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A novel deletion mutation in the TUSC3 gene in a consanguineous Pakistani family with autosomal recessive nonsyndromic intellectual disability
https://doi.org/10.1186/1471-2350-12-56
© Khan et al; licensee BioMed Central Ltd. 2011
Received: 7 January 2011
Accepted: 22 April 2011
Published: 22 April 2011
Abstract
Background
Intellectual disability (ID) is a serious disorder of the central nervous system with a prevalence of 1-3% in a general population. In the past decades, the research focus has been predominantly on X-linked ID (68 loci and 19 genes for non syndromic X linked ID) while for autosomal recessive nonsyndromic ID (NSID) only 30 loci and 6 genes have been reported to date.
Methods
Genome-wide homozygosity mapping with 500 K Nsp1 array (Affymetrix), CNV analysis, PCR based breakpoint mapping and DNA sequencing was performed to explore the genetic basis of autosomal recessive nonsyndromic ID in a large Pakistani family.
Results
Data analysis showed linkage at 8p23 locus with common homozygous region between SNPs rs6989820 and rs2237834, spanning a region of 12.494 Mb. The subsequent CNV analysis of the data revealed a homozygous deletion of 170.673 Kb which encompassed the TUSC3 gene.
Conclusion
We report a novel deletion mutation in TUSC3 gene which is the second gene after TRAPPC9 in which mutation has been identified in more than one family with autosomal recessive NSID. The study will aid in exploring the molecular pathway of cognition.
Keywords
Background
Intellectual disability (ID), also frequently referred to as Mental Retardation or cognitive impairment (CI), is a condition where intelligence quotient is less than 70, there is deficiency in at least two adaptive skills like communication, reading, writing, self care etc and onset before 18 year of age [1]. Assuming a population mean IQ of 100, ID is subcategorized as mild (50-55 to 70), moderate (35-40 to 50-55), severe (20-25 to 35-40) and profound (below 20-25) [1, 2].
Generally it is believed that ~25% of genetic ID patients are thought to have autosomal recessive mode of inheritance [3]. To date 30 loci including six known genes have been reported to be involved in autosomal recessive NS-ID (ARNS-ID) [4]. These include PRSS12 (Protease, Serine, 12 or Neurotrypsin; MIM# 606709) [5], CRBN (Cereblon; MIM# 609262) [6], CC2D1A (Coiled-coil and C2 domain containing protein 1A; MIM# 610055) [7], GRIK2 (Glutamate receptor, ionotropic, kainite 2; MIM#138244) [8], TUSC3 (Tumor suppressor candidate 3; MIM# 601385) [9, 10], TRAPPC9 (Trafficking protein particle complex subunit 9; MIM# 611966) [11–13].
Chromosome 8 is considered an average chromosome with respect to size (146.364 Mb), number of genes (1198), repeat content and degree of segmental duplication [14]. But its p arm showed high degree of sequence variations, particularly within its distal-most ~15 megabase region. This region is believed to be of prime importance in the human genome because of the high expression pattern of nervous system related genes, and has recently been touted as a "hub" for neuropsychiatric developmental disorders [15]. Many genomic imbalances on 8p locus, such as duplication of 8p23.1-8p22.2, are associated with learning disability [16]. Also one gene for microcephaly (MCPH1) and one gene for NS-ID, namely TUSC3, have been identified on 8p.
In this study we present the clinical and molecular analysis of a consanguineous Pakistani family with autosomal recessive NS-ID, and report a novel mutation comprising deletion of the entire TUSC3 gene (except for the promoter and 1st exon) and its down stream region at the 8p23 locus.
Methods
A: Sampling and DNA extraction
The family was recruited from a rural part of Sindh province of Pakistan, after getting prior approval from Institutional Review Board (Quaid-I-Azam U IRB#1- Biomedical; IORG0002926; IRB00003532), and blood samples were taken from available affected and unaffected family members and DNA was extracted from whole blood by following the standard proteinase K/phenol/chloroform isolation method. Written informed and photography consents (Translated in local Urdu language) were obtained from the parents/guardians of patients participating in this study, which conforms to Helsinki Declaration and local legislation.
B: Clinical Assessments
Affected family members were evaluated with the help of standard questionnaire (translated and amended version of Wechsler Intelligence Scale in Urdu) for severity of disease and IQ assessments. Photographs of affected members were taken with written consent to study and publish their facial features. Two individuals from two different loops of the pedigree were selected for cranial CT scan (IV-10 and IV-14) to screen for abnormal brain anatomical features. For information on disease onset, parents were interviewed about the prenatal, perinatal and neonatal medical history of the proband.
C: Molecular Assessments
(i) Genome-wide scan
Whole Genome scan was performed by using Genechip Mapping 500K array NspI chip (Affymetrix) for four affected and 2 unaffected family members and the data was analyzed using dChip and HomozygosityMapper software for homozygosity mapping and copy number analysis [17, 18]. After fine mapping the region in complete family data was analyzed with Merlin for two-point and multipoint LOD score calculations. For this purpose pedigree was divided in to two loops to overcome size limitations of the Merlin.
(ii) PCR based deletion break-point mapping
To map the deletion breakpoints, primers were designed (for both upstream and downstream regions) between the deleted and intact SNPs identified from the microarray data, using Primer3 software (version 0.4.0) [19]. PCR was performed for both sets of primers, and only those sets (from both the distal and proximal regions) were selected that lie closest to the deleted SNPs and showed amplification. PCR was performed using a forward primer from the distal set and reverse primer from the proximal set to amplify the junction fragment. The junction fragment was then sequenced, and the exact physical co-ordinates were determined using the BLAT tool of the UCSC Genome Browser to align the sequence to the genome [20].
Results
Clinical description
Pedigree of a Pakistani family segregating AR-NSID. Square represent male and circles female while black symbols represents affected individuals and clear symbols unaffected individuals. Autozygosity mapping was done for individuals IV-11, IV-12, IV-13 and IV-14. All affected individuals share a homozygous haplotype in the chromosome 8p23.1 region, which is shown in boxes. Markers with physical map distances are shown on the left side.
Facial pictures of patients with front and side pose; Individual IV-10 has a minor ophthalmic issue, whereas individuals IV-11, and IV-14 reveal no apparent dysmorphism.
Summary of the clinical data of affected individuals.
Clinical Findings | IV-12 | IV-14 | IV-13 | IV-6 | IV-10 | IV-11 |
---|---|---|---|---|---|---|
Sex | Female | Female | Female | Male | Male | Male |
Age on assessment | 15 years | 10 years | 13 years | 18 years | 10 years | 11 years |
Developmental delay | + | + | + | + | + | + |
Head Circumference | 52 | 51 cm | 51 | Not Available | 52 cm | 50 cm |
Speech Development | + | + | + | + | + | + |
Dysmorphic feature | - | - | - | - | - | - |
Skeletal Problem | - | - | - | - | - | - |
Ophthalmological problem | - | - | - | - | - | - |
Epilepsy | - | + | - | - | - | - |
Mental retardation | Severe | Severe | Severe | Severe | Severe | Severe |
Growth | Normal | Normal | Weak | Normal | Normal | Normal |
Schooling | - | - | - | - | - | - |
Learning Disability | + | + | + | + | + | + |
Muscular dystrophy | - | - | - | - | - | - |
Self biting | - | - | - | - | - | - |
Molecular studies
(i) Genome-wide Homozygosity mapping and CNV analysis
Graphical representation of the homozygous by decent (HBD) regions identified by homozygosity mapper. The red bar indicates the HBD identified on chromosome 8.
Two point and multipoint LOD score between identified HBD and chromosome 8 markers.
Markers | Genetic Position in cM (Rutgers map, build 36) | Physical Position in bp {Feb.2009 (GRCh37/hg19} | Two point LOD Score | Multipoint LOD Score |
---|---|---|---|---|
D8S1781 | 6.8 | 3678400 | 2.2342 | 4.2286 |
D8S262 | 7.13 | 3777275 | 1.0814 | 4.2143 |
D8S518 | 9.88 | 4587958 | 0.5706 | 1.456 |
rs6989820 | - | 5041417 | -3.8284 | -4.0196 |
D8S1140 | 11.96 | 5617059 | 3.1114 | 2.9368 |
D8S277 | 14.92 | 6616946 | 2.9565 | 5.0000 |
D8S351 | 18.94 | 8877155 | 3.0286 | 5.1617 |
D8S1469 | 19.38 | 9090104 | 1.523 | 5.1693 |
D8S1721 | 19.7 | 10240822 | 1.8241 | 5.1717 |
D8S550 | 20.85 | 10981913 | - Infinity | - Infinity |
D8S552 | 24.76 | 12842458 | 2.9823 | 5.1646 |
D8S1106 | 24.76 | 12936149 | 3.2113 | 5.1646 |
D8S1790 | 26.35 | 13166462 | 3.2843 | 5.1229 |
D8S1731 | 27.95 | 15338590 | 1.3825 | 4.5748 |
rs2237834 | - | 17479493 | -3.8284 | -4.4686 |
D8S1145 | 32.78 | 18452816 | - Infinity | - Infinity |
D8S298 | 40.11 | 21862145 | - Infinity | - Infinity |
(ii) Deletion breakpoint mapping
Ideogram depicting the 12.3 Mb homozygous region on 8p23 locus. Red bar is representing total region of deletion which contains TUSC3 gene shown in blue color bar. The dotted line from red bar over the sequence chromatogram indicates the junction point.
Discussion and Conclusion
Autosomal recessive NSID accounts for ~25% of genetic ID cases and may be more common than X linked cases, however, the molecular basis of AR-NSID is relatively poorly known because of clinical and genetic heterogeneity, and the absence of distinguishing clinical criteria. In non-syndromic ID, cognitive deficit is the sole clinical feature among the patients. It is suggested that ID may be caused by the disruption of the biological and molecular processes in the nervous system such as neuronal differentiation and synaptic plasticity, synaptic vesicles cycling and gene expression, regulation profiling etc [22]. There are number of co-translational and post-translational modifications required for protein stability and proper protein folding into its 3D structure, which are essential for normal protein function. One such post-translational modification, N-glycosylation, has previously reported to be involved in non-syndromic X-linked ID at a TUSC3 paralogous gene, IAP [9].
In the current study we analyzed a four generation Pakistani family with 6 affected individuals having severe psychomotor developmental delay, segregating autosomal recessive mode of inheritance. The major clinical presentations of all patients were normal except for the cognitive dysfunction. However comparison of the clinical data (Biometric and neurological data) of our family with the patients of earlier reports did not reveal any significant difference in the phenotypic expression [9, 10]. TUSC3 is the 5th gene in autosomal recessive NSID and the 2nd in which a 3rd mutation has been identified, after TRAPPC9. Our study will aid in diagnostic assessment of AR-NSID individuals.
Declarations
Acknowledgements
We wish to thank all the members of this family for their voluntary participation and cooperation with this study. MAK was supported by Indigenous Scholarship and International Research Support Program (IRSIP) from the Higher Education Commission (HEC) of Pakistan. JBV is a National Alliance for Research on Schizophrenia and Depression Independent Investigator. This research was supported by grants from the HEC (NRPU-1118), and the Canadian Institutes of Health Research (#MOP-102758).
Authors’ Affiliations
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Pre-publication history
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