Chromosome analyses

The classical chromosomal analysis in the context of postnatal diagnostics is one of the most fundamental investigations in genetic analysis. It includes the examination of cultured peripheral blood lymphocytes and miscarriage tissue. Chromosomal analysis, in combination with genetic counselling, is advisable in the following cases:

Newborns with congenital malformations
Children with developmental delays and/or behavioural abnormalities
Relatives of individuals with structural chromosomal anomalies
Men with small testes and/or gynaecomastia
Newborns with hypospadias or intersex genitalia
Suspected chromosomal syndrome (e.g., Down syndrome, Cri-du-Chat syndrome, )

Reproductive genetics (couples with fertility concerns)

Couples with infertility prior to undergoing IVF or ICSI
Couples with two or more spontaneous miscarriages or stillbirths
Relatives of individuals with structural chromosomal anomalies
Women with primary or secondary amenorrhoea or premature menopause
Men with azoospermia or oligozoospermia
Men with small testes and/or gynaecomastia

Standard examination material consists of 2–5 ml of sodium- or lithium-heparinized whole blood. Results are typically available within 2–3 weeks, or within 1 week in urgent cases. The examination of miscarriage tissue is primarily aimed at clarifying the cause of the miscarriage and assessing the risk for subsequent pregnancies. In addition to placental tissue, fetal tissue such as skin, umbilical cord, or fascia lata should also be sent in sterile physiological saline solution. The evaluation of all cell cultures is conducted after the preparation and staining of chromosome specimens. For this purpose, cells in the process of division are first enriched by adding the spindle fibre toxin colchicine. Subsequently, the cells undergo specific treatments (hypotonic shock, fixation) and the cell suspension is applied onto slides. After staining the preparations (GTG staining), they are analysed under a microscope using digital imaging techniques on a computer monitor.

Chromosomal aberrations are responsible for 0.5–1% of all congenital malformations. In general, chromosomal aberrations refer to changes visible under a light microscope. These changes involve either the number of chromosomes (numerical) or their structure (structural chromosomal aberrations). If a chromosomal aberration is found in all body cells, it is referred to as a constitutional disorder. If only specific body cells are affected, it is termed a somatic disorder.

Symptom / disorder	Relative frequency
Miscarriages / stillbirths Miscarriages 5th-11th week of pregnancy Miscarriages 12-24 weeks gestation Miscarriages > 24th week of pregnancy	Total ca. 30% 50% 25% 5%
Congenital complex malformations	4-8%
Congenital heart defects	13%
Mental retardation IQ < 20 IQ 20-49 IQ 50-69	3-10% 12-35% 3%
Infertility	2%
Azoospermia	15%
True hermaphroditism	25%
Primary ovarian insufficiency (incl. string gonads)	65%

Information / syndrome	Trisomy 21 (Down syndrome)	Trisomy 18 (Edwards syndrome)	Trisomy 13 (Pätau syndrome)
Karyotype	47,+21	47,+18	47,+13
	Frequency	Frequency	Frequency
Free trisomy	92%	80%	75%
Translocation	5%	10%	20%
Mosaic	3%	10%	5%
Incidence	1:650	1:3.000 (f:m = 4:1)	1–2:10.000 (f:m = 4:3)
Symptoms	1) Mental retardation 2) Facial dysmorphia (e.g., Macroglossia, oblique eyelid axes, auricular dysplasia, epicanthus, four-finger furrow) 3) Organ malformations (heart defects, duodenal atresia)	1) Severe psychomotor retardation 2) Facial dysmorphia (e.g., microcephaly, auricular dysplasia) 3) Organ malformations (e.g., heart defects, horseshoe kidney, contractures)	1) Severe psychomotor retardation 2) Facial dysmorphia (e.g., microcephaly, coloboma, cleft palate, auricular dysplasia) 3) Organ malformations (e.g., heart defects, omphalocele, hexadactyly, renal cysts)

Information / syndrome	Turner syndrome	Triple X syndrome	Klinefelter syndrome	Jacobs (double Y) syndrome
Karyotype	45,X 55% 45,X; 45% mosaic and structural anomalies	47,XXX 98% 47,XXX; 2% mosaic	47,XXY 80% 47,XXY; 20% other X-polysomies or mosaics	47,XYY Mainly pure 47,XYY; also X- and Y polysomies
Frequency	1:2.500 female newborns 1:100 conceptions	1:1.000 female newborns	1:1.000–1:500 male newborns	1:1.000 male newborns
Phenotype	Short stature, primary amenorrhea, constrictive gonads, IQ normal	Very variable (2/3 of the cases without clear phenotype), partly tall stature, partly IQ lower normal range- subnormal	High growth, gynecomastia, hypogonadism, azoospermia, IQ approx. 10 points below family average	High stature, normal fertility, IQ normal to slightly subnormal, possibly emotional disorders
Prognosis/therapy	Substitution of estrogen and growth hormone, for 45,X/46,XY mosaic prophylactical gonadectomy (risk of degeneration)	As a rule, no specific therapy indicated	Developmental support in childhood, substitution of testosterone	Psychological-pedagogical support for learning difficulties and for the prevention of behavioral disorders

Structural chromosomal aberrations arise from one or more breaks, leading to a reorganization either within a single chromosome (intrachromosomal) or between multiple chromosomes (interchromosomal). The chromosomal set resulting from this reorganization can be balanced (without loss and/or gain of chromosomal material) or unbalanced (with loss and/or gain). While balanced chromosomal sets are generally not associated with clinical symptoms, unbalanced rearrangements typically result in a chromosomal syndrome characterized by the classical triad of symptoms: psychomotor retardation, craniofacial dysmorphic features, and malformations of internal organs. The phenotypic expression and severity are highly variable and usually cannot be predicted.

The most important structural aberrations are

Reciprocal translocation: This involves a breakage event in two chromosomes with mutual exchange of the acentric fragments. In affected patients, gametes with unbalanced chromosomal sets can form during meiosis, depending on how the chromosomes separate, leading to a risk of clinically noticeable offspring or miscarriages. The frequency in newborns is approximately 1 in 700 (500–1,000).

Robertsonian translocation: Caused by a break in two acrocentric chromosomes (chromosomes 13, 14, 15, 21, and 22) in the centromere region, the two centric fragments fuse together. In these cases, compensable, acentric p-arm fragments are lost, whose loss is currently not known to have any clinical consequences. Depending on how chromosomes are distributed during meiosis, gametes with unbalanced chromosomal sets can form. The most significant Robertsonian translocation is translocation trisomy 21 (“hereditary” Down syndrome). The frequency in newborns is about 1 in 1,000.

Deletion: Loss of a chromosomal segment, either at the end (terminal deletion) or within a chromosome (interstitial deletion). The best-known deletion syndromes are Cri-du-Chat syndrome (deletion of 5p-) and Wolf-Hirschhorn syndrome (deletion of 4p-). The frequency of deletions is approximately 0.9 in 10,000 newborns.

Inversion: An inversion arises from two breaks in a chromosome, with a 180° rotation of the segment between the breakpoints. If the centromere lies within this segment, it is called a pericentric inversion; otherwise, it is referred to as a paracentric inversion. Due to recombination events during meiosis, gametes with unbalanced chromosomal sets can form in the case of pericentric inversion. Frequency in newborns: approximately 1.3 in 10,000.

Insertion: This refers to the insertion of a chromosomal segment at a different location within the genome.

Duplication: Duplication of a chromosomal segment.

Ring chromosome: Ring chromosomes arise through the loss of the ends of a chromosome and the fusion of the two breakpoints.

Marker chromosome: This refers to an additional, structurally altered chromosome whose origin can often only be determined using specialized methods (M-FISH or array-CGH). Newly formed marker chromosomes whose chromosomal origin remains unclear pose a significant challenge for prenatal diagnostics, as only very vague statements can be made regarding potential clinical symptoms. Frequency in newborns (including mosaics) is about 3 in 10,000.

Prenatal Diagnosis

The most important invasive prenatal diagnostic methods include chromosomal analysis from chorionic villi, amniotic cells, and umbilical cord blood (cordocentesis). The chosen method depends on the specific diagnostic question.

A chorionic villus sampling (CVS) can be performed as early as the 10th week of pregnancy (gestational week). Around 10-30 mg of chorionic villus tissue is typically collected transabdominally under ultrasound guidance. From this tissue, a direct preparation (result available by the next day at the latest), a short-term culture (result in 1 day), and a long-term culture (1-2 weeks) are prepared. The miscarriage risk following CVS is approximately 0.1% in experienced centers. Amniocentesis (AC) is carried out as an early amniocentesis in the 13th to 15th week of pregnancy and as a classical amniocentesis in the 15th to 17th week of pregnancy. Under ultrasound guidance, 10-20 ml of amniotic fluid is removed transabdominally with a fine needle. Several long-term cultures are established from the amniotic fluid cells, and the evaluation results are available after about 2 weeks. The miscarriage risk is approximately 0.1%, slightly higher for early amniocentesis. Cordocentesis can only be performed starting from the 20th week of pregnancy, and the results are available within one week. The miscarriage risk for cordocentesis is estimated to be between 0.5% and 1%.

Tumor Cytogenetics

Since neoplastic cells are often characterized by specific chromosomal aberrations, chromosomal banding analysis is still considered the gold standard in leukemia and lymphoma diagnostics, despite the advent of many new laboratory diagnostic methods. The detection of gross structural changes plays a crucial role in both the initial diagnosis and the progression of the disease, as well as in the WHO classification.