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METHODS AND TECHNOLOGIES

Chromosome Analyses

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 counseling, is advisable in the following cases:

Newborns with congenital malformations

Children with developmental delays and/or behavioral abnormalities

Relatives of individuals with structural chromosomal anomalies

Men with small testes and/or gynecomastia

Newborns with hypospadias or intersex genitalia

Suspected chromosomal syndrome (e.g., Down syndrome, Cri-du-Chat syndrome, Prader-Willi 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 amenorrhea or premature menopause

Men with azoospermia or oligozoospermia

Men with small testes and/or gynecomastia

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 fiber 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 analyzed 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 malformations4-8%
Congenital heart defects
13%
Mental retardation
IQ < 20
IQ 20-49
IQ 50-69

3-10%
12-35%
3%
Infertility2%
Azoospermia15%
True hermaphroditism25%
Primary ovarian insufficiency
(incl. string gonads)
65%
Couples with recurrent miscarriages2-5%
Information/ syndrome Trisomy 21
(Down syndrome)
Trisomy 18
(Edwards syndrome)
Trisomy 13
(Pätau syndrome)
Karyotype47,+2147,+1847,+13
FrequencyFrequencyFrequency
Free trisomy92%80%75%
Translocation5%10%20%
Mosaic3%10%5%
Incidence1:6501:3.000 (f:m = 4:1)1–2:10.000 (f:m = 4:3)
Symptoms1) 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 syndromeTriple X syndromeKlinefelter syndromeJacobs (double Y) syndrome
Karyotype45,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
Frequency1:2.500 female newborns
1:100 conceptions
1:1.000 female newborns1:1.000–1:500 male newborns1:1.000 male newborns
PhenotypeShort 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/therapySubstitution 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.

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