DISEASED METABOLISM OF

NUCLEIC ACIDS
Nucleic Acids Metabolism
Nucleic acid Synthesis
 Purine Anabolism

Purine Catabolism
Salvage Pathway of Purines
Salvage Pathway of Pyrimidine
 Salvage Pathway of Nucleotides

 Nucleotides (purine and pyrimidine) are synthesized from intermediates in their

degradative pathway.

Nucleotide salvage pathways are used to recover bases and nucleosides that are formed

during degradation of RNA and DNA.

This is important in some organs because some tissues cannot undergo de novo synthesis

The salvaged products can then be converted back into nucleotides

Required less energy than de novo pathways.

Accounts for 90% of daily nucleotide biosynthesis

Disorders of Nucleic acid
Metabolism
 Disorders of Pyrimidine Metabolism

Orotic Acidurias

 Due to inability of mitochondria to utilize carbonyl phosphate

which then become available to cytosolic overproduction of Orotic acid

Lesch-Nyhan Syndrome
 Molecular Basis of
Lecsh-Nyhan Syndrome
Molecular basis of HPRT deficiency in Italian Lesch-Nyhan patients: identification of
nine novel mutations

Performed biochemical and molecular genetic studies on 28 Italian patients from

25 families. They all had absent HPRT activity.

Genetic analysis identified 24 HPRT mutations, 9 of which had not been

previously reported: 74C>G (P25R), IVS2þ1G>C, 194^195delTC,
329^332delCAAC insTCTs, IVS91G>A, 506insC,
IVS81G>C,606G>T(L202F),418G>C(G140R).

 The virtual complete absence of HPRT activity was related to deletions,

nonsense, or missense mutations leading to nonconservative amino acid changes.
 1) Diagnosis of HPRT Deficiency

 HPRT and APRT (adenine phosphoribosyl transferase) activities were

determined on red blood cell lysates by (HPLC)-linked methods.

Conversion of the radiolabelled bases [14C]hypoxanthine and

[14C]adenine into the respective nucleotides by intact red blood cells
incubated in a PPribP-producing medium was also measured.
 2) Genetic Study

Genomic DNA was extracted from venous blood using kit

Primers were designed to amplify by PCR of all 9 HPRT gene exons and flanking
intronic sequences, on the basis of the known genomic sequence.

PCR products were purified using DNA Purification Kit.

Sequenced on the sense and antisense strands utilizing the sequencing Kit.

The sequencing reactions were analysed.

The presence of mutations found in affected individuals was also checked in possible
healthy carriers.
 3) Selective-Medium Test For HPRT
Mutant cells

The test of lymphocyte growth in 6-thioguanine-enriched medium (selective

for HPRT-mutant cells) was performed in families.

Lymphocytes are separated from blood and harvested in culture medium.

 3 out of 96 wells in a plate are loaded with 200ml of medium with added 6-
thioguanine and 2000 cells per well.

After 3 days of culture, cells that have multiplied are counted and an index
representing the frequency of mutation is calculated (MF).
Point Mutation
FASTA Sequence of HPGRT Protein
Nomenclature for the description of mutations and other sequence variations
Thank
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