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ASE REPORT

A Novel �-Galactosidase A Mutant (M42L) Identified in a RenalVariant of Fabry Disease

David Rosenthal, MD, Yeong-Hau H. Lien, MD, PhD, Donna Lager, MD, Li-Wen Lai, PhD,Shuhua Shang, MD, Nelson Leung, MD, and Fernando C. Fervenza, MD, PhD

A 65-year-old man presented to our institution for workup of proteinuria. His serum creatinine level was 1.7 mg/dL130 �mol/L), and he had proteinuria with protein of almost 5 g/24 h. Fabry disease was diagnosed by means ofidney biopsy and low serum and leukocyte levels of �-galactosidase A. Review of his history, family history,hysical examinations, and diagnostic studies did not show other findings typical of this disease. His renal functionontinued to decline, and he eventually underwent a living unrelated renal transplantation 5 years later. Three yearsfter transplantation, his creatinine level is 1.7 mg/dL (130 �mol/L), and corrected iothalamate clearance is3 mL/min/1.73 m2. Genetic studies showed that he has a novel missense mutation (M42L) in exon 1. Methionine atodon 42 is highly conserved in eukaryotic �-galactosidase A orthologues. This genotype predicts a minorisfolding of �-galactosidase A because of a small difference in hydrophobicity between methionine and leucine.is mutation resulted in a very low, but detectable, serum level of �-galactosidase A (0.002 U/L; normal range, 0.016

o 0.2 U/L). Cases of Fabry disease that present with predominantly renal manifestations are rare and require a highndex of suspicion for diagnosis. Because treatment for Fabry disease recently has become available, it is importantor clinicians to be aware of this disease and pursue the diagnosis in cases of otherwise unexplained renalysfunction. Am J Kidney Dis 44:E85-E89.2004 by the National Kidney Foundation, Inc.

NDEX WORDS: Fabry disease; �-galactosidase A; genotype; phenotype; mutation analysis.

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ABRY DISEASE is a rare X-linked recessiveglycosphingolipid storage disease caused by

utations in the �-galactosidase A gene.1 �-Galac-osidase A is a lysosomal enzyme essential foremoval of globotriaosylceramide from cells. In thebsence of this enzyme, globotriaosylceramide andigalactosylceramide accumulate mainly in endo-helial cells from renal, cardiac, and nervous sys-em tissue. These substances also accumulate inenal epithelial cells, neurons, myocardial cells,nd vascular smooth muscle cells. Removal of 3exose residues from globotriaosylceramide is es-ential for lysosomal processing of this molecule.ost patients with classic Fabry disease have a

omplete lack of enzyme function of �-galactosi-ase A and present with a combination of cardiac,phthalmological, neurological, dermatological, andenal problems. Manifestations include cardiomy-pathy, valvular heart disease, small macules andapules in the bathing trunk region (angiokerato-as), episodic pain in a stocking-glove distribution

acroparesthesias), hypohidrosis, cornea verticil-ata, cerebrovascular disease, and proteinuria withhronic renal failure.2

Many different mutations of the �-galactosi-ase gene have been reported.3 Patients withutations that leave them with some residual

nzyme function may have only 1 or 2 manifesta-

ions and are referred to as oligosymptomatic.

merican Journal of Kidney Diseases, Vol 44, No 5 (November), 2

ery few of these cases have been reported in theiterature. Most of these patients have had at least

other manifestation of Fabry disease, such aseft ventricular (LV) hypertrophy4-6 or angiokera-omas.7 We report a case of a renal variant ofabry disease caused by a new missense muta-

ion in the �-galactosidase A gene.

CASE REPORT

A 65-year-old white man was referred to our institutionor evaluation of long-standing proteinuria. He first becameroteinuric approximately 20 years ago, but no cause hadver been discovered. He did not have a personal or familyistory of kidney disease or diabetes. His father died at age06, and his mother died at age 86. He had hypertension,hich had been diagnosed 7 years earlier and treated with an

From the Departments of Medicine; and Pathology, Mayolinic and Foundation, Rochester, MN; and the Universityf Arizona, Tucson, AZ.Received May 3, 2004; accepted in revised form July 1,

004.The genetic diagnosis of Fabry disease was supported in

art by a grant from Dialysis Clinic Inc, a nonprofit organi-ation (Y.-H.H.L. and L.-W.L.).

Address reprint requests to Fernando Fervenza, MD,hD, Division of Nephrology and Hypertension, Mayolinic, 200 First St SW, Rochester, MN 55905. E-mail:

ervenza.fernando@mayo.edu© 2004 by the National Kidney Foundation, Inc.0272-6386/04/4405-0028$30.00/0

doi:10.1053/j.ajkd.2004.07.018

004: E85-E89 e85

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ngiotensin-converting enzyme inhibitor. Blood pressuresad been around 130/80 mm Hg in the months beforeresentation.Urinalysis showed proteinuria and normal microscopic

ndings. Twenty-four–hour urine collection showed proteinxcretion of 4,940 mg/24 h, with a creatinine clearance of 69L/min/1.73 m2. Serum and urine protein electrophoresisere negative for monoclonal immunoglobulins. His serum

lbumin level was within normal limits at 3.9 g/dL (39 g/L).erum lipid profile showed a total cholesterol level of27 mg/dL (5.9 mmol/L), triglyceride level of 190 mg/dL2.1 mmol/L), low-density lipoprotein cholesterol level of49 mg/dL (3.9 mmol/L), and high-density lipoprotein cho-esterol level of 40 mg/dL (1.0 mmol/L). Other test resultsere normal or negative, including complement, antineutro-hil cytoplasmic antibody, anti-DNA antibody, and hepatitiserological results.

To ascertain the cause of his proteinuria, a renal biopsyas performed. On light microscopy, focal and segmentallomerulosclerosis, as well as tubulointerstitial atrophy, wereeen. Immunofluorescence findings were unremarkable. Elec-ron microscopy showed the effacement of podocyte footrocesses that would be expected given the degree of protein-ria present. None of these findings pointed to a specificiagnosis. However, electron microscopy also showed lami-ated myelin figures (zebra bodies) in glomerular visceralpithelial cells (Fig 1). These are seen in patients with suchysosomal storage diseases as Fabry disease. A diagnosis ofabry disease was confirmed by measurement of his serum-galactosidase level, which was detectable at 0.002 U/L,ut less than the lower limit of normal (normal range, 0.016o 200 U/L). Leukocyte �-galactosidase activity also wasow at 0.22 U/1010 cells (normal range, 0.6 to 3.63 U/1010

ells).These findings prompted a search for other manifestations

f Fabry disease, such as cardiac, ophthalmological, derma-ological, or neurological abnormalities. Chest radiographbtained at the time of diagnosis did not show signs ofardiac enlargement. An electrocardiogram showed first-egree atrioventricular block (PR interval, 246 milliseconds)nd left anterior hemiblock, but no signs of LV hypertrophy.chocardiogram showed upper-normal LV size with normalV wall thickness. Parameters are ventricular septum thick-ess, 11 mm (normal, 11 to 14 mm); posterior wall thick-ess, 11 mm (normal, 11 to 14 mm); LV diastolic diameter,6 mm (normal, 41 to 55 mm); LV systolic diameter, 27 mmnormal, 25 to 37 mm); LV mass, 249 g; LV mass index,04 g/m2; and ejection fraction, 60% to 65%. There were noalvular abnormalities. He had a skin rash around the ankle,hich had waxed and waned for approximately 2 years.orphological characteristics and location of this rash were

nconsistent with the angiokeratomas normally found in thewimming-trunk distribution in patients with Fabry disease,nd he did not recall having rashes in the past. Ophthalmo-ogical examination, which included slit-lamp microscopy,howed no corneal or lenticular abnormalities. He denied aistory of acroparesthesias or decreased sweating.After diagnosis, his proteinuria and progressive renal insuffi-

iency were managed conservatively for a number of years byeans of protein restriction and aggressive management of

lood pressure and electrolyte and fluid balance. Despite this E

herapy, his renal insufficiency continued to progress, and 3ears after diagnosis, his serum creatinine level had increasedo 3.9 mg/dL (345 �mol/L) and iothalamate clearance hadecreased to 22 mL/min. Two years later, he underwent auccessful preemptive living unrelated renal transplantation.e has been maintained on triple immunosuppression therapyith tacrolimus, mycophenolate mofetil, and prednisone. His

urrent serum creatinine level is 1.7 mg/dL (150 �mol/L), withcorrected iothalamate clearance of 53 mL/min/1.73 m2.Because of his unique presentation, genetic diagnosis was

erformed by using polymerase chain reaction sequencing,s described previously.8 We identified an A↔C transver-ion in codon 42 (Fig 2), which results in substitution ofeucine for methionine (M42L). This mutation has not beeneported previously and was not found in 100 normal chro-osomes; thus, a polymorphism is unlikely. After his diag-

osis, screening for Fabry disease was offered to otheramily members. His 43-year-old daughter was found toave the same mutation, but with a low-normal leukocyte-galactosidase level (0.94 U/1010 cells; normal range, 0.6

o 3.63 U/1010 cells).

DISCUSSION

Fabry disease presenting solely with renal in-olvement is very rare, and only a few cases haveeen reported in the literature.4,9,10 Our patient isnique in that he developed end-stage renal diseaseESRD) at the age of 70 years and there are noypical Fabry stigmata, including angiokeratoma,croparesthesia, cornea verticillata, hypohidrosis,nd hypertrophic cardiomyopathy. Molecular diag-osis showed a novel missense mutation in the-galactosidase A gene. Methionine at codon 42 isighly conserved. It is conserved in 85% of eukary-tic and 100% of mammalian �-galactosidase Arthologues.11 Two different missense mutationsffecting this amino acid have been reported previ-usly: M42T and M42V. Both are associated withlassic Fabry disease.11-13 These mutations predictignificant misfolding of the enzyme because of anmino acid substitution that results in significanthanges in hydrophobicity.14 As for the M42Lutation, both methionine and leucine are aliphatic

onpolar amino acids with a minimal difference inydrophobicity.15 It is possible that because of thisinor difference, M42L does not result in major

hanges in the structure of �-galactosidase A.hether this is the basis of the unique clinical

resentation of our case remains to be investigated.Microscopic involvement of the kidney is seen

n virtually all patients with Fabry disease, androteinuria and/or renal failure are present in ap-roximately 69% to 74% of patients. Most reach

SRD around the fifth decade of life.16-18 Patients

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ith Fabry disease with only 1 or 2 manifestationsf the disease are referred to as oligosymptomatic.he most common oligosymptomatic variant re-orted has been the cardiac variant. These patientsenerally present with a mild late-onset diseaseonfined to myocardial tissue, with no involvementf vascular endothelium.2 Most eventually progresso severe cardiomyopathy, often requiring transplan-ation.

Renal variants of Fabry disease are rare. Na-

Fig 1. Electron micrograph showing laminated

ao et al4 screened 514 unselected Japanese male i

atients with ESRD treated with long-term hemo-ialysis and identified 6 patients with Fabryisease (occurrence rate, 1.2%). The prevalenceate of the renal variant in the dialysis populationaries from 0% to 0.47% in other countries.19

mong the cases reported by Nakao et al,4 1atient had classic Fabry disease that was misdi-gnosed, 4 patients had LV hypertrophy, andnly 1 patient had a normal echocardiogram. Itppears that the kidney and heart frequently are

n figures in glomerular visceral epithelial cells.

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re overlapping symptoms between cardiac andenal variants.4-6 Strictly speaking, in the seriesf Nakao et al,4 only the case without LV hyper-rophy is considered the renal variant. Our pa-ient has predominantly renal manifestation, withossible minor involvement of the cardiac con-uction system. Two other previously reportedases of the renal variant had proteinuria only.9,10

he genotypes of these 4 cases are E66Q, M42L,301Q, and S247C. Among them, E66Q and301Q are associated with the cardiac vari-nt.20,21 It is possible that the renal and cardiacariants may share the same genotype, and clini-al manifestations are determined by other ge-etic modifiers or unknown factors.Until recently, recognition of Fabry disease did

ot impact on the patient’s prognosis because noreatment was available. However, the availabilityf recombinant �-galactosidase has made it pos-ible for these patients to be treated. One random-

Fig 2. Sequencing traces from wild type, homozy-ote (proband), and heterozygote (daughter). An A↔Cransversion (ATG↔CTG) is indicated by arrows.

zed trial documented improvement in creatinine N

learance, but not inulin clearance or proteinuria, inypical patients with Fabry disease treated with-galactosidase A every 2 weeks for 6 months.22

ecause only typical patients with Fabry diseaseere included in these trials, there is a paucity ofata regarding the treatment of oligosymptomaticatients. Although recent reviews recommendedreatment for all patients with Fabry disease,23 theptimal timing of treatment and long-term benefitsre not yet known. Because this patient had ESRDnow status post kidney transplantation) as the soleanifestation of Fabry disease and had no other

ymptoms, it was decided not to recommend thate undergo enzyme replacement therapy. In addi-ion, there is no evidence that patients with ESRDith the Fabry disease renal variant will benefit

rom enzyme replacement therapy.19

In conclusion, we report a novel missense muta-ion of the �-galactosidase A gene found in antypical renal variant of Fabry disease. This M42Lutation predicts a minor change in protein struc-

ure, which correlates with residual enzyme activitynd clinical pictures. The present case illustrateshat Fabry disease can present late in life with renalysfunction as the sole abnormality. We suggesthat Fabry disease be considered in the differentialiagnosis of male patients presenting with an other-ise unexplained renal failure.

REFERENCES

1. Brady RO, Gal AE, Bradley RM, Martensson E, WarshawL, Laster L: Enzymatic defect in Fabry’s disease: Ceremide

rihexosidase deficiency. N Engl J Med 276:1163-1167, 19672. Desnick RJ, Ioannou YA, Eng CM: �-Galactosidase A

eficiency: Fabry disease, in Scriver CR BA, Sly WAS,alle D (eds): The Metabolic and Molecular Basis of Inher-

ted Diseases. New York, NY, McGraw-Hill, 2001, pp 3733-7743. Pastores GM, Lien YHH: Biochemical and molecular

enetic basis of Fabry disease. J Am Soc Nephrol 13:S130-133, 2002 (suppl 2)4. Nakao S, Kodama C, Takenaka T, et al: Fabry disease:

etection of undiagnosed hemodialysis patients and identifi-ation of a “renal variant” phenotype. Kidney Int 64:801-07, 20035. Meehan SM, Junsanto T, Rydel JJ, Desnick RJ: Fabry

isease: Renal involvement limited to podocyte pathologynd proteinuria in a septuagenarian cardiac variant. Patho-ogic and therapeutic implications. Am J Kidney Dis 43:164-71, 20046. Clarke JTR, Knaack J, Crawhall JC, Wolfe LS: Cer-

mide trihexosidosis (Fabrys disease) without skin lesions.

Engl J Med 284:233-235, 1971

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7. Ko YH, Kim HJ, Roh YS, Park CK, Kwon CK, ParkH: Atypical Fabry’s disease—An oligosymptomatic vari-

nt. Arch Pathol Lab Med 120:86-89, 19968. Yang CC, Lai LW, Whitehair O, Hwu WL, Chiang SC,

ien YHH: Two novel mutations in the alpha-galactosidasegene in Chinese patients with Fabry disease. Clin Genet

3:205-209, 20039. Sawada K, Mizoguchi K, Hishida A, et al: Pointutation in the alpha-galactosidase A gene of atypical Fabry

isease with only nephropathy. Clin Nephrol 45:289-294,99610. Germain DP, Shabbeer J, Cotigny S, Desnick RJ:

abry disease: Twenty novel alpha-galactosidase A muta-ions and genotype-phenotype correlations in classical andariant phenotypes. Mol Med 8:304-310, 200211. Shabbeer J, Yasuda M, Luca E, Desnick RJ: Fabry

isease: 45 novel mutations in the alpha-galactosidase A geneausing the classical phenotype. Mol Genet Metab 76:23-30,002

12. Davies JP, Eng CM, Hill JA, et al: Fabry disease:ourteen alpha-galactosidase A mutations in unrelated fami-

ies from the United Kingdom and other European countries.ur J Hum Genet 4:219-224, 199613. Topaloglu AK, Ashley GA, Tong BZ, et al: Twenty

ovel mutations in the alpha-galactosidase A gene causingabry disease. Mol Med 5:806-811, 1999

14. Garman SC, Garboczi DN: The molecular defect lead- r

ng to Fabry disease: Structure of human alpha-galactosidase. Jol Biol 337:319-335, 200215. Creighton T: Proteins: Structures and molecular prop-

rties. New York, NY, Freeman, 198416. Alroy J, Sabnis S, Kopp JB: Renal pathology in Fabry

isease. J Am Soc Nephrol 13:S134-S138, 2002 (suppl 2)17. Branton MH, Schiffmann R, Sabnis SG, Murray GJ,

t al: Natural history of Fabry renal disease: Influence oflpha-galactosidase A activity and genetic mutations onlinical course. Medicine (Baltimore) 81:122-138, 2002

18. Galanos J, Nicholls K, Grigg L, Kiers L, Crawford A,ecker G: Clinical features of Fabry’s disease in Australianatients. Intern Med J 32:575-584, 200219. Grunfeld JP: How to improve the early diagnosis of

abry’s disease? Kidney Int 64:1136-1137, 200320. Lien YHH, Lai LW, Lui CY: Unexpected diagnosis of

abry disease in an 80-year-old man with syncope. Cardiol-gy 96:115-116, 200121. Nakao S, Takenaka T, Maeda M, et al: An atypical

ariant of Fabrys disease in men with left-ventricular hyper-rophy. N Engl J Med 333:288-293, 1995

22. Schiffmann R, Kopp JB, Austin HA, et al: Enzymeeplacement therapy in Fabry disease—A randomized con-rolled trial. JAMA 285:2743-2749, 2001

23. Desnick RJ, Brady R, Barranger J, et al: Fabryisease, an under-recognized multisystemic disorder: Expertecommendations for diagnosis, management, and enzyme

eplacement therapy. Ann Intern Med 138:338-346, 2003