Evaluating the clinical efficacy of a long-read sequencing-based approach for carrier screening of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is the second most common fatal genetic disease in infancy. It is caused by deletion or intragenic pathogenic variants of the causative gene SMN1, which degenerates anterior horn motor neurons and leads to progressive myasthenia and muscle atrophy. Early treatment improves motor function and prognosis in patients with SMA, but drugs are expensive and do not cure the disease. Therefore, carrier screening seems to be the most effective way to prevent SMA birth defects. In this study, we genetically analyzed 1400 samples using multiplex ligation-dependent probe amplification (MLPA) and quantitative polymerase chain reaction (qPCR), and compared the consistency of the results. We randomly selected 44 samples with consistent MLPA and qPCR results for comprehensive SMA analysis (CASMA) using a long-read sequencing (LRS)-based approach. CASMA results showed 100% consistency, visually and intuitively explained the inconsistency between exons 7 and 8 copy numbers detected by MLPA in 13 samples. A total of 16 samples showed inconsistent MLPA and qPCR results for SMN1 exon 7. CASMA was performed on all samples and the results were consistent with those of resampling for MLPA and qPCR detection. CASMA also detected an additional intragenic variant c.-39A>G in a sample with two copies of SMN1 (RT02). Finally, we detected 23 SMA carriers, with an estimated carrier rate of 1/61 in this cohort. In addition, CASMA identified the "2 + 0" carrier status of SMN1 and SMN2 in a family by analyzing the genotypes of only three samples (parents and one sibling). CASMA has great advantages over MLPA and qPCR assays, and could become a powerful technical support for large-scale screening of SMA.

DNA methylation as a new tool for the differential diagnosis between T-LBL and lymphocyte-rich thymoma.

T-lymphoblastic lymphoma (T-LBL) and thymoma are two rare primary tumors of the thymus deriving either from T-cell precursors or from thymic epithelial cells, respectively. Some thymoma subtypes (AB, B1, and B2) display numerous reactive terminal deoxynucleotidyl transferase-positive (TdT+) T-cell precursors masking epithelial tumor cells. Therefore, the differential diagnosis between T-LBL and TdT+ T-lymphocyte-rich thymoma could be challenging, especially in the case of needle biopsy. To distinguish between T-LBL and thymoma-associated lymphoid proliferations, we analyzed the global DNA methylation using two different technologies, namely MeDIP array and EPIC array, in independent samples series [17 T-LBLs compared with one TdT+ lymphocyte-rich thymoma (B1 subtype) and three normal thymi, and seven lymphocyte-rich thymomas compared with 24 T-LBLs, respectively]. In unsupervised principal component analysis (PCA), T-LBL and thymoma samples clustered separately. We identified differentially methylated regions (DMRs) using MeDIP-array and EPIC-array datasets and nine overlapping genes between the two datasets considering the top 100 DMRs including ZIC1, TSHZ2, CDC42BPB, RBM24, C10orf53, and MACROD2. In order to explore the DNA methylation profiles in larger series, we defined a classifier based on these six differentially methylated gene promoters, developed an MS-MLPA assay, and demonstrated a significant differential methylation between thymomas (hypomethylated; n = 48) and T-LBLs (hypermethylated; n = 54) (methylation ratio median 0.03 versus 0.66, respectively; p < 0.0001), with MACROD2 methylation status the most discriminating. Using a machine learning strategy, we built a prediction model trained with the EPIC-array dataset and defined a cumulative score taking into account the weight of each feature. A score above or equal to 0.4 was predictive of T-LBL and conversely. Applied to the MS-MLPA dataset, this prediction model accurately predicted diagnoses of T-LBL and thymoma. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Next generation sequencing panel as an effective approach to genetic testing in patients with a highly variable phenotype of neuromuscular disorders.

Neuromuscular disorders (NMDs) include a wide range of diseases affecting the peripheral nervous system. The genetic diagnoses are increasingly obtained with using the next generation sequencing (NGS). We applied the custom-design targeted NGS panel including 89 genes, together with genotyping and multiplex ligation-dependent probe amplification (MLPA) to identify a genetic spectrum of NMDs in 52 Polish patients. As a result, the genetic diagnosis was determined by NGS panel in 29 patients so its diagnostic utility is estimated at 55.8%. The most pathogenic variants were found in CLCN1, followed by CAPN3, SCN4A, and SGCA genes. Genotyping of myotonic dystrophy type 1 and 2 (DM1 and DM2) as a secondary approach has been performed. The co-occurrence of CAPN3 and CNBP mutations in one patient as well as DYSF and CNBP mutations in another suggests possibly more complex inheritance as well as expression of a phenotype. In 7 individuals with single nucleotide variant found in NGS testing, the MLPA of the CAPN3 gene was performed detecting the deletion encompassing exons 2-8 in the CAPN3 gene in one patient, confirming recessive limb-girdle muscular dystrophy type 1 (LGMDR1). Thirty patients obtained a genetic diagnosis (57.7%) after using NGS testing, genotyping and MLPA analysis. The study allowed for the identification of 27 known and 4 novel pathogenic/likely pathogenic variants and variants of uncertain significance (VUS) associated with NMDs.In conclusion, the diagnostic approach with diverse molecular techniques enables to broaden the mutational spectrum and maximizes the diagnostic yield. Furthermore, the co-occurrence of DM2 and LGMD has been detected in 2 individuals.

The course of cytokine and chemokine gene expression in clinically suspect arthralgia patients during progression to inflammatory arthritis.

OBJECTIVES: Autoantibody responses increase years before the onset of inflammatory arthritis (IA) and are stable during transitioning from clinically suspect arthralgia (CSA) to IA. Cytokine and chemokine levels also increase years before IA onset. However, the course in the at-risk stage of CSA during progression to disease or non-progression is unknown. To increase the understanding of processes mediating disease development, we studied the course of cytokine, chemokine and related receptors gene expression in CSA patients during progression to IA and in CSA patients who ultimately did not develop IA. METHODS: Whole-blood RNA expression of 37 inflammatory cytokines, chemokines and related receptors was determined by dual-colour reverse transcription multiplex ligation-dependent probe amplification in paired samples of CSA patients at CSA onset and either at IA development or after 24 months without IA development. ACPA-positive and ACPA-negative CSA patients developing IA were compared at CSA onset and during progression to IA. Generalised estimating equations tested changes over time. A false discovery rate approach was applied. RESULTS: None of the cytokine/chemokine genes significantly changed in expression between CSA onset and IA development. In CSA patients without IA development, G-CSF expression decreased (P = 0.001), whereas CCR6 and TNIP1 expression increased (P < 0.001 and P = 0.002, respectively) over a 2 year period. Expression levels in ACPA-positive and ACPA-negative CSA patients who developed IA were similar. CONCLUSION: Whole-blood gene expression of assessed cytokines, chemokines and related receptors did not change significantly from CSA to IA development. This suggests that changes in expression of these molecules may not be related to the final process of developing chronicity and may have occurred preceding CSA onset. Changes in gene expression in CSA patients without IA development may provide clues for processes related to resolution.

Systematic molecular analyses for 115 karyotypically normal men with isolated non-obstructive azoospermia.

STUDY QUESTION: Do copy-number variations (CNVs) in the azoospermia factor (AZF) regions and monogenic mutations play a major role in the development of isolated (non-syndromic) non-obstructive azoospermia (NOA) in Japanese men with a normal 46, XY karyotype? SUMMARY ANSWER: Deleterious CNVs in the AZF regions and damaging sequence variants in eight genes likely constitute at least 8% and approximately 8% of the genetic causes, respectively, while variants in other genes play only a minor role. WHAT IS KNOWN ALREADY: Sex chromosomal abnormalities, AZF-linked microdeletions, and monogenic mutations have been implicated in isolated NOA. More than 160 genes have been reported as causative/susceptibility/candidate genes for NOA. STUDY DESIGN, SIZE, DURATION: Systematic molecular analyses were conducted for 115 patients with isolated NOA and a normal 46, XY karyotype, who visited our hospital between 2017 and 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied 115 unrelated Japanese patients. AZF-linked CNVs were examined using sequence-tagged PCR and multiplex ligation-dependent probe amplification, and nucleotide variants were screened using whole exome sequencing (WES). An optimized sequence kernel association test (SKAT-O), a gene-based association study using WES data, was performed to identify novel disease-associated genes in the genome. The results were compared to those of previous studies and our in-house control data. MAIN RESULTS AND THE ROLE OF CHANCE: Thirteen types of AZF-linked CNVs, including the hitherto unreported gr/gr triplication and partial AZFb deletion, were identified in 63 (54.8%) cases. When the gr/gr deletion, a common polymorphism in Japan, was excluded from data analyses, the total frequency of CNVs was 23/75 (30.7%). This frequency is higher than that of the reference data in Japan and China (11.1% and 14.7%, respectively). Known NOA-causative AZF-linked CNVs were found in nine (7.8%) cases. Rare damaging variants in known causative genes (DMRT1, PLK4, SYCP2, TEX11, and USP26) and hemizygous/multiple-heterozygous damaging variants in known spermatogenesis-associated genes (TAF7L, DNAH2, and DNAH17) were identified in nine cases (7.8% in total). Some patients carried rare damaging variants in multiple genes. SKAT-O detected no genes whose rare damaging variants were significantly accumulated in the patient group. LIMITATIONS, REASONS FOR CAUTION: The number of participants was relatively small, and the clinical information of each patient was fragmentary. Moreover, the pathogenicity of identified variants was assessed only by in silico analyses. WIDER IMPLICATIONS OF THE FINDINGS: This study showed that various AZF-linked CNVs are present in more than half of Japanese NOA patients. These results broadened the structural variations of AZF-linked CNVs, which should be considered for the molecular diagnosis of spermatogenic failure. Furthermore, the results of this study highlight the etiological heterogeneity and possible oligogenicity of isolated NOA. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Grants from the Japan Society for the Promotion of Science (21K19283 and 21H0246), the Japan Agency for Medical Research and Development (22ek0109464h0003), the National Center for Child Health and Development, the Canon Foundation, the Japan Endocrine Society, and the Takeda Science Foundation. The results of this study were based on samples and patient data obtained from the International Center for Reproductive Medicine, Dokkyo Medical University Saitama Medical Center, Koshigaya, Japan. The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Prenatal diagnosis of a trisomy 7 mosaic case: CMA, CNV-seq, karyotyping, interphase FISH, and MS-MLPA, which technique to choose?

OBJECTIVE: This study aims to perform a prenatal genetic diagnosis of a high-risk fetus with trisomy 7 identified by noninvasive prenatal testing (NIPT) and to evaluate the efficacy of different genetic testing techniques for prenatal diagnosis of trisomy mosaicism. METHODS: For prenatal diagnosis of a pregnant woman with a high risk of trisomy 7 suggested by NIPT, karyotyping and chromosomal microarray analysis (CMA) were performed on an amniotic fluid sample. Low-depth whole-genome copy number variation sequencing (CNV-seq) and fluorescence in situ hybridization (FISH) were used to clarify the results further. In addition, methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was performed to analyze the possibility of uniparental disomy(UPD). RESULTS: Amniotic fluid karyotype analysis revealed a 46, XX result. Approximately 20% mosaic trisomy 7 was detected according to the CMA result. About 16% and 4% of mosaicism was detected by CNV-seq and FISH, respectively. MS-MLPA showed no methylation abnormalities. The fetal ultrasound did not show any detectable abnormalities except for mild intrauterine growth retardation seen at 39 weeks of gestation. After receiving genetic counseling, the expectant mother decided to continue the pregnancy, and follow-up within three months of delivery was normal. CONCLUSION: In high-risk NIPT diagnosis, a combination of cytogenetic and molecular genetic techniques proves fruitful in detecting low-level mosaicism. Furthermore, the exclusion of UPD on chromosome 7 remains crucial when NIPT indicates a positive prenatal diagnosis of trisomy 7.

First Report of Filipino ß0-Thalassemia/ß-Thalassemia in a Chinese Family.

A pregnant woman living in Fujian Province, southeastern China, presented due to a risk of having a baby with ß-thalassemia major, during her second pregnancy, since she and her husband were suspected as ß-thalassemia carriers and their affected daughter was a transfusion-dependent patient. Using the common α-thalassemia and ß-thalassemia genotypes test, the pregnant woman was diagnosed as a ß-thalassemia carrier with ßIVS-2 - 654 (C→T)/ßN genotype and her daughter had a homozygosity for IVS - 2 - 654 (C→T) mutation, however, no abnormalities were detected in her husband. SMRT identified a Filipino ß0-deletion in her husband, and MLPA also revealed an unknown deletion in the HBB gene. Electrophoresis showed approximately 350 bp of the PCR product, and the ß-Filipino genotype presented novel fracture fragments ranging from 5,112,884 to 5,231,358 bp, and lacked a 118,475 bp fragment relative to the wild-type sequence. The daughter was therefore diagnosed with the ßIVS-2 - 654 (C→T)/ßFilipino genotype. Prenatal diagnosis with umbilical cord blood at 27th week of gestation showed heteroztgosity for IVS - 2 - 654 (C→T) mutation in the fetus and continued pregnancy was recommended. In conclusion, we identified the Filipino ß0-deletion in a Chinese family, from Fujian area, for the first time, during prenatal screening.

Phenotypic and Genotypic Characterization of Hereditary Angioedema in Saudi Arabia.

Hereditary angioedema (HAE) is a potentially life-threatening autosomal dominant disorder affecting roughly 1:50,000 individuals. It is commonly characterized by swelling of the larynx, gastrointestinal tract, extremities, and skin. There is growing genetic heterogeneity associated with this disease but more than 95% of mutations are found in SERPING1, the gene which encodes complement 1 inhibitor (C1-INH). HAE cohorts from several populations have been published but no large scale study has been reported from the Arab world to date. Here we document the clinical and genetic findings of HAE patients from a single Saudi institution, which is a major referral center at the national level. A total of 51 patients across 17 unrelated families were recruited including two large multi-generational families, of which one contained an in-frame exonic deletion that was resolved through MLPA. Two cases were negative for all the genes we tested (including F12, PLG, ANGPT1, MYOF, KNG1, and HS3ST6). The predominant HAE subtype in our cohort was type I, at 76%. We were able to uncover a mutation in 49 patients (96%). No type III (normal C1-INH) patients were encountered in the clinic, suggesting that this subtype does not play a major role in HAE pathogenesis in Saudi Arabia. Additionally, the existence of four patients with consistently normal complement 4 (C4) levels alongside abnormal C1-INH profiles highlights the utility of dual screening for both proteins in suspected patients.

Combined Reverse-Transcriptase Multiplex Ligation-Dependent Probe Amplification and Next-Generation Sequencing Analyses to Assign Unclassified BCL2-/BCL6- Nonrearranged Small B-Cell Lymphoid Neoplasms as Follicular or Nodal Marginal Zone Lymphoma.

Distinguishing between follicular lymphoma (FL) and nodal marginal zone lymphoma (NMZL) can be difficult when morphologic and phenotypic features are unusual and characteristic cytogenetic rearrangements are absent. We evaluated the diagnostic contribution of ancillary techniques-including fluorescence in situ hybridization (FISH)-detected 1p36 deletion; reverse-transcriptase, multiplex, ligation-dependent probe amplification (RT-MLPA); and next-generation sequencing (NGS)-for tumors that remain unclassified according to standard criteria. After review, 50 CD5-negative small B-cell lymphoid neoplasms without BCL2 and BCL6 FISH rearrangements were diagnosed as FLs (n = 27), NMZLs (n = 5), or unclassified (n = 18) based on the 2016 World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues. FISH helped identify the 1p36 deletion in 3 FLs and 1 unclassified tumor. Most classified FLs had an RT-MLPA germinal center B-cell (GCB) signature (93%) or were noncontributive (7%). Classified NMZLs had an RT-MLPA activated B-cell signature (20%), had an unassigned signature (40%), or were noncontributive (40%). Among unclassified tumors, the RT-MLPA GCB signature was associated with mutations most commonly found in FLs (CREBBP, EZH2, STAT6, and/or TNFRSF14) (90%). An RT-MLPA-detected GCB signature and/or NGS-detected gene mutations were considered as FL identifiers for 13 tumors. An activated B-cell signature or NOTCH2 mutation supported NMZL diagnosis in 3 tumors. Combining the RT-MLPA and NGS findings successfully discriminated 89% of unclassified tumors in favor of one or the other diagnosis. NGS-detected mutations may be of therapeutic interest. Herein, we detected 3 EZH2 and 8 CREBBP mutations that might be eligible for targeted therapies.

Copy number variations in SPAST and ATL1 are rare among Brazilians.

Copy number variations (CNV) may represent a significant proportion of SPG4 and SPG3A diagnosis, the most frequent autosomal dominant subtypes of hereditary spastic paraplegias (HSP). We aimed to assess the frequency of CNVs in SPAST and ATL1 and to update the molecular epidemiology of HSP families in southern Brazil. A cohort study that included 95 Brazilian index cases with clinical suspicion of HSP was conducted between April 2011 and September 2022. Multiplex Ligation Dependent Probe Amplification (MLPA) was performed in 41 cases without defined diagnosis by different massive parallel sequencing techniques (MPS). Diagnosis was obtained in 57/95 (60%) index cases, 15/57 (26.3%) being SPG4. Most frequent autosomal recessive HSP subtypes were SPG7 followed by SPG11, SPG76 and cerebrotendinous xanthomatosis. No CNVs in SPAST and ATL1 were found. Copy number variations are rare among SPG4 and SPG3A families in Brazil. Considering the possibility of CNVs detection by specific algorithms with MPS data, we consider that this is likely the most cost-effective approach to investigate CNVs in these genes in low-risk populations, with MLPA being reserved as an orthogonal confirmatory test.