Download Newborn screening programs

SLE
• HLA class I, class II, and class III are associated
with SLE, but only classes I and II contribute
independently to increased risk of SLE
• Other genes which contain risk variants for SLE
are IRF5, PTPN22, STAT4, CDKN1A, ITGAM,
BLK, TNFSF4 and BANK1. Some of the
susceptibility genes may be population
specific
1
screening
• Firstly, there is screening of individuals and
couples known to be at significant or high risk
because of a positive family history-sometimes
referred to as targeted, or family, screening
because it focuses on those most likely to benefit.
This includes carrier, or heterozygote, screening,
as well as presymptomatic testing.
• Secondly, there is the screening offered to the
general population, who are at low risk-this is
sometimes referred to as community genetics.
2
Screening
• Population screening involves the offer of genetic
testing on an equitable basis to all relevant
individuals in a defined population.
• Its primary objective is to enhance autonomy by
enabling individuals to be better informed about
genetic risks and reproductive options.
• A secondary goal is the prevention of morbidity
due to genetic disease and alleviation of the
suffering that this would impose.
3
SCREENING THOSE AT HIGH RISK
• If it was easy to recognize carriers of
autosomal and X-linked recessive disorders
and persons who are heterozygous for
autosomal dominant disorders which show
reduced penetrance or a late age of onset,
much doubt and uncertainty would be
removed when providing information in
genetic counseling.
4
CARRIER TESTING FOR AUTOSOMAL
RECESSIVE AND X-LINKED DISORDERS
• In a number of autosomal recessive disorders, such as some of the
inborn errors of metabolism, e.g. Tay-Sachs disease, and the
hemoglobinopathies, e.g. sickle-cell disease, carriers can be
recognized with a high degree of certainty using biochemical or
hematological techniques such that DNA analysis is not necessary.
• In other single-gene disorders, it is possible to detect or confirm
carrier status by biochemical means in only a proportion of carriers,
e.g. the presence of abnormal coagulation studies in a woman at
risk of being a carrier for hemophilia.
• A significant proportion of obligate carriers of hemophilia will have
normal coagulation, however, so that a normal result in a woman at
risk does not exclude her from being a carrier.
5
Occasionally, carriers for certain disorders can have mild clinical
manifestations of the disease
6
BIOCHEMICAL ABNORMALITIES IN
CARRIERS
• In some disorders the biochemical abnormality seen is
a direct product of the gene and the carrier status can
be tested for with confidence. For example, in carriers
of Tay-Sachs disease the range of enzyme activity
(hexosaminidase) is intermediate between levels found
in normal and affected persons.
• In many inborn errors of metabolism, however, the
enzyme activity levels in carriers overlap with the
normal range, so that it is not possible to reliably
distinguish between heterozygote carriers and those
who are homozygous normal.
7
Indirect
• A grossly elevated serum creatine kinase (CK)
often confirms the diagnosis of DMD in a boy
presenting with features of the disorder.
• Obligate female carriers of DMD have, on
average, serum CK levels that are elevated
compared to those of the general female
population. There is, however, a substantial
overlap of CK values between normal and
obligate carrier females.
8
9
Random inactivation of the X
chromosome
• There is another reason for difficulty with
carrier testing in the case of X-linked recessive
disorders. Random inactivation of the X
chromosome in females means that many,
often the majority, of female carriers of Xlinked disorders cannot be reliably detected by
biochemical methods.
10
PRESYMPTOMATIC DIAGNOSIS OF
AUTOSOMAL DOMINANT DISORDERS
• Many autosomal dominant single-gene disorders
either have a delayed age of onset or exhibit
reduced penetrance.
• The results of clinical examination, specialist
investigations, biochemical studies and family
DNA studies can allow one to predict whether a
person has inherited the gene before the onset of
symptoms or signs. This is known as
presymptomatic diagnosis or predictive testing
11
CLINICAL EXAMINATION
• It is not unusual to examine an apparently unaffected relative of
someone with NF1 who has had no medical problems, only to
discover that they have sufficient numbers of a diagnostic feature,
such as café-au-lait spots or cutaneous neurofibromas, to confirm
that they are affected. However, NF1 is a relatively rare example of
a dominantly inherited disorder that is virtually 100% penetrant by
the age of 5 or 6 years, with visible external features. With many
other disorders clinical examination presents greater challenges.
• Reaching a diagnosis in Marfan syndrome can be notoriously
difficult because of the variable features and the overlap with other
joint hypermobility disorders, even though very detailed diagnostic
criteria have been established.
• Poly cystic kidney
• Tuberous sclerosis
12
• In TSC imaging studies of the brain by CT scan
to look for intracranial calcification is a more
or less routine investigation, as well renal
ultrasound to identify the cysts known as
angiomyolipoma(ta). Use of these relatively
non-invasive tests in relatives of persons with
TSC can detect evidence of the condition in
asymptomatic persons.
13
SPECIALIST INVESTIGATION (Intracranial calcification
(arrowed) in an asymptomatic person with tuberous sclerosis
14
• It is important to point out, however, that the
absence of these findings on clinical or
specialist investigation does not always
exclude the diagnosis of the disorder being
tested for but does reduce the likelihood of
the person concerned having inherited the
gene.
15
BIOCHEMICAL TESTS
• In a number of autosomal dominant disorders
biochemical tests can determine whether or
not a person at risk has inherited a gene.
Examples include the use of serum cholesterol
levels in persons at risk for familial
hypercholesterolemia and assay of the
appropriate urinary porphyrins or the specific
enzyme deficiency in the various dominant
porphyrias
16
Autosomal disorders that show a delayed age of onset or exhibit reduced
penetrance in which linked DNA markers or specific mutational analysis can
be used to offer presymptomatic diagnosis
17
Familial adenomatous polyposis
• in persons at risk for familial adenomatous
polyposis, colonoscopy looking for the
presence of colonic polyps can be offered as a
regular screening procedure to those who
have been shown to be at high risk of
developing colonic cancer by molecular
studies. Conversely, individuals who have
been shown not to have inherited a mutation
in the APC gene do not need to be screened.
18
• In contrast, for persons at risk for HD, in which
there is not yet any effective treatment to delay
the onset or progression of the disorder, the
benefit of predictive testing is not immediately
obvious.
• It is possible that employers, life insurance
companies and society in general will put indirect
and, on occasion, direct pressure on persons at
risk for inherited disorders to have such testing.
19
POPULATION SCREENING
20
CRITERIA FOR A SCREENING PROGRAM
21
Sensitivity: a / (a + c)-proportion of true
positives; specificity: d / (d + b)-proportion of
true negatives
22
Sensitivity: 96 / (96 + 4) = 96%;
specificity is 510 100 / (510 100 +
4980) = ∼99%
23
• Table 20.5 explains this further. Of great
interest too is the positive predictive value of a
screening test, which is the proportion of
positive tests that are true positives, and this
is illustrated in Table 20.6.
24
Newborn screening programs
• Newborn screening programs have been
introduced on a widespread basis for
phenylketonuria, galactosemia and congenital
hypothyroidism.
• In all of these disorders early treatment can
prevent the development of learning
disabilities.
25
• The case of newborn screening for Duchenne
muscular dystrophy also deviates from the
screening paradigm because there is no
effective treatment.
• In this situation the indication is to try to
identify families for whom genetic counseling
could be offered with a view to alerting
relevant females to their possible carrier
status.
26
27
PHENYLKETONURIA
• Routine biochemical screening of newborn infants for
phenylketonuria was recommended by the Ministry of
Health in the UK in 1969 after it had been shown that a
low-phenylalanine diet could prevent the severe learning
disabilities that previously had been a hallmark of this
condition
• The screening test, which is sometimes known as the
Guthrie test, is carried out on a small sample of blood
obtained by heel-prick at age 7 days.
• An abnormal test result is further investigated by repeat
analysis of phenylalanine levels in a venous blood sample. A
low-phenylalanine diet is extremely effective in preventing
learning
28
Newborn screening programs
• . Any woman with phenylketonuria who is
contemplating pregnancy should adhere to a
strict low-phenylalanine diet both before and
during pregnancy to minimize the risk of brain
damage to her unborn child
29
GALACTOSEMIA
• Classic galactosemia affects approximately 1 in 50
000 newborn infants and usually presents with
vomiting, lethargy and severe metabolic collapse
within the first 2 or 3 weeks of life.
• Newborn screening is based on a modification of
the Guthrie test with subsequent confirmation by
specific enzyme assay. The early introduction of
appropriate dietary restriction can prevent the
development of serious complications such as
cataracts, liver failure and learning disabilities.
30
CONGENITAL HYPOTHYROIDISM
• Screening for congenital hypothyroidism was first
introduced in the USA in 1974 and is now
undertaken in most parts of the developed world.
• treatment with life-long thyroxine replacement is
extremely effective in preventing the severe
developmental problems associated with the
classic picture of 'cretinism'. The most common
cause of congenital hypothyroidism is absence of
the thyroid gland rather than an inborn error of
metabolism
31
CYSTIC FIBROSIS
• It is based on the detection of an elevated
blood level of immunoreactive trypsin (IRT),
which is a consequence of blockage of
pancreatic ducts in utero, supplemented by
DNA analysis
• The rationale for screening is that it is hoped
that early treatment with physiotherapy and
antibiotics will improve the long-term
prognosis.
32
SICKLE-CELL DISEASE AND
THALASSEMIA
• Newborn screening based on hemoglobin
electrophoresis is undertaken in many
countries with a significant Afro-Caribbean
community.
• . In the case of sickle-cell disease, treatment
involves the use of oral penicillin to reduce the
risk of pneumococcal infection resulting from
immune deficiency secondary to splenic
infarction
33
POPULATION CARRIER SCREENING
• Widespread screening for carriers of autosomal
recessive disorders in high-incidence populations
was first introduced for the hemoglobinopathies
and has been extended to several other
disorders.
• The rationale behind these programs is that
carrier detection can be supported by genetic
counseling so that carrier couples can be
forewarned of the 1 in 4 risk that each of their
children could be affected.
34
POPULATION CARRIER SCREENING
• In Cyprus in 1974 the birth incidence of βthalassemia was 1 in 250 (carrier frequency 1 in
8). Following the introduction of a comprehensive
screening program to determine the carrier
status of young adults, which had the support of
the Greek Orthodox Church, the incidence of
affected babies declined by over 95% within 10
years
• Similar programs in Greece and Italy have seen a
drop in the incidence of affected homozygotes of
over 50%.
35
POSITIVE AND NEGATIVE ASPECTS OF
POPULATION SCREENING
36
TECHNIQUES USED IN PRENATAL
DIAGNOSIS
37
38
Amniocentesis
• Amniocentesis involves the aspiration of 10-20ml of
amniotic fluid through the abdominal wall under
ultrasound guidance
• This is usually performed around the 16th week of
gestation. The sample is spun down to yield a pellet of cells
and supernatant fluid.
• The fluid can be used in the prenatal diagnosis of neural
tube defects by assay of α-fetoprotein
• The availability of PCR has meant that direct DNA analysis is
usually possible without the need for culture.
• When a couple is considering amniocentesis as an option
they should be informed of the 0.5-1% risk of miscarriage
associated with the procedure
39
A diagram of the technique of
amniocentesis
40
CHORIONIC VILLUS SAMPLING
• In contrast to amniocentesis, chorionic villus
sampling (CVS), which was first developed in
China, enables prenatal diagnosis to be
undertaken during the first trimester.
• This procedure is usually carried out at 11-12
weeks gestation under ultrasound guidance by
either transcervical or, more usually,
transabdominal aspiration of chorionic villi
41
• Chromosome analysis can be undertaken on
chorionic villi either directly, looking at
metaphase spreads from actively dividing
cells, or following culture.
• Direct chromosomal analysis of chorionic villi
usually allows a provisional result to be given
within 24h.
42
CVS
• although it has the disadvantage that even in
experienced hands this procedure conveys a
1-2% risk of causing miscarriage.
• There is also evidence that this technique can
cause limb abnormalities in the embryo if
carried out before 9-10 weeks gestation - for
this reason CVS is not now performed before
11 weeks gestation.
43
CHORIONIC VILLUS SAMPLING
44
ULTRASOUND
• ULTRASOUND can be used not only for obstetric
indications, such as placental localization and the
diagnosis of multiple pregnancies, but also for the
prenatal diagnosis of structural abnormalities that are
not associated with known chromosomal, biochemical
or molecular defects.
• For example, a search can be made for polydactyly as a
diagnostic feature of a multiple abnormality syndrome,
such as one of the autosomal recessive short-limb
polydactyly syndromes that are associated with severe
pulmonary hypoplasia and are invariably lethal.
Similarly,
45
polydactyly
46
Micrognathia (small jaw)
47
• however, detailed 'fetal anomaly' scanning is
being offered routinely to all pregnant women at
around 18 weeks gestation as a screening
procedure for structural abnormalities such as
neural tube defects or cardiac anomalies.
• In addition, the observation that increased nuchal
translucency (NT) is seen in fetuses who are
subsequently born with Down syndrome, has
resulted in the introduction of measurements of
nuchal pad thickness in the first and second
trimesters as a screening test for Down syndrome
48
FETOSCOPY
• Fetoscopy involves visualization of the fetus by
means of an endoscope. Increasingly, this
technique is being superseded by detailed
ultrasound scanning, although occasionally
fetoscopy is still undertaken during the second
trimester to try to detect the presence of
subtle structural abnormalities that would
point to a serious underlying diagnosis.
49
• Fetoscopy has also been used to obtain
samples of tissue from the fetus that can be
analyzed as a means of achieving the prenatal
diagnosis of several rare disorders like
epidermolysis bullosa (skin) and ornithine
transcarbamylase deficiency (liver)
• Unfortunately, fetoscopy is associated with a
3-5% risk of miscarriage.
50
CORDOCENTESIS
• Although fetoscopy can also be used to obtain a small
sample of fetal blood from one of the umbilical cord
vessels in the procedure known as cordocentesis,
improvements in ultrasound have enabled
visualization of the vessels in the umbilical cord,
allowing transabdominal percutaneous fetal blood
sampling.
• Fetal blood sampling is routinely used in the
management of rhesus isoim-munization and can be
used to obtain samples for chromosome analysis to
resolve problems associated with possible
chromosomal mosaicism in chorionic villus or
amniocentesis samples.
51
RADIOGRAPHY
• The fetal skeleton can be visualized by
radiography from 10 weeks onwards and this
technique has been used in the past to
diagnose inherited skeletal dysplasias.
52
PRENATAL SCREENING
• MATERNAL SERUM SCREENING
• maternal serum screening is offered for neural
tube defects and Down syndrome using a
blood sample obtained from the mother at 16
weeks gestation. In this way up to 75% of all
cases of open neural tube defects and 60-70%
of all cases of Down syndrome can be
detected.
53
NEURAL TUBE DEFECTS
• In 1972 it was recognized that many pregnancies in
which the baby had an open neural tube defect (NTD)
could be detected at 16 weeks' gestation by assay of a
protein in maternal serum known as α-fetoprotein
(αFP). αFP is the fetal equivalent of albumin and is the
major protein in fetal blood.
• If the fetus has an open NTD, the level of αFP is
elevated in both the amniotic fluid and maternal serum
as a result of leakage from the open defect. Open NTDs
fulfil the criterion of being serious disorders, as
anencephaly is invariably fatal,
54
• The curves for the levels of maternal serum
αFP in normal and affected pregnancies
overlap, so that in practice an arbitrary cut-off
level has to be introduced below which no
further action is taken.
• This is usually either the 95th centile or 2.5
multiples of the median (MoM), and as a
result around 75% of screened open spina
bifida cases are detected.
55
Women with a value on or above 2.5 multiples
of the median are offered further investigations
56
Causes of elevated maternal serum
αFP
57
• Other contributory factors are a general
improvement in diet and the introduction of
periconceptional folic acid supplementation
58
Maternal risk factors for Down syndrome
59
• This latter approach is based on the discovery that, at
16 weeks gestation, maternal serum αFP and
unconjugated estriol levels tend to be lower in Down
syndrome pregnancies than in normal pregnancies,
whereas the level of maternal serum human chorionic
gonadotropin is usually elevated. None of these
parameters gives absolute discrimination, but taken
together they provide a means of modifying a woman's
prior age-related risk to give an overall probability that
the unborn baby is affected.
• When this probability exceeds 1 in 250, invasive
testing in the form of amniocentesis or placental
biopsy is offered.
60
61
• In trisomy 18 all the biochemical parameters
are low, including hCG.
• It has recently been shown that another
biochemical marker, inhibin-A, is also
increased in maternal serum in Down
syndrome pregnancies. Quadruple test
62
Ultrasonography
• Almost all pregnant women are routinely offered a 'dating'
scan at around 12 weeks gestation
• Nuchal translucency (NT) applies to Down syndrome, the
other autosomal trisomy syndromes, i.e. trisomy 13 and
trisomy 18, Turner syndrome, triploidy, as well as a wide
range of other fetal abnormalities and rare syndromes.
• The risk of Down syndrome correlates with absolute values
of NT as well as maternal age but, as NT also increases with
gestational age, it more usual now to relate the risk to the
centile value for any given gestational age. In one study,
80% of Down syndrome fetuses had NT above the 95th
centile.
63
• A chromosome abnormality is found in 50% of
fetuses with an exomphalos identified at 18
weeks, and a rocker-bottom foot is a very
characteristic finding in babies with trisomy
18, who are also invariably growth retarded.
64
65
ADVANCED MATERNAL AGE
• Most centers routinely offer amniocentesis or
CVS to women aged 37 years or over, and the
option is often discussed with women from
the age of 35 years onwards.
66
PREVIOUS CHILD WITH A
CHROMOSOME ABNORMALITY
• recurrence risk figures, for couples who have had a
child with Down syndrome due to non-disjunction or a
de novo unbalanced Robertsonian translocation, the
risk in a subsequent pregnancy is usually given as the
mother's age-related risk plus approximately 0.5%.
• If one of the parents has been found to carry a
balanced chromosomal rearrangement, such as a
chromosomal translocation or pericentric inversion,
which has caused a previous child to be born with
serious problems due to an unbalanced chromosome
abnormality, then the recurrence risk is likely to be
between 1-2% and 15-20%.
67
FAMILY HISTORY OF A NEURAL TUBE
DEFECT
• In high-risk situations amniocentesis with
assay of the chemical α-fetoprotein has been
used for prenatal diagnosis in the past.
Ultrasound examination of the fetus in
conjunction with assay of maternal serum αfetoprotein has proved equally reliable.
68
FAMILY HISTORY OF OTHER CONGENITAL
STRUCTURAL ABNORMALITIES
• If the risk to a pregnancy is increased, detailed
ultrasound examination looking for the
specific structural abnormality can be offered
at around 16-18 weeks gestation. Midtrimester ultrasound will detect most serious
cranial, cardiac, renal and limb malformations.
69
OTHER HIGH-RISK FACTORS
• These include parental consanguinity, a poor obstetric
history and certain maternal illnesses.
• . A poor obstetric history, such as recurrent
miscarriages or a previous unexplained stillbirth, could
indicate an increased risk of problems in a future
pregnancy and would be an indication for detailed
ultrasound monitoring.
• A history of three or more unexplained miscarriages
should be investigated by parental chromosome
studies to exclude a chromosomal rearrangement such
as a translocation or inversion
70
• Maternal illnesses, such as poorly controlled
insulin-dependent diabetes mellitus or
epilepsy treated with anticonvulsant
medications such as sodium valproate, would
also be indications for detailed
ultrasonography.
71
SPECIAL PROBLEMS IN PRENATAL
DIAGNOSIS
• FAILURE TO OBTAIN A SAMPLE OR CULTURE
FAILURE
 Fortunately, the risk of either of these events
occurring is less than 1%
• AN AMBIGUOUS CHROMOSOME RESULT
• In approximately 1% of cases, CVS shows
evidence of apparent chromosome mosaicism,
i.e. the presence of two or more cell lines with
different chromosome constitutions. This can
occur for several reasons:
72
• The sample is contaminated by maternal cells.
• culture artifact
• If mosaicism is present in only one culture then it
is probably an artifact and does not reflect the
true fetal karyotype.
• The mosaicism is limited to a portion of the
placenta, or what is known as confined placental
mosaicism.
• The mosaicism is limited to a portion of the
placenta, or what is known as confined placental
mosaicism.
73
3 levels of mosaicism
• If a single abnormal cell is identified in only one culture
this is assumed to be a culture artifact, or what is
termed level 1 mosaicism (pseudomosaicism)
• If the mosaicism extends to two or more cells in two or
more cultures this is taken as evidence of true
mosaicism, or what is known as level 3 mosaicism
• The most difficult situation to interpret is when
mosaicism is present in two or more cells in only one
culture, which is termed level 2 mosaicism.
• There is up to a 20% chance that the mosaicism is real
and will be present in the fetus
74
The presence of a marker
chromosome
• marker chromosome, is a small chromosomal
fragment the specific identity of which cannot be
determined by conventional cytogenetic
techniques
• If this is found to be present in one of the
parents, then it is unlikely that it will be of any
significance to the fetus.
• If, on the other hand, it is a de novo finding, there
is up to a 15% chance that the fetus will be
phenotypically abnormal.
75
Marker chromosome
• The risk is lower if the marker chromosome
contains satellite material, or is largely made
up of heterochromatin
76
ULTRASOUND 'SOFT' MARKERS
• choroid plexus cysts are sometimes seen in the
developing cerebral ventricles in mid-trimester.
Initially, it was thought that these were invariably
associated with the fetus having trisomy 18.
• It is now known that choroid plexus cysts occur
frequently in normal fetuses, although if they are
very large and do not disappear spontaneously
they can be indicative of a chromosome
abnormality.
77
TERMINATION OF PREGNANCY
• The presence of a serious abnormality in a
fetus in the majority of developed countries is
an acceptable legal indication for termination
of pregnancy.
78
PGD
79
PGD
• In the procedure the female partner is given hormones to
induce hyperovulation, and oocytes are then harvested
transcervically, under sedation, with ultrasound guidance.
• Motile sperm from a semen sample are added to the
oocytes in culture (in-vitro fertilization (IVF) - the same
technique as developed for infertility) and incubated to
allow fertilization to occur.
• If genetic analysis is to be undertaken on DNA from a single
cell (blastomere) from the early embryo (blastocyst) at the
eight-cell stage on the third day, fertilization is achieved
using intracytoplasmic sperm injection (ICSI) of a single
sperm to avoid the presence of extraneous sperm.
80
PGD
• Choosing a desired sex (boy or girl)
• Choosing a normal cell with matched HLA with
his or her affected siblings
• Micromanipoulation: The nucleus of the
oocyte from the genetic mother (who carried
the mitochondrial mutation) was removed
and inserted into a donor oocyte from which
the nucleus had been removed. This is cell
nuclear replacement (CNR) technology.
81
IVF and ICSI
• In these methods the risk of congenital
abnormalities was basically the same as for
the general population conceiving in the
normal way.
• However, concern has begun to emerge that
there might be a small but abnormal increase
in the incidence of conditions due to defective
genomic imprinting
82
DONOR INSEMINATION
• screening of sperm donors for cystic fibrosis mutations and
chromosome rearrangements has become routine practice in
many countries
83
DETECTION OF FETAL CELLS IN THE
MATERNAL CIRCULATION
• Using antibodies raised to antigens specific to
the fetal trophoblast, there is evidence that
cells of fetal origin are present in the maternal
circulation in the first trimester.
• For Rh compatibility (embryonic RBC)
84
PRENATAL TREATMENT
• A possible model for successful prenatal treatment is
provided by the autosomal recessive disorder congenital
adrenal hyperplasia (CAH) .
• There is evidence that in a proportion of cases the
virilization can be prevented if the mother takes a powerful
steroid known as dexamethasone in a very small dose from
4 to 5 weeks gestation onwards.
• Specific prenatal diagnosis of CAH can be achieved by DNA
analysis of chorionic villi. If this procedure confirms that the
fetus is both female and affected, then the mother
continues to take low doses of dexamethasone throughout
pregnancy, which suppresses the fetal pituitary - adrenal
axis and can prevent virilization of the female fetus.
85
86