Objective:To explore the genetic basis of eighteen patients with tetrahydrobiopterin deficiency (BH4D) from Gansu Province.Methods:Eighteen patients diagnosed with BH4D at Gansu Provincial Maternal and Child Health Care Hospital from January 2018 to December 2021 were selected as the study subjects. Whole exome sequencing was carried out, and candidate variants were verified by Sanger sequencing.Results:All of the thirty-six alleles of the eighteen patients were successfully determined by molecular genetic testing. Sixteen patients were found to harbor variants of the PTS gene, and two had harbored variants of the QDPR gene. Ten variants were detected in the PTS gene, with the most common ones being c. 259C>T (34.38%) and c. 286G>A (15.63%). Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c. 259C>T was classified as a pathogenic variant, whilst the c. 286G>A, c. 166G>A, c. 200C>T, c. 272A>G, c. 402A>C, c. 421G>T, c. 84-291A>G and c. 317C>T were classified as likely pathogenic variants. A novel c. 289_290insCTT variant was classified as likely pathogenic (PM1+ PM2_Supporting+ PM3+ PP3+ PP4). The two variants (c.478C>T and c. 665C>T) detected in the QDPR gene were both classified as variants of uncertain significance (PM1+ PM2_Supporting+ PP3+ PP4). Conclusion:Genetic testing has clarified the pathogenic variants in these BH4D patients, which has enabled timely and accurate clinical intervention and treatment, and provided a reference for genetic counseling and reproductive guidance for their families.

Objective:To explore the value of whole exome sequencing for the inferential analysis of recessive genetic disease carrier status for couples with a child died of Primary immunodeficiency (PID).Methods:Clinical data was collected from four couples with a childbearing history of PID who had sought genetic counseling and undergone genetic testing at Henan Provincial People′s Hospital from February 2017 to December 2021. Whole exome sequencing (WES) was performed on both partners of each couple, and candidate variants were validated by Sanger sequencing and fluorescent quantitative PCR. Prenatal diagnosis was conducted on fetuses of these couples after confirming the variants.Results:A total of six variants were detected in four genes including IL2RG, BTK, CYBB, and DUOX2. Among these, the c.1265G>A and c.3329G>A variants of the DUOX2 gene and the c. 676C>T variant of the IL2RG gene were previously known as pathogenic variants. On the other hand, the Exon5_8del variant of the IL2RG gene, the c. 184_185delAC variant of the BTK gene, and the c. 472A>T variant of the CYBB gene were unreported previously. Based on the guidelines from the American College of Medical Genetics and Genomics, the IL2RG: Exon5_8del, BTK: c. 184_185delAC and CYBB: c. 472A>T variants were classified as likely pathogenic (PVS1+ PM2_Supporting+ PP4).Prenatal diagnosis was conducted for three couples during their subsequent pregnancies, and the results revealed that the fetuses had the wild-type genotypes at the c. 184_185 position of the BTK gene, the c. 472 position of the CYBB gene, and the c. 676 position of the IL2RG gene. Follow-up examinations one year after birth has found no abnormality in the infants. Conclusion:WES is an important tool to infer and analyze the carryier status for couples who had given births to children died of PID and improve the positive detection rate.

Objective:To analyze the clinical and genetic characteristics of ten Chinese pedigrees affected with 7q11.23 duplication syndrome.Methods:From December 2017 to January 2022, ten pedigrees diagnosed with 7q11.23 duplication syndrome at the First Affiliated Hospital of Zhengzhou University were enrolled as the study subjects. Clinical data of all subjects were collected, and some had subjected to copy number variation sequencing or single nucleotide polymorphism array to analyze the pattern of inheritance.Results:The probands had included six fetuses and four adolescents. Four of the six prenatal cases showed abnormal ultrasound indicators, including three with soft indicators and one with abnomal feta structural development. The clinical phenotype of the four adolescent cases had included mental retardation, delayed language development, and attention deficit hyperactivity disorder. The size of the copy number variations had ranged from 1.31 to 1.42 Mb, involving the classic region of 7q11.23 duplication syndrome. Of these, five cases had undergone parental origin testing, three cases were de novo, and two were hereditary. Conclusion:Individuals with 7q11.23 duplication syndrome may show substantial clinical phenotypic heterogeneity, hence the affected families should be provided with pre-pregnancy consultation and reproductive guidance.

Objective:To explore the pregnancy outcome and postpartum clinical phenotype of LCR22B/C~D central 22q11.2 deletion syndrome.Methods:For fetuses diagnosed with central 22q11.2 deletion by chromosomal microarray analysis (CMA) at the Prenatal Diagnosis Center of the Third Affiliated Hospital of Zhengzhou University from January 2019 to April 2022, their prenatal imaging, parental CMA verification, pregnancy outcomes and postpartum clinical phenotype were analyzed.Results:Eight cases of central 22q11.2 deletion syndrome were included, including six cases with LCR22B~D 22q11.2 deletions and two with LCR22C~D 22q11.2 deletions. Among the six cases with LCR22B~D type 22q11.2 deletions, three had shown cardiovascular malformations (right aortic arch, ventricular septal defect, mild tricuspid regurgitation), one had shown urinary defect (right kidney heterotopia). Two cases with LCR22C~D 22q11.2 deletions showed nonspecific ultrasonographic findings, including oligohydramnios with growth restriction and nuchal skin thickening. The CMA verification showed that six cases were inherited from their parents, and five couples had chosen to continue with the pregnancy. Postpartum follow-up showed that the physical and intellectual development of all children were normal. One couple had opted to terminate the pregnancy considering the ectopic fetal right kidney. Two remaining cases had decided to terminate their pregnancies without parental verification.Conclusion:The central 22q11.2 deletion syndrome of the LCR22B/C~D type is different from the classical types. Its genetic information mainly comes from parents. Prenatal imaging has mainly shown cardiovascular and urinary abnormalities. Postnatal growth and intellectual development have been normal. Therefore, the couples should be provided with suffice prenatal genetic counseling.

Objective:To assess the prognostic value of methylation of interferon regulatory factor 6 ( IRF6) gene promoter in patients diagnosed with Kidney renal clear cell carcinoma (KIRC). Methods:The primary lesions of fifty KIRC patients who were diagnosed at the First Affiliated Hospital of Nanjing Medical University from January 2016 to January 2020 were collected. The expression of IRF6 protein was determined with an immunohistochemical method. The correlation between the level of IRF6 expression and survival and/or metastasis status was analyzed. The mRNA and protein levels of the IRF6 in KIRC and normal renal tissues were compared by using bioinformatic tools. The difference in the methylation rate of the IRF6 gene promoter between tumor and adjacent tissues was analyzed by searching the online databases. Statistical analysis was carried out for the methylation status of the IRF6 gene promoter region to select those negatively correlated with the overall survival (OS) among the patients. In vitro experiments were conducted with cell lines to verify the correlation between the status of promoter methylation and transcription level of the IRF6 gene. Results:The mRNA and protein levels of the IRF6 gene in KIRC tissues were significantly lower than those of the normal controls, and this was more prominent in patients who had died or developed metastasis. The extent of IRF6 gene promoter methylation in the KIRC tissues was much higher compared with that of the adjacent normal renal tissues. There was a significant negative correlation between the methylation of the IRF6 gene promoter and mRNA level of the IRF6 ( R= -0.52). The higher methylation degree in the IRF6 gene promoter regions cg12034118 and cg16030177, the shorter the OS and worse prognosis in the patients. Only twenty CpG sites in cg12034118 were confirmed to be highly methylated in KIRC cell lines. The transcription level of the IRF6 gene was upregulated in a time- and dose-dependent manner after the treatment with demethylation reagent 5-azadeoxycytidine. Conclusion:The methylation of IRF6 gene promoter in the renal tissues of KIRC patients is closely correlated with the OS. Cg12034118 may provide a promising biomarker for laboratory detection, and its high methylation rate has certain reference value for the prognosis.

Objective:To assess the association between the polymorphisms of integral protein α4 ( ITGA4) and intercellular adhesion molecule 1 ( ICAM-1) genesand the risk and clinicopathological characteristics of Crohn′s disease (CD) among Chinese patients. Methods:From January 2010 to January 2021, a total of 215 CD patients and 529 gender- and age-matched healthy controls were enrolled from the Second Affiliated Hospital of Wenzhou Medical University as the study subjects. Genotypes of ITGA4 (rs6740847, rs7562325) and ICAM-1 (rs5498) were determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Harvey-Bradshaw Index (HBI) was applied to assess the disease activity of CD, and the patients were further divided into subgroups based on the Montreal Classification Criteria of CD. Unconditional logistic regression was employed to analyze the distribution of ITGA4 (rs6740847, rs7562325) and ICAM-1 (rs5498) polymorphisms between the patients and healthy controls and their association with the clinicopathological characteristics of the patients. Results:The frequencies of T allele and CT+ TT genotypes of ITGA4 (rs7562325) were higher in CD patients than the healthy controls (40.70% vs. 31.57%, P=0.001; 62.79% vs. 54.36%, P=0.042). The G variant and AG+ GG genotypes of ITGA4 (rs6740847) were less common in patients with moderately to severely active CD compared with those with mildly active CD (31.18% vs. 51.72%, P=0.002; 55.91% vs. 75.86%, P=0.042). However, the opposite conclusion was drawn for the G allele (G) and AG+ GG genotypes of ICAM-1 (rs5498) (31.45% vs. 17.24%, P=0.027; 54.30% vs. 31.04%, P=0.020). Compared with patients with terminal ileal or ileocolic CD, G allele and AG+ GG genotypes of ITGA4 (rs6740847) were more prevalent in patients with colonic CD (55.26% vs. 29.38%, P<0.000 1; 84.21% vs. 53.11%, P<0.000 1). The same conclusion could also be drawn for the G allele and AG+ GG genotypes of ICAM-1 (rs5498) (42.11% vs. 26.84%, P=0.008; 73.69% vs. 46.33%, P=0.002). The frequency of homozygous GG genotype of ICAM-1 (rs5498) was lower in patients with stricturing and penetrating CD than those with non-stricturing and non-penetrating CD (0.00% vs. 12.32%, P=0.001). The G allele and AG+ GG genotypes of the ITGA4 (rs6740847) were more common in patients with perianal lesions than those without (40.28% vs. 30.77%, P=0.049; 72.22% vs. 51.75%, P=0.004). Conclusion:Variants of the ITGA4 (rs7562325) may be a risk factor for CD, whilst those of the ITGA4 (rs6740847) may be associated with the decline of disease activity and risk for colon involvement and perianal lesions. Variants of the ICAM-1 (rs5498) may increase the risk of disease activity and colonic involvement in CD patients, however, it may be a protective factor for stenosis and penetration. In addition, variants of the ITGA4 (rs6740847) and ICAM-1 (rs5498) may be associated with the early onset of CD.

Objective:To explore the genetic etiology of two children with Spinal muscular atrophy with respiratory distress type 1 (SMARD1), and prevent the recurrence of birth defects.Methods:Two unrelated families who had visited the Obstetrics and Gynecology Medical Center of Drum Tower Hospital from August to November 2021 were selected as the study subjects. Copy number of SMN1 gene exon 7 for the probands and their parents was detected by multiple ligation-dependent probe amplification (MLPA). and whole exome sequencing (WES) was carried out to screen the variants in the probands. Sanger sequencing was used to validate the variants within the families. Pathogenecity of the variants were predicted by bioinformatic analysis. Based on the results, prenatal diagnosis was performed for the fetuses. Results:Both probands were found to harbor compound heterozygous variants of the IGHMBP2 gene, which were inherited from their parents. Among these, c. 1144C>T, c. 866delG and c. 1666C>G were previously unreported and respectively classified as pathogenic variant (PVS1+ PM2_Supporting+ PP3+ PP4), likely pathogenic variant (PM1+ PM2_Supporting+ PM4+ PP3+ PP4) and likely pathogenic variant (PM1+ PM2_Supporting+ PP2+ PP3+ PP4) based on the ACMG guidelines. Through preimplantation genetic testing for monogenic (PGT-M) and interventional prenatal diagnosis, transmission of the variants within the families was successfully blocked. Conclusion:The SMARD1 in both children may be attributed to the compound heterozygous variants of the IGHMBP2 gene, which has facilitated the genetic diagnosis and counselling, and provided reference for delineating the molecular pathogenesis of this disease.

Objective:To explore the clinical and genetic characteristics of five children with epilepsies due to variants of SCN8A gene. Methods:Clinical data of five children (four males and one female) admitted to Linyi People′s Hospital due to hereditary epilepsies between August 2015 and August 2022 were collected. Whole exome sequencing was carried out for these children, and candidate variants were verified by Sanger sequencing.Results:All of the five children were found to harbor variants of the SCN8A gene. Case 1, who had benign familial infantile epilepsy, inherited a known pathogenic c. 4840A>G variant from his father with similar symptoms. Cases 2 to 4 had presented with intermediate epilepsy. Among these, case 2 has harbored a de novo c. 3967G>A variant which was rated as pathogenic (PS1+ PS2+ PM1+ PM2_Supporting+ PP3) based on the guidelines from the American College of Medical Genetics and Genomics. Cases 3 and 4 were found to respectively harbor a de novo c. 415A>T and a c. 4697C>T variant, which were both rated as likely pathogenic (PS2+ PM1+ PM2_Supporting+ PP3). Case 5, who had early-onset infantile epileptic encephalopathy transformed into Lennox Gastaut-like syndrome, has harbored a de novo c. 5615G>A variant, which was known to be pathogenic. The children had their age of onset ranging from 2 to 14 months, and all had focal seizures and generalized tonic clonic seizures. Four children (cases 1, 2, 3 and 5) had cluster seizures, four (cases 1 to 4) had become seizure-free after single or dual treatment and showed normal growth and development, whilst case 5 was drug-resistant and showed severe developmental retardation. Conclusion:The five children had new features such as cluster seizures, occasional benign seizures in adulthood, and intermediate epilepsy which are prone to relapse after discontinuation of medication, which may be attributed to the pathogenic variants of the SCN8A gene.

Objective:To analyze the clinical characteristics of three patients with Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome.Methods:Three patients with IPEX syndrome diagnosed at the Children′s Hospital of Fudan University from January 24, 2013 to July 29, 2019 were selected as the study subjects. Their clinical features, laboratory investigations and results of genetic testing were summarized. Treatment and prognosis were also explored.Results:All of the three children had developed the disorder during infancy. One child had initial features including diabetes and diabetic ketoacidosis, whilst the other two had initiated by diarrhea. All patients had gastrointestinal involvement, and one was diagnosed as very early onset inflammatory bowel disease by colonoscopy and biopsy. Two children also had endocrine glands involvement. One child had manifested type 1 diabetes and positivity for thyroglobulin and thyroid peroxidase antibodies, though his thyroid function had remained normal. Another one had hypothyroidism and was treated by levothyroxine. Genetic testing revealed that all children had harbored missense variants of the FOXP3 gene, including c. 1222G>A (p.V408M), c. 767T>C (p.M256T) and c. 1021A>G (p.T341A). The clinical symptoms of one patient were alleviated following allogeneic hematopoietic stem cell transplantation. One patient was stable after treatment with infliximab plus insulin, and one child had died of refractory septic shock and multiple organ dysfunction syndrome at 3 months old. Conclusion:FOXP3 gene variant-associated IPEX syndrome may have very early onset and diverse clinical manifestations. For male patients with infantile onset chronic diarrhea, multiple endocrine or multiple system involvement, genetic testing is recommended, which may facilitate early diagnosis, treatment and genetic counseling.

Objective:To explore the clinical manifestations and pathogenic variant in a family with epilepsy, developmental delay and brain deformity.Methods:Clinical data of the child and his family members who had visited the Department of Pediatrics, Linyi People's Hospital on July 2, 2022 were collected. The child, his sister and parents were subjected to high-throughput sequencing, and the result was verified by Sanger sequencing.Results:The child was a 6-year-old boy with developmentally delay and had epileptic seizures with fever sensitivity for four years. Cranial imaging showed brain dysplasia, while the video electroencephalogram showed abnormal discharge. High-throughput sequencing showed the child has harbored a heterozygous c. 5G>T (p.Arg2Leu) variant of TUBB2A gene, which was unreported previously. His sister also carried the variant and had similar clinical manifestations, whilst his parents were of the wild-type and had normal clinical phenotypes. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as pathogenic (PS2+ PM2_Supporting+ PM5+ PP1+ PP2+ PP3). Conclusion:The heterozygous c. 5G>T (p.Arg2Leu) variant of the TUBB2A gene, in the form of gonadal mosaicism, probably underlay the disorders in this family.