Cytogenetics is a fascinating field of study that analyzes the number and structure of chromosomes. Today, cytogenetic analyses are typically performed during pregnancy to determine if a fetus is at risk of aneuploidies. But how did we get from the first observation of chromosomes to helping families?

The study of cytogenetics has a long and rich history that began nearly two centuries ago, and it continues to play a vital role in research and diagnostic labs where clinicians are not only finding answers, but are also using this technology to direct treatments. This piece is by no means exhaustive, but rather a small attempt to identify the major landmarks that have aided in the advancement of this field. The term karyotyping denotes the visualization of the human genome as a diploid set of chromosomes in a eukaryotic nucleus. Heinrich W.G. von Waldeyer-Hartz is credited for coining this term in 1888, although Karl von Nageli had first observed chromosomes in flowering plants in 1842. Today, a trained cytogeneticist is well versed in the structure and function of cells including karyotyping- the sole methodology that ruled for decades before molecular cytogenetics (FISH and microarray) started addressing and complementing its limitations.

Of significance in any historical commentary on the field of cytogenetics should be acknowledging the involvement of cell and tissue culture. Sydney Ringer, a British physiologist, is credited for the “Ringer’s Lactate” solution- one of the early experiments (1882) describing in vitro salt solutions to maintain animal organs outside of the body. Wilhelm Roux (1885) and Ross G. Harrison (1907-1910) then established the principle and methodology of tissue culture, respectively.

POLYTENE AND LAMPBRUSH CHROMOSOMES: EDOUARD-GERARD BALBIANI (1823-1899) AND WALTHER FLEMMING (1843-1905)

E. Balbiani, a microbiologist-turned-embryologist proposed the theory of germ cell autonomy in Chironomus. The identification of polytene chromosomes was a critical step in the identification of the now well-recognized chromosomal banding patterns that assists in the identification of structural rearrangements and genomic alterations. The ability to visualize transcriptionally active chromatin, also known as Balbiani rings or chromosome puffs (~200µm in size) was another substantial advancement. Lampbrush chromosomes, identified by Walther Flemming in 1882, elucidated the active transcription of several genes in the diplotene phase of meiotic prophase I of amphibians, birds and insects. Flemming is also credited for creating the terms chromatin and mitosis.

GIEMSA BANDING: GUSTAV GIEMSA (1867-1948)

Giemsa-banding, or G-banding, was named after the German chemist and bacteriologist, Gustav Giemsa, and set the pace for the multiple staining techniques that are utilized today in most laboratories that employ cell culture methodologies. The first report on the Giemsa stain was published in 1904. Giemsa stain, a mixture of azure B, eosin and methylene blue, has been historically used for histopathological staining of malarial and other blood parasites such as, Trypanosoma and Chlamydia. Giemsa stain was classically used to stain blood and bone marrow smears to identify and differentiate erythrocytes, lymphocytes, monocytes and platelets. However, it was the specificity of this stain for attaching to phosphate groups of DNA at the adenine-thymine (A-T) bonding that allowed for karyotyping of condensed metaphase chromosomes. Even today, various modifications of G-banding are utilized in traditional cytogenetics laboratories. Banding of chromosomes has been particularly useful not only in identifying numerical abnormalities (trisomies of chromosomes 13, 18 and 21, Turner syndrome, Klinefelter syndrome, etc.) but also for the identification of terminal microdeletions (Cri-du-chat syndrome, Wolff-Hirschhorn syndrome), and structural chromosomal anomalies such as landmark translocations in neoplastic specimens. Currently, digestion of proteins on the chromatin by trypsin followed by Giemsa staining is utilized by laboratories to significantly improve the quality and resolution of the chromosome spreads for analysis and interpretation.

HYPOTONIC SOLUTION: T.C. HSU (1917-2003)

The serendipitous discovery of hypotonic solution by T.C. Hsu, a Chinese American cell biologist, revolutionized mammalian and human cytogenetics. His historic paper, “Mammalian chromosomes in vitro – the karyotype of man,” published in 1952, describes in detail the pretreatment method for spreading chromosomes which improved the methodology and led to the accurate identification that 23 pairs of chromosomes exist in human somatic cells. He is aptly known as the “Father of Mammalian Cytogenetics” for his salient contributions.

Alka Chaubey_quoteHUMAN GENOME COMPLEMENT=46: JOE H. TJIO (1919-2001) AND ALBERT LEVAN (1905- 1998)

Cytogeneticist Joe Hin Tjio, while visiting the Institute of Genetics of the University of Lund in Sweden in 1955, is the first reported person to recognize the correct number of human diploid chromosomes. Tijo published the Hereditas report in 1956, coauthored by Albert Levan, which corrected the human diploid genome complement from 48 to 46. This study was performed by optimizing Hsu’s technique of hypotonic solution treatment on human embryonic lung fibroblasts to improve the quality of the chromosome spreads.

X-INACTIVATION: MARY FRANCES LYON (1925-2014)

In 1960, S. Ohno and T. Hauschka provided early evidence regarding X chromosome allocycly in female mice cells. Mary F. Lyon, an English geneticist, built on this early work to formulate mice studies that would show the process of X-chromosome inactivation (Xi). At that time only sex chromosome linked ocular albinism had been identified in humans, with males lacking retinal epithelial pigment and heterozygous females showing an irregular pigmentation pattern. Lyon discovered Xi while working on radiation hazards and made significant contributions to mammalian genetics. Her hypothesis suggesting the process of Xi as a dosage compensation method became an important landmark in understanding the effect of Xi on both genetic disorders as well as normal female development.

DOWN SYNDROME AND TRISOMY 21: JEROME LEJEUNE (1926-1994) AND MARIE GAUTHIER (B.1925)

Despite the controversy regarding who should get the primary credit for the identification of trisomy 21 (Down syndrome), it is important to note that this was the first example of a defect in intellectual development that was associated with a chromosomal anomaly. Jerome Lejeune, a French pediatrician and geneticist, co-authored the key C R Hebd Seances Acad Sci paper with M. Gauthier and R. Turpin in 1959. In addition to trisomy 21, Lejeune’s group was also credited with the identification of several other genetic syndromes including Cri-du-Chat syndrome (1964), 18q minus syndrome (1966), and trisomies of chromosomes 9 (1970) and 8 (1971).

PATAU SYNDROME AND TRISOMY 13 : KLAUS PATAU (1908-1975)

Around the time Lejeune’s group was busy identifying other chromosomal deletions and trisomies, Klaus Patau, a German-born American geneticist, reported the first case of trisomy 13, also known as Bartholin-Patau syndrome, in 1960. Thomas Bartholin had reported the first clinical description of this syndrome which includes intellectual disability, malformed ears, clefting and polydactyly in 1656. It was later elucidated to occur due to nondisjunction of chromosomes during meiosis.

EDWARDS SYNDROME AND TRISOMY 18: JOHN H. EDWARDS (1928- 2007)

In 1960, John H. Edwards, a British medical geneticist, identified the genetic cause of a syndrome with multiple congenital malformations, now widely recognized as trisomy 18. He reported in a 1960 Lancet paper that the extra chromosome belonged to the E group. However, it was reported as trisomy 17, largely due to the lack of banding and low resolution of the chromosome spreads. Another of Edwards’ major contributions includes placental sampling (initially introduced to detect Rhesus negative fetuses), which would later revolutionize the prenatal diagnosis of trisomies. Edwards also reported on a series of 20 cases of Cornelia de Lange syndrome and developed the “Oxford grid”- a map to compare gene sequences in different animal species and humans, thereby forming the concept of synteny.

FRAGILE X SYNDROME: HERBERT LUBS AND FREDERICK HECHT

In 1969, an American medical geneticist, Herbert Lubs observed an unusual “marker X chromosome” with a constriction near the end of the long arm of the X chromosome in four male individuals with intellectual disability. This constriction that appeared to be a “broken” X chromosomes was later named the “fragile-site” in 1970 by Frederick Hecht. Lubs received numerous accolades for his work on the Fragile X Syndrome, reported as the most common inherited cause of intellectual disability in males. He later went on to describe at least 20 other X-linked genes associated with intellectual disability. The first prenatal cytogenetic diagnoses and chromosome abnormalities in cancer were also carried out in Lubs’s laboratory. In 1970, Hecht identified another fragile site on chromosome 16 and hypothesized that to be the haptoglobin locus in man.

MEIOTIC CROSSING OVER, GENETIC TRANSPOSITION, TELOMERES AND CENTROMERES: BARBARA MCCLINTOCK (1902-1992)

Barbara McClintock is regarded as one of the world’s most distinguished cytogeneticists, earning the 1983 Nobel Laureate in Physiology and Medicine with major contributions in the field of maize cytogenetics. She studied maize reproduction in the 1920’s and utilized microscopy to demonstrate fundamental genetic concepts. Her work led to the discovery of genetic recombination by crossing over during meiosis, a mechanism by which chromosomes exchange information and maintain variability. The first genetic map of maize was produced in her lab whereby the physical traits in maize were linked to the chromosome regions. She also elucidated the role of telomeres and centromeres in the conservation of genetic information.

PHILADELPHIA CHROMOSOME AND LEUKEMIA: JANET ROWLEY (1925-2013)

As constitutional cytogenetics was slowly evolving over the 1950’s-1970s, the biggest breakthrough came in cancer cytogenetics with the identification of the Philadelphia chromosome, a balanced translocation involving the BCR gene (chr 22) and ABL1 gene (chr 9). Published in 1973 by American geneticist Janet Rowley, this was the first report to identify a chromosomal translocation in the etiology of leukemia and other cancers. Rowley also identified the t(8;21) in acute myelogenous leukemia and t(15;17) in acute promyelocytic leukemia. The identification of these translocations opened the door to the development of therapeutic wonder drugs (Gleevac, ATRA) for leukemia patients harboring these translocations. Little did she know, forty years ago, that she was paving the way for the term “precision medicine”!

SMITH-MAGENIS SYNDROME: R. ELLEN MAGENIS (1925-2014) AND ANN C.M. SMITH

In 1986, Ellen Magenis, a pediatric geneticist and a board certified cytogeneticist, along with Smith, a genetic counselor, identified and reported the first nine patients with a recognizable syndrome that would become known as the Smith-Magenis syndrome caused by a deletion of chromosome 17p11.2. Magenis’ first major research project, which remarkably preceded the human genome project, involved the linking of the haptoglobin locus to the chromosome 16q fragile site (along with Hecht and Lovrien).

CHROMOSOMAL CAUSES OF INFERTILITY: ORLANDO (JACK) MILLER AND DOROTHY (SANDY) MILLER

Jack Miller, trained in obstetrics and gynecology and genetics, reported the first XXY/trisomic 21 male in 1959 and the first XXYY male in 1961. Joining Columbia University in 1960, Jack collaborated with Roy Breg at Yale University to identify several chromosomal causes of infertility and intellectual disability. In 1964, Miller’s wife, Sandy, joined his group. By 1971, the Miller team, as they were famously called, had pioneered the use of chromosomal banding techniques in interspecific somatic cell hybrids to assign a gene to a specific autosome. They also identified each mouse chromosome via banding techniques and developed a simple way to identify the centromeric end of mouse linkage groups. Another significant cytogenetic demonstration by the Miller team was the association of the absence of human ribosomal RNA and lack of silver staining with the loss of one or more acrocentric human chromosomes. They also provided the first evidence of existence of tumor suppressor genes, and homogeneously stained regions (HSRs). Additionally, Sandy led the comparative genomic studies on great ape chromosomes (1977), human and mouse satellite DNA sequences (1990, 1991) and comparative mapping of human and marsupial chromosomes by in situ hybridization (1994).

IN SITU HYBRIDIZATION (ISH): JOSEPH G. GALL AND MARY L. PARDUE

In 1969, Gall and Pardue first reported the detection of RNADNA hybrid molecules by autoradiography. These cytological preparations from the oocytes of Xenopus illustrated the hybridization of ribosomal RNA to the amplified ribosomal genes. This was a landmark study toward the development of novel cytogenetics tools. Pardue also performed crucial work on telomeres (the ends of chromosomes) and elucidated their role in chromosomal replication, cell division and in maintaining the architecture of interphase nuclei. 

INTERPHASE CYTOGENETICS: THOMAS CREMER

Thomas Cremer, a German professor of human genetics and anthropology, had a keen eye for molecular cytogenetics and 3D/4D analyses of nuclear structure. In 1980, the science of interphase cytogenetics gained credence where normal and aberrant chromosomal regions could be directly visualized in the nucleus. However, it was not until 1990 that he conceptualized the technique of comparative genomic hybridization (CGH) with Peter Lichter. In 2005, Cremer was recognized for his pioneering work on higher order chromatin organization utilizing laser-UV micro-irradiation and fluorescence-based molecular cytogenetics. Cremer’s team continues their work on the topography of chromosomes in the nucleus with a focus on nuclear architecture.

FLUORESCENCE IN SITU HYBRIDIZATION (FISH): DANIEL PINKEL

Pinkel advanced Cremer’s work, and in 1988, along with his team, utilized FISH for the detection of trisomy 21 and chromosome 4 translocations. He then published a landmark report in 1992 where the technique of CGH was utilized as a method to cytogenetically analyze solid tumors.

SOUTHERN BLOTTING AND MICROARRAY: UWE MASKOS AND EDWIN M. SOUTHERN

Southern blotting, named after Ed Southern who worked with Sir Alec Jeffreys, was one of the techniques that revolutionized molecular genetics. However, in 1992, Southern and his German student, Uwe Maskos, described the first report of in situ oligonuceotide hybridizations performed on glass supports. This work was the primary scientific methodology that would lead to the development of microarray technology as we know today. Affymetrix was the first company to fabricate DNA microarrays using photolithography called “GeneChip” and released the first HIV genotyping GeneChip product in 1994. Southern founded Oxford Gene Technology in 1996 and won a 1999 patent infringement lawsuit based on his patent holdings in microarray technology. Despite the fact that microarray was included in the standard guidelines for the evaluation of individuals with intellectual disability, autism, developmental delay and congenital anomalies, it wasn’t until 2014 that Affymetrix received the FDA clearance of the first whole genome microarray – CytoScan Dx Assay.

THE FUTURE: NEXT-GENERATION CYTOGENOMICS (NGC)

Now that the human genome has been sequenced and sequencing is becoming more accessible – the question arises- what’s next? We now have access to the variants within our genome without knowing what it all means. How we understand the functional consequences is one of the biggest challenges. Karyotyping, microarray, sequencing – each has its strengths and limitations. Next Generation Sequencing, while a revolutionizing technology, is still unable to routinely identify structural variations. While innovators are busy designing algorithms to address these limitations, the researchers and the scientific community are left with a handful of options to understand the precise role of all the components of the human genome: chromosome capture conformation (3C) modifications such as 4C and 5C, chromosome territories/boundaries and their role in cellular function, oligopaint FISH, de novo genome mapping and analysis of structural variation using long reads (Bionanogenomics), role of nuclear matrix in disease pathogenesis (e.g. laminopathies). Which one of these will open the future of Next Generation Cytogenomics is yet to be seen.