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Biochemical geneticsNewborn screeningPopulation screening
Biochemical geneticsNewborn screeningPopulation screening
1902Sir Archibald Garrod
Alkaptonuria (black urine disease)
“Chemical individualities”
Inborn error of metabosim
Alkaptonuria X
Enzymopathies : deficiency or complete loss of enzyme activity
> 5000 genesMostly autosomal recessive and just a few are X-
linkedSubstrate
Metabolic disorders (mostly mutations in genes encoding
enzymes)
If enzyme is defective: Accumulation of substrate Absence of the end product Alternative pathway product
Alternate
productsProduct
Enzyme
Substrate
ProductEnzyme
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
Carrier
The majority of biochemical and enzymatic tests are
not reliable for carrier detection
An ideal test discriminates three genotypes (healthy-non carrier, healthy carrier, affected)
Health
y
Affecte
d
Health
y
Carrier
Affecte
d
Most enzymopathies are autosomal recessive (AR) and
just a few are X-linked
Fabry disease Lysosomal storage disease Hunter syndrome* (Mucopolysaccharidoses
type II) Lysosomal storage diseaseLesch-Nyhan syndrome
Disorder of purine metabolismOrnithine transcarbamylase deficiency
Urea cycle disorders
Some X-linked metabolic disorders
* Distinguish Hurler syndrome and Hunter syndrome
Hurler syndrome (Mucopolysaccharidoses type I) is autosomal recessive
Hunter syndrome (Mucopolysaccharidoses type II) is X-linked
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 deficiencyAutosomal recessiveClinical phenotype:
Mental retardationCoarse faciesRetinal degenerationCorneal cloudingCardyomyiopathyHepatosplenomegaly
Hunter syndrome (Mucopolysaccharidoses type II) Iduronate 2-sulfatase deficiencyX-linkedClinical phenotype similar to MSP1 but milder
Optional slide
Hurler syndrome (MPS type I)Autosomal recessive
If parents are carrier the risk for a new baby to be affected is ¼The 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.
X-linkedOverproduction and accumulation of uric acid due to mutation the HPRT1 gene encoding hypoxanthine phosphoribosyltransferase 1Some clinical feature:
Self-injury, including biting and head banging Gouty arthritis (arthritis caused by an
accumulation of uric acid in the joints),Kidney stonesBladder stonesAbnormal involuntary muscle movements such as
flexing, jerking, and flailing
Optional slide Lesch-Nyhan syndrome
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 baby’s 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
Name of disease Inheritance Enzyme
Phenylketonuria AR Phenylalanine hydroxylase
Alkaptonuria AR Homogentisic acid oxidase
Oculocutaneous albinism AR Tyrosinase
Homocystinuria AR Cystathione α-synthase
Maple syrup urine disease AR Branced-chain-β-ketoacid decarboxylase
Some disorders of amino acid metabolism
Phenylketonuria (PKU): autosomal recessive
Mutation in phenylalanine hydroxylase (PAH) geneclassic PKU – strict dietvariant PKU (milder form) – less strict dietnon-PKU hyperphenylalaninemia - normal diet
Mutation in genes encoding enzymes of tetrahydrobiopterin (BH4)* metabolism (less common)
PAH (phenylalanine hydroxylase)Phenylalanine Tyrosine
BH4 (tetrahydrobiopterin)
* BH4 is cofactor not only for PAH but for two other hydroxylases (tyrosine and tryptophan)
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
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
Clinical manifestation of classic PKU
Low-phenylalanine diet started soon after the birth prevents brain damage
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
USLME
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 geneticsNewborn screeningPopulation screening
A disease that can be missed clinically at birthA high enough frequency in the populationA delay in diagnosis will induce irreversible damages to the babyA simple and reasonably reliable test existsA treatment or intervention that makes a difference if the disease is detected early
Newborn screening:Common considerations in determining whether to
screen for disorders
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.
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
Newborn screening for phenylketonuria (PKU)Introduced in Scotland by Robert
Guthrie in 1963. Neonatal screening for phenylketonuria became nationwide in 1969-70.
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
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)
PhenylketonuriaCongenital hypothyroidismGalactosemiaCongenital 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 geneticsNewborn screeningPopulation screening
Thalassemia screening in Mediterranean region
Tay-Sachs disease in Ashkenazi Jews
Sardinia
Voluntary screening for thalassemia carriersIf a carrier is found then testing of the extended family is offeredPremarital decisionPrenatal diagnosis and termination of the pregnancy if fetus is affected
Thalassemia screening in Sardinia
approximately 11% of population was screened before screening : 100 newborns with thalassemia per yearafter implementation of screening: 5 newborns with thalassemia per year
Result of thalassemia screening in Sardinia
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 recessivemutation in the HEXA gene on
chromosome15q
1 in 27 is a carrier of Tay-Sachs mutation
Incidence of the Tay-Sachs disease is 100 times higher than in other population
Tay-Sachs disease in Ashkenazi Jews
Mutation frequency among carriersmutations Ashkenazi Jews Other ethnic origin 4 bp insertion 80% 16-20%Exon 2 splice junction 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 Tay-Sachs 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.
“cherry-red” spot
Tay-Sachs disease
Optional slide
Carrier screening for Tay-Sachs mutation in Ashkenazy
JewsIt 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
Healthy individuals with very low enzyme activity (level similar to Tay-Sachs patients) have been identifiedThey carry a “ real” Tay-Sachs mutation in one gene and a“pseudodeficient” mutation in another gene . Pseudodeficient mutations do not result in disease , but can lead to wrong interpretation of enzymatic assayThe 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
Optional slideUnexpected finding of carrier
screening for Tay-Sachs 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
Q
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
A