Genetics And Fetal Development

Genetics and Fetal Development Michael Emerson, M.D.

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I.

Genetic Basis of Pregnancy A.

Chromosome abnormalities during pregnancy 1.

Origin: gametogenesis, fertilization, post-zygotic cleavage

2.

Consequences: preimplantation death, implantation failure, spontaneous abortion

B.

Classes of chromosome aberrations (CA) 1.

Aneuploidy

2.

Polyploidy

3.

Structural rearrangements

C. Aneuploidy during pregnancy 1. Aneuploid oocytes: 15-30% 2. Aneuploid spermatozoa: 3% 3. Fertilization-related CAs: 8%

There are three sources of chromosome abnormalities that occur during the course of pregnancy. Primarily during the formation of oocytes and spermatozoa during gametogenesis, during the course of fertilization with the fusion of the oocyte and the sperm and in the postzygotic period. The consequences of any kind of chromosome abnormality usually is on a preimplantation death, implantation failure, spontaneous abortion, stillbirth or an infant born with birth defects. 15% of the population identified in a clinically identified pregnancy spontaneously aborts of which 60-70% are due to chromosome abnormalities. We have now begun to define the incidence of chromosome abnormalities prior to the third week of gestation and have found surprisingly that a significant number of pregnancies are lost following implantation. It is estimated that as many as 70% of all conceptions that are identified through in vitro fertilization programs, for example, may be carrying a chromosome abnormality. The significance of that is such that one of my colleagues remarked that "it is a miracle that any one of us is in this room." Following the 12th week of pregnancy, in the second and third periods of gestation, the incidence of stillbirths may be 5-10% and again chromosome abnormalities play a significant role in each of these stages.

4. Aneuploid preimplantation embryos: 24% 5. Aneuploid first trimester embryos: 15-25% 6. Aneuploid 2nd and 3rd trimester fetuses: 7% 7. Aneuploid newborns: 0.3-0.5% 8. Structural congenital malformations: 4% 9. Mental retardation: 12%

There are three classes of chromosome abnormalities that we will briefly discuss with you. Aneuploidy which is defined as a gain or loss of single whole chromosomes. Polyploidy in which you have an additional set or sets of chromosomes. In the human, the chromosome complement consists of 23 different kinds of chromosomes so we have speak of triploidy with 69 chromosomes and tetraploidy with 92 chromosomes. Then there are instances where chromosomal rearrangements occur. The chromosomes physically break and heal or restitute in new forms or arrangements.

10. Congenital heart defects: 13% This is a normal karyotype in which to emphasize to you that indeed there are 46 chromosomes, 22 pairs of non-sex chromosomes or autosomes and an X and a Y constituting a male. It is possible for us using various kinds of stains to identify not only individual chromosomes from one another, but gains and losses of specific segments of chromosomes as well. Then you are looking at what constitutes G-banding in which they use trypsin and a Giemsa staining.

D. Types of CAs during pregnancy 1.

Aneuploidy a. 45, X: 28% b. Trisomy 16: 31% c. Trisomy 18: 5%

In the case of aneuploidy, and the next set of slides will briefly illustrate each of the classes of chromosome aberrations, there are three chromosomes and the total count is now 47 instead of the normal number of 46. This is the characteristic karyotype associated with trisomy 21 in which there is in all of the cells presumably an extra chromosome 21.

d. Trisomy 21: 8% e. Trisomy 22: 10% 2. Polyploidy a. Triploidy: 20% b. Tetraploidy: 8.6% 3. Structural rearrangements: 5.1% E.

Risk of trisomy conception following abortion of unknown karyotype 1. Risk of subsequent trisomy abortion: 4.5% 2. Risk of subsequent trisomic liveborn: 0.45%

F. II.

Risk of trisomic offspring subsequent to trisomic abortion: 0.5%

Chromosome Aberrations as a Cause of Congenital Malformations A.

Autosomal chromosome aberrations 1. Trisomy 21 (Down) a.

Incidence: 1 in 600-800 live births

b.

Risk increases with advancing maternal age

c.

Mechanism

of

origin:

nondisjunction and

nontranslocation d.

Triploidy. Each chromosome is represented 3 times throughout the entire complement and so the total chromosome number is 69 and this is an example of polyploidy. Another example of polyploidy is shown in this lower segment in which the total chromosome count is 92, an example of tetraploidy. Each chromosome has its own particular pair. Tetraploidy characteristically arises after the first postzygotic cleavage in which the chromosomes divide but the cytoplasm does not, so the chromosome number immediately goes from 46 to 92. The cell fails to divide, to form two daughter cells. You still have only one parental cell remaining but now the chromosome number has been immediately doubled. Invariably, this will lead to a missed abortion early in the first trimester although again, for all of the statements we make, there are exceptions. There have been a few, a small number of examples of diploid, tetraploid mosaic infants born with birth defects presumably associated with the fact that a portion of their cells now have a doubling of their chromosome number. Breakage of the chromosome at two particular sites on either of the two arms and the broken ends of the long part healing and forming this ring. It also means, of course, that pieces or segments of genetic material have been lost in the formation of this ring chromosome thereby leading to chromosome and genetic imbalance and will be associated with either spontaneous abortions, stillbirth or an infant born with a genetic abnormality.

Clinical findings: hypotonia, characteristic facies, cardiac malformations, duodenal atresia

Depending upon the nature and the appearance of the oocytes, it has been

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e.

Recurrence risks 1) In trisomic Down syndrome 2) In translocation Down syndrome

2. Trisomy 13 a.

Incidence: 1/2,000-4,000 live births

b.

Clinical findings: cleft lip and/or cleft palate, microphthalmia, polydactyly, parietal scalp lesions, cardiac and renal anomalies

3.

Trisomy 18 a.

Incidence: 1/2,000-4,000 live births

b.

Clinical findings: SGA, hypertonia, contractures, characteristic facies, short rib cage, overlapping 1st and 5th fingers, rocker-bottom feet, dorsiflexion of hallux

B.

Sex chromosome aberrations 1.

Gonadal dysgenesis (Turner) a.

Cytogenetic

findings:

4 5 , X , mosaicism,

isochromosome, and structural abnormalities b.

Clinical findings: short stature, webbed neck, low hairline, shield-like chest, increased carrying angle

2.

Syndromes which appear normal during gestation a. Triplo-X, 47,XXX b. Kleinfelter, 47,XXY c. YY males

C. Structural rearrangements 1.

Translocations: balanced and unbalanced

2.

Deletions and duplications: cri-du-chat and 5p-

3.

Inversions

D. Uniparental disomy (UD) and imprinting (I) 1.

UD: both chromosomes of a pair derived from one parent

2.

I: maternal and paternal genes differentially altered during meiosis

3.

Clinical implications: Beckwith Weidemann syndrome

III. Gene Mutations as a Cause of Congenital Malformations A. Pedigree analysis 1.

Autosomal dominant: vertical transmission

2.

Autosomal recessive: horizontal pattern of familial transmission

3.

X-linked: oblique pattern of familial transmission

B. Characteristics of autosomal dominant traits 1.

Variable in penetrance and expression due to: a.

Genomic imprinting

b.

Anticipation due to unstable DNA: myotonic dystrophy

c.

Mosaicism: osteogenesis imperfecta

d.

Somatic mutation: familial cancer

reported that at least 15-30% of oocytes carry a gain or a loss of a single whole chromosome or aneuploidy. In certain studies, this number could literally be doubled if one selects, and there is ascertainment bias in these studies, oocytes that morphologically appear to be more abnormal than the general population of oocytes. Spermatozoa, you will note, carry quite a significantly fewer percentage of chromosomally abnormal sperm. We have to recognize that the large contribution of chromosome abnormalities in the aneuploid class arise through the maternal line. During the course of fertilization and preimplantation of embryos… again you note the significant contribution that single gain or single loss of chromosomes constitute to embryonic development and these numbers drop off such that we know in the newborn period that 1 out of 200, at least, newborns is carrying a chromosomal abnormality that is going to significantly impair the quality and the quantity in many cases of the life of that newborn. When we begin to break down aneuploidy into the various kinds of aneuploidy, you will note certain features. For example, the most common chromosome abnormality is the loss of one of the sex chromosomes, either the X or the Y. Indeed, the major source of this loss, interestingly enough, is through the spermatozoa. The failure, about 75% of the cases is of a 45,X gonadal dysgenesis in the liveborn or Turner's syndrome. About 75% of those cases, interestingly enough, are through the spermatozoa. The failure of the X and the Y to be included in a spermatozoa. A chromosome abnormality that rarely, if ever, is seen in the liveborn population is trisomy 16. This is probably the highest particular form of a chromosome abnormality and not related to age as opposed to trisomy 21 and many of the other chromosome abnormalities. 45,X also is not related to the age of the mother in this particular case. On the other hand trisomy 18 and trisomy 21 and 22 are for the most part related to maternal age and also contribute to a significant portion of the population of chromosomally abnormal embryos. Triploidy is the second largest or third largest class. So if one were to try to classify which are the three most common chromosome abnormalities, one would have to say trisomy 16 an extra 16 with 47 chromosomes, 45,X in which one of the sex chromosomes are missing and triploidy. Later in this presentation, I will describe to you the origin and the breakdown of the origin of triploidy. Tetraploidy will constitute almost 9% of the different kinds of chromosome aberrations that exist in the chromosomally abnormal abortion population. So these are percentages of the 60 or 70% that occur with chromosome abnormalities in the first, second and third trimesters of pregnancy and about 5% will be these structural rearrangements. From a practical point of view I wanted to emphasize the following. Suppose one is counseling a woman who has had a spontaneous loss but chromosomal abnormalities were not performed. You do not know the chromosome constitution of that particular previous pregnancy and she asked the following question. What is the risk of a trisomy if I get pregnant a second time? The answer is two-fold. First, if it occurs, the next pregnancy is a spontaneous abortion, there is about a 4.5% chance that that second pregnancy which aborted, having had one previous one, is carrying a chromosome abnormality, specifically an extra chromosome. However, if she asks what is the chance having had a miscarriage before but not knowing the karyotype that she will have a liveborn with trisomy? I want to emphasize to you that that risk is 0.5% which in reality is not different from the risk to the general population. Suppose you know what the chromosome constitution was in that first pregnancy. She had a loss, a spontaneous abortion, karyotypes were performed and she is now asking you what is the risk of a trisomy in the subsequent pregnancy in the offspring and the answer again is 0.5%. When we talk to women who have had a liveborn with a chromosome aberration because now the data is different. If the woman had a liveborn with a chromosome abnormality and she was over 35, her risk is related to her age and not to the fact that she has had a trisomy offspring. However,

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C. Characteristics of autosomal recessive traits 1.

2.

Risk of being a carrier based on ethnicity: a.

Tay Sachs disease: 1 in 30 Ashkenazi Jews

b.

Sickle cell disease: 1 in 10 African Americans

c.

Cystic fibrosis: 1 in 20 in Caucasians

d.

Thalassemia: Greek and Italians

Carrier testing a.

Tay Sachs disease: hexosaminidase activity levels

b.

Sickle cell disease: hemoglobin electrophoresis

c.

Cystic fibrosis: DNA testing (up to 64 of >500 mutations)

d. 3.

Thalassemia: CBC and MCV profile

Prenatal diagnosis available for TSD, SS, CF and thalassemia (alpha, beta)

D. X-linked inheritance 1.

Fragile X mental retardation (FHR) syndrome a.

Most common genetic form of MR in males

b.

Atypical pattern of inheritance

c.

1)

20% of male carriers unaffected

2)

50% of female carriers affected

Gene defect identified on Xq27 1)

Increase in trinucleotide repeats, CGG

2)

All males and 50% of females with full mutation (>200 repeats) are mentally retarded

d.

a.

Diagnosis now by DNA analysis (not cytogenetics)

b.

Requires considerable genetic counseling

Multifactorial inheritance 1.

Characteristics: a.

Pedigrees do not follow Mendelian expectations

b.

Sex effect often observed: pyloric stenosis

c.

Recurrence risk depends on sex of affected patient and number of affected relatives

2.

d.

Relationship to affected relatives

e.

Severity of defect in relatives

Neural tube defects (anencephaly and spina bifida) a.

Factors in expression: genetic predisposition (role of ethnicity), environmental insult, and time

b.

Environmental insult appears to involve either abnormal folate metabolism or dietary deficiency

c.

Time: neural tube formation between 21 and 28 days post-conception

3.

Implications of multifactorial inheritance a.

Chromosome abnormalities. Chromosome aberrations as a cause of congenital malformations. This we should be able to go through in quick order. Your notes certainly are relatively complete and I will make certain specific comments to add to those notes. Basically, we are talking initially of autosomal chromosome aberrations. These do not involve the sex chromosomes and so we are speaking of trisomy 21, 13 and trisomy 18. These are the classic trisomies that occur in the abortion and stillbirth and liveborn population. Facies you see in Down syndrome have oblique palpebral fissures, triangulation of the mouth, the protruding tongue which is a reflection of underdevelopment of the cheeks. Hypoplasia of the processes that give rise to the oral cavity so that the tongue which is normal in size - it is not a large tongue - does not have enough space in the oral cavity to support itself. You are all familiar with the relationship between advancing maternal age and Down syndrome. It is interesting to point out that despite our intense effort of maternal serum screening and prenatal diagnosis, the incidence of Down syndrome is actually increasing in the liveborn population and there are several reports in the last few years documenting this. So the dotted curve is the distribution, if you will, for the general population and the shift to your right indicates the risk. So if the overall risk of Down syndrome is 1 in 800, then at age 30, where this is potentially achieved, by age 35 the risk has gone up almost 3-fold and certainly has gone up 10-fold by age 40. This again is distribution at the infant or neonatal period. The actual incidence, at the time of amniocentesis, is higher and the time at CVS it is even higher. So a woman's risk of Down syndrome is actually changing during the course of her pregnancy depending upon the time that you are doing your particular evaluation.

Permutation is first step before full mutation

2. Prenatal diagnosis of fragile X

E.

if she is a woman who had a trisomy offspring and is now coming to you for consultation with respect to a second pregnancy and she is less than 35 years of age, now her risk will be on the order of 1-2%. We are talking in general terms, not about specific chromosome abnormalities. So it does depend on the age of the woman when you are counseling with respect to this particular factor. Over 35, it remains what her original age-related risk of a chromosome abnormality. Less than 35, the risk is different.

Most cases sporadic

These are risk figures that have been developed empirically by simply observing groups of women of varying maternal ages and determining what their risk of a fetus is. So that at age 35-37, the risk of a chromosome abnormality, not just Down syndrome, approaches 1%. It is this figure that one introduces when one discusses cost benefit ratio or risk benefit ratios to women. Because the risk of amniocentesis is usually quoted, for example, at 1 in 200 and therefore the risk to a 35-year-old, at least the risk of a chromosome abnormality is at least 2 times the risk of losing the pregnancy as a direct consequence of the procedure itself. For age 40, the risk now is 2% and so if the risk of the procedure is 0.5% , there is indeed a 4 times likelihood of finding a chromosome abnormality as opposed to causing the loss of a pregnancy. It is these kinds of comparisons that one uses in counseling patients for prenatal diagnosis. The origin of these chromosome abnormalities is primarily nondisjunction. That is to say the normal chromosome number in a somatic cell is 46 and each chromosome is paired. During gametogenesis, be it oogenesis or spermatogenesis, this chromosome number is reduced in half from 46 to 23. The paired condition becomes unpaired and fertilization of a gamete by a second gamete containing the same number restores the diploid number, 46 in a human cell, and the fact that each chromosome again is represented twice. In approximately 95% of the cases of trisomy 21, this is what happens. During the course of gametogenesis, during myosis, the paired condition is retained and it actually happens primarily in myosis-1. If you recall, myosis is a two-step event and so the chromosomes fail to separate from one another and both members of the pair of chromosomes are incorporated into this gamete so that the chromosome number in the gamete is 24, not

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b.

When counseling, never say "never" or 1 in a million chance of recurrence

IV. Screening for Genetic Diseases A.

Carrier screening in pregnancy according to ethnicity

B.

Maternal serum alpha fetoprotein (MSAFP) 1.

Neural tube defects affect 1-2/1,000 pregnancies

2.

Prenatal diagnosis is possible in 95% of cases a.

Requires ultrasonography and amniotic fluid AFP analysis

b. 3.

Applied directly to high risk pregnancies

MSAFP screening available to all pregnant women a.

2-3% of MSAFP tests positive and 10% of these actually affected with open neural tube defect

b.

False positive associated with incorrect gestation, twinning, omphalocele, cystic hygroma, fetal demise, congenital nephrosis

c.

Protocol for MSAFP screening 1)

Best time: 16-18 weeks gestation

2)

Values >2.5 MoM requires ultrasound and amniocentesis

C. Multiple serum marker screening 1.

Down syndrome a.

AFP (low), unconjugated estriol (low), and human chorionic gonadotrophin (hCG)(high)

b.

Combine with maternal age, weight, race and diabetes status

c.

From 5-7% of multiple marker screening are positive

d.

Amniocentesis recommended when risk of Down syndrome >1 in 250 (which equals risk to 35 year old)

e.

Over 60% of cases of trisomy 21 detected

2.

False positive associated with adverse pregnancy outcome

3.

Trisomy 18 using multiple serum markers a.

0.75 MoM for AFP, 0.6 MoM for unconjugated estradiol, and 0.5 MoM for hCG

b.

Odds of being affected given positive result is 14 to 1, ie, in 14 such cases, one trisomy 18 detected

4.

5.

Triploidy (69 chromosomes) and multiple marker screening a.

Origins of extra set of chromosomes

b.

Low hCG (<0.5 MoM) indicative of maternal origin

c.

High hCG (>2.0 MoM) indicative of paternal origin

Low (<0.08 MoM) unconjugated estradiol suggestive of Xlinked ichthyosis

D. Ultrasonography in the detection of fetal malformations 1.

Trisomy 21

23, and fertilization will introduce the third member and hence the term trisomic. This is a classic example, of course, of aneuploidy. Trisomy 13. This occurs less frequently - 1 in 2,000 to 1 in 4,000 in the live birth. The primary reason for this is that it is a larger chromosome containing many more genetic elements that causes greater disturbance in embryonic development and hence the loss is significantly higher. Characteristically, they present with abnormal helix formation. Microphthalmia, cleft lip and palate, holoprosencephaly, polydactyly with hyperconvex nails, micropenis and cryptorchidism - undescended testes, classic example of rocker bottom feet and the possibility of looking at the scalp region and observing these parietal scalp lesions that are quite unique to trisomy 13. I point these features out because you have a responsibility in examining fetal development as well as liveborn to make certain observations. Many times, quite frankly, those performing evaluations of these fetuses fail to recognize something of this significant nature. Trisomy 18 results in a myriad of structural defects because we are dealing with a chromosome that now contains thousands of genes. So it is not unusual to find that these individuals have a very typical facies including a very prominent occiput, a pixie-like face, small ribcage, limited abduction of the hips, an unusual posturing of the hands in which the nails are hypoplastic and overlap in a characteristic fashion. There is dorsiflexion of the hallux of the big toe and also dermatoglyphics. The sweat pore pattern is unique that it is in the form of arches like arches in a door instead of the whirls and loops that are characteristic of a chromosomally abnormal pregnancy. You are observing again a montage of chromosome abnormalities. Emphasizing to you again the extra chromosome of 21 for Down syndrome, the extra chromosome for 18 in trisomy 18, the extra chromosome in trisomy 13 and also in this particular segment of the screen, the 45,X, the Triplo-X with 47 chromosomes and 3 X. The individual with an XYY sex chromosome constitution and XXY. I segregated these because they play relatively little role with the exception of the 45,X in the pregnancy population. You will not see these three characteristic chromosome abnormalities in the pregnancy population. You will see them in the liveborn and I want to emphasize if you do see them that is basically because they had a certain frequency of chance like anyone else but they are not present in any numbers in excess. So the sex chromosome aberrations, the focus is that of gonadal dysgenesis in which you can have a number of different chromosome constitutions all leading to gonadal dysgenesis of 45,X chromosomes. Mosaicism is very characteristic and in the 45,X, 46,XX individual about 25% of these will menstruate and have the potential of becoming pregnant. So it is important to distinguish these two classes. Isochromosomes in which the centromere divides in a way in which the two arms of the chromosome are identical with one another. So in an isochromosome, you either have duplication of the short arm or duplication of the long arm. It is not an uncommon chromosome abnormality but the chromosomes, instead of dividing longitudinally, divide horizontally and create two chromosome abnormalities, one with duplication of the short arm and one with duplication of the long arm. I want to emphasize again that there are a number of chromosome abnormalities which will appear normal during gestation and again I emphasize the Triplo-X, the 47,XXY Klinefelter and the so-called double males. Those individuals with two Y chromosomes. Gonadal dysgenesis and the appearance, the short stature, the webbing of the neck, the wide carrying angle, the lack of sexual development in terms of breasts and the fact that shortly after birth, gonadal dysgenesis occurs. So a zygote, a fertilized egg, containing 46 chromosomes and a pair of chromosomes may have a normal mitotic process occurring with a formation of two daughter cells each with 46 chromosomes. But then one of these daughter cells may have misdivision taking place so that one of the daughter cells now only has 45 chromosomes and one member of the pair

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2.

3.

a.

Nuchal thickening in first trimester

b.

Pelviectasis in second trimester

c.

Echogenic bowel

Trisomy 18 a.

Omphalocele

b.

Choroid plexus cysts

c.

Echogenic bowel

Trisomy 13 a. Holoprosencephaly

whereas this second daughter cell has the extra chromosome. So you immediately set up three cell lines. One in which the chromosome number is normal as shown here to the right with 46 chromosomes in a human and each pair and two daughter cells with chromosome abnormality gain and loss of a chromosome. In many cases, this will be lethal and so you will only see two cell lines but if this happens to involve the X-chromosome, then it is not unusual to find cells with 45,X, cells with 46,XX and cells with 47 Triplo-X if you will. Depending on the time in which this error occurs that will determine the distribution of the cells with the chromosome abnormality and potentially the impact. Whatever impact they do have will be modified by the presence of the normal cells in terms of 46 chromosomes and each chromosome is represented twice.

b. Cleft lip/palate c. Kidney anomalies 4.

V.

45, X a.

Nuchal thickening

b.

Cardiac malformation

Teratology and Fetal Maldevelopment A.

Definition of a teratogen

B.

Teratogenic effects include SAB, congenital malformation and behavioral dysfunction

C. Periods of greatest sensitivity to teratogen 1.

"All or none" period: up to 18 days post conception

2.

Period of greatest sensitivity: 21 to 56 days

3.

CNS sensitivity exists throughout pregnancy

D. Recognized human teratogens 1.

Maternal infections: CMV, rubella, varicella, toxoplasmosis

2.

Drugs: retinoic acid, valproic acid, thalidomide

3.

Radiation exposure: >5 rads

4.

Maternal disease: alcohol, diabetes, maternal PKU

D. Nonteratogens: Agent orange, caffeine, LSD, video display terminals, anesthetic gases

The lower corner of a structural change leading to gonadal dysgenesis in which there has been loss of the genetic material in the short arm of the Xchromosome. It appears as if there are genetic elements along the entire length of the X-chromosome, both in the short and the long arm, that affect normal gonadal formation. So it appears not to matter with respect to that aspect of development whether it involves the short arm or the long arm. However, there are differences in the phenotype of individuals who have short arm deletions versus those who have long arm deletions. These are individuals with gonadal dysgenesis. This is an individual with Triplo-X. Such individuals usually appear normal. They may be slightly taller. They have a higher incidence of learning disabilities and psychosocial problems in adolescence and adulthood but otherwise, the majority of them function very well in society. This particular individual also is phenotypically normal. Has four X chromosomes but unfortunately, such individuals are institutionalized, the few that have been described, because this is associated with developmental delays and retardation. 47,XXY, and the major feature is seminiferous tubule dysgenesis. I want to emphasize however that these are not found usually in the pregnancy population that you as obstetricians would be dealing with unless this would occur through a prenatal diagnosis. The third category is that of structural rearrangements. I have classified them into three groups. Translocations, in which genetic material has been moved from one place to another. Usually these are characterized as being balanced. So although there has been a shift in the genetic material, the total gene content is unchanged and hence this individual would be clinically normal. But if the shift has occurred and as a consequence of that, there is loss or gain of genetic material through this translocation, then it reverts back to the effects seen with aneuploidy in gains of whole chromosomes, loss of whole chromosomes. In this particular case, you have again an unbalanced karyotype but we are talking about segments of genetic material. There are instances of deletions and duplications which are self defining as well as inversions in which the order of the genetic material has been changed.

Let me present to you an example of Down syndrome in which the chromosome number is normal with 46 chromosomes. When one looks at the karyotype, one only sees two 21 chromosomes. However, on more careful inspection, one sees that chromosome 14 does not match the members of the pair and that there appears to be extra genetic material on the short arm of chromosome 14. This is a classic example of a translocation involving chromosomes 21 and 14. So this particular karyotype represents an individual with Down syndrome because they have three doses of the genetic material on chromosome 21, two as separate entities and the third in association with chromosome 14. The problem is that this particular chromosome rearrangement of 14 and 21 can be carried by one or the other parent. Here is a karyotype of a perfectly normal individual, normal in terms of their physical and mental development, but at risk for Down syndrome. Because as you can appreciate, there is only one 21 and the second 21 is attached to chromosome 14. This individual is balanced, has all the genetic material a normal cell has but the genetic material has been rearranged. This individual is normal but at reproductive risk for passing on this structurally altered 14 in 6

association with this chromosome 21 and that is the nature of these structural rearrangements. They place the prospective parent at reproductive risk in terms of the next generation. There has been breakage of genetic material in chromosome 14 and 21 and if you look carefully, they actually have two centromeres, one from 14 and one from 21. Apparently, genetically one of these centromeres is silenced and is functioning in terms of chromosome movement but basically it is a fusion of genetic material of the long arms of 14 and 21. The short arm, if any material is lost, apparently does not contain genetic information that is of any clinical significance. This contributes and attempts to explain why an individual carrying such a chromosome rearrangement is termed balanced and does not have any anomalies, be it physical or functional, associated with that chromosome rearrangement. There are a number of possibilities that can occur and I am simply going to outline them for you very quickly in this cartoon here. It is possible for this normal 14 to go with the normal 21 as is shown here. The reciprocal of that will be the structurally altered 14,21 that is shown here. It is possible for the structurally altered 14 carrying the 21 to go with the 21. Now you have a gamete in which you have a double dose as opposed to the normal circumstances, a single dose. A double dose is shown here of chromosome material from 21 and that sets up the unbalanced translocation Down syndrome pregnancy. The other possibility is that the two 14s go with one another as shown here and the other possibility is that the 21 would be by itself. So there are six possibilities. There are actually others but these are the six major possibilities. This last sequence, where the two 14s go together and the 21 is by itself, is extremely rare and I would like you to focus on and I emphasize to you that from a clinical perspective, these are the four major gametes that we are concerned about. Immediately, this particular gamete that lacks a 21, only has a 14 and is going to result in a loss and actually clinically a pregnancy that is not going to be clinically identified. In essence, we are really dealing with three clinical possibilities. One-third, in theory, should be a normal pregnancy. Normal in terms of one 14 and one 21 fertilized by a sperm, if this were carried by a female, for example, also carrying one 14 and a 21. So one-third of the live born, theoretically, should be normal physically and chromosomally, mentally and chromosomally. A third of the pregnancies should carry the balanced rearrangement like the parent. They should have only 45 chromosomes but be normal because there will be only two doses of 21 and two doses of 14 when fertilization takes place. So this particular gamete has all the genetic material present in this particular gamete except that they are together. A third of the pregnancies, theoretically, should have Down syndrome. Why? Because in this particular gamete, there are two doses of 21 genetic material. One- third, one-third, one-third. In reality, that does not occur. In reality, it does depend if the mother or the father is carrying this rearrangement. If it is the mother, then instead of 33% of Down syndrome, we only see about 10 or 11%. There is seemingly a selection pressure towards the formation of carriers and a reduction in the expectation from one-third to about 10-11% if it is a female. If it is a male, it turns out it is about 4 or 5% so these are certainly much higher than the general population but the theoretical expectation is not met. There is indeed a selection pressure, either at the gametic level or the zygotic level or the postimplantation period, we are not really sure where, against these chromosomally abnormal pregnancies and an enhancement, when we look at the distribution, of the carrier frequency. If one does chromosome analysis on women and their partners who have experienced three or more spontaneous abortions with or without normal liveborn, the possibility of detecting a structural rearrangement such as a 14,21 that I just described to you is about 4.7%. If the couple comes to you and have experienced two abortions, with or without normal liveborn, the incidence is about 2.5%. So it does change depending upon the history of the couple coming to you. But one should counsel them that there is approximately a 1 in 20 chance that one will find a chromosomal rearrangement that places them at increased reproductive risk for the formation of unbalanced gametes and the formation of unbalanced embryos and the possibility of liveborn with birth defects. Just to again illustrate the fact that chromosome abnormalities can involve segments of genetic material with significant 7

consequences, this is a dysmorphic child. You can appreciate certain features in terms of the overall appearance, for example. The filtrum, the space between the nose and the lips is increased as well as epicanthus is certainly emphasized. This is an individual that has a deletion in the short arm of chromosome 5 and these present in the newborn period with a characteristic cry. The cry of the cat - the cri-du-chat. But these can be picked up because there are changes in the formation of the larynx in association with this chromosome abnormality that appears to change the nature of their crying pattern. This is simply to emphasize to you that it takes two chromosome breaks, not a single break, to cause a reciprocal translocation of the filled and unfilled chromosomes to exchange genetic material. Broken chromosomes are sticky and they heal and many times they heal back in their original configuration. But if these two chromosomes are lying in juxtaposition to one another, they can exchange genetic material. For an inversion to occur, again, there must be two breaks and now the segment of genetic material must undergo a 180º rotation. You can appreciate that what they are demonstrating in this illustration is a change in the position of the centromere from being off center to being what we term metacentric. One way to identify the possibility that such an inversion is taking place. Most of the time, the inversions do not have any clinical consequences and when these are reported from the cytogenetics lab, it is important that one provides a clinical interpretation, not just a chromosome interpretation. Finally, in this sequence of events, I want to emphasize to you the role of a relatively new non-Mendelian pattern of inheritance. Again, characteristically, it does not matter if genetic material comes through the maternal or the paternal line. It matters that there is a mutation present and it does not usually matter if it comes from the mother or the father. What I am going to illustrate to you is two particular biological phenomena that violate that principle, if you will. One is called uniparental disomy and if we look at those words it simply means that two of the chromosomes have come from one parent. You have a pair of chromosomes, indeed, but somehow the pair came from only the mother or the father. So both chromosomes that appear come from one parent. How can that be? Well, presumably, these particular pregnancies began as trisomy pregnancies and if the extra chromosome was from the maternal line, then you had two chromosomes from the mother, one from the father and hence the trisomy. If there is a loss of one of these chromosomes, it is possible that the chromosome that is lost came from the parent that only contributed one of those three chromosomes. In this particular example that I am citing, it is from the father. This is called a fetal rescue. The term is beginning to be introduced into the literature and you end up with 46 chromosomes but through various genetic analyses, you can demonstrate that the two members of a pair of chromosomes came from one parent. It matters which chromosome we are talking about. If the extra set involves chromosome 15, you can have various kinds of syndromes, Prader-Willi and Angelman's syndrome depending upon whether it is maternal or paternal. If it is chromosome 21 and you have two maternal chromosomes 21, for the most part you may get some intrauterine growth retardation and small for gestational age, but in essence, you may get a perfectly normal individual. Imprinting, a second phenomenon, means it does matter where these genes come from. It does matter if they came from the mother or the father because as chromosomes pass through myosis and gametogenesis, the genetic information is being processed and certain maternal genes are being turned on and others being turned off and in a similar fashion, certain paternal genes. These same genes are being turned on and turned off. So at the time of conception and during embryonic development, the maternal and paternal genes are not equal. They may be actually complementary to one another. This is a child born with Beckwith Weidemann syndrome. This is a syndrome associated with gigantism - overgrowth so they have larger livers and spleens. Omphalocele - large tongues and large for birthweight. When we look at these particular children, it has become apparent that a significant number of them have two sets of genetic information from chromosome 11P15 meaning the 11th chromosome on the short arm… P for the short arm, petite if you will… and they do not have genetic information from the mother. The way in which they arise, I have mentioned before. Through trisomy or fetal rescue if you will. They may well have started out with three doses of genetic material from the parent. 8

Two from the father, one from the mother. The mother's chromosomes are not… the genetic segments in this area are not working and hence the filled in portion. The functional genetic information is from the father. Hence, you have too much information, if you will, in those simplistic terms and you end up with Beckwith Weidemann syndrome. What happens if you have two doses from the maternal line? It appears just to be a complement of that. You have dwarfism and there are mouse models that seem to mimic this particular situation and the attempts now are to identify the specific gene or genes that are involved. The point I am simply making to you is that genetic information through the mother and the father are not necessarily equal. This is not true of all of the genetic material, segments of genetic material. To have normal development requires that one of the genes, in this particular case, from the father is functioning and from the mother it is not. When you disturb that balance, you disturb the normal course of development and this is a form of imprinting and there are a series of genetic conditions associated with imprinting. Gene mutation as a cause of genital malformation and I want to emphasize to you pedigree analysis and certain characteristics of different patterns of inheritance. There is always a possibility that you may be presented with a history and one of the features of that is to attempt to determine what pattern of inheritance might be involved. This is an attempt to summarize that in simplistic terms. Autosomal dominant, the term simply means this. That both males and females are affected. Males by the filled-in square, females by the filled-in circle. Autosomal, meaning again both males and females are affected. Dominant means that you see the particular trait generation after generation. There is indeed a vertical transmission of the trait under consideration. In autosomal recessive conditions, you see the following. That the previous generation and subsequent generations are normal and so the affected individuals, you see a horizontal pattern of inheritance. You see two normal individuals who are carriers producing unaffected offspring, in this particular case a male, but autosomal means it could be a female. In X-linked recessive, the pattern of the pedigree is oblique so that one can see carrier females and affected males. So again, depending upon how the pedigree looked, one has not a vertical or a horizontal but an oblique pattern of transmission. These are major characteristics which are quickly useful, I believe, in making a determination of the possible patterns of inheritance. The term penetrance means that the gene is present and will or will not express itself. Penetrance means the degree or percent to which a gene which is present actually presents itself. So there are conditions like Marfan's in which if you had 100 known carriers of the gene, possibly only 90% of them would actually show the clinical features of the Marfan's syndrome. Different genes have different degrees of penetrance. Expressivity refers to the degree to which this particular gene expresses itself. Take a condition like polydactyly. Polydactyly is an autosomal dominant involving both males and females. The penetrance of that may also be about 7590% but there are individuals who have six fingers, there are individuals who have seven toes and that is an expression of the expressivity of this gene. That it varies in its expression and so people have different numbers of fingers and toes if you will. Osteogenesis imperfecta. Almost all of the cases of osteogenesis imperfecta are autosomal dominant. The expectation then is that we are going to see an affected with an affected offspring and it doesn't always work that way. As you can appreciate, the accordion-like appearance of the bones from numerous fractures and the beaded appearance of the ribs and the beaded appearance of the skull. So our expectation is that generation after generation will be affected with osteogenesis imperfecta and there is a 50% risk that an affected parent will have an affected offspring be it a male or a female. Germinal mosaicism. This particular parent in the germ line is carrying a mutation that produces osteogenesis imperfecta and this individual has up to a 50% risk of having an affected offspring even though this individual is normal. We estimate that about in 6% of the cases of osteogenesis imperfecta we are going to see normal parents. Our expectation 9

again is that generation after generation we will this. But this is an exception, if you will, but a defined exception. So it is possible that you cannot counsel on the basis of autosomal recessive. This is an autosomal recessive pattern or pedigree. Unaffected generation and a horizontal pattern to the affected. But this again, there are alternative explanations and one of them of course is the germ line mosaicism. In the autosomal recessive conditions, we are concerned about the carrier risk of gene mutations based upon ethnicity and you are familiar with sickle-cell disease and we will elaborate on that in a moment. Concern about carrier testing. Who should we be testing and how do we counsel them and who do we offer prenatal diagnosis, if you will? The carrier risk for various ethnic groups should be known to you. In the case of Tay Sachs disease among Ashkenazi Jews it is 1 in 30. Among non-Ashkenazi Jews it is 1 in 300. Sickle cell disease among African-Americans is approximately 1:10 and one can, sitting with a paper and pencil, figure out immediately what the incidence of this disorder is. Two persons of African-American ethnicity, each have a 1 in 10 chance so couples are at risk 1 in 100, 1 in 10 times 1 in 10. Since it is a recessive disorder, if they are both carriers, they have a 1 in 4 chance so we can quickly estimate that 1 in 400 individuals of African-American ancestry or ethnicity will be affected with sickle cell disease. You can apply these same approaches or calculations to Tay Sachs and cystic fibrosis. One in 20 among Caucasians, is a carrier for cystic fibrosis. One in 20 times 1 in 20 is 1 in 400. One in every 100 couples of Caucasian background is at reproductive risk for cystic fibrosis. Since they are both carriers, they have a 1 in 4 risk and therefore we would estimate in the newborn period that there will be 1 in 1,600. The number comes closer to 1 in 2,000 because they use a figure of 1 in 25. I deliberately use the 1 in 20. I think it is a more correct figure. Among individuals of Greek and Italian background from the Mediterranean area, we also must be concerned about thalassemia. Tay Sachs. As an example of a normal appearing child interacting with its environment at three to four months, gradually over the next few months, for reasons that are not known, it loses its ability to have contact with the environment and eventually at age 4 and 5, it is hospitalized because of recurrent infections, seizures and eventually dies. There is no treatment for this and we know the pattern of inheritance. Both parents are carriers and there is a 1 in 4 chance. Half of their offspring, of course, will be carriers like themselves. We can do carrier testing such that we can measure the value or level of hexosaminidase A, one of the forms of hexosaminidase, and we can demonstrate different values for noncarriers in heterozygotes and Tay Sachs disease. So effective has this screening pattern been among Ashkenazi Jews that the majority of newborns with Tay Sachs are of non-Jewish background. In the United States, about 25-30 children are born each year with Tay Sachs and virtually none of them are of Ashkenazi background even though their risk is 10 times higher than the non-Ashkenazi background. This 1 in 300 is actually obtained. One in 300 times 1 in 300 times 1 in 4 is about 1 in 360,000 and there are about 4.4 million births. So nonscreening among the non-Ashkenazi is the result of that. So one then is obligated to test the couple in which only one member is of Ashkenazi background and I think you do have that kind of responsibility. The problem with Tay Sachs is that the disease manifests itself three months, four months, after conception. In utero, the damage is already occurring in the central nervous system. This is an accumulation of the ganglioside associated with Tay Sachs disease so therapy is going to be virtually impossible with the technologies that we have available to us today. Since there is no treatment and they invariably die, prenatal diagnosis is characteristically applied. So this is a defined population. The testing is diagnostic. It is accurate. It is inexpensive. There is no treatment. Prenatal diagnosis is available and cost savings are an issue here in terms of hospitalization. These children spend a significant portion of their reduced life in the hospital. You are familiar with sickle cell disease. It is a defined population - the African-American population primarily but not exclusively. It's diagnostic. It is accurate. It is inexpensive but this is not a fatal disease. Prenatal diagnosis is available but not very frequently used. So one asks whether or not this would be cost effective in terms of a prenatal diagnosis. Certainly, I think most of you, if not all of you, are aware of the great harm that has been done because of the 10

stigmatization and the misinterpretation of the clinical significance of the carrier versus the affected status. Primarily, we have DNA technologies available to us. We know everything that is to be known about sickle cell disease but how to cure it. We know the molecular change, the single nucleotide base pair, we know the change in the amino acid, we know the change in the protein, we know how it brings about its affect. We can diagnose it as shown here by virtue of DNA technology, but we don't know how to cure it. Cystic fibrosis presents a similar problem. It is the most common, lethal disorder among Caucasians. The incidence, as I mentioned, is about 1 in 1,600 to 1 in 2,500. This number varies depending upon the ethnicity of the people being tested. It is estimated that 1 in 25 may be a carrier. Think about it. In the United States, there are more than 8 million carriers of cystic fibrosis. There are now nearly 600 different mutations in the CFG. How do you test for each of these 600 mutations? So most of the facilities providing the service will tell you that they will test 12 or 30, and although I don't want to do any commercials, there is a company that now does 70 of these. But what is the problem? You test for 70 and you get a negative result. This does not mean that that person tested is not a carrier. They may be for a CF mutation that you simply did not test for. Does that present a problem? It really does present a problem and that problem I am going to try to illustrate to you in this fashion. Suppose for example you do not do any testing for CF and in a sense neither parent is positive. It is a 1 in 2,500 risk that I have given to you before. Suppose you do testing and here is the dilemma. One of the parents turns out to be a carrier and the other one does not. Here you went ahead to try to "do the right thing". One turns out to be a carrier. What you have done is increased the risk to these parents and you have no way to resolve the nature of that risk. For example, if you were able to detect 75% of the mutations and one parent is positive, they have a risk now of about 1 in 400 of an offspring of CF. You have increased their risk from 1 in 2,500 to 1 in 400 by doing that carrier screening and you don't have a way out. That is the problem with carrier screening for CF. Fragile X chromosome. Is becoming a very important topic in obstetrics and gynecology. It turns out that the fragile X is the most common form of mental retardation in males and there is an atypical pattern of inheritance. It is not the classic X-linked recessive inheritance pattern that you are familiar with. Twenty percent of the male carriers are unaffected. These are nonexpressing males. They carry the gene mutation, if you will, and they are clinically normal. What happens here is that they can pass their genetically altered but clinically nonpenetrant chromosome to their daughters who now become obligate carriers and these nonexpressing males will have grandchildren with a fragile-X chromosome. That is part of the problem. Fifty percent of female carriers are affected. You are familiar with X-linked and carrier females with hemophilia and color blindness. They are not supposed to be affected. Duchenne muscular dystrophy for the most part. No clinical affect. Not true here and females will be affected. Basically what we have found in the majority but not all of the cases of fragile X is trinucleotide repeats. The elements making up the DNA code, you have CGG repeats in these individuals as I will show in a moment what this means, and there are degrees of repeats. These individuals present with a characteristic facies. Long facies, long nose, prominent ears. These are three brothers from the same family and they were originally described from a cytogenetic perspective. We don't use cytogenetics anymore but if you can appreciate where the circles are in this and this part you will see it is as if a piece of the long arm of the X chromosome is separated. As if it were broken off from the X chromosome and hence the name fragile. There are other fragile sites. This was the first one. Here is the normal event in which you have only 30 repeats. In the person who is affected with the full mutation, this sequence of CGG is repeated an enormous number of times, probably in excess of 200 and maybe several thousand times. Individuals who are carrying the mutation may not be affected, some are, but they are at reproductive risk for passing on this particular chromosome in which the number of repeats has been increased. So these individuals for the most part are unaffected but in the formation of egg or sperm, primarily egg - oocyte formation - a tremendous expansion of the CGG repeats occurs as is illustrated here. For some reason, this expansion is destabilizing in terms of normal development. 11

Again, I am not sure that you will appreciate this but the first column is a male, the second column is a female and what is shown to your right is the expansion as shown here. The numbers representing the increase in the number of nucleotide units. This even shows it more significantly where there are three classes. This is small in numbers of the units making up that particular gene. This is the full mutation indicating expansion and this is the premutation in which expansion has occurred but not fully. Just to demonstrate the numbers that are involved, here is an individual female who is a carrier. One of her X's has 74, one has 30 of these units and the affected male has greater than 700. Here is a male with 79 who is unaffected and has a female who seemingly also is unaffected but has 81 and therefore is at risk. Another carrier with less than 100 units having a number of children with less than 100 but here is an individual with 90 and expansion takes place. We don't know or understand this process. We can count the units by virtue of the DNA technology. When it occurs, where it occurs, how it brings about its effect is currently being studied. But we know that if you have 60 or less of these units in the maternal line that the chances of expansion is less than 1%. But if you have more than 90 of these units, these repeat CGGs, then the expansion is also almost 100%. That means almost 100% of the time they will have an affected male if they have a male and it means almost 100% of the time those females carrying the expansion will also be similarly affected. There is now a whole series of diseases in which disease expression is a consequence of expansion of trinucleotide repeats. Huntington's disease, for example, is a repeat of the CAG unit repeated many times to cause the disease and expansion occurs in these particular cases. Myotonic dystrophy is another disorder that you may be familiar with. Multifactorial inheritance. Again, we have to recognize that dominant inheritance, recessive inheritance, frequently as it occurs, there are numerous exceptions and there are pedigrees that do not follow Mendelian expectations. There are certain conditions in which one sex is affected more so than another. First born males characteristically present in pyloric stenosis so I would quickly ask you, suppose you see a female with pyloric stenosis. What does that mean in terms of the risk to that family? It turns out that the risk to that specific family is much higher than the risk to a family with a male with pyloric stenosis. Why? Because it is the wrong sex. If it is the wrong sex, it means that this couple is carrying more than the average amount of genetic information that is going to contribute to pyloric stenosis. If you will, it is harder for a female to have pyloric stenosis so if you do observe it, it really means is that this family is closer to the threshold. I wanted to emphasize not only the sex but recurrence risks. The counseling that you provide is determined by how many individuals are affected. The more individuals that are affected, the higher the risk of recurrence. The relationship. Is it to sibling, first degree, second degree relative meaning cousins, aunts and uncles and also the severity of the defect. For example, in cleft lip, is it unilateral? Bilateral? Is it the sex? Cleft lip is more common among males. Two-thirds of the time it is on the left side. So if it is bilateral in a female, it is the wrong sex, it is very severe, the risk to that family is much higher and it does change. These characteristics have to be taken into account when one is counseling. Again, I don't know how well this illustrated but this is the affected population with males and females to illustrate that this is non-Mendelian inheritance. This is the distribution in first degree relatives, siblings for the most part, if it is a male offspring. The distribution will be different depending upon the condition if it is a female offspring. So the risk of recurrence is usually in the order of 1-5% but certainly taking into consideration the sex, the degree of severity, the number of individuals, that risk can go as high as 25%. One striking example of multifactorial inheritance is that of the neural tube defects and this is to dramatize this. This is myeloschisis, if you will, in which both the posterior and anterior neuropores fail to close and the entire spinal canal has been exposed. We know that there are three elements responsible for multifactorial conditions such as an open neural tube defect. There is a genetic predisposition. There are certain groups of people that seem to carry more than the average number of genes. For example, we will focus in on neural tube defects, those from Wales and Ireland, northern Europeans. There is an environmental insult and you will hopefully have been reading about the role of folic acid and folate metabolism in the etiology of neural tube defects as well as the possibility that it has a role in congenital malformations and in cardiovascular disease as well. 12

The third element is time because the neural tube closes by day 28 following conception. It originates at approximately day 18 and is completed in its development by day 28. So if there is a genetic predisposition, then that insult acting on that genetic predisposition has to act at a certain point in time. If the insult occurs before or after 18 to 28 days, it should not have any impact. This is very, very important in terms of giving you a model by which counseling around congenital malformations have to operate. Maternal serum alpha fetoprotein and multiple marker screening as well as ultrasonography. Neural tube defects in the United States affect somewhere on the order of 1 to 2 per 1,000 pregnancies and it does vary among ethnic groups. It is characteristically much lower among African-Americans, much higher among people from Wales and Ireland and prenatal diagnosis, as you know, is possible. In terms of neural tube defects, when one performs an analysis for alpha fetoprotein, one finds that about 2-3% of those values are elevated and of those that are positive, approximately 10% are actually affected so one has to deal with false positives and false negatives. In terms of false positives, what contributes to an elevation of AFP that is not associated with a neural tube defect? The wrong time. Why the wrong time? Because alpha fetoprotein in the maternal serum is rising throughout the pregnancy until about 28 weeks gestation and then begins to drop. If you think the pregnancy is at 16 weeks when it is at 20 weeks, you have a certain expectation of that value at 16 weeks. But if indeed the pregnancy is 20 weeks, it is going to give you a false elevation because alpha fetoprotein again is rising during the course of the pregnancy. This is a quantitative test so twins and multiple gestation will increase the value of alpha fetoprotein in the maternal serum. It is estimated now that half of all twin pregnancies are detected through maternal serum alpha fetoprotein programs. Fetal demise and cystic hydroma. Alpha fetoprotein is produced in the liver, circulates in the fetal bloodstream and gets out through the skin and through urination. But if there is a fetal demise, the compartments now, these various fetal compartments, the amniotic fluid compartment have altered relationships and this stuff, AFP, simply leaks out in enormous quantities. The best time for screening is between 16 and 18 weeks. Why? I will show you in a moment and the question is, if the value is greater than 2.5 MoMs it requires at least offering ultrasound and amniocentesis and we can talk about that. Now, MSAFP is a screening test. A diagnostic test is a yes or a no but this is a screening test because there is overlap between the affected populations and the unaffected populations. We arbitrarily draw a line, and in this particular case and deliberately for points of examination, we drew the line at 2 MoM. We say that any value of AFP in the maternal serum greater than 2 will be considered at risk for a neural tube defect. Any value less than 2 will be considered as not at risk. What is the fallout? Well, if you draw this line arbitrarily, and it is arbitrarily at 2, you have a false positive rate of 4%. That means that 1 out of every 25 women that walk into your office will be considered at risk for a neural tube defect. The detection rate using 2 as a cutoff is 80%. This also means that you are going to miss 20% of the cases of spina bifida or of anencephaly just using this test alone. So in good conscious, you say to me, "Let's change the rules of this game. I don't like this 2 so let's move to 2. 5 where we are today." What is the fallout, if you will, of shifting the cutoff at 2.5. The false positive rate goes down to 2%. That part I think you like. That means only 1 out of every 50 women will be identified at increased risk. But what is the detection rate? It goes from 80% down to 70%. You are not going to miss two now, you are going to miss three. That point has to be gotten across not only to the practitioner but to the patient that you are counseling. I have to tell you that that dynamic has had a positive change. Two or three years ago this was not the case and prospective parents would come in hysterical because they were told or understood that they had a neural tube defect and offering ultrasound and amniocentesis was simply going to confirm that. That has changed and very much to the positive and I think that is because of the counseling that the obstetrician has been able to provide. Why do this test at 16 to 18 weeks? Why not at 12 weeks? Why not at 20 weeks? You can, but here it shows you the 13

empiric data why. The solid lines are the unaffected and neural tube defects and this is the overlap. That is 16-18 weeks, but if you do this test at 18-20 weeks, look at the dotted line. You can appreciate the difference between that part of the population, that overlap, and you are going to get, performing this screening test at 18-20 weeks, a greater degree of false positives. So it is to your advantage to do this screening at 16-18 weeks. I know that you don't control the patients admissions and entry to you but this is the data as to why you want to do this between 16 and 18 weeks. The differentiation between the affected and the unaffected population is better but not complete. That is why it is still a screening test, but it is better at this particular point in time. This shows you why ultrasound can be helpful because the dotted line is with ultrasound. The solid line is data based on the last menstrual period. Notice that if you use the first day of the last menstrual period, you again have a greater degree of overlap between the affected and unaffected populations and you have greater separation when you use ultrasound. So that is the first step. I will tell you in our experience that about half of the time when patients come in because they are told they are at increased risk for a neural tube defect, they had the wrong data and this is where ultrasound can be helpful. We use ultrasound, not the dates, as the final determiner. So multiple marker screening, if I can conclude this portion of the presentation with you, has been applied to Down syndrome, trisomy 18, triploidy, X-linked ichthyosis and adverse pregnancy outcome. Now I am going to talk about extending beyond alpha fetoprotein to the use of unconjugated estriol and human chorionic gonadotropin. This is an evolving story. We started with neural tube defects, then it was Down syndrome, now it is trisomy 18 and triploidy, Xlinked ichthyosis and adverse pregnancy outcome which hasn't been fully defined. I don't think that we have seen the end of the value of the multiple marker screening. You are familiar with this, I hope. Namely that when you look at alpha fetoprotein for Down syndrome, there is a shift to the left and that the median of this, if you draw a line and divide the affected population, is about 0.7 versus 1 for the normal population. This is the spina bifida. So a shift, a reduction in alpha fetoprotein has been associated with Down syndrome. It has also been found that unconjugated estriol has a similar distribution. The reason that we can use unconjugated estriol is because alpha fetoprotein is produced in the liver. Unconjugated estriol is originally synthesized in the adrenal glands and processed in the placenta but it is an independent organ system that has been involved and it too has this shift to the left as shown here. Let me say in terms of words that the hCG is increased in Down syndrome so that the median value is about 2.2 for that population. hCG turns out to be the most accurate predictor of Down syndrome if you had to use any one of the three. So basically there are seven elements that go into characterizing the risk of Down syndrome to women and this is usually, as you know, applied to women less than 35 years of age. It is alpha fetoprotein, unconjugated estriol and hCG which is a placentally derived biochemical parameter and you combine that with maternal age, because the risk of Down syndrome increases with advancing maternal age with the weight. Thin women concentrate, heavy women dilute, particularly, alpha fetoprotein. For reasons that we are not aware of or can explain, African-American women have about a 10% natural increase in alpha fetoprotein. So, you either have to have two curves or you have to have a correction factor. Women who are insulin dependent also have alterations in alpha fetoprotein and you introduce a correction factor. About 5-7% of the tests are positive and we recommend that when you combine all of these seven parameters, if the risk is greater than 1 in 250, you offer them amniocentesis. We can with this approach detect somewhere on the order of 60% of the cases of trisomy 21. Again emphasizing that 40% of the time, you are not going to detect this and it is not unusual, unfortunately, for a woman to have this screening test and possibly deliver a child with Down syndrome. There have been lawsuits with respect to this. Fortunately, the judges have understood the nature of the screening tests. A second application of multiple marker screening is to take certain multiples of the median, because they are predictive of trisomy 18, and these are fixed. Alpha fetoprotein of 0.75, 0.6 for unconjugated estriol and 0.5 for hCG. Then the OAPR, the odds of being affected given a positive result, is 14 to 1. This means you would do 14 amniocenteses and 14

get one case of trisomy 18. That is a better OAPR than when you apply amniocentesis to 35 year old women for Down syndrome. The OAPR there is 100 to 1. You do 100 amnios and you detect one case of trisomy 21 prenatally. Here, the OAPR is 14 to 1 and if, as is being developed, variable MoMs are applied for AFP, unconjugated estriol and hCG, the OAPR will be 10 to 1. You will only do ten amnios and get 1 case. Triploidy. Triploidy arises in three major ways. You can have two sperm entering an oocyte simultaneously - dispermy and so you have 69 chromosomes. You can have an unreduced sperm in which the chromosome number in that sperm is 46, not 23, and it appears as larger morphologically. That occurs about 25% of the time and about 10% of the time you have an unreduced oocyte. Myosis or oogenesis fails and the oocyte has 46 chromosomes. Now it matters where the extra set of chromosomes come from in terms of these values. Here is a triploidy that resulted in a spontaneous abortion. A very small reduced embryo, a very large hydropic placenta and this has 69 chromosomes. Triploidy is a very large embryo with a very small placenta. The level of hCG indicates the origin of the extra set of chromosomes. If the hCG is low, then the extra set is maternal in origin. If the hCG is high, then the extra set is paternal in origin. This is the classic example of imprinting. It is the same genetic material from the mother and the father but it has been processed and the information and the expression of the genetic information from the father is contributing to placenta formation. The genetic information coming from the maternal line is contributing to the embryonic development. If you have too much, you have an extra set of genetic information from the paternal that is contributing to the placental formation, you get too much, too large a placenta. If you have extra genetic material coming from the maternal line, the maternal line emphasizing embryonic development, you get too much of the embryo and too little of the placenta. So the levels are telling you not only the possibility of triploidy, but they are also telling you where the extra set may well have come from. The relationship of genetic disorders in ultrasonography. It plays a role much broader than genetics. But trisomy 21, when one observes nuchal thickening in the first trimester, pelviectasis in the second trimester - controversial, an echogenic bowel, one has to raise with the couple the possibility of trisomy 21. Omphalocele is an easy observation to make but controversial is the role of choroid plexus cysts and there are controversies in the field as to the significance of choroid plexus cysts in association with trisomy 18. Holoprosencephaly, cleft lip and palate. These are relatively easy to distinguish with ultrasonography and raise the issue of trisomy 13. Nuchal thickening and any cardiac malformation raises the issue of 45,X. These are increased in the nuchal thickening, pelviectasis and our data, in terms of data for pelviectasis, is almost 2% now as opposed to other studies showing only a risk of 1 in 385. Echogenic bowel is very important. You have to raise three elements with echogenic bowel. Chromosome aberration up to 25%, cystic fibrosis up to 25% rounding off and intrauterine fetal infection, CMV and toxoplasmosis and finally an adverse outcome in about one-third of the pregnancies associated with echogenic bowel. The major cause of echogenic bowel is the fetus swallowing blood. We have observed about 4 pregnancies out of 100 in which there were no other ultrasound findings except choroid plexus cysts in trisomy 21 and trisomy 18. But we also counseled the patient that this is what has not been observed and I don't know how you get around that particular conflict at this particular point in time. Types of teratogens. Drugs taken by mothers, infection, radiation is a big problem and you have to monitor what the level of exposure is. Most of the time, despite the fact that there may be a series of x-rays, they never exceed 5 rads and therefore the pregnancy is not at increased risk. Hypoxia and hypothermia. Temperature of 102º for more than three days has been associated with neural tube defects so it matters when the exposure takes place. What was the length of the fever? What was the degree of the fever? The timing of where defects occur is very important and to explain relationships between limb and heart defects that occur simultaneously in an affected fetus or embryo is because the developmental sequence for these two independent organ systems is occurring at the same time. Many agents that we are concerned about are not teratogens. Not teratogens. That doesn't mean that they don't have an effect on the individual who is exposed but coffee, aspirin, diagnostic radiation and particular video displays, but that 15

they are not associated with birth defects, despite reports to the contrary.

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