Chapter 4 Candidate gene discovery using whole exome sequencing for
4.4 A candidate gene identified from analysis of de novo variants
4.4.1 Family ND9
4.4.1.1 Clinical Phenotype
Family ND9 is a non-consanguineous family, with no previous family history. Individual JT753 presented symptoms at very early age. He had partial left third nerve palsy and left sixth nerve palsy, as well as right sided ptosis and lower limb dystonia. It was reported that he has unusual movements of his legs and had hyperekplexia in both lower limbs. He also presented ocular motility problems. The clinical diagnosis was one of Complex Moebius syndrome. MRI scans showed right perisylvian polymicrogyria and slight callosal dysmorphism. Figure 4-18 oultines the family pedigree and figure 4-19 shows sections of the MRI scans.
Figure 4-18 Pedigree of family ND9
Pedigree outlining two generations of the family ND9. DNA samples of individuals with JT numbers were available, but only individuals marked with an asterisks (*) have been used for NGS genetic investigations.
I
II
JT748
JT749
JT750
JT751
JT753
JT752
JT754
*
*
*
*
Figure 4-19 MRI scans from individual JT753.
MRI scans of individual JT753 showing brain malformations. Right perisylvian polymicrogyria as demonstrated by the indistinct grey/white matter interface; (Blue arrow). Slight callosal dysmorphism, with a bulky anterior limb and constricted splenium; (White arrows).
4.4.1.2 Candidate gene
An in house bioinformatics pipeline was used for data analysis (section 2.2.10.3), in the first instance, assuming de novo incidence as only a single individual was affected in the family and there was no history of consanguinity. Analysis for a recessive mode of inheritance was also performed, identifying both homozygous and compound heterozygous variants. As there was no history of consanguinity, autozygosity mapping was not performed for this family. Filtering of biallelic variants did not reveal any potential
T2 Axial T1 Coronal
T1 Coronal T1 Sagital
pathogenic variant, however an interesting de novo variant was identified. Table 4-11 summarizes the de novo variants identified in family ND9 with a CADD score above 15.
Furthermore, CNV analysis was also carried out for this family (see section 2.2.10.7) to identify potential CNVs that could cause the phenotype. CNV analysis did not reveal any variants that could be interpreted as pathogenic, increasing our confidence in the TUBA1A variant summarised in table 4-11.
Table 4-11 De novo variant identified in family ND9.
De novo variant in TUBA1A (NM_006009.4) was annotated based on the human genome
assembly GRCh37/hg19.
4.4.1.3 Novel de novo variant in TUBA1A
Alpha and beta tubulins form the heterodimers that comprise the major components of the microtubules, a major constituent of the cytoskeleton. Microtubules are essential for various cellular processes such as mitosis and intracellular transport (Forth and Kapoor, 2017). Nine α-tubulin and nine β-tubulin isotypes have been identified (Gadadhar et al., 2017) with tissue specific expression variability. Tubulin genes are also known to play a key role in central nervous system development, particularly in axonal guidance and neuronal migration, and mutations in various α- or β-tubulins genes have linked to developmental disorders with brain malformations. These malformations include defects in cortical development, polymicrogyria, gyral disorganisation, and agenesis or abnormalities of the midline commissural structures, such as the corpus callosum. Mutated tubulin genes known to cause these disorders include TUBA1A, TUBA8, TUBB2A, TUBB4A, TUBB, TUBB2B and TUBB3 (Romaniello, 2015).
Gene Location Variant Protein change
TUBA1A 12:49579706 c.443C>T p.G148E
Condel Polyphen2 SIFT CADD score
deleterious (0.873) probably_damaging (0.983) deleterious_low_ confidence (0) 26.8 MAF
gnomAD Protein Function OMIM
In Homozygous region
Absent Major components of microtubules Lissencephaly 3 N/A
Variant Information
Pathogenicity prediction
TUBA1A is a highly-conserved gene that expresses predominantly in the developing brain, and decreases in postnatal and adult periods (Gardner et al., 2018). Heterozygous mutations in TUBA1A have been mainly associated with lissencephaly type 3 (MIM number: 611603). Most of the cases reported consist of patients with de novo variants in TUBA1A (Keays et al., 2007), but a hereditary form has been identified where in two sisters who inherited a heterozygous TUBA1A variant from their mother, who had somatic mutation in the gene (Jansen et al., 2011).
Recent genetic studies have expanded the clinical spectrum of TUBA1A-associated disorders, with cases presenting overlapping brain malformations but not necessarily lissencephaly. An interesting study reporting a de novo TUBA1A p.R2H variant identified in four unrelated patients but without presenting lissenchephaly. The patients described in this study had mild phenotypic variability, but shared phenotypes included developmental delay, microcephaly, dysplasia or thinning of the corpus callosum, dysmorphic basal ganglia and hypoplasia of the cerebellar vermis (Gardner et al., 2018). Two of the patients also had bilateral perisylvian polymicrogyria, resembling the phenotypes observed in family ND9.
Another recently published study describes a different de novo variant in TUBA1A (p.D127E) but the described patient does not have lissenchephaly. Clinical features of this case include motor delay, occipital polymicrogyria, hypoplasia of the corpus callosum, cerebellar hypoplasia, dysplastic brainstem, on-separative basal ganglia and volume loss of the white matter (Sato et al., 2018b).
Sato et al. outline two variants (p.R2H and p.D127E) that appear to have a minimal change of amino acid residue in terms of physicochemical properties, yet sufficient to cause a pathogenic phenotype. It is important to appreciate that a highly conserved protein such as TUBA1A has critical functions in cell cycle and maintenance that could be disrupted even with subtle changes of amino acid residues. Such changes could possibly affect the way in which α- and β-tubulins form heterodimers and polymerise to form the microtubule network. Disruption in the microtubule formation or stability could potentially affect downstream cellular mechanisms that are key to development. It is essential to establish a new genotype-phenotype correlation with variants in TUBA1A, as many recent studies are identifying variants that cause a wide range of brain malformations that may or may not include lissencephaly.