L17 Biochem

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


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!


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

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L17 Biochem