Homozygosity for a gross partial gene deletion of the C-terminal end of ATP7B in a Wilson patient with hepatic and no neurological manifestations.код для вставкиСкачать
American Journal of Medical Genetics 138A:340 –343 (2005) Homozygosity for a Gross Partial Gene Deletion of the C-Terminal End of ATP7B in a Wilson Patient With Hepatic and no Neurological Manifestations Lisbeth Birk Møller,1 Peter Ott,2 Connie Lund,1 and Nina Horn1* 1 The Kennedy Institute, Gl. Landevej 7, DK 2600 Glostrup, Denmark Department of Hepato-gastroenterology V, Aarhus University Hospital, DK 8000 Aarhus C, Denmark 2 We identified a partial gene deletion of ATP7B in a patient with Wilson disease with hepatic onset. The deletion covered exon 20 including major parts of the flanking introns. The breakpoints were identified and the size of the deletion determined to be 2144 bp. The deletion is predicted to lead to a mutated protein product containing 45 aberrant amino acids after transmembrane domain 7, and lacking the transmembrane domain 8 as well as the entire C-terminal cytoplasmic tail. This is the first time a partial gene deletion has been demonstrated in ATP7B. The patient presented at age 10 with hepatic manifestations, including severe jaundice, hepato-splenomegaly, ascites, and spider naevi. The liver biopsy showed fibrosis and early signs of cirrhosis. There was a Kayser–Fleischer ring but no neurological manifestations. All symptoms disappeared with penicillamine therapy. This suggests that the C-terminal cytoplasmatic tail of ATP7B, is not essential for its neurological function. Large deletions in ATP7B may be an overlooked cause of Wilson disease. Patients that are homozygotes for deletions may be valuable for the understanding of the function of various regions of the ATP7B protein. ß 2005 Wiley-Liss, Inc. KEY WORDS: Wilson disease; hepatic type; partial gene deletion; ATP7B; molecular diagnosis INTRODUCTION Wilson disease (WND) [OMIM 277900] is an autosomal recessive disturbance of copper transport characterized by copper accumulation in the liver and subsequently in other organs, mainly brain, kidneys, and cornea, followed by hepatic and/or neurologic symptoms due to copper toxicity. The defective gene ATP7B codes for an energy-requiring pump that is necessary for the incorporation of copper into ceruloplasmin and for biliary excretion of the metal. Grant sponsor: Novo Nordisk Foundation; Grant sponsor: Foundation of 1870; Grant sponsor: Ludvig and Sara Elsass Foundation; Grant sponsor: Apotekerfonden, The Lundbeck Foundation; Grant sponsor: Ovita and Jeppe Juhl’s Foundation. *Correspondence to: Nina Horn, Ph.D., D.MSc., The Kennedy Institute, Gl. Landevej 7, 2600 Glostrup, Denmark. E-mail: email@example.com Received 6 May 2005; Accepted 17 August 2005 DOI 10.1002/ajmg.a.30977 ß 2005 Wiley-Liss, Inc. A broad range of disease-causing mutations has been reported in ATP7B. Non-conservative missense mutations are prevalent while other types of mutations such as small insertions or deletions causing frameshift, nonsense mutations, and splice-site mutations are less frequent [Hsi and Cox, 2004]. In a substantial number of patients, no or only one mutation has been identified. A reason could be that partial gene deletions of one or more exons are overlooked by current methods. Gross deletions of ATP7B have not yet been described, except for an interstitial deletion in the long arm of chromosome 13 that encompassed the WND and RB1 loci in a boy showing both Wilson disease and retinoblastoma [Riley et al., 2001]. However, in that case, the deletion was not homozygous and the authors postulate that the co-occurrence of retinoblastoma and Wilson disease was the consequence of an acquired somatic mutation at the retinoblastoma locus and an inherited mutation at the Wilson disease locus of the maternally derived chromosome 13, superimposed on the hemizygosity associated with the paternally derived deletion. We now describe a patient with Wilson disease who is homozygous for a gross partial gene deletion in the 30 end of the gene. CLINICAL REPORT The propositus was born 1970 in Turkey, the third of four children. The parents are first cousins. The first child, a girl, born 1964, died at the age of 3 months of unrelated causes. The second child, a boy born in 1965, presented in 1976 with jaundice of 2–3 month duration, vomiting, and ascites. He died of coagulopathy before a suspicion of Wilson disease could be confirmed. A younger brother born in 1983 was healthy at examination in 2003. At age 10 years, the boy presented with hepato-splenomegaly, ascites, jaundice, and spider naevi. A Kayser–Fleischer ring and undetectable levels of ceruloplasmin established the diagnosis of Wilson disease. After administration of penicillamine at a final dose of 1 g/day, jaundice disappeared within a few weeks, and ascites vanished over a year. In 1990, the liver biopsy still showed fibrosis and elevated copper content (300 ng/mg (w/w); normal <15) and the 24 hr urine copper during penicillamine treatment (9.1 mmol/day; normal range: 0.3–1.3) was also elevated even after 10 years of treatment. In 1990, the Kayser–Fleischer ring had disappeared. Since 2002, he has been treated with zinc (50 mg elementary Zn t.i.d). At the latest examination (May 2003) he was well, without clinical signs of liver disease. He has never had neurological or psychological symptoms. MATERIALS AND METHODS Isolation of Genomic DNA Genomic DNA was extracted from peripheral blood lymphocytes or cultured fibroblasts using the NaCl method [Grimberg et al., 1989]. Partial Gene Deletion in a Wilson Patient 341 TABLE I. DNA Primers Used in PCR Reactions Name WND8U WND8L WND17I WND19U WND19L WND20U WND20L WND21U WND21L Sequence (50 –30 ) Direction Location aacccttcactgtccttgtc aggcagctcttttctgaac aatcgcagacgctgtcaagc ggcagaccccttcctcac cctgggagagagaagccttt ctaggtgtgagtgcgagtt cagcatttgtcccaggt aatggctcagatgctgtt gcttgtggtgagtggaggcaag Forward Reverse Forward Forward Reverse Forward Reverse Forward Reverse Upstream exon 8 Downstream exon 8 Within exon 17 Upstream exon 19 Downstream exon 19 Upstream exon 20 Downstream exon 20 Upstream exon 21 Downstream exon 21 PCR DNA or cDNA was mixed with 200 nM of each primer, 50 mM of each dNTP, and 0.04 U/ml of AmpliTaq polymerase. Amplification was carried out for 40 cycles of 958C for 30 sec, 558C for 1 min, 728C for 2 min, and a final extension at 728C for 7 min. Multiplex PCR of two exons, the exon analyzed for a deletion and a non-deleted control exon, were investigated simultaneously in a single PCR reaction. Combinations of primer sets 8U/8L, 19U/19L, 20U/20L, and 21U/21L were used (Table I). Spanning of the Deletion Using Flanking Primers Selective amplification of the mutated ATP7B allele was performed using 19U and 21L primers flanking the deleted exons. Amplification of genomic DNA was performed using 150 nM of each primer, 37.5 mM of each dNTP, and 0.04 U/ml of AmpliTaq polymerase. The PCR cycling conditions were 40 cycles of 958C for 1 min, 558C for 1 min, 728C for 4 min, and a final extension at 728C for 7 min. Sequencing of the amplified fragment with the same primers identified the deletion breakpoints. Reverse Transcription (RT-PCR) Total RNA was isolated from 104 –106 Epstein–Barr virustransformed lymphocytes or cultured skin fibroblasts with the Rneasy Mini Kit (QIAgen, Bothell, WA). Single-stranded cDNA was synthesized using Superscript II Rnase H-Reverse Transcriptase (Life Technologies (Gibco BRL), Gaithersburg, MD, USA) and a mixture of random hexamer primers (Amersham Biosciences, Buckinghamshire, UK). The cDNA was amplified by PCR as described above. Sequence Analysis Purified PCR products were sequenced with the dideoxynucleotide chain termination method [Sanger et al., 1977] using 32 P-labeled primer and ThermoSequenase (United States Biochemical Corp (USB), Cleveland, OH, USA). RESULTS We identified a partial gene deletion in the ATP7B gene in a sample of genomic DNA obtained from our Wilson patient. Amplification of exons showed that two consecutive exons, 20 and 21 could not be amplified. The presence of a partial gene deletion was confirmed by multiplex PCR where one of the exons of interest and a non-deleted exon were tested for amplification in a single PCR reaction (Fig. 1A). Using the primer set, 19U/21L, located upstream exon 19 and downstream exon 21, respectively, we were able to span the deletion and amplify a PCR product of about 600 bp from the index patient (Fig. 1B). As expected a PCR product of the same size was demonstrated in both parents (Fig. 1B) but also in the younger healthy brother (not shown). Sequencing of the amplified fragments showed that the deletion covered the fragment starting 87 bp downstream exon 19 and ending 1 bp upstream exon 21. Thus the major part of intron 19, the entire exon 20 and the entire intron 20 except for one base pair were deleted [c.4021 þ 87_41252del]. Comparison with the genomic ATP7B sequence (U11700, http:// Fig. 1. A: Identification of deletion by multiplex PCR. Genomic DNA from the index patient (I) was tested for the amplification of selected exons on a 2% agarose gel. DNA from a control person (C), was included to verify the compatibility of each pair of primers. The sizes of the amplified exons are 299 bp (exon 8), 215 bp (exon 19), 253 bp (exon 20), and 361 bp (exon 21). The marker (L) is fX174DNA HaeIII digest. B: PCR amplification of the junction fragment: PCR amplification on genomic DNA from the index patient (I), the father (F), the mother (M), and a control person (C) was performed using the primer-pair 19U/ 21L (see Table I). The PCR products were separated on a 2% agarose gel. The marker (L) is fX174DNA HaeIII digest. C: Amplification of aberrant transcripts by RT-PCR: cDNA from the index patient (I), the father (F), the mother (M), and a control person (C) was amplified using the primer-pair 17I/21L (see Table I). The PCR products were separated on a 2% agarose gel. The marker (L) is Hyperladder IV (Bioline). The identities of the resulting products were confirmed by DNA sequencing. 342 Møller et al. Normal intron 19 50309317 Patient Normal intron20/exon 21 50307175 tgggggttagtgagtggctcactcactggctggctggctagag IIIIIIIIIIIIIIIIIIIII tgggggttagtgagtggctcagCTATAAGAAGCCTGACCTGG IIIIIIIIIIIIIIIIIIIII ctgttgcgttcctgctttccagCTATAAGAAGCCTGACCTGG Fig. 2. Exact localization of the breakpoints. The PCR amplified products covering the deletion junction from the index patient were sequenced to identify the deletion breakpoints. The nucleotide sequence of the recombination joint identified in the patient is shown. Vertical lines mark identical base pairs. The deletion was from 50309338 in intron 19 to position 50307195 in intron 20 (reference sequence: U11700, http://genome.UCSC.edu). genome.UCSC.edu) documented that the size of the genomic deletion was 2,144 bp [g.50309338_50307195del] (Fig. 2). Using the primer set, 17I/21L, a product of about 800 bp was obtained from the index patient, his parents, and a normal control person (Fig. 1C). Sequencing disclosed that the products were not identical. The product from the index patient contained exon 18, exon 19, the first part of intron 19, and the last bp of intron 20. Exon 18 was correctly spliced, whereas the non-deleted parts of intron 19 (86 bp) and intron 20 (1 bp) were included in the cDNA sequence. The protein product encoded by the resulting cDNA is predicted to contain the normal 1,341 amino acids encoded by exon 1–19 plus an addition of 45 aberrant residues. As exon 20 contains 103 bp, the obtained product from the control person is differing only slightly in size from the product obtained from the index patient. DISCUSSION We here report the finding of a homozygous gross deletion in the C-terminus of ATP7B in a patient with the hepatic type of WND. This is the first report of this type of mutation in ATP7B. This is surprising since in Menkes disease, partial gene deletions account for a substantial proportion of mutations in the homologous gene, ATP7A [Tümer et al., 2003]. Despite extensive screening of the coding region of ATP7B using various techniques, the highest mutation detection rate is about 90%, even after sequencing of all exons [Waldenström et al., 1996; Curtis et al., 1999]. Our results suggest that partial gene deletions in ATP7B may be the causative mutation in some of the uncharacterized alleles. At present, it is impossible to estimate the relative contribution of gross deletions in ATP7B in WND. Of the nearly 60 gross gene deletions reported in ATP7A [Tümer et al., 2003] none are identical, and they are spread over the entire gene including promotor and terminator regions. There are several reasons why partial gene deletions of ATP7B may have been overlooked. Patients with Wilsons disease that carry a partial deletion of one or more exons on one allele may appear to be homozygous for a point mutation on the opposite allele, if the latter occur within the deleted region. Therefore, a suspected homozygosity of a point mutation should be confirmed by analyzing both parents of the Wilson patient. If the mutation cannot be found in both parents and paternity is confirmed, this could indicate the presence of a gross deletion. In the present case, the deletion in the index patient was found because it occurred in the homozygous state. The identification of both breakpoints and definition of primers to span the deletion allowed direct demonstration of the carrier state in both parents as well as in the younger brother. Partial gene deletions in the heterozygous state are not detectable by exon amplification because the normal allele will mask its presence. Alternative methods will be required, for example, quantitative techniques to detect a reduced dosage of an exon. Development of a method for a large-scale search for deletions, also in the heterozygous state, is ongoing. In our patient, the partial gene deletion leads to a protein product lacking the C-terminus after the transmembrane segment 7 but enlarged by 45 aberrant amino acids (p.1341_1465del45ins). The deletion removed several signals including a di-leucine motif. A homologous di-leucine in ATP7A [Hsi et al., 2004] has been shown to be important for the localization of ATP7A in the trans Golgi network [Petris et al., 1998], and, by analogy, the deletion in our patient may disrupt the normal trafficking of ATP7B to and from the trans-Golgi network implicating inadequate loading of ceruloplasmin. A critical role for the C-terminal of ATP7B in copper loading of ceruloplasmin was also indicated in experiments in yeast ccc2 where expression of an ATP7B mutant lacking a part of the Cterminus (p.1371_1465del) prevented copper loading of the ceruloplasmin homolog, Fet3p [Hsi et al., 2004]. In agreement with these observations, ceruloplasmin was below the detection limit in the patient. This defect is likely to be caused by instability of the protein due to improper folding [Hsi et al., 2004]. The clinical case fulfilled the Sternlieb criteria for Wilson disease [Sternlieb, 1990]. The diagnosis is further supported by the death of the older brother with a very similar clinical picture. The two affected brothers presented early, both at the age of 10. The disease was characterized as an aggressive hepatitis with clinical signs of cirrhosis and hepatic decompensation at the age of 10. In larger populations, it has been difficult to establish a clear relationship between the site of the mutation and the presentation of the disease, even in homozygous patients. However, the lack of neurological symptoms in our patient suggests that the C-terminal part of ATP7B is not essential for its neurological functions. This is in accordance with the observations in the LEC rat, an animal model of Wilson disease, characterized by a large gene deletion resulting in loss of half of the ATP binding domain and the last two transmembrane domains plus the C terminus [Wu et al., 1994]. Even in the homozygous state, the deletions primarily cause hepatic and not neurological disease [Santon et al., 2003]. Thus, studies of patients who are homozygous for deletions in the ATP7B gene are valuable for the further understanding of the relation between structure and function of this protein. ACKNOWLEDGMENTS This study was supported by The Novo Nordisk Foundation, The Foundation of 1870, Ludvig and Sara Elsass Foundation, Apotekerfonden, The Lundbeck Foundation, and Ovita and Jeppe Juhl’s Foundation. REFERENCES Curtis D, Durkie M, Balac (Morris) P, Sheard D, Goodeve A, Peake I, Quarrell O, Tanner S. 1999. A study of Wilson disease mutations in Britain. Hum Mut 14:304–311. Grimberg J, Nawoschik S, Belluscio L, McKee R, Turck A, Eisenberget A. 1989. A simple and efficient non-organic procedure for the isolation of genomic DNA from blood. Nucleic Acids Res 17:8390. HGMD. 2005. HGMD entry for ATP7B. Partial Gene Deletion in a Wilson Patient Hsi G, Cox DW. 2004. A comparison of the mutation spectra of Menkes disease and Wilson disease. Hum Genet 114:165–172. Hsi G, Cullen LM, Glerum DM, Cox DW. 2004. Functional assessment of the carboxy-terminus of the Wilson disease copper-transporting ATPase, ATP7B. Genomics 83:473–481. OMIM. 2005a. 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