La tunisie Medicale - 2011 ; Vol 89 ( n°05 ) : 479 - 484
[ 19985 times seen ]
Summary

Background: Mental retardation is one of the most frequent major handicap, with a 1-3 % frequency in the general population, it appear a major problem of public health. The recent progress of molecular biology and cytogenetic allowed to identify new genes for non syndromic autosomal recessive mental retardation (NSAR-MR).
Aim: Genetic analysis of NSAR-MR: the GRIK2 gene (6q16.3-q21) and the TUSC3 gene (8p22).
Methods: Four Tunisian families with NSAR-MR were included in this study. Genotyping was made using polymorphic microsatellite markers and statistical analysis was validated using the Fast Link programme of the Easy linkage software (V4:00beta).
Results: Genotyping and linkage analysis excluded linkage of the GRIK2 gene and TUSC3 gene.
Conclusion: Our results confirm the extreme genetic heterogeneity of NSAR-MR.

Key - Words
Article

Mental retardation (MR) is one of the major handicaps in general population. It is defined as a generalized disorder, characterized by significantly impaired cognitive functioning and deficits in two or more adaptative behaviors (such as social skills and communication), with onset before the age of 18 years [1]. Moderate to severe mental retardation (IQ‹50) was estimated to affect 0.4-0.5% of the population [2]. These statistics oscillate among the different epidemiological studies. MR is characterized by high heterogeneous etiology [3], as an important number of studies indicate that the MR may result from genetic impairment [4, 5, 6].
The research from the genetic of non syndromic mental retardation has progressed during last years. More than 20 genes associated with non syndromic X-linked mental retardation have been identified so far [7, 8], while the role of autosomal genes remains limited. To date, only six genes have been reported in NSAR-MR [9, 10]. These are PRSS12 (MRT1: OMIM 606709) [11, 12], CC2D1A ( MRT3: OMIM 608443) [13, 14], CRBN ( MRT2: OMIM 607417) [15, 16, 17], GRIK2 ( MRT6: OMIM 611092) [18], TUSC3 (MRT7: OMIM 601093) [19] and TRAPPC9 ( MRT13:OMIM 613192) [10].
GRIK2 gene encodes a Kainate receptor subunit involved in synaptic transmission [20]. TUSC3 encodes one subunit of the oligosaccharyltransferase (OTase) complex that catalyzes the transfer of an oligosaccharide chain on nascent proteins [21].
In the present study we report the genetic analysis of GRIK2 and TUSC3 in four Tunisian families with NSAR-MR.

MATERIALS AND METHODS

MR families
Four Tunisian families including at least two affected children were selected in a cohort for families with MR, and recruited at the department of congenital and hereditary disorders.
Consanguinity was present in two families (MR-S and MR-D).
A total of 9 patients (5 males and 4 females) and their parents were considered for linkage analysis. The pedigrees are shown in figures 1 and 2. All affected children were born from healthy parents with normal delivery. Clinical examination did not reveal facial dysmorphism, congenital malformations and neurological disturbance. Head circumferences, body heights and weights were normal. The degrees of mental retardation were ranged from mild to severe (Table 1). Biological investigation revealed normal karyotype and normal metabolic screening. Resonance magnetic imaging (MRI) of the brain for all patients did not reveal abnormalities.
Genotyping and haplotypes analysis
After obtaining informed consent from patients and parents for minors, DNA was extracted from peripheral blood of all 21 individuals. A genetic Linkage analysis was carried out for the 4 families using two intragenic polymorphic microsatellite markers for GRIK2 gene: D6S449 (AFM 296 ze5) and
D6S1543 (AFMa 111zf5). For TUSC3, four extragenic microsatellites markers were selected covering the locus:
D8S1827 (AFM107ya1), D8S1731 (AFMa311wd1) and D8S549 (AFM303zc1), D8S261 (AFM123xg5) available in Ensembl database (http://www.ensembl.org) (Tel-D8S1827- D8S1731-D8S549-D8S261-Cent) (Table 2). The genetic

Table 1 : Clinical Features of the 9 affected patients
Voir Tableau 1

Table 2 : Lod Score of GRIK2 gene linkage analysis in families MR-B, MRD and MR-A
Voir Tableau 2

distance between the flanking markers D8S1827 and D8S261 was 6.55cM according to the version of the Genethon map [22].
All PCR reactions were carried out in 50μl aliquot containing 150 ng of genomic DNA, one aliquot of PCR buffer, dNTPs mix (0.2mM each), 1.5 mM MgCl2, 1 μM of each forward and reverse primer, 0.5 unit of Taq DNA polymerase (Invitrogen, Foster City, CA, USA). The cycling conditions were: 1 cycle at 96°C for 5 min, 30 cycles at 96°C for 30s, specific annealing temperature for 30s, and 72°C for 30s, and one final cycle of extension at 72°C for 7 min. PCR reactions were carried out in a Perkin Elmer 2400 thermocycler . Amplified markers were electrophoresed on an ABI Prism 3130 DNA capillary sequencer (Applied Biosystems, Foster City, USA) and were analyzed with Gene Mapper software (Applied Biosystems).

Statistical and Linkage analysis

Informativeness of markers
A marker was considered as informative, if at least one of the two parents was heterozygous for the tested marker.
Linkage analysis
Two point parametric Lod scores between the disease loci and markers were calculated using the Fast Link programme of the easy Linkage Plus software package (v4.00 beta) [23].

Figure 1 : Pedigrees and haplotypes analysis for GRIK2 gene. All black circles and squares indicate confirmed affected status.
Voir Figure1

RESULTS

Informativeness of the markers:
GRIK2 gene: Haplotypes analysis revealed only one non informative family (MR-S) for the two tested markers (D6S449 and D6S1543).
TUSC3 gene: All families were subtyped with two polymorphic markers (D8S549 and D8S1731), in order to increase informativeness, two other microsatellites markers (D8S1827 and D8S261) were added to families MR-A and MR-D. All the studied families were informative for all tested markers.
Haplotypes analysis
GRIK2 gene: For the three studied families, haplotypes analysis allowed us to exclude linkage to GRIK2 gene; indeed this analysis revealed that different haplotypes segregated with NSARMR in two families (MR-D and MR-B). For family MRB, affected and unaffected individuals shared a common haplotype with unaffected individual (Fig 1).
TUSC3 gene: Haplotypes analysis for families MR-B and MRS showed that different haplotypes segregated with NSAR-MR phenotype. Families MR-D and MR-A were genotyped in the first step with two polymorphic microsatellites markers D8S1731 and D8S549. Haplotypes analysis showed that affected individuals shared the same haplotypes, this giving evidence for linkage. Two additional microsatellites markers D8S1827 and D8S261 covering TUSC3 region were used; new haplotypes were shared by affected individuals but with absence of homozygosity (Fig. 2).
Lod scores analysis
Two point parametric Lod scores were calculated for three informative families MR-D, MR-A and MR-B between GRIK2 gene and the two tested markers. For TUSC3 Lod scores were calculated for all tested markers in all families. No significant values were obtained in all cases (Tables 3 - 4).

DISCUSSION


NSARMR is a clinically and genetically heterogeneous disease which is reported from different populations. To date only six genes has been identified [9, 10]. Motazacker et al. [21] confirmed linkage to the GRIK2 gene in a large Iranian family

Figure 2 : Pedigrees and haplotypes analysis for TUSC3 gene. Black circles and squares indicate confirmed affected status.
Voir Figure 2

and identified a novel mutation within this gene, in the same way Garshasbi et al. [19] and Molinari et al. [21] concluded linkage to the TUSC3 gene in one Iranian and one European families and reported two different novel mutations. In the present study we reported the linkage analysis of GRIK2 and TUSC3 genes in Tunisian families. The pedigrees were consistent with an autosomal recessive inheritance pattern of the disease. The current strategy for identification of a new defect gene in NSAR-MR affected families remains positionalcloning. For consanguineous families, the strategy of linkage analysis is usually based on homozygosity mapping method.
The power of homozygosity mapping is to localize a disease gene, using a small number of patients. Affected individuals in a sibling should share a common homozygous haplotype transmitted by heterozygous parents [25]. In our study genotyping, haplotypes analysis and lod score calculation concluded the exclusion of the GRIK2 gene in the three informative families. For TUSC3 gene, haplotypes analysis for family MR-D could conclude to linkage but the absence of homozygozity on patients’ haplotype in consanguineous family

Table 3 : Lod Score of TUSC3 gene linkage analysis in families MR-D, MR-B, MR-S, MR-A.
Voir Tableau 3

and reduced lod scores lead us to exclude the TUSC3 gene linkage. While for family MR-A linkage to TUSC3 may be a candidate gene which has to be confirmed by gene sequencing.
These results are in accordance with previous papers revealing that NSAR-MR is very heterogeneous disease [9]. Genetic analysis in the same families with 3 genes (PRSS12, CRBN, CC2D1A) confirmed exclusion of linkage (personal data). It is noteworthy that other genes were implicated in our pathology, indeed Inlow and Restifio [24] estimated that the total number of genes defects causing autosomal recessive MR could run into the thousands. Linkage analysis in these families is often complemented by the genome wide scan; this approach has been proven to be successful in the identification of new genes causing NSAR-MR.

Acknowledgement:
We thank patients and their family members for their participation in this study. We also thank the department of hereditary and congenital disease Charles Nicolle hospital.

Reference
  1. American Psychiatric Association (1995).Diagnostic and Statistical Manual of Mental Disorders, 4th revised edition DSMIV (Washington,D.C : APA ).
  2. Ropers HH. X-linked mental retardation: many genes for a complex disorder. Curr Opin Genet Dev. 2006; 6: 260-9.
  3. Hou JW, Wang TR, Shang SM. An epidemiological and aetiological study of children with intellectual disability in Taiwan. J Intellct Disabili Res. 1998; 42: 137-44.
  4. Lucy Raymond F, Patrick T. The genetics of mental retardation. Hum Molec Genet. 2006; 15: 110-16.
  5. Arveiler B, Alembik Y, Hanauer A. Linkage analysis suggests at least two loci for X-linked non-specific mental retardation. Am J Med Genet. 1988; 30: 473-83.
  6. Bahi-Buisson N, Chelly J, DesPortes V. Actualités sur la génétique des retards mentaux liés au chromosome X. Rev Neurol (Paris) 2006; 62: 952-63.
  7. Ropers HH. X-linked mental retardation: many genes for a complex disorder. Curr Opin Genet Dev. 2006; 16: 260-9.
  8. Castelvi-Bell S, Mila M. Genes responsible for mental retardation. Mol gen Met. 2001; 72: 104-8.
  9. Basel-Vanagaite L. Genetics of autosomal recessive nonsyndromic mental retardation: recent advances. Clin Genet. 2007; 72: 167-74.
  10. Asif M, Lianna K, Abdul N et al. Identification of Mutations in TRAPPC9, which Encodes the NIK- and IKK-b- Binding Protein, in Non syndromic Autosomal-Recessive Mental Retardation. Am J hum Genet. 2009; 85: 1-7.
  11. Molinari F, Rio M, Meskenaite V et al. Truncating neurotrypsin mutation in autosomal recessive non syndromic mental retardation. Science. 2002; 298: 1779-81.
  12. Didelot G, Molinari F, Tchenio P. Tequila, a neurotrypsin ortholog, regulates long- term memory formation in Drosophila. Science. 2006; 313: 851-3.
  13. Basel-Vanagaite L, Attia R, Yahav M. The CC2D1A, a member of a new gene family with C2 domains, is involved in autosomal recessive non syndromic mental retardation. J Med Genet. 2006; 43: 203-10.
  14. Basel-Vanagaite L, Alkelai A, Straussberg R et al. Mapping of a new locus for autosomal recessive non-syndromic mental retardation in the chromosomal region 19p13.12-p13.2: further genetic heterogeneity. J Med Genet. 2003; 40: 729-32.
  15. Higgins J, Pucilowska J, Lombardi R, Rooney J. Candidate genes for recessive non- syndromic mental retardation on chromosome 3p (MRT2A). Clin Genet. 2004; 65: 496-500.
  16. Higgins J, Pucilowska J, Lombardi R, Rooney J. A mutation in a novel ATP- dependent Lon protease gene in a kindred with mild mental retardation. Neurology. 2004; 63: 1927-31.
  17. Higgins J, Rosen D, Loveless J, Clyman J, Grauy M. A gene for non syndromic mental retardation maps to chromosome 3p25- pter. Neurology. 2000; 55: 335-40.
  18. Najmabadi H, Kuss AW. A defect in the ionotropic glutamate receptor 6 gene (GRIK2) is associated with autosomal recessive mental retardation. Am J Hum Genet. 2007; 81: 792-8.
  19. Garshasbi M, Hadavi V, Habibi H et al. A defect in the TUSC3 gene is associated with autosomal recessive mental retardation. Am J Hum Genet. 2008; 82: 1158-64.
  20. Motazacker M, Benjamin RR, Tim Het al. A defect in the ionotropic Glutamate Receptor 6 Gene (GRIK2) is associated with Autosomal Recessive Mental Retardation. Am J Hum Genet. 2007; 81: 792-8.
  21. Molinari F, Foulquier F, Tarpey PS et al. Oligosaccharyltransferasesubunit mutations in non syndromic mental retardation. Am J Hum Genet. 2008; 82: 1150-7.
  22. Dib C, Faure S, Fizames C et al. A comprehensive genetic map of the human genome based on 5,264 microsatellites. Nature. 1996; 380:152-4.
  23. Ott P. Computer simulation methods in human linkage analysis. Proc Nat Acad Sci USA. 1989; 86:4175-8.
  24. Inlow JK, Restifo LL. Molecular and comparative genetics of mental retardation. Genetics 2004; 166:835-81.
  25. Lander ES, Botstein D. Homozygosity mapping: a way to map human recessive traits with the DNA of inbred children. Science 1987; 236: 1567-70.
Login
E-mail :
Password :
Remember Me Forgot password? Sign UP
Archives
2021
January
February
March
April
May
June
July
August
September
October
November
December
Keywords most used
treatment Child diagnosis surgery prognosis Tunisia Children Crohn’s disease Breast cancer screening Cancer epidemiology Ulcerative colitis Risk factors tuberculosis
Newsletter
Sign up to receive our newsletter
E-mail :
Stay in Touch
Join Us! !