Molecular definition of a region of chromosome 21 that causes features of the Down syndrome phenotype

Down syndrome (DS) is a major cause of mental retardation and heart disease. Although it is usually caused by the presence of an extra chromosome 21, a subset of the diagnostic features may be caused by the presence of only band 21q22. We now present evidence that significantly narrows the chromosomal region responsible for several of the phenotypic features of DS. We report a molecular and cytogenetic analysis of a three-generation family containing four individuals with clinical DS as manifested by the characteristic facial appearance, endocardial cushion defect, mental retardation, and probably dermatoglyphic changes. Autoradiograms of quantitative Southern blots of DNAs from two affected sisters, their carrier father, and a normal control were analyzed after hybridization with two to six unique DNA sequences regionally mapped on chromosome 21. These include cDNA probes for the genes for CuZn-superoxide dismutase (SOD1) mapping in 21q22.1 and for the amyloid precursor protein (APP) mapping in 21q11.2-21.05, in addition to six probes for single-copy sequences: D21S46 in 21q11.2-21.05, D21S47 and SF57 in 21q22.1-22.3, and D21S39, D21S42, and D21S43 in 21q22.3. All sequences located in 21q22.3 were present in three copies in the affected individuals, whereas those located proximal to this region were present in only two copies. In the carrier father, all DNA sequences were present in only two copies. Cytogenetic analysis of affected individuals employing R and G banding of prometaphase preparations combined with in situ hybridization revealed a translocation of the region from very distal 21q22.1 to 21qter to chromosome 4q. Except for a possible phenotypic contribution from the deletion of chromosome band 4q35, these data provide a molecular definition of the minimal region of chromosome 21 which, when duplicated, generates the facial features, heart defect, a component of the mental retardation, and probably several of the dermatoglyphic changes of DS. This region may include parts of bands 21q22.2 and 21q22.3, but it must exclude the genes S0D1 and APP and most of band 21q22.1, specifically the region defined by S0D1, SF57 and D21S47.     

Cloning a new human gene from chromosome 21q22.3 encoding a glutamic acid-rich protein expressed in heart and skeletal muscle

The identification and functional characterization of genes on chromosome 21 is a necessary step to understand the pathogenesis of the various phenotypic anomalies that affect Down syndrome patients. Using direct cDNA selection we have identified a new gene, SH3BGR, that maps to 21q22.3, proximal to HMG14, and is differentially expressed in heart and skeletal muscle. SH3BGR encodes a novel protein that is characterized by the presence of a proline-rich region containing the consensus sequence for a SH3-binding domain and by an acidic carboxyl-terminal region containing a glutamic acid-rich domain predicted to assume a coiled coil. The presence of two functional domains involved in protein-protein interactions suggests that SH3BGR could be part of a multimeric complex. Its overexpression might alter specific functions of muscular tissue and therefore take part in the pathophysiology of muscular hypotonia in Down syndrome. Received: 12 August 1996 / Revised: 22 October 1996     

Heart morphogenesis is not affected by overexpression of the<Emphasis Type="Italic"> Sh3bgr</Emphasis> gene mapping to the Down syndrome heart critical region

Congenital heart disease (CHD) is the most common birth defect in humans and is present in 40% of newborns affected by Down syndrome (DS). The SH3BGR gene maps to the DS-CHD region and is a potential candidate for the pathogenesis of CHD, since it is selectively expressed in cardiac and skeletal muscle. To determine whether overexpression of Sh3bgr in the murine heart may cause abnormal cardiac development, we have generated transgenic mice using a cardiac- and skeletal-muscle-specific promoter to drive the expression of a Sh3bgr transgene. We report here that heart morphogenesis is not affected by overexpression of Sh3bgr.C.S. and R.D.L. contributed equally to this work     

Heterotrisomy, a significant contributing factor to ventricular septal defect associated with Down syndrome?

Down syndrome (DS; trisomy 21) is associated with a wide range of variable clinical features, one of the most common being congenital heart defects (CHD). We used molecular genetic techniques to study the inheritance of genes on chromosome 21 in children with DS and CHD. Polymorphic markers on the long arm of chromosome 21 were analysed in 99 families who had a child with DS. Of these, 60 children had a CHD and 39 children had no CHD. Heterotrisomy describes the inheritance of an allele from each of three different grandparents. In some cases heterotrisomy will involve the inheritance of three different alleles. Heterotrisomic regions were defined as those showing retention of non-disjoining parental heterozygosity at polymorphic loci in the non-disjoined chromosomes of children with DS. Using polymorphic non-coding markers, we identified a consistent 9.6-cM minimum region (D21S167-HMG14) of heterotrisomy in children with DS and ventricular septal defect (VSD). Comparing individuals with DS and VSD to all others with DS (those either with no CHD or with any other CHD combined) shows the individuals with DS and VSD to have significantly more non-reduction or heterotrisomy in this region (P=0.006, Fisher's exact test, two-tailed). We postulate that heterotrisomy for a gene or genes in this region is a contributing factor to the pathogenesis of VSD in trisomy 21 either through the presence of three different specific alleles or through the presence of specific combinations of alleles.     

Down syndrome: molecular mapping of the congenital heart disease and duodenal stenosis.

Down syndrome (DS) is a major cause of congenital heart and gut disease and mental retardation. DS individuals also have characteristic facies, hands, and dermatoglyphics, in addition to abnormalities of the immune system, an increased risk of leukemia, and an Alzheimer-like dementia. Although their molecular basis is unknown, recent work on patients with DS and partial duplications of chromosome 21 has suggested small chromosomal regions located in band q22 that are likely to contain the genes for some of these features. We now extend these analyses to define molecular markers for the congenital heart disease, the duodenal stenosis, and an "overlap" region for the facial and some of the skeletal features. We report the clinical, cytogenetic, and molecular analysis of two patients. The first is DUP21JS, who carries both a partial duplication of chromosome 21, including the region 21q21.1-q22.13, or proximal q22.2, and DS features including duodenal stenosis. Using quantitative Southern blot dosage analysis and 15 DNA sequences unique to chromosome 21, we have defined the molecular extent of the duplication. This includes the region defined by DNA sequences for APP (amyloid precursor protein), SOD1 (CuZn superoxide dismutase), D21S47, SF57, D21S17, D21S55, D21S3, and D21S15 and excludes the regions defined by DNA sequences for D21S16, D21S46, D21S1, D21S19, BCE I (breast cancer estrogen-inducible gene), D21S39, and D21S44. Using similar techniques, we have also defined the region duplicated in the second case occurring in a family carrying a translocation associated with DS and congenital heart disease. This region includes DNA sequences for D21S55 and D21S3 and excludes DNA sequences for D21S47 and D21S17.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of the congenital heart disease 5/tryptophan rich basic protein homologue gene during heart development in Medaka fish,Oryzias latipes

The congenital heart disease 5 (CHD5)/tryptophan rich basic protein (WRB) is a protein containing a tryptophan‐rich carboxy‐terminal region, which was discovered in the human fetal heart. In humans, this CHD5/WRB is located between the markers ACTL5‐D21S268 within the Down syndrome (DS) Region‐2 at chromosome 21. Congenital heart disease is commonly linked to DS patients. The functions of this gene product are unknown. To identify the functions of CHD5/WRB in heart formation during embryogenesis, the medaka CHD5 cDNA (mCHD5) was isolated and its gene expression pattern and the localization of its gene product were investigated. The obtained mCHD5 belongs to the CHD5 superfamily, whose members include coiled‐coil proteins. The mCHD5 gene was found to be expressed in the developing heart after stage 28 at which the chamber (ventricle and atrium) differentiation in the heart tube is initiated in the embryo. Its gene product was also detected in the developing heart at embryonic stage 28 and 35. Knocking‐down of mCHD5 function caused severe cardiac disorder, including abnormal chamber differentiation, abnormal looping and ocular abnormality such as Cyclops. Our results provide the mCHD5 gene expression pattern as well as its physiological role during heart formation in a vertebrate model system.     

Linkage mapping of the AML1 gene on human chromosome 21 using a DNA polymorphism in the 3' untranslated region.

We have detected a polymorphism in the 3' untranslated region of the AML1 gene, which is located at the breakpoint on chromosome 21 in the t(8;21)(q22;q22.3) translocation often associated with patients with acute myeloid leukemia. Informative CEPH families were genotyped for this polymorphism and used to localize the gene on the linkage map of human chromosome 21. The AML1 gene is located between the markers D21S216 and D21S211, in chromosomal band 21q22.3.     

Human chromosome 21q22.2-qter carries a gene(s) responsible for downregulation of mlc2a and PEBP in Down syndrome model mice

Congenital heart disease (CHD) is a major clinical manifestation of Down syndrome (DS). We recently showed that chimeric mice containing a human chromosome 21 (Chr 21) exhibited phenotypic traits of DS, including CHD. Our previous study showed that myosin light chain-2a (mlc2a) expression was reduced in the hearts of chimeric mice and DS patients. We found that phosphatidylethanolamine binding protein (PEBP) was also downregulated in Chr 21 chimeras in this study. As mlc2a is involved in heart morphogenesis, and PEBP controls the proliferation and differentiation of different cell types, these genes are candidates for involvement in DS-CHD. The DS-CHD candidate region has been suggested to span between PFKL and D21S3, which is the STS marker near the ETS2 loci. To identify gene(s) or a gene cluster on Chr 21 responsible for the downregulation of mlc2a and PEBP, we fragmented Chr 21 at the EST2 loci, by telomere-directed chromosome truncation in homologous recombination-proficient chicken DT40 cells. The modified Chr 21 was transferred to mouse ES cells by microcell-mediated chromosome transfer (MMCT), via CHO cells. We used ES cell lines retaining the Chr 21 truncated at the ETS2 locus (Chr 21E) to produce chimeric mice and compared overall protein expression patterns in hearts of the chimeras containing the intact and the fragmented Chr 21 by two-dimensional electrophoresis. While mouse mlc2a and PEBP expression was downregulated in the chimeras containing the intact Chr 21, the expression was not affected in the Chr 21E chimeras. Therefore, we suggest that Chr 21 gene(s) distal from the ETS2 locus reduce mouse mlc2a and PEBP expression in DS model mice and DS. Thus, this chromosome engineering technology is a useful tool for identification or mapping of genes that contribute to the DS phenotypes.     

Further localization of a gene for paroxysmal dystonic choreoathetosis to a 5-cM region on chromosome 2q34

Paroxysmal dystonic choreoathetosis (PDC) is a rare neurological disorder characterized by episodes of involuntary movement, involving the extremities and face, which may occur spontaneously or be precipitated by caffeine, alcohol, anxiety, and fatigue. PDC is transmitted as an autosomal dominant trait with incomplete penetrance. A gene implicated in this paroxysmal disorder has been mapped to a 10–15 cM region on chromosome 2q31–36 in two families. We describe a third family with PDC. Two-point linkage analyses with markers linked to the candidate PDC locus were performed. A maximum two-point LOD score of 4.20 at a recombination fraction of zero was obtained for marker D2S120, confirming linkage to the distal portion of chromosome 2q. The anion exchanger gene, SLC2C, maps to this region, but the family was poorly informative for polymorphic markers within and flanking this candidate gene. Haplotype analysis revealed a critical recombination event that confines the PDC gene to a 5-cM region bounded by the markers D2S164 and D2S377. We compared the haplotype in our family with that in another chromosome 2-linked PDC family, but did not detect a region of shared genotypes. However, identifying a third family whose disease maps to the same region and narrowing the critical region will facilitate identification of the 2q-linked PDC gene. Received: 10 June 1997 / Accepted: 17 September 1997     

Characterization of a Translocation-Associated Deletion Defines the Candidate Region for the Gene Responsible for Branchio-Oto-Renal Syndrome

Fluorescencein situhybridization analysis of an 8q translocation breakpoint, dir ins(8)(q24.11;q13.3;q21.13), carried by an individual presenting with Branchio-Oto-Renal (BOR) syndrome, resulted in the identification of an associated deletion. The generation of a YAC contig and the isolation of overlapping recombinant P1 and λ phage clones from the region allowed further characterization of this deletion. Its size was estimated to be between 470 and 650 kb, and it was flanked by the two polymorphic markers D8S1060 and D8S1807. This mapping led us to reevaluate the localization of the gene responsible for BOR syndrome and has now focused the search for the BOR gene to within the limits of this deletion.     

De novo satellited 21q associated with corpus callosum dysgenesis, colpocephaly, a concealed penis, congenital heart defects, and developmental delay

De novo satellited 21q associated with corpus callosum dysgenesis, colpocephaly, a concealed penis, congenital heart defects, and developmental delay: We present clinical and cytogenetic data on an infant with de novo satellited 21 q. A 3-month-old boy was found to have microcephaly, developmental delay, hypertelorism, down-slanting palpebral fissures, large low-set ears, a prominent nose, a broad philtrum, a concealed penis, interventricular septal defects, corpus callosum dysgenesis, colpocephaly, ventriculomegaly, and a de novo karyotype of 46,XY,21qs. Standard Ag-NOR staining and FISH studies confirmed a satellite and a deletion on the long arm of a chromosome 21. Quantitative-fluorescent polymerase chain reaction using the polymorphic small tandem repeat markers specific for chromosome 21 determined a maternal origin of the deletion and the breakpoint between D21S156 (21q22.1) (present) and D21S53 (21q22.3) (absent), centromeric to the known minimal holoprosencephaly critical region, D21S13-21qter. The present case provides evidence of the correlation of a distal region of chromosome 21 to the phenotypic effects of monosomy 21.     

Identification and molecular characterization of de novo translocation t(8;14)(q22.3;q13) associated with a vascular and tissue overgrowth syndrome

Klippel-Trenaunay syndrome (KTS) is a disorder primarily characterized by capillary-venous vascular malformations associated with altered limb bulk and/or length. We report the identification of a balanced translocation involving chromosomes 8q22.3 and 14q13 in a patient with a vascular and tissue overgrowth syndrome consistent with KTS. We demonstrated that translocation t(8;14)(q22.3;q13) arose de novo. These data suggest that a pathogenic gene for a vascular and tissue overgrowth syndrome (KTS) may be located at chromosome 8q22.3 or 14q13. Fluorescence in situ hybridization (FISH) analysis was used to define the breakpoint on chromosome 8q22.3 to a <5-cM interval flanked by markers AFMA082TG9 and GATA25E10, and the 14q13 breakpoint within a 1-cM region between STSs WI-6583 and D14S989. This study provides a framework for the fine-mapping and ultimate cloning of a novel vascular gene at 8q22.3 or 14q13.     

The gene for cystathionine beta-synthase (CBS) maps to the subtelomeric region on human chromosome 21q and to proximal mouse chromosome 17

The human gene for cystathionine beta-synthase (CBS), the enzyme deficient in classical homocystinuria, has been assigned to the subtelomeric region of band 21q22.3 by in situ hybridization of a rat cDNA probe to structurally rearranged chromosomes 21. The homologous locus in the mouse (Cbs) was mapped to the proximal half of mouse chromosome 17 by Southern analysis of Chinese hamster X mouse somatic cell hybrid DNA. Thus, CBS/Cbs and the gene for alpha A-crystalline (CRYA1/Crya-1 or Acry-1) form a conserved linkage group on human (HSA) chromosome region 21q22.3 and mouse (MMU) chromosome 17 region A-C. Features of Down syndrome (DS) caused by three copies of these genes should not be present in mice trisomic for MMU 16 that have been proposed as animal models for DS. Mice partially trisomic for MMU 16 or MMU 17 should allow gene-specific dissection of the trisomy 21 phenotype.     

Exceptional complex chromosomal rearrangement and microdeletions at the 4q22.3q23 and 14q31.1q31.3 regions in a patient with azoospermia

In this report we describe the first patient ever found to have azoospermia in association with both exceptional complex chromosomal rearrangements and microdeletions at two translocation breakpoints. A 36-year-old male who had been suffering from male factor infertility was admitted to our clinic. The patient also displayed mild dysmorphia. An analysis of the patient's semen revealed azoospermia. GTG banding revealed the presence of an exceptional complex chromosomal rearrangement involving chromosomes 1, 4, 10 and 14. Using subtelomeric FISH analysis, the patient's karyotype was designated as 46,XY,t(1;10)(q43q44;q21q26.1)(CEB108/T7+,D1S3738-;10PTEL006+,D10S2290+, D1S3738+), ins(14;4) (q31.3;q23q33)(D14S1420+; D4S3359+, D4S2930+). Array-CGH analysis revealed two microdeletions at the 4q22.3q23 and 14q31.1q31.3 chromosomal regions. We suggest that microdeletions at the 4q22.3q23 and 14q31.1q31.3 chromosomal regions associated with both an exceptional complex chromosomal rearrangement and the Homo sapiens chromosome 4 open reading frame 37 (C4orf37) gene located at the 4q22.3q23 region might be associated with male factor infertility.