NEW YORK (GenomeWeb) – By combining autozygome mapping and exome sequencing, an Alfaisal University-led team of researchers has homed in on gene mutations behind the primary microcephaly and primordial dwarfism affecting three families.
Alfaisal's Fowzan Alkuraya and his colleagues first applied their approach to a Yemeni family with three affected sons. As they reported in the American Journal of Human Genetics today, they uncovered a mutation in RTTN that had been missed in previous genetic analyses. This then prompted the researchers to look for other families with similar phenotypes, of which they found two — one from Saudi Arabia and the other from Canada — that harbored other RTTN mutations.
"[W]e show that RTTN is a gene that is mutated in severe primary microcephaly with associated growth deficiency and complex brain malformations including simplified gyration," Alkuraya and his colleagues wrote in their paper. "Our finding of a non-canonical splicing mutation that evaded detection by a clinical exome laboratory is a reminder that commonly used filters can miss causal mutations."
The first family — a multiplex consanguineous family — had three sons with primary microcephaly and growth deficiency. The parents and two daughters were healthy.
The index patient, the eldest son, underwent aCGH panel testing for primary microcephaly and whole-exome sequencing, but the results were negative.
This led the researchers to suspect that an undescribed gene might be behind this family's condition, and they recruited the entire family for genetic analysis. They first performed genome-wide genotyping on the family and mapped runs of homozygosity, finding two ROHs that were only shared by the affected family members. However, these ROHs didn't overlap with any known primary microcephaly loci.
Alkuraya and his colleagues then turned to exome sequencing, generating at least 50X coverage for their samples. In these two regions, the researchers found no novel coding or canonical splicing variants, but they did uncover an intronic variant in exon 23 of RTTN.
In silico predictions indicated that the variant leads to a cryptic donor site, and RT-PCR of blood samples found that the intervening seven basepairs of intron 23 were retained, introducing a premature stop codon.
The researchers also noted that this variant was absent from the 1,000 Genome, the ExAC Browser, and 650 in-house ethnically matched exome datasets, and that it segregated with the family’s phenotype as an autosomal recessive trait.
This finding, Alkuraya and his colleagues said, suggests that such a mutation could have been missed in other families. They queried an internal database to uncover a second consanguineous family exhibiting a similar phenotype of severe primary microcephaly and growth retardation.
The researchers noted that their initial analysis of the index patient from this family had generated a short list of two variants, one in RTTN and one in ETV1. Re-analysis in light of their more recent findings led them to home in on the RTTN variant as the likely causal one. Sanger sequencing confirmed that the index patient was homozygous for the RTTN (c.3190A>C) variant, while his healthy parents and sisters were heterozygous.
Further, a combined linkage analysis drawing on these two families generated a single significant linkage peak in the region spanning RTTN.
Alkuraya and his colleagues then cast a wider net to search for other families, finding one in Canada that had two affected sons. Sequencing of a known microcephaly and primordial dwarfism-linked gene and aCGH testing was normal, and whole-exome sequencing of the eldest sibling uncovered biallelic RTTN mutations affecting highly conserved amino acids. Sanger sequencing confirmed that both affected children had the mutations.
RTTN, the researchers noted, encodes rotatin and has a role in the basal body and cilia.
In 2012, two RTTN missense mutations were linked to two families with a recessive form of polymicrogyria, a developmental brain malformation. This new finding, Alkuraya and his colleagues said, expands the spectrum of RTTN-related phenotypes.
"We propose that the rather limited phenotype associated with the two missense mutations reported in 2012 might have been allele specific and that the true clinical spectrum of RTTN-related phenotypes entails microcephalic primordial dwarfism and a complex brain phenotype that encompasses simplified gyration and other malformations," the researchers said. "This expanded phenotypic spectrum suggests a role for RTTN in cellular proliferation and neuronal migration."