Authors: Tian Wang, Wu Ren, Fangfang Fu, Hairong Wang, Yan Li, Jie Duan
Categories: Research Article, CHARGE syndrome, CHD7, Idiopathic hypogonadotropic hypogonadism, Intrafamilial phenotypic variability, Oligogenicity, SMCHD1
Source: Heliyon
Authors: Tian Wang, Wu Ren, Fangfang Fu, Hairong Wang, Yan Li, Jie Duan
CHARGE syndrome is a congenital hereditary condition involving multiple systems. Patients are easily misdiagnosed with idiopathic hypogonadotropic hypogonadism (IHH) due to the overlap of clinical manifestations. An accurate clinical diagnosis remains challenging when the predominant clinical manifestation resembles hypogonadotropic hypogonadism.
This original research is conducted based on the genetic finding and analysis of clinical cases. Whole-exome sequencing (WES) and in-silico analyse were performed on two sisters to investigate the pathogenesis in this family. Homology modelling was conducted to evaluate structural changes in the variants.
WES and Sanger sequencing revealed two siblings carrying a nonsense mutation (NM_017780.4: c.115C > T) in exon 2 of CHD7 inherited from a mildly affected mother and a missense mutation (NM_015295.3: c.2582T > C) in exon 20 of SMCHD1 inherited from an asymptomatic father. The nonsense mutation in CHD7 was predicted to generate nonsense-mediated decay, whereas the missense mutation in SMCHD1 decreased protein stability.
We identified digenic CHD7 and SMCHD1 mutations in IHH-associated diseases for the first time and verified the synergistic role of oligogenic inheritance. It was also determined that WES is an effective tool for distinguishing diseases with overlapping features and establishing a molecular diagnosis for cases with digenic or oligogenic hereditary disorders, which is beneficial for timely treatment, and family genetic counseling.
CHARGE syndrome is a complex congenital hereditary condition involving multiple systems, including the ocular, cardiac, aural, reproductive, and endocrine systems. The manifestations of CHARGE syndrome are vary and include colobomas, heart defects, choanal atresia, developmental delay, genital hypoplasia, ear anomalies and deafness [1]. Hypogonadotropic hypogonadism, is characterized by the insufficient synthesis and secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) synthesis and secretion, due to disturbances or dysfunction of the hypothalamic–pituitary–gonadal (HPG) axis [2,3]. Hypogonadotropic hypogonadism is a prominent clinical feature of CHARGE syndrome, that may result in delayed sexual maturation, failure to develop secondary sexual characteristics, and abnormal genitalia. Additionally, it may result in infertility, resulting enormous psychological and physiological challenges for affected individuals and their families [4]. Hypogonadotropic hypogonadism presents in several different genetic disorders, such as idiopathic hypogonadotropic hypogonadism (IHH), Waardenburg syndrome (WS), Bardet-Biedl syndrome (BBS) and Turner Syndrome [1].
IHH is a group of rare reproduction-related genetic disorders attributed to the in-sufficiency of gonadotropin-releasing hormone (GnRH). The major clinical features of IHH include absent or delayed puberty, underdeveloped secondary sexual characteristics, and variable non-reproductive features [5]. The non-reproductive features of the IHH include cleft lip/palate, conductive deafness, ear anomalies, congenital heart disease, renal anomalies, dental agenesis, visual impairment and intellectual disabilities, and so on [5,6]. There are two subtypes of IHH: Kallmann syndrome (KS) with anosmia or hyposmia and normosmic idiopathic hypogonadotropic hypogonadism (nIHH) with a normal sense of smell [7].
In addition to the overlapping characteristics, CHARGE syndrome and IHH also share the causal genes, CHD7. CHD7 gene encodes an integral membrane protein. It belongs to the chromatin remodeling family, which manipulates chromatin structure to activate and repress transcription [8]. The CHD7 mutations have been reported in more than 90 % of patients with CHARGE syndrome [9] and 3–10 % of patients with IHH [[10], [11], [12]]. In addition to CHD7 gene, more than 100 genes [13] known to involving in various developmental processes, such as migration, secretion, fate specification and olfactory axon guidance [5,10], have been reported to be associated with IHH.
Given the overlapbetween CHARGE syndrome and IHH, as well as the variable penetrance and expressivity of the pathogenic gene [14], it is challenging to deter-mine which specific genetic disorder it belongs to, especially when hypogonadotropic hypogonadism is a leading symptom of the patient. Hencs, genetic testing is a valuable tool to distinguish diseases with overlapping features. Moreover, some additional features have been discovered in patients with IHH by following careful examination of their genetic results. Therefore, whole-exome sequencing (WES) may help improve therapeutic and management strategies for affected individuals and families [5,15].
SMCHD1 belongs to the SMC gene superfamily, a family of proteins that involved in chromosome condensation and cohesion, genome maintenance and gene regulation. SMCHD1 has been implicated in X staining inactivation and epigenetic gene silencing through CpG island methylation modification. Researchers have found that SMCHD1 could play important role in the development of autosomal dominant inherited diseases such as facioscapulohumeral muscular dystrophy [16]. Herein, we report a Chinese family comprised of three patients who manifested variable features among the family members, mainly exhibiting hypogonadotropic hypogonadism with novel digenic disease-causing mutations in CHD7 and SMCHD1 by WES.
This study was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (TJ-IRB20211016). Written informed consent was obtained from all participants.
The proband and her older sister underwent WES. DNA was extracted from the peripheral blood of the patients using Qiagen DNA Blood Midi/Mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol. Then, 50 ng genomic DNA was interrupted to generate around 200 bp fragments, which were then subjected to end-repair, deoxyadenosine addition at the 3′ends, and adaptors ligation. Subsequently, DNA fragments were hybridized and captured using Berry's NanoWES Human Exome V1.0 (Berry Genomics, Beijing, China) following the manufacturer's instructions. The amplified and qualified library was sequenced on the Novaseq 6000 platform (Illumina, San Diego, USA) with an average depth of 100-fold coverage, and raw sequence reads were generated as 150bp paired end. Sequencing reads were aligned to the human genome assembly hg38 (GRCh38) using the Burrows–Wheeler Aligner (BWA) tool after removing adaptor sequences. Variant calling was performed with GATK (https://software.broadinstitute.org/gatk/) and Verita Trekker® Variants Detection System by Berry Genomics. Furthermore, variant annotation was performed using ANNOVAR [13] and the Enliven® Variants Annotation Interpretation System (Berry Genomics). The rare variants with minor allele frequencies (MAF) < 1 % in protein-coding regions in population databases (dbSNP, gnomAD, 1000 Genomes, and Shenzhou Genome Database of the Berry Genomics) were taken for further inter-pretation. In total, four prediction tools (SIFT, Polyphen2, MutationTaster, CADD) were used to assess variant pathogenicity, and the conservation scores were evaluated using GERP and PhyloP. The interpretation pathogenicity of variants was classified based on the guideline of American College of Medical Genetics and Genomics (ACMG).
The two candidate variants identified by WES were verified in the two siblings and their parents using Sanger sequencing. The primers were designed using Primer3 and the products were analyzed using an ABI 3730XL DNA Analyzer.
Protein domains of the CHD7 were generated by Illustrator for Biological Sequences (IBS) software [17]. Conservative predictions of CHD7 and SMCHD1 were conducted using UGENE software [18]. The three-dimensional (3D) structure of SMCHD1 was analyzed by PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC), using AF-A6NHR9-F1-model_v4 as the template, which was generated by Al-phaFold (https://alphafold.ebi.ac.uk). Prediction of protein stability was carried out with DUET (http://biosig.unimelb.edu.au/duet/stability) and I-Mutant (http://gpcr.biocomp.unibo.it/cgi/predictors/I-Mutant3.0/I-Mutant3.0.cgi), respectively. The potential phosphorylation site was predicted with NetPhos-3.1 (http://www.cbs.dtu.dk/services/NetPhos/).
We searched the PubMed to investigate the cases of oligogenic inheritance together with CHD7 gene. The key words are as CHD7, Kallmann syndrome, hypogonadotropic hypogonadism, digenic and oligogenic. The chromosomal locations of the partner genes with CHD7 were determined using tools on the NCBI (http://www.ncbi.nlm.nih.gov/genome/tools/gdp). The protein-protein interactions were predicted with Genemania tool (https://genemania).
A 15-year-old Chinese girl was referred to gynecologic department of state hospital in October 2018 with primary amenorrhea and the developmental retardation of secondary sexual characteristics. She was born in April 2003 by an uneventful spontaneous delivery from a non-consanguineous family (Fig. 1A). The patient presented with delayed puberty, hyposmia, short stature (height: 150 cm), protruding ears and mild mental retardation (Fig. 1B) (Table 1). She self-reported the congenital deafness and underwent cochlear implantation at a young age. An olfactory test revealed that she was unable to identify alcohol, vinegar or water. The clinical characteristics of the proband were indicative of KS.Fig. 1Pedigrees, clinical characteristics, and detection of variants in the family with IHH-associated diseases. (A) Pedigree of the affected family. Arrows indicate the probands. (B): Ear and breast of the proband. (C) Sanger sequencing of two variants in the affected family (upper c.115C > T (p.Gln39Ter); lower c.2582T > C (p.Ile861Thr)).22Y07415: proband; 22Y07415FU0: father; 22Y07415MU0: mother; 22Y07415JJ0: sister.Fig. 1Table 1Clinical manifestations of three affected individuals in the family.Table 1Diagnostic criteriaaProbandSisterMotherIncidence in CS patients Incidence in CS patients [19]Age (years)192452MajoraColobomaAbsentAbsentAbsent66 %Choanal atresia or cleft lip or palateAbsentAbsentAbsent47 %Abnormal external, middle, or inner ears, including hypoplastic semicircular canalsProtruding earsAbsentAbsent94 %Pathogenic CHD7 variant✓✓✓MinoraCranial nerve dysfunction including hearing lossAnosmia, hearing lossAbsentAnosmia69–90 %Dysphagia/feeding difficultiesAbsentAbsentAbsent82 %Structural brain anomaliesAbsentAbsentAbsent50 %Developmental delay/intellectual disabilities/autismMild intellectual disabilitiesAbsentAbsent88 %Hypothalamo-hypophyseal dysfunction and genital anomaliesPrimary amenorrhea, delayed pubertyPrimary amenorrhea, delayed pubertySpaniomenorrhea68 %Cardiac anomaliesAbsentTetralogy of fallot, atrial septal defectsAbsent78 %Renal anomaliesSkeletal/limb anomaliesAbsentAbsentAbsent46–80 %aAccording to diagnostic criteria by Hale [19]. CHARGE syndrome (CS): 2 majors + any number of minor criteria.
The patient's breast development had not initiated, and a swab could be inserted into the vagina up to approximately 8 cm. Subsequently, an endocrine test revealed inappropriately low levels of follicle stimulating hormone (FSH: 0.34 IU/L) and luteinizing hormone (LH: <0.1 IU/L) (Table 2). However, the patient's karyotype was normal (46, XX). Therefore, the patient was continuously treated with oral estradiol valerate for 21 days and progesterone for 10 days per cycle starting in November 2018. Following the administration of the hormone therapy, the patient experienced withdrawal bleeding. Regular reviews of liver and kidney function indicated continuously elevated alkaline phosphatase levels and treatment for liver protection was administered simultaneously. Nine months after initial treatment, she was switched to oral estradiol tablets/estradiol and dydrogesterone tablets due to side effects on the liver.Table 2Basal pituitary hormones’ assessment and gynecological transrectal ultrasonography results of siblings.Table 2MeasuresProbandSister2018.102019.052020.082021.022022.012023.032017.052020.09FSH(IU/L)0.34/<0.10<0.10/<0.05<0.1<0.1LH(IU/L)<0.10/<0.10<0.10/0.03<0.1<0.1E2(pg/mL)5.12/16.7911.15/204.847.887.24PRL(ng/mL)12.14/9.858.96/11.988.8711.40T(ng/mL)<0.025/0.1110.041/0.2500.0450.063P(ng/mL)//<0.050.224/0.100.435<0.05Size of uterine (cm)2.21.91.21.91.73.32.82.1//3.93.42.6//Anteroposterior diameter of cervix(cm)1.31.1//1.72.5//Endometrial thickness(cm)0.290.19//0. 60.9//Size of left ovary(cm)unclear1.40.8//1.80.8///Size of right ovary(cm)2.21.41.30.9//1.5*0.8///Antral follicle count////////“/” indicates not detected.
Regular reviews of basal pituitary hormone levels and gynecological transrectal ultrasonography results are shown in Table 2. At the age of 18, physical examination indicated a loss of breast development, sparse pubic or axillary hair, naive vulva, and a height of 144 cm in March 2021. Additionally, bone's age (15 years old) lagged behind her chronological age (18 years old). After another 4 months (Jul 2021), her breast development had progressed (Tanner stage 3), pubic or axillary hair was visible, and height had increased to 148 cm. In addition, FSH and LH levels were both lower than 0.1 IU/L at the subsequent follow-ups (Table 2).
The patient was later evaluated at the age of 19. Her height and weight were 150 cm and 38 kg, respectively. Moreover, FSH and LH were still less than 0.1 IU/L. Notably, pubertal development corresponded to Tanner stage 3. Computed tomography (CT) of the temporal bone revealed normal semicircular canals. Additionally, tests of eyes, teeth, extremities and joints showed normal results. Furthermore, ultrasound of thyroid, liver, gallbladder, spleen, pancreas, kidney and heart revealed no significant abnormalities. Finally, adrenocorticotropic hormone (ACTH) and growth hormone levels were <1.00 (normal 7.2–63.3 pg/ml) and 0.53 ng/ml, respectively.
Patient 2 was the older sister of the proband. She exhibited persistent cyanosis of the lips and distal extremities at birth (Aug 1997). At the age of 4 years, she underwent cardiac surgery as the ultrasound results indicated tetralogy of fallot and atrial septal defects. She presented with delayed puberty and was diagnosed with hypogonadotropic amenorrhea at 18 years of age (Table 1). Subsequently, the patient received hormone replacement therapy and induced menstrual cycles. Her breasts developed quickly, and her height increased significantly one month after the therapy. In addition, she had normal senses of smell and exhibited no other clinical deformities. The sub-sequent follow-ups of the basal pituitary hormones are listed in Table 2.
The proband's mother was 52 years old (height: 150 cm, menopause at 48 years). She had a history of spaniomenorrhea with menstrual cycle of 3–4 months and experienced menopause at 48 years of age. She also exhibited olfactory dysfunction (Table 1).
Among the family members, the third sibling had a normal pregnancy history. However, the patient developed cerebral palsy and osteomalacia. He died at the age of approximately 8 months. Notably, the paternal grandparents exhibited bilateral congenital blindness, whilethe father (height: 170 cm) and other family members had no significant history of delayed puberty, anosmia, hearing impairment, congenital blindness, cardiac anomalies, or other anomalies.
The proband and her older sister were subjected to WES. The coverage at a depth of 20X as 98.50 % and 98.27 % for the proband and their sister, respectively.
Two heterozygous variants were revealed and shared by two siblings, which were inherited from the mildly affected mother and asymptomatic father respectively (Fig. 1C). A maternally inherited variant at the start of exon 2 in CHD7 (NM_017780.4: c.115C > T; p.Gln39Ter) generated a substitution of glutamine with a stop codon at position 39. As a result, the truncated CHD7 lost all functional domains (Fig. 2A and B). Furthermore, multiple functional con-sequence annotation algorithms predicted that this mutation would be deleterious (Table S1). The wild Gln39 in this sequence was evolutionarily conserved across various species (Fig. 2C). This variant was neither recorded by any human public database, nor described in previous literature. Accordingly, it was classified as pathogenic based on the following null variant with very strong evidence of pathogenicity (PVS1), absence in control population databases (PM2), and co-segregation with the disease in three affected members (PP1).Fig. 2Structure and functional analysis of c.115C > T; p.Gln39Ter mutation in CHD7.. (A) The premature termination codon arise from c.115C > T(p.Gln39Ter). (B) Truncated protein of CHD7 arise from c.115C > T(p.Gln39Ter). (C) Conservation of the Gln39 across various species.Fig. 2
The other identified variant in the patients was a paternally inherited variant (NM_015295.3: c.2582T > C; p.Ile861Thr) that induced a novel missense substitution in exon 20 of the SMCHD1 gene. This variant has been found at an extremely low frequency in the eastern populations within gnomAD (0.0005823) and ExAC (0.0001) databases. Moreover, the p.Ile861Thr mutation in SMCHD1 was predicted to be damaging, possibly damaging, disease-causing, tolerable, conserved and conserved, using SIFT, Polyphen2, Mutation Taster, CADD, GERP and phyloP tools respectively (Table S1). Multiple sequence alignment of SMCHD1 amino acid sequences with UGENE demonstrated the c.2582T > C (p.Ile861Thr) variant was in a conserved region among species (Fig. 3A). Homology modeling of SMCHD1 protein was performed using AlphaFold and visualized using PyMOL (Fig. 3B). The results indicated the mutant Thr861 formed a new hydrogen bond with adjacent Pro909, unlike Ile861 (Fig. 3C and D), which could create a significant conformational alteration and potentially destabilize the protein structure.Fig. 3Schematic representation of c.2582T > C; p.Ile861Thr mutation in SMCHD1 protein models. (A) Conservation of the ILE861 across various species. (B) The red arrow points to the position of ILE861 in the SMCHD1 protein model. (C) Stick models shows the amino acids around ILE861. (D) A new hydrogen bond between Thr861 and Pro909 (orange arrow) was created in mutant type. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)Fig. 3
We predicted the stability of the mutation protein using DUET and the result indicated decreased protein stability (ΔΔG value = −1.285 kcal/mol). The other prediction tool, I-Mutant also supported the decreased stability of the protein (ΔΔG value = −1.63 kcal/mol). NetPhos-3.1 (http://www.cbs.dtu.dk/services/NetPhos/) suggested the mutation p.Ile861Thr introduced a potential Thrphosphorylation site which phosphorylated by protein kinase DNAPK and cdc2. Collectively, this variant was listed as having un-known significance (VUS) (PM2, PP2) based on ACMG criteria.
We compiled the literature and found that CHD7 gene has been associated with CS or KS/nIHH in a digenetic or oligogenic manner with other known 24 IHH-causing genes in 36 cases (including the current study) (Table 3). Notably, The majority of the cases were diagnosed as KS or nIHH. Almost 66.67 % (24/36) of the participants were Chinese in origin. The most frequent co-existence partner genes were PROKR2 and FGFR1, followed by NSMF, RELN, SEMA3E and SEMA3A. The chromosomal localizations of each gene are shown in Fig. 4A. The PPI networks with 45 nodes and 902 links based on GeneMANIA illustrated strong connections among these 25 IHH genes (Fig. 4B). The functions of these genes were significantly enriched in signaling pathways of neuron and axon guidance, growth factor receptor regulation and cell migration. Co-expression represented the largest percentage of the interactions (71.12 %), followed by predicted (14.21 %) and physical interactions (9.95 %). Additionally, CHD7 and SMCHD1 showed co-expression relationships which were recorded in GSE17920 and GSE70461. Finally, the PPI networks of CHD7 and SMCHD1 demonstrated that CHD8 exhibited the most significant association with these genes (Fig. 4C).Table 3Overview of the coexistence of CHD7 and other known IHH-associated gene in digenic or oligogenic mode with IHH-related diseases in previous literature.Table 3No.SexDiseaseRaceGenec.DNA and amino acid changeStateResourceACMGReference1MKSChineseCHD7c.2824A > G p.Thr942AlaHetNALB[20]SEMA3Ec.760G > C p.Glu254GlnHetNANANSMFc.188C > T p.Pro63LeuHetNANA2MKSChineseCHD7c.8250T > G p.Phe2750LeuHetNALBFGFR1c.736C > T p.Arg246TrpHetNANA3MKSChineseCHD7c.3247A > G p.Thr1083AlaHetNANACHD7c.6379G > A p.Ala2127ThrHetNAVUSHS6ST1c.1177G > A p.Asp393AsnHetNANA4MKSChineseCHD7c.7912A > G p.Ile2638ValHetNANAFGF17c.580C > G p.Gln194GluHetNANA5MnIHHChineseCHD7c.6955C > T p.Arg2319CysHetNANAPROKR2c.533G > C p.Trp178SerHetNANA6MnIHHChineseCHD7c.2656C > T p.Arg886TrpHetNANASOX2c.6955C > A p.Thr 232AsnHetNANA7MKSCaucasianCHD7c. 2840G > A p.Arg947GlnHetPaternalVUS[21]SEMA3Ac. 618_619delCC Ile206MetfsTer5HetMaternalLP8MCSChineseCHD7c.4033C > T p.Arg1345CysHetNAVUS[22]SEMA3Ac.196C > T p.Arg66TrpHetNAVUS9MCSChineseCHD7c.2189C > T p.Thr730IleHetNAVUSNSMFc.533C > A p.Thr178AsnHetNAVUS10MCSChineseCHD7c.4549_4563del p.S1517_T1521delHetNAVUSSEMA3Ec.760G > C p.Glu254GlnHetNAVUS11MKSCaucasianCHD7c.3088A > C p.Asn1030HisHetNAP[23]FGFR1c.853C > T p.Arg285TrpHetNALP12MKS/CHD7c.2548A > C p.Lys850GlnHetNALPLHX4c.913G > T p.Gly305TrpHetNALP13MKSCaucasianCHD7c.2053_2058dupGCAAAA p.Lys683_Thr684insAlaLysHetNAVUSGNRHRc.416G > A p.Arg139HisHetNAPGNRHRc.30_31delinsAA p.Asn10_Gln11delinsLysLysHetNALP14MKSChineseCHD7c.7235A > T p.Glu2412ValHetMaternalNA[13]PROKR2c.533G > C p.Trp178SerHetMaternalNA15MAnosmiaChineseCHD7c.5980T > G p.Trp1994GlyHetPaternalNACDONc.2905G > A p.Val969IleHetMaternalNA16MsKSChineseCHD7c.5980T > G p.Trp1994GlyHetPaternalNACDONc.2905G > A p.Val969IleHetMaternalNA17MKSChineseCHD7c.8773G > A p.Ala2925ThrHetMaternalVUS[24]PROKR2c.337T > C p.Tyr113HisHetMaternalPFEZF1c.748C > G p.Arg250GlyHetMaternalVUS18MKS/CHD7c.6107C > T p.Pro2036LeuHetNANA[25]PCSK1c.239G > A p.Arg80GlnHetNANA19FnIHHCaucasianCHD7c.1565G > T p.Gly522ValHomNANA[26]MCM9c.911A > G p.Asn304SerHomNANA20MKSChineseCHD7c.3311T > C p.Ile1104ThrHetNANA[27]RELNc.4441A > C p.Lys1481GlnHetNANA21MKSChineseCHD7c.3568C > G p.Leu1190ValHetNANARELNc.4441A > C p.Lys1481GlnHetNANA22/nIHHChineseCHD7c.4516G > A p.Gly1506SerHetNANANSMFc.877A > C p.Thr293ProHetNANA23MKSChineseCHD7c.4516G > A p.Gly1506SerHetNANAEGFc.1813G > C p.Val605LeuHetNANA24MnIHHChineseCHD7c.2656C > T p.Arg886TrpHetNANAPLXNA1c.2803G > A p.Ala935ThrHetNANA25MnIHHChineseCHD7c.2189C > T p.Thr730IleHetNANADLX5c.593A > C p.Asn198ThrHetNANA26MKSChineseCHD7c.4516G > A p.Gly1506SerHetNANAERBB4c.1972A > T p.Ile658PheHetNANA27MKSChineseCHD7c.30del p.Phe10LeufsTer25HetNANANRP2c.1333A > C p.Ile445LeuHetNANAPLXNB1c.4740C > A p.His1580GlnHetNANA28MKSChineseCHD7c.1727C > T p.Pro576LeuHetNAVUSRELNc.4441A > C p.Lys1481GlnHetNANAMTORc.889G > A p.Asp297AsnHetNANA29FKSFinnishCHD7c.8188G > A p.Ala2730ThrHetNAVUS[28]FGFR1p.Arg365LysfsTer5HetNANA30FKSFinnishCHD7c.4847A > G p.Tyr1616CysHetNAVUSSEMA3Ac.196C > T p.Arg66TrpHetNANA31MnIHHFinnishCHD7c.2613+5G > AHetNAVUSFGFR1p.Ser436PhefsTer3HetNANA32MKSChineseCHD7c.6955C > T p.Arg2319CysHetNAP[29]PROKR2c.533G > C p.Trp178SerHetNANA33MnIHHCaucasianCHD7c.3245C > T p.Thr1082IleHetPaternalNA[30]FGFR1c.12G > T p.Trp4CysHetPaternalNAPROKR2c.802C > T p.Arg268CysHetMaternalNA34MKS/CHD7c.3056T > G p.Phe1019CysHetNANA[31]SEMA3Ec.1855C > T p.Arg619CysHetNANA35FKS/CHD7c.2440C > T p.Gln814TerHetNAP[32]KAL1c.1627G > A p.Val543IleHetNANA36FCSChineseCHD7c.115C > T p.Gln39TerHetMaternalPThis studySMCHD1c.2582T > C p.Ile861ThrHetPaternalVUSF: Female; M: Male; KS: Kallmann syndrome; nIHH: nIHH; CS: CHARGE syndrome; LB: NA: Not available.Fig. 4Genes linked to oligogenic inheritance with CHD7 gene in IHH individuals. (A) The chromosomal location of the partner genes; (B) Protein-protein interactions of the partner genes; (C) CHD7 and SMCHD1 protein interaction.Fig. 4
In the present study, we describe a family who were initially diagnosed with KS. The proband was reclassified as having CHARGE syndrome after genetic testing revealed CHD7 nonsense mutations that met the criteria by Hale [19]. However, the affected sister was diagnosed with nIHH, as their condition did not fulfill any of the diagnostic criteria for CHARGE syndrome recommended by Blake [33], Verloes [34], and Hale [19].
CHD7 gene encodes an integral membrane protein. It belongs to the chromatin remodeling family, which manipulates chromatin structure to activate and repress transcription [8]. The CHD7 gene is located on 8q12 and consists of 38 exons encoding chromodomain helicase DNA binding (CHD)protein 7 [35,36]. CHD7 protein is a highly conserved gene widely expressed in various tissues, containing 2997 amino acids and locating in the nucleoplasm and nucleolus. As a member of the chromosomal domain helicase family, CHD7 is an ATP-dependent chromatin remodeling enzyme that regulates DNA accessibility in a tissue-specific manner. The CHD7 protein belongs to a member of CHD7 family which contains four major two chromodomains, an SNF2 family N-terminal domain, a helicase ATP binding domain, and two BRK do-mains [37]. CHD7 has been shown to play a critical role in chromatin remodeling, apoptosis, cell cycle regulation, transcription, and embryonic stem cell differentiation [38,39]. Pathogenic CHD7 mutations are often de novo nonsense or frameshift mutations, which may lead to the loss of function of protein products due to translational termination, resulting in CHD7 haploid deficiency [40]. Of note, deleterious loss-of function CHD7 mutations interrupt the regulation of gene expression and result in disordered neural crest development [41]. CHD7 gene mutations are considered to be the genetic cause of over 90 % of patients with typical CHARGE syndrome [[42], [43], [44]]. Therefore, identifying each novel pathogenic mutation of CHD7 gene, expanding the mutational spectrum, and incorporating it into the database is beneficial for the diagnosis of CHARGE syndrome and estimating the prevalence of the disease [45,46].
In this study, we identified a nonsense variant p. Gln39Ter in exon 2 of CHD7 gene. Exon 2, which is the first and largest coding exon of the CHD7 gene, encodes 555 amino acid. All the nonsense variants identified in this exon abolish all functional domains of the protein or are predicted to be degraded by nonsense-mediated decay (NMD), leading to haploinsufficiency. More than 74 nonsense mutations have been recorded in the HGMD, Clinvar and CHD7 databases. Among these, 62 mutations were associated with CHARGE syndrome, 2 with KS, and 8 lacked clinical records (Table S2). Among prior cases, two patients with whole CHD7 gene deletion and deletion of exon 3–38, exhibited typical CHARGE syndrome [20]. Among all these nonsense mutations, ap-proximately 73 % of them involve the mutations in the amino acid Gln (glutamine) to a premature termination codon in the exon 2 of CHD7 gene. The nonsense mutation p.Gln39Ter was considered deleterious and potentially caused the phenotype observed in our three patients, consistent with haploinsufficiency due to NMD. Collectively, we speculated that variant p.Gln39Ter is deleterious and may be a causative factor in the observed phenotype in our three patients.
Clinical symptoms of CHD7 mutations present a wide spectrum, from severe CHARGE syndrome to mild self-limited delayed puberty [47]. The CHD7 mutations are reported in more than 90 % of patients with CHARGE syndrome [9] and 3–10 % of patients with KS/nIHH [[10], [11], [12]]. CHARGE syndrome is a well-known genetic disorder with multisystem congenital malformations, characterized mainly by eye coloboma, heart deformities, choanal atresia, retardation of growth, genital hypoplasia, and ear defects) [48]. Notably, CHARGE syndrome and KS/nIHH share some phenotypic similarities, such as genital hypoplasia, hearing loss, cleft lip and palate, and renal abnormalities [12].
In CHARGE syndrome patients, truncating CHD7 mutations are predominantly observed, whereas missense variants are frequently observed in IHH patients [[49], [50]]. Additionally, CHD7 mutations have also been reported in affected individuals initially diagnosed with KS/nIHH and then re-evaluated for CHARGE syndrome [12,47]. Similarly, the proband in our study was re-diagnosed with CHARGE syndrome after identifying the CHD7 nonsense mutation. The result further supported the notion that KS/nIHH are the milder allelic variant of CHARGE syndrome [1,11]. The term “CHD7-related disorder” was proposed to encompass the overall clinical spectrum of deleterious CHD7 mutations [19]. These findings highlight the importance of a com-prehensive clinical assessment by a specialized multidisciplinary team for patients with KS/nIHH with null CHD7 mutation, to distinguish between CHARGE syndrome and KS/nIHH [51].
The majority of CHARGE syndrome cases are sporadic and familiar CHARGE syndrome cases are relatively uncommon [52]. A previous review article indicated that 17 known CHARGE syndrome families exhibit significant intrafamilial clinical variability. These families included parent-child, siblings, and monozygotic twin pairs. Notably, the parent-child pair showed that the parents of a severely affected patient were usually mildly affected, and this became apparent only after the detection of the CHD7 mutation [9]. Meanwhile, in a Finnish family, the mildly affected father transmitted p.Gln1599* mutation in CHD7 to his two more severely affected sons. In agreement with the previous observations, the moderately affected mother of the proband in our study presented with spaniomenorrheaand mild olfactory dysfunction, without other CHARGE-associated symptoms. However, the symptoms of two siblings were more severe than those of their mothers.
The striking clinical interfamilial variability of CHD7 mutation indicates that additional pathogenic mutations may also be present in the affected family in digenetic or oligogenic inheritance. For instance, the siblings in our study carried an additional heterozygous SMCHD1 mutation (p.Ile861Thr) which inherited from the unaffected father. The SMCHD1 protein contains two a GHKL-type ATPase domain, a SMC hinge domain. The variant c.2582T > C (p.Ile861Thr) is located at the junction between the GHKL-type ATPase and SMC hinge domain. Heterozygous loss-of-function SMCHD1 mutations are known to involve in facioscapulohumeral muscular dystrophy type 2. Heterozygous gain-of-function missense SMCHD1 mutations are associated with Bosma arhinia microphthalmia syndrome (BAMS), which is characterized with absence of the nose, microphthalmia, and IHH [53]. This suggests that there issignificant phenotypic overlap between the SMCHD1-related diseases and IHH, such as hypoplasia of corpus callosum, hearing impairment, HH, and ocular abnormalities.
SMCHD1 is an epigenetic repressor which is overexpressed in the human olfactory epithelium and 97 % of the patients had hypogonadal features in 41 cases [54]. SMCHD1 has also been observed in some patients with IHH without nasal abnormalities [55]. The mutation p.Ile861Thr was predicted to be deleterious and enhanced contactwith adjacent amino acids 908 and 909. It also generated a new potential Thrphosphorylation site, which is phosphorylated by protein kinases DNAPK and cdc2. Collectively, we speculated p.Ile861Thr mutation increased SMCHD1's activity and resulted in IHH through the gain-of-function mechanism.
The multigenerational families harboring SMCHD1 mutations were found with broad clinical features, presenting with only mild dysmorphism or anosmia. Thus, the authors speculated that coexistence of oligogenic genes with SMCHD1 gene influences phenotypic variability. We hypothesized that SMCHD1 p.I861 mutation interacts as a modifier gene to underlie the phenotypic heterogeneity in the family harboring the CHD7 pathogenic mutation. However, the pathogenicity of SMCHD1 p.Ile861Thr requires further analysis through in vivo and in vitro experimental studies.
CHD7 gene has been described to contribute to CHARGE syndrome or KS/nIHH in a digenetic or oligogenic manners with other 24 known IHH-causing genes in 36 cases. The majority of the cases were diagnosed as KS ornIHH. The most frequent co-existence partner gene with CHD7 was PROKR2, which reported in four KS/nIHH cases with disrupted GnRH neuron development [13,20,24,29]. In our analysis, the PPI networks demonstrated significant interactions between these genes. In addition, CHD7 and SMCHD1 were found to be co-expressed and shared connections with CHD8, providing further evidence of synergistic pathogenic role of CHD7 and SMCHD1. However, most inherited oligogenic partner genes are typically discovered within families with limited sample sizes. Therefore, additional functional validation is re-quired to clarify the molecular mechanisms underlying the oligogenic genetic contribution to pathogenesis of IHH, given the complexity of disease symptoms.
Since most patients with CHARGE syndrome exhibit features of hypogonadotropic hypogonadism, which is typical characteristics of IHH, it is crucial to consider the possibility of CHARGE syndrome when clinically diagnosing IHH. In addition, Multiple Pituitary Hormones Deficiency (MPHD) also needs to be distinguished during the diagnosis. In this context, WES, as an un-biased genomic approach, could provide a more precise and comprehensive analysis of the molecular causes of diseases [56]. In the realm of IHH, most patients require life-long sex steroid replacement and fertility induction with GnRH [5]. Furthermore, particularly in case of CHARGE syndrome, attention should bepaid to the treatment of other potentially affected organs, in addition to gonads.
When CHD7 mutations are detected, a thorough clinical history and physical examination of both the patient and family members are essential for optimize medical management. This is particularly relevant for girls carrying CHD7 mutations, as they may not initially present with distinctive clinical features; nevertheless, they could exhibit developmental abnormalities later during adolescence. In this situation, timely intervention plays a crucial role in promoting the development of secondary sexual characteristics. For individuals with the hypogonadotropic hypogonadism phenotype, fertility can often be restored through GnRH therapy. However, disease-causing mutations may potentially transmit into future generations and may exhibit more severe symptoms in off-spring [9]. The prenatal diagnosis of CHARGE syndrome is quite difficult. Prenatal ultrasound has certain limitations in the diagnosis of this syndrome. The main clinical defects of this syndrome, such as eye defects, posterior nostril occlusion, and hearing impairment, are difficult to detect on prenatal ultrasound. Once the CHD7 pathogenic variant has been identified in an affected family member, prenatal and preimplantation genetic testing are possible. Therefore, it is crucial to emphasize the risk of transmitting the disease to all IHH-related family members and consider prenatal diagnosis or preimplantation genetic diagnosis [57]. Genetic detection is helpful for the timely intervention in promoting the development of secondary sexual characteristics and beneficial for guiding the treatment recommendations of pregnancy in CHARGE syndrome. Gene targeted therapy may be a new trend in the future development of CHARGE syndrome. In addition, WES increases the diagnostic yield of disorders with genetic or oligogenic inheritance, further reinforcing the importance of WES in elucidating complex inheritance mechanisms [13].
However, there are still some limitations in this study. First, we did not find the genetic cause of the third child in this family who died after birth because no biological samples were retained. Second, the possible molecular mechanisms underlying the oligogenic genetic contribution to pathogenesis of IHH need to be further explored in future work. In addition, our study involves only three patients, the existing results still need to be further validated in a larger population in the future.
We identified digenic CHD7 and SMCHD1 mutations in IHH-associated diseases for the first time and verified the synergistic role of oligogenic inheritance. Our findings indicate the significance of a thorough clinical assessment and follow-up for CHARGE features, as well as individualized genetic counseling for patients with CHD7 mutation. These results could broad the evidence of oligogenic inheritance in IHH-related diseases to some extent. Additionally, we report that WES serves as an effective tool for genetic testing for IHH-associated diseases, which can aid in the diagnosis, prognosis, and genetic counseling of patients.
The study was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (TJ-IRB20211016).
Written informed consent was obtained from all the participants.
This work was supported by the Natural Science Foundation of Hubei (grant number 2021CFB411). The funders played no role in the study design, data collection or analysis, the decision to publish, or manuscript preparation.
The data that has been used is confidential.
Tian Wang: Conceptualization, Formal analysis, Methodology, Writing – original draft, Writing – review & editing, Data curation. Wu Ren: Formal analysis, Methodology. Fangfang Fu: Methodology, Validation. Hairong Wang: Formal analysis, Methodology. Yan Li: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing. Jie Duan: Conceptualization, Project administration, Supervision, Writing – review & editing.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.