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Autosomal recessive polycystic kidney disease (ARPKD) is characterized by progressive cystic degeneration of the kidneys resulting in kidney failure and congenital hepatic fibrosis (CHF) often complicated with portal hypertension1–3. Renal cysts in ARPKD originate from dilated collecting ducts. Most ARPKD patients present perinatally or in childhood; a minority present in adulthood.
ARPKD is caused by mutations in the PKHD1 gene that encodes fibrocystin/polyductin, a protein that functions on primary non-motile cilia4,5. ARPKD is the most common childhood-onset ciliopathy with an estimated frequency of 1 in 20, 000. The carrier frequency for pathogenic PKHD1 mutations is estimated to be approximately 1 in 706. Given the recessive pattern of inheritance, parents of ARPKD patients are not expected to develop clinical features. However, this has not been systematically studied.
We performed ultrasound (USG) evaluations on 110 parents and a maternal grandmother from 64 independent ARPKD families. We identified various abnormalities, including multiple liver cysts and increased renal medullary echogenicity. Here we present, the imaging, clinical and molecular findings in this group of ARPKD family members.
All patients and their parents were enrolled in the protocol, “Clinical Investigations into the Kidney and Liver Disease in Autosomal Recessive Polycystic Kidney Disease/Congenital Hepatic Fibrosis and other Ciliopathies” (www.clinicaltrials.gov, trial NCT00068224), approved by the NHGRI Institutional Review Board and gave written informed consent. Patients who carried a clinical diagnosis of ARPKD made by a nephrologist were qualified to enroll. Evaluations of the index cases at the NIH Clinical Center included complete abdominal standard probe and high-resolution ultrasound (HR-USG) and magnetic resonance imaging (MRI), comprehensive biochemical testing of blood and urine and sequencing of the PKHD1 gene. The clinical diagnosis of ARPKD for each index case at NIH was based upon established criteria2,7 including typical kidney and liver involvement on imaging and/or biopsy and autosomal recessive inheritance. All parents were evaluated by standard and HR-USG; abdominal MRI and magnetic resonance cholangiopancreatography (MRCP) were performed on parents with abnormalities on standard probe USG.
Complete sequencing of PKHD1 was performed in all patients. When DNA samples were available, mutation analysis in parents was performed to determine the parental origin of the mutations identified in the proband. Complete sequencing of the PKHD1 gene was performed in selected parents with imaging abnormalities.
For the longest open reading frame of PKHD1, coding exons (2–67) and their intronic boundaries were sequenced in two directions using a Beckman CEQ 8000 system (Beckman Coulter, Inc., Fullerton, CA) and a contract with Agencourt (Beverly, MA). Sequencher Software (GeneCodes, Ann Arbor, MI) was used to analyze DNA variants. The pathogenicity of missense variants was evaluated as described8 using segregation analysis, general population frequencies, 3 computational prediction tools (Align GVGD [http://agvgd.iarc.fr/agvgd_input.php]; PolyPhen [http://coot.embl.de/PolyPhen/]; and SNAP [http://cubic.bioc.columbia.edu/services/SNAP/]) and the splice variant interpretation software NetGene2 Server (http://www.cbs.dtu.dk/services/NetGene2/).
Complete abdominal USG evaluations were performed using standard (4 MHz) and high resolution (7 MHz) ultrasonographic probes by a single technologist (K.T.D.) (AVI Sequoia Inc, Mountain View, CA). Magnetic resonance imaging (MRI) was performed on a 1.5 or 3 Tesla machine (Philips Medical Systems, NA, Bothell, Washington; General Electric Healthcare, Waukesha, WI, USA) without intravenous contrast media. USG, MRI and MRCP images were evaluated by a team of 2 radiologists (B.I.T and P.C).
In 93 patients the clinical diagnosis of ARPKD was confirmed after NIH evaluations. One family contributed 4 affected siblings, 7 families contributed 2 siblings, and one family contributed an aunt and niece pair leaving 82 independent families. In 73 patients from 63 families the diagnosis was confirmed molecularly; clinical and mutational data from these 73 patients were previously published8,9.
A total of 110 parents (62 mothers and 48 fathers) and a maternal grandmother from 63 independent ARPKD families were available for evaluation. Eighty five parents were from molecularly confirmed families and 25 were from ARPKD families in which the diagnosis was made based on clinical criteria. Mothers’ ages ranged from 27 to 66 years (39 ± 7.2), fathers’ ages ranged from 29 to 58 years (40 ± 6.3) and ages of the total group ranged from 27 to 66 years (39.5 ± 6.8).
Kidney USG showed increased medullary echogenicity in 6 mothers (5.5 %) whose ages ranged from 30 to 62 years (38.8 ± 11.8) (Table 1, Figure 1A–G). Multiple small liver cysts were identified in 10 parents (9 %) including 5 mothers and 5 fathers (Figure 2A–L). The ages of the parents with multiple liver cysts ranged from 36 to 49 years (44.2 ± 4.1). These liver cysts were mostly small and peripherally located. The findings of increased medullary echogenicity and liver cysts in all patients were detected by both standard and high resolution ultrasound probes. MRCP images were inconclusive in terms of the connectivity of the cysts to the biliary system (Figure 2A–F).
A 44 years old father with multiple liver cysts (Table 1, parent no: 16, Figure 2A) also had unilateral multiple parapelvic kidney cysts (Figure 1H). Multiple unilateral kidney cysts were detected in another parent (#17). A moderately increased liver echo pattern associated with coarse texture suggestive of CHF, was detected in 4 parents (table 1, parents 12, 14, 18 and 19, Figure 2L) including 2 with multiple liver cysts (parents 12 and 14) and two with splenomegaly (parents 14 and 19)10. A total of 9 parents had mild splenomegaly (spleen lengths 15–16 cm); in 5, this was in the absence of fatty changes of the liver. Incidentally, fatty changes in the liver suggested by increased echogenicity in association with smooth echo texture were detected in 20 parents10.
Two other parents with multiple liver cysts (Table 1, parent no 11 and 15) at ages 41 and 46, also had 2 kidney cysts each. Five other fathers ages 38 to 46 had single liver cysts. Two mothers at ages 41 and 46 and a father at age 36 had 2 renal cysts and single renal cysts were detected in 7 parents ages 36 to 49.
Other findings, which may be purely coincidental included gall bladder polyps in 3 parents, liver hemangioma in 3 mothers and a father, left duplicated collecting system in one mother and gall stones in one mother. The remainder of the abdominal USG examinations, including pancreas, were normal in all parents.
A 76 year-old maternal grandmother was evaluated because she was documented to have CHF on liver biopsy performed at age 75 prompted by the incidental CT finding of abnormal liver parenchyma. USG imaging at NIH (Figure 2M) showed markedly increased liver echogenicity with moderately coarse echotexture; spleen volume was normal at 100 ml and kidneys were unremarkable.
Liver and kidney-related serum and urine chemistries including serum cystatin C, PTH, and measurements of calcium and creatinine in 24 hour urines were performed in 6 patients including 3 mothers with increased medullary echogenicity (Table 1, parent no: 1, 3 and 5), 2 mothers with multiple liver cysts (Table 1, parents #10 and 11), and the maternal grandmother. All were within normal limits except for elevated alanine aminotransferase (44 U/L, normal 6–41), aspartate aminotransferase (43 U/L, normal 9–34) and gamma glutamyl transferase (151 U/L, normal 7–38) in the grandmother. Specifically, there was no evidence for hypercalciuria, hyperparathyroidism, or renal tubular acidosis.
Complete sequencing of the coding exons of PKHD1 was performed on 16 parents with abnormal imaging findings and the maternal grandmother in an affort to identify additional mutations. These sequences confirmed the mutation transmitted to the proband but did not reveal any other pathogenic variants. The maternal grandmother was heterozygous for the p.Thr36Met mutation in PKHD1.
Our findings on 110 ARPKD parents and a maternal grandmother suggest that carrier status for PKHD1 mutations creates a predisposition to multiple liver cysts and renal involvement associated with increased medullary echogenicity on USG.
The ultrasound finding of increased medullary echogenicity is commonly reported as “nephrocalcinosis”, a term that describes the deposition of radiologically-demonstrable calcium salts in renal parenchyma11,12. The differential diagnosis of “nephrocalcinosis” diagnosed radiologically includes true histopathological nephrocalcinosis and medullary sponge kidney (MSK), which itself can be associated with calcium depositions (Table 2)11,13. Most cases of MSK are sporadic; the presence of rare dominantly inherited forms of MSK, and its association with different malformation syndromes, suggest that MSK is an inherited developmental disorder14–16. The molecular basis of MSK remains largely unknown, two sequence variants of the GDNF gene were recently reported in a few MSK patients14. While intravenous pyelography is the gold standard for the diagnosis of MSK, computerized tomography has become the preferred diagnostic modality. MSK is clinically symptomatic in only a subset of patients and, therefore, is probably under-diagnosed15. The prevalence of MSK is estimated at 5 in 10 000 – 100 00014,15. Based upon our findings, we speculate that carrier (heterozygous) status for PKHD1 mutations may be one underlying molecular cause of MSK. Supporting this hypothesis, the microscopic pathology of MSK resembles that of ARPKD; both are characterized by dilated collecting ducts, although MSK involves only the precalyceal collecting ducts14,15. Furthermore, MSK has been reported in patients with autosomal dominant polycystic kidney disease17 and congenital hepatic fibrosis18, most commonly caused by homozygous mutations in PKHD1. Given that the estimated carrier frequency for PKHD1 mutations is 1 in 70 and 5 % of ARPKD parents have increased medullary echogenicity, our data suggest that the frequency of PKHD1-related MSK would be approximately 7 in 10 000.
Polycystic liver disease (PLD) occurs most commonly as a part of ADPKD. PLD without renal cysts, often referred to as isolated PLD, is genetically distinct from ADPKD19,20. Clinical diagnostic criteria for PLD are available only for at-risk individuals; 21 in patients younger than 40 years, the presence of any liver cysts is considered to be diagnostic of PLD, while in those older than 40, 4 or more cysts are required to differentiate PLD from simple liver cysts20. These criteria, however, need to be further vetted in a larger cohort of PCLD patients. Isolated PLD is genetically heterogeneous19; approximately 20 to 30% of the isolated cases are due to mutations in either PRKCSH22 or SEC63; the molecular cause of the remainder of isolated PLD is unknown. PRKCSH encodes the beta-subunit of glucosidase II, an ER glucosidase that is involved in quality control of newly synthesized glycoproteins. SEC63 encodes Sec63, an integral ER membrane protein that is a part of the multi-protein translocon, the translocation machinery for integral membrane and secreted proteins. It was recently shown that glucosidase IIβ and Sec63p are required in mice for adequate expression of a functional complex of the polycystic kidney disease gene products, polycystin-1, polycystin-2 and fibrocystin/polyductin encoded by Pkhd123, 24. This is supported by the fact that the histopathological features of isolated PLD are very similar to those of the PLD in ADPKD; in both disorders cysts originate from biliary microhamartomas, also termed von Meyenburg’s complexes25,26. However, the reason why defects in glucosidase IIβ and Sec63 do not cause renal cystic disease is unknown.
The majority of PLD patients are clinically asymptomatic, with normal liver enzymes and intact synthetic function21,27. The incidence of isolated PLD in autopsy series26,28,29 (2–3 in 10 000) is higher than its estimated clinical incidence (< 1 in 10 000), suggesting that most cases remain undetected. Our data suggest that heterozygous carriers of PKHD1 mutations are predisposed to PLD. In all ARPKD parents with multiple liver cysts, these cysts were initially detected by standard probe ultrasound; in every case the number of cysts on initial evaluation exceeded 4. Based on the estimated carrier frequency for PKHD1 mutations of 1 in 70, and our finding that 9 % of ARPKD parents have PLD, the frequency of PKHD1-related PLD would be approximately 1 in 1000. Given the small size of the liver cysts and normal liver-related blood chemistries in ARPKD parents, this condition is more likely to remain undiagnosed, explaining the discrepancy between disease frequencies.
In addition to CHF, ARPKD patients might have liver cysts that are in continuity with the biliary tree, differing form the isolated cysts in PLD. MRCP images of the ARPKD parents were not conclusive in terms of the connectivity of the cysts to biliary ducts. However, the small size and peripheral location of these cysts suggest that they are isolated from the biliary tree, similar to the PLD cysts. This difference in the characteristics of liver cysts between children with ARPKD and their parents is surprising. The increased liver echogenicity associated with coarse echotexture identified in some parents and the biopsy proven CHF in the maternal grandmother presented suggest some PKHD1 carriers are also prone to CHF.
To exclude the possibility that these parents had two PKHD1 mutations, we performed complete sequencing of the gene. However, we found no additional PKHD1 mutations in these individuals. Other genes, including those encoding ciliary proteins, may contribute to cyst formation through synergistic heterozygosity30,31; this could explain why only a subset of PKHD1 mutation carriers develop these liver and or kidney findings.
In summary, our data suggest that carrier status for ARPKD is a predisposition to renal involvement associated with increased medullary echogenicity on USG (possibly MSK), and liver involvement in the form of asymptomatic PLD and CHF in some cases. All these PKHD1 mutation carriers with abnormal kidney and/or liver imaging were clinically asymptomatic. It remains to be determined if some of these individuals might become symptomatic as they age.
We thank the ARPKD Alliance for their extensive support and the patients and their families who generously participated in this investigation. Supported by the Intramural Research Programs of the National Human Genome Research Institute, National Cancer Institute and the NIH Clinical Center.
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