Testes genéticos em diagnóstico pré-natal: onde estamos, para onde vamos / Prenatal genetic testing: where we are at, where we are heading to

Este texto tem como objetivo apresentar uma revisão acerca do estado da arte da citogenética convencional e molecular aplicada ao diagnóstico pré-natal, discutindo as aplicações, vantagens e desvantagens dos diferentes métodos, em suas bases teóricas e históricas. Desde 1960, a citogenética convencional, com a análise microscópica dos cromossomos em divisão, vem sendo utilizada como padrão ouro. Entretanto, mesmo adotando essa abordagem, para uma significativa parcela de casos não é possível estabelecer diagnóstico sindrômico definitivo em cerca de metade dos pacientes que apresentam cariótipo normal, na presença de malformações. Para esse grupo, as técnicas moleculares que envolvem estudos em nível genômico poderiam permitir a identificação de novos microarranjos cromossômicos possivelmente responsáveis pelo fenótipo anormal, contribuindo para a caracterização molecular e estabelecimento de um diagnóstico mais preciso, uma abordagem perinatal mais adequada e um aconselhamento genético mais detalhado. Destaca-se o advento das técnicas de FISH, SKY, CGH e array CGH como promissoras aliadas, de forma complementar ao cariótipo convencional

This paper aims at presenting a review of the state of the art of conventional and molecular cytogenetics applied to prenatal diagnosis, the applications, pros and cons of different techniques and their historical and theoretical background. Since 1960, conventional cytogenetics, based on the analysis of chromosomes has been used as a gold standard. However, for a significant proportion of cases it is not possible to establish definitive syndromic diagnosis in about half of the patients with normal karyotype in the presence of malformations. For this group, molecular techniques at the genomic level might allow the identification of new chromosomal areas potentially responsible for the abnormal phenotype, contributing to the molecular characterization and establishment of a more accurate diagnosis and the most appropriate perinatal approach, including a more detailed genetic counseling. The advent of FISH techniques, SKY, CGH and array CGH will be discussed as promising tools to complement cytogenetic diagnosis based on conventional karyotyping

Spectral karyotyping of seven prenatally detected marker chromosomes and complex chromosome aberrations / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To perform spectral karyotyping (SKY), fluorescence in situ hybridization (FISH) and conventional karyotyping on prenatally detected marker chromosomes and complex chromosomal aberrations.</p><p><b>METHODS</b>Five marker chromosomes and 2 complex chromosome aberrations diagnosed by G banding were collected. SKY was performed to verify the composition of marker chromosomes. FISH was used to confirm the diagnosis when necessary. In certain cases, C or N banding technique was employed to verify the composition of chromosomes. Results of ultrasonography and pregnancy outcome were reviewed.</p><p><b>RESULTS</b>Among the 5 marker chromosomes, 2 were large and 3 were medium in size, 4 were de novo and one was inherited from the father. By SKY analysis, 2 marker chromosomes have originated from non-acrocentric chromosomes (4 and 9), whilst the other two have originated from acrocentric chromosomes (21 and 22). The remainder was derived from X chromosome. The SKY results were confirmed by FISH in 3 cases. Four cases have chosen to terminate the pregnancy after genetic counseling. A fetus with inherited paternal marker chromosome was delivered at term, and showed normal development during the first year of life. As for the other 2 cases with complex chromosome aberrations, by SKY examination, one had duplication in chromosome 8 and the other had chromosome rearrangements derived from translocation between chromosomes 2 and 6. In the latter case the fetus was delivered at term but showed developmental retardation at 6 months.</p><p><b>CONCLUSION</b>SKY in combination with FISH can facilitate identification of the origins of marker chromosomes as well as complex chromosomal aberrations. With combined information from ultrasonography, SKY and FISH, effective counseling may be offered to the patients.</p>

Combined spectral karyotyping and microarray-based comparative genomic hybridization for the diagnosis of a case with ring chromosome 15 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To assess the value of spectral karyotyping (SKY) combined with microarray-based comparative genomic hybridization (array-CGH) for the diagnosis of complex ring chromosome aberration.</p><p><b>METHODS</b>For an 8-year-old boy featuring growth retardation, G-banding analysis has indicated a 46,XY,r(15)? karyotype, which was delineated by SKY in combination with array-CGH.</p><p><b>RESULTS</b>The ring chromosome has originated from chromosome 15 according to SKY analysis. Position of the breakpoint (15q26.3) and a 594 kb deletion were revealed by array-CGH.</p><p><b>CONCLUSION</b>Molecular cytogenetic technologies are efficient tools for clarifying complex chromosomal abnormality, which has provided a powerful tool for conventional cytogenetic analysis.</p>

Combined use of molecular cytogenetic techniques to detect a small chromosomal translocation / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Comprehensive use of molecular cytogenetic techniques for the detection of 1 case of small chromosome translocation.</p><p><b>METHODS</b>Following conventional chromosome preparation, G-banding karyotype analysis, spectral karyotyping (SKY), whole chromosome painting, two-color fluorescence in situ hybridization (FISH) and subtelomeric probe FISH were performed.</p><p><b>RESULTS</b>G-banded karyotype was 46, XX, ?(22q11.3), SKY karyotype analysis was 46, XX, der (4)t(4;6) and found no abnormalities on chromosome 22, staining signal was not found with any abnormalities on chromosome 6. Two-color FISH indicated a chromosomal translocation segment of 22q13.3 to one end of the short arm of chromosome 4. Subtelomeric FISH probe showed the end of the long arm of chromosome 22 and the end of the short arm of chromosome 4 reciprocal translocation. High resolution G-banding and FISH result indicated 46, XX, t(4;22)(p15.3;q13.2).</p><p><b>CONCLUSION</b>The testing of small chromosomal translocation should be combined with clinical information and integrated use of molecular cytogenetic techniques to improve the accuracy of diagnosis of chromosomal diseases.</p>

Application of spectral karyotyping to cytogenetic analysis in acute myeloid leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the value of spectral karyotyping (SKY) in cytogenetic analysis of acute myeloid leukemias (AML).</p><p><b>METHODS</b>Nine AML patients were analyzed by R-banding and SKY. MLL, PML-RARalpha, AML1-ETO fusion genes were detected by dual fusion- fluorescence in situ hybridization (D-FISH).</p><p><b>RESULTS</b>All 9 samples were successfully hybridized. SKY identified structural aberrations including 9q -, t(15;17) and ins(10;17) (q22;p11p12) ; and some numeral abnormalities. The results of SKY confirmed those of R-band karyotyping and D-FISH; with more accurate localization.</p><p><b>CONCLUSION</b>SKY appears to be fairly stable, accurate and sensitive, for AML cytogenetic study.</p>

Comparative genomic hybridization: the profile of chromosomal imbalances in rhabdomyosarcoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To characterize the profile of chromosomal imbalances of rhabdomyosarcoma(RMS).</p><p><b>METHODS</b>Comparative genomic hybridization (CGH) was used to investigate genomic imbalances in 25 cases of primary RMS including 10 cases of alveolar rhabdomyosarcoma (ARM), 12 cases of embryonic rhabdomyosarcoma (ERMS), 3 cases of polymorphic rhabdomyosarcoma (PRMS) and 2 RMS cell lines (A240 originated from ARMS and RD from PRMS), with correlation to histological type, pathologic grading, clinical staging, gender and age, respectively.</p><p><b>RESULTS</b>All twenty-five rhabdomyosarcomas showed evidence of increased or decreased DNA sequence copy numbers involving one or more regions of the genome. (1) The frequently gained chromosome regions in RMS were 2p, 12q, 6p, 9q, 10q, 1p, 2q, 6q, 8q, 15q, 18q, and the frequently lost chromosome regions were 3p, 11p, 6p. (2) The frequently gained chromosome arms in ARMS were 12q, 2p, 6, 2q, 4q, 10q, 15q. The frequently lost chromosome arms were 3p, 6p, 1q, 5q. The frequently gained chromosome regions in ERMS were 7p, 9q, 2p, 18q, 1p, 8q. The frequently lost chromosome arms in ERMS were 11p. (3) The frequently gained chromosome arms in translocation associated RMS were 12q, 2, 6, 10q, 4q and 15q (> 30%), 3p, 6p, 5q (> 30%) were the frequently loss chromosome arms. The frequently gained chromosome regions in non-translocation associated RMS were 2p, 9q, 18q (> 30%), and 11p, 14q (> 30%) were the frequently loss chromosome regions. Gain of 12q was significantly correlated with the translocation-associated tumors (P < 0.05). (4) Gains of 9q was significantly correlated with clinical staging (P < 0.05).</p><p><b>CONCLUSIONS</b>Gain of 2p, 12q, 6p, 9q, 10q, 1p, 2q, 6q, 8q, 15q, 18q and loss of 3p, 11p, 6p may be involved in the tumorigenesis of RMS. Gains of 12q may be correlated with gene fusion/chromosomal translocation in ARMS. Gains of 9q may be correlated with an early tumor stage of RMS.</p>

The clinical application of spectral karyotyping in the analysis of chromosomal abnormalities / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To evaluate value of spectral karyotyping (SKY) in the detection of chromosomal abnormalities.</p><p><b>METHODS</b>A total of 17 metaphase chromosome samples were investigated by SKY, including 10 normal and 5 balanced translocation samples of peripheral blood lymphocytes, one der(Y) sample of peripheral blood lymphocytes and one marker chromosome sample of amniotic fluid cells. The results were compared with those of G-banding diagnosis.</p><p><b>RESULTS</b>Ten normal and 5 balanced translocation samples were diagnosed successfully by SKY in accordance with the results of G-banding; furthermore, SKY analysis revealed that the der(Y) fragment originated from p-arm of chromosome 21 while the marker chromosome originated from chromosome 5.</p><p><b>CONCLUSION</b>SKY is a very sensitive and specific whole genome analysis tool for chromosomal abnormality diagnosis, and exceedingly valuable in the diagnosis on complex chromosomal abnormalities that can not be determined by G-banding.</p>

Application of spectral karyotyping in diagnosis of complex chromosome aberration / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the value of spectral karyotyping (SKY) to identify the complex chromosome aberration.</p><p><b>METHODS</b>Four cases were selected that can not be identified by standard cytogenetic techniques. The chromosome specimens were detected by the routine SKY method, and the results were analyzed by the SKY View software.</p><p><b>RESULTS</b>By using SKY a case of complex chromosome rearrangements and two cases of chromosome duplication were identified. However it could not identify the chromosome inversion and the breakpoint of chromosome aberration.</p><p><b>CONCLUSION</b>SKY may be a valuable tool in identification of complex chromosome translocation, rearrangement, minute aberration and unknown derivative chromosomes. Though SKY can not replace the standard cytogenetic techniques, but it will be the benefit supplementary.</p>

The value of multiplex fluorescence in situ hybridization in the detection of complex karyotypic abnormalities of acute myeloid leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the value of multiplex fluorescence in situ hybridization (FISH) in the detection of complex karyotypic abnormalities of acute myeloid leukemia (AML).</p><p><b>METHODS</b>Multiplex FISH was used in combination with conventional cytogenetics (CC) and interphase FISH to study 14 cases of AML with complex karyotypic abnormalities.</p><p><b>RESULTS</b>In the 14 cases of AML studied, conventional cytogenetics detected 23 numerical and 56 structural chromosome abnormalities. Among them 4 gained whole chromosome and 4 lost whole chromosome which were confirmed by multiplex FISH. Twelve chromosome losses detected by CC were revised as derivative chromosomes resulted from various structural aberrations, and 26 derivative and 19 marker chromosomes were characterized precisely by multiplex FISH. Most of them were resulted from unbalanced translocations, including 2 complex 8; 21 translocations, which have not been reported previously: t (8; 21), der (8) t (8; 21) (8pter --> 8q22::21q22 --> 21qter), der (21) t (8; 21; 8) (8qter --> 8q22:: 21p13 --> 21q22::8q22 --> 8qter) and t (21; 8; 18; 1), der (8) t (8; 21) (8pter --> 8q22:: 21q22 --> 21qter), der (21) t (21; 8; 18; 1) (21p13 --> 21q22?::8q22 --> 8q24 ?:: 18??::1q??q??). The complex karyotypic abnormalities involved nearly all chromosomes, of which the chromosomes 17, 7 and 5 were more involved than the rest.</p><p><b>CONCLUSION</b>Multiplex FISH in combination with conventional cytogenetics may characterize the complex chromosomal abnormalities more precisely. Introduction of this technique to the study of AML with complex chromosomal abnormalities is warranted.</p>

Application of spectral karyotyping in leukemia--review / 中国实验血液学杂志

Spectral karyotyping (SKY) is a novel cytogenetic technique, has been developed to unambiguously display and identify all 24 human chromosomes at one time without a priori knowledge of any abnormalities involved. SKY discerns the aberrations that can not be detected very well by conventional banding technique and fluorescent in situ hybridization (FISH). So SKY is hyper-accurate, hypersensitive, and hyper-intuitional. In this paper the basic principle of SKY technique and its application in leukemia cytogenetics were reviewed.

Study on the mechanism of oocyte aneuploidy by spectral karyotyping / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish the method of detecting oocyte aneuploidy by spectral karyotyping (SKY).</p><p><b>METHODS</b>The unfertilized oocytes were fixed 1-2 days after oocyte retrieval. Spectral karyotyping was performed according to the protocol.</p><p><b>RESULTS</b>64% of oocytes were normal, 36% of oocytes were aneuploidy, of which 22% were due to nondisjunction and 14% unbalanced predivision.</p><p><b>CONCLUSION</b>SKY is an effective method for detecting oocyte aneuploidy. Both nondisjunction and unbalanced predivision are involved in oocyte aneuploidy formation.</p>