A diagnostic approach to identifying submicroscopic 7p21 deletions in Saethre-Chotzen syndrome: fluorescence in situ hybridization and dosage-sensitive Southern blot analysis.

PURPOSE: To report on the use of fluorescence in situ hybridization (FISH) and dosage-sensitive Southern blot analysis in the molecular diagnosis of patients with Saethre-Chotzen syndrome. METHODS: FISH and dosage-sensitive Southern blot analysis utilizing TWIST gene probes were performed on patients with Saethre-Chotzen syndrome but without an identifiable TWIST sequence variation. RESULTS: Four unrelated patients with a deletion of the TWIST gene were identified by Southern blot; one of them had a complex chromosomal rearrangement involving 7p21 and no apparent deletion by FISH, suggesting a smaller deletion in the region including the TWIST gene. A fifth patient had an abnormal TWIST gene fragment on Southern blot analysis that segregated with the disease in the family; FISH was normal in this patient, suggesting a partial deletion or rearrangement in or near the gene. CONCLUSION: FISH and dosage-sensitive Southern blot analysis are useful diagnostic tools in Saethre-Chotzen syndrome without TWIST sequence variation.

Monodactylous limbs and abnormal genitalia are associated with hemizygosity for the human 2q31 region that includes the HOXD cluster.

Vertebrates have four clusters of Hox genes (HoxA, HoxB, HoxC, and HoxD). A variety of expression and mutation studies indicate that posterior members of the HoxA and HoxD clusters play an important role in vertebrate limb development. In humans, mutations in HOXD13 have been associated with type II syndactyly or synpolydactyly, and, in HOXA13, with hand-foot-genital syndrome. We have investigated two unrelated children with a previously unreported pattern of severe developmental defects on the anterior-posterior (a-p) limb axis and in the genitalia, consisting of a single bone in the zeugopod, either monodactyly or oligodactyly in the autopod of all four limbs, and penoscrotal hypoplasia. Both children are heterozygous for a deletion that eliminates at least eight (HOXD3-HOXD13) of the nine genes in the HOXD cluster. We propose that the patients' phenotypes are due in part to haploinsufficiency for HOXD-cluster genes. This hypothesis is supported by the expression patterns of these genes in early vertebrate embryos. However, the involvement of additional genes in the region could explain the discordance, in severity, between these human phenotypes and the milder, non-polarized phenotypes present in mice hemizygous for HoxD cluster genes. These cases represent the first reported examples of deficiencies for an entire Hox cluster in vertebrates and suggest that the diploid dose of human HOXD genes is crucial for normal growth and patterning of the limbs along the anterior-posterior axis.

Precise localisation of 3p25 breakpoints in four patients with the 3p-syndrome.

In patients with the 3p-syndrome, hemizygous deletion of 3p25-pter is associated with profound growth failure, characteristic facial features, and mental retardation. We performed a molecular genetic analysis of 3p25 breakpoints in four patients with the 3p- syndrome, and a fifth patient with a more complex abnormality, 46,XY,der(3)t(3;?)(p25.3;?). EBV transformed lymphoblasts from each of the patients were initially characterised using fluorescent in situ hybridisation (FISH) and polymorphic microsatellite analyses. The 3p-chromosome from each patient was isolated from the normal chromosome 3 in somatic cell hybrid lines and subsequently analysed with polymorphic and monomorphic PCR amplifiable markers from 3p25. The analysis clearly shows that all five breakpoints are distinct. Furthermore, we have identified yeast artificial chromosomes that cross the 3p25 breakpoints of all four 3p-patients. Two of the patients were deleted for the von Hippel-Lindau (VHL) tumour suppressor gene, although neither has yet developed evidence of VHL disease. The patient with the most centromeric breakpoint, between D3S1585 and D3S1263, had the most severe clinical phenotype including an endocardial cushion defect that was not observed in any of the four patients who had more telomeric breakpoints. This study should provide useful insights into critical regions within 3p25 that are involved in normal human growth and development.

Molecular and cytogenetic studies of an X;autosome translocation in a patient with premature ovarian failure and review of the literature.

We have identified a patient with premature ovarian failure (POF) and a balanced X;autosome translocation: 46,X,t(X;6)(q13.3 or q21;p12) using high-resolution cytogenetic analysis and FISH. BrdU analysis showed that her normal X was late-replicating and translocated X earlier-replicating which is typical of balanced X;autosome rearrangements. Molecular studies were done to characterize the breakpoint on Xq and to determine the parental origin. PCR probes of tetranucleotide and dinucleotide repeat polymorphisms, and genomic probes were used to study DNA from the patient, her chromosomally normal parents and brother, and somatic cell hybrids containing each translocation chromosome. The translocation is paternally derived and is localized to Xq13.3-proximal Xq21.1, between PGK1 and DXS447 loci, a distance of 0.1 centimorgans. A "critical region" for normal ovarian function has been proposed for Xq13-q26 [Sarto et al., Am J Hum Genet 25:262-270, 1973; Phelan et al., Am J Obstet Gynecol 129:607-613, 1977; Summitt et al., BD:OAS XIV(6C):219-247, 1978] based on cytogenetic and clinical studies of patients with X;autosome translocations. Few cases have had molecular characterization of the breakpoints to further define the region. While translocations in the region may lead to ovarian dysfunction by disrupting normal meiosis or by a position effect, two recent reports of patients with premature ovarian failure and Xq deletions suggest that there is a gene (POF1) localized to Xq21.3-q27 [Krauss et al., N Engl J Med 317:125-131, 1987; Davies et al., Cytogenet Cell Genet 58:853-966, 1991] or within Xq26.1-q27 [Tharapel et al., Am J Hum Genet 52:463-471, 1993] responsible for POF.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of human chromosome region 3p14.2-21.3-specific YAC clones using Alu-PCR products from a radiation hybrid.

Deletion of DNA sequences from at least three different regions on the short arm of human chromosome 3 (3p13-14, 3p21 and 3p25) are frequently observed during the development of many solid tumors, including lung cancers and renal cell carcinomas. In order to physically characterize the 3p21 region, we previously identified a radiation fusion hybrid that contained about 20 megabases of DNA from chromosome region 3p14.2-p21.3. In this study total Alu-PCR products from this hybrid were used as a probe to isolate 86 yeast artificial chromosomes (YAC) clones from a 620-kb average insert YAC library (ICRF). Sixty-nine Alu-PCR markers, generated from the YACs, and seven PCR primers were used to screen for overlaps between individual clones. Seven contigs were identified encompassing 32 YAC clones. Based on previous information about localization of the PCR primers, the three largest contigs could be assigned to smaller subregions between 3p14.2 and 3p21.3. By this work a large proportion of the 3p14.2-21.3 region is covered with large-insert YAC clones.

Recurrent cytogenetic abnormalities in squamous cell carcinomas of the head and neck region.

We characterized the breakpoints, gains, and losses of chromosome material in squamous cell carcinomas of the head and neck region from 29 patients. Cell lines were karyotyped in 1/3 of cases, direct preparations or early in vitro harvests in 1/3, and both in 1/3 of cases. GTG-banding was employed in all cases, as were C-banding and RBG- and AgNOR-staining in most. Some tumors were near-diploid and others near-tetraploid, but many had mixed populations, with diploid, tetraploid, and octoploid subclones representing essentially the same karyotypic pattern. The most frequent changes were deletions. Losses affecting 3p13-p24, 5q12-q23, 8p22-p23, 9p21-p24, and 18q22-q23 ranged in frequency from 40% to 60% of tumors. Loss of the short arm of the inactive X occurred in 70% of tumors from female patients, and loss or rearrangement of the Y occurred in 74% of tumors from male patients. Loss of 18q appeared to be associated with short survival, as did the presence of multiple deletions. There was gain (2-5 extra copies) of 3q21-qter, 5p, 7p, 8q, and 11q13-q23 in 28-38% of tumors. Three tumors had an hsr involving 11q13-q21. Gain of material at 11q13 is postulated to be associated with amplification of the PRADI/CCND gene at that locus. A translocation between proximal 1p and either an acrocentric short arm or proximal 8p or 9p was observed in squamous cell carcinomas of the head and neck region but not in female genital tract tumors. No other abnormalities appeared to be site specific, suggesting a pattern of genetic evolution in squamous cell carcinoma that is independent of anatomic site.

Cytogenetic findings in pituitary adenoma: results of a pilot study.

Pituitary adenomas are the most common tumors of the sellar region and are, with rare exception, benign neoplasms. The natural history of these lesions is only poorly understood and, although histologic, immunocytologic, and ultrastructural characteristics have been well established, predicting the natural history of individual tumors is, at best, unreliable. In an effort to better characterize pituitary adenomas, we have analyzed the karyotypes of 18 surgical specimens and attempted to correlate with the morphologic appearance and the clinical data, for example, endocrinologic subtypes, histology, and tumor invasiveness. Most hormonal subtypes were studied including seven growth hormone-prolactin (GH-PRL), two Prolactin (PRL), two Adrenocorticotrophin (ACTH), seven nonsecretory (NULL). No correlations with morphology or invasiveness could be made. Of 7 null cell adenomas, five (71%) had normal karyotypes, whereas of 11 hormone-secreting adenomas three (28%) were normal. Of seven tumors with mixed GH-PRL activity, six had abnormal karyotypes. At least three chromosomes harbored abnormalities shared by more than two tumors. The results demonstrate that chromosome abnormalities are also found in benign tumors. These findings, however, suggest that hormone-secreting adenomas may be more likely to be associated with karyotypic abnormalities especially those of the GH-PRL variety. Genetic abnormalities associated with chromosomes 1, 4, 7, and 19 were common and warrant further investigation.