Fig. 1.19. MSAFP level in the normal and NTD populations
Table of Contents- Lesson 1 ) (Next)
(Glossary)
CLINICAL DIAGNOSIS
There is a large number of multiple congenital anomaly
(MCA) syndromes whose diagnosis is based on gestalt-a clinical
impression that quickly comes to mind on seeing the patient. The
diagnosis of achondroplasia, for instance, rests solely on the
clinical examination that may include anthropometric measurements
and x-rays. There are no biochemical tests needed for confirmation.
When making a clinical diagnosis, problems arise
in situations where there is a continuum between the normal and
abnormal phenotypes. Autosomal dominant traits can be mild, moderate,
or severe. On the mild end of the spectrum, the diagnosis can
be questionable. For this reason, criteria have been established
for the diagnosis of certain disorders. As an example, the diagnosis
of neurofibromatosis requires two or more of the following
features: six or more cafe-au-lait spots greater than 5 mm in
diameter, axillary freckling, two or more neurofibromas or one
plexiform neurofibroma, optic glioma (tumor within the eye), Lisch
nodules (small tumors on the iris), bone lesions, or a first degree
relative with neurofibromatosis. Often observation over time is
required to establish a diagnosis. Included in Appendix D is a
fact sheet on NF discussing diagnosis, genetic counseling and
follow-up. Fact sheets are useful for patient reference and for
understanding the need for follow-up care.
SCREENING TESTS
When available, laboratory tests can be invaluable
in establishing a diagnosis or identifying individuals who carry
genes coding for adult onset dominant or recessive genetic conditions.
Screening tests for specific genetic disorders have as
their primary goal early diagnosis which makes timely treatment
or prevention possible. Newborn screening for PKU
is a well known example of a screening test. Phenylketonuria has
an incidence of 1 in 10,000 to 1 in 15,000. If untreated, 95%
of affected individuals will develop moderate to severe mental
retardation. With dietary restriction of phenylalanine, virtually
all patients with PKU will be mentally normal.
The Guthrie test (bacterial inhibition assay) is
one of the tests used to determine the phenylalanine level in
the blood. Blood on filter paper is placed on agar plates with
a strain of bacillus subtilis that requires phenylalanine for
growth. The presence of growth is indicated by a halo surrounding
the filter paper. If positive, blood phenylalanine and tyrosine
levels are determined, and if elevated, a confirmatory assay for
phenylalanine hydroxylase is done. Since phenylalanine is an essential
amino acid (not produced by the body and comes from dietary sources),
it is essential for a baby to be fed prior to testing. Ideally
this test should be done on the second or third day of life. It
is important to realize that this test is a screening test done
to identify elevated phenylalanine levels. It is not diagnostic,
as hyperphenylalaninemia can be reversible, transient, or secondary
to an elevated level of tyrosine, or other variants unrelated
to classic PKU. In PKU the phenylalanine level is usually 20-40
mg/dl in comparison with normal levels of 4-6 mg/dl. On the average,
1 in 20 babies with positive screens will have classic PKU. PKU
is one of the single gene disorders that can be diagnosed based
on the presence of an elevated precursor protein or substrate.
Newborn screening programs are usually sponsored
by the state and include tests for such conditions as PKU (Guthrie
test), galactosemia (transferase assay), congenital hypothyroidism
(T4, TSH determination), and hemoglobinopathies such as sickle
cell anemia (isoelectric focusing or DNA diagnosis).
Prenatal screening, specifically maternal serum alpha
fetoprotein (MSAFP) screening or amniotic fluid chromosome analysis,
seeks to identify fetal pathology. The level of MSAFP is elevated
in neural tube defects (NTDs) and low in certain chromosomal disorders.
Since around 95% of NTDs occur in families with a negative family
history and since NTDs and chromosome anomalies are serious disorders,
MSAFP screening is now considered standard of care. This is further
discussed in Lesson 3 on Genetic Services.
Alpha fetoprotein is made in the fetal liver and
finds its way through the placenta to the maternal circulation.
MSAFP determination is done in mid-trimester (15-20 weeks gestation).
The level of MSAFP increases with gestational age, so the test
result is expressed in multiples of the median (MoM) with 1 MoM
considered normal, 0.5 MoM half of normal, and 2.5 MoM 2 1/2 times
normal.
It is important to emphasize that MSAFP is a screening
test. The increase in MSAFP that occurs with NTDs is presumably
dependent upon the size of the lesion. For this reason, the MSAFP
level in the normal population overlaps the MSAFP level in the
NTD population.
Fig. 1.19. MSAFP level in the normal and NTD populations
An arbitrary cutoff point has been drawn at 2.5 MoM.
Values of 2.5 MoM or more are considered elevated (to the right
of the dashed line). Any value less than 2.5 MoM is considered
normal (to the left of the dashed line). Clearly, problems arise
where the two populations overlap resulting in false positive
(and patient anxiety) and false negative (and false reassurance)
results.
Sensitivity refers to
the proportion of individuals who are diagnosed as affected among
the total population of affected individuals (true positive divided
by true positive plus false negative). With a sensitivity of 90%
for anencephaly, 90 will be to the right of the cutoff point and
correctly diagnosed, 10 will be to the left of the cutoff point
and misdiagnosed. Specificity refers to the proportion
of individuals diagnosed as normal over the total population of
normal individuals (true negative divided by true negative plus
false positive).
Sensitivity and specificity are opposite concepts.
If the cutoff point (the dashed line) is moved to the left, to
2 MoM, this would increase the sensitivity by decreasing the number
of false negative tests. However, this would lower the specificity
by increasing the number of false positives. The national consensus
appears to be a cutoff point at 2.5 MoM. This concept of sensitivity
and specificity is important in the understanding of the inherent
false positives that can lead to increased patient cost and anxiety,
and false negatives that can lead to misdiagnosis.
This concept of sensitivity and specificity is important
in practically all testing situations. In PKU, where a misdiagnosis
has grave consequences, the screening test is set up so that the
sensitivity is 98% when the test is performed at two days of age,
following a regular diet, and approaches 100% at two to four weeks
of age. In contrast, the sensitivity is 84% when the test is done
at less than 24 hours of age.
CARRIER TESTS
Carrier testing for a
gene disorder has as its primary goal to establish the carrier
status of an individual or a couple so that they can exercise
reproductive choice. These tests are often directed at particular
ethnic groups (e.g., Jews for Tay-Sachs (TS) disease, Yupik
Eskimos for congenital adrenal hyperplasia (CAH)), who by virtue
of their ethnic background are at high risk for being carriers
of these conditions. Among Ashkenazi Jews, 1 in 30 are carriers
for TS; and among the Yupik Eskimos, 1 in 10 are carriers for
CAH. Carrier testing is also instigated by a positive family
history such as Duchenne muscular dystrophy, fragile X syndrome,
or certain chromosomal disorders.
The methods utilized to test for the above conditions include: biochemical tests for Tay-Sachs disease (hexosaminidase A), and congenital adrenal hyperplasia (21 hydroxylase); DNA testing for Duchenne muscular dystrophy (direct DNA mutation analysis), and fragile X syndrome (Southern blot hybridization or PCR); and karyotype analysis for carriers of chromosome abnormalities.
Knowing the carrier status of one or both parents
can be critical to reproductive choice. There are a number of
options available to couples who carry abnormal genes or chromosomes,
including adoption or choosing not to have children. Some couples
may choose to have children and take the chance that they will
be affected. Others may opt for prenatal diagnosis and pregnancy
termination. Some couples may choose prenatal diagnosis so they
can make arrangements to deliver at a hospital where immediate
treatment is available. Artificial insemination with a normal
donor egg or sperm is another option some at-risk couples may
choose.
PRESYMPTOMATIC DIAGNOSIS
A third type of genetic testing is presymptomatic
diagnosis which has as its primary goal to identify the presence
of an abnormal gene in at-risk, but otherwise healthy, adults.
These individuals come to clinical attention because of a positive
family history of a genetic disorder. A classic example is Huntington
disease (HD). HD is an autosomal dominant trait with an average
age of onset between the fourth and fifth decades. The gene has
been mapped to the short arm of chromosome 4p16.3. The HD gene
has also been cloned and the abnormality identified as an expansion
of a trinucleotide repeat sequence. The sequence of bases, CAG,
is repeated in tandem 11 to 34 times in normal individuals. Thirty-nine
or more copies of CAG are present in HD individuals. Individuals
at risk can seek presymptomatic diagnosis prior to starting a
family.
Presymptomatic diagnosis is also available to families
at risk for autosomal dominant breast cancer. Again, these
individuals come to clinical attention because of a positive family
history of breast and/or ovarian cancer. The BRCA1 breast cancer
gene has been identified on chromosome 17q. The gene for BRCA2
is on chromosome 13q. Individuals who carry the abnormal gene
have an 80-90% lifetime risk of developing breast/ovarian cancer.
Individuals may seek presymptomatic testing so that those carrying
the gene can be more vigilant with breast examination and mammography,
or alternatively opt for prophylactic mastectomy or oophorectomy.
The diagnosis of Huntington disease and breast cancer involves
DNA studies which will be discussed in the next section.
Presymptomatic diagnosis and, to a lesser extent,
carrier testing, have numerous psychological, social, ethical,
legal, and financial repercussions and need to be approached with
caution. For this reason, there is a national consensus protocol
for presymptomatic diagnosis of Huntington disease. This is a
multi-step process requiring four to five clinic visits with involvement
by geneticists, psychiatrists, neurologists, psychologists and
social workers (Appendix D).
Finally, confirmatory diagnosis is provided to the individuals seeking consultation. This can involve chromosomal, biochemical, or molecular DNA studies, as indicated by the individual cases.
SUMMARY
Some genetic disorders, such as achondroplasia,
can be diagnosed based solely on the results of a physical examination.
Other disorders are diagnosed because affected individuals have
elevated or decreased levels of certain proteins or enzymes. More
recently, techniques have been developed to analyze DNA segments
and genes.
In addition to establishing a diagnosis, some
laboratory tests are used for the purpose of screening the general
population and identifying those persons who might benefit from
further studies. In serum alpha fetoprotein testing during pregnancy,
only women with abnormally high or low levels of MSAFP are referred
on for further testing.
In a few cases, tests are available to determine
carrier status for a particular recessive genetic condition such
as Tay-Sachs disease, CF or fragile X. DNA studies have also been
helpful in identifying individuals at risk for adult onset disorders
prior to the onset of symptoms.
Carrier testing and presymptomatic diagnosis are
not without risks. Verifying a person's carrier status for a particular
disease may change his or her outlook on life, family relationships,
reproductive choices, employment opportunities, access to insurance,
etc.
PRACTICE ACTIVITY 6
1. List 3 reproductive choices available to couples
who carry genes coding for
Tay-Sachs disease.
2. List 2 possible drawbacks to presymptomatic testing
for Huntington disease.
PRACTICE ACTIVITY 6: ANSWERS
1. The list may include any of the following: not
having children, adoption, choosing to have children and taking
the chance they will be affected, opting for prenatal diagnosis
and terminating a pregnancy with an affected fetus, opting for
prenatal diagnosis and preparing to deliver at a hospital where
immediate treatment is available, or artificial insemination with
a normal donor egg or sperm.