Novel method for the genomic analysis of PKD1 mutation in autosomal dominant polycystic kidney disease
Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease. Although next-generation sequencing (NGS) technology can be used to sequence tens of thousands of DNA molecules simultaneously. It has poor capture efficiency for the six PKD1 pseudogenes and GC-rich re...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2023-01-01
|
| Series: | Frontiers in Cell and Developmental Biology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fcell.2022.937580/full |
| _version_ | 1828068688701423616 |
|---|---|
| author | Shunlai Shang Shunlai Shang Shunlai Shang Chao Wang Chao Wang Lang Chen Wanjun Shen Yuansheng Xie Wenge Li Qinggang Li |
| author_facet | Shunlai Shang Shunlai Shang Shunlai Shang Chao Wang Chao Wang Lang Chen Wanjun Shen Yuansheng Xie Wenge Li Qinggang Li |
| author_sort | Shunlai Shang |
| collection | DOAJ |
| description | Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease. Although next-generation sequencing (NGS) technology can be used to sequence tens of thousands of DNA molecules simultaneously. It has poor capture efficiency for the six PKD1 pseudogenes and GC-rich regions. Multiplex ligation-dependent probe amplification (MLPA) technology can detect consecutive deletions of exons, but it is less sensitive for single-base mutations. However, pathogenic genes might not be detected in some patients, even when using the above methods. Improving the detection rate of pathogenic genes is an important technical problem hindering clinical diagnosis of ADPKD. Four pedigrees of ADPKD patients with mutation sites not identified by NGS were examined by other methods. First, MLPA was performed. Then, pedigrees in which MLPA did not identify pathogenic genes were subjected to multiplex polymerase chain reaction (MPCR) and targeted region sequencing. Finally, the detected mutation sites were verified by Sanger sequencing. The results showed that MLPA detected the following PKD1 exonic deletion mutations in three pedigrees: PKD1-18 nt–290 nt, PKD1-up-257 nt, PKD1-up-444 nt and PKD1-3 nt–141 nt. A new mutation site was identified through targeted region sequencing in one pedigree: PKD1 NM_001009944: c.151T > C at the protein level, described as p. Cys51Arg. In summary, we established a system of genetic detection and analytical methods, from NGS to MLPA to targeted region sequencing and finally to Sanger sequencing. We combined MPCR and targeted region sequencing for the first time in ADPKD diagnosis, which further improved diagnosis accuracy. Moreover, we identified one new missense mutation and four new deletion mutations. |
| first_indexed | 2024-04-11T00:07:34Z |
| format | Article |
| id | doaj.art-488afc8334894b12b56242724d083551 |
| institution | Directory Open Access Journal |
| issn | 2296-634X |
| language | English |
| last_indexed | 2024-04-11T00:07:34Z |
| publishDate | 2023-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cell and Developmental Biology |
| spelling | doaj.art-488afc8334894b12b56242724d0835512023-01-09T10:29:55ZengFrontiers Media S.A.Frontiers in Cell and Developmental Biology2296-634X2023-01-011010.3389/fcell.2022.937580937580Novel method for the genomic analysis of PKD1 mutation in autosomal dominant polycystic kidney diseaseShunlai Shang0Shunlai Shang1Shunlai Shang2Chao Wang3Chao Wang4Lang Chen5Wanjun Shen6Yuansheng Xie7Wenge Li8Qinggang Li9Department of Nephrology, China-Japan Friendship Hospital, Beijing, ChinaDepartment of Nephrology, Chinese PLA General Hospital, Medical School of Chinese PLA, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing, ChinaSchool of Medicine, Nankai University, Tianjin, ChinaDepartment of Nephrology, Chinese PLA General Hospital, Medical School of Chinese PLA, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing, ChinaClinical Medical School, Guangdong Pharmaceutical University, Guangzhou, ChinaDepartment of Nephrology, Chinese PLA General Hospital, Medical School of Chinese PLA, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing, ChinaDepartment of Nephrology, Chinese PLA General Hospital, Medical School of Chinese PLA, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing, ChinaDepartment of Nephrology, Chinese PLA General Hospital, Medical School of Chinese PLA, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing, ChinaDepartment of Nephrology, China-Japan Friendship Hospital, Beijing, ChinaDepartment of Nephrology, Chinese PLA General Hospital, Medical School of Chinese PLA, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing, ChinaAutosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease. Although next-generation sequencing (NGS) technology can be used to sequence tens of thousands of DNA molecules simultaneously. It has poor capture efficiency for the six PKD1 pseudogenes and GC-rich regions. Multiplex ligation-dependent probe amplification (MLPA) technology can detect consecutive deletions of exons, but it is less sensitive for single-base mutations. However, pathogenic genes might not be detected in some patients, even when using the above methods. Improving the detection rate of pathogenic genes is an important technical problem hindering clinical diagnosis of ADPKD. Four pedigrees of ADPKD patients with mutation sites not identified by NGS were examined by other methods. First, MLPA was performed. Then, pedigrees in which MLPA did not identify pathogenic genes were subjected to multiplex polymerase chain reaction (MPCR) and targeted region sequencing. Finally, the detected mutation sites were verified by Sanger sequencing. The results showed that MLPA detected the following PKD1 exonic deletion mutations in three pedigrees: PKD1-18 nt–290 nt, PKD1-up-257 nt, PKD1-up-444 nt and PKD1-3 nt–141 nt. A new mutation site was identified through targeted region sequencing in one pedigree: PKD1 NM_001009944: c.151T > C at the protein level, described as p. Cys51Arg. In summary, we established a system of genetic detection and analytical methods, from NGS to MLPA to targeted region sequencing and finally to Sanger sequencing. We combined MPCR and targeted region sequencing for the first time in ADPKD diagnosis, which further improved diagnosis accuracy. Moreover, we identified one new missense mutation and four new deletion mutations.https://www.frontiersin.org/articles/10.3389/fcell.2022.937580/fullADPKDPKD1mPCRMLPA (multiplex ligation-dependent probe amplification)targeted region sequencing |
| spellingShingle | Shunlai Shang Shunlai Shang Shunlai Shang Chao Wang Chao Wang Lang Chen Wanjun Shen Yuansheng Xie Wenge Li Qinggang Li Novel method for the genomic analysis of PKD1 mutation in autosomal dominant polycystic kidney disease Frontiers in Cell and Developmental Biology ADPKD PKD1 mPCR MLPA (multiplex ligation-dependent probe amplification) targeted region sequencing |
| title | Novel method for the genomic analysis of PKD1 mutation in autosomal dominant polycystic kidney disease |
| title_full | Novel method for the genomic analysis of PKD1 mutation in autosomal dominant polycystic kidney disease |
| title_fullStr | Novel method for the genomic analysis of PKD1 mutation in autosomal dominant polycystic kidney disease |
| title_full_unstemmed | Novel method for the genomic analysis of PKD1 mutation in autosomal dominant polycystic kidney disease |
| title_short | Novel method for the genomic analysis of PKD1 mutation in autosomal dominant polycystic kidney disease |
| title_sort | novel method for the genomic analysis of pkd1 mutation in autosomal dominant polycystic kidney disease |
| topic | ADPKD PKD1 mPCR MLPA (multiplex ligation-dependent probe amplification) targeted region sequencing |
| url | https://www.frontiersin.org/articles/10.3389/fcell.2022.937580/full |
| work_keys_str_mv | AT shunlaishang novelmethodforthegenomicanalysisofpkd1mutationinautosomaldominantpolycystickidneydisease AT shunlaishang novelmethodforthegenomicanalysisofpkd1mutationinautosomaldominantpolycystickidneydisease AT shunlaishang novelmethodforthegenomicanalysisofpkd1mutationinautosomaldominantpolycystickidneydisease AT chaowang novelmethodforthegenomicanalysisofpkd1mutationinautosomaldominantpolycystickidneydisease AT chaowang novelmethodforthegenomicanalysisofpkd1mutationinautosomaldominantpolycystickidneydisease AT langchen novelmethodforthegenomicanalysisofpkd1mutationinautosomaldominantpolycystickidneydisease AT wanjunshen novelmethodforthegenomicanalysisofpkd1mutationinautosomaldominantpolycystickidneydisease AT yuanshengxie novelmethodforthegenomicanalysisofpkd1mutationinautosomaldominantpolycystickidneydisease AT wengeli novelmethodforthegenomicanalysisofpkd1mutationinautosomaldominantpolycystickidneydisease AT qinggangli novelmethodforthegenomicanalysisofpkd1mutationinautosomaldominantpolycystickidneydisease |