The Uromodulin C744G mutation causes MCKD2 and FJHN in ... - Core

original article

http://www.kidney-international.org & 2007 International Society of Nephrology

The Uromodulin C744G mutation causes MCKD2 and FJHN in children and adults and may be due to a possible founder effect MTF Wolf1,2, BB Beck1, F Zaucke3, A Kunze3, J Misselwitz4, J Ruley5, T Ronda1, A Fischer6, F Eifinger1, C Licht1, E Otto7,8, B Hoppe1 and F Hildebrandt7,8 1

Department of Pediatric Nephrology of the University Children’s Hospital, University of Cologne, Cologne, Germany; 2Department of Human Genetics, University of Cologne, Cologne, Germany; 3Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany; 4Department of Pediatric Nephrology, University Children’s Hospital, Jena, Germany; 5Department of Pediatric Nephrology, INOVA Hospital for Children, Kidney Center, Annandale, Virginia, USA; 6Department of Nephrology, Kantonsspital Lucerne, Lucerne, Switzerland; 7Department of Pediatrics, University of Michigan, Ann Arbor, USA and 8Department of Human Genetics, University of Michigan, Ann Arbor, USA

Autosomal dominant medullary cystic kidney disease type 2 (MCKD2) is a tubulo-in terstitial nephropathy that causes renal salt wasting, hyperuricemia, gout, and end-stage renal failure in the fifth decade of life. This disorder was described to have an age of onset between the age of 20–30 years or even later. Mutations in the Uromodulin (UMOD) gene were published in patients with familial juvenile hyperuricemic nephropathy (FJHN) and MCKD2. Clinical data and blood samples of 16 affected individuals from 11 different kindreds were collected. Mutational analysis of the UMOD gene was performed by exon polymerase chain reaction (PCR) and direct sequencing. We found the heterozygous C744G (Cys248Trp) mutation, which was originally published by our group, in an additional four kindreds from Europe and Turkey. Age of onset ranged from 3 years to 39 years. The phenotype showed a variety of symptoms such as urinary concentration defect, vesicoureteral reflux, urinary tract infections, hyperuricemia, hypertension, proteinuria, and renal hypoplasia. Haplotype analysis showed cosegragation with the phenotype in all eight affected individuals indicating that the C744G mutation may be due to a founder effect. Moreover, we describe a novel T229G (Cys77Gly) mutation in two affecteds of one kindred. Three of the affected individuals were younger than 10 years at the onset of MCKD2/FJHN. Symptoms include recurrent urinary tract infections compatible with the published phenotype of the Umod knockout mouse model. This emphasizes that MCKD2 is not just a disease of the young adult but is also relevant for children. Kidney International (2007) 71, 574–581. doi:10.1038/sj.ki.5002089; published online 24 January 2007 KEYWORDS: MCKD2; FJHN; Uromodulin; Tamm–Horsfall protein

Correspondence: F Hildebrandt, Department of Pediatrics and Communicable Diseases, University of Michigan, 8220C MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0646, USA. E-mail: [email protected] Received 23 June 2006; revised 30 October 2006; accepted 29 November 2006; published online 24 January 2007 574

Medullary cystic kidney disease (MCKD) is a progressive tubulo-interstitial nephropathy with autosomal dominant inheritance, which leads to end-stage renal disease (ESRD) in adulthood.1 Medullary cystic kidney disease type 2 (MCKD2) (OMIM 603860) includes clinical features of reduced urinary concentration, salt wasting, and end-stage renal failure. In contrast to MCKD1, in MCKD2 a more severe phenotype concerning hyperuricemia and gout has been described. Moreover, in MCKD2, an earlier median age of onset has been reported (median age of onset in MCKD2 is 32 years of age compared to 62 years in MCKD1).2 Otherwise, these two diseases are clinically undistinguishable. In MCKD small corticomedullary cysts are common, but not always detected. Kidney size is normal or only slightly reduced. MCKD shows a renal histologic triad of (1) tubular basement disintegration, (2) tubular atrophy with cyst development at the corticomedullary border, and, (3) interstitial cell infiltration associated with fibrosis.3 Neither imaging results, nor pathological findings are pathognomonic for MCKD2. The condition shares clinical and morphological similarities to recessive juvenile nephronophthisis (NPH).4 In contrast to juvenile onset in NPH, end-stage renal failure in MCKD occurs in adulthood. Moreover, NPH is inherited in an autosomal recessive pattern, whereas MCKD is autosomal dominant. MCKD2 was localized on chromosome 16p12 in an Italian family2 and the responsible gene has been identified.5 Missense mutations in the uromodulin (UMOD) gene have been detected in four MCKD kindreds first by Hart et al.5 It was then confirmed in 34 additional MCKD kindreds by others including our group.6–8 Surprisingly, homozygous UMOD mutations were described too, causing a more severe phenotype in the affected individuals.9 Besides MCKD2, UMOD mutations have also been reported in familial juvenile hyperuremic nephropathy (FJHN) and in autosomal dominant glomerulocystic kidney disease.10 Kidney International (2007) 71, 574–581

original article

MTF Wolf et al.: The Uromodulin C744G mutation causes MCKD2 and FJHN

UMOD mutations alter the encoded Tamm–Horsfall protein (THP), the most abundant urine protein in humans. THP is expressed primarily at the luminal side of renal epithelial cells of the thick ascending loop of Henle and of early distal convoluted tubules. The vast majority of mutations were found in exon 4, which encodes for a calcium-binding EGF (epidermal growth factor) domain and change cysteine amino acids, which form disulfide bonds. Decreased urinary UMOD excretion and retention of the misfolded UMOD protein in the endoplasmatic reticulum, resulting in an increased rate of apoptosis, was described.8 UMOD is a transmembrane protein, which can be secreted into the urine by cleavage of the GPI anchor.11 Functional roles of THP have been described as a protective factor in urinary tract infections, in binding to complement factors, in myeloma kidney, and in nephrolithiasis.12–15 Recently, Hodanova et al.16 published an additional chromosomal locus for uromodulin-associated kidney

disease on chromosome 1q41. Further evidence of genetic heterogeneity in FJHN/MCKD2 was shown by Vylet0 al et al.,17 who identified only six UMOD mutations in 19 kindreds. We here report the first mutation in UMOD, which is found in five different kindreds. This finding may be owing to a founder effect. We observe that the same mutation causes FJHN in some patients and MCKD2 in other patients. In addition, we present a novel UMOD mutation in exon 4. RESULTS Clinical data

The five families presenting with UMOD mutations included eight living individuals affected with MCKD/FJHN. Four of six also suffered from hyperuricemia (Table 1). The patients presented with hyperuricemia between 3 and 39 years of age (Table 1). ESRD developed between 6 and 52 years. Imaging by ultrasound revealed in all families pathologic results

Table 1 | Synopsis of the clinical data, imaging results, renal histology of UMOD patients with a C744G (Cys248Trp) mutation Family Age at Age at number Sex presentation ESRD

PCra (mg/dl)

GFRa PUAa 2 (ml/min/1.73m ) (mg/dl) Clinical symptoms

Imaging

Histology NA Interstitial fibrosis, atrophic tubuli, sclerosed glomeruli, thickened basement membrane NA

F739 I-1 II-1

M M

39 18

45 NA

8.4m 1.9m

NA 63k

5.3 6.6m

CRI CRI, hyperuricemia

US (small kidneys) US (small kidneys, reduced parenchyma, one cyst)

II-2

F

17

NA

NA

NA

NA

CRI, hyperuricemia

NA

K7 I-1

F

9

18

1.4m

70k

5.4

Enuresis nocturna, US (small kidneys, NA VUR, multiple UTI, echogenic, diminished proteinuria, CRI, mild cortico-medullary hyperuricemia, NTX, differentiation, multiple cysts in right kidney) NTX rejection

K8 I-2 II-2

F M

42 9

NA NA

0.6 1.7m

NA 63k

4.1 6.4m

Hypertension CRI, hyperuricemia, renal hypoplasia of right kidney, proteinuria

K10 I-1

M

3

6

0.9m

62k

7.5m

Focal dysplastic renal CRI, hyperuricemia, US (small kidneys, urinary concentration diminished parenchyma, atrophic defect, hypertension, corticomedullary tubuli, interstitial fibrosis mild proteinuria differentiation, no cysts)

K11 I-1

F

46

52

1.5m

58k

5.4

CRI, hypertension, mild proteinuria, mild hyperuricemia, dialysis

US (small right kidney, NA both kidneys with echogenic parenchyma, no cysts)

US (normal size of kidney, 4–5 cortical cysts in each kidney, up to 3 cm in diameter)

Tubulo-interstitial sclerosis and fibrosis with interstitial lymphocytic cell infiltration, atrophic tubuli, thickened basement membrane, staining of THP under Bowman capsule

CRI, chronic renal insufficiency; GFR, glomerular filtration rate; NTX, renal transplant; PCr, plasma creatinine; PUA, plasma uric acid; NA, not available; US, ultrasound; UTI, urinary tract infection; VUR, vesico-ureteral reflux; THP, Tamm–Horsfall protein; age at presentation and age at end-stage renal disease (ESRD) are given in years. a Arrows denote increase vs decrease in relation to age-dependent normal values. Kidney International (2007) 71, 574–581

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Table 2 | Synopsis of the clinicial data, imaging results, renal histology of UMOD patients with a T229G (Cys77Gly) mutation. Family Age at Age at number Sex presentation ESRD

PCra (mg/dl)

GFR a (ml/min/1.73m2)

PUAa (mg/dl) Clinical symptoms

A214 I-1

M

39

45

NA

NA

NA

II-1

M

13

NA

1.2

89

8.2

Imaging

Histology

CRI, hyperuricemia, US (small kidneys) hemodialysis CRI, hyperuricemia, US (small kidneys, urinary concentration reduced parenchyma, defect, duplicated one cyst) urethral opening

‘Chronic glomerulonephritis’ Interstitial fibrosis, microcysts, thickened Bowman capsule, tubular atrophy, thickened basement membrane

CRI, chronic renal insufficiency; GFR, glomerular filtration rate; PCr, plasma creatinine; PUA, plasma uric acid; NA, not available; US, ultrasound; age at presentation and age at end-stage renal disease (ESRD) is given in years. a Arrows denote increase vs decrease in relation to age-dependent normal values.

K8

K11 I-2

I-1

K7 I-1

II-1

I-2

II-3

II-2 (237)

(257)

257

237

D16S3045

(250) (244)

(?)

250

(252)

(?) (?)

244

244 (?)

(160) (?)

160 181 280

D16S3099 D16S749 D16S3036 D16S3041

(183) (152) (179) (278)

250 240 (?) 164 175 285

(?)

(183)

152 187 282

II-2

II-1

237 250

257 256 240 185 160 175 280

D16S3076 D16S3045 D16S3054 D16S3099 D16S749 D16S3036 D16S3041

I-1

257 250 240 185 160 175 280

237 250 240 183 152 179 278

II-4

D16S3076 D16S3054

237

D16S3076 257 D16S3045 256 D16S3054 240 D16S3099 (185) D16S749 160 D16S3036 175 D16S3041 280

240 183 152 179 278

I-2

II-1

II-2

II-3 237

D16S3076 D16S3045 D16S3054 D16S3099 D16S749 D16S3036 D16S3041

II-4

250

257 250

244

244

183 152 179

183 160 181

278

280

III-1 III-1

III-2

D16S3076

237

257

D16S3045

250

D16S3054

244 183

250 244

D16S3099 D16S749 D16S3036 D16S3041

152 179 278

K10

160 181 280

II-1

IV-1

IV-2

I-1

1-2

I-1

183

III-2

F739

D16S3076

237

257

D16S3045

250

250

D16S3054

244

244

D16S3099

183

183

D16S749

152

160

D16S3036

179

181

D16S3041

278

276

D16S3076 D16S3045 D16S3054 D16S3099 D16S749 D16S3036 D16S3041

I-2 257 250 240 187

237 250 240 183 152 179 278

152 181 276

257 250 240 189 160 179 276

II-2

II-1 D16S3076 D16S3045 D16S3054 D16S3099 D16S749 D16S3036 D16S3041

237 250 240 187 164 181 278

237 250 240

257 237 250 250 240 240

183 152 179 278

189 160 179 276

183 152 179 278

II-3 257 250 240 189 164 181 278

257 250 240 187 152 181 276

237 250 240 187 164 181 278

Figure 1 | Pedigrees and results of the haplotype analysis in all kindreds with the C744G (Cys248Trp) mutation. Circles denote female patients, squares denote male patients. Filled symbols denote affected individuals, crosshatches denote deceased individuals. Roman numerals denote generations. Arrows denote individuals in whom DNA was available for haplotype analysis. Inferred haplotypes are shown in parenthesis. Differently shaded bars symbolize haplotypes. Paternal haplotypes are drawn to the left, maternal haplotypes to the right. Marker positions are indicated with left individuals. In two kindreds (K10 and K11), no DNA samples of the parents were available. Therefore, in these two individuals, the alleles could not clearly assigned to one haplotype. Under the hypothesis of a shared haplotype, the haplotype colored in black showed cosegregation with the phenotype in all eight living affected individuals of the five different kindreds. Only the microsatellite D16S3054 shows different alleles in K8 and F739 (240 bp) compared to K7, K10, and K11 (244 bp). This difference can be attributed to a mutation in the microsatellite repeat.

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original article


including small kidneys, decreased parenchyma, or cysts. Renal histology in four cases was compatible with MCKD (Tables 1 and 2). F739 has been presented in Wolf et al.6 The pedigrees of all kindreds, except A214, are shown in Figure 1. K7. This female patient is now 24 years old. She is of Italian descent. A paternal sister, the twin brother of her father and her paternal grandmother suffered from ESRD, starting in their early twenties. Her aunt and her uncle received a kidney transplant. Her father suffered from hypertension, but was otherwise healthy. At the age of nine years, she developed enuresis nocturna and the diagnosis of vesico-ureteral reflux was made. The initial serum creatinine was 1.4 mg/dl. Surgical correction by ureteroplasty was performed. After surgery, she continued to suffer from recurrent urinary tract infections. Renal ultrasound revealed reduced bilateral kidney size. At the age of 13 years, her renal function deteriorated (serum creatinine 1.8 mg/dl). She presented with a non-selective gross proteinuria (1.3 g/24 h) and became hypertensive (140/70 mm Hg). Unfortunately, there are only scant data about her plasma uric acid available. The plasma uric acid at the age of 18 years was 5.4 mg/dl while on intermittent hemodialysis. At this age, she had developed symptoms of ESRD and was started on hemodialysis. At the age of 21 years, she received a living-related donor kidney from her mother. Five months later, she experienced an acute transplant rejection and hemodialysis was reinitiated. At the age of 23 years, she became pregnant under dialysis and delivered twins in the 30th week of gestation age. K8. This is a boy of 13 years of age and of Turkish descent. There is no family history of renal disease. At the age of nine years, the boy underwent emergency surgery because of a perforated appendix. Blood analysis revealed an increased creatinine value of 1.7 mg/dl. During follow-up, right renal hypoplasia was diagnosed by i.v. scintigraphy with relative renal functions of 26% on the right-hand side and 74% on the left-hand side. In the ultrasound, no renal cysts but a small, echogenic, right kidney was noted. At that time, his creatinine clearance was 63 ml/min/1.73m2. Hyperuricemia was noted at the age of 10 years (6.4 mg/dl). Selective glomerular proteinuria (1.26 g/24 h) and reduced urinary excretion of uric acid (Fe in Urate ¼ 3.85%) were detected at the age of 12 years. Uric acid increased to 8.8 mg/dl. The proteinuria is currently improving under treatment with ramipiril (0.35 g/24 h). Urinary excretion of UMOD was markedly reduced (Figure 2). By mutational analysis, his mother was identified as the carrier of the mutation. Except for hypertension, she was otherwise healthy (Table 1). She refused further clinical diagnostics. K10. This is a boy of 14 years who is of German descent. His family is from Thuringia. As an infant, he showed symptoms of renal tubular acidosis and reduced urinary concentration capacity with a maximum urinary osmolality of 460 mosmol/kg H2O. At the age of 3 years, he presented with an increased serum creatinine of 0.9 mg/dl and hyperuricemia (7.5 mg/dl). His creatinine clearance was already markedly reduced (62 ml/min/1.73m2). Renal biopsy Kidney International (2007) 71, 574–581

kDa

1

Coomassie 2 3

4

1

UMOD 2 3

4 kDa

97

97

66

66

45

45

30

30 20

20

Figure 2 | Urinary UMOD excretion in relation to total urinary protein excretion. The first four lanes on the left-hand side show Coomassie urinary protein staining for individual K8 (lane 1), healthy controls (lanes 2 and 3), and individual K11 (lane 4), indicating additional bands of urinary protein in K11. The band with a size of 66 kDa corresponds to albumin. On the right-hand side, the corresponding Western blot for urinary UMOD staining is shown. In lanes 2 and 3, strong staining for UMOD in the healthy controls was found. In lane 1 (K8) and lane 4 (K11), UMOD staining revealed almost no band for K8 and only a weak band for K11.

showed focal dysplastic renal parenchyma, atrophic tubuli, and interstitial fibrosis. Renal ultrasound revealed small kidneys, diminished corticomedullary differentiation but no cysts. At the age of 6 years, he developed persistent proteinuria and at the age of 10 years he presented with hypertension. Since 3 years of age, he has been treated with allopurinol, which seems to have only a limited effect on preservation of his renal function. The social situation of the family is very tense and the parents refused further collaboration. K11. This is a 53-year-old female patient from Switzerland. There was no family history of renal disease. She first presented with hypertension and mild proteinuria (460 mg/ day) at the age of 46 years. At that time, her creatinine was 1.5 mg/dl and her uric acid was normal at 5.4 mg/dl. Renal ultrasound showed four to five cortical cysts in each kidney with a diameter of 1–3 cm. Kidney size was normal. Renal biopsy was performed at the age of 49 years when renal function was further deteriorating with a creatinine value of 2.4 mg/dl. Renal biopsy revealed tubulo-interstitial sclerosis and fibrosis with interstitial lymphocytic cell infiltration, atrophic tubuli, thickened basement membrane, and staining of Tamm–Horsfall protein under the Bowman capsule. Urinary excretion of UMOD was markedly reduced (Figure 1). At the age of 52 years, peritoneal dialysis was initiated. A214. This is a 21 years old male from the United States of America (Table 2). Family history is positive, as his father suffers from ESRD and receives hemodialysis. Two renal biopsies in the father were described as showing chronic glomerulonephritis (Table 2). At the age of 13 years, the patient’s blood chemistry showed an elevated creatinine value of 1.2 mg/dl and hyperuricemia (8.2 mg/dl). Renal ultrasound showed small kidneys, reduced parenchyma and one cyst. He was normotensive but showed a urinary concentration defect 577

original article

with a specific gravity of 1.008. His glomerular filtration rate was 89 ml/min/1.73m2. In the following month, his serum uric acid increased to 9.8 mg/dl, his creatinine rose to 1.4 mg/ dl, and the glomerular filtration rate declined to 76 ml/min/ 1.73m2. Renal biopsy at the age of 18 years showed interstitial fibrosis, microcysts, thickened Bowman capsule, tubular atrophy, and thickened basement membrane. His serum creatinine rose to 2.1 mg/dl. Mutation analysis

We performed mutational analysis in the five affected individuals described above by exon polymerase chain reaction (PCR) and direct sequencing of all UMOD exons. In families K7, K8, K10, and K11, the affected individuals were analyzed and a C744G mutation was detected, causing the amino-acid exchange Cys248Trp (Figure 3a). This mutation was previously found in F739.6 In this publication, nucleotide numbers were referred to the published mRNA resulting in the annotation C849G (Cys248Trp). Now we have changed the annotation starting with nucleotide number one at the first amino acid coding nucleotide triplet resulting in the annotation C744G (Cys248Trp). In family A214, a novel nucleotide exchange T229G was found, resulting in the amino-acid exchange Cys77Gly (Figure 3b). The mutation was identified in the affected father and son. The previously published C744G mutation and the newly discovered T229G mutation are both located in exon 4. None of the mutations were found in 100 healthy Caucasian controls. Therefore, our detected mutations are unlikely to be common polymorphisms and most likely disease causing. Haplotype analysis

Haplotype analysis with seven polymorphic microsatellites was performed in all individuals with the C744G (Cys248Trp) mutation and their relatives as far as available. In two kindreds (K10 and K11), no DNA samples of the parents were available. Therefore, in these two individuals, the alleles could not clearly assigned to one haplotype. Under the hypothesis of a shared haplotype, the haplotype colored in black (Figure 1) showed cosegregation with the phenotype in all eight living affected individuals of the five different kindreds. Only the microsatellite D16S3054 showed different alleles in K8 and F739 (240 bp) compared to K7, K10, and K11 (244 bp). This difference can be attributed to an ancestral mutation in the microsatellite repeat. Therefore, we conclude that the C744G mutational hotspot in the five kindreds may be due to a founder effect. DISCUSSION

Evaluation of genotype–phenotype correlations in MCKD2 has long been impossible due to the limited number of patients and the individual character of each mutation for a kindred. We identified another four kindreds with the same C744G mutation, which we have published in the German kindred F739.6 We here present in eight individuals from five 578


kindreds carrying the same C744G mutation the broad variation of clinical courses. The five kindreds, including F739, are from all over Europe and Turkey. Genotype– phenotype correlation was performed not including the individuals of kindred A214, because of the different mutation. Renal cysts were found by renal ultrasound in three of seven affected individuals, supporting the diagnosis of MCKD2. A family history compatible with autosomal dominant inheritance was only found in three kindreds (K7, K8, and F739). The mother of K8 was only hypertensive. In two other affected individuals, no family history of renal disease or hyperuricemia was found. In four affected individuals, ESRD was diagnosed. The age of onset of ESRD varied between 6 and 52 years of age. None of the patients presented with gout. Hyperuricemia was found in all patients. Interestingly, uric acid was only slightly elevated in K7, K8, and K11. Including the previously described kindred F739,6 three of the eight affected individuals show symptoms by the age of 10 years (one with MCKD2 and two with FJHN). Only two of eight patients entered renal insufficiency in the fifth decade of life, emphasizing that the UMOD-associated diseases should also be taken into consideration in the field of pediatric nephrology. Haplotype analysis was performed in all available individuals. Under the hypothesis of haplotype sharing, cosegregation of the haplotype colored in black with the phenotype in all eight living affected individuals was shown (Figure 1). Only the microsatellite D16S3054 showed different alleles in K8 and F739 (240 bp) compared to K7, K10, and K11 (244 bp). The different alleles for D16S3054 can be attributed to an ancestral mutation in the repeat of the microsatellite marker. We conclude that the high frequency of the C744G mutation in the five kindreds may be due to a founder effect. However, the patients with the C744G mutation come from four different countries from central Europe and Turkey. Taking this wide distribution into consideration, we assume that this mutation may have a much higher frequency and may be responsible for a larger portion of FJHN/MCKD patients. Mutations in UMOD have been found to result in MCKD2, FJHN, and glomerulocystic kidney disease, indicating a broad variety of clinical symptoms (Table 3).8 FJHN presents with hyperuricemia between childhood and early adult life (age of onset: 3–17 years) sometimes causing gout.18 A typical characteristic of FJHN is a reduced urinary fractional excretion of urate (FEUrat). Different hypotheses concerning reduced FEUrat are discussed.19 Reference values for FEUrat differ for male patients (8.173.2%), female patients (12.872.9%), and children (12–30%).18,20 The other characteristics of FJHN and MCKD2 are shown in Table 3. According to these criteria, we tried to assign the correct diagnosis to the five kindreds. We diagnosed FJHN in K8 and K10 and MCKD2 in K7 and K11. F739 would have been compatible with either MCKD2 or FJHN. All affected individuals presented with the same C744G mutation. Therefore, we are able to confirm the hypothesis of Dahan et al.,21 by molecular genetics, who Kidney International (2007) 71, 574–581

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a

WT

AGC

CGC

Ser

Arg Lys

AAG

AGC

CGC AAG

GCC

TGC GCG

Ala Cys Ala

CAC

AGC

GGC

His Trp Ser

TGG

Gly

MUT: F739 K7 K8 K10 K11 GCC

TGC

GCG CAC

TGG

AGC GGC

G

Ser Arg Lys Ala Cys/Trp Ala His

b

Trp Ser Gly

WT

CCT GGA GCT CAC AAC TGC

TCC

GCC AAC

AGC

Pro Gly

Ala His Asn Cys Ser

Ala Asn Ser

CCT

GGA

GCT CAC AAC TGC G

Pro

Gly Ala His Asn Cys/Gly Ser Ala Asn Ser

MUT: A214

TCC

GCC AAC

AGC

Figure 3 | Mutations in the UMOD gene. The upper sequence shows the wild-type (WT) sequence and the lower sequence reveals the mutation (MUT) in each family. The resulting amino acids are indicated below nucleotide sequences. Both mutations are located in exon 4. The altered nucleotide and amino acids are shown in red. (a) In affected individuals of families F739, K7, K8, K10, and K11, a heterozygous C744G substitution was detected resulting in a Cys248Trp amino-acid exchange. (b) Affected individuals in family A214 showed a heterozygous T229G substitution, causing a Cys77Gly change.

suspected on the basis of linkage analysis that FJHN and MCKD2 are facets of the same disease. K7 was diagnosed with MCKD2 due to renal cysts and enuresis nocturna, indicating polyuria. Unfortunately there are scant data about her uric acid levels. Under hemodialysis, she had serum uric acid levels of 5.4 mg/dl, indicating that she may not have suffered from severe hyperuricemia. However, with an age of onset of 9 years, she would have been very young for the development of MCKD2. Only one report about MCKD2 in childhood has been published yet. Bleyer et al.22 described a kindred with MCKD2, in which the youngest affected patient with an UMOD mutation was just 7 years. Although the author calls the described disease MCKD2, the clinical characteristics would fit better to FJHN, as the affected patient showed severe hyperuricemia, a young age of onset and no renal cysts were described. Hart et al.5 also described a kindred with MCKD2, in which the vast majority but not all affected individuals have hyperuricemia. In addition, they showed renal cysts justifying the diagnosis of MCKD2. This underlines the heterogeneity of the phenotype. Interestingly, patient K7 became symptomatic with recurrent urinary tract infections. An Umod knockout mouse model with an increased rate of urinary tract infections was described.23 Nevertheless, this patient also suffered from vesico-ureteral reflux. So it is difficult to decide whether infections were caused by vesico-ureteral reflux or the decreased UMOD secretion. Recurrent urinary tract infections even after surgery may hint to a potential responsibility of UMOD for the infections. Unfortunately, there seems to be incomplete penetrance in K7. Obviously, the father of K7 was not severely affected, presenting only with hypertension, but his twin brother and his sister received a kidney transplant. Incomplete penetrance was also reported by Bleyer et al., with a mother only suffering from hyeruricemia without renal impairment but an affected child with impaired renal function.22 Incomplete or age-related penetrance should be taken into consideration when a pedigree is drafted. Incomplete penetrance and the broad variety of phenotyps may hint at the ‘two hit’ hypothesis in MCKD2 as it was already shown for ADPKD.24–25 In addition, ‘transheterozyote’ mutations in MCKD1 and MCKD2 in analogy to ADPKD, showing heterozygous

Table 3 | Comparison of the characteristics of familial hyperuricemic juvenile nephropathy (FJHN) and medullary cystic kidney disease type 2 (MCKD2)

Age at presentation (years) Age at ESRD (years) Symptoms

Ultrasound Histopathology

FJHN

MCKD2

Childhood, adolescence, early adult life 20–40 Hyperuricemia, sometimes gout, renal hypoexcretion of urate (FEurato12% in children), hypertension inconsistently, absent or minimal proteinuria Echogenic kidneys

Early to later adult life (B32 years)

Tubular atrophy, fibrosis, interstitial cell infiltration, segmentally sclerosed glomeruli, thickening of the basement membrane

Polyuria, salt loss, hyperuricemia, sometimes gout, No data about FEurat in MCKD2, hypertension, no data about proteinuria in MCKD2 Small, echogenic kidneys, often with small medullary cysts, but not necessary for diagnosis Like in FJHN plus microcysts and tubular dilation

FEurat, fractional excretion of urate. Kidney International (2007) 71, 574–581

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mutations in PKD1 and PKD2, could also result in a more diverse spectrum of symptoms.24–25 The THP knockout mouse showed that the mere absence of THP is not sufficient to cause histological changes associated with MCKD2/FJHN.26 Patient K10 was diagnosed with FJHN but also showed symptoms of a urinary concentration defect, which is untypical for FJHN.20 Moreover, this patient has a very early age of onset with severe clinical symptoms. The youngest patient with FJHN described was 4 years of age.18 Interestingly, treatment with allopurinol did not preserve renal function despite its suggestion as an effective treatment.27 Although proteinuria is not a main criterion for the diagnosis of FJHN or MCKD2, four of the eight patients showed proteinuria (two with FJHN and two with MCKD2). Proteinuria ranged from 0.46 g/24 h to 1.3 g/24 h. One patient was treated with an angiotensin-converting enzyme, reducing proteinuria significantly. Taking the described characteristics of FJHN and MCKD2 into account and trying to apply these to the patients with the C744G mutation, it becomes obvious that no clearcut differentiation between FJHN and MCKD2 is possible (e.g. given the atypical early onset of MCKD2 in K7, renal hypoplasia in K8, renal concentration defect in K10 with FJHN). More discrepancies have been published by Hart et al.5 and Bleyer et al.22 Recently, it was published that the THP knockout mouse does not develop cysts.28 Possibly, the cysts may develop in some UMOD patients due to secondary effects. As the molecular pathogenesis is the same for FJHN and MCKD2, it seems questionable, whether a further differentiation between FJHN and MCKD2 is meaningful. Bleyer et al.22 has already suggested the term uromodulin-associated kidney disease, which could be subdivided into early-onset and lateonset uromodulin-associated kidney disease. However, the molecular basis for the broad clinical variety remains elusive. The Cys248Trp mutation is located in a recently described new D8C domain.29 The cysteine at position 248 is also conserved in D8C domains of other genes as LZP variants, LZP and GP-2. The D8C domain consists of eight conserved cysteine residues that are probably involved in disulfide bond formation. Cys248 is supposed to form a disulfide bridge with Cys 256.29 The T229G mutation alters the amino-acid Cys77Gly in A214. The same amino acid has already been published to be changed in FJHN – but by a different mutation (G230A, Cys77Tyr).30 Both mutations (Cys77Gly and Cys248Trp) involve cysteine residues, which are highly conserved in the UMOD protein throughout evolution. The high cysteine content of THP (48 cysteine residues forming 24 disulfide bonds) and correct formation of the disulfide bonds were suspected to be the rate-limiting step for the export of the premature THP out the endoplasmatic reticulum, which regulates the efficiency of THP maturation.31 This results in decreased urinary UMOD excretion and retention of the misfolded UMOD protein in the endoplasmatic retention, causing an increased rate of apoptosis. Interestingly, this effect was reversed in cell culture by treatment with 580


colchicine or a chaperone (sodium 4-phenylbutyrate).32 Colchicine is a common treatment for gout. Further studies will have to show whether this will be a future therapeutical option for patients with MCKD/FJHN too. MATERIALS AND METHODS Patients We ascertained 16 MCKD individuals from 11 different families. Seven families are from Germany, one comes from Switzerland, one from Italy, one from the USA, and one from Turkey. Age at diagnosis, age at onset of ESRD, hyperuricemia, imaging data, and biopsy results were reviewed if available. Diagnostic criteria were normal or small-sized kidneys with occasional small corticomedullary cysts and renal insufficiency. At least one of the optional criteria as hypertension, a family history of renal disease, reduced fractional urinary excretion of uric acid, or hyperuricemia was demanded. Hyperuricemia was defined as serum uric acid concentration 41 SD higher than the normal values for age and gender.33 The study was approved by the ethics committees of the Albert-Ludwigs-University Freiburg and the University Hospital Cologne. All participating family members provided informed consent. Haplotype analysis of the UMOD gene To analyze linkage to the MCKD2 locus, we performed haplotype analysis with seven polymorphic microsatellite markers: cen – D16S3076 – D16S3045 – D16S3054 – D16S3099 – D16S749 – D16S3036 – D16S3041 – tel. Fluorescently labeled PCR products were detected by a Genetic Analyzer 3100TM (Applied Biosystems, Foster City, CA, USA) and were analyzed by the GENOTYPERTM software. Mutational analysis of the UMOD gene Mutational analysis was performed by exon PCR of the Uromodulin gene. Primer sequences were used as described by Wolf et al.6 PCR products were purified using the Marligen Rapid PCR Purification System. Purified PCR products were sequenced, using a Genetic Analyzer 3700 (Applied Biosystems) and resulting sequences were evaluated with the SequencherTM Software. Urinary excretion of UMOD Western blot analysis for UMOD staining was performed according to Rampoldi et al.8 ACKNOWLEDGMENTS

We thank all members of the MCKD families for their participation. We thank Professor R Witzgall (University of Regensburg) for providing us the UMOD antibody. The technical assistance of Steffi Schneider is gratefully acknowledged. Sources of support: Professor Hildebrandt: the Fritz-Thyssen-Stiftung (1999–2001) and DFG-Fu202/ 3–2, Germany. Dr Wolf: the Koeln Fortune Program Faculty of Medicine, University of Cologne (184/2004), Deutsche Nierenstiftung, and the German Research Foundation (DFG WO 1229/2–1), Germany.

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