Copyright
©The Author(s) 2021.
World J Diabetes. Dec 15, 2021; 12(12): 2058-2072
Published online Dec 15, 2021. doi: 10.4239/wjd.v12.i12.2058
Published online Dec 15, 2021. doi: 10.4239/wjd.v12.i12.2058
Table 1 List of primer pairs used for polymerase chain reaction
| Patient | Gene | Template ID | Forward Primer | Reverse Primer | Amp Size (bp) |
| Patient 1 | IGSF10 | chr4:39435930-39435950 | ACATTTCCGCCCACATCAGAAG | TCAGCTGTGCCTCTCATCTCAT | 246 |
| Patient 2 | IGSF10 | chr3:151161270-151161285 | TAACAGGTGGTGCTGCAATGAC | AAGCACTGTGGAACTGAAGTGC | 251 |
| Patient 3 | KLB | chr3:151164660-151164670 | AGCAATGTCAGCTTTGGGGAAG | GCTTTGGGAGGCAGAGGAAAAT | 260 |
| Patient 4 | ANOS1 | chrX:8565100-8565108 | TGTGACACTGCATGTGTCTTCAC | TGACCAGCTGTGAGTTCCTCAA | 236 |
Table 2 Gonadotropin-releasing hormone stimulation test result
| Dose (mIU/ml) | 0 | 15 min | 30 min | 60 min | 120 min | |
| Patient 1 | FSH | 0.88 | 1.33 | 1.72 | 2.47 | 3.07 |
| LH | 0.66 | 0.98 | 1.22 | 2.31 | 2.67 | |
| Patient 2 | FSH | 0.69 | 1.19 | 1.26 | 1.61 | 3.07 |
| LH | 0.23 | 0.32 | 0.38 | 0.64 | 2.61 | |
| Patient 3 | FSH | 0.85 | 1.29 | 1.64 | 1.88 | 2.42 |
| LH | 0.63 | 0.84 | 1.02 | 1.36 | 1.53 | |
| Patient 4 | FSH | 0.78 | 1.30 | 1.44 | 1.84 | 2.02 |
| LH | 0.68 | 0.94 | 1.29 | 1.33 | 1.63 |
Table 3 List of gene related to Kallmann syndrome and normosmic hypogonadotropic hypogonadism
| Disease | Gene (Phenotype MIM number) | Count |
| Kallmann syndrome and normosmic hypogonadotropic hypogonadism | FGFR1(147950), FGF8 (612702), PROK2 (610628), CHD7 (612370), WDR11 (614858) | 5 |
| Normosmic hypogonadotropic hypogonadism | LEP (614962), LEPR (614963), NR0B1 (300200), SRA1, GNRHR (146110), GNRH1 (614841),KISS1R (614837), KISS1 (614842), TACR3 (614840), TAC3 (614839), NR5A1, HESX-1, LHX3, SOX2, FSHB (229070), LHB (228300), PC1, PNPLA6 (215470), RNF216, OTUD4, STUB1, POLR3A (607694), POLR3B (614381), RAB3GAP1, RAB3GAP2, RAB18, TBCID20, DMXL2, KISS1R(614837), NDNF (618841) | 30 |
| Kallmann syndrome | ANOS1 (308700), FGF17 (615270), IL17RD (615267), DUSP6 (615269), SPRY4 (615266), FLRT3 (615271),, KLB, PROKR2 (244200), SEMA3A (614897), SEMA3E, SOX10, HS6ST1 (614880), CCDC141, FEZF1 (616030), IGSF10, SMCHD1, NELF (614838), SOX3 | 18 |
Table 4 Sequence alignment and sequencing depth
| Sample | Patient 1 | Patient 2 | Patient 3 | Patient 4 |
| Raw reads (PEM) | 36.83373 | 39.8752 | 39.856 | 39.975 |
| reads mapping rate (%) | 99.59 | 99.58 | 99.64 | 99.65 |
| Target duplication rate (%) | 14.15 | 14.32 | 14.59 | 14.75 |
| Target mean depth | 130.55 | 139.46 | 151.43 | 145.43 |
| T 10X coverage rate (%) | 99.57 | 99.6 | 99.45 | 99.27 |
| T 20X coverage rate (%) | 99.04 | 99.13 | 99.26 | 99.04 |
| T 30X coverage rate (%) | 97.98 | 98.23 | 98.57 | 98.35 |
Table 5 Distribution of single-nucleotide polymorphisms
| Sample | Patient 1 | Patient 2 | Patient 3 | Patient 4 |
| Total | 82986 | 84748 | 84579 | 84731 |
| dbsnp, n (%) | 82205 (99.06) | 83887 (98.98) | 83752 (99.02) | 83835 (98.94) |
| 1000g_EAS, n (%) | 77404 (93.27) | 78717 (92.88) | 78737 (93.09) | 78824 (93.03) |
| ExAC_EAS, n (%) | 45558 (54.90) | 45630 (53.84) | 45773 (54.12) | 45936 (54.21) |
| GnomAD_exome_EAS, n (%) | 45617 (54.97) | 45698 (53.92) | 45866 (54.23) | 46025 (54.32) |
| GnomAD_genome_EAS, n (%) | 81924 (98.72) | 83549 (98.59) | 83441 (98.65) | 83536 (98.59) |
| Exonic, n (%) | 22847 (27.53) | 23016 (27.16) | 23100 (27.31) | 22921 (27.05) |
| Splicing, n (%) | 239 (0.29) | 246 (0.29) | 251 (0.30) | 259 (0.31) |
| UTR3, n (%) | 3103 (3.74) | 3161 (3.73) | 3152 (3.73) | 3084 (3.64) |
| UTR5, n (%) | 2234 (2.69) | 2310 (2.73) | 2305 (2.73) | 2333 (2.75) |
| Intronic, n (%) | 48754 (58.75) | 50084 (59.10) | 49683 (58.74) | 50121 (59.15) |
| Intergenic, n (%) | 1902 (2.29) | 2108 (2.49) | 2184 (2.58) | 2145 (2.53) |
| Upstream, n (%) | 816 (0.98) | 881 (1.04) | 882 (1.04) | 827 (0.98) |
| Downstream, n (%) | 371 (0.45) | 379 (0.45) | 379 (0.45) | 366 (0.43) |
| Ncrna_exonic, n (%) | 764 (0.92) | 726 (0.86) | 730 (0.86) | 790 (0.93) |
| Ncrna_splicing, n (%) | 6 (0.01) | 8 (0.01) | 4 (0.00) | 3 (0.00) |
| Ncrna_intronic, n (%) | 1888 (2.28) | 1764 (2.08) | 1848 (2.18) | 1823 (2.15) |
| Synonymous SNV, n (%) | 11529 (13.89) | 11613 (13.70) | 11559 (13.67) | 11518 (13.59) |
| Nonsynonymous SNV, n (%) | 10755 (12.96) | 10727 (12.66) | 10815 (12.79) | 10749 (12.69) |
| Stopgain, n (%) | 85 (0.10) | 92 (0.11) | 94 (0.11) | 84 (0.10) |
| Stoploss, n (%) | 10 (0.01) | 10 (0.01) | 11 (0.01) | 8 (0.01) |
| Unknown, n (%) | 483 (0.58) | 589 (0.70) | 635 (0.75) | 576 (0.68) |
Table 6 Indel distribution
| Sample | Patient 1 | Patient 2 | Patient 3 | Patient 4 |
| Total | 13495 | 13931 | 13760 | 13794 |
| dbsnp, n (%) | 12301 (91.15) | 12675 (90.98) | 12538 (91.12) | 12551 (90.99) |
| 1000g_EAS, n (%) | 8269 (61.27) | 8454 (60.68) | 8378 (60.89) | 8410 (60.97) |
| ExAC_EAS, n (%) | 5560 (41.20) | 5547 (39.82) | 5562 (40.42) | 5637 (40.87) |
| GnomAD_exome_EAS, n (%) | 5306 (39.32) | 5284 (37.93) | 5286 (38.42) | 5355 (38.82) |
| GnomAD_genome_EAS, n (%) | 12567 (93.12) | 12991 (93.25) | 12760 (92.73) | 12812 (92.88) |
| Exonic, n (%) | 708 (5.25) | 733 (5.26) | 707 (5.14) | 694 (5.03) |
| Splicing, n (%) | 199 (1.47) | 182 (1.31) | 201 (1.46) | 192 (1.39) |
| UTR3, n (%) | 669 (4.96) | 676 (4.85) | 667 (4.85) | 661 (4.79) |
| UTR5, n (%) | 375 (2.78) | 384 (2.76) | 364 (2.65) | 373 (2.70) |
| Intronic, n (%) | 10469 (77.58) | 10845 (77.85) | 10712 (77.85) | 10767 (78.06) |
| Intergenic, n (%) | 297 (2.20) | 312 (2.24) | 309 (2.25) | 307 (2.23) |
| Upstream, n (%) | 160 (1.19) | 187 (1.34) | 170 (1.24) | 184 (1.33) |
| Downstream, n (%) | 51 (0.38) | 65 (0.47) | 66 (0.48) | 68 (0.49) |
| Ncrna_exonic, n (%) | 103 (0.76) | 93 (0.67) | 102 (0.74) | 99 (0.72) |
| Ncrna_splicing, n (%) | 0 (0.00) | 2 (0.01) | 4 (0.03) | 1 (0.01) |
| Ncrna_intronic, n (%) | 407 (3.02) | 398 (2.86) | 400 (2.91) | 391 (2.83) |
| Frameshift insertion, n (%) | 94 (0.70) | 103 (0.74) | 96 (0.70) | 100 (0.72) |
| Frameshift deletion, n (%) | 135 (1.00) | 124 (0.89) | 132 (0.96) | 137 (0.99) |
| Nonframeshift insertion, n (%) | 198 (1.47) | 216 (1.55) | 200 (1.45) | 181 (1.31) |
| Nonframeshift deletion, n (%) | 217 (1.61) | 215 (1.54) | 202 (1.47) | 203 (1.47) |
| Stopgain, n (%) | 7 (0.05) | 8 (0.06) | 9 (0.07) | 10 (0.07) |
| Stoploss, n (%) | 1 (0.01) | 1 (0.01) | 0 (0.00) | 1 (0.01) |
| Unknown, n (%) | 99 (0.73) | 106 (0.76) | 111 (0.81) | 103 (0.75) |
Table 7 Specific information about IGSF10, KLB, ANOS1 mutation
| Type | Patient 1 | Patient 2 | Patient 3 | Patient 4 |
| Chr.Start.End | chr3.151161279.151161279 | chr3.151164665.151164665 | chr4.39435942.39435942 | chrX.8565101.8565101 |
| Vcf_mut | T/C | C/G | C/T | C/A |
| GT | 0/1 | 0/1 | 0/1 | 1/1 |
| AD | 51/53 | 30/31 | 34/47 | 0/86 |
| AAChange.HGVS | IGSF10:NM_178822.4:5/6:c.5456A>G:p.(Lys1819Arg) | IGSF10:NM_178822.4:4/6:c.3104G>C:p.(Arg1035Thr) | KLB:NM_175737:exon2:c.C938T:p.T313M | ANOS1:NM_000216:exon4:c.G515T:p.C172F |
| cytoBand | 3q25.1 | 3q25.1 | 4p14 | Xp22.31 |
| InterVar_automated | Uncertain significance | Uncertain significance | Uncertain significance | Uncertain significance |
| ACMG(missense only) | PM1,PM2,BP4 | . | PM1,BP4 | PM1,PM2,PP3 |
| gnomAD_exome_ALL | . | 0.0004 | 0.0001 | 0 |
| SIFT_pred | T | D | T | D |
| Polyphen2_HDIV_score | 0.011 | 0.981 | 0.036 | 1 |
| Polyphen2_HDIV_pred | B | D | B | D |
| Polyphen2_HVAR_score | 0.056 | 0.69 | 0.016 | 1 |
| Polyphen2_HVAR_pred | B | P | B | D |
| LRT_score | 0.039 | 0 | 0.59 | 0 |
| LRT_pred | N | D | N | D |
| MutationTaster_score | 0.808 | 1 | 1 | 1 |
| MutationTaster_pred | D | N | N | D |
| MutationAssessor_score | 0.15 | 2.47 | 2.535 | 4.455 |
| MutationAssessor_pred | N | M | M | H |
| FATHMM_score | -0.27 | -0.65 | 1.43 | -5.61 |
| FATHMM_pred | T | T | T | D |
| CADD_raw | 0.585 | 1.94 | -0.287 | 6.358 |
| CADD_phred | 8.054 | 15.84 | 0.711 | 29.4 |
| fathmm-MKL_coding_score | 0.039 | 0.257 | 0.068 | 0.967 |
| fathmm-MKL_coding_pred | N | N | N | D |
| GERP++_RS | 0.193 | 5.46 | -7.48 | 4.51 |
- Citation: Sun SS, Wang RX. Molecular diagnosis of Kallmann syndrome with diabetes by whole exome sequencing and bioinformatic approaches. World J Diabetes 2021; 12(12): 2058-2072
- URL: https://www.wjgnet.com/1948-9358/full/v12/i12/2058.htm
- DOI: https://dx.doi.org/10.4239/wjd.v12.i12.2058