PMC4383199_01
PMC4383199_02
Facial, dental, and hand profiles of male and female probands. Frontal (a, d) and lateral (b, e) views of the male proband at 17 (a, b) and 22 (d, e) years of age. Frontal (g, l) and lateral (h, m) views of the female proband at 13 (g, h) and 18 (l, m) years of age. Facial features comprise a long triangular face, frontal bossing, broad and high nasal bridge with bulbous nasal tip, large ears, macrodontia of central incisors (f, n), retrognathia, and a pointed chin. (c, i) Small and short hands were identified in both sibs.
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PMC4383199_02_13039_2015_126_Fig1_HTML.jpg
Fig1
The probands were referred to our centre at the ages of 17 (boy) and 13 (girl) (Figure 1). Clinical examination revealed facial dysmorphisms (Figure 1), including long triangular face, frontal bossing, arched and bushy eyebrows (with slight synophrys in the boy), large and prominent ears, broad and high nasal bridge with bulbous nasal tip, anteverted nares, long philtrum, macrodontia of central incisors (Figure 1f and 1n), and a nasal voice. Skeletal anomalies included brachymetacarpia (Figure 1c and 1i). Ratios of peak areas for individual alleles showed that both siblings inherited two copies of the maternal allele and one copy of the paternal allele (Additional file 1: Figure S1).
ANKRD11
intragenic duplication in the probands and their mother. (a) High-resolution array CGH profiles reveal in both sibs a duplication affecting part of the ANKRD11 coding region. Both parents' profiles are normal. (b) Physical map of 16q24.3 showing the exon/intron structure of ANKRD11 (dark blue), the BAC probe CTD-3200N1 used in i-FISH (red), the deleted region (blue), and its hypothetical maximum size (dotted line). (c, d) i-FISH of peripheral blood lymphocytes from patient 1 (c) and his mother (d). BAC probe CTD-3200N1 yields two hybridization signals, one of which is enlarged, in all nuclei of patient 1 (c) and in 5% of maternal nuclei (d) (red arrow).
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Fig2
Array CGH revealed that both siblings were heterozygous for a novel microduplication of 89 kb in 16q24.3 (chr16:89,350,931-89,439,639, hg19), so far unreported in the Database of Genomic Variants (DGV, http://projects.tcag.ca/variation/project.html) (Figure 2a). The duplication extends from ANKRD11 intron 2 to exon 9 or intron 10, depending on the minimum and maximum size of the duplication inferred from reference transcript NM_013275.5 (Figure 2b). Thus, the proximal breakpoint occurred between oligonucleotides A_16_P03198579 and A_14_P119531, and the distal breakpoint between oligonucleotides A_16_P03198760 and A_16_P20559017 (Figure 2b). Parental molecular karyotypes were normal (Figure 2a). The mother's attenuated phenotype and microsatellite typing suggested low-degree maternal mosaicism of the duplication; this suspicion was confirmed by i-FISH with BAC probe CTD-3200N1, which partially covers the duplication (Figure 2b). FISH validated the microarray finding, confirming an ANKRD11 intragenic tandem duplication in both siblings (Figure 2c). Scoring of 500 nuclei in both parents and a normal control revealed that ~5% of maternal cells carried the duplication (Figure 2d); the father exhibited no mosaicism (data not shown).
Molecular characterization of the
ANKRD11
intragenic duplication. (a) Relative expression of ANKRD11 mRNA in lymphocytes of the patients' mother (pink), male patient (blue), and female patient (lilac), compared to a pool of 10 normal controls (gray), whose value was set to 1, using Taqman probes for exon junctions 2-3, 7-8, and 12-13. *, p < 0.01. (b) Graphic representation of wild-type and mutated ANKRD11 mRNAs and corresponding proteins. Non-coding exons are in pink, and coding exons in light pink. Gray arrows show the duplication of exons 3-8, oriented in tandem. Black arrows indicate positions of primers used to sequence the duplication junction. The electropherogram of the mutant ANKRD11 transcript is shown, with exon 8 in yellow and exon 3 in blue; the amino-acid sequence is above the nucleotide sequence. Juxtaposition of exon 3 with exon 8 creates a premature termination codon. GenBank accession numbers of the splice-variant transcripts are KM670019 and KM670020. The truncated protein translated from the mutant ANKRD11 transcript is shown at bottom.
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Fig3
For exon junctions 3-4 and 7-8, included in the duplicated region, PCR product levels were elevated in probands, whereas for exon junction 12-13, outside the duplication, PCR product levels were comparable in probands and controls (Figure 3a). Consistent with this, direct sequencing of ANKRD11 cDNA, using primers at the end of exon 8 and the beginning of exon 3, revealed two mutant ANKRD11 transcripts (Figure 3b). The duplication produces a splice site and so two splice variants were present, one with exon 8 sequence followed by the duplicated exon 3, and the other with deletion of the first codon of the duplicated exon 3 (CAG) (Figure 3b). In both variants, juxtaposition of exon 3 with exon 8 caused a frameshift resulting in premature termination (Figure 3b). The resultant hypothetical truncated protein contains only the ankyrin repeats (Figure 3b).