Mutation variability at polycystic kidney disease detected by NGS

Поликистозная болезнь почек (ПП) является клинически и генетически гетерогенной группой заболеваний, может наследоваться как аутосомно-доминантно (АД), так и аутосомно-рецессивно (АР). К развитию АР ПП приводят мутации в гене PKHD1. Большинство мутаций при АД ПП находят в гене PKD1 (80-85%). Примерно в 15% случаев мутации выявляют в гене PKD2. Клиническое и генетическое разнообразие ПП требует поиска мутаций в нескольких генах, поэтому он является трудоемким, дорогостоящим и требует много времени. Метод массового параллельного секвенирования (МПС) позволяет проводить поиск мутаций в нескольких генах одновременно независимо от их размера. Проведен поиск мутаций в 254 семьях с ПП методом МПС с использованием панели, включающей гены PKHD1, PKD1, PKD2, HNF1B и GANAB. Два варианта в гене PKHD1 было идентифицировано в 49 семьях (19%), один вариант найден в 9 случаях (3,5%); в гене PKD1 обнаружено 62 варианта (24,5%), в гене PKD2 - 6 вариантов (2,5%), в гене HNF1B - 9 вариантов (3,5%). В 119 семьях, что составило 47%, мутации найдены не были. У больных из семей с генеалогически установленным АД типом наследования в большинстве случаев (39 из 66; 59%) выявлены варианты в гене PKD1, приводящие к ПП. Из 59 изолированных случаев ПП в 17% (10 человек) идентифицированы 2 варианта в гене PНKD1, в 20% (12 человек) - в гене PKD1. При неизвестном типе наследования (129 случаев) в 29,5% (38 чел.) найдены 2 варианта в гене PНKD1, в 8,5% (11 чел.) - в гене PKD1, в 3% (4 чел.) - в гене PKD2, в 4% (5 чел.) - в гене HNF1B. Таким образом, МПС относительно быстро позволяет проводить молекулярно-генетический анализ одновременно в нескольких генах у больных с признаками ПП. Polycystic kidney disease is a heterogeneous group of autosomal dominant or autosomal recessive disorders with age of manifestation varying from prenatal period to adulthood. Autosomal recessive polycystic kidney disease is caused by mutations in the PKHD1 gene. Approximately 85% of all autosomal dominant polycystic kidney disease cases are caused by mutations in the PKD1 gene, and around 15% - by mutations in the PKD2 gene. All these genes are large, and mutations were found to be scattered throughout the genes without any clustering. Therefore, mutation detection requires a lot of time, money, and effort. Due to clinical and genetic diversity of polycystic kidney disease, the search for mutations has to be carried out in several genes. Mass parallel sequencing (MPS) allows to analyze several genes simultaneously regardless of their size. 254 families with polycystic kidney disease were examined using mass parallel sequencing with a gene panel that included PKHD1, PKD1, PKD2, HNF1B and GANAB. Two variants in PKHD1 were found in 49 families (19%), one variant - in 9 families (3.5%); in PKD1 62 variants were detected (24.5%), in PKD2 - 6 variants (2.5%), in HNF1B - 9 variants (3.5%). In 119 families (47%) there were no mutations in the target genes. Among 66 patients from families with autosomal dominant polycystic kidney disease, 39 patients (59%) had mutations in the PKD1 gene. Out of 59 sporadic cases, 10 patients (17%) had 2 variants in PНKD1, 12 patients (20%) - in PKD1. 38 patients (29.5%) out of 129 patients with unknown type of inheritance had 2 variants in PНKD1, 11 patients (8.5%) - in PKD1, 4 patients (3%) - in PKD2, 5 patients (4%) - in HNF1B. Mass parallel sequencing allows to carry out relatively rapid molecular genetic analysis of several genes simultaneously for patients with symptoms of polycystic kidney disease.

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

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.

Prevalence of PKD1 gene mutation in cats in Turkey and pathogenesis of feline polycystic kidney disease

Polycystic kidney disease (PKD) is one of the most common hereditary diseases in cats, with high prevalence in Persian and Persian-related cats. PKD is caused mainly by an inherited autosomal dominant (AD) mutation, and animals may be asymptomatic for years. We screened 16 cats from various breeds exhibiting a renal abnormality by ultrasound examination and genotyped them for the c.10063C>A transversion on exon 29 of the polycystin-1 ( PKD1) gene, by PCR–restriction fragment length polymorphism (PCR-RFLP). Among these cats, a Siamese nuclear family of 4 cats with ancestral hereditary renal failure were screened by whole-genome sequencing (WGS) to determine novel variations in genes associated with both AD and autosomal recessive PKD in humans. During the study period, one cat died as a result of renal failure and was forwarded for autopsy. Additionally, we screened 294 cats asymptomatic for renal disease (Angora, Van, Persian, Siamese, Scottish Fold, Exotic Shorthair, British Shorthair, and mixed breeds) to determine the prevalence of the mutation in cats in Turkey. Ten of the symptomatic and 2 of the asymptomatic cats carried the heterozygous C → A transversion, indicating a prevalence of 62.5% and 0.68%, respectively. In the WGS analysis of 4 cats in the Siamese nuclear family, novel variations were determined in the fibrocystin gene ( PKHD1), which was not compatible with dominant inheritance of PKD.

Identification of missense and synonymous variants in Iranian patients suffering from autosomal dominant polycystic kidney disease

Background: Autosomal dominant polycystic kidney disease (ADPKD), the predominant type of inherited kidney disorder, occurs due to PKD1 and PKD2 gene mutations. ADPKD diagnosis is made primarily by kidney imaging. However, molecular genetic analysis is required to confirm the diagnosis. It is critical to perform a molecular genetic analysis when the imaging 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 frequency of PKD1 gene mutations in Iranian patients with ADPKD diagnosis. Methods: Genomic DNA was extracted from blood samples from 22 ADPKD patients, who were referred to the Qaem Hospital in Mashhad, Iran. By using appropriate 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 DNA sequencing in the patients has shown that exons 35, 36 and 37 were non- polymorphic, and that most mutations had occurred in exons 44 and 45. In two patients, an exon-intron boundary mutation had occurred in intron 44. Most of the variants were missense and non-synonymous types. Conclusion: In the present study, we have shown the occurrence of nine novel missense or synonymous variants in PKD1 gene. These data could contribute to an improved diagnostic and genetic counseling in clinical settings.

Prevalence of autosomal dominant polycystic kidney disease in Persian and Persian-related cats in Brazil

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disease in cats. However, scarce data on its prevalence are available in Brazil. Persian cats and Persian-related breeds were assessed by molecular genotyping for a C to A transversion in exon 29 of PKD1 gene to determine ADPKD prevalence in a Brazilian population. Genomic DNA extracted from peripheral whole blood or oral swabs samples was used to amplify exon 29 of PKD1 gene employing a PCR-RFLP methodology. From a total of 616 animals, 27/537 Persian and 1/17 Himalayan cats showed the single-nucleotide variant (C to A) at position 3284 in exon 29 of feline PKD1. This pathogenic variation has been identified only in heterozygous state. The prevalence of ADPKD in Persian cats and Persian-related breeds was 5.03% and 1.6%, respectively. There was no significant association between feline breed, gender or age with ADPKD prevalence. Of note, the observed ADPKD prevalence in Persian cats and Persian-related breeds in Brazil was lower than the ones reported in other parts of the world. This finding may be related to genetic counseling and consequent selection of ADPKD-free cats for reproduction.

Identification of a mutation in the polycystin 1 gene in a patient with intracranial aneurysm

Интракраниальная аневризма - заболевание соединительной ткани многофакторной природы, приводящее к спонтанным субарахноидальным кровоизлияниям. Обнаружено ранее неописанное изменение нуклеотидной последовательности гена PKD1 в гетерозиготном состоянии c.6847G>A с патогенной значимостью у пациентки с субарахноидальным кровоизлиянием. Intracranial aneurysm is a multifactorial connective tissue disease leading to spontaneous subarachnoid hemorrhages. It was identified undescribed heterozygous nucleotide sequence change of the PKD1 gene c.6847G>A in patient with subarachnoid hemorrhage with pathogenic significance.