Genetic Diagnosis and Identification of a Novel De Novo RELA Variant in Familial Behçet-like Autoinflammatory Syndrome Type 3: A Case Report.

To explore the clinical and molecular basis for a Chinese pedigree affected with Complete androgen insensitivity syndrome (CAIS). A CAIS pedigree presented at Tianjin Medical University General Hospital between 2019 and 2021 was selected as the study subject. Clinical data of the proband was collected, along with peripheral blood samples from the proband and her family members. Chromosomal karyotyping, sex-determining region of the Y chromosome (SRY) testing, and next-generation sequencing (NGS) were carried out for the proband, and candidate variant was verified by Sanger sequencing of her family members. Prenatal diagnosis was provided for the sister of the proband. This study was approved by the Tianjin Medical University General Hospital (Ethics No. IRB2023-WZ-070). The 18-year-old proband, who has a social gender of female, underwent laparoscopic examination, which showed no presence of uterus and ovaries. The karyotype of peripheral blood sample was 46,XY, with SRY gene detected. NGS indicated that the proband has harbored a heterozygous c.1988C>G (p.Ser663Ter) variant of the AR gene. Sanger sequencing confirmed that her mother and sister had both harbored the same variant, whilst her father and younger sister were of the wild-type. Prenatal diagnosis revealed that her sister's first fetus had harbored carried the same variant, which had led to termination of pregnancy. Her second fetus did not carry the variant, and a healthy boy was born. Based on guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as likely pathogenic (PM2_Supporting+PM4+PP3_Moderate+PP4). The c.1988C>G (p.Ser663Ter) variant of the AR gene probably underlay the CAIS in the proband. The accurate diagnosis of sex development disorders will rely on the physicians' thorough understanding of the clinical symptoms and pathogenic genes. Genetic testing and counseling can enable precise diagnosis, prenatal diagnosis, and guidance for reproduction.

Should We Diagnose Autosomal Dominant Alport Syndrome When There Is a Pathogenic Heterozygous COL4A3 or COL4A4 Variant?

Homozygosity for the hyperunstable hemoglobin variant Hb Agrinio (HBA2:c.89T>C) leads to severe antenatal anemia: Eight new cases in three families.

Objectives To provide genetic counseling and prenatal molecular diagnosis for two families with megalencephalic leukoencephalopathy with subcortical cysts (MLC). Methods Two MLC patients (probands 1 and 2) were admitted to the Department of Pediatrics of Peking University First Hospital in June 2011 and June 2009, respectively. Peripheral blood was collected and DNA sequencing was performed for genetic analysis for the two MLC patients and their parents. Amniotic fluid and villus of two fetuses (fetus 1 and 2) were collected at 21+4 and 12+3 weeks of gestational age from their mothers when they were pregnant again. The genomic DNA of the two fetuses was extracted and corresponding sites of MLC1 gene were sequenced. Haplotype analysis using a combination of 3 microsatellite markers (AR, DXS6807 and DXS6797) on chromosome X and sex-determining region of Y chromosome was performed to detect maternal cell contamination. Verification of the prenatal molecular diagnosis and follow-up study after birth were conducted for both fetuses. Results Macrocephaly, motor development delay and typical findings on brain MRI were identified in the two probands, and were clinically diagnosed with MLC. Compound heterozygous mutations were detected in proband 1 [c.353C>T (p.T118M) and c.803C>G (p.T268R)] and proband 2 [c.353C>T (p.T118M) and c.836T>C(p.L279P)], respectively. MLC was genetically diagnosed. Heterozygous variation in c.353[c.353C>T (p.T118M)] and wild c.803C were identified in fetus 1, and both wild c.353C and c.836T were found in fetus 2. No maternal cell contamination was detected in both fetuses. Sequencing the corresponding sites after birth confirmed the prenatal diagnosis, and the head circumference and motor development were normal in fetus 1 at 5 months old. No macrocephaly was found and no DNA sequencing was done in fetus 2 at one month old. Conclusions Genetic counseling and prenatal molecular diagnosis for MLC families combined with clinical and genetic diagnosis are important in preventing MLC. Haplotype analysis with a combination of three microsatellite markers on chromosome X and sex-determining region of Y chromosome is useful in detecting maternal cell contamination and avoiding its influence on prenatal diagnosis, and confirming the reliability of prenatal diagnosis. Key words: Megalencephalic leukoencephalopathy with subcortical cysts; Genetic leukoencephalopathy; Membrane proteins; Prenatal diagnosis

Objective To provide experimental evidence for genetic counseling and prenatal molecular diagnosis by analyzing the clinical characteristics and screening for pathogenic genes of a five-generation suspected multiple epiphyseal dysplasia (MED) family (17 patients). Methods The family members' medical history, general physical examination and hip joint X-ray examination were collected. Peripheral blood samples of the family members were collected and DNA were extracted from these samples. The exons of clinical genes from probands' DNA were sequenced by High throughput sequencing method. Next Gene software was used to compare and analyze the sequence and INGENUITY software was further used to annotate the mutations in order to find the pathogenic mutations in probands. The suspicious mutations were confirmed in pedigree members by PCR and Sanger sequencing. Results The family consisted of 5 generations and 38 members. Pedigree analysis was consistent with autosomal dominant inheritance. There were 17 patients in the family, and their clinical manifestations showed abnormal walking posture in childhood, pain in hip and knee joints, and typical pathological changes of epiphyseal dysplasia on X-ray. Cartilage oligomeric matrix protein (COMP) gene c.1153G>A (p.Asp385Asn) missense heterozygous mutation was screened in proband, which was genotypically and phenotypically segregated in the pedigree. Conclusion A missense mutation of the comp gene has been identified in a pedigree affected with MED which was the first reported in a big family. Our result is conducive to the further diagnosis and treatment and also provides a molecular basisfor the future prenatal diagnosis. Key words: Osteochondrodysplasias; Pedigree; DNA, intergenic; DNA mutational analysis

Prenatal exome sequencing for fetuses with structural abnormalities: the next step.

Sex determining region Y box transcription factor 2 (SOX2) mutations lead to bilateral anophthalmia with autosomal dominant human inheritance. SOX2 mutations could result in severe ocular phenotypes usually associated with variable systemic defects. Most patients described with SOX2 anophthalmia syndrome possessed de novo mutations in this gene. In this case report, we describe 2 brothers with mental retardation and bilateral anophthalmia caused due to SOX2 germline mosaicism in unaffected parents. Next-generation DNA sequencing was carried out to determine the family's possible cause of genetic mutation. Sanger sequencing was performed on the patients and their parents. Prenatal diagnosis was done in both pregnancies of the older brother's wife via chorionic villus sampling. A novel heterozygous pathogenic frameshift deletion variant (exon1:c.58_80del:p.G20fs) was identified in the SOX2 gene, which was confirmed by Sanger sequencing in both affected brothers and did not exist in healthy parents, indicating germline mosaicism. Most SOX2 mutations known look to arise de novo in probands and are diagnosed through anophthalmia or microphthalmia. Prenatal diagnosis should be offered to healthy parents with a child with SOX2 mutation every pregnancy.

Background and purpose: Intellectual disability-7 (MRD7) is a subtype disorder of intellectual disability (MRD) involving feeding difficulties, hypoactivity, and febrile seizures at an age of early onset, then progressive intellectual and physical development deterioration. We purposed to identify the underlying causative genetic factors of three individuals in each Chinese family who presented with symptoms of intellectual disability and facial dysmorphic features. We provided prenatal diagnosis for the three families and genetic counseling for the prevention of this disease.Methods: We collected retrospective clinical diagnostic evidence for the three probands in our study, which included magnetic resonance imaging (MRI), computerized tomography (CT), electroencephalogram (EEG), and intelligence tests for the three probands in our study. Genetic investigation of the probands and their next of kin was performed by Trio-whole exome sequencing (WES). Sanger sequencing or quantitative PCR technologies were then used as the next step to verify the variants confirmed with Trio-WES for the three families. Moreover, we performed amniocentesis to explore the state of the three pathogenic variants in the fetuses by prenatal molecular genetic diagnosis at an appropriate gestational period for the three families.Results: The three probands and one fetus were clinically diagnosed with microcephaly and exhibited intellectual developmental disability, postnatal feeding difficulties, and facial dysmorphic features. Combining probands’ clinical manifestations, Trio-WES uncovered the three heterozygous variants in DYRK1A: a novel variant exon3_exon4del p.(Gly4_Asn109del), a novel variant c.1159C>T p.(Gln387*), and a previously presented but rare pathogenic variant c.1309C>T p.(Arg437*) (NM_001396.5) in three families, respectively. In light of the updated American College of Medical Genetic and Genomics (ACMG) criterion, the variant of exon3_exon4del and c.1159C>T were both classified as likely pathogenic (PSV1+PM6), while c1309C>T was identified as pathogenic (PVS1+PS2_Moderate+PM2). Considering clinical features and molecular testimony, the three probands were confirmed diagnosed with MRD7. These three discovered variants were considered as the three causal mutations for MRD7. Prenatal diagnosis detected the heterozygous dominant variant of c.1159C>T p.(Gln387*) in one of the fetuses, indicating a significant probability of MRD7, subsequently the gestation was intervened by the parents’ determination and professional obstetrical operation. On the other side, prenatal molecular genetic testing revealed wild-type alleles in the other two fetuses, and their parents both decided to sustain the gestation.Conclusion: We identified two novel and one rare mutation in DYRK1A which has broadened the spectrum of DYRK1A and provided evidence for the diagnosis of MRD7 at the molecular level. Besides, this study has supported the three families with MRD7 to determine the causative genetic factors efficiently and provide concise genetic counseling for the three families by using Trio-WES technology.

Global burden of breast cancer is expected to increase to >2 million new cases every year by 2030 and 10% of these are likely to have hereditary breast and ovarian cancer syndrome. Identifying these individuals by pedigree and BRCA1/2 mutation analyses will enable us to offer targeted mutation testing and appropriate counseling. This study from a tertiary care hospital showed that of the 127 breast cancer patients on treatment during 2014-2015, 24 of them fulfilled the criteria of hereditary breast and ovarian cancer syndrome after detailed verbal autopsy and pedigree analysis, and BRCA1 and 2 next-generation sequencing done after pre-test counseling revealed mutations in 13 cases (54%), these included 9 BRCA1 mutations (69%) and 4 BRCA2 mutation (31%). Subsequent post-test counseling recommended targeted mutation analysis for 64 high-risk members in these 13 families with pathogenic mutations, which will help in surveillance for early detection, appropriate management, and prevention of the disease by decreasing the burden to both family and nation. Results from this preliminary study highlight the importance of genetic counseling, pedigree analysis, and genetic testing. It can be recommended that all oncology units should have a genetic counseling service for providing appropriate support to oncologists, patients, and families to prevent unnecessary testing; however, breast cancer screening program is incomplete without evaluating for hereditary breast and ovarian cancer syndrome.

P109 Validation of genotyping method for human leukocyte antigen based on next generation sequencing technology

Objective To analyze gene mutations and the etiology of six fetuses with osteogenesis imperfecta detected by prenatal ultrasonography. Methods From March 2016 to May 2017, six gravidas of singleton pregnancy and their fetuses that were diagnosed with osteogenesis imperfecta by prenatal ultrasonography were enrolled in this study. Gravida 1 came to the Center of Prenatal Diagnosis of the First Affiliated Hospital of Zhengzhou University for prenatal diagnosis, while the other five were referred to that center after termination to identify genetic defects with their fetal tissues. Next-generation sequencing technology was carried out for exome sequencing in the genomes of six fetuses. Suspected mutations were confirmed by polymerase chain reaction and Sanger sequencing. Two hundred unrelated healthy individuals were analyzed with Sanger sequencing for validation of novel mutations. Results Fetus 1 carried a heterozygous mutation in collagen, typeⅠ, alpha-1 (COL1A1) gene, c.724G>C(p.Gly242Arg), which was found in the mother and brother but not in the father. Fetus 2 carried a known heterozygous mutation in COL1A1 gene, c.2461G>A(p.Gly821Ser), which was found in the mother but not in the father. Four heterozygous mutations, c.2282G>A(p.Gly761Asp), c.1002+5G>A in COL1A1 gene, c.1774G>A(p.Gly592Ser) and c.3277G>T(p.Gly1093Cys) in collagen, typeⅠ, alpha-2 (COL1A2) gene, were respectively carried by fetuses 3 to 6, but not by their parents. Mutations of c.724G>C(p.Gly242Arg), c.2282G>A (p.Gly761Asp) and c.1002+5G>A in COL1A1 gene and c.3277G>T (p.Gly1093Cys) in COL1A2 gene were four novel mutations, which were not found in the 200 unrelated healthy individuals. The mother of fetus 1 who was highly suspected with osteogenesis imperfecta selected to continue the pregnancy because the family members had mild symptoms. After delivery, cord blood was collected for genetic test and the result was consistent with that of prenatal genetic diagnosis. Fetus 1 had no fractures during a six-month follow-up after birth. Conclusions Mutations in the COL1A1 and COL1A2 genes may be the etiology of osteogenesis imperfecta in these six fetuses. Results of this study could enrich the data on COL1A1 and COL1A2 mutations relating to osteogenesis imperfecta, and provide a basis for genetic counseling. Key words: Osteogenesis imperfecta; Collagen typeⅠ; Mutation; Prenatal diagnosis; Genetic testing

BackgroundPrezygotic de novo mutations may be inherited from parents with germline mosaicism and are often overlooked when the resulting phenotype affects only one child. We aimed to identify paternal germline mosaicism in an index family and provide a strategy to determine germline mosaicism.‘MethodsWhole‐exome sequencing was performed on an Alport syndrome‐affected child. Variants were validated using Sanger sequencing in the pedigree analysis. An apparent de novo mutation was tested by next‐generation sequencing (NGS) following chromosome microdissection of the mutant region (MicroSeq) to clarify its homologous chromosome source. Mosaic mutation in sperm samples was detected using targeted next‐generation sequencing (TNGS). Self‐prepared mosaic DNA samples of the 3% and 0.1% mutant fractions were used to evaluate the TNGS detection sensitivity.ResultsTwo novel heterozygous variants, maternally inherited c.1322delT (p.Ile441Thrfs*17) and the de novo mutation c.2939T>A (p.Leu980Ter), in the COL4A3 gene were discovered in the propositus. MicroSeq identified c.2939T>A in the paternal chromosome, which was in trans with c.1322delT. The frequency of c.2937A was 2.65% in the father's sperm sample. We also showed that a 500X depth coverage may detect a mosaic mutation with an allele frequency as low as 2%–3% using TNGS.ConclusionMicroSeq is a valuable tool to identify the allele source of de novo mutations in a single patient. TNGS can be used to assess the mosaic ratios of known sites. We provided a systematic algorithm to detect germinal mosaicism in a single patient. This algorithm may have implications for genetic and reproductive counseling on germline mosaicism.

Molecular characterization of human adenoviruses in urban wastewaters using next generation and Sanger sequencing

Studies on the acceptance of prenatal diagnosis and termination of pregnancy for single gene disorders within Islamic societies in the Middle East are limited. A few have examined the attitudes toward pregnancy termination for fetal indications, but a dearth of published data exists on actual behavior and uptake. This study reports on all prenatal diagnosis requests for single gene disorders, from the Sultanate of Oman, over 9 years. A retrospective study was conducted during which the medical records of all women who performed prenatal diagnoses for single gene disorders were reviewed. A total of 148 invasive procedures were performed for 114 families. The total number of yearly requests for prenatal diagnosis increased exponentially from three in 2012 to 21 in 2020. Sixty-four different diagnoses were tested for with the majority being autosomal recessive in nature. Seventy-one percent (28/39) of cases where an affected pregnancy was identified were terminated. Fifty-two of the 114 women (45.6%) repeated prenatal diagnosis in a future pregnancy. Seventy-two couples (63%) were consanguineous parents related as second cousins or closer. The majority of tests performed were for couples from Muscat (27%), Albatinah (27%), and Alsharqiya (20.3%) governorates in Oman. The findings of this study provide evidence that prenatal diagnosis is an acceptable reproductive option to prevent the occurrence of genetic disorders that meet termination eligibility criteria as outlined by the Islamic Jurisprudence (Fiqh) Council Fatwa, among Omani Muslim couples.

Background: von Willebrand Disease (VWD) is the most common hereditary coagulation disorder. Generally, coagulation disorders are more prevalent in communities with high rate of consanguinity. Due to segregation of multiple alleles, combinations of abnormal variants causing unique clinical presentations can occur. Aims: We aim to present our single centre experience of VWD in pediatrics. Methods: Retrospective data analysis of all pediatric patients diagnosed with (VWD) throughout a period of 10 years, dating from January 2012 till December 2021. In addition, molecular testing using next generation sequencing (NGS) of a family, with a spectrum of bleeding phenotypes was done. Results: Out of 455 cases with hereditary coagulation disorders, 219 (48%) were diagnosed with VWD throughout the study period (141 girls and 78 boys), with an age of presentation ranging from 18 months to 14 years. Significant bleeding episodes were encountered in 42/219 patients (19%). History of consanguinity was documented in 175 cases (80%). The most common presenting feature was mucocutaneous bleeding, followed by menorrhagia and then post-traumatic/post-surgical hemorrhage. Factor replacement therapy was used in 34 cases and desmopressin (DDAVP) in 22 cases. Hormonal therapy was offered to 18 girls presenting with menorrhagia. However, only 7 of them were compliant. The index case is a 4-year-old girl, presented with profuse post-adenotonsillectomy hemorrhage. Her past history was negative for any significant bleeding, but no previous surgical challenges. Parents are cousins, and family history was irrelevant. Routine preoperative investigations didn’t reveal any abnormalities apart from raised mean platelet volume (14 fL), and a platelet count of 180 × 10ˆ9/L. Bleeding didn’t respond optimally to FFP, but controlled with multiple platelet transfusions and recombinant activated factor VII. Molecular studies using NGS revealed two different heterozygous variants in the CD36 gene. The first heterozygous variant was NM_001001547.2:c338_339del (p.Ser113Phefs*20), a frameshift variant which results in loss of function of the protein product of CD36 gene. The second was deletion of exons 2 and 3 (NM_001001547.2:c.1_120del), which results in loss of a significant portion of the coding region of CD36 gene. To our knowledge, both novel variants have not been reported in the literature. Biallelic mutations in CD36 are associated with platelet glycoprotein IV deficiency (PG4D), characterized by macrothrombocytopenia with variable bleeding phenotype (PubMed: 7533783; OMIM: 173510). In addition, A heterozygous variant in the VWF gene, NM_000552.4:c.3692A>G (p.Asn1231Ser), was identified. This heterozygous variant was reported in a case with von Willebrand disease type 1 (PubMed: 28971901). The distinctive combination of these three variants explains the severe bleeding phenotype. NGS identified the same heterozygous frameshift variant of CD36 in the mother, while the father carries both the deletional variant of CD36 and the missense variant of VWF gene. Two sisters (ages: 8 & 15 years) harbor the heterozygous VWF gene variant. On further enquiries, the eldest was found to have mild to moderate menorrhagia, that didn’t require any medical advice. Family pedigree is shown in figure 1. Image:Summary/Conclusion: Genotype-phenotype correlation is mandatory in coagulation disorders, presenting with significant bleeding episodes.