Dihydropyrimidine Dehydrogenase Deficiency: Homozygosity for an Extremely Rare Variant in DPYD due to Uniparental Isodisomy of Chromosome 1

JIMD Reports DOI 10.1007/8904_2018_138 RESEARCH REPORT Dihydropyrimidine Dehydrogenase Deficiency: Homozygosity for an Extremely Rare Variant in DPYD due to Uniparental Isodisomy of Chromosome 1 André B. P. van Kuilenburg • Judith Meijer • Rutger Meinsma • Belén Pérez-Dueñas • Marielle Alders • Zahurul A. Bhuiyan • Rafael Artuch • Raoul C. M. Hennekam Received: 23 July 2018 / Revised: 17 August 2018 / Accepted: 20 August 2018 / Published online: 23 October 2018 # Society for the Study of Inborn Errors of Metabolism (SSIEM) 2018 Abstract Dihydropyrimidine dehydrogenase (DPD) deficiency is a rare autosomal recessive disorder of the pyrimidine degradation pathway and can lead to intellectual disability, motor retardation, and seizures. Genetic variations in DPYD have also emerged as predictive risk factors for severe toxicity in cancer patients treated with fluoropyrimidines. We recently observed a child born to nonconsanguineous parents, who demonstrated seizures, cognitive impairment, language delay, and MRI abnormalities and was found to have marked thymine-uraciluria. No residual DPD activity could be detected in peripheral blood mononuclear cells. Molecular analysis showed that the child was homozygous for the very rare c.257C > T (p. Pro86Leu) variant in DPYD. Functional analysis of the recombinantly expressed DPD mutant showed that the DPD mutant carrying the p.Pro86Leu did not possess any Communicated by: Jörn Oliver Sass A. B. P. van Kuilenburg (*) : J. Meijer : R. Meinsma : M. Alders : R. C. M. Hennekam Amsterdam UMC, University of Amsterdam, Departments of Clinical Chemistry, Genetics and Pediatrics, Amsterdam Gastroenterology & Metabolism, Amsterdam, The Netherlands e-mail: a.b.vankuilenburg@amc.uva.nl B. Pérez-Dueñas : R. Artuch Departments of Neuropediatrics and Clinical Biochemistry, Institut de Recerca Sant Joan de Déu, CIBERER-ISCIII, Barcelona, Spain B. Pérez-Dueñas Vall d’Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain Z. A. Bhuiyan Service de Médecine Génétique, Laboratoires de Médecine Génétique, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland residual DPD activity. Carrier testing in parents revealed that the father was heterozygous for the variant but unexpectedly the mother did not carry the variant. Microsatellite repeat testing with markers covering chromosome 1 showed that the DPD deficiency in the child is due to paternal uniparental isodisomy. Our report thus extends the genetic spectrum underlying DPYD deficiency. Introduction Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme of the pyrimidine degradation pathway, catalyzing the reduction of uracil and thymine to 5,6dihydrouracil and 5,6-dihydrothymine, respectively. In patients with a complete DPD deficiency (MIM 274270), a considerable variation in the clinical presentation has been observed ranging from severely (neurologically) affected to symptomless. Therefore, a DPD deficiency is probably a necessary, but not a sole prerequisite for the onset of a clinical phenotype (Fleger et al. 2017; van Kuilenburg et al. 1999). Delayed cognitive and motor development and convulsive disorders are relatively frequent manifestations, whereas growth retardation, microcephaly, dysmorphia, autism, hypotonia, and ocular abnormalities are less frequently observed (Chen et al. 2014; Enns et al. 2004; van Kuilenburg et al. 1999, 2002a, 2009). In addition, patients with a DPD deficiency have a strongly reduced capacity to degrade the widely used chemotherapeutic drug 5-fluorouracil and, therefore, an increased likelihood of suffering from severe and sometimes fatal multi-organ toxicity (Johnson and Diasio 2001; van Kuilenburg 2004). 66 DPYD is present as a single copy gene on chromosome 1p21.3 and consists of 23 exons (Wei et al. 1998). A large number of variants have been described in DPYD including large genomic deletions and amplifications (van Kuilenburg et al. 2009). The identification of novel disease-causing genomic aberrations is important to allow analysis of genotype-phenotype relationships in DPD-deficient patients and screening of cancer patients at risk. Our study identified a novel genetic mechanism underlying DPD deficiency, and we present the first patient with a complete DPD deficiency due to paternal uniparental isodisomy of chromosome 1. Materials and Methods Sequence analysis of DPYD, including analysis of intragenic rearrangements, was carried out essentially as described before (van Kuilenburg et al. 2017). Analysis of pyrimidine metabolites was performed using reversedphase HPLC combined with electrospray tandem-mass spectrometry (van Lenthe et al. 2000). Functional expression of a DPYD mutation in mammalian HEK293 Flp-In cells and subsequent analysis of recombinantly expressed DPD protein levels and DPD activity were performed as described before (van Kuilenburg et al. 2017). Twenty six microsatellite repeat markers spreading over the full length of chromosome 1 were used for haplotype analysis. These included 21 markers from ABI-Prism Linkage Mapping Set MD panels 1 and 2 (PE Biosystems, Foster City, CA, USA) and 5 additional markers: D1S2775, D1S2719, D1S2793, D1S415, and D1S2753 (NCBI, UniSTS). After PCR, the amplified fragments were separated using the ABI Prism 377 automatic DNA sequencer (PE Biosystems, Foster City, CA, USA), and the length of the fragments was analyzed with GeneMapper software (PE Biosystems, Foster City, CA, USA). Results Case Report The female patient was the first child of non-consanguineous Portuguese parents. Developmental delay was noticed during the second year of life: she walked unassisted at the age of 20 months and showed language delay. At the age of 3 years, she started to have seizures. Despite treatment with valproic acid and carbamazepine, she continued to have seizures every few weeks to months. A neuropsychological study at 5 years and 8 months using the McCarthy Scales of Children’s Abilities (MSCA) showed significantly reduced scores [verbal, 22; perceptual performance, 22; quantitative, 22; memory, 25; motor, 24 (controls: mean  standard deviation 50  10); and general cognitive index, 50 JIMD Reports (controls: mean  standard deviation 100  15)]. Neurological examination at the age of 7 years revealed a nondysmorphic child with normal growth and head circumference and poor fine and gross motor coordination. She was socially engaging and showed cognitive impairment and language delay. Magnetic resonance imaging (MRI) demonstrated symmetrically enlarged lateral ventricles and a thin corpus callosum. Cerebral white matter signal was normal. EEG showed generalized slow wave discharges with maximal amplitude in frontal lobes and poor organization of background activity. At 10 years, the Peabody Picture Vocabulary Test-IV (PPVT-IV) revealed markedly low verbal abilities (verbal age 4 years and 4 months). An attempt to withdraw valproic acid at 10 years increased epileptic activity. Currently, the patient is 12 years old, and she has adapted to a mainstream school with the support of special education teachers and speech therapy. She suffers from occasional partial and secondarily generalized tonicclonic seizures. Background activity on EEG recording has normalized, and no paroxysms are registered. Biochemical and Genetic Studies As part of a screening for inborn errors of metabolism, purines and pyrimidines were analyzed in urine and plasma. Strongly elevated concentrations of uracil and thymine were observed in urine and plasma which suggested that the patient had a DPD deficiency (Table 1). Subsequent analysis showed no residual DPD activity in peripheral blood mononuclear cells. Sequence analysis of DPYD showed that the patient was homozygous for the c.257C > T (p.Pro86Leu) variant (Table 1). Expression of the mutant DPYD construct containing the c.257C > T (p.Pro86Leu) variant in HEK293 Flp-In cells showed that the DPD mutant carrying the Pro86Leu variant possessed hardly any residual activity (0.7%) compared to the wild-type enzyme (Fig. 1). To exclude the possibility that the lack of DPD activity was the result of an inability to produce the mutant DPD protein in HEK293 Flp-In cells, the DPD protein expression levels were analyzed by immunoblotting. Figure 1 shows that the mutant DPD protein, carrying the Pro86Leu variant, was expressed in a comparable amount as the wild-type protein. Thus, the lack of DPD activity of the mutant DPD enzyme in HEK293 Flp-In cells is not due to rapid degradation of the mutant DPD protein in the HEK293 Flp-In lysates. DNA sequence analysis in the father demonstrated that he was heterozygous for the c.257C > T variant in DPYD, but in the mother the variant could not be detected. DPYD is prone to acquire genomic rearrangements due to the presence of an intragenic fragile site FRA1E, but MLPA analysis showed no intragenic deletions or amplifications of DPYD in the patient or parents. Haplotype analyses with 26 JIMD Reports 67 Table 1 Biochemical and genetic analysis of a DPD-deficient patient Urine (mmol/mmol creatinine) Plasma (mM) Subject Uracil Thymine Uracil Thymine DPD activity [nmol/(mg total protein
h)] DPYDa Patient 236 131 13.4 15.2 <0.025 c.257[C > T];[C > T] Father n.a. n.a. n.a. n.a. n.a. c.257[C > T];[¼] Mother n.a. n.a. n.a. n.a. n.a. c.257C¼ 9.9  2.8 (n ¼ 54)b Controls Median 5 <1 0.19 0.04 Range 1–35 (n ¼ 112) <1 (n ¼ 112) 0.08–0.36 (n ¼ 100) 0.02–0.09 (n ¼ 100) n.a. not available Nomenclature according to http://varnomen.hgvs.org/ b Data taken from (van Kuilenburg et al. 2002b) a Fig. 1 DPD activity and immunoblot analysis of recombinantly expressed wild-type and mutant DPD enzymes. The results represent the relative DPD activity (mean + SD, n ¼ 3) of the DPD mutant carrying the Pro86Leu variant compared to wild-type DPD enzyme. The insert shows the immunoblot analysis of the expressed wild-type and mutant DPD enzyme microsatellite repeats distributed over chromosome 1 to probe for homozygosity for the c.257C > T variant in DPYD by uniparental isodisomy for chromosome 1 demonstrated the patient to be homozygous for all 26 markers (Fig. 2). Fourteen markers were uninformative since they could have been inherited from either parent. For one marker only paternal uniparental disomy (UPD) could be proven. Paternal isodisomy was observed for 11 markers (Fig. 2). phenotype typically observed in clinically affected patients with DPD deficiency (van Kuilenburg et al. 1999, 2002a, 2009). The MRI findings in the present patient are nonspecific and have been reported infrequently in DPDdeficient patients (Chen et al. 2014; Enns et al. 2004). Chromosome 1 is not known to contain imprinted areas or imprinted genes, so UPD of chromosome 1 is not expected to cause a phenotype by a disturbed methylation. The frequency of UPD in newborn is considered to be 1 in 3,500–5,000 (Liehr 2010). Chromosomes 7, 11, 14, 15, and 16 are most often involved in uniparental isodisomy formation, and for chromosome 1 only, a moderate frequency of uniparental isodisomy has been observed (Liehr 2010). The c.257C > T variant (rs568132506) is extremely rare in the general population (allele frequency 5.4  10 5 in gnomAD; http://gnomad.broadinstitute.org/ variant/1-98206012-G-A). So far, this variant has been described in only one patient with a complete DPD Discussion Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterized by thymineuraciluria in homozygous-deficient patients. Here, we present the first case of DPD deficiency due to uniparental isodisomy. The phenotype in the patient, i.e., cognitive impairment, language delay, and seizures, is similar to the 68 JIMD Reports Fig. 2 Genotype analysis of chromosome 1 using 26 microsatellite repeats. The patient was homozygous for all 26 markers and showed paternal uniparental isodisomy for 11 markers and paternal UPD for 1 marker, and 14 markers were uninformative. The presence of the c.257C > T variant (M) or wild-type sequence (WT) of DPYD is indicated for the patient and parents deficiency (van Kuilenburg et al. 2002a). Analysis of the crystal structure of DPD showed that Pro86 is in close proximity to one of the iron-sulfur clusters in the N-terminal domain (van Kuilenburg et al. 2002a). The introduction of a leucine at this position would interfere with the binding of the iron-sulfur cluster, thereby inhibiting electron transport and thus activity (van Kuilenburg et al. 2002a). The elucidation of genetic mechanisms underlying DPD deficiency is increasingly being appreciated since DPD deficiency has been recognized as an important determinant of fluoropyrimidine-associated toxicity in cancer patients (van Kuilenburg et al. 2017; van Kuilenburg 2004). To date, many pathogenic variants have been described in DPYD, and additional rare variants may collectively explain an appreciable fraction of patients with DPD deficiency. Therefore, the identification of novel genetic mechanisms underlying DPD deficiency will not only allow analysis of genotype-phenotype relationships in DPDdeficient patients but also screening of cancer patients at risk. Our study showed that uniparental isodisomy should be considered in DPD-deficient patients with only one parent being a carrier for a pathogenic variant in DPYD. Synopsis The c.257C > T (p.Pro86Leu) variant in DPYD results in a mutant DPD enzyme without residual activity, and uniparental isodisomy should be considered in DPD-deficient patients with only one parent being a carrier for a pathogenic variant in DPYD. Compliance with Ethics Guidelines Conflict of Interest André van Kuilenburg, Judith Meijer, Rutger Meinsma, Belén Pérez-Dueñas, Marielle Alders, Zahurul A. Bhuiyan, Rafael Artuch, and Raoul Hennekam declare that they have no conflict of interest. Details of Ethical Approval The study (W16_179 # 16.210) was approved by the Medical Ethics Committee of the Academic Medical Center. JIMD Reports Patient Consent Statement All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional) and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from the parents of the child included in this study for publication. Authors’ Contribution André van Kuilenburg and Raoul Hennekam: study design, data analysis, and drafting of the article Judith Meijer, Rutger Meinsma, Marielle Alders, and Zahurul A. Bhuiyan: experimental data acquisition and data analysis Belén Pérez-Dueñas and Rafael Artuch: patient care and drafting of the article Guarantor and Corresponding Author André B.P. van Kuilenburg accepts full responsibility for the work and conduct of the study, had access to the data, and controlled the decision to publish. 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