Mutations in the AHI1 gene, encoding jouberin, cause Joubert syndrome with cortical polymicrogyria

Abstract

Joubert syndrome (JS) is an autosomal recessive disorder marked by agenesis of the cerebellar vermis, ataxia, hypotonia, oculomotor apraxia, neonatal breathing abnormalities, and mental retardation. Despite the fact that this condition was described >30 years ago, the molecular basis has remained poorly understood. Here, we identify two frameshift mutations and one missense mutation in the AHI1 gene in three consanguineous families with JS, some with cortical polymicrogyria. AHI1, encoding the Jouberin protein, is an alternatively spliced signaling molecule that contains seven Trp-Asp (WD) repeats, an SH3 domain, and numerous SH3-binding sites. The gene is expressed strongly in embryonic hindbrain and forebrain, and our data suggest that AHI1 is required for both cerebellar and cortical development in humans. The recently described mutations in NPHP1, encoding a protein containing an SH3 domain, in a subset of patients with JS plus nephronophthisis, suggest a shared pathway.

Figure A1

Additional brain images of patient 144-2, showing polymicrogyria (arrows). The MTM is not visible in these planes.

Figure 1

Mutations in AHI1, which encodes Jouberin, lead to cerebellar vermis aplasia, the MTM, and cortical polymicrogyria. A, Genotypes of the three consanguineous families displaying evidence of linkage to the JBTS3 locus. Affected members of family MTI (molar-tooth on brain imaging) 010 and MTI 115 display homozygosity for markers across the entire region (double red bar), whereas the affected individual in family MTI 144 shows homozygosity for only a single marker (D6S292), which suggests a double recombination. Triangles with question marks (?) represent unknown sex and affection status. B, Magnetic resonance imaging analysis of each of the JBTS3-linked families corresponding to the pedigrees above. Top row, Axial images showing the MTI (blackened arrows), frontal polymicrogyria (unblackened arrows), and thin corpus callosum (arrow with C). Bottom row, Midline sagittal images showing horizontal superior cerebellar peduncle (arrowheads). The asterisk (*) represents mega cisterna magna. Additional scans showing polymicrogyria in patient 144-2 can be found in figure A1 (online only). C, Sequence chromatograms corresponding to the families above. Family MTI 010 displays a single-base insertion at position 787 in exon 8, family MTI 115 displays a T1328A point mutation in exon 10, and family MTI 144 displays a 2-base deletion at positions 1188–89, each of which leads to deleterious mutations in the Jouberin protein.

Figure 2

Exonic structure of AHI1 and the Jouberin protein, including patient mutations. A, The gene encoded in ∼215 kb of genomic DNA and that contains 31 exons, 2 of which are alternatively spliced, producing shorter isoforms. Patient mutations are indicated by arrows and occur in exon 8 and exon 10. B, The predicted full-length human Jouberin protein gives a 1,196-aa message with seven WD40 repeats and an SH3 domain.

Figure 3

Conservation of amino acids surrounding the mutation (V443D) in family 115. The mutated valine (V) is highlighted, and the boxed area shows the sequence conservation among diverse species.

Figure 4

Northern-blot analysis of Ahi1 (Jouberin) expression patterns in mice. A, Total mouse embryonic (E) 7–17 polyA mRNA was probed with a fragment of Ahi1 corresponding to exons 6–10. Expression was first evident at E7 and remained strong at E11, E15, and E17. There is a single band at ∼4.7 kb (arrow) corresponding to the mouse Ahi1 gene from the Human Genome Browser. B, Total mRNA from mouse organs, aged P0, probed with the same Ahi1 fragment. Strongest expression was shown in the hindbrain and forebrain, with two bands visualized at 4.7 kb and 3.5 kb (arrows), corresponding to the two major splice variants (fig. 3). Total mRNA loading control is shown below.

Figure 5

RT-PCR of Ahi1 (Jouberin) expression in mouse whole brain, cerebellum, and cortex, at different time points. Band-intensity quantification (standardized against G3PDH controls) revealed that Jouberin showed increasing and then decreasing expression as development progressed. Maximum expression occurred in the cerebellum at E18 and P0, and maximum expression occurred in the cortex at E16 and E18. Decreased but persistent expression in the adult was also evident. G3PDH controls are shown below. C = negative control.