Ultra-Rare Variants Identify Biological Pathways and Candidate Genes in the Pathobiology of Non-Syndromic Cleft Palate Only
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Collection
2.2. Sequencing and Variant Detection
- Any gene should have <20% of bases covered <20× in each sample;
- Any gene should not differ by >10% in mean coverage between cases and controls.
2.3. Gene-Set Enrichment Analysis (GSEA)
2.4. Gene-Set Burden and Variants Analysis
3. Results
3.1. Quality Checks
3.2. GSEA Results
3.3. Gene-Set Burden Test Results
3.4. Clinical Variants
4. Discussion
- GSEA highlighted genes with ultra-rare variants cluster into gene ontology sets that are highly relevant to nsCPO pathobiology (cytoskeleton rearrangement, cell-adhesion, ECM-cell interaction);
- GCA identified two top-ranking genes (COL2A1 and GLI3) significantly overlapping in the list of genes relevant to nsCPO pathobiology;
- Pathogenic and likely pathogenic variants in genes, accounting for autosomal dominant orofacial cleft syndromes (CDON, COL2A1, IRF6, SNRPB), have been identified in four cases (10.8%).
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Category | Term | Nominal p * | Adjusted p * | Genes |
---|---|---|---|---|
GO: MF | cytoskeletal protein binding (GO:0008092) | 1.43 × 10−7 | 1.57 × 10−4 | RAB11B, XIRP1, ABL2, MAP1A, OBSCN, PARVB, ADGRV1, TUBGCP5, FMNL2, TTN, AP1G1, FSCN2, TAOK1, KIF17, UTRN, FKBP4, FLII, FLNB, VPS18, ANK3, CLIP2, APC, PPARG, CLSTN1, CLIP3, SETD2, PARVA, VPS41, MAST1, MYH15, MYBPC3, MYH2, MYH3, MYH4, TTLL9, EML2, PANX1, ACE, FRMD4B, IQGAP1, MAPK8IP3, TTBK2, CYFIP1, BAIAP2L1, NAV3, KCNQ2, MYO18B, GNB3, TBCD, KPNB1, NRCAM, SYNM, SYNE1, NUMA1, CLIP4, ARL4C, FARP1, MACF1, KIRREL1, DAAM2, RFLNA, MYO15A, TTLL13 |
GO: MF | microtubule plus-end binding (GO:0051010) | 1.92 × 10−5 | 1.06 × 10−2 | CLIP2, APC, CLIP3, TTBK2, NUMA1, CLIP4 |
GO: MF | cell adhesion molecule binding (GO:0050839) | 3.97 × 10−5 | 1.24 × 10−2 | RAB11B, ABCF3, CDHR3, TRPC4, OBSCN, FMNL2, TENM2, TAGLN2, UTRN, FLNB, ANK3, ITGA10, APC, NTNG1, AHSA1, TENM3, TNN, PARVA, TES, ADAM15, ITGB7, IQGAP1, PTPRB, PTPRH, BAIAP2L1, PTPRZ1, NRCAM, DOCK9, LAMA3, LAMB2, GRIN2A, GRIN2B, MACF1, KIRREL1, CDH7 |
GO: MF | protein-containing complex binding (GO:0044877) | 4.52 × 10−5 | 1.24 × 10−2 | XIRP1, NOG, ABL2, MAP1A, PDK2, TSHR, ARID1A, FMNL2, TTN, HIRA, ARID1B, AP1G1, FCGR2A, OTOF, AEBP1, MIF, FSCN2, PLCB2, CNGB1, UTRN, NCOA2, FLII, ANK3, ABI3BP, FOS, ANXA11, ITGA10, APC, CPD, CDC42BPB, PEX26, MTOR, TNN, PARVA, MYH15, CTSK, GAD1, MYH2, MYH3, MYH4, SPHK2, ADAM15, VPS33A, PANX1, ITGA2B, ITGB7, IQGAP1, ITPR1, MED24, CYFIP1, SACS, PTPRZ1, GLI3, GNB3, KPNB1, DOCK2, HNF1B, CARMIL2, SYNM, SYNE1, NUMA1, LAMA3, GRB2, LAMB2, LAMB3, GRIN2A, GRIN2B, LIN28A, RELL2, H1-0, H1-5, LRP2, MACF1, MYO15A, CPEB1 |
Pathway | ECM-receptor interaction (M7098) | 7.03 × 10−6 | 1.69 × 10−2 | COL2A1, COL4A2, COL5A2, ITGA10, TNN, LAMA1, ITGA2B, ITGB7, LAMA3, LAMA4, LAMB2, LAMB3 |
Pathway | Integrin signaling pathway (P00034) | 1.07 × 10−5 | 1.28 × 10−2 | PARVB, MAP3K4, COL2A1, COL4A2, COL5A2, FLNB, COL7A1, ITGA10, PARVA, MAP2K3, LAMA1, ITGA2B, ITGB7, LAMA3, LAMA4, LAMB2, LAMB3 |
Pathway | Ensemble of genes encoding core extracellular matrix including ECM glycoproteins, collagens and proteoglycans (M5884) | 2.77 × 10−5 | 1.86 × 10−2 | CRIM1, AEBP1, COL2A1, COL4A2, COL5A2, COL7A1, ABI3BP, COL28A1, EFEMP2, NTNG1, ZP1, SNED1, TNN, LAMA1, TINAGL1, IGSF10, PXDN, VWA5B1, LAMA3, LAMA4, LAMB2, LAMB3 |
Pathway | Axon guidance (1270303) | 5.21 × 10−5 | 2.42 × 10−2 | EPHB3, SCN11A, ERBB3, ABL2, RPS6KA2, TRPC4, SCN10A, FGF6, ALCAM, PLXNB1, RASAL2, COL4A2, ANK3, RASA3, CNTNAP1, ITGA10, RAPGEF2, PSMA5, LAMA1, FRS2, ITGA2B, IQGAP1, KSR1, KCNQ2, CACNB1, TRPC7, NRCAM, CAMK2G, KSR2, GRB2, SRGAP2, GRIN2A, GRIN2B, ROBO1 |
Cases | Controls | ||||
---|---|---|---|---|---|
GENES | Variants | No_Variants | Variants | No_Variants | p-Value |
ZFYVE26 | 7 | 28 | 0 | 38 | 0.0041 |
THSD7B | 6 | 29 | 0 | 38 | 0.0095 |
SPATC1 | 6 | 29 | 0 | 38 | 0.0095 |
IGHG1 | 6 | 29 | 0 | 38 | 0.0095 |
EXO1 | 0 | 35 | 7 | 31 | 0.0119 |
JAKMIP3 | 9 | 26 | 2 | 36 | 0.0210 |
AHNAK | 9 | 26 | 2 | 36 | 0.0210 |
PLEKHN1 | 5 | 30 | 0 | 38 | 0.0216 |
NBEAL2 | 5 | 30 | 0 | 38 | 0.0216 |
USP42 | 5 | 30 | 0 | 38 | 0.0216 |
OR4K1 | 5 | 30 | 0 | 38 | 0.0216 |
STARD9 | 5 | 30 | 0 | 38 | 0.0216 |
UNC13A | 5 | 30 | 0 | 38 | 0.0216 |
OLFML2B | 4 | 31 | 0 | 38 | 0.0481 |
MROH9 | 4 | 31 | 0 | 38 | 0.0481 |
SLC4A5 | 4 | 31 | 0 | 38 | 0.0481 |
VWA3B | 4 | 31 | 0 | 38 | 0.0481 |
CAND2 | 4 | 31 | 0 | 38 | 0.0481 |
USP4 | 4 | 31 | 0 | 38 | 0.0481 |
LRP2BP | 4 | 31 | 0 | 38 | 0.0481 |
ERAP2 | 4 | 31 | 0 | 38 | 0.0481 |
DOPEY1 | 4 | 31 | 0 | 38 | 0.0481 |
GLI3 | 4 | 31 | 0 | 38 | 0.0481 |
TRBV6-7 | 4 | 31 | 0 | 38 | 0.0481 |
LOXL2 | 4 | 31 | 0 | 38 | 0.0481 |
APOBEC1 | 4 | 31 | 0 | 38 | 0.0481 |
COL2A1 | 4 | 31 | 0 | 38 | 0.0481 |
LIMA1 | 4 | 31 | 0 | 38 | 0.0481 |
HAL | 4 | 31 | 0 | 38 | 0.0481 |
RAI1 | 4 | 31 | 0 | 38 | 0.0481 |
MPP3 | 4 | 31 | 0 | 38 | 0.0481 |
RSAD1 | 4 | 31 | 0 | 38 | 0.0481 |
CILP2 | 4 | 31 | 0 | 38 | 0.0481 |
ZNF600 | 4 | 31 | 0 | 38 | 0.0481 |
LILRA4 | 4 | 31 | 0 | 38 | 0.0481 |
TUBB1 | 4 | 31 | 0 | 38 | 0.0481 |
UTRN | 6 | 29 | 1 | 37 | 0.0497 |
NLRC3 | 6 | 29 | 1 | 37 | 0.0497 |
URB1 | 6 | 29 | 1 | 37 | 0.0497 |
Gene | Genomic Position * | Variant Type | Variant Class | CADD Score | Protein Consequence | Population Frequency | Population |
---|---|---|---|---|---|---|---|
COL2A1 | 12-48377883-G-T | SNV | missense | 28 | p.Pro643His | 7.16 × 10−6 | Iranian |
COL2A1 | 12-48369250-C-T | SNV | missense | 9 | p.Gly1246Ser | 0.000598 | Italian |
COL2A1 | 12-48373812-G-A | SNV | Stop gain | 46 | p.Arg887Ter | 0 | Italian |
COL2A1 | 12-48376305-C-T | SNV | missense | 19 | p.Ala761Thr | 8.77 × 10−6 | Italian |
GLI3 | 7-42007446-C-T | SNV | missense | 27.5 | p.Gly727Arg | 0.007131 | Italian |
GLI3 | 7-42063171-C-G | SNV | missense | 23.5 | p.Gly465Arg | 0.003811 | Iranian |
GLI3 | 7-42187851-C-T | SNV | missense | 28.4 | p.Arg114Lys | 0.002434 | Italian |
GLI3 | 7-42187885-G-A | SNV | missense | 24.7 | p.Pro103Ser | 4.03 × 10−6 | Italian |
Genes | Genomic Position * | Protein Consequence | HGVS Nomenclature | ACMG Classif. | OMIM Disease and Inheritance | ClinVar Accession | Genotype |
---|---|---|---|---|---|---|---|
CDON | 11-125875899-G-A | p.Gln536Ter | NM_001243597.2: c.1606C>T | LP | Holoprosencephaly 11; AD | Het | |
COL2A1 | 12-48373812-G-A | p.Arg887Ter | NM_001844.5: c.2659C>T | P | Stickler syndrome, type I; AD | VCV000817513.4 | Het |
IRF6 | 1-209974677-A-C | p.Trp28Gly | NM_006147.4: c.82T>G | LP | {Orofacial cleft 6}; AD | VCV000464464.1 | Het |
SNRPB | 20-2442575-T-A | p.Ter232Cysext *?§ | NM_198216.2: c.686-136A>T | LP | Cerebrocostomandibular syndrome; AD | Hom |
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Iovino, E.; Scapoli, L.; Palmieri, A.; Sgarzani, R.; Nouri, N.; Pellati, A.; Carinci, F.; Seri, M.; Pippucci, T.; Martinelli, M. Ultra-Rare Variants Identify Biological Pathways and Candidate Genes in the Pathobiology of Non-Syndromic Cleft Palate Only. Biomolecules 2023, 13, 236. https://doi.org/10.3390/biom13020236
Iovino E, Scapoli L, Palmieri A, Sgarzani R, Nouri N, Pellati A, Carinci F, Seri M, Pippucci T, Martinelli M. Ultra-Rare Variants Identify Biological Pathways and Candidate Genes in the Pathobiology of Non-Syndromic Cleft Palate Only. Biomolecules. 2023; 13(2):236. https://doi.org/10.3390/biom13020236
Chicago/Turabian StyleIovino, Emanuela, Luca Scapoli, Annalisa Palmieri, Rossella Sgarzani, Nayereh Nouri, Agnese Pellati, Francesco Carinci, Marco Seri, Tommaso Pippucci, and Marcella Martinelli. 2023. "Ultra-Rare Variants Identify Biological Pathways and Candidate Genes in the Pathobiology of Non-Syndromic Cleft Palate Only" Biomolecules 13, no. 2: 236. https://doi.org/10.3390/biom13020236