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PubMed2026

Unraveling the genomic blueprint of the Indian black soldier fly: From genome assembly to evolutionary insights.

The black soldier fly (BSF) (Hermetia illucens) has been renowned for its sustainable bioconversion capabilities, resulting in smart protein production with wide applications in animal feed, bioenergy, and biofertilizer. However, the genetic mechanisms underlying efficient bioconversion and productivity remain poorly understood. To advance strain-specific applications and strengthen genetic resource availability, we present the whole genome sequencing (WGS) data for an Indian isolate of black soldier fly. The assembled genome was 1.46 Gb with a scaffold N50 of 172.7 Mb, and a GC content of 42.6%. Furthermore, 64.17% of genomic sequences were masked as repeated, and 14,317 protein-coding sequences were identified. Variant analysis against the reference genome identified 34.44 million variants (∼33.25 million SNPs and ∼ 1.18 million INDELs), with the majority (99.3%) classified as MODIFIER, 0.54% as LOW impact, 0.14% as MODERATE, and only 0.003% as HIGH impact. Comparative genomic analysis with other related species revealed expansions of gene families in BSF associated with Immune effector (Antimicrobial peptides (AMPs), Lysozymes, and Peptidoglycan Recognition Protein (PGRP) and Detoxification (cytochrome P450 enzymes). Notably, AMPs in the Indian isolate showed enhanced copy number variation in defensin (27) and PGRP (40) compared to reference BSF, suggesting potential regional adaptations to pathogen exposure. Collectively, this genomic data provides an improved resource for evolutionary studies, functional genomics, and targeted genetic improvement of BSF for sustainable bioconversion applications.

Comparative genomics↗

PubMed2000

Synergistic effect of alpha-adducin and ACE genes causes blood pressure changes with body sodium and volume expansion.

BACKGROUND: The genetic dissection of a polygenic, multifactorial, quantitative disease such as arterial hypertension is hampered by a large environmental variance and by genetic heterogeneity. METHODS: To reduce the environmental variance, we measured the pressor response to a saline load (PRSL) and the basal plasma renin activity (PRA) under very controlled conditions in 145 essential hypertensive patients, as they may have the most direct clinical expression of the putative genetic alteration in renal Na handling and blood pressure (BP) regulation caused by the alpha-adducin and angiotensin-converting enzyme (ACE) polymorphism. RESULTS: PRSL was smaller in patients homozygous for the wild-type (Gly460) variant of alpha-adducin compared with that of patients bearing at least one copy of the 460Trp variant (2.5 +/- 0.6 vs. 7.0 +/- 0.9 mm Hg, P = 0.0001), whereas the ACE genotype was not associated with differences in PRSL. Both alpha-adducin and ACE affect PRA, with lower values correlated with the number of 460Trp or D alleles (P = 0.019 and 0.017, respectively). Most important, alpha-adducin and ACE interact epistatically in determining the PRSL, doubling the variance explained when epistasis is taken into account (variance from 7.7 to 15.5%). CONCLUSION: These findings support the involvement of ACE and alpha-adducin in PRSL and PRA control, which are of paramount importance in setting the BP level and its response to therapy.

Adult↗

PubMed1999

Differences between hepatitis C virus 5' untranslated region quasispecies in serum and liver.

It is unclear whether the sequence populations of hepatitis C virus (HCV) quasispecies in the liver and in serum are different, as a variety of studies on this subject provide conflicting results. In the current study, the populations of HCV 5' untranslated region (5' UTR) sequences in paired serum and liver samples from six patients with chronic hepatitis were analysed. Liver-derived, negative-strand viral RNA was amplified with a highly strand-specific Tth-based assay, and extensive measures, including accounting for template copy number, were undertaken to lower the risk of sporadic artefactual polymorphism. Amplified sequences were compared by single-strand conformation polymorphism analysis and by direct sequencing of identified differences. In four patients, liver samples were found to contain variants within the quasispecies which were not found in serum or negative-strand viral RNA, while in the remaining two patients, low virus titre prevented a reliable quasispecies analysis. These results suggest the presence in the same individual of HCV variants differing in the 5' UTR and possibly replicating with different kinetics.

5' Untranslated Regions↗

PubMed2003

Large-scale variation among human and great ape genomes determined by array comparative genomic hybridization.

Large-scale genomic rearrangements are a major force of evolutionary change and the ascertainment of such events between the human and great ape genomes is fundamental to a complete understanding of the genetic history and evolution of our species. Here, we present the results of an evolutionary analysis utilizing array comparative genomic hybridization (array CGH), measuring copy-number gains and losses among these species. Using an array of 2460 human bacterial artificial chromosomes (BACs) (12% of the genome), we identified a total of 63 sites of putative DNA copy-number variation between humans and the great apes (chimpanzee, bonobo, gorilla, and orangutan). Detailed molecular characterization of a subset of these sites confirmed rearrangements ranging from 40 to at least 175 kb in size. Surprisingly, the majority of variant sites differentiating great ape and human genomes were found within interstitial euchromatin. These data suggest that such large-scale events are not restricted solely to subtelomeric or pericentromeric regions, but also occur within genic regions. In addition, 5/9 of the verified variant sites localized to areas of intrachromosomal segmental duplication within the human genome. On the basis of the frequency of duplication in humans, this represents a 14-fold positional bias. In contrast to previous cytogenetic and comparative mapping studies, these results indicate extensive local repatterning of hominoid chromosomes in euchromatic regions through a duplication-driven mechanism of genome evolution.

Animals↗

PubMed1996

Molecular and evolutionary analyses of a variable series of genes in Borrelia burgdorferi that are related to ospE and ospF, constitute a gene family, and share a common upstream homology box.

In this study we report on the molecular characterization of a series of genes that constitute a gene family related to ospE and ospF. Some members of this family appear to represent recombined or variant forms of ospE and ospF. Variant ospE and ospF genes were found in several Borrelia burgdorferi isolates, demonstrating that their occurrence is not a phenomenon relevant to only a single isolate. Hybridization analyses revealed that the upstream sequence originally identified 5' of the full-length ospEF operon exists in multiple copies ranging in number from two to six depending on the isolate. This repeated sequence, which we refer to as the upstream homology box (UHB), carries a putative promoter element. In some isolates, UHB elements were found to flank copies of ospE and ospF that exist independently of each other. We refer to this group of UHB-flanked genes collectively as the UHB gene family. The evolutionary relationships among UHB gene family members were assessed through DNA sequence analysis and gene tree construction. These analyses suggest that some UHB-flanked genes might actually represent divergent forms of other previously described genes. Analysis of the restriction fragment length polymorphism patterns of the UHB-flanked genes among B. burgdorferi isolates demonstrated that these patterns are highly variable among isolates, suggesting that these genes are not phylogenetically conserved. The variable restriction fragment length polymorphism patterns could indicate recombinational activity in these sequences. The presence of numerous copies of the UHB elements and the high degree of homology among UHB-flanked genes could provide the necessary elements to allow for homologous recombination, leading to the generation of recombination variants of UHB gene family members.

Amino Acid Sequence↗

PubMed1997

Tissue-specific selection for different mtDNA genotypes in heteroplasmic mice.

Mammalian mitochondrial DNA (mtDNA) is a highly polymorphic, high-copy-number genome that is maternally inherited. New mutations in mtDNA segregate rapidly in the female germline due to a genetic bottleneck in early oogenesis and as a result most individuals are homoplasmic for a single species of mtDNA. Sequence variants thus accumulate along maternal lineages without genetic recombination. Most of the extant variation in mtDNA in mammalian populations has been assumed to be neutral with respect to selection; however, comparisons of the ratio of replacement to silent nucleotide substitutions between species suggest that the evolution of mammalian mtDNA is not strictly neutral. To test directly whether polymorphic mtDNAs behave as neutral variants, we examined the segregation of two different mtDNA genotypes in the tissues of heteroplasmic mice. We find evidence for random genetic drift in some tissues, but in others strong, tissue-specific and age-related, directional selection for different mtDNA genotypes in the same animal. These surprising data suggest that the coding sequence of mtDNA may represent a compromise between the competing demands of different tissues and point to the existence of unknown, tissue-specific nuclear genes important in the interaction between the nuclear and mitochondrial genomes.

Age Factors↗

PubMed2006

CYP17, SRD5A2, CYP1B1, and CYP2D6 gene polymorphisms with prostate cancer risk in North Indian population.

To investigate the involvement of the CYP17, SRD5A2, CYP1B1, and CYP2D6 variants with prostate cancer, a case-control study of 100 patients and an equal number of age-matched control men was conducted. There appears to be a nonsignificant increase with risk of prostate cancer for individuals carrying one copy of the CYP17 A2 allele (OR, 1.80; 95% CI, 0.99-3.29, P=0.05). The risk was increased in individuals having two A2 alleles (OR; 2.81, 95% CI, 1.06-7.40, P=0.03). Compared with men having the VV genotype of SRD5A2 gene, there was no significant association between the VL genotype and the risk of prostate cancer (OR; 0.54, 95% CI; 0.29-1.03, P=0.06). There was no difference in the occurrence of the genotype LL between controls and prostate cancer patients (OR; 0.90, 95% CI; 0.43-1.89, P=0.79). There was a nonsignificant increased risk of prostate cancer for individuals carrying the CYP1B1Leu/Val genotype (OR, 1.70, 95% CI, 0.91-3.17, P =0.09), which was increased in those having the Val/Val allele (OR, 3.38; 95% CI, 1.13-10.07, P=0.02). Relative to men homozygous for the wild-type allele in CYP2D6 gene, those heterozygous for the B allele had an odds ratio of 1.78 (95% CI, 0.76-4.17, P=0.18) for patients, and for homozygous individuals, it was 1.95 (0.55-6.93, P=0.30). These observations have suggested that the CYP17 A2/A2, CYP1B1 Val/Val, and CYP2D6 genotypes may be associated with an altered risk of prostate cancer, while the CYP2D6 and SRD5A2 V89L polymorphism have no association with its risk in the North Indian population.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1999

Optimization of plasmid maintenance in the attenuated live vector vaccine strain Salmonella typhi CVD 908-htrA.

The broad objective of the research presented here is to develop a noncatalytic plasmid maintenance system for the stabilization of multicopy expression plasmids encoding foreign antigens in a Salmonella typhi live-vector vaccine strain such as CVD 908-htrA. We have enhanced the maintenance of expression plasmids at two independent levels. First, we removed dependence upon balanced-lethal maintenance systems that involve catalytic enzymes expressed from multicopy plasmids; we accomplished this through incorporation into expression plasmids of a postsegregational killing system based on the noncatalytic hok-sok plasmid addiction system from the antibiotic resistance factor pR1. We also included at least one naturally occurring plasmid partition function in our expression plasmids, which eliminates random segregation of these plasmids, thereby enhancing their inheritance and stability; to accomplish this, we incorporated either the par locus from pSC101, the parA locus from pR1, or both. We monitored the stability of optimized expression plasmids within CVD 908-htrA by quantitating expression of a variant of green fluorescent protein (GFPuv) by using flow cytometry. In this report, we demonstrate the utility of this novel plasmid maintenance system in enhancing the stability of our expression plasmids and go on to show that as the copy number of stabilized plasmids increases, the toxicity of GFPuv synthesis also increases. The implications of these observations for the rational design of immunogenic and protective bacterial live vector vaccines are discussed.

Antigens, Bacterial↗

PubMed1990

Closely related DNA sequences specify distinct patterns of developmental expression in Drosophila melanogaster.

Three short synthetic DNA sequences, which are closely related to one another, confer three distinct patterns of developmental expression on the heat shock hsp70 gene in transgenic Drosophila melanogaster lines. These results show that small variations or even single base pair changes in a repeated element of a regulatory sequence can create promoters that display new specificities of tissue and developmental regulation. Interestingly, the three patterns of developmental expression conferred by the synthetic DNAs resemble in part those of the known developmental genes: glucose dehydrogenase (Gld), Dopa decarboxylase (Ddc), and salivary gland secretory proteins (Sgs), respectively. In each case, the defined regulatory region of the known developmental gene contains multiple sequences that are similar or identical to the synthetic sequence that confers a similar pattern of developmental expression on the hsp70 gene. Thus, these results are congruent with the view that short sequence elements in multiple copies can confer either simple or relatively complex patterns of developmental expression on a receptive promoter like that of hsp70. Furthermore, the fact that the three variants tested produced three distinct patterns of expression in transgenic animals suggests that the number of different elements is large.

Animals↗

PubMed1988

Suppression of chromosomal mutations affecting M1 virus replication in Saccharomyces cerevisiae by a variant of a viral RNA segment (L-A) that encodes coat protein.

For the maintenance of "killer" M1 double-stranded RNA in Saccharomyces cerevisiae, more than 30 chromosomal genes are required. The requirement for some of these genes can be completely suppressed by a cytoplasmic element, [B] (for bypass). We have isolated a mutant unable to maintain [B] (mab) and found that it is allelic to MAK10, one of the three chromosomal MAK genes required for the maintenance of L-A. The heat curing of [B] always coincided with the loss of L-A. To confirm that [B] is located on L-A, we purified viral particles containing either L-A or M1 from strains with or without [B] activity and transfected these purified particles into a strain which did not have either L-A or M1. The transfectants harboring L-A and M1 from a [B] strain showed the [B] phenotype, but the transfectants with L-A and M1 from a [B-o] strain did not show the [B] phenotype. Furthermore, the transfectants having L-A from a [B] strain and M1 from a [B-o] strain also showed the [B] phenotype. Therefore, we concluded that [B] is a property of a variant of L-A. In the transfection experiment, we also proved that the superkiller phenotype of the [B] strain is a property of L-A and that L-A with [B] activity can maintain a higher copy number of M1 regardless of the source of M1 viruslike particles. These data suggest that MAK genes whose mutations are suppressed by [B] are concerned with the protection of M1 (+) single-stranded RNA or the formation of M1 viruslike particles and that an L-A with more efficient production of M1 viruslike particles can completely dispense with the requirement for those MAK genes.

Chromosomes↗

PubMed1989

Chromosome heteromorphism quantified by high-resolution bivariate flow karyotyping.

Maternal and paternal homologues of many chromosome types can be differentiated on the basis of their peak position in Hoechst 33258 versus chromomycin A3 bivariate flow karyotypes. We demonstrate here the magnitude of DNA content differences among normal chromosomes of the same type. Significant peak-position differences between homologues were observed for an average of four chromosome types in each of the karyotypes of 98 different individuals. The frequency of individuals with differences in homologue peak positions varied among chromosome types: e.g., chromosome 15, 61%; chromosome 3, 4%. Flow karyotypes of 33 unrelated individuals were compared to determine the range of peak position among normal chromosomes. Chromosomes Y, 21, 22, 15, 16, 13, 14, and 19 were most heteromorphic, and chromosomes 2-8 and X were least heteromorphic. The largest chromosome 21 was 45% larger than the smallest 21 chromosome observed. The base composition of the variable regions differed among chromosome types. DNA contents of chromosome variants determined from flow karyotypes were closely correlated to measurements of DNA content made of gallocyanin chrome alum-stained metaphase chromosomes on slides. Fluorescence in situ hybridization with chromosome-specific repetitive sequences indicated that variability in their copy number is partly responsible for peak-position variability in some chromosomes. Heteromorphic chromosomes are identified for which parental flow karyotype information will be essential if de novo rearrangements resulting in small DNA content changes are to be detected with flow karyotyping.

Bisbenzimidazole↗

PubMed1985

Cryptic satellites rich in inverted repeats comprise 30% of the genome of a hermit crab.

One major very highly repeated (VHR) DNA (approximately 7 X 10(6) copies/genome; repeat unit = 156 base pairs (bp)), a family of three minor VHR DNAs (approximately 2.8 X 10(6) copies/genome; repeat units = 71-74 bp), and a number of trace components account for almost 30% of the genome of a hermit crab. The repeat units of the three minor variants are defined by identical 14-bp G + C-rich inverted repeats that might form cruciforms. Two copies of the repeat unit (CCTA) of one of two patent satellites of this crab (Skinner, D. M., and Beattie, W. G. (1974) Biochemistry 13, 3922-3929; Skinner, D. M., Beattie, W. G., Blattner, F. R., Stark, B. P., and Dahlberg, J. E. (1974) Biochemistry 13, 3930-3937) occur at the center of one in seven of the G + C-rich inverted repeats; copies of the other patent satellite (Chambers, C. A., Schell, M. P., and Skinner, D. M. (1978) Cell 13, 97-110) are found in main component DNA. The sequences of both the major and minor VHR DNAs are characterized by short tracts of An and/or Tn (n = 4-7) residues whose presence would permit the formation of perfectly matched stems separated by loops of 8-16 bp. The An and/or Tn tracts are interspersed with segments of G + C-rich DNA and are arranged differently in the major and minor VHR DNAs. Although the repeat units of the major and the three minor VHR DNAs are arranged in tandem, the composition and sequence of their bases are such that they do not form distinct bands in CsCl gradients; they are cryptic satellites.

Animals↗

PubMed2006

Extense variant gene family repertoire overlap in Western Amazon Plasmodium falciparum isolates.

In order to find a molecular basis for observations of relatively fast developing immunity to malarial infections in the Western Amazon region, the partial var, stevor and rif gene repertoires of nine different Plasmodium falciparum isolates collected in 1985 and 2000-2004 were evaluated. In contrast to previous results from South East Asia, the variant gene repertoire in Brazilian isolates is rather small and redundant. While the individual var repertoire sizes of Brazilian strains did not differ from Southeast Asian/African isolates, we found an over three times higher overlap of var sequence repertoires in Amazonian strains which was also conserved over time, suggesting the ongoing circulation of a similar var gene repertoire. Coincidently, almost 40% of the sequences identified herein showed the highest degree of similarity to var genes from either Brazilian or Venezuelan isolates, indicating a limited var repertoire of P. falciparum in the Amazon Basin as a whole. The intrastrain similarities of var genes were slightly but significantly lower than in Southeast Asian/African samples suggesting a higher selective pressure for diversification in Amazonian isolates. Despite of higher copy numbers per genome, rif genes also showed a significant repertoire overlap. stevor genes, which share the same predominant subtelomeric localization as var and rif genes, showed a still higher repertoire overlap and were highly similar to 3D7 stevor genes, indicating stronger functional conservation than var and rif genes. This is the first study that reveals that P. falciparum variant gene repertoires of certain areas can be limited. This has important implications for the strain-specific immunity against variant antigens occurring in these areas.

Animals↗

PubMed2002

Aloe spp.--plants with vertebrate-like telomeric sequences.

Chromosome termini of most eukaryotes end in tracks of short tandemly repeated GC-rich sequences, the composition of which varies among different groups of organisms. Plant species predominantly contain (TTTAGGG)n repeats at their telomeres. However, a few plant species, including members of Alliaceae and Aloe spp. (Asphodelaceae) were found to lack such Arabidopsis-type (T3AG3)n telomeric repeats. Recently, it has been proposed that the lack of T3AG3 telomeric repeat sequences extends to all species forming the Asparagales clade. Here, we analysed the composition of Aloe telomeres by single-primer PCR and fluorescence in-situ hybridization (FISH) with directly labelled Arabidopsis-type (TTTAGGG)28-43 DNA probe, and with vertebrate-type (TTAGGG)33-50 DNA and a (C3TA2)3 peptide nucleic acid (PNA) probe. It was found that Nicotiana tabacum contained Arabidopsis-type telomeric repeats, while Aloe telomeres lacked the corresponding FISH signals. Surprisingly, FISH with the highly specific vertebrate-type (C3TA2)3 PNA probe resulted in strong T2AG3-specific FISH signals at the ends of chromosomes of both Aloe and Nicotiana tabacum, suggesting the presence of T2AG3 telomeric repeats in these species. FISH with a long (TTAGGG)33-50 DNA probe also highlighted Aloe chromosome ends, while this probe failed to reveal FISH signals on tobacco chromosomes. These results indicate the presence of vertebrate-like telomeric sequences at the telomeres of Aloe spp. chromosomes. However, single-primer PCR with (TAG3)5 primers failed to amplify such sequences in Aloe, which could indicate a low copy number of T2AG3 repeats at the chromosome ends and/or their co-orientation and interspersion with other repeat types. Our results suggest that telomeres of plant species, which were thought to lack GC-rich repeats, may in fact contain variant repeat types.

Aloe↗

PubMed1986

Evolutionarily selected replication origins: functional aspects and structural organization.

A selective replicative pressure occurs during the evolution of simian virus 40 variants. When the replication origin is duplicated as an inverted repeat, there is a dramatic enhancement of replication. Having regulatory sequences located between the inverted repeat of ori magnifies their enhancing effect on replication. A passage 20 variant and a passage 45 variant containing three pairs of an inverted repeat of ori replicated more efficiently than a passage 13 variant containing nine copies of ori arranged in tandem. A 69-base-pair cellular sequence inserted between inverted repeats of ori of both passage 40 and 45 variants enhanced simian virus 40 DNA replication. Differences in replication efficiencies became greater as the total number of replicating species was increased in the transfection mixture, under conditions where T antigen is limiting. In a competitive environment, sequences flanking the replication origin may be inhibitory to replication.

Animals↗

PubMed2006

Genetic polymorphisms of human ABC transporter ABCG2: development of the standard method for functional validation of SNPs by using the Flp recombinase system.

The vector-mediated introduction of cDNA into mammalian cells by calcium phosphate co-precipitation or permeation with lipofectamine is widely used for the integration of cDNA into genomic DNA. However, integration of cDNA into the host's chromosomal DNA occurs randomly at unpredictable sites, and the number of integrated recombinant DNAs is not controllable. To investigate the effect of genetic polymorphisms of ABCG2 on the protein expression and the drug resistance profile, we developed the Flp-In method to integrate one single copy of ABCG2 variant-cDNA into FRT-tagged genomic DNA. More than 20 metaphase spreads were examined for both fluorescence in situ hybridization (FISH) mapping and multicolor-FISH analysis, and it has been revealed that ABCG2 cDNA was incorporated into the telomeric region of the short arm on one of chromosomes 12 in Flp-In-293 cells. Based on the currently available SNP data for human ABCG2, we have created a total of seven variants by site-directed mutagenesis and stably expressed them in Flp-In-293 cells. While mRNAs of those integrated ABCG2 variants and wild type were evenly expressed in Flp-In-293 cells, the protein expression levels of F208S and S441N variants were found to be markedly low. It is suggested that the protein instability due to enhanced degradation resulted in the low levels of their protein expression. Thus, the Flp recombinase system would provide a useful tool to validate the effect of nonsynonymous SNPs on the protein stability and post-translational modification of ABCG2.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2001

Prenatal RHD gene determination and dosage analysis by PCR: clinical evaluation.

BACKGROUND: Use of the polymerase chain reaction (PCR) for detection of the RHD gene can measure the RHD gene status for unborn babies at risk for hemolytic disease of the newborn (HDN). The occurrence of D gene variants has led to errors in prenatal typing. Previous reports have highlighted the danger of assigning a positive fetus as negative, resulting in intrauterine fetal deaths. OBJECTIVE: To evaluate the effectiveness of a testing strategy whereby PCR was not only performed to determine the presence/absence of the RHD gene, but also used to assess the D gene copy number (zero, one or two RHD genes) in family studies for at risk pregnancies. METHODS: Samples comprising maternal (57) and paternal (42) peripheral blood samples, amniotic fluid (64), and matching cord blood (64) were collected. Rhesus (Rh) serotyping was performed on all blood samples. For RHD genotyping, DNA was extracted from all samples except for 28 cord samples, where only serotyping was performed (total 199 DNA genotyping). RHD gene PCR amplified exon 4 and exon 7 regions of the RHD gene. The dosage of RHD gene was determined by comparing the intensity of the RHD gene to that of the RHCE gene. RESULTS: A total of 197/199 samples showed concordance between exon 4 and exon 7 PCR results. Two discrepant results occurred in one family: the father carried one normal D gene and one D gene variant where PCR was tested to be positive using exon 4 but negative using exon 7. One of a pair of dizygotic twins inherited this abnormal D gene and was mildly affected by HDN. This was correctly identified antenatally and the pregnancy successfully managed. The concordance rate between serotypes and genotypes for 135 blood samples was 100%. Amongst the family groups, 8/14 heterozygous fathers transmitted the D gene and 26/26 homozygous fathers transmitted the D gene to the babies. The concordance rate between RHD genotypes from amniotic fluid and Rh D serotypes from cord blood was also 100%. CONCLUSION: The present study demonstrates the effectiveness of using PCR in a clinical setting. It verifies the importance of testing more than one region of the gene, and also the need for a testing strategy where both maternal and paternal testing for RHD gene dosages are performed.

Amniotic Fluid↗

PubMed2021

Detection of PKD1 and PKD2 Somatic Variants in Autosomal Dominant Polycystic Kidney Cyst Epithelial Cells by Whole-Genome Sequencing.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by the development of multiple cysts in the kidneys. It is often caused by pathogenic mutations in PKD1 and PKD2 genes that encode polycystin proteins. Although the molecular mechanisms for cystogenesis are not established, concurrent inactivating germline and somatic mutations in PKD1 and PKD2 have been previously observed in renal tubular epithelium (RTE). METHODS: To further investigate the cellular recessive mechanism of cystogenesis in RTE, we conducted whole-genome DNA sequencing analysis to identify germline variants and somatic alterations in RTE of 90 unique kidney cysts obtained during nephrectomy from 24 unrelated participants. RESULTS: Kidney cysts were overall genomically stable, with low burdens of somatic short mutations or large-scale structural alterations. Pathogenic somatic "second hit" alterations disrupting PKD1 or PKD2 were identified in 93% of the cysts. Of these, 77% of cysts acquired short mutations in PKD1 or PKD2 ; specifically, 60% resulted in protein truncations (nonsense, frameshift, or splice site) and 17% caused non-truncating mutations (missense, in-frame insertions, or deletions). Another 18% of cysts acquired somatic chromosomal loss of heterozygosity (LOH) events encompassing PKD1 or PKD2 ranging from 2.6 to 81.3 Mb. 14% of these cysts harbored copy number neutral LOH events, while the other 3% had hemizygous chromosomal deletions. LOH events frequently occurred at chromosomal fragile sites, or in regions comprising chromosome microdeletion diseases/syndromes. Almost all somatic "second hit" alterations occurred at the same germline mutated PKD1/2 gene. CONCLUSIONS: These findings further support a cellular recessive mechanism for cystogenesis in ADPKD primarily caused by inactivating germline and somatic variants of PKD1 or PKD2 genes in kidney cyst epithelium.

Humans↗