The keeshond defect in cardiac conotruncal development is oligogenic1

Earlier studies in the keeshond breed of dogs established that isolated conotruncal defects (CTDs) are a group of genetically and embryologically related cardiac malformations, including sub-clinical defects of the conal septum, conal ventricular septal defects, tetralogy of Fallot, and persistent truncus arteriosus. The same spectrum occurs in some human families. In both species, inheritance of non-syndromic CTDs is usually complex and multifactorial inheritance has been assumed. Previous studies in the keeshond suggested that susceptibility to CTD is an autosomal recessive trait, with alleles at modifying loci affecting severity. Here we report results of a genome-wide scan for CTD linked loci in a keeshond × beagle F1 backcross pedigree in which 46 of 101 offspring had CTDs. Two-point linkage analysis identified regions of suggestive linkage on each of three chromosomes CFA2, CFA9, and CFA15. No single locus accounted for segregation of CTDs in the pedigree, ruling out a single autosomal susceptibility locus. Multipoint analysis with Genehunter resulted in a corrected LOD score of 3.7 at the locus on CFA9 and supported linkage to the loci on CFA2 and CFA15 (LOD scores of 2.71 and 3.03). Genehunter Twolocus analysis suggested that CTD-predisposing alleles of these three loci are necessary, at least in pairs, to produce CTD. The canine CTD-linked chromosome regions are orthologous to human regions HSA5q11-13, HSA5q31, HSA17q11-24, and HSA4q31. We excluded from the linked regions in the dog, a number of genes known to have a role in the etiology of CTDs and predict that continuing studies will identify CTD-predisposing genes not previously recognized.Electronic Supplementary Material Supplementary material is available for this article at ¹ Nucleotide sequence data reported here are available at GenBank under accession numbers: AY438631, AY438632, AY438633, AY438634, AY438635, AY438636, AY438630     

Familial non-syndromic conotruncal defects are not associated with a 22q11 microdeletion

Molecular studies have shown microdeletions in region q11 of chromosome 22 in nearly all patients with DiGeorge, velocardiofacial and conotruncal anomaly face syndromes (DGS, VCFS and CTAFS, respectively) and in a high percentage of non-syndromic familial cases of conotruncal defects (CTD). CTD account for roughly a fourth to a third of all non-syndromic congenital heart defects (CHD), thus, 22q11 could harbor a major genetic factor of CHD. We searched for a 22q11 microdeletion in familial cases of non-syndromic CTD. Thirty-six cases of various isolated CTD, that is without history of hypocalcemia, immune deficiency, absent thymus, and dysmorphic appearance, were selected. With 48178, a cosmid probe localized in the smallest deleted region of the DiGeorge critical region (DGCR), we found no deletions by fluorescence in situ hybridization in these 36 affected individuals of 16 families with recurrent CTD. Moreover, D22S264, a microsatellilte localized at the distal part of the largest deleted region, was used to genotype the patients. Thirty-two patients out of 37 were heterozygous and hence not deleted at this locus, whereas 5 were uninformative. In conclusion, there are no large deletions in familial cases of various CTD, whether these defects are identical or not within a family. This result does not rule out other minor anomalies in this chromosomal region.     

Molecular studies of DiGeorge syndrome.

DiGeorge Syndrome (DGS) is often associated with loss of a portion of the proximal long arm of chromosome 22. Using a probe for the D22S9 locus, we have examined DNA from eight DGS cell lines and from one balanced-translocation carrier parent of a DGS proband. The D22S9 locus is deleted in four DGS patients, with deletion of 22pter----q11 because of unbalanced translocation. The locus is not deleted from three DGS probands with normal chromosomes or from two DGS probands with interstitial deletions of 22q11. The interstitial deletion DGS probands are also heterozygous for D22S43, another proximal 22q11 locus. This suggests that D22S9 and D22S43 are in a flanking but not critical region for DGS. One of the interstitial deletion DGS probands is monosomic for BCRL2 but has two copies of the flanking BCRL4 and BCR loci. Thus, the region critical to DGS (DGCR) may be in proximity to the BCRL2 locus.     

RXRA and HSPA5 map to the telomeric end of dog chromosome 9

Previous results showed that loci from human chromosome 17q (HSA17q) map to the centromeric two-thirds of dog chromosome 9 (CFA9). In these studies fluorescence in situ hybridization (FISH) using a human total chromosome 17 painting probe, indicated that the telomeric one-third of CFA9 must have homology to one or more human chromosomes other than HSA17. Here we report that this distal part of CFA9 contains a segment syntenic to the telomeric end of HSA9q and mouse chromosome 2 (MMU2). The gene loci encoding retinoid X receptor, alpha (RXRA) and heat shock protein 5 (HSPA5 or GRP78), which are found on HSA9q34 and MMU2, occupy a region on CFA9 distal to NF1 and CRYBA1. FISH of a canine specific genomic cosmid clone for RXRA demonstrated the more telomeric localization of this locus to NF1 on CFA9. A linkage map developed for the distal region of CFA9 included: NF1-(2·7 CM )-CRYBA1-(6·5 CM )-RXRA-(22 CM )-HSPA5. The next best order, RXRA-NF1-CRYBA1-HSPA5 with a difference in the log odds of 1·43 does not correspond to our findings with FISH. The most probable map order places HSPA5 distal to RXRA on CFA9 whereas in humans it lies centromeric of RXRA on HSA9q34.     

Identification of a human chromosome-specific interstitial telomere-like sequence (ITS) at 22q11.2 using double-strand PRINS

Interstitial telomeric sequences (ITSs), telomere-like repeats at intrachromosomal sites, are common in mammals and consist of tandem repeats of the canonical telomeric repeat, TTAGGG, or a repeat similar to this. We report that the ITS in human chromosome region 22q11.2 is, in the sequenced genome database, 101 tandem repeats of the sequence TTAGGGAGG. Using the primed in situ labeling (PRINS) technique and primers against the canonical telomeric repeat (TTAGGG), we illuminated telomeric sites for all chromosomes and an ITS locus at 22q11.2. Using the TTAGGGAGG sequence, we designed PRINS primers that efficiently and specifically illuminate the 22q11.2 ITS locus without illuminating telomeric and other ITS loci. The 22q11.2 locus has more repeat units than other ITSs loci enabling an unprecedented high detection frequency for this interstitial telomere locus. The 22q11.2 is associated with hot spots for disease-related chromosome breaks for multiple disorders, such as DiGeorge syndrome and chronic myeloid leukemia. We describe our findings that the ITS at 22q11.2 is in the same area of, and proximal to the common rearrangement region of multiple disorders. We suggest that the ITS might be involved in DNA repair processes in this area to protect the chromosome from more serious damage.     

Interstitial deletions in DiGeorge syndrome detected with microclones from 22q11

DiGeorge syndrome in humans is charaterized by immunodeficiency, heart defects, mental retardation and facial dysmorphism; cytogenetic analysis has shown that deletions at 22q11 occur in approximately 25% of cases. To generate DNA markers from this region, we have microdissected and microcloned band q11 of human Chromosome (Chr) 22. Nineteen thousand clones were obtained from material dissected from 20 chromosome fragments. Seventeen of 61 clones analyzed (28%) were repetitive, 27 (44%) gave no signal, and 17 (28%) detected single copy sequences of which ten mapped to Chr 22. Two of these were found to be deleted in patients with DiGeorge syndrome and either monosomy for 22q11-pter or visible interstitial deletions of 22q11. These two markers are also hemizygous in patients with no visible chromosomal abnormality, demonstrating that submicroscopic deletions are common in DiGeorge syndrome patients.     

A genetic etiology for DiGeorge syndrome: consistent deletions and microdeletions of 22q11.

DiGeorge syndrome (DGS), a developmental field defect of the third and fourth pharyngeal pouches, is characterized by aplasia or hypoplasia of the thymus and parathyroid glands and by conotruncal cardiac malformations. Cytogenetic studies support the presence of a DGS critical region in band 22q11. In the present study, we report the results of clinical, cytogenetic, and molecular studies of 14 patients with DGS. Chromosome analysis, utilizing high-resolution banding techniques, detected interstitial deletions in five probands and was inconclusive for a deletion in three probands. The remaining six patients had normal karyotypes. In contrast, molecular analysis detected DNA deletions in all 14 probands. Two of 10 loci tested, D22S75 and D22S259, are deleted in all 14 patients. A third locus, D22S66, is deleted in the eight DGS probands tested. Physical mapping using somatic cell hybrids places D22S66 between D22S75 and D22S259, suggesting that it should be deleted in the remaining six cases. Parent-of-origin studies were performed in five families. Four probands failed to inherit a maternal allele, and one failed to inherit a paternal allele. On the basis of these families, and of six maternally and five paternally derived unbalanced-translocation DGS probands in the literature, parent of origin or imprinting does not appear to play an important role in the pathogenesis of DGS. Deletion of the same three loci in all 14 DGS probands begins to delineate the region of chromosome 22 critical for DGS and confirms the hypothesis that submicroscopic deletions of 22q11 are etiologic in the vast majority of cases.     

Mouse and Human CRKL Is Dosage Sensitive for Cardiac Outflow Tract Formation

The human chromosome 22q11.2 region is susceptible to rearrangements during meiosis leading to velo-cardio-facial/DiGeorge/22q11.2 deletion syndrome (22q11DS) characterized by conotruncal heart defects (CTDs) and other congenital anomalies. The majority of individuals have a 3 Mb deletion whose proximal region contains the presumed disease-associated gene TBX1 (T-box 1). Although a small subset have proximal nested deletions including TBX1, individuals with distal deletions that exclude TBX1 have also been identified. The deletions are flanked by low-copy repeats (LCR22A, B, C, D). We describe cardiac phenotypes in 25 individuals with atypical distal nested deletions within the 3 Mb region that do not include TBX1 including 20 with LCR22B to LCR22D deletions and 5 with nested LCR22C to LCR22D deletions. Together with previous reports, 12 of 37 (32%) with LCR22B–D deletions and 5 of 34 (15%) individuals with LCR22C–D deletions had CTDs including tetralogy of Fallot. In the absence of TBX1, we hypothesized that CRKL (Crk-like), mapping to the LCR22C–D region, might contribute to the cardiac phenotype in these individuals. We created an allelic series in mice of Crkl, including a hypomorphic allele, to test for gene expression effects on phenotype. We found that the spectrum of heart defects depends on Crkl expression, occurring with analogous malformations to that in human individuals, suggesting that haploinsufficiency of CRKL could be responsible for the etiology of CTDs in individuals with nested distal deletions and might act as a genetic modifier of individuals with the typical 3 Mb deletion.     

A novel, single nucleotide polymorphism-based assay to detect 22q11 deletions

Velocardiofacial syndrome, DiGeorge syndrome, and conotruncal anomaly face syndrome, now collectively referred to as 22q11deletion syndrome (22q11DS) are caused by microdeletions on chromosome 22q11. The great majority ( approximately 90%) of these deletions are 3 Mb in size. The remaining deleted patients have nested break-points resulting in overlapping regions of hemizygosity. Diagnostic testing for the disorder is traditionally done by fluorescent in situ hybridization (FISH) using probes located in the proximal half of the region common to all deletions. We developed a novel, high-resolution single-nucleotide polymorphism (SNP) genotyping assay to detect 22q11 deletions. We validated this assay using DNA from 110 nondeleted controls and 77 patients with 22q11DS that had previously been tested by FISH. The assay was 100% sensitive (all deletions were correctly identified). Our assay was also able to detect a case of segmental uniparental disomy at 22q11 that was not detected by the FISH assay. We used Bayesian networks to identify a set of 17 SNPs that are sufficient to ascertain unambiguously the deletion status of 22q11DS patients. Our SNP based assay is a highly accurate, sensitive, and specific method for the diagnosis of 22q11 deletion syndrome.     

Molecular studies of a translocated (X;22) DiGeorge patient using somatic cell hybridization.

In order to better characterize the chromosomic rearrangement of an unbalanced 45XX t(X;22) (q28;q11) DiGeorge patient, a somatic hybrid clone segregating the translocated chromosome was constructed and investigated using X and 22 linked markers. Our study demonstrated that this de novo translocation was from paternal origin. The breakpoint was assigned between DXS296 and IDS loci at Xq28 and between D22S9 and BCRL2 at 22q11. This observation and published data allow to locate a "critical region" for DiGeorge syndrome between these two last loci on 22q11. Our hybrid clone may be a useful tool for mapping new probes arising in this region.     

Clinical, cytogenetic, and molecular characterization of seven patients with deletions of chromosome 22q13.3.

We have studied seven patients who have chromosome 22q13.3 deletions as revealed by high-resolution cytogenetic analysis. Clinical evaluation of the patients revealed a common phenotype that includes generalized developmental delay, normal or accelerated growth, hypotonia, severe delays in expressive speech, and mild facial dysmorphic features. Dosage analysis using a series of genetically mapped probes showed that the proximal breakpoints of the deletions varied over approximately 13.8 cM, between loci D22S92 and D22S94. The most distally mapped locus, arylsulfatase A (ARSA), was deleted in all seven patients. Therefore, the smallest region of overlap (critical region) extends between locus D22S94 and a region distal to ARSA, a distance of > 25.5 cM.     

Investigations with fluorescence in situ hybridization (FISH) demonstrate loss of the telomeres on the reciprocal chromosome in three unbalanced translocations involving chromosome 15 in the Prader-Willi and Angelman syndromes

Two patients with classical features of Angelman syndrome (AS) and one with Prader-Willi syndrome (PWS) had unbalanced reciprocal translocations involving the chromosome 15 proximal long arm and the telomeric region of chromosomes 7, 8 and 10. Fluorescence isitu hybridization (FISH) was used for the detection of chromosome 15(q11-13) deletions (with probes from the PWS/AS region) and to define the involvement of the telomere in the derivative chromosomes (with library probes and telomere-specific probes). The 15(q11-13) region was not deleted in one patient but was deleted in the other two. The telomere on the derivative chromosomes 7, 8 and 10 was deleted in all three cases. Thus, these are true reciprocal translocations in which there has been loss of the small satellited reciprocal chromosome (15) fragment.     

Linkage disequilibrium of three polymorphic RFLP markers in the apolipoprotein AI-CIII gene cluster on chromosome 11

A panel of glial tumors consisting of 11 low grade gliomas, 9 anaplastic gliomas, and 29 glioblastomas were analyzed for loss of heterozygosity by examining at least one locus for each chromosome. The frequency of allele loss was highest among the glioblastomas, suggesting that genetic alterations accumulate during glial tumor development. The most common genetic alteration detected involved allele losses of chromosome 10 loci; these losses were observed in all glioblastomas and in three of the anaplastic gliomas. In order to delineate which chromosome 10 region or regions were deleted in association with glial tumor development, a deletion mapping analysis was performed, and this revealed the partial loss of chromosome 10 in eight glioblastomas and two of the anaplastic gliomas. Among these cases, three distinct regions of chromosome 10 were indicated as being targeted for deletion: one telomeric region on 10p and both telomeric and centromeric locations on 10q. These data suggest the existence of multiple chromosome 10 tumor suppressor gene loci whose inactivation is involved in the malignant progression of glioma.     

Excess of deletions of maternal origin in the DiGeorge/Velo-cardio-facial syndromes. A study of 22 new patients and review of the literature

We have determined the parental origin of the deleted chromosome 22 in 29 cases of DiGeorge syndrome (DGS) using a CA-repeat mapping within the commonly deleted region, and in one other case by using a chromosome 22 short arm heteromorphism. The CA-repeat was informative in 21 out of 29 families studied and the deleted chromosome was of maternal origin in 16 cases (72%). When these data are pooled with recent results from the literature, 24 de novo DGS, velo-cardio-facial syndrome (VCFS) and isolated conotruncal cardiac disease deletions are found to be of maternal origin and 8 of paternal origin, yielding a ² of 8 with a probability level lower than 0.01. These data, and review of the literature on familial DGS/VCFS and isolated conotruncal cardiopathies suggest that there is a strong tendency for the 22q11.2 deletions to be of maternal origin.     

A single major-gene defect underlying cardiac conotruncal malformations interferes with myocardial growth during embryonic development: studies in the CTD line of keeshond dogs.

The common forms of isolated congenital heart disease are usually not inherited in a Mendelian pattern, and most are considered multifactorial threshold traits. A large subset consisting of a group of malformations of the ventricular outflow region, termed "conotruncal defects" (CTDs), include subarterial ventricular septal defects, tetralogy of Fallot, and persistent truncus arteriosus. Similar aggregations of CTDs have been reported in human families and in the keeshond breed of dog. The results of our early breeding experiments utilizing noninbred keeshonds were not consistent with any hypothesis of a fully penetrant monogenic inheritance. Here we report a recent series of genetic and embryologic studies conducted after more than 10 generations of selective inbred matings between affected-CTD-line dogs. The results are now consistent with a defect at a single autosomal locus, the Mendelian pattern of transmission having been obscured prior to selective inbreeding by genetic background. On the basis of morphometric embryologic studies, the mutant CTD allele causes conotruncal malformations in homozygous animals by interfering with myocardial growth in the conotruncus during the critical window when the conotruncal cushions fuse to form the conotruncal septum.     

Deletion mapping by FISH with BACs in patients with partial monosomy 22q13

Patients with deletions in 22q13 are known to have phenotypic features that include normal or accelerated growth, large hands and feet, hypotonia, delayed psychomotor development and mild facial dysmorphism. To date, very few cases have been investigated by detailed molecular genetic analysis. We have analyzed three new patients with terminal deletions in 22q. We compared the cytogenetic observations with molecular data assessed by fluorescence in situ hybridization and an array of characterized bacterial artificial chromosome recombinants. The shortest region of deletion overlap is localized in 22q13.2–qter distal to the marker D22S94, but the telomeric repeat in the deleted chromosome appears to remain intact. When parental alleles were investigated in two of the three patients, the aberrant homolog was found to be of paternal origin in both cases. Although the deleted region still spans >20 cM, molecular analysis of additional patients and screening for new genes might help in elucidating candidate genes connected with the dysmorphisms defined by deletions of 22q13. Received: 14 August 1997 / Accepted: 27 January 1998     

Deletions of the long arm of chromosome 10 in progression of follicular thyroid tumors

Previous studies of follicular thyroid tumors have shown loss of heterozygosity (LOH) on the short arm of chromosome 3 in carcinomas, and on chromosome 10 in atypical adenomas and carcinomas, but not in common adenomas. We studied LOH on these chromosomal arms in 15 follicular thyroid carcinomas, 19 atypical follicular adenomas and 6 anaplastic (undifferentiated) carcinomas. Deletion mapping of chromosome 10 using 15 polymorphic markers showed that 15 (37.5%) of the tumors displayed LOH somewhere along the long arm. Thirteen of these tumors showed deletions involving the telomeric part of chromosome 10q, distal to D1OS 187. LOH on chromosome 3p was found in 8 (20%) cases. Seven of these also showed LOH on chromosome 10q. In eight cases LOH was seen on chromosome 10q but not 3p. In comparison, the retinoblastoma gene locus at chromosome 13q showed LOH in 22% of the tumors. Most of these also had deletions on chromosome 10q. The results indicate that a region at the telomeric part of 10q may be involved in progression of follicular thyroid tumors.     

Physical mapping of probes within 14q32, a subtelomeric region showing a high recombination frequency

The genetic linkage map of chromosome 14q32 contains 11 loci which span a distance of more than 60 cM. We have assigned 10 of these loci and the AKT1 proto-oncogene to segments of 14q32, using breakpoints derived from four independent chromosomal deletions or rearrangements. The most telomeric breakpoint was found in a proband (HSC 6) carrying a ring-14 chromosome. HSC 6 is monosomic for the distal part of 14q32, which contains the immunoglobulin heavy-chain locus (IGH), and random markers D14S20, D14S19, and D14S23. Two other chromosomal breakpoints, found in probands HSC 121 and HSC 981, could not be distinguished from each other using DNA probes, although the cytogenetic breakpoints appeared to be different at 14q32.32 and 14q32.31, respectively. The region between the breakpoints of HSC 6 and HSC 121 contains AKT1, D14S1, D14S17, and D14S16. The entire telomeric band 14q32 is assumed to contain about 10% of chromosome 14, or approximately 10 Mb. The 8 most telomeric loci, including D14S1, map to 14q32.32-qter, which measures only several megabases. However, these loci span a genetic distance of 23 cM. The high recombination frequency contrasts with the observation that two of the gamma genes in the IGH constant region show a high degree of linkage disequilibrium, though 180 kb apart. This finding suggests that a telomeric localization per se does not lead to a higher recombination frequency and favors the hypothesis that the higher recombination frequency at the telomeres may be due to specific "hot spots" for recombination.