Modification of PCR Conditions and Design of Exon-Specific Primers for the Efficient Molecular Diagnosis of PKD1 Mutations

Liu, Weiqiang; Chen, Min; Wei, Jun; He, Wenyin; Li, Zhihua; Sun, Xiaofang; Shi, Yongyu

doi:10.1159/000368464

Cited by 5 publications

(2 citation statements)

References 36 publications

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“…Subsequently, the long-range PCR ampli cation product was used as a template for the second round of PCR ampli cation. Primer sequences and PCR ampli cation system was followed as mentioned in a previous study [17]. Sanger sequencing analysis of the PKD1 gene ampli cation product was performed on an ABI 3730 machine.…”

Section: Pkd1 Sequencing With Gdnamentioning

confidence: 99%

Single-cell Sequencing-based Pre-implantation Genetic Testing-M (PGT-M) of the Heterozygous Mutations of PKD1 Gene

Miao

et al. 2020

Preprint

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Objectives: Polycystic kidney disease (PKD) is a common autosomal monogenic genetic disease. PKD1 mutation accounts for about 85% of ADPKD patients. Pre-implantation genetic testing-M (PGT-M for monogenic) is an important approach to prevent the transmission of genetic diseases from parents to the offspring. Design: In this study, We have identified the family linkage and mutation site in embryos with NGS-based SNP phasing and Sanger Sequencing.Methods: Multiple Annealing and Looping Based Amplification Cycles (MALBAC) method was employed to amplify the whole genome of trophoblast cells. Copy Number Variant (CNV), and single nucleotide polymorphism (SNP) were used to assess the embryo state. Results: In the eight embryos, Embryo 02 and Embryo 04 were removed from further analysis because of the Multiple chromosomes abnormal (2 of 8, 25%). Embryo 05, Embryo 06, Embryo 07, and Embryo 08 were judged as 46,XN,-15q(q23→qter,~31M,×1,mos*), 45,XN,-16(×1), 47,XN,+2(×3),-7p(pter→p14.3,~35M,×1,mos*), and 46,XN, +16(×3,mos*),-20p(pter→p11.23,~23M,×1,mos*),+22(×3,mos*), respectively (4 of 8, 50%). Meanwhile, Embryo 01 and Embryo 03 were judged as 46, XN (2 of 8, 25%). The results of SNP phasing and Sanger Sequencing suggested that Embryo 01 and Embryo 05 had none of PKD1 gene mutation. Limitations: Up to now, PGT-M is complicated and expensive. Meanwhile, PGT-M obtains the final diagnosis through invasive manipulation of embryos, which may bring adverse effects on offspringConclusion: NGS-based single-cell sequencing combined with CNV, Sanger Sequencing, and SNP phasing is a reliable testing system for PGT-M application. This work presented here would provide a detailed understanding of the NGS-based single-cell sequencing application in ADPKD.

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Section: Pkd1 Sequencing With Gdnamentioning

confidence: 99%

Single-cell Sequencing-based Pre-implantation Genetic Testing-M (PGT-M) of the Heterozygous Mutations of PKD1 Gene

Miao

et al. 2020

Preprint

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“…Relevant diagnostic studies about ADPKD are summarized in table 1[7,9,10,11,12,17,18,19,20,21,22,23,24,25,26]. Our center started to detect PKD gene mutations and set up the screening system in the late 1990s.…”

Section: Molecular Diagnosismentioning

confidence: 99%

The Clinical Manifestation and Management of Autosomal Dominant Polycystic Kidney Disease in China

Xue

Zhou

et al. 2016

Kidney Dis

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Background: Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic hereditary kidney disease characterized by progressive enlargement of renal cysts. The incidence is 1-2‰ worldwide. Mutations in two genes (PKD1 and PKD2) cause ADPKD. Currently, there is no pharmaceutical treatment available for ADPKD patients in China. Summary: This review focused on advances in clinical manifestation, gene diagnosis, risk factors, and management of ADPKD in China. There is an age-dependent increase in total kidney volume (TKV) and decrease in renal function in Chinese ADPKD patients. ADPKD is more severe in males than in females. Great progress has been made in molecular diagnosis in the last two decades. Nephrologists found many novel PKD mutations in Chinese ADPKD patients early through polymerase chain reaction, and then through liquid chromatography in 2000s, and recently through next-generation sequencing. Major predictive factors for ADPKD progression are age, PKD genotype, sex, estimated glomerular filtration rate (eGFR), and TKV. With respect to the management of ADPKD, inhibitors targeting mTOR and cAMP are the focus of clinical trials. Triptolide has been used to treat ADPKD patients in clinical trials in China. Triptolide significantly protected eGFR of ADPKD patients compared with placebo. Key Messages: ADPKD affects about 1.5 million people in China. An additional PKD gene besides PKD1 and PKD2 was not found in the Chinese. The prevalence of intracranial aneurysm in Chinese ADPKD patients was 12.4%. The predictive factors for eGFR decrease in Chinese ADPKD patients are TKV, proteinuria, history of hypertension, and age. The treatment strategies in clinical trials for ADPKD patients in China are similar to those in the West except for triptolide. Facts from East and West: (1) ADPKD is diagnosed globally by ultrasound detection of kidney enlargement and presence of cysts. Recent analyses of variants of the PKD1 and PKD2 genes by next-generation sequencing in Chinese and Western ADPKD patients might lead to the development of reliable genetic tests. (2) Besides lifestyle changes (low-salt diet, sufficient fluid intake, and no smoking), blood pressure control is the primary nonspecific treatment recommended by Kidney Disease - Improving Global Outcomes (KDIGO) for ADPKD patients. How low the blood pressure target should be and what the means of achieving it are remain open questions depending on the severity of chronic kidney disease and the age of the patients. In a recent Chinese study, diagnostic needle aspiration and laparoscopic unroofing surgery successfully improved infection, pain, and hypertension. Peritoneal dialysis was found to be a feasible treatment for most Chinese ADPKD patients with end-stage renal disease. In most Western centers, patients without contraindication are selected for peritoneal dialysis. Kidney transplantation with concurrent bilateral nephrectomy was successful in relieving hypertension and infection in Chinese ADPKD patients. In Western countries, sequential ...

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Identifying gene mutations of Chinese patients with polycystic kidney disease through targeted next‐generation sequencing technology

Wang

Shang

et al. 2019

Molec Gen & Gen Med

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Background Polycystic kidney disease (PKD) is the most common hereditary kidney disease. The main mutational genes causing autosomal dominant polycystic kidney disease (ADPKD) are PKD1 and PKD2 as well as some rare pathogenic genes. Unilateral PKD is rare in clinics, and its association with gene mutations is unclear. Methods Targeted next‐generation sequencing (NGS) was performed to detect the renal ciliopathy‐associated genes (targeted NGS panel including 63 genes) in PKD patients. Results Forty‐eight PKD1 and PKD2 mutation sites were detected in 44 bilateral PKD patients, of which 48 were PKD1 mutation sites (87.5%) and six were PKD2 mutation sites (12.5%). All of which exhibited typical ADPKD. Furthermore, we detected HNF1B heterozygous mutations in three families. Although these three patients showed HNF1B heterozygous mutations, their clinical characteristics differed and showed phenotypic heterogeneity. Conclusions Targeted NGS panel was helpful in detecting typical ADPKD patients and even in non‐typical PKD patients. Macromutation in HNF1B may lead to bilateral PKD. The 16 novel PKD gene mutation sites and two novel PKD2 gene mutation sites discovered in this study have some significance in genetic counseling for ADPKD patients, and increase the number of studied families and expand the mutation database of ADPKD.

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Modification of PCR Conditions and Design of Exon-Specific Primers for the Efficient Molecular Diagnosis of PKD1 Mutations

Cited by 5 publications

References 36 publications

Single-cell Sequencing-based Pre-implantation Genetic Testing-M (PGT-M) of the Heterozygous Mutations of PKD1 Gene

Single-cell Sequencing-based Pre-implantation Genetic Testing-M (PGT-M) of the Heterozygous Mutations of PKD1 Gene

The Clinical Manifestation and Management of Autosomal Dominant Polycystic Kidney Disease in China

Identifying gene mutations of Chinese patients with polycystic kidney disease through targeted next‐generation sequencing technology

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