PMC4188067_01
PMC4188067_02
Facial phenotype of the patient. The frontal view of patient was shown chronologically at the age of 3 years showing strabismus (A), at the age of 12 years after the surgery to correct strabismus. She had normal teething before taking anticonvulsant (B), at the age of 18 years showing the side effects of damaged teeth and thickened gum from antiepileptic drugs (C), and at the age of 30 years after the exodontias surgery of the rest of decayed teeth with facial dysmorphism characterized by long and thin face with prognathia and high nasal bridge (D). She also had severe flaccid flatfeet (E, F), which were successfully corrected by an orthopedic surgery lengthening the calcaneus with a homologuous graft from the iliac crest at the age of 16 years (G, H). At the age of 31 years, she had fungal infection on the skin of her both hands that provoked a severe dermatitis (I, J).
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F1
Her facial appearances from 3 years to 30 years of age are shown chronologically in Figures 1A-D. In addition, orthopedic surgery for her severe flaccid flatfeet was also successfully performed by lengthening of the calcaneus with a homologous graft from her iliac crest at 16 years of age (Figures 1E-H).In her latest evaluation, the 30-year-old subject still suffers from intractable epilepsy. Additionally, she has global muscular atrophy, hypotonia, joint hyperflexibility (except for her elbows and knees which were rigid), scoliosis, and facial dysmorphism characterized by a long thin face with prognathia and high nasal bridge (Figure 1D). The mouth is usually open with sialorrhea, a full lower lip and a thin upper lip, showing thickened gums (Figure 1C), possibly related to antiepileptic medications. In addition, she has a fungal infection on the skin of her hands exclusively that has provoked a severe dermatitis (Figures 1I and J). TCF7L1 and USP39 were not different among patient (II-1), her mother (I-2), or her sister (II-2) by qRT-PCR using two primer pairs from each gene (Table 1, data not shown). The mRNA expression levels of CAPG, VAMP8, and RNF181 were doubled in the duplication patient compared to her two normal sister and mother by qRT-PCR using two primer pairs designed within CAPG and one primer pair from VAMP8 and RNF181, respectively (Figures 3D-F, Table 1). No amplicon of the putative fusion gene USP39/TCF7L1 was obtained from RT-PCR, suggesting it is not expressed (Figure 4, Table 1).
The brain MRI views without contrast at the age of 14 years. They showed cortical dysplasia and asymmetric lateral ventricles with an enlarged right ventricle.
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F2
Brain MRI without contrast revealed cortical dysplasia and asymmetric lateral ventricles with an enlarged right ventricle at the age of 14 years (Figure 2).
Three candidate genes showing overexpression in qRT-PCR among 15 genes within the 2p11.2 duplication identified by array CGH and confirmed by qPCR. (A) Array CGH showing the internal boundaries of minimal 390 kb heterozygous interstitial duplication at 2p11.2 showing 15 annotated genes involved. Three positional candidate genes, CAPG with recurrent infection and VAMP8 as well as RNF181 with intellectual disability, were depicted in red. Vertical arrows show duplicated region in array CGH and transcription direction is indicated by an arrow for each gene. (B) Pedigree of the family. Patient (II-1) with a multisystem developmental disorder and 2p11.2 microduplication shared the chromosome translocation with her asymptomatic mother (I-2) and younger sister (II-2). (C) The qPCR with genomic DNAs of the patient (II-1), her healthy sisier (II-2), and healthy mother (I-1) showed the duplication at 2p11.2 in the patient only from the primer pairs #2 and #3 of the duplicated region. (D, E, F) qRT-PCR showing the overexpression of three genes. The mRNA expression of each gene was increased by 1.7-2.1 folds in the patient compared to the unaffected mother and the sister.
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F3
Therefore a deletion or uniparental disomy in the critical imprinted region of Prader-Will and Angelman syndromes was highly unlikely (data not shown) However, array CGH analysis revealed a heterozygous 390 kb interstitial duplication (Chr2: 85,474,356-85,864,257 [hg 19]) in band 2p11.2 with a maximum size of 399 kb (Chr2: 85,469,151-85,868,355 [hg 19]) (Figure 3A). The duplicated region was detected in the patient only and confirmed by real-time quantitative PCR (qPCR) (Figure 3C). TCF7L1 and USP39 were not different among patient (II-1), her mother (I-2), or her sister (II-2) by qRT-PCR using two primer pairs from each gene (Table 1, data not shown). The mRNA expression levels of CAPG, VAMP8, and RNF181 were doubled in the duplication patient compared to her two normal sister and mother by qRT-PCR using two primer pairs designed within CAPG and one primer pair from VAMP8 and RNF181, respectively (Figures 3D-F, Table 1).
Prediction of a putative fusion gene USP39/TCF7L1 due to the duplication. If the distal and proximal duplication breakpoints truncate two genes, TCF7L1 and USP39, it will produce a putative fusion gene USP39/TCF7L1, because the transcription direction of both genes is same. In order to amplify the putative fusion gene, a forward primer Fusion-USP39-F and a reverse primer Fusion-TCF7L1-R were used (Table 1).
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F4
No amplicon of the putative fusion gene USP39/TCF7L1 was obtained from RT-PCR, suggesting it is not expressed (Figure 4, Table 1).