3.1. Laboratory data
In patient 1, the level of 25-hydroxyvitamin D was found to be 11.78 ng/mL. Liver function, renal function, erythrocyte sedimentation rate, and the levels of electrolytes, calcium, phosphate, parathyroid hormone, and C-reactive protein were normal. Blood osmotic pressure was 297 mOsm/kg·H20 and urine osmotic pressure was 620 mOsm/kg·H20. The growth hormone level was found to be 0.895 ng/mL. No abnormalities were observed in the thyroid function test. The cortisol circadian rhythm pattern was as follows: 08:00 120 ng/mL; 16:00 106.6 ng/mL; 00:00 13.93 ng/mL. The 24-h urine-free cortisol level was 188.40 µg/24 h. Color Doppler ultrasound examination revealed that the uterus size was small. The levels of the following sex hormones were measured: blood prolactin, 94.67 µIU/mL; estradiol, 13.7 pg/mL; progesterone, 0.303 ng/mL; testosterone 0.13 ng/mL; LH <0.1 mIU/mL; FSH 0.894 mIU/mL.
The GnRH stimulation test revealed that the peak values of LH and FSH exceeded 1 mIU/mL, which indicated stimulation, see Table 1 . MRI scan of the pituitary and CT scan of the adrenal glands revealed no abnormalities. No abnormal lesions were observed in the uterus and breasts.
The following observations were made in patient 2: ACTH 08:00 91.32 pg/mL; alanine aminotransferase 12.58 IU/L; aspartate aminotransferase 12.81 IU/L; blood creatinine 43.75 μmol/L; urine osmotic pressure 898 mOsm/kg·H20; blood osmotic pressure 291 mOsm/kg·H20.
The GnRH stimulation test revealed that the basal values of LH and FSH secretion were low. Both peaked at 120 min, but the values did not exceed 1 mIU/ml, see Table 1. The levels of the following sex hormones were tested: blood prolactin, 201.1 µIU/mL; estradiol, 14.12 pg/mL; progesterone, 0.479 ng/mL; testosterone 0.361 ng/mL; LH <0.1 mIU/mL; FSH 0.257 mIU/mL. The cortisol rhythm was found to be normal, and MRI scan of the pituitary and CT scan of the bilateral adrenal glands revealed no obvious abnormalities.
In patient 3, Urine protein 2+; urine glucose 3+, ketone body 3+, blood ketone 2.8mmol/L, the growth hormone level was found to be 0.301 ng/mL. No abnormalities were observed in the thyroid function test. The cortisol circadian rhythm pattern was as follows: 08:00 103.1 ng/mL; 16:00 29.67 ng/mL; 00:00 37.39 ng/mL. The levels of the following sex hormones were measured: blood prolactin, 350.6 µIU/mL; estradiol, 21.17 pg/mL; progesterone, 1.07 ng/mL; testosterone 0.46 ng/mL; LH <0.1 mIU/mL; FSH 0.61 mIU/mL. THE islet function test, see Table 2
The GnRH stimulation test revealed that the peak values of LH and FSH exceeded 1 mIU/mL, which indicated stimulation, see Table 1
In patient 4, the growth hormone level was found to be 0.175 ng/mL. No abnormalities were observed in the thyroid function test. The cortisol circadian rhythm pattern was normal. The levels of the following sex hormones were measured: blood prolactin157.2 µIU/mL; estradiol, 5 pg/mL; progesterone, 0.13 ng/mL; testosterone 0.46 ng/mL; LH <0.1 mIU/mL; FSH 0.91 mIU/mL.
The GnRH stimulation test revealed that the peak values of LH and FSH exceeded 1 mIU/mL, which indicated stimulation, see Table 1
3.2. Genetic testing
Quality control of raw WES data
Quality control analysis of the raw WES data (using FastQC) of the two samples is illustrated in Figure 1. The average quality of the bases was greater than 30 (accuracy greater than 99.9%), and the sequence quality was satisfactory.
3.2.1. Sequence alignment and sequencing depth
Exome sequencing of the two samples yielded 39M paired-end reads, of which 99% (mapped reads) sequences could be matched to the human reference genome, and the proportion of duplicate reads was approximately 15%. The average sequencing depth (mean depth) exceeded 130X. see Table 3.
3.2.2. Extent of variation
The bioinformatics analysis revealed that the sample from patient 1 had 82,986 SNPs and 13,495 INDELs. Through dbSNP annotation, 99.06% of the SNPs and 91.15% of the INDELs could be annotated.
Patient 2 had 84,748 SNPs and 13,931 INDELs. Through dbSNP annotation, 98.98% of SNPs and 90.98% of the INDELs could be annotated.
Patient 3 had 84,579 SNPs and 13,760 INDELs. Through dbSNP annotation, 99.02% of SNPs and 90.99% of the INDELs could be annotated.
Patient 4 had 84,731 SNPs and 13,794 INDELs. Through dbSNP annotation, 98.94% of SNPs and 90.99% of the INDELs could be annotated. see Table 4 and Table 5
3.2.3. Analysis of candidate gene mutations
The gene mutations were filtered according to the following criteria: (1) the mutation should be located in the exon; (2) the mutation should not be synonymous; (3) population frequency should be greater than 0.001.
After filtering, only two mutation sites were detected (one each in IGSF10, and CHD72 genes) for patient 1, whereas four mutation sites were detected (one each in SOX6, DMXL2, IGSF10, and ANOS1 genes) for patient 2. One mutation sites were detected in the KLB for patient 3,one mutation sites were detected in the ANOS1 for patient 4. Mutations in the IGSF10 gene were common to both patients. Furthermore, the literature search revealed that the genes CHD72, ANOS1, IGSF10, and DMXL2 were also related to IHH.
3.2.4. Verification of candidate sites
To verify the pathogenic sites in the four patients, we compared the parental genotypes and found that IGSF10 (p.Lys1819Arg), KLB p.T313M and ANOS1 p.C172F may harbor the pathogenic site, Population data did not reveal the presence of a mutation at this site, and the mutation frequency of p.Arg1035Thr in the gnomAD database was found to be 0.0004. and the mutation frequency of p.T313M in the gnomAD database was found to be 0.0001. and the mutation frequency of p.C172F in the gnomAD database was found to be 0. which is illustrated in Table 6 and Figure 2. List of primer pairs used for PCR, see Table 7.
MutationTaster predicted Lys1819Arg to be a harmful mutation, whereas SIFT_pred and Polyphen2_HDIV predicted Arg1035Thr to be a harmful mutation. whereas SIFT_pred predicted p.C172F to be a harmful mutation.The two mutations of IGSF10 are located in the region encoding the immunoglobulin I-set domain and in the nondomain region. The mutations of ANOS1 are located in the region encoding the WAP domain,the mutations of KLBare are located in the region encoding the Glyco_hydro_1 domain. (see Figure 3)