Abstract
Objective: To establish genetic linkage between polymorphic microsatellite loci and a disease locus responsible for an autosomal recessive type of nonsyndromic mental retardation (MR).
Background: Although MR is the most common developmental disability in the United States, the etiologies of most nonsyndromic cases are not known.
Methods: A genealogic database provided information to reconstruct the relationships between 32 individuals from five nuclear families in a single pedigree with 10 affected individuals with nonsyndromic MR. To find a MR disease locus in this population, we performed a genome-wide search using genetic loci spaced at 10- to 20-cM intervals. Pairwise linkage analysis, multipoint linkage analysis, and haplotype reconstruction were used to localize the disease gene.
Results: Genetic linkage between a MR disease locus and locus D3S3050 on chromosome 3p25-pter was established with a Zmax = 9.18 at θ = 0.00. Fine mapping this region delimited a 13.47-cM candidate interval defined by key recombinants at loci D3S3525 and D3S1304. Multipoint linkage analysis refined the critical region to a 6.71-cM interval flanked by loci D3S3525 and D3S1560. Evidence that a gene for MR resides in this location is supported by previous breakpoint deletion mapping studies performed in the chromosome 3p− syndrome.
Conclusions: These results suggest that a gene on the subtelomeric region of chromosome 3p contributes to general intelligence. The genes for the cell adhesion L1-like molecule (CALL), the inositol triphosphate receptor (ITPR1), and the AD neuronal thread protein (AD7c-NTP) are leading positional candidates because of their role in brain development, neuronal signaling, and structure.
Mental retardation (MR) is the most common developmental disability in the United States, affecting approximately 1% to 3% of the general population.1-3 Its initial presentation in childhood, developmental delay, is a significant clinical and educational problem in the pediatric population.4-7 The etiologies of MR are diverse and include chromosomal anomalies, recognizable malformation syndromes, structural brain abnormalities, environmental factors or teratogens, and hereditary factors. In some estimates, genetic or inherited metabolic etiologies are found in 66% of cases.8 An autosomal recessive mode of inheritance may account for nearly one fourth of mentally retarded individuals who do not have major physical stigmata, chromosomal anomalies, or fragile X syndrome.9-13
Visible and submicroscopic terminal (subtelomeric) chromosomal rearrangements are found more frequently in individuals with syndromic and idiopathic MR.14,15 The involvement of the terminal regions for cryptic chromosomal anomalies in MR patients is illustrated in several disorders such as cri du chat syndrome,16 α-thalassemia MR,17 and Miller–Dieker18 and Wolf–Hirschhorn syndromes.19 The most common type of syndromic MR in males, fragile X syndrome, also involves the subtelomeric region of the long arm of the X chromosome.20,21 The frequency of these terminal chromosomal sites as genetic causes of MR syndromes has led investigators to screen for telomeric deletions and rearrangements by fluorescent in situ hybridization22 and DNA polymorphism analysis.14,15 The results of these studies suggest that subtelomeric chromosomal abnormalities may be the cause of MR in 5% to 10% of children with dysmorphic features.15 However, it is not clear if these chromosomal anomalies are present more frequently in nondysmorphic cases.14
The unavailability of large family pedigrees with nonsyndromic, autosomal recessive MR has limited the use of genetic linkage analysis to identify causative genes. In this study, we used a private genealogic database to reconstruct the relationship between five nuclear families with 10 members with nonsyndromic MR. The results of a genome-wide search in these families provide evidence that a mutation in a gene on the telomeric region of chromosome 3p can cause a type of nonspecific MR. A comparison of our mapping data with previous breakpoint deletion studies in an MR malformation syndrome (3p− syndrome)23-27 affirms that the cell adhesion L1-like molecule (CALL),28 the inositol triphosphate receptor (ITPR1),29 and the AD neuronal thread protein (AD7c-NTP)30 genes are excellent positional candidates because of their role in brain development, neuronal signaling, and structure.
Subjects and methods.
Genealogic assessment.
The New York State Department of Health and the Center for the Disabled Institutional Review Boards approved this research study. Informed consent was obtained before clinical and genetic testing. The family provided private genealogy books that documented their origins and established the lineage of the study participants (figure 1). A founder couple arrived in America in 1849 from Trippstadt, a town near the modern city of Kaiserslautern in the Rhineland-Palatinate region of Germany. The Cyrillic 2.1.3 software program (Cherwell Scientific Publishing Ltd., Oxford, UK) was used to draw the pedigree.
Figure 1. Abridged pedigree of a family with nonsyndromic mental retardation. Black circles (women) and squares (men) represent affected individuals with mental retardation (n = 10). Unaffected individuals are not shaded. Consanguineous marriages are joined by double bars uniting the individuals. Reconstructed haplotypes for the five chromosome 3p markers D3S3525, D3S3050, D3S1515, D3S1560, and D3S1304 are shown below individuals. Haplotypes segregating with the disease are boxed. Individuals 18, 21, and 29 are recombinant at locus D3S3525, and individual 23 is recombinant at locus D3S1304. For reasons of confidentiality, the order and gender of at-risk individuals are changed.
Clinical evaluations.
A detailed dysmorphologic evaluation, mental status assessment, and neurologic examination were performed on all study participants. The dysmorphologic evaluation included inspection for craniofacial, thoracoabdominal, digital, and limb malformations, and a comparison of anthropologic measurements of the head, eyes, ears, philtrum, limbs, and digits to published standards.31 The results of scholastic achievement and formal intelligence tests were reviewed in affected individuals. A clinical assessment of higher cognitive function was based on Stanford–Binet and Wechsler Intelligence Scales or by testing an individual’s age-adjusted fund of knowledge, problem solving ability, social awareness and judgment, and abstract thinking.32 The neurologic examination included a detailed evaluation of the cranial nerves, deep tendon reflexes, and motor and sensory systems.
Genotyping and linkage analyses.
High molecular weight genomic DNA was isolated from whole-blood lysate by methods described previously.33 A screening set of highly polymorphic, fluorescently labeled microsatellite loci (CHLC/Weber Human Screening Set Version 8, Research Genetics Inc., Huntsville, AL) were used at 10- to 20-cM intervals throughout the entire genome. Additional telomeric loci were identified from the Marshfield Genetic Database (http://www.marshmed.org). Fifty nanograms of genomic DNA were used in a PCR in a total volume of 15 μL with 0.8 μM of each primer, 1.5 mM MgCl2, 200 μM deoxynucleoside triphosphates (dNTPs), 50 mM KCl, 10 mM of Tris HCl (pH = 8.3), 0.01% gelatin, and 0.5 units of Taq DNA polymerase (Perkin Elmer–Cetus, Foster City, CA). Reactions were performed in a 96-well microtiter plate and amplification was carried out for 30 cycles (45 seconds denaturation at 95 °C, 45 seconds annealing at 55 °C, and 45 seconds extension at 72 °C) on a Genius thermocycler (Techne Inc., Princeton, NJ). The last extension step was 7 minutes at 72 °C. Four microliters of the reaction mixture were electrophoresed on an EASIgel (6.0% acrylamide/0.3% bis-acrylamide [7 M urea]) (Scotlab, Strathclyde, Scotland). The PCR products were visualized on a FMBIO II (Hitachi Software Engineering Co., South San Francisco, CA) scanning unit. Genotypes were determined blind to diagnosis. All assumptions regarding phenotype definition and genetic parameters (mode of inheritance, penetrance, and frequency of the susceptibility allele) were made a priori without any information about the genetic marker phenotypes.
Pairwise and multipoint linkage analyses were performed using the MLINK and LINKMAP programs of the FASTLINK package (version 4.0P).34-37 The lod score calculations assumed an autosomal recessive mode of inheritance, no gender differences, and a mutant gene frequency of 1/10,000. Allele frequencies were set at 1/n, where n is equal to the number of published alleles for each locus (http://gdb.infobiogen.fr). To avoid prohibitive calculation times, the pedigree was abridged and modified to break the consanguinity loops by doubling individuals in inbred marriages. The recombination fractions and the order of genetic markers were set as reported in the Marshfield Genetic Database. A genetic map of the telomeric region of chromosome 3p was reconstructed using this database, GeneMap’99 (http://www.ncbi.nlm.nih.gov), and previous deletion mapping data.24
Results.
Phenotypic features.
The initial clinical presentation in all 10 affected individuals was developmental delay during early childhood. Language and social skills were more involved than motor skills. There was no history of pre- or postgestational complications in affected individuals. None of the affected individuals had peri- or postnatal infections, afebrile seizures, toxic exposures, or significant head trauma. Hearing and vision screening were normal. All affected individuals had articulation disturbances (dyslalia) with childish speech but no autistic features. The mean ± SD age of affected individuals was 32.4 ± 10.13 years (range, 9 to 46). Full scale or composite IQ in six affected individuals indicated that they were functioning in the mild to moderate MR range (IQ 51.83 ± 11.96; range, 38–70) as assessed by the Stanford–Binet and Wechsler Intelligence Scales. A bedside clinical assessment33 of higher cognitive function in the remaining four affected individuals estimated that they had mild to moderate MR (IQ, 40–60). The nine affected adults, aged 29 to 46, had limited adaptive skills in communication, home living, and self-care, and could not live independently. The psychoeducational profile of an affected girl, age 9, demonstrated mild MR (IQ = 70, Stanford–Binet) and limited adaptive skills in communication (age equivalent = 4; chronological age = 9; Peabody Picture Vocabulary Test-R) and functional academics. The general appearance of the mentally retarded individuals was normal except for their facial expression, which appeared blank and inappropriate for the social circumstance. One man had pectus carinatum. There were no significant dysmorphic features in the other individuals. Results of general physical and neurologic examinations in all study participants were normal. All affected individuals had normal cytogenetic studies and newborn screening examinations. Individuals 17 and 18 (see figure 1) had normal results on the following: T1- and T2-weighted brain MRI; high-resolution (550 band resolution Giemsa stain) cytogenetic studies; DNA testing for a CCG repeat expansion in fragile X; fasting plasma amino acids, urine amino acids, and organic acids; and pyruvate, lactate, and ammonia. Analysis of the family pedigree (see figure 1) revealed a characteristic pattern for autosomal recessive inheritance.
Genetic analyses.
We genotyped 32 individuals (9 affected, 23 unaffected) at 172 microsatellite markers evenly spaced at 10- to 20-cM intervals throughout the genome. Only loci in the subtelomeric region of chromosome 3p yielded significant lod scores >3.0. A maximum lod score (Zmax) of 9.18 at a recombination fraction (θ) of 0.00 was obtained at locus D3S3050 (figure 2). Genotyping four additional loci in this region supported the assignment of the disease locus to chromosome 3p25-pter (table). Pairwise linkage analysis yielded lod scores that indicated that the disease locus was within the interval defined by the following chromosome 3p loci: D3S3525-5.60 cM-D3S3050-3.92 cM-D3S1515-0.59 cM-D3S1560-3.36 cM-D3S1304. The Human Gene Nomenclature Committee (http://www.gene.ucl.ac.uk) assigned MRT2A as the disease locus.
Figure 2. Genotypes using the tetranucleotide repeat locus D3S3050 on the subtelomeric region of chromosome 3p. A 6% polyacrylamide gel demonstrates the homozygous segregation of the “6” allele in affected individuals with mental retardation. PCR was performed using fluorescein-labeled oligonucleotide primers and the products were visualized on a fluorescent image-scanning unit.
Pairwise lod scores (Z) between chromosome 3p markers and the MRT2A disease locus
Haplotype data in this genetic region (see figure 1) defined the MRT2A locus in a 13.47-cM interval between loci D3S3525 and D3S1304 by demonstrating a lack of homozygosity for these loci in individuals 18, 21, 23, and 29. Recombinations in the heterozygous individuals 22 and 24 confirmed the observations in homozygous individuals.
Multipoint analysis (figure 3) between D3S3525, D3S3050, D3S1515, D3S1560, and D3S1304 and the disease locus placed the disease gene in the 13.47-cM region between loci D3S3525 and D3S1304 with an estimated Zmax = 9.5. The Zmax−1 calculation placed the disease gene 3.4 cM proximal to D3S3535 in a 6.71-cM interval between loci D3S3525 and D3S1560.
Figure 3. Graph of multipoint lod scores. The positions of the marker loci are indicated above the graph. Distances are measured in centiMorgans from locus D3S3525.
Genetic and physical mapping data.
The order- and gender-averaged distances of the loci for the MRT2A locus and the locations of the CALL, AD7c-NTP, ITPR1, a brain expressed sequence (KIAA0212), and differentiated human embryo chondrocyte (DEC1) genes are shown in figure 4. A distal deletion breakpoint in an individual with the 3p− syndrome confirms the localization of gene(s) for MR distal to locus D3S1304.24 The relative positions of the CALL, AD7c-NTP, ITPR1, KIAA0210, and DEC1 genes indicate that they are involved in the MR, cerebral dysgenesis, growth retardation, and skeletal abnormalities in 3p− patients.38
Figure 4. An integrated genetic map of chromosome 3p25-pter. The sex-averaged distances in centiMorgans between loci are shown in relationship to the top of chromosome 3. The relative positions of the cell adhesion L1-like (CALL), AD neuronal thread protein (AD7c-NTP), inositol triphosphate receptor (ITPR1), a brain-expressed sequence (KIAA0212), and differentiated human embryo chondrocyte (DEC1) genes are shown.
Discussion.
The results of this study support four important observations. First, the terminal ends of human chromosomes are “hot spots” in the etiopathogenesis of MR. Second, breakpoint mapping in individuals with the 3p− syndrome confirms that a single susceptibility gene for MR resides within our linked candidate interval. Third, several positional candidate genes in the region encode for proteins that are crucial for normal brain development, neuronal adhesion, and molecular signaling. Fourth, a gene for a recessive form of nonsyndromic MR exists on the subtelomeric region of chromosome 3p.
The 3p− syndrome provides crucial information about the regions surrounding our candidate interval.24,27,39 It is important to realize that the 3p− syndrome phenotype is distinctly different from the nonsyndromic type of MR described in this study. Besides severe MR, this syndrome has concomitant growth failure, cerebral dysgenesis, microcephaly, deafness, and craniofacial and skeletal abnormalities.26 Because genetic material is deleted on the telomere in the 3p− syndrome, it is presumed that an undefined number of genes are missing in the region. Fluorescent in situ hybridization studies and analysis of polymorphic DNA markers demonstrate that patients with the most extensive deletions have a more severe phenotype.24-27 Unlike patients with the typical 3p− syndrome, a single individual with a larger proximal deletion had cyanotic heart disease during infancy with a complete endocardial cushion defect.39 Because of the cardiac anomalies in this patient, other investigators mapped two candidate genes (Caveolin-3 and PMCA2)40,41 involved in normal cardiac development within the extended deleted region. In contrast to patients with the 3p− syndrome, the affected individuals in our study had no evidence of a cytogenetic deletion on the subtelomeric region of chromosome 3p and recombinants in polymorphic loci delimited a small interval that did not contain the Caveolin-3 and PMCA2 genes (figure 4). There are several physical features that distinguish our nonsyndromic patients from individuals with the 3p− syndrome, including the absence of cardiac valvular disease or other features of the 3p− syndrome such as growth failure, microcephaly, deafness, and craniofacial and skeletal abnormalities. In two affected patients in our study, there was no evidence of hydrocephalus or migration abnormalities on brain MRI.
The CALL, ITPR1, and AD7c-NTP genes are intriguing positional candidates for the nonsyndromic MR phenotype in our patients. The CALL gene is a particularly interesting candidate based on its putative role in neurogenesis.28 The CALL glycoprotein belongs to an immunoglobulin family of neural cell adhesion molecules that are primarily expressed on the axons of postmitotic neurons and involved in neuronal migration and neurite outgrowth during development.42,43 Heterogeneous missense mutations in a homologous gene on the X chromosome, the cell adhesion molecule L1 (L1CAM), cause hydrocephalus, spastic paraparesis, and the MASA (mental retardation, aphasia, shuffling gait, and adducted thumbs) syndrome. Mutations in genes encoding for neural cell adhesion molecules may be a frequent cause of MR because some individuals from families with these syndromes only have MR and do not have the other associated features.
Less is known about the role of the ITPR1 or the AD7c-NTP genes in human disease states because mutations have not yet been described. The ITPR1 gene is predominantly expressed in cerebellar Purkinje cells but is also concentrated in neurons in the hippocampal CA1 region, caudate, putamen, and cerebral cortex.29 It shares sequence and functional homology with the ryanodine receptor. Both the ITPR1 and the ryanodine receptor trigger the release of calcium from intracellular stores. The AD7c-NTP gene is overexpressed in brains of AD patients.30
The results of this study localized a gene for nonsyndromic MR to the subtelomeric region of chromosome 3p. Already there is evidence from the 3p− syndrome that this region harbors an MR-related gene. Future studies analyzing the above positional candidate genes for mutations in affected MR individuals and physical mapping efforts to identify new genes in the region may profoundly affect our understanding of the genetics of MR. Identifying new genes within the candidate interval that are involved in normal brain development and intelligence will provide the basis for studies focused on searching for the causes of MR. Although the prevalence of the type of MR phenotype described in this study in unknown, the identification of a gene for general intelligence will advance our understanding of the developmental neurobiology of MR and open avenues for better interventions and effective treatments.
Acknowledgments
Acknowledgment
The authors thank Leah Schraga for her assistance during the initial stages of this project.
Footnotes
-
See also page 328
-
A New Investigator Award (J.J.H.) from the Wadsworth Center provided the funding for genotyping in this research study. Clinical investigations were supported by the Center for the Disabled.
- Received February 17, 2000.
- Accepted March 31, 2000.
References
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