Pathogenicity analysis of novel variations in Chinese Han patients with polycystic kidney disease

Accepted Manuscript Pathogenicity analysis of novel variations in Chinese Han patients with polycystic kidney disease

Zishui Fang, Shiyan Xu, Yonghua Wang, Liwei Sun, Yi Feng, Yibin Guo, Hongyi Li, Weiying Jiang PII: DOI: Reference:

S0378-1119(17)30403-1 doi: 10.1016/j.gene.2017.05.046 GENE 41946

To appear in:

Gene

Received date: Revised date: Accepted date:

22 February 2017 24 April 2017 22 May 2017

Please cite this article as: Zishui Fang, Shiyan Xu, Yonghua Wang, Liwei Sun, Yi Feng, Yibin Guo, Hongyi Li, Weiying Jiang , Pathogenicity analysis of novel variations in Chinese Han patients with polycystic kidney disease, Gene (2017), doi: 10.1016/ j.gene.2017.05.046

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ACCEPTED MANUSCRIPT Pathogenicity analysis of novel variations in Chinese Han patients with Polycystic Kidney Disease Zishui Fang1, Shiyan Xu1,2, Yonghua Wang1, Liwei Sun1, Yi Feng1, Yibin Guo1, Hongyi Li1* , Weiying Jiang1* 1. Department of Medical Genetics, ZhongShan School of Medicine, Sun Yat-sen University,

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Guangzhou 510080, China. 2. ShenZhen People’s Hospital

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*Corresponding author: Weiying Jiang. Tel:+(86) 20 87331928; fax: +(86) 20 87331928; E-mail

87331928; E-mail address: [email protected]

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address: [email protected] and Hongyi Li. Tel:+(86) 20 87331928; fax: +(86) 20

Zishui Fang and Shiyan Xu contributed equally to this work.

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Abstract

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Objective Locus and allellic heterogeneity in polycystic kidney disease (PKD) is a great challenge in precision diagnosis. We aim to establish

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comprehensive methods to distinguish the pathogenic mutations from the

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variations in PKD1, PKD2 and PKHD1 genes in a limited time and lay the foundation for precisely prenatal diagnosis, preimplantation genetic

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diagnosis and presymptom diagnosis of PKD. Methods Nested PCR

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combined with direct DNA sequencing were used to screen variations in PKD1, PKD2 and PKHD1 genes. The pathogenicity of de novel variations was assessed by the comprehensive methods including clinic data and literature review, databases query, analysis of co-segregation of the variants with the disease, variant frequency screening in the population, evolution conservation comparison, protein structure analysis and splice sites predictions. Results 17 novel mutations from 15 Chinese 1

ACCEPTED MANUSCRIPT Han families were clarified including 10 mutations in PKD1 gene and 7 mutations in PKHD1 gene. The novel mutations were classified as 4 definite pathogenic, 2 highly likely pathogenic, 4 likely pathogenic, 7 indeterminate by the comprehensive analysis. The results were verified

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the truth by the follow-up visits. Conclusions The comprehensive

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methods may be useful in distinguishing the pathogenic mutations from

and presymptom diagnosis of

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the variations in PKD1, PKD2 and PKHD1 genes for prenatal diagnosis PKD. Our results also enriched PKD

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genes mutation spectrum and evolved possible genotype-phenotype

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correlations of Chinese Han population.

Keyword: Polycystic kidney disease; Autosomal dominant polycystic

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kidney disease; Autosomal recessive polycystic kidney disease；Novel

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varaitions; Pathogenicity prediction

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Abbreviations：PKD, polycystic kidney disease. ADPKD, autosomal dominant polycystic kidney disease. ARPKD, autosomal recessive polycystic kidney disease. DHPLC, denaturing high performance liquid chromatography; SSCP, single strand polymorphism analysis; PGD, preimplantation genetic diagnosis; ASSP ,alternative splice site predictor.

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1. Introduction

Polycystic kidney disease (PKD) including autosomal dominant polycystic kidney (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD) is the most prevalent, potentially lethal, monogenic disorders that result in renal cyst development (Sandro et al. 2007; Peter et al. 2009).

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ACCEPTED MANUSCRIPT ADPKD, also known as an adult renal cystic disease, is the most frequently inherited renal cystic disorder with an incidence between 1 in 400 to 1 in 1000. It is a systemic disorder characterized by multiple, progressive bilateral development and enlargement of cysts in kidneys,

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which typically cause end-stage renal disease (ESRD) in adult life

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(Gabow et al. 1992). ADPKD is genetically heterogeneous with

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mutations of the PKD1 gene accounting for approximately 85% of all cases of ADPKD and the PKD2 gene in most of the remainder ( Reeders

located

on

chromosome

16p13.3,

encodes

an

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(MIM601313)

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et al. 1985; Kimberling et al. 1993; Peters et al. 1993). PKD1 gene

approximately 14kb transcript with 46 exons extending to 50kb of the

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genomic DNA. The 5’ part of PKD1 gene covering exons 1-33 is

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duplicated three or more times proximally on chromosome 16(The European Polycystic Kidney Disease Consortium. 1994; Mochizuki et al.

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1996). PKD2 gene (MIM173910) located on chromosome 4q21, encodes

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a 3kb open reading frame with 15 exons and extends to a 70kb genomic area (Chaowen et al. 2011). The protein products of PKD1 and PKD2, polycystin-1(approximate 460kDa) (Hughes et al. 1995; International Polycystic Kidney Disease Consortium. 1995) and polycystin-2 (approximate 110kDa) (Mochizuki et al. 1996; Hayashi et al. 1997) are membrane proteins that probably form a functional complex (Qian et al.

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ACCEPTED MANUSCRIPT 1997; Tsiokas et al. 1997; Qian et al. 2002; Chauvet et al. 2004; Low et al. 2006). ARPKD, also known as an infantile polycystic kidney disease, invariably associated with congenital hepatic fibrosis (CHF), is the most common

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childhood-onset ciliopathy, with an estimated frequency of 1 in

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7000-20,000 live births (Meral et al. 2010). Approximately 1 of 70

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individuals is a carrier of an ARPKD mutant allele (Zerres et al. 1998). The neonatal disease is characterized by bilateral fusiform dilation of the

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collecting ducts, often leading to massive kidney enlargement. Due to the

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occurrence of pulmonary hypoplasia because of oligohydramnios, approximately 30% of patients die in the perinatal period (Roy et al.

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1997). One known gene, PKHD1 (MIM 606702) located on chromosome

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6p12, was considered to cause the disease (Bergmann et al. 2003). The longest open reading frame (ORF) of PKHD1 is 12.2kb and contains 67

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exons that encode a transmembrane receptor protein called fibrocystin or

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polyductin (Onuchic et al. 2002; Ward et al. 2002). In the past several decades, several groups have performed mutation detection on the transcript encoding the largest open reading frame by gene linkage analysis (Turco et al. 1995) or denaturing high-performance liquid chromatography (DHPLC) analysis (Rossetti et al. 2002; Furu et al. 2003) or single strand polymorphism analysis (SSCP) (Bergmann et al. 2003; Shuzhong et al. 2005) or direct sequencing (Gunay et al. 2010; 4

ACCEPTED MANUSCRIPT Sandro et al. 2012) or next-generation sequencing (Zhang et al. 2005) etc. However, the large size and complexity of polycystic kidney disease (PKD) gene particularly PKD1 gene as well as marked allelic heterogeneity are obstacles to molecular testing by direct DNA analysis

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for clinical diagnostic purposes (Furu et al. 2003). So far, genetic testing

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of PKD1, PKD2，PKHD1 gene is the only useful method for diagnosis

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and prognosis of PKD, particularly for asymptomatic individuals, or those without a family history, which is helpful to make a firm diagnosis,

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and facilitate prenatal diagnostics and preimplementation genetic

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diagnostics (PGD).

In the present study, we focused on how to run a comprehensive analysis

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to identify a pathogenic mutation for the patient diagnosis. And a group

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of novel variations of PKD gene were clarified from 15 Chinese Han families using DNA sequencing. Subsequently, a series of bioinformatics

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methods were used to predict the pathogenicity of these novel variations.

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These new information not only contribute to the molecular diagnosis of PKD which is of great importance to determine the risk for future offspring and siblings but also allow us to compare Han mutational patterns with other populations.

2. Materials and Methods 2.1 Patients

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ACCEPTED MANUSCRIPT Patients and family members in this study were from the First Affiliated Hospital of Sun Yat-sen University. Family history and clinical information were collected from all pedigrees. Probands in the study were carried out comprehensive biochemical and imaging examinations

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as well as pathological observations. Asymptomatic at-risk individuals

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were also examined by ultrasonography. Ethical approval was obtained

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from the Sun Yat-sen University ethical committee. Informed consent was obtained and blood was drawn from each participant. Genomic DNA

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was isolated from peripheral blood lymphocytes using Blood Kit (Magen,

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Canton, China) according to the manufacturer’s instructions, and if variations were needed to verify if they resulted in the change of splicing

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site, the RNA of these samples were isolated also. The coding region and

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intron-exon boundaries region of the PKD1, PKD2, PKHD1 genes were screened for mutations using direct sequencing. In addition, 100 healthy

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blood samples from the donors, without kinship, were collected for

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normal controls.

2.2 PCR amplification Given the complexity of PKD1, long-range (LR) PCR was employed for initial amplification of the PKD1 duplicated region. PCR conditions and DNA sequencing primers were initially taken from some existing literatures (Zhang et al. 2005; Zhang et al. 2006; Audrézet et al. 2012; Sandro et al. 2012). For the purpose of optimization, some primers are 6

ACCEPTED MANUSCRIPT redesigned. For the duplicated region, the PKD1 specific first-round products were used as the template, while for the remainder of PKD1, PKD2 and PKHD1 amplicons were amplified directly from genomic DNA. All PCR products from these amplifications were electrophoresed

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on 2% agarose gels to confirm amplification of right-sized fragments.

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Then, the PCR products were sequenced by standard methods. cDNA was

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generated with the Superscript III cDNA synthesis kit (Invitrogen) to verify the change of splicing site.

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2.3 Pathogenicity Assessment Methods

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In this study, frame-shifting insertions or deletions, nonsense mutation and typical splicing site change were defined as pathogenic mutations.

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Several methods were used to evaluate the pathogenicity of novel variants:

Database

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(1) A series of databases were queried including the ADPKD Mutation (http://pkdb.mayo.edu/),

ARPKD

Mutation

Database

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(http://www.humgen.rwth-aachen.de/index.php), the Human Genome

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Mutation Database (HGMD) (http://www. hgmd. cf. ac. uk/ac/index.php), single nucleotide sequence polymorphism (dbSNP) database ( http:// www.ncbi.nlm.nih.gov/snp) and the previously published PKD mutation detection articles to ascertain whether the variant was novel or not; (2) The segregation of characterized mutations in all family members were analyzed; (3) The population frequency of novel missense mutations were estimated by analyzing 100 unrelated normal control; (4) Amino acids in 7

ACCEPTED MANUSCRIPT mutation

position

were

examined

for

their

conservation

（ http://www.ebi.ac.uk/Tools/msa/clustalw2/) in ten species including mouse, rat, chimpanzee, pig, dog, frog, monkey, chicken, zebrafish and bovine; (5) Novel variations in introns that may disrupt the original

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canonical splice sites were evaluated by the splice variant interpretation

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software SplicePort and Alternative Splice Site Predictor (ASSP) or

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confirmed by RT-PCR; (6) Missense mutations were evaluated by web-based computational pathogenicity prediction tools including Align

PolyPhen-2

(http://genetics.bwh.harvard.edu/pph2/);

(7)

The

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and

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GVGD (http://agvgd. iarc. fr/agvgdinput.php), SIFT (http:// sift. jcvi. org/)

structure of protein was predicted by SWISS-MODEL website

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(http://swissmodel. expasy. org/ interactive) and the possible impact of an

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amino acid substitution on the structure and function of the protein were estimated by DS Visualizer 1.7 and PyMol software.

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2.4 Pathogenicity Category of Novel Variation

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All novel variations analyzed by these web-based software programs were finally sorted into six categories: 1）Definite pathogenic mutation should satisfy the following conditions: First, novel variation was validated to co-segregate between patients and normal family members; Second, allele frequency did not exceed 1% in the population; Third, evolution conservation was high; Fourth, the mutation resulted in the change of protein structure or splicing aberration; Final, no other definite 8

ACCEPTED MANUSCRIPT pathogenic variations were found in the patient. 2 ） Highly likely pathogenic mutation should satisfy the following conditions: First, novel variation was predicted to be deleterious by Align-GVGD，SIFT, and Poly-Phen-2 unanimously or to affect splicing by SplicePort and ASSP

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software unanimously; Second, allele frequency did not exceed 1% in the

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population; Third, the characteristic variation was segregated between

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patients and normal family members; Fourth, no other definite pathogenic variations were found in the patient. 3) Likely pathogenic mutation

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should satisfy the following conditions: First, novel variation was

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predicted to be deleterious by Align-GVGD，SIFT, and Poly-Phen-2 inconsistently or to affect splicing by SplicePort and ASSP inconsistently

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or several novel variations were found simultaneously in the patient;

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Second, allele frequency did not exceed 1% in the population; Third, the characteristic variation was segregated between patients and normal

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family members; Fourth, no other definite pathogenic variations were

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found in the patient. 4) Indeterminate should satisfy novel variations coexisting with definite pathogenic variation in the patient. 5) Polymorphisms should satisfy the following conditions: First, novel variations were scored as benign or predicted to have no effect on splicing; Second, the novel variation should be found in unrelated normal controls; Third, allele frequency was over 1%. 6) Otherwise, they were sorted into “Probable polymorphisms”. 9

ACCEPTED MANUSCRIPT 3. Results In our study, 15 families with PKD were screened using direct DNA sequencing, and 17 novel mutations were clarified including 10 ones in PKD1 gene and 7 ones in PKHD1 gene (Table 1), which were never

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encountered before in the Chinese Han population. What’s more,

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consistent with some previous reports, no prevalent mutation has been

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noted. The pedigree maps in this study were shown in Figure 1. All genotype and phenotype information of available family members were

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summarized in the Table 2.

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In family 3, a heterozygous mutation c.602+5G>A in the intron of PKHD1 gene was found in the mother of proband. PKHD1 gene did not

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transcript in lymphocytes, only expressed in renal epithelial cells.

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Therefore, splicing site change cannot be verified by RT-PCR. Both of the prediction software showed that the mutation resulted in splicing

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aberration. Combining the genotypes and phenotypes analysis of the

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family members, we considered that the mutation (c.602+5G>A) was a pathogenic

mutation.

In

family

5,

a

heterozygous

mutation

c.11157-29G>T in the intron of PKD1 gene was found in the mother of proband. The cDNA sequencing proved that the mutation did not cause the change of splicing site (The result not shown) and the allele frequency of this mutation was 0% in the population. We could not rule out the possibility that this mutation might be a functional gene locus. Therefore, 10

ACCEPTED MANUSCRIPT we classified this mutation as indeterminate. In family 7, a heterozygous mutation c.11156+5G>A in the intron of PKD1 gene was found in the mother of proband. In addition, no definite pathogenic mutation was found in the parents of proband. By cDNA sequence, we identified that

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the mutation led to a splicing aberration in the PKD1 gene which resulted

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in the loss of exon 38 of PKD1 gene, as shown in Figure 2. Therefore, we

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classified this mutation as definite pathogenic mutation.

In our study, we also have performed prenatal diagnosis for two families

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in our study. Family 8 has 2 consecutive sick fetuses. The results of

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prenatal diagnosis had shown that the proband, the first fetus, had carried two mutations, c.2341C>T (p.R781X) in the PKHD1 gene from father

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and the novel c.10058T>G(p.L3353R) in PKHD1 gene from mother.

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According to our classification method, the novel mutation c.10058T>G was classified as likely pathogenic. In the second pregnancy, the second

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fetus also carried the two mutations. B-mode ultrasound images showed

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that bilateral kidney enlargement of the fetus and oligohydramnios. The parents underwent prenatal diagnosis consultation. After serious consideration, they decided to terminate the pregnancy. Now the family are using the preimplantation genetic diagnosis technology to bear healthy offspring. For another family 13, the proband was aborted due to the abnormal kidney development. Unfortunately, the DNA of the proband was not available. Subsequently, the PKD genes of parents were 11

ACCEPTED MANUSCRIPT screened. In a second pregnancy, we performed a prenatal diagnosis for the fetus. The fetus also carried several novel mutations , the indeterminate mutation c.12046G>A (p. G4016S) in PKD1 gene and the likely pathogenic mutation c.10058T>G (p. L3353R) in PKHD1 gene as

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well as the indeterminate mutation c.7445G>A (p.C2482F). But the result

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of B ultrasound showed that the kidneys of the fetus were normal at the

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gestation. The parents decided to continue the pregnancy. Now the fetus has been born, so far the kidneys of the child developed normally. A

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long-term follow-up of the child is also under way.

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For novel missense mutations, the pathogenicity were predicted through multiple softwares (PolyPhen-2, SIFT, Align-GVGD). The prediction

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criteria were shown in Figure 3 and the prediction results were shown in

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Table 3. The change of protein structure is also an important reference for pathogenic classification. The substitution of amino acid can change the

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spatial structure of the protein, which is often manifested through

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affecting the stability of α-helix, β sheet-layer and random coil as well as the number of hydrogen bonds to destroy the function of protein. The structure of protein was predicted by SWISS-MODEL website and the possible impact of amino acid substitution on the structure and function of the protein were estimated by DS Visualizer 1.7 and PyMol software. The schematic diagram of the change of the protein structure was shown in Figure 4, and the results of protein change were presented in Table 3. 12

ACCEPTED MANUSCRIPT For allele frequency of novel mutations, they were validated through the analysis of 100 normal controls，and the allele frequency of mutations were shown in Table 1. In addition, an important reference in determining whether a missense mutation was likely pathogenic or not was the

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conservation degree in different species. In our study, we compared

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human being with other 9 species. Schematic diagram of species

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conservation was shown in Figure 5，and the results of conservative comparison were shown in Table 1. Recurrence of a variant in two or

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more patients with no other clear mutation also strongly supported a

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pathogenic likelihood. Additionally, segregation analysis also contributed to identifying pathogenic likelihood.

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We synthesized the above analysis results, and classified the

displayed

in

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pathogenicity of these novel variations, and the classification results were Table

1.

Thereinto,

c.6018G>C,

c.6442delG,

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c.7146_7153insCTCACTTC, c.11156+5 G>A in PKD1 gene were

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considered definite pathogenic, c.602+5 G>A, c.7717C>T in PKHD1 gene were considered highly likely pathogenic, c.12313A>C in PKD1 gene and c.230G>T, c.10058T>G, c.11246C>C/T in PKHD1 gene were considered likely pathogenic, c.751C>A, c.934G>A, c.3913A>G, c.12046G>A, c.11157-29G>T , c.12313A>C in PKD1 gene and c.2873A>T, c.7445 G>A in PKHD1 gene were considered indeterminate. 4. Discussion 13

ACCEPTED MANUSCRIPT In the present study, we established the comprehensive methods to distinguish the pathogenic mutations from the variations in PKD1, PKD2 and PKHD1 genes in a limited time and lay the foundation for prenatal diagnosis, preimplantation genetic diagnosis and presymptom diagnosis

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of PKD.

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We gave a much clearer view of the type and pattern of mutations

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associated with PKD, revealed some novel changes, and provided a hint of genotype/phenotype associations. In the absence of a family history,

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bilateral renal enlargement and cysts or the presence of multiple bilateral

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cysts with hepatic cysts together with the absence of other manifestations suggesting a different renal cystic disease provide presumptive evidence

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for the diagnosis. The diagnosis of PKD is often ambiguous in patients

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especially for younger at-risk individuals, where renal sonography may not be conclusive or when the family history is unknown (Nicolau et al.

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1999). What’s more, no hot mutations in PKD genes have been reported,

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which means mutations are usually private, highly variable and spread throughout the entire gene. So far, genetic testing of PKD1, PKD2， PKHD1 gene is the only useful method for diagnosis and prognosis of PKD. The ADPKD disease has a delayed clinical onset, some patients have already passed the defective gene to the young generation before experiencing any kind of symptom. The comprehensive methods in the 14

ACCEPTED MANUSCRIPT present study is available for the pre-symptomatic diagnosis. For example, the family 1 had a negative family history with ADPKD. The proband’s son and daughter both presented with normal renal image by ultrasounds at 23 years and 28 years respectively. However, the mother was patient

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with PKD and died of renal failure after diagnosis at 53 years old. The

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putative missense mutation c.6018G>C (p.Trp2006Cys) in exon 15 of

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PKD1 gene was identified in the proband. Based on our method, we can deduce that the p.Trp2006Cys of PKD1 was a pathogenic mutation.

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Unfortunately, the mutation has been inherited by her son. We advised

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him to take his blood pressure, do a renal ultrasound, renal function exam and urine routine every three months to real-time monitor his health

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condition. For 1 year following-up, he presented with hypertension and

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bilateral renal cysts. Although there is no cure for PKD, pre-symptomatic diagnosis can contribute to slowing progression to end-stage renal failure

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effectively and carrying out timely prevention and treatment by

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controlling blood pressure and proteinuria etc. In addition, it is difficult to clinically distinguish ARPKD and early-onset cases of ADPKD as well as other childhood causes of renal cystogenesis (Cobben et al. 1990; Sujansky et al. 1990; Gabow PA. 1993; De et al. 2005; Shamshirsaz et al. 2005; Reed et al. 2008). In our study, family 4 and 7 had continuous multi fetuses with renal enlargement and cysts. The gene of ARPKD was thoroughly screened and no pathogenic mutation 15

ACCEPTED MANUSCRIPT was found. Subsequently, we screened the genes of ADPKD, where we indeed found pathogenic mutations in PKD1 gene. Similarly, in family 12, the proband presented bilateral kidney enlargement and abnormal echo at the gestation of 23 weeks. We didn’t find any pathogenic mutations in

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PKHD1 gene of the proband, however, a novel missense mutation

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c.12313A>C/A in PKD1 gene which came from the mother of the

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proband was found. Despite the fact that ADPKD is often considered as a disease occurring in adulthood, it is clear that the disease begins in

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infancy in these cases. Therefore, from our research point of view, the

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traditional view that ADPKD onset only occurred in the adult stage should be modified. To avoid a misdiagnosis, there is a strong demand for

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the prenatal diagnosis to detect the mutations located in the PKD1, PKD2

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and PKHD1 genes for the fetus with PKD.

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When a novel missense mutation or more than one novel variations were found in the same patient, it is difficult to determine accurately whether

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the novel or which one mutation is the pathogenic variation. Additionally, because of the high prevalence of polymorphisms and private mutations, particularly in PKD1, it is difficult to determine whether a specific genetic change is a mutation or a polymorphism (Chaowen et al. 2011). For those families that found novel mutations through prenatal diagnosis of PKD, it is not possible to carry out functional verification experiments for those novel mutations due to time constraint. Therefore, the 16

ACCEPTED MANUSCRIPT comprehensive methods screening and identifying pathogenic mutations in PKD1, PKD2 and PKHD1 genes could offer great promise in the diagnosis and treatment of PKD patients. In the current study, all novel variations were first detected in family

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members and unrelated normal controls. Then the pathogenicity of

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variations was analyzed by web-based software applications including

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SIFT, PolyPhen-2, Align-GVGD, SWISS MODEL . Long term follow up was also performed at the same time. All analyses have finally sorted

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those variations into the corresponding categories and our results

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demonstrated the utility of bioinformatics evaluation of gene variations in PKD genes. Of note, in some affected individuals, there is no prior

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family history, suggesting the presence of de novo PKD gene mutations.

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And in some families, according to the analysis of clinical symptoms and genetic pattern of disease, no pathogenic mutations on the corresponding

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presumptive gene were found. This may be due to missed mutations such

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as deep intronic changes that affect splicing or gene promoter change not detected by current exon-based screening methods (Ying et al. 2011). Alternatively, it is possible that the disease in these patients is caused by a third gene (M.C. Daoust et al. 1995; S. de Almeida et al. 1995). Analysis of the variability in renal function between monozygotic twins and siblings lends support to the role of genetic modifiers (Persu et al. 2004). It was also possible that hypermethylation of CpG islands in promoter or 17

ACCEPTED MANUSCRIPT other region of PKD1 gene could also inactive the PKD1 gene and cause the disease (Lan et al. 2002). Moreover, other mutation mechanisms, e.g., gross deletions or genomic rearrangements are not detectable. In addition, significant intrafamilial variability in the severity of renal and extrarenal

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manifestations suggested genetic and environmental modifying factors

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(Vicente et al. 2001). Hormonal influences, especially associated with

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more severe liver disease in female individuals, indicate a role for non-genetic factors (Sandro et al. 2007). The severity of symptoms, the

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age of onset, and the rates of progression to end-stage renal failure or

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death vary widely among the PKD patients, which has a great challenge for the patient’s diagnosis. Besides, the transcription of PKHD1 is not

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available in peripheral blood. Though the prediction software contributed

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to predicting original splice sites changes, its functional studies are rather limited. Therefore, this kind of variations should be interpreted carefully

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when utilized in clinical diagnosis. In general, truncated mutation can

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result in the earlier onset age and more severe disease phenotype. However, some still did not be onset at about 30 years old. One possible reason accounting for this is individual heterogeneity. All the aforementioned challenges and limitations do add the difficulty of finding the cause of the disease. Because no conclusion can be drawn about the correlation between the types of mutations and phenotypes, more data related to the genotypes 18

ACCEPTED MANUSCRIPT and phenotypes of PKD should be accumulated. Undoubtedly, the identification of more common mutations, especially in particular populations, will aid molecular diagnostics in these districts. Additionally, the novel mutation analysis will also lead to a useful DNA-based

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diagnostic test. The definition of further mutations will accompanied by

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the establishment of a more clear process for the identification of disease

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associated changes and polymorphisms , and the prospects for gene-based diagnostics will improve.

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5. Conclusion

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We established the comprehensive methods to distinguish the pathogenic mutations from the variations in PKD1, PKD2 and PKHD1 genes in a

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limited time. The comprehensive methods may be greatly useful for the

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prenatal diagnosis and pre-symptomatic diagnosis of PKD. Total of 17 novel mutations were clarified including 10 mutations in PKD1 gene and

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7 mutations in PKHD1 gene in the Chinese Han population. Moreover,

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we revealed that ADPKD is not only an adult-onset form, but also an infancy-onset form. It is the traditional view that ADPKD onset only occurred in the adult stage should be modified.

Acknowledgements We thank those patients and their families for taking part in our investigation. This work was supported by the National Natural Science Foundation of China grants No.31171214 and U1132606 as well as 19

ACCEPTED MANUSCRIPT Provincial Science and Technology Project of Guangdong Province 2014A020213020.

Conflict of interest disclosure The authors declare that they have no conflict of interest.

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Reference

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Audrézet MP1, Cornec-Le Gall E, Chen JM, Redon S, Quéré I, Creff J, Bénech C, Maestri S, Le Meur Y, Férec C (2012) Autosomal dominant polycystic kidney disease: comprehensive mutation analysis of PKD1 and PKD2 in 700 unrelated patients. Hum Mutat 33:1239-50 Bergmann C, Senderek J, Sedlacek B, Pegiazoglou I, Puglia P, Eggermann T, Rudnik-Schöneborn S, Furu L, Onuchic LF, De Baca M, Germino GG, Guay-Woodford L, Somlo S, Moser M, Büttner R, Zerres K (2003) Spectrum of mutations in the gene for autosomal recessive polycystic kidney disease (ARPKD/PKHD1). J Am Soc Nephrol 14: 76-89 Cobben JM, Breuning MH, Schoots C, Kate LP, Zerres K (1990) Congenital hepatic fibrosis in autosomal dominant polycystic kidney disease. Kidney Int 38: 880-885 Chauvet V, Tian X, Husson H, et al (2004) Mechanical stimuli induce cleavage and nuclear translocation of the polycystin-1 C terminus. J Clin Invest 114: 1433-1443 Chaowen Yu, Yuan Yang, Lin Zou, Zhangxue Hu, Jing Li, Yunqiang Liu, Yongxin Ma, Mingyi Ma, Dan Su and Sizhong Zhang (2011) Identification of novel mutations in Chinese Hans With autosomal dominant polycystic kidney. BMC Medical Genetics 12:164 De Rycke M, Georgiou I, Sermon K, et al (2005) PGD for autosomal dominant polycystic kidney disease type 1. Mol Hum Reprod 11: 65-71 Furu L, Onuchic LF, Gharavi A, Hou X, Esquivel EL, Nagasawa Y, Bergmann C, Senderek J, Avner E, Zerres K, Germino GG, Guay-Woodford LM, Somlo S (2003) Milder presentation of recessive polycystic kidney disease requires presence of amino acid substitution mutations. J Am Soc Nephrol 14:2004-2014 Gabow PA, Johnson AM, Kaehny WD, Kimberling WJ, Lezotte DC, Duley IT, Jones RHGabow PA, Johnson AM, Kaehny WD, Kimberling WJ, Lezotte DC, Duley IT, Jones RH (1992) Factors affecting the progression of renal disease in autosomal-dominant polycystic kidney disease. Kidney Int 41:1311-1319 Gabow PA (1993) Autosomal dominant polycystic kidney disease. N Engl J Med 329:332-342 Gunay-Aygun M, Tuchman M, Font-Montgomery E, Lukose L, Edwards H, Garcia A, Ausavarat S, Ziegler SG, Piwnica-Worms K, Bryant J, Bernardini I,Fischer R, Huizing M, Guay-Woodford L, Gahl WA(2010) PKHD1 Sequence Variations in 78 Children and Adults with Autosomal Recessive Polycystic Kidney Disease and 20

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Congenital Hepatic Fibrosis. Mol Genet Metab 99:160-73 Hughes J, Ward CJ, Peral B, et al (1995) The polycystic kidney disease 1 (PKD1) gene encodes a novel protein with multiple cell recognition domains. Nat Genet10: 151-160 Hayashi T, Mochizuki T, Reynolds DM, Wu G, Cai Y, Somlo S (1997) Characterization of the exon structure of the polycystic kidney disease 2 gene (PKD2). Genomics 44: 131-36 International Polycystic Kidney Disease Consortium (1995) Polycystic kidney disease: the complete structure of the PKD1 gene and its protein. Cell 81: 289-298 Jang DG, Chae H, Shin JC, Park IY, Kim M, Kim Y (2011) Prenatal diagnosis of autosomal recessive polycystic kidney disease by molecular genetic analysis. J Obstet Gynaecol Res 37:1744-1747 Kimberling WJ, Kumar S, Gabow PA, Kenyon JB, Connolly CJ, Somlo S (1993) Autosomal dominant polycystic kidney disease: localization of the second gene to chromosome 4q13-q23. Genomics 18:467-472 Lan Ding, Sizhong Zhang, Weimin Qiu, Cuiying Xiao, Shaoqing Wu, Ge Zhang, Lu Cheng, Sixiao Zhang (2002) Novel mutations of PKD1 gene in Chinese patients with autosomal dominant polycystic kidney disease. Nephrol Dial Transplant 17:75-80 Low SH, Vasanth S, Larson CH, et al (2006) Polycystin-1, STAT6, and P100 function in a pathway that transduces ciliary mechanosensation and is activated in polycystic kidney disease. Dev Cell 10: 57-69 M.C. Daoust, D.M. Reynolds, D.G. Bichet, S. Somlo (1995) Evidence for a third genetic locus for autosomal dominant polycystic kidney disease. Genomics 25 : 733-736 Mochizuki T, Wu G, Hayashi T, Xenophontos SL, Veldhuisen B, Saris JJ, Reynolds DM, Cai Y, Gabow PA, Pierides A, Kimberling WJ, Breuning MH, Deltas CC, Peters DJ, Somlo S (1996) PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein. Science 272:1339-42 Meral Gunay-Aygun, Maya Tuchman, Esperanza Font-Montgomery, Linda Lukose, Hailey Edwards, Angelica Garcia, Surasawadee Ausavarat, Shira G. Ziegler, Katie Piwnica-Worms, Joy Bryant, Isa Bernardini, Roxanne Fischer, Marjan Huizing, Lisa Guay-Woodford, and William A. Gahl (2010) PKHD1 Sequence Variations in 78 Children and Adults with Autosomal Recessive Polycystic Kidney Disease and Congenital Hepatic Fibrosis. Mol Genet Metab 99: 160 Nicolau C, Torra R, Badenas C, Vilana R, Bianchi L, Gilabert R, Darnell A, Brú C (1999) Autosomal dominant polycystic kidney disease types 1 and 2: assessment of US sensitivity for diagnosis. Radiology 213:273-6 Onuchic LF, Furu L, Nagasawa Y, Hou X, Eggermann T, Ren Z, Bergmann C, Senderek J, Esquivel E, Zeltner R, Rudnik-Schöneborn S, Mrug M, Sweeney W, Avner ED, Zerres K, Guay-Woodford LM, Somlo S, Germino GG (2002) PKHD1, the polycystic kidney and hepatic disease 1 gene, encodes a novellarge protein containing multiple immunoglobulin-like plexin-transcription-factor domains and parallel beta-helix 1 repeats. Am J Hum Genet 70:1305-1317 21

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Peters DJ, Spruit L, Saris JJ, Ravine D, Sandkuijl LA, Fossdal R, Boersma J, van Eijk R, Norby S, Constantinou-Deltas CDPeters DJ, Spruit L, Saris JJ, Ravine D, Sandkuijl LA, Fossdal R, Boersma J, van Eijk R, Norby S, Constantinou-Deltas CD et al (1993) Chromosome 4 localization of a second gene for autosomal dominant polycystic kidney disease. Nat Genet 5(4):359-362 Persu A, Duyme M, Pirson Y, et al (2004) Comparison between siblings and twins supports a role for modifier genes in ADPKD. Kidney Int 66: 2132-2136 Peter C. Harris and Vicente E. Torres (2009) Polycystic Kidney Disease. Annu Rev Med 60:321-337 Qian F, Germino FJ, Cai Y, Zhang X, Somlo S, Germino GG(1997) PKD1 interacts with PKD2 through a probable coiled-coil domain. Nat Genet 16: 179-183 Qian F, Boletta A, Bhunia AK, et al (2002) Cleavage of polycystin-1 requires the receptor for egg jelly domain and is disrupted by human autosomal-dominant polycystic kidney disease 1- associated mutations. Proc Natl Acad Sci USA 99: 16981-16986 Reeders ST, Breuning MH, Davies KE, Nicholls RD, Jarman AP, Higgs DR, Pearson PL, Weatherall DJReeders ST, Breuning MH, Davies KE, Nicholls RD, Jarman AP, Higgs DR, Pearson PL, Weatherall DJ (1985) A highly polymorphic DNA marker linked to adult polycystic kidney disease on chromosome 16. Nature 317:542-544 Roy S, Dillon MJ, Trompeter RS, Barratt TM (1997) Autosomal recessive polycystic kidney disease: long-term outcome of neonatal survivors. Pediatr Nephrol 11(3):302-306 Rossetti S, Chauveau D, Walker D, Saggar-Malik A, Winearls CG, Torres VE, Harris PC(2002) A complete mutation screen of the ADPKD genes by DHPLC. Kidney Int 61:1588-1599 Reed BY, Mc Fann K, Bekheirnia MR, Nobakhthaghighi N, Masoumi A, Johnson AM, Shamshirsaz AA, Kelleher CL, Schrier RW (2008) Variation in age at ESRD in autosomal dominant polycystic kidney disease. Am J Kidney Dis 51:173-183 Sujansky E, Kreutzer SB, Johnson AM, Lezotte DC, Schrier RW, Gabow PA (1990) Attitudes of at-risk and affected individuals regarding presymptomatic testing for autosomal dominant polycystic kidney disease. Am J Med Genet 35: 510-515 S. de Almeida, E. de Almeida, D. Peters, J.R. Pinto, I. Tavora, J. Lavinha, M. Breuning, M.M. Prata (1995) Autosomal dominant polycystic kidney disease: evidence for the existence of a third locus in a Portuguese family. Hum Genet 96:83-88 Shuzhong Zhang, Changlin Mei, Dianyong Zhang, Bing Dai, Bing Tang, Tianmei Sun, Haidan Zhao, Yukun Zhou, Lin Li, Yumei Wu, Wenjing Wang, Xuefei Shen, Ji Song (2005) Mutation Analysis of Autosomal Dominant Polycystic Kidney Disease Genes in Han Chinese, Nephron Exp Nephrol 100:63-76. Sharp AM, Messiaen LM, Page G, Antignac C, Gubler MC, Onuchic LF, Somlo S, Germino GG, Guay-Woodford LM (2005) Comprehensive genomic analysis of PKHD1 mutations in ARPKD cohorts. J Med Genet 42:336-349 Shamshirsaz AA, Reza Bekheirnia M, Kamgar M, Johnson AM, Mc Fann K, Cadnapaphornchai M, Nobakhthaghighi N,Schrier RW (2005) 22

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Autosomal-dominant polycystic kidney disease in infancy and childhood: progression and outcome. Kidney Int 68:2218-2224 Sandro Rossetti and Peter C. Harris (2007) Genotype-Phenotype Correlations in Autosomal Dominant and Autosomal Recessive Polycystic Kidney Disease. J Am Soc Nephrol. 18: 1374-1380 Sandro Rossetti, Katharina Hopp, Robert A. Sikkink, Jamie L. Sundsbak, Yean Kit Lee, Vickie Kubly, Bruce W. Eckloff, Christopher J. Ward, Christopher G. Winearls, Vicente E. Torres, and Peter C. Harris (2012) Identification of Gene Mutations in Autosomal Dominant Polycystic Kidney Disease through Targeted Resequencing. J Am Soc Nephrol 23:915-933 The European Polycystic Kidney Disease Consortium (1994) The polycystic kidney disease1 gene encodes a 14kb transcript and lies within a duplicated region on chromosome16. Cell 77:881-894 Turco AE, Padovani EM, Peissel B, et al (1995) Gene linkage analysis and DNA based detection of autosomal dominant polycystic kidney disease (ADPKD) in a newborn infant. Case report. J Perinat Med 23:205-212 Tsiokas L, Kim E, Arnould T, Sukhatme VP, Walz G (1997) Homo- and heterodimeric interactions between the gene products of PKD1 and PKD2. Proc Natl Acad Sci USA 94: 6965-6970 Vicente E Torres, Peter C Harris, Yves Pirson (2007) Autosomal dominant polycystic kidney disease. Lancet 369: 1287-1301 Ward CJ, Hogan MC, Rossetti S, Walker D, Sneddon T, Wang X, Kubly V, Cunningham JM, Bacallao R, Ishibashi M, Milliner DS, Torres VE, Harris PC (2002) The gene mutated in autosomal recessive polycystic kidney disease encodes a large, receptor-like protein. Nat Genet 30:259-69 Ying-Cai Tan , Jon Blumenfeld , Hanna Rennert (2011) Autosomal dominant polycystic kidney disease: Genetics, mutations and microRNAs. Biochimica et Biophysica Acta 1812 : 1202-1212 Ying-Cai Tan, Alber Michaeel, Jon Blumenfeld, Stephanie Donahue, Tom Parker, Daniel Levine,and Hanna Rennert (2012) A Novel Long-Range PCR Sequencing Method for Genetic Analysis of the Entire PKD1 Gene. The Journal of Molecular Diagnostics 14:305-313 Zerres K, Rudnik-Schoneborn S, Steinkamm C, Becker J, Mucher G(1998) Autosomal recessive polycystic kidney disease. J Mol Med (Berl) 76:303-309 Zhang S, Mei C, Zhang D, et al(2005) Mutation analysis of autosomal dominant polycystic kidney disease genes in Han Chinese. Nephron Exp Nephrol 100:63-76 Zhang YH, Zhang DY (2006) Mutation detection of ADPKD PKD1 gene in Hans by denaturrmance liquid chromatography. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 23:283-288

Figures and Tables Figure 1. The pedigree maps in the study. The arrow indicated the proband.

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Figure 2. (A-1) The linear sketch map of PKD1 gene exon 38 after normal splicing showed the normal transcription. (A-2)The linear sketch map of PKD1 gene exon 38 after abnormal splicing due to the mutation c.11156+5 A>G showed the loss of exon 38 (140bp) of the transcription. (B) Reverse transcription–PCR (RT-PCR) and cDNA sequencing confirmed this abnormal splicing. RT-PCR showed skipping of exon 38(140bp). The reference sequence is NC_000016.9 and CCDS 32369.1. Figure 3. Predictive criteria for pathogenicity of multiple software. For PolyPhen-2, the classifiers ordered from least likely to interfere with function to most likely from left to right, that is, the higher the score gets, the more serious the pathogenicity; For SIFT, the lower the score gets, the more serious the pathogenicity; For Align-GVGD, the higher the class gets, the more serious the pathogenicity. Figure 4. The schematic diagram of the change of protein structure caused by the change of amino acid. (A) Normal protein structure; (B) Protein structure after amino acid change at a certain site. The position of the arrow indicated the loss of one α-helix and the increase of one β sheet-layer. Figure 5. Schematic diagram of conservation of species. Arrows indicated the site p.958Asp (D) of fibrocystin. In all of the ten species, the site of the amino acid is not conservative only in the chicken, which indicated the site is highly conserved among species. Table 1 The variations of PKD genes in our study Table 2 PKD gene mutation and clinical symptoms of patients and family members in this study Table 3 Pathogenicity prediction and change of protein structure of novel missense mutation

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Figure 1

25

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Figure 2

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PT

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Figure 3

27

RI

PT

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Figure 4

28

PT

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Figure 5

29

PKD1

Gene

30

Arg2477His

Asp3368Asn

Thr3510Met

c.7430G>A

c.10102G>A

c.10529C>T

CACTTC

Ser2382fs*2X

Glu2148fs*13X

c.6442delG

c.7146_7153insCT

Trp2006Cys

c.6018G>C

Missense

Missense

Missense

Insertion

Deletion

Missense

Missense

Missense

Ala1311Thr

Val1604Met

Missense

Missense

0%

y 0%

frequenc

Allele

2.9%

rare

rare

0%

0%

0%

0.16%

rare

Highly conservation

monkey and zebrafish

Not conserved only in cattle，

Highly conservative

chicken ，frog and zebrafish

Not conserved only in

NA

NA

Highly conservative

PT

indeterminate

likely pathogenic

highly likely pathogenic

definite pathogenic

definite pathogenic

definite pathogenic

highly likely pathogenic

indeterminate

indeterminate

indeterminate

indeterminate

Pathogenic classification

RI

SC

Highly conservative

NU

frog and zebrafish

Not conserved in dog，chicken,

MA

0%

Not conserved in all species

monkey

Conservated only in cattle and

Evolution conservation

The variations of PKD genes in our study

D

PT E

Missense

Mutation type

Ile1305Val

Ala312Thr

Pro251Thr

CE

change

AC

Amino acid

c.4810G>A

c.3931G>A

c.3913A>G

c.934G>A

c.751C>A

Nucletide change

Table 1

Audrézet et al. 2012

Zhang et al. 2005

Zhang et al. 2006

novel

novel

novel

Audrézet et al. 2012

Zhang et al. 2005

novel

novel

novel

Previous description

ACCEPTED MANUSCRIPT

NA

Gly4016Ser

Ile4105Leu

c.11157-29G>T

c.12046G>A

c.12313A>C

PKHD1

PKD2

NA

c.11156+5 G>A

31

NA

Lys626Arg

Arg781X

Asp958Val

Cys2482Tyr

Arg2573Cys

Leu3353Arg

Pro3749Leu

c.1877A>G

c.2341C>T

c.2873A>T

c.7445 G>A

c.7717C>T

c.10058T>G

c.11246C>C/T

Cys77Phe

c.230G>T/G

c.602+5 G>A

Val516Leu

c.1546G>T

Missense

Mutation type

0%

IVS Silent

Missense

Missense

Missense

Missense

Missense

Truncating

Missense

Splice site

Missense

Missense

Missense

Missense

D

0%

0%

0%

0%

0%

0%

5%

NA

0%

1.4%

0%

0%

PT E

0%

0.9%

novel

likely pathogenic

PT indeterminate

highly likely pathogenic likely pathogenic likely pathogenic

Highly conservative Highly conservative Highly conservative

indeterminate

novel

novel

novel

novel

novel

Sharp et al. 2005 definite pathogenic

Highly conservative

Highly conservative

NA

Jang DG et al. 2011

novel polymorphism

RI

SC

NU

Low conservative degree

NA

Highly conservative

highly likely pathogenic

Chaowen et al. 2011

likely pathogenic

Highly conservative

novel

likely pathogenic

Low conservative degree

novel

novel

novel

Zhang et al. 2005

Previous description

indeterminate

indeterminate

definite pathogenic

likely pathogenic

Pathogenic classification

Highly conservative

NA

NA

and zebrafish

Not conserved only in frog

Evolution conservation

MA

Allele frequency

Splice site

CE

AC

Gly3560Arg

c.10678G>A

PKD1

Amino acid change

Nucletide change

Gene

ACCEPTED MANUSCRIPT

32

4

3

2

1

Family

c.6018G>C/G*

None

Proband

Husband of proband

PKD2 None

PKHD1

None

None

c.10529C>T/C, c.10678G>A/G

None

None

None

NA

Daugter of proband

Proband

Proband

Father of proband

Mother of proband

Proband

NA

None

None

None

None

c.6018G>C/G*

Son of proband None

None

NA

c.602+5G>A/G*

c.7717C>T/C*

PT

Double kidney enlargement and cyst at the gestation period

Normal

Normal

RI

Intrahepatic bile duct expansion and hepatosplenomegaly ,hypertension at the age of 1.

c.7717C>T/C*, c.602+5G>A/G*

SC

Bilateral renal enlargement and cyst, renal calculus, hypertension , oligozoospermia at the age of 28

Normal

NU

MA

Hypertension from the age of 16,bilateral renal cyst, oligozoospermia at the age of 24.

Normal

Died of ESRD at the age 53

Clinical Symptoms

None

None

D

None

None

PT E

c.1546G>T /G

CE

PKD1

Members of family

AC

Table 2 PKD gene mutation and clinical symptoms of patients and family members in this study

3 consecutive sick fetuses

Note

ACCEPTED MANUSCRIPT

33

8

7

6

5

4

Family

c.7430G>A/G

Father of proband

c.10678G>A/G, c.11157-29G>T/G*

Proband

None

None

None

PKD2

None None

None

c.11156+5G>A/G*

None

None

c.3931G>A/G, c.10102G>A/G

Father of proband

Mother of proband

Proband

Father of proband

Mother of proband

None

None

None

NA

NA

None

NA

Proband

NA

Proband

None

c.4306C>T/C

c.11157-29G>T/G*

Mother of proband

None

Son of proband

c.10678G>A/G

Father of proband

SC

c.10058T>G/T*

c.2341C>T/C

Normal

Normal

Bilateral kidney enlargement at the gestation period

c.10058T>G/T*, c.2341C>T/C

PT

RI

Normal

Normal

renal cysts by histopathological examination

Oligospermia, renal transplantation at the age of 27

NU

Died of ESRD

None

None

NA

None

NA

MA

Normal

None

D

Bilateral kidney enlargement and cysts of the fetus at the gestation of 27 week

Normal

Normal

Clinical Symptoms

Normal

c.1877A>G/A

None

None

PKHD1

c.1877A>G/A

PT E

c.10529 C>T/C

Mother of proband

CE

AC

PKD1

Members of family

2 consecutive sick fetuses

2 consecutive sick fetuses

29 years old

Note

ACCEPTED MANUSCRIPT

34

12

11

10

9

Family

c.6442delG

None

Proband

Son of proband

c.12313A>C/A*

None

Old sister of proband

None

Father of proband

Mother of proband

c.12313A>C/A*

Proband

None

None

None

None

None

None

None

None

NA

c.1546G >T/G

None

Proband

None

None

c.3931G>A/G, c.10678G>A/G

None

NA

D

Mother of proband

NA

None

None

NA

Proband

None

PT E

None

None

None

Bilateral kidney enlargement and cyst at the gestation of 31 weeks

Normal

Normal

Single kidney transplantation due to renal failure

Clinical Symptoms

PT

RI

SC

Normal

Normal

Normal

Bilateral kidney enlargement gestation of 23 weeks

and abnormal echo at the

Bilateral kidney enlargement and diffusely increased echogenicity,liver cyst

Normal

NU

Normal

Bilateral kidney enlargement at the gestation period

MA

PKHD1

None

None

Grandson or granddaughter of proband

None

None

None

PKD2

Father of proband

None

Daugter of proband

CE

AC

PKD1

Members of family

36 years old

39 years old

28 years old

2 consecutive sick fetuses

Note

ACCEPTED MANUSCRIPT

35

15

14

13

Family

None None

c.4810G>A/G None c.7146-7153 ins CTCACTTC* None

Grandfather of proband

Grandmother of proband

Proband

Father of proband

None

None

None

None

Mother of proband

None

NA

c.3913A>G/A*, c.4810G>A/G

Proband

None

Father of proband

NA

Brother of proband

None

None

NA

PKD2

None

None

None

None

c.230G>T/G*

PT

RI

SC

Normal

Kidney cyst,multiple renal calculi, hypertension, asthenospermiaoligospermia

Normal

Left kidney stone, left kidney cyst

Normal

Suspected right kidney stone

Enlarged kidneys, poor corticomedullary differentiation, right kidney increased echogenicity , oligohydramnios at the gestation of 23 weeks

Normal

Double kidney stone, liver cyst

Normal

Enlarged echogenic kidneys, oligohydramnios at the gestation of 27 weeks

Clinical Symptoms

NU

MA

c.11246C>C/T*

NA

D

c.2873A>A/T*, c.7445 G>A/G* c.10058T>T/G* , c.7445 G>A/G*

c.10058T>T/G*

NA

PKHD1

PT E

c.12046G>G/A *

Mother of proband

c.12046G>G/A * c.751C>C/A*, c.934G>G/A*

NA

CE

PKD1

AC

Father of proband

Proband

Members of family

24 years old

67 years old

69 years old

28 years old

39 years old

1 years old

27 years old

27 years old

Note

ACCEPTED MANUSCRIPT

Sister of proband

Mother of proband

PKD1

D

PT E

CE

c.7146-7153 ins CTCACTTC*

NA

AC

Members of family

* represent novel mutation; NA, No Available ;

15

Family

None

NA

PT

RI

Kidney cyst

Died of ESRD

Clinical Symptoms

SC

PKHD1

NU

MA

None

NA

PKD2

Note

ACCEPTED MANUSCRIPT

36

ACCEPTED MANUSCRIPT

Nucleotide

Amino acid

change

change

c.751C>A

Pro251Thr

c.934G>A

Ala312Thr

PolyPhen-2

SIFT

(prediction

(prediction

result , score)

result ,score)

BENIGN,

TOLERATED,

Class

0.005

0.61

C35

BENIGN ,

TOLERATED,

Class

0.011

0.08

C55

TOLERATED,

Class

POSSIBLY c.3913A>G

Ile1305Val

DAMAGING, 0.774

0.26

PKD1

Trp2006Cys

DAMAGING, 1.0

PT E Asp958Val

AC

CE

c.2873A>T

Cys77Phe

c.7445 G>A

NU

become shorter, 1 β sheet-layer adds, 3 hydrogen bonds reduce 1 random coil

0.002

0.84

C0

add

DAMAGING, 0.997

POSSIBLY DAMAGING, 0.71

reduce,6 hydrogen bonds reduce

2 β sheet-layers

AFFECT PROTEIN

Class

become longer, 3

FUNCTION,

C65

hydrogen bonds

0.0

add

AFFECT PROTEIN

Class

1 hydrogen bond

FUNCTION,

C65

add

0.03

DAMAGING,

1 α-helix add, 2 β

AFFECT PROTEIN

Class

FUNCTION,

C65

0.00

DAMAGING, 0.65

DAMAGING, 0.995

AFFECT PROTEIN

Class

FUNCTION,

C65

random coil add, 52 hydrogen bonds

2 β sheet-layers loss, 25 hydrogen bonds add 1 α-helix loss, 1 β

AFFECT PROTEIN

Class

sheet-layers add,

FUNCTION,

C65

12 hydrogen bonds

0.00 37

sheet-layers add, 1

add

0.00

PROBABLY Leu3353Arg

sheet-layers

4 hydrogen bonds

POSSIBLY

c.10058T>G

become longer, 2 β

Class

0.999

Arg2573Cys

C65

1 β sheet-layer

TOLERATED,

PKHD1

c.7717C>T

FUNCTION,

add

BENIGN,

PROBABLY Cys2482Tyr

Class

2 hydrogen bonds

C55

PROBABLY

c.230G>T

PROTEIN

None

0.07

DAMAGING,

D

Ile4105Leu

AFFECT

1 α-helix loss

Class

0.999

c.12313A>C

change

TOLERATED,

MA

Gly4016Ser

GVGD

0.0

PROBABLY c.12046G>A

Protein Structure

C25

SC

PROBABLY c.6018G>C

Align

PT

Gene

Pathogenicity prediction and change of protein structure of novel missense mutation

RI

Table 3

add

ACCEPTED MANUSCRIPT

PROBABLY c.11246C>T

Pro3749Leu

DAMAGING, 0.999

AFFECT PROTEIN

Class

1 β sheet-layers

FUNCTION,

C65

become shorter

AC

CE

PT E

D

MA

NU

SC

RI

PT

0.00

38