Download Prenatal diagnosis of phenylketonuria

Indian J Med Res 122, November 2005, pp 400-403
Prenatal diagnosis of phenylketonuria
Sudha Kohli, Renu Saxena, Elizabeth Thomas, Pradeep Rao* & I.C.Verma
Department of Genetic Medicine, Sir Ganga Ram Hospital, New Delhi & *Genetic Laboratory & Research Centre,
Bangalore, India
Received January 18, 2005
We report prenatal diagnosis of phenylketonuria by linkage analysis of the markers linked to the
phenylalanine hydroxylase (PAH) gene. Three markers comprising STR (TCTAT)n in intron 3,
VNTR (30bp long cassette) in the 3' UTR and Xmn1 RFLP were ascertained in the affected child,
the parents and the chorionic villi sample. The foetus was confirmed to be heterozygous for the
mutant allele. The diagnosis that the foetus was unaffected was confirmed by biochemical tests in
the newborn.
Key words Molecular studies - phenylalanine hydroxylase (PAH) gene - phenylketonuria (PKU) - prenatal diagnosis
affected child desire prenatal diagnosis in the next
pregnancy. However, prenatal diagnosis is not
possible by PAH enzyme assay because the enzyme
is only expressed in the liver. Prenatal diagnosis of
PKU is feasible only by molecular studies.
Phenylketonuria (PKU) is an autosomal recessive
disorder of amino acid metabolism, caused by a
deficiency of the hepatic enzyme phenylalanine
hydroxylase (PAH), the key enzyme controlling
L-phenylalanine catabolism. PKU is the commonest
genetic disorder leading to mental retardation in the
West, however, it is less common in India. Kaur
et al 1 screened 4451 cases for inborn errors of
metabolism in Delhi and detected PKU in 4 (0.08%)
cases. A higher incidence of PKU has been reported
in south India 2,3. Appaji Rao 4 during screening of
172,369 newborns in Bangalore, detected six cases
of PKU (1 in 28728 screened). PKU induced mental
retardation can be prevented by a phenylalanine
restricted diet, the special diet is difficult to obtain
in India, and is expensive. It is not surprising
therefore, that in India, most parents having an
The human PAH gene covers 100 kb of genomic
DNA on chromosome 12, band region q22-24.1. It
has 13 exons and a complex 5’ untranslated region
containing cis-acting, trans-activated regulatory
elements. A large number of mutations, 498 at last
count on 11.05.05, has been identified 5. There are
no recurrent common mutations, therefore the
pathological mutation cannot be identified by simple
detection methods. Sequencing of the PAH gene is
required for establishing the causative mutation,
costing about US $1000 (personal communication,
400
KOHLI et al: PRENATAL DIAGNOSIS OF PHENYLKETONURIA
401
Dr Flemming Guttler, Denmark). For prenatal
diagnosis, an alternative approach to mutation
analysis of PAH deficiency is by linkage analysis of
polymorphic markers associated with the PAH locus,
provided the affected child is available and a firm
diagnosis of PKU has been established. We report
here prenatal diagnosis of PKU by linkage analysis
of the markers linked with PAH gene.
A consanguineously married couple, who had a
daughter with PKU, attended genetic clinic in
Bangalore in 2002, desiring prenatal diagnosis in
current pregnancy. The daughter had severe mental
retardation, light coloured skin and hair. Her serum
phenylalanine was 663 µmol/l, (normal range 77±18
µmol/l) while the tyrosine was 85 µmol/l (normal
range 81±23 µmol/l). Phenylpyruvic acid was
increased in the urine. Ferric chloride test and
dinitrophenylhydrazine (DNPH) test on the urine
were positive 6. PKU was diagnosed in the child at
the age of one year. The patient showed response
to low phenylalanine diet. The parents could not
afford the commercial formulations with low
phenylalanine content and the child therefore was
only on a modified dietary therapy (low protein
therapy).
DNA was extracted from peripheral blood
lymphocytes of parents, the affected child and
chorionic villi sample (CVS) aspirated at 12 wk
of gestation by proteinase K/sodium dodecyl
sulphate (SDS) digestion followed by salt
precipitation method 7 and phenol chloroform
e x t r a c t i o n s 8, r e s p e c t i v e l y . D N A s a m p l e s o f
parents, affected child and CVS were amplified for
short tandem repeats (STR) (TCTAT) n in intron 3,
variable number of tandem repeats (VNTR; 30 bp
long cassette in the 3' UTR) and restriction
fragment length polymorphism (RFLP) XmnI
alleles as per the protocol of Eisensmith et al 9. The
amplified product for RFLP was digested with
Xmn1.The polymorphism of STR sequences were
analyzed on 4 per cent polyacrylamide gel and
visualized by silver staining. The VNTR and Xmn1
polymorphisms were viewed on ethidium bromide
stained 3 per cent agarose gels.
Fig. 1. Ethidium bromide stained 3 per cent agarose gel showing
variable number of tandem repeats (VNTR) alleles. Lane 1: mother;
Lane 2: father; Lane 3: proband; Lane 4: foetus; Lane M: 500 bp DNA
ladder.
Fig. 2. Ethidium bromide stained 3 per cent agarose gel showing
Xmn1 restricted fragment length polymorphism (RFLP) alleles.
Lane M: qX 174 Hae III digest DNA ladder; Lane U: Undigest;
Lane 1: mother (+/+); Lane 2: father (-/+); Lane 3: proband (+/+);
Lane 4: foetus (+/+).
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INDIAN J MED RES, NOVEMBER 2005
In VNTR marker gel (Fig. 1) the mother showed
two alleles (allele 1 and allele 2). The daughter with
PKU inherited allele 1, demonstrating allele1 to be
carrying the mutant gene. The father was
homozygous for this marker and was therefore
uninformative at this locus. The foetus had inherited
the normal allele (allele 2) from the mother.
In Xmn1 marker analysis (Fig. 2) the mother was
homozygous (+/+) and was uninformative at this
locus. The father was heterozygous (-/+). The
daughter with PKU was homozygous (+/+),
indicating that the mutant allele inherited from the
father was +. The CVS DNA was +/+ indicating the
foetus had inherited the affected allele (+) from the
father.
Both the mother and father were heterozygous for
the STR marker and showed two alleles ‘a’ and ‘b’.
The daughter with PKU was homozygous for allele
‘a’ and thus demonstrating ‘a’ as the mutant gene.
The CVS DNA carried both the alleles ‘a’ and ‘b’,
indicating that the foetus had inherited the affected
allele (a) from only one parent and normal allele (b)
from the other.
The parents were consanguineous and hence the
affected daughter was homozygous for all three loci
studied. All the three markers taken together
indicated that the foetus had inherited the normal
allele from the mother and the mutant allele from
the father. The foetus was thus diagnosed to be a
carrier of PKU. The unaffected status of the foetus
was confirmed by biochemical tests (serum
phenylalanine 0.5 mg/dl) in the new born.
There are reports of prenatal diagnosis of PKU
by linkage analysis10-13. Daiger et al10 established the
usefulness of RFLPs for prenatal diagnosis and
carrier detection of PKU in 34 families having atleast
one affected child. Huang et al 11 analyzed
polymorphism information content of STR sequences
and performed prenatal diagnosis by linkage analysis
in two Chinese families at risk for PKU. Fan et al12
carried out prenatal diagnosis in five Chinese PKU
families, by screening for mutation in exons 3 and 7
of the PAH gene, in combination with STR linkage
analysis. Romano et al 13 has reported on the use of
STR and VNTR markers for prenatal diagnosis in
two Italian families at risk for PKU. Relationship
between standardized linkage disequilibria and
physical map distances of the polymorphic sites
indicates that there is no recombination hotspot in
the human PAH gene, since the recombination rate
within the locus appears to be uniform and likely to
be occurring at a rate similar to that within HLA gene
cluster14.
Prenatal diagnosis of PKU is done ideally, after
identifying the mutation in the affected child by
sequencing the gene. If diagnosis in the affected
child is definite, prenatal diagnosis can be done by
linkage analysis of STR, VNTR and Xmn1 RFLP
markers. This approach is easy to perform,
inexpensive, quick and can be used in all PKU
families, once the diagnosis is certain.
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Reprint requests: Dr I. C. Verma, Senior Consultant & Head, Department of Genetic Medicine, Sir Ganga Ram Hospital
Rajinder Nagar, New Delhi 110060, India
e-mail: [email protected]
[email protected]