The SPCH1 Region on Human 7q31: Genomic Characterization of the Critical Interval and Localization of Translocations Associated with Speech and Language Disorder

Family KE

In 1987, the KE family (see fig. 1) was referred for genetic counseling by the director of a school for children with speech and language problems, at which many family members had been pupils. The condition was characterized as a developmental verbal dyspraxia (Hurst et al. 1990).

Patients Bearing Chromosome Translocations

CS is a 5.5-year-old boy with language impairment and verbal dyspraxia. He has a de novo balanced reciprocal translocation t(5;7)(q22;q31.2) which was identified prior to his birth via amniocentesis. Examination at birth showed no abnormalities, but he was referred back to the genetics team at age 2 years because of concerns of delayed speech and mild motor delay. Subsequent assessment at age 3 years 6 mo, performed by means of the Bailey scale, gave an overall mental development in the mildly delayed range. Although nonverbal skills were in the normal range, there was impairment in both understanding and expression of speech. A diagnosis was made of oral dyspraxia. By 4 years of age, CS was able to put two words together, and his understanding had progressed. Fine and gross motor development had also improved, although there was still evidence of mild impairment. There is no history of speech and language disorder in the family of CS, and none of his siblings (one full sibling and three half-siblings) have any language problems. His mother reports that he has never been able to laugh spontaneously or to sneeze.

BRD is an 8-year-old boy with a history of receptive and expressive language problems, accompanied by behavioral difficulties and low-range intellectual abilities, despite normal physical/motor development. He continues to show difficulties following verbal instructions in school, and has word-finding and sequencing problems accompanied by poor articulation. An MRI scan at age 6 years 11 months detected a small dysembryoplastic neuroepithelial tumor in his right temporal lobe. A more detailed description of the clinical phenotype of BRD is given in Warburton et al. (2000). Cytogenetic analysis previously revealed a de novo balanced reciprocal translocation, t(2;7)(p23;q31.3) (Warburton et al. 2000).

Construction of Human-Hamster Somatic Cell Hybrids

The Chinese hamster mutant cell line a23, deficient in thymidine kinase, was cultured in 5% CO₂ as a monolayer in Dulbecco's modification of Eagle medium (DMEM, Life Technologies) supplemented with 10% fetal calf serum (FCS). White cells were separated from peripheral blood from three affected members of the KE family (II-2, II-9, and III-20) using Histopaque (Sigma). Each sample was combined in equal proportions with trypsinized a23 cells and was placed in serum-free medium. After the cells were spun down, the medium was aspirated. The cell mixture was resuspended in 50% polyethylene glycol (PEG, molecular weight 1450, Sigma), was washed, and was set up in culture with DMEM and 10% FCS at low cell density. After 24 h, the culture medium was replaced with the same medium containing hypoxanthine, aminopterin, and thymidine (HAT, Life Technologies). Once hybrid colonies reached 2–4 mm in diameter (14 d after fusion), they were picked into 24-well plates. DNA prepared from samples of 1×10⁵–5×10⁵ cells was tested by PCR for chromosome 7–specific markers.

Genotyping and Linkage Analysis

Primers flanking novel polymorphic repeats were designed using the PRIMER program, accessed through the United Kingdom Human Genome Mapping Project resource center. Fluorescence-based semiautomated genotyping was performed as described (Fisher et al. 1998). Linkage analyses were run under the assumption that the disorder in the KE pedigree is due to a single autosomal dominant locus with full penetrance, as described (Fisher et al. 1998).

Fluorescence In Situ Hybridization (FISH)

PHA-stimulated T lymphocytes or lymphoblastoid cells lines were harvested by conventional techniques, and fixed suspensions were dropped onto slides. Slides were denatured at 70°C in 70% formamide/2×SSC for 2 min 30 s, were incubated in cold 2×SSC, and were serially dehydrated in 70%, 90%, and 100% (twice) ethanol at room temperature. Probe DNA was labeled by nick translation with biotin (Gibco BRL BioNick Labeling System) or Digoxigenin (DIG; Roche) following manufacturers’ protocols. FISH of BACs and PACs was performed as described (Millwood et al. 1997). Biotinylated probes were visualized with two layers of FITC-conjugated streptavidin (green; Vector Labs) and biotinylated goat anti-streptavidin (Vector Labs). DIG-labeled probes were visualized with mouse anti-DIG antibodies (Roche), followed by Cy5-conjugated rabbit anti-mouse and goat anti-rabbit antibodies (pseudocolored blue). Chromosomes were counterstained with Vectorshield containing propidium iodide (Vector Labs). The slides were viewed on a Nikon Optiphot, and images were captured with a Bio-Rad MRC 1024 laser-scanning confocal microscope and Lasersharp software.

Determination of Genomic Organization of CAGH44

Exon/intron boundaries for exons 1–2 of CAGH44 were identified by comparison of the reported mRNA sequence (accession U80741) to completed BAC sequence. Boundaries for exons 3–6 were determined using either long-range PCR or vectorette (Munroe et al. 1994). For the former, we amplified products from human genomic or BAC DNA, using the Expand Long Template System (Boehringer Mannheim), with primers designed from CAGH44 mRNA. Products were sequenced using BigDye Terminator Cycle Sequencing kits (PE Applied Biosystems). For the vectorette method, libraries were made by complete digestion of BAC DNA using frequent-cutting restriction enzymes followed by ligation to annealed vectorette bubble anchors. Fragments containing exon/intron boundary sequences were amplified from vectorette libraries by PCR with the NotI-A primer in combination with specific primers derived from available CAGH44 sequences.

Construction of a BAC-/PAC-Based Sequence Map Spanning the SPCH1 Interval of 7q31

Preliminary analysis of chromosome 7 physical-mapping data indicated that 152 STSs map to the original SPCH1 interval, between D7S2459 and D7S643, specifically within four YAC contigs, designated N, O, P, and Q (Bouffard et al. 1997). Although many BAC and PAC clones from this region have been sequenced and deposited as separate records in the public databases, much of the sequence information is not fully cross-referenced and integrated with other related sequences or with other mapping data. Therefore, we compared the sequence for all 152 STSs mapping between D7S2459 and D7S643 to the “non-redundant,” “high-throughput genome sequence,” and WU-GSC human sequencing project-specific databases using the BLAST algorithm (Altschul et al. 1997). At the time of the most recent analyses (March 2000), 132 of these STSs were found in fully sequenced BACs/PACs, whereas an additional 4 were detected in clones for which partial sequence was available. Thus, only 16 STSs were not found in a sequenced clone.

BLAST analysis of the insert ends of sequenced clones facilitated the establishment of overlaps among adjacent clones and orientation of various sequence blocks. These electronic analyses allowed us to assemble a detailed BAC-/PAC-based sequence map of 7q31 consisting of several blocks of contiguous sequence, the largest of which exceeds 1.5 Mb in length (fig. 2). Within these sequenced regions, we have been able to accurately determine marker order and intermarker distances. In a number of cases, the marker order differs from that previously established in physical-mapping studies. We estimate that the D7S2459–D7S643 interval currently contains >7.75 Mb of completed sequence. No sequence has been obtained, as yet, from the region covered by YAC contig P (sWSS1095-sWSS3263), and this region is estimated from physical mapping studies to span ∼1.25 Mb (Bouffard et al. 1997). Therefore, these analyses indicate that the D7S2459–D7S643 interval is likely to be ⩾9 Mb in size. Of note, all pairs of adjacent STSs within assembled sequence blocks are separated by <220 kb.

Transcript Map of 7q31

GeneMap ’99 includes 147 ESTs that have been mapped to the interval between anchor markers D7S2459 and D7S480 using radiation-hybrid panels G3 and GB4. Analysis of these ESTs suggested that they cluster into <96 transcripts, including 18 genes that have been fully sequenced and/or encode proteins of known function. Electronic PCR and BLAST searches revealed that the majority (>85%) of the ESTs map to sequenced BACs/PACs. A total of 50 of the anonymous transcripts and 13 of the known genes from GeneMap ’99 could be localized precisely within the D7S2459–D7S643 sequence contigs. (Eleven anonymous transcripts and three known genes are contained in sequenced clones that map proximal or distal to the D7S2459–D7S643 interval; the remaining 19 transcripts were not found in BAC/PAC sequences.) An additional six genes not present in the GeneMap ’99 interval (PDS, LAMB1R, MDG1, CAGH44, TSA806, and KCND2) were identified and were localized within the D7S2459–D7S643 contigs via annotations in BAC/PAC sequence records. Finally, one of the STSs (sWSS3217) residing within an as-yet-unsequenced region was found, by homology screening, to correspond to the HF.12 gene. Figure 2 shows the relative positions of all the known genes detected, with additional summary information provided in table 1.

Search for a 7q31 Microdeletion in Family KE

Previously, cytogenetic analyses of affected members of the KE family failed to detect any chromosomal abnormality. However, these studies did not rule out a microdeletion within the SPCH1 interval. We therefore chose to screen affected individuals for the absence of STSs within the critical region. This analysis required the prior separation of the chromosome 7 harboring the mutation from the normal homologue. Somatic hybrid cell lines containing human chromosome 7 on a hamster background were derived from three affected individuals of the KE family (see Methods). DNA from these cell lines was genotyped with the Généthon markers D7S692 and D7S522, previously shown to be polymorphic in the affected individuals. In each case, one allele precisely cosegregates with SPCH1 (Fisher et al. 1998). This allowed identification of hybrid cell lines containing only an affected chromosome 7, those containing only a normal chromosome 7, and those containing both copies of chromosome 7. Hybrid cell lines harboring only the affected chromosome 7 were tested for the presence of 120 STSs mapping within the D7S2459–D7S643 interval (see fig. 2). The average distance between these STSs is <100 kb. In all cases, the expected PCR product was generated from the hybrid cell lines but not from hamster DNA controls, indicating that all 120 STSs are present on the affected chromosome 7 in the KE family.

Generation of Novel Polymorphic Markers in 7q31 and Fine Mapping of the SPCH1 Locus

Our previous linkage study of the KE family with all the Généthon markers from 7q31 identified critical recombinations in affected female III-12 and unaffected male III-3 that defined D7S2459 and D7S643 as proximal and distal limits for SPCH1, respectively (Fisher et al. 1998) (see fig. 3). Within this ∼5.6-cM region, we demonstrated that the ∼3.6-cM interval between D7S692/D7S2425 and CFTR cosegregates perfectly with the disorder (see figs. 2 and 3). We therefore used data from the BAC/PAC sequence contigs to develop novel polymorphic markers from the D7S2459–D7S692 and CFTR–D7S643 intervals, in order to refine further the positions of the III-12 proximal and III-3 distal recombination breakpoints, respectively. Note that the only Généthon markers from these intervals (D7S2456, D7S655, and D7S2487) were uninformative with respect to the disorder in the KE family.

Finished sequence data from these intervals was searched for stretches of unbroken tandem dinucleotide repeats with copy numbers of ⩾16, which would be predicted to be polymorphic (Weber 1990). PCR primers flanking these repeats were used for semiautomated genotyping of the KE family. The results were analyzed with parametric linkage programs and haplotypes were determined as in our previous linkage study. Positions of new markers relative to the STS map could be accurately established from the sequence data (see fig. 2).

Genotyping with four novel markers at the proximal end (table 2) revealed that the recombination breakpoint in individual III-12 maps between 013A and 062B-369B-369C. This refines the SPCH1 interval by a few hundred kb at the proximal end, and excludes three known genes as candidates: PDS, DRA, and DLD (fig. 2). At the distal end of the SPCH1 interval, physical mapping data indicated that a polymorphic tetranucleotide repeat, D7S2847, lies between CFTR and D7S643. Genotyping of the KE family with D7S2847 demonstrated that the recombination in individual III-3 maps proximal to this marker. Investigation of four new markers generated from the sequence data between CFTR and D7S2847 (table 2) revealed that the III-3 recombination breakpoint is localized between 363B-084A and 330B. (084B was uninformative with respect to the disorder.) This result excludes a region of >2.65 Mb, containing the KCND2 gene, from the distal end of the SPCH1 interval. In addition, it limits the SPCH1 locus to within YAC contigs N and O, with only one uncloned gap in the region. Haplotypes of the relevant markers for critical individuals from the KE family are shown in figure 3.

Two Translocation Breakpoints in 7q31 Associated with Speech and Language Disorder

We have investigated de novo balanced translocations involving 7q31 in two unrelated patients with speech and language disorder, CS 46,XY t(5;7)(q22;q31.2) and BRD 46,XY t(2;7)(p23;q31.3) (see Subjects and Methods for clinical descriptions). The 7q31 breakpoints were localized using two-color FISH to metaphase spreads, with BACs/PACs selected from proximal and distal ends of the 7q31 contigs. The breakpoints of both patients were found to map in the D7S2459–D7S643 interval (fig. 4), suggesting that the translocations might be relevant to the etiology of speech and language disorder.

Metaphase FISH with a series of further clones identified from the contigs mapped the CS breakpoint between RG250D13, which consistently hybridized to the derivative 7 [der(7)] and RG308B22, which consistently hybridized to the derivative 5 [der(5)]. Two BACs have been identified on the basis of fingerprint data to span the gap between these clones (fig. 2), and these are in the process of being sequenced. Whereas clone NH0208M04 mapped to der (5), NH0563O05 gave signals on both der(7) and der(5), suggesting that this BAC crosses the 7q31 breakpoint (fig. 4).

A recent study using FISH with YAC clones reported that the BRD translocation mapped between CFTR and D7S643 (Warburton et al. 2000). We have been able to confirm this and to localize the breakpoint at higher resolution using BAC/PAC clones. These analyses indicate that the BRD breakpoint maps within GS180J15, which gives signals on both der(7) and der(2) (fig. 4). Therefore, the 7q31 breakpoints of CS and BRD are separated by ⩾3.75 Mb of DNA, as estimated from completed sequence and the size of YAC contig P (fig. 2). Furthermore, the BRD translocation maps ⩾1.45 Mb distal to the newly refined SPCH1 interval, as established from the most recent linkage analyses of the KE family reported above (fig. 2).

Candidate Gene CAGH44, in the Vicinity of the CS Translocation

CAGH44 mRNA was originally isolated from human brain in an attempt to identify novel CAG-repeat-containing cDNAs whose expansion might be implicated in the etiology of neuropsychiatric disorders (Margolis et al. 1997). The CAGH44 product contains a stretch of 40 consecutive glutamine residues, which is the longest polyglutamine tract found thus far in an unexpanded protein. This is encoded by a combination of CAG and CAA codons, such that there are never more than five consecutive CAGs. There is a second polyglutamine stretch, containing only 10 glutamines, encoded by (CAG)₇(CAA)(CAG)(CAA), which is separated from the first stretch by eight amino acids. Currently, there is only partial cDNA sequence reported for this gene, covering 912 bases of the coding region, and no information about its genomic structure.

Although Margolis et al. (1997) previously localized CAGH44 to 6q14-15 by radiation-hybrid mapping, the chromosome 7 physical map and sequence data indicates that, in fact, it resides in 7q31, close to the CS translocation breakpoint (fig. 2). Specifically, bioinformatic analyses found that RG250D13, the BAC hybridizing to der(7) adjacent to the CS translocation breakpoint, includes the first 258 bases of the partial CAGH44 coding sequence, organized into two exons. Analysis of sequence data from clones linking RG250D13 to RG308B22 enabled us to orient the completed RG250D13 sequence (including the two 5′ CAGH44 exons) relative to the sequence contigs. This indicated that the more 3′ exons of CAGH44 should lie on the distal side of RG250D13. Using long-range PCR and the vectorette method on BAC DNA, we were able to confirm this and to determine the genomic organization of the CAGH44 3′ coding region (table 3), thereby demonstrating the presence of four 3′ exons (3, 4, 5, and 6) in clone NH0563O05, in the interval between sWSS2794 and sWSS1765. This coincides exactly with the region containing the CS breakpoint (fig. 2). Bioinformatic analysis of genomic sequence from BAC RG250D13, upstream of the proposed CAGH44 coding region, confirms that the first ATG in the reported cDNA sequence is very likely to correspond to the start of the open reading frame. Therefore, exon 1 is indeed the first coding exon of this gene, although there may be additional exons upstream of this containing 5′ untranslated sequence. However, the ORF probably extends beyond the 3′ end of the currently reported cDNA sequence, since no in-frame stop codon has yet been reached. In addition, our PCR and vectorette analyses indicate that the last 43 bases (870–912) in the cDNA sequence reported by Margolis et al. (1997) do not come from the CAGH44 gene, but are likely to be an artifact of the original cloning procedure. This hypothesis is supported by the presence of an EcoRI site at 871–876 and by the observation that bases 878–912 are highly homologous (94% identity) to human ribosomal protein S16 mRNA.

Given that CAGH44 maps to the same region as the CS breakpoint and encodes a brain-expressed polyglutamine repeat, we decided to analyze this gene in family KE. We did not detect any expansion of the regions encoding the polyglutamine stretches in members of the family. Furthermore, sequencing of the currently known CAGH44 coding region did not reveal any variant cosegregating with the disorder.

1. Electronic PCR screening, http://www.ncbi.nlm.nih.gov/STS
2. GeneMap ’99, http://www.ncbi.nlm.nih.gov/genemap (for radiation-hybrid map data)
3. HGMP, http://www.hgmp.mrc.ac.uk (for PRIMER program)
4. NCBI BLAST, http://www.ncbi.nlm.nih.gov/BLAST/ (for homology searches of sequence data)
5. NHGRI chromosome 7–mapping data, http://genome.nhgri.nih.gov/chr7
6. UniGene, http://www.ncbi.nlm.nih.gov/UniGene/ (for clustering of ESTs)
7. WU-GSC chromosome 7 sequencing data and BLAST searches, http://genome.wustl.edu/gsc

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