Avirachan T. Tharapel

Apparently balanced de novo translocations in patients with abnormal phenotypes: report of 6 cases.

Six patients have been ascertained because of abnormal phenotypes but with apparently balanced de novo translocations. Five of them were mentally retarded with multiple congenital anomalies. The sixth patient had normal mental development but revealed ambiguous genitalia and multiple congenital anomalies. No syndromal diagnosis was possible in any of the six cases. The appearance of apparently balanced reciprocal translocations in association with abnormal phenotype may be coincidental, or the two may be causally related. If the latter is true, the causal relationship may be based upon: I) a submicroscopic chromosomal loss, 2) position effect, or 3) a mutation at the site of the break in one or both translocated chromosomes.

Localization of SRY by primed in situ labeling in XX and XY sex reversal

Primed in situ labeling (PRINS) can be used to localize DNA segments too small to be detected by fluorescence in situ hybridization. By PRINS we identified the SRY gene in two XX males, a woman with XY gonadal dysgenesis, and an azoospermic male with Xp-Yp interchange. Because PRINS has been used generally in the study of repetitive sequences, we modified the technique for study of the single copy 2. 1-kb SRY sequence. SRY signals were identified at band Yp11.31p11.32 in normal XY males and in the woman with XY gonadal dysgenesis. SRY signals were identified on Xp22 in one XX male but not in the other. They were identified in the corresponding region (Xp22) of the der(X) in the azoospermic male with Xp-Yp interchange. SRY signals were not observed in normal XX females. Presence of SRY in DNA samples from the various subjects was confirmed by polymerase chain reaction. We conclude that PRINS is ideal for rapid localization of single copy genes and small DNA segments in general.

Minor chromosome variations and selected heteromorphisms in 200 unclassifiable mentally retarded patients and 200 normal controls.

SummaryLymphocyte chromosome preparations from 200 mentally retarded children and 200 normal adult controls were analyzed by G-, Q-, and C-banding techniques for minor chromosome variations (G and Q) and selected heteromorphisms (G and C). Minor variations scored included inv(9), prominent or decreased short arms and/or satellite on acrocentric chromosomes, and 17ph. C heteromorphisms analyzed included those involving 1qh, 9qh, and 16qh regions. Length variations of Yq were scored on G-banded karyotypes. No significant differences in frequencies of scored minor variations or heteromorphisms were noted between the retarded and control populations.

Validation of Primed In Situ Labeling (PRINS) for Interphase Analysis: Comparative Studies with Conventional Fluorescence In Situ Hybridization and Chromosome Analyses

Primed in situ labeling (PRINS) is a rapidly developing new technology with wide ranging clinical applications. To assess the sensitivity, specificity, and accuracy of PRINS, we carried out a retrospective study on cultured bone marrow cells to detect aneuploidy for chromosomes 7, 8, and 12. The results were then compared to the results of previous fluorescence in situ hybridization (FISH) and chromosome analyses (CA). In patients who showed aneuploidy with CA, both FISH and PRINS confirmed the aneuploidy in interphase cells. FISH and PRINS also showed excellent correlation with conventional cytogenetic analysis for the detection of mosaic aneuploidies. However, both FISH and PRINS showed significantly higher sensitivity in the detection of abnormal clones compared to CA. In 9 of the 17 cases, there were no significant differences in the detection rates between the two methods. Based on our studies, we conclude that PRINS is as sensitive as FISH in most cases for aneuploidy detection; and that PRINS, like FISH, is more sensitive than conventional CA for aneuploidy detection.

A cytogenetic survey of 200 unclassifiable mentally retarded children with congenital anomalies and 200 normal controls

SummaryA cytogenetic survey was carried out on 200 patients with mental retardation and multiple congenital anomalies, and on 200 normal adult controls. Patients with a known syndrome were excluded from the survey. Chromosome analyses were carried out on ‘blind-coded’ slides using the ASG banding technique as the routine stain. After the initial analyses (at least 15 cells per person) the slides were decoded, destained and reused for C and Q band polymorphism studies.Five major chromosome abnormalities were detected in the patient group during the survey. They included three patients with de novo, apparently balanced, reciprocal translocations, karyotypes 46,XY,rcp(3;16)(q21;p12); 46,XX,rcp(5;8)(p15;q22); and 46,XX,rcp(5;12)(p11;q24); one with karyotype 47,XX,+mar and one with karyotype 46,XX,der(13),t(13;?)(q34;?). One additional patient whose karyotype in lymphocytes was 46,XX,inv(9)(p11;q13) was found to have a mosaic karyotype 46,XX,inv(9)(p11;q13)/46,XX,inv(9) (p11;q13),der(12),t(12;?)(p13;?) in cultured skin fibroblasts. None of the 200 controls had a major chromosome abnormality.From the combined results of this and previous surveys it is now apparent that about 6.2% of the unclassifiable mentally retarded patients with three or more congenital anomalies and about 0.7% of the controls reveal major chromosome abnormalities.

Tissue limited mosaicism for unbalanced autosomal translocation in a child with congenital anomalies and mental retardation.

We studied a patient with a sporadic mental retardation/multiple congenital anomalies syndrome. Chromosome analysis showed a 46,XX, inv(9)(p11;q13) karyotype in all lymphocytes. Fibroblasts from two separate skin biopsies revealed a mosaic karyotype. Some 22.5% of fibroblasts had a karyotype like that of the lymphocytes, while 77.5% of fibroblasts had a karyotype 46,XX,inv(9)(p11;q13),der(12),t(12;?)(p13;?). The data in this case emphasize the drawbacks of confining cytogenetic analysis to lymphocytes.

PRINS for mapping single-copy genes.

Primed in situ labeling (PRINS) is a sensitive and specific method that can be used for the localization of single copy genes and sequences too small for detection by conventional fluorescence in situ hybridization. By the use of PRINS, the human SRY gene was localized to Yp11.31-p11.32 and the SOX3 gene to Xq26-q27. In other studies, we localized specific deletions of RBM and DAZ, candidate genes for AZF (azoospermia factor) to proximal Yq11.2, the AZF region, in an infertile male. Locus-specific oligonucleotide probes (PRINS primers) were annealed to chromosomal DNA in situ and extended on preparations fixed on glass slides in the presence of dATP, dCTP, dGTP, dTTP, biotin-16-dUTP, and Taq DNA polymerase. After addition of avidin-conjugated fluorophore, signals were visualized by fluorescence microscopy in metaphase spreads from patients and controls. With further development, the PRINS method may prove useful for localization of single-copy genes, in general, and for the detection of gene deletions.

Philadelphia chromosome-positive chronic myelocytic leukemia with a supplementary t(4;9)(q21;p22) and long survival.

A Ph-positive chronic myelocytic leukemia (CML) patient was clinically and cytogenetically evaluated during a 12-year period. She acquired a supplementary chromosome abnormality, t(4;9)(q21;p22), at least 5 years prior to transformation to blastic phase. Her blast crisis was accompanied by characteristic chromosome changes, such as trisomy 1, trisomy 17, and multiple Ph chromosomes.

Human Chromosome Nomenclature

Advancements in methodology and discovery of the diploid human chromosome number invigorated further research in human cytogenetics (1,2). The eventful years that followed witnessed the birth of a new specialty—human cytogenetics—which provided answers to many intriguing phenomena in medicine. Little was known at the time that human cytogenetics would form the backbone of present-day “human genetics,” providing answers to questions regarding human reproduction, behavior, aging, and disease while generating knowledge that could be applied to the treatment and prevention of many disorders.

PRINS for the Detection of Gene Deletions in Cancer

The chromosomal regions 13q14 and 17p13 often are found rearranged in hematopoietic tumors in humans, but the rearrangements can be subtle and can escape detection on gross cytogenetic analysis. For example, submicroscopic perturbations of the RB1 and p53 tumor suppressor genes, located in 13q14 and 17p13, respectively, frequently occur in leukemias; this has been confirmed by molecular methods such as fluorescence in situ hybridization (FISH). Our group modified the primed in situ labeling (PRINS) method to study RB1 and p53 in cultured bone marrow cells from leukemia patients with known deletions within the 13q14 or 17p13 regions. Locus-specific oligonucleotide probes (PRINS primers) were annealed to chromosomal DNA on glass slides and extended in the presence of the four trinucleotide precursors, biotin-16-dUTP, and Taq DNA polymerase. After addition of avidin-conjugated fluorophores, the resulting signals could be visualized by fluorescence microscopy in metaphase spreads and interphase nuclei of controls but were absent in the corresponding preparations from patients. The results of these and similar studies suggest that with further development, PRINS might be used as a convenient and rapid alternative to FISH in the delineation of deletions involving single genes or unique sequences.