Chromosome analysis. There are several conditions, some relatively common and some rare, that result from either abnormal numbers of chromosomes, defects in size or configuration of certain single chromosomes, or abnormal composition of the chromosome group that determines sexual characteristics. Laboratory diagnosis, at present, takes three forms. First, chromosome charts may be prepared on any individual by culturing certain body cells, such as WBCs from peripheral blood or bone marrow, and by introducing a chemical such as colchicine, which kills the cells at a specific stage in mitosis when the chromosomes become organized and separated, and then photographing and separating the individual chromosomes into specific groups according to similarity in size and configuration. The most widely used system is the Denver classification. The 46 human chromosomes are composed of 22 chromosome pairs and, in addition, 2 unpaired chromosomes, the sex chromosomes (XX in the female and XY in the male). In a Denver chromosome chart (karyotype) the 22 paired chromosomes are separated into 7 groups, each containing 2 or more individually identified and numbered chromosomes. For example, the first group contains chromosomes 1 to 3, the seventh group contains chromosomes 21 to 22. In addition, there is an eighth group for the two unpaired sex chromosomes. Chromosome culture requires substantial experience and care in preparation and interpretation. Material for chromosome analysis can be obtained in the first trimester of pregnancy by means of chorionic villus biopsy.
Barr body test. The other, more widely used technique provides certain useful information about the composition of the sex chromosome group. Barr found that the nuclei of various body cells contain a certain stainable sex chromatin mass (Barr body) that appears for each X chromosome more than one that the cell possesses. Therefore, a normal male (XY) cell has no Barr body because there is only one X chromosome, a normal female (XX) cell has one Barr body, and a person with the abnormal configuration XXX has two Barr bodies. The most convenient method for Barr body detection at present is the buccal smear. This is obtained by scraping the oral mucosa, smearing the epithelial cells thus collected onto a glass slide in a monolayer, and, after immediate chemical fixation, staining with special stains. Comparison of the results, together with the secondary sex characteristics and genitalia of the patient, allows presumptive diagnosis of certain sex chromosome abnormalities. The results may be confirmed, if necessary, by chromosome karyotyping.
Specimens for buccal smear should not be obtained during the first week of life or during adrenocorticosteroid or estrogen therapy, because these situations falsely lower the incidence of sex chromatin Barr bodies. Certain artifacts may be confused with the nuclear Barr bodies. Poor slide preparations may obscure the sex chromatin mass and lead to false negative appearance. Only about 40%-60% of normal female cells contain an identifiable Barr body. The buccal smear by itself does not reveal the true genetic sex; it is only an indication of the number of female (X) chromosomes present. Many labs no longer do this test.
The third method is nucleic acid probe, more sensitive than either Barr body or standard chromosome analysis. However, the chromosome abnormality must be known and a probe must be available for that specific gene or chromosome area.
Klinefelter’s syndrome. In this condition the patient looks outwardly like a male, but the sex chromosome makeup is XXY instead of XY. The external genitalia are usually normal except for small testes. There is a tendency toward androgen deficiency and thus toward gynecomastia and decreased body hair, but these findings may be slight or not evident. There also is a tendency toward mental deficiency, but most affected persons have perfectly normal intelligence. Patients with Klinefelter’s syndrome are almost always sterile. Testicular biopsy used to be the main diagnostic method, with histologic specimens showing marked atrophy of the seminiferous tubules. A buccal smear can be done; it shows a “normal female” configuration with one Barr body (due to the two XX chromosomes). In the presence of unmistakably male genitalia, this usually is sufficient for clinical diagnosis. Since 10% of cases have a mosaic cell pattern, chromosome karyotyping is now the procedure of choice.
Turner’s syndrome (ovarian agenesis). Turner’s syndrome is the most frequent chromosomal sexual abnormality in females, just as Klinefelter’s syndrome is in males. In Turner’s syndrome there is a deletion of 1 female (X) chromosome so that the patient has only 45 chromosomes instead of 46 and only 1 female sex chromosome instead of 2. Typically the affected female has relatively short stature but normal body proportions. There is deficient development of secondary sex characteristics and small genitalia, although body hair usually is female in distribution. Some affected persons have associated anomalies such as webbing of the neck, coarctation of the aorta, and short fingers. They do not menstruate and actually lack ovaries. A buccal smear should be “sex-chromatin negative,” since Barr bodies appear only when the female sex chromosomes number more than one. If the buccal smear is “chromatin positive,” a chromosome karyotype should be ordered, because some patients with Turner’s syndrome have mixtures of normal cells and defective cells (mosaicism). Some investigators believe that in patients with short stature only, chromosome karyotyping should be done without a buccal smear, since most of the “nonphenotypic” Turner’s syndrome patients have mosaicism rather than XO genotype. Most geneticists karyotype without buccal smear due to smear interpretation problems.
Down’s syndrome. Down’s syndrome is a relatively frequent disorder associated with two different chromosome abnormalities. Most patients (about 92%) have an extra number 21 chromosome in the number 21-22 chromosome group (therefore having 3 chromosomes in this group instead of 2, a condition known as trisomy 21). These patients have a total of 47 chromosomes instead of 46. The chromosome abnormality has nothing to do with the sex chromosomes, which are normal. This type of Down’s syndrome apparently is spontaneous, not inherited (i.e., there is no family history of Down’s syndrome and there is very little risk the parents will produce another affected child). This nonfamilial (sporadic) type of Down’s syndrome occurs with increased frequency when the mother is over age 35. About 5% of patients have familial Down’s syndrome; the patient has an extra 21-type chromosome, but it is attached to one of the other chromosomes, most often in the 13-15 group (called the “D group” in some nomenclatures). This type of arrangement is called a “translocation.” The translocation attachment is most frequent on the number 14 chromosome, but it may attach elsewhere. The translocation abnormality can be inherited; it means that one parent has a normal total number of chromosomes, but one of the pair of number 21 chromosomes was attached to one of the number 14 chromosomes. The other number 21 and the other number 14 chromosome are normal. The two-chromosome (14 + 21) cluster behaves in meiosis as though it were a single number 14 chromosome. If the abnormal chromosome cluster is passed to a child, two situations could result: a child with clinical Down’s syndrome who received the translocated 14 + 21 chromosome plus the normal number 21 chromosome from one parent (and another number 21 chromosome from the other parent, making a total of three number 21 chromosomes), or a carrier who received the translocated 14 + 21 chromosome but did not receive the other (normal) number 21 chromosome from the same parent (the translocated 14 + 21 chromosome plus a number 21 chromosome from the other parent make a total of two number 21 chromosomes). The translocation Down’s syndrome patient has a total of 46 chromosomes (the two-chromosome unit counts as a single chromosome).
Clinically, an infant or child with Down’s syndrome usually has some combination of the following: prominent epicanthal folds at the medial aspect of the eyes, flattened facies, flat bridge of the nose, slanted lateral aspect of the eyes, mental retardation or deficiency, broad hands and feet, and a single long transverse crease on the palm instead of several shorter transverse creases. Other frequent but still less common associated abnormalities are umbilical hernia, webbing of the toes, and certain types of congenital heart disease. There also is an increased incidence of acute leukemia.
Diagnosis usually can be made clinically, but chromosome karyotyping is a valuable means of confirmation and of diagnosis in equivocal cases. It probably is advisable to do chromosome karyotyping in most children with Down’s syndrome, because the type of chromosome pattern gives an indication of the prognosis for future children.
Prenatal diagnosis can be made in the first trimester by chorionic villus biopsy with chromosome analysis. Screening for Down’s syndrome can be done using maternal serum during the 16th to 18th gestation week. If the maternal alpha-fetoprotein serum level is lower than normal, the unconjugated estriol (E3) lower than normal, and the beta human chorionic gonadotropin (beta-hCG) higher than normal, this suggests possible Down’s syndrome. This would have to be confirmed with fetal cells obtained by amniocentesis (chorionic villus biopsy is not done after the 12th week of pregnancy).
Fragile X chromosome. The fragile X chromosome refers to a narrowing in the X chromosome, at which point the chromosome breaks more easily than usual when cultured in a medium that is deficient in thymidine and folic acid. The syndrome is said to be second only to Down’s syndrome as a cause of hereditary mental retardation. The fragile X abnormality is reported to be associated with 30%-50% of cases of X-linked mental retardation as part of a syndrome which also includes certain mild facial changes. About 30%-35% of female carriers may have mild mental retardation, which is unusual for heterozygotic status in most genetic illnesses and very unusual for an X-linked inherited disorder (in which the carrier female seldom has clinical symptoms). In addition, about 20% of males with the chromosome defect are asymptomatic and not detectable by standard chromosome analysis. Male offspring of a (heterozygous) carrier female would have a 50% chance of developing the syndrome. Unfortunately, only about 30%-56% of heterozygotic females demonstrate the fragile X defect using current laboratory methods. Sensitivity of these methods is age dependent, and best detection rates occur testing women less than 30 year old. There have also been reports of some affected men with normal range IQ who would qualify as carriers. It has been estimated that as many as 20% of male offspring with normal IQS born to female carriers actually are themselves carriers. DNA probe methods are now available that can often detect fragile X presence when standard chromosome analysis is equivocal or negative.
Adult polycystic kidney disease (PKD-1). This autosomal dominant condition is reported to be present in 1 of 1,000 live births. Multiple cysts form in the kidney and eventually enlarge, destroying nearby renal parenchyma and in many cases eventually resulting in renal failure. The genetic abnormality is located on chromosome 16. DNA probes are used that bracket the gene area (gene linkage analysis using restriction fragment length polymorphism).
Other chromosomal disorders. A wide variety of syndromes, usually consisting of multiple congenital deformities and anomalies, are now found to be due to specific chromosomal abnormalities. The most common of these involve trisomy in the 13-15 (D) group and in the 16-18 (E) group. Some patients with repeated spontaneous abortions have abnormal karyotypes. Various tumors have yielded abnormal chromosome patterns, but no one type of tumor is associated with any consistent pattern (except for chronic myelogenous leukemia).
Commonly accepted indications for buccal smear. These include the following:
1. Ambiguous or abnormal genitalia
2. Male or female infertility without other known cause
3. Symptoms suggestive of Turner’s syndrome or Klinefelter’s syndrome, such as primary amenorrhea
Indications for chromosome karyotyping
These include the patients in the buccal smear groups just described for confirmation or initial diagnosis and the following:
1. Down’s syndrome infants or possible carriers
2. Mentally defective persons
3. Persons with multiple congenital anomalies
