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Obstet Gynecol Clin N Am 35 (2008) 435–458

Prenatal Diagnosis and Genetic ScreeningdIntegration into Prenatal Care Valerie J. Rappaport, MD Division of Maternal Fetal Medicine, Dept of Ob/Gyn, University of New Mexico School of Medicine, MSC 10 5580, Albuquerque, NM 87131, USA

Prenatal care was originally developed to protect the health of the mother. Evaluation of the health and development of the baby has been a more recent purpose of prenatal care. Birth defects, while recognized since ancient times, were felt to be due to a whim of nature, exposure of the mother to adverse events, or perhaps to the inferior character of the mother or father. The process of fetal development was largely hidden from view and understanding. In the last 3 decades, perinatal medicine has made tremendous advances in scientific knowledge and in the successful application of this knowledge toward understanding the fetal aspects of pregnancy. These advances include technology that allows us both direct and indirect access to the fetal compartment. The rapid transition of new genetic knowledge and DNA analysis from bench-top to commercial applications has greatly enhanced the ability to diagnosis genetic disorders before birth. Refinement of invasive fetal diagnostic techniques has increased the safety of these procedures and has opened the possibility of in utero therapy. Ultrasound in particular has revolutionized the field of obstetrics by providing detailed imaging of the developing baby. This ability to view the fetus is not only important in the medical evaluation of the pregnancy, but has also become one of the central features of the prenatal experience for the pregnant couple. These advances have brought about a dramatic change in how the fetus is conceptualized. No longer is the fetus perceived as only a part of the pregnant woman, but is increasingly perceived as a distinct entity that can be the independent focus of diagnostic testing and individual therapy. Evaluation of the health of the fetus and screening for birth defects

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has become an important part of prenatal care. All women in the United States are now offered some form of prenatal diagnosis or genetic screening during pregnancy. Overview of birth defectsdscope of the problem Any practitioner who engages in the care of pregnant women will also, sooner or later, be involved in the care of a pregnancy complicated by a fetus with a birth defect or developmental disorder. Congenital abnormalities are a frequent occurrence in human reproduction. It is estimated that more than 50% of first-trimester spontaneous abortions and 6% to 12% of stillborn infants have chromosomal abnormalities. About 25% to 35% of stillbirths are associated with intrinsic anomalies at autopsy. These include single malformations (40%), multiple malformations (40%), and deformations or dysplasia (20%) [1]. Approximately 3% of live births are complicated by a major congenital anomaly or genetic disorder. Half of these anomalies and disorders are detectable before or at birth. According to estimates, about 7.9% of individuals are diagnosed with a genetic condition or birth defect by the age of 25. Causes of congenital anomalies Congenital anomalies can be categorized into three groups based on etiology: (1) polygenic or multifactorial causes; (2) genetic causes, including chromosomal and single-gene disorders; and (3) environmental causes, including such external influences as maternal medical disorders and teratogens. The most common causes of structural birth defects are polygenic or multifactorial. Polygenic disorders are conditions caused by a combination of genetic influences as well as a coalescence of other factors, including maternal environmental factors. About 65% to 80% of birth defects are classified as multifactorial. Included in this group are many of the most common structural birth defects, such as cleft lip, neural tube defects, gastroschisis, and clubfoot. These disorders do not follow classic Mendelian inheritance patterns but may show some degree of family clustering. Most commonly they tend to occur sporadically without previous family occurrences. The recurrence risk in general is highest in first-degree relatives, and then rapidly tapers off, so that the risk level reaches population baseline for third-degree relatives. From 10% to 25% of birth defects are felt to be due to classical genetic abnormalities. This group includes inherited recessive disorders such as cystic fibrosis; X-linked disorders, such as muscular dystrophy; autosomal-dominant disorders; and mitochondria inheritance. Genetic abnormalities can also occur as a result of alterations in contiguous genes, typically microdeletions. One of the most common microdeletion syndromes is del22q11 resulting in a variety of clinical outcomes, including DiGeorge



syndrome and velo-cardio-facial syndrome. Also in this group are disorders resulting from duplications or deletions of entire chromosomes, such as Down syndrome (trisomy 21) and Turner’s syndrome (monosomy X). Although these disorders are genetic in origin, familial occurrences may not be present. Many recessive genes remain silent in a family until the chance pairing of a couple with the same recessive gene. In addition, because only one quarter of offspring will be affected, the condition may not show up until there are already many healthy children in the family. Autosomal-dominant conditions in the fetus can result from de novo mutations in the absence of affected parents. Once the mutation in present, the affected individual has a 50% possibility of transmitting the condition to future generations. A minority of birth defectsdless than 10%dare felt to be caused by environmental agents. Included in this group of defects are those related to conditions affecting the fetal environment, such as maternal diabetes; maternal use of alcohol, prescription drugs, and nonprescription drugs; congenital infections; and mechanical constraint problems. These agents affect an embryo that would otherwise be destined to develop normally. Although environmental agents cause a minority of congenital anomalies, this is an important group in that many of these extrinsic exposures are potentially preventable. Prenatal detection of congenital anomalies The increasing availability of prenatal genetic diagnosis has led to an important role of the obstetrician/gynecologist in identifying couples at risk to have a baby with an inherited disorder or congenital anomaly. This process of risk identification or screening is intended to identify pregnancies where there is an increased possibility of congenital anomalies. A variety of screening approaches are used, including patient medical and exposure history, family history, population-based carrier screening, maternal serum screening, and, finally, ultrasound, which can be both a screening tool and a diagnostic tool. Family history Obtaining a family history at the onset of prenatal care is both costeffective and critical as an initial screen for genetic risk. Gathering information for first-degree relatives (parents, siblings and offspring), second-degree relatives (uncles, aunts, grandparents, nieces, and nephews), and third-degree relatives (cousins) is recommended. Of particular concern is any history of a genetic diagnosis, recurrent pregnancy loss, birth defects, developmental delay, stillbirth, or other adverse reproductive outcomes. In addition, inquiring about a history of certain adult-onset disorders, such as thrombosis, familial cancers, infertility, psychiatric illness, autism,



and behavioral disorders may be of signiďŹ cance. Information regarding ethnic background, consanguinity, and potential teratogen exposures is also collected as part of the genetic family screening interview. In clinical practice, drawing out a full pedigree and conducting a full genetic interview are both tedious and time-consuming. An eective way to integrate genetic screening into clinical practice is through the use of questionnaires or checklists. A number of commercially available genetic screening tools are designed for the prenatal care setting. One example of a detailed history form is available through the March of Dimes Web site (http://www. Another widely used form in clinical practice can be obtained through the American College of Obstetricians and Gynecologists (ACOG). Parental ages should be recorded as part of the initial family history. Advancing maternal age has a well-known association with increasing risk of chromosome aneuploidy, in particular autosomal trisomies (Table 1). Maternal age, however, is not associated with an increase in other

Table 1 Maternal age and risk of chromosome abnormalities at birth Maternal age at delivery (y)

Down syndrome

All chromosome abnormalities

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

in in in in in in in in in in in in in in in in in in in in in in in in in

1250 1190 1111 1031 935 840 741 637 535 441 356 281 217 166 125 94 70 52 40 30 24 19 16 14 13

in in in in in in in in in in in in in in in in in in in in in in in in in

476 476 455 435 417 385 385 323 286 244 179 149 123 105 81 63 49 39 31 24 19 15 11 9 7

Data from Hook, EB. Chromosome abnormalities and spontaneous fetal death following amniocentesis: further data and associations with maternal age. Am J Hum Genet 1983; 35:110–6.



congenital anomalies in the absence of concurrent conditions, such as pregestational diabetes. Advanced paternal age has not been conclusively shown to be associated with an increase in aneuploidy risk. However, several population-based studies have suggested that advancing paternal age over 45 is associated with an increased risk of a variety of structural and single-gene disorders. Reported birth defects include congenital heart defects, tracheoesophageal fistula, musculoskeletal anomalies, skeletal dysplasias, autism, schizophrenia, and such single-gene disorders as Marfan’s syndrome, neurofibromatosis, osteogenesis imperfecta, and achondroplasia [2]. Although the risk for these disorders as a group may be as much as four to five times higher than baseline, the absolute risk is small. It is estimated that paternal age effects add less than 1% risk to the background risk for a given pregnancy. Prenatal testing for the hundreds of potentially involved loci is not clinically available. However, targeted ultrasound may be a useful screen to exclude structural birth defects. Carrier screening for recessive conditions with increased prevalence in certain ethnic groups has become a standard part of prenatal care. Ethnic origin should be recorded as part of the initial screen. Determining ethnicity can be challenging because many patients do not have a clear idea of their family origins. In addition, many individuals may identify multiple ethnicities in their background. A specific checklist that allows patients to list multiple ethnicities is useful in this screening and is a part of most prenatal genetic screening questionnaires. A positive response on the screening questionnaire indicates a possible risk that needs further exploration (Box 1). Recurrent spontaneous abortions, stillbirths, and anomalous liveborns in the family history may indicate a familial chromosomal balanced translocation. Previous siblings with congenital anomalies or a family history of anomalies may indicate a specific risk that would require targeted testing. For example, a family history of congenital heart disease would suggest the need for a fetal echocardiography. A maternal family history of thrombosis, stroke, and recurrent pregnancy loss may indicate an inherited thrombophilia that could affect pregnancy outcome. Genetic counseling and targeted ultrasound are also generally recommended if there is a family history of structural birth defects or a genetic disease. Referral for genetic consultation may also be warranted for potential teratogen exposures. Such exposures could be related to medications, infections, or maternal medical disease, such as pregestational diabetes.

Prenatal screening for specific genetic disorders The ACOG currently recommends universal access to cystic fibrosis–carrier screening as well as other carrier screening in specific high-risk groups. The purpose of carrier screening is to identify asymptomatic individuals



Box 1. Family and maternal history indications for genetic counseling Family history indications for genetics consultation Personal or family history of a known or suspected genetic disorder, birth defect, chromosome disorder, or metabolic disorder Family history of mental retardation Familial chromosome rearrangement Known carrier or family history of a genetic disorder Unexplained infertility, multiple pregnancy losses, or unexplained previous stillbirth Congenital absence of the vas deferens Premature ovarian failure or elevated follicle-stimulating hormone under the age of 40 Family history of early-onset cancer or multiple family members affected with early- or late-onset cancer Personal or family history of stroke or blood clots before age 50 or known thrombophilia allele in other family members Maternal medical indications for genetic consultation Known maternal genetic disorder Advanced maternal or paternal reproductive age Maternal history of congenital anomalies Pregestational diabetes Maternal history of acute fatty liver Maternal seizure disorder Exposure to drugs with known fetal risk (eg, isotretinoin, warfarin lithium, tetracycline, seizure medications, statins, angiotensin-converting enzyme inhibitors) Significant alcohol exposure (binge drinking or over two drinks a day) Occupational exposure to lead, mercury Significant radiation exposure Certain infectious diseases (eg, primary cytomegalovirus, rubella, toxoplasmosis), primary varicella infection, parvovirus, sustained hyperthermia >2 days

who are heterozygous carriers for common genetic disorders. For a condition to be considered appropriate for population-based carrier screening, several criteria should be met. The natural history of the disorder should be well understood and carry a potential for signiďŹ cant morbidity and mortality. The detection rate for disease-causing mutations should be greater



than 90% or an allele frequency greater than 1% in the target population [3]. In addition, there must be adequate laboratory capacity to allow ready access to testing and there must be adequate resources for follow-up of positive results. Cystic fibrosis Cystic fibrosis is the most common severe autosomal-recessive disease in individuals of Northern European background, affecting about 30,000 individuals in the United States [4]. Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane regulator gene leading to impaired chloride conductance across cell membranes affecting primarily the pulmonary, digestive, and male reproductive systems. The disease is characterized by chronic pulmonary infections with progressive deterioration of lung function. Early intervention and aggressive therapy to prevent deterioration of lung function is the cornerstone of treatment. Lung transplant has been used increasingly for end-stage pulmonary disease. Pancreatic insufficiency occurs in about 85% of individuals with classic cystic fibrosis resulting in poor weight gain and chronic malabsorption. In men, cystic fibrosis transmembrane regulator gene mutations are associated with congenital bilateral absence of the vas deferens and infertility. Women with cystic fibrosis are less likely to have infertility and are increasingly able to successfully complete pregnancy. With aggressive treatment to preserve lung function and pancreatic enzyme replacement, the life span for affected individuals is now into the 30s and it is estimated that individuals born in the most recent decade may live into their 40s [5]. About 15% of individuals with cystic fibrosis have a milder disease course and a life span of about 56 years [6]. Over half of cystic fibrosis in the Northern European population is caused by a single mutation known as the delta F508 allele. However, over 1000 disease-causing mutations have now been described [5]. Cystic fibrosis occurs worldwide and has been described in individuals of every ethnic background. Although cystic fibrosis is often described as a disease of Northern European populations, the incidence varies markedly from 1 in 2500 for people of Northern European descent to 1 in 25,000 for individuals from Finland [7]. One of the highest incidences worldwide is found in Native Americans of Zuni Pueblo background, where the disease frequency is estimated at 1 in 333 [8]. The ability to detect mutations varies greatly between ethnic groups and even within ethnic groups. Given this, the result of cystic fibrosis screening must always be described with the residual risk for the patient’s particular ethnic background in mind (Table 2). Prenatal screening for cystic fibrosis has been viewed as a model for the integration of genetic testing into routine medical care [5]. In 2001, the ACOG and the American College of Medical Genetics (ACMG) recommended incorporating cystic fibrosis screening into clinical practice for both prenatal and preconception screening for individuals of Northern European



Table 2 Carrier risks for cystic fibrosis before and after mutation testing with ACOG recommended panel Estimated carrier risk Racial or ethnic group

Detection rate

Before testing

After negative test

Northern European or Caucasian Ashkenazi Jewish Hispanic American African American Asian American

88% 94% 72% 64% 49%

1 1 1 1 1

1 1 1 1 1

in in in in in

25 24 58 61 93

in in in in in

200 385 205 165 185

Data from American College of Medical Genetics. Technical standards and guidelines for CFTR mutation testing. 2006 edition. Available at Activities/stds-2002/cf.htm. Accessed June 12, 2008.

and Ashkenazi background. In 2005, the ACOG issued revised guidelines based on a review of the program to date. The current ACOG guidelines recommend offering cystic fibrosis screening to all patients regardless of ethnic background. They also recommend making the patient aware of their residual risk based on a best estimate of ethnic background. A critical aspect of cystic fibrosis screening is to provide appropriate counseling to patients so that they are aware of the limitations of the testing and that a small but real residual risk for cystic fibrosis exists even after tests show negative for carrying cystic fibrosis (see Table 2). Patient information pamphlets describing cystic fibrosis screening are available from a variety of sources including the ACOG, the March of Dimes, and commercial laboratories. These written materials are useful in clinical practice to reinforce information and counseling points. Generally, prenatal testing is initiated with the pregnant woman followed by testing of the partner if a mutation is identified. In cases where there are time constraints, concurrent testing of both the partners may be indicated. Initial ACOG/ACMG guidelines recommended a panel for identifying 25 pan-ethnic mutations that were present in at least 0.1% of patients with cystic fibrosis. In the 2005 guidelines, two mutations were eliminated, leaving a recommended panel of 23 mutations. Many commercial laboratories, however, offer panels for detecting considerably more mutations. These expanded panels may be appropriate to enhance mutation detection in certain ethnic groups. For example, using an expanded 86-mutation panel in the Hispanic population may increase the detection rate to 78%, as opposed to 72% with the standard panel. The same expanded panel in the African American population increases the detection rate to 81%, as opposed to 65% with the standard panel [9,10]. In obstetric practices with significant numbers of patients of non-European background, screening with an enhanced panel may beneficial. Complete sequence analysis of the cystic fibrosis transmembrane regulator gene is possible although not recommended for carrier screening. This technology is useful in evaluation of an individual



with cystic fibrosis, a family history of cystic fibrosis, or a male with congenital bilateral absence of the vas deferens where a mutation cannot be detected on an expanded panel [5]. If a parent is found to carry a cystic fibrosis mutation, testing of the partner should be performed. If the partner’s test for carrying cystic fibrosis is negative, the risk of an affected baby is very small but not zero. In addition, patients should be aware of the clinical consequences of the particular mutation they carry since not all cystic fibrosis mutations result in classical cystic fibrosis. Genetic consultation is recommended when a patient is found to carry a cystic fibrosis mutation to address the issues of genotype/phenotype correlations and residual risk as well as to address other issues regarding notification of relatives, prenatal diagnostic testing, and the availability of newborn screening. Tay-Sachs disease Tay-Sachs disease is a lysosomal storage disease caused by a deficiency of the enzyme hexosaminidase A. The result of this enzyme deficiency is the accumulation of GM2 gangliosides throughout the body, leading to a severe progressive neurologic disorder. Infants affected with Tay-Sachs disease appear normal at birth. However, by 5 to 6 months of age, their muscle tone deteriorates, they fall short of developmental milestones, they develop mental retardation, and they become blind. They generally die by 6 years of age. There is no effective treatment for this disorder [11]. Tay-Sachs disease is an autosomal-recessive disorder and was one of the initial disorders for which carrier screening was first clinically available. The carrier rate in Ashkenazi Jewish individuals is 1 in 30 as compared with the background rate of 1 in 300. Successful application of carrier screening programs in the Ashkenazi Jewish population, initiated in the 1970s, have resulted in a more than 90% decreased incidence of this disorder in the North American Jewish community. Today in North America, the vast majority of children born with Tay-Sachs are of non-Jewish parents. Individuals of French Canadian and Cajun background have also been shown to have higher carrier rates than those of the general population. The ACOG recommends that screening be offered to high-risk populations [11]. It has been suggested that this screening should be expanded to the general population because of the availability of a highly accurate, relatively inexpensive enzyme screen and the fact that the majority of children currently born with Tay-Sachs disease are of non-Jewish parents [12]. Carrier testing can be performed by either DNA analysis or biochemical analysis. Clinical screening is often performed using both of these techniques. In the Jewish population, DNA analysis for the three most common mutations detects 94% of carriers. Biochemical analysis for hexosaminidase activity will detect 98% of carriers. In the non-Jewish population, mutation testing may detect fewer than 50% of carriers. Therefore, enzyme testing is



recommended for screening of non-Jewish individuals. Of note, pregnancy and oral contraceptives result in decreased levels of hexosaminidase serum levels. Serum screening in pregnancy may result in misclassification of women as carriers. Therefore, if biochemical screening is performed in women who are pregnant or taking oral contraceptive, peripheral blood leukocyte testing must be used [11]. A further complication in Tay-Sachs disease screening is that up to one third of the mutations identified in non-Jewish individuals are associated with a pseudodeficiency state in which individuals have a low activity level of hexosaminidase A when tested on conventional artificial substrate. However, these individuals are able to catalyze the breakdown of natural substrate GM2 ganglioside and are not at risk of having a child with Tay-Sachs disease. Additional biochemical and DNA analysis is needed to clarify the results. Referral for genetic consultation to review any ambiguous or positive screening results is highly recommended. Other genetic disorders in individuals of Eastern European Jewish descent Subsequent to Tay-Sachs screening programs, a number of disorders have been identified in the Ashkenazi populations that meet criteria for screening. These include disorders with a carrier rate of at least 1% in the Ashkenazi population and a small number of common mutations leading to carrier detection rates of over 90%. The disease incidence of these disorders ranges from 1 in 900 to 1 in 40,000 (Table 3). While individually these are rare disorders, it is estimated that about one in five individuals of Ashkenazi descent carries a mutation for at least one of these disorders. Current ACOG/ACMG guidelines suggest offering screening for four of these disorders to couples where at least one partner is of Ashkenazi ancestry.

Table 3 Recessive disorders in individuals of Eastern European Jewish descent Disorder

Disease incidence

Carrier frequency

Detection rate


1 in 3000

1 in 30

Canavan disease Cystic fibrosis Familial dysautonomia Fanconi anemia group C Neimann-Pick disease, A Mucolipidosis IV Bloom syndrome Gaucher’s disease

1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1

98% hexosaminidase A; 94% DNA based 98% 97% 99% 99% 95% 95% 95%–97% 95%

in in in in in in in in

6400 2500 3600 32,000 32,000 65,000 40,000 900

in in in in in in in in

40 29 32 89 90 127 100 15

Data from March of Dimes genetic screening pocket facts. White Plains (NY): March of Dimes; 2001.



These four conditions are Tay-Sachs disease, cystic fibrosis, Canavan disease, and familial dysautonomia [13]. Carrier screening tests are also available commercially for a number of other disorders that are less prevalent in Ashkenazi populations but that meet screening criteria. These disorders include Fanconi anemia, Neimann-Pick disease, mucolipidosis IV and Bloom syndrome. All of these carrier tests have a very high sensitivity in the Jewish population. However, the detection rate in the non-Jewish population is unknown. When both parents are of Ashkenazi descent and one has a positive carrier screen while the other parent is screen negative, the possibility of an affected child can be excluded with high probability. In mixed couples, however, the ability to detect a carrier state in the non-Jewish partner is unknown, leading to an uncertain ability to exclude the possibility of having a child with the condition. Couples of mixed background should be counseled regarding this uncertainty before embarking on screening for these disorders. Gaucher’s disease is another disorder frequently included in commercially available Jewish genetic disorders panels. While the carrier frequency is high for this disorderd1 in 15dthe clinical course of the disease is quite variable, ranging from a severe childhood disease to mild or unapparent disease in an adult. Effective treatment is available through enzyme-replacement therapy. Given the clinical spectrum, some couples may choose to decline Gaucher’s disease screening even if they opt for the other carrier screens. Couples of Ashkenazi Jewish ancestry should be made aware of these extended testing options either through educational material or genetic counseling. However, current ACOG standards do not recommend routinely offering this extended testing. Due to the complexity of this testing and the many options to consider, referral for genetic counseling may be of benefit for couples considering extended testing [13]. Hereditary disorders of hemoglobin synthesis Hereditary disorders of hemoglobin synthesis represent the most common single-gene disorders in the world. It is estimated that more than 270 million persons worldwide are heterozygous carriers of hereditary disorders of hemoglobin, and at least 300,000 affected homozygotes or compound heterozygotes are born every year. The disorders include mutations causing structurally abnormal hemoglobin, such as sickle cell disease, as well as disorders resulting in quantitative hemoglobin abnormalities, such as a- and b-thalassemia. The carrier frequency varies markedly worldwide. The highest carrier rates are in individuals of Southeast Asian, African, or Mediterranean descent. Persons of northern European, Japanese, Native American, Inuit, or Korean descent are considered to be at low risk. Carrier screening programs for the hemoglobinopathies have been widely implemented in many high-risk populations with variable success. The first nationwide population-based carrier screening program in the United States was for sickle



cell anemia and is widely viewed as a failure [14]. Eventually this program was abandoned. The experience, however, provides insights into the potential pitfalls of implementing population-wide carrier screening programs. In contrast, carrier screening for b-thalassemia in Cyprus, while socially controversial, has resulted in the virtual elimination of affected homozygote newborns in that region [15,16]. In the United States, identification of high-risk couples and voluntary screening are standard aspects of prenatal screening. High-risk groups include those of African American, Southeast Asian, Chinese, or Mediterranean ancestry. The appropriate screening test for individuals identified as high risk for hemoglobin structural disorders is hemoglobin electrophoresis [17]. The hemoglobin S solubility testing to screen for sickle cell trait in persons of African background is not recommended because this will miss other structural hemoglobin variants as well as the thalassemia disorders, both of which are in increased prevalence in this ethnic group. Mean corpuscular volume (MCV) as an initial screen should be obtained for patients at increased risk for b- or a-thalassemia. Those who have an MCV level less than 80 mm3 maybe a carrier of one of the thalassemia traits, and should undergo hemoglobin electrophoresis. Elevated HbF and HbA2 greater than 3.5% are associated with b-thalassemia. a-thalassemia carrier status can only be detected through molecular genetic testing. When an MCV is below normal, iron deficiency anemia has been excluded, and hemoglobin electrophoresis is not consistent with b-thalassemia trait, molecular genetic testing should be offered to detect a-globin gene deletions characteristic of a-thalassemia [17]. Couples identified as carriers of structural or quantitative hemoglobin disorders should be referred to a prenatal genetics center for counseling and possible diagnostic testing. Fragile X syndrome Fragile X was originally reported by Lubs [18] in 1969 as a syndrome of X-linked male mental retardation. The condition is characterized by a marker or fragile site on the X chromosome, which can be visualized when the cells are grown in a low-folate cell culture environment. Subsequently the gene responsible for this condition was identified as the fragile X mental retardation1 gene located on the X chromosome at Xq27.3. The gene is characterized by a repetitive CGG trinucleotide sequence at the 5’ promoter region. In the general population, the CGG sequence is repeated from 6 to 50 times. In premutation carriers, the CGG sequence repeats from 55 to 200 times. In females, premutations are unstable and can undergo expansion during oogenesis or postzygotic mitosis. This expansion results in CGG sequences of 200 or more, which are considered full mutations and are clinically expressed as fragile X syndrome. Expansion does not occur in male-to-male transmission and to date there have been no children with a full mutation inherited from a parent with a normal size allele [19].



In an affected male, fragile X syndrome is classically associated with distinctive facial features, such as large ears, a long face, a prominent forehead, prognathism, high arched palate, and, occasionally, cleft palate. The disorder includes developmental delay and mild to severe mental retardation, attention deficit hyperactivity disorder, speech and language delay, anxiety, hand flapping, and autistic spectrum disorders. Fragile X syndrome is not confined to males. Female full-mutation carriers have milder features than males but they also exhibit a similar range of physical characteristics and cognitive impairment. About 50% to 70% of females with full mutations exhibit IQs that are borderline or within the mentally retarded range. Female full-mutation carriers who have normal-range IQs may have learning disabilities or emotional problems, such as social anxiety, shyness, hyperactivity, or impulsive behaviors [19]. Individuals with expanded repeat lengths varying from 50 to 200 repeats do not exhibit the classical fragile X syndrome phenotype, but are considered fragile X premutation carriers. Recently, it has been recognized that premutation carriers may present with a spectrum of clinical findings. Mild manifestations of fragile X syndrome have been seen in male premutation carriers. In addition, males over 50 may have a progressive neurodegenerative disorder characterized by tremor, ataxia, Parkinsonism, and peripheral neuropathy known as fragile X–associated tremor/ataxia syndrome. Women with premutations are usually unaffected intellectually and physically. However, they do have an increased risk for premature ovarian failure or ovarian dysfunction. Premutation alleles have been identified in about 2% of women with idiopathic premature ovarian failure and in 14% of women with a family history of premature ovarian failure and no known history of fragile X [20]. The prevalence of fragile X syndrome ranges from 1 in 4000 for males and 1 in 6000 for females. In a recent large study looking at over 40,000 asymptomatic women, the premutation carrier frequency was 1 in 154 [21]. Given the prevalence of the premutations in the general population, the accuracy of detecting full mutations by DNA analysis, and the seriousness of the clinical syndrome, some have suggested that general population screening for fragile X be offered in the United States. However, a number of complexities in this screening make population-based implementation problematic. Among the difficulties in offering population-based screening are the complex multigenerational inheritance patterns, the variable phenotype of full-mutation carriers, the fact that 50% of females with full mutations have normal IQs and may have only subtle cognitive features, the phenomena of contraction or reversion when an individual who carries an expanded allele transmits a smaller allele to her offspring, and the significant number of women who would be identified with intermediate alleles. About 1 in 52 women would likely carry high-intermediate alleles with 45 to 60 CGG repeats. Although alleles containing fewer than 55 repeats are considered stable, exceptions have been reported where expansion has occurred,



making counseling very difficult for this potentially large group of women. Currently, fragile X screening is recommended in the preconception or prenatal setting based on family history indications. This would include a known family history of fragile X as well as a family history of unexplained mental retardation, developmental delay, autistic spectrum disorders, attention deficit disorders, and mental illness–personality disorders. A family history of premature ovarian failure, women under the age of 40 with elevated follicle-stimulating hormone levels, and a family history with late-onset tremor or ataxia of unknown origin should also raise clinical suspicion of fragile X [20]. Given the complexity of the phenotype of this disorder in relationship to DNA results, referral to a genetic counseling center experienced in prenatal genetics is recommended for this testing.

The role for preconception genetic screening Carrier screening for single-gene disorders is often or perhaps always best performed before pregnancy. In addition, evaluation of couples with known or possible genetic risk factors is also best undertaken before pregnancy. The identification of carrier status or other genetic risk can be very emotionally distressing, especially when this occurs during an ongoing pregnancy. In some families, assessment of genetic risk may involve obtaining medical records from affected individuals and genetic testing of multiple family members. The process of contacting family members, obtaining records, and analyzing DNA mutations can be very time-consuming and complex, easily taking 2 to 3 months to complete. When the evaluation is undertaken during pregnancy, the results of this testing may not be available until the late third trimester if at all. Resolution of these issues is much easier and less hurried without the time limits, stress, and emotional turmoil of an advancing pregnancy. Preconception carrier screening allows carrier couples to consider the fullest range of reproductive options. Knowledge of the risk of having an affected child may influence a carrier couple’s decision to conceive. Some couples, after appropriate risk assessment and counseling, may decide that reproductive risks are too high to undertake pregnancy. They would have the option of avoiding pregnancy and adopting as an alternative to undergoing pregnancy. Preimplantation genetic diagnoses are increasingly available. Using this technique with in vitro fertilization, embryos can be screened for the identified disorder so that only unaffected embryos are implanted. The technique, while expensive because of the need for in vitro fertilization, offers a possibility for couples at risk to have healthy children while avoiding the need for abortion [22]. Other options include receiving gamete donations (sperm or egg donor), pursuing adoption, seeking a standard prenatal diagnosis, or accepting the genetic risk without further testing.



Universal screening for birth defects in prenatal care Universal screening for birth defects is now a standard aspect of prenatal care. The two primary screening techniques are ultrasound and maternal serum screening. Recently, screening has focused on using an approach that combines these two modalities.

Chromosome aneuploidy screening Chromosome disorders affect approximately 1 in 500 newborns. Down syndrome is the most common chromosome abnormality at birth and the second most common birth defect requiring hospital care at birth in the United States [23]. Prenatal chromosome analysis is highly accurate in detecting chromosome disorders before birth using such diagnostic testing procedures as chorionic villus sampling in the first trimester and amniocentesis in the second and third trimester. These procedures, however, are expensive, invasive to the mother, and incur some degree of risk to the pregnancy. Given these factors, multiple strategies have been developed over the years to identify a population ‘‘at risk’’ that would have the most benefit from diagnostic testing. The association of Down syndrome with maternal age has been recognized since the early 1900s [24]. In the 1970s, amniocentesis became technically feasible in the second trimester and the risk was felt to be low enough to offer this testing to women age 35 and over. This age cutoff was chosen to represent 5% of the pregnant population, a 5% screen-positive rate (SPR). Maternal age, however, as a screen for Down syndrome has a low detection rated30%dand is much less effective than any of the current serum or ultrasound screening algorithms [25]. Despite this, the concept of maternal age as the primary screen for Down syndrome has become firmly imbedded in both the medical community and the lay community. Diagnostic testing with chorionic villus sampling or amniocentesis for women over the age of 35 represents the standard of care in many communities. In the 1980s, open neural tube defect screening with maternal serum alpha fetoprotein (MSAFP) was introduced to the United States and was officially recommended by the ACOG as a component of prenatal care in 1985. The association of low MSAFP with Down syndrome was recognized early in this screening program and led to the exploration of many different serum and urinary markers for Down syndrome screening. From 1986 to 1995, addition of the beta subunit of human chorionic gonadotropin (b-hCG), unconjugated estriol (uE3), and inhibin resulted in the double marker test (MSAFP and b-hCG), the triple marker test (MSAFP, b-hCG, and uE3), and quad marker test (MSAFP, b-hCG, uE3, and inhibin). Each additional biochemical marker increased the detection rates for Down syndrome, although adding both cost and complexity to the biochemical screening.



While the initial focus in biochemical screening was on the second trimester, a variety of first-trimester markers have recently become available. The most widely studied markers in the first trimester are free b-hCG, total b-hCG, and pregnancy-associated plasma protein A (PAPP-A). PAPP-A, a glycoprotein produced by the trophoblast, is reduced in Down syndrome and trisomy 18 (Table 4). This marker is only useful as a quantitative screen in the first trimester. In contrast, b-hCG is the one marker that has been found to be useful in both the first and second trimester. A major change in paradigm has been the use of ultrasound as a quantitative tool for first-trimester screening. Studies in the early and mid-1990s revealed a strong association between increasing nuchal translucency with increasing risk of Down syndrome and other chromosome abnormalities [26–28]. The nuchal translucency is an area of fluid collection at the back of the neck of the embryo that can be observed in the first trimester and quantitatively measured from the 11th week to the completion of 13 weeks. Quantitative measurements must be performed according to standardized techniques to achieve reproducible detection rates. Certification of competency and an ongoing quality assurance program are critical for practitioners performing quantitative nuchal translucency (NT) screening. Seventy-five percent of fetuses with Down syndrome were found to have a nuchal translucency greater than the 95th percentile [26]. When nuchal translucency measurements are expressed as multiple of the mean they can be incorporated into a screening protocol much like other biochemical markers.

Screening algorithms The availability of both first- and second-trimester serum markers for Down syndrome and the incorporation of ultrasound as a quantitative marker have resulted in a proliferation of strategies and algorithms that could potentially be used for clinical screening. Several large multicenter trials, now completed, attempt to address this issue and to establish optimal approach to screening (Table 5) [29–31].

Table 4 Relative changes in serum markers and nuchal translucency in trisomy 21, trisomy 18, and open neural tube defects

PAPP-A Free b-hCG Nuchal translucency Total b-hCG uE3 Alpha-fetoprotein Inhibin

Trisomy 21

Trisomy 18

Open neural tube defects

Y [ [ [ Y Y [

Y Y [ Y Y Y d

d d d d d [ d



Table 5 Efficacy of various Down syndrome screening protocols at a 5% screen-positive rate Screening test by trimester First trimester Maternal age Nuchal translucency measurement Combined nuchal translucency, PAPP-A, free or total b-hCG Second trimester Triple marker: MSAFP, uE3, total b-hCG Quad marker: MSAFP, uE3, total b-hCG, inhibin First and second trimester Integrated screen First trimester: nuchal translucency, PAPP-A (not reported) Second trimester: b-hCG, uE3, MSAFP, inhibin Serum integrated screen First trimester: PAPP-A (not reported) Second trimester: b-hCG, uE3, MSAFP, inhibin Stepwise sequential First trimester: nuchal translucency, PAPP-A, free or total b-hCG (reported) Second trimester: integrated screen Contingent sequential First trimester: nuchal translucency, PAPP-A, free or total b-hCG (reported) Second trimester: screening only for intermediate-risk group (no further testing for low-risk group)

Detection rate for trisomy 21 30% 64%–70% 82%–87%

69% 81% 94%–96%




Data from American College of Obstetricians and Gynecologists. ACOG practice bulletin. Clinical management guidelines for obstetricians-gynecologists. Screening for fetal chromosomal abnormalities. Obstet Gynecol 2007;109(1):217–27.

First-trimester only screening Screening in the first trimester using nuchal translucency alone detects 75% of Down syndrome fetuses using a 5% SPR. If nuchal translucency is combined with serum markers PAPP-A and free b-hCG, the detection rate for Down syndrome is higherd82% to 87% [30]. The detection rate for other aneuploidies, such as T18, T13, and 45X, is about 78%. These detection rates are similar or slightly higher than those for quad screening. The advantages of first-trimester screening are earlier diagnosis in affected cases and earlier reassurance for patients who are anxious. Another advantage of first-trimester screening is accurate pregnancy dating. False-positive screens due to dating errors do not occur in first-trimester screening programs, whereas dating issues are common in second-trimester screening programs and may double the false-positive rate in clinical practices where routine dating ultrasound is not standard. Limitations of first-trimester screening are that nuchal translucency measurements cannot always be obtained in all pregnancies, with maternal habitus most often being the limiting factor. Access to nuchal translucency–certified sonographers may also be a problem is some areas.



Nuchal translucency screening is the only screening methodology that evaluates each fetus individually. This is important in multiple gestations. In other screening methods, a single serum level is used to estimate risks for two or more fetuses. In dizygotic twins with one affected fetus, the serum marker production masks the abnormalities in the affected fetus. The estimated efficacy of second-trimester screening in twin gestations using a 5% SPR is only 47% [32]. In contrast, a recent study of 535 twin gestations using an SPR of 5% showed a detection rate of 83% for Down syndrome and 67% for trisomy 18 using nuchal translucency alone. When using nuchal translucency plus serum markers, 100% of Down syndrome and trisomy 18 cases were detected [33]. First-trimester screening with nuchal translucency and serum markers should be considered for twin gestations. For higher-level multiples, nuchal translucency alone is the only available screening modality available [34,35]. Abnormal first-trimester screening in chromosomally normal fetuses may have other implications for the pregnancy. Increased nuchal translucency has been associated with congenital heart defects [36], skeletal dysplasia, and other genetic disorders, as well increased risk of pregnancy loss, growth restriction, and stillbirth [34,37]. Follow-up of pregnancies with increased nuchal translucency and normal chromosomes includes detailed fetal structural ultrasound and echocardiogram, close follow-up for fetal growth, and careful examination of the neonate at birth. Low PAPP-A has also been associated with increased risk of stillbirth, particularly due to disorders associated with placental dysfunction, such as abruption and growth restriction [38]. Although clinical studies are lacking in terms of appropriate obstetric management, following closely for growth, hypertensive disorders, and fetal well-being may be reasonable. Second-trimester serum screening Second-trimester serum screening is performed between 15 and 21 weeks and most commonly involves measurement of MSAFP, uE3, and hCG (triple screen); or those three markers plus inhibin (quad screen). Recent prospective trials have demonstrated increased detection rates with the quad screen [31]. Therefore, for patients who initiate prenatal care after 14 weeks’ gestation, quad screening rather than triple screening is currently recommended [35]. Algorithms combining first- and second-trimester screening While first- or second-trimester screening alone have advantages in some select populations, several large studies have demonstrated that using an approach that integrates first- and second-trimester screening markers results in optimal detection rates while minimizing the SPR [30,31]. Various options for this type of combined first- and second-trimester screening have been proposed and are now commercially available.



Integrated screen Integrated screening involves combining two parameters from the first trimesterdnuchal translucency and PAPP-Adwith four parameters from the second trimesterdalpha-fetoprotein, hCG, uE3, and inhibin. These six parameters are analyzed using the maternal age as the a priori risk level. The screening result is not given until after the second-trimester blood draw. This algorithm provides the highest detection rate with the lowest screen-positive rate, 94% to 96% at 5% SPR. In areas where nuchal translucency measurement is not available or cannot be obtained because of technical factors, a serum-only integrated screen can be used. Serum-only integrated screening has been trialed in clinical practice and shown to have a Down syndrome detection rate of 79% to 87%. However, 13% of first-trimester samples were not usable because of dating errors resulting in 13% of the population receiving quad marker results only [39]. The major criticism of integrated screening relates to the lack of a first-trimester result. The majority of Down syndrome and trisomy 18 conceptions are identified in the initial component of the screening. Withholding a risk estimate in the first trimester prevents women from accessing first-trimester diagnosis. Access to pregnancy termination is increasingly unavailable in the second trimester and the procedure has increasing medical risks later into the second trimester. In addition, studies indicate increased maternal anxiety and lack of bonding to the pregnancy until testing results are known. It has also been suggested that withholding first-trimester information without the explicit consent of the patient may violate ethical principles involved in medical informed consent [40]. Sequential screening Sequential screening strategies are intended to address the issue of providing first-trimester results while maintaining the high detection and low SPR achieved by integrated screening. Stepwise sequential screening involves two steps. First-trimester nuchal translucency, PAPP-A, and free b-hCG are obtained. A first-trimester risk level is calculated. If the risk is very high after the first-trimester result, generally defined as 2% or greater, diagnostic testing is discussed and no further serum screening is performed. Less than 1% of the population would be in this group. For the remainder of the population, the first-trimester result is discussed. If they elect to continue with screening, a second serum sample is collected at 15 to 20 weeks for MSAFP, uE3, total b-hCG, and inhibin. Serum levels are then analyzed in conjunction with the first-trimester screen results (integrated screen analysis), and a final risk estimate is obtained. Stepwise sequential screening differs from integrated screening in that b-hCG is collected in both the first and second trimesters. This allows calculation of a first-trimester risk. Stepwise sequential also differs from simply ordering a first-trimester screen followed



by a quad marker screen in that the ďŹ rst- and second-trimester markers are analyzed together with a integrated computer algorithm. In quad marker screening, the a priori risk level is automatically assigned as the maternal age–related risk. However, this is not the correct risk in a population where ďŹ rst-trimester screening has already excluded most of the cases of Down syndrome. The patient’s actual a priori risk after ďŹ rst-trimester screening is not equivalent to her age-related risk. Ordering a quad marker test after ďŹ rst-trimester screening results in an inaccurate risk assessment and increased screen-positive rates [41]. Contingency screening While stepwise sequential screening has been shown to be highly eective with an acceptable SPR, the process of obtaining two sets of laboratory tests and two sets of calculations for each patient is expensive and timeconsuming. In addition, retesting patients with a very low risk level after the ďŹ rst-trimester screen may not signiďŹ cantly increase the detection rate for Down syndrome. Contingent sequential screening is an algorithm in which the second-trimester follow-up is contingent upon the ďŹ rst-trimester results. Contingency screening uses the same process as stepwise sequential. After ďŹ rst-trimester results are obtained, the results are discussed with the patient. In the very high risk group, diagnostic testing is discussed and no further serum testing is indicated. In contingent sequential screening a very low risk group is also deďŹ ned. In this group, the results of the ďŹ rst-trimester risk assessment are discussed and the patient is given the option of diagnostic testing. However, no further serum testing is performed. Depending on the low-risk cuto used, this group would encompass 70% to 75% of the population. For patients in the middle range, representing 25% to 30% of the population, serum is obtained in the second trimester for an integrated screening result. This approach has been suggested to be optimal in terms of screening costs and outcomes [42]. However, clinical studies are still pending to look at the feasibility of applying this somewhat complicated algorithm in routine clinical practice.

American College of Obstetricians and Gynecologists guidelines for chromosome screening Implementing chromosome screening into routine practice is challenging given the array of options and lack of consensus on the ideal screening protocol. In January 2007, the ACOG released updated clinical guidelines to address these uncertainties and suggest appropriate steps for implementing new algorithms in clinical practice. Some of these suggested guidelines for clinical practice are as follows:  All women who initiate care before 20 weeks’ gestation should be oered screening, regardless of maternal age.



 It is not necessary to oer all screening options to all patients. Each practice should identify the strategy that best meets the needs of its patients and uses resources available in the community.  Women who are seen in the ďŹ rst trimester should be oered a strategy that combines both ďŹ rst- and second-trimester screening. Women ďŹ rst seen in the second trimester should be oered quad screening.  Regardless of the testing selected, patients should receive information about detection rates, false-positive rates, advantages, and limitations so they can make an informed decision.  Risk levels should be communicated as a numeric risk rather than a positive/negative result.  Both screening and diagnostic testing should be available to all women who present for care before 20 weeks’ gestation, regardless of risk levels or maternal age [35]. Screening for neural tube defects Universal screening for neural tube defects was the original purpose of maternal serum screening programs and remains an important aspect of prenatal screening. MSAFP has been used as a universal screen for neural tube defects since the 1980s. The detection rate of MSAFP screening programs is 75% to 80%. The majority of encephaloceles and 30% to 40% of myelomeningoceles are missed. High-resolution ultrasound has been proposed as an alternative and better screen for neural tube defects. The sensitivity of ultrasound for the detection of neural tube defects has been reported to be as high 94% to 100% [41]. While the detection rate is expected to be higher in a perinatal referral center, one report looking at outcomes with routine ultrasound in a scanning center found a 96% detection rate [43]. Oering screening for neural tube defects is recommended for all prenatal patients [35]. High-resolution ultrasound is an alternative to MSAFP in practices where this is available. Diagnostic testing The goal of screening is to identify women who would beneďŹ t most from diagnostic testing and to decrease the need for invasive procedures, resulting in decreased cost and procedure-related losses. However, the concept of using set thresholds for oering diagnostic testing has been questioned. Screening does not detect all fetuses with aneuploidy and will not detect most fetuses with other types of chromosome disorders. The risk level for invasive testing is much lower in recent studies than has been traditionally reported [44,45]. In addition, women perceive their own risk as well as the risk of having an aected baby dierently and individually. This has led to questions regarding the ethics of using threshold-based cutos in that this may undermine the individual autonomy of the pregnant woman to



make her own best decision based on her personal perceived risk [46]. In recognition of these concerns, the ACOG currently recommends that all patients be informed of the option of diagnostic testing as an alternative to screening, regardless of maternal age of risk levels [35]. Future directions The rapid transition of genetic research from laboratory to clinical applications, driven in part by an aggressive biomedical industry, assures that the number of options available for directed and universal screening in preconception and pregnancy care will continue to expand. Discussions are currently underway for consideration of universal screening for fragile X, spinal muscular atrophy, and Tay-Sachs disease. The use of ultrasound as a screening and diagnostic tool will continue to expand. Other first-trimester and second-trimester ultrasonographic markers have been proposed and are being assessed for clinical utility. New laboratory techniques may enhance the diagnostic capabilities of chorionic villus sampling and amniocentesis. Comparative genomic hybridization (CGH) is being used increasingly as an adjuvant to standard karyotype to diagnosis microdeletion/duplication disorders or as a general screen in fetuses with anomalies where standard chromosome testing is normal. CGH testing, which is already being used postnatally, detects significantly more and smaller changes in the amount of chromosomal material in significantly less time than a standard chromosome karyotype would take and may eventually replace standard techniques for cytogenetic analysis. Finally, advances in in utero therapy and specialized delivery planning time are starting to provide options for selected disorders [47,48]. These advances may bring about a transition in the future from an abortion-based mindset to realistic treatment-based options for fetuses affected with congenital disorders. References [1] Silver RM, Varner MW, Reddy U, et al. Work-up of stillbirth: a review of the evidence. Am J Obstet Gynecol 2007;196(5):433–44. [2] Lazarou S, Morgentaler A. The effect of aging on spermatogenesis and pregnancy outcomes. Urol Clin North Am 2008;35(2):331–9. [3] Gross SJ, Pletcher BA, Monaghan KG. Carrier screening in individuals of Ashkenazi Jewish descent. Genet Med 2008;10(1):54–6. [4] Foundation CF. Annual Patient Registry Database. 2002. [5] Langfelder-Schwind E, Kloza E, Sugarman E. Cystic fibrosis prenatal screening in genetic counseling practice: recommendations of the National Society of Genetic Counselors. J Genet Couns 2005;14(1):1–15. [6] ACOG Committee Opinion. Number 325, December 2005. Update on carrier screening for cystic fibrosis. Obstet Gynecol 2005;106(6):1465–8. [7] Kere J, Estivill X, Chillon M, et al. Cystic fibrosis in a low-incidence population: two major mutations in Finland. Hum Genet 1994;93(2):162–6.



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Prenatal Diagnosis and Genetic Screening--Integration into P