Chromosomal Abnormalities

Robyn C. Reed, M.D., Ph.D.

Michelle M. Dolan, M.D.

Raj P. Kapur, M.D., Ph.D.

Joseph R. Siebert, Ph.D.

Advances in genetic testing have blurred the distinction between the cytogenetic and molecular classifications of disease, and with continuing evolution of laboratory methods, “genomic” disease is becoming the preferred term. The foundational study in cytogenetics, G-banding, is still widely used, but the development of increasingly sensitive methods for investigating genomic DNA, such as fluorescence in situ hybridization (FISH), array-based oligonucleotide comparative genomic hybridization (a-CGH), and single nucleotide polymorphism (SNP) arrays, has expanded the scope of cytogenetic testing to include changes too small to detect by karyotype (Table 3.1, Figure 3-1). Although newer genomic technologies such as next-generation sequencing now permit screening the entire genome to the level of the nucleotide, conventional and molecular cytogenetic techniques continue to play an important role in pediatric and surgical pathology. In this chapter, we focus on constitutional abnormalities, those conditions that are present from conception or at birth and that typically have significant clinicopathologic findings. Related topics not covered in this chapter include chromosomal rearrangements associated with pediatric neoplasms, single-gene mutations, and mutations in mitochondrial DNA. Cytogenetic changes characteristic of childhood tumors are introduced as part of the discussion of specific neoplasms in other chapters and have been the subject of several excellent reviews (1,2,3). For information about the mitochondrial genome and related diseases, the reader is referred to the review by Schapira (4). Because knowledge about cytogenetic abnormalities and their clinicopathologic correlations is growing rapidly, the most up-to-date information about many such abnormalities can be found in online resources and current publications; several excellent resources are listed in Table 3.2.

The evolution of genomic techniques has resulted in numerous diagnostic testing options, each with advantages and disadvantages. In complex cases, a team approach to diagnosis is helpful. Cytogeneticists, molecular geneticists, clinical geneticists, and genetic counselors can provide valuable assistance to clinicians and pathologists in establishing a differential diagnosis and selecting and interpreting an appropriate sequence of tests (5). Some common indications for cytogenetic testing in pediatric pathology are listed in Table 3.3.

CYTOGENETIC TECHNIQUES

Conventional Cytogenetic Analysis (G-Banding)

G-banding is performed on metaphase (dividing) cells and permits the evaluation of the entire chromosome complement. The need for metaphase cells requires that specimens be cultured for up to several days; this is a major contributing factor to the time- and labor-intensive nature of conventional cytogenetic testing. A variety of tissue types can be cultured to yield metaphase cells for analysis, including peripheral and cord blood, chorionic villi, amniotic fluid, bone marrow, lymph nodes, and solid tumors. Technical considerations for postmortem tissue sampling are listed in Table 3.4. Analyses of peripheral and cord blood, chorionic villi, and amniotic fluid are typically performed to identify and characterize constitutional abnormalities (i.e., those present at birth and typically found in every cell), whereas analyses of bone marrow, lymph nodes, and tumors are performed to identify and characterize abnormalities associated with neoplastic disorders. Although sampling the correct area of the specimen is most critical in the case of neoplastic disorders (in which only part of the sample may be involved), it is also important in some constitutional studies. For example, placental specimens may contain cells of both fetal origin (chorionic stroma, amniocytes) and maternal origin (decidualized endometrium). Imprecise sampling may therefore result in the preferential growth of maternal cells, rather than the fetal cells of interest.

The goal of tissue culture is to optimize the conditions of cell culture media, temperature, pH, and sterility to stimulate cells to proceed through the cell cycle to mitosis. After the cells are arrested in mitosis, they are harvested, fixed, and dropped onto glass slides. The slides are then treated with a proteolytic enzyme and stained with Wright-Giemsa stain, resulting in a series of alternating light and dark bands

(G-bands) characteristic of each of the 22 pairs of autosomes and 2 sex chromosomes. Darkly staining (positive) G-bands are rich in adenine (A) and thymine (T) residues and have relatively few genes, in contrast with lightly staining G-bands, which are rich in guanine (G) and cytosine (C) residues and are relatively gene rich.

TABLE 3.1 CYTOGENETIC METHODS

	Karyotype	Array CGH	SNP Array	FISH	Cell-Free Fetal DNA
Appropriate samples	Chorionic villi Amniocytes Cord blood lymphocytes Peripheral blood lymphocytes Skin or soft tissue fibroblast culture	Chorionic villi Amniocytes Cord blood lymphocytes Peripheral blood lymphocytes Skin or soft tissue fibroblast culture	Chorionic villi Amniocytes Cord blood lymphocytes Peripheral blood lymphocytes Skin or soft tissue fibroblast culture	Chorionic villi Amniocytes Cord blood lymphocytes Peripheral blood lymphocytes Skin or soft tissue fibroblast culture Formalin-fixed, paraffinembedded tissue Air-dried touch preps	Maternal peripheral blood
Turnaround time^a	1-2 wk	1-2 wk	1-2 wk	1-2 d	1-2 wk
Method	Stain prophase or metaphase chromosomes, and examine banding patterns	Hybridize patient and control DNA with probes (e.g., oligonucleotides) affixed to glass slide.	Hybridize patient DNA with DNA oligomers on microarray chip.	Hybridize fluorescently labeled DNA probes to patient DNA affixed to slide	Massively parallel DNA sequencing, computer analysis
Resolution limit^b	3-5 Mb	˜50-100 kb	50-100 kb	50-400 kb	Currently, whole chromosome (21, 18, 13, X, and Y), and additional select chromosomal regions.
Notes	Requires viable, mitotically active cells. If tissue is cultured (i.e., not a direct prep), may select for biased cell subsets. Best method for detecting balanced translocations and inversions	Sensitive method for detecting small duplications and deletions. Interpretation can be complicated by copy number variants (CNV). Testing parental DNA may help determine if a CNV is benign or disease causing	Sensitive method for detecting small duplications and deletions. Can detect uniparental isodisomy	Targeted method for detecting small duplications, deletions, and translocations. Must select probes directed to specific DNA sequences.	Screening test only. Results must be confirmed with analysis of chorionic villi, amniocytes, or cord or peripheral blood. False positives can occur with CPM or maternal mosaicism (e.g., 45,X/46,XX mosaic). False negatives can occur with confined fetal mosaicism
^aTurnaround times are approximate and dependent on a number of factors; it is critical to contact the laboratory for specific details. ^bLimit of resolution depends on band-level resolution (G-banding) and array design and configuration (microarray).

FIGURE 3-1 • Maximum resolution of cytogenetic and molecular laboratory methods. Ranges are approximate, and different laboratories may have different limits of detection and reportable ranges.

G-banding enables the detection across the entire genome of both numerical (gain or loss of a chromosome) and structural (e.g., translocation, deletion, inversion) abnormalities. These abnormalities are described using a specialized nomenclature provided in the International System for Human Cytogenetic Nomenclature (ISCN) (6). See Table 3-5 for a glossary of cytogenetic terms commonly encountered in the practice of pediatric pathology, including examples of commonly used ISCN nomenclature.

Molecular Cytogenetic Analysis (FISH)

Although G-banding provides a rapid way of screening the entire genome for numerical or structural chromosomal abnormalities, it is hampered by the need for dividing (meta-phase) cells and by relatively low resolution (approximately 3 to 5 Mb). A major advantage to FISH is the ability to evaluate nondividing (interphase) cells, although it can also be performed on metaphase cells. Thus, FISH can be performed on specimens that cannot be cultured or that do not yield metaphase cells after culturing. FISH is a method that can be readily adapted to a variety of specimen types including cell suspensions, touch imprints, disaggregated tissues such as tumors, and paraffin-embedded tissues. Of note, it is typically not successful in specimens that have undergone decalcification. A large number of interphase cells (usually at least 200) can be rapidly evaluated by FISH, making it much faster and more sensitive than G-banding analysis.

TABLE 3.2 ONLINE RESOURCES FOR CYTOGENETICS AND MOLECULAR GENETICS

GeneReviews: genereviews.org

OMIM, Online Mendelian Inheritance in Man: omim.org

DECIPHER, Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources: decipher.sanger.ac.uk

ECARUCA, European Cytogeneticists Association Register of Unbalanced Chromosome Aberrations: ecaruca.net

Briefly, FISH is performed by applying fluorescently labeled probes (DNA sequences typically several hundred kb in length and complementary to known sequences) to cells that are affixed to a glass slide. The DNA of the probe and specimen are concurrently heat denatured for several minutes, and the slide is incubated at 37°C for 6 to 14 hours to allow the probe to bind to its target sequence. After washing and the addition of a nuclear counterstain such as 4′6′-diamidino-2-phenylindole (DAPI), the cells are examined under a fluorescence microscope.

Numerous different probe types are commercially available or can be developed within the laboratory. The types most frequently used to evaluate pediatric constitutional abnormalities are enumeration probes (directed against the pericentromeric region of chromosomes and used to detect chromosomal gains or losses) and locus-specific probes (directed against specific genes or loci and used to detect deletions or duplications of those regions).

Chromosomal Microarray

Array-based genomic technologies are routinely used in cytogenetic laboratories, and for the evaluation of patients with developmental delay, they are now considered the

first-line test (9,10). Unlike G-banding and FISH, which use metaphase and interphase cells as the substrate, microarrays evaluate DNA that has been extracted from the specimen. These assays can detect copy number gains or losses with a markedly increased resolution (typically <100 kb, depending on the array configuration). Like FISH, arrays evaluate specimen DNA with fluorescent labeled probes. However, unlike FISH, in which a single probe set is applied to patient DNA affixed to a slide, in array-based testing, the patient DNA is applied to a slide to which thousands of oligonucleotide probes are affixed. The level of resolution of these arrays is dependent on the number of these probes in the array, the distance between probes on a chromosome, and the degree to which certain chromosomal regions are “targeted” (i.e., sampled by a larger number of probes than other regions).

TABLE 3.3 INDICATIONS FOR PRENATAL GENOMIC TESTING

Structural fetal anomalies

Fetal growth restriction

Recurrent miscarriage or fetal loss

Known parental genomic abnormality

TABLE 3.4 TISSUE SAMPLING FOR CYTOGENETIC STUDIES: TECHNICAL CONSIDERATIONS

KARYOTYPE: Viable, mitotically active cells

Neonate:

Umbilical cord blood, peripheral blood lymphocytes (if infant has not been transfused), skin fibroblasts

Recent fetal demise:

Fibroblasts and chondrocytes remain viable the longest.
Sample fascia, cartilage, or lung
Use sterile blade; do not include epidermis, which may be contaminated. Avoid contacting formalin with blade.

Macerated fetus:

Fibroblasts may still be viable. Sample fascia, tendon, or cartilage
Chances of successful culture: ˜70% within 3 days of death; case reports of success 6 days after death
Also sample fetal surface of the placenta.

a-CGH AND SNP ARRAY: High-quality DNA

Fresh or fresh frozen tissue when possible (liver, placenta, cord or peripheral blood, other).
Formalin-fixed, paraffin-embedded tissue may be possible, but DNA tends to be more fragmented and may give less information.

FISH: Interphase or metaphase cells

Air-dried touch prep slides and fresh tissue are ideal; metaphase analysis requires suspension of cultured cells.
Formalin-fixed, paraffin-embedded tissue possible; high level of nuclear truncation due to slide preparation
Unacceptable: frozen tissue; decalcified specimens

TABLE 3-5 GLOSSARY OF TERMS USED FREQUENTLY IN CYTOGENETICS

Aneuploidy—gain or loss of all of one or more chromosomes (e.g., trisomies, monosomies). Does not include ploidy abnormalities (e.g., haploidy, triploidy, tetraploidy). Example: 47,XX,+21—female karyotype with 47 chromosomes, including three copies of chromosome 21

Chimerism—the presence in one zygote of a cell line derived from another zygote

Deletion—loss of part of a chromosome. Example: 46,XY,del(15)(q11.2q13)—deletion within the short arm of a chromosome 15, encompassing bands 15q11.2 through 15q13

Duplication—gain of part of a chromosome. Example: 46,XX,dup(22)(q13.31q13.33)—duplication of the long arm of a chromosome 22, encompassing bands 22q13.31 through 22q13.33

Haploidy—half (23) of the normal diploid (46) chromosome complement

Insertion—intercalation of part of one chromosome into another chromosome (interchromosomal) or into a different location within the same chromosome (intrachromosomal)

Inversion—180-degree rotation of an intrachromosomal segment

Pericentric—the inverted segment includes the centromere (it involves both the short and long arms of the chromosome)

Paracentric—the inverted segment does not include the centromere (it involves only one chromosome arm).

Mosaicism—the presence of two or more cell lines derived from a single zygote

Nondisjunction—failure of homologous chromosomes or sister chromatids to segregate properly during meiosis or mitosis

Parental imprinting—differential expression of alleles based on the parent of origin

Polyploidy—one or more complete extra set of chromosomes. Examples: triploidy (69,XXX), tetraploidy (92,XXYY)

Translocation—recombination of nonhomologous parts of two chromosomes

Balanced—reciprocal translocation with no net gain or loss of chromosomal material. Example: 46,XX,t(9;22)(q34;q11.2)—translocation between the long arm of a chromosome 9 at band 9q34 and the long arm of a chromosome 22 at band 22q11.2

Unbalanced—the presence of only one of two translocation partners, resulting in net gain and loss of translocated portions of the involved chromosomes. Example: 46,XX,der(22)t(9;22)(q34;q11.2)—the presence of only the derivative (abnormal) chromosome 22 from the translocation above, resulting in net gain of material from 9q and net loss of material from 22q

Robertsonian—translocation involving fusion of the long arms of two acrocentric chromosomes (13, 14, 15, 21, or 22) with resultant loss of their short arms

Uniparental disomy—both chromosomes of a homologous pair are derived from the same parent

Heterodisomy—UPD in which the two homologues differ (due to nondisjunction in meiosis I)

Isodisomy—UPD in which the homologues are identical (due to failure of sister chromatids to separate in meiosis II)

TABLE 3-6 INCIDENCE OF ANEUPLOIDY DURING DEVELOPMENT

Gestation (Weeks)^a			0	6-8	20	40
Gestation (Weeks)^a	Sperm	Oocytes	Preimplantation Embryos	Spontaneous Abortions	Stillbirths	Live Births
Incidence of aneuploidy	1%-2%	˜20%	˜20%	35%-54%	4%-6%	0.3%-0.6%
Most common aneuploidies	Various	Various	Various	45, X, +16, +21, +22, polyploidy	45,X, +13, +18, +21, polyploidy	45, X, +13, +18, +21, XXX, XXY, XYY
^aData pooled from multiple references (13, 71, and two new references: Boue J, Boue A Lazar P. Retrospective and prospective epidemiological studies of 1500 karyotyped spontaneous abortuses. Teratology, 1975;12:11-26.
Menasha J, Levy B, Hirschhorn K, et al. Incidence and spectrum of chromosome abnormalities in spontaneous abortions: new insights from a 12-year study. Genet Med, 2005;7:251-63.).

Two major types of arrays are in routine clinical use. a-CGH identifies regions of copy number gain or loss by hybridizing a mixture of an equal concentration of patient DNA and normal control DNA. By contrast, SNP arrays do not use a concurrent control DNA sample but rather compare the patient DNA to a well-characterized reference genome. Like a-CGH, SNP arrays can identify copy number changes but can also identify copy number neutral loss of heterozygosity, in which two copies of a chromosomal region are present, but both are derived from a single parent. Hybrid arrays containing both oligonucleotides and SNPs are also routinely used. Compared to G-banding and FISH, array-based techniques more precisely determine the size of aberrant regions as well as their gene content. A limitation of chromosomal microarrays is their ability to detect only unbalanced rearrangements leading to gain or loss of DNA; balanced rearrangements cannot be detected.

In cases where a fetal structural abnormality is identified by ultrasound, a-CGH is more sensitive than G-banding for detecting unbalanced rearrangements resulting in gain or loss of DNA (e.g., deletions, duplications). In a recent meta-analysis, a-CGH detected pathogenic abnormalities in 10% of cases with a normal karyotype (11). Similar results are found in first-trimester spontaneous abortions, where a-CGH detects abnormalities in 13% of cases with a normal karyotype (12). However, G-banding can detect some abnormalities (e.g., balanced rearrangements, polyploidy) that cannot be detected by chromosomal microarray.

CONSTITUTIONAL ABNORMALITIES

Constitutional chromosomal abnormalities are present at birth and affect all cell lines. Such abnormalities are common: for example, it is estimated that as many as 25% of conceptions are aneuploid, of which 99% are spontaneously aborted (13). The rates and types of chromosomal abnormalities detected in spontaneous abortions differ throughout gestation, with a higher rate detected in early gestation (Table 3-6). The pathology of early embryonic loss and its poor correlation with cytogenetic findings are discussed in Chapter 2.

Aneuploidy, defined as gain or loss of all of one or more chromosomes, is one of the most common types of cytogenetic abnormality found in prenatal specimens, such as chorionic villi, amniotic fluid, or spontaneous abortions (Table 3-7). The rate of aneuploidy among all liveborn infants is approximately 0.5%, although the rate in malformed infants is significantly higher (21). Most aneuploidy occurs as a result of nondisjunction, the failure of homologous chromosomes (in meiosis) or sister chromatids (in mitosis) to segregate appropriately into daughter cells (22,23,24). When nondisjunction occurs in meiosis, the result is an aneuploid gamete; this is the basis for most nonmosaic aneuploid conceptuses (25). Nondisjunction of autosomes (chromosomes 1 to 22), which is far more common during oogenesis than spermatogenesis, typically occurs during the first meiotic division, when homologous chromosomes segregate. In contrast, sex chromosome nondisjunction occurs more commonly during male gametogenesis in the second meiotic division, when sister chromatids separate.

TABLE 3-7 CHROMOSOMAL ANOMALIES IN SPONTANEOUS ABORTIONS

Abnormality	Reported Rate of Occurrence (%)^a
Autosomal trisomy	50-60
Polyploidy	20-25
Monosomy X	10-20
Translocations	2-5
^aPooled results from references (15,16,18,19,20).

Mitotic nondisjunction, occurring after a zygote is formed, may result in mosaicism, that is, the presence of two distinct cell lines both derived from a single zygote. Examination of human embryos conceived by in vitro fertilization suggests that rates of spontaneous postzygotic nondisjunction are very high (20% to 50% of preimplantation embryos) (13,26). Although genetic or pharmacologic alterations that disrupt meiotic and mitotic checkpoints predispose to nondisjunction and aneuploidy (25), most instances of nondisjunction are sporadic. In one population-based study, spontaneous abortion due to aneuploidy was not associated with an increased risk of a similar event in subsequent pregnancies (27).

Autosomal Trisomies

Most nonmosaic autosomal trisomies arise from errors during the first meiotic division in oogenesis (28,29). Oogenesis in humans begins during fetal development. In the second trimester, oocytes arrest in late prophase of the first meiotic division and remain in a “dormant” state until one to five decades after birth. During this period of meiotic arrest, recombination between homologous chromosomes occurs (13). The physical sites of recombination, termed chiasmata, stabilize the chromosomal pairs through metaphase (30). It is believed that the prolonged meiotic arrest increases the risk of nondisjunction, possibly due to age-related loss of chiasmata.

Trisomies 13, 18, and 21 are the only autosomal trisomies compatible with survival to term. Their features are summarized in Tables 3-8, 3-9, and 3-10 and Figures 3-2, 3-3, and 3-4. Two alternative theories have been developed to explain how a trisomy results in a specific clinical phenotype (31). The “amplified developmental instability” hypothesis suggests that increased expression of hundreds of genes on the trisomic chromosome disrupts the global balance of gene expression and/or protein stoichiometry during
development, leading to abnormalities of development. This is supported by the presence of common features between trisomies and segmental chromosomal duplications, including craniofacial abnormalities, structural cardiac abnormalities, and cognitive impairment. The “gene dosage effect” hypothesis asserts that a small set of genes, expressed at 1.5-fold greater than normal levels, is responsible for many of the phenotypic findings. Based on phenotypic mapping studies of individuals with duplications of part of chromosome 21 and clinical features of Down syndrome, a 1.6 to 5 Mb region of the long arm of chromosome 21 has been proposed to be the “Down syndrome critical region,” three copies of which are thought to account for many of the features of Down syndrome (7,65). In mice, duplication of part of chromosome 16 (homologous to human chromosome 21) results in phenotypic features similar to those in Down syndrome, including craniofacial alterations, changes in cerebellar architecture, and impaired learning and memory. Similar critical regions have been proposed for trisomy 13 (14) and trisomy 18 (66). Selective overexpression of smaller regions or single candidate genes in these homologous regions in the mouse may provide an experimental basis for understanding how trisomies influence development.

TABLE 3-8 MALFORMATIONS AND POSTNATAL DISORDERS ASSOCIATED WITH TRISOMY 21/DOWN SYNDROME (DS)

Malformation		% of DS^a	Postnatal Disorder		% of DS^a
Craniofacial^b			Central nervous system
	Upslanted palpebral fissures	>50		Cognitive impairment	100
	Ear anomalies	>50		Early-onset Alzheimer dementia	>50^c
	Epicanthal folds	>50		Seizures
	Flat midface	>50	Cancer (relative risk)
	Hypertelorism	25-50		Acute lymphoblastic leukemia (31)
	Other: brachycephaly, midline parietal hair whorl, mild microcephaly, choanal stenosis, cleft palate without cleft lip			Acute myeloid leukemia (26) Lymphoma (3) Colon (3)
Cardiovascular		>50		Testicular (21)
	Atrioventricular canal	10-25	Transient abnormal myelopoiesis		5-10
	Patent ductus arteriosus	10-25	Autoimmune (relative risk)
	Tricuspid valve defects	10-25		Crohn disease (3)
	Ventricular septal defect	5-10		Ulcerative colitis (3)
	Atrial septal defect	5-10		Celiac disease (5)	1-5
	Tetralogy of Fallot	1-5		Early-onset diabetes mellitus (3)
	Other: coronary valve defects, hypoplastic right heart, hypoplastic left heart, anomalies of coronary circulation, coarctation of the aorta, other aortic anomalies, pulmonary artery stenosis, anomalies of great veins, single umbilical artery			Thyroiditis (32) Autoimmune hepatitis (33) Psoriasis (4)
			Musculoskeletal
				Hypotonia Joint hyperextensibility	>50
Digestive tract		10-25	Other		>50
	Duodenal stenosis	5-10		Testicular microlithiasis
	Hirschsprung disease Anal atresia/stenosis	1-5 1-5		Enlarged thymic Hassall corpuscles	25-50
	Other: tracheoesophageal fistula, esophageal atresia/stenosis, nonduodenal intestinal atresia/stenosis, intestinal malrotation, ectopic anus, annular pancreas			Abnormal lymphocyte subsets Ocular: Glaucoma Strabismus Nystagmus	10-25 10-25 10-25 5-10 25-50
Respiratory				Scoliosis
	Anomalies of larynx, trachea, or bronchi Pulmonary anomalies			Hearing loss^d
Genitourinary
	Obstructive defects of renal pelvis, ureter, bladder neck, or urethra	1-5
	Cryptorchidism	5-10
	Hypospadias/epispadias	1-5
Central nervous system
	Hypoplastic superior temporal gyrus Flat frontal poles, retarded myelination Hydrocephalus
Limb
	Clinodactyly (fifth finger)	25-50
	Single transverse palmar crease	25-50
	Syndactyly	1-5
	Other: clubfoot, polydactyly, limb reduction defects, rhizomelic shortening
Ocular
	Brushfield spots	1-5
	Cataract	5-10
	Keratoconus	5-10
^aData pooled from multiple references (34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52). ^bMany of the craniofacial features are less distinct in fetuses than in infants or children. ^cOnset of Alzheimer dementia is age dependent. One hundred percent have pathologic changes by age 40 years and >50% have clinical findings by age 50 years. ^dThe incidence of hearing loss varies with age and aggressive treatment of middle ear infections.

Most nonmosaic autosomal trisomies lead to early embryonic demise. Although trisomies 13, 18, and 21 are compatible with survival to term, each has a high rate of embryonic and fetal loss (Tables 3-6, 3-7, and 3-11). A retrospective examination found that 10% of fetuses with trisomy 21 and 32% with trisomy 18 diagnosed by amniocentesis subsequently died spontaneously in utero; deaths occurred at a constant rate throughout the second and third trimesters (76). Analysis of placentas from fetuses and infants with apparently nonmosaic trisomy 13 or 18 suggests that those surviving to term have placental mosaicism (79). It is possible that survival to term requires that at least a subset of placental cells be diploid and that nonmosaic placental trisomy 13 or 18 is always lethal. Although trisomies for each of the autosomes have been identified in spontaneous abortions, the pattern is not random; for example, trisomies 16, 21, and 22 are particularly common in the first trimester. Most trisomic abortuses manifest as highly disorganized embryos (see Chapter 2).

Prenatal diagnosis and elective termination of pregnancy also impact the rate of liveborn trisomies. Definitive prenatal diagnosis requires chorionic villus sampling, amniocentesis, or other invasive procedure to obtain tissue for genomic studies. To reduce unnecessary risks and cost associated with invasive prenatal diagnosis, noninvasive screening methods have been developed that identify pregnancies at greatest risk for aneuploidy. Sequencing of maternal plasma cell-free fetal DNA (cfDNA)
is currently the most sensitive noninvasive screening method for detecting trisomy 21, with a detection rate of 98% to 100% and a false-positive rate of 0% to 0.3% (80,81). cfDNA testing is also in clinical use to detect trisomies 13 and 18; the sensitivity for detection of these aneuploidies ranges from 78% to 100%, with greater sensitivity for trisomy 18 than for trisomy 13 (80). Because cfDNA is mostly derived from placental trophoblast cells, one potential source of false-positive and false-negative results is confined placental or fetal mosaicism, estimated to affect 1 in 1000 to 1 in 4000 pregnancies (82). Therefore, despite the high specificity, confirmatory invasive diagnostic testing (by chorionic villus sampling or amniocentesis) is required

before a decision is made to terminate the pregnancy (83). Another current testing option is an “integrated test” that includes quantitative measurement of pregnancy-associated plasma protein A, alpha-fetoprotein, unconjugated estriol, free β-human chorionic gonadotropin, and/or inhibin A in maternal serum, early second-trimester ultrasound evaluation of nuchal translucency, and maternal age (Table 3-11). This approach affords an 80% to 85% detection rate of trisomy 21, with a false-positive rate of 1% to 5% (84,85). Similar sensitivity and specificity have been reported for trisomy 18 based on a two-stage screening approach using maternal serum markers.

TABLE 3-9 MALFORMATIONS ASSOCIATED WITH TRISOMY 18

Malformation		% of Cases^a
General
	Intrauterine growth restriction	25-50^b
	Fetal hydrops	5-10
Craniofacial
	Microcephaly	25-50^b
	Choroid plexus cyst Other: triangular facies, abnormal calvarial shape (“strawberry” skull), hydrocephalus, micrognathia, hypotelorism, cleft lip/palate, small ears, wide fontanelles, narrow nasal bridge, microstomia, short sternum
Cardiovascular
	Ventricular septal defect	>50
	Atrioventricular canal defect	25-50
	Other: ectopia cordis (pentalogy of Cantrell), hypoplastic left or right heart, overriding aorta, single umbilical artery, patent ductus arteriosus, tetralogy of Fallot, double-outlet right ventricle, transposition of the great arteries, mitral valvular disease
Digestive tract
	Omphalocele	10-25
	Meckel diverticulum	>50
	Other: anorectal atresia, esophageal atresia, pyloric stenosis, ectopic pancreas, abnormal liver lobation
Respiratory
	Abnormal lung lobation, tracheal stenosis, tracheoesophageal fistula
Genitourinary
	Abnormal genitalia, cloacal exstrophy, obstructive uropathy, horseshoe kidney, renal aplasia/hypoplasia, renal/ureteral duplication, cryptorchidism, bifid uterus
Central nervous system
	Cerebellar and pontine hypoplasia	>50
	Meningomyelocele ± Chiari malformation	10-25
	Other: anencephaly, craniorachischisis, hippocampal dysplasia, agenesis of the corpus callosum, neural migration defects
Limb
	Clenched hand with overlapping digits	>50
	Radial ray defects	5-10
	Rocker-bottom feet	25-50
	Other: arthrogryposis, polydactyly, phocomelia, syndactyly, hypoplastic nails, ectrodactyly
Ocular
	Coloboma, cataract, cloudy cornea, retinal hypopigmentation, microphthalmia, iridial hypoplasia
Musculoskeletal
	Other: diaphragmatic defect, absent 12th ribs, malformed occipital bones
Other viscera
	Hypoplasia of adrenals, thymus, thyroid, and/or gallbladder, accessory spleen
Placenta/cord
	Umbilical cord cysts Small placenta
^aData pooled from multiple references (15,17,53,54,55,56,57,58,59,60,61,62). ^bFrequency as a second-trimester ultrasound finding.

TABLE 3-10 MALFORMATIONS ASSOCIATED WITH TRISOMY 13

Malformation		% of Cases^a
General
	Intrauterine growth restriction	10-50^b
	Fetal hydrops	5-10
Craniofacial
	Microcephaly	10-50
	Holoprosencephalic facies (cyclopia, ethmocephaly, cebocephaly, premaxillary agenesis/dysgenesis)	>50
	Cleft lip/palate (midline/bilateral > unilateral) Hypotelorism Other: malformed ears, absent ear canal, aplasia cutis of scalp, choanal stenosis or atresia; hemangiomas, receding forehead, sparse curled eyelashes, natal teeth, micrognathia
Cardiovascular		>50
	Ventricular septal defect	25-50
	Patent ductus arteriosus	25-50
	Echogenic intracardiac foci (myocardial calcifications)	10-25
	Other: dextrocardia, tetralogy of Fallot, atrial septal defect, truncus arteriosus, aortic coarctation, pulmonary atresia/stenosis, bicuspid aortic valve, single umbilical artery
Digestive tract
	Pancreatic-splenic fusion Appendiceal diverticulum Other: omphalocele, abnormal liver lobation, intestinal atresia, Meckel diverticulum
Respiratory
	Abnormal lung lobation
Genitourinary
	Obstructive dysplasia	25-50
	Renal/ureteral duplications	25-50
	Other: cryptorchidism, double vagina, bicornuate uterus, abnormal fallopian tubes, small penis, abnormal scrotum
Central nervous system
	Holoprosencephaly	25-50
	Arhinencephaly	>50
	Cerebellar malformations	25-50
	Other: anencephaly, meningomyelocele, agenesis of the corpus callosum, hydrocephaly, hippocampal dysplasia, neural migratory defects, choroid plexus cyst
Limb
	Postaxial polydactyly	˜50
	Other: syndactyly, rocker-bottom feet, hypoplastic nails, clubbed feet, hypoplastic nails, single transverse palmar crease, radial aplasia
Ocular
	Microphthalmia	25-50
	Coloboma of iris or retina	25-50
	Other: retinal dysplasia, aniridia, anophthalmia, cataract, premature vitreous body, hypoplasia of optic nerve
Musculoskeletal
	Dysplastic/fused lumbosacral ± thoracic vertebra, absent 12th ribs, hypoplastic sphenoid bone, diaphragmatic defect	>50
Hematologic
	Irregular neutrophil nuclei Increased fetal and Gower-2 hemoglobin
^aData pooled from multiple references (16,55,60,61,63,64). ^bReported rates of IUGR appear to be higher in populations that were studied later in gestation.

FIGURE 3-2 • Trisomy 21. A: 35-week fetus with typical late-gestation facies (epicanthal folds, broad nose, bulging tongue). B: Similar facial changes are apparent in 2-month-old infant (note increased slant of palpebral fissures). C: Single palmar crease. D: Lateral view of brain showing small superior temporal gyrus and enlarged middle temporal gyrus. E: Duodenal atresia. F: Atrioventricular canal, with large primum atrial septal defect, large ventricular septal defect in position of AV canal, and cleft septal leaflet of the tricuspid valve.

FIGURE 3-3 • Trisomy 18. A: Late-gestational fetus with omphalocele and rocker-bottom feet. B: Young infant with widely separated eyes and mild trigonocephaly. C: Infant shown in (B), with dysplastic ear and micrognathia. D: Infant with triangular facies, ocular hypertelorism, and bilateral cleft lip. E: Overlapping digits in pattern common to trisomy 18, with distal polydactyly. F: Horseshoe kidney (with ureters and urinary bladder).

FIGURE 3-4 • Trisomy 13. A: Infant with cebocephaly (ocular hypotelorism and single nostril nose), one of the facial changes associated with holoprosencephaly. B: Aplasia cutis of the scalp. C: Postaxial polydactyly. D: Alobar holoprosencephaly. E: Appendiceal diverticula (“dinosaur tail”) are pathognomonic for trisomy 13, although not present in every case. F: Fusion of spleen (left) and tail of pancreas (note tiny splenic islands within the pancreas).

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