Abstract：
BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of multiple renal cysts causing kidney enlargement and end-stage renal disease (ESRD) in half the patients by 60 years of age. The aim of the study was to determine the genetic aetiology in Maltese patients clinically diagnosed with ADPKD and correlate the clinical features.
METHODS: A total of 60 patients over 18 years of age clinically diagnosed with ADPKD were studied using a customized panel of genes that had sufficient evidence of disease diagnosis using next generation sequencing (NGS). The genes studied were PKD1, PKD2, GANAB, DNAJB11, PKHD1 and DZIP1L. Selected variants were confirmed by bidirectional Sanger sequencing with specifically designed primers. Cases where no clinically significant variant was identified by the customized gene panel were then studied by Whole Exome Sequencing (WES). Microsatellite analysis was performed to determine the origin of an identified recurrent variant in the PKD2 gene. Clinical features were studied for statistical correlation with genetic results.
RESULTS: Genetic diagnosis was reached in 49 (82%) of cases studied. Pathogenic/likely pathogenic variants PKD1 and PKD2 gene were found in 25 and in 23 cases respectively. The relative proportion of genetically diagnosed PKD1:PKD2 cases was 42:38. A pathogenic variant in the GANAB gene was identified in 1 (2%) case. A potentially significant heterozygous likely pathogenic variant was identified in PKHD1 in 1 (2%) case. Potentially significant variants of uncertain significance were seen in 4 (7%) cases of the study cohort. No variants in DNAJB11 and DZIP1L were observed. Whole exome sequencing (WES) added the diagnostic yield by 10% over the gene panel analysis. Overall no clinically significant variant was detected in 6 (10%) cases of the study population by a customized gene panel and WES. One recurrent variant the PKD2 c.709+1G > A was observed in 19 (32%) cases. Microsatellite analysis showed that all variant cases shared the same haplotype indicating that their families may have originated from a common ancestor and confirmed it to be a founder variant in the Maltese population. The rate of decline in eGFR was steeper and progression to ESRD was earlier in cases with PKD1 variants when compared to cases with PKD2 variants. Cases segregating truncating variants in PKD1 showed a significantly earlier onset of ESRD and this was significantly worse in cases with frameshift variants. Overall extrarenal manifestations were commoner in cases segregating truncating variants in PKD1.
CONCLUSIONS: This study helps to show that a customized gene panel is the first-line method of choice for studying patients with ADPKD followed by WES which increased the detection of variants present in the PKD1 pseudogene region. A founder variant in the PKD2 gene was identified in our Maltese cohort with ADPKD. Phenotype of patients with ADPKD is significantly related to the genotype confirming the important role of molecular investigations in the diagnosis and prognosis of polycystic kidney disease. Moreover, the findings also highlight the variability in the clinical phenotype and indicate that other factors including epigenetic and environmental maybe be important determinants in Autosomal Dominant Polycystic Kidney Disease.
METHODS: A total of 60 patients over 18 years of age clinically diagnosed with ADPKD were studied using a customized panel of genes that had sufficient evidence of disease diagnosis using next generation sequencing (NGS). The genes studied were PKD1, PKD2, GANAB, DNAJB11, PKHD1 and DZIP1L. Selected variants were confirmed by bidirectional Sanger sequencing with specifically designed primers. Cases where no clinically significant variant was identified by the customized gene panel were then studied by Whole Exome Sequencing (WES). Microsatellite analysis was performed to determine the origin of an identified recurrent variant in the PKD2 gene. Clinical features were studied for statistical correlation with genetic results.
RESULTS: Genetic diagnosis was reached in 49 (82%) of cases studied. Pathogenic/likely pathogenic variants PKD1 and PKD2 gene were found in 25 and in 23 cases respectively. The relative proportion of genetically diagnosed PKD1:PKD2 cases was 42:38. A pathogenic variant in the GANAB gene was identified in 1 (2%) case. A potentially significant heterozygous likely pathogenic variant was identified in PKHD1 in 1 (2%) case. Potentially significant variants of uncertain significance were seen in 4 (7%) cases of the study cohort. No variants in DNAJB11 and DZIP1L were observed. Whole exome sequencing (WES) added the diagnostic yield by 10% over the gene panel analysis. Overall no clinically significant variant was detected in 6 (10%) cases of the study population by a customized gene panel and WES. One recurrent variant the PKD2 c.709+1G > A was observed in 19 (32%) cases. Microsatellite analysis showed that all variant cases shared the same haplotype indicating that their families may have originated from a common ancestor and confirmed it to be a founder variant in the Maltese population. The rate of decline in eGFR was steeper and progression to ESRD was earlier in cases with PKD1 variants when compared to cases with PKD2 variants. Cases segregating truncating variants in PKD1 showed a significantly earlier onset of ESRD and this was significantly worse in cases with frameshift variants. Overall extrarenal manifestations were commoner in cases segregating truncating variants in PKD1.
CONCLUSIONS: This study helps to show that a customized gene panel is the first-line method of choice for studying patients with ADPKD followed by WES which increased the detection of variants present in the PKD1 pseudogene region. A founder variant in the PKD2 gene was identified in our Maltese cohort with ADPKD. Phenotype of patients with ADPKD is significantly related to the genotype confirming the important role of molecular investigations in the diagnosis and prognosis of polycystic kidney disease. Moreover, the findings also highlight the variability in the clinical phenotype and indicate that other factors including epigenetic and environmental maybe be important determinants in Autosomal Dominant Polycystic Kidney Disease.
摘要：
背景：常染色体显性多囊肾病（ADPKD）的特征是在60岁的患者中，有一半的患者出现多发性肾囊肿，导致肾脏肿大和终末期肾病（ESRD）。该研究的目的是确定临床诊断为ADPKD的马耳他患者的遗传病因并关联临床特征。
方法：对60名年龄超过18岁的临床诊断为ADPKD的患者进行了研究，使用下一代测序（NGS）对一组具有疾病诊断充分证据的定制基因进行了研究。研究的基因是PKD1，PKD2，GANAB，DNAJB11、PKHD1和DZIP1L。通过使用特定设计的引物的双向Sanger测序来确认所选择的变体。然后通过全外显子组测序(WES)研究通过定制基因组鉴定没有临床显著变体的情况。进行微卫星分析以确定PKD2基因中鉴定的复发变体的起源。研究了临床特征与遗传结果的统计相关性。
结果：在49例（82%）的研究病例中达到了基因诊断。分别在25例和23例中发现了致病/可能的致病变体PKD1和PKD2基因。基因诊断PKD1:PKD2病例的相对比例为42:38。在1例（2％）中鉴定出GANAB基因的致病性变异。在1例(2%)的PKHD1中鉴定出潜在显著的杂合子可能致病变体。在研究队列的4例（7％）病例中发现了潜在的不确定意义的显着变体。在DNAJB11和DZIP1L中未观察到变体。全外显子组测序(WES)使诊断产量比基因组分析增加10%。通过定制的基因组和WES,在研究群体的6例(10%)中总体上没有检测到临床上显著的变异。在19例（32％）病例中观察到一种复发性变异PKD2c.7091G>A。微卫星分析表明，所有变异病例都具有相同的单倍型，表明它们的家族可能起源于共同的祖先，并证实它是马耳他人口的创始人变异。与PKD2变异病例相比，PKD1变异病例的eGFR下降速度更快，ESRD进展更早。在PKD1中分离截短变体的病例显示ESRD的发作明显较早，在移码变体的病例中，这种情况明显更糟。在PKD1中分离截断变体的情况下，肾外表现更为普遍。
结论：这项研究有助于表明，定制的基因面板是研究ADPKD患者和WES患者的首选方法，WES增加了对PKD1假基因区域中存在的变异的检测。在我们患有ADPKD的马耳他队列中鉴定出PKD2基因的创始人变体。ADPKD患者的表型与基因型显着相关，证实了分子研究在多囊肾病的诊断和预后中的重要作用。此外，研究结果还强调了临床表型的变异性，并表明包括表观遗传和环境在内的其他因素可能是常染色体显性多囊肾病的重要决定因素。
方法：对60名年龄超过18岁的临床诊断为ADPKD的患者进行了研究，使用下一代测序（NGS）对一组具有疾病诊断充分证据的定制基因进行了研究。研究的基因是PKD1，PKD2，GANAB，DNAJB11、PKHD1和DZIP1L。通过使用特定设计的引物的双向Sanger测序来确认所选择的变体。然后通过全外显子组测序(WES)研究通过定制基因组鉴定没有临床显著变体的情况。进行微卫星分析以确定PKD2基因中鉴定的复发变体的起源。研究了临床特征与遗传结果的统计相关性。
结果：在49例（82%）的研究病例中达到了基因诊断。分别在25例和23例中发现了致病/可能的致病变体PKD1和PKD2基因。基因诊断PKD1:PKD2病例的相对比例为42:38。在1例（2％）中鉴定出GANAB基因的致病性变异。在1例(2%)的PKHD1中鉴定出潜在显著的杂合子可能致病变体。在研究队列的4例（7％）病例中发现了潜在的不确定意义的显着变体。在DNAJB11和DZIP1L中未观察到变体。全外显子组测序(WES)使诊断产量比基因组分析增加10%。通过定制的基因组和WES,在研究群体的6例(10%)中总体上没有检测到临床上显著的变异。在19例（32％）病例中观察到一种复发性变异PKD2c.7091G>A。微卫星分析表明，所有变异病例都具有相同的单倍型，表明它们的家族可能起源于共同的祖先，并证实它是马耳他人口的创始人变异。与PKD2变异病例相比，PKD1变异病例的eGFR下降速度更快，ESRD进展更早。在PKD1中分离截短变体的病例显示ESRD的发作明显较早，在移码变体的病例中，这种情况明显更糟。在PKD1中分离截断变体的情况下，肾外表现更为普遍。
结论：这项研究有助于表明，定制的基因面板是研究ADPKD患者和WES患者的首选方法，WES增加了对PKD1假基因区域中存在的变异的检测。在我们患有ADPKD的马耳他队列中鉴定出PKD2基因的创始人变体。ADPKD患者的表型与基因型显着相关，证实了分子研究在多囊肾病的诊断和预后中的重要作用。此外，研究结果还强调了临床表型的变异性，并表明包括表观遗传和环境在内的其他因素可能是常染色体显性多囊肾病的重要决定因素。
