Expanding the TUBB3‐Related Phenotypic Landscape: Fetal Diagnosis of Novel TUBB3 Variant Linked With Phenotypic Variability Within a Single Family

What's already known about this topic? Tubulinopathies are a heterogenous group of complex brain malformations. While TUBB3-related brain malformations can be mild, the degree of phenotypic variability among familial carriers is still poorly characterized. What does this study add? Our family exhibits a remarkably high degree of intra-familial variability. This variability has not been previously documented in the literature. Expanding the pre- and post-natal phenotypic spectrum associated with inherited tubulinopathies can improve prenatal counseling in these disorders. The male fetus (Table 1, fetus 1) was the first pregnancy of unrelated parents. First trimester screening was normal. Second trimester Ultrasound (US) at 22 then 25 weeks of gestation (WG) showed distortion of interhemispheric fissure (DIHF) with left lateral ventriculomegaly, dysmorphic frontal horn, thin corpus callosum, and enlarged left sylvian fissure. 26 WG MRI confirmed distortion of interhemispheric and frontal lobe asymmetry, short and thin corpus callosum < 5th percentile. Thin corpus callosum HP: 0033725 Short corpus callosum HP:0200012 Enlarged sylvian cistern HP:0100952 Abnormal pons morphology HP:0007361 Aplasia of the pyramids of the medulla oblongata HP:0034973 Anterior distortion of the interhemispheric fissure (no HPO) Distortion of the interhemispheric fissure (no HPO) Abnormal left ventricle morphology HP:0001711 Thin corpus callosum HP:0033725 Abnormal basal ganglia morphology HP:0002134 Delayed ability to walk HP:0031936 Delayed fine motor development HP:0010862 – An affected girl, normal delivery of at 23 years old. – TOP of an affected fetus (fetus 1) – TOP of an affected fetus (fetus 2) Healthy parents with normal cerebral MRI. One healthy brother with normal cerebral MRI. Distortion of the interhemispheric fissure (no HPO) Motor delay HP:0001270 Gait disturbance HP:0001288 Bilateral talipes equinovarus HP:0001776 Language impairment HP:0002463 Global developmental delay HP:0001263 The mother (Table 1, mother) had a history of fine motor delays and speech therapy. MRI showed DIHF, abnormal left ventricular morphology, asymmetric cerebral peduncles, thin corpus callosum and dysmorphic basal ganglia. Her cognitive skills were unremarkable, with satisfactory social and vocational performance. She also has a daughter (Table 1, daughter) from her previous union. This 8-year-old child has a neurodevelopmental disorder with delayed milestones (sitting at 9 months, walking 21 months), axial hypotonia, balance issues, language delay and required special education. Cognitive testing was heterogenous. MRI findings were consistent with tubulinopathy: anterior DIHF, left ventriculomegaly, and basal ganglia anomalies initially interpreted as perinatal stroke. Following genetic counseling and informed consent. Trio whole exome sequencing (tWES) was performed on fetal tissue (fetus 1) and parents' blood lymphocytes. Exonic sequences were enriched using AGILENT SureSelect All Exon V7 kits and sequenced on a NextSeq 550 from Illumina. The bioinformatic pipeline was performed using BWA MEM algorithms for alignment (hg19), GATK and FreeBayes for variant calling. ANNOVAR and ALAMUT for annotation. Relevant variants are carefully reviewed by molecular geneticists and discussed with clinicians to determine the significance of the findings. Sanger sequencing was performed on uncultured amniocytes for fetus 2 and blood lymphocytes for other relatives. tWES performed in fetus and parents led to the identification of a heterozygous missense variant in TUBB3 gene NM_006086.4(TUBB3):c.259C>T p.(Pro87Ser) that was maternally inherited (Table 2). This gene is known to be responsible of an autosomal dominant form of tubulinopathy: cortical dysplasia complex with other brain malformations 1 (OMIM: 614039). This variant was absent from control databases and has not been reported in the literature, occurs in a missense-intolerant gene (gnomAD [v4.0.0] Z-score = 5.67), with multiple lines of computational evidence in favor of a deleterious effect on gene (CADD: 25.8; SIFT: damaging; REVEL: 0.741; AlphaMissense: Likely Pathogenic) and with a strong genotype phenotype correlation. The family investigation was extended to the mother's first-degree relatives: her only brother and father have normal MRI and do not carry the family TUBB3 variant, while her mother has a normal MRI but could not be tested. It was classified as probably pathogenic (Class 4) following the American College of Medical Genetics classification. No other pathogenic or likely pathogenic variants were identified. Facing neurodevelopmental prognostic uncertainty associated with this complex cerebral malformation, the couple decided to proceed to termination of pregnancy (TOP) at 28 WG. Fetal pathology examination showed multiple cerebral anomalies including (Figure 1) DIHF, enlarged left sylvian fissure, abnormal morphology of the pons, bulbs, and pyramids. No clear anomaly in neuronal migration was found. There were no microscopic abnormal findings. First and second rows: Pre- and postnatal imaging findings (Ultrasound and MRI) suggestive of tubulinopathy in three individuals. Fetus1 (III.4): Ultrasound imaging at 22 WG showing in coronal plane, frontal lobe asymmetry related to the anterior DIHF, in sagittal plane a short and thin corpus callosum. Fetal MRI at 26 WG shows in the axial plane an enlarged left lateral ventricle (∆), and a discrete enlargement of the left sylvian fissure (*). The mother's (II.3) brain MRI at 31 years shows in axial plane a discrete DIHF (↕), a dysmorphic enlarged left lateral ventricle (∆), and in frontal plane a dysmorphic left basal ganglia (←). The daughter's (III.3) brain MRI at 8 years shows in frontal plane anterior DIHF (↕), dysmorphic appearance of the left lateral ventricle (∆), and in axial plane a dysmorphic left basal ganglia (←). Third row: Fetal autopsy findings in Fetus 1 (III.4) suggestive of tubulinopathy. Asymmetric sylvian fissures: The left sylvian valley (*) is more enclosed. Anterior distortion of interhemispheric fissure (↕), asymmetrical reliefs of the pons and bulbs, and pyramids of the medulla oblongata (]). The mother had another pregnancy the following year, prenatal diagnosis revealed this familial variant in a female fetus (fetus 2) at 12 WG, the couple decided to proceed to TOP in the face of prognostic uncertainty. Tubulinopathies represent a diverse group of complex brain malformations, with pathogenic variants in the TUBB3 gene associated with relatively milder malformations [1], albeit the adult phenotype being under-reported. This family harbor a likely pathogenic novel TUBB3 variant and exhibits highly intrafamilial phenotypic heterogeneity among postnatal carriers. All carriers displayed the distinctive DIHF [2] with various lateralized malformations such as asymmetric ventriculomegaly, dysmorphic basal ganglia, and uniquely, asymmetric sylvian fissures—a finding not previously reported postnatally for TUBB3-related tubulinopathies. But manifestations ranged from normal cognition in the mother to mild intellectual disability and motor delays in her daughter, an unprecedented variability for TUBB3-related spectrum. Therefore, it is not possible to predict the severity of neurological damage in the future children based on the MRI alone. Faced with this prognostic uncertainty and the range of possible neurological outcomes, the parents requested a medical abortion and then prenatal diagnosis for subsequent pregnancies. Although only one of the grandparents could be tested, their normal MRI could suggest that this TUBB3 variant may have arisen de novo in the mother. While causing a broad spectrum of brain malformations [3, 4], TUBB3 pathogenic variants demonstrated unpredictable expressivity here, highlighting challenges for prenatal prognostication. Integrating multimodal data from imaging, molecular diagnosis, and pathology is crucial for optimally counseling families prenatally given TUBB3's heterogeneous presentation. We extend our profound gratitude to the patients for their invaluable participation, and we would like to express our sincere acknowledgment to the Department of Cytogenetics, specifically Erika Launay and Sylvie Jaillard, for their expert assistance in processing fetal samples. The authors have nothing to report. Consent has been obtained from the mother and daughter for this publication (see Patient consent form). The authors declare no conflicts of interest. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy and ethical restrictions.