Genetic Studies -Direct Detection of Abnormal Genes by DNA Testing Many genetic diseases continue to be detected by the effects

they produce in body structure, function, or chemistry. With the elucidation of gene structure and the cataloging of human gene mutations, direct detection of hundreds of mutations is possible. For known genetic disorders for which specific mutational analysis is not available, indirect analysis using varied techniques, including protein expression, may be applicable. A database of medical genetics information resources for physicians and other health care providers and researchers maintains daily updates of available testing worldwide at http:// www.genetests.org. Registration is required.

Genetic testing for diagnostic purposes requires patient education and consent, physician request, and coordination of sample collections. Diagnostic tests must be done in a Clinical Laboratory Improvement Act (CLIA)approved laboratory. Research labs cannot provide this service and should not be contacted for clinical testing. Sensitivity of testing in genetic disease needs to be addressed because many diseases may have different causes, and many genetic tests are not capable of finding all mutations in complex genes. For example, nearly 1,000 gene mutations have been linked to cystic fibrosis, but still an estimated 3% to 10% of mutations cannot be found. Interpretation of results can be a challenge also, especially in situations in which a gene change can be demonstrated, but it is not known whether it is a harmless change or not. An example of this is polymorphism in the BRCA-1 gene. In such situations, interpretation may rely on comparison to gene changes found in known affected relatives. A variety of morbid genetic changes have been discovered, including gain or loss of a single base pair or larger group of base pairs as well as repetitive sequences that get copied over and over so many times that they disable the function of the gene. Detection strategies are tailored to the type of mutation present or suspected. Procedure

Establish availability and sensitivity of clinical testing and inform the patient of the benefits, limitations, and consequences of testing (see Genetic Counseling). Informed consent may be required. Prepayment may be required. Test results may take weeks or months. Obtain samples or specimens of body fluids or tissues as specified by the receiving laboratory. Overnight shipment must usually be arranged.

Clinical Implications

Improved diagnosis of types of cancer may have therapeutic implications. Discovery of hereditary disease or cancer may have implications for other family members. Precise DNA tests can be done for some inherited diseases (eg, cystic fibrosis, Duchenne's and Becker's muscular dystrophy, some polycystic kidney diseases). Paternity identity testing and forensic testing Identification of microbes in infectious diseases (eg, chlamydia, cytomegalovirus) Prediction of progression in neuromuscular disorders (eg, Huntington's disease, myotonic dystrophy, cerebellar ataxia) Identification of comorbid disease risks (eg, progressive kidney failure in some hearing-loss syndromes) Identification of reproductive risks Explanation of miscarriage and stillbirth Potential associations with common diseases of aging (eg, cardiovascular disease, Alzheimer's disease)

Cytogenetics -Chromosomal Analysis The karyotype, a study of chromosome distribution for an individual, determines chromosome numbers and chromosome structure (Chart 11.4); alterations in either of these can produce problems. The standard karyotype can be a diagnostic precursor to genetic counseling. Additional or missing pieces of most chromosomal material cause

developmental problems. Despite much speculation, it is not known exactly how these abnormalities translate into structural or functional anomalies. Predictions almost always depend on comparisons with clinical findings from other similar cases that present the same evidence. Standard chromosome studies can be helpful in evaluation of the following clinical situations:

Multiple malformations of structure and function Failure to thrive Mental retardation Ambiguous genitalia or hypogonadism Recurrent miscarriages Infertility Primary amenorrhea or oligomenorrhea Delayed onset of puberty Stillbirths or miscarriages (particularly with associated malformations) Prenatal diagnosis of potential or actual abnormalities related to chromosome disorders (eg, Down syndrome, especially in offspring of mothers >35 years of age) Detection of parents with chromosomal mosaicism or translocations, who may be at high risk for transmitting genetic abnormalities to their children Sex determination Selected cancers and leukemias in which abnormalities of the chromosomes may reveal prognosis or disease stage

Reference Values Normal

46 chromosomes Women: 44 autosomes + 2 X chromosomes (karyotype 46,XX) Men: 44 autosomes + 1 X and 1 Y chromosome (karyotype 46,XY) A photograph of representative karyotype is included with report.

Procedure Specimens for chromosome analyses are generally obtained as follows, using aseptic procedures and special kits and containers:

Be aware that heparinized venous blood leukocytes from peripheral vascular blood samples are used most frequently because they are the most easily obtained. Preparation of the cells takes at least 3 days. The time required is directly proportional to the complexity of the analytic process. Collect bone marrow in a green-topped tube, at least 5 mL in a heparinized syringe (2025 units of heparin). Biopsies can sometimes be completed within 24 hours. Bone marrow analysis is often done to diagnosis certain categories of leukemias. Remember that fibroblasts from skin or other surgical specimens can be grown and preserved in long-term culture mediums for future studies. Growth of a sufficient amount of the specimen for studies usually requires at least 1 week. These specimens are especially helpful in detecting mosaicism (different chromosome constitutions in different tissues) and in the study of stillbirths, neonatal death, and spontaneous abortion. Be aware that amniotic fluid in the prenatal period obtained through amniocentesis and stored in a sterile container requires at least 1 week to produce a sufficient amount of cell growth for analysis. These studies are often done for prenatal detection of chromosomal abnormalities (see Chapter 15). Remember that chorionic villus sampling (CVS) can be done at earlier stages of pregnancy (about 9 weeks) than can amniocentesis. Some initial CVS studies can be done almost immediately after conception. Occasional falsepositive results represent mosaicism of the placenta (the presence of several cell lines, some of which may not be found in the fetus). These studies need confirmation of findings through long-term culture (see Chapter 15).

Grow cells from fetal tissue or from early-trimester products of conception to determine causes of spontaneous abortion. Cells from the fetal surface of the placenta may be easiest to grow and are the most likely to be successful. Take the buccal smear, for detecting sex chromosomes, from the inner cheek and use fluorescent in situ hybridization with probes specific for the X or Y chromosome. Take dried blood spot from heel of newborn. Place specimens of lymph nodes or solid tumors in sterile containers. Remember that chromosome analysis is often performed using other specimens, such as skin, fascia, lung tissue, kidney, or the placenta. At least 2 mm of volume is needed for an adequate specimen. See Chapter 1 guidelines for intratest care.

Clinical Implications Many chromosomal abnormalities can be placed into one of two classes; some examples follow:

Abnormalities of number o Autosomal: Trisomy 21 (Down syndrome) Trisomy 18 (Edwards' syndrome) Trisomy 13 (Patau's syndrome) o Sex chromosome syndromes: Ullrich-Turner syndrome (45, single X)short stature, webbed neck, and renal and C anomalies Klinefelter's syndrome (47, XXXY)hypogonadism, infertility, learning disabilities, undeveloped secondary characteristics XXY; 47, XXYtall, increased risk for behavior problems Triple XXXincreased risk for infertility and behavior problems Abnormalities of structure o Deletions: Cri du chat/cat's cry syndrome: the distal part of the chromosome 5 short arm is deleted. Missing short arm of chromosome 18: 18p- is deleted. Prader-Willi syndrome: 15 Q is deleted in some cases. o Duplications: extra material from the second band in the long arm of the third chromosome: 3q2 trisomy (Cornelia de Lange's syndrome resemblance) o Translocations: translocation of chromosomes 11 and 22: t(11;22) or 14 and 21 o Isochromosomes: a single chromosome with duplication of the long arm of the X chromosome: i(Xq) (a variant of Turner's syndrome) o Ring chromosomes: a chromosome 13 with the ends of the long and short arms joined together, as in a ring: r(13) o Mosaicism: two cell lines, 1 normal female and the other for Turner's syndrome: 46, X; 45, X

Interventions Pretest Patient Care

Provide information and referrals for appropriate genetic counseling and treatment if necessary. Explain the purpose, procedure, and limitations of the genetic test together with the known risks and benefits. This education process should be done by a genetic counselor. Obtain informed, signed, and witnessed consent. This is required for most genetic tests. Follow Chapter 1 guidelines for safe, effective, informed pretest care.

Posttest Patient Care

If an amniotic fluid specimen or CVS is obtained for analysis, follow the same precautions as listed in Chapter 15.

Provide timely information and compassionate support and guidance for parents, children, and significant others. See Chapter 1 for guidelines for safe, effective, informed posttest care.

Clinical Alert

Occasionally, it is possible to line up a certain chromosomal pattern with specific genes and to then understand the clinical picture from analyzing these results. However, for the most part, the association between specific chromosomal abnormalities and specific sets of findings is not yet well understood. Interpretations from karyotype studies usually come from correlations with similar cases rather than from any theoretical considerations. Therefore, because many variables exist, predictions must be made cautiously and judiciously. Most laboratories provide interpretations of results. However, it may be necessary to talk directly with laboratory personnel to fully understand the meaning of an unusual karyotype.

Special Chromosomal Studies Fragile X syndrome is one of the most common genetic causes of mental retardation. An X-linked trait, it is more commonly seen in males. Females may carry this gene without exhibiting any of its characteristics; however, they can also be as severely affected as males. This syndrome takes its name from the small area on the long arm of the X chromosome that looks like a break in the arm (although it actually is not). The cells need to be grown in a special medium to reveal this pattern; a regular karyotype will miss it. Even with the special medium, not all cells show the characteristic. In female carriers of this trait, the syndrome becomes harder to detect as the woman ages. Accurate detection of fragile X syndrome at a molecular level is now available. Rare conditions such as excess chromosome breakage (Fanconi's anemia) or abnormal centromeres (Roberts' syndrome) merit special analytic processes and procedures. Chromosome and molecular analyses are done using a venous blood sample (5 mL with EDTA tube) to identify the fragile X mental retardation syndrome and possible carrier status.