Hereditary renal amyloidosis associated with variant lysozyme in a large English family

Julian D. Gillmore, David R. Booth, S. Madhoo, Mark B. Pepys and Philip N. Hawkins

Immunological Medicine Unit, Division of Medicine, ICSM, Hammersmith Hospital, London, UK

Correspondence and offprint requests to: J. D. Gillmore, Immunological Medicine Unit, Division of Medicine, ICSM, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK.



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Two kindreds with hereditary systemic amyloidosis caused by the first two mutations to be described in the human lysozyme gene were discovered recently and study of the variant lysozyme has been powerfully informative about mechanisms of amyloid fibrillogenesis. However, the clinical manifestations in these families, additional members of which have lately been identified, have not previously been reported in detail.

Methods. The proband presented with proteinuria aged 50 and a family history of amyloidosis, and underwent renal biopsy, whole-body serum amyloid P component (SAP) scintigraphy, and sequencing of the lysozyme gene. Her family history and the phenotype of hereditary lysozyme amyloidosis were thoroughly documented and compared with the presentation and natural history of all other known patients with this condition.

Results. The proband belonged to an extended English family other members of which were known to have hereditary lysozyme amyloidosis. Those with amyloid in previous generations presented with renal involvement, frequently developed complications due to gastrointestinal amyloid, and died before age 60. All amyloid deposits were composed of lysozyme and complete concordance was established between amyloid and heterozygosity for a point mutation in the lysozyme gene, encoding the previously reported Asp67His substitution in the mature protein.

Conclusion. The phenotype, reported for the first time in this extended kindred, contrasts with that of an apparently unrelated family carrying the same mutation who presented with spontaneous hepatic haemorrhage and rupture, and with the manifestations in a family with the lysozyme Ile56Thr variant who presented with dermal petechiae before proceeding to fatal visceral amyloidosis. A remarkably wide spectrum of disease can be caused by the same amyloid fibril protein, although renal involvement predominates in all cases except those dying of hepatic rupture.

Keywords: amyloid; amyloidosis; hereditary lysozyme amyloidosis; lysozyme



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Hereditary non-neuropathic systemic amyloidosis is a very rare autosomal dominant condition that causes serious morbidity and is usually fatal [1]. There is widespread deposition of amyloid in the tissues but the major clinical manifestations are related to renal, cardiac, and hepatic involvement. In different kindreds, the condition is caused by mutations in the genes encoding apolipoprotein A-I (apoA-I) [25], fibrinogen {alpha}-chain [6,7] and lysozyme [8].

Variant lysozyme was first discovered to be an amyloid fibril protein associated with hereditary systemic amyloidosis in 1993 [8]. Since the structure of human lysozyme was known to atomic resolution [9], and the folding has been extensively studied, this discovery provided a powerful model for understanding fibrillogenesis [10]. However, the clinical manifestations of hereditary lysozyme amyloidosis have not previously been reported in detail. We report here a kindred with hereditary lysozyme amyloidosis found to be part of a previously reported family, detailing the clinical manifestations for the first time. In addition, we report the correct amyloid type and phenotype in a family originally reported to have apoA-I amyloidosis [11] and review the clinical manifestations of hereditary lysozyme amyloidosis in all of three kindreds known to have this condition.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Proband and kindred
A 51-year-old Caucasian English woman with a family history of renal disease was found to have proteinuria during a routine examination when aged 50 years. She underwent extensive investigation to elucidate the cause of her proteinuria. The family history was reviewed in detail and several members of the kindred were similarly investigated.

Scintigraphy with iodinated serum amyloid P component
Serum amyloid P component (SAP) binds avidly and specifically to all types of amyloid fibrils and radiolabelled pure SAP is a quantitative in vivo probe for detecting and monitoring amyloid deposits [12]. SAP was labelled with 123I. Each of eight patients investigated received an intravenous injection of 100 MBq of 123I-SAP and underwent anterior and posterior whole-body gamma-camera scanning 24 h later.

Histology and immunohistochemistry
Formalin-fixed paraffin-embedded renal biopsy specimens were available from the proband (III8), her non-identical twin sister (III9), and relatives III2, III3, III4 and III6; lung and gastrointestinal tissue from III2 were also available. No tissues were available to us from the proband's older brother or mother but amyloid had previously been identified in their renal biopsies at other UK centres. Amyloid was identified by its pathognomonic green birefringence in 6-µm sections stained with Congo red and viewed in crossed polarized light [13]. Immunohistochemical staining for lysozyme in 6-µm tissue sections was performed exactly as previously described, using monospecific anti-lysozyme antiserum [8]. Specificity of staining was established by its complete abolition following absorption of the antibody with pure human lysozyme (Sigma). Sections were also stained with monospecific antiserum to human apoA-I.

Direct DNA sequencing
DNA was extracted from the blood of the proband, her sister (III9), and individuals III3, IV4, IV5, III5, III6 and III1. Exon 2 of the lysozyme gene was amplified by polymerase chain reaction (PCR) and the nucleotide sequence determined as previously reported [8].



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Proband
The proband was clinically well at presentation, with blood pressure 140/80 and no other physical abnormality. Serum creatinine was 104 µmol/l, albumin 35 g/l, and 24-h urine protein 0.5 g; liver function tests were normal. A renal biopsy at presentation revealed amyloid. Electrocardiogram, echocardiogram, and nerve conduction studies were normal, and there was no evidence of peripheral or autonomic neuropathy. Proteinuria and renal function have remained stable in the year since presentation.

Kindred
A family tree of the kindred is shown in Figure 1Go. The proband has two daughters (IV6, IV7) aged 26 and 25, respectively, who are both clinically well. The proband's elder brother (III7) developed renal failure, received a transplant aged 49, and died of congestive cardiac failure aged 57. Her non-identical twin sister (III9) presented aged 48 with anaemia for which no cause was found, but 2 years later her creatinine was 160 µmol/l and a renal biopsy revealed amyloid. Renal function has remained stable in the 1 year since then, but she developed hypochromic, microcytic anaemia associated with melaena. She unfortunately refused endoscopic investigation following a normal barium meal. She has six children between the ages of 22 and 36 years, all of whom are clinically well but have not been investigated further. The proband's younger brother (III10) is clinically well at 48 years of age, but has not been investigated further. The proband's mother (II13) presented with anaemia aged 56 and died within a year with end-stage renal failure due to biopsy-proven amyloidosis.



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Fig. 1. Family tree of the affected kindred. The proband is indicated by the arrow. Dead individuals are indicated by a diagonal line through the symbol. The symbols are filled to show the following information:

Renal impairment or proteinuria

Amyloid confirmed histologically

SAP scintigraphy positive for amyloid

Lysozyme mutation present

Normal renal function, SAP scan and genotype

Symbols or part of symbols left blank indicate that tests have not been done and/or information is not available on these individuals.

 
The proband's maternal grandfather (I4), who died aged 38, was one of 12 siblings and had a cousin of the same name (I2) who was known to our Unit. She (I2) was married twice and children from both marriages were found to have amyloidosis. Although she herself was well until her sudden death from unknown causes aged 60 years, she must have been heterozygous for the lysozyme mutation. Of the affected children from the first marriage, II2 died aged 43 years with end-stage renal failure and II3 died with `uncharacterized carcinoma' in his 40s. He had three children, all of whom had biopsy-proven amyloidosis. Patient III2 presented at the age of 33 years with hypertension and deteriorating renal function. He became dialysis dependent aged 41 years at which time there was miliary mottling on the chest radiograph and lung biopsy revealed amyloidosis. At the age of 49 years he received a renal transplant but died post-operatively of extensive bleeding from gastric amyloid. A cholecystectomy specimen from III3 contained amyloid; however, he and his three sons (IV1, IV2, IV3) are clinically well.

Patient III4 presented with hypertension and renal impairment aged 45 years [14]. She became dialysis dependent and died at the age of 48 years of sepsis. Of the affected children from I2's second marriage, II5 died in renal failure aged 38 years, and II6 presented with hypertension and renal impairment aged 33 years and died 3 months later following rapid progression of his renal failure. His daughter, aged 43 years, remains clinically well, but his son (III6) presented to our Unit in 1981 aged 23 years, with renal impairment (serum creatinine 136 µmol/l) and dry, gritty eyes. We diagnosed hereditary non-neuropathic amyloidosis and reported the case, although we had not then identified the amyloid fibril protein [15]. His renal impairment has deteriorated remarkably slowly in a non-linear fashion and he remains dialysis independent 18 years later with a serum creatinine of 218 µmol/l, but has intermittent rectal bleeding, and retrosternal burning pain relieved by omeprazole.

Serum amyloid P component scintigraphy
The scans (Fig. 2AGo) demonstrated extensive amyloid deposits in the kidneys, spleen, and liver of the proband. A similar distribution was seen in her non-identical twin sister (III9), III6 and III3. Interestingly, III6 has now been followed up for 10 years with serial SAP scintigraphs, during which time there has been no evidence of accumulation of amyloid deposits (Figure 2BGo). SAP scintigraphy in III5, IV4, IV5 and III1 was normal.



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Fig. 2. (A) Posterior whole-body scintigraphic images after intravenous injection of 123I-human SAP. There is heavy amyloid deposition in the liver, spleen and kidneys of the proband (left). The image on the right from patient III5 shows no amyloid and is normal, with tracer distributed throughout the blood-pool only. (B) Anterior whole-body SAP scintigraphs taken 10 years apart in patient III6. There is heavy amyloid deposition within the liver and spleen with little tracer remaining in the background blood-pool. No change in distribution or load is evident between the two scans.

 
Histology and immunohistochemistry
Extensive amyloid deposits were found in the renal biopsy specimens of the proband, her sister (III9), III2, III3, III4 and III6, and all stained specifically with antibodies to lysozyme although with variable intensity, possibly reflecting different fixation methods. The staining was completely abolished by prior absorption of the antiserum with an excess of pure human lysozyme (Sigma). Lysozyme is thus the major component of the amyloid fibrils. There was no staining with antibodies to apoA-I.

Lysozyme gene mutation
Sequencing of exon 2 of the lysozyme gene from the proband, III9, III6, and III3 revealed that they were heterozygous for a single base substitution that altered the codon at position 67 of the mature protein from that for Asp to His (Fig. 3Go). The remainder of the gene in those individuals was identical to the published sequence of the human lysozyme gene [16]. Amplification of exon 2 of the lysozyme gene of III5, IV4, IV5 and III1 revealed wild-type sequence. The concordance of the mutation with clinical, histological and scintigraphic evidence of amyloid is shown in Table 1Go.



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Fig. 3. (A) Partial DNA sequence of the lysozyme gene of the proband. A substitution of guanidine by cytosine results in a change at codon 67 from GAT (aspartic acid) to CAT (histidine) (mutation arrowed). (B) This substitution introduces an N1aIII restriction enzyme site. Lane 1, uncut lysozyme exon 2 PCR product, lanes 2 and 3 are the normal and His67 variant PCR products respectively, digested with N1aIII..

 

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Table 1. Concordance of lysozyme mutation and amyloidosis
 


   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The demonstration that the amyloid deposits in affected members of this kindred were composed of lysozyme and the complete concordance between presence of the lysozyme gene mutation and development of amyloidosis indicate that the mutation is the cause of disease in this family. We have previously reported part of this kindred [8,15], and it is one of only three known families with hereditary lysozyme amyloidosis. The others are an English family, which we have been unable to link to our present kindred, even though they carry the identical Asp67His lysozyme mutation [17], and a kindred with the Ile56Thr lysozyme mutation [8], previously incorrectly reported by another group to have apoA-I amyloidosis [11]. This was based upon immunohistochemical staining but omitting the critical specific antigen absorption control.

A renal presentation was almost universal among affected members of the present kindred although the age at which it was detected varied from 23 to 50 years. The renal impairment progressed at rates differing widely between members of the kindred. The serum creatinine in III6, the proband in our original report [15], has only increased from 136 to 218 µmol/l over 18 years, whilst his father (II6) had rapidly progressive renal failure and died within 3 months of presentation. Two members of the kindred have received renal transplants, with one post-operative death due to gastric bleeding and with the other graft functioning successfully for 8 years.

Despite massive hepatic and splenic amyloid deposits in all affected members of the kindred, none of them exhibited adverse clinical effects related to this organ involvement. This differs sharply from the other family with Asp67His lysozyme amyloidosis, in whom the three affected members in different generation all presented with massive, and usually fatal, hepatic haemorrhage between the ages of 15 and 50 [17]. The reason for the contrasting phenotype in these kindreds is unknown. The single affected survivor from this family underwent emergency liver transplantation but has subsequently developed gastrointestinal bleeding associated with macroscopic amyloid deposits throughout the gastrointestinal tract. It is noteworthy that the proband's sister (III9) is currently undergoing investigation for gastrointestinal blood loss, that III2 died of bleeding from extensive gastric amyloid, and that III6 has had intermittent blood loss per rectum and retrosternal burning relieved by omeprazole. Indeed, all upper gastrointestinal biopsies have shown very extensive amyloid, and gross mucosal lesions have been evident at endoscopy in most symptomatic individuals.

The peripheral and autonomic nervous systems were apparently spared in all affected members of the present kindred. The proband, III9, III6, and III3 were carefully screened by electrocardiography and echocardiography, but showed no evidence of cardiac amyloid deposition. Similarly, the surviving member of the other kindred with Asp67His lysozyme amyloidosis does not have cardiac involvement or peripheral neuropathy. This contrasts with hereditary systemic amyloidosis due to variant transthyretin in which the heart and nerves are invariably involved, and renal apoA-I amyloidosis in which this occurs in some kindreds [5,18].

The only other known lysozyme variant, Ile56Thr, also caused systemic amyloidosis but presented differently [11], with dermal petechiae in all affected subjects as well as major visceral amyloid. The proband of the single reported family had multiple amyloid nodules in a resected section of small bowel, in addition to amyloidotic hepatosplenomegaly and proteinuria. The proband's mother died of renal failure aged 52, 1 year after the discovery of proteinuria. The proband's sister developed melaena and renal impairment aged 24, and over 10 years progressed very gradually to end-stage renal failure, from which she died. At autopsy the liver, spleen and abdominal lymph nodes were all enlarged with heavy amyloid deposition. An affected cousin of the proband presented aged 27 with hepatomegaly and left-sided abdominal pain requiring splenectomy. He subsequently developed renal impairment and a chronic cough, and the chest radiograph showed prominent miliary mottling, similar to that in subject III2 of the present Asp67His kindred.

Lysozyme is the major secreted product of macrophages and is produced by several cell types in the gastrointestinal tract (possibly accounting for the gastrointestinal amyloid involvement) as well as by hepatocytes. Elimination of the supply of variant lysozyme by orthotopic liver transplantation, analogous to that used to treat hereditary transthyretin amyloidosis [19,20], is therefore not possible. The only therapy available for lysozyme amyloidosis at present is supportive management. It is interesting, however, that our patient III6, who has had very slowly progressive renal impairment and remains clinically well, has had no evidence of accumulation of amyloid deposits in 10 years. Our extensive experience with serial SAP scintigraphy in over 1000 patients with amyloidosis suggests that many individuals with hereditary systemic amyloidosis attain a steady-state equilibrium between deposition and mobilization. This mechanism may explain the lack of progression of amyloid in our case and several other cases of hereditary apoA-I amyloidosis in which the total body amyloid load was also large [21].

In conclusion, the phenotype of hereditary lysozyme amyloidosis is very variable, both within and between families. It is generally associated with renal dysfunction, which in the present family is the usual mode of presentation, but the age of onset and rate of progression of renal failure are highly variable. Marked hepatic and splenic amyloidosis are universal but may have few clinical effects, although all cases in one family with the Asp67His variant presented with rupture of the liver. Amyloid deposition in the gastrointestinal tract is common and may cause haemorrhage. In marked contrast to hereditary amyloidosis caused by other amyloidogenic variant proteins, the heart and peripheral nerves are spared. Pulmonary amyloid, indicated by radiographic miliary mottling, occurs occasionally but has not been clinically important. The prognosis is variable with some patients surviving for 20 years from diagnosis and others dying within a year. To date, no known affected patient has survived beyond the age of 60 years (see Note added in proof).

The mutation is penetrant in all subjects studied so far in the present Asp67His lysozyme family. It is therefore likely that affected descendants of this large kindred may have presented with renal amyloidosis and the diagnosis of amyloid been missed, or else its type mis-identified, probably as AL (formerly known as primary amyloidosis), possibly leading to inappropriate treatment with cytotoxic chemotherapy. Thorough investigation of the family history is therefore essential in all patients presenting with renal amyloid disease.



   Acknowledgments
 
This work was supported by MRC Programme Grant G979/005l0 to MBP and PNH. We thank the referring physicians and the patients and their families, for making this study possible.



   Notes
 
Note added in proof

We have lately discovered subclinical lysozyme Asp67His amyloidosis in a 74-year-old lady. Her relationship to the present kindred is at present unknown. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Pepys MB. Amyloidosis. In: Frank MM, Austen KF, Claman HN, Unanue ER, eds. Samter's Immunologic Diseases, 5th edn. Little, Brown & Co, Boston, 1994; 637–655
  2. Jones LA, Harding JA, Cohen AS, Skinner M. New USA family has apolipoprotein AI (Arg26) variant. In: Natvig JB, Førre Ø, Husby G et al., eds. Amyloid and Amyloidosis 1990. Kluwer Academic Publishers, Dordrecht, 1991, 385–388
  3. Soutar AK, Hawkins PN, Vigushin DM et al. Apolipoprotein AI mutation Arg-60 causes autosomal dominant amyloidosis. Proc Natl Acad Sci USA 1992; 89: 7389–7393[Abstract]
  4. Vigushin DM, Gough J, Allan D et al. Familial nephropathic systemic amyloidosis caused by apolipoprotein AI variant Arg26. Q J Med 1994; 87: 149–154[Medline]
  5. Booth DR, Tan SY, Booth SE et al. A new apolipoprotein AI variant, Trp50Arg, causes hereditary amyloidosis. QJ Med 1995; 88: 695–702[Abstract]
  6. Benson MD, Liepnieks J, Uemichi T, Wheeler G, Correa R. Hereditary renal amyloidosis associated with a mutant fibrinogen {alpha}-chain. Nature Genet 1993; 3: 252–255[ISI][Medline]
  7. Uemichi T, Liepnieks JJ, Benson MD. Hereditary renal amyloidosis with a novel variant fibrinogen. J Clin Invest 1994; 93: 731–736[ISI][Medline]
  8. Pepys MB, Hawkins PN, Booth DR et al. Human lysozyme gene mutations cause hereditary systemic amyloidosis. Nature 1993; 362: 553–557[ISI][Medline]
  9. Artymiuk PJ, Blake CCF. Refinement of human lysozyme at 1.5Å resolution analysis of non-bonded and hydrogen-bond interactions. J Mol Biol 1981; 152: 737–762[ISI][Medline]
  10. Booth D, Sunde M, Bellotti V et al. Instability, unfolding, and aggregation of human lysozyme variants underlying amyloid fibrillogenesis. Nature 1997; 385: 787–793[ISI][Medline]
  11. Zalin AM, Jones S, Fitch NJS, Ramdsden DB. Familial nephropathic non-neuropathic amyloidosis: clinical features, immunohistochemistry and chemistry. Q J Med 199l; 81: 945–956
  12. Hawkins PN, Pepys MB. Imaging amyloidosis with radiolabelled SAP. Eur J Nucl Med 1995; 22: 595–599[ISI][Medline]
  13. Puchtler H, Sweat F, Levine M. On the binding of Congo red by amyloid. J Histochem Cytochem 1962; 10: 355–364[ISI]
  14. Feest TG, Wallis JP. Familial nephropathic amyloidosis associated with indomethacin responsive fever. Proc EDTA-ERA 1985; 21: 683–685
  15. Lanham JG, Meltzer ML, de Beer FC, Hughes GRV, Pepys MB. Familial amyloidosis of Ostertag. Q J Med 1982; 51: 25–32[Medline]
  16. Peters CWB, Kruse U, Pollwein R, Grzeschik K-H, Sippel AE. The human lysozyme gene. Sequence organization and chromosomal localization. Eur J Biochem 1989; 182: 507–516[Abstract]
  17. Harrison RF, Hawkins PN, Roche WR et al. `Fragile' liver and massive hepatic haemorrhage due to hereditary amyloidosis. Gut 1996; 38: 151–152[Abstract]
  18. Benson MD, Uemichi T. Transthyretin amyloidosis. Amyloid. Int J Exp Clin Invest 1996; 3: 44–56
  19. Holmgren G, Steen L, Ekstedt J et al. Biochemical effect of liver transplantation in two Swedish patients with familial amyloidotic polyneuropathy (FAP-met30). Clin Genet 1991; 40: 242–246[ISI][Medline]
  20. Rydh A, Suhr O, Hietala S et al. Serum amyloid P component scintigraphy in familial amyloid polyneuropathy: regression of visceral amyloid following liver transplantation. Eur J Nucl Med 1998; 25: 709–713[ISI][Medline]
  21. Persey MR, Booth DR, Booth SE et al. A new deletion mutation of the apolipoprotein AI gene causing hereditary amyloidosis. Clin Sci 1996; 90: 33
Received for publication: 8. 2.99
Accepted in revised form: 31. 5.99