RTTN Mutations Cause Primary Microcephaly and Primordial Dwarfism in Humans

Abstract

Primary microcephaly is a developmental brain anomaly that results from defective proliferation of neuroprogenitors in the germinal periventricular zone. More than a dozen genes are known to be mutated in autosomal-recessive primary microcephaly in isolation or in association with a more generalized growth deficiency (microcephalic primordial dwarfism), but the genetic heterogeneity is probably more extensive. In a research protocol involving autozygome mapping and exome sequencing, we recruited a multiplex consanguineous family who is affected by severe microcephalic primordial dwarfism and tested negative on clinical exome sequencing. Two candidate autozygous intervals were identified, and the second round of exome sequencing revealed a single intronic variant therein (c.2885+8A>G [p.Ser963(∗)] in RTTN exon 23). RT-PCR confirmed that this change creates a cryptic splice donor and thus causes retention of the intervening 7 bp of the intron and leads to premature truncation. On the basis of this finding, we reanalyzed the exome file of a second consanguineous family affected by a similar phenotype and identified another homozygous change in RTTN as the likely causal mutation. Combined linkage analysis of the two families confirmed that RTTN maps to the only significant linkage peak. Finally, through international collaboration, a Canadian multiplex family affected by microcephalic primordial dwarfism and biallelic mutation of RTTN was identified. Our results expand the phenotype of RTTN-related disorders, hitherto limited to polymicrogyria, to include microcephalic primordial dwarfism with a complex brain phenotype involving simplified gyration.

Keywords: ciliopathy; lissencephaly; primary cilium; primordial dwarfism; rotatin.

Figure 1

Identification of Three Families Affected by Primary Microcephaly, Lissencephaly, and Dwarfism (A) Pedigree of family 1 and representative facial photos of the three affected siblings. Note the severe microcephaly and related facial dysmorphism. (B) Pedigree of family 2 and representative facial photos and brain MRI of the index individual. Note the very small brain volume and lissencephaly. (C) Pedigree of family 3 and representative brain MRI of II:4. Note the severe cerebral and cerebellar hypoplasia, agenesis of the corpus callosum, reduced sulcation, deformed ventricles, and large cerebrospinal-fluid intensity areas occupying the majority of supratentorial compartments bilaterally.

Figure 2

Identification of a Primary-Microcephaly-Associated Locus Defined by RTTN Mutations (A) An exome filtering scheme of the two studied families shows that RTTN is the only commonly mutated gene. (B) Linkage analysis of the two families revealed a linkage peak (minimum LOD score of 3.9) that spans RTTN (circled in red), as shown below. Note that this is a minimum LOD score because we inputted family 1 as first rather than second cousins for computational limitation.

Figure 3

Identification of Four Mutations in RTTN (A) Schematic of RTTN shows the four variants on the corresponding genic regions. Multispecies alignment is shown to highlight the strong conservation of the three missense variants. (B) Schematic of RTTN shows the four variants on the corresponding domains.