Preliminary Screening of Genetic Variants in Each Group
After preliminary sequencing screening, 309–431 genes harbouring variants, including SNPs and indels, were predicted to be harmful per single patient in each group. After eliminating the variants detected in the NC group and comparing the three remaining groups, the number of genes identified per single patient in the IHH, VC, and SN groups was approximately 98–135. Six hundred and forty-five genetic variants were shared between the nIHH and VC groups, and 116 genetic variants were shared by the nIHH, VC, and SN groups (Fig. 2).
Genetic Variants In nIHH Patients
We compared the genetic variants in the nIHH group, after excluding those identified in the NC group, with previously identified IHH pathogenic variants (Tables 2 and 3). Variants were detected in 10 loci and nine genes in nine patients. CHD7 gene variants were identified in patients nIHH2 and nIHH4. Variants in the remaining genes, ANOS1, FGFR1, SEMA7A, OTUD4, AMH, KLB, GATA2, and POLR3B, were all detected once.
Table 2
Common mutated genes in group nIHH
| Num. | nIHH1 | nIHH2 | nIHH3 | nIHH4 | nIHH5 | nIHH6 | nIHH7 | nIHH8 | nIHH9 |
PRRC2A | 4 | | | PRRC2A | | | PRRC2A | PRRC2A | | PRRC2A |
WNK2 | 3 | | | | | | | WNK2 | WNK2 | WNK2 |
UTP20 | 3 | UTP20 | | UTP20 | UTP20 | | | | | |
THSD1 | 3 | | | THSD1 | | THSD1 | THSD1 | | | |
SMPD1 | 3 | | | SMPD1 | | SMPD1 | SMPD1 | | | |
SLC16A3 | 3 | | | SLC16A3 | | | | SLC16A3 | SLC16A3 | |
PIK3C2B | 3 | | PIK3C2B | | | PIK3C2B | | | | PIK3C2B |
PBRM1 | 3 | PBRM1 | | | | | | PBRM1 | PBRM1 | |
KRT13 | 3 | | | | | KRT13 | KRT13 | | KRT13 | |
KPNA2 | 3 | | KPNA2 | | KPNA2 | | | | KPNA2 | |
KDM2B | 3 | | | KDM2B | | | | KDM2B | KDM2B | |
HGFAC | 3 | | | | | HGFAC | HGFAC | HGFAC | | |
GPR158 | 3 | GPR158 | | GPR158 | | | GPR158 | | | |
CYP2C8 | 3 | | CYP2C8 | CYP2C8 | CYP2C8 | | | | | |
CYB5D2 | 3 | | CYB5D2 | | | CYB5D2 | | CYB5D2 | | |
COL12A1 | 3 | | COL12A1 | COL12A1 | | | | COL12A1 | | |
COL11A2 | 3 | COL11A2 | COL11A2 | | COL11A2 | | | | | |
BICC1 | 3 | | BICC1 | | BICC1 | | | | | BICC1 |
ANK1 | 3 | | ANK1 | | | | ANK1 | ANK1 | | |
ZNF595 | 2 | | | | ZNF595 | | ZNF595 | | | |
ZNF335 | 2 | ZNF335 | | | | | | | ZNF335 | |
ZNF236 | 2 | | | ZNF236 | | | | | | ZNF236 |
ZNF225 | 2 | | | ZNF225 | | | ZNF225 | | | |
ZFYVC26 | 2 | | | ZFYVC26 | | | | | | ZFYVC26 |
ZCCHC4 | 2 | | | ZCCHC4 | | | | | | ZCCHC4 |
VPS37C | 2 | | | VPS37C | | VPS37C | | | | |
VIT | 2 | | | VIT | | | | | | VIT |
VAV2 | 2 | | VAV2 | | | | | | | VAV2 |
USP2 | 2 | | USP2 | USP2 | | | | | | |
TXNDC5 | 2 | | TXNDC5 | | | | | | | TXNDC5 |
TTLL8 | 2 | | | TTLL8 | | TTLL8 | | | | |
TSPAN16 | 2 | | | TSPAN16 | | | | TSPAN16 | | |
TRPM5 | 2 | | | TRPM5 | | | | TRPM5 | | |
TRAF2 | 2 | TRAF2 | | | | TRAF2 | | | | |
TRA2B | 2 | | | TRA2B | | | TRA2B | | | |
TPP1 | 2 | | | | TPP1 | TPP1 | | | | |
TOP2B | 2 | | TOP2B | | TOP2B | | | | | |
TNFRSF1B | 2 | | | TNFRSF1B | | | TNFRSF1B | | | |
After removing the NC group data, part of the mutant genes in the nIHH group (from high to low frequency). |
Table 3
The presence of IHH pathogenic gene has been determined in group nIHH
| nIHH1 | nIHH2 | nIHH3 | nIHH4 | nIHH5 | nIHH6 | nIHH7 | nIHH8 | nIHH9 |
CHD7 | | ⚪ | | ⚪ | | | | | |
ANOS1 | | | | ⚪ | | | | | |
FGFR1 | | | ⚪ | | | | | | |
SEMA7A | | | ⚪ | | | | | | |
OTUD4 | | ⚪ | | | | | | | |
AMH | | | | | | | | | ⚪ |
KLB | | | | ⚪ | | | | | |
GATA2 | | | | | | | ⚪ | | |
POLR3B | | | | | | | ⚪ | | |
After excluding the NC group data, the number of mutant genes related to IHH pathogenesis identified in previous studies included in the mutant genes in the nIHH group. |
Subsequently, we performed an analysis based on the ACMG criteria and summarised the results. There were 41 variants were identified as pathogenic or likely pathogenic based on the ACMG criteria. Variants in ADAMTS6 and COL12A1 were identified in two patients. The ADAMTS6 variant identified in patients nIHH7 and nIHH8 was 64747447A > C. Only the FGFR1 variant in nIHH patient 3 was a previously identified IHH pathogenic variant (Table 4).
Table 4
| nIHH1 | nIHH2 | nIHH3 | nIHH4 | nIHH5 | nIHH6 | nIHH7 | nIHH8 | nIHH9 |
P | PDZD4 | WDPCP | FGFR1 | NCAPG2 | CCNO | CFH | CLASP1 | ADAMTS6 | MRC2 |
| PBRM1 | EYA1 | | TANC2 | ABCB4 | IL36RN | ATP13A3 | MC4R | ZNF236 |
| SRP54 | | | | | UBR3 | ADAMTS6 | | |
| | | | | | | CACNB1 | | |
| | | | | | | ELL | | |
LP | DSP | | PLEKHO1 | SLC4A11 | RYR2 | SECISBP2 | GLYCTK | EIF2B5 | PYCR2 |
| BRF1 | | COL12A1 | KDM6B | KLHL3 | | COL12A1 | PGAM2 | CPA6 |
| | | Figure 4 | | PRKCH | | PLG | | NARS2 |
| | | TMPRSS3 | | | | | | LENG8 |
| | | TMPRSS3 | | | | | | |
| | | NCF4 | | | | | | |
P: pathogenic; LP: likely pathogenic. Blackened and italicized mutation gene appears twice or more. |
An analysis of variants that were repeated in the nIHH group showed that variants in THSD1, SMPD1, SCL16A3, and KDM2B were each identified in three patients. These genes showed the highest repetition rate in the nIHH group, but no variants in these genes were found in any other group.
In the pathway enrichment analysis of the nIHH group, after excluding variants detected in the NC group, each nIHH patient was found to have 12–41 enriched pathways, and 12 pathways were enriched in more than two patients. Of these, the cell division pathway (GO: 0051301) was enriched in three patients: nIHH3, nIHH4, and nIHH9 (Table 5).
Table 5
Enrichment pathways of nIHH
| Num | nIHH1 | nIHH2 | nIHH3 | nIHH4 | nIHH5 | nIHH6 | nIHH7 | nIHH8 | nIHH9 |
GO:0051301: cell division | 3 | | | ⚪ | ⚪ | | | | | ⚪ |
GO:0090066: regulation of anatomical structure size | 2 | ⚪ | | | | ⚪ | | | | |
M5880: NABA ECM AFFILIATED | 2 | ⚪ | | | | | | ⚪ | | |
hsa04142: Lysosome | 2 | | | ⚪ | | ⚪ | | | | |
GO:0009410: response to xenobiotic stimulus | 2 | | | | | | ⚪ | ⚪ | | |
R-HSA-2672351: Stimuli-sensing channels | 2 | | | | | | | ⚪ | ⚪ | |
GO:0009582: detection of abiotic stimulus | 2 | | ⚪ | ⚪ | | | | | | |
GO:0030111: regulation of Wnt signaling pathway | 2 | | ⚪ | | | | | ⚪ | | |
GO:0035176: social behavior | 2 | | | | ⚪ | ⚪ | | | | |
GO:0050905: neuromuscular process | 2 | | | | ⚪ | ⚪ | | | | |
R-HSA-6811442: Intra-Golgi and retrograde Golgi-to-ER traffic | 2 | | | | ⚪ | | | | ⚪ | |
GO:0045197: establishment or maintenance of epithelial cell apical/basal polarity | 2 | | | | | ⚪ | | ⚪ | | |
After screening of enrichment pathways, some enrichment pathways included in the nIHH group (from high to low frequency). |
Co-analysis Of The Three Patient Groups
Nine nIHH patients, 19 VC patients, and five SN patients shared 116 variants, with 28 variant-harbouring genes detected in five or more patients. PRRC2A was the gene most commonly involved, with variants in this gene detected in one SN patient (SN3) and eight nIHH and VC patients. AKAP13 variants were detected in eight patients; MICAL variants were detected in seven patients; and PBRM1, RECQL5, DOCK8, DCAF13, and SLC26A4 variants were detected in six patients. RECQL5 variants were detected in six patients, five of whom had had splice-site variants at locus 73626919 (Table 6). The genetic variants detected in each group are presented in supplementary materials.
In the enrichment pathway analysis, only nine pathways were identified in all three groups of patients. “M5880: NABA ECM AFFILIATED”, “R-HSA-9675108: nervous system development”, and “GO:0090066: regulation of anatomical structure size” were identified in five patients; “R-HSA-9716542: signalling by Rho GTPases”, “Miro GTPases”, and “RHOBTB3” were identified in four patients; and the other pathways were identified in three patients (Table 7). The channels of enrichment analysis in each group are presented in supplementary materials.
Comparison Of Different Variant Sites In The Same Gene
We screened 17 variant-harbouring genes with the highest frequency among patients in the nIHH group (TTN, IST1, NEFH, CCDC177, TRIP10, FAM174B, USH2A, PCLO, CASQ2, BPTF, MUC19, ALMS1, PLEC, NT5DC4, MUC17, RIC8A, and OBSCN) and determined the status of these 17 genes in all patients (Table 8). Variants in two genes, TTN and IST1, were detected in 83% (38/46) and 98% (45/46) of patients, respectively. Variants in NEFH, CCDC177, TRIP10, FAM174B, and USH2A were detected in more than 50% of patients, and the selected 17 genes accounted for more than 25% of the variants detected.
Table 8
Overview of common mutations in four groups
Gene | Proportion(nIHH) | Proportion (SN) | Proportion (VC) | Proportion (NC) | Proportion (Total) |
TTN | 9/9 | 4/5 | 15/19 | 10/13 | 38/46 |
IST1 | 9/9 | 4/5 | 19/19 | 13/13 | 45/46 |
NEFH | 8/9 | 2/5 | 9/19 | 7/13 | 26/46 |
CCDC177 | 8/9 | 0/5 | 4/19 | 4/13 | 16/46 |
TRIP10 | 6/9 | 3/5 | 3/19 | 0/13 | 12/46 |
FAM174B | 6/9 | 2/5 | 10/19 | 7/13 | 19/46 |
USH2A | 6/9 | 0/5 | 6/19 | 4/13 | 16/46 |
PCLO | 5/9 | 0/5 | 2/19 | 0/13 | 7/46 |
CASQ2 | 5/9 | 1/5 | 6/19 | 0/13 | 12/46 |
BPTF | 4/9 | 5/5 | 10/19 | 7/13 | 26/46 |
MUC19 | 4/9 | 3/5 | 5/19 | 2/13 | 14/46 |
ALMS1 | 4/9 | 2/5 | 4/19 | 3/13 | 13/46 |
PLEC | 4/9 | 3/5 | 9/19 | 2/13 | 18/46 |
NT5DC4 | 4/9 | 2/5 | 9/19 | 5/13 | 20/46 |
MUC16 | 3/9 | 4/5 | 8/19 | 8/13 | 23/46 |
RIC8A | 3/9 | 2/5 | 6/19 | 5/13 | 19/46 |
OBSCN | 2/9 | 3/5 | 7/19 | 4/13 | 16/46 |
The sequence of mutant genes is from more to less according to the number of repeats in group nIHH |
For specific variant sites and exons, we present the details of some genes for each group. Of the NEFH variants detected, seven nIHH patients had variants in exon 4; one nIHH patient had a variant in exon 1; and the remaining two SN patients, nine VC patients, and seven NC patients had variants in exon 4. In addition, 21 variants in the NEFH gene were detected in seven nIHH patients, whereas only six NEFH variants were detected in the SN group, 12 in the VC group, and 11 in the NC group.
Seven nIHH patients had a CCDC177 variant located in exon 1, and all of these were at locus 70039793. There were three and four CCDC177 variants detected in the VC and NC groups, respectively. These variants were all located in exon 1 but were not all at the same site as the variant detected in the nIHH patients. PCLO variants were almost exclusively found in nIHH patients, and five of the six PCLO variants were in exon 5.
For OBSCN, two variants were detected in the nIHH group, three were detected in the SN group, seven were detected in the VC group, and four were detected in the NC group. The OBSCN variants were scattered across 16 exons. There were 19 TTN variants in the nIHH group, seven in the SN group, 33 in the VC group, and 23 in the NC group, scattered across 47 exons. Exon 276 was the most frequent site for TTN variants, with four in the nIHH group, one in the SN group, two in the VC group, and four in the NC group. The details of the variant sites are provided in supplementary materials .