Prenatal diagnosis of triosephosphate isomerase deficiency

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               Prenatal Diagnosis of Triosephosphate Isomerase Deficiency
         By Roopen Arya, Michel R.A. Lalloz, Kypros H. Nicolaides, Alastair J. Bellingham, and D. Mark Layton

First-trimester prenatal diagnosis was undertaken by chori-          clinically unaffected. Prenatal diagnosis in the second case
onic villus DNA analysis in two unrelated families with the          showed the fetus to be homozygousfor the codon 104 muta-
inherited glycolytic disorder triosephosphate isomerase              tion and thus affected by TPI deficiency.This representsthe
(TPI) deficiency. The propositus in each family was shown            first molecular diagnosis during early pregnancy of a human
to be homozygous for a missense mutation (GAG + GAC)                 glycolytic enzyme disorder.
at codon 104 of the TPI gene. In the first case the fetus was        0 1996 by The American Society of Hematology.
heterozygous for the codon 104 mutation and therefore

                                                                     (father 421, mother 413 EU/g Hb at 30°C) and sister (542 EUlg Hb
F    IRST DESCRIBED in 1965, deficiency of triosephos-
      phate isomerase (TPI) is a rare recessive inbom error
of the Embden-Meyerhof pathway.’ TPI catalyzes the inter-
                                                                     at 30°C) were consistent with heterozygosity for TPI deficiency.
                                                                     Screening for mutations within the TPI gene identified a G      +   C
                                                                     transversion at nucleotide 315 (codon 104), for which the propositus
conversion of dihydroxyacetone phosphate (DHAP) and
                                                                     was homozygous and parents heterozygous. Soon after the initial
glyceraldehyde-3-phosphate and is found in all somatic cells.        diagnosis the mother of the propositus became pregnant. After ge-
The human TPI gene spans 3.5 i<b of DNA located on the               netic counseling,the family requested prenatal diagnosis. Transabdomi-
short arm of chromosome 12 (12~13) comprises 7 exons
                                        and                          nal Chorionic villus samplig was performed at 13 weeks’ gestation.
encoding a 1.2-kb mRNA that is translated into a 248-amino              Family 2. The affected child is a 2-year-old boy with nonsphero-
acid              Homozygous TPI deficiency classically re-          cytic hemolytic anemia, recurrent infections, and symmetrical lower
sults in a multisystem disorder characterized by hemolytic           motor neurone weakness of the lower limbs. At presentation his Hb
anemia, progressive neuromuscular dysfunction, cardiomy-             level was 9.5 g/dL and reticulocyte count was 28%. RBC TPI activity
opathy, and increased susceptibility to infection: In affected       was markedly reduced at 138 EU/g Hb at 30°C with 47-fold elevation
individuals erythrocytes show reduced TPI activity accom-            in DHAP (1,750 nmoVg Hb). The parental RBC TPI activities (father
panied by metabolic block in glycolysis with elevation of            585, mother 594 EU/g Hb at 30°C) were consistent with heterozygos-
                                                                     ity for TPI deficiency. DNA analysis showed the propositus to be
DHAP. No specific therapy is available for the neuropathic           homozygous and his parents heterozygous for the codon 104 muta-
manifestations of the disease and most severely affected chil-       tion. In a subsequent pregnancy prenatal diagnosis was performed
dren fail to survive beyond the age of 5 years.                      by chorionic villus sampling at 12 weeks’ gestation.
   In 1989 we reported the feasibility of prenatal diagnosis
for TPI deficiency by biochemical analysis of enzyme activ-          Mutation Analysis
ity and glycolytic intermediates on fetal blood obtained by
                                                                        Genomic DNA was extracted from blood and chorionic villus
ultrasound-guided cordocentesis in mid-ge~tation.~     Though        samples, obtained with informed consent, by standard methods. The
reliable, this procedure can only be performed from 20 weeks         codon 104 mutation was identified using the restriction endonuclease
gestation and thus carries the prospect of late abortion in the      Ple I (New England Biolabs UK Ltd, Hitchin, UK) which specifically
event a pregnancy is affected.6Early diagnosis by biochemi-          recognizes both normal and mutant sequence. A 146-bp DNA frag-
cal analysis of trophoblast or amniocyte cannot unambigu-            ment was amplified by the polymerase chain reaction (PCR) method
ously distinguish heterozygous and homozygous deficiency             from 1.0 pg chorionic villus DNA using 200 ng each of primers T1
states in the fetus on the basis of DHAP levels because              and T2 (T1:5’CTGAACCTTGGC’ITCATC3’;T25‘GACATCCCT-
of alternative catabolism of DHAP by a-glycerophosphate              TATCTTCTCTC3’), complementary to intronic sequence flanking
dehydrogenase in these               Therefore, the ability to       exon 3 of the TPI gene. DNA was initially denatured at 94°C for 3
                                                                     minutes followed by 30 cycles of PCR: 94°C denaturation for 30
perform early prenatal diagnosis by genotypic analysis is
                                                                     seconds, annealing at 54°C for 1 minutes, and primer extension at
highly desirable. We describe first-trimester prenatal diagno-       72°C for 1 minute. Twenty microliters of PCR product was incubated
sis for TPI deficiency by chorionic villus DNA analysis in two       with Ple I (2 U) at 37°C ovemight. DNA fragments were visualized
previously unreported nonconsanguineous Northem European             by UV-transillumination after separation by electrophoresis in 4%
families, in each of which the affected child was shown to
be homozygous for a missense mutation (GAG;Glu -+
GAC;Asp) at codon 104 of the TPI gene.                                  From the Department of Haematological Medicine and the Harris
                                                                     Birthright Research Centre for Fetal Medicine, King’s College Hos-
                 MATERIALS AND METHODS                               pital, London, UK.
                                                                        Submitted October 4, 1995; accepted March 8, 1996.
Patient History and Laboratory Findings                                 R.A. is supported by the Medical Research Council, UK.
   Family I . The propositus presented at the age of 3 months with      Address reprint requests to Roopen Arya, MO, Department of
unexplained hemolytic anemia (hemoglobin [Hb] level, 5.6 g/dL;       Haematological Medicine, King’s College Hospital, Denmark Hill,
41% reticulocytes) but no neurodevelopmental abnormality. The di-    London SE5 9RS, UK.
agnosis of TPI deficiency was confirmed by reduced red blood cell       The publication costs of this article were defrayed in part by page
(RBC) enzyme activity (566 EU/g Hb; mean normal 1,244 EU/g Hb        charge payment. This arficle must therefore be hereby marked
at 30°C) and 70-fold elevation in DHAP (2,565 nmoVg Hb; mean         “advertisement” in accordance with 18 U.S.C. section 1734 solely to
normal 37 nmoVg Hb) indicative of metabolic block. RBC TPI           indicate this fact.
activity was higher than is usual in homozygous deficiency because      0 1996 by The American Socieg of Hematology.
of marked reticulocytosis. The RBC TPI activities of both parents       0006-4971/96/87I1-0060$3.00/0

Blood, Vol 87, No 11 (June l ) , 1996: pp 4507-4509                                                                                   4507
4508                                                                                                                                    ARYA ET AL

agarose containing 0.5 pglmL ethidium bromide. The G 4 C trans-
version at nucleotide 315 modifies the site of Plr I cleavage, yielding
a DNA fragment which differs in size by 14 bp from that generated
by the normal allele (Fig I). DNA extracted from a plasmid clone
of the human TPI gene containing the codon 104 mutation (a gift
                                                                                               1:l            I          1:2
from Dr Lynne Maquat, Department of Human Genetics, Roswell
Park Cancer Institute, Buffalo, NY) served as a positive control for
Plr I restriction. Double-stranded DNA sequencing, incorporating
primer TI 5'-end-labeled with y"P-ATP (3.000 Cilmmol; Amer-
sham International plc, Little Chalfont, UK), was performed by di-
deoxy cycle sequencing (fmol DNA Sequencing System; Promega,
Southampton, UK). DNA sequence analysis verified the presence of                   11:1                    II:2                      II:3
the G -, C transversion at nucleotide 315 in the affected families.
                                  RESULTS                                               P     N      M I:l      1:2 II:1 I : 11:3
   In family I (Fig 2), Ple I restriction analysis of PCR                      I                                                            1      bp

product showed the fetus (113) to be heterozygous for the
                                                                                                                                              - 146
codon 104 mutation. All four subjects heterozygous for the                                                                                    - 111
                                                                                                                                              - 97
mutation (I: I , 1:2, 11: 1, and 11:3) exhibit an identical pattern
of bands which incorporate those seen in normal (N) and
mutant (M) controls. In addition, these four lanes also show
                                                                                                                                              -    49
                                                                                                                                              -    35
a prominent 146-bp band corresponding to undigested PCR
product (P). This is due to heteroduplex formation between
normal and mutant DNA resulting in the abolition of the Ple                       Fig 2. Codon 104 mutation analysis in family 1: Agarose gel elec-
I recognition site preventing cleavage (see Fig I ) . Because                  trophoresis of PCR product after digestion with Ple 1. The propositus
the heteroduplex accounts for half the total PCR product,                      (11:Z) shows 97- and 49-bp DNA fragments confirming homozygosity
the digested DNA fragments appear less intense than the                        for the codon 104 mutation. The heterozygous parents (I:1 and 1 2 1
                                                                               and sister (11:l) show fragments originating from both normal and
corresponding fragments in normal or mutant DNA samples.                       mutant alleles. Similarly, the fetus (11:3)is heterozygous for the codon
At the family's request, confirmatory fetal blood sampling                     104 mutation and therefore clinically unaffected. P, undigested PCR
was performed at 20 weeks' gestation. Fetal RBC TPI activ-                     product; N, normal genomic DNA; M, plasmid DNA of 315C TPI mu-
ity was reduced at 820 EU/g Hb at 30°C (mean normal,                           tant; 6x174 Hid I molecular size marker is shown in the first and
 1,420 EU/g Hb) with a normal DHAP, consistent with het-                       last lanes.
erozygous TPI deficiency and concordant with the results of
DNA analysis. A healthy male infant was delivered at term
and the results of prenatal diagnosis confirmed postnatally                    and often death in early childhood. Characterization of the
by phenotypic and molecular analysis (data not shown).                         molecular basis of TPI deficiency in two families enabled
   In family 2 (Fig 3), the severely affected propositus (11: I )              prenatal diagnosis early in pregnancy, providing the couples
is, as in family 1, homozygous for the codon 104 mutation.                     at risk with informed reproductive choice. In both families
DNA analysis showed the fetus (II:3) to be homozygous for                      the propositus was homozygous for the codon 104 mutation
the mutation and therefore affected by TPI deficiency. Based                   (GAG;Glu 4 GAC;Asp), which allowed rapid and specific
on this result the family elected to abort the pregnancy. The                  prenatal diagnosis by Ple I restriction analysis of PCR prod-
diagnosis was confirmed by analysis of trophoblast tissue.                     uct amplified from genomic DNA. This report represents the
                                                                               first described molecular diagnosis of a human glycolytic
                                                                               disorder in the first trimester of pregnancy.
   TPI deficiency is the most severe of the erythroenzymopa-                      The codon 104 mutation has now been reported in I 1 of
thies, associated with progressive neuromuscular impairment                     12 families with TPI deficiency.')'" Only two other defects,
                                                                               a missense mutation at codon 240 (TTC;Phe --t CTC;Leu)
        n o m 1                              Digamted PCR product              and nonsense mutation at codon 189 (CGA;Arg TGA;s-         +
5'. -             AQT AGAT   GAGgttag..3'        1 1 bp
                                                  1               35 bp        top), each identified in single kindreds have been de-
3'..GAAACCCCTCAGTCTAC          TCcaatc..5'                   b-
                                                                               scribed."." The reason for predominance of a single geno-

                                             --                                type is not known. The codon 104 mutation does not occur
S'..CATGTCT       TTGGGGACTCAGATGAG..3'          97   m           49   m       at the site of any recognized mutation hotspot, raising the
3'..GTACAGAA       ACCCWTCTACTC..S'                                            possibility that this mutation may have originated in a com-
                                                                               mon founder. This homogeneity will facilitate prenatal diag-
   Fig 1. Molecular diagnosis of the TPI codon 104 (GAG GAC)
mutation by Ple I restriction analysis of PCR product amplified from           nosis by the approach described in the majority of affected
genomic DNA. Normal and mutant sequences are shown: upper case,                families.
coding sequence; lower case, intron sequence. The specific restric-
tion enzyme recognition sequence (GAGTCN.) is underlined. The G
C transversion (shown in bold) results in cleavage 14 bp upstream
                                                                           -      The importance of residue 104 to TPI structure and func-
                                                                               tion is implied by its conservation in all species characterized
of the cutting site in the wild-type allele. Restriction o the 146-bp
                                                          f                    to date, from Escherichia coli to humans. Substitution of
PCR product yields fragments of 111 and 35 bp in t h e normal allele           aspartate for glutamate at residue 104 has been predicted to
and 97 and 49 bp in the mutant allele.                                         disrupt counterbalancing of charges in the (YIDbarrel struc-
PRENATAL DIAGNOSIS OF TPI DEFICIENCY                                                                                                       4509

                                                                              We thank Dr Lynne Maquat for generously providing a plasmid

               I: 1            I           1:2
                                                                           clone of the 315C human TPI gene and David McGonigle for bio-
                                                                           chemical studies. We are grateful to Prof John Lilleyman and Dr
                                                                           Andrew Will for referral of patients.


11:1                      k k
        P N M I:l I:2 II:1 II:2 II:3
                                                                             1. Schneider AS, Valentine WN, Hattori M, Heins HL: Hereditary
                                                                           hemolytic anemia with triosephosphate isomerase deficiency. N Engl
                                                                           J Med 272:229, 1965
                                                                               2. Brown JR, Daar IO. Krug JR, Maquat LE: Characterisation of
                                                                           the functional gene and several processed pseudogenes in the human
                                                                           triosephosphate isomerase gene family. Mol Cell Biol 5: 1694, 1985
                                                                               3. Maquat LE, Chilcote R. Ryan PM: Human triosephosphate
                                                                           isomerase cDNA and protein structure. J Biol Chem 2603748. 1985
                                                                               4. Schneider AS, Valentine WN. Baughan MA, Paglia DE, Shore
                                                                  bP       NA, Heins HL: Triosephosphate isomerase deficiency. A multisys-
                                                               - 146       tem inherited enzyme disorder: Clinical and genetic aspects, in Beu-
                                                               - 111       tler E (ed): Hereditary disorders of erythrocyte metabolism. New
                                                               -   97      York, NY, Grune & Stratton, 1968, p 265
                                                               -   49          5. Bellingham AJ, Lestas AN, Williams LHP. Nicolaides KH:
                                                                           Prenatal diagnosis of a red cell enzymopathy: Triosephosphate iso-
                                                               -   35      merase deficiency. Lancet 2:4 19, I989
                                                                               6. Pekrun A, Neubauer BA, Eber SW, Lakomek M. Seidel H,
                                                                           Schroter W: Triosephosphate isomerase deficiency: Biochemical and
   Fig 3. Prenatal diagnosis of homozygous TPI deficiency due to           molecular genetic analysis for prenatal diagnosis. Clin Genet 47: 175,
the codon 104 mutation in family 2. The propositus (11:l)is homozy-
gous and parents (I:1 and 1:2) heterozygous for t h e mutation. Prenatal
diagnosis of a subsequent pregnancy at 12 weeks' gestation showed              7. Clark ACL. Szobolotsky MA: Triosephosphate isomerase de-
t h e fetus (11:3) to be homozygous for the codon 104 mutation. (P, N,     ficiency: Prenatal diagnosis. J Pediatr 106:417, 198.5
M, and the DNA size marker are a s described in Fig 2 .  1                     8. Dallapicolla B, Novelli G, Cuoco C, Porro E: First trimester
                                                                           studies of a fetus at risk for triosephosphate isomerase deficiency.
                                                                           Prenat Diag 7:289, 1987
                                                                               9. Daar IO, Artymiuk PJ, Phillips DC, Maquat LE: Human trio-
ture of the TPI molecule, promoting unfolding of the enzyme                sephosphate isomerase deficiency: A single amino acid substitution
and accounting for the decreased thermal stability of the                  results in an unstable enzyme. Proc Natl Acad Sci USA 83:7903,
mutant enzyme observed in vitro." To date, all homozygotes
                                                                               IO. Schneider A, Westwood B, Yim C, Prchal J. Berkow R, La-
for the codon 104 mutation have exhibited a severe clinical                botka R, Warrier R, Beutler E: Triosephosphate isomerase defi-
phenotype. This may not be the case for all genotypes, and                 ciency: Repetitive Occurrence of point mutation in amino acid 104
recently a single kindred in which two affected siblings ex-                in multiple apparently unrelated families. Am J Hematol 50:263.
hibit discordant clinical phenotypes has been reported.'l.I3                 I995
The possibility of phenotypic variation should be borne in                      1 I . Chang M, Artymiuk PJ, Wu X, Hollan S . Lammi A, Maquat
mind when counseling families at risk about the likely clini-               LE: Human triosephosphate isomerase deficiency resulting from mu-
cal effects in an affected child.                                           tation of Phe-240. Am J Hum Genet 52: 1260. 1993
   Prospects for treatment of severe TPI deficiency are lim-                    12. Daar 10. Maquat LE: Premature translation termination medi-
                                                                            ates triosephosphate isomerase mRNA degradation. Mol Cell Biol
ited by the need to correct the enzyme deficiency in neural                 8:8O2, 1988
and muscle cells. In the long term this may be achieved by                      13. Hollan S , Fujii H, Hirono A, Hirono K, Karro H, Miwa S,
gene therapy. Until then the ability to offer early prenatal                Harsanyi V, Gyodi E, Inselt-Kovacs M: Hereditary triosephosphate
diagnosis to families at risk for this often lethal disease will            isomerase deficiency: Two severely affected brothers one with and
remain an important option.                                                 one without neurological symptoms. Hum Genet 92:486, 1993

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