Identification of Novel Nonsense Mutations in Iranian patients suffering from autosomal dominant polycystic kidney disease

Background: Autosomal dominant polycystic kidney disease (ADPKD) is the predominant type of inherited kidney disorder, which occurs due to PKD1 and PKD2 gene mutations. ADPKD diagnosis is made primarily by kidney imaging; however, molecular genetic analysis needs to be implicated to confirm the diagnosis. It is critical to perform a molecular genetic analysis when the diagnosis is uncertain, particularly in simplex cases (i.e., a single occurrence in a family), in people with remarkably mild symptoms, or in individuals with atypical presentations. The main aim of this study is to determine the likelihood of PKD1 gene mutations in Iranian patients with ADPKD diagnosis. Methods: Genomic DNA was isolated from blood samples from 26 ADPKD patients, who were referred to the Qaem Hospital in Mashhad, Iran. By using suitable primers, 16 end exons of PKD1 gene that are regional hotspots, were replicated with PCR. Then, PCR products were subjected to DNA directional Sanger sequencing. Results: The results of DNA sequencing in the patients showed that exons 35, 36 and 37 were non- polymorphic, while most mutations had occurred in exons 44 and 45. Only in two patients, exon-intron boundary mutation had occurred in intron 44. Most of the variants were missense and non-synonymous types. Conclusion: In this study, we present nine novel mutations/polymorphisms in PKD1. These data will contribute to an improved diagnostic and genetic counseling in clinical settings.

One of the most prevalent inherited kidney disorders that involves both kidneys is Autosomal dominant polycystic kidney disease (ADPKD). It leads to a progressive loss of kidney function and possible kidney failure (1). About one to two infants in 1000 live at birth are affected by this disorder and approximately 10% of people who undergo dialysis; suffer from this disease (2,3). ADPKD occurs in two types: type I and type II caused by PKD1 and PKD2 mutations, respectively (4,5).
PKD2 mutations lead to end-stage kidney or renal disease at age 74 and occur in 10-15% of cases; on the other hand, PKD1 mutations lead in the average to endstage kidney or renal disease at age 54.3 and occur in 80-90 % of total cases. The latter is the more severe form of the disease (1,3,5). Patients having end-stage kidney or renal disease should receive renal replacement therapy (RRT) support including dialysis and renal transplantation. Dialysis may be the only available modality in these patients. After all, dialysis has its limitations, including sometimes lack of vascular access, risks of vascular thrombosis and infections, diminished quality of life, and loss of biosynthetic functions of the kidney (6). ADPKD patients who have diagnosed before age 30 and hypertension and hematuria before age 35 have a worse renal outcome than those who do not have (7). ADPKD diagnosis is done typically by kidney ultrasound imaging, computed tomography scan or magnetic resonance imaging; however, considering the ADPKD similarity to other cystic kidney disorders, conventional imaging methods do not often lead to a definite and accurate diagnosis (1,2). In this condition, genetic methods can be effective in an accurate and careful diagnosis. Besides, molecular diagnosis methods have an important role in the confirmation of definite diagnosis, especially in young kidney donors, patients with negative family history, people who presented ADPKD with unusual symptoms in childhood and patients who have relatives suffering from this disorder (8,9).
ADPKD is the most frequent genetic kidney pathology (frequency of about 0.1%), which results in 5%-8% of end-stage renal diseases (ESRDs). The ailment is progressive, ending in polycystic enlarged kidneys. This typically results in ESRD in late middle age (5). Polycystin-1, large multidomain protein, is the protein encoded by PKD1. It has domains and regions that are homologous with a number of different proteins (10). International Polycystic Kidney Disease Consortium 1995 (11,12).
Most cases of ADPKD leading to ESRD, are caused by PKD1 mutations (16).
Nevertheless, the genetic determination of the locus mutation has advanced slowly, due to the fact that (1) PKD1 contains a 12,906-bp coding sequence divided into 46 exons (2) and, the 5′ region of the gene, from upstream of exon 1 to exon 33, is inserted in a complex genomic area and repeated more than 4 times, on the same chromosome (European Polycystic Kidney Disease Consortium 1994). These PKD1like homologous genes (HG) have revealed a number of specific deletions and a low level of substitutions (about 2%), in comparison with PKD1 (17). HG loci have made the analysis of PKD1 highly difficult. Thus, the quantity of identified PKD1 mutations is still incomplete, with 82 modifications described in the Online Human Gene Mutation Database (HGMD) (18). A number of methods have been used to screen the repeated region (19)(20)(21)(22)(23), however, the 3′ area has received insufficient attention, with 57.3% of all mutations found in the single-copy area covering 20% of the coding region. PKD2 (a less-complex gene) has revealed 41 mutations with potential effects of truncating and possibly inactivating the translated protein (24). A discrete number of missense changes have also been described (19,(23)(24)(25)(26).
Since numerous somatic mutations are needed to explain the formation of multiple cysts and the notion of a significant rate of formation of novel germline mutations (19), it has been proposed that infrequent mechanisms promote a high rate of PKD1 mutations. First, a long polypyrimidine region in IVS21, which could theoretically form triplex DNA structures (27,28), has been considered as a possible cause for creating mutations in downstream exons (22). Later on, these multiple substitutions and other modifications were described to match HG sequences, possibly indicating a gene conversion with the remotely located HG loci (21,29).

Patient selection
Twenty-two ADPKD patients were collected from regional city hospital; Ghaem hospital (Mashhad, Iran) between April 2012 to March 2013; they were included at diagnosis and disease characteristics of the ADPKD. The present study was approved by Mashhad University of Medical Sciences ethics committee, and before the blood sample was collected, all patients provided their informed consent.
We excluded clinically patients suffering from Von Hippel-Lindau disease and Tuberous Sclerosis. In addition, patients without symptoms of polycystic kidney disease or those who had other syndromes were also excluded in this study.

Amplification assay
Genomic DNA was isolated from 22 whole-blood samples using the standard saltingout method according to the manufacturer's instructions and it was quantified by NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA). Eight-specific primers within the area of the exon 31-46 were designed (Table1). using the Primer 3 software. and all sequences were checked for self-or inter-molecular annealing with nucleic-acid-folding software (OligoAnalyzer 3.1). We performed local-alignment analyses with the BLAST program to confirm the specificity of the designed primers PCR products were analyzed by electrophoresis in a 1.5% agarose gel stained with ethidium bromide for identifying the PKD-specific products for use as a template in sequencing reactions.

Sanger sequencing
Sequencing products were run on an ABI 3130XL Genetic Analyzer (Macrogene Company South Korea), according to the manufacturer's guidelines.
Data analysis was performed with Chromas software version 2.6.5 (Technelysium, South Brisbane, Australia).

Results
Twenty-two patients with an average age of 36.69 ±7.30 years, who suffered from ADPKD, were studied. The sequencing results of the patients were reported in Table   1 and Figure 1

Conclusion
In this study, we present nine novel mutations/polymorphisms in PKD1. These data will contribute to an improved diagnostic and genetic counseling in clinical settings.

Ethics approval and consent to participate
The study was approved by the Ethics Committee of Mashhad University of Medical Sciences. An informed consent was obtained from each individual participating in this study.

Consent for publication
Our study is not a case report, and identifying images or other personal or clinical detail of participants that compromise anonymity are not included. Consent to publish from the patients, or in case of minors, the patients' guardians is "Not Applicable".

Availability of data and materials
All data are included in this published article. Any additional information related to this study is available from the author for correspondence upon reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This study was financially supported by grants from Vice-Chancellor of Research, Mashhad University of Medical Sciences, Mashhad, Iran, (Grant# 901006 ).
The funders of this study do not have any roles in study design, data collection, analysis, result interpretation, writing and the decision to submit the manuscript for publication.

Authors' contributions
FK performed the experiments. MAK, AT, participated in the study design and scientific discussion of the data. MAK supervised the study. All authors contribute to the writing, read and approved the final manuscript.  Figure 1