Xiao Ning Chen

Moderate expansion of a normally biallelic trinucleotide repeat in spinocerebellar ataxia type 2

The gene for spinocerebellar ataxia type 2 (SCA2) has been mapped to 12q24.1. A1.1–megabase contig in the candidate region was assembled in P1 artificial chromosome and bacterial artificial chromosome clones. Using this contig, we identified a CAG trinucleotide repeat with CAA interruptions that was expanded in patients with SCA2. In contrast to other unstable trinucleotide repeats, this CAG repeat was not highly polymorphic in normal individuals. In SCA2 patients, the repeat was perfect and expanded to 36–52 repeats. The most common disease allele contained (CAG)37, one of the shortest expansions seen in a CAG expansion syndrome. The repeat occurs in the 5′–coding region of SCA2 which is a member of a novel gene family.

VI. Genome Structure and Cognitive Map of Williams Syndrome

Williams syndrome (WMS) is a most compelling model of human cognition, of human genome organization, and of evolution. Due to a deletion in chromosome band 7q11.23, subjects have cardiovascular, connective tissue, and neurodevelopmental deficits. Given the striking peaks and valleys in neurocognition including deficits in visual-spatial and global processing, preserved language and face processing, hypersociability, and heightened affect, the goal of this work has been to identify the genes that are responsible, the cause of the deletion, and its origin in primate evolution. To do this, we have generated an integrated physical, genetic, and transcriptional map of the WMS and flanking regions using multicolor metaphase and interphase fluorescence in situ hybridization (FISH) of bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs), BAC end sequencing, PCR gene marker and microsatellite, large-scale sequencing, cDNA library, and database analyses. The results indicate the genomic organization of the WMS region as two nested duplicated regions flanking a largely single-copy region. There are at least two common deletion breakpoints, one in the centromeric and at least two in the telomeric repeated regions. Clones anchoring the unique to the repeated regions are defined along with three new pseudogene families. Primate studies indicate an evolutionary hot spot for chromosomal inversion in the WMS region. A cognitive phenotypic map of WMS is presented, which combines previous data with five further WMS subjects and three atypical WMS subjects with deletions; two larger (deleted for D7S489L) and one smaller, deleted for genes telomeric to FZD9, through LIMK1, but not WSCR1 or telomeric. The results establish regions and consequent gene candidates for WMS features including mental retardation, hypersociability, and facial features. The approach provides the basis for defining pathways linking genetic underpinnings with the neuroanatomical, functional, and behavioral consequences that result in human cognition.

The genetic architecture of Down syndrome phenotypes revealed by high-resolution analysis of human segmental trisomies

Down syndrome (DS), or trisomy 21, is a common disorder associated with several complex clinical phenotypes. Although several hypotheses have been put forward, it is unclear as to whether particular gene loci on chromosome 21 (HSA21) are sufficient to cause DS and its associated features. Here we present a high-resolution genetic map of DS phenotypes based on an analysis of 30 subjects carrying rare segmental trisomies of various regions of HSA21. By using state-of-the-art genomics technologies we mapped segmental trisomies at exon-level resolution and identified discrete regions of 1.8–16.3 Mb likely to be involved in the development of 8 DS phenotypes, 4 of which are congenital malformations, including acute megakaryocytic leukemia, transient myeloproliferative disorder, Hirschsprung disease, duodenal stenosis, imperforate anus, severe mental retardation, DS-Alzheimer Disease, and DS-specific congenital heart disease (DSCHD). Our DS-phenotypic maps located DSCHD to a <2-Mb interval. Furthermore, the map enabled us to present evidence against the necessary involvement of other loci as well as specific hypotheses that have been put forward in relation to the etiology of DS—i.e., the presence of a single DS consensus region and the sufficiency of DSCR1 and DYRK1A, or APP, in causing several severe DS phenotypes. Our study demonstrates the value of combining advanced genomics with cohorts of rare patients for studying DS, a prototype for the role of copy-number variation in complex disease.

The human lumican gene: Organization, chromosomal location, and expression in articular cartilage

A human lumican cDNA sequence was derived by polymerase chain reaction techniques from RNA obtained from intestine, placenta, and articular cartilage. A contiguous sequence of 1729 bases was obtained corresponding to an observed message size of 1.8 kilobases (kb). The cDNA sequence consists of an 80-base pair (bp) 5′-untranslated region, a 1014-bp coding sequence, and a 618-bp 3′-untranslated region terminating in a 17-bp poly(A) tail. The deduced lumican protein sequence has 338 amino acids, including a putative 18-residue signal peptide. The human lumican gene was shown to be spread over about 7.5 kb of genomic DNA and to be located on chromosome 12q22. The gene consists of 3 exons separated by introns of 2.2 and 3.5 kb. The shorter 5′-intron resides 21 bases prior to the translation initiation codon, and the 3′-intron resides 152 bases prior to the translation termination codon. The lumican message is expressed at high levels in adult articular chondrocytes but at low levels in the young juvenile. This age-related trend in message level is not, however, common to all tissues in which the lumican gene is expressed. Lumican is present in the extracellular matrix of human articular cartilage at all ages, although its abundance is far greater in the adult. In the adult cartilage lumican exists predominantly in a glycoprotein form lacking keratan sulfate, whereas the juvenile form of the molecule is a proteoglycan.

Down syndrome congenital heart disease: a narrowed region and a candidate gene.

Purpose: Down syndrome (DS) is a major cause of congenital heart disease (CHD) and the most frequent known cause of atrioventricular septal defects (AVSDs). Molecular studies of rare individuals with CHD and partial duplications of chromosome 21 established a candidate region that included D21S55 through the telomere. We now report human molecular and cardiac data that narrow the DS-CHD region, excluding two candidate regions, and propose DSCAM (Down syndrome cell adhesion molecule) as a candidate gene.Methods: A panel of 19 individuals with partial trisomy 21 was evaluated using quantitative Southern blot dosage analysis and fluorescence in situ hybridization (FISH) with subsets of 32 BACs spanning the region defined by D21S16 (21q11.2) through the telomere. These BACs span the molecular markers D21S55, ERG, ETS2, MX1/2, collagen XVIII and collagen VI A1/A2. Fourteen individuals are duplicated for the candidate region, of whom eight (57%) have the characteristic spectrum of DS-CHD.Results: Combining the results from these eight individuals suggests the candidate region for DS-CHD is demarcated by D21S3 (defined by ventricular septal defect), through PFKL (defined by tetralogy of Fallot).Conclusions: These data suggest that the presence of three copies of gene(s) from the region is sufficient for the production of subsets of DS-CHD. This region does not include genes located near D21S55, previously proposed as a “DS critical region,” or the genes encoding collagens VI and XVIII. Of the potential gene candidates in the narrowed DS-CHD region, DSCAM is notable in that it encodes a cell adhesion molecule, spans more than 840 kb of the candidate region, and is expressed in the heart during cardiac development. Given these properties, we propose DSCAM as a candidate for DS-CHD.

Williams syndrome deficits in visual spatial processing linked to GTF2IRD1 and GTF2I on Chromosome 7q11.23

Purpose: To identify the relationship between specific genes and phenotypic features of Williams syndrome. Methods: Subjects were selected based on their deletion status determined by fluorescence in situ hybridization using a panel of 24 BACs and cosmids spanning the region commonly deleted and single gene analysis using Southern blotting. From the cohort of subjects, three had atypical deletions. Physical examinations and cognitive tests were administered to the three subjects and the results were compared to those from a cohort of typical WS subjects. Results: The molecular results indicate smaller deletions for each subject. In all three cases, typical Williams facies were absent and visual spatial abilities were above that of full deletion WS subjects, particularly in the qualitative aspects of visual spatial processing. Conclusions: Combining the molecular analysis with the cognitive results suggest that the genes GTF2IRD1 and GTF2I contribute to deficits on visual spatial functioning.

Is it Williams Syndrome? GTF2IRD1 implicated in Visual-Spatial Construction and GTF2I in Sociability Revealed by High Resolution Arrays

Genetic contributions to human cognition and behavior are clear but difficult to define. Williams syndrome (WS) provides a unique model for relating single genes to visual–spatial cognition and social behavior. We defined a ∼1.5 Mb region of ∼25 genes deleted in >98% of typical WS and then rare small deletions, showing that visual–spatial construction (VSC) in WS was associated with the genes GTF2IRD1 and GTF2I. To distinguish the roles of GTF2IRD1 and GTF2I in VSC and social behavior, we utilized multiple genomic methods (custom high resolution oligonucleotide microarray, multicolor FISH and somatic cell hybrids analyzed by PCR) to identify individuals deleted for either gene but not both. We analyzed genetic, cognitive and social behavior in a unique individual with WS features (heart defects, small size, facies), but with an atypical deletion of a set of genes that includes GTF2IRD1, but not GTF2I. The centromeric breakpoint localized to the region 72.32–72.38 Mb and the telomeric breakpoint to 72.66 Mb, 10 kb downstream of GTF2IRD1. Cognitive testing (WPPSI‐R, K‐BIT, and PLS‐3) demonstrated striking deficits in VSC (Block Design, Object Assembly) but overall performance 1.5–3 SD above WS means. We have now integrated the genetic, clinical and cognitive data with previous reports of social behavior in this subject. These results combine with previous data from small deletions to suggest the gene GTF2IRD1 is associated with WS facies and VSC, and that GTF2I may contribute to WS social behaviors including increased gaze and attention to strangers.

A comprehensive large-insert yeast artificial chromosome library for physical mapping of the mouse genome

A yeast artificial chromosome (YAC) library with large insert size and deep coverage is an essential resource for the construction of physical maps of mammalian genomes. Two large-insert YAC libraries of the mouse genome have previously been reported. Larin and associates (199l) constructed a threefold coverage library with average insert size of 700 kb, using the mouse strain C3H. Kusumi and colleagues (1993) constructed a library with the larger-insert portion providing 3.6-fold with an average insert size of 680 kb, using the strain C57Bl/6J. Other mouse YAC libraries with smaller inserts were constructed by Chartier and coworkers (1992) and Rossi and associates (1992). These libraries are excellent resources for positional cloning, but none is ideal for construction of a physical map of the entire mouse genome. Here, we report the construction and availability of a mouse YAC library providing roughly 10-fold coverage with an average insert size of 820 kb. This library provides the basis for our current effort to construct a complete physical map anchored to the mouse genetic map (Dietrich et al. 1996), with our smaller previous mouse library from the same strain (Kusumi et al. 1993) providing additional coverage as required. The library was constructed with a YAC vector different from the traditionally used vector pYAC4. The vector (Spencer et al. 1993) consists of two arms carried on different plasmids. The pRMLI vector arm carries a TRP1 selectable marker with a complete promoter element, and the pRML2 arm carries the URA3 marker. Among its advantages, the vector allows simultaneous selection for both Trp + and Ura + transformants. By contrast, use of pYAC4 vector requires single selection for Ura § followed by screening for Trp +, as its TRP1 promoter is weak. The vector also contains T3 and T7 promoters flanking the cloning site to facilitate production of probes from the insert DNA. Additionally, pRML1 carries a yeast centromere with an adjacent GALl promoter and a heterologous thymidine kinase gene. Growth on galactose to inactivate the centromere, plus selection for thymidine kinase expression, increases the copy number of the YAC. The YAC library was prepared with genomic DNA from C57BL/6J female mice according to Foote and Denny (1994) with several modifications. DNA was isolated from kidney nuclei as described by Strauss and colleagues (1992) and partially digested by EcoRI-EcoRI methylase competition. The products of this digestion were size selected by pulsed field gel electrophoresis to be larger than 800 kb, by using conditions under which DNA of this size migrates in the zone of limiting mobility. This DNA was

Mammalian DSCAMs: roles in the development of the spinal cord, cortex, and cerebellum? ☆

Central nervous system (CNS) development involves neural patterning, neuronal and axonal migrations, and synapse formation. DSCAM, a chromosome 21 axon guidance molecule, is expressed by CNS neurons during development and throughout adult life. We now report that DSCAM and its chromosome 11 paralog DSCAML1 exhibit inverse ventral-dorsal expression patterns in the developing spinal cord and distinct, partly inverse, expression patterns in the developing cortex, beginning in the Cajal-Retzius cells. In the adult cortex, DSCAM predominates in layer 3/5 pyramidal cells and DSCAML1 predominates in layer 2 granule cells. In the cerebellum, DSCAM is stronger in the Purkinje cells and DSCAML1 in the granule cells. Finally, we find that the predicted DSCAML1 protein contains 60 additional N-terminal amino acids which may contribute to its distinct expression pattern and putative function. We propose that the DSCAMs comprise novel elements of the pathways mediating dorsal-ventral patterning and cell-fate specification in the developing CNS.

Cryptic translocation identification in human and mouse using several telomeric multiplex FISH (TM-FISH) strategies

Experimental data published in recent years showed that up to 10% of all cases of mild to severe idiopathic mental retardation may result from small rearrangements of the subtelomeric regions of human chromosomes. To detect such cryptic translocations, we developed a “telomeric” multiplex fluorescence in situ hybridization (M-FISH) assay, using a set of previously published and commercially available subtelomeric probes. This set of probes includes 41 cosmid/PAC/P1 clones located from less than 100 kilobases to approximately 1 megabase from the end of the chromosomes. Similarly, a published mouse probe set, comprised of BACs hybridizing to the closest known marker toward the centromere and telomere of each mouse chromosome, was used to develop a mouse-specific “telomeric” M-FISH. Three different combinatorial labeling strategies were used to simultaneously detect all human subtelomeric regions on one slide. The simplest approach uses only three fluors and can be performed in laboratories lacking sophisticated imaging equipment or personnel highly trained in cytogenetics. A standard fluorescence microscope equipped with only three filters is sufficient. Fluor-dUTPs and labeled probes can be custom made, thus dramatically reducing costs. Images can be prepared using imaging software (Adobe Photoshop) and analysis performed by simple visual inspection.