Autosomal recessive polycystic kidney disease (ARPKD, MIM 263200) is an inherited disorder of the kidney that typically presents in the neonatal period and has an incidence of 1/20 000 to 1/40 000.1 Its principle manifestations involve the fusiform dilation of the renal collecting ducts and bile ducts dilation and fibrosis around the portal vein. ARPKD is caused by mutations in the PKHD1 gene, which is mapped to chromosome region 6p12, extending over a genomic region of 472 kb, with 66 coding exons and a transcript of 16 235 bp. The product of this gene, fibrocystin, predominantly expressed in kidneys, also in the pancreas and liver, is predicted to contain 4074 amino acids with a molecular weight of 447 kDa.2 The conserved domains and predicted structure of the fibrocystin suggest that it may act as a receptor that plays a role in the collecting duct and biliary differentiation.3-5
Recently, nearly 10 cases were reported in China, but there was a lack of a comprehensive molecular genetics analysis. Here, we reported the clinical characteristics of a case with ARPKD and analyze the genetic features of this patient as well as of his father using targeted exome sequencing and Sanger sequencing.
A 15-year-old young boy was accidentally diagnosed of “nephrocalcinosis” in both kidneys and elevation of parathyroid hormone (PTH). The patient was admitted to the Department of Endocrinology in the Peking Union Medical College Hospital. He had been in his usual state of health until five months ago when he caught a cold and underwent abdominal ultrasonography that indicated diffuse hyperechoic spots in the kidneys. Blood tests conducted in the local hospital showed that the level of PTH was elevated to 62.4 ng/dl (normal range <20 ng/dl). The patient had no symptoms and signs on physical examination. His blood pressure was 110/65 mmHg, pulse rate was 78 beats per minute. Laboratory results demonstrated slightly elevated serum creatinine and PTH, low level of urinary calcium, and vitamin D insufficiency (Table 1). Glomerular filtration rate was 85.4 ml/min. Ultrasonography and technetium-99m sestamibi 131I subtraction scanning of the parathyroid gland showed no abnormality. Abdominal ultrasound demonstrated a plump configuration of the kidneys, multiple cysts, hyperechoic spots in the bilateral kidneys, and splenomegaly. CT urography (CTU) showed both kidneys with multiple cysts with calcinosis on the wall and multiple bile duct dilatations in the liver (Figure 1). There was no family history of genetic diseases in either parent. His parents had no consanguinity. Ultrasonography in the father revealed multiple liver cysts but no renal cysts, while the mother had no liver or renal cysts. His mother recalled the process of pregnancy and delivery was normal.
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Table 1. Laboratory results of the patient
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Figure 1. CTU images of the patient. A: Coronal plane. B: Cross section. The CT scanning showed different sizes of multiple cysts and scattered calcinosis in bilateral kidneys and dilation of bile ducts.
Genomic DNA was extracted from the peripheral blood leukocytes of the patient, his father, and 10 healthy individuals using Axygen blood genomic extraction kit (Axygen Biosciences, USA). Informed consent was obtained from each individual subject. The mother of the patient refused DNA analysis for personal reasons.
Targeted exome sequencing was performed by BGI Biotechnology Company (Beijing, China). Briefly, 3 g of genomic DNA was sheared into 150–200-bp fragments randomly, ligated to Illumina sequencing adapters to prepare the hybrid library. Exome capture was carried out using Agilent SureSelect Human All Exon Kit following the manufacturer’s protocol (Agilent Technologies, USA). Each captured library was then loaded on Hiseq2000 platform and paired-end sequencing was performed, with read lengths of 90 bp, providing at least 50 average depths for each sample. Raw image files were processed by Illumina Basecalling Software 1.7 for base calling with default parameters. Sequence reads in each individual were aligned to the human reference genome builds hg18 using SOAPaligner 2.20.
For Sanger sequencing, two pairs of primers were designed to amplify exons 14 and 37 of the PKHD1 gene where mutations were suspected through targeted exome sequencing (Table 2). About 1.5 ml of each primer, 1.5 ml of DNA, 15 ml of PCR mix (2´EasyTaq PCR SuperMix, TransGen Biotech, China) and 10.5 ml of distilled deionized H2O were added up to a total reaction volume of 30 ml. PCR amplification was performed at 95°C for 4 minutes, followed by 30 cycles at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 45 seconds, and a final extension at 72°C for 10 minutes. PCR products were sequenced using a commercial service provided by a biotechnology company (Beijing Tianyi Huiyuan Life Science and Technology Inc., China).
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Table 2. Primers of exons 14 and 37 of PKHD1 gene
Multiple renal cysts with calcinosis accompanied with dilation of bile ducts and splenomegaly were found by ultrasonography and CT scanning for this case. This evidence suggested that diagnosis should be one of renal cystic disease that may be inherited, developmental, or acquired. Acquired renal cysts were excluded for patients who had no underlying disease. Simple renal cysts could not explain such extensive cysts in kidneys and bile duct dilation in the liver. No “bouquet of flowers”, which is a typical manifestation of medullary spongy kidney, was observed in the CTU image. Therefore, the diagnosis was limited to the inherited cystic disease of the kidney. The majority of cases with autosomal dominant polycystic kidney disease (ADPKD), which was identified in adults with a positive family history of ADPKD, had a loss of normal kidney configuration. However, in our case, the patient had a relatively early onset, normal kidney configuration and no family history which suggested a diagnosis of ARPKD.
Two Heterozygous mutations p.N327D(AAT->GAT) and p.G1979R(GGA->AGA) in PKHD1 gene were identified after targeted exome sequencing with 100× average depths and 100% coverage. Point mutations of other encoding regions were identified as single nucleotide polymorphisms based on NCBI dbSNP database.
Compound heterozygous PKHD1 gene mutations, A979G and G5935A, were identified by Sanger sequencing for the patient. These mutations lead to the substitution of an asparagine with an aspartate at amino acid 327 (N327D) and a glycine with an arginine at amino acid 1979 (G1979R) respectively. His father was identified to be a heterozygote for A979G mutation only, while no mutations were detected at exon 14 or 37 of PKHD1 gene in the 10 healthy individuals (Figure 2).
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Figure 2. DNA sequence of exons 14 (left) and 37 (right) of PKHD1 gene. A: Patient. B: Patient’s father. C: Healthy person. Compound heterozygous PKHD1 gene mutations, A979G and G5935A, were identified for the patient. His father was a heterozygote for A979G mutation only. In 10 healthy persons, no mutations were detected at exons 14 and 37 of the PKHD1 gene.
Hereditary renal cystic disease should be differentiated from other commonly seen cystic diseases of the kidney in clinical situations including renal cysts and diabetes syndrome.6 Hereditary renal cystic disease is characterized by the multicystic and symmetrically enlarged kidneys resulted in the changes of configuration and function of kidneys. Different from ADPKD, ARPKD may manifest at an early age, and is often accompanied with ectatic portal bile ducts or portal hepatic fibrosis. ARPKD shows a wide spectrum of clinical presentations, with the majority having significant renal disease, ranging from massively enlarged and cystic kidneys in utero, causing neonatal death in approximately 30% of cases, to neonatal survivors with a significant renal phenotype that may result in end-stage renal disease (ESRD).7 Most patients consulting in the clinic are adolescents or adults. Dias et al8 reported that the median age at diagnosis was 61.45 months (0–336.5 months). Guay-Woodford and Desmond9 analyzed the clinical features of 209 patients with ARPKD and found that the rate of disease progression correlated with the age at the time of ARPKD diagnosis. Those patients who were diagnosed at the age >14 years had a slower progression. Hypertension and chronic renal insufficiency are the most complications. Adeva et al10 reported that among 65 patients with ARPKD, the presenting feature in the neonates (<1 year) was typically associated with renal enlargement, but in the older groups (1-20 years and >20 years) more often involved manifestations of liver disease, including hepatosplenomegaly, hypersplenism, and cholangitis. During follow-up, 22 patients developed renal insufficiency and eight developed ESRD, mostly from the neonatal group. The likelihood of being alive without ESRD differed significantly with 36%, 80%, and 88% survival rates in the three groups respectively, 20 years of after diagnosis. While according to the follow-up of 164 neonatal survivors, Bergmann et al7 pointed out that chronic renal failure was first detected at a mean age of 4 years; actuarial renal survival rates were 86% at 5 years, 71% at 10 years, and 42% at 20 years. For this patient, occult abnormal configuration of kidneys is occasionally detected by abdominal ultrasound examination. Although not usually considered a feature of ARPKD, the frequent occurrence of nephrocalcinosis in ARPKD has been described previously.11 However, the elevated level of creatinine and PTH and relatively low level of serum calcium indicate early renal insufficiency. In addition, ectatic bile ducts and splenomegaly is also seen in this patient. Therefore, this patient should be closely followed-up for his liver and kidney function.
The molecular diagnostics for ARPKD is still a challenge due to the large size and possible alternative splicing of PKHD1 gene, plus marked allelic heterogeneity. Currently, several techniques available for detecting PKHD1 gene mutations include PCR-SSCP (polymerase chain reaction combined with single-strand conformation polymorphism) analysis and PCR combined with denaturing high performance liquid chromatography (DHPLC).12,13 To identify PKHD1 deletions, multiplex ligation-dependent probe amplification is a sensitive and rapid method.14 Those techniques are complicated, time consuming, and not suitable for use in clinical settings. As one of the major breakthroughs in technology, exome sequencing is being used in detecting pathogenic genes of rare single-gene diseases.15,16 By the means of targeted exome sequencing for screening and Sanger sequencing to confirm, we found that the compound heterozygous PKHD1 gene mutations are the underlying molecular basis for patients with ARPKD. Screening by targeted exome sequencing followed by confirming by Sanger sequencing is a cost-effective method for PKHD1 gene mutation detection.
There are more than 500 mutations in exons of PKHD1 gene based on ARPKD Mutation Database (http://www.humgen.rwth-aachen.de/). The types of change include 311 missense mutations (202 probably pathogenic, 26 SNP), 56 nonsense mutations, 88 frameshift mutations, and 56 silent mutations. The majority of the type of mutations is compound heterozygotes. These mutations almost distribute throughout PKHD1 gene; there is no obvious hot spots or clustering phenomenon. Eight mutations reported in more than five origins include p.L1966fs, p.T36M, p.I222V, p.A1254fs, p.I2957T, p.D3230fs, p.R781X, and p.S2861G, which may be due to an ancestor effect. The two mutations we detected lie on the structure domain of fibrocystin which is IPT/TIG3 and G8 1 respectively. As fibrocystin is a large protein, the crystal structure has not been disclosed. It is hard to do the protein tertiary structure prediction through bioinformatics. However the amino acid substitutions on structure domain would change the function of fibrocystin.
In this case, exons 14 and 37 of PKHD1 gene of the patient’s father was also amplified and sequenced. We detected the same mutation in exon 14 in the father and son, while no mutations were found in exon 37 of the father. Because the mother of the patient refused gene analysis for personal reasons, in line with Mendel’s laws of inheritance, we speculated that the mother may have the same mutation in exon 37 as that of the son. Multiple allelism and the high rate of different compound heterozygotes hamper genotype-phenotype correlations. In a previous study, preliminary genotype-phenotype correlations could be drawn for the type of mutation rather than for the site of the individual mutation.12,17 Patients with two truncating mutations consistently have a severe renal presentation that results in neonatal death, while patients with at least one mutation typically have a milder renal presentation.7,18 Milder presentation of recessive polycystic kidney disease requires presence of amino acid substitution mutations.19 Most patients enrolled in Adeva’s study mentioned above were relatively mild and carried missense mutations; no patients had two truncating mutations.10 But sib-pairs with the same PKHD1 mutations may exhibit phenotype of markedly discordant severity. The variability of the disease presentation and course most likely reflects the importance of modifier genes in this disease as well as environmental influences. In our case, the phenotype of compound heterozygous PKHD1 gene mutations is mild. No similar mutations were reported in the literature so far. As severity of PKHD1 mutations did not determine kidney size or function,20 the prognosis of this patient with two missense mutations remains to be awaited.
In summary, the clinical diagnosis of ARPKD was confirmed and compound heterozygous PKHD1 gene mutations were identified in the patient in our study. We conclude that targeted exome sequencing is suitable for genetic diagnosis of single-gene inherited diseases like ARPKD in which the pathogenic gene is a large.
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