Biochemical genetics

Newborn screening
Population screening

Biochemical genetics
Newborn screening
Population screening

1902
Sir Archibald Garrod
Alkaptonuria (black urine disease)
Chemical individualities

Inborn error of metabosim

Alkaptonuria

Metabolic disorders
(mostly mutations in genes encoding enzymes)
Enzymopathies :
deficiency or complete loss of enzyme activity
> 5000 genes

Mostly autosomal recessive and just a few are X-linked

Substrate
Enzyme

Product

Substrate
Enzyme

Alternate
products

Product
If enzyme is defective:
Accumulation of substrate
Absence of the end product
Alternative pathway product

Laboratory diagnosis of enzymopathies

Biochemical assays: measurement of concentration
in blood or urine
substrate
end product
alternative pathway product
Enzymatic assays : measurement of enzyme
activity or quantity in blood, serum, amniotic fluid or
cells (cultured skin fibroblasts, erythrocytes,
amniotic cells etc)
Molecular analysis of the disease-causing gene
(DNA from blood, chorionic villi, amniotic cells, etc)

Laboratory diagnosis of enzymopathies

Biochemical and enzymatic tests are used for diagnosis
in affected individuals. With a few exceptions, these
tests are not reliable for carrier detection

Molecular testing can be used for carrier

detection, as well as for prenatal and
preimplantation diagnosis

An ideal test discriminates three genotypes

(healthy-non carrier, healthy carrier, affected)

The majority of biochemical and enzymatic tests are

not reliable for carrier detection

Most enzymopathies are autosomal

recessive (AR) and just a few are

X-linked

Some X-linked metabolic disorders

Fabry disease Lysosomal storage disease

Hunter syndrome* (Mucopolysaccharidoses type II)

Lysosomal storage disease

Lesch-Nyhan syndrome

Disorder of purine metabolism

Ornithine transcarbamylase deficiency

Urea cycle disorders

* Distinguish Hurler syndrome and Hunter syndrome

Hurler syndrome (Mucopolysaccharidoses type I) is
autosomal recessive
Hunter syndrome (Mucopolysaccharidoses type II) is
X-linked

Optional
slide

Mucopolysaccharidoses (MPS) :
Deficiency of enzymes needed to break down
glycosaminoglycans.
At least 7 types of MSP are known each
caused by a deficiency of a specific lysosomal
enzyme

Hurler syndrome (Mucopolysaccharidoses type I)

Alpha-L-iduronidase deficiency
Autosomal recessive

Clinical phenotype:
Mental retardation
Coarse facies
Retinal degeneration
Corneal clouding
Cardyomyiopathy
Hepatosplenomegaly
Hunter syndrome (Mucopolysaccharidoses type II)

Iduronate 2-sulfatase deficiency

X-linked
Clinical phenotype similar to MSP1 but milder

Hurler syndrome (MPS type I)

Autosomal recessive
If parents are carrier the risk for a new baby
to be affected is
The risk of healthy sibs of affected proband to
be a carrier is 2/3
Hunter syndrome (MPS type II) X-linked
If family have one affected son, the risk for
another boy is 1/2. The risk of the sister of
affected proband to be a carrier is 1/2.

Optional
slide

Lesch-Nyhan syndrome

X-linked
Overproduction and accumulation of uric acid due to
mutation the HPRT1 gene encoding hypoxanthine
phosphoribosyltransferase 1
Some clinical feature:
Self-injury, including biting and head banging
Gouty arthritis (arthritis caused by an accumulation
of uric acid in the joints),
Kidney stones
Bladder stones
Abnormal involuntary muscle movements such as
flexing, jerking, and flailing

Two examples of USMLE-type questions on

metabolic disorders

A 2-year-old boy with mental retardation has chewed the tips of his
fingers on both hands and a portion of his lower lip. His serum uric
acid concentration is increased, and he has a history of uric acid
renal calculi. His 5-year-old brother has similar findings. Which of
the following abnormal enzyme activities is the most likely cause
of these findings?
(A) Decreased adenine phosphoribosyltransferase
(B) Decreased adenosine deaminase
(C) Decreased hypoxanthine-guanine phosphoribosyltransferase
(D) Increased phosphoribosylpyrophosphate synthetase
(E) Increased xanthine oxidase

A 2-year-old boy with mental retardation has chewed the tips of his
fingers on both hands and a portion of his lower lip. His serum uric
acid concentration is increased, and he has a history of uric acid
renal calculi. His 5-year-old brother has similar findings. Which of
the following abnormal enzyme activities is the most likely cause
of these findings?
(A) Decreased adenine phosphoribosyltransferase
(B) Decreased adenosine deaminase
(C) Decreased hypoxanthine-guanine phosphoribosyltransferase
(D) Increased phosphoribosylpyrophosphate synthetase
(E) Increased xanthine oxidase

A 2-year-old boy with mental retardation has chewed the tips

of his fingers on both hands and a portion of his lower lip. His
serum uric acid concentration is increased, and he has a history
of uric acid renal calculi. His 5-year-old brother has similar

findings. The mother is currently pregnant. What is the risk

that a new baby is also affected with a disease?
(A) 25%

(B) 50%
(C) 75%
(D) 100%
(E) Virtually 0

A 2-year-old boy with mental retardation has chewed the tips

of his fingers on both hands and a portion of his lower lip. His
serum uric acid concentration is increased, and he has a history
of uric acid renal calculi. His 5-year-old brother has similar

findings. The mother is currently pregnant. What is the risk

that a new baby is also affected with a disease?
(A) 25%

(B) 50%
(C) 75%
(D) 100%
(E) Virtually 0

If the sex of new baby is unknown then the risk

is 0.5 x 0.5 = 0.25 or 25% (probability of getting
mutant allele from mother x probability that the
babys sex is a male)
If the sex of a new baby is known than the
answer is
50% to be affected for boy
50% to be a carrier for girl

Some disorders of amino acid metabolism

Name of disease

Inheritance

Enzyme

Phenylketonuria

AR

Phenylalanine hydroxylase

Alkaptonuria

AR

Homogentisic acid oxidase

Oculocutaneous albinism AR

Tyrosinase

Homocystinuria

Cystathione -synthase

AR

Maple syrup urine disease AR

Branced-chain--ketoacid decarboxylase

Phenylketonuria (PKU): autosomal recessive

PAH (phenylalanine hydroxylase)
Phenylalanine

Tyrosine
BH4 (tetrahydrobiopterin)

* BH4 is cofactor not only for PAH but for two

other hydroxylases (tyrosine and tryptophan)

Mutation in phenylalanine hydroxylase (PAH) gene

classic PKU strict diet

variant PKU (milder form) less strict diet

non-PKU hyperphenylalaninemia - normal diet

Mutation in genes encoding enzymes of tetrahydrobiopterin (BH4)*

metabolism (less common)
Phenylalanine
Tyrosine
Metabolites : phenylacetate, phenylpyruvate, phenethylamine
Elevated levels of phenylalanine in the blood and detection of
phenylketones in the urine is diagnostic
Defect in BH4 metabolism (rare):
pterins in urine sample
dihydropteridine reductase activity in blood

Clinical manifestation of classic PKU

Infants appear normal until they are a few months old.
Without treatment with a special low-phenylalanine diet,
these children develop permanent intellectual disability

Seizures, delayed development, behavioral problems

Untreated individuals may have a musty or mouse-like odor
Lighter skin and hair than unaffected family members

Low-phenylalanine diet started soon after the

birth prevents brain damage

USLME
A 9-month-old girl has had two seizures in the past month. She

was born at home and received no state-mandated newborn

screening. She has developmental delays. Her skin is fair and her
hair is a lighter color than that of other family members. Her
diapers have a musty odor. Which of the following is most likely
to have an increased concentration in this infant's urine?
(A) Homocysteine
(B) Homogentisic acid
(C) Isoleucine
(D) Isovaleric acid
(E) Phenylacetic acid

USLME
A 9-month-old girl has had two seizures in the past month. She

was born at home and received no state-mandated newborn

screening. She has developmental delays. Her skin is fair and her
hair is a lighter color than that of other family members. Her
diapers have a musty odor. Which of the following is most likely
to have an increased concentration in this infant's urine?
(A) Homocysteine
(B) Homogentisic acid
(C) Isoleucine
(D) Isovaleric acid
(E) Phenylacetic acid

During normal screening for phenylketonuria, a male newborn has a

serum phenylalanine concentration of 35 mg/dL (greater than 20

mg/dL is considered a positive test). Signs of tyrosine deficiency

also are apparent. Enzymatic analysis using cultured fibroblasts,
obtained after circumcision, shows normal activity of phenylalanine
hydroxylase.
A possible explanation for these findings is a deficiency in function
of which of the following coenzymes?
(A) Adenosylcobalamin

(B) Biopterin
(C) Dihydroquinone
(D) Pyridoxal phosphate
(E) Tetrahydrofolic acid

During normal screening for phenylketonuria, a male newborn has a

serum phenylalanine concentration of 35 mg/dL (greater than 20

mg/dL is considered a positive test). Signs of tyrosine deficiency

also are apparent. Enzymatic analysis using cultured fibroblasts,
obtained after circumcision, shows normal activity of phenylalanine
hydroxylase.
A possible explanation for these findings is a deficiency in function
of which of the following coenzymes?
(A) Adenosylcobalamin

(B) Biopterin
(C) Dihydroquinone
(D) Pyridoxal phosphate
(E) Tetrahydrofolic acid

Inborn error of metabolism, the enzymes

which deficiency leads to these disorders,
clinical manifestation etc are subjects of
Biochemistry and Pathology courses.
Just be aware that this topic is quite popular
in USMLE questions

Biochemical genetics
Newborn screening
Population screening

Newborn screening:
Common considerations in determining whether to
screen for disorders
A disease that can be missed clinically at birth
A high enough frequency in the population
A delay in diagnosis will induce irreversible damages to the baby
A simple and reasonably reliable test exists
A treatment or intervention that makes a difference if the
disease is detected early

Newborn screening for phenylketonuria (PKU)

Introduced in Scotland by Robert
Guthrie in 1963.
Neonatal screening for phenylketonuria
became nationwide in 1969-70.
Newborn screening for PKU is based on
detection of hyperphenylalaninemia using
the Guthrie microbial or other assays on
a blood spot obtained from a heel prick
Robert Guthrie (1916 - 1995)
The "Father of Newborn Screening."
Robert Guthrie (1916 - 1995)
The "Father of Newborn
Screening."

A piece of card onto which neonatal

blood from a heel-prick is
impregnated as several discrete spots

Guthrie card bacterial inhibition assay

(BIA), a time-tested, inexpensive, simple,
and reliable test (many false positive)
Tandem mass spectrometry (MS)
produces fewer false positive test results
than the BIA. MS can be used to identify
numerous other metabolic disorders on
the same sample.

The first test to be universally mandated

across the U.S. was the Guthrie test for PKU
The newborn blood test is often erroneously
referred to as a "PKU test", even though all
states now universally test for congenital
hypothyroidism, galactosemia, and increasing
numbers of other diseases as well

Newborn screening and early

intervention with low-phenylalanine diet
allow individual with PKU genotype to
have normal life, including education,
professional jobs, marriage etc,
Babies born to mothers with PKU who no longer
follow a low-phenylalanine diet
Their own PAH activity is normal , but exposure to very
high levels of phenylalanine before birth result in
Intellectual disability
Low birth weight
Microcephaly
Behavioral problems

A 24-ycar-oId woman with phenylketonuria (PKU) gives birth to her

first child. Although there is no history of PKU in the father's family,
the couple could not afford genetic testing of the father or
consistent prenatal care. At birth, the child is small, microcephalic,
and has elevated blood phenylalanine. What is the most likely
explanation for this neonate s symptoms?
(A) Father is a carrier of PKU
(B) Maternal translocation with unbalanced segregation in meiosis I
(C) Maternal translocation with unbalanced segregation in meiosis II
(D) Maternal uniparental disomy
(E) Phenylalanine was not adequately restricted from the mother's
diet during pregnancy

A 24-ycar-oId woman with phenylketonuria (PKU) gives birth to her

first child. Although there is no history of PKU in the father's family,
the couple could not afford genetic testing of the father or
consistent prenatal care. At birth, the child is small, microcephalic,
and has elevated blood phenylalanine. What is the most likely
explanation for this neonate s symptoms?
(A) Father is a carrier of PKU
(B) Maternal translocation with unbalanced segregation in meiosis I
(C) Maternal translocation with unbalanced segregation in meiosis II
(D) Maternal uniparental disomy
(E) Phenylalanine was not adequately restricted from the mother's
diet during pregnancy

Newborn screening has been adopted in many countries

around the world, though the lists of screened diseases

vary widely (from 1 to 100)
Phenylketonuria

Congenital hypothyroidism
Galactosemia
Congenital deafness (HEAR)
Various biochemical, endocrine, blood and other
disorders are now considered as a target for newborn
screening
A lot of countries do not have newborn screening!

Biochemical genetics
Newborn screening
Population screening

Thalassemia screening in Mediterranean region

Tay-Sachs disease in Ashkenazi Jews

Thalassemia screening in Sardinia

Sardinia

Voluntary screening for thalassemia carriers

If a carrier is found then testing of the extended
family is offered
Premarital decision
Prenatal diagnosis and termination of the pregnancy if
fetus is affected

Result of thalassemia screening in Sardinia

approximately 11% of population was screened

before screening : 100 newborns with thalassemia

per year
after implementation of screening: 5 newborns with
thalassemia per year

Screening for Tay-Sachs disease in Ashkenazi

Jews in UAS, Canada and Israel
Ashkenazi Jews (those originating from the Western and

Eastern Europe diaspora), who make up more than 80

percent of world Jews and are believed to be descended
from about 1,500 Jewish families dating back to the 14th
century
Tay Sachs disease
(deficiency of hexosaminidase A) :
autosomal recessive
mutation in the HEXA gene on
chromosome15q

Tay-Sachs disease in Ashkenazi Jews

1 in 27 is a carrier of Tay-Sachs mutation
Incidence of the Tay-Sachs disease is 100 times higher than in
other population
Mutation frequency among carriers
mutations
4 bp insertion
Exon 2 splice junction

Ashkenazi Jews

Other ethnic origin

80%

16-20%

10-15%

<1%

Tay-Sachs disease is a rare inherited

disorder that progressively destroys
neurons in the brain and spinal cord.
The most common form of Tay-Sachs
disease becomes apparent in infancy.
Infants with this disorder typically
appear normal until the age of 3 to 6
months, when their development slows
and muscles used for movement weaken.
Affected infants lose motor skills such
as turning over, sitting, and crawling.
They also develop an exaggerated
startle reaction to loud noises. As the
disease progresses, children with TaySachs disease experience seizures,
vision and hearing loss, intellectual
disability, and paralysis. An eye
abnormality called a cherry-red spot,
which can be identified with an eye
examination, is characteristic of this
disorder. Children with this severe
infantile form of Tay-Sachs disease
usually live only into early childhood.

Tay-Sachs disease

cherry-red spot

Optional
slide

Carrier screening for Tay-Sachs

mutation in Ashkenazy Jews

It is a rare example when enzymatic

assay allows reliable carrier
detection
Assay for hexosaminidase A which is
deficient in Tay-Sachs patients and
decreased in carriers

Unexpected finding of carrier screening

for Tay-Sachs mutation:

Optional
slide

Healthy individuals with very low enzyme activity (level

similar to Tay-Sachs patients) have been identified
They carry a real Tay-Sachs mutation in one gene
and apseudodeficient mutation in another gene .
Pseudodeficient mutations do not result in disease , but
can lead to wrong interpretation of enzymatic assay
The enzyme assay may be invalid because of
differences between the natural substrate and an
artificial substrate used in testing
Molecular analysis is required to exclude
pseudodeficient mutation

In the United States and Canada, the

incidence of Tay-Sachs disease in the
Jewish population had declined by more
than 90% since the advent of genetic

screening

For which of the following diseases has

genetic screening (at the population level)
been most effective?

a. Sickle-cell disease
b. Cystic fibrosis
c. Tay-Sachs disease
d. Hemochromatosis
e. alpha 1-antitrypsin deficiency

For which of the following diseases has

genetic screening (at the population level)
been most effective?

a. Sickle-cell disease
b. Cystic fibrosis
c. Tay-Sachs disease
d. Hemochromatosis
e. alpha 1-antitrypsin deficiency