Application of Whole Exome-trio Analysis Reveals Rare Variants Associated With Congenital Pouch Colon

doi:10.21203/rs.3.rs-1235311/v1

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Research Article

Application of Whole Exome-trio Analysis Reveals Rare Variants Associated With Congenital Pouch Colon

https://doi.org/10.21203/rs.3.rs-1235311/v1

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posted 13 Jan, 2022

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Anorectal malformations (ARMs) are individually common, but congenital pouch colon (CPC), a rare anorectal anomaly, causes a dilated pouch in the genitourinary tract. In this work, we attempted to identify de novo heterozygous missense variants and further discovered variants of unknown significance (VUS), which could provide insights into CPC manifestation and its etiology. From whole exome sequencing (WES) performed earlier, the trio exomes were analyzed from those who were admitted to J.K. Lon Hospital, SMS Medical College, Jaipur, India between 2011-2017. The proband exomes were compared with the unaffected sibling/family members, and we sought to ask whether any variants of significant interest are associated with the CPC manifestation. The WES data from a total of 64 samples, including 16 affected neonates with their parents and unaffected siblings, were used for the study. Although all samples had unaffected sibling samples and data, we restricted our pool of analyses to all probands (11 male and 5 female) and unaffected parents/siblings. We previously attempted to understand the genetic makeup of CPC and identified genes responsible for the disease using WES. We examined the role of rare allelic variation associated with CPC in a 16 proband/parent trio family comparing the mutations to those of their unaffected parents/siblings. Our study across 16 probands revealed extremely rare variants, viz. TAF1B, MUC5B and FRG1. The variants were further validated to reveal disease-causing mutations associated with CPC and genitourinary diseases that could close the gaps of surgery by bringing intervention in therapies.

whole exome sequencing

trio exome

missense variants

congenital pouch colon

Whole-exome sequencing (WES) has been an invaluable and cost-effective approach to identify genetic variants responsible for both Mendelian and polygenic diseases¹. In the recent past, it has allowed the detection of clinically relevant genomic regions spanning known unknown regions, disease-associated sites and untranslated regions (UTRs)². In addition to the well-known diseases, prenatal abnormalities, structural anomalies and congenital defects were studied, demonstrating a good diagnostic yield^3,4. While WES approaches are abundant, they are limited if the disease in question is characteristically rare and medically inconclusive. This could be a deterrent because of the challenges in variant discovery, including rare and low-frequency mutations using next-generation sequencing (NGS) technologies. A recent decrease in the cost of WES and the accuracy of NGS has enabled researchers to study a large number of WES samples, but in the case of rare diseases, studying exome trios (proband/parents) or quads with the addition of siblings to discover single nucleotide variations (SNVs) and indels has proven to be a major landmark in the discovery of rare disease variants. For example, Boycott et al., 2013; Gahl et al., 2012; Jacob et al., 2013 employed characteristic trio-exome analysis to infer candidate or driver mutations in rare diseases^5–7. With human disease and genetic variation studies largely driven by NGS, a paramount challenge would be to explore de novo mutations, i.e., those not inherited from either father or mother. To check this, parent–child trios/quad WES analysis could be a powerful approach, although biases impede the identification of potential de novo mutations. Nevertheless, a majority of mutations may not transcend from parent to offspring, making comprehensive genetic variants to be analyzed to confirm them as causal⁸. Trio-based exome sequencing has provided a benefit for identifying de novo variants in rare diseases, attributing them to largely heterozygous/causal mutations⁹. For rare diseases, although WES analysis often makes assumptions regarding disease inheritance (de novo vs. recessive), variant frequency and genetic heterogeneity, it has opened the path toward improved disease management or prognosis and effective therapies. For example, WES trios in schizophrenia patients for recessive genotypes were studied, with rare mutations in voltage gated sodium ion channels contributing to the disorder¹⁰. In another study, Jin et al. (2017) identified pathogenic mutations with an increased rate of de novo mutations in early-onset high myopia (EOHM) patients¹¹. Recently, Quinlan-Jones et al. correlated proband–parent trios to determine the clinical utility of molecular autopsy underlying the etiology of structural anomalies¹².

Congenital pouch colon (CPC) is a rare type of high anorectal malformation wherein a part of or the entire colon becomes dilated in the form of a pouch with a fistula connecting the genitor-urinary tract¹³. Most of the incidences have been reported from India, with cases common to other countries accounted for, although males are prone to be largely affected, i.e., male to female ratio of 4:1¹⁴. From WES approaches, we previously identified mutations that are causal to CPC and reported candidate missense mutations¹⁵. In another study, we inferred the role of long noncoding RNAs (lncRNAs) from WES and identified lnc-EPB41-1-1, located in the intergenic regions of EPB41 that are known to interact with KIF13A¹⁶. In this extended WES study of CPC, we examined the role of rare allelic variation associated with CPC in a 16 proband/parent trio family comparing the mutations to those of their unaffected parents/siblings. Keeping in view of understanding the genetic basis of CPC that could possibly delve into variation, an attempt was made to discover variants contributing to phenotypic heterogeneity.

Trio selection, sample collection and ethical approval

The CPC subjects were recruited from the J.K. Lon Hospital, SMS Medical College, Jaipur, India, in accordance with a protocol approved by the institutional ethics committee (IEC), SMS Medical College and Hospital, Jaipur, Rajasthan. The informed consent was obtained from all subjects, i.e., from parents for themselves (because blood from parents was also taken along with their children) and also for their children. All methods were performed in accordance with the relevant guidelines and regulations as per IEC, SMS Medical College and Hospital coalescing Helsinki guidelines. Blood samples were collected from all the probands, parents and unaffected siblings if any. The WES data from a total of 64 samples, including 16 affected neonates with their parents and unaffected siblings, were used for the study. Although all samples had unaffected sibling samples and data, we restricted our pool of analyses to all probands (11 male and 5 female) and unaffected parents/siblings. The methods and pipeline leading to family/quad analyses are summarized in Figure 3 (Supplementary information).

Variant annotation, filtering and quality control

The details of sequencing and variant calling in CPC subjects have been previously described¹⁵. Briefly, WES was performed on an Illumina multiplexed sequencer with paired-end chemistry and 110x effective coverage. Using our in-house developed pipeline⁵⁶, all unmapped sequence reads were aligned to the human reference genome (hg38), and variants were called. The mutations from the WES study were manually checked to discover the variants, if any, across the samples (Figure 1). First, all variants with MAF <0.05 were interpreted using ‘grep’ and awk liner commands, if present in probands but absent in their respective parents (Supplementary information) and healthy siblings. After manually checking the variants, we confined the prioritization of variants with filters set to an average depth of 250 and MAF <0.01 and MAF<=0.01% across all the trio samples. Further checking with the dbSNP¹⁷, ClinVar¹⁸, GnomAD¹⁹ and COSMIC²⁰ databases, we used SNP-Nexus²¹ to filter mutations listed in a cohort of databases, viz. SIFT¹, PolyPhen-2²², Ensembl Variant Effect Predictor²³, MutationTaster²⁴, CADD²⁵ and GERP²⁶ prioritize pathogenic mutations if they are deleterious in nature. The CNVs and variants of unknown significance (VUS) were inferred by mapping the final list of variants to the SNPnexus. As a final check in reaching consensus for the variants present in all the probands, we checked the variants with multiple bioinformatics tools to find bona fide variants at the union of intersection of these methods, which we construe them to be causal.

Downstream analyses

We used the vcftools package (https://vcftools.github.io), and the mutations in probands were further checked with their relative parents/siblings for heterozygosity transmission. We also looked into the homozygous variants and considered them causal for CPC, where the proband was found to be homozygous and their respective parents and unaffected siblings if any were heterozygous for the specific allele. Enrichment analysis of data was performed to calculate the inclusiveness of parameters such as binomial probability and hypergeometric distribution²⁷. After high-throughput screening, we undertook a candidate gene-set analysis based upon significantly enriched sets or rare mutations specifically in colon-related disorders. Seeking novel insights into the disorder, we used pathway analysis based upon gene ontology (GO) derived from PANTHER ontology²⁸ (http://pantherdb.org/tools) and EnrichR²⁹ (https://amp.pharm.mssm.edu/Enrichr/) annotation terms. Gene Ontology-based annotations included the biological network gene ontology tool (BinGO)³⁰, a plug-in for ontology annotation in Cytoscape³¹ used for ontological analysis in the form of biological, cellular and metabolic processes.

Identification of transcripts for comparative screening

From RNA-Seq, a quality check was ensued after total RNA was isolated and cDNA double strand synthesis was performed on a pair of CPC type-4 samples. RNA-Seq was performed on an Illumina HiSeq 2000 platform with 2x100 bp paired-end sequencing chemistry, which generated ca. 32 million read pairs. The pair of samples was run through differential gene expression analysis using Cufflinks³² and DESeq³³ pipelines, and a consensus was reached. To infer the role of lncRNAs, UVA FASTA software (https://fasta.bioch.virginia.edu/fasta_www2/fasta_list2.shtml#) (v36. 6.8 version) and NONCODE FASTA repository³⁴ were downloaded, and the intergenic regions of the genes from WES samples were queried. The lncRNA NONHSAT002007 was identified based on a query coverage e-value < 0.01. The sequences were carefully checked for bidirectional blast hits, and the lncRNA was visualized using an Ensembl genome browser for bona fidelity.

SNP Genotyping

For Sequenom genotyping, a multiplexed iPLEX assay was designed for 20 ng of DNA per sample to determine SNP calls using the Agena biosciences assay design suite. MALDI-TOF-MS analysis was performed using the Agena biosciences massArray analyzer platform on 29 SNPs in 37 DNA samples (16 probands and 21 controls). This method consists of five steps: PCR amplification, shrimp alkaline phosphatase treatment, single base extension, nanodispensing, and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry³⁵. Data acquisition was automatically performed, and the mass window of analyte peak observation was set at 4500-9000 Da. Call frequencies, expressed as percentages, were calculated for each SNP.

We achieved a mean read depth of 80 in the targeted regions for an average depth of coverage 110x. We generated an average of 840667 total variants comprising 775262 SNPs and 65405 indels per exome in probands compared to 736820 SNPs and 63418 indels in unaffected samples from a total of 777216 variants (Figure 2; see methods). Missense variants constituted the largest variation followed by loss-of-function variants in cases when compared to unaffected samples, with an overall 0.35% missense, 0.02% frameshift, 0.004% stop gain and 0.0009% stoplost. The downstream analysis leading to variant calling was performed carefully to yield the list of the final number of variants. This was also checked with gene density and high linkage disequilibrium (LD) regions, even as MAF <=0.01 was sought for with SNPsnap having no candidate matches, thereby confirming that these variants are extremely rare. Given the rare phenotype, the frequency of the prioritized variants was checked for in agreement with that of 1000 Genomes, GnomAD and ExAC databases. From the reported familial history, relatedness tests were not felt necessary, as the pedigree confirmed correct parenthood for all affected/unaffected samples (Supplementary Table 1). From the final list of segregated variants (Table 1), we identified three mutations in theMUC5B, FRG1, andTAF1B genes, which we deem to be extremely rare variants (Table 2a). Furthermore, we also found an AK9 copy number variant (CNV) that was run through SNPsnap³⁶to assess whether rare allelic variation was enriched for particular biological annotations (Supplementary Table 6). Finally, a set of candidate variants inferred from all samples was checked for validation using Sequenom array/plex (Table 3).

We found two independent lines of evidence from various tools and reached consensus in detecting variants from each sample with all filtering steps. For example, the pathogenic mutations contributing to each relevant proband were screened first wherein three variants, viz. FRG1 (4-189957414), TAF1B (2-9904885) and MUC5B (11-1238987) were confirmed through ClinVar and GnomAD. While the FRG1 mutation happens to be a CNV-associated missense seen in Z15 and Z99 samples, it is further augmented by the fact that low expression of FRG1 is associated with tumor progression in the colon⁵⁵. The TAF1B mutation is highly associated with colorectal tissues, as we found these stopgain/missense mutations to be highly prolific in some of the aggressive CPC probands, viz. Z15, Z19, Z34, Z42, Z46, Z54, Z62, Z66, Z74. TAF1B is known to be the second largest subunit of the TATA box-binding protein (TBP)-containing promoter selectivity factor TIF-1B/SL1 and is connected with ribosomal transcription⁵¹. We found that the number of detected variants was associated with colon, rectal and genitourinary tissues and the presence of vivid disease occurrence. The density of these pouch-related tissues with missense/stop gain mutations can be attributed to aggressive CPC type IV.

Dissecting the genetic architecture of a rare disease is certainly an arduous task. Our CPC exome analysis was intended to fill this gap by detecting SNVs and CNVs affecting the focal genes/loci. Assessing these variants in CPC has provided ample evidence of strata coherent to CPC traits. In our study, at least three other genes from trio-exome analysis were reported in colon-related ailments, viz. DLC1, HAVCR1 and GBA3, but their allele frequencies are not bona fide and comparable. While we aimed to characterize the variants by employing different approaches, a polygenic model was assumed. This could be compounded with two assumptions: (a) capturing exomes and identifying deleterious mutations from a high depth of coverage exomes and (b) identifying large cohorts of mutations that fall in a low depth of coverage exomes. Although we observed both classes of genetic variation contributing to the etiology of the disease, inferring proband-parent trios and detecting de novo and transmitted genetic variants is quite a challenge. By considering extremely rare variants and adopting a strategy of identifying them in high-depth exomes, we validated all 16 trios. Nevertheless, we could not compare the detection yield inherent to this spectrum of patients owing to lack of CPC phenotype and trio-exome studies of similar design. Although previous studies have shown relatively similar methods, they detected medically relevant variants in the majority of the diseased phenotypes¹¹. Our findings are in agreement with a large number of reports for rare variants, suggesting that the cumulative contribution of variants across different genes is associated with distinct phenotypes. In addition, an important challenge for researchers and clinicians currently in investigating rare disorders involves predicting pathogenicity for VUS. With several guidelines mentioned for predicting the pathogenicity of variants^8,18,39, molecular investigators face a daunting task in considering a rare variant as benign or pathogenic and inferring them to be pathogenic. In explaining the germline/heritability of complex variants, based on the rare-variant hypothesis, we argue that the extremely rare variants are associated with phenotype sampling⁵⁴. Next, we showed how we can influence and prioritize these extremely rare variants and further proposed an optimization procedure to check the variants called between MAF <0.01 and MAF 0.01%. To address this, we discarded many variants that have MAF<0.01 and finally expanded the current annotation and prioritization to accommodate the CPC framework.

In rare diseases, where only a minority of the population is affected and prevalent in a specific geographical location, identifying and considering VUS will require thoughtful consideration. A notable among them, viz. The sequestome (SQSTM1 or P62) gene encodes a multifunctional scaffolding protein involved in multiple cellular processes^40–41 in addition to showing mitochondrial integrity, import and dynamics as a discriminating autophagy receptor⁴². In addition, p62/SQSTM1 is ubiquitously expressed in various cell types, such as the cytoplasm, nucleus and lysosomes^43, and is known to be overexpressed in various human genitourinary diseases, including colon cancer⁴⁴, hepatocellular carcinoma⁴⁵ and prostate cancer⁴⁶ (Supplementary Table 2). While KCNJ12 was also among the bona fide variants, our variant classification did not compel us to be considered extremely rare (not shown). It is known to initiate transcription by RNA polymerase I and acts as a channel for regulatory signals, while KCNJ12 encodes ATP-sensitive inward rectifier potassium channel 12 and is subtly associated with repolarization of channels^52–53. A list of segregated variants and those that were not extremely rare variants in the form of SQSTM1 and KCNJ12 were also considered for validation. The SQSTM1 mutation is invariably inherited from the father in the case of the Z12 index case and from the mother in the case of Z54. As the aforementioned variants were considered extremely rare variants, we also found AK9 (6-109528998.109528999 C|-) to be consistently seen across all probands. This deletion is invariably associated with nucleotide metabolism pathways and maintains homeostasis of cellular nucleotides³⁸. Although AK9 (deletion) was phenomenally seen in all probands, we considered it a rare variant candidate for validation. Although multiple lines of evidence suggest that apart from VUS, mutations yielding somatic copy number alterations (SCNA) could not be ruled, as we found some uncommon missense mutations with MAF <0.05 in C7orf57, C9orf84, ORF5AR1, FGFR4, HLA-DRB5, NOTCH2NLA, and MUC5B genes that could be nonpathogenic/causal to CPC.

One of the interesting findings that emerged from our study is the role of the known unknowns or hypothetical genes that could be predecessors of noncoding; hence, their establishing roles in diseases such as CPC is limited⁴⁷. Notably, the C10orf120 gene harbors CTCF binding sites, as these mutations remain undefined for most disease types, including cancer⁴⁸. We observed that there was significant enrichment of CNVs and indels associated with intestinal/colon-related-specific genes, as they are widespread in tissues showing chromosomal instability, cooccur with neighboring chromosomal aberrations and are frequent in colon, rectum and gastrointestinal tumors but rare in other diseases. We argue that this mutational disruption associated with CTCF binding sites could be associated with pathogenesis, as it appears to be conserved in a majority of CPC probands (Supplementary Table 3). Another orphan ORF, viz. C7orf31 also harbors CTCF binding sites, which in fact showed significant enrichment for biological processes associated with regulatory, cellular and metabolic pathways (Table 4). Another C10orf120 has somatic variants subtly contributing to CPC pathogenesis and the maximum number of gains and losses observed in the form of CNVs in the CDC27, HLA-DRB5 and MST1 L genes (Table 2b). Although some of these ORFs' maternal association and pathogenicity cannot be ruled out, we construe that there are candidate genes that could be promising biomarkers as precursors of CPC, which is beyond the scope of this canonical hypothesis (Supplementary Table 4). In addition, we screened our variants from the Indian Genome Variant Database (IGVDB) and found that they are already reported in the Indian subpopulation^49, and this stratification allowed us to review the patterns influencing common and rare variants. In principle, rare variants were found to have stronger patterns than common variants. Thus, there is an inherent need to study the mutations in the known unknown regions, which would possibly delve into understanding rare diseases.

To gain insights into the role of lncRNAs, we revisited our hypothesis from our previous study¹⁶ and reconfirmed whether NONHSAT002007 was inferred in WES samples with predictions from the NONCODE database. While we did not find mutations in lncRNAs from trio-exome analyses, we argue that the mutations in essential genes tend to be causal for rare diseases, paving the way for driver mutations with the mutations in noncoding genes suppressed for selective pressure. On a different note, we aimed to evaluate exome enrichment to that of the transcriptome and anticipated that any of the variants seen from our WES study could be associated with the differentially expressed genes (DEGs). From the transcriptome pair of CPC type-4 (proband and its unaffected parent), we observed several transcripts, alternative splice variants and fusion genes, but none of them could be associated with the causal genes inferred from the exome study (Supplementary Table 5). Although we found RGPD2 and RGPD4 genes known to be significantly associated with bowel/colon as among the top enriched, nevertheless, it is hypothetical to infer global gene expression from just a pair of datasets. This approach, if studied on all samples, we believe, could identify transcripts present at low levels, which in fact could be associated with the pathogenesis of CPC. As CPC cases emerge, it is difficult to classify the clinical significance of pathogenic variants without trio analysis, especially the interpretation of de novo variants. The trio exome data could provide insights into whether the variant could be inherited and whether the carrier mutation is transcended to the progeny. In conclusion, we argue that the genetic variability in CPC has had remarkable significance not only with the parents but also within each proband. With genetic diseases leading causes of death in infants, rapid clinical/trio exome sequencing could provide early diagnoses that could impact decision making in critically ill pediatric patients. Although more efficient early diagnosis could be made with intervention using chromosomal microarray (CMA), it was shown that the clinical utility of trio/exome/whole genome sequencing is more than the CMA⁵⁰. The discovery of causal mutations could provide insights into developmental disorders/anorectal malformations, such as CPC, and their etiology, which closes the gaps of surgery, taking forward to precision therapy.

Competing interests:

The authors declare no competing interests.

Authors’ contributions:

S.G. and P.S. wrote the initial draft and analyzed the data. A.K performed Sequenom analysis on discussion with K.M., P.M. ORB and P.S.; K.M., O.R.B and P.S. conceived the project. O.R.B and P.S. proofread the manuscript before all authors agreed to it.

Funding:

P.M., and K.M. gratefully acknowledge the support of Indian Council of Medical Research toward Grant #5/41/11/2012 RMC.

Acknowledgments:

S.G. acknowledges the support of senior research fellowship from the Council of Scientific and Industrial Research, Government of India (Award #09/892(0002)2015-EMR-I). K.M. and P.S. gratefully acknowledges the support of BTIS-Net, Government of India and Advanced Bioinformatic Centre, Government of Rajasthan toward the support of Bioinformatics Infrastructure Facility at Birla Institute of Scientific Research (BISR), Jaipur.

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Due to technical limitations, tables xlsx is only available as a download in the Supplemental Files section.

No competing interests reported.

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Application of Whole Exome-trio Analysis Reveals Rare Variants Associated With Congenital Pouch Colon

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