Function of PHEX mutations p.Glu145* and p.Trp749Arg in families with X-linked hypophosphatemic rickets by the negative regulation mechanism on FGF23 promoter transcription.

X-linked hypophosphatemic rickets (XLH) is characterized by increased circulating fibroblast growth factor 23 (FGF23) concentration caused by PHEX (NM_000444.5) mutations. Renal tubular resorption of phosphate is impaired, resulting in rickets and impaired bone mineralization. By phenotypic-genetic linkage analysis, two PHEX pathogenic mutations were found in two XLH families: c.433 G > T, p.Glu145* in exon 4 and c.2245 T > C, p.Trp749Arg in exon 22. Immunofluorescence showed that the localization of p.Glu145* and p.Trp749Arg mutant and secretory PHEX (secPHEX) changed, with decreased expression. In a HEK293T cell model co-transfected with PHEX, secPHEX, and FGF23, wild-type PHEX, secPHEX, and FGF23 proteins were distributed in the cell membrane or endoplasmic reticulum, while the mutant was located in the nuclear membrane and cytoplasm. qPCR of p.Glu145* revealed decreased PHEX and secPHEX mRNA expression in cells, with no difference in mRNA expression of p.Trp749Arg. Both mutations decreased intracellular PHEX endopeptidase activity. Western blot analysis showed decrease in mutant and secPHEX protein expression and no FGF23 protein expression in single-transfected PHEX and secPHEX cells. In cells co-transfected with FGF23, PHEX and secPHEX mutation promoted FGF23 expression. Dual-luciferase reporter gene was used to detect the effect of PHEX on FGF23 promoter. The dual-luciferase reporter gene showed that after PHEX overexpression, the activity of mutant firefly luciferase was significantly higher than that of wild type. The regulatory mechanism between PHEX and FGF23 is still unclear, but we found that PHEX is a direct transcriptional inhibitor of FGF23 and affects the expression of FGF23. This study verified the pathogenicity of the two variants and revealed the possible regulatory mechanism between PHEX and FGF23.

Genetic Analysis and Functional Study of a Pedigree With Bruck Syndrome Caused by PLOD2 Variant.

Background: Bruck syndrome (BS) is a rare autosomal recessive inherited osteogenesis imperfecta disease characterized by increased bone fragility and joint contracture. The pathogenic gene of type I BS is FKBPl0, whereas that of type II BS is PLOD2. No significant difference has been found in the clinical phenotype between the two types of BS. In this study, we performed genetic analysis of a BS pedigree caused by PLOD2 variant and studied the corresponding cellular function. Methods: Serum biochemistry, parathyroid hormone (PTH), 25-hydroxyvitamin D [25-(OH) D], osteocalcin, and 24-h urinary calcium levels of a family member with BS was assessed. The genes of the proband were analyzed by second-generation sequencing and exon capture techniques. Sanger sequencing was also performed for the suspected responsible variant of the family member. Wild- and variant-type lentivirus plasmids were constructed by gene cloning and transfected into HEK293T cells. Cell function was verified by real-time quantitative polymerase chain reaction, western blotting, and immunofluorescence detection. Results: In this pedigree, the proband was found to have a homozygous variant c.1856G > A (p.Arg619His) in exon 17 of PLOD2 (NM_182943.3). His consanguineous parents and sisters were p.Arg619His heterozygous carriers. The mRNA expression of PLOD2 in the constructed p.Arg619His variant cells was significantly upregulated, while the expression of PLOD2 and collagen I protein in the cell lysate was significantly downregulated. Immunofluorescence revealed that the wild-type PLOD2 was mainly located in the cytoplasm, and the expression of the PLOD2 protein after c.1856G > A variant was significantly downregulated, with almost no expression, aligning with the western blot results. The serum sodium, potassium, calcium, phosphorus, magnesium, alkaline phosphatase, PTH, 25-(OH) D, osteocalcin, and 24 h urinary calcium levels of the proband, his parents, and sisters were normal. Conclusion: Through gene and cell function analyses, PLOD2 Arg619His missense variant was preliminarily confirmed to cause BS by reducing protein expression.

Prevalence, Risk Factors, and Prognostic Factors of Primary Malignant Bone Neoplasms with Bone Metastasis at Initial Diagnosis: A Population-Based Study.

Background: Bone metastasis (BM) has been proven to be responsible for the poor prognosis of primary malignant bone neoplasms (PMBNs). We aimed to identify the prevalence, risk factors, and prognostic factors for PMBNs patients with BM based on the Surveillance, Epidemiology, and End Results (SEER) database. Methods: 4,758 patients diagnosed with PMBNs from 2010 to 2018 were selected from the SEER database. All patients were divided into two groups: the BM group or the non-BM group. Pearson's chi-square test and Fisher's exact method were used to assess baseline characteristics, and logistic regression analysis was applied to assess risk factors. In addition, a nomogram was constructed based on the results of Cox regression analysis among 227 patients with BM. The good performance and clinical applicability of the nomogram were tested by the concordance index, operating characteristic curve, area under the curve, calibration curves, and decision curve analysis. Results: 227 (4.8%) patients had metastasis to bone at diagnosis. Primary site outside the extremities (axial: odds ratio, OR = 1.770; others: OR = 1.951), Ewing sarcoma (OR = 2.845), larger tumor size (5-8 cm: OR = 3.403; >8 cm: OR = 5.562), tumor extension beyond the periosteum (OR = 2.477), and regional lymph node metastasis (OR = 2.900) were associated with a higher risk of BM at the initial diagnosis of PMBNs. Five independent prognostic factors were found in the survival analysis: pathological type (chondrosarcoma vs. osteosarcoma: hazard ratio, HR = 0.342; Ewing sarcoma vs. osteosarcoma: HR = 0.592; and chordoma vs. osteosarcoma: HR = 0.015), marital status (HR = 2.457), pulmonary metastasis (HR = 1.934), surgery at the primary site (HR = 0.164), and chemotherapy (HR = 0.084). A nomogram based on these prognostic factors could be a good predictor of cancer-specific survival. Conclusions: We identified the prevalence, risk factors, and prognostic factors correlated with BM in PMBNs patients. The related nomogram could be a practical tool for therapeutic decision-making and individual counseling.

A novel compound heterozygous variant linked to hematuria in a family with hereditary factor VII deficiency.

BACKGROUND: Hereditary factor VII deficiency (FVIID) is a rare congenital autosomal recessive bleeding disorder. In clinical manifestations, its onset is caused by variant of the F7 gene (NM_019616) with strong heterogeneity. We identified a family with hematuria caused by a novel F7 compound heterozygous variant and investigated the FVIID-dependent mechanism impacted by these variants. METHODS: Coagulation factors in the proband were functionally verified. We located pathogenic variants in relevant genes using next-generation sequencing after target enrichment and verified them by Sanger sequencing. We examined the coagulation activity and secretion pattern of recombinant FVII variants expressed in cells and observed their location and stability by immunofluorescence. RESULTS: We found a missense variant c.1207G>A (p.Gly403Ser) and a frameshift variant c.154_155del (p.Arg53fs) in the F7 gene of the proband. FVII activity tests showed that the variants significantly decreased its presence in the cell culture supernatant. Moreover, the R53fs mutant lacked the FVII functional domain and had no detectable activity. Immunofluorescence indicated that the p.Gly403Ser variant was distributed to the cell membrane and cytoplasm, whereas the FVII R53fs variant was not detected. Deficient FVII protein function and severe coagulation disorder are the likely causes of hematuria and other bleeding symptoms in the proband. CONCLUSIONS: The newly discovered F7 gene variants enrich the spectrum of hereditary FVII deficiency and provide a new foundation for the diagnosis and treatment of this type of coagulation disorder.

Different phenotypes of neurological diseases, including alternating hemiplegia of childhood and rapid-onset dystonia-parkinsonism, caused by de novo ATP1A3 mutation in a family.

BACKGROUND: The spectrum of neurological diseases related to ATP1A3 gene mutations is highly heterogeneous and exhibits different phenotypes. Phenotype overlaps, including alternating hemiplegia of childhood (AHC), early infantile epileptic encephalopathy, and rapid-onset dystonia-parkinsonism (RDP), can also occur at extremely low incidences. Currently, over 90 types of pathogenic mutations have been identified in ATP1A3. PATIENTS AND METHODS: The family of a 2-year-11-month-old proband with AHC was recruited for this clinical investigation. The proband was screened for candidate mutation gene sites using next-generation sequencing and target-region capture technology. Sanger sequencing was used to identify carriers among family members. RESULTS: The mother of the proband with AHC was diagnosed with dystonia (later diagnosed as RDP). The biochemical and immune indices of the proband and the mother were not abnormal. Moreover, brain imaging of the proband revealed no significant abnormalities. However, the electroencephalogram of the mother was mildly abnormal, with no spike wave discharge. Brain MRI revealed slight cerebellar atrophy. Electromyography revealed neurogenic damage, with a decrease in the conduction velocity of the left ulnar and radial nerves. Based on the sequencing data, both the proband and her mother carried c.823G > C p. (Ala275Pro) heterozygotes; other family members were not identified as carriers. With a PolyPhen-2 score of 0.997 and SIFT score of 0.001, this mutation can be considered damaging. CONCLUSION: Family genotype-phenotype correlation analysis revealed that the phenotype and gene mutation were co-segregated, suggesting that it may be a pathogenic mutation.

Review and Analysis of Two Gitelman Syndrome Pedigrees Complicated with Proteinuria or Hashimoto's Thyroiditis Caused by Compound Heterozygous SLC12A3 Mutations.

Gitelman syndrome (GS) is an autosomal recessive inherited salt-losing renal tubular disease, which is caused by a pathogenic mutation of SLC12A3 encoding thiazide-sensitive Na-Cl cotransporter, which leads to disturbance of sodium and chlorine reabsorption in renal distal convoluted tubules, resulting in phenotypes such as hypovolemia, renin angiotensin aldosterone system (RAAS) activation, hypokalemia, and metabolic alkalosis. In this study, two GS families with proteinuria or Hashimoto's thyroiditis were analyzed for genetic-phenotypic association. Sanger sequencing revealed that two probands carried SLC12A3 compound heterozygous mutations, and proband A carried two pathogenic mutations: missense mutation Arg83Gln, splicing mutation, or frameshift mutation NC_000016.10:g.56872655_56872667 (gcggacatttttg>accgaaaatttt) in exon 8. Proband B carries two missense mutations: novel Asp839Val and Arg904Gln. Both probands manifested hypokalemia, hypomagnesemia, hypocalcinuria, metabolic alkalosis, and RAAS activation; in addition, the proband A exhibited decreased urinary chloride, phosphorus, and increased magnesium ions excretion, complicated with Hashimoto's Thyroiditis, while the proband B exhibited enhanced urine sodium excretion and proteinuria. The older sister of proband B with GS also had Hashimoto's thyroiditis. Electron microscopy revealed swelling and vacuolar degeneration of glomerular epithelial cells, diffuse proliferation of mesangial cells and matrix, accompanied by a small amount of low-density electron-dense deposition, and segmental fusion of epithelial cell foot processes in proband B. Light microscopy showed mild mesangial hyperplasia in the focal segment of the glomerulus, hyperplasia, and hypertrophy of juxtaglomerular apparatus cells, mild renal tubulointerstitial lesions, and one glomerular sclerosis. So, long-term hypokalemia of GS can cause kidney damage and may also be susceptible to thyroid disease.

Genetic diagnosis history and osteoarticular phenotype of a non-transfusion secondary hemochromatosis.

BACKGROUND: It is not easy to identify the cause of various iron overload diseases because the phenotypes overlap. Therefore, it is important to perform genetic testing to determine the genetic background of patients. AIM: To investigate the genetic background of a patient with hemochromatosis complicated by psoriasis on both lower extremities. METHODS: Ten years ago, a 61-year-old male presented with iron overload, jaundice, hemolytic anemia and microcytic hypochromic anemia. Computed tomography of the left knee joint showed enlargement of the tibial medullary cavity and thinned bone cortices. Magnetic resonance imaging showed hepatic hemochromatosis, extensive abnormal signals from bone marrow cavities and nodular lesions in the lateral medullary cavity of the upper left lateral tibia. Single photon emission computed tomography showed radial dots of abnormal concentration in the upper end of the left tibia and radial symmetry of abnormal concentrations in joints of the extremities. The patient showed several hot spot mutations of the HFE and G6PD genes detected by next-generation sequencing, but no responsible gene mutation was found. The thalassemia gene was detected by gap-PCR. RESULTS: The patient was found to carry the -α4.2 and --SEA deletion mutations of the globin gene. These two mutations are common causes of Southeast Asian α-thalassemia, but rarely cause severe widespread non-transfusion secondary hemochromatosis osteoarthropathy. The simultaneous presence of an auxiliary superposition effect of a rare missense mutation of the PIEZO1 gene (NM_001142864, c.C4748T, p.A1583V) was considered. Moreover, several rare mutations of the IFIH1, KRT8, POFUT1, FLG, KRT2, and TGM5 genes may be involved in the pathogenesis of psoriasis. CONCLUSION: The selection of genetic detection methods for hemochromatosis still needs to be based on an in-depth study of the clinical manifestations of the disease.

Immunohistochemical identification of osteoclasts and multinucleated macrophages.

Osteoclasts are bone-resorbing multinuclear cells derived from hematopoietic stem cells which are specialised to carry out lacunar bone resorption. The immunophenotype of giant cell-containing bone lesions in a wide range of osteoclast-like giant cells was similarly assessed. Both multinucleated macrophages and osteoclasts were found to express CD68. Multinucleated macrophages, but not osteoclasts, expressed GrB and Ki67. CD13+/CD14+/CD68+/GrB-/Ki67-/CD56- all giant-cell lesions noted in giant cells of bone. Giant cells have an osteoclast phenotype in most giant cell-rich lesions of bone, which do not express the macrophage-associated antigens GrB and Ki67. Our results indicate that they are formed from osteoclast precursors of mononuclear phagocyte.