W. J. Kleijer

Genotype and phenotype in patients with dihydropyrimidine dehydrogenase deficiency

Abstract Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterised by thymine-uraciluria in homozygous deficient patients and has been associated with a variable clinical phenotype. In order to understand the genetic and phenotypic basis for DPD deficiency, we have reviewed 17 families presenting 22 patients with complete deficiency of DPD. In this group of patients, 7 different mutations have been identified, including 2 deletions [295–298delTCAT, 1897delC], 1 splice-site mutation [IVS14+1G>A)] and 4 missense mutations (85T>C, 703C>T, 2658G>A, 2983G>T). Analysis of the prevalence of the various mutations among DPD patients has shown that the G→A point mutation in the invariant splice donor site is by far the most common (52%), whereas the other six mutations are less frequently observed. A large phenotypic variability has been observed, with convulsive disorders, motor retardation and mental retardation being the most abundant manifestations. A clear correlation between the genotype and phenotype has not been established. An altered β-alanine, uracil and thymine homeostasis might underlie the various clinical abnormalities encountered in patients with DPD deficiency.

Prenatal diagnosis of genetic disorders.

Three hundred and fifty pregnancies were monitored by transabdominal amniocentesis in the fourteenth to sixteenth week of gestation followed by karyotyping or biochemica assays of cultured amniotic fluid cells and analysis of alpha-fetoprotein in the amniotic fluid supernatant. The pregnancy was interrupted in 36 cases (10%) either becasue of a fetal abnormality or the presence of a male fetus in pregnancies at risk for an X-linked disease. Four chromosomal aberrations were found in 87 pregnancies tested because of advanced maternal age. In 101 pregnancies with a recurrence risk of Downs syndrome, 2 fetuses with an abnormal karyotype were detected. In 11 cases, in which 1 parent was a carrier of a balanced translocation, 2 unbalanced fetal karyotypes were found. Fetal chromosome studies in 43 pregancies at risk for an X-linked disease indicated the presence of a male fetus in 21 cases. Prenatal diagnosis of 11 different metabolic diseases was performed in a total of 34 cases. Microchemical techniques were used to allow completion of the diagnosis of seven different enzyme deficiencies within 9 to 22 days after amniocentesis. Alpha-fetoprotein assay in the amniotic fluid supernatant of 47 pregnancies at risk for an open neural tube defect resulted in the detection of 3 anencephalic fetuses during the second half of pregnancy. The safety and reliability of amniocentesis and the possible effects on the outcome of pregnancy are evaluated. Prenatal diagnosis offers a promising alternative for parents who are at risk of having a child with a genetic disease which can be detected in amniotic fluid or in cultured amniotic fluid cells.

Stable isotope dilution analysis of N-acetylaspartic acid in CSF, blood, urine and amniotic fluid: Accurate postnatal diagnosis and the potential for prenatal diagnosis of canavan disease

SummaryA sensitive and selective analytical technique is described for the determination ofN-acetylaspartic acid in body fluids using stable isotope dilution in combination with positive chemical ionization mass spectrometry with selected ion monitoring.Control mean and ranges have been established: in urine 19.5 and 6.6–35.4 µmol/mmol creat.; in plasma 0.44 and 0.17–0.81 µmol/L; in cerebrospinal fluid 1.51 and 0.25–2.83 µmol/L; and in amniotic fluid 1.27 and 0.30–2.55 µmol/L.In a patient with Canavan disease,N-acetylaspartic acid concentration was elevated 80-fold in urine and 20-fold in plasma compared to the control means. A subsequent pregnancy of the mother was monitored and theN-acetylaspartic acid concentration in the amniotic fluid was within the control range and a healthy child was born.

Juvenile Hyaline Fibromatosis: Clinical Heterogeneity in Three Patients

Background: Systemic hyalinoses are genetic generalized fibromatoses characterized by an accumulation of hyalin in the dermis. Two distinctive syndromes are recognized in the literature: infantile systemic hyalinosis (ISH) and juvenile hyaline fibromatosis (JHF). ISH and JHF are sometimes difficult to separate since they show significant overlap. Observations: We report on 3 children from two unrelated families suffering from JHF. The first child is severely handicapped by joint contracture, massive hyperplasia of the gingivae, diffuse skin papules and subcutaneous nodules occupying the scalp, face, perianal area, palms, soles and chest. At the same age, the second child only shows pearly skin papules on the face, groin and perianal area and gingival hyperplasia without joint stiffness or any other subjective complaint. The third patient, a brother of the second child, developed mild skin abnormalities by the end of the first year. The occurrence in siblings and consanguinity in the second family suggests autosomal recessive inheritance. Histological skin examination in the 3 cases showed hyaline deposition in the dermis and abnormal ultrastructure of fibroblasts. Biochemical findings showed mucopolysaccharide abnormalities in both families. Conclusion: Our patients do not only illustrate the different expressions of JHF but also show some overlap with ISH, suggesting a common cause for both disorders. Genetic studies will finally answer this question.

Prenatal diagnosis of sialidosis with combined neuraminidase and ø-galactosidase deficiency

Recently a combined deficiency of neuraminidase and P-galactosidase was reported in two patients (Wenger et al. 1978, O’Brien 1978), who had earlier been described as having variant types of Gq-gangliosidosis (Justice et al. 1977, Koster et al. 1976). We have found similar deficiencies in the second of two children who both died at birth with severe hydrops fetalis (Niermeijer et al. unpublished). The parents are consanguineous, of Turkish origin, and living in The Netherlands. Cultured skin fibroblasts and liver, obtained at autopsy, showed a deficiency of p-galactosidase with a residual activity of 5-10 % of normal. In addition, we found an almost complete deficiency of neuraminidase activity in the fibroblasts and an increased level of sialic acid in both fibroblasts and liver (3 and 23 times the normal levels, respectively). We now wish to report the results of the prenatal diagnosis which was offered to the parents during the third pregnancy. Amniocentesis was done in the 17th week (Dr. R. Vosters, University Hospital, Rotterdam). After 18 days of growth, the amniotic fluid cells were harvested in their first passage for enzyme assays. P-galactosidase and neuraminidase activities were determined using the fluorogenic 4-methyl-umbelliferyl substrates and micromethods, as described earlier for p-galactosidase in the prenatal analysis for GM1-gangliosidosis (Kleijer et al. 1976). The results are shown in Table 1. Both B-galactosidase and neuraminidase activity are strongly reduced in the amniotic fluid cells from the pregnancy at risk, as was the case in fibroblasts of the index patient. These results indicated that the fetus was affected with the variant type of sialidosis. Further evidence was obtained 1 week later by the demonstration of a 2-fold elevated sialic acid content of the amniotic fluid cells (44.3 nmol/mg protein, control: 22.1 nmoYmg). The parents decided on termination of the pregnancy, which was done by prostaglandin induction in the 21st week of pregnancy. The prenatal diagnosis was confirmed by the demonstration of reduced activities in the freshly obtained fetal liver and brain for P-galactosidase (5-10 % of normal activity) and neuraminidase (undetectable). The sialic acid content was 20-fold

α-N-acetylgalactosaminidase deficiency, a new lysosomal storage disorder

SummaryA new lysosomal storage disease with autosomal recessive inheritance is described in two male siblings of 5 1/2 and 4 years of age. Clinical manifestations started after 9 months of age with neurological symptoms, followed by progressive psychomotor deterioration. Urinary oligosaccharide excretion was abnormal and showed a characteristic pattern on chromatography. Enzyme assays showed a profound deficiency of lysosomal α-N-acetylgalactosaminidase in cultured fibroblasts, leukocytes and plasma from the patients and reduced activity in material from the parents. The defiiciency was demonstrated both with an artificial substrate and a natural one, the blood group A trisaccharide. Excessive intra-lysosomal storage of α-N-acetylgalactosamine-containing material was demonstrated in cultured fibroblasts from the patients, using the lectin fromHelix pomatia which is specific for terminal α-N-acetylgalactosamine residues.

Mucopolysaccharidosis type IIIB (Sanfilippo B): identification of 18 novel α-N-acetylglucosaminidase gene mutations

Mucopolysaccharidosis type IIIB (MPS IIIB or Sanfilippo B disease) is an autosomal recessive storage disorder caused by deficiency of the lysosomal enzyme α-N-acetylglucosaminidase. Mutation screening was performed on a group of 22 patients using a combination of SSCP/heteroduplex analysis of amplified genomic fragments and direct sequencing of cDNA fragments. Twenty-one different mutations were identified, 18 of them novel. Together they account for 82% of the disease alleles. The mutation spectrum consists of two small insertions, two small deletions, three nonsense mutations, and 14 different missense mutations, one of them (M1L) affecting the initiation codon. The vast genetic heterogeneity seen in this disorder is reflected by the fact that only three of the mutations were identified in more than one patient.

β-Mannosidase deficiency: Heterogeneous manifestation in the first female patient and her brother

Summaryβ-Mannosidase deficiency was demonstrated in fibroblasts of a girl who showed severe psychomotor retardation, bone deformities and gargoylism and recurrent skin and respiratory infections and who died at 20 years of age from bronchopneumonia. This first demonstration of a female patient confirms the autosomal recessive inheritance of β-mannosidosis. Further investigation of this gipsy family revealed β-mannosidosis in an older brother with a milder manifestation of gargoyl facial dysmorphology, mental retardation, hearing impairment and recurrent infections. β-Mannosidase activity was completely deficient in his cultured skin fibroblasts, leukocytes and plasma. In urine a characteristic disaccharide was present. Heterozygote levels of β-mannosidase were found in fibroblasts and/or plasma of the parents and one sister.

Somatic cell hybridisation studies showing different gene mutations in Niemann-Pick variants

SummaryCultured skin fibroblasts from patients with different clinical types of Niemann-Pick disease were hybridized and sphingomyelinase activities were measured in the heterokaryon cell population. Both the natural substrate (3H-choline) sphingomyelin and the chromogenic analogue hexadecanoylamino-4-nitrophenylphosphorylcholine were used in the complementation analysis. In fusions between cells from type C Niemann-Pick disease with those from type A or B a clear restoration of sphingomyelinase activity occurred, whereas no complementation was found in other fusion combinations. The results indicate that at least two different genes are involved in the mutations leading to the different Niemann-Pick variants.

A new simple enzyme assay for pre- and postnatal diagnosis of infantile neuronal ceroid lipofuscinosis (INCL) and its variants

Palmitoyl-protein thioesterase (PPT) deficiency was recently shown to be the primary defect in infantile neuronal ceroid lipofuscinosis (INCL). The available enzyme assay is complicated and impractical for diagnostic use and is, in practice, unavailable. We have developed a new fluorimetric assay for PPT based on the sensitive fluorochrome 4-methylumbelliferone. This PPT assay is simple, sensitive, and robust and will facilitate the definition of the full clinical spectrum associated with a deficiency of PPT. PPT activity was readily detectable in fibroblasts, leucocytes, lymphoblasts, amniotic fluid cells, and chorionic villi, but was profoundly deficient in these tissues from INCL patients. Similarly, a deficiency of PPT was shown in patients with the variant juvenile NCL with GROD. These results show that rapid pre- and postnatal diagnosis can be performed with this new enzyme assay for PPT.