Molecular Genetics & Genomic Medicine最新文献

Background: When the SRY gene is present in a 46,XX fetus, some degree of testicular development is expected. Our laboratory performed prenatal genetic testing for a fetus that had screened positive for Y chromosome material by noninvasive prenatal screening (NIPS) but that had apparently typical female development by ultrasound imaging. The aim of this study was to determine the clinical relevance of the NIPS results.

Methods: We analyzed fetal material obtained via amniocentesis procedure by G-banding, microarray, and fluorescence in situ hybridization (FISH). Optical genome mapping (OGM) was also performed.

Results: G-band analysis revealed a normal 46,XX karyotype. Microarray and FISH analyses together detected an SRY+ gain of 5.7 Mb from terminal Yp that was translocated to terminal Xq, with a loss of 1.6 Mb from terminal Xq. The final karyotype was 46,X,der(X)t(X;Y)(q28;p11.2). Prenatal ultrasound and postnatal physical examination revealed apparently typical female genitalia. The Xq deletion encompassed a gene, IKBKG, that is sensitive to loss of function, suggesting that preferential inactivation of the derivative X chromosome allowed for typical female development. OGM software did not directly identify this translocation.

Conclusion: This case demonstrates how the SRY gene may be present in a 46,XX biological female without differences of sexual development.

Objective: Mammalian Diaphanous-Related Formin (mDia1), which is encoded by the DIAPH1 gene, serves as essential for the regulation of cell morphology and cytoskeletal organization. The role of DIAPH1 in brain development has been extensively established. This study aims to evaluate the clinical, neuroradiological, and genetic characteristics of patients with DIAPH1-related disease and determine probable genotype-phenotype relationships.

Methods: In the current study, exome sequencing was performed to identify the genetic basis of the clinical presentation in an Iranian 7-year-old boy. Validation of the detected variant was done by Sanger sequencing. Furthermore, we performed a comprehensive review of the literature.

Results: Here, we detected a novel homozygous c.1285C> T (p.Gln429*) pathogenic variant in the patient. In silico analysis with prediction software tools identified this variant as a probable source of damage. Twenty cases from seven studies were found after a review of the literature. The patients' main symptoms were a developmental delay, microcephaly, and seizures. The mean age of onset for patients in the group of 20 patients with a known age of onset was 2.3 months (SD = 1.6). Of the variants identified, c.2769del, c.684+1G>A, and c.2332C> T were identified in 72% of the patients.

Conclusion: Considering the variant's position in the gene and the encoding protein, a pathogenic effect is predicted for the variant. So, the patient's clinical manifestation is probably caused by this pathogenic variant. Moreover, by studying clinical manifestations in all molecularly confirmed reported cases, provided a comprehensive overview of clinical presentation, and attempted to find a genotype-phenotype correlation.

Background: Targeted next generation sequence analyses in a cohort of 961 previously described patients with clinically suspected Duchene muscular dystrophy (DMD) revealed that 145/961 (15%) had variants in genes associated with other muscular dystrophies (OMDs).

Methods: NGS was carried out in DMD negative patients after deletion/duplication analysis followed by WES for No variant cases.

Results: The majority of patients with OMDs had autosomal recessive diseases that included Limb-Girdle Muscular Dystrophies (LGMDs), Bethlem, Ullrich congenital Myopathies and Emery-Driefuss muscular dystrophy. 3.5% of patients were identified with other disorders like Charcot-Marie Tooth and Nemaline myopathy. A small percentage of patients, 0.6% remain undiagnosed. Of a total of 78 genetic variants identified, 44 were found to be novel. Interestingly, a third of patients with OMDs were found to have LGMD2E/R4, a severe form of LGMD that afflicts young children with clinical symptoms similar to DMD. Almost one third of the unrelated LGMD2E/R4 patients had the same point mutation (c.544A>C) in the SGCB gene, suggestive of a founder effect, described here for the first time in India.

Conclusion: This study underscores the need for a complete genetic work up to precisely diagnose patients and to initiate appropriate counseling programs, disease management and prevention strategies.

Background: Next-generation sequencing (NGS) technology enables sample multiplexing for interrogation of multiple regions of interest (ROI). Leveraging this, together with access to affordable NGS platforms, we explored the practicality of moving capillary electrophoresis (CE), noncapillary electrophoresis and single-gene testing to NGS. In this work, we evaluated the iSeq 100's capacity to validate 89 samples at once.

Methods: Genomic DNA was extracted from 89 archival samples of varying specimen types. Polymerase chain reaction (PCR) was done with in house primers, library preparation with the Nextera XT Library Preparation Kit and cleaning up with paramagnetic beads. The sequencing was performed on one Illumina iSeq 100 flow cell.

Results: With our workflow, 88 out of 89 samples were accurately sequenced with variant alleles identified. One sample of the 88 samples was initially discordant because the primers used were in a heterozygous deletion region. Upon redesigning of primers, the sample proved concordant.

Conclusions: The iSeq-Nextera workflow proved accurate. However, variant allele frequencys generated by the Nextera are not precise.

Background: Pathogenic variants in the mismatch repair genes are associated with an elevated lifetime risk of colorectal cancer (CRC). We previously identified two variants of uncertain significance (VUS) in the MLH1 gene, c.696_698del, p.(Cys233del) and c.1919C > G, p.(Pro640Arg), in Danish families with numerous occurrences of CRC.

Methods: To reclassify the variants we collected clinical data, initiated tumor and co-segregation analysis, and performed RNA splicing analysis, subcellular localization, and protein stability studies.

Results: The functional analysis revealed that the c.696_698del, p.(Cys233del) variant had an effect at the RNA level, on subcellular localization, and on protein stability, while the c.1919C > G, p.(Pro640Arg) variant showed decreased expression in localization studies and decreased protein stability. These results suggest both variants disrupt DNA mismatch repair.

Conclusion: By applying all collected data and functional results we propose to reclassify the c.696_698del, p.(Cys233del) and the c.1919C > G, p.(Pro640Arg) variants as likely pathogenic (class 4) using MMR gene-specific ACMG/AMP guidelines. Consequently, the two MLH1 variants can now be used for risk assessment of variant carriers, while family members without the variants can be excluded from intensified cancer surveillance and follow population recommendations.

Background: The main features of Axenfeld-Rieger Syndrome (ARS) are ocular, auditory, neurological, and morphological brain abnormalities. Mutations in forkhead box protein C1 (FOXC1) are among the responsible genes causing ARS, but neuropsychiatric features have rarely been reported. The case of an ARS patient (a 77-year-old man) with delusions of jealousy and impairment of working memory, in addition to the main clinical features, glaucoma and leukoencephalopathy, is presented.

Methods: The mutation in the patient's genome was found with whole exome sequencing and in silico analysis using PolyPhen-2 and SIFT. Furthermore, AlphaFold2 and PyMOL were used to predict the protein structure based on the mutation.

Results: A novel mutation at the forkhead domain of FOXC1 gene (c.408C>A, p.Phe136Leu) was found and confirmed in the patient's family, and it was predicted to cause protein damage; the SIFT score was 0, meaning deleterious, and the PolyPhen2 result also indicated damaging (score: 0.997). The predicted protein structure based on the novel mutation was different from that of the native structure. In the literature review, 6 of 95 (6.3%) cases showed neuropsychiatric features. Of them, 5 of 6 (83.3%) mutations were located in the forkhead domain.

Conclusion: A novel mutation was found in the FOXC1 gene (c.408C>A, p.Phe136Leu), which possibly induces delusions of jealousy and impairment of working memory, as well as features of ARS, by changing the protein structure. Mutations in that domain of the FOXC1 gene may be important not only for ocular abnormalities but also for brain function.

Background: Congenital muscular dystrophies (CMDs) are diverse early-onset conditions affecting skeletal muscle and connective tissue. This group includes collagen VI-related dystrophies such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM), caused by mutations in the COL6A1, COL6A2 and COL6A3 genes. We report a consanguineous Malian family with three siblings affected by UCMD due to a novel homozygous splice site variant in the COL6A1 gene.

Methods: After obtaining consent, three affected siblings and their relatives underwent physical examinations by specialists and laboratory tests where possible. DNA was extracted from peripheral blood for genetic testing, including Whole Exome Sequencing (WES). Putative variants were confirmed through Sanger Sequencing and assessed for pathogenicity using in silico tools.

Results: The three siblings and their healthy parents, from a consanguineous marriage, presented with early-onset progressive muscle weakness, walking difficulty, proximal motor deficits, severe muscle atrophy, hypotonia, skeletal deformities, joint hyperlaxity, ankyloses at the elbows and knees, keloid scars and dental crowding. No cardiac involvement was detected and creatine kinase (CK) levels were normal. All had low serum calcium levels, treated with oral supplements. Needle myography indicated myopathic patterns. WES identified a novel splice site variant in the first intron of COL6A1 (c.98-1G>C), which segregated with the disease within the family. This variant is predicted to cause exon 2 skipping in COL6A1, with a high CADD score of 33 and Splice AI predicting it as deleterious.

Conclusion: We identified a novel COL6A1 variant in a consanguineous family, highlighting the need for further studies in larger African cohorts to enhance genetic epidemiology and prepare for future therapeutic research.

Background: Impaired myocardial function and arrhythmia are important manifestations of Marfan syndrome (MFS). Studies assessing myocardial fibrosis in relation to these manifestations are scarce.

Methods: This cross-sectional, single-center study assessed ventricular volumes, ventricular function, and myocardial fibrosis by cardiac magnetic resonance imaging (CMR) in patients with MFS harboring a (likely) pathogenic FBN1 variant. The presence and extent of fibrosis were assessed by late gadolinium enhancement (LGE) and extracellular volume measurement (ECV). Data on 24-h Holter monitoring and clinical data were extracted from electronic patient records.

Results: The study included 32 unselected patients with MFS (median age 38 years [range 10-69], 41% women). No focal myocardial fibrosis was detected. Six patients (21%) had diffuse fibrosis (ECV > 29%). No association was found between the presence of diffuse fibrosis and clinically relevant myocardial dysfunction. Five patients (16%) had reduced left ventricular ejection fraction (LVEF < 55%). While all of these exhibited mitral annular disjunction (MAD), only two had ECV > 29%. Patients with MAD had increased indexed LV volumes (median end-diastolic volume, 92 mL/m² [IQR, 78-100] vs. 78 mL/m² [IQR, 71-87]; median end-systolic volume, 31 mL/m² [IQR, 23-46] vs. 22 mL/m² [IQR, 21-28]), also after adjusting for the presence of mitral and aortic valve regurgitation. No differences in ECV were seen between patients with and without MAD.

Conclusions: In this cohort of patients with MFS, focal myocardial fibrosis was not detected using CMR. Although diffuse fibrosis was observed in 21% of patients, no evident connection to clinically relevant myocardial dysfunction was found. Further studies should evaluate the impact of diffuse fibrosis on clinical outcome prediction.

Background: Sotos syndrome (SS) is a rare disorder characterized by overgrowth, distinctive facial features, and intellectual disability that is primarily caused by NSD1 pathogenic variants or 5q35 microdeletions.

Methods: We retrospectively analyzed the clinical characteristics and 339 anthropometric measurements over an average of 4.3 years of follow-up in 57 Korean children with SS. Sex-specific percentile curves for height, weight, and head circumference were developed using a generalized additive model that included factors such as location, scale, and shape.

Results: Males with SS demonstrated higher height before the age of 12.0, greater weight before 10.0, and larger head circumference before 15.5 compared to age- and sex-matched controls. Females with SS displayed higher height before 17.0, greater weight before 10.5, and larger head circumference before 12.0 compared to controls. Bone age was advanced compared to chronological age in 40% of males and 8% of females at their last visit. The predicted and target adult heights were not significantly different between groups. In subgroup analysis, the intragenic variant group (n = 48) showed a higher mean standard deviation score of height and weight in males, and head circumference in females compared to the microdeletion group (n = 9).

Conclusions: Korean children with genetically confirmed SS exhibited overgrowth in height, weight, and head circumference. Overgrowth phenotypes were more prominent in patients with NSD1 intragenic variants than in those with microdeletions. This is the first study to provide reference data on the growth of Korean children with SS.

Background: High sequence homology between CYP21A2 and CYP21A1P poses challenges to genetic diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD). Traditional genetic testing is unable to provide an accurate diagnosis due to the genetic complexity of CAH.

Methods: Deletions, duplications, and recombination breakpoints were precisely identified by long-read sequencing (LRS).

Results: This study presented a pregnant woman, a 21-OHD carrier detected by MLPA, and her husband, a normal subject also detected by MLPA. The fetus was suspected of having 21-OHD based on clinical presentations such as enlarged adrenal glands, atypical external genitalia and karyotyping of 46, XX. LRS further identified the fetus as having the most severe salt-wasting (SW) form of 21-OHD with a compound heterozygote genotype. One allele was TNXA/TNXB CH-2, while the other allele was CYP21A1P/CYP21A2 CH-8. LRS precisely determined the genotypes of the fetus's father and grandmother with duplications, which misdiagnosed by MLPA. The multidisciplinary team recommended immediate glucocorticoid and mineralocorticoid treatment for the child after birth to prevent life-threatening adrenal crisis.

Conclusions: LRS provides precise diagnosis for family members with CYP21A2 deletion or duplication, improving disease management and preventing potential adrenal crises. When used in pre-pregnancy genetic testing, LRS can indicate high genetic risk and guide the appropriate therapy during pregnancy and immediately after birth.