GENOMICS IN OBSTETRICS
ABNORMALITIES OF CHROMOSOME NUMBER
ABNORMALITIES OF CHROMOSOME STRUCTURE
MODES OF INHERITANCE
CHROMOSOMAL MICROARRAY ANALYSIS
FETAL DNA IN THE MATERNAL CIRCULATION
Genetics is the study of genes, heredity, and the variation of inherited characteristics. Medical genetics deals with the etiology and pathogenesis of human diseases that are at least partially genetic in origin, along with their prediction and prevention. Thus, it is closely linked to genomics, which is the study of how genes function and interact. In addition to chromosomal, mendelian, and nonmendelian genetic conditions reviewed in this chapter, medical genetics includes preimplantation and prenatal diagnosis, gene therapy, and newborn screening, which are discussed in Chapters 14, 16, and 32, respectively.
Genetic disease is common. Between 2 and 3 percent of newborns have a recognized structural defect. In another 3 percent of individuals, a defect is diagnosed by age 5, and another 8 to 10 percent are discovered by age 18 to have one or more functional or developmental abnormalities. An astonishing two thirds of the population will experience a disease with a genetic component during their lifetime. Advances in genomics are used increasingly to provide information regarding susceptibility to genetic diseases, and there is every indication that this field will reshape prenatal diagnosis (Bodurtha, 2012).
GENOMICS IN OBSTETRICS
Completed in 2003, the Human Genome Project identified nearly 25,000 human genes and led to rapid expansion of genomic research to better understand disease biology (Bodurtha, 2012; Feero, 2010; McKusick, 2003). One example is the International HapMap (Haplotype Map) Project, which studies the effects of genetic variation (National Human Genome Research Institute, 2012). HapMap investigates the nearly 10 million single nucleotide polymorphisms that comprise 0.5 percent of our DNA. Researchers look at how groups of common polymorphisms affect factors such as propensity to particular diseases and response to treatment. Another example is dbGaP, the database of Genotypes and Phenotypes, which is maintained by the National Center for Biotechnology Information (NCBI) (2013a). This database includes studies of genotype and phenotype interactions, such as genome-wide association studies and medical diagnostic assays. It is hoped that data from dbGaP will be used to develop tests or products that address public health needs.
The NCBI also maintains several genetic and genomic databases useful in obstetrics and maternal-fetal medicine practice. These include Online Mendelian Inheritance in Man (OMIM), GeneTests, and the Genetics Home Reference. Each is freely accessible to clinicians and researchers. Of these, OMIM is a comprehensive catalog of human genes and phenotypes originally created by the National Library of Medicine in collaboration with Johns Hopkins University. Clinicians can use OMIM to gain detailed information regarding particular syndromes and their genetic basis. Or, if a syndrome is suspected but the diagnosis is unclear, it may aid formulation a differential diagnosis by searching for syndromes that include particular traits or abnormalities. As of 2013, OMIM included more than 14,000 different genes with known sequences and nearly 4000 mendelian or mitochondrial conditions—phenotypes—with a known molecular basis (Johns Hopkins University, 2013).
Another database, GeneTests, provides information on genetic conditions, the benefits and limitations of available tests for a given disorder, and how to send a specimen to a particular laboratory. As of 2013, the GeneTests website contained 575 clinical reviews and more than 3000 genetic tests, and it provided contact information for more than 600 laboratories. Additional information regarding laboratories that perform genetic tests is available from the NCBI’s Genetic Testing Registry (2013c). The NCBI (2013b) has also established a database of genetic information intended for patients, the Genetics Home Reference. This database contains information on more than 2000 genetic conditions and genes.
Chromosomal abnormalities figure prominently in genetic disease. They are present in approximately 50 percent of spontaneous abortions, 5 percent of stillbirths, and 0.5 percent of liveborn infants (Parker, 2010; Schwartz, 2012). In the European Surveillance of Congenital Anomalies (EUROCAT) network of population-based registries, chromosomal abnormalities were recognized in 0.4 percent of pregnancies, with Down syndrome comprising more than half of cases (Dolk, 2010).
In humans, the 22 pairs of autosomes and one pair of sex chromosomes may be affected by various abnormalities. Karyotypes are described using the International System for Human Cytogenetic Nomenclature, a standardized format agreed upon by the genetics community (Shaffer, 2009). Abnormalities fall into two broad categories—those of chromosome number, such as trisomy, and those of chromosome structure, such as a deletion or translocation. Each chromosome has a short arm, termed the “p” or petit arm, and a long arm known as the “q” arm, selected because it is the next letter of the alphabet. The two arms are separated by the centromere.
When reporting a karyotype, the total number of chromosomes is listed first, corresponding to the number of centromeres. This is followed by the sex chromosomes, XX or XY, and then by a description of any structural variation. Specific abnormalities are indicated by standard abbreviations, such as del (deletion) and inv (inversion). The affected region or bands of the p or q arms are then designated, so that the reader will know both the exact abnormality location and the way in which the chromosomal complement is abnormal. Some examples of standard karyotype nomenclature are shown in Table 13-1.
TABLE 13-1. Examples of Karyotype Designations Using the International System for Human Cytogenetic Nomenclature (2009)
Abnormalities of Chromosome Number
The most easily recognized chromosomal abnormalities are numerical. Aneuploidy is inheritance of either an extra chromosome—resulting in trisomy, or loss of a chromosome—monosomy. These differ from polyploidy, which is an abnormal number of haploid chromosome sets, such as triploidy. The estimated incidence of various numerical chromosomal abnormalities is shown in Table 13-2.
TABLE 13-2. Frequency of Numerical Chromosomal Abnormalities
Trisomy accounts for approximately half of all chromosomal abnormalities. In most cases, it results from nondisjunction, which is failure of normal chromosomal pairing and separation during meiosis. Nondisjunction may occur if the chromosomes: (1) fail to pair up, (2) pair up properly but separate prematurely, or (3) fail to separate.
The risk of any autosomal trisomy increases steeply with maternal age, particularly after age 35 (Fig. 13-1). Aging is thought to break down the chiasmata that keep the paired chromosomes aligned. Oocytes are held suspended in midprophase of meiosis I from birth until ovulation, in some cases for 50 years. Following completion of meiosis at the time of ovulation, nondisjunction will result in one gamete having two copies of the affected chromosome, leading to trisomy if fertilized. The other gamete, receiving no copy of the affected chromosome, will be monosomic if fertilized. Between 10 and 20 percent of oocytes are aneuploid secondary to meiotic errors, compared with 3 to 4 percent of sperm. Although each chromosome pair is equally likely to have a segregation error, it is rare for trisomies other than 21, 18, or 13 to result in a term pregnancy. As shown in Figure 13-2, many fetuses with autosomal trisomy will be lost before term.
FIGURE 13-1 Maternal age-related risk for selected aneuploidies. (Redrawn from Nicolaides, 2004, with permission.)
FIGURE 13-2 Gestational-age-related risk for selected chromosomal abnormalities, relative to the risk at 10 weeks’ gestation. (Redrawn from Nicolaides, 2004, with permission.)
Following a pregnancy with an autosomal trisomy, the risk for any autosomal trisomy in a future pregnancy is approximately 1 percent until the woman’s age-related risk exceeds this. Accordingly, invasive prenatal diagnosis is offered in subsequent pregnancies (Chap. 14, p. 297). Parental chromosomal studies are not indicated unless Down syndrome was due to an unbalanced translocation.
Trisomy 21—Down Syndrome. In 1866, J. L. H. Down described a group of mentally retarded children with distinctive physical features. Nearly 100 years later, Lejeune (1959) demonstrated that Down syndrome is caused by an autosomal trisomy. The trisomy 21 karyotype is shown in Figure 13-3. Trisomy 21 is the etiology of 95 percent of Down syndrome cases, whereas 3 to 4 percent is due to a robertsonian translocation. The remaining 1 to 2 percent is secondary to an isochromosome or mosaicism. The nondisjunction that results in trisomy 21 occurs during meiosis I in almost 75 percent of cases. The remaining events occur during meiosis II.
FIGURE 13-3 Abnormal male karyotype with trisomy 21, consistent with Down syndrome (47,XY,+21). (Photograph contributed by Dr. Frederick Elder.)
Down syndrome is the most common nonlethal trisomy. Its prevalence is approximately 1 per 500 recognized pregnancies, including abortuses, stillbirths, and liveborn infants (Dolk, 2010). There is a significant fetal loss rate, as shown in Figure 13-2. Approximately 30 percent of fetuses with Down syndrome are lost between 12 and 40 weeks, and 20 percent between 16 and 40 weeks (Snijders, 1999). As a result, Down syndrome is found in 1 per 740 live births in the United States or 13.5 per 10,000. This represents an increase of approximately 33 percent compared with the rate in the late 1970s (Parker, 2010; Shin, 2009). The rise in prevalence is explained by the increase in maternal age distribution during this period.
Adult women with Down syndrome are fertile, and a third of their offspring will have Down syndrome (Scharrer, 1975). Contraceptive options are discussed in Chapter 38 (p. 695). Males with Down syndrome are almost always sterile because of markedly decreased spermatogenesis.
Clinical Findings. It is estimated that 25 to 30 percent of second-trimester fetuses with Down syndrome will have a major malformation that can be identified sonographically (Vintzileos, 1995). Approximately 40 percent of liveborn infants with Down syndrome are found to have cardiac defects, particularly endocardial cushion defects and ventricular septal defects (Figs. 10-22 and 10-23, p. 210). Gastrointestinal abnormalities develop in 7 percent and include duodenal atresia, esophageal atresia, and Hirschsprung disease (Fig. 10-28, p. 214) (Rankin, 2012).
Characteristic features of Down syndrome are shown in Figure 13-4. Typical findings include brachycephaly; epicanthal folds and up-slanting palpebral fissures; Brushfield spots, which are grayish spots on the periphery of the iris; a flat nasal bridge; and hypotonia. Infants often have loose skin at the nape of the neck, short fingers, a single palmar crease, hypoplasia of the middle phalanx of the fifth finger, and a prominent space or “sandal-toe gap” between the first and second toes. Some of these findings are sonographic markers for Down syndrome, which are reviewed in Chapter 14 (p. 292).
FIGURE 13-4 Trisomy 21—Down syndrome. A. Characteristic facial appearance. B. Redundant nuchal tissue. C. Single transverse palmar crease. (Photographs contributed by Dr. Charles P. Read and Dr. Lewis Waber.)
Health problems more common in children with Down syndrome include hearing loss in 75 percent, severe optical refractive errors in 50 percent, cataracts in 15 percent, thyroid disease in 15 percent, and an increased incidence of leukemia (American Academy of Pediatrics, 2001). The degree of mental impairment is usually mild to moderate, with an average intelligence quotient (IQ) score of 35 to 70. Social skills in affected children are often higher than predicted by their IQ scores.
Recent data suggest that approximately 95 percent of liveborn infants with Down syndrome survive the first year. The 10-year survival rate is at least 90 percent overall and is 99 percent if major malformations are absent (Rankin, 2012; Vendola, 2010). A number of organizations offer education and support for prospective parents faced with diagnosis of a Down syndrome fetus. These include the March of Dimes, National Down Syndrome Congress (www.ndsccenter.org), and National Down Syndrome Society (www.ndss.org).
Trisomy 18—Edwards Syndrome. This constellation of abnormalities and their association with another autosomal trisomy was first described by Edwards (1960). In population-based series, prevalence of trisomy 18 is approximately 1 per 2000 recognized pregnancies—including abortuses, stillbirths, and live births, and approximately 1 per 6600 liveborn infants (Dolk, 2010; Parker, 2010). The difference in prevalence is explained by the high in-utero lethality of the condition, as 85 percent of trisomy 18 fetuses are lost between 10 weeks’ gestation and term (see Fig. 13-2). Perhaps not surprisingly, survival of liveborn infants is likewise bleak. More than half die within the first week, and the 1-year survival rate is only approximately 2 percent (Tennant, 2010; Vendola, 2010). The syndrome is three- to fourfold more common in females (Lin, 2006; Rosa, 2011). Unlike Down and Patau syndromes, which involve acrocentric chromosomes and thus may stem from a robertsonian translocation, it is uncommon for Edwards syndrome to result from a chromosomal rearrangement.
Clinical Findings. Virtually every organ system can be affected by trisomy 18. Common major anomalies include heart defects in almost 95 percent—particularly ventricular septal defects, as well as cerebellar vermian agenesis, enlarged cisterna magna, myelomeningocele, diaphragmatic hernia, omphalocele, imperforate anus, and renal anomalies such as horseshoe kidney (Lin, 2006; Rosa, 2011; Yeo, 2003). Sonographic images of several of these are shown in Chapter 10 (p. 200).
Cranial and extremity abnormalities are also particularly common and include a prominent occiput, posteriorly rotated and malformed ears, micrognathia, small mouth, clenched hands with overlapping digits, radial aplasia, hypoplastic nails, and rockerbottom or clubbed feet. Characteristic sonographic findings include a “strawberry-shaped” cranium and choroid plexus cysts (Fig. 13-5). In otherwise low-risk pregnancies, the risk for trisomy 18 is increased only when a choroid plexus cyst is associated with other abnormalities. Alone, this cyst may be considered a normal variant.
FIGURE 13-5 Trisomy 18–Edwards Syndrome. A. This transventricular sonographic view shows fetal choroid plexus cysts and a “strawberry-shaped” (unusually angulated) skull. Although not shown here, the fetal profile typically demonstrates micrognathia, with a very small, recessed mandible. B. This three-dimensional (3-D) sonographic image shows the characteristic hand position of clenched fists with overlapping digits. C. 3-D sonographic image displays a rockerbottom foot.
Pregnancies with trisomy 18 that reach the third trimester often develop fetal-growth restriction, and the mean birthweight is less than 2500 grams (Lin, 2006; Rosa, 2011). Mode of delivery should be discussed in advance, because abnormal fetal heart rate tracings are common during labor. In older reports, more than half of undiagnosed fetuses underwent cesarean delivery for “fetal distress” (Schneider, 1981).
Trisomy 13—Patau Syndrome. This constellation of fetal abnormalities and their association with yet another autosomal trisomy was described by Patau and colleagues (1960). The prevalence of trisomy 13 is approximately 1 per 12,000 live births and 1 per 5000 recognized pregnancies, which includes abortuses and stillbirths (Dolk, 2010; Parker, 2010). As with trisomy 18, trisomy 13 is highly lethal, and most affected fetuses are lost between 10 weeks and term (see Fig. 13-2).
Approximately 80 percent of pregnancies with Patau syndrome result from trisomy 13. The remainder are caused by a robertsonian translocation involving chromosomes 13 and 14, der(13;14)(q10;q10). This translocation is the most common structural chromosomal rearrangement. It is carried by approximately 1 in 1300 individuals, although the risk of an affected liveborn infant is less than 2 percent (Nussbaum, 2007).
Clinical Findings. Trisomy 13 is associated with abnormalities of virtually every organ system. One characteristic finding is holoprosencephaly. This is present in approximately two thirds of cases and may be accompanied by microcephaly, hypotelorism, and nasal abnormalities that range from a single nostril to a proboscis (Solomon, 2010). Cardiac defects are found in up to 90 percent of fetuses with trisomy 13 (Shipp, 2002). Other abnormalities that suggest trisomy 13 include neural-tube defects—particularly cephalocele, microphthalmia, cleft lip- palate, omphalocele, cystic renal dysplasia, polydactyly, rockerbottom feet, and areas of skin aplasia (Lin, 2007). For the fetus or infant with a cephalocele, cystic kidneys, and polydactyly, the differential diagnosis includes trisomy 13 and the autosomal-recessive Meckel-Gruber syndrome, which is lethal. Sonographic images of several of these abnormalities are shown in Chapter 10 (p. 201).
Few trisomy 13 fetuses survive until birth. Of those that do, the 1-week survival is approximately 40 percent, and 1-year survival is only about 3 percent (Tennant, 2010; Vendola, 2010). Counseling regarding prenatal diagnosis and management options is similar to that described with trisomy 18.
For the mother, trisomy 13 is the only aneuploidy linked with an increased risk for preeclampsia. Hyperplacentosis and preeclampsia develop in up to half of pregnancies with trisomy 13 carried beyond the second trimester (Tuohy, 1992). Chromosome 13 contains the gene for soluble fms-like tyrosine kinase-1, called sFlt-1, which is an antiangiogenic protein associated with preeclampsia. Investigators have documented overexpression of the sflt-1 protein by trisomic 13 placentas and in serum of women with preeclampsia (Bdolah, 2006; Silasi, 2011). The role of antiangiogenic growth factors in the etiopathogenesis of preeclampsia is discussed in Chapter 40 (p. 735).
Other Trisomies. In the absence of mosaicism, which is discussed below, it is rare for other autosomal trisomies to result in a live birth. There are case reports of live births with trisomy 9 and with trisomy 22 (Kannan, 2009; Tinkle, 2003). Trisomy 16 is the most common trisomy found with first-trimester losses, accounting for 16 percent, but it is not identified later in gestation. Trisomy 1 has never been reported.
Nondisjunction creates an equal number of nullisomic and disomic gametes. As a rule, missing chromosomal material is more devastating than having extra chromosomal material, and almost all monosomic conceptuses are lost before implantation. The one exception is monosomy for the X chromosome, Turner syndrome, which is discussed subsequently. Despite the strong association between maternal age and trisomy, there is no association between maternal age and monosomy (see Fig. 13-1).
This is an abnormal number of complete haploid chromosomal sets. Polyploidy accounts for approximately 20 percent of spontaneous abortions but is rarely encountered later in gestation.
Triploid pregnancies have three haploid sets or 69 chromosomes. To have three haploid chromosomal sets, one parent must contribute two sets, and the phenotypic presentation differs according to the parent of origin. In diandric triploidy, also known as type I triploidy, the extra chromosomal set is paternal, resulting from fertilization of one egg by two sperm or by a single diploid—and thus abnormal—sperm. Diandric triploidy produces a partial molar pregnancy, which is discussed in Chapter 20 (p. 396). Diandric triploidy accounts for most triploid conceptions, but the first-trimester loss rate is extremely high. As a result, two thirds of triploid pregnancies identified beyond the first trimester are caused instead by digynic triploidy (Jauniaux 1999). In a digynic triploid pregnancy, also known as type II triploidy, the extra chromosomal set is maternal, and the egg fails to undergo the first or second meiotic division before fertilization. Digynic triploid placentas do not develop molar changes. However, the fetus often displays asymmetric growth restriction (Jauniaux, 1999).
Triploidy is a lethal aneuploidy, and more than 90 percent of fetuses with either the diandric or digynic form have multiple structural anomalies. These include abnormalities of the central nervous system, heart, face, and extremities, as well as severe growth restriction (Jauniaux, 1999). Counseling, prenatal diagnosis, and delivery options are similar to those for trisomies 18 and 13. The recurrence risk for a woman whose triploid fetus survived past the first trimester is 1 to 1.5 percent, and thus prenatal diagnosis is offered in future pregnancies (Gardner, 1996).
Tetraploid pregnancies have 4 haploid sets or 92 chromosomes. Four sets of chromosomes results in either 92,XXXX or 92,XXYY. This suggests a postzygotic failure to complete an early cleavage division. The conceptus invariably succumbs, and the recurrence risk is minimal.
Sex Chromosome Abnormalities
45,X—Turner Syndrome. This is the only monosomy compatible with life. However, it is also the most common aneuploidy in abortuses and accounts for 20 percent of first-trimester losses. The prevalence of Turner syndrome is approximately 1 per 5000 live births or 1 per 2500 girls (Cragan, 2009; Dolk, 2010). The missing X chromosome is paternally derived in 80 percent of cases (Cockwell, 1991; Hassold, 1991).
Monosomy X encompasses three distinct phenotypes. Approximately 98 percent of these conceptuses are so abnormal that they abort early in the first trimester. In a second group, large cystic hygromas are identified in either the first or second trimester, frequently accompanied by hydrops (Fig. 10-16, p. 206 and Chap. 15, p. 315). In such cases, fetal demise almost invariably results. Only the third and least-common phenotype has the potential for postnatal survival. Affected fetuses may have small cystic hygromas visible in the first or second trimester, which do not result in hydrops, and they often have other major abnormalities. One reason for the wide range of Turner syndrome phenotype is that only half of liveborn infants actually have monosomy X. Approximately one fourth have mosaicism, such as 45,X/46,XX or 45,X/46,XY. Another 15 percent have isochromosome X, that is, 46,X,i(Xq)(Milunsky, 2004; Nussbaum, 2007).
Abnormalities associated with Turner syndrome include a major cardiac malformation—such as coarctation of the aorta or bicuspid aortic valve—in 30 to 50 percent; renal anomalies, particularly horseshoe kidney; and hypothyroidism. Other features include short stature, broad chest with widely spaced nipples, congenital lymphedema, webbed posterior neck (resulting from cystic hygromas), and minor bone and cartilage abnormalities. Intelligence is generally in the normal range, although affected individuals are more likely to have visual-spatial organization deficits and difficulties with nonverbal problem solving and interpretation of social cues (Jones, 2006). Growth hormone is typically administered in childhood to ameliorate short stature (Kappelgaard, 2011). More than 90 percent have ovarian dysgenesis and require estrogen repletion beginning just before adolescence. An exception is if a mosaicism involves a Y chromosome. Such cases are at risk for germ cell neoplasm—regardless of whether the child is phenotypically male or female, and eventual prophylactic bilateral gonadectomy is indicated (Cools, 2011; Schorge, 2012).
47,XXX. Approximately 1 in 1000 female infants has an additional X chromosome—47,XXX. The extra X is maternally derived in more than 90 percent of cases (Milunsky, 2004). Pubertal development and fertility are usually normal, although premature ovarian failure has been reported (Holland, 2001). Tall stature is common. The overall major malformation rate is not increased with 47,XXX. That said, atypical phenotypic features have been described in some individuals and include epicanthal folds, clinodactyly, hypotonia, genitourinary problems, and seizure disorders (Tartaglia, 2010). Attention deficit disorder and delays in language development and motor skills have also been reported (Linden, 2002). It is estimated that because of the variability in presentation and subtlety of abnormal findings, only 10 percent of affected children are ascertained clinically.
Females with two or more extra X chromosomes—48,XXXX or 49,XXXXX—are likely to have physical abnormalities apparent at birth. These abnormal X complements are associated with varying degrees of mental retardation. For both males and females, the IQ score is lower with each additional X chromosome.
47,XXY—Klinefelter Syndrome. This is the most common sex chromosome abnormality. It occurs in approximately 1 per 600 male infants. The additional X chromosome is maternally or paternally derived with equal propensity (Jacobs, 1995; Lowe, 2001). There is also a slight association with either advanced maternal age or advanced paternal age (Milunsky, 2004).
Infants with XXY appear phenotypically normal and usually do not have an increased incidence of anomalies. As children, boys are typically tall and have normal prepubertal development. However, they have gonadal dysgenesis, do not undergo normal virilization, and require testosterone supplementation beginning in adolescence. They may develop gynecomastia. In general, IQ scores are within the normal range but slightly below those of siblings, and delays in speech, reading, and motor skills are not uncommon (Girardin, 2011).
47,XYY. This aneuploidy occurs in approximately 1 in 1000 male infants. There is no association with paternal age, anomaly rates are not increased, and there are no unusual phenotypic features. These boys tend to be tall, they have normal puberty, and fertility is unimpaired. They are at increased risk for oral and written language impairments, but intelligence is generally normal (Ross, 2009). A commonly held misconception was that XYY karyotype was associated with criminal or violent behavior. However, these early reports have been refuted.
Males with more than two Y chromosomes—48,XYYY—or with both additional X and Y chromosomes—48,XXYY or 49,XXXYY—have obvious physical abnormalities and significant mental retardation.
Abnormalities of Chromosome Structure
Structural chromosomal abnormalities include deletions, duplications, translocations, isochromosomes, inversions, ring chromosomes, and mosaicism (see Table 13-1). Their overall birth prevalence is approximately 0.3 percent (Nussbaum, 2007). Identification of a structural chromosomal abnormality raises two primary questions. First, what phenotypic abnormalities or later developmental abnormalities are associated with this finding? Second, is evaluation of parental karyotype indicated—specifically, are the parents at increased risk to carry this abnormality? If so, what is their risk to have future affected offspring?
Deletions and Duplications
A chromosomal deletion indicates that a portion of a chromosome is missing, and a duplication means that a portion has been included twice. Deletions involving DNA segments large enough to be seen with standard cytogenetic karyotyping are identified in approximately 1 per 7000 births (Nussbaum, 2007). Common deletions may be referred to by eponyms—for example, del 5p is called cri du chat syndrome.
Most deletions and duplications occur during meiosis and result from malalignment or mismatching during the pairing of homologous chromosomes. When this happens, the misaligned segment may be deleted (Fig. 13-6). Or, if the mismatch remains when the two chromosomes recombine, it may result in a deletion in one chromosome and a duplication in the other. When a deletion or duplication is identified in a fetus or infant, the parents should be offered karyotyping to determine if either carries a balanced translocation—as this would significantly increase the recurrence risk.
FIGURE 13-6 A mismatch during pairing of homologous chromosomes may lead to a deletion in one chromosome and a duplication in the other. del = deletion; dup = duplication.
Microdeletion Syndromes. A chromosomal deletion smaller than 3 million base pairs may not be detectable with standard karyotyping. Termed microdeletions, these may require molecular cytogenetic techniques for identification. Despite the relatively small size, a microdeletion may involve a stretch of DNA that contains multiple genes—causing a contiguous gene syndrome, which can include serious but unrelated phenotypic abnormalities (Schmickel, 1986). When a specific microdeletion syndrome is suspected, it is usually confirmed using fluorescence in situ hybridization (p. 276). Examples of common microdeletion syndromes are listed in Table 13-3.
TABLE 13-3. Some Microdeletion Syndromes Detectable by Fluorescence In Situ Hybridization (FISH)
The region of DNA that is deleted in a microdeletion syndrome (or duplicated in a microduplication) is termed a genomic copy number variant when applied to chromosomal microarray analysis discussed on page 277. Use of array-based technology has identified copy number variants that result in previously uncharacterized microdeletion syndromes—including single gene and intragenic deletions (Mikhail, 2011; Schwartz, 2012). It is likely that continued expansion of this technology will dramatically advance our knowledge of the genetic basis of disease.
22q11 Microdeletion Syndrome. This syndrome is also known as DiGeorge syndrome, Shprintzen syndrome, and velocardiofacial syndrome. It is the most common microdeletion, with prevalence of 1 per 2000 to 7000 births (Shprintzen, 2008). Although it is inherited in an autosomal dominant fashion, most cases arise from de novo mutations. The full deletion includes 3 million base pairs, encompasses 40 genes, and may include 180 different features—thus posing some counseling challenges (Shprintzen, 2008). It was once thought that different constellations of features characterized the DiGeorge and Shprintzen phenotypes, but it is now accepted that they represent the same microdeletion (McDonald-McGinn, 2011).
Associated abnormalities include conotruncal cardiac anomalies in more than 75 percent of affected individuals, such as tetralogy of Fallot, pulmonary atresia, truncus arteriosus, interrupted aortic arch, and ventricular septal defects. Immune deficiency, such as T-cell lymphopenia, also develops in approximately 75 percent. More than 70 percent have velopharyngeal insufficiency or cleft palate. Other manifestations include learning disabilities and mental retardation, hypocalcemia, renal anomalies, esophageal dysmotility, hearing loss, behavioral disorders, and psychiatric illness. Short palpebral fissures, bulbous nose tip, micrognathia, short philtrum, and small or posteriorly rotated ears are characteristic facial features (McDonald-McGinn, 2011).
Microduplication Syndromes. These syndromes are caused by duplication of DNA regions smaller than 3 million base pairs. In some cases, a microduplication may involve the exact DNA region that causes a recognized microdeletion syndrome. Examples of these include the velocardiofacial, Smith-Magenis, and Williams-Beuren syndromes (Hassed, 2004; Potocki, 2000; Somerville, 2005).
These are DNA rearrangements in which a segment of DNA breaks away from one chromosome and attaches to another. The rearranged chromosomes are called derivative (der) chromosomes. There are two types—reciprocal and robertsonian translocations.
Reciprocal Translocations. A double-segment or reciprocal translocation develops when there are breaks in two different chromosomes and the broken fragments are exchanged, so that each affected chromosome contains a fragment of the other. If no chromosomal material is gained or lost in this process, the translocation is considered balanced. The prevalence of reciprocal translocations is approximately 1 per 600 births (Nussbaum, 2007). Although transposition of chromosomal segments can cause abnormalities—due to repositioning of specific genes—the balanced carrier is usually phenotypically normal. The risk of a major structural or developmental abnormality in an apparent balanced translocation carrier is approximately 6 percent. Interestingly, using microarray-based studies, as many as 20 percent of individuals who would otherwise appear to have a balanced translocation may have missing or redundant DNA segments that are below the resolution of a standard karyotype (Manning, 2010).
Balanced translocation carriers are at risk to produce unbalanced gametes that result in abnormal offspring. As shown in Figure 13-7, if an oocyte or sperm contains a translocated chromosome, fertilization results in an unbalanced translocation—monosomy for part of one affected chromosome and trisomy for part of the other. The observed risk of a specific translocation can often be estimated by a genetic counselor. In general, translocation carriers identified after the birth of an abnormal child have a 5- to 30-percent risk of having liveborn offspring with unbalanced chromosomes. Carriers identified for other reasons, for example, during an infertility evaluation, have only a 5-percent risk. This is probably because the gametes are so abnormal that conceptions are nonviable.
FIGURE 13-7 A carrier of a balanced translocation may produce offspring who are also carriers of the balanced rearrangement (B), offspring with unbalanced translocations (C, D), or offspring with normal chromosomal complements (A).
Robertsonian Translocations. These involve only acrocentric chromosomes, which are chromosomes 13, 14, 15, 21, and 22. In an acrocentric chromosome, the p arm is extremely short. In a robertsonian translocation, the q arms of two acrocentric chromosomes fuse at one centromere to form a derivative chromosome. Also, one centromere and the p arms of each chromosome are lost. The p arms contain the satellite regions, which contain only genes coding for ribosomal RNA. As these are present in multiple copies on other acrocentric chromosomes, the translocation carrier is usually phenotypically normal. Because the number of centromeres determines the chromosome count, a robertsonian translocation carrier has only 45 chromosomes.
Robertsonian translocations are found in approximately 1 per 1000 newborns. Balanced carriers have reproductive difficulties for a number of reasons. If the fused chromosomes are homologous, from the same chromosome pair, the carrier can produce only unbalanced gametes. Each egg or sperm contains either both copies of the translocated chromosome, which would result in trisomy if fertilized, or no copy, which would result in monosomy. If the fused chromosomes are nonhomologous, four of the six possible gametes would be abnormal.
The most common robertsonian translocation is der(13;14)(q10;q10), which may result in Patau syndrome, discussed on page 263. The observed incidence of abnormal offspring is approximately 15 percent if a robertsonian translocation is carried by the mother and 2 percent if carried by the father. Robertsonian translocations are not a major cause of miscarriage and are found in fewer than 5 percent of couples with recurrent pregnancy loss. When a fetus or child is found to have a translocation trisomy, both parents should be offered karyotype analysis. If neither parent is a carrier, the recurrence risk is extremely low.
These abnormal chromosomes are composed of either two q arms or two p arms of one chromosome fused together. Isochromosomes are thought to arise when the centromere breaks transversely instead of longitudinally during meiosis II or mitosis. They can also result from a meiotic error in a chromosome with a robertsonian translocation. An isochromosome containing the q arms of an acrocentric chromosome behaves like a homologous robertsonian translocation, and such a carrier can produce only abnormal unbalanced gametes. When an isochromosome involves nonacrocentric chromosomes, with p arms containing functional genetic material, the fusion and abnormal centromere break results in two isochromosomes. One is composed of both p arms, and one is composed of both q arms. It is likely that one of these isochromosomes would be lost during cell division, resulting in the deletion of all the genes located on the lost arm. Thus, a carrier is usually phenotypically abnormal and produces abnormal gametes. The most common isochromosome involves the long arm of the X chromosome, i(Xq), which is the etiology of 15 percent of Turner syndrome cases.
When there are two breaks in the same chromosome, and the intervening genetic material is inverted before the breaks are repaired, the result is a chromosomal inversion. Although no genetic material is lost or duplicated, the rearrangement may alter gene function. There are two types—pericentric and paracentric.
Pericentric Inversion. If there are breaks in both the p and q arms of a chromosome, such that the inverted material includes the centromere, the inversion is pericentric (Fig. 13-8). This causes problems in chromosomal alignment during meiosis and confers significant risk for the carrier to produce abnormal gametes and abnormal offspring. In general, the observed risk of abnormal offspring in a pericentric inversion carrier is 5 to 10 percent if ascertainment was made after the birth of an abnormal child. However, the risk is only 1 to 3 percent if prompted by another indication. An important exception is a pericentric inversion on chromosome 9—inv(9)(p11q12), which is a normal variant present in approximately 1 percent of individuals.
FIGURE 13-8 Mechanism of meiosis in the setting of either pericentric inversion (one involving the centromere) or paracentric inversion (not involving the centromere). Individuals with pericentric inversions are at increased risk to produce offspring with a duplication/deletion. Those with paracentric inversions are at increased risk for early pregnancy loss.
Paracentric Inversion. If there are two breaks within one arm of a chromosome, and the inverted material does not include the centromere, the inversion is paracentric (see Fig. 13-8). The carrier makes either normal balanced gametes or gametes that are so abnormal as to preclude fertilization. Thus, although infertility may be a problem, the risk of having an abnormal offspring is extremely low.
If deletions occur at both ends of the same chromosome, the ends may come together to form a ring chromosome. The regions at the end of each chromosome are called telomeres and contain specialized nucleoprotein complexes that stabilize chromosomes. If only telomeres are lost, all necessary genetic material is retained, so the carrier is essentially balanced. With deletions extending more proximally than the telomeres, the carrier is likely to be phenotypically abnormal. An example of this is a ring X chromosome, which may result in Turner syndrome.
Ring chromosome carriers have reproductive difficulties. The ring prevents normal chromosome alignment during meiosis and thus produces abnormal gametes. It also disrupts cell division, which may cause abnormal tissue growth and lead to short stature, borderline to moderate mental deficiency, and minor dysmorphisms. A ring chromosome may form de novo or may be inherited from a carrier parent. Parent-to-child transmission is always maternal, possibly because of compromised spermatogenesis.
A mosaic individual has two or more cytogenetically distinct cell lines that are derived from a single zygote. Phenotypic expression of mosaicism depends on several factors, including whether the cytogenetically abnormal cells involve the placenta, the fetus, part of the fetus, or some combination. For example, mosaicism found in cells from amnionic fluid culture does not always reflect the fetal chromosome complement. The different levels of mosaicism and their clinical significance are presented in Table 13-4. When the abnormal cells are present in only a single flask of amnionic fluid, the finding is likely pseudomosaicism, caused by cell-culture artifact (Bui, 1984; Hsu, 1984). When abnormal cells involve multiple cultures, however, true mosaicism is more likely, and further testing of fetal blood or skin fibroblasts may be warranted. A second cell line is verified in 60 to 70 percent of these fetuses (Hsu, 1984; Worton, 1984).
TABLE 13-4. Types of Mosaicism Encountered in Amnionic Fluid Cultures
Confined Placental Mosaicism. According to studies of chorionic villus sampling (CVS), 2 percent of placentas are mosaic, even though the associated fetus is usually normal (Henderson, 1996). The mechanism underlying confined placental mosaicism may be either mitotic nondisjunction or partial correction of a meiotic error, and the mechanism appears to be chromosome-specific (Robinson, 1997). Fifteen to 20 percent of cases are associated with an adverse pregnancy outcome, such as miscarriage, fetal-growth restriction, or stillbirth (Reddy, 2009).
Fetal-growth restriction from placental mosaicism may arise in one of two ways. If the placenta has a population of aneuploid cells, impaired placental function may affect the growth of a cytogenetically normal fetus (Kalousek, 1983). Alternatively, if the fetus receives two otherwise normal copies of one chromosome, but both copies are from the same parent—uniparental disomy, then abnormal growth may result (p. 273).
In some cases, survival of cytogenetically abnormal fetuses may be due to placental mosaicism. Examples are trisomy 13 and 18 fetuses, who survive to term because of early “trisomic correction” in some cells that become trophoblasts (Kalousek, 1989).
Gonadal Mosaicism. Mosaicism confined to the gonads likely arises from a mitotic error in cells destined to become the gonad and results in a population of abnormal germ cells. Because spermatogonia and oogonia divide throughout fetal life, and spermatogonia continue to divide throughout adulthood, gonadal mosaicism may also follow a meiotic error in previously normal germ cells. Gonadal mosaicism may explain de novo autosomal dominant mutations in the offspring of normal parents. It may cause autosomal dominant diseases such as achondroplasia and osteogenesis imperfecta, as well as X-linked diseases such as Duchenne muscular dystrophy. Gonadal mosaicism may also explain the recurrence of such diseases in more than one child in a previously unaffected family. The potential for gonadal mosaicism explains the 6-percent recurrence risk after the birth of a child with a disease caused by a “new” mutation.
MODES OF INHERITANCE
Monogenic (Mendelian) Inheritance
A monogenic disorder is caused by a mutation or alteration in a single locus or gene in one or both members of a gene pair. Monogenic disorders are also called mendelian to signify that their transmission follows the laws of inheritance proposed by Gregor Mendel. Types of mendelian inheritance include autosomal dominant, autosomal recessive, X-linked, and Y-linked. Other monogenic patterns of inheritance are mitochondrial inheritance, uniparental disomy, imprinting, and trinucleotide repeat expansion, which is also termed anticipation. By age 25, approximately 0.4 percent of the population exhibits an abnormality attributed to a monogenic disorder, and 2 percent will have at least one such disorder during their lifetime. Some common single-gene disorders are listed in Table 13-5.
TABLE 13-5. Some Common Single-Gene Disorders
Acute intermittent porphyria
Adult polycystic kidney disease
Antithrombin III deficiency
BRCA1 and BRCA2 breast and/or ovarian cancer
Familial adenomatous polyposis
Hereditary hemorrhagic telangiectasia
Hypertrophic obstructive cardiomyopathy
Long QT syndrome
Neurofibromatosis type 1 and 2
von Willebrand disease
Congenital adrenal hyperplasia
Androgen insensitivity syndrome
Chronic granulomatous disease
Fragile X syndrome
Hemophilia A and B
Muscular dystrophy—Duchenne and Becker
Ocular albinism type 1 and 2
Relationship between Phenotype and Genotype
When considering inheritance, it is the phenotype that is dominant or recessive, not the genotype. With a dominant disease, the normal gene may direct the production of normal protein, but the phenotype is abnormal because it is determined by protein produced by the abnormal gene. With a recessive disease, a heterozygous carrier may produce detectable levels of an abnormal gene product but have no features of the condition because the phenotype is directed by the product of the normal co-gene. For example, erythrocytes from carriers of sickle-cell anemia contain approximately 30 percent hemoglobin S, but because the other 70 percent is hemoglobin A, these cells do not sickle in vitro.
Heterogeneity. Genetic heterogeneity explains how different genetic mechanisms can result in the same phenotype. Locus heterogeneity indicates that a specific disease phenotype can be caused by mutations in different genetic loci. It also explains why some diseases appear to follow more than one type of inheritance. An example is retinitis pigmentosa, which may develop following mutations in at least 35 different genes or loci and may result in autosomal dominant, autosomal recessive, or X-linked forms.
Allelic heterogeneity describes how different mutations of the same gene may affect presentation of a particular disease. For example, although only one gene has been associated with cystic fibrosis—the cystic fibrosis conductance transmembrane regulator gene (CFTR)—more than 1000 mutations in this gene have been described and result in varying disease severity. This is discussed in Chapter 14 (p. 295).
Phenotypic heterogeneity explains how different disease states can arise from different mutations in the same gene. For example, mutations in the fibroblast growth factor receptor 3 (FGFR3) gene may result in several different skeletal disorders, including achondroplasia and thanatophoric dysplasia, both of which are discussed in Chapter 10 (p. 217).
Autosomal Dominant Inheritance
If only one member of a gene pair determines the phenotype, that gene is considered to be dominant. Carriers have a 50-percent chance of passing on the affected gene with each conception. A gene with a dominant mutation generally specifies the phenotype in preference to the normal gene. That said, not all individuals will necessarily manifest an autosomal dominant condition the same way. Factors that affect the phenotype of an autosomal dominant condition include penetrance, expressivity, and occasionally, presence of codominant genes.
Penetrance. This term describes whether or not a dominant gene is expressed at all. A gene with recognizable phenotypic expression in all individuals has 100-percent penetrance. If some carriers express the gene but some do not, then penetrance is incomplete. This is quantitatively expressed by the ratio of those individuals with any phenotypic characteristics of the gene to the total number of gene carriers. For example, a gene that is expressed in some way in 80 percent of individuals who have that gene is 80-percent penetrant. Incomplete penetrance explains why some autosomal dominant diseases appear to “skip” generations.
Expressivity. Individuals with the same autosomal dominant trait—even within the same family—may manifest the condition differently. Genes with such variable expressivity can produce disease manifestations from mild to very severe. Examples include neurofibromatosis, tuberous sclerosis, and adult polycystic kidney disease.
Codominant Genes. If two different alleles in a gene pair are both expressed in the phenotype, they are considered to be codominant. Blood type, for example, is determined by expression of dominant A and B red-cell antigens that can be expressed simultaneously. Another example is the group of genes responsible for hemoglobin production. An individual with one gene directing production of hemoglobin S and the other directing production of hemoglobin C will produce both S and C hemoglobin.
Advanced Paternal Age. Paternal age older than 40 is associated with increased risk for spontaneous genetic mutations, particularly single base substitutions. This may result in offspring with new autosomal dominant disorders or X-linked carrier states. The risk is greater for some conditions than for others. In particular, advanced paternal age has been associated with mutations in the fibroblast growth factor receptor 2 (FGFR2) gene, which may cause craniosynostosis syndromes such as Apert, Crouzon, and Pfeiffer syndromes; mutations in the FGFR3 gene, which may result in achondroplasia and thanatophoric dysplasia; and mutations in the RET proto-oncogene, which may cause multiple endocrine neoplasia syndromes (Jung, 2003; Toriello, 2008). Because these disorders are uncommon, the actual risk for any individual condition is low.
Advanced paternal age has also been associated with a slightly increased risk for Down syndrome and for isolated structural abnormalities (Grewal, 2011; Toriello, 2008; Yang, 2007). It is not generally considered to pose an increased risk for other aneuploidies, probably because the aneuploid sperm cannot fertilize an egg.
Autosomal Recessive Inheritance
A recessive trait is expressed only when both copies of the gene function in the same way. Thus, autosomal recessive diseases develop only when both gene copies are abnormal. Heterozygous carriers are usually undetectable clinically but may have biochemical test abnormalities. Many enzyme deficiency diseases display autosomal recessive inheritance, and enzyme activity in the carrier is approximately half of normal. Although this reduction usually does not cause clinical disease, it provides a phenotypic alteration that can be used for carrier screening. Other recessive conditions can be identified only by molecular genetic testing.
Unless carriers are screened for a specific disease, such as cystic fibrosis, they usually are recognized only after the birth of an affected child or the diagnosis of an affected family member (Chap. 14, p. 295). If a couple has a child with an autosomal recessive disease, the recurrence risk is 25 percent for each subsequent pregnancy. Thus, 1/4 of offspring will be homozygous normal, 2/4 will be heterozygous carriers, and 1/4 will be homozygous abnormal. In other words, three of four children will be phenotypically normal, and 2/3 of phenotypically normal siblings are actually carriers.
A heterozygous carrier of a recessive condition is only at risk to have affected children if his or her partner is heterozygous or homozygous for the disease. Genes for rare autosomal recessive conditions have low prevalence in the general population. Thus, the likelihood that a partner will be a gene carrier is low—unless there is consanguinity or the partner is a member of an at-risk group (American College of Obstetricians and Gynecologists, 2009b). This is discussed further in Chapter 14 (p. 294).
Inborn Errors of Metabolism. Most of these autosomal recessive diseases result from absence of a crucial enzyme, leading to incomplete metabolism of proteins, lipids, or carbohydrates. The metabolic intermediates that build up are toxic to a variety of tissues and may result in mental retardation or other abnormalities.
Phenylketonuria. This classic example of an autosomal recessive disease is caused by mutations in the phenylalanine hydroxylase (PAH) gene. PAH is needed to metabolize phenylalanine to tyrosine, and homozygotes have diminished or absent enzyme activity. This leads to abnormally high levels of phenylalanine, resulting in progressive intellectual impairment, autism, seizures, motor deficits, and neuropsychological abnormalities (Blau, 2010). Also, because phenylalanine competitively inhibits tyrosine hydroxylase—which is essential for melanin production, affected individuals have hair, eye, and skin hypopigmentation.
Approximately 3000 reproductive-aged women in the United States have phenylketonuria (PKU). The carrier frequency is approximately 1 in 60, and the disease affects 1 in 10,000 to 15,000 white newborns (American College of Obstetricians and Gynecologists, 2009a). PKU is one of the few metabolic disorders for which there is treatment. Early diagnosis and limitation of dietary phenylalanine beginning in infancy are essential to prevent neurological damage. Accordingly, all states and many countries now mandate newborn screening for PKU, and approximately 100 cases per million births are identified worldwide. The special diet should be continued indefinitely, as those who abandon the phenylalanine-restricted diet have a significantly lower IQ and neuropsychological impairments (Blau, 2010).
Affected women who do not adhere to a phenylalanine-free diet are at risk to have otherwise normal (heterozygous) offspring who sustain in utero damage as a result of being exposed to toxic phenylalanine concentrations. Phenylalanine is actively transported to the fetus, and hyperphenylalaninemia increases the risk for miscarriage and for PKU embryopathy. This is characterized by mental retardation, microcephaly, seizures, growth impairment, and cardiac anomalies. Among women on unrestricted diets, the risk to have a child with mental retardation may exceed 90 percent, and as many as 1 in 8 children have cardiac defects (Lenke, 1980). The Maternal Phenylketonuria Collaborative Study, which included 572 pregnancies followed more than 18 years, reported that maintenance of serum phenylalanine levels between 160 and 360 μmol/L (2 to 6 mg/dL) significantly reduced the fetal abnormality risk (Koch, 2003; Platt, 2000). Women who achieved optimal phenylalanine levels before 10 weeks’ gestation had children with mean IQ scores in the normal range when assessed at age 6 to 7 years (Koch, 2003). Preconceptional counseling is recommended with a goal of maintaining an optimal phenylalanine concentration from 3 months before conception and continuing this throughout pregnancy (American College of Obstetricians and Gynecologists, 2009a).
Consanguinity. Two individuals are considered consanguineous if they have at least one recent ancestor in common. First-degree relatives share half of their genes, second-degree relatives share a fourth, and third-degree relatives—cousins—share one eighth. Because of the potential for shared deleterious genes, consanguinity confers an increased risk to have offspring with otherwise rare autosomal recessive diseases or multifactorial disorders. First cousins have a twofold increased risk—4 to 6 percent overall, in the absence of a family history of genetic disease.
Incest is defined as a sexual relationship between first-degree relatives such as parent-child or brother-sister and is universally illegal. Progeny of such unions carry the highest risk of abnormal outcomes, and up to 40 percent of offspring are abnormal as a result of recessive and multifactorial disorders (Freire-Maia, 1984; Nadiri, 1979).
X-Linked and Y–Linked Inheritance
Most X-linked diseases are recessive. Common examples include color blindness, hemophilia A and B, and Duchenne and Becker muscular dystrophy. When a woman carries a gene causing an X-linked recessive condition, each of her sons has a 50-percent risk of being affected, and each daughter has a 50-percent chance of being a carrier.
Males with an X-linked recessive gene are usually affected because they lack a second X chromosome to express the normal dominant gene. A male with an X-linked disease cannot have affected sons because they cannot receive his X chromosome. Women with an X-linked recessive gene are generally unaffected by the disease it causes. In some cases, however, the random inactivation of one X chromosome in each cell—termed lyonization—is skewed, and female carriers may have features of the condition. For example, approximately 10 percent of female carriers of hemophilia A display factor VIII levels less than 30 percent of normal, and a similar proportion of female hemophilia B carriers have factor IX levels less than 30 percent. With either type of hemophilia, the female carrier is at increased risk for abnormal bleeding at the time of delivery (Plug, 2006). Similarly, because female carriers of Duchenne or Becker muscular dystrophy are at increased risk for cardiomyopathy, periodic evaluation for cardiac dysfunction and neuromuscular disorders is recommended (American Academy of Pediatrics, 2005).
X-linked dominant disorders mainly affect females, because they tend to be lethal in males. Two examples are vitamin D-resistant rickets and incontinentia pigmenti. One exception is fragile X syndrome, which is discussed subsequently.
The prevalence of Y-linked chromosomal disorders is low. This chromosome carries genes important for sex determination and a variety of cellular functions related to spermatogenesis and bone development. Deletion of genes on the long arm of Y results in severe spermatogenic defects, whereas genes at the tip of the short arm are critical for chromosomal pairing during meiosis and for fertility.
Human cells contain hundreds of mitochondria, each with its own genome and associated replication system. Oocytes contain approximately 100,000, but sperm contain only about 100, and these are destroyed after fertilization. Each mitochondrion has multiple copies of a 16.5-kb circular DNA molecule that contains 37 genes. Mitochondrial DNA encodes peptides required for oxidative phosphorylation, as well as ribosomal and transfer RNAs.
Mitochondria are inherited exclusively from the mother. Thus, although males and females both can be affected by a mitochondrial disorder, transmission is only through the mother. When a cell replicates, mitochondrial DNA sorts randomly into each of the daughter cells, a process termed replicative segregation. A consequence of replicative segregation is that any mitochondrial mutation will be propagated randomly into the daughter cells. Because there are multiple copies of mitochondrial DNA in each cell, the mitochondrion may contain only normal or only abnormal DNA—homoplasmy, or it may contain both normal and abnormally mutated DNA—heteroplasmy. If a heteroplasmic oocyte is fertilized, the relative proportion of abnormal DNA may affect whether the individual manifests a given mitochondrial disease. It is not possible to predict the potential degree of heteroplasmy among offspring, and this poses a challenge for genetic counseling.
As of 2013, 28 mitochondrial diseases or conditions with known molecular basis were described in OMIM (Johns Hopkins University, 2013). Examples include myoclonic epilepsy with ragged red fibers (MERRF), Leber optic atrophy, Kearns-Sayre syndrome, Leigh syndrome, several forms of mitochondrial myopathy and cardiomyopathy, and susceptibility to both aminoglycoside-induced deafness and chloramphenicol toxicity. Even aging is considered a mitochondrial disease!
DNA Triplet Repeat Expansion—Anticipation
Mendel’s first law is that genes are passed unchanged from parent to progeny, and barring new mutations, this is true for many genes or traits. However, certain genes are unstable, and their size, and thus function, may be altered during parent-to-child transmission. This is manifested clinically by anticipation—a phenomenon in which disease symptoms seem to be more severe and to appear at an earlier age in each successive generation. Examples of some DNA triplet (trinucleotide) repeat diseases are shown in Table 13-6.
TABLE 13-6. Some Disorders Caused by DNA Triplet Repeat Expansion
Dentatorubral pallidoluysian atrophy
Fragile X syndrome
Kennedy disease—spinal bulbar muscular atrophy
Fragile X Syndrome. This is the most common inherited form of mental retardation and affects approximately 1 in 3600 males and 1 in 4000 to 6000 females (American College of Obstetricians and Gynecologists, 2010). Fragile X syndrome is caused by expansion of a repeated trinucleotide DNA segment—cytosine-guanine-guanine (CGG)—at chromosome Xq27. When the CGG repeat number reaches a critical size—the full mutation—the fragile X mental retardation 1 (FMR1) gene becomes methylated. Methylation inactivates the gene, which halts expression of FMR1 protein.
Although transmission of the syndrome is X-linked, both the sex of the affected individual and the number of CGG repeats determine whether offspring are affected and to what degree. Intellectual disability is generally more severe in males, in whom average IQ scores are 35 to 45 (Nelson, 1995). Affected individuals may have speech and language problems and attention deficit-hyperactivity disorder. Fragile X syndrome is also the most common known cause of autism. Associated phenotypic abnormalities become more prominent with age and include a narrow face with large jaw, prominent ears, connective tissue abnormalities, and macroorchidism in postpubertal males. Clinically, four groups have been described (American College of Obstetricians and Gynecologists, 2010):
• Full mutation—more than 200 repeats
• Premutation—55 to 200 repeats
• Intermediate—45 to 54 repeats
• Unaffected—fewer than 45 repeats
When a full mutation is present, males typically have significant cognitive and behavioral abnormalities and phenotypic features. In females, however, random X-inactivation results in variable expression, and the disability may be much less severe.
For individuals with a premutation, evaluation and counseling are more complex. A female with the fragile X premutation is at risk to have offspring with the full mutation. The likelihood of expansion to a critical full mutation depends on the current number of maternal repeats. The risk of a full mutation in an offspring is 5 percent or less if the CGG repeat number is below 70 but exceeds 95 percent with 100 to 200 CGG repeats (Nolin, 2003). Expansion is extremely unlikely in a male premutation carrier, but all of his daughters will carry the premutation. Among women with no risk factors, approximately 1 in 250 carries a fragile X premutation, and the risk is about 1 in 90 in those with a family history of mental retardation (Cronister, 2008). Premutation carriers may themselves experience significant health consequences. Males with the premutation are at increased risk for the fragile X tremor ataxia syndrome (FXTAS). Females are less likely to have FXTAS, although they have a 20-percent risk for fragile X-associated primary ovarian insufficiency.
The American College of Obstetricians and Gynecologists (2010) recommends testing for women with a family history of fragile X syndrome; individuals with unexplained mental retardation, developmental delay, or autism; and women with premature ovarian insufficiency. Prenatal diagnosis can be accomplished by amniocentesis or CVS (Chap. 14, p. 297). Specimens obtained by either can be used to accurately determine the CGG repeat number, although CVS may not accurately determine FMR1 gene methylation status. Thus, DNA-based molecular testing with Southern blot and polymerase chain reaction are preferred.
This occurs when both members of a chromosome pair are inherited from the same parent. Often, uniparental disomy does not have clinical consequences. However, if chromosomes 6, 7, 11, 14, or 15 are involved, offspring are at increased risk for an abnormality because of parent-of-origin differences in gene expression (Shaffer, 2001). Although several genetic mechanisms may cause uniparental disomy, the most common is trisomic rescue, shown in Figure 13-9. After a nondisjunction event produces a trisomic conceptus, one of the three homologues may be lost. This will result in uniparental disomy for that chromosome in approximately one third of cases.
FIGURE 13-9 Mechanism of uniparental disomy arising from trisomic “rescue.” A. In normal meiosis, one member of each pair of homologous chromosomes is inherited from each parent. B. If nondisjunction results in a trisomic conceptus, one homologue is sometimes lost. In a third of cases, loss of one homologue leads to uniparental disomy.
Isodisomy is the unique situation in which an individual receives two identical copies of one chromosome in a pair from one parent. This mechanism explains some cases of cystic fibrosis, in which only one parent is a carrier but the fetus inherits two copies of the same abnormal chromosome from that parent (Spence, 1988; Spotila, 1992). It also has been implicated in abnormal growth related to placental mosaicism.
A gene may be inherited in a transcriptionally silent state—inherited but not expressed—depending on whether it is inherited from the mother or father. The phenotype of the individual varies according to the parent of origin. Imprinting affects gene expression by epigenetic control, that is, gene activity regulation by modification of genetic structure other than alteration of the underlying nucleotide sequence. For example, methyl group addition may alter gene expression and thereby affect the phenotype without changing the genotype. Importantly, the effect may be reversed in a subsequent generation, because a female who inherits an imprinted gene from her father will pass it on in her oocytes with a maternal—rather than paternal—imprint, and vice versa.
Selected diseases that can involve imprinting are shown in Table 13-7. A useful example includes two very different diseases that may be caused by microdeletion, uniparental disomy, or imprinting for the 15q11-q13 DNA region. First, Prader–Willi syndrome is characterized by obesity and hyperphagia; short stature; small hands, feet, and external genitalia; and mild mental retardation. In more than 70 percent of cases, Prader-Willi syndrome is caused by microdeletion or disruption for the paternal 15q11.2-q13. The remaining cases are due to maternal uniparental disomy or due to maternal gene imprinting with the paternal gene inactivated.
TABLE 13-7. Some Disorders That Can Involve Imprinting
In contrast, Angelman syndrome includes severe mental retardation; normal stature and weight; absent speech; seizure disorder; ataxia and jerky arm movements; and paroxysms of inappropriate laughter. In approximately 70 percent of cases, Angelman syndrome is caused by microdeletion for the maternal 15q11.2-q13. In 2 percent, the syndrome is caused by paternal uniparental disomy, and another 2 to 3 percent are due to paternal gene imprinting with the maternal genes inactivated.
There are other examples of imprinting important to obstetrics. Complete hydatidiform mole, with a paternally derived diploid chromosomal complement, is characterized by abundant placental growth with no fetal structures (Chap. 20, p. 396). Conversely, an ovarian teratoma, with a maternally derived diploid chromosomal complement, is characterized by the growth of various fetal but no placental tissues (Porter, 1993). It thus appears that paternal genes are vital for placental development, maternal genes essential for fetal development, and that both are necessary for normal fetal development.
Traits or diseases are considered to have multifactorial inheritance if they are determined by the combination of multiple genes and environmental factors. Polygenic traits are determined by the combined effects of more than one gene. Most congenital and acquired conditions, as well as common traits, display multifactorial inheritance. Examples include malformations such as clefts and neural-tube defects, diseases such as diabetes and heart disease, and features or traits such as head size or height. Abnormalities that display multifactorial inheritance tend to recur in families, but not according to a mendelian pattern. If a couple has had a child with a multifactorial birth defect, their empiric risk to have another affected child is 3 to 5 percent. This risk declines exponentially with successively more distant relationships. Some characteristics of multifactorial conditions are shown in Table 13-8.
TABLE 13-8. Characteristics of Multifactorial Diseases
There is a genetic contribution:
No mendelian pattern of inheritance
No evidence of single-gene disorder
Nongenetic factors are also involved in disease causation:
Lack of penetrance despite predisposing genotype
Monozygotic twins may be discordant
Familial aggregation may be present:
Relatives are more likely to have disease-predisposing alleles
Expression more common among close relatives:
Becomes less common in less closely related relatives—fewer predisposing alleles
Greater concordance in monozygotic than dizygotic twins
Adapted from Nussbaum, 2007.
Multifactorial traits that have a normal distribution in the population are termed continuously variable. A measurement that is more than two standard deviations above or below the population mean is considered abnormal. Continuously variable traits tend to be less extreme in the offspring of affected individuals, because of the statistical principle of regression to the mean.
Threshold Traits. Some multifactorial traits do not appear until a threshold is exceeded. Genetic and environmental factors that create propensity or liability for the trait are themselves normally distributed, and only individuals at the extreme of the distribution exceed the threshold and exhibit the trait or defect. Phenotypic abnormality is thus an all-or-none phenomenon. Examples include cleft lip-palate and pyloric stenosis.
Certain threshold traits have a clear male or female predominance. If an individual of the less common gender has the characteristic or defect, the recurrence risk is greater in his or her offspring (Fig. 13-10). An example is pyloric stenosis, which is approximately four times more common in males (Krogh, 2012). A female with pyloric stenosis has likely inherited more predisposing genetic factors than are necessary to produce the defect in a male, and the recurrence risk for her children or siblings is thus higher than the expected 3 to 5 percent. Her male siblings or male offspring would have the highest liability because they not only will inherit more than the usual number of predisposing genes but also are the more susceptible gender.
FIGURE 13-10 Schematic example of a threshold trait, such as pyloric stenosis, which has a predilection for males. Each gender is normally distributed, but at the same threshold, more males than females will develop the condition.
The recurrence risk for threshold traits is also greater if the defect is severe. An example is that the recurrence risk after the birth of a child with bilateral cleft lip and palate is approximately 8 percent, but it is only about 4 percent following a child with unilateral cleft lip alone.
Cardiac Defects. Structural cardiac anomalies are the most common birth defects, with a birth prevalence of 8 per 1000. More than 100 genes believed to be involved in cardiovascular morphogenesis have been identified, including those directing production of various proteins, protein receptors, and transcription factors (Olson, 2006; Weismann, 2007).
The risk to have a child with a cardiac anomaly is approximately 5 to 6 percent if the mother has the defect and 2 to 3 percent if the father has the defect (Burn, 1998). Selected left-sided lesions, including hypoplastic left heart syndrome, coarctation of the aorta, and bicuspid aortic valve, may have recurrence risks four- to sixfold higher (Lin, 1988; Lupton, 2002; Nora, 1988). Observed recurrence risks for specific cardiac malformations are listed in Table 49-4 (p. 977).
Neural-Tube Defects. This is the second most common class of birth defects after cardiac anomalies. Their sonographic features and prenatal diagnosis are described in Chapters 10 (p. 201) and 14 (p. 283), respectively, and their prevention with folic acid is discussed in Chapter 9 (p. 181).
Neural-tube defects (NTDs) are classic examples of multifactorial inheritance. Their development may be influenced by hyperthermia, hyperglycemia, teratogen exposure, ethnicity, family history, fetal gender, and various genes. Selected risk factors are more strongly associated with specific NTD location. Hyperthermia has been associated with anencephaly risk; pregestational diabetes with cranial and cervical-thoracic defects; and valproic acid exposure with lumbosacral defects (Becerra, 1990; Hunter, 1984; Lindhout, 1992).
Almost 50 years ago, Hibbard and Smithells (1965) postulated that abnormal folate metabolism was responsible for many NTDs. For a woman with a prior affected child, the recurrence risk of 3 to 5 percent is decreased by at least 70 percent—and potentially by as much as 85 to 90 percent—with periconceptional folic acid supplementation at a dosage of 4 mg/day (Grosse, 2007; MRC Vitamin Study Research Group, 1991). However, most NTD cases do not occur in the setting of maternal folic acid deficiency, and it has become clear that the gene-nutrient interactions underlying folate-responsive NTDs are complex. The NTD risk may be affected by genetic variation in folate transport or accumulation, impaired folate utilization via secondary nutrient deficiencies such as vitamin B12 or choline deficiency, and genetic variation in activity of folate-dependent metabolic enzymes (Beaudin, 2009).
The two most common prenatal genetic tests, cytogenetic analysis and fluorescence in situ hybridization (FISH), are used primarily for aneuploidy detection. For the diagnosis of a specific disease in which the genetic basis is known, DNA-based tests are often employed, typically using polymerase chain reaction (PCR) for rapid amplification of DNA sequences. A new technology that has become clinically available is chromosomal microarray analysis (CMA), which allows the entire genome to be screened for differences in small DNA sequences that characterize genetic disease. Traditionally, each of the above tests has been performed on amnionic fluid or chorionic villi. Recently, however, attention has focused on use of cell-free fetal DNA found in the maternal circulation. A technique known as massively parallel sequencing has enabled researchers to identify trisomy 21 and other aneuploidies using cell-free fetal DNA from maternal blood, and there is potential for cell-free fetal DNA to be used to diagnose a wide range of genetic conditions in the future.
Any tissue containing dividing cells or cells that can be stimulated to divide is suitable for cytogenetic analysis. The dividing cells are arrested in metaphase, and their chromosomes are stained to reveal light and dark bands. The most commonly used technique is Giemsa staining, which yields the G-bands shown in Figure 13-3. Each chromosome has a unique banding pattern that permits its identification as well as detection of deleted, duplicated, or rearranged segments. The accuracy of cytogenetic analysis increases with the number of bands produced. High-resolution metaphase banding routinely yields 450 to 550 visible bands per haploid chromosome set. Banding of prophase chromosomes generally yields 850 bands.
Because only dividing cells can be evaluated, the rapidity with which results are obtained correlates with the rapidity of cell growth in culture. Fetal blood cells often produce results in 36 to 48 hours. Amnionic fluid, which contains epithelial cells, gastrointestinal mucosal cells, and amniocytes, usually yields results in 7 to 10 days. If fetal skin fibroblasts are evaluated postmortem, stimulation of cell growth can be more difficult, and cytogenetic analysis may take 2 to 3 weeks.
Fluorescence In Situ Hybridization
This tool provides a rapid method for determining numerical changes of selected chromosomes and confirming the presence or absence of a specific gene or DNA sequence. FISH is particularly useful for the rapid identification of a specific aneuploidy and for verification of suspected microdeletion or duplication syndromes. Speed is important in some instances because these findings may alter pregnancy management.
To perform FISH, cells are fixed onto a glass slide, and fluorescent-labeled chromosome or gene probes are hybridized to the fixed chromosomes, as shown in Figures 13-11 and 13-12. Each probe is a DNA sequence that is complementary to a unique region of the chromosome or gene being investigated. If the DNA sequence of interest is present, hybridization is detected as a bright signal, visible by microscopy. The number of signals indicates the number of chromosomes or genes of that type in the cell being analyzed. Findings are probe-specific. Thus, FISH does not provide information on the entire chromosomal complement but merely the chromosomal or gene region of interest.
FIGURE 13-11 Steps in fluorescence in situ hybridization (FISH).
FIGURE 13-12 Interphase fluorescence in situ hybridization (FISH) using α-satellite probes for chromosomes 18, X, and Y. In this case, the three light blue signals, two green signals, and absence of red signals indicate that this is a female fetus with trisomy 18. (Image contributed by Dr. Frederick Elder.)
The most common prenatal application of FISH involves testing interphase chromosomes with DNA sequences specific to chromosomes 21, 18, 13, X, and Y. Shown in Figure 13-12 is an example of interphase FISH using α-satellite probes for chromosomes 18, X, and Y to confirm trisomy 18. In a review of more than 45,000 cases, the concordance between FISH analysis and standard cytogenetic karyotyping was 99.8 percent (Tepperberg, 2001). The American College of Medical Genetics (2000) recommends that clinical decision making based on FISH also incorporate consistent clinical information or confirmatory chromosomal analysis.
This technique, named after Edward Southern, allows identification of one or several DNA fragments of interest from among the million or so typically obtained by enzyme digestion of the entire genome. As illustrated in Figure 13-13, a restriction endonuclease enzyme digests the DNA, resulting in fragments that are then separated using agarose gel electrophoresis. This is followed by transfer to a nitrocellulose membrane that binds DNA. Probes homologous for the DNA segment of interest are then hybridized to the DNA bound to the membrane, using a marker that permits their identification. Basic principles of the Southern blot technique also can be applied to RNA—known as Northern blotting—and to proteins—Western blotting.
FIGURE 13-13 Southern blotting analysis. Genomic DNA is isolated from leukocytes or amniocytes and digested with a restriction enzyme. This procedure yields a series of reproducible fragments that are separated by agarose gel electrophoresis. The separated DNA fragments are then transferred (“blotted”) to a nitrocellulose membrane that binds DNA. The membrane is treated with a solution containing a radioactive single-stranded nucleic acid probe, which forms a double-stranded nucleic acid complex at membrane sites when homologous DNA is present. These regions are then detected by autoradiography.
Polymerase Chain Reaction
This tool enables the rapid synthesis of large amounts of a specific DNA sequence or gene. To do this, either the entire gene sequence must be known or the DNA sequences at the beginning and end of the gene must be known. PCR involves three steps that are repeated many times. First, double-stranded DNA is denatured by heating. Then, oligonucleotide primers corresponding to a target sequence on each separated DNA strand are added and become annealed to either end of the target sequence. Finally, a mixture of nucleotides and heat-stable DNA polymerase is added to elongate the primer sequence, and new complementary strands of DNA are synthesized. The procedure is repeated multiple times to permit exponential amplification of the DNA segment.
Real-time PCR is used to amplify a specific gene while simultaneously quantifying the target gene. This allows accurate quantification of gene expression. Massively parallel genomic sequencing or “shotgun sequencing” uses random small DNA fragments, which are amplified using millions of sequence tags. These small amplified sequences are computer-aligned, allowing the entire genome sequence to be ascertained. The number of unique sequences can then be counted and expressed as a percentage. This permits relative quantification of specific genes or, in the case of aneuploidy determination, chromosomes.
If a specific disease-causing gene has not been identified, then linkage analysis may be used to estimate the likelihood that an individual or fetus has inherited the abnormal trait. This technique is used to estimate the location of different genes and their approximate distance from each other.
Specific scattered markers are selected for study based on the suspected location of the gene responsible for the condition. DNA from each family member is analyzed to determine whether any of the selected markers have been transmitted along with the disease gene. If individuals with the disease have the marker but individuals without the disease do not, the gene causing the disease is said to be linked to that marker. This suggests that they lie close to each other on the same chromosome. Limitations of this technique are that it is imprecise, that it depends on family size and availability of family members for testing, and that it relies on the presence of informative markers near the gene.
Chromosomal Microarray Analysis
This testing uses the principles of PCR and nucleic acid hybridization to screen DNA for many different genes or mutations simultaneously. Doing so can identify deletions and duplications as small as 1 kilobase—termed genomic copy number variants—whereas the resolution of standard karyotyping is approximately 3 megabases. Two types of arrays are used clinically: (1) comparative genomic hybridization (CGH) arrays, which detect microdeletions and microduplications in DNA and (2) single-nucleotide polymorphism (SNP) arrays, in which the variation may involve just one nucleotide. As shown in Figure 13-14, the CGH microarray platform contains DNA fragments of known sequence. DNA from the individual (or fetus) to be tested is labeled with a fluorescent dye and then exposed to the DNA fragments fixed on the chip. Normal control DNA is labeled with a different fluorescent probe. Finally, the intensity of fluorescent probe signals is determined with a laser scanner. Use of SNP arrays is similar, except that the DNA is compared with known sequence variants, allowing determination of whether the fetus is heterozygous or homozygous for a mutation.
FIGURE 13-14 Chromosomal microarray analysis. A. Actual microarray chip size. B. Each chip contains thousands of cells (squares). C & D. Each cell contains thousands of identical oligonucleotides on its surface, and each cell is unique in its nucleotide content. E. During genetic analysis, a mixture containing tagged fetal DNA is presented to the chip. Complementary DNA sequences bind. F. If a laser is shined on the chip, DNA sequences that have bound will glow. This identifies a matching sequence. (From Doody, 2012, with permission.)
Arrays may be genome-wide or may be targeted to known genetic syndromes. Genome-wide arrays are used in research settings, for example, to identify novel microdeletion syndromes in individuals with intellectual disability (Slavotinek, 2008). In the prenatal setting, only targeted arrays are used. A major drawback of either type is the detection of copy number variants of uncertain clinical significance(Manning, 2010). When an abnormal pregnancy is found to have a variant that has not previously been associated with an abnormality, it may not be possible to determine whether the variant is benign or pathological.
CMA is used clinically for prenatal diagnosis and for stillbirth evaluation. The technique is expected to identify autosomal trisomies, sex chromosomal abnormalities, and other unbalanced chromosomal rearrangements visible with standard karyotype analysis. However, balanced chromosomal rearrangements such as translocations and inversions may not be identified. In addition, CGH arrays will not detect triploidy, mosaicism below 20 percent, or some marker chromosomes (Bui, 2011). A potential benefit of SNP arrays is the detection of triploidy. SNP arrays may also demonstrate loss of heterozygosity in a specimen, important for detection of uniparental disomy and consanguinity.
In pediatrics, CMA is considered a first-tier diagnostic test for children with mental retardation, congenital abnormalities, or dysmorphic features but with a normal karyotype. In such cases, CMA identifies an abnormality in up to 15 percent (Manning, 2010; Miller, 2010).
For stillbirth evaluation, CMA is more likely to provide a genetic diagnosis than standard karyotyping, in part because it does not require dividing cells. The Stillbirth Collaborative Research Network found that when karyotyping was uninformative, approximately 6 percent of cases had either aneuploidy or a pathogenic copy number variant identified with CMA (Reddy, 2012). Overall, CMA yielded results nearly 25 percent more often than standard karyotyping alone.
Targeted arrays are performed in the prenatal setting using chorionic villi or amnionic fluid. In a multicenter trial of more than 4000 pregnancies, Wapner and coworkers (2012) found that CMA identified all cases of common aneuploidies and unbalanced chromosomal rearrangements seen with standard karyotyping. When standard karyotyping results were normal, a microdeletion or microduplication of known or likely clinical significance was found in 6 percent if the indication was a fetal structural abnormality and in nearly 2 percent if performed for advanced maternal age or abnormal serum aneuploidy screening. Similar results have been reported by others (Hillman, 2011). Of concern is that variants of uncertain clinical significance are also identified in approximately 2 percent, which poses a challenge for counseling (Dugoff, 2012; Wapner, 2012). When copy number variants of uncertain clinical significance are encountered, parental testing is indicated.
Recently, the American College of Obstetricians and Gynecologists (2013) endorsed offering CMA testing when prenatal sonography identifies major fetal abnormalities. If the abnormalities suggest trisomy 21, 18, or 13, then karyotype analysis or FISH may be the initial test, and CMA can also be considered. Comprehensive genetic counseling is required before and after CMA testing. Also, depending on the platform used, it may need to include not only the potential to detect findings of uncertain clinical significance, but also adult-onset disease, consanguinity, and even non-paternity (American College of Obstetricians and Gynecologists, 2013).
Fetal DNA in the Maternal Circulation
Fetal cells are present in maternal blood at a very low concentration, only 2 to 6 cells per milliliter (Bianchi, 2006). And, some intact fetal cells may persist in the maternal circulation for decades following delivery. Persistent fetal cells may result in microchimerism, which has been implicated in maternal autoimmune diseases such as scleroderma, systemic lupus erythematosus, and Hashimoto thyroiditis. For prenatal diagnosis, the use of intact fetal cells is limited by their low concentration, persistence into successive pregnancies, and difficulties in distinguishing fetal from maternal cells. Cell-free fetal DNA overcomes these limitations.
Cell-Free Fetal DNA
This is released from apoptotic placental trophoblast—rather than actual fetal cells—and can be reliably detected in maternal blood after 7 weeks’ gestation (Bodurtha, 2012). It comprises 3 to 6 percent of the circulating cell-free DNA in maternal plasma, with proportions increasing as gestation advances (Lo, 1998). Unlike intact fetal cells, cell-free fetal DNA is cleared within minutes from maternal blood (Lo, 1999). In the research setting, cell-free fetal DNA has been used to detect numerous single-gene disorders through paternally inherited alleles. These include myotonic dystrophy, achondroplasia, Huntington disease, congenital adrenal hyperplasia, cystic fibrosis, and β-thalassemia (Wright, 2009). Clinical applications of cell-free fetal DNA include determination of Rh (CDE) genotype, determination of fetal gender, and detection of autosomal trisomies (Fig. 13-15).
FIGURE 13-15 Cell-free fetal DNA is actually derived from apoptotic trophoblast. The DNA is isolated from maternal plasma, and real-time quantitative PCR may be used to target specific regions or sequences. This may be used for Rh D genotyping, identification of paternally inherited single-gene disorders, or fetal sex determination. Using a technique called massively parallel genomic sequencing, screening for trisomies 21, 18, and 13 may be performed.
Rh D Genotype Evaluation. Fetal Rh D genotype assessment from maternal blood offers several potential benefits. Administration of anti-D immune globulin to an Rh D-negative pregnant woman carrying an Rh D-negative fetus can be eliminated. In the setting of Rh D alloimmunization, early identification of an Rh D-negative fetus can avoid unnecessary amniocentesis and/or serial fetal middle cerebral artery Doppler assessment. Rh D genotype evaluation using cell-free fetal DNA is done using real-time PCR to target multiple exons of the RHD gene. In a metaanalysis of more than 3000 pregnancies by Geifman-Holtzman and associates (2006), the average diagnostic accuracy approximated 95 percent, and only 3 percent of samples had inconclusive results. Subsequent studies have described accuracies of 99 to 100 percent (Minon, 2008; Tynan, 2011). Rh D genotyping using cell-free fetal DNA is used routinely in Europe. However, as of 2013, it has not been widely adopted in the United States. A theoretical concern is that women with false-negative test results would not receive anti-D immune globulin, leading to a potential increase in Rh D alloimmunization (Szczepura, 2011).
Fetal Sex Determination. From the standpoint of genetic disease, fetal sex determination may be clinically useful if the fetus is at risk for an X-linked disorder. It may also be beneficial if the fetus is at risk for congenital adrenal hyperplasia because maternal corticosteroid therapy may be avoided if the fetus is male. In a metaanalysis of more than 6000 pregnancies by Devaney and colleagues (2011), the sensitivity of cell-free fetal DNA testing for fetal sex determination approximated 95 percent between 7 and 12 weeks’ gestation, improving to 99 percent after 20 weeks. The test specificity was 99 percent at both time periods, suggesting that cell-free fetal DNA is a reasonable alternative to invasive testing in selected cases.
Aneuploidy Screening. Fetal Down syndrome and other autosomal trisomies can be detected from maternal plasma using massively parallel sequencing or by targeted (selective) sequencing of chromosome-specific regions (Chiu, 2008; Fan, 2008; Sparks, 2012). By simultaneously sequencing millions of DNA fragments, investigators can identify whether the proportion or ratio of fragments from one chromosome is higher than expected. Because sequences of fetal DNA are specific to individual chromosomes, samples from those with Down syndrome have a larger proportion of DNA sequences from chromosome 21. This technology has been termed noninvasive prenatal testing (NIPT).
Recent trials of NIPT in high-risk pregnancies have yielded detection rates for trisomies 21, 18, and 13 of approximately 98 percent, at a false-positive rate of 0.5 percent or less (American College of Obstetricians and Gynecologists, 2012; Bianchi, 2012; Palomaki, 2011, 2012). NIPT has recently become clinically available as a screening test, but it is not currently considered a replacement diagnostic test. Pretest counseling is recommended, with formal genetic counseling if an abnormal result is identified. Recommendations for its use are discussed in Chapter 14 (p. 292).
Limitations. There are several important limitations to using cell-free fetal DNA testing in its present form (Benn, 2012; Geifman-Holtzman, 2006). Because placental cells are being evaluated, confined placental mosaicism may yield abnormal results that do not reflect fetal karyotype. Similarly, results may not be as accurate in a multifetal gestation or with a vanishing twin (Chap. 45, p. 892). There may be false-negative results if fetal DNA levels are insufficient in the sample. This theoretically results in failure to detect an Rh-negative fetus. In the case of aneuploidy, there may be inability to differentiate trisomy from unbalanced translocation (Benn, 2012; Geifman-Holtzman, 2006). Finally, because the technology identifies differences in the relative proportion of chromosomal fragments, triploidy may not be identified.
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