Genetic study of the CDKN2A and CDKN2B genes in renal cell carcinoma patients.

Objectives: While recent studies have demonstrated several genetic alterations are associated with pathogenesis of RCC, the significance of cyclin-dependent kinase inhibitor 2A (CDKN2A) and cyclin-dependent kinase inhibitor 2B (CDKN2B) in tumorigenesis of RCC is less clear. We investigate the distribution of CDKN2A and CDKN2B mutations in patients with RCC and analyze the impact of CDKN2A and CDKN2B mutations on RCC. Methods: A pathological examination was conducted using thirty fresh renal tissue samples with renal masses that had undergone partial or radical nephrectomy. Multiplex ligation-dependent probe amplification (MLPA) was used to detect genetic aberrations of CDKN2A and CDKN2B in genomic DNA isolated from samples. Subsequently, CDKN2A and CDKN2B mutations were confirmed using chromosomal microarray technique. Results: Twenty-one patients were diagnosed with RCC, eight with benign diseases, including angiomyolipoma (AML) and oncocytoma, and one with mucinous adenocarcinoma of renal pelvis. Two of twenty-one patients (9.5 %) with clear-cell RCC were positive for CDKN2A and CDKN2B gene deletions. Interestingly, patients with CDKN2A and CDKN2B mutations were associated with sarcomatoid patterns of RCC (2 out of 4, 50 %). In contrast, no CDKN2A or CDKN2B deletions were detected in samples from benign renal tumors, papillary RCC, or other kidney cancers. Conclusions: This study demonstrated the potential use of CDKN2A and CDKN2B as biomarkers for the prognostic and molecular classification of renal cancer. CDKN2A and CDKN2B mutations may be associated with RCC development and sarcomatoid changes. Further research is needed to understand the underlying molecular mechanisms of CDKN2A and CDKN2B in the pathogenesis of RCC.

Genetic insights into fetal kidney development: Variants in HNF1A and PKHD1 genes.

The orchestration of fetal kidney development involves the precise control of numerous genes, including HNF1A, HNF1B and PKHD1. Understanding the genetic factors influencing fetal kidney development is essential for unraveling the complexities of renal disorders. This study aimed to search for disease-causing variants in HNF1A, HNF1B, PKHD1 genes, among fetus and babies or via parental samples, using sanger sequencing, NGS technologie and MLPA. The study revealed an absence of gene deletions and disease-causing variants in the HNF1B gene. However, five previously SNPs in the HNF1A gene were identified in four patients (patients 1, 2, 3, and 4). These include c.51C > G (Exon1, p. Leu17=), c.79A > C (Exon1, p. Ile27Leu), c.1375C > T (Exon7, p. Leu459=), c.1460G > A (Exon7, p. Ser487Asn), and c.1501 + 7G > A (Intron7). Additionally, in addition to previously SNPs identified, a de novo heterozygous missense mutation (p.E508K) was detected in patient 4. Furthermore, a heterozygous mutation in exon 16 (p. Arg494*; c.1480C > T) was identified in both parents of patient 5, allowing predictions of fetal homozygosity. Bioinformatic analyses predicted the effects of the c.1522G > A mutation (p.E508K) on splicing processes, pre-mRNA structures, and protein instability and conformation. Similarly, the c.1480C > T mutation (p. Arg494*) was predicted to introduce a premature codon stop, leads to the production of a shorter protein with altered or impaired function. Identification of variants in the HNF1A and in PKHD1 genes provides valuable insights into the genetic landscape of renal abnormalities in affected patients. These findings underscore the heterogeneity of genetic variants contributing to renal disorders and emphasize the importance of genetic screening.

Droplet digital PCR analysis of CDH13 methylation status in Slovak women with invasive ductal breast cancer.

Identifying novel epigenetic biomarkers is a promising way to improve the clinical management of patients with breast cancer. Our study aimed to determine the methylation pattern of 25 tumor suppressor genes (TSG) and select the best methylation biomarker associated with clinicopathological features in the cohort of Slovak patients diagnosed with invasive ductal carcinoma (IDC). Overall, 166 formalin-fixed, paraffin-embedded (FFPE) tissues obtained from patients with IDC were included in the study. The methylation status of the promoter regions of 25 TSG was analyzed using semiquantitative methylation-specific MLPA (MS-MLPA). We identified CDH13 as the most frequently methylated gene in our cohort of patients. Further analysis by ddPCR confirmed an increased level of methylation in the promoter region of CDH13. A significant difference in CDH13 methylation levels was observed between IDC molecular subtypes LUM A versus HER2 (P = 0.0116) and HER2 versus TNBC (P = 0.0234). In addition, significantly higher methylation was detected in HER2+ versus HER2- tumors (P = 0.0004) and PR- versus PR+ tumors (P = 0.0421). Our results provide evidence that alteration in CDH13 methylation is associated with clinicopathological features in the cohort of Slovak patients with IDC. In addition, using ddPCR as a methylation-sensitive method represents a promising approach characterized by higher precision and technical simplicity to measure the methylation of target CpGs in CDH13 compared to other conventional methods such as MS-MLPA.

Genomic Characterization of Partial Tandem Duplication Involving the KMT2A Gene in Adult Acute Myeloid Leukemia.

BACKGROUND: Gene rearrangements affecting KMT2A are frequent in acute myeloid leukemia (AML) and are often associated with a poor prognosis. KMT2A gene fusions are often detected by chromosome banding analysis and confirmed by fluorescence in situ hybridization. However, small intragenic insertions, termed KMT2A partial tandem duplication (KMT2A-PTD), are particularly challenging to detect using standard molecular and cytogenetic approaches. METHODS: We have validated the use of a custom hybrid-capture-based next-generation sequencing (NGS) panel for comprehensive profiling of AML patients seen at our institution. This NGS panel targets the entire consensus coding DNA sequence of KMT2A. To deduce the presence of a KMT2A-PTD, we used the relative ratio of KMT2A exons coverage. We sought to corroborate the KMT2A-PTD NGS results using (1) multiplex-ligation probe amplification (MLPA) and (2) optical genome mapping (OGM). RESULTS: We analyzed 932 AML cases and identified 41 individuals harboring a KMT2A-PTD. MLPA, NGS, and OGM confirmed the presence of a KMT2A-PTD in 22 of the cases analyzed where orthogonal testing was possible. The two false-positive KMT2A-PTD calls by NGS could be explained by the presence of cryptic structural variants impacting KMT2A and interfering with KMT2A-PTD analysis. OGM revealed the nature of these previously undetected gene rearrangements in KMT2A, while MLPA yielded inconclusive results. MLPA analysis for KMT2A-PTD is limited to exon 4, whereas NGS and OGM resolved KMT2A-PTD sizes and copy number levels. CONCLUSIONS: KMT2A-PTDs are complex gene rearrangements that cannot be fully ascertained using a single genomic platform. MLPA, NGS panels, and OGM are complementary technologies applied in standard-of-care testing for AML patients. MLPA and NGS panels are designed for targeted copy number analysis; however, our results showed that integration of concurrent genomic alterations is needed for accurate KMT2A-PTD identification. Unbalanced chromosomal rearrangements overlapping with KMT2A can interfere with the diagnostic sensitivity and specificity of copy-number-based KMT2A-PTD detection methodologies.

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.

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.

Mutation analysis and clinical profile of South African patients with Neurofibromatosis type 1 (NF1) phenotype.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic condition with complete age-dependent penetrance, variable expressivity and a global prevalence of ∼1/3,000. It is characteriszed by numerous café-au-lait macules, skin freckling in the inguinal or axillary regions, Lisch nodules of the iris, optic gliomas, neurofibromas, and tumour predisposition. The diagnostic testing strategy for NF1 includes testing for DNA single nucleotide variants (SNVs), copy number variants (CNVs) as well as RNA analysis for deep intronic and splice variants, which can cumulatively identify the causative variant in 95% of patients. In the present study, NF1 patients were screened using a next-generation sequencing (NGS) assay targeting NF1 exons and intron/exon boundaries for SNV and NF1 multiple ligation-dependent probe amplification (MLPA) analysis for CNV detection. Twenty-six unrelated Southern African patients clinically suspected of having NF1, based on the clinical diagnostic criteria developed by the National Institute of Health (NIH), were included in the current study. A detection rate of 58% (15/26) was obtained, with SNVs identified in 80% (12/15) using a targeted gene panel and NF1 gene deletion in 20% (3/15) identified using MLPA. Ten patients (38%) had no variants identified, although they met NF1 diagnostic criteria. One VUS was identified in this study in a patient that met NF1 diagnostic criteria, however there was no sufficient information to classify variant as pathogenic. The clinical features of Southern African patients with NF1 are similar to that of the known NF1 phenotype, with the exception of a lower frequency of plexiform neurofibromas and a higher frequency of developmental/intellectual disability compared to other cohorts. This is the first clinical and molecular characterisation of a Southern African ancestry NF1 cohort using both next-generation sequencing and MLPA analysis. A significant number of patients remained without a diagnosis following DNA-level testing. The current study offers a potential molecular testing strategy for our low resource environment that could benefit a significant proportion of patients who previously only received a clinical diagnosis without molecular confirmation.

Detection and differentiation of seven porcine respiratory pathogens using a multiplex ligation-dependent probe amplification assay.

Respiratory diseases due to viral or bacterial agents, either alone or in combination, cause substantial economic burdens to the swine industry worldwide. Rapid and reliable detection of causal pathogens is crucial for effective epidemiological surveillance and disease management. This research aimed to employ the multiplex ligation-dependent probe amplification (MLPA) assay for simultaneous detection of seven distinct pathogens causing respiratory problems in swine, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine respiratory coronavirus (PRCV), porcine circovirus type 2 (PCV2), Pasteurella multocida, Actinobacillus pleuropneumoniae, and Glässerella parasuis. The results indicated no probe cross-reactivity among the seven target agents with other swine pathogens. The detection limits ranged from 5 to 34 copies per assay for the target organisms. The MLPA assay was evaluated with 88 samples and compared to real-time or multiplex PCR for the target pathogens. The MLPA assay demonstrated high relative test sensitivities (100 %) and reasonable to good relative specificities at 62.5 %, 95.1 %, 86.8 %, and 97.6 % for PRRSV, P. multocida, G. parasuis, and PCV2, respectively, relative to comparator PCR assays. In 71 samples where MLPA and comparator PCR assays matched exactly, infections were detected in 64 samples (90.1 %), with PRRSV being the most commonly found virus and 50.7 % of the samples showing co-infection with two to five of the pathogens. This approach serves as a valuable tool for conducting differential diagnoses and epidemiological investigations of pathogen prevalence within swine populations.

Low-level physical activity predictors among adults living with HIV in Ethiopia's southern region, focusing on work, transportation, and recreation domains: unmatched case-control study.

BACKGROUND: Low-level physical activity (LLPA) is crucial for the well-being of adults living with HIV (PLWHs). However, many do not engage in enough physical activity, leading to adverse health outcomes. Identifying the determinants of LLPA can aid in developing effective interventions. Despite this, Ethiopia lacks evidence on this topic. This study aimed to identify predictors of LLPA among PLWHs in the Gedeo zone, located in southern Ethiopia. METHODS: An unmatched case-control study was conducted on PLWHs in the Gedeo zone who visited two hospitals and healthcare institutions between December 29th, 2017 and January 22nd, 2019. Respondents were classified into three categories based on their total physical activity levels: high, moderate, and low. Cases were defined as those meeting the criteria for LLPA, while controls were those who did not fall under the cases category. Data was collected using the WHO Stepwise surveillance tool and analyzed using Epidata v3.1 templates and SPSS v22. Predictor variables with a P-value < 0.25 in bivariable analysis and < 0.05 with a 95% confidence interval in multivariable analysis were selected. RESULTS: The study involved 633 HIV-positive adults, with a response rate of 92.41%. Most participants were under 34 years old, with an average age of 36.47±(9.055) for cases and 36.38±(8.389) for controls. The multivariable analysis revealed that educational status (AOR = 4.85, P = 0.02, 95%CI (1.28-18.44)), sex (AOR = 0.24, P = 0.04, 95%CI (0.07-0.90)), duration on ART being exposed for 1-4 Years (AOR = 0.12, P < 0.001, 95%CI (0.03-0.44)) and being exposed for 5-9 Years (AOR = 0.03, P < 0.001, 95%CI (0.01-0.16)), and former alcohol use (AOR = 0.11, P < 0.01, 95%CI (0.02-0.56) were significant predictors of LLPA performance. CONCLUSIONS: The study concluded that educational status, sex, ART duration, and past alcohol use are key determinants of LLPA performance among PLWHs in southern Ethiopia. This suggests that policymakers should implement public health campaigns to promote healthy habits, particularly low-level physical activity, among PLWHs.

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.

Case report: A family of atypical hemolytic uremic syndrome involving a CFH::CFHR1 fusion gene and CFHR3-1-4-2 gene duplication.

Mutations in the complement factor H (CFH) gene are associated with complement dysregulation and the development of atypical hemolytic uremic syndrome (aHUS). Several fusion genes that result from genomic structural variation in the CFH and complement factor H-related (CFHR) gene regions have been identified in aHUS. However, one allele has both CFHR gene duplication and CFH::CFHR1 fusion gene have not been reported. An 8-month-old girl (proband) presented with aHUS and was treated with ravulizumab. Her paternal grandfather developed aHUS previously and her paternal great grandmother presented with anti-neutrophil cytoplasmic antibody-associated vasculitis and thrombotic microangiopathy (TMA). However, the proband's parents have no history of TMA. A genetic analysis revealed the presence of CFH::CFHR1 fusion gene and a CFHR3-1-4-2 gene duplication in the patient, her father, and her paternal grandfather. Although several fusion genes resulting from structural variations of the CFH-CFHR genes region have been identified, this is the first report of the combination of a CFH::CFHR1 fusion gene with CFHR gene duplication. Because the CFH-CFHR region is highly homologous, we hypothesized that CFHR gene duplication occurred. These findings indicate a novel pathogenic genomic structural variation associated with the development of aHUS.

Novel 4.18 Mb deletion resulting in 2q37 microdeletion syndrome combined with PTH resistance found in one Chinese patient.

BACKGROUND: 2q37 microdeletion syndrome is a rare clinical condition characterized by a series of physical abnormalities. Its Albright hereditary osteodystrophy (AHO)-like manifestations and possible complication of biochemical abnormalities indicating PTH resistance greatly increased the likelihood of misdiagnosis with classic pseudohypoparathyroidism (PHP) caused by GNAS mutation or methylation alteration, even though there have only been six reports of such clinical occasions. PURPOSE: to investigate the underlying genetic defect in a male patient presenting hypocalcemia, elevated PTH and with a history of kyphosis. METHOD: clinical information was collected, while the DNA was extracted from peripheral blood and subjected to methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and exome sequencing. RESULT: Physical characteristics featuring short stature, obesity, round face, short neck, and shortened 4th metacarpal and laboratory examination of the patient suggested the presence of PTH resistance, which is indicative of PHP. MS-MLPA did not reveal methylation alterations or deletions of GNAS, STX16 or other monogenetic alterations responsible for iPPSDs, but WES revealed a long-range deletion of approximately 4.18 Mb of the 2q37 region that spanned AGAP1 to NDUFA10, indicating that the patient had 2q37 microdeletion syndrome with PTH resistance. CONCLUSION: After undergoing MS-MLPA and exome sequencing, a novel deletion spanning 4.18 Mb on the 2q37 region was identified in one male patient, clarifying the diagnosis of 2q37 microdeletion syndrome with PTH resistance. The new genetic discovery added to our understanding of the molecular defects that cause inactivating PTH/PTH-related protein signaling disorders (iPPSDs).

Identification of two novel large deletions in FBN1 gene by next-generation sequencing and multiplex ligation-dependent probe amplification.

BACKGROUND: Mutations in fibrillin-1 (FBN1) are known to be associated with Marfan syndrome (MFS), an autosomal dominant connective tissue disorder. Most FBN1 mutations are missense or nonsense mutations. Traditional molecular genetic testing for the FBN1 gene, like Sanger sequencing, may miss disease-causing mutations in the gene's regulatory regions or non-coding sequences, as well as partial or complete gene deletions and duplications. METHODS: Next-generation sequencing, multiplex ligation-dependent probe amplification and gap PCR were conducted on two MFS patients to screen for disease-causing mutations. RESULTS: We identified two large deletions in FBN1 from two MFS patients. One patient had a 0.23 Mb deletion (NC_000015.9:g.48550506_48779360del) including 5'UTR-exon6 of FBN1. The other patient harbored a 1416 bp deletion (NC_000015.9:g.48410869_48412284del) affecting the last exon, exon 66, of the FBN1 gene. CONCLUSION: Our results expanded the number of large FBN1 deletions and highlighted the importance of screening for large deletions in FBN1 in clinical genetic testing, especially for those with the classic MFS phenotype.

Detection of PTCH1 Copy-Number Variants in Mosaic Basal Cell Nevus Syndrome.

Basal cell nevus syndrome (BCNS) is an inherited disorder characterized mainly by the development of basal cell carcinomas (BCCs) at an early age. BCNS is caused by heterozygous small-nucleotide variants (SNVs) and copy-number variants (CNVs) in the Patched1 (PTCH1) gene. Genetic diagnosis may be complicated in mosaic BCNS patients, as accurate SNV and CNV analysis requires high-sensitivity methods due to possible low variant allele frequencies. We compared test outcomes for PTCH1 CNV detection using multiplex ligation-probe amplification (MLPA) and digital droplet PCR (ddPCR) with samples from a BCNS patient heterozygous for a PTCH1 CNV duplication and the patient's father, suspected to have a mosaic form of BCNS. ddPCR detected a significantly increased PTCH1 copy-number ratio in the index patient's blood, and the father's blood and tissues, indicating that the father was postzygotic mosaic and the index patient inherited the CNV from him. MLPA only detected the PTCH1 duplication in the index patient's blood and in hair and saliva from the mosaic father. Our data indicate that ddPCR more accurately detects CNVs, even in low-grade mosaic BCNS patients, which may be missed by MLPA. In general, quantitative ddPCR can be of added value in the genetic diagnosis of mosaic BCNS patients and in estimating the recurrence risk for offspring.

Validation of SMA screening kits with SMN1 gene analysis in a Turkish cohort.

OBJECTIVE: It is crucial to start early treatment in Spinal Muscular Atrophy (SMA) with available drugs to stop the progression of the disease, therefore making SMA screening preferable. This study assessed Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) compared to Multiplex Ligation-dependent Probe Amplification (MLPA) for detecting Spinal Muscular Atrophy (SMA) through SMN1 gene copy number analysis in a Turkish cohort. METHODS: We analyzed 249 DNA samples, previously tested for SMN1 and SMN2 gene deletion via MLPA, using qRT-PCR kits from three different companies. Accuracy, sensitivity, and specificity of qRT-PCR in identifying deletions of SMN1 copy number variations. RESULTS: High accuracy (96.2-98.7%) achieved with qRT-PCR for detecting homozygous deletions, heterozygous deletions, and copy number variations in the SMN1 gene. Minor discrepancies between qRT-PCR and MLPA were observed, possibly due to single nucleotide polymorphisms affecting primer binding. CONCLUSIONS: The qRT-PCR method proved to be a rapid, cost-effective, and accurate technique, aligning well with the demands of routine SMA screening, suggesting its general suitability for application in SMA screening programs. This research highlights the importance of improving molecular methodologies and the value of collaborations between government and relevant sectors to overcome rare diseases, particularly through the enhancement of screening initiatives which is the first and most effective strategy to protect the public health.

Combination of minimal residual disease on day 15 and copy number alterations results in BCR-ABL1-negative pediatric B-ALL: A powerful tool for prediction of induction failure.

The current genomic abnormalities provide prognostic value in pediatric Acute Lymphoblastic Leukemia (ALL). Furthermore, Copy Number Alteration (CNA) has recently been used to improve the genetic risk stratification of patients. This study aimed to evaluate CNA profiles in BCR-ABL1-negative pediatric B-ALL patients and correlate the data with Minimal Residual Disease (MRD) results after induction therapy. We examined 82 bone marrow samples from pediatric BCR-ABL1-negative B-ALL using the MLPA method for the most common CNAs, including IKZF1, CDKN2A/B, PAX5, RB1, BTG1, ETV6, EBF1, JAK2, and PAR1 region. Subsequently, patients were followed-up by multiparameter Flow Cytometry for MRD (MFC-MRD) assessment on days 15 and 33 after induction. Data showed that 58.5 % of patients carried at least one gene deletion, whereas 41.7 % of them carried more than one gene deletion simultaneously. The most frequent gene deletions were CDKN2A/B, ETV6, and IKZF1 (30.5 %, 14.6 %, and 14.6 %, respectively), while the PAR1 region showed predominantly duplication (30.5 %). CDKN2A/B and IKZF1 were related to positive MRD results on day 15 (p = 0.003 and p = 0.007, respectively). The simultaneous presence of more than one deletion was significantly associated with high induction failure (p = 0.001). Also, according to the CNA profile criteria, the CNA with poor risk (CNA-PR) profile was statistically associated with older age and positive MRD results on day 15 (p = 0.014 and p = 0.013, respectively). According to our results, the combined use of CNAs with MRD results on day 15 can predict induction failure and be helpful in ameliorating B-ALL risk stratification and treatment approaches.

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.

Application of a Multiplex Ligation-Dependent Probe Amplification-Based Next-Generation Sequencing Approach for the Detection of Pathogenesis of Duchenne Muscular Dystrophy and Spinal Muscular Atrophy Caused by Copy Number Aberrations.

Both Duchenne muscular dystrophy (DMD; OMIM no. 310200) and spinal muscular atrophy (SMA; OMIM no. 253300/253550/253400/271150) are genetic disorders characterized by progressive muscle degeneration and weakness. Genetic copy number aberrations in the pathogenetic genes DMD and SMN1 lead to alterations in functional proteins, resulting in DMD and SMA, respectively. Multiplex ligation-dependent probe amplification (MLPA) has become a standard method for the detection of common copy number aberrations (CNAs), including DMD and SMN1 deletions, both of which are associated with poor clinical outcomes. However, traditional MLPA assays only accommodate a maximum of 60 MLPA probes per test. To increase the number of targeted sequences in one assay, an MLPA-based next-generation sequencing (NGS) assay has been developed that is based on the standard MLPA procedure, allows high-throughput screening for a large number of fragments and samples by integrating additional indices for detection, and can be analyzed on all Illumina NGS platforms.

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.