A novel lysozyme mutation Phe57Ile associated with hereditary renal amyloidosis

Kidney International, Vol. 63 (2003), pp. 1652–1657

A novel lysozyme mutation Phe57Ile associated with hereditary renal amyloidosis MASAHIDE YAZAKI, SANDRA A. FARRELL, and MERRILL D. BENSON Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana; Department of Laboratory Medicine, the Credit Valley Hospital, Mississauga, Ontario, Canada; and Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis, Indiana

A novel lysozyme mutation Phe57Ile associated with hereditary renal amyloidosis. Background. Variant forms of lysozyme, a ubiquitous bacteriolytic enzyme, are known to lead to hereditary non-neuropathic renal amyloidosis and, so far, three different mutations of the lysozyme gene have been reported. In this study, we report a novel lysozyme variant, Phe57Ile, associated with renal amyloidosis in three patients in one Italian Canadian family. Methods. The proband was a 52-year-old woman who developed renal failure at the age of 42 years. Renal biopsy demonstrated replacement of glomeruli by amyloid. Her younger sister and her younger daughter who underwent renal transplantation also had renal amyloidosis. The proband’s older daughter and her niece were in good health. To elucidate pathogenesis of this hereditary renal amyloidosis, DNA analyses of the lysozyme gene, including single strand confirmation polymorphism, direct DNA sequence, and restriction fragment length polymorphism analyses, were performed. Results. DNA analyses of the lysozyme gene revealed a T to A transversion at the first position of codon 57 of the lysozyme gene in the proband, her sister, and her affected and unaffected daughters, indicating a replacement of Phe by Ile at residue 57. In addition, DNA sequencing demonstrated a C to A transversion at the second position of codon 70, denoting a replacement of Thr by Asn at residue 70, in the proband’s sister and her niece. Thus, the proband’s sister is compound heterozygous for the Phe57Ile and Thr70Asn alleles. Conclusion. Distinctive clinical features in patients of this family are nephropathy due to renal amyloidosis. Our results indicate that the novel lysozyme variant Phe57Ile is associated with renal amyloidosis in this family. From our results, a clear relation between the Thr70Asn polymorphism and renal amyloidosis could not be demonstrated.

Hereditary renal amyloidosis may be caused by mutations in a number of amyloid precursor proteins, including transthyretin (TTR), apolipoprotein A-I (apoA-I), gelsolin, fibrinogen A ␣-chain, lysozyme, and apolipoKey words: hereditary amyloidosis, lysozyme variant, nephropathy, polymorphism, compound heterozygosity. Received for publication July 9, 2002 and in revised form November 4, 2002 Accepted for publication December 10, 2002

 2003 by the International Society of Nephrology

protein A-II (apoA-II) [1–6]. Each type of amyloidosis is inherited as an autosomal-dominant disease and is associated with a structurally altered protein that aggregates to form amyloid fibrils. These proteins may be the result of single nucleotide change, deletion, or insertion in the gene coding for the amyloid precursor protein. Lysozyme is a ubiquitous bacteriolytic enzyme present in external secretions and in polymorphonuclear leukocytes and macrophages. Variant lysozyme is known to cause non-neuropathic renal amyloidosis and, so far, three mutations in the lysozyme gene, which are associated with renal amyloidosis, have been described [5, 7]. Herein, we report a novel lysozyme variant (Phe57Ile) associated with renal amyloidosis in a Caucasian family. CASE REPORT: THE PROBAND The proband (II-1 in Fig. 1) was a 52-year-old Italian Canadian woman. She had been in good health until age of 39 years, and she developed renal failure at the age of 42 years. Renal biopsy demonstrated replacement of glomeruli by amyloid deposits with the characteristic green birefringence under polarized light with Congo red staining (Fig. 2). Dialysis was initiated, and she underwent renal transplantation at the age of 44 years. However, she developed ischemic heart disease in her late forties and she died of postoperative complications at the age of 52 years, following triple coronary bypass surgery. She did not show any overt signs of hepatosplenomegaly, neuropathy, amyloid cardiomyopathy, or skin petechiae. DNA analysis of a number of genes that are associated with renal amyloidosis, including TTR, apoA-I, apoA-II, and fibrinogen A ␣-chain failed to reveal any abnormalities. Her father (I-1 in Fig. 1) reportedly died of heart attack at the age of 57. Her mother died of gastric cancer when she was in her 80s. Both parents came from large families, with no recognized history of renal failure. A daughter (III-2 in Fig. 1) of the proband developed hypertension at the age of 17 years. A renal biopsy demonstrated severe amyloid deposition in glomeruli and arterioles. She was started on hemodialysis at the age of

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Fig. 1. Pedigree and Tsp509 I restriction site analysis of lysozyme exon 2 polymerase chain reaction (PCR) products. Solid symbols denote affected patients and an inner solid circle denotes an unaffected mutant gene (lysozyme Phe57Ile) carrier. Asterisks indicate a carrier of a Thr70Asn allele. An arrow indicates the proband. In restriction fragment length polymorphism (RFLP) analysis, the T to A transversion at the first position of codon 57 of the lysozyme gene creates a Tsp509 I restriction site within the 236 bp exon2 PCR amplified fragment. Enzyme digestion gives fragments of 128 bp and 108 bp for the mutant allele. The PCR-amplified fragments (236 bp) of the proband (II-1), her affected sister (II-2), and her unaffected (III-1) and affected daughter (III-2) were incompletely digested, indicating heterozygosity for the mutation. Abbreviations are: M, 100 bp molecular ruler DNA size standard (Bio-Rad); C, normal control.

Fig. 2. Histopathologic findings of biopsied kidney of the proband showing amyloid deposition in glomeruli and vascular walls (arrows). (A) Congo red staining. (B) Apple green birefringence typical of amyloid under polarized light.

22 years and underwent renal transplantation at the age of 23 years. She has been well for 13 years. The oldest daughter of the proband (III-1), at the age of 42 years, is clinically well. II-2, a younger sister of the proband had proteinuria at the age of 35 years, although her blood pressure was normal. Renal biopsy revealed amyloid deposition in glomeruli, vessel walls, and tubular basement membranes. She underwent renal transplantation at the age of 43 years and has been in good health since then. The proband’s niece (III-3) is clinically well at the age of 33 years. METHODS DNA isolation After full informed consent was obtained, genomic DNA samples of the proband (II-1), the proband’s sister

(II-2), two daughters (III-1 and III-2), and her niece (III-3) were isolated from peripheral blood leukocytes using standard phenol/chloroform method. Single-strand conformation polymorphism (SSCP) analysis All four exons of the lysozyme gene [8] were examined by SSCP analysis using oligonucleotide primers as indicated in Table 1. Polymerase chain reaction (PCR) was done in a total volume 50 ␮L, containing 5 ␮L of 10 ⫻ PCR buffer (100 mmol/L Tris HCl, pH 8.3, 500 mmol/L KCl, 12 mmol/L MgCl2), 8 ␮L of 1.25 mmol/L desoxynucleoside triphosphate (dNTP), 15 pmol of each primer, 2.5 units of Taq polymerase (Sigma Chemical Co., St. Louis, MO, USA) and 100 ng of genomic DNA. Amplification was performed using a Perkin-Elmer Thermal

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Fig. 3. DNA sequence analysis of exon 2 of the lysozyme gene. Both thymine and adenine (arrows) are present at the first position of codon 57 of the lysozyme gene in the DNA sequence of the proband (II-1) and her affected sister (II-2). Furthermore, both cytosine and adenine (*) are present at the second position of codon 70 in the proband’s sister (II-2) and her unaffected niece (III-3) who does not have the T to A transversion at the first position of codon 57.

Cycler (Norwalk, CT, USA) for 35 cycles consisting of denaturing at 94⬚C for 1 minute, annealing at 60⬚C for 1 minute, and extension at 72⬚C for 1 minute. SSCP analysis was performed using 6 ␮L of the PCR product as previously described [9]. To confirm that the lysozyme Phe57Ile mutation is not a polymorphism that is observed in healthy people, SSCP analysis of lysozyme exon 2 was performed on DNA from 100 Caucasian volunteers. Direct DNA sequencing Exon 2 of the lysozyme gene of the family members (II-1, II-2, III-1, III-2, and III-3), and exons 1, 3, and 4 of III-1 and III-2 were examined by direct DNA sequencing. PCR was done as indicated above. PCR products were purified by QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) according to the manufacture’s protocol. Sequencing reaction was performed by using 1.0 ␮L of purified PCR product as template and the Thermo Sequence [␣-33P]-ddNTP-Radiolabeled Terminator Cycle Sequencing Kit (USB Cleveland, OH, USA). Samples were electrophoresed on a 6% polyacrylamide gel with 7 mol/L urea at 45W for 3 hours using a glycerol-tolerant gel buffer, dried, and exposed to Kodak X-Omat film (Rochester, NY, USA). Restriction fragment length polymorphism (RFLP) analysis A 236 bp subsequence of exon 2 of the lysozyme gene was amplified using oligonucleotide primers Lyz E2-1

Table 1. Olignucleotide primers for human lysozyme gene Lyz Lyz Lyz Lyz Lyz Lyz Lyz Lyz

E1-1: E1-2: E2-1: E2-2: E3-1: E3-2: E4-1: E4-2:

5⬘-CAGTCA ACA TGA AGG CTC TCA T-3⬘ 5⬘-GTT CCA TAC GTA GCT AAT TCT CT-3⬘ 5⬘-CAC TTA GTG TTG CTG TTT ATT ACT-3⬘ 5⬘-AGA TTG GTC AAA TAT TAG CTT GTC-3⬘ 5⬘-ATT CCT TAC CAC CTG TCT TTC A-3⬘ 5⬘-TGC TCT CAT TGT ATA TAT CAA CAG-3⬘ 5⬘-AAC CTT TCA CCA TTT GCT TCA TC-3⬘ 5⬘-ATG TGA GAG AGA CAA AAT GAG CT-3⬘

and Lyz E2-2 presented in Table 1. PCR products were digested with the endonuclease Tsp509 I (New England Biolabs, Beverly, MA, USA) at 65⬚C for 4 hours, and then electrophoresed through a 3% (weight/volume) Nusieve GTG agarose gel (BMA, Rockland, ME, USA). RESULTS SSCP analysis of exon 2 of the lysozyme gene revealed three patterns of abnormally migrating bands in the family members compared to normal controls. One is seen in the proband (II-1), her unaffected daughter (III-1), and her affected daughter (III-2). The second pattern is seen in her affected sister (II-2). The third is seen in an unaffected niece (III-3). No other abnormalities were detected in exons 1, 3, and 4 by SSCP analysis. Direct DNA sequencing of exon 2 in the proband (II-1) showed a single base transversion at the first nucleotide of codon 57 in the lysozyme gene [8] (TTT to ATT), indicating a replacement of Phe by Ile at residue 57 (Fig. 3). This

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Yazaki et al: Renal amyloidosis with lysozyme Phe57Ile Table 2. Five kindreds with lysozyme amyloidosis Families

1

2

3

Mutation (exon) Ethnic background Residence Clinical features

His56Thr (exon 2) English UK Nephropathy, petechiae

Phe57Ile (exon 2) Italian Canada Nephropathy

Organs where amyloid deposits were proven Reference

Kidney, liver, spleen, skin, lymph nodes

Kidney

Trp64Arg (exon 2) French France Nephropathy, intestinal obstruction, Sicca syndrome Kidney, GI tract, salivary glands

[5, 12]

Present study

[7]

4

5

Asp67His (exon 2) English UK Nephropathy, GI bleeding

Asp67His (exon 2) English UK Liver rupture

Kidney, liver, spleen, GI tract

Liver, spleen, lymph nodes, rectum

[5, 13, 14]

[11]

GI stands for gastrointestinal.

nucleotide change was detected in the proband’s daughters (III-1 and III-2) and her sister (II-2). In addition, DNA sequencing showed a single base transversion at the second position of codon 70 (ACC to AAC), indicating a replacement of Thr by Asn at residue 70 in the proband’s sister (II-2) and her niece (III-3). Other mutations were not seen in exons 1, 3, and 4 of III-1 or III-2 by the DNA sequence analysis, consistent with the result of SSCP analysis (data not shown). RFLP analysis of the 236 bp PCR product from LyzE2-1 and LyzE2-2 primers revealed that the proband had the Tsp509 I recognition site associated with the T to A mutation at the first position of codon 57 of the lysozyme gene. Electrophoresis of the digested PCR product in the proband (II-1), her daughters (III-1 and III-2), and sister (II-2) gave bands of 128 bp and 108 bp in addition to the normal 236 bp band, indicating heterozygosity for the Phe57Ile mutation (Fig. 1). By SSCP analysis of exon 2 lysozyme gene in 100 unrelated persons (200 alleles), 12 persons had the abnormal migration pattern as seen in the proband’s niece (III-3). Direct DNA sequence analysis of exon 2 lysozyme gene revealed that none of these 12 persons had the Phe57Ile mutation. All of them were heterozygous for ACC (Thr) and AAC (Asn) at codon 70 (data not shown). Therefore, the allele frequency of Thr70Asn of lysozyme gene in this study was 6.0% (12 alleles/200 alleles). DISCUSSION Hereditary renal and nonneuropathic amyloidosis is known as Ostertag type amyloidosis [10], and, by recent molecular and biochemical techniques, the pathogenesis of this type of amyloidosis has been gradually elucidated. To date, several types of amyloidosis, including fibrinogen A ␣-chain, lysozyme, and apoA-II have been shown to cause this Ostertag-type amyloidosis. In the present study, our results indicate that renal amyloidosis in this kindred is associated with the newly discovered lysozyme mutation Phe57Ile. During the clinical courses of all patients in this family, the most notice-

able clinical manifestation was nephropathy caused by renal amyloidosis. In particular, the distinctive feature of amyloidosis in this family is that affected individuals do not have significant dysfunction due to amyloid deposition in other visceral organs, including the heart, liver, spleen, peripheral nerve, and skin. Amyloidosis associated with lysozyme mutations is a very rare autosomal-dominant disorder; only four families have been so far reported [5, 7, 11]. Two lysozyme mutations (Ile56Thr and Asp67His) were first described in two English kindreds in 1993 [5], and since then, an additional mutation (Trp64Arg) has been reported in a French family [7]. A cardinal clinical feature of the amyloidosis caused by these three mutations is nephropathy due to amyloid deposition in kidney and peripheral neuropathy is not noticeable. The clinical phenotype, however, may be variable with each lysozyme mutation or even with the same mutation (Table 2). With the lysozyme Ile56Thr mutation [5, 12], all patients had skin petechiae in addition to nephropathy, and with Trp64Arg [7], intestinal obstruction and sicca syndrome were shown. In lysozyme Asp67His, the clinical pictures were sharply different between the first reported family and the second reported family [11]. In the first reported family with the Asp67His variant, patients had few clinical symptoms related to liver and spleen dysfunction [5, 13, 14]. Nonetheless, they had marked hepatic and splenic amyloidosis [5, 13, 14]. On the other hand, in the second reported family, all three affected members in three generations presented with massive hepatic hemorrhage due to hepatic amyloid deposition between the ages of 15 and 50 years [11]. Of interest is that no patients in this family had renal amyloidosis on histopathologic examination [11]. Our results showed that two members (II-2 and III-3) of the family presented here have a Thr70Asn allele and the proband’s affected sister (II-2) is compound heterozygous for the Phe57Ile variant allele and the Thr70Asn allele. Lysozyme Thr70Asn substitution reported by Booth et al [15, 16] is now recognized as a polymorphism in the lysozyme gene. Although it is hypothesized that the hydrogen bonds in the lysozyme molecule that make an

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important contribution to the stability of the ␤-domain would be disturbed in both the Thr70Asn substitution and in the Asp67His variant [15–17], a relationship between the Thr70Asn polymorphism and amyloid fibril formation has not been demonstrated. Additionally, in many genetic disorders, some polymorphisms are known to play a very important role in severity of the disease. In TTR amyloidosis, some polymorphisms of the TTR gene were known to have an impact on the clinical phenotype. While Terazaki et al [18], reported a compound heterozygous patient with TTR Val30Met and Arg104His, the clinical picture of that patient was significantly milder than that of patients with TTR Val30Met and wild-type TTR in the same family [18]. Moreover, it was recently reported that TTR Thr119Met molecules increase the stability of the TTR tetramer with TTR Val30Met molecules compared to the tetramer composed of TTR Val30Met and wild-type TTR [19]. However, in the study described herein, the clinical picture of the proband’s sister (II-2) was almost the same as that of proband. Furthermore, the proband’s niece (III-3), who carries the Thr70Asn allele, inherited it from her mother (II-2) and 12 out of 100 unrelated Caucasian volunteers who have this allele do not have any symptoms associated with lysozyme amyloidosis. In addition, the onset of disease in the proband’s affected daughter (III-2), who does not have the polymorphism allele, was obviously earlier that that of II-2. Therefore, no clear association between the Thr70Asn and lysozyme amyloidosis could be found in this study. While the proband’s older daughter (III-1) also has the Phe57Ile variant allele, she has not shown any symptoms of by amyloidosis. At this time, it is very difficult to say whether she is an unaffected gene carrier or her disease is late onset. However, in many genetic disorders, especially in TTR amyloidosis, it is not unusual that clinical variability, including difference of the onset and the symptoms, is observed even in the same kindred. Holmgren et al [20] reported a pair of monozygotic twins with TTR Val30Met. At age of 55, one brother had already had symptoms of TTR amyloidosis (Val30Met) for 7 years, while another was in good health [20]. Hence, in addition to the genetic mutation, other factors could play an important role in the onset of the amyloidosis. Although it is not clear how the lysozyme Phe57Ile variant leads to amyloid fibril formation, the mechanisms of fibril formation have been investigated in the two mutations, Ile56Thr and Asp67His [17, 21, 22]. X-ray crystallography of these lysozyme variants indicated that structure is either identical to the wild-type lysozyme (Ile56Thr) or slightly rearranged (Asp67His), as mentioned above [17]. In addition, these variants were still enzymatically active [17, 21]. These data suggest that the mutations do not alter the normally folded structure so as to directly facilitate fibril formation. However, these

variants were less stable with respect to denaturation than wild-type lysozyme [17, 21]. Thus Booth et al [17] suggested that partly folded intermediates, which were described as “molten globule-like form” are involved in fibrillogenesis. On the other hand, Canet et al [22] suggested that the mechanisms causing the instability of these lysozyme variants could be different from each other. The location of substitution of the Ile56Thr in the variant lysozyme is at the base of ␤-domain, which has an influence on the folding of the ␤-domain [22]. The location of the His67Asp in the variant lysozyme is in an outer loop of the ␤-domain, which gains native structure only toward the end of the refolding process [22]. Hence, Canet et al [22] suggested that, in the Ile56Thr variant, the origin of the instability of the intermediates is primarily caused by a reduction in the folding rate, compared to an increase in the unfolding rate in the Asp67His variant, which is 160 times greater than that of the wildtype. Therefore, the mechanism of amyloidogenesis in the Phe57Ile may be similar to that in the Ile56Thr variant, as suggested above [22], since the Phe57Ile variant is also located at the base of the ␤-domain. ACKNOWLEDGMENTS This work was supported by NIH grants PHS AG 10133, DK42111, and RR-00750, Veterans Affairs Medical Research, the Marion E. Jacobson Fund, and the Machado Family Research Fund. Reprint requests to Merrill D. Benson, M.D., Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, 975 West Walnut Street, IB-503, Indianapolis, Indiana 46202. E-mail: [email protected]

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