Conserved gene structure and genomic linkage for D-dopachrome tautomerase (DDT) and MIF.

Macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (DDT) are small proteins, which are related both by sequence and by in vitro enzyme activity. Here we show that the gene for DDT in human and mouse is identical in exon structure to MIF. Both genes have two introns that are located at equivalent positions, relative to a twofold repeat in protein structure. Although in similar positions, the introns are in different phases relative to the open reading frame. Other members of this superfamily exist in nematodes and a plant, and a related gene in C. elegans shares an intron position with MIF and DDT. In addition to similarities in structure, the genes for DDT and MIF are closely linked on human Chromosome (Chr) 22 and mouse Chr 10.

Molecular cloning and characterization of the bovine and human tuftelin genes.

The bovine tuftelin gene was cloned and its structure determined by DNA sequence analysis and comparison to bovine tuftelin cDNA. The analyses demonstrated that the cDNA contains a 1014 bp open reading frame encoding a protein of 338 residues with a calculated molecular weight of 38,630 kDa and an isoelectric point of 5.85. Although similar, these results differ from those previously published [Deutsch et al. (1991) J. Biol. Chem. 266, 16021-16028] which contained a different conceptual amino acid sequence for the carboxy terminal region and identification of a different termination codon because of the absence of a single guanine residue in the published sequence. The protein does not appear to share homology or domain motifs with any other known protein. The bovine gene consists of 13 exons ranging in size from 66 to 1531 bp, the latter containing the encoded carboxy terminal and 3' untranslated regions. These exons are embedded in greater than 28 kbp of genomic DNA and codons are generally not divided at exon/intron borders. Sequence analysis of the cDNA and products produced by reverse transcriptase/polymerase chain reaction demonstrated that exons 2, 5 and 6 are alternatively spliced. The 3' portion of the human gene was also isolated and characterized by DNA sequencing, which demonstrated agreement between the bovine and human sequences in the segment in question. The difference between the presently reported sequence and that of the previously published one suggests the possibility of an unusual type of polymorphism which would result in markedly different amino acid sequences at the carboxy terminal region of the protein. The human tuftelin gene was localized to chromosome 1q21 by in situ hybridization.

The DiGeorge syndrome minimal critical region contains a goosecoid-like (GSCL) homeobox gene that is expressed early in human development.

The majority of patients with DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS) have deletions of chromosomal region 22q11.2. The abnormalities observed in these patients include conotruncal cardiac defects, thymic hypoplasia or aplasia, hypocalcemia, and characteristic facial features. To understand the genetic basis of these disorders, we have characterized genes within the region that is most consistently deleted in patients with DGS/VCFS, the minimal DiGeorge critical region (MDGCR). In this report, we present the identification and characterization of a novel gene, GSCL, in the MDGCR, with homology to the homeodomain family of transcription factors. Further, we provide evidence that this gene is expressed in a limited number of adult tissues as well as in early human development. The identification of GSCL required a genomic sequence-based approach because of its restricted expression and high GC content. The early expression, together with the known role of homeobox-containing proteins in development, make GSCL an outstanding candidate for some of the abnormalities seen in DGS/VCFS.

Disruption of the clathrin heavy chain-like gene (CLTCL) associated with features of DGS/VCFS: a balanced (21;22)(p12;q11) translocation.

The smallest region of deletion overlap in the patients we have studied defines a DIGeorge syndrome/velocardiofacial syndrome (DGS/VCFS) minimal critical region (MDGCR) of approximately 250 kb within 22q11. A de novo constitutional balanced translocation has been identified within the MDGCR. The patient has some features which have been reported in individuals with DGS/VCFS, including: facial dysmorphia, mental retardation, long slender digits and genital anomalies. We have cloned the breakpoint of his translocation and shown that it interrupts the clathrin heavy chain-like gene (CLTCL) within the MDGCR. The breakpoint of the translocation partner is in a repeated region telomeric to the rDNA cluster on chromosome 21p. Therefore, it is unlikely that the patient's findings are caused by interruption of sequences on 21p. The chromosome 22 breakpoint disrupts the 3' coding region of the CLTCL gene and leads to a truncated transcript, strongly suggesting a role for this gene in the features found in this patient. Further, the patient's partial DGS/VCFS phenotype suggests that additional features of DGS/VCFS may be attributed to other genes in the MDGCR. Thus, haploinsufficiency for more than one gene in the MDGCR may be etiologic for DGS/VCFS.

Type I oculocutaneous albinism associated with a full-length deletion of the tyrosinase gene.

Type I oculocutaneous albinism is an autosomal recessive disorder in which the biosynthesis of melanin is reduced or absent in skin, hair, and eyes because of deficient activity of tyrosinase (EC 1.14.18.1). Type I oculocutaneous albinism is caused by mutations in the tyrosinase structural gene, TYR; however, no large TYR gene deletions have been identified previously in humans. Here we report a patient with type IB oculocutaneous albinism who is a compound heterozygote for TYR allele containing a mutation that is likely to affect pre-RNA splicing and a paternally inherited allele in which the TYR gene is completely deleted, the first such allele described to date. Aside from the albinism in the proband, his phenotype and that of his normally pigmented father is otherwise normal, suggesting that this TYR deletion does not involve other functionally important contiguous genes.

Cloning a balanced translocation associated with DiGeorge syndrome and identification of a disrupted candidate gene.

DiGeorge syndrome (DGS), a developmental defect, is characterized by cardiac defects and aplasia or hypoplasia of the thymus and parathyroid glands. DGS has been associated with visible chromosomal abnormalities and microdeletions of 22q11, but only one balanced translocation--ADU/VDU t(2;22)(q14;q11.21). We now report the cloning of this translocation, the identification of a gene disrupted by the rearrangement and the analysis of other transcripts in its vicinity. Transcripts were identified by direct screening of cDNA libraries, exon amplification, cDNA selection and genomic sequence analysis using GRAIL. Disruption of a gene in 22q11.2 by the breakpoint and haploinsufficiency of this locus in deleted DGS patients make it a strong candidate for the major features associated with this disorder.

Classical Noonan syndrome is not associated with deletions of 22q11.

Deletions of 22q11 cause DiGeorge sequence (DGS), velo-cardio-facial syndrome (VCFS), conotruncal anomaly face syndrome, and some isolated conotruncal heart anomalies. Demonstration of a 22q11 deletion in a patient with manifestations of DGS and Noonan syndrome (NS) has raised the question of whether NS is another of the chromosome 22 microdeletion syndromes. This prompted us to evaluate a cohort of patients with NS for evidence of 22q11 deletions. Five of 6 NS propositi studied in our laboratory with marker N25 (D22S75) did not have a 22q11 deletion. A 2-month-old infant with several findings suggestive of NS did have a 22q11 deletion, suggesting that a small number of 22q11 deletion propositi may present with a NS-like picture. However, most cases of NS must have another cause.

Prenatal diagnosis of the derivative chromosome 22 associated with cat eye syndrome by fluorescence in situ hybridization.

Cytogenetic studies of cultured amniocytes demonstrated a karyotype of 46,XX/47,XX, +mar. A bisatellited, dicentric, distamycin-DAPI negative, NOR-positive marker was present in 76 per cent of the metaphases examined. Similar markers have been associated with cat eye syndrome (CES). We report on the utilization of fluorescence in situ hybridization (FISH) with a 14/22 alpha-satellite probe and a chromosome 22-specific cosmid for locus D22S9 to determine the origin of the prenatally detected supernumerary marker chromosome. FISH studies demonstrated that the marker is a derivative of chromosome 22 and enabled us to provide the family with additional prognostic information.

Molecular characterization of the marker chromosome associated with cat eye syndrome.

Cat eye syndrome (CES) is associated with a supernumerary bisatellited marker chromosome which is derived from duplicated regions of 22pter-22q11.2. In this study we have used dosage and RFLP analyses on 10 CES patients with marker chromosomes, by using probes to five loci mapped to 22q11.2. The sequences recognized by the probes D22S9, D22S43, and D22S57 are in four copies in all patients, but the sequences at the more distal loci, D22S36 and D22S75, are duplicated only in some individuals. D22S36 is present in three copies in some individuals, and D22S75 is present in two copies in the majority of cases. Only three individuals have a duplication of the most distal locus examined (D22S75), and these individuals have the largest marker chromosomes identified in this study. From the dosage analysis it was found that the marker chromosomes are variable in size and can be asymmetric in nature. There is no obvious correlation between the severity of the phenotype and the size of the duplication. The distal boundary of the CES critical region (D22S36) is proximal to that of DiGeorge syndrome, a contiguous-gene-deletion syndrome of 22q11.2.

Prevalence of 22q11 microdeletions in DiGeorge and velocardiofacial syndromes: implications for genetic counselling and prenatal diagnosis.

Deletions of chromosome 22q11 have been seen in association with DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS). In the present study, we analysed samples from 76 patients referred with a diagnosis of either DGS or VCFS to determine the prevalence of 22q11 deletions in these disorders. Using probes and cosmids from the DiGeorge critical region (DGCR), deletions of 22q11 were detected in 83% of DGS and 68% of VCFS patients by DNA dosage analysis, fluorescence in situ hybridisation, or by both methods. Combined with our previously reported patients, deletions have been detected in 88% of DGS and 76% of VCFS patients. The results of prenatal testing for 22q11 deletions by FISH in two pregnancies are presented. We conclude that FISH is an efficient and direct method for the detection of 22q11 deletions in subjects with features of DGS and VCFS as well as in pregnancies at high risk for a deletion.

Cytogenetic and molecular investigation of a balanced Xq13q translocation in a patient with retinoblastoma.

We report on a 4-year-old girl with retinoblastoma and de novo balanced translocation [46,X,t (X;13) (q23;q13)]. Unilateral retinoblastoma was discovered at age 9 months along with developmental delay and several manifestations of Turner syndrome. Chromosome studies showed an X/13 translocation and an X inactivation pattern showing the translocated X chromosome active in all 50 cells examined. Standard Southern blot analysis and pulsed field gel electrophoresis using a 3.8 kb EcoR1 fragment of the cDNA probe to the 3' end of the RB1 locus demonstrated a normal genomic pattern. The results of the cytogenetic and molecular analysis suggests that the RB1 locus has not been disrupted by the chromosome rearrangement. This case is the fifth report of an X/13 translocation associated with a retinoblastoma.

Localization of the rhabdomyosarcoma t(2;13) breakpoint on a physical map of chromosome 13.

Previous investigations of the pediatric soft tissue tumor alveolar rhabdomyosarcoma have identified a characteristic translocation t(2;13)(q35;q14). We have employed a physical mapping strategy to localize the site of this translocation breakpoint on chromosome 13. Using a panel of somatic cell hybrid and lymphoblast cell lines with deletions and unbalanced translocations involving chromosome 13, we have mapped numerous probes from the 13q12-q14 region and demonstrate that this region is divisible into five physical intervals. These probes were then mapped with respect to the t(2;13) rhabdomyosarcoma breakpoint by quantitative Southern blot analysis of an alveolar rhabdomyosarcoma cell line with two copies of the derivative chromosome 13 and one copy of the derivative chromosome 2. Our findings demonstrate that the t(2;13) breakpoint is localized within a map interval delimited by the proximal deletion breakpoints in lymphoblast lines GM01484 and GM07312. Furthermore, the breakpoint is most closely flanked by loci D13S29 and TUBBP2 within this map interval. These findings will facilitate chromosomal walking strategies for cloning the regions disrupted by the alveolar rhabdomyosarcoma translocation. In addition, this physical map will permit rapid determination of the proximity of new cloned sequences to the translocation breakpoint.

Isolation and regional localization of 35 unique anonymous DNA markers for human chromosome 22.

Thirty-five new, unique, DNA probes have been isolated and each has been assigned to one of five regions on chromosome 22. The distribution of probes along the chromosome is what would be expected based on the estimated size of each region with the exception of the short arm (22p). RFLP analysis was performed using 13 different restriction enzymes and over 50% of the probes were found to have useful polymorphisms. Probes mapping to 22q11 were further characterized by pulsed-field gel analysis and it has been possible to link several on large restriction fragments. These 35 new probes will be useful reagents for producing genetic and physical maps of chromosome 22.

Molecular and cytogenetic analysis of chromosomal arms 2q and 13q in alveolar rhabdomyosarcoma.

We present cytogenetic and molecular genetic analyses of two cases of alveolar rhabdomyosarcoma. The characteristic translocation between chromosomes 2 and 13, t(2;13)(q35;q14), has been identified in both cases. Using cell lines derived from these tumor specimens, we have performed Southern blot analysis to investigate the possibility of rearrangement of 14 candidate genes mapping to the relevant regions of 2q and 13q. These candidate genes can be divided into 5 groups: signal transduction proteins (RB1, inhibin alpha, FLT1, and HOX4B), muscle-specific products [myosin light chain, desmin, and nicotinic cholinergic receptor subunits gamma and delta (CHRNG and CHRND)], extracellular matrix proteins (collagen type VI alpha 3 chain, elastin, and fibronectin), transformation-associated products (intestinal alkaline phosphatase and L-plastin), and other genes (esterase D). Conventional gel electrophoresis followed by Southern blot analysis indicated no evidence of rearrangement within or near these genes except for a rearrangement in the CHRNG-CHRND locus, which occurred only in a subpopulation of the late recurrence tumor cells of one patient. In addition, we employed pulsed-field gel electrophoresis-Southern blot analysis to demonstrate the absence of detectable rearrangements within a larger region around each of these genes.

Comparative mapping of the constitutional and tumor-associated 11;22 translocations.

The reciprocal t(11;22)(q23;q11) is the most common non-Robertsonian constitutional translocation in humans. The tumor-associated 11;22 rearrangement of Ewing sarcoma (ES) and peripheral neuroepithelioma (NE) is cytologically very similar to the 11;22 constitutional rearrangement. Using immunoglobulin light-chain constant region, ETS1 probes, and the technique of in situ hybridization, we previously were able to show that the constitutional and ES/NE breakpoints are different. As a first step toward isolating these translocation junctions and to further distinguish between them, we have made somatic cell hybrids. Cells from a constitutional 46,XX,inv(9),t(11;22) carrier and from an ES cell line with a t(11;22) were separately fused to a hypoxanthine-guanine phosphoribosyltransferase-deficient Chinese hamster cell line (RJK88). Resulting clones were screened with G-banding and Southern hybridization. Hybrid clones derived from the constitutional t(11;22) were established which contained the der(22) and both the der(22) and the der(11). Hybrid clones derived from the ES cell line containing the der(11) were isolated. Using the technique of Southern hybridization we have sublocalized the loci; ApoA1/C3, CD3D, ETS1, PBGD, THY1, D11S29, D11S34, and D11S147 to the region between the two breakpoints on chromosome 11 and V lambda I, V lambda VI, V lambda VII, and D22S10 to the region between the breakpoints on chromosome 22. Using anonymous DNA probes, we found that D22S9 and D22S24 map proximal to the constitutional breakpoint and that D22S15 and D22S32 map distal to the ES breakpoint on chromosome 22.

Clinical, cytogenetic, and pedigree findings in 18 cases of Aicardi syndrome.

Eighteen girls with Aicardi syndrome were identified through a survey of neurologists, geneticists, and ophthalmologists. All had infantile seizures, developmental delay, agenesis of the corpus callosum (complete: 72%, partial: 28%), and characteristic chorioretinal lacunar lesions. Costovertebral defects including hemivertebrae, scoliosis, and absent or malformed ribs were present in 39%, cortical heterotopias were present in 50%, and microphthalmia was identified in a third. Cytogenetic investigation was carried out in all families. An unbalanced X;3 translocation, 46,X,der(X)t(X;3)(p22.3;p23)mat, was discovered in a girl with chorioretinal lacunar lesions characteristic of Aicardi syndrome, developmental delay, and infantile seizures. However, this child had a normal appearing corpus callosum on CT and magnetic resonance imaging scans and therefore did not meet the criteria for inclusion in the study. Chromosomes of all other patients and parents were normal. Findings at birth, age of seizure onset, treatment, and prognosis are discussed. The pedigree data from these 18 families demonstrated an unaffected male:female sib ratio of 1:1.7 and a 14% spontaneous abortion rate. The findings of this study support the contention that Aicardi syndrome is an X-linked dominant disorder with early embryonic lethality in hemizygous males and that all cases represent new mutations.