diagnostic assessments were performed, including an MRI (Magnetic Resonance Imaging) of the brain and cervical spinal cord, which showed mild hyperintensity of the white matter next to the anterior horns of the lateral ventricles (presumably of vasculopathy origin). Laboratory tests encompassing white and red cell counts, erythrocyte sedimentation rate, antiphospholipid antibodies, serum protein electrophoresis, assessment of liver, renal, and thyroid function, vitamin B12, folic acid, glucose levels, and urine analysis resulted in normal. Additionally, genetic testing for mutations of the SPAST genes was performed, yielding negative results.

NGS (Next-Generation Sequencing) studies for inherited cases of spastic paraplegia (see Appendix 1 for the gene list) revealed a novel variant of unknown significance (VUS) (c.1249A > G, p.D417N), in TUBB4A (CADD score 25.80; CADD score is a tool for scoring the deleteriousness of single nucleotide variants). To the best of our knowledge, this variant has not been reported in the literature or any databases in association with these diseases, although two pathogenic variants have been identified in the same exon [6,7,8].

In silico studies suggested a 99% probability that p.D417N was pathogenic (with a sensitivity and specificity of 14% and 99%, respectively) based on PolyPhen-2, Provean, Mutation Taster, and UMD-Predictor Pro site prediction [9, 10].

Whole exome sequencing was conducted on the patient's DNA using the NextSeq500 Illumina platform as reported elsewhere. The interpretation of variants followed the guidelines of the American College of Medical Genetics (ACMG). Variants with a frequency exceeding 1% in the gnomAD database, those with CADD scores < 20, classified as benign/probably benign according to ACMG criteria, and those of uncertain significance in autosomal recessive genes are filtered out [11, 12].