Hereditary Hemochromatosis Caused by SUGP2 and DENND3 Mutation in China: A Case Report

Hereditary hemochromatosis (HH) is an iron overload disease that is caused by mutations in genes regulating iron homeostasis, leading to excessive absorption and toxic accumulation of iron in the liver, pancreas, skin, heart, joints, and pituitary gland [1]. HH is a multisystemic disease with typical clinical manifestations including cirrhosis, diabetes, and skin pigmentation [1]. With the development of genetic testing methods and detection of affected individuals in different regions, rare gene mutations have been discovered [2-4]. A 53-year-old man was admitted to our hospital in July 2022. Twenty-four years prior, the patient gradually exhibited fatigue and decreased labor endurance without obvious induction. Twenty years ago, the patient experienced dizziness, lack of clarity in thinking, and memory loss. Seven years ago, these symptoms worsened and the patient's response slowed and gradually stopped working. He began to experience various bodily discomforts, including tinnitus, deafness, dry eyes, dizziness, chest tightness, palpitation, shortness of breath, belching, stomach fullness, and weakness of bowel and urine. The patient was moody, primarily depressed. He had abnormal perceptions, such as feelings of the upper body being hot and the legs being swollen and cool. He had taken a variety of drugs, including mood-regulating medications (e.g., duloxetine, paroxetine, sertraline, citalopram, lorazepam, and magnesium valproate) and traditional Chinese medicines (e.g., white Astragalus membranaceus, Cassia twig, and Radix bupleuri). However, no significant improvements were observed. Six months previously, the symptoms had worsened again. He felt that life was worse than death. A written Informed consent was signed by the patient. Within 3 h of admission, neurologists completed an assessment to document clinical symptoms and signs. Blood tests, such as complete blood count, liver and kidney function tests, a 12-item cytokine panel, ferritin levels, and folate levels, were processed within 24 h. Neuropsychological scales and imaging tests, including brain magnetic resonance imaging (MRI), liver MRI, and abdominal ultrasound, were finalized within 3 days of admission. Bone marrow and liver biopsies were performed by experienced physicians utilizing techniques like ultrasound and were completed within 7 days. The peripheral blood was collected in the morning. Clinical exome sequencing analysis was accomplished by MyGenostics Inc., Beijing, China. Genomic DNA was extracted from peripheral blood cells. The exonic regions and flanking splicing or intronic junctions of the whole genome were captured and sequenced using DNBSEQ-T7. The FASTQ files were mapped to the human reference genome (hg19). The identified variants were annotated using ANNOVAR, associated with 1000 genomes, Exome Aggregation Consortium, and the Human Gene Mutation Database, and further predicted by MutationTaster, SIFT, PolyPhen-2 and GERP++. Except for black skin on the face and neck, no other abnormalities were found upon physical examination. Patient was conscious and fluent. His both pupils were circular, equal in size, and reactive to light; both eyeballs moved fully in all directions, and no nystagmus was observed. The nasolabial sulci were symmetrical and there was no tongue deviation upon protrusion. The muscle strength and tension and tendon reflexes of the limbs were normal. The neck was soft. The Kirschner's and Brinell's signs were negative. The Bartholinus sign was not elicited. Cognitive function examination revealed normal orientation and signs of memory impairment, specifically an inability to recall what was eaten just 2 h after a meal, as well as calculation difficulties, exemplified by errors in performing calculations such as 100 - 7 and inability to calculate the answer to 93 -7 correctly. The patient underwent a comprehensive examination, and the significant diagnostic results are outlined below. The results of iron metabolism indicated that serum ferritin (SF) was over 1500.0 ng/mL (23–336 ng/mL), unsaturated iron binding capacity was 0.9 μmol/L (31–51 μmol/L), serum iron was 33.3 μmol/L (9–32 μmol/L), transferrin was 1.4 g/L (2–4 g/L), total iron binding capacity was 34.2 μmol/L (45–75 μmol/L) and transferrin saturation was 97.3% (20%–55%). Liver MRI revealed diffuse low signal intensity in the hepatic parenchyma (Figure 1A). Liver biopsy showed visible iron deposition in the cytoplasm of the hepatocytes (Figure 1B). Bone marrow biopsy revealed iron deposition primarily within histiocytes among the hematopoietic cells (Figure 1C). Additional testing revealed mild liver damage, diabetes, myocardial injury, and mild cognitive impairment. Based on these findings, the patient was excluded from having major acquired diseases. The elevated levels of SF and transferrin saturation coupled with the low T2-weighted signal on liver MRI, indicated the possibility of hemochromatosis. The potential pathogenic variations were confirmed by Sanger sequencing. Whole-exome sequencing revealed that the patient had variations in two loci: SUGP2 (NM_001017392) c.1916G > A (p.R639Q) and DENND3 (NM_014957) c.1149C > T (p.F383=) (Figure 1D). Additionally, all variants were heterozygous. Based on ACMG variant classification guidelines, the pathogenicity of SUGP2 locus is rated as Likely benign. The DENND3 locus is listed in the Clinvar database and is rated as Benign. We conducted further genetic tests on the patient's relatives. The family pedigree is depicted in Figure 1E. His mother and two daughters also carried mutations in either SUGP2 c.1G > A (p.R639Q), DENND3 c.1C > T (p. F383=) or both. Furthermore, the patient's eldest daughter showed abnormal SF and liver diffusion-weighted imaging findings. On the basis of these findings, the patient was diagnosed with HH. The patient was administered a low-iron diet, phlebotomy, and deferasirox. We recommended that the eldest daughter regularly reassess ferritin levels and, if necessary, seek early treatment to remove excess iron. The patient was followed up every two month and is currently undergoing follow-up. All symptoms, including neurological signs, have gradually improved and SF levels gradually decreased. We report a Chinese family with HH with novel compound heterozygous mutations, SUGP2 c.1916 G > A (p.R639Q) and DENND3 c.1149 C > T (p.F383=). The latter was a newly discovered site mutation. These gene mutations are autosomally recessive. In contrast to Western countries, such as Europe and America, where homeostatic iron regulator (HFE) p.C282Y or p.H63D mutations are dominant, China is dominated by non-HFE gene mutations, and this patient is consistent with them, including the newly discovered SUGP2 p.R639Q and DENND3 p.L708V [2, 3]. The SUGP2 variant reported here aligns with previously identified mutations in China [3]. However, the mutation in DENND3 at this specific site is novel, with only five prior cases of DENND3 p.L708V mutation reported [2]. The mutations of SUGP2 or DENND3 are pathogenic [2, 3]. Although the SUGP2 and DENND3 variants in this patient are classified as benign, their pathogenicity is uncertain. However, the patient has already developed pathogenic manifestations, suggesting that the mutation may exhibit different pathogenic potentials in different individuals. Future studies will also be devoted to evaluating the functional research and experimental verification of the pathogenicity of SUGP2 and DENND3 mutations. SUGP2 is a chromatin-associated protein that is abundant in the nucleus of male germ cells and plays a role in mRNA splicing [5]. A Chinese study showed that inhibiting SUGP2 expression downregulated p-Smad1/5 and hepatic antimicrobial protein (HAMP) expression, suggesting that SUGP2 may play a new role in downregulating the BMP/Smad pathway and hepcidin expression [3]. Human hepcidin is encoded by the HAMP gene. It binds closely to ferritin, resulting in decreased iron absorption in the duodenum and a reduced release of iron from the spleen during erythrocytic phagocytosis, thus regulating iron metabolism [6]. Based on the above studies, it is reasonable to speculate that the mutation of SUGP2 gene, resulting in the decrease of SUGP2 expression, leads to the increase of hepcidin, and finally the increase of iron deposition in liver, spleen and other organs. The small guanosine triphosphate enzyme Rab12 and its upstream activator DENND3 regulate structural degradation of the transferrin receptor (TFR) [7]. TFR is involved in maintaining intracellular iron homeostasis, therefore, DENND3 may also play a role in iron homeostasis. A study in China also showed that the overexpression of DENND3 and activation of mutation DENND3 p.L708V may affect p-Smad1/5 levels and the expression of TFR2 and HAMP [3]. Therefore, we speculate that DENND3 may also be involved in regulating iron homeostasis. When HH is diagnosed, it is important to consult with and screen other family members, especially first-degree relatives (parents, siblings, and children) [8, 9]. We performed genetic testing on the patient and his family and found that his mother and two daughters had mutations in one or both. His oldest daughter had also developed hemochromatosis. These variants may be inherited from his mother. A study exploring the clinical features of non-HFE-associated HH showed that 40% of patients with HH with SUGP2 or DENND3 mutations suffered from cirrhosis, 20% suffered from skin hyperpigmentation, and 10% suffered from both diabetes and hypogonadism [2]. In our case, the patient presented with alopecia, myocardial damage, arrhythmia, arthralgia, diabetes, skin pigmentation, and other symptoms, which are consistent with the systemic manifestations that can be involved in HH. However, patients with HH rarely complain of memory loss and seldom visit the neurology department first. Studies on the cognitive impairment caused by hemochromatosis are rare. In a recent study, Veronica et al. explored the interaction and synergy between common single- nucleotide polymorphisms of the major iron homeostasis genes (HFE, FPN1, HAMP, TF) and the major lipoprotein transporter gene (APOE) in patients with dementia of various etiologies (Alzheimer's disease, vascular dementia, and mild cognitive impairment). Established genetic risk factors may be regulated by a specific genetic background, making patients differently susceptible for managing iron overload, and resulting in diverse clinical phenotypes [10]. The Mini-Mental State Examination and Montreal Cognitive Assessment scores for our patient decreased. Hippocampal atrophy was of Grade 2. However, no iron deposition was observed in the brain. The mechanism underlying cognitive impairment in patients with HH remain unclear. Changes in cognitive function were focused on during follow-up. Cognitive function improved after active treatment, which is consistent with the findings of other studies [11]. To sum up, this report describes a Chinese family with HH and mutations in SUGP2 and DENND3. HH exhibits a range of clinical features, physical signs, imaging abnormalities, and biological characteristics. We propose that all gene mutations discovered thus far should be included in the database. Based on clinical observations, HH should be suspected if a patient exhibits abnormally high ferritin levels and complex manifestations of multisystem injuries. After ruling out acquired causes of disease, HH related genetic testing should be conducted to prevent disturbances caused by iron accumulation in the body. Huiqing Qiu: data curation, writing – original draft. Mengwei Yuan: writing – original draft. Ziyu Guo: data curation. Jiayi Liang: supervision. Yang Li: supervision. Yan Gao: supervision. Sha He: writing – review and editing. Xiaowei Ma: conceptualization, writing – review and editing. The authors have nothing to report. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.