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Case report
This proband was referred for child psychiatric assessment for problems in social development, lifelong severe emotional lability, poor development of play, fantasy and problem-solving, and primary
encopresis. He is the second child of non-consanguineous parents. There was no family history of
developmental disorders. Pregnancy and delivery at 42 weeks gestational age were uneventful. He
was born with a unilateral coloboma of the eye. Early milestones of psychomotor and language
development were within normal range. He was a very active toddler with poor development of
symbolic play. Attention has always been a problem. Until primary school he remained more
interested in sensopathic toys than age-appropriate games. Socializing with peers has always been a
problem. He has always been oversensitive for loud noises. He had problems to adjust to changes.
Pediatric assessment of the encopresis did not show any somatic reasons for the problem. He had
marked anxieties about toilets and the encopresis seemed a consequence of toilet avoidance. Clinical
assessment showed a unilateral coloboma and no other dysmorphic signs. Social interactions lacked
reciprocity and were characterized by interpretation problems, often leading to unexpected emotional
reactions. Receptive and productive vocabulary and syntaxis were within normal range but there were
marked impairments in language pragmatics, e.g. understanding figurative meaning, puns and irony.
Non-verbal communication, e.g. emotion recognition, was impaired. Fast mood changes and anxiety
were induced by often irrelevant triggers. His attention was poor. He showed perseverations and was
abnormally dependent on routines. Intelligence testing with Wechsler Intelligence Scale for Children
Revised (WISC-R) showed a full scale IQ of 71, in the borderline range. Theory-of-mind testing with
the Strange Stories Test showed marked deficits in Theory-of-Mind. According to DSM-IV criteria, and
he fulfilled the criteria for pervasive developmental disorder and for primary encopresis.
Study of gene expression using single nucleotide polymorphisms (SNPs)
Primers
used
for
amisyn
(ex6,
rs105248):
for
gDNA
amplification,
aaaatctaagactgctgtttttccc-3’
5’-aaactgcctatcctggtgactcttc-3’
5’-agaaaaatgcaccttcttccagatc-3’
and
and
5’5’-
aaaatctaagactgctgtttttccc-3’ and the nested primer set 5’-attaactgccaatccaaaattatgg-3’ and 5’tttcccaataaattcaattgttttc-3’
for
cDNA
amplification,
5’-tattaagcaggaaggcattttaatg-3’
tttcccaataaattcaattgttttc-3’ for gDNA and cDNA sequencing.
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and
5’-
Primers
used
for
KIAA1305
(ex9,
rs801737):
5’-attgtcaccaatgagcagattcacatcctg-3’
and
5’-
gaatctcttcatggtgcctgatgacccc-3’ and the nested primer set 5’-gaatctcttcatggtgcctgatgacccc-3’ and 5’atcatggaggctgaggcaccgca-3’
for
cDNA
amplification,
5’-cactgacattggcaacttcctgaag-3’
and
5’-
aacactgacgccagcgaagagt-3’ for gDNA amplification and gDNA and cDNA sequencing.
Study of gene expression and copy-number variation (CNV) using rtQ-PCR
rtQ-PCR using SYBR Green chemistry on the Roche Lightcycler 480 apparatus was performed (a) to
study the expression pattern of Amisyn in different mouse tissues, (b) to confirm aCGH data
concerning the CNV of proximal chromosome 14, and (c) to analyse relative expression of amisyn
(syntaxin-binding protein 6, STXBP6) and KIAA0323 in the patient and five male controls. DNA (from
mouse tissues and from patient and controls) and cDNA (from patient and controls) was obtained as
described in ‘material and methods’ section, and cDNA was diluted 1:15. For quality control, nonamplification control (NAC) samples were further processed in parallel with the cDNA samples but
addition of the Superscript III enzyme was omitted. No NAC samples showed amplification in the rtQPCR analysis. All analyses were performed twice and with a technical replicate. Primers were
designed using the online Roche probefinder software.
(a) Primers used to study Amisyn expression in different mouse tissues were: AmEx2Up 5’tctgcctgtcagtgacaaacaa-3’
and
AmEx3Low
5’-ggagccctcaaactgtttga-3’,
and
AmEx4Up
5’-
cacagacaggaagccagagttt-3’ and AmEx5Low 5’-gctgaatggaggatgctgtt-3’, and primers used for
normalization using housekeeping gene beta-Actin (Actb) were: ActbUp 5’-acccacactgtgcccatctac-3’
and ActbLow 5’-agccaagtccagacgcagg-3’.
(b) Primers used for detection of genomic CNV of the proximal part of chromosome 14 were 298I9Up
5’-aactcccatcctttctggaaca-3’ and 298I9Low 5’-aaaggagaatcaatgaccttggaa-3’, and primers for the 81base pair region within the p53 gene for normalization were: p53Up 5'-cccaagcaatggatgatttga-3' and
p53Low 5'-gagcttcatctggacctgggt-3'.
(c) KIAA0323 was chosen as a control gene both for its location on 14q11.2, proximal to the
breakpoint and its high expression level in lymphoblasts. Expression of amisyn and KIAA0323 was
quantified relative to the mean of three housekeeping genes, beta-actin (ACTB), CLK2 and GUSB.
Primers used to study amisyn expression in EBV transformed lymphocytes of patient and controls
were: am1Up 5’-aatccaaaattatgggaggaaac-3’ and am1Low 5’-ggcttgccttctgcactg-3’, am2Up 5’-
2
agcgtcagaaaaatgcacct-3’ and am2Low 5’-agctgaatggaggatgctgt-3’, primers used for KIAA0323 were:
KIAA0323Up 5’-gagtcatggatggcaagagg-3’ and KIAA0323Low 5’-cggaactggtcattggagac-3’, primers
used for normalization using housekeeping genes beta-actin, CLK2 and GUSB were: ACTBUp 5’ccaaccgcgagaagatga-3’ and ACTBLow 5’-tccatcacgatgccagtg-3’, CLK2Up 5’-tcgttagcaccttaggagagg-3’
and CLK2Low 5’-tgatcttcagggcaactcggg-3’, and GUSBUp 5’-agagtggtgctgaggattgg-3’ and GUSBLow
5’-ccctcatgctctagcgtgtc-3’.
Mutation analysis
We investigated 227 patients with autism. All individuals met the criteria for autism as defined by the
DSM-IV. Patients were selected for an IQ in the range of 2 standard deviations +/- the IQ of the index
patient. Genomic DNA was isolated from white blood cells using standard procedures. Mutation
analysis (heterozygous variations) was performed on all coding exons and exon-intron boundaries of
the amisyn gene using DHPLC technology (Transgenomic WAVE® 3500HT DNA Fragment Analysis
System (DNASep HT), Transgenomic Inc.). PCR conditions and column temperatures for fragment
analysis were calculated with the Navigator Software (Transgenomic Inc.). Specific primers used for
PCR and sequencing are listed as supplementary data. Sequencing of samples with abnormal DHPLC
profiles was carried out using ABI PRISM BigDye® Terminator v3.1 Cycle sequencing kit on an
ABI3130XL automated DNA sequencer (Applied Biosystems).
Primers used for the amplification of genomic DNA fragments containing the exons and exon-intron
boundries of the amisyn gene analysed by DHPLC and subsequent sequencing:
5’-
tgttgtcaattccaactttcc-3’ and 5’-cctggatacctcagaatgaact-3’ (exon 2), 5’-ccaacttgcaggtgcactaa-3’ and 5’tgtgaaaagaatgcctttgct-3’
(exon
3),
5’-gcccttataaatcttttgacagga-3’
and
5’-
tgagtctaaagaaacaggatgactaaa-3’ (exon 4), 5’-tcagctgagccagtttctga-3’ and 5’-aacaccccagagctgagaag3’ (exon 5), 5’-agggagcagcacagtgaaat-3’ and 5’-ttcttgtaaaaactgctgaacaaac-3’ (exon 6).
Electronic-Database Information
UCSC Genome Browser (Kent WJ 2002), http://genome.ucsc.edu/ (for physical map of human region
14q11.2-q12)
National Center for Biotechnology Information (NCBI),
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- NCBI-Entrez Nucleotide, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide (for BAC
clones AL163052, AL132827, AL079352 at 14q12 and AL132780 at 14q11.2; for mRNAs BC067278,
BC009499, AL834346, AK096957 and AF161505 (amisyn [NM_014178]) and U04840 (NOVA1 gene
[NM_006489 and NM_002515]) at 14q12, M17016 (GZMB [NM_004131]), BC027974 (GZMH
[NM_033423]), BC014460 (CTSG [NM_001911]), M69136 (CMA1 [NM_001836]), AB037726
(KIAA1305 [XM_370756]) and BC042554 (KIAA0323 [NM_015299]) at 14q11.2, and housekeeping
genes BC053603 (human CLK2 [NM_003993]), BC014142 (human GUSB [NM_000181]), BC002409
(human ACTB [NM_001101]) and X03672 (mouse Actb [NM_213719]))
- NCBI-Entrez Genome, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Genome (for in silico
analysis of AL132827 and AL132780 on chromosome 14q)
- NCBI dbSNP, http://www.ncbi.nlm.nih.gov/SNP/ (for known SNPs located in the coding sequences of
amisyn [rs1052484 (G/C)], KIAA1305 [rs8017377 (G/A)], GZMB, GZMH, CTSG, CMA1, and NOVA1 at
14q12)
The Institute for Genomic Research (TIGR), http://www.tigr.org/tdb/tgi/ (for in silico design of the
complete cDNA sequence amisyn, CMA1, NOVA1 and KIAA1305)
PromoterInspector
(Genomatix),
http://genomatix.gsf.de/cgi-bin/promoterinspector
/promoterinspector.pl (for promoter analysis of amisyn)
Open Regulator Annotation database (ORegAnno), http://www.oreganno.org/oregano/Index.jsp (for
the curation of known regulatory elements from scientific literature).
Roche probefinder, https://qpcr2.probefinder.com/input.jsp?organism=h_sap (for design of rtQ-PCR
primersets).
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